sa@eci 4. hrvatskog kongresa medicinskih ...biochemia medica god. 13, prilog broju 1–2, 2003. 3...

UDK 612.348.7/575.1 ISSN 1330-0962

BIOCHEMIA MEDICAÂSOPIS HRVATSKOGA DRU[TVA MEDICINSKIH BIOKEMIÂRATHE JOURNAL OF THE CROATIAN SOCIETY OF MEDICAL BIOCHEMISTS

Biochemia Medica godi{te 13 prilog broju 1–2 str. 1–149 Zagreb 2003.Biochemia Medica Vol. 13 supplement No. 1–2 pp. 1–149 Zagreb 2003

SA@ECI4. HRVATSKOG KONGRESA

MEDICINSKIH BIOKEMIÂRA

Abstracts4th Croatian Congress

of Medical Biochemists

Savjet – Editorial CouncilI. ÊPELAK, [. DVORNIK, M. FLOEGEL-MR[I], G. IVANKOVI], D. JURETI], K. LIPOVAC,

A. ROGULJI], A. VALÎ], J. WAGNER

Predsjednik uredni~kog odbora – Editor-in-ChiefE. SUCHANEK

Urednici – EditorsE. TOPI], N. VRKI], M. [TEFANOVI]

Uredni~ki odbor – Editorial BoardD. ^VORI[]EC, K. \URI], M. MARADIN, B. KUNOVI], B. GRAHOVAC, D. KRALJ, A. NAZOR,

@. REBAC, D. ROGI], A. STAVLJENI]-RUKAVINA, E. TOPI], D. TURNER

Lektor za hrvatski i engleski jezik – Croatian and English Language EditorA. BEZI] REDOVNIKOVI]

Osniva~ i izdava~ – PublisherHRVATSKO DRU[TVO MEDICINSKIH BIOKEMIÂRA

Tisak – Print“KIKA-GRAF” – Zagreb, Horva}anska 28/I

Naklada – Edition1000 PRIMJERAKA

âsopis izlazi dva puta godi{nje u obliku dvobroja. Rukopisi i ~lanci {alju se Uredni{tvu ~asopisa Biochemia Medica, Zavod za klini~kubiokemiju i biologiju reprodukcije, Zagreb, Petrova 13, telefon (01) 4604-751 i telefaks (01) 4633-512. Godi{nja pretplata za pravneosobe iznosi 200 kn, za fizi~ke osobe 100 kn, za inozemstvo 50 DEM. Za redovito primanje ̂ asopisa potrebno je svaku promjenu adresejaviti Uredni{tvu. âsopis se {alje svim ~lanovima Hrvatskoga dru{tva medicinskih biokemi~ara. @iro ra~un HDMB je 2360000-1101318371.

âsopis je oslobo|en pla}anja poreza na promet na temelju ~lanka 19. to~ka 14. stavak 2 Zakona o porezu na promet proizvoda i usluga,mi{ljenjem Ministarstva prosvjete i kulture Republike Hrvatske.

âsopis Biochemia Medica Upisan je u evidenciju javnih glasila pod brojem 1365 koju vodi Ministarstvo informiranja. Izdavanje ~asopisapodupire Ministarstvo znanosti Republike Hrvatske.

Biochemia medica is published two times per year.

Subscription rate: an annual volume in the country is 200 HRK for institutions and 100 HRK for individuals, all other countries 25EUR, payable to the account ZABA HR** 2500-3296504.

SA@ECI4. HRVATSKOG KONGRESA

MEDICINSKIH BIOKEMI^ARA

Abstracts4th Croatian Congress

of Medical Biochemists

Zadar, 24.-28. rujna 2003.Zadar, September 24-28, 2003

UPUTE AUTORIMA

Biochemia medica (u nastavku teksta BM)je ~asopis Hrvatskoga dru{tva medicinskihbiokemi~ara, namijenjen ~lanovima Dru{tvakao i svima ~iji je stru~ni i znanstveni interespovezan s ovim podru~jem. Zada}a je ~asopisaBM objavljivanje znanstvenih, stru~nih i pre-glednih ~lanaka iz podru~ja medicinske bio-kemije i laboratorijske dijagnosrike, pisamauredni{tvu, prikaza knjiga.

Svi radovi se {alju Uredni{tvu ~asopisa, aupute za oblikovanje prikazane su u skladu stekstom: International Committee of MedicalJournal: Uniform Requirements for Manu-scripts Submitted to Biomedical Journals. AnnIntern Med 1988; 1008: 258-65. Radovi koji ni-su u skladu s uputama, bit }e vra}eni auto-rima.

1. TEKST RADA

îtav rad s prilozima treba poslati u triprimjerka, otisnut dvostrukim proredom napapiru standardne veli~ine (A4). Preporu~a seposlati kona~nu ina~icu rukopisa (nakon pri-hva}anja za tisak) i na disketi (3.5’’), u jednomod uobi~ajenih programa, primjerice MicrosoftWord, uz napomenu o kojem se programu radi.

Sadràj rada treba prilagoditi temi koja seobra|uje, a savjetujemo sljede}i redoslijed:

1.1. Naslov ~lanka treba biti {to kra}i ijasniji. Imena autora navode se punim imenomi prezimenom, a posebno se za svakog autorapojedina~no navodi to~an naziv ustanove. Nadnu stranice treba napisati adresu i telefonskibroj autora kojem se Uredni{tvo moè obratiti.

1.2. Uvod je kratak i jasno prikazuje po-dru~je koje se obra|uje, uz spominjanje radovakoji su s tim izravno povezani.

1.3. Materijal, ispitanici i metode koje su uradu primijenjene prikazuju se jezgrovito i ja-sno za razumijevanje sadràja opisanog istraì-vanja. Metode poznate iz literature se ne opi-suju, ve} se navodi izvorni literaturni podatak.

1.4. Rezultati rada prikazuju se jasno i pre-cizno, a u prikazima mjerenja treba upotre-bljavati SI sustav. Vjerodostojnost rezultatatreba obraditi statisti~kim metodama.

1.5. Rasprava i zaklju~ci. Svrha je raspravetuma~enje rezultata i njihova usporedba s po-

stoje}im spoznajama na tom podru~ju, iz ~egamogu proiza}i jasni zaklju~ci.

1.6. Saètak s klju~nim rije~ima i naslovommora biti napisan na hrvatskom i engleskomjeziku.

2. TABLICE I SLIKE

Rezultate treba popratiti razumnim brojemtablica i slika.

2.1. Tablice. Svaka se tablica pi{e na po-sebnom listu papira, a mora imati naslov iredni broj koji je povezuje s tekstom (u radu senavode kao Tablica 1, itd.). Naslov tablice iglavni dijelovi moraju biti napisani i na engle-skom jeziku. U tablicama se ne smiju upotre-bljavati kratice osim uobi~ajenih mjernih je-dinica.

2.2. Slike. Svaka slika mora biti ozna~enanaslovom i rednim brojem, prema redoslijedukojim se pojavljuje u tekstu rada. Opisi slikatiskaju se na posebnom listu papira na hrvat-skom i engleskom jeziku, a sadràjno morajubiti dostatni za tuma~enje. Kao slike prilaù seizvorne crno-bijele fotografije na sjajnom pa-piru (ozna~ene brojem na pole|ini) te izvornicrteì izra|eni tu{em na bijelom ili paus papi-ru, ili slike otisnute na laserskom pisa~u.

3. LITERATURA

Popis literature pi{e se na posebnom papi-ru, s rednim brojevima prema redoslijedu ko-jim se citati pojavljuju u tekstu. âsopise trebaprikazivati skra}enicom koja se upotrebljava uIndex Medicusu, a literatura se citira kakonavodi Lije~ni~ki vjesnik.

Primjerice:

Garrnero P, Shih WJ, Gineyts E, Karpf DB,Delmas PD. Comparison of new biochemicalmarkers of bone turnover in late postmeno-pausal osteoporotic women in response toalendronate treatment. J Clin EndocrinolMetab 1994;79:1693-700.

Delmas PD. Biochemical markers of boneturnover in osteoporosis. U: Riggs BL, MeltonLJ III, ur. Osteoporosis etiology, diagnosticsand management. New York: Raven Press,1988:297-316.

INSTRUCTIONS TO AUTHORS

Biochemia Medica (further: BM) is a jour-nal issued by the Croatian Medical Biochemis-try Society for the benefit of its members andall those with scientific and professional inter-est in this field. The intention of the journal isto publish scientific, professional nad reviewarticles dealing with the field of medical bio-chemistry and laboratoy diagnosis, letters tothe editor, book reviews.

All manuscripts should be sent to the editorof the journal and prepared according to in-structions based on the following: Interna-tional Committee of Medical Journals: UniformRequirements for Manuscripts Submitted toBiomedical Journals. Ann Intern Med 1988;1008: 258-65. The manuscripts not complyingwith the instructions will be returned to au-thors.

1. THE TEXT OF A MANUSCRIPT

Complete manuscript is submitted togetherwith two copies and printed double-spaced on astandard A4 paper format. Forwarding the fi-nal version of the manuscript (after accep-tance) on a 3.5’’ diskette is recommended, witha note on the application used (e.g. MicrosoftWord). Diskettes are returned to authors. Thecontent of the manuscript is organized with re-gard to the studied topic, but the following or-der is suggested:

1.1 The title is short and clear. Authors’names and surnames and surnames are citedin full, with precise mention of affiliating insti-tutions for each author. Corresponding author’saddress and phone number is stated at the bot-tom of the title page.

1.2 Introduction is brief and clearly statesthe topic, including directly associated and rel-evant references.

1.3. Study material, subjects and methodsapplied in the study are presented concisely inorder to allow understanding of the study per-formed. The methods known from the litera-ture are not described but original reference iscited instead.

1.4 Results are presented precisely andclearly, with SI system used when referring tomeasurements. Significance is demonstratedby statistical methods.

1.5 Discussion and results. The aim of dis-cussion is interpretation of results and theircomparison with established data, leading toclearly defined conclusions.

1.6 Abstract does not contain more than 150words including the title. Key words should beadded.

2. TABLES AND FIGURES

Results are accompanied by a reasonablenumber of tables and figures.

2.1 Tables. Each table should be submittedon a separate page and include a title and anordinary number relating it to the text (tablesare referred to in the text as able 1, etc.).Apart from common measurement units, ab-breviations should not be used in tables.

2.2 Figures. Each figure contains a title andits number following the order of its referencein the text. Legends are submitted on a sepa-rate page and contain an interpretation neces-sary to understand the figure. Laser printedfigures or original black and white photographson a glossy paper numbered on the back aresubmitted.

3. REFERENCES

References are submitted on separate pagesin the numbered sequence following their men-tion in the text. Publications and journals arecited according to abbreviations proposed in In-dex Medicus.

Examples:

Garnero P. Shih WJ, Gineyts E, Karpf DB,Delmas PD. Comparison of new biochemicalmarkers of bone turnover in late postmeno-pausal osteoporotic women in response toalendronate treatment. J Clin EndocrinolMetab 1994;79:1693-700.

Delmas PD. Biochemical markers of boneturnover in osteoporosis. In: Riggs BL, MeltonLJ III, editors. Osteoporosis etiology, diagnos-tics and management. New York: Raven Press,

1988; 297-316.

SADR@AJ

Po~asno predavanje . . . . . . . . . . . 113

Plenarna predavanjaPL-I-III Plenarna predavanja . . . . . . . . 115

Simpozijska predavanjaS1 Okoli{ i zdravlje . . . . . . . . . . . . . 119S2 Nova dostignu}a u onkologiji . . . . . . 115S3 Molekularna laboratorijska medicina . . 120S4 Nove tehnologije u laboratorijskoj

medicini . . . . . . . . . . . . . . . . . 128S5 Laboratorijska medicina temeljena

na dokazima . . . . . . . . . . . . . . . 134S6 Hitna laboratorijska dijagnostika . . . . 141

PosteriP1 Bolesti hematopoetskog sustava 149P2 Bolesti jetre . . . . . . . . . . . . . . . 159P3 Bolesti kardiovaskularnog sustava

i dijagnostika hitnih stanja . . . . . . . 164P4 Bolesti ko{tanog i vezivnog sustava . . . 180P5 Endokrinolo{ke bolesti . . . . . . . . . 185P6 Neurodegenerativne bolesti . . . . . . . 189P7 Procjena analiti~kih tehnika i metoda . . 191P8 Laboratorijski informati~ki sustavi . . . 107P9 Razno . . . . . . . . . . . . . . . . . . 111

Znanstvene radioniceZR1 Farmakogenetika . . . . . . . . . . . 135ZR2 Autoimune bolesti . . . . . . . . . . . 140

Industrijske radioniceIR2 Procjena analiti~kih tehnika i metoda . 145

Indeks autora . . . . . . . . . . . . . . . 147

TABLE OF CONTENTS

Honorary lecture . . . . . . . . . . . . . 113

Plenary lecturesPL-I-III Plenary lectures . . . . . . . . . . 115

Symposium lecturesS1 Environment and health . . . . . . . . . 119S2 New advances in oncology . . . . . . . 115S3 Molecular laboratory medicine . . . . . 120S4 New technologies in laboratory medicine 28S5 Evidence based laboratory medicine . . 134S6 Emergency laboratory diagnosis . . . . 141

PostersP1 Diseases of hematopoietic system . . . . 149P2 Liver diseases . . . . . . . . . . . . . . 159P3 Cardiovascular diseases and emergency

diagnosis . . . . . . . . . . . . . . . . 164P4 Bone and connective tissue diseases . . . 180P5 Endocrine diseases . . . . . . . . . . . 185P6 Neurodegenerative diseases . . . . . . . 189P7 Evaluation of analitical techniques

and methods . . . . . . . . . . . . . . . 191P8 Laboratory information systems . . . . . 107P9 Miscellanea . . . . . . . . . . . . . . . 111

Scientific workshopsZR1 Pharmacogenetics . . . . . . . . . . . 135ZR2 Autoimmune diseases . . . . . . . . . 140

Industry workshopsIR2 Evaluation of analytical techniques

and methods . . . . . . . . . . . . . . 145

Author Index . . . . . . . . . . . . . . . 147

BIOCHEMIA MEDICA god. 13, PRILOG broju 1–2, 2003. 3

POÂSNO PREDAVANJE Saèci 4. hrvatskog kongresa medicinskih biokemi~ara

Po~asno predavanjeHonorary lecture

PPPo~asno predavanje

Honorary lecture

PP

UTJECAJ GLOBALIZACIJE NA RAZVOJZDRAVSTVENIH SUSTAVA I ZDRAVLJE

Stavljeni}-Rukavina A.

Klini~ki zavod za laboratorijsku dijagnostiku Medicinskog

fakulteta i Klini~kog bolni~kog centra “Zagreb”, Zagreb

Danas se sve više u svijetu postavlja pitanje ukojoj mjeri procesi globalizacije utje~u nazdravlje ljudi i razvoj zdravstvenih sustava. Podpojmom globalizacije ve}ina ljudi najprijepodrazumijeva pojam op}ih politi~kih prava, ras-tu}u ekonomsku liberalizaciju, porast razmjenekapitala, kretanje ljudi u potrazi za zaposlenjem,rast velikih multinacionalnih kompanija, te pri-mjenu informacijskih tehnologija u uspostavisveukupnih odnosa ekonomije, razmjeni infor-macija i znanja u cijelom svijetu. No, u biti glo-balizacija je više od ekonomije; ona uz politi~keima zna~ajne socijalne i kulturološke posljedicekoje uvelike utje~u na zdravlje ljudi i razvojzdravstvenih sustava. Rizici od odre|enih zaraz-nih bolesti (HIV), promjena sadràja ozona, pro-mjene na~ina ìvota, porast prekograni~nih prije-nosa bolesti hranom (BSE), širenje rezistencijena lijekove (antibiotici, tuberkulostatici) zahti-jevaju ja~anje me|unarodne suradnje u zakono-davnoj regulaciji u cilju sveop}e zaštite zdravljaljudi koje ne poznaje granica. Poèljne i pozitivnepromjene koje globalizacija donosi u podru~juznanja, stjecanja profesionalnih vještina i op}aedukacija zdravstvenih struka vodi k op}em po-kretu slobode profesionalnog rada u zdravstvu usvijetu. Zato prostorna dimenzija globalizacijepredstavlja izazov zdravstvenim strukama dadjeluju širom svijeta, a ne samo unutar nacio-nalnog zdravstvenog sustava. Vremenska dimen-

PP

IMPACT OF GLOBALIZATION ON HEALTHCARE SYSTEM DEVELOPMENT AND

HUMAN HEALTH

A. Stavljeni}-Rukavina

Clinical Institute of Laboratory Diagnosis, Zagreb Univer-sity Hospital Center, Zagreb University School of Medicine,

Zagreb

The issue of the extent to which the processesof globalization influence human health and de-velopment of health care system has been attract-ing ever more attention worldwide. For the ma-jority of people, the term globalization primarilyimplies general political rights, growing eco-nomic liberalization, increased capital turnover,migration of people in search for employment,further growth of large multinational companies,and use of computer technologies in the establish-ment of overall economy relationships, and infor-mation and knowledge exchange all over theworld. However, globalization is more than pureeconomy; besides political, it will also entail con-siderable social and cultural sequences that willgreatly influence human health and developmentof health care systems. The risk of particular in-fectious diseases (HIV), modified ozone composi-tion, lifestyle changes, increased trans-border di-etary transmission of diseases (BSE), spread ofdrug resistance (antibiotics, tuberculostatics), etc.require tight international collaboration in thefield of legal provisions aiming at universal hu-man health protection knowing no borders. Thedesirable and favorable changes brought by glo-balization in the fields of knowledge, acquiringprofessional skills and general education of medi-cal professions lead towards a general movement

4 BIOCHEMIA MEDICA god. 13, PRILOG broju 1–2, 2003.

Saèci 4. hrvatskog kongresa medicinskih biokemi~ara POÂSNO PREDAVANJE

zija globalizacije traì od zdravstva stalnu prila-godbu rada i institucionalne promjene koje odgo-varaju tehnološkim dostignu}ima vremena. Teh-nološka dostignu}a poti~u trajnu edukaciju i stje-canje novih vještina u struci, organizaciji i uprav-ljanju zdravstvenim sustavima. Kako se doìv-ljavaju pozitivni i negativni u~inci globalizacijena pojedinca u zdravstvenom sustavu i društvuje pitanje na~ina na koji mislimo i doìvljavamopromjene. Informacijske i komunikacijske teh-nologije su omogu}ile globalnu mreù kojom seizmjenjuju i šire ideje, znanja i omogu}avaju brzepromjene u lije~enju i prevenciji bolesti. Poten-cijalni pozitivni u~inci su upravo u tom segmentuod najve}eg zna~enja za op}e zdravlje ljudi širomsvijeta. Stvaranje banaka podataka, na~ela dobreklini~ke prakse, telemedicina, upotreba inova-tivnih tehnologija, virtualne institucije za eduka-ciju i lije~enje, te edukacija op}e populacije ozdravlju i bolesti samo su dio kognitivne dimen-zije globalnog zdravlja. Kritike koje prate glo-balizacijske procese su usmjerene na pitanje ho}eli oni pove}ati ve} postoje}u nejednakost u mo-gu}nostima korištenja pozitivnih u~inaka poputnovih tehnologija i lijekova, ho}e li pove}ati do-minaciju bogatijih nad siromašnijim dijelom svi-jeta, ho}e li omogu}iti sve ve}i utjecaj klju~nihme|unarodnih institucija na kreiranje zdrav-stvene politike i zaštite zdravlja i okoliša. Svenavedeno je istodobno nov izazov zdravstvenimstrukama da iskoriste pozitivne u~inke op}egaznanja i znanosti u cilju stvaranja strategije ko-jom }e se unaprijediti zdravlje pojedinca i za-jednica širom svijeta.E-mail: [email protected]

of professional freedom in health care worldwide.Therefore, the spatial dimension of globalizationimplies a challenge to health professions to be-come active all around the world rather thanwithin the national health system alone. The tem-poral dimension of globalization calls for con-tinuous adjustment of health care activities andinstitutional modifications that will correspondto the technologic advances of the time.Technologic advances in turn stimulate continu-ous education and acquiring new skills in theprofession, organization and management ofhealth care systems. How the favorable andunfavorable impacts of globalization on the indi-vidual in a health care system are perceived, is amatter of our reasoning and experiencing thesechanges. The computer and communication tech-nologies have enabled a global network to ex-change and disseminate ideas and knowledge,which then leads to fast modifications in diseasemanagement and prevention. The potentialfavorable effects in this very segment are recog-nized as most important for human health ingeneral. The establishment of databases, princi-ples of Good Clinical Practice, telemedicine, useof innovative technologies, virtual institutions foreducation and treatment, and education of thegeneral population on health and disease are onlya part of the cognitive dimension of global health.The criticism accompanying the processes of glo-balization refers to fear that they may increasethe existing inequity in the availability of thefavorable effects such as novel technologies andmedication, and the predominance of the richover the poor regions of the world; and whetherthey will favor an ever greater influence of the keyinternational institutions on designing healthpolicy, health care and environmental protection.All these issues at the same time present a newchallenge for health professions to make use ofthe favorable impacts of the general knowledgeand science to create a strategy for promotion ofboth individual and community health all overthe world.E-mail: [email protected]


PLENARNA PREDAVANJA Saèci 4. hrvatskog kongresa medicinskih biokemi~ara

PL-1

ULOGA FUNKCIONALNE GENOMIKEU ONKOLOGIJI

Paveli} K.

Institut “Ru|er Boškovi}”, Zagreb

Posljednje su godine obiljeène dramati~nimutjecajem novog konceptualnog pristupa brzim isveobuhvatnim analiti~kim metodama u širokojprimjeni u biomedicini, koje }e nesumnjivo do-nijeti velike promjene u našoj svakodnevnoj me-dicinskoj praksi. Ovaj pristup nazvan funk-cionalnom genomikom predstavlja temeljit za-okret u znanstvenoj strategiji: od istraìvanjapotaknutih pretpostavkama prema formulacijizasnovanoj na otkri}u i naknadnim testiranjemove pretpostavke. Funkcionalna genomika obu-hva}a 3 znanstvene discipline: transkriptomiku,proteomiku i bioinformatiku. Jedna od najva`-nijih primjena transkriptomike (microarraysDNK) je ispitivanje promjena u ekspresiji genakoje prate promjene u fiziologiji stanica tijekomrazvoja, progresije stani~nog ciklusa, medika-mentnog lije~enja ili progresije bolesti. Brojne sustudije pokazale kako se povezani fenotipoviuglavnom odraàvaju u povezanim uzorcima sta-ni~nih transkripata, što zna~i da se stani~no sta-nje moè obiljeìti i klasificirati prema uzorkugenske ekspresije. Ova je tehnologija dovela doširokog spektra primjene, kao što su potvr|ivanjecilja odre|enog lijeka, disekcija putanje, otkri-vanje genske funkcije i anotacija humanog ge-noma. U istraìvanju raka postoje dvije kate-gorije proteomike: proteomika ekspresije i funk-cionalna proteomika. Proteomika ekspresije po-maè u promatranju proteina koji se razli~itoprikazuju u doti~nom tkivu, tjelesnoj teku}ini ili

Plenarna predavanjaPlenary lectures

PL-I–III.Plenarna predavanja

Plenary lectures

PL-1

ROLES OF FUNCTIONAL GENOMICS INONCOLOGY

K. Paveli}

Rudjer Boškovi} Institute, Zagreb

Recent years have been marked by a dramaticimpact of a new conceptual approach to mass-scale, rapid and global analysis methods in bio-medicine which will undoubtedly bring majorchanges in our daily medical practice. This ap-proach called functional genomics represents afundamental shift in scientific strategy: from hy-pothesis-driven research to discovery-based for-mulation and subsequent testing of the hypoth-esis. Functional genomics includes 3 scientificdisciplines: transcriptomics, proteomics andbioinformatics. One of the most important uses oftranscriptomics (DNA-microarrays) is to studychanges in gene expression that accompanychanges in cell physiology during development,cell cycle progression, drug treatment or diseaseprogression. A number of studies have shown thatrelated phenotypes are generally reflected in re-lated patterns of cellular transcripts, implyingthat the cellular state can be characterized andclassified by the gene-expression pattern. Thistechnology has led to a broad spectrum of appli-cations such as drug target validation, pathwaydissection, discovery of gene function and annota-tion of the human genome. In cancer research,there are two categories of proteomics: expressionproteomics and functional proteomics. Expres-sion proteomics helps look at the proteins that aredifferently displayed in a given tissue, body fluidor serum. This holds promise for identifying dis-ease markers important in early detection/diag-


Saèci 4. hrvatskog kongresa medicinskih biokemi~ara PLENARNA PREDAVANJA

serumu. To nudi izglede za identificiranje biljegabolesti va`nih za rano otkrivanje/dijagnozu, kao iza pra}enje terapijske u~inkovitosti, što bi nakoncu trebalo dovesti do novih, ’pametnih lije-kova’. Funkcionalna proteomika pomaè raspo-znati kako proteini djeluju mu|usobno u ìvu}emsustavu te kako ìvu}e stanice odgovaraju narazli~ite signale. Funkcionalna proteomika }enedvojbeno pove}ati naše razumijevanje biologijestanica raka. Autor }e razraditi i raspraviti sli-jede}e teme u svjetlu funkcionalne genomike:prijesimptomatsku dijagnostiku, potvr|ivanjeprognoze, klasifikaciju tumora, te identifikacijugenske ekspresije i ciljeva specifi~nih za pojedinubolest na temelju proteina.E-mail: [email protected]

nosis, and in monitoring therapeutic efficacy,leading eventually to the design of novel ’smartdrugs’. Functional proteomics helps determinehow proteins interact in the living system andhow living cells respond to different signals.Functional proteomics will undoubtedly increaseour understanding of the biology of cancer cells.The author will elaborate and discuss the follow-ing topics in the light of functional genomics:presymptomatic diagnosis, prognostic validation,tumor classification, and identification of gene-expression and protein-based disease-specific tar-gets.E-mail: [email protected]

PL-2

MIKROTEHNOLOGIJA I NANO-TEHNOLOGIJA: UTJECAJ NA KLINI^KO

LABORATORIJSKE PRETRAGE

Kricka L.

University of Pennsylvania Medical Center, Philadelphia,

PA, SAD

Tehnologija mikro~ipova (mikrofluidni ~ipovi,bio~ipovi, bioelektronski ~ipovi), tehnologija mi-croarrays (proteinski, peptidni, oligosaharidni,oligonukleotidni i cDNK ~ipovi) i nanotehnologija(nanocjev~ice, nano~estice) spadaju me|u naj-aktivnija podru~ja istraìvanja i razvoja u bio-analiti~kim znanostima. One omogu}avaju pro-vedbu brzih i pojednostavljenih pretraga (npr.tzv. ure|aji ’laboratorija na ~ipu’), istodobno te-stiranje više analita visokog kapaciteta, te nudeput ka sofisticiranijim prenosivim analiti~kimure|ajima lakšim za uporabu koji bi se moglirabiti na svakom mjestu uz bolesnika ili ~akpostati osobnim laboratorijima. Njihov potpunu~inak na klini~ko laboratorijske pretrage tektreba shvatiti, no mogu}e je nazrijeti koristi što}e ih one donijeti u budu}nosti. Najuzbudljivijavrst pretraga što }e ih one omogu}iti vjerojatno}e proizi}i iz trenutno intenzivnih i široko pro-vo|enih ispitivanja humanog genoma, proteoma iinteraktosoma. Na trìštu su se ve} pojaviliure|aji s mikro~ipovima, primjerice ~ipovi za ka-pilarnu elektroforezu, proteinske i DNK mi-croarrays, te ’patch-clamp’ ~ipovi. Ure|aji nanoveli~ine još su daleka budu}nost za rutinski la-boratorij, ali se zlatne i srebrne ~estice nanoveli~ine ve} godinama rabe za obiljeàvanje u

PL-2

MICROTECHNOLOGY ANDNANOTECHNOLOGY: IMPACT ON

CLINICAL LABORATORY TESTING

L. Kricka

University of Pennsylvania Medical Center,PA, USA

Microchip technology (microfluidic chips,biochips, bioelectronic chips), microarray technol-ogy (protein, peptide, oligosaccharide, oligonucle-otide and cDNA chips) and nanotechnology(nanotubes, nanoparticles) are among the mostactive areas of research and development in thebioanalytical sciences. They are enabling rapidand simplified assays (e.g., so-called ’lab-on-a-chip’ devices), high-throughput simultaneousmulti-analyte testing, and offer a route to moresophisticated and easier-to-use portable analyti-cal devices that could be used at the point-of-careor even become personal laboratories. Their fullimpact on clinical laboratory testing is yet to berealized, but it is possible to glimpse the benefitsthat they will provide in the future. The most ex-citing type of testing that they will enable is likelyto emerge from the current intense and wide-spread investigations of the human genome,proteome, and interactosome. Already, some mi-crochip devices, such as capillary electrophoresischips, protein and DNA microarrays, and patch-clamp chips, have been commercialized. Nano-sized devices are still a remote prospect for theroutine laboratory, but nano-sized particles ofgold and silver have been in use for a number ofyears as labels in binding assays. One type of test-


PLENARNA PREDAVANJA Saèci 4. hrvatskog kongresa medicinskih biokemi~ara

testovima vezanja. Jedna vrst pretraga koja bi nakoncu mogla rezultirati iz kombinirane primjenemikro- i nanotehnologije su na stanicama teme-ljene pretrage. Analizatori veli~ina kojih se sma-njuje sve bliè veli~ini stanice ve} su se pokazalikorisnima, npr. nedavno u ’patch-clamp’ ~ipovimaza testove ionskih kanala na pojedina~nim stani-cama te u razli~itim ure|ajima s mikroelek-trodama za manipuliranje i izoliranje stanica.Velika uspješnost mikro~ipova u mnoštvu raz-li~itih primjena u istraìvanjima (npr. analizaproteina, genetski testovi, odabir stanica, imunotestovi, otpuštanje uzorka u masenoj spektralnojanalizi) daje snaàn poticaj razvoju ovih vrstaure|aja za primjenu u rutinskom laboratoriju, au ovom }e se predavanju propitati utjecaj nano- imikrotehnologije na klini~ki laboratorij.E-mail: [email protected]

ing that may eventually be enabled by a combina-tion of micro- and nano-technology is cell-basedtesting. Shrinking analyzers nearer to the size ofcells have already been shown to be beneficial, asin the recent patch-clamp chips for ion-channelassays on single cells, and in various microelec-trode devices for cell manipulation and isolation.The broad success of microchips in many differ-ent research applications (e.g., protein analysis,genetic tests, cell selection, immunoassay, sampledelivery in mass spectral analysis) provides astrong impetus to development of these types ofdevices for use in routine laboratories, and thistalk will explore how nano- and microtechnologywill impact the clinical laboratory.E-mail: [email protected]

PL-3

RAZBORITA UPORABALABORATORIJSKIH TESTOVA

Müller M.

Kaiser Franz Joseph Hospital, Institute of Laboratory

Diagnostics, Be~, Austrija

U laboratorijskoj su medicini smislena, to~na iprecizna rutinska mjerenja bitna za dijagnozu,procjenu rizika, lije~enje i pra}enje bolesnika.Dijagnosti~ki laboratoriji danas primjenjujuupravljanje kvalitetom kako bi postigli ove ci-ljeve i poboljšali kvalitetu rezultata pretraga.Me|utim, ve}ini od ovih sustava upravljanja kva-litetom nedostaje medicinski dio laboratorijskemedicine povezan s bolesnikom; ovi su sustaviusredoto~eni na klasi~no analiti~ko osiguranjekvalitete i optimiranje procesa. Laboratorijskinalazi su bitni gledaju}i laboratorijsku dijagno-stiku kao medicinsku disciplinu te kao potporaodjelnim lije~nicima pri donošenju odluka. Kakobi se postigli ovi ciljevi i lije~nicima osiguralikvalitetni laboratorijski nalazi, treba razmotritiili provesti slijede}e: racionalan odabir pretragaza pojedinu bolest; pripremu bolesnika; uzorko-vanje; rukovanje uzorcima prije analize; bara-tanje rezultatima pretraga poslije analize; tuma-~enje rezultata pretraga i prihva}anje odgovor-nosti; i konzultaciju s lije~nicima u svezi s po-jedinim bolesnikom. Dijagnosti~ka podru~ja kaošto su koagulacija, pra}enje lijekova, interakcijelijekova, endokrinologija, imunofenotipiziranjestanica, priro|ene grješke metabolizma, pra}enjemaligniteta i virologija poglavito zahtijevaju la-

PL-3

RATIONAL USE OFLABORATORY TESTING

M. Müller

Kaiser Franz Joseph Hospital, Institute of LaboratoryDiagnostics, Vienna, Austria

In laboratory medicine meaningful, accurateand precise routine measurements are essentialfor the diagnosis, risk assessment, treatment andfollow-up of patients. To achieve these goals andto improve the quality of their test results, diag-nostic laboratories have nowadays being imple-menting quality management. However, most ofthese quality management systems lack the medi-cal, patient related part of laboratory medicine;the systems are focused on classical, analyticalquality assurance and process optimization. Con-sidering laboratory diagnosis as a medical disci-pline and to support decision making by physi-cians at the wards, interpretation of laboratoryreports is essential. To reach these goals and toprovide physicians with quality oriented labora-tory reports the following points have to be con-sidered or implemented: rational, disease ori-ented test selection; patient preparation; sam-pling procedure; preanalytical handling of sam-ples; postanalytical handling of test results; inter-pretation of test results and accepting responsibil-ity; and patient oriented consultation with clini-cians. Diagnostic areas such as coagulation, drugmonitoring, drug interactions, endocrinology,immunophenotyping of cells, inborn errors of me-tabolism, malignancy monitoring, and virology


Saèci 4. hrvatskog kongresa medicinskih biokemi~ara PLENARNA PREDAVANJA

boratorijsko tuma~enje i potporu pri donošenjuklini~kih odluka. Prikazati }e se primjeri iz razli-~itih podru~ja laboratorijske medicine. Procesinterdisciplinskog klini~kog konzilija proširiti }eobveze laboratorija dovode}i ih sve bliè bole-sniku. Osoblje dijagnosti~kog laboratorija pro-voditi }e skrb o bolesniku, jer }e dijagnosti~kaekspertiza u budu}nosti biti partner jednak kli-ni~aru. Uz aktivne interakcije dijagnosti~ki }elaboratorij preìvjeti u okruènju usmjerenomprema bolesniku, koje }e zahtijevati interdis-ciplinska znanja o vode}im na~elima fiziologije ipatofiziologije, kao i spremnost za prihva}anjeodgovornosti za tako donešene odluke.E-mail: [email protected]

especially require data interpretation and labora-tory support on clinical decision making. Exam-ples of various areas in laboratory medicine willbe presented. The process of interdisciplinaryclinical consultation will expand laboratory obli-gations nearer to the patient. The personnel of adiagnostic laboratory will be providing patientcare, as diagnostic expertise will be an adequatepartner to the clinicians in the future. With activeinteractions, diagnostic laboratory will survive ina patient oriented environment that demandsinterdisciplinary knowledge of the great princi-ples of physiology and pathophysiology as well asreadiness to accept responsibility for the decisionsthus made.E-mail: [email protected]


SIMPOZIJSKA PREDAVANJA Saèci 4. hrvatskog kongresa medicinskih biokemi~ara

S-1Okoli{ i zdravlje

Environment and health

S1-1

DODACI U PREHRANI - LIJEKOVI ILIHRANA (PRIMJER: LAKTOZNA

INTOLERANCIJA)

Luka~-Bajalo J.

Farmaceutski fakultet, Sveu~ilište u Ljubljani, Ljubljana,

Slovenija

Zrak, voda i hrana iz okoliša predstavljajuconditio sine qua non za ìvot ljudi. Oni mogu bitidobri, manje dobri ili ~ak štetni, ovisno o egzo-genim ili/i endogenim uzrocima. Postoje mnogaendogena stanja u ljudi u kojima hrana rekla-mirana kao ’dobra’ zapravo nije dobra za odre-|enu osobu. Uzimanje odre|ene hrane ili sasto-jaka hrane izaziva mnoge probavne bolesti. Ta-kav je primjer nepodnošenje laktoze (NL) kaoposljedica niske razine laktaze u crijevima, gdjemlijeko ili mlije~ni proizvodi koji sadrè laktozumogu izazvati blaè ili ~ak teške probavne idruge simptome. NL je normalno stanje za odra-sle sisavce i javlja se u otprilike 90% crnih i dale-koisto~nih populacija. U bjela~kim populacijamasituacija je obrnuta, pa ve}ina odraslih osoba(70%-90%) mogu probaviti laktozu u glukozu igalaktozu. Cilj ovoga prikaza je pokazati kako seNL moè samodijagnosticirati i kako ìvjeti s NL.Na koncu èlimo naglasiti kako su prehrana tedodaci hrani s niskim sadràjem laktoze obve-zatni kod NL i sli~nih stanja.E-mail: [email protected]

S1-1

FOOD ADDITIVES - DRUGS OR FOOD(EXAMPLE: LACTOSE INTOLERANCE)

J. Luka~-Bajalo

Faculty of Pharmacy, University of Ljubljana, Ljubljana,Slovenia

Air, water and food from the environment areconditio sine qua non of our lives. They can begood, less good or even harmful, depending on ex-ogenous or/and endogenous causes. There aremany endogenous conditions in humans in whichfood advertised as ’good’ is not really good for aparticular person. Many digestive disorders areprovoked by consumption of a particular food orfood ingredients. Lactose intolerance (LI) as aconsequence of low intestinal lactase is such anexample, where milk or milk products containinglactose can provoke mild or even severe digestiveand other symptoms. LI is a normal condition foradult mammals and occurs in approximately90% of black and Far East populations. In Cau-casian populations the situation is opposite, andmost of adults (70%-90%) can digest lactose toglucose and galactose. The aim of this presenta-tion is to illustrate how to self-diagnose LI andhow to live with it. Finally, we would like to stressthat low lactose diet and food additives are ob-ligatory in LI and similar conditions.E-mail: [email protected]

Simpozijska predavanjaSymposium lectures


Saèci 4. hrvatskog kongresa medicinskih biokemi~ara SIMPOZIJSKA PREDAVANJA

S1-2

STANI^NI MEHANIZMI TOKSI^NOSTITEŠKIH METALA

Herak-Kramberger C. M., Saboli} I.

Institut za medicinska istraìvanja i medicinu rada, Zagreb

Geološki i biološki procesi na Zemlji odràvajuravnote`ni odnos metala u prirodi. Uporabommetala u industriji i poljoprivredi ~ovjek remetiovu prirodnu ravnoteù i time ugroàva ljudskozdravlje. Pojedini teški metali, kao što su kadmij(Cd), ìva (Hg), olovo (Pb), arsen (As) i platina(Pt), vrlo su štetni za zdravlje sisavaca. Glavniizvori teških metala za ~ovjeka su zrak, hrana ivoda. Nakon apsorpcije metali izazivaju razli~itetoksi~ne u~inke u organizmu, a glavni ciljni orga-ni su im bubrezi, jetra i spolni organi, pa je pore-me}aj njihove funkcije naj~eš}a posljedica takvogtrovanja. U ljudi i pokusnih ìvotinja nefrotok-si~nost Cd, Hg i Pt o~ituje se poreme}enom re-apsorpcijom i sekrecijom tvari u proksimalnimkanali}ima (PK). Glavni simptomi nefropatije uz-rokovane teškim metalima su poliurija, prote-inurija, fosfaturija, aminoacidurija i glukozurija,koji ukazuju na ošte}enu funkciju razli~itih pri-jenosnih mehanizama smještenih u ~etkastojmembrani stanica PK. U pokusima na ìvoti-njama trovanim kadmijem utvr|eno je da po-reme}aj funkcije ~etkaste membrane moè na-stati zbog: a) izravne inhibicije specifi~nih prije-nosnika, b) skra}enja i ošte}enja mikrovila i c)gubitka specifi~nih prijenosnika u membrani. Cdizravno inhibira aktivnost vakuolne H+-ATPaze ismanjuje njezinu ekspresiju u stani~nim orga-nelama, te tako ometa unutarstani~ni promet(endocitozu i egzocitozu) proteina posredovan ve-zikulama, što rezultira smanjenjem broja speci-fi~nih prijenosnika u ~etkastoj membrani. Nada-lje, za normalno odvijanje prometa prijenosnikanu`na je strukturna i funkcijska cjelovitost ci-toskeleta. Pokazano je da Cd, Hg i Pt ošte}ujustrukturu, ure|enost i ekspresiju citoskeleta, tetako dodatno ometaju ugradnju prijenosnika u~etkastu membranu. Pokusi na štakorima uka-zuju na to da su sli~ni stani~ni mehanizmi od-govorni i za toksi~ne u~inke teških metala uepitelu muških spolnih organa, zbog ~ega nastajuporeme}aji u sekreciji i fertilitetu.E-mail: [email protected]

S1-2

CELL MECHANISMS OF HEAVY METALTOXICITY

C. M. Herak-Kramberger, I. Saboli}

Institute of Medical Research and Occupational Medicine,Zagreb

Metals are natural constituents of the environ-ment and their balance is achieved by both geo-logic and biologic cycles. Their utilization by hu-mans in industry and agriculture has trans-formed many metals into potential health haz-ards. Some metals such as cadmium (Cd), mer-cury (Hg), lead (Pb), arsenic (As) and platinum(Pt) (often termed as heavy metals) are found tobe very toxic to mammals. The main sources ofexposure to these metals are air, food and water.Following absorption, they cause a variety oftoxic effects in the kidney, liver and reproductivesystem, their major target organs, that result intheir dysfunction. Nephrotoxicity in humans andexperimental animals due to exposure to Cd, Hgand Pt is primarily manifested by impaired reab-sorption and secretion in proximal tubules (PT).The main symptoms of heavy metal nephrotoxic-ity include polyuria, proteinuria, phosphaturia,aminoaciduria and glucosuria, indicating in-volvement of various brush-border membrane(BBM) transporters. As found in experimental Cdnephrotoxicity in rats, impaired functional capac-ity of the BBM in heavy metal nephrotoxicity mayresult from: (a) direct inhibition of BBM trans-porters; (b) shortening and loss of microvilli; and(c) loss of specific transporters from the mem-brane. The loss of BBM transporters may becaused by impaired vesicle-mediated recycling(endo- and exocytosis) of proteins in PT cells dueto diminished expression of the vacuolar H+-AT-Pase and direct inhibition of its activity in intrac-ellular organelles. In addition, Cd, Hg and Ptwere found to affect the structure, polymerizationstate and abundance of cytoskeleton in PT cells,thus contributing to the impaired intracellularvesicle trafficking and preventing functional inte-gration of proteins into the BBM. The experimen-tal evidence in rats show that similar cellularmechanisms of heavy metal toxicity may be in-volved in the male reproductive system causingdysfunctions of the epithelium and impaired fer-tility.E-mail: [email protected]



S1-3

STRESNI PROTEINI NA RELACIJISTANICA - OKOLIŠ

Bariši} K.

Farmaceutsko-biokemijski fakultet Sveu~ilišta u Zagrebu,

Zagreb

Proteini toplinskog šoka (hsps) bitni su zaodràvanje stani~ne homeostaze te za prilagod-bene funkcije stanice. Prva funkcija koja je pri-druèna ovim iznimno konzerviranim proteinimabila je termotolerancija, tj. zaštita od slijede}egizlaganja toplinskom šoku. Zaštitna uloga hspsodnosi se i na zaštitu od drugih oblika stresa ipovezana je s njihovim svojstvom molekularnihpratitelja koji, kontroliraju}i strukturiranje, sor-tiranje, degradaciju, translokaciju i zdruìvanjeproteina, igraju va`nu ulogu u prijenosu signala,organizaciji stani~nog skeleta, apoptozi, prezen-taciji antigena te stani~noj migraciji, proliferacijii adheziji. Hsps su va`ni u adaptaciji stanice namnoge druge stresore, primjerice infekciju, slo-bodne radikale ili mehani~ki stres, pruàju}i sta-nici zaštitu od štetnih utjecaja iz okoliša i pro-vociraju}i autoimuni odgovor kri`nom reakcijomizme|u hsp mikroorganizama i svojih stani~nihkomponenata. Prema molekularnoj masi svrsta-ni su u nekoliko obitelji: mali, hsp40-60, hsp70,hsp90 i hsp110. Ponajbolje je karakterizirana obi-telj hsp70 koju ~ine konstitutivni ili hsc (heatshock cognate) 73, inducibilni hsp72, 78 kDa pro-tein reguliran glukozom (grp78) ili BiP, smještenu lumenu endoplazmatskog retikuluma, te grp75mitohondrijski oblik hsp70 koji se još nazivamito-BiP. Hsp70 je induciran razli~itim stresnimstimulansima. Naša su istraìvanja in vivo i invitro pokazala promijenjene koncentracije hsp70u akutnoj i subkroni~noj intoksikaciji okratok-sinom A. Ekspresija hsp70 regulirana je na tran-skripcijskoj razini pomo}u ~imbenika toplinskogšoka (HSF). Fenomen kri`ne zaštite kod odgo-vora na toplinski šok predstavlja osnovu za pri-mjenu hsps u medicini, primjerice, kod kardio-vaskularnih bolesti te u zaštiti organa za tran-splantaciju. Kako se u posljednje vrijeme isti~euloga hsps u imunološkim procesima, oni bi mo-gli biti iskorišteni za dizajniranje novih lijekovaza lije~enje autoimunih i malignih bolesti.E-mail: [email protected]

S1-3

STRESS PROTEINS BETWEEN CELLAND ENVIRONMENT

K. Bariši}

Zagreb University School of Pharmacy and Biochemistry,Zagreb

Heat shock proteins (hsps) are essential for cel-lular homeostasis and adaptive functions. Thefirst function to be attributed to these extraordi-narily conserved proteins was thermotolerance,i.e. protection from further exposure to heatshock. The protective role of hsps has been ex-tended to other stresses and generally correlateswith their properties as molecular chaperonescontrolling protein folding, sorting, degradation,translocation and assembly, and playing an im-portant role in cell signaling, cytoskeletal organi-zation, apoptosis, antigen presentation, cell mi-gration, proliferation and adhesion. Hsps alsoplay major roles in the processes of adaptation toa number of cellular stresses, such as infection,high temperature, free radicals, or mechanicalstress, providing cells with protection from envi-ronmental insults and evoking autoimmune re-sponses by cross-reaction between the hsp of mi-croorganisms and cellular ’self ’ components.Hsps are divided into families based on their mo-lecular mass: small, hsp40-60, hsp70, hsp90,hsp110. The best characterized family is hsp70family consisting of hsp73 or hsc73 (from heatshock cognate), constitutive member of hsp70family, hsp72, stress inducible hsp70 familymember, 78 kDa glucose-regulated protein, Grp78or BiP, located within the ER lumen, and mito-chondrial hsp70, known as Grp75 or mito-BiP.Hsp70 is induced by a number of stress stimuli.Our in vitro and in vivo investigations haveshown changes in the hsp70 concentration uponacute and subchronic intoxication with ochra-toxin A. Hsp70 expression is regulated at tran-scriptional level by the heat shock factor (HSF).The cross-protection phenomenon of heat shockresponse is the basis for many potential medicalapplications of hsps, for example, for interventionin cardiovascular diseases or in organ preserva-tion and transplantation. Since hsps are involvedin immune processes, they have a potential to beused in the development of immune system-basedtherapeutics targeted at cancer and autoimmunediseases.E-mail: [email protected]



S1-4

OKSIDATIVNI STRES I STANI^NA SMRT

Rumora L., @ani}-Grubi{i} T.

Farmaceutsko-biokemijski fakultet, Zavod za medicinskubiokemiju i hematologiju, Zagreb

Razli~iti poticaji iz okoline mogu utjecati napreìvljavanje i umiranje stanica u organizmu.Reaktivni produkti kisika predstavljaju jednu odnajja~ih i naj~eš}ih prijetnja za sve ìve orga-nizme. Reaktivni spojevi kisika, poput super-oksidnog radikala, vodikovog peroksida, singlet-nog kisika, hidroksilnog radikala i peroksidnogradikala, mogu se nakupljati unutar stanice usli-jed normalnih metaboli~kih procesa te razli~itihtoksi~nih procesa. Ti spojevi mogu poremetitiprirodne antioksidativne obrambene sustave, štorezultira ošte}enjem svih glavnih skupina biološ-kih makromolekula, poput nukleinskih kiselina,proteina, ugljikohidrata i lipida. Oksidativnistres se definira kao poreme}aj u ravnoteì iz-me|u prooksidansa i antioksidansa, što dovodi dopotencijalnog ošte}enja stanice. Smatra se da jeoksidativni stres jedan od uzro~nika brojnih bio-loških i patoloških procesa poput starenja, upale,karcinogeneze, ishemije-reperfuzije, kao i ~itavogniza bolesti kao što su dijabetes, ateroskleroza i/ili neurodegenerativne bolesti. Smatra se da je uzoksidativni stres i proces apoptoze uklju~en upatogenezu navedenih bolesti. Apoptoza je na~inumiranja stanica do kojega ~esto dolazi u fizio-loškim uvjetima tijekom razvoja, ali i zbog pato-loških poticaja, a omogu}ava strogu kontrolubroja stanica i veli~ine tkiva, kao i zaštitu odstanica koje mogu poremetiti homeostazu. Kodapoptoze dolazi do smanjenja volumena stanice,kondenzacije kromatina, internukleosomne frag-mentacije DNK, te do fragmentiranja stanice utzv. apoptoti~na tjelešca. U procesu apoptoze su-djeluju ~lanovi nekoliko razli~itih proteinskihobitelji: neki od njih ga poti~u, a neki inhibiraju.Spojevi peroksovanadija poznati su kao sna`niinzulinomimetici i inhibitori tirozinskih fosfa-taza. Zbog svoje stabilnosti i djelovanja ovi spo-jevi postaju privla~ni kao potencijalni farmako-loški agensi u terapiji dijabetesa, ali je prethodnopotrebno ispitati njihovu mogu}u toksi~nost zastanice. Raspravljati }e se o djelovanjima pero-ksovanadijevog spoja bpV (phen) na RINm5Fstanice inzulinoma štakora.E-mail: [email protected]

S1-4

OXIDATIVE STRESS AND CELL DEATH

L. Rumora, T. @ani}-Grubi{i}

Department of Medical Biochemistry and Hematology,School of Pharmacy and Biochemistry, University of Zagreb,

Zagreb

The intrinsic balance between life and deathcan be influenced by a number of environmentalstresses. Reactive products of oxygen are amongstthe most potent and omnipresent threats faced bythe living body. Intracellular accumulation of re-active oxygen species such as superoxide anion,hydrogen peroxide, singlet oxygen, hydroxyl radi-cal, and peroxy radical, can arise from toxic in-sults or normal metabolic processes. These speciesmay perturb the cell’s natural antioxidant defensesystems, resulting in damage to all of the majorclasses of biological macromolecules, includingnucleic acids, proteins, carbohydrates and lipids.Oxidative stress has been defined as a distur-bance in the prooxidant-antioxidant balance, re-sulting in potential cell damage. It has been im-plicated in several biologic and pathologic proc-esses like aging, inflammation, carcinogenesis,ischemia-reperfusion, and in diseases includingdiabetes mellitus, atherosclerosis, and/or neuro-degenerative diseases. Apoptosis has also beenlinked to these diseases, suggesting that bothprocesses might be involved in these pathologies.Apoptosis is a form of cell death that occurs dur-ing several physiologic and pathologic situationsin multicellular organisms and constitutes acommon mechanism of cell replacement, tissueremodeling, and removal of damaged cells.Apoptosis is a complex process characterized bycell shrinkage, chromatin condensation, inter-nucleosomal DNA fragmentation, and formationof apoptotic bodies. Several protein families areimplicated in apoptosis: some in the induction ofthe process and some in the attenuation of theprocess. Peroxovanadium compounds are potentinsulinomimetic agents and protein tyrosinephosphatase inhibitors. Stability and potency ofthese compounds renders them attractive agentsin diabetes mellitus, but their possible citotoxicitycould jeopardize their therapeutic use. The effectsof peroxovanadium compound bpV (phen) on ratinsulinoma RINm5F cell survival will be dis-cussed.E-mail: [email protected]



S1-5

ALERGENI IZ OKOLIŠA - UZRO^NICIBOLESTI I MOGU]NOSTI LIJEÊNJA

Samarìja I.

Zavod za farmakologiju, Farmaceutsko-biokemijski fakultetSveu~ilišta u Zagrebu, Zagreb

Postoji mnoštvo štetnih inhalacijskih i nein-halacijskih tvari u našem vanjskom i unutarnjemokolišu koje mogu izazvati alergijsku reakciju. Tomogu biti nespecifi~ni (anorganske ili organsketvari) ili specifi~ni agensi (pelud, insekti, plijesni,bakterije, virusi, prašina, epitel i dlake ìvotinja,neki lijekovi, pa i samo sunce). Takove tvari kojenas okruùju tijekom cijele godine ili samosezonski za pojedince predstavljaju antigen kojiizaziva alergijsku reakciju poput alergijske ast-me, sezonskog alergijskog rinitisa (peludna groz-nica), vazomotornog rinitisa, ekceme na koì (ur-tikarijski osip), reakciju na o~ima (alergijski ko-njunktivitis), solarne alergije itd. Zbog oteànihmogu}nosti utvr|ivanja ovih alergena za poje-dinca va`no je provoditi preventivne mjere tepratiti biometereološku prognozu. Me|utim, pripojavi simptoma neophodno je primijeniti odgo-varaju}e lijekove. To su antihistaminici koji ~ineosnovni farmakološki pristup, a uz njih se kodteìh oblika alergija rabe i glukokortikoidi. Na-stanak alergijske reakcije vezan je za aktiviranjemastocita i pove}ano osloba|anje medijatoraupale. Stoga antihistaminici djeluju selektivnokompetitivnim antagonizmom na histaminskeH1-izoreceptore i time onemogu}uju u~inke hi-stamina na perifernim ciljnim stanicama. Anti-histaminici registrirani u Hrvatskoj su: difen-hidramin (Dimidril), kloropiramin (Synopen), ce-tirizin (Letizen), ketotifen (Dihalar), loratadin(Claritine, Contral, Flonidan, Rinolan) i fekso-fenadin (Telfast). Loratadin i feksofenadin seuvelike rabe kod sezonskih alergija (alergijskogrinitisa), osloba|anja peludi pri oprašivanju,alergijskih ko`nih bolesti, kao i u profilaksi sun-~anih alergija. Ne izazivaju sedaciju niti antiko-linergi~ni u~inak zbog slabe liposolubilnosti, au~inak im je brz i traje 24 sata. Difenhidramin jeipak naju~inkovitiji kod cjelogodišnjih alergijskihreakcija (uzrokovanih ku}nom prašinom, ìvo-tinjskom dlakom i sl.), premda izaziva sedaciju.Zbog pove}ane u~estalosti alergijskih reakcijaneophodno je posvetiti ve}u pozornost edukacijišire populacije kako bi se uspostavio pravilan na-~in ìvota, sprije~ila i snizila u~estalost alergija,te smanjila uporaba lijekova.E-mail: [email protected]

S1-5

ALLERGENS FROM THE ENVIRONMENT -DISEASE AGENTS AND TREATMENT

OPTIONS

I. Samarìja

Department of Pharmacology, School of Pharmacy andBiochemistry, University of Zagreb, Zagreb

There are many inhaling or noninhaling sub-stances in our outdoor and indoor environmentthat can cause allergic reactions. These can benonspecific (inorganic or organic substances) orspecific agents (pollen, insect stings, mold, bacte-ria, viruses, dust, animal epithelium and hair,some drugs, and sun). These substances presentin our environment throughout the year or sea-sonally represent antigens that can induce aller-gic reaction such as allergic asthma, seasonal al-lergic rhinitis (hay fever), vasomotor rhinitis, ec-zema on the skin (urticarial rash), reactions onthe eyes (allergic conjunctivitis), solar allergy, etc.As it is difficult to identify allergens on an indi-vidual basis, it is important to perform preventivemeasures and monitor biometeorologic reports.However, the onset of symptoms requires the useof appropriate drugs, i.e. antihistaminics, whichare the first-line pharmacological approach, withthe addition of glucocorticoids in more severeforms of allergy. The onset of allergic reaction isrelated to the activation of mastocytes and in-creased release of inflammatory mediators.Therefore, antihistaminics act selectively by com-petitive antagonism on H1-isoreceptors, thus pre-venting the action of histamines on peripheraltarget cells. The following antihistaminics havebeen registered in Croatia: diphenhydramine(Dimidril), chlorpiramine (Synopen), cetirizine(Letizen), ketotifen (Dihalar), loratadine (Clari-tine, Contral, Flonidan, Rinolan) and fexophe-nadine (Telfast). Loratadine and fexophenadineare mostly used for season allergy: allergic rhini-tis associated with pollen release during pollina-tion, allergic skin reactions, and as a prophylaxisof solar allergy. They neither cause drowsinessand psychomotor impairments nor anticholiner-gic actions because of their low liposolubility.Their effect is quick and lasts for 24 hours.Diphenhydramine is the most potent drug for per-ennial allergy (caused by home dust, animal hair,etc.), although it has a sedative effect. The everincreasing prevalence of allergic reactions callsfor better education of the population at large asthe only way to adopt healthier lifestyle, to pre-vent or reduce the rate of allergies, and to de-crease the use of drugs.E-mail: [email protected]



S1-6

BIOKEMIJSKI MEHANIZMINEFROTOKSI^NOSTI MIKOTOKSINA

Petrik J., êpelak I.

Zavod za medicinsku biokemiju i hematologiju,Farmaceutsko-biokemijski fakultet, Zagreb

Mikotoksini su sekundarni metaboliti poje-dinih vrsta plijesni, koji kontaminiraju hranubiljnog i ìvotinjskog podrijetla. Vrlo su stabilni inakupljaju se u tkivima ljudi i ìvotinja. Bubrezisu glavni ciljni organ za toksi~no djelovanjeokratoksina A (OTA), citrinina, vomitoksina tezearalenona. Uz nefrotoksi~ni u~inak ovi toksinimogu djelovati i hepatotoksi~no, genotoksi~no,kancerogeno te imunosupresivno. Etiologija imehanizam nastanka endemske nefropatije teoko 50% slu~ajeva tubulointersticijskog nefritisanisu razjašnjeni. Temeljem brojnih istraìvanjau~inaka mikotoksina, poglavito OTA, pretpo-stavlja se njihova zna~ajna uloga u etiološkommozaiku navedenih bolesti. Smatra se tako|er davomitoksin moè uzrokovati glomerulonefritis,budu}i da utje~e na regulaciju stvaranja IgA. Nastani~noj razini navedeni mikotoksini, ovisno okoncentraciji, mogu poja~ati stvaranje reaktivnihkisikovih radikala, citokina, aktivnost endonu-kleaza, kaspaza te stvaranje DNK ’adukata’,stimulirati apoptozu, mijenjati stani~nu prolife-raciju, inhibirati transportne sustave za organ-ske anione i katione, te inhibirati sintezu pro-teina i DNK. U okviru opse`nih in vivo i in vitroispitivanja nefrotoksi~nih u~inaka OTA odredilismo neke od spomenutih mehanizama na šta-korima soja Wistar tijekom 60 dana, ali uz re-lativno niske koncentracije OTA (120 µg/kg tje-lesne mase/dan). Elektroforetska analiza DNKnije pokazala znakovitu fragmentaciju (DNAladdering). Apoptoti~ne stanice su vizualiziranetehnikom TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling) ibojene in situ hematoksilinom i eozinom. Brojapoptoti~nih stanica kod pokusnih ìvotinja kojesu bile izloène djelovanju OTA tijekom 10 danapove}ao se 5 puta, nakon 30 dana 6,4 puta, anakon 60 dana ~ak 12,7 puta. Koncentracija li-pidnih peroksida kod skupine ìvotinja koje subile izloène OTA kroz 60 dana zna~ajno jeporasla (36%) uz istodobno smanjenje kataliti~nekoncentracije superoksid dismutaze (26%) u bu-bre`nom tkivu štakora. Rezultati ovoga radapokazuju da OTA ve} u malim koncentracijamaaktivira procese koji stimuliraju apoptozu i oksi-dativni stres u bubre`nim stanicama.

E-mail: [email protected]

S1-6

BIOCHEMICAL MECHANISMS OFMYCTOTOXIN NEPHROTOXICITY

J. Petrik, I. êpelak

Department of Medical Biochemistry and Hematology,School of Pharmacy and Biochemistry, University of Zagreb,

Zagreb

Mycotoxins are secondary metabolites producedby several species of fungi that contaminate food ofplant and animal origin. They are exceptionallystable and accumulate in human and animal tis-sues. Kidney is the main target organ for toxic ef-fects of ochratoxin A (OTA), citrinin, vomitoxin andzearalenone. These mycotoxins can also act as hepa-totoxic, genotoxic and carcinogenic agents, and theyaffect normal immune response. The etiology andmechanism of development of endemic nephropathyand approximately 50% of other tubulointerstitialnephritides are not completely understood. Basedon numerous studies of mycotoxin toxicity, espe-cially OTA, their significant role in the etiologicmosaic of the mentioned diseases has been as-sumed. Moreover, vomitoxin can cause glome-rulonephritis by inducing dysregulation of IgA pro-duction. At the cellular level, the concentration-de-pendent effects of mycotoxins have been observed:induction of reactive oxygen species and cytokineproduction, enhancement of endonuclease andcaspase activities and DNA adduct formation,stimulation of apoptosis, modulation of cell prolif-eration, and inhibition of transport systems for or-ganic anions and cations as well as inhibition ofprotein and DNA synthesis. In our extensive in vivoand in vitro studies of the nephrotoxic effects ofOTA, we examined some of these effects in Wistarrats exposed to very low OTA concentrations (120mg OTA/kg body weight daily) for 60 days. OTAtreatment caused an increase in the number ofapoptotic epithelial renal cells. DNA analysis didnot show characteristic fragmentation (DNAladdering). The apoptotic cells were visualized us-ing TUNEL (terminal deoxynucleotidyl trans-ferase-mediated dUTP nick end labeling) assay andstained with hematoxylin and eosin in situ. Thenumber of apoptotic cells in OTA treated rats for10, 30 and 60 days increased 5-, 6.4- and 12.7-fold,respectively, as compared to control cells. The con-centration of lipid peroxides showed an increase(36%), however, SOD activity decreased (26%) inrats treated for 60 days. The results of this studyshowed that the processes stimulating apoptosisand oxidative stress in renal cells could be acti-vated by exposure to even very low concentrations ofOTA.E-mail: [email protected]



S2-1

TELOMERI, TELOMERAZA I KARCINOM

Ivankovi} G.

Op}a bolnica Vara`din, Vara`din

Opse`na istraìvanja proteklih nekoliko go-dina donijela su golem napredak u razumije-vanju klju~nih komponenata i sloène regulacijetelomeraze. Ova jedinstvena stani~na reverznatranskriptaza je bitna za odràvanje stabilnostitelomera i potrebna je za neograni~enu prolife-raciju gotovo svih malignih stanica. Identifika-cija komponenata telomeraze i sposobnost rekon-stitucije aktivnosti telomeraze u normalnim ljud-skim stanicama omogu}ava ispitivanje i defini-ranje uloge telomeraze u razli~itim stani~nimprocesima, kao što su stani~no starenje, starenje,razgradnja/obnova DNK, stani~na imortalizacijai karcinom. Ektopi~na ekspresija hTERT koja od-re|uje aktivnost telomeraze moè imortaliziratirazli~ite humane normalne stani~ne tipove. Te-lomeraza je potrebna za dugotrajnu proliferacijumalignih stanica, što dokazuje njenu ulogu ustani~noj imortalizaciji i onkogenezi.E-mail: [email protected]

S2Nova dostignu}a u onkologiji

New advances in oncology

S2-1

TELOMERES, TELOMERASE AND CANCER

G. Ivankovi}

Vara`din General Hospital, Vara`din

Significant research efforts in the past fewyears have tremendously increased the under-standing of the key components and complexregulation of telomerase. This unique cellular re-verse transcriptase is essential for maintainingtelomere stability and is required for the unlim-ited proliferation of almost all cancer cells. Iden-tification of the core components of telomeraseand the ability to reconstitute telomerase activityin normal human cells have allowed researchersto experimentally test and define the role oftelomeres in diverse cellular processes, such ascellular senescence, aging, DNA damage/repair,cellular immortalization and cancer. Demonstra-tions that ectopic expression of hTERT, the rate-limiting determinant of telomerase activity, canimmortalize diverse normal human cell types andthat telomerase is required for the longterm pro-liferation of cancer cells strongly support the roleof telomerase in cell immortalization and onco-genesis.E-mail: [email protected]

S2-2

NOV PRISTUP RANOM OTKRIVANJUMALIGNIH STANICA U PERIFERNOJ

KRVI

Albert W.H., Gutierrez B., Böcher O.

AdnaGen AG, Langenhagen, Njema~ka

Pojava tumorskih stanica u perifernoj krviosoba koje boluju od karcinoma moè posluìtikao rana indikacija koja ukazuje na širenje tu-mora izvan izvornog mjesta nastanka. Usprkosobrambenim mehanizmima organizma pojedinestanice karcinoma mogu se nastaniti u udaljenimpodru~jima i stvoriti kolonije kao po~etni korak unastanku metastaza. Krvlju prenešena udaljenametastaza vode}i je uzrok smrti povezanih s ra-kom. Stoga se rano otkrivanje metastatskog po-

S2-2

NEW APPROACH TO EARLY DETECTIONOF MALIGNANT CELLS IN PERIPHERAL

BLOOD

W.H. Albert, B. Gutierrez, O. Böcher

AdnaGen AG, Langenhagen, Germany

The occurrence of tumor cells in peripheralblood of individuals suffering from cancer mayserve as an early indication that primary tumorhas evaded from its tissue of origin. Despitedefense mechanisms of the body, individual can-cer cells may attach in distant regions and formcolonies as an initial step of metastasis forma-tion. A blood-borne distant metastasis is the lead-ing cause of cancer-related deaths. Hence, earlydetection of the metastatic potential can be esti-



tencijala moè procijeniti otkrivanjem tumorskihstanica u krvotoku. Osjetljivsot i specifi~nost suglavni ciljevi svake metode koja se rabi u tu svr-hu. Me|utim, ve}ina opisanih visoko osjetljivihtehnika udruèna je sa sve ve}im problemomspecifi~nosti zbog pozadinskog signaliziranja iznelegalne transkripcije. Naša je grupa razvilametodu za otkrivanje tumorskih stanica i njihovuspecifi~nu identifikaciju i analizu s niòm grani-com otkrivanja od jedne tumorske stanice na 5mL krvi. Da bismo to postigli, kombinirali smooboga}ivanje tumorskih stanica uz primjenu spe-cifi~no izra|ene mješavine protutijela s RT-mul-tipleks PCR tehnikama za otkrivanje mRNK kojekodiraju za tumor specifi~ne biljege. Privremenirezultati ispitivanja slu~ajeva pokazali su da po-java tumorskih stanica u krvi bolesnika s karci-nomom ukazuje na mogu}i recidiv tumora, u ne-kim slu~ajevima nekoliko mjeseci prije porastatumorskih biljega u serumu. U zaklju~ku, uspo-stavljena je osjetljiva i specifi~na metoda za ot-krivanje diseminiranih tumorskih stanica u peri-fernoj krvi bolesnika s karcinomom testisa, dojkei kolona/rektuma. Ova nova metoda predstavljaopciju za klini~are u predvi|anju razvoja meta-staza i moè omogu}iti ispravan odabir bolesnikaza adjuvantnu terapiju.E-mail: [email protected]

mated by detecting tumor cells in the circulation.Sensitivity and specificity are the main objectivesof any method used for this purpose. However,most of the highly sensitive techniques describedare associated with an increasing problem ofspecificity due to background signaling from ille-gitimate transcription. Our group has establisheda method of tumor cell selection and their specificidentification and analysis with a lower detectionlimit of one tumor cell in 5 mL blood. To achievethis we combined tumor cell enrichment using aspecifically designed antibody mixture with RT-multiplex PCR techniques for the detection ofmRNAs encoding for tumor specific markers. Pre-liminary results of case studies showed the occur-rence of tumor cells in blood of carcinoma pa-tients to indicate a potential tumor relapse, insome cases several months prior to the elevationof serum tumor markers. In conclusion, a sensi-tive and specific method to detect disseminatedtumor cells in peripheral blood of testicular,breast and colorectal carcinoma patients hasbeen established. This innovative method is anoption for clinicians as a predictive tool with re-spect to metastasis formation and may result inan appropriate selection of patients for adjuvanttherapy.E-mail: [email protected]

S2-3

ANTIGEN SPECIFIÂN ZA PROSTATUI RANO OTKRIVANJE RAKA

PROSTATE

Stieber P.

Institute of Clinical Chemistry, Klinikum Grosshadern,University of Munich, München, Njema~ka

Antigen specifi~an za prostatu (PSA) je serinproteaza što ju proizvodi prostata i otpušta u krvu raznim bolestima prostate. Tako je, za razlikuod ve}ine drugih onkoloških biomarkera, PSA(skoro) specifi~an za doti~ni organ. Me|utim, kaoi kod drugih biomarkera u onkologiji, još je izra-èniji nedostatak specifi~nosti za tumor. Rak pro-state jednako kao dobro}udne bolesti prostate,fizi~ka trauma prostate te ~imbenici poremetnjeili razli~itih utjecaja na `lijezdu mogu dovesti dozna~ajnog porasta PSA u serumu. Kako se rakprostate uglavnom javlja u starijih muškaracakoji istodobno boluju od dobro}udne hiperplazijeprostate, problem diferencijalne dijagnoze pove-}ava se, a tumorska specifi~nost se smanjuje sdobi. Godinama je ve} poznato da su visoke razi-

S2-3

PROSTATE-SPECIFIC ANTIGENAND EARLY DETECTION OF PROSTATE

CANCER

P. Stieber

Institute of Clinical Chemistry, Klinikum Grosshadern,University of Munich, Munich, Germany

Prostate-specific antigen (PSA) is a serine pro-tease produced by the prostate gland and releasedinto the blood in various prostatic disorders;thus, in contrast to most other oncologicbiomarkers, PSA is (almost) organ-specific. Nev-ertheless, there is an even more pronounced lackin tumor specificity as for other biomarkers in on-cology. Prostate cancer as well as benign prostaticdiseases, physical trauma to the prostate and in-terfering or influencing factors can lead to sig-nificant increases in serum PSA. As prostate can-cer generally occurs in elderly men at the sametime suffering from benign hyperplasia of theprostate, the problem of differential diagnosis in-creases and tumor specificity decreases with age.High total PSA levels have been known for years



ne ukupnog PSA dobar pokazatelj prisutnostiraka prostate. U~estala uporaba odre|ivanjaPSA ~ak je dovela do pomaka u dijagnostici pre-ma raku prostate ograni~enom na taj organ. Ot-prije nekoliko godina dodatno odre|ivanje slo-bodnog PSA i primjena omjera PSA poboljšali sudijagnosti~ku u~inkovitost PSA i njegovih izofor-ma kod raka prostate, uglavnom u rasponu odzna~ajnog preklapanja vrijednosti PSA uzroko-vanih dobro}udnim i zlo}udnim bolestima pros-tate. Na osnovi naših klini~kih ispitivanja utvr-|ena je sveukupna dodatna korist u cjelokupnomrasponu preklapanja ukupnog PSA izme|u 2 i 20ng/ml (Abbott, MEIA, AxSYM), no ~esto primje-njivane prijelomne (cut-off) vrijednosti svode ovuvisoku dijagnosti~ku korist na minimum. Stogasmo u rutinskoj primjeni PSA i slobodnog PSAukinuli uporabu fiksnih prijelomnih vrijednosti,pa rezultate tuma~imo za svakog bolesnika poje-dina~no izra~unavanjem individualne vjerojat-nosti za razvoj raka prostate (odnosno odgova-raju}e pozitivnosti biopsije). Nakon po~etnog od-re|ivanja PSA i slobodnog PSA u obzir se uzimasamo brzina porasta PSA u svakog pojedinog bo-lesnika. Znanstvene procjene provedene posljed-njih godina pokazale su kako razumna primjenaPSA i slobodnog PSA te pa`ljivo tuma~enje re-zultata mo`e rezultirati stalnim pove}anjem kli-ni~ke zna~ajnosti ovih biljega. Prilagodbom re-zultata stanju svakog pojedinog bolesnika moguse izbje}i nepotrebne biopsije ili pak poduprijetiodluku o potrebi biopsije.

to be a good indicator of the presence of prostatecancer. The frequent use of PSA determinationhas even caused a shift to diagnosing more or-gan-confined prostate cancers. Additional deter-mination of free PSA and use of PSA ratio havefor several years now improved the diagnostic ef-ficiency of PSA and its isoforms in prostate can-cer mainly in the range of significant overlap ofPSA values caused by benign and malignantprostatic disorders. Based on our clinical investi-gations the overall additional advantage in thewhole overlapping range of total PSA between 2and 20 ng/ml (Abbott, MEIA, AxSYM) is obvious,but the often used fixed cut off values reduce thishigh diagnostic advantage to a minimum. There-fore, in our routine utilization of PSA and freePSA we put an end to the use of fixed cut off val-ues, and interprete the results of each individualpatient by calculating the individual probabilityof prostate cancer (i.e. the corresponding rate ofbiopsy positivity). Following initial determina-tion of PSA and free PSA, only the rate of PSAincrease in the individual patient is taken in con-sideration. Scientific evaluation in recent yearshas demonstrated that a judicious use of PSAand free PSA, and careful interpretation of theresults can continuously increase the clinical sig-nificance of these markers. By adapting the re-sults to the patient’s individual situation, unnec-essary biopsies can be avoided and decision onthe necessity to perform a biopsy can be sup-ported.

S2-4

MOLEKULARNA CITOGENETIKA UOTKRIVANJU BOLESTI

Davidovi}-Mrsi} S.

Klini~ki zavod za laboratorijsku dijagnostiku, Klini~kibolni~ki centar “Zagreb”, Zagreb

Tehnike molekularne citogenetike danas ima-ju gotovo jednaku va`nost kao i tehnike klasi~necitogenetike. Ve}ina tehnika molekularne cito-genetike temelji se na na~elima fluorescentne insitu hibridizacije (FISH). FISH je tehnika kojaomogu}ava vizualizaciju i time izravnu identifi-kaciju specifi~nih DNA i m-RNA sekvenca (dije-lova gena, specifi~nih kromosomskih regija i ci-jelih kromosoma) u stanici. Pomo}u ove metodemogu se iskorištavati ne samo metafazni kromo-somi, nego i interfazne jezgre. FISH se danasrutinski primjenjuje u prenatalnoj dijagnostici,

S2-4

MOLECULAR CYTOGENETICSIN DISEASE DETECTION

S. Davidovi}-Mrsi}

Clinical Institute of Laboratory Diagnosis, Zagreb Univer-sity Hospital Center, Zagreb

Today molecular cytogenetic techniques havethe same role as classic cytogenetic techniques.Most molecular cytogenetic techniques are basedon fluorescent in situ hybridization (FISH). FISHis a technique that enables visualization andidentification of specific DNA and m-RNA se-quences (gene parts, specific chromosomal regionsand whole chromosomes) in cell. With thismethod, not only metaphase chromosomes butalso interphase nuclei can be used. FISH is rou-tinely used in prenatal diagnosis, postnatal clini-cal cytogenetics, cancer cytogenetics, chimeric fol-



S2-5

DIJAGNOSTIKA KARCINOMA ŠTITNJAÊ

Juki} T., Lukinac Lj., Franceschi M., Mateša N.,

Kusi} Z.

Klinika za onkologiju i nuklearnu medicinu KB “Sestremilosrdnice”, Zagreb

Klini~ka obrada bolesnika s ~vorom štitnja~eobuhva}a uzimanje anamneze, fizikalni pregled,odre|ivanje serumske vrijednosti TSH, scinti-grafiju i ultrazvuk (UZV) štitnja~e te citološkupunkciju ~vora pod kontrolom UZV, primjenakojega omogu}ava raniju dijagnozu maligne na-ravi ~vora. Ve}ina ~vorova štitnja~e su benigni, akarcinomi se nalaze u manje od 5% ~vorova. Tu-mori štitnja~e su rijetki i ~ine 0,74% tumora umuškaraca i 2,3% tumora u èna. Karcinomi foli-kularnog epitela (KFE) su naj~eš}i tumori štit-nja~e, od kojih papilarni ~ine 80%, a folikularni10%. Ve}ina KFE su neinvazivne naravi i imajudobru prognozu, ali 5%-10% ovih tumora poka-zuje agresivnije ponašanje uz lokalno širenje iudaljene metastaze. Osnova lije~enja je kirurškoodstranjenje štitnja~e, nakon ~ega slijedi radio-jodna (131I) terapija i hormonska supresijska te-rapija L-tiroksinom. Pra}enje ovih bolesnikauklju~uje mjerenje serumske koncentracije ti-reoglobulina (Tg), scintigrafiju cijelog tijela s ra-diojodom (131I WBS) i UZV vrata. Ve}ina KFEnakuplja jod i mogu se lije~iti pomo}u 131I. Po-višen Tg i negativna 131I WBS upu}uju na re-cidiv tumora ili metastaze koje ne nakupljaju jod.Nuklearno-medicinske (NM) pretrage otkrivanja131I negativnih tumora uklju~uju scintigrafiju s201Talij i 99mTc-MIBI i pozitronsku emisijskutomografiju (PET). Radiološke pretrage uklju-~uju RTG plu}a, kompjutoriziranu tomografiju(CT) i nuklearnu magnetsku rezonanciju (NMR).Medularni karcinomi (MKŠ) ~ine 5% karcinomaštitnja~e. Potje~u od parafolikularnih C stanica, ajavljaju se kao sporadi~ni karcinomi (80%) ili senaslje|uju (20%) u tri klini~ka sindroma: MEN2A, MEN 2B i obiteljski MKŠ. Mutacija proto-onkogena-ret uzrok je nasljednog oblika bolesti.Osnovno lije~enje svih MKŠ je operacija. Neop-hodno je utvrditi proširenost bolesti i isklju~itinasljedni oblik MKŠ. Obrada i pra}enje bole-

postnatalnoj klini~koj citogenetici, citogeneticitumora, pra}enju kimerizma nakon transplan-tacije alogene koštane srì, pra}enju populacijeizloène ioniziraju}em zra~enju itd.E-mail: [email protected]

low-up after allogeneic bone marrow transplanta-tion, follow-up of population exposed to ionizingradiation, etc.E-mail: [email protected]

S2-5

DIAGNOSIS OF THYROID CARCINOMA

T. Juki}, Lj. Lukinac, M. Franceschi, N. Mateša, Z.Kusi}

University Department of Oncology and Nuclear Medicine,Sestre milosrdnice University Hospital, Zagreb

Evaluation of a patient with thyroid noduleincludes history, physical examination, serumTSH level measurement, thyroid scan, thyroid ul-trasonography (US), and US-guided fine needleaspiration biopsy (FNAB) of thyroid nodules. Theintroduction of FNAB has significantly contrib-uted to the earlier diagnosis of malignant nod-ules. Most of the thyroid nodules are benign,whereas carcinoma is found in less than 5% ofthese nodules. Thyroid cancer is a rare malig-nancy, accounting for 0.74% of cancer in men and2.3% in women. Carcinoma of follicular epithe-lium (CFE) is the most common thyroid malig-nancy, 80% being papillary and 10% follicularcancer. Most of CFE cases have a favorable prog-nosis, however, 5%-10% of these tumors presentaggressive nature with local invasion and distantmetastases. Surgical removal of the thyroid glandfollowed by radioiodine (131I) therapy and L-thy-roxine suppression therapy is the main therapeu-tic approach for CFE. The follow-up of these pa-tients includes serum thyroglobulin (Tg) determi-nation, 131I whole body scan (131I WBS), andUS of the neck. Most of the CFE accumulate io-dine and can be treated with 131I. Elevated Tgand negative 131I WBS indicate the presence oftumor recurrence or metastases without the abil-ity to accumulate iodine. Nuclear medicine (NM)imaging studies in detection of 131I negativetumors include 201Thallium and 99mTc MIBIscintigraphy and positron emission tomography(PET). Other imaging studies include chest x-ray,computed tomography (CT) and nuclear mag-netic resonance imaging (NMR). Medullary thy-roid cancer (MTC) accounts for 5% of all thyroidcancers and is derived from parafollicular or Ccells. Tumors may occur sporadically (80%) or infamilies (20%) as part of three clinical syn-dromes: MEN 2A, MEN 2B and familial MTC.Mutation of ret proto-oncogene is the origin of



snika s MKŠ uklju~uje UZV vrata i abdomena,RTG plu}a, CT/NMR prsnog koša, odre|ivanjetumorskih biljega kalcitonina (Ct) i karcinoem-brijskog antigena (CEA) u serumu. Scintigrafijacijelog tijela s 111Indij oktreotidom indicirana jeu slu~aju povišenog Ct, a anti-CEA scintigrafija uslu~aju povišenog CEA. Druge NM pretrage ot-krivanja metastaza MKŠ uklju~uju 99mTc DMSAi 131I MIBG scintigrafiju cijelog tijela. Anapla-sti~ni karcinom je rijedak karcinom štitnja~e inajagresivniji karcinom u ~ovjeka, do danas bezuspješne terapije.

heritable MTC. Surgical therapy is the primarytreatment for MTC. It is necessary to determinethe extent of the disease and evaluate for heritableMTC. Diagnostic evaluation and follow-up of pa-tients with MTC consist of US of the neck and ab-domen, x-ray and CT/NMR of the chest, serumcalcitonin (Ct) and carcinoembryonic antigen(CEA) determination. 111In octreoscan is indi-cated in case of elevated Ct, and anti-CEA scan incase of elevated CEA. Other NM imaging studiesinclude 99mTc DMSA and 131I MIBG scan. Ana-plastic carcinoma is a rare type of thyroid cancerand the most aggressive cancer in humans, so farwithout successful treatment modality.

S2UP-1 (usmeno priop}enje)

PREDVI\ANJE UÎNKOVITOSTIKEMOTERAPIJE POMO]U SERUMSKOGCA 15-3 U BOLESNICA S RAKOM DOJKE

Nekulova M.1, Simickova M.1, Pecen L.2, Valik D.1

1 Masaryk Memorial Cancer Institute; 2 Computer Instituteof Academy of Science, Brno, Republika ê{ka

Cilj studije je bio utvrditi odraàvaju li serum-ske razine tumorskih biljega procijenjene pomo-}u novoga algoritma vjerno odgovor na kemote-rapiju u bolesnica s rakom dojke. Skupina od 122bolesnice s ponovljenim rakom dojke, medijandobi 64,4±13,1 (raspon 38-76) godina, lije~ena jekemoterapijom. Serumske razine CA 15-3 (MEIA,Abbott) mjerene su tijekom sveukupno 854 ci-klusa kemoterapije. Matemati~ki algoritam jeprimijenjen za procjenu koncentracija prije i tije-kom lije~enja (c0 ...cn), relativnih promjena tu-morskih biljega nakon prvog i drugog ciklusakemoterapije (rc1=c0/c1, rc2=c1/c2, rcn=cn-1/cn) iomjera ovih promjena (rc1/rc2 ... rcn/rcn+1) radiizra~unavanja kretanja biljega. Najbolji rezultatpostupnog multivarijantnog Coxova modela zaprocjenu preìvljenja bez bolesti dobiven je zarc1=c0/c1, rc2=c1/c2 i omjer rc1/rc2. U Coxovumodelu je primijenjena metoda analize preìvlje-nja, uz uporabu Wilcoxonova i log-rank testova.Terapija se smatrala u~inkovitom (bolesnice s od-govorom) ako je postignuta potpuna ili djelomi~-na remisija prema kriterijima UICC. Odgovor jepostignut u 52 bolesnice, sa stopom odgovora od42,62% (95% CI: 33,85-51,40), dok je progresija(PB) nastupila u 70 bolesnica - 57,37% (95% CI:48,10-66,15), tj. bolesnice u kojih je odgovor izo-stao. Vrijeme do progresije (VDP) kretalo se od 4do 38 mjeseci i još se procjenjuje u 17 bolesnica

S2UP-1 (oral presentation)

PREDICTION OF CHEMOTHERAPYEFFICACY USING SERUM CA 15-3 IN

BREAST CANCER PATIENTS

M. Nekulova1, M. Simickova1, L. Pecen2, D. Valik1

1 Masaryk Memorial Cancer Institute; 2 Computer Instituteof Academy of Science, Brno, Czech Republic

The aim of our study was to determinewhether changes in serum levels of tumor mark-ers as evaluated by a novel algorithm closely re-flected response to chemotherapy in breast cancerpatients. A group of 122 breast cancer patientswith recurrent disease, median age 64.4±13.1(range 38-76) years were treated with chemo-therapy. Serum levels of CA 15-3 (MEIA, Abbott)were determined during a total of 854 chemo-therapy cycles. A mathematical algorithm wasused to evaluate pretreatment and subsequentconcentrations (c0...cn), relative changes in tumormarkers following first and second chemotherapycycle (rc1=c0/c1, rc2=c1/c2, rcn=cn-1/cn) andratios of these changes (rc1/rc2...rcn/rcn+1) tocompute a marker trend. The best result of astepwise multivariate Cox model for disease-freesurvival estimate was obtained for rc1=c0/c1,rc2=c1/c2 and ratio rc1/rc2. The method of sur-vival analysis was used in Cox model, andWilcoxon and log-rank tests were performed.Therapy was considered effective (responders) ifcomplete or partial remission was achieved ac-cording to UICC criteria. Response was achievedin 52 patients - response rate was 42.62% (95%CI: 33.85-51.40), whereas progression (PD) oc-curred in 70 patients - 57.37% (95% CI: 48.10-66.15) - nonresponders. Time to progression(TTP) varied from 4 to 38 months and is still be-



S3-1

PRENATALNA MOLEKULARNADIJAGNOSTIKA DANAS I SUTRA

Serti} J.

Klini~ki zavod za laboratorijsku dijagnostiku, Medicinskifakultet Sveu~ilišta u Zagrebu i Klini~ki bolni~ki centar

“Zagreb”, Zagreb

Program molekularne dijagnostike genskihbolesti uklju~uje nekoliko kategorija: 1) bolesnikei ~lanove obitelji s visoko rizi~nim nasljednimporeme}ajima; 2) osobe fertilne dobi prije i zavrijeme trudno}e; 3) bolesnike centara za IVF; 4)osobe u prevenciji poligenskih multifaktorskihporeme}aja. Prenatalna dijagnostika uklju~ujeultrazvu~ne biokemijske i molekularnogenskeaspekte. Automatizirana genska analitika omo-gu}uje brzu prenatalnu molekularnu dijagnozugenskih promjena primjenom fluorescentne hi-bridizacije, pri ~emu se DNK izdvaja izravno iz0,1 ml fetalne krvi, 2 ml amnijske teku}ine ili 2mg korionskih resica. Biološki materijal dobivase amniocentezom ili koriocentezom izme|u 12. i17. tjedna trudno}e, a nalaz se šalje klini~aruistoga ili slijede}eg dana. Molekularna dijagno-stika genskih bolesti pojedinih zemalja uklju~enaje u sheme europskih centara izvrsnosti kojiputem European Molecular Genetics QualityNetwork i Reference Institute for Bioanalytics ofDeutsche Gesellschaft für klinische Chemie nad-ziru kvalitetu rada (www.emqn.org, www.dgkc-online.de). Danas se prenatalna molekularna di-

S3-1

PRENATAL MOLECULAR DIAGNOSISTODAY AND TOMORROW

J. Serti}

Clinical Institute of Laboratory Diagnosis, ZagrebUniversity Hospital Center, Zagreb

The program of molecular diagnosis of genediseases includes several categories: 1) patientsand family members with high risk hereditarydisorders; 2) persons at fertile age before and dur-ing pregnancy; 3) patients from IVF centers; and4) individuals requiring prevention of polygenicmultifactorial disorders. Prenatal diagnosis in-cludes ultrasound biochemical and moleculargenic aspects. Automated gene analyses enablerapid prenatal molecular diagnosis of gene al-terations by fluorescent hybridization, with DNAisolated directly from 0.1 ml of fetal blood, 2 ml ofamniotic fluid or 2 mg of chorionic villi. Biologicmaterial is obtained by amniocentesis orchoriocentesis between 12th and 17th week of ges-tation, and the result is forwarded to the clinicianon the same or next day. Molecular diagnosis ofgene diseases is in some countries included inevaluation schemes of European quality centers,which monitor performance quality through Eu-ropean Molecular Genetics Quality Network andReference Institute for Bioanalytics of DeutscheGesellschaft für klinische Chemie (www.emqn.org, www.dgkc-online.de). Prenatal molecular di-agnosis is presently performed for autosomal re-

S3Molekularna laboratorijska medicina

Molecular laboratory medicine

(cenzurirane bolesnice). Regresijska analiza kon-centracija CA 15-3 podijelila je bolesnice s odgo-vorom u dvije podskupine nakon drugog ciklusakemoterapije: i) bolesnice s dobrim odgovoromkoje su ispunjavale slijede}i kriterij: rc1/rc2>1,31i rc1>1,6 i rc2>1,26; ova je skupina uklju~ila 51bolesnicu (41,8%; 95% CI: 33,05-50,56); i ii)bolesnice s manje povoljnim odgovorom koje nisuispunjavale nijedan od gore navedenih uvjeta(predvi|eno VDP: p<0,0002 primjenom Wilcoxo-nova testa i p<0,0036 primjenom log-rank testa).Ispitivanje je dijelom dobilo potporu od Scientificand Research Scheme MZ00020980501.E-mail: [email protected]

ing assessed in 17 patients (censored patients).Regression analysis of CA15-3 concentrations cat-egorized responders into two subgroups after sec-ond chemotherapy cycle: i) good responders ful-filling the criterion: rc1/rc2>1.31 and rc1>1.6and rc2>1.26 - the group of good responders in-cluded 51 patients (41.8%, 95% CI: 33.05-50.56);and ii) less favorable responders not fulfillingany of the above conditions (predicted TTP was:p<0.0002 using Wilcoxon test and p<0.0036 usinglog-rank test). The study was in part supported bythe Scientific and Research SchemeMZ00020980501.E-mail: [email protected]



jagnostika provodi za autosomno recesivne pore-me}aje, kao što su cisti~na fibroza, spinalnamiši}na atrofija, zatim X vezane poreme}aje kaošto su Duchenneova miši}na distrofija, sindromfragilnog X kromosoma, hemofilija A, te za auto-somno dominantne poreme}aje kao što je Hun-tingtonova bolest. Prenatalna molekularna dija-gnostika se ne preporu~a za nasljedne bolesti(hemokromatoza) kod kojih se klini~ka slikarazvija tek u odrasloj dobi, a koje uz pravodobnolije~enje imaju dobru prognozu. Prenatalna dija-gnostika provodi se i biokemijskim metodama zaneke poreme}aje kao što je sindrom Smith-Lemli-Opitz. Prenatalna molekularna dijagno-stika u budu}nosti }e sigurno biti usmjerena nakarcinom i rje|e nasljedne poreme}aje kao što jesindrom trojnog A. Molekularna dijagnostika }ese razvijati unutar opcije manje invazivnih i ma-nje riskantnih zahvata. Izdvajanje fetalnih stani-ca iz maj~ine cirkulacije predstavlja obe}avaju}ipristup k neinvazivnoj prenatalnoj dijagnozi. Zavjerovati je stoga da }e budu}nost molekularnedijagnostike obiljeìti prijeimplantacijska dija-gnostika.E-mail: [email protected]

cessive disorders such as cystic fibrosis, spinalmuscle atrophy, X-linked disorders like Duchennemuscular dystrophy, fragile X chromosome syn-drome, hemophilia A, and autosomal dominantdisorders like Huntington’s disease. Prenatal mo-lecular diagnosis is not recommended for heredi-tary diseases (hemochromatosis) where clinicalpicture does not show progression until adult ageand where prognosis is good providing timelytherapy. Prenatal diagnosis is also performed bybiochemical methods for some disorders such asSmith-Lemli-Opitz syndrome. In the future, pre-natal molecular diagnosis will certainly be ori-ented to treatment of cancer and rare inheriteddisorders like triple A syndrome. Molecular diag-nosis will develop within the option of less inva-sive procedures and those involving lesser risk.Fetal cell isolation from maternal circulation is apromising approach to noninvasive prenatal di-agnosis. We may therefore believe that the futureof molecular diagnosis will be marked bypreimplantational diagnosis.E-mail: [email protected]

S3-2

HEMOKROMATOZA I WILSONOVABOLEST

ûkovi}-âvka S., Vuceli} B.

Klini~ki bolni~ki centar “Zagreb”, Zagreb

Nasljedna hemokromatoza (NH) je autosomnorecesivni poreme}aj metabolizma èljeza koji re-zultira apsorpcijom prekomjerne koli~ine èljezaiz crijevne sluznice. Višak èljeza nakuplja se ujetri, koì, guštera~i, srcu, zglobovima i testisima.Rani klini~ki simptomi bolesti su pojava bolova utrbuhu, slabost, letargija i gubitak na teìni. Kodnelije~enih osoba u kasnijem tijeku bolesti pri-sutna je bron~ana boja koè, še}erna bolest, kar-dijalno popuštanje ili aritmije, artritis i hipogo-nadizam. Dijagnoza se postavlja prema vrijedno-stima testa zasi}enja transferina, koncentracijiferitina u serumu, koncentraciji èljeza u jetre-nom tkivu i nalazu geneti~kog testiranja mu-tacije C282Y i H63D gena HFE (kromosomskilokus 6p21). Uobi~ajeni rizik za bra}u i sestreindeksnog ispitanika iznosi 25%. Ve}ina bolesni-ka lije~i se rutinskom terapijskom venepunkci-jom. Ranim postavljanjem dijagnoze i lije~enjembolesnika prije razvoja klini~kih simptoma mo-

S3-2

HEMOCHROMATOSIS AND WILSON’SDISEASE

S. ûkovi}-âvka, B. Vuceli}

Zagreb University Hospital Center, Zagreb

Hereditary hemochromatosis (HH) is an auto-somal recessive disorder which is characterizedby inappropriately high absorption of iron by thegastrointestinal mucosa, resulting in excessivestorage of iron, particularly in the liver, skin,pancreas, heart, joints and testes. Abdominalpain, weakness, lethargy and weight loss arecommon early symptoms. Other findings in un-treated individuals include progressive increasein skin pigmentation, diabetes mellitus, conges-tive heart failure or arrhythmias, arthritis, andhypogonadism. The diagnosis is typically basedon screening for serum transferrin-iron satura-tion, serum ferritin concentration and confirma-tory tests such as histologic assessment of hepaticiron stores on liver biopsy and/or molecular ge-netic testing for the C282Y and H63D mutationsin the HFE gene (chromosomal locus 6p21). Usu-ally the risk of having HH for sibs of a proband is25%. The usual therapy is removal of excess iron



gu}e je sprije~iti razvoj potencijalnih kompli-kacija. Wilsonova bolest je autosomno recesivnabolest koja dovodi do preoptere}enja organizmabakrom, a o~ituje se jetrenim, neurološkim ipsihijatrijskim poreme}ajima ili njihovom kom-binacijom. Dijagnoza se temelji na niskim kon-centracijama bakra i ceruloplazmina u serumu,pove}anoj koli~ini izlu~enog bakra u mokra}i ipove}anoj koli~ini bakra u suhom tkivu jetre.Zna~ajnu ulogu u dijagnozi ima geneti~ko testi-ranje gena ATP7B (kromosomski lokus 13q14.3-q21.1), jer rezultati testiranja metabolizma bakra~esto nisu dovoljni za dijagnozu. Geneti~ko testi-ranje osobito je va`no za odre|ivanje genetskogstatusa ro|aka. Sestre i bra}a indeksnog bole-snika imaju rizik od 25% za naslje|ivanje bolesti.Bolest se lije~i lijekovima koji odstranjuju višakbakra iz organizma (kelatori - penicilamin, tri-entin i amonijak tetratiomolibdat) ili cinkom.Primjena kelatora ili cinka moè kod asimp-tomatskih osoba sprije~iti razvoj klini~kih simp-toma, a kod bolesnika ih smanjiti.E-mail: [email protected]

by routine phlebotomy. Early diagnosis and treat-ment to normalize serum iron before the symp-toms develop prevent the potential complicationsof HH. Wilson’s disease (WD) is an autosomal re-cessive disorder of copper overload that canpresent with hepatic, neurologic or psychiatricdisturbances, or a combination of these. The diag-nosis depends in part on the detection of low se-rum copper and ceruloplasmin concentrations,increased urinary copper excretion and high he-patic tissue copper concentration. Molecular ge-netic testing of the ATP7B gene (chromosomal lo-cus 13q14.3-q21.1) is playing an increasingly im-portant role in the diagnosis, as copper studiesare frequently equivocal. Molecular genetic test-ing is also important to determine the genetic sta-tus of at-risk sibs. Sibs of an affected individualhave a 25% chance of having WD. Treatment withcopper chelating agents (penicillamine, trientine,ammonium tetrathiomolybdate) or zinc can pre-vent the development of clinical findings inasymptomatic affected individuals and reducefindings in many symptomatic patients.E-mail: [email protected]

S3-3

ZNAÊNJE UPORABEMOLEKULARNO-BIOLOŠKIH METODA

U HEMATOONKOLOGIJI

Zadro R.

Klini~ki zavod za laboratorijsku dijagnostiku Medicinskogfakulteta Sveu~ilišta u Zagrebu i KBC “Zagreb”, Zagreb

Podjela malignih hematoloških bolesti premaSvjetskoj zdravstvenoj organizaciji uklju~uje po-znate genetske poreme}aje u leukemijama i lim-fomima te njihovo prognosti~ko zna~enje. Zbogtoga su razvoj i primjena klini~ki korisnih teh-nika molekularne biologije od velike va`nosti uanalizi malignih hematoloških bolesti: lan~anareakcija polimerazom (PCR), multipleks PCR iPCR u stvarnom vremenu, analiza po Southernu,fluorescentna in situ hibridizacija, a u novije vri-jeme profil ekspresije gena tehnologijom mikro-zraka koja je postala va`na za razumijevanjesloènih bioloških procesa i njihove uloge u pa-tologiji bolesti na molekularnoj razini. U akut-nim limfati~nim leukemijama u djece identifi-ciran je profil ekspresije gena koji dobro korelirasa šest razli~itih kromosomskih abnormalnosti,dok je u akutnim mijeloi~nim leukemijama uodraslih pokazana korelacija izme|u triju speci-fi~nih kromosomskih translokacija i profila eks-

S3-3

ROLE OF MOLECULAR BIOLOGYMETHODS IN HEMATO-ONCOLOGY

R. Zadro


The classification system of hematologic ma-lignancies according to World Health Organiza-tion acknowledges the key role of genetics in thedefinition of specific entities as well as the prog-nostic heterogeneity of disease entities. Over thepast few years, a spectrum of molecular biologictechniques including polymerase chain reaction(PCR), multiplex PCR and real time PCR, South-ern blotting and fluorescent in situ hybridizationhave entered into the mainstream of hemato-on-cology. Lately the analysis of gene expression pat-terns with microarray technology is becoming in-creasingly important to our understanding ofcomplex biological processes and their role in dis-ease pathology at the molecular level. In pediatricB-cell acute lymphoblastic leukemia, gene expres-sion signatures have been identified that correlatewith six different chromosomal abnormalities.Likewise, in adult acute myeloid leukemia, agene-expression-based predictor has been createdthat can identify three different chromosomal



presije gena. Takav znakovit ’molekularni potpis’moè predvidjeti bolesnike u kojih }e do}i dorelapsa bolesti ili do postizanja dugotrajne potpu-ne remisije. Tako|er se pojava sekundarne akut-ne mijeloi~ne leukemije moè predvidjeti na te-melju geneti~kog otiska primarnog tumora. Mo-lekularno-geneti~ke metode va`na su dopuna ru-tinske evaluacije malignih hematoloških bolestikoja uklju~uje morfološku, histološku, imunofe-notipsku i citogenetsku analizu; njihova posebnavrijednost je u rasvjetljavanju nejasne dijagnoze,otkrivanju skrivenih abnormalnosti, klasifikacijiprividno homogenih bolesti, identifikaciji speci-fi~nih molekularnih meta za ciljanu terapiju,utvr|ivanju molekularnih profila koji predvi|ajuishod bolesti, kvantificiranje minimalne ostatnebolesti u pra}enju iš~ezavanja malignog klona,predvi|anju relapsa i utvr|ivanju uspješnosti sa-kupljanja mati~nih stanica za autolognu trans-plantaciju, kao i u situacijama kada razli~iti teh-ni~ki ~imbenici onemogu}avaju izvo|enje i/ilituma~enje konvencionalne citogenetike.E-mail: [email protected]

translocations with a high rate of accuracy. Thischaracteristic molecular signature at the time ofdiagnosis can provide information that could pre-dict which patients would relapse and whichwould remain in continuous complete remission.Secondary acute myeloid leukemia could also bepredicted on the basis of gene-expression profilingof primary tumor. Molecular genetic testing pro-vides important data that are not always evidentfrom routine pathologic evaluation, such as mor-phology, histology, immunophenotyping andkaryotyping. Their primary role is diagnostic ambi-guity; detection of cryptic abnormalities; prognosticstratification; identification of specific moleculartargets that are amenable to targeted therapy; iden-tification of molecular signatures that predict out-come; quantification of minimal residual diseaseafter therapy, early detection of relapse, and evalua-tion of adequacy of purging of autologous stem cellharvests; and when various technical factors pre-clude the performance and/or interpretation of con-ventional karyotypic studies.E-mail: [email protected]

S3-4

TESTIRANJE DNK I KONTROLAKVALITETE U LABORATORIJU ZA

KLINI^KU DIJAGNOSTIKU

Neumaier M.

Institute for Clinical Chemistry, University HospitalMannheim of the University Heidelberg, Mannheim,

Njema~ka

Njema~ko društvo za klini~ku kemiju i la-boratorijsku medicinu osnovalo je 1996. godineslu`benu Radnu grupu za molekularnu dija-gnostiku u laboratorijskoj medicini. Cilj Grupe jebio pruìti potporu uspostavi metoda moleku-larne biologije u klini~kom laboratoriju. Posebniciljevi u okviru ovoga zadatka bili su slijede}i: 1)primjena i proširenje okvira vanjske procjenekvalitete (EQA), 2) uspostava mreè za napredaku struci i baze podataka izme|u suradnih labo-ratorija i organizacija, 3) programi izobrazbe i 4)suradnja s drugim radnim grupama koje rade nasrodnim temama (npr. WG Preanalytics). Od us-postave programa EQA broj suradnih laborato-rija se pove}ao s 40 na 200 zahvaljuju}i sve-europskoj suradnji s drugim nacionalnim znan-stvenim društvima i organizacijama EQA. Proši-rio se je i izbor ispitivanih pokazatelja, pa se sadanude faktor V., faktor II., HFE, ApoE, ApoB100,MTHFR, inhibitor proteinaze 1, UGT-1a, TPMT,

S3-4

DNA TESTING AND QUALITY CONTROLIN THE CLINICAL DIAGNOSTIC

LABORATORY

M. Neumaier

Institute for Clinical Chemistry, University HospitalMannheim of the University Heidelberg, Mannheim,

Germany

In 1996, the German Society for Clinical Chemis-try and Laboratory Medicine (DGKL) has estab-lished an official Working Group on Molecular Diag-nosis in Laboratory Medicine. The Group´s objectiveswere to support the establishment of molecular biol-ogy methods in the clinical laboratory. Specific aimsfor this task were: 1) implementation and extensionof external quality assessment (EQA) schemes; 2) es-tablishment of a proficiency network and databasebetween participating laboratories and organiza-tions; 3) educational training programs; and 4) coop-eration with other working groups active in relatedtopics (e.g., WG Preanalytics). Since the implementa-tion of the EQA program, the number of participat-ing laboratories has increased from 40 to 200 owingto Europe-wide collaboration with other national sci-entific societies and EQA organizations. Also, theportfolio of parameters tested has increased now of-fering Factor V, Factor II, HFE, ApoE, ApoB100,MTHFR, proteinase inhibitor 1, UGT-1a, TPMT,



Cyp450 2D6, a i dalje se širi prema zahtjevima.Uz ove dijagnosti~ke EQA, provode se tehni~kinadzori razli~itih faza u procesu geneti~kog ispi-tivanja, npr. priprava DNK, procjena i izvješ}e zaprodukt PCR, sekvencioniranje DNK i probir namutacije. Zasad bi se moglo zaklju~iti slijede}e:prijeanaliti~ki ~imbenici (kvaliteta materijala,vrijeme i uvjeti prijevoza, inhibitori itd.) kriti~nisu za kvalitetu rezultata molekularnog testira-nja. Molekularne metode koje se rabe za amplifi-kaciju u testovima genotipiziranja vrlo su mo}niglede njihove tehni~ke izvedbe. Jednostavne me-tode jednako su vrijedne kao i nove tehnike. Zadoti~ni test nema korelacije izme|u sofisticira-nosti metode i kvalitete dijagnosti~ke procjene.Zapaèno je da vrijednost rezultata testa nagloopada u prisutnosti manjih kontaminacija šablo-ne (1/8 to 1/16) u uzorku. Ve}ina grješaka nijeuzrokovana krivim primarnim podacima, negoposlijeanaliti~kim tuma~enjem. ^lanovi Radnegrupe isto su tako uklju~eni u razli~ite me|una-rodne radne grupe EC4 i IFCC-a kako bi dopri-nijeli sve ve}oj uspješnosti laboratorijskog testi-ranja DNK u klini~ko kemijskim laboratorijima,npr. kroz organiziranje i provedbu te~ajeva izo-brazbe. Sveukupno, rezultati našega jednogo-dišnjeg iskustva opravdavaju stalne napore kojise ulaù u budu}u provedbu programa vanjskekontrole kvalitete i sheme ispitivanja stru~nosti,s naglaskom na sve-europskoj suradnji.E-mail: [email protected]

Cyp450 2D6, and is constantly being expanded ondemand. In addition to these diagnostic EQAs, tech-nical surveyed exercises have been performed ad-dressing various steps in the genetic testing processlike DNA preparation, PCR product evaluation andreporting, DNA sequencing and mutational screen-ing. Tentatively, we can draw the following conclu-sions. Preanalytical factors (material quality, trans-portation time and modalities, inhibitors, etc.) arecritical for the quality of the molecular test result.Molecular methods used for the amplification ingenotyping assays appear to be very robust with re-spect to their technical performance. Simple methodsare as valid as new techniques. For a given test, thereis no correlation between the sophistication of themethod and the quality of diagnostic evaluation. Va-lidity of test results has been observed to steeply de-cline with minor template contaminations (1/8 to 1/16) present in the sample. Most mistakes are notcaused by faulty primary data, but by postanalyticalinterpretation. Members of the Working Group arealso involved in different international workinggroups of the EC4 and IFCC to contribute to increas-ing the proficiency in DNA laboratory testing inclinical chemistry laboratories, e.g., by organizingand performing teaching courses. Taken together, theresults from our year-long experience justify the con-tinued efforts to implement external quality controlprograms and proficiency testing schemes in the fu-ture, with emphasis on Europe-wide collaboration.E-mail: [email protected]

S3-5

LIZOSOMSKE BOLESTI ODBIOKEMIJSKE OSNOVE DO LIJEÊNJA

Fumi} K.


Lizosomi su organele u kojima se u kiselomokruènju odvija najve}i dio razgradnje sloènihmakromolekula. Za te procese odgovorno je višeod 50 razli~itih lizosomskih hidrolaza uz odgo-varaju}e aktivacijske proteine. Kod lizosomskihbolesti nakupljanja nedostaje ili je zna~ajno sni-èna aktivnost nekog od tih proteina, što za po-sljedicu ima nakupljanje nerazgra|enog mate-rijala u organeli, ošte}enje stanice i okolnog tki-va. Dosad je poznato više od 40 razli~itih lizosom-skih bolesti nakupljanja s ukupnom zastuplje-noš}u od jednog slu~aja u pribli`no 7500 ìvoro-|enih. Premda se na prisutnost lizosomskih bole-

S3-5

LYSOSOMAL DISEASES FROMBIOCHEMICAL BASIS TO TREATMENT

K. Fumi}

Clinical Institute of Laboratory Diagnosis, Zagreb Univer-sity Hospital Center, Zagreb

Lysosomes are organelles where most degrada-tion of complex macromolecules takes place inacid conditions. More than 50 different lysosomalhydrolases are responsible for this process, to-gether with appropriate activation proteins. Theactivity of some of these proteins is either missingor decreased in lysosomal storage diseases, whichresults in the accumulation of undegraded mate-rial in the organelle and damage to the cell andsurrounding tissue. Up to the present, more than40 different lysosomal storage diseases have beenidentified, with an overall prevalence of 1 per ap-proximately 7500 live births. Although the pres-



S3-6

ANALIZA STANI^NOG CIKLUSA STANICACD133+ I CD133- IZDVOJENIH IZ KRVI

UZETE IZ PUPKOVINE

Grškovi} B., Ruì~ka K., Qujeq D., Müller M.M.

Institute for Laboratory Diagnosis, Kaiser Franz JosephHospital, Be~, Austrija

Krvne stanice uzete iz pupkovine (mati~nestanice/prastanice) pokazuju znatnu proliferacij-sku sposobnost, što dovodi do velike ekspanzijeovih stanica u odgovaraju}im uvjetima stani~nihkultura. Cilj ovoga rada bio je analizirati stani~niciklus stanica CD133+ i CD133- metodom pro-to~ne citometrije ovisno o razli~itim uvjetimakultivacije kao što su dodatak seruma, ~imbenikamati~nih stanica (stem cell factor, SCF), inter-leukina 3 (IL-3) i interleukina 6 (IL-6). Za izdva-janje stanica rabili smo imunomagnetski sustav.Stanice CD133+ i CD133- nasadili smo u Isco-veov modificirani Dulbeccov medij (IMDM) s

sti nakupljanja moè posumnjati na temelju kli-ni~ke slike, zbog preklapanja fenotipova obi~no jeteško postaviti nedvojbenu dijagnozu, pa se onazasniva na biokemijskoj analizi. Krajnji cilj jeidentifikacija lizosomskog enzima ili nedostatkaproteina koji su u osnovi poreme}aja. Poznavanjeporeme}aja enzima ili proteina nije neophodnosamo za kona~nu dijagnozu, nego je i preduvjetza provedbu prenatalnih dijagnosti~kih pretraga.Gdjegod je to mogu}e treba pokušati prona}imutaciju ne samo radi potvr|ivanja dijagnoze,nego i da bi se omogu}ilo pouzdano odre|ivanjeheterozigota kod pojedinaca koji zatraè genetskisavjet. Prijedlog da se lizosomske bolesti nakup-ljanja lije~e nadomjestkom ošte}enog normalnimenzimom prvi je iznio Christian de Duve 1964.godine. Me|utim, nadomjesna enzimska terapijapostala je stvarnost tek ranih devedesetih godinaprošloga stolje}a kada se dokazala sigurnom iu~inkovitom kod lije~enja Gaucherove bolestitipa 1. Sada je takva terapija mogu}a i za Fabry-jevu bolest, Pompeovu bolest i mukopolisahari-dozu tipa 1. Takvu vrst terapije trenutno prima 9bolesnika u Hrvatskoj. Budu}e mogu}nosti lije-~enja u bolesnika s lizosomskim, ali i drugim na-sljednim metaboli~kim bolestima obuhva}aju in-hibiciju sinteze supstrata, repopulaciju jetre,transplantaciju mati~nih stanica i gensku tera-piju.E-mail: [email protected]

ence of a lysosomal storage disease can often besuspected on the basis of clinical picture, a defi-nite diagnosis is usually difficult to make becauseof phenotype overlapping and therefore rests withbiochemical analysis. The ultimate goal, however,is identification of the lysosomal enzyme or pro-tein deficiency that is at the root of the disorder.Knowledge of the enzyme or protein defect is notonly necessary for definitive diagnosis but is alsoa prerequisite for prenatal diagnostic tests.Whenever possible, identification of the causativemutation by appropriate molecular genetic test-ing should be attempted not only to confirm thediagnosis but also to enable reliable heterozygotedetermination in individuals seeking geneticcounseling. The suggestion that lysosomal stor-age diseases could be treated by replacing the de-fective enzyme with its normal counterpart wasfirst posed by Christian de Duve in 1964. How-ever, it was not until the early 1990s that enzymereplacement therapy became a reality with thedemonstration of its safety and effectiveness intype 1 Gaucher disease. This therapy is nowavailable also for Fabry disease, Pompe diseaseand mucopolysaccharidosis type I. Future possi-bilities for therapy in patients with lysosomal butalso with other inherited metabolic diseases in-volve inhibition of substrate synthesis, liverrepopulation, stem cell transplantation and genetherapy.E-mail: [email protected]

S3-6

CELL CYCLE ANALYSIS OF CD133+ ANDCD133- CELLS ISOLATED FROM

UMBILICAL CORD BLOOD

B. Grškovi}, K. Ruì~ka, D. Qujeq, M.M. Müller

Institute for Laboratory Diagnosis, Kaiser Franz JosephHospital, Vienna, Austria

Umbilical cord blood cells (stem/progenitorcells) exhibit high proliferative capacities leadingto large expansion of cells in appropriate cell cul-ture conditions. The aim of this study was toevaluate by flow cytometry the cycling status ofCD133+ and CD133- cells depending on variousculture conditions, such as addition of serum,stem cell factor (SCF), interleukin 3 (IL-3) andinterleukin 6 (IL-6). An immunomagnetic systemwas used for cell separation. CD133+ and CD133-cells were seeded in Iscove’s modified Dulbecco’smedium (IMDM) with different serum concentra-tions and were stimulated with SCF (100 ng/ml),



razli~itim koncentracijama seruma te stimuliraliSCF-om (100 ng/ml), IL-3 (50 ng/ml) i IL-6 (50ng/ml). Rezultati našega istraìvanja pokazali suda se nakon izdvajanja stanica CD133+ i CD133-96,0±0,6% i 98,8±0,2% nalazi u fazi G0/G1, te2,9±0,4% i 0,6±0,05% u fazi S stani~nog ciklusa.Nakon tjedan dana uzgoja u mediju IMDM obo-ga}enom konjskim serumom (25%) utvrdili smoda je 25,0±3,5% stanica CD133+ i 30,7±1,3% sta-nica CD133- u fazi S. Kombinacijom konjskog ifetalnog tele}eg seruma (12,5%-tni svaki) uo~enoje 30,0±0,6% stanica CD133+ te 27,0±5,2% sta-nica CD133- u fazi S. Dokazali smo da se stani~niciklus stanica CD133+ ne mijenja dodatkom12,5%-tnog seruma, me|utim, ima za posljedicusmanjenje udjela stanica CD133- u fazi S stani~-nog ciklusa. Na temelju rezultata istraìvanjamoèmo zaklju~iti da podrijetlo i koncentracijaseruma potrebnog za uzgoj mati~nih stanica/prastanica ima u~inak na njihovu ekspanziju uuvjetima kulture in vitro. Obilje`ja stani~nogciklusa stanica CD133+ i CD133- me|usobno serazlikuju.E-mail: [email protected]


LDL-RECEPTOR IHIPERKOLESTEROLEMIJA

Zrinski-Topi} R.1, Gabela O.2, Serti} J.1, Floegel M.2,Stavljeni}-Rukavina A.1

1 Klini~ki zavod za laboratorijsku dijagnostiku, Medicinskifakultet Sveu~ilišta u Zagrebu i Klini~ki bolni~ki centar“Zagreb”; 2 Zavod za biokemiju i molekularnu biologiju,

Farmaceutsko-biokemijski fakultet Sveu~ili{ta u Zagrebu,Zagreb

Hiperkolesterolemija se razvija kao posljedicajednog ili istodobno više prisutnih poreme}aja prikojima se mijenja katabolizam aterogenih lipo-proteina. Obiteljska hiperkolesterolemija (FH) jemonogenska, autosomno dominantna bolest uz-rokovana mutacijama u genu za receptor kojiveè lipoprotein male gusto}e (LDL-receptor).Poznato je više od 700 razli~itih mutacija kojeutje~u na funkcijska svojstva LDL-receptora. Di-jagnoza FH temelji se na klini~kim i biokemij-skim zna~ajkama. Prehrambeni ~imbenici kao idrugi genski lokusi koji utje~u na razinu kole-sterola u krvi mogu ometati dijagnosti~ki pri-stup. Cilj je bio utvrditi postojanje i u~estalostmolekularnih promjena u genu za LDL-receptoru bolesnika s hiperkolesterolemijom. Ispitana jeskupina hiperkolesterolemi~nih osoba (N=249)izabrana prema vrijednostima za LDL-kolesterol

IL-3 (50 ng/ml) and IL-6 (50 ng/ml). Our experi-ments demonstrated that immediately upon sepa-ration 96.0±0.6% of CD133+ cells and 98.8±0.2%of CD133- cells were in the G0/G1 phase, while2.9±0.4% and 0.6±0.05% were in the S-phase, re-spectively. After one-week cultivation in IMDMsupplemented with horse serum (25%) 25.0±3.5%of CD133+ and 30.7±1.3% of CD133- cells weredetected in the S-phase. A combination of horseand fetal calf serum (12.5% each) yielded30.0±0.6% and 27.0±5.2% of CD133+ and CD133-cells in the S-phase, respectively. Serum concen-trations of 12.5% did not change cell cycle charac-teristics in CD133+ cells while resulting in lowerproportions of CD133- cells in the S-phase. Inconclusion, our data indicate that the source andconcentration of the serum used for cultivation ofstem/progenitor cells have an impact on in vitroexpansion. The cell cycle properties of CD133+and CD133- cells seem to be different.E-mail: [email protected]


LDL-RECEPTOR ANDHYPERCHOLESTEROLEMIA

R. Zrinski-Topi}1, O. Gabela2, J. Serti}1, M. Floegel2,A. Stavljeni}-Rukavina1

1 Clinical Institute of Laboratory Diagnosis, ZagrebUniversity Hospital Center, 2 Department of Biochemistry

and Molecular Biology, School of Pharmacy andBiochemistry, University of Zagreb, Zagreb

Hypercholesterolemia progresses as a conse-quence of one or more concurrent disorders whichalter atherogenic lipoprotein metabolism. Familialhypercholesterolemia (FH) is a common monogenicautosomal dominant disorder caused by mutationsin the low density lipoprotein receptor (LDL-receptor) gene. Until now, more than 700 differentmutations have been identified in the LDL-receptorgene. The diagnosis of FH is generally based onclinical and biochemical features. However, manyfactors including diet and variations at other geneloci may modulate blood cholesterol levels and af-fect the diagnostic pathway. The aim of the studywas to establish the incidence and frequency of mo-lecular changes in the LDL-receptor gene in pa-tients with hypercholesterolemia. A total of 249hypercholesterolemic patients with LDL-cholesterolabove 4.0 mmol/L were selected. The presence ofmolecular changes in the promotor region and 18



>4,0 mmol/L. Pretraìvanje promotora i svih 18eksona gena za LDL-receptor provedeno je meto-dom konformacijskog polimorfizma jednolan~aneDNK (PCR-SSCP). Otkrivene promjene u poje-dinim eksonima potvr|ene su sekvencioniranjemulomaka DNK i metodom polimorfizma duljinerestrikcijskih ulomaka (PCR-RFLP). Molekular-ne promjene u genu za LDL-receptor bile suprisutne u 68% bolesnika s hiperkolesterolemi-jom. Šest razli~itih mutacija otkriveno je u 30(12%) bolesnika. Mutacijama zahva}eni eksonikodiraju za dvije od ukupno pet funkcijskih do-mena LDL-receptorskog proteina: domenu kojaveè ligand i domenu homolognu s prekursoromepidermnog ~imbenika rasta, uloga kojega jenu`na za proces recikliranja LDL-receptora. Mu-tacija Cys127Arg (FH-Zagreb) je novootkriveniporeme}aj gena za LDL-receptor. U preostalihbolesnika bili su prisutni slijede}i polimorfizmi:81 T>C, 1413 G>A, 1773 T>C i 1959 C>T, koji nemijenjaju aminokiselinski slijed, ali se mogu o~i-tovati promijenjenom aktivnoš}u proteina. U26% bolesnika bila je prisutna kombinacija dvajuili više polimorfizama. Zaklju~eno je kako vrst iu~estalost molekularnih promjena u genu zaLDL-receptor doprinose boljem poznavanju pato-geneze hiperkolesterolemije te pomaù u iznala-ànju najpogodnijeg dijagnosti~kog pristupa zagenski uvjetovanu hiperkolesterolemiju.E-mail: [email protected]


DJELOMI^NA DUPLIKACIJA 16Q:KLINI^KO I CITOGENETSKO ISPITIVANJE

Petkovi} I., Bariši} I., Sansovi} I.

Klinika za dje~je bolesti Zagreb, Klinika za pedijatriju, Zagreb

Duplikacije dugog kraka kromosoma 16 suveoma rijetka pojava. Spoznaje o klini~kim po-sljedicama su zbog malog broja opisanih bole-snika oskudne i predmet su znanstvenog intere-sa. Prikazujemo rezultate klini~kih i citogenet-skih ispitivanja u 10-godišnje djevoj~ice. Klini~-kim pregledom otkrivena je dizmorfija i to epi-kantus, displasti~ne, nisko poloène uške, puniobrazi, tanke usnice i mikrognatija. Tjelesni ra-zvoj bio je uredan, a intelektualni grani~an. Pri-sutne su bile i poteško}e u u~enju, uglavnom kaoposljedica problema ponašanja, agresivnosti ineposlušnosti. Citogenetsko ispitivanje probandai obaju roditelja izvedeno je na pripravcima do-bivenim kratkotrajnom kulturom stanica peri-ferne krvi. Oba su roditelja imala uredan kario-


PARTIAL DUPLICATION 16q: CLINICALAND CYTOGENETIC STUDY

I. Petkovi}, I. Bariši}, I. Sansovi}

Zagreb Children’s Hospital, Zagreb

Duplication 16q is a rare condition in live-born infants and its clinical presentation has notyet been well defined. We present results of clini-cal and cytogenetic studies in a 10-year-old girl.Clinical examination showed dysmorphic traitsincluding hypoplastic supraorbital ridges, epi-canthus, dysplastic and low set ears, full cheeks,thin lips and micrognathia. Her somatic develop-ment was normal, however, she experienced learn-ing difficulties, mostly due to behavioral prob-lems, aggressiveness and disobedience. Cytoge-netic analysis was performed on peripheral bloodculture of the patient and her parents. Both par-ents presented with normal karyotype, while cy-togenetic analysis in the proband identified anextra GTG-positive band in the long arm of chro-

exons of the LDL-receptor gene was examined by themethod of single strand conformation polymor-phism (PCR-SSCP). Detected changes were con-firmed by DNA sequencing and method of restric-tion fragment length polymorphism (PCR-RFLP).Molecular changes in the LDL-receptor gene weredetected in 68% of patients with hypercholeste-rolemia. Six different mutations were demonstratedin 30 (12%) patients. Exons with mutations encodetwo of five functional domains of the LDL-receptorprotein: the ligand binding domain and a regionthat shares sequence identity to the human epider-mal growth factor. This domain mediates receptorrecycling. The Cys127Arg (FH-Zagreb) mutationhas not yet been reported. Genetic polymorphisms81 T>C, 1413 G>A, 1773 T>C and 1959 C>T weredetected in the remaining patients. These sequencechanges do not alter amino acids and are likely tobe markers of other changes causing defective pro-tein. In our group of patients, 26% had two or moregenetic polymorphisms. The type and frequency ofmolecular changes in the LDL-receptor gene werefound to provide a better insight into thepathogenesis of hypercholesterolemia; they couldalso facilitate selection of the most appropriate di-agnostic approach for genetic hypercholesterolemia.E-mail: [email protected]



tip, dok je kod djevoj~ice identificirana ve}a du-ìna dugog kraka kromosoma 16. Metodom FISHustanovljeno je da je aberantan kromosom u ci-jelosti gra|en od materijala kromosoma 16. Pre-ciznom analizom rasporeda GTG-pruga pretpo-stavljena je duplikacija regije 16q13-q22. Rezul-tati dosad provedenih ispitivanja ukazuju na toda je duplikacija distalnog dijela dugoga krakakromosoma 16 udruèna s tipi~nom klini~kommanifestacijom uklju~uju}i i kratak ìvotni vijek.Usporedba rezultata klini~kih i citogenetskih is-pitivanja naše bolesnice s dostupnim prikazima uliteraturi ukazala je na to da je trisomija proksi-malnog dijela dugoga kraka udruèna s blagomklini~kom slikom, dok su agresivnost i problemiponašanja dominantna manifestacija trisomije16q13.E-mail: [email protected]

mosome 16. FISH with chromosome 16 paintingprobe stained the aberrant chromosome com-pletely. The analysis suggested duplication of thelong arm of chromosome 16, most probably in-volving the 16q13-q22 region. It has been pro-posed that duplication of the distal long arm seg-ment may cause typical features of trisomy 16q,including short survival. On the other hand, com-parison of the findings recorded in our patient tothe cases reported elsewhere revealed that trisomyfor the proximal segment of the long arm of chro-mosome 16 may be associated with mild clinicalpresentation with behavioral problems as a majormanifestation of trisomy 16q13.E-mail: [email protected]

S4Nove tehnologije u laboratorijskoj medicini

New technologies in laboratory medicine

S4-1

“MICROARRAY” TEHNOLOGIJA KAONOVO SREDSTVO U SUVREMENOJ

LABORATORIJSKOJ DIJAGNOSTICI

Ferrari M.1,2, Foglieni B.1, Ferrari F.1, Stenirri S.1,

Cremonesi L.1

1 Unit of Genomics for Diagnosis of Human Pathologies,IRCCS Ospedale San Raffaele, i 2 Diagnostica e Ricerca San

Raffaele S.p.A., Milano, Italija

Posljednjih godina laboratorijska se medicinaubrzano mijenja s uvo|enjem visoko razvijenihtehnologija za testiranje DNK. Sadašnje metodo-logije zasnovane na proteinima zamjenjuju seanaliti~kim metodama zasnovanim na DNK/RNK za pra}enje humanih patoloških stanja. Je-dan od najva`nijih izazova je uvo|enje sustavavisokog kapaciteta kao što su ’DNK ~ipovi’ udijagnosti~ke laboratorije. Pomo}u minijaturnetest platforme tehnologije utemeljene na mikro-~ipu omogu}avaju brzu analizu genetskih poda-taka u velikim populacijskim uzorcima, ~ime seskra}uje vrijeme i ru~na obrada. Najva`nije pri-mjene bio~ipova uklju~uju identifikaciju genskeekspresije i genetske varijacije, od kojih objemogu imati utjecaja na humanu molekularnu di-

S4-1

MICROARRAY TECHNOLOGY AS A NEWTOOL IN MODERN LABORATORY

DIAGNOSIS

M. Ferrari1,2, B. Foglieni1, F. Ferrari1, S. Stenirri1,L. Cremonesi1

1 Unit of Genomics for Diagnosis of Human Pathologies,IRCCS Ospedale San Raffaele, and 2 Diagnostica e Ricerca

San Raffaele S.p.A., Milan, Italy

In recent years, laboratory medicine has beenchanging rapidly with the introduction of highlyadvanced technologies for DNA testing. Currentprotein-based methodologies are being replacedby DNA/RNA-based analytical methods formonitoring of human pathologies. One of themost important challenges is the introduction ofhigh throughput systems such as ’DNA chips’ intodiagnostic laboratories. Through a miniaturizedtest platform, microchip-based technologies allowfor rapid analysis of genetic information in largesample populations thus reducing time andmanual work. The most important biochip appli-cations include gene expression and genetic vari-ation identification, and both may have an effecton human molecular diagnosis and functional



jagnostiku i funkcionalnu genomiku, kao i naidentificiranje patogena, analizu okoliša i sudskumedicinu. Štoviše, objavljeno je kako najnovijeprimjene tehnologije mikro~ipova poboljšavajuprotokole zasnovane na proteinima: razvijeni su~ipovi protutijela i antigena kao nova sredstva zaanalizu proteoma. îpovi DNK, oligonukleotidivisoke gusto}e ili molekule cDNK u~vrš}ene zakrutu podlogu prvotno su razvijeni za ispitivanjediferencijalne genske ekspresije uz microarraycDNK za identificiranje gena biomarkera kod ra-ka i drugih bolesti. Za otkrivanje mutacija u os-novi su se rabila dva pristupa: ugradnja za alelspecifi~nog nukleotida (ekstenzija primera, eks-tenzija jednog nukleotida /SNE/ ili ekstenzija je-dne baze /SBE/) i za alel specifi~na hibridizacija.Od ovih ~ini se da primjena mikroelektronike bo-lje odgovara potrebama molekularne dijagnosti-ke. Mi smo se usredoto~ili na tehnologiju nano-gena koja pruà mogu}nosti višestruke primje-ne, kao što je analiza usidrene in situ ampli-fikacije, priprave DNK/RNK iz bakterija za pro-bir patogena, imuno testiranja, analize jedno-stavnih ponovljenih sekvenca te otkrivanje SNP/mutacije. Na osnovi ove tehnologije razradili smotestove za identificiranje nekih ~estih talijanskihmutacija gena za beta-globin što uzrokuje beta-talasemiju, u genu ABC transportera specifi~nogza mre`nicu (ABCA4) uklju~enom u Stargardto-voj bolesti, te u elementu L-feritina koji reagirana èljezo (IRE), što dovodi do nasljedne hiper-feritinemije - sindroma katarakta.

genomics as well as on pathogen identification,environmental analysis and forensic medicine.Moreover, recent applications of the microchiptechnology have been reported to improve protein-based protocols: antibody and antigen chips arebeing developed as new tools for proteome analy-sis. DNA chips, high-density oligonucleotides orcDNA molecules attached to a solid support wereinitially developed to study differential gene ex-pression with cDNA microarray for the identifica-tion of biomarker genes in cancer and other dis-eases. For mutation detection two approacheshave been used basically: allele specific nucle-otide incorporation (primer extension, single nu-cleotide extension /SNE/ or single base extension/SBE/) and allele specific hybridization. Amongthese, the use of microelectronics seems to betterfit the needs of molecular diagnosis. We have fo-cused on the Nanogen technology, which has thepotential for several applications such as analy-sis of anchored in situ amplification, preparationof DNA/RNA from bacteria for pathogen screen-ing, immunoassays, analysis of simple sequencerepeats and SNP/mutation detection. Based onthis technology, we have developed assays for theidentification of some common Italian mutationsin the beta-globin gene causing beta-thalassemia,in the retina-specific ABC transporter (ABCA4)gene involved in Stargardt disease, and in theiron responsive element (IRE) of L-ferritin lead-ing to hereditary hyperferritinemia - cataractsyndrome.

S4-2

PROTEOMIKA: ISPITIVANJE TERAPIJSKEREZISTENCIJE U STANICAMA RAKA

Sinha P.

Institut für Medizinische und Chemische Labordiagnostik,Klagenfurt, Austrija

Kemorezistencija ostaje neriješenim proble-mom u klini~koj onkologiji. Stoga je va`no iden-tificirati molekularne ~imbenike koji }e dovestido razumijevanja mehanizama rezistencije na li-jekove u stanicama raka. Na razini ekspresijeproteina to se moè u~initi pomo}u proteomikekoja je u proteklom desetlje}u dospjela u središtezanimanja i istraìvanja. Analizu proteoma naj-~eš}e se provodi kombinacijom dvodimenzijskeelektroforeze na poliakrilamid gelu (2D-PAGE) imasene spektrometrije (MS), što omogu}ava vi-soko kapacitetno identificiranje i kvantificiranjeproteina što ih izraàvaju stanice, tkiva ili orga-

S4-2

PROTEOMICS: A STUDY OF THERAPYRESISTANCE IN CANCER CELLS

P. Sinha

Institut für Medizinische und ChemischeLabordiagnostik, Klagenfurt, Austria

Chemoresistance remains an unresolved prob-lem in clinical oncology. Therefore it is importantto identify molecular factors that lead to under-standing of the mechanisms of drug resistance incancer cells. At the level of protein expression, thiscan be done using proteomics that has become thefocus of significant interest and research over thepast decade. Proteome analysis is most commonlyaccomplished by a combination of two-dimen-sional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS), and allowsfor identification and quantification of the pro-teins expressed by cells, tissues or an organism in



S4-3

TEHNIKA MIKRODIJALIZE U PRA]ENJUTKIVNIH PROMJENA KOD BOLESTI U

LJUDI

Palicka V., Zivny P., Hruba P., Fertek D., Zivna H.

Charles University, Hradec Kralove, Republika ê{ka

Mikrodijaliza je tehnika prikladna za pra}e-nje krvnog protoka i koncentracija analita uintersticijskom prostoru raznih tjelesnih tkiva.Primjenom malih tankih kapilarnih cjev~ica uve-

nizam. Model sustava za ispitivanje pojava rezi-stencije je primjena kemorezistentnih stani~nihlinija raka. Mi smo uspostavili stani~ne linije ra-ka (èluca i guštera~e, melanoma, neuroblasto-ma) koje pokazuju kemorezistentna svojstva pre-ma razli~itim lijekovima protiv raka kako bismoispitali terapijsku rezistenciju. U tom kontekstusmo razvili poseban sustav za duge imobiliziranepH gradijente (48 cm) kako bismo poboljšali re-zoluciju. U sustavu je kombiniran reproducibilnigel kalup uz pomo} kompjutorski vo|ene mjerneposude s primjenom vrlo visokih napona tijekomizoelektri~nog fokusiranja. Ovi ~imbenici omogu-}avaju znatno pove}anu snagu razdvajanja uzasve pogodnosti tehnologije IPG. Uz to je razvijenpostupak bojenja srebrom sukladan masenojspektrometriji MALDI-ToF. Uz primjenu ovogaprotokola koli~ina proteina moè se smanjiti na10 µg po gelu. Iako je bojenje srebrom oko 100puta osjetljivije od bilo koje uobi~ajene Cooma-ssie boje, postoje i neki nedostaci u svezi s iden-tificiranjem proteina. Postupak zahtijeva naj-stroù ~isto}u zbog problema one~iš}enja kera-tinom, a ~epovi gela moraju se izrezati odmahnakon bojenja. Za ispitivanje poslijetranslacij-skih modifikacija optimirali smo protokol za imu-noblot nakon izoelektri~nog fokusiranja. Blotira-nje proteina nakon frakcioniranja u imobilizira-nim pH gradijentima uvijek je izazivalo stanoviteprobleme. Opisujemo postupak s trakama IPGodljevenim na Net-Fix kao unutarnju podlogukoja je propusna za elektri~nu struju. Postupakfokusiranja moè se provesti u uobi~ajenim su-stavima IPG, npr. IPGphor (Amersham Bioscien-ces), koji omogu}avaju elektri~no potpomognuturehidraciju. Za potrebe me|unarodne znanstvenerazmjene razra|ujemo našu stranicu na Inter-netu, www.chemoresistance.net, kako bismo us-postavili nove baze podataka za tumore koje }e sesastojati od 2-D gela s identificiranim protein-skim to~kama.

a high throughput manner. A model system tostudy resistance phenomena is the use ofchemoresistant cancer cell lines. We have estab-lished cancer cell lines (gastric and pancreaticcarcinoma, melanoma, neuroblastoma) exhibit-ing chemoresistant properties against various an-ticancer drugs to study therapy resistance. In thiscontext, we have developed a special system forlong immobilized pH gradients (48 cm) to im-prove spot resolution. The system combines repro-ducible gel casting using computer-driven bu-rettes with the use of very high voltages duringisoelectric focusing. These factors enable greatlyincreased separation power with all advantagesof IPG technology. Additionally, a silver stainingprocedure compatible with MALDI-ToF massspectrometry has been developed. Using this pro-tocol, protein amount can be reduced to 100 µgper gel. While silver staining is about 100 timesmore sensitive than any common Coomassie-stain, there are also a few disadvantages in con-nection with protein identification. The procedurerequires stringent cleanliness because of keratincontamination problems and gel plugs have to beexcised directly after staining. To study post-translational modifications, we optimized a pro-tocol for immunoblotting after isoelectric focus-ing. Blotting of proteins after fractionation in im-mobilized pH gradients has always caused someproblems. We describe a procedure using IPGstrips cast on Net-Fix as an internal support thatis permeable to electric current. The focusing pro-cedure can be carried out in commonly used IPGsystems, e.g., IPGphor by Amersham Biosciences,where electrically assisted rehydration can beperformed. For international scientific exchange,we are expanding our Internet page www.chemoresistance.net to establish new tumor data-bases consisting of 2-D gels with identified pro-tein spots.

S4-3

MICRODIALYSIS TECHNIQUE IN THEMONITORING OF TISSUE CHANGES IN

HUMAN DISEASE

V. Palicka, P. Zivny, P. Hruba, D. Fertek, H. Zivna

Charles University, Medical Faculty Hradec Kralove,Czech Republic

Microdialysis is a technique appropriate formonitoring blood flow and analyte concentra-tions in interstitial space of different body tissues.



denih u tkivo moè se mjeriti krvni protok iodre|eni analiti. Sondu za mikrodijalizu ~ini ka-pilarna cjev~ica s polupropusnom membranom.Tvari iz intersticijske teku}ine prolaze kroz poreu membrani, a odabir se provodi razli~itim veli-~inama membranskih pora (obi~no do mole-kularne teìne od 20 kDa). Male molekule moguizlaziti i ulaziti, pa se mjere ~iste promjene. Nadinamiku protoka analita iz tkiva u otopinu unu-tar sonde za mikrodijalizu utje~u brojni ~imbe-nici (tj. promjene u tkivnoj koncentraciji analita,hidrostatski tlak, tkivna perfuzija, osmolalnostunutar i izvan sonde, protok otopine za mikro-dijalizu i dr.). Uza standardizaciju ovih uvjetamikrodijaliza bi se mogla primjenjivati za: mje-renje promjena tkivnog protoka krvi uz dodatakpravog biljega protoka u perfuzijsku otopinu;mjerenje koncentracija specifi~nih analita u tkiv-nom intersticiju - naj~eš}e mlije~ne kiseline, glu-koze, glicerola i ureje; mjerenje tkivne koncen-tracije lijekova; usporedbu rezultata mjerenjakoncentracije u tkivu i sistemskim teku}inama;predvi|anje promjena sistemske cirkulacije; us-poredbu tkivnih promjena s razvojem bolesti iprognozom za bolesnika. Zasad je mikrodijalizametoda koja obe}ava, koja je minimalno inva-zivna i moè se bez problema rabiti ~ak i zapra}enje promjena u ~vrstim organima (jetra,bubrezi te tkivo središnjeg ìv~anog sustava).Objavljena su neka iskustva u podru~ju pro-mjena krvotoka u jetri štakora nakon djelomi~nehepatektomije ili nefrektomije. Mikrodijaliza mo-è osigurati dodatne podatke o tkivnom meta-bolizmu u bolesnika koji se podvrgavaju operacijina otvorenom srcu te o razvoju šoka. Mikrodi-jaliza je tehnika koja obe}ava i otvara nova po-dru~ja u biokemiji tkiva.E-mail: [email protected]

By using small, thin capillary tubes inserted intothe tissue, one can measure blood flow and par-ticular analytes. Microdialysis probe is a capil-lary tube with semipermeable membrane. Sub-stances from the interstitial fluid diffuse acrosspores in the membrane. Selection is made by dif-ferent size of the membrane pores (usually up to amolecular weight of 20 kDa). Small moleculescan pass in and out, and net changes are meas-ured. The dynamics of the analyte tissue-to-solu-tion flow within the microdialysis probe is influ-enced by numerous factors (i.e. changes in theanalyte tissue concentration, hydrostatic pres-sure, tissue perfusion, osmolality both within andbeyond the probe, flow rate of microdialysis solu-tion, etc.). By standardization of these conditions,microdialysis could be used to: measure changesin tissue blood flow using proper flow markeradded to the perfusion fluid; measure concentra-tions of specific analytes in tissue interstitium,with lactic acid, glucose, glycerol and urea beingthe most common analytes; measure drug concen-trations in the tissue; compare results of tissueand systemic fluid concentrations; predictchanges in systemic circulation; and compare tis-sue changes with the disease development andprognosis of the patient. Up to now, microdialysisis a promising method, which is minimally inva-sive and can be used without problems even formonitoring changes in solid organs (liver, kidney,and central nervous tissue). Some experienceshave been published in the field of blood flowchanges in rat liver after partial hepatectomy ornephrectomy. Microdialysis can add informationon tissue metabolism in patients undergoingopen-heart surgery and shock development.Microdialysis is a promising technique whichopens new fields in tissue biochemistry.E-mail: [email protected]

S4-4

GLIKANSKE STRUKTURE - IZVOR NOVIHINFORMACIJA U DIJAGNOSTICI

Dumi} J., Flögel M.

Zavod za kemiju i molekularnu biologiju, Farmaceutsko-biokemijski fakultet Sveu~ili{ta u Zagrebu, Zagreb

Glikozilacija je najzastupljenija posttransla-cijska modifikacija, a glikokonjugati igraju neza-mjenjivu ulogu u brojnim biološkim procesima,uklju~uju}i oplodnju, imune reakcije, replikacijuvirusa, infekciju mikroorganizmima, stani~nirast, adheziju i upalu. Ipak, glikanske su se

S4-4

GLYCAN STRUCTURES - A SOURCE OFNEW DIAGNOSTIC INFORMATION

J. Dumi}, M. Flögel

Department of Biochemistry and Molecular Biology, School ofPharmacy and Biochemistry, University of Zagreb, Zagreb

Glycosylation is the most abundant post-translational modification. The importance ofglycoconjugates for many biological processes in-cluding fertilization, immune defense, viral repli-cations, microbial infection, cell growth, adhesionand inflammation is well documented, yet glycan



strukture još donedavno rijetko rabile kao izvorinformacija u dijagnostici, primarno zbog golemeindividualne varijabilnosti glikana (mikrohete-rogenosti) te zbog nedostatka pristupa~nih, lakoizvedivih analiti~kih metoda. Unato~ poteško}a-ma, analiza se glikana pokazala klju~nom za di-jagnostiku nasljednih bolesti uzrokovanih pore-me}ajem metabolizma ugljikohidrata, primjericeTay-Sachsove bolesti, Gaucherove bolesti, konge-nitalne miši}ne distrofije tipa Fukuyama te kon-genitalnih poreme}aja glikozilacije. No, u po-sljednje vrijeme brojni dokazi o povezanosti spe-cifi~nih glikozilacijskih promjena i ste~enih bo-lesti, kao što su dijabetes, alergije, tumori, auto-imune, kardiovaskularne i neurološke bolesti,potaknuli su silan razvoj analiti~kih alatka i po-stupaka za analizu glikanskih struktura (MALDI-MS, HPAEC, FACE). Razli~itost individualnih’glikanskih profila’ silno se odraàva u fiziološkojraznolikosti, pa }e za individualizaciju terapijebiti nu`no identificirati glikanske biomarkere ko-ji su jedinstveni za odre|enu populaciju bolesni-ka. ’Glikanski profili’ biti }e pokazatelji individu-alnih odgovora na razli~ite lijekove u smislu nji-hove u~inkovitosti ili toksi~nosti (farmakogliko-mika). Tehnologija koja najviše obe}ava je GlycoChip® array-mikrozrake kompleksnih ugljikohi-drata, kojom je mogu}e pouzdano analizirati in-terakcije proteina i glikanskih struktura. Mnogesu bolesti, uz promjene glikana, povezane i spromjenama lektina, fizioloških receptora gliko-konjugata. U našem su laboratoriju osmišljene isintetizirane nove analiti~ke alatke Glycopro-bes®, pomo}u kojih je prvi puta mogu}e odreditilektinsku aktivnost u kompleksnim biološkimuzorcima. Odre|ivanje lektinske aktivnosti pru-à zna~ajno preciznije i vrijednije podatke odmjerenja same koli~ine lektina. Poznavanje spe-cifi~nih promjena glikozilacije i lektinskih aktiv-nosti u skoroj }e budu}nosti postati nezaobilazankorak za dijagnozu, prognozu i lije~enje mnogihbolesti.E-mail: [email protected]

structures were until recently seldom used to pro-vide diagnostic information, mainly because oftwo reasons: enormous individual glycan vari-ability (microheterogeneity) and lack of userfriendly analytical methods. In spite of difficul-ties, the analysis of glycans has proved to be es-sential in diagnosing hereditary disorders basedon defective carbohydrate metabolism, e.g., Tay-Sachs disease, Gaucher disease, muscle-eye-braindisease, Fukuyama-type congenital musculardystrophy (FCMD) and congenital disorders ofglycosylation (CDG). However, tremendous en-hancement of analytical tools and procedures forglycan analysis (MALDI-MS, HPAEC, FACE) hasbeen initiated by the growing evidence that manyacquired diseases (e.g., cancer, diabetes, allergies,autoimmune, cardiovascular, neurologic and gas-tric disorders) could also be correlated to specificglycosylation changes, and that such changescould either cause the pathology or be a conse-quence of the disease. The variability of indi-vidual glycan profiles is reflected in physiologicaldiversity. Thus, individualized therapy will re-quire identified glycan biomarkers, unique to spe-cific patient populations. ’Glycomic profiles’ willbe indicative of individual responses to differentdrugs in terms of efficacy and toxicity (pharmaco-glycomics). The most promising relevant tool isGlycoChip® technology - a microarray of complexcarbohydrates, allowing for accurate and efficientanalysis of protein-glycan interactions. Not onlyglycans but also their specific physiologicalreceptors - lectins are affected in many diseases.Glycoprobes® have been designed and synthe-sized in our lab as a valuable new tool to monitorlectin activity in complex biological samples. Lec-tin activities provide substantially more accurateand informative data than the measurements oflectin content. Knowing the specific changes ofglycosylation and lectin activities will become in-dispensable for the diagnosis, prognosis andtreatment of many diseases in the near future.E-mail: [email protected]

S4-5

SMJERNICE STARD ZA PRIKAZ REZULTATAANALIZE DIJAGNOSTI^KE TO^NOSTI

Šimundi} A.M.

Klini~ki zavod za kemiju, Klini~ka bolnica “Sestremilosrdnice”, Zagreb

Tijekom proteklih desetak godina znakovito jeporastao broj radova objavljenih u citiranim pu-blikacijama koji sadrè sve sloèniju statisti~ku

S4-5

THE STARD INITIATIVE FOR REPORTINGSTUDIES OF DIAGNOSTIC ACCURACY

A.M. Šimundi}

Clinical Institute of Chemistry, Sestre milosrdniceUniversity Hospital, Zagreb

A significant increase in the number of paperswith statistical methods of high complexity hasbeen observed in widely cited journals over the



analizu. Usporedno s porastom broja objavljenihradova pojavila se i potreba standardiziranjauvjeta izvo|enja istraìvanja i objavljivanja re-zultata. Osnovni cilj takve standardizacije obu-hva}a razumljivost, usporedivost i ponovljivostsvakog znanstvenog rada. Do danas su objavljenebrojne smjernice i preporuke za mnoge granebiomedicinskog istraìvanja: za izvještavanjerandomiziranog klini~kog pokusa (CONSORTStatement), za provo|enje epidemiološke analize,za statisti~ku analizu u medicinskim publika-cijama, za provo|enje i objavljivanje rezultatamulticentri~nih studija, za objavljivanje rezul-tata ispitivanja iz podru~ja psihologije, onkolo-gije, molekularne biologije i dr. Osobito zanim-ljiva za medicinske biokemi~are je nedavna po-java smjernica za potpuno i to~no izvještavanjerezultata analiza dijagnosti~ke to~nosti (TheStandards for Reporting of Diagnostic Accuracy,inicijativa STARD). Dijagnosti~ka to~nost je po-jam koji opisuje potencijal nekog dijagnosti~kogpostupka (naj~eš}e laboratorijskog testa) da raz-lu~i izme|u dvaju stanja. Naj~eš}e se radi o izbo-ru izme|u zdravlja i bolesti, no u obzir dolazi isvaki drugi izbor izme|u dvaju stanja. Metodo-logija analize dijagnosti~ke to~nosti laboratorij-skog testa obuhva}a ispitivanje njegove klini~keosjetljivosti i specifi~nosti, prediktivnih vrijed-nosti, u~inkovitosti te ROC analizu, a dobivenerezultate trebalo bi izvještavati poštuju}i smjer-nice inicijative STARD. Inicijativu STARD su pri-hvatili urednici mnogih ~asopisa i tendencija jeda one postanu standard, te da njihovo poštiva-nje bude obveza autora. Ovo }e predavanje datipregled smjernica STARD i njihovo tuma~enje.E-mail: [email protected]

past decade. This phenomenon has generated apush towards standardizing the design, perform-ance and reporting of research studies. The maingoals of such standardization are comprehensive-ness, comparability and reproducibility of thestudy. Many guidelines and recommendations fora variety of scientific fields in biomedicine havebeen published to date: CONSORT statement forpublishing results of controlled clinical trials,guidelines for epidemiologic studies, for statisti-cal reporting in articles intended for medicaljournals, for multicenter trials as well as for tri-als in psychology, oncology, molecular biology,etc. Of special interest to the readers in the field ofclinical chemistry is a recent publication ofstandards for complete and accurate reporting ofstudies of diagnostic accuracy (The Standards forReporting of Diagnostic Accuracy, STARD Initia-tive). Diagnostic accuracy refers to the potentialof a diagnostic procedure (most commonly labo-ratory test) to discriminate between two condi-tions. Most commonly it is the choice betweenhealth and disease, however, any other dichoto-mous choice is possible. Diagnostic accuracy of alaboratory procedure can be analyzed and ex-pressed in a number of ways, including clinicalsensitivity, specificity, predictive values, efficacyand ROC analysis. Results should be reportedfollowing the guidelines of STARD Initiative. TheInitiative has already been adopted by many edi-torial boards. The tendency is to make them astandard mandatory for authors. This lectureaims to explain and highlight the most importantparts of the STARD Initiative.E-mail: [email protected]


DOKAZIVANJE HERPES SIMPLEXVIRUSA TEHNIKOM PCR U STVARNOM

VREMENU

Kozi} S., Vince A., I{~i}-Be{ J., Ba}a-Vrakela I.,Kutela N.

Klinika za infektivne bolesti “Dr. Fran Mihaljevi}”,Zagreb

Herpes simpleks virus (HSV) tipa 1 i 2 uzro-kuje niz klini~kih simptoma središnjeg ìv~anogsustava (S@S). HSV infekcija u imunokompro-mitiranih osoba i novoro|en~adi moè dovesti doozbiljnih komplikacija sa smrtnim ishodom, akose terapija ne primijeni na vrijeme. Referentnametoda za dokazivanje HSV infekcije S@S-a je


DETECTION OF HERPES SIMPLEX VIRUSBY REAL-TIME PCR

S. Kozi}, A. Vince, J. I{~i}-Be{, I. Ba}a-Vrakela, N.Kutela

Dr. Fran Mihaljevi} University Hospital for InfectiousDiseases, Zagreb

Herpes simplex virus (HSV) types 1 and 2cause a variety of clinical symptoms in the cen-tral nervous system (CNS). HSV infection oftenoccurs in immunocompromised patients andneonates leading to serious complications anddeath unless treated on time. Molecular detectionof HSV DNA has become a reference standard as-say for CNS infections caused by HSV. The aim of



molekularno otkrivanje HSV DNK. Cilj rada bio jeuvo|enje osjetljivog, specifi~nog i brzog labora-torijskog testa za otkrivanje HSV DNK, te razli-kovanje HSV 1 i HSV 2 tipa virusa. Analiziranoje 50 likvora uzetih od bolesnika s encefalitisomili meningoencefalitisom. DNK je izolirana iz li-kvora brzom ekstrakcijskom tehnikom. OtkrivanjeHSV DNK izvedeno je pomo}u ure|aja LightCycler (LC) koji omogu}ava otkrivanje PCR pro-dukata u stvarnom vremenu. Otkrivanje se osnivana na~elu prijenosa energije fluorescentne rezo-nance (FRET, fluorescence resonance energytransfer). Dodatna analiza krivulje topljivosti(melting curve) omogu}ava razlikovanje HSV 1 iHSV 2 tipa virusa. Kontrola kvalitete rada osi-gurana je uporabom unutarnjih kontrola, te ne-gativnih i pozitivnih kontrola tijekom svakeanalize. Od ukupno 50 uzoraka likvora dokazalismo HSV 1 tip virusa u 4 uzorka. Dva pozitivnauzorka pripadala su novoro|en~adi, dok su dvabila uzeta od odraslih osoba. Niti jedan uzoraknije bio pozitivan na HSV 2 tip virusa. PCR ustvarnom vremenu je osjetljiva, specifi~na i brzametoda za otkrivanje HSV DNK u likvoru. S ob-zirom na to da se ovakva analiza moè izvesti zamanje od dva sata nakon punktiranja, u slu~ajupozitivnog nalaza moè se dovoljno rano primi-jeniti terapija.E-mail: [email protected]

the study was to introduce a sensitive, specificand rapid laboratory test for HSV DNA detectionand differentiation of HSV 1 and HSV 2. A totalof 50 cerebrospinal fluid (CSF) samples obtainedfrom patients with encephalitis or meningoen-cephalitis were analyzed. DNA was extractedfrom CSF by the rapid extraction protocol. HSVDNA detection was performed by a newly devel-oped LightCycler (LC) instrument that enablesreal-time detection of PCR products (continuousmonitoring of amplicon development). Detectionwas based on the fluorescence resonance energytransfer (FRET) principle. Melting curve analysisenabled the HSV 1 and HSV 2 strains to distin-guish. Quality control was assured by using in-ternal controls (IC), and negative and positivecontrols. Using LC PCR we detected 4 HSV 1positive samples out of 50, two from newbornsand adults each. None of the samples was HSV 2positive. Real-time PCR is a sensitive, specificand rapid method for HSV DNA detection in CSFsamples. Analysis can be done in less than twohours, which is essential for early therapy initia-tion.E-mail: [email protected]

S5Laboratorijska medicina temeljena na dokazima

Evidence based laboratory medicine

S5-1

BOLESTI DVADESETOGA STOLJE]A:PROBLEMI DVADESETPRVOGA

STOLJE]A?

Oreškovi} S.

Škola narodnog zdravlja “Andrija Štampar”, Zagreb

Ne moèmo ne ~uditi se nad skokovitim na-pretkom medicinskih tehnologija od 1900. godinedo današnjega dana. Naši se djedovi još sje}ajuku}nih posjeta negdašnjih lije~nika. Glavni in-strumenti koje su lije~nici toga doba imali na raspo-laganju bili su znanje i dosjetljivost te nekolikoosnovnih instrumenata ili bo~ica koje bi lije~nikuvijek nosio sa sobom. Danas ljudi ìve u pro-sjeku 30 godina duè od svojih predaka s po~etka

S5-1

TWENTIETH CENTURY DISEASES:TWENTY-FIRST CENTURY PROBLEMS?

S. Oreškovi}

“Andrija [tampar” School of Public Health, Zagreb

One cannot help but wonder at the leap ofprogress made in medical technologies from 1900up to the present day. Our grandparents still re-member home visits made by old time doctors.The main instruments that a doctor disposed ofat that time were knowledge and quick-wittedness as well as several basic instruments orvials which a doctor would always carry withhim. Nowadays people live on an average 30years longer than their ancestors at the beginning



20. stolje}a. Na cijeli taj proces utjecali su mno-gobrojni ~imbenici, poglavito razvoj industrijein`injeringa, ~ime su otvorene mogu}nosti zaneprocjenjive i krajnje slo`ene inovacije u me-dicini. Me|utim, tradicionalni pristupi su ozbilj-no podcjenjivali teret mentalnih bolesti kao štosu depresija, ovisnost o alkoholu i shizofrenija,vode}i ra~una samo o smrtnosti, ali ne i inva-lidnosti. Projekcije za budu}nost pokazuju kakobi se 2020. godine udio psihijatrijskih i neuro-loških stanja u sveukupnom optere}enju mogaopove}ati za gotovo polovicu, s 10,5% ukupnogaoptere}enja na skoro 15%. To je ve}i propor-cionalni porast negoli onaj za kardiovaskularnebolesti. O~ekuje se da }e do 2020. godine opte-re}enje bolestima uzrokovanim prenosivim, ma-terinskim i perinatalnim stanjima te prehram-benim nedostacima pasti na petinu sveukupnogoptere}enja. Isto se tako o~ekuje da }e do 2020.godine optere}enje bolestima koje se mogu pri-pisati duhanu nadmašiti ono izazvano bilo kojompojedinom boleš}u. O~ekuje se da }e udio duhanaporasti s razine od 2,6% sveukupnog svjetskogoptere}enja bolestima 1990. godine na tek neštoispod 9% ukupnog optere}enja u 2020. godini, uusporedbi s malo ispod 6% za ishemijsku bolestsrca, za koju se pretpostavlja da }e biti vode}abolest u svijetu. Duhan i psihijatrijske bolestinisu samo svjetski, nego isto tako nacionalni iregionalni hitan zdravstveni problem s kojim }ese tek morati suo~iti mnoge vlade, pa tako i vladaRepublike Hrvatske.

of the 20th century. Numerous factors influencedthe whole process, especially the development ofthe engineering industry, which opened up oppor-tunities for invaluable and extremely complex in-novations in medicine. However, the burdens ofmental illnesses, such as depression, alcohol de-pendence and schizophrenia, have been seriouslyunderestimated by traditional approaches thattake account only of deaths and not disability.The projections show that psychiatric and neuro-logic conditions could increase their share in thetotal global burden by almost half, from 10.5 percent of the total burden to almost 15 per cent in2020. This is a bigger proportionate increase thanthat for cardiovascular diseases. By 2020, the dis-ease burden due to communicable, maternal andperinatal conditions and nutritional deficienciesis expected to fall to a fifth of the total. Also, by2020, the burden of disease attributable to to-bacco is expected to outweigh that caused by anysingle disease. From its 1990 level of 2.6 per centof all disease burden worldwide, tobacco is ex-pected to increase its share to just under 9 percent of the total burden in 2020, compared withjust under 6 per cent for ischemic heart disease,the leading projected disease. Tobacco andpsyciatric disorders are not only global, but alsonational and regional health emergency thatmany governments, including the Government ofCroatia, have yet to confront.

Table. Murray C, Lopez A. Global burden of disease. Harvard University Press, 1999.



S5-2

LABORATORIJSKA MEDICINATEMELJENA NA DOKAZIMA - UVODNA

RIJE^

Rogi} D.

Klini~ki zavod za laboratorijsku dijagnostiku Klini~kogbolni~kog centra “Zagreb” i Medicinskog fakulteta

Sveu~ili{ta u Zagrebu, Zagreb

Jednostavna proizvodnja monoklonskih pro-tutijela, automatizacija svih klasi~nih grana la-boratorijske medicine, proteomika, nanotehno-logije... to su samo neke od pokreta~kih sila kojestoje iza brzog porasta broja i vrsta laborato-rijskih pretraga kojima smo svjedoci u današnjevrijeme. Laboratorijske pretrage naru~uju se sasvrhom donošenja ili isklju~enja dijagnoze, pro-cjene prognoze, ali i zbog toga “što se kod tihbolesnika te pretrage uvijek rade”. Dijagnosti~kestudije koje se bave u~inkovitoš}u i stvarnomkorisnoš}u odre|ene laboratorijske pretrage naj-~eš}e su nedovoljno dobro dizajnirane i zbog togamogu stvoriti krivi dojam o stvarnom doprinosuodre|ene pretrage u klini~kom zbrinjavanju bo-lesnika. Posljedi~no, sustavni pregledi dijagno-sti~kih studija tako|er uglavnom sa`imaju po-grešne rezultate, pa su i zaklju~ci takvih studijanerijetko krivo usmjereni. Strogi proces pro-cjenjivanja dijagnosti~kih pretraga prije uvo|e-nja u klini~ku praksu ne samo da bi sprije~iopogrešne zaklju~ke glede dijagnostike i lije~enja,nego bi smanjio i cijenu zdravstvene skrbi bri-sanjem nepotrebnih pretraga. Optimalan dizajnstudije podrazumijeva usporedbu ishoda u ran-domiziranoj skupini bolesnika u kojih je na-pravljena pretraga koja se ispituje naspram onihu kojih je u~injena standardna pretraga ili nijeu~injena nikakva pretraga. Gotovo jedini primjertakvog pristupa dijagnosti~kim pretragama jesustudije koje uspore|uju ishod (duljina boravka ubolnici, smrtnost) u bolesnika kojima je u okviruobrade kod prijma zbog boli u prsima u~injentroponin I. naspram skupine bolesnika kojima suu~injene standardne enzimske pretrage. U tomsmislu se sustavnim pristupom i radom na di-zajnu dijagnosti~kih studija otvaraju vrata pre-ma dobivanju pravih informacija o dometima iograni~enjima pojedine dijagnosti~ke pretrage ukontekstu sumnje na odre|enu bolest. Na taj sena~in ne poboljšava samo kvaliteta skrbi za bole-snika, nego se isto tako zaustavlja zna~ajan anepotreban odljev financijskih sredstava unutarzdravstvenog sustava.E-mail: [email protected]

S5-2

EVIDENCE-BASED LABORATORYMEDICINE - INTRODUCTORY REMARKS

D. Rogi}


The ease of monoclonal antibody production,automation in all classic fields of laboratorymedicine, proteomics, nanotechnologies ... theseare just some of the driving forces behind therapid expansion in the number and variety oflaboratory tests being performed nowadays.Laboratory tests are generally ordered to confirmor exclude a dignosis, to estimate prognosis, butalso because “we always order these tests in suchpatients”. Diagnostic studies dealing with the ef-ficacy and real utility of a certain laboratory testfrequently are quite inappropriately designed,thus generating a wrong impression on the clini-cal utility of the test in question. Consequently,systematic reviews of diagnostic studies just sum-marize wrong results leading to wrong conclu-sions. A process of strict evaluation before a labo-ratory test enters the clinical arena should elimi-nate such events and at the same time save sub-stantial health resources. An optimal study de-sign consists of outcome comparison between tworandomized groups of patients, i.e. one undergo-ing the diagnostic test evaluated, and the otherundergoing standard protocol (another test or notesting at all). Some of the rare examples of suchstudies are those on troponin I testing performedin chest pain patients and evaluated according tothe length of stay and mortality. Correct diagnos-tic study design is the only scenario to obtain rel-evant information on the discriminating power ofa laboratory test within a specific disease context.Such approach not only increases the quality ofpatient care, but also improves the cost-efficiencyof health services.E-mail: [email protected]



S5-3

OSNIVANJE PODRU@NICE COCHRANECOLLABORATION U HRVATSKOJ

Mastilica M.

Katedra za medicinsku sociologiju i ekonomiku zdravstva,Škola narodnog zdravlja “Andrija Štampar” Medicinskog

fakulteta Sveu~ilišta u Zagrebu, Zagreb

Suvremena medicina kao znanost i umije}elije~enja uspješnost svoje prakse nu`no temeljina pra}enju i primjeni rezultata znanstvenih me-dicinskih istraìvanja. Razvoj medicinske prakseutemeljene na znanstvenim dokazima (evidencebased medicine) danas je op}enito prihva}en kaozna~ajan pokazatelj kvalitete zdravstvene zašti-te. Problem je što se medicinsko znanje tako brzomijenja i pove}ava da sama mogu}nost pra}enjanovih znanstvenih spoznaja postaje od presudneva`nosti za u~inkovitost rada lije~nika prakti~a-ra, ali i medicinskih znanstvenika i zdravstvenihplanera. Lije~nici se danas suo~avaju sa sve ve-}im obiljem informacija koje proizlaze iz oko30.000 biomedicinskih znanstvenih ~asopisa i17.000 novih biomedicinskih knjiga izdanih nagodinu. Archie Cochrane (1900.-1988.), engleskiepidemiolog, ve} je 1970-ih godina upozorio napotrebu ’kriti~kih saètaka’ svih relevantnih re-zultata klini~kih istraìvanja, podijeljenih premaspecijalnostima i dostupnih svima kako bi seomogu}ilo uspješnije i znanstveno utemeljeno do-nošenje medicinskih odluka, na što je 1993. go-dine osnovana Cochrane Collaboration, me|una-rodna znanstvena organizacija dobrovoljno okup-ljenih medicinskih stru~njaka (oko 5500 iz cijelo-ga svijeta), koja ima za svrhu promicanje zdrav-stvenog odlu~ivanja putem pripremanja, prikup-ljanja i osiguravanja dostupnosti sustavnih znan-stvenih pregleda o u~incima zdravstvenih inter-vencija. Nastojanje Cochrane Collaboration jeu~initi svakom dostupnim najnovije i pouzdaneznanstvene dokaze/informacije o medicinskimu~incima u prevenciji, lije~enju ili rehabilitacijiodre|enog zdravstvenog problema ili skupineproblema. Sustavni znanstveni pregledi Cochra-ne kao temeljni proizvodi Cochrane Collabora-tion predstavljaju sinteze rezultata znanstvenihistraìvanja (randomiziranih kontroliranih kli-ni~kih pokusa i drugih istraìvanja) naj~eš}e ra-|ene metodom meta-analize, a objavljuju se uelektronskom obliku (u obliku CD-ROM-a ili naInternetu) u Cochrane Database of SystematicReviews, što zajedno s bazom podataka rando-miziranih klini~kih pokusa ~ini glavni dio Coc-hrane Library (1147 sustavnih znanstvenih pre-gleda i 311.024 klini~ka pokusa u izdanju 2001.).U Hrvatskoj je skupina nastavnika pri Medicin-skom fakultetu u Zagrebu, Školi narodnog zdrav-

S5-3

ESTABLISHMENT OF A COCHRANECOLLABORATION BRANCH IN CROATIA

M. Mastilica

Department of Medical Sociology and Health Economics,Andrija Štampar School of Public Health, Zagreb

University School of Medicine, Zagreb

In modern medicine as a science and treat-ment skill, the efficiency of practice is inevitablybased on the follow-up and implementation of thescientific medical research results. The develop-ment of evidence based medicine has currentlybeen generally accepted as a significant indicatorof health care quality. The problem is that medi-cal knowledge changes and increases so quicklythat the very opportunity of following novel scien-tific concepts has become crucial for the efficiencyof medical practitioners as well as of medical sci-entists and medical planning officers. At present,the physicians face an ever growing body of infor-mation deriving from some 30,000 biomedical sci-entific periodicals and 17,000 new biomedicinebooks published a year. As early as the 1970s,Archie Cochrane (1900-1988), an English epide-miologist, pointed to the need of ’critical ab-stracts’ of all relevant results of clinical studies,categorized according to specialties and generallyavailable, thus to allow for more efficient and sci-entifically based decision making in medicine.This initiative resulted in the establishment ofCochrane Collaboration, an international scien-tific organization of medical professionals gath-ered on a voluntary basis (around 5500 from allover the world), with the aim to promote medicaldecision making through preparation, collectionand ensuring availability of systematic scientificsurveys on medical intervention effects. The pur-pose of Cochrane Collaboration is to make the lat-est and reliable scientific evidence/informationon medical effects in the prevention, treatmentand rehabilitation of a particular health problemor group of problems widely available. The sys-tematic Cochrane scientific surveys as the mainCochrane Collaboration products provide a syn-thesis of research results (randomized controlledclinical trials and other studies), mostly per-formed by the method of meta-analysis and pub-lished in the form of electronic data (CD-ROM oron the Internet) in the Cochrane Database of Sys-tematic Reviews which, along with the databaseon randomized clinical trials make the mainbody of Cochrane Library (1147 systematic scien-tific reviews and 311,024 clinical trials in the2001 issue). A group of the School of Medicineand Andrija Štampar School of Public Healthfaculty and expert clinicians in collaborationwith the Ministry of Health have launched activi-



S5-4

LABORATORIJSKA MEDICINATEMELJENA NA DOKAZIMA I ŠE]ERNA

BOLEST

Sandberg S.1, Horvath A.R.2

1 Haukeland University Hospital, Bergen, Norve{ka i2 University of Szeged, Szeged, Ma|arska

Uporaba pretraga u laboratorijskoj medicini ustalnom je porastu. Stalno se uvode nove pre-trage, dok se ’stare’ pretrage rijetko ukidaju. Tobi, zajedno s ograni~enim javnim sredstvima zazdravstvo, trebalo predstavljati izazov za stru~-njake iz laboratorijske medicine da pribave do-kaze za iskorištenje razli~itih pretraga. Kako bi-smo mi kao struka potkrijepili vrijednost i u~in-kovitost klini~kih odluka te široko obznanili re-zultate prakse utemeljene na dokazima, trebamoosigurati slijede}e: 1) dijagnosti~ki to~na primar-na istra`ivanja visoke kvalitete; 2) sustavne vi-soko kvalitetne preglede klini~ke korisnosti di-jagnosti~kih pretraga; 3) razvoj i provedbu smjer-nica zasnovanih na dokazima za primjenu dijag-nosti~kih pretraga; IFCC je odlu~io usredoto~iti

lja “Andrija Štampar” i stru~njaka iz klini~ke me-dicine u suradnji s Ministarstvom zdravstva po-krenula inicijativu za razvoj aktivnosti CochraneCollaboration u našoj sredini. Aktivnosti bi se uprvom redu ogledale u širenju dostupnosti i upo-rabi elektronske Cochrane baze sustavnih znan-stvenih pregleda u odlu~ivanju u medicinskojpraksi, što bi bio zna~ajan doprinos njenoj u~in-kovitosti i kvaliteti. Zasad je baza podatakaCochrane dostupna korisnicima Ovida, me|utim,svaka bi zdravstvena ustanova (bolnica, domzdravlja) trebala imati mogu}nost pristupa ovojbazi klini~kih smjernica. Druga razina aktivnostibila bi okupljanje zainteresiranih stru~njaka izsvih specijalnosti koji bi bili registrirani surad-nici Cochrane Collaboration i radili na razvojusustavnih znanstvenih pregleda klini~kih istra-`ivanja na svom podru~ju. Ovim putem tako|erpozivamo sve zainteresirane da nam se jave. Ko-na~an cilj tih nastojanja bio bi osnivanje Hrvat-skoga Cochrane Collboration centra koji bi bio upravom smislu središnjica za širenje dostupnostii izradu medicinskih smjernica utemeljenih naznanstvenim pokazateljima. U svijetu se sustav-ni znanstveni pregledi-smjernice Cochrane sveviše prepoznaju kao najbolji izvor znanstveno do-kazane medicinske prakse i nadamo se da }e i uHrvatskoj doprinijeti boljoj, kvalitetnojoj i u~in-kovitijoj zdravstvenoj zaštiti. Sve dodatne infor-macije o Cochrane Collaboration mogu se na}i naInternet stranici www.cochrane.de.E-mail: [email protected]

ties aimed at the development of Cochrane Col-laboration activities in Croatia. These activitieswill be primarily focused on extending the avail-ability and utilization of the Cochrane databaseof systematic scientific reviews in the Croatianmedical practice and decision making, whichshould greatly improve their quality and effi-ciency. For the time being, the Cochrane databasehas been available to Ovid users, however, theidea is that every health institution (hospital,medical center) should have access to this data-base of clinical guidelines. The anticipated activi-ties should also include assembling of interestedmedical specialists who will be registeredCochrane Collaboration collaborators and workon the development of systematic scientific re-views of clinical studies in the respective field ofmedicine. This is an opportunity to invite allthose interested to contact us. The ultimate goal isthe foundation of the Croatian Cochrane Collabo-ration Center, which will function as a genuinecenter for the development and dissemination ofscientific evidence based medical guidelines. TheCochrane systematic scientific reviews - guide-lines have been ever more recognized worldwideas the supreme source of evidence based medicalpractice, and we do hope that they will contributeto better, upgraded and efficient health care alsoin Croatia. Any additional information on Coch-rane Collaboration is available at the websitewww.cochrane.de.E-mail: [email protected]

S5-4

EVIDENCE BASED LABORATORYMEDICINE AND DIABETES MELLITUS

S. Sandberg1, A.R. Horvath2

1 Haukeland University Hospital, Bergen, Norway, and2 University of Szeged, Szeged, Hungary

In laboratory medicine, the use of tests is onan increase. New tests are constantly introducedby enthusiasts, whereas ’old’ tests are seldom re-moved from the repertoire. This, together withlimited public funds for health care, should un-derline the challenge for laboratory medicine pro-fessionals to provide evidence for the utility of dif-ferent tests. Therefore, in order to support the va-lidity and effectiveness of clinical decisions, andto disseminate the results of evidence-based prac-tice, we as a profession need to provide: 1) goodquality primary research of diagnostic accuracy;2) good quality systematic reviews on the clinicalusefulness of diagnostic tests; and 3) developmentand implementation of evidence-based guidelineson the use of diagnostic tests; the IFCC has de-



se na temu Laboratorij i še}erna bolest, te je utom smislu uspostavio radnu grupu. Ova }e Rad-na grupa pribaviti znanstvene dokaze o tomekako dijagnosticirati i pratiti še}ernu bolest po-mo}u laboratorijskih pokazatelja, te }e ove doka-ze upotrijebiti u stru~noj izobrazbi. To~nije, Rad-na }e grupa provesti slijede}e: a) ispitati najboljudijagnosti~ku strategiju za dijagnosticiranje še-}erne bolesti; b) pregledati i ispitati sadašnjuuporabu laboratorijskih pretraga, te obrazovatilije~nike i bolesnike u tuma~enju laboratorijskihpretraga koje se primjenjuju u dijagnosticiranju ipra}enju še}erne bolesti; c) pru`iti kvalitetnuspecifikaciju za analite koji se rabe u dijagno-sticiranju i pra}enju še}erne bolesti; d) dati pre-poruke za minimalne kriterije pri uporabi gluko-metara; i e) nastojati se povezati s drugim grupa-ma ili pojedincima (uklju~uju}i proizvo|a~e) koji}e sura|ivati u poslu koji treba obaviti. U preda-vanju }e se prikazati laboratorijska medicina za-snovana na dokazima u svezi s aktivnostimaGlobalne kampanje za še}ernu bolest.E-mail: [email protected]

cided to focus on the Laboratory and DiabetesMellitus and has therefore established a taskforce to address this topic. The Task Force willprovide scientific evidence on how to diagnoseand monitor diabetes mellitus by laboratory pa-rameters and will use this evidence to educate theprofession. In more detail, the Task Force will: (a)examine the best diagnostic strategy for diagnos-ing diabetes mellitus; (b) review and study thecurrent use of laboratory tests, and educate doc-tors and patients in the interpretation of labora-tory tests used in diagnosing and monitoring ofdiabetes mellitus; (c) provide quality specifica-tions for analytes used to diagnose and monitordiabetes mellitus; (d) give recommendations onthe minimum criteria for use of glucometers; and(e) try to associate with other groups or persons(including manufacturers) who will co-operate onthe work to be done. The lecture will present evi-dence-based laboratory medicine in relation tothe activities of the Global Campaign on DiabetesMellitus.E-mail: [email protected]

S5-5

BILJEZI ALKOHOLIZMA - PRISTUPZASNOVAN NA DOKAZIMA

Simonsson P.

Department of Clinical Chemistry, Malmö UniversityHospital, Malmö, Švedska

Biokemijski biljezi mogu se rabiti kao sred-stvo za otkrivanje zlouporabe alkohola i, što jenajva`nije, za pra}enje lije~enja od alkoholizma.Me|utim, ovom pitanju valja pristupiti kriti~ki,vode}i ra~una o klini~kim i biokemijskim aspek-tima. Iz klini~ke perspektive alkoholizam je ši-roko definirano stanje gdje nedostaje dijagno-sti~ki zlatni standard i izra`ava se u razli~itimoblicima u razli~itim populacijama. Stoga se i di-jagnosti~ke potrebe klini~ara znatno razlikuju.To treba imati na umu pri utvr|ivanju biljegazlouporabe alkohola za pojedinu zemlju. Idealnibi biljeg trebao biti visoko osjetljiv i specifi~an zazlouporabu alkohola. Osjetljivost tradicionalnihbiljega (GT i MCV) kre}e se u rasponu od 25% do75%, no specifi~nost im je niska, jer su im po-višene vrijednosti ~este u razli~itim bolestima.Razli~iti biljezi razlikuju se i po vremenu u kojemsu korisni. Uglavnom, u klini~koj kemiji potreb-na je kombinacija biljega. Transferin s nedostat-kom ugljikohidrata (CDT) pojavio se kao spe-cifi~an biljeg zlouporabe alkohola i postao najva`-nijim biljegom alkoholizma u švedskoj klini~kojpraksi. Na njega ne utje~u lijekovi, kao ni pra-te}a medicinska stanja, uz neke iznimke. Me-|utim, povišene razine CDT-a na|ene su u nekih

S5-5

MARKERS OF ALCOHOLISM - EVIDENCEBASED APPROACH

P. SimonssonDepartment of Clinical Chemistry, Malmö University

Hospital, Malmö, Sweden

Biochemical markers can be used as tools todetect alcohol abuse and, most importantly, to fol-low up treatment of alcoholism. However, the sub-ject has to be approached critically, keeping bothclinical and biochemical aspects in mind. From aclinical perspective, alcoholism is a diffusely de-fined condition lacking a diagnostic gold stand-ard and it is expressed in several forms in differ-ent populations. The diagnostic need of the clini-cian therefore varies considerably. This has to beconsidered when providing state markers of alco-hol abuse. The ideal marker should be highly sen-sitive and specific to alcohol abuse. Traditionalmarkers (GT and MCV) have a sensitivity in therange of 25% to 75% but exhibit low specificity aselevated levels are common in a variety of disor-ders. Different markers also have different timespans during which they are useful. Generally, acombination of markers has to be provided inclinical chemistry. Carbohydrate-deficient trans-ferrin (CDT) has emerged as a specific marker ofalcohol abuse and has become the most importantmarker of alcoholism in Swedish clinical prac-tice. It is not influenced by medication or, withsome exceptions, concurrent medical conditions.However, elevated levels have been found in some



S5-6

POBOLJŠANJE RADNOG PROTOKA IISKORIŠTENOSTI U LABORATORIJU

SVEUÎLIŠNE BOLNICEGriesmacher A.

University Hospital of Innsbruck, Central Institute of Medical andChemistry Laboratory Diagnosis, Innsbruck, Austrija

Sveu~ilišna bolnica u Innsbrucku ima 1500kreveta i nudi sve suvremene medicinske speci-jalnosti. Središnji zavod za medicinsku i kemij-sku laboratorijsku dijagnostiku (CIMCL) je odgo-voran za laboratorijsku dijagnostiku Bolnice, a u2002. godini proveo je 4.800.000 analiza. Zavod jeodgovoran za slijede}e pretrage: hematološke,koagulacijske, klini~ko kemijske, terapijsko pra-}enje lijekova/toksikološke, proteine, endokrino-loške i serološke. Na raspolaganju je i 24-satnahitna slu`ba sa suènim rasponom pretraga. Što-više, Zavod je uklju~en i u brojna klini~ka ispi-tivanja. CIMCL je pneumatskim portalom spojens ~itavom Bolnicom. Mjerenja iz klini~ke kemije,proteina, hormona, lijekova i serologije provodese na 9 razli~itih analizatora. U najbliòj se bu-du}nosti planira provedba konsolidacijskog pro-grma kako bi se na najmanju mjeru svelo cije-panje uzoraka i smanjio broj tehni~ara potrebnihza rad na svim ovim analizatorima. Glavni je ciljove konsolidacije stvaranje tzv. Laboratorijskejezgre, gdje }e se rutinski rad odvijati na dvjemasuvremenim analiti~kim platformama. Time }ese smanjiti cijepanje uzoraka i vrijeme proteklood prijma uzorka do izdavanja nalaza, i omogu-}iti u~inkovitije iskorištenje ljudskih potencijala.Uz to, algoritmi razborite postupne dijagnostikeprovode se za funkcijske testove štitnja~e, trom-bofiliju, hemofiliju i hematologiju, kako bi se po-boljšao dijagnosti~ki doprinos laboratorija. Tu-ma~enjem laboratorijskih nalaza bolesnika i di-jagnosti~kih algoritama odgovornost se pomi~eprema klini~kom laboratoriju i prisiljava osobljelaboratorija da prihvati pristup sve bliì samombolesniku.E-mail: [email protected]

patients with liver diseases. Genetic variants oftransferrin are found in 1%-2% of the populationand may cause false negative and false positiveresults. Congenital disorders of glycosylation areassociated with markedly increased levels. CDTis analyzed by ion exchange chromatography, ei-ther on minicolumns or using HPLC. As carbohy-drate-deficient forms of transferrin exist as a-,mono-, di- and trisialotransferrin, it is essentialthat a consensus is reached on the molecular defi-nition of CDT. Work is now in progress by an in-ternational group with the aim to standardizeand calibrate CDT.E-mail: [email protected]

bolesnika s jetrenom boleš}u. Genetske varijantetransferina nalaze se u 1%-2% populacije i moguuzrokovati la`no negativne i la`no pozitivne re-zultate. Priro|eni poreme}aji glikozilacije udru-èni su sa znatno povišenim razinama CDT-a.CDT se analizira kromatografijom ionske izmje-ne, na minikolonama ili pomo}u HPLC. Kakooblici transferina s nedostatkom ugljikohidratapostoje kao a-, mono-, di- i trisialotransferin, bit-no je posti}i konsenzus o molekularnoj definicijiCDT-a. Jedna me|unarodna grupa upravo radina standardiziranju i kalibriranju CDT-a.E-mail: [email protected]

S5-6

IMPROVING THE WORKFLOW ANDUSAGE OF AN UNIVERSITY HOSPITAL

LABORATORYA. Griesmacher

University Hospital of Innsbruck, Central Institute of Medical andChemistry Laboratory Diagnosis, Innsbruck, Austria

The University Hospital of Innsbruck has1500 beds and is offering all modern medicalspecialties. The Central Institute of Medical andChemistry Laboratory Diagnosis (CIMCL) is re-sponsible for the Hospital laboratory diagnosisand performed 4,800,000 analyses in the year2002. The Institute is responsible for the follow-ing examinations: hematology, blood coagulation,clinical chemistry, therapeutic drug monitoring/toxicology, proteins, endocrinology, and serology.A 24-hour emergency service with a reduced spec-trum is also available. Moreover, the Institute isinvolved in a number of clinical studies. A pneu-matic post is connecting the entire Hospital withCIMCL. Nine analyzers are used for the measure-ments in clinical chemistry, proteins, hormones,drugs and serology. In order to minimize thesplitting of samples and to reduce the number oftechnicians necessary for running all theseanalyzers, a consolidation program will be imple-mented in the nearest future. The aim of this con-solidation is mainly the creation of the so-calledCore Laboratory concentrating the routineworkflow on two modern analytical platforms.This will reduce sample-splitting and turn-around time, and will enable a more efficient useof human resources. In addition, algorithms for arational stepwise diagnosis have been imple-mented for thyroid function tests, thrombophilia,hemophilia and hematology in order to improvethe diagnostic input of the laboratory. The inter-pretation of laboratory patient reports and diag-nostic algorithms shifts responsibility towardsclinical laboratory and forces laboratory staff tomore patient-oriented approaches.E-mail: [email protected]



S6-1

PRETRAGE UZ BOLESNIKA: PREGLED IPOGLED U BUDU]NOST

Rumenjak V.

Zavod za medicinsko laboratorijsku dijagnostiku, Op}abolnica “Sveti Duh”, Zagreb

Pretrage uz bolesnika su podru~je medicinskobiokemijske dijagnostike koje zauzima sve va`ni-je mjesto u klini~ko laboratorijskoj dijagnostici.U usporedbi s medicinsko biokemijskim labora-torijem, pretrage uz bolesnika su podru~je koje setehnološki najbr`e razvija. Istodobno pretrage uzbolesnika ostaju podru~je mnogobrojnih nedo-umica, osobito stoga što i primijenjena tehno-logija mijenja konvencionalan pristup medicin-sko laboratorijskoj dijagnostici. Tijekom 1980-tihpretrage uz bolesnika postale su pristupa~ne bol-nicama kao alternativa centralnom laboratorijuza izvo|enje brzih laboratorijskih testova. Zbogsve sna`nijeg pritiska za kra}im vremenom obra-de i razvoja ure|aja s ve}im mogu}nostima pre-trage uz bolesnika postaju sve popularnije. Ipak,mnoge od tih tehnologija uvedene su bez znanjamedicinsko biokemijskog laboratorija i bez orga-nizacijskog plana osiguranja kontrole kvalitete.Pretrage uz bolesnika definirane su kao svakoispitivanje koje nije provedeno u laboratoriju,nego se poduzima uz bolesnika, a rezultati kojegamogu utjecati na medicinsko lije~enje bolesnika.Pretrage uz bolesnika razlikuju se od analizaizvedenih u centralnom laboratoriju po tome štone zahtijevaju laboratorijski prostor, nego se la-boratorij nalazi na mjestu pru`anja medicinskeintervencije: uzimanje uzorka, provedba testa(analize) i tuma~enje rezultata odvijaju se uzbolesnika. Razlikovanje pretraga uz bolesnika odcentralnog laboratorija je donekle arteficijelno,ali sa stajališta upravljanja nu`no, jer pretrageuz bolesnika uklju~uju ne-laboratorijsko osobljekoje rabi posebne tehnologije prilago|ene pri-mjeni uz bolesnika. Pretrage uz bolesnika imajusvoj po~etak prije nekoliko desetlje}a. Me|utim,kroz mnoge godine ove su pretrage ostale ogra-ni~ene na samo nekoliko testova. Zahvaljuju}ipristupu koji nije zahtijevao brigu o troškovimalije~enja i boravka bolesnika u jedinicama inten-zivne skrbi rijetko je bilo potrebe za brzim testo-vima. Ovakav pristup po~eo se mijenjati kada sutroškovi lije~enja postali sastavni dio procjene

S6Hitna laboratorijska dijagnostikaEmergency laboratory diagnosis

S6-1

POINT OF CARE TESTING: AN OVERVIEWAND LOOK TO THE FUTURE

V. Rumenjak

Institute of Medical Laboratory Diagnosis, Sveti DuhGeneral Hospital, Zagreb

Point of care testing (POCT) has attracted con-siderable interest in medical laboratory diagno-sis. Compared with testing performed at medicalbiochemistry laboratory, POCT is one of the mostrapidly growing areas of the diagnostic industry.At the same time, POCT remains controversial,and some of these technologies challenge the con-ventional approach to performing laboratory test-ing. During the 1980s, POCT became available tohospitals as an alternative to central laboratoryfor providing rapid laboratory testing. Due to in-creasing pressures for faster turnaround timeand the development of a broader menu of de-vices, POCT has been growing in popularity.However, some of these technologies were intro-duced at hospitals without the knowledge of theclinical laboratory and in the absence of an or-ganizational plan to ensure quality control.POCT has been defined as all testing not under-taken at a laboratory but close to the patient, itsresults possibly leading to change in the respec-tive patient care. POCT differs from a centrallaboratory testing in that it does not require alaboratory, since the specimen is obtained, the testperformed and the result reviewed at the very sitewhere care is provided. Differentiating POCTfrom central laboratory is somewhat artificial,but is useful from the management point of viewbecause it involves testing by nonlaboratory per-sonnel using specialized technologies adapted forPOCT testing. The beginnings of POCT datemany decades back. During these years, POCTwas limited to a few tests. There was not muchconcern about the costs of health care and lengthof stay at intensive care units, so there was littleneed of POCT. This line of reasoning began tochange when health care cost became part of themedical care quality assessment. This change hasresulted in reassessment of the role of clinicallaboratory and POCT. On the other hand, as theresult of this reassessment, POCT is occasionallyoverutilized with little benefit for the patient.E-mail: [email protected]



kvalitete lije~enja. Ove promjene rezultirale suponovnom procjenom uloge klini~kog laboratorijai mogu}nosti pretraga uz bolesnika. S drugestrane, kao rezultat ovakvog pristupa u nekim jeslu~ajevima došlo do zna~ajnog pove}anja pre-traga uz bolesnika, ali s malom koristi za bole-snika.E-mail: [email protected]

S6-2

OSIGURANJE KVALITETE PRETRAGA UZBOLESNIKA

Perkov S., Flegar-Meštri} Z.

Zavod za klini~ku kemiju, Klini~ka bolnica “Merkur”,Zagreb

Osiguranje kvalitete mjerenja analita iz punekrvi ima odre|ene specifi~nosti koje je potrebnopoznavati pri definiranju kriterija za kvalitetu,odre|ivanju analiti~ke pouzdanosti mjerenja iizboru strategije provo|enja kontrole kvalitetekoja }e osigurati dobivanje pouzdanih rezultatamjerenja prema zadanim kriterijima. Zahtjevi zakvalitetom analita prve kategorije hitnosti tre-bali bi se temeljiti na velil~ini biološke varijacije.Problem je, me|utim, što je vrlo malo podataka oveli~ini biološke varijacije za parametre acido-baznog statusa, odnosno uop}e je nema za par-cijalni tlak kisika u arterijskoj krvi. U tom slu-~aju mogu se rabiti kriteriji prihvatljivosti rezul-tata temeljeni na veli~ini varijacije distribucijerezultata bolesnika. Mnogo zna~ajniji problem je~injenica da veli~ina varijacije pri analizi uzora-ka pune krvi na multiparametarskim acidobaz-nim analizatorima ne proizlazi iz poteško}a uanaliti~kom procesu, nego poteško}e naj~eš}e na-staju zbog nemogu}nosti kontrole predanaliti~-kih ~imbenika. U odre|ivanju analiti~ke pouzda-nosti mjerenja analita iz pune krvi name}e seproblem sljedljivosti me|unarodno prihva}enihreferentnih metoda i materijala zbog nedostatkaodgovaraju}eg kontrolnog materijala koji bi posvom sastavu bio što sli~niji punoj krvi, bio do-stupan u dovoljnim koli~inama, imao stabilnostkroz dulje razdoblje i pokrivao cijelo koncentra-cijsko podru~je. Preporuka je stoga za odre|i-vanje analiti~ke nepreciznosti i neto~nosti rabitikomercijalne kontrolne uzorke proizvo|a~a ana-lizatora koji zadovoljavaju kriterije sljedljivostime|unarodno prihva}enih referentnih materija-la i metoda u proizvodnji kalibratora i kontrolnihuzoraka. Zbog toga je prije puštanja u rad multi-parametarskog analizatora za pretrage uz bole-

S6-2

POINT OF CARE QUALITY ASSURANCE

S. Perkov, Z. Flegar-Meštri}

Institute of Clinical Chemistry, Merkur University Hospital,Zagreb

Assurance of the quality of measurement ofanalytes from whole blood implies certain specificfeatures which should be well recognized andtaken into account in defining the quality crite-ria, determining analytical reliability of themeasurement, and selecting a quality assurancestrategy which will provide reliable results ac-cording to the required criteria. The requirementsfor the quality of first category urgency analytesshould be based on the size of biological varia-tion. The problem arises from the fact that fewdata are available on the size of biological varia-tion for the parameters of acid-base status, i.e.there are no data at all for arterial oxygen partialpressure. In this case the criteria of acceptabilityof the results based on the size of distributionvariability of patient results can be used. The ma-jor issue is that variability in multi-profile bloodgas testing of whole blood does not relate to diffi-culties in the analytical process but rather lies inthe difficulties in controlling preanalytical fac-tors. The determination of analytical reliability ofmeasurement of analytes from whole blood im-poses a problem of compliance with the interna-tionally accepted reference methods and materi-als due to the lack of control material that wouldhave a matrix similar to whole blood, be avail-able in an adequate quantity, have stability for along period, and span the medically significantrange. Therefore, for the determination of ana-lytical inaccuracy and imprecision the use ofcommercial control samples provided by themanufacturer of the analyzer meeting the require-ments of compliance with the internationally ac-cepted reference materials and methods in manu-facturing calibrators and control samples is rec-ommended. For this reason, before putting amulti-parameter analyzer into operation for the



snika vrlo va`no u~initi analiti~ku procjenu kojauklju~uje odre|ivanje analiti~ke nepreciznosti,neto~nosti, linearnosti, te komparativna mjere-nja respektabilnim rutinskim metodama. Kom-parativna mjerenja rutinskim metodama ne sa-mo što omogu}avaju uskla|ivanje rezultata, negoneizravno omogu}avaju provo|enje vanjske kon-trole kvalitete, ako je respektabilni rutinski ana-lizator uklju~en u program vanjske procjene kva-litete. Standardizacija predanaliti~ke faze, izo-brazba o mogu}im predanaliti~kim pogreškamauz redovno provo|enje kontrole kvalitete i us-kla|ivanje rezultata s referentnim analizatorimaosigurati }e dobivanje pozdanih rezultata mjere-nja analita prve kategorije hitnosti na multipa-rametarskim analizatorima uz bolesnika.E-mail: [email protected]

determination of bedside tests it is particularlyimportant to perform analytical assessment, in-cluding determination of analytical imprecision,inaccuracy, linearity, as well as comparativemeasurements using respectable routine methods.In addition to correlating the results, compara-tive measurements using routine methods also in-directly allow for the external quality control if arespectable routine analyzer has been included inthe external quality assessment program. Stand-ardization of the preanalytical stage, educationon potential preanalytical errors together withregular quality control and correlation of resultswith reference analyzers will provide realiable re-sults of measurement of the analytes of the firstcategory of urgency on multi-parameter bedsideanalyzers.E-mail: [email protected]

S6-3

DIJAGNOSTI^KA VA@NOST BRZOGFENOTIPIZIRANJA SERUMSKE

BUTIRILKOLINESTERAZE

Simeon-Rudolf V.1, Šurina B.2

1 Institut za medicinska istraìvanja i medicinu rada,2 Zavod za klini~ku kemiju, Klini~ka bolnica “Merkur”,

Zagreb

Suksametonij je kratkodjeluju}i miši}ni relak-sans koji se daje bolesnicima neposredno prijekirurškog zahvata u svrhu trahealne intubacije.Obi~na serumska butirilkolinesteraza (BChE,EC 3.1.1.8) brzo ga razgra|uje. Uz onu obi~nu(U) BChE u ljudskom serumu postoji u nekolikogeneti~kih varijanata: atipi~na (A), fluorid-rezi-stentna (F), tiha (S), Kalow (K) i J varijanta. Bio-kemijskim metodama moè se identificirati pet-naestak fenotipova ovoga enzima u serumimaljudi. Neke geneti~ke varijante (A, F, S) imajusmanjen afinitet za suksametonij i sporije garazgra|uju nego obi~na BChE, pa je njegova kon-centracija prilikom primjene dovoljna da se veèna acetilkolinski receptor i blokira prijenos im-pulsa izme|u ìvca i miši}a. Stoga nastupa pro-duèna paraliza miši}a, a u nekim krajnjim slu-~ajevima to moè završiti smr}u. Otkrivanje va-rijanata BChE potrebno je da bi se identificiraleosobe koje mogu biti osjetljive na kratkodjeluju}imiši}ni relaksans, kako bi se njima i njihovimbliskim ro|acima mogao dati pravodobni stru~nisavjet. U Zavodu za klini~ku kemiju Klini~kebolnice “Merkur” osnovana je Jedinica za fenoti-piziranje BChE s podacima o obiteljima kod kojih

S6-3

DIAGNOSTIC ROLE OF RAPID SERUMBUTYRYLCHOLINESTERASE

PHENOTYPING

V. Simeon-Rudolf1, B. Šurina2

1 Institute of Medical Research and Occupational Health,2 Institute of Clinical Chemistry, Merkur University

Hospital, Zagreb

Suxamethonium (succinyldicholine) is ashort-acting muscle relaxant that is administeredto patients undergoing surgery to facilitate tra-cheal intubation. In most cases the drug isquickly hydrolyzed by usual serumbutyrylcholinesterase (BChE, EC 3.1.1.8). How-ever, several genetic variants of this enzyme havelow affinity for the drug and prolong the time ofhydrolysis. Therefore, the drug blocks acetylcho-line receptors, impulse transmission betweennerve and muscle is inhibited, and prolongedmuscle paralysis ensues. In some extreme casesprolonged apnea may even result in cardiac ar-rest and death. BChE in human serum exists inseveral genetic forms; these include the usual (U),atypical (A), fluoride resistant (F), silent (S),Kalow (K) and J variants, giving rise to fifteenphenotypes which can be identified by standardbiochemical methods coupled with family studies.Individuals who have inherited A or/and F vari-ants are unable to quickly hydrolyze suxame-thonium at pharmacological concentrations. De-tection of BChE variants is necessary in order toidentify patients prone to experiencing sensitivityto short-acting muscle relaxants and to counsel



su ustanovljene uro|ene varijante BChE sa sla-bim afinitetom prema suksametoniju. Ustanov-ljene su ’Kartice upozorenja’ kao informacija odva`ne medicinske naravi kako bi se upozoriloanesteziologa na mogu}nost ekspresije prolongi-rane respiracijske apneje kod primjene kratko-djeluju}ih miši}nih relaksansa. Uvedena je me-toda za biokemijsku identifikaciju fenotipova ko-ja se temelji na mjerenju aktivnosti enzima istupnja inhibicije hidrolize butiriltiokolina dibu-kainom, natrijevim fluoridom, ureom i dimetil-nim karbamatom Ro 02-683. Postupak je prila-go|en za analizator VP Abbott i testiran u su-djelovanju suradnika Jedinice za fenotipiziranjeu Me|unarodnom programu kontrole kakvo}emjerenja aktivnosti i odre|ivanja fenotipovaBChE. Na taj je na~in mogu}e brzo prijeopera-cijski odrediti ima li bolesnik varijantu BChEkoja sporo razgra|uje suksametonij, te je takomogu}e izbje}i komplikacije pri operacijskom za-hvatu.E-mail: [email protected]

them and their relatives who may be similarly af-fected. The Cholinesterase Unit for detection ofindividuals, carriers of inherited suxamethoniumsensitive BChE variants has been formed at theInstitute of Clinical Chmistry, Merkur ClinicalHospital, Zagreb, Croatia. The method has beenestablished for identification of BChE phenotypesin human serum. It comprises the measurementof the activity of the enzyme and the inhibition ofbutyrylthiocholine hydrolysis by dibucaine, so-dium fluoride, urea and the dimethylcarbamateRo 02 0683. The procedure has been adapted forVP Abbott analyzer and tested during participa-tion in the International Proficiency ProgrammeNo. 4 for quality control of measurements andBChE phenotyping. Cholinesterase Unit is issu-ing Warning Cards to carriers of inherited serumBChE variants in order to avoid prolonged respi-ratory apnea that suxamethonium may cause.E-mail: [email protected]

S6-4

ZNA^ENJE LABORATORIJSKEDIJAGNOSTIKE U MO@DANOM UDARU

Vukasovi} I.


Mo`dani udar, prvi uzrok smrtnosti i invalid-nosti u Republici Hrvatskoj, nastaje zbog intra-cerebralnog ili subarahnoidnog krvarenja (15%-20%) ili zbog mo`dane ishemije uzrokovane he-modinamski, trombozom ili embolijom (80%-85%). Predstavlja akutno stanje koje zahtijevahitnu intervenciju uklju~uju}i i hitnu laborato-rijsku obradu uzoraka krvi, likvora i mokra}e.Iako se današnja dijagnostika mo`danog udaratemelji prete`ito na tzv. metodama slikovnog pri-kaza doplerske sonografije, ekstrakranijskogobojenog doplera, transkranijskog doplera tekompjutorizirane tomografije (CT), ipak je u dife-rencijalnoj dijagnostici i pra}enju tijeka bolestineophodna analiza likvora. Odre|ivanjem hema-togenih pigmenata u likvoru mogu}e je razlu~itikrvarenja uza skeletne strukture, intracerebral-nu hemoragiju, subduralnu hemoragiju, cere-bralni ili subduralni hematom te krvarenja izaneurizama. Razina glikemije predstavlja pre-diktivnu vrijednost za razvoj pogoršanja i hemo-ragijsku transformaciju. Novije studije upu}ujuna povezanost koncentracije homocisteina, lipo-

S6-4

THE ROLE OF LABORATORY DIAGNOSISIN STROKE

I. Vukasovi}


Stroke is the leading cause of death and dis-ability in Croatia. Stroke occurs due to intracer-ebral or subarachnoid hemorrhage (15% - 20%)or cerebral ischemia caused by hemodynamic im-pairment, thrombosis or embolism (80% - 85%).Stroke is an acute state that requires immediateintervention including emergency laboratory test-ing of blood, cerebrospinal fluid (CSF) and urinesamples. Although current diagnosis of stroke ismostly based on neuroimaging methods ofdoppler sonography, extracranial color doppler,transcranial doppler and computed tomography,CSF analysis is necessary in differential diagno-sis and follow-up of the course of disease.Hemorrhages adjacent to skeletal structures, in-tracerebral hemorrhage, subdural hemorrhage,cerebral or subdural hematoma and aneurysmalhemorrhage can be differentiated by determina-tion of hematogenic pigments in CSF. The level ofglycemia has a predictive value for exacerbationand hemorrhagic transformation. Recent studiespoint to correlation of homocystein, lipoprotein(a) and total antioxidative status with the sever-



proteina (a), totalnog antioksidativnog statusa sjakoš}u cerebrovaskularne bolesti. Proteine spe-cifi~ne za središnji ìv~ani sustav, neuron speci-fi~nu enolazu (NSE), S-100 protein i mijelinskibazi~ni protein (MBP) nakon mo`danog udaranalazimo u serumu. Osloba|aju se iz ošte}enihnervnih stanica, prolaze krvno-mo`danu barijerui dospijevaju u cirkulaciju. NSE predstavlja os-jetljiv biljeg postishemijskog i hemoragijskog oš-te}enja mozga. S-100 protein nakon 8 sati do 4.dana od mo`danog udara korelira s veli~inom in-farkta te se rabi u predvi|anju klini~kog ishoda.Vršna vrijednost MBP 4-5. dana nakon udara ko-relira s veli~inom lezije. Godine 1992. zapo~eli suprvi kontrolirani pokusi o djelotvornosti rekom-binantnog tkivnog plazminogena (rtPA) u lije~e-nju mo`danog udara 3-6 sati od po~etka simpto-ma. Lije~enje se provodi prema to~no odre|enomprotokolu, a svako odstupanje donosi velik rizikza komplikacije, poglavito hemoragijsku trans-formaciju. Kumulativne meta-analize potvrdilesu pozitivan u~inak ove terapije unato~ brojnimrizicima. Istraìvani su i biokemijski pokazateljirizika u primjeni ove terapije. Prije i tijekomprovo|enja terapije potrebne su koagulacijske ihematološke pretrage. Više autora izvijestilo je ozna~enju odre|ivanja koncentracije metalopro-teinaze matriksa 9 (MMP-9) prije po~etka tera-pije. Ipak, ostaje nejasno doprinose li pove}anekoncentracije MMP-9 u plazmi stvaranju trombarezistentnog na trombolizu ili je to epifenomennastao zbog drugih ~imbenika.E-mail: [email protected]

ity of cerebrovascular disease. Neuron specificenolase (NSE), S-100 protein and myelin basicprotein (MBP), the central nervous system specificproteins, are found in serum of stroke patients.These proteins are released from damaged neu-rons, cross the blood-brain barrier and reach thecirculation. NSE is a sensitive marker ofpostischemic and hemorrhagic brain lesion. At 8hours to 4 days post-stroke, S-100 protein corre-lates with infarct size and is used to predict clini-cal outcome. On day 4-5 post-stroke, the peakMBP value correlates with lesion size. Controlledstudies of the efficiency of recombinant tissueplasminogen (rtPA) in the management of stroke3-6 hours from the onset of symptoms werelaunched in 1992. The treatment is performed ac-cording to a strict protocol, whereby any depar-ture is associated with a high risk of complica-tions, especially hemorrhagic transformation.Cumulative meta-analyses have confirmed thefavorable effect of this therapy in spite of numer-ous risks. Biochemical parameters of the risk as-sociated with this therapy have also been investi-gated. Coagulation and hematology tests are re-quired before and during therapy. The role ofpretherapeutic matrix metalloproteinase 9 (MMP-9) determination has been reported by several au-thors. However, it remains to clarify whether el-evated plasma concentration of MMP-9 contrib-utes to the formation of thrombus resistant tothrombolysis or it is an epiphenomenon due tosome other factors.E-mail: [email protected]

S6-5

DIJAGNOSTIKA SUSTAVA ZGRUŠAVANJAI FIBRINOLIZE U PORODNIŠTVU

Rai} B.

Zavod za hematologiju Klinike za unutarnje bolesti,Klini~ka bolnica “Sestre milosrdnice”, Zagreb

U humanoj reprodukciji bitno je odrànje maj-~ine i fetalne cirkulacije. Za to su odgovorni su-stav zgrušavanja i sustav fibrinolize. Tijekomnormalne trudno}e aktivira se sustav zgruša-vanja, a fibrinoliti~na se aktivnost smanjuje. Vri-jednosti fibrinogena su pove}ane zbog pove}anogvolumena plazme i pove}ane sinteze fibrinogena.Koncentracija ~imbenika zgrušavanja je pove}a-na, pogotovo FV i FVIII, te FVII i FX. AktivnostF XIII pada i ima za posljedicu odlaganje fibrinau uteroplacentnoj cirkulaciji. Inhibitori ATIII iPC se smanjuju. Smanjena fibrinoliti~na aktivnost

S6-5

COAGULATION AND FIBRINOLYSISDIAGNOSIS IN OBSTETRICS

B. Rai}

Department of Hematology, University Department ofMedicine, Sestre milosrdnice University Hospital, Zagreb

In human reproduction, maintenance of thematernal and fetal circulation is crucial. The sys-tems of coagulation and fibrinolysis are responsi-ble for it. During normal pregnancy, the coagula-tion system is activated, whereas the fibrinolyticactivity is reduced. The values of fibrinogen areelevated because of the increased plasma volumeand fibrinogen synthesis. The concentration of co-agulation factors is increased, especially of FVand FVIII as well as FVII and FX. The activity ofFXII decreases, resulting in fibrin deposition inthe uteroplacental circulation. The ATIII and PC



se tijekom normalne trudno}e ne mijenja. Ubr-zava se tek nakon poroda. Zna~ajan je porast pla-zminogena i PAI. Razina razgradnih proizvodafibrina/fibrinogena je niska. Zna~ajan je i umje-reni pad trombocita (do donje granice normale).Potkraj trudno}e volumen trombocita poraste.Promjene u laboratorijskim testovima u trudno}i,porodu i babinju mogu izazvati teška krvarenja itromboembolijske bolesti (DVT, plu}na embolija).Sindrom antifosfolipidnih protutijela obiljeèn jesklonoš}u pojavi arterijske ili venske tromboze.Zbog navedenih promjena tijekom normalne tru-dno}e neophodna je kontrola laboratorijskih tes-tova sustava zgrušavanja i fibrinolize. Me|utim,tijekom trudno}e nastaju i poreme}aji u smisluDIK-a i nastaju dodatne komplikacije kod: 1) ab-rupcije placente, 2) embolije plodnom vodom, 3)septi~nog poba~aja, 4) rupture uterusa, 5) produ-ljenog šoka, 6) eklampsije i preeklampsije, 7)sindroma HELLP. Eklampsija i preeklampsija sustanja koja nastaju tijekom trudno}e, te je neo-phodno laboratorijski otkriti promjene u smislupada trombocita, smanjenja fibrinogena, potroš-nje ~imbenika zgrušavanja, eventualne poja~anefibrinoliti~ne aktivnosti, te porast FDP. U ènakojima je ve} prije trudno}e dijagnosticirana ne-ka bolest zbog ovih ve} navedenih promjena nu`-na je laboratorijska kontrola sustava zgrušava-nja i fibrinolize. No, još ve}e zna~enje ova labo-ratorijska dijagnostika ima u mogu}im izne-nadnim, dodatnim komplikacijama tijekom i na-kon poroda, te u babinju.

inhibitors decrease. The suppressed fibrinolyticactivity remains unchanged during normal gesta-tion, and accelerates only after delivery. There isa considerable increase in plasminogen and PAI.The level of fibrin/fibrinogen degradation prod-ucts is low. There is also a moderate platelet de-crease (to the lower normal limit). Towards theend of gestation, the platelet volume increases.Changes in the laboratory tests during preg-nancy, delivery and puerperium can cause severebleeding and thromboembolic disorders (DVT,pulmonary embolism). The syndrome ofantiphospholipid antibodies is characterized byproneness to arterial or venous thrombosis. Thesechanges that may occur during normal preg-nancy require control laboratory testing of coagu-lation and fibrinolysis. However, disorders interms of DIC may also occur during pregnancy,entailing additional complications in: 1) abruptioplacentae, 2) amniotic fluid embolism, 3) septicabortion, 4) uterus rupture, 5) delayed shock, 6)eclampsia and preeclampsia, and 7) HELLP syn-drome. Eclampsia and preeclampsia are condi-tions occurring during gestation, thus changes interms of platelet decline, fibrinogen decrease, co-agulation factor utilization, possible enhanced fi-brinolytic activity, and FDP increase should bedetected by laboratory testing. The women inwhom a disease associated with these changeshas already been diagnosed before pregnancy re-quire laboratory control of coagulation and fibri-nolysis systems. However, this laboratory diagno-sis plays an even greater role in the possible sud-den, additional complications intra- andpostpartally as well as in the puerperium.


VA@NOST TIMSKOG PRISTUPA UDIJAGNOSTICI UZ BOLESNIKA (POINT

OF CARE)

Brklja~i} V., Juri~i} Lj., Rogi} D.


Odre|ivanje uz bolesnika (point of care test-ing, POCT) danas zauzima posebno mjesto uklini~ko laboratorijskoj praksi. Brze i pravodobneanalize doprinose racionalizaciji i ubrzavanju te-rapijskih postupaka. U KZZLD KBC “Zagreb”cjelovit svakodnevni nadzor odre|ivanja uz bole-snika obavlja mali stru~ni tim koji je zaduèn i zapoduku osoba koje }e ga neposredno provoditi.Ukupno je pet ure|aja Nova Biomedical Stat


THE ROLE OF TEAM APPROACH INPOINT OF CARE DIAGNOSIS

V. Brklja~i}, Lj. Juri~i}, D. Rogi}


Point of care testing currently has a specialplace in the clinical laboratory practice. Rapidand timely analyses contribute to rationalizationand acceleration of therapetic procedures. Com-prehensive daily monitoring of point of care test-ing has been performed at the Clinical Institute ofLaboratory Diagnosis, Zagreb University Hospi-tal Center, by a small expert team also responsiblefor training of persons involved in direct monitor-ing. A total of five Nova Biomedical Stat Profile



Profile M smješteno u Hitni laboratorij te u jedi-nice intenzivne skrbi Op}e, Kardijalne kirurgije,Neurokirurgije i Dje~jeg odjela. Pouzdanost ure-|aja dnevno u zadanim vremenskim razmacimate prema potrebi provjeravaju za to zadu`eni me-dicinski biokemi~ari pomo}u komercijalnih kon-trola i humanih uzoraka (heparinizirana krv).Ovakva organizacija rada navedenih ure|aja istatisti~ki podaci daju uvid u kvalitetu i isprav-nost rada ure|aja, ali i laboratorijskog i medi-cinskog osoblja, te isto tako ukazuju na mogu}epropuste i mjesta gdje i kako treba djelovati radipoboljšanja svih analiza koje se izvode na ure|a-jima uz bolesnika. U radu je prikazana polugo-dišnja pohrana i statisti~ka obrada dobivenih re-zultata te izrada prate}e dokumentacije pomo}uosobnog ra~unala i komercijalnog programa. Sta-tisti~ka obrada podataka komercijalne vodenekontrole (χ, SD i CV) je pokazala zadovoljavaju}upreciznost i to~nost, kao i dobru korelaciju nauzorcima heparinizirane krvi na svih pet ure|a-ja. Tijekom ispitivanog razdoblja (6 mjeseci) dobi-veni CV (%) iznosili su do 5% za sve analite. Uskoroj budu}nosti u planu je povezivanje svihure|aja i integriranje u laboratorijski informa-cijski sustav i elektroni~ko poslovanje laborato-rija.E-mail: [email protected]

M instruments have been installed at the Emer-gency Laboratory and intensive care units at De-partments of Surgery, of Cardiac Surgery, of Neu-rosurgery, and of Pediatrics. Instrument reliabil-ity is verified daily at defined periods and when-ever necessary by responsible medical biochemistsusing commercial controls and human specimens(heparinized blood). Such organization and dis-tribution of the instruments and statistical dataprovide evidence on the quality and proper per-formance of the instruments as well as of thelaboratory and medical staff; it also reveals thepossible oversights and points where improve-ments are necessary in the analyses performed aspoint of care testing. Semi-annual collection andstatistical processing of the results obtained, andproduction of accompanying documentation byuse of personal computer and commercial pro-gram are presented. Statistical data processingon commercial water control (χ, SD and CV)showed satisfactory precision and accuracy aswell as good correlation on heparinized bloodsamples on all five instruments. During the pe-riod of observation (6 months), the CVs (%) re-corded were up to 5% for all analytes. A plan inthe near future involves connection of all instru-ments and integration into the laboratory compu-ter system and electronic laboratory operations.E-mail: [email protected]


POSTERI Sa`eci 4. hrvatskog kongresa medicinskih biokemi~ara

P1Bolesti hematopoetskog sustavaDiseases of hematopoietic system

P1-1

UTJECAJ ERITROPOETINA NAVRIJEDNOSTI KRVNE SLIKE KODHEMODIJALIZIRANIH BOLESNIKA

Bošnjak N., Sapunar A., Dujmov I.

Klini~ka bolnica “Split”, Odjel za medicinsku laboratorijskudijagnostiku, Split

Eritropoetin je glikoprotein koji se proizvodi ustanicama bubrega i izlu~uje u plazmi kao reak-cija bubrega na prisutnu hipoksiju. Bolesnici sanemijom imaju u plazmi visoke vrijednosti eri-tropoetina koje su obrnuto proporcionalne kon-centraciji hemoglobina. Bolesnici sa zatajenjembubrega u uremiji imaju poreme}enu proizvod-nju eritropoetina i stoga relativno nisku koncen-traciju u plazmi. Klini~ku primjenu eritropoetinnalazi u lije~enju anemije zbog nedostatne pro-izvodnje eritropoetina te kako bi se normalna eri-tropoeza podigla na višu razinu. Obradili smo 61bolesnika na dugogodišnjoj dijalizi (36 muška-raca prosje~ne dobi 55 godina i 25 `ena prosje~nedobi 62 godine). Usporedili smo njihove vrijed-nosti broja eritrocita, hemoglobina, prosje~nogvolumena eritrocita (MCV), postotka hipokrom-nih eritrocita, retikulocita i koncentraciju hemo-globina u retikulocitima s onima u kontrolnojskupini od 30 zdravih ispitanika sli~ne prosje~nedobi. Vrijednosti smo odre|ivali na hematološ-kom broja~u tvrtke Bayer Technicon H*3. Hemo-dijalizirani bolesnici podijeljeni su u ~etiri skupi-ne prema dozi eritropetina. U svim skupinamaodre|ivali smo navedene testove ponovno nakon~etiri tjedna davanja eritropoetina. Kod ovihbolesnika s anemijom primjena rekombinatnogeritropoetina nije dovela do zna~ajnog poboljša-nja anemije, ali su oni trebali manje transfuzija

P1-1

EFFECT OF ERYTHROPOIETIN ONBLOOD TEST RESULTS INHEMODIALYSIS PATIENTS

N. Bošnjak, A. Sapunar, I. Dujmov

Department of Medical Laboratory Diagnosis,Split University Hospital, Split

Erythropoietin is a glycoprotein hormone pro-duced primarily by the kidneys in response to hy-poxia and is secreted into the plasma. Therefore,anemic patients have increased plasma erythro-poietin levels that are inversely proportional totheir hemoglobin level. In contrast, patients withuremia usually have considerably impairederythropoietin production and thus relatively lowplasma levels. Clinical application of erythropoi-etin is in the treatment of anemias due to inad-equate production of endogenous erythropoietinand for stimulation of physiologic erythropoiesisto supranormal levels. We studied 61 patients (36male, mean age 55 years and 25 female, mean age62 years) with chronic renal insufficiency onhemodialysis. The values of red blood cells(RBC), hemoglobin, mean corpuscular volume(MCV), percentage of hypochromic RBC,reticulocytes and hemoglobin concentration inreticulocytes were determined on a Technicon H*3blood counter (Bayer) and compared with thosein the control group of patients (n=30).Hemodialysis patients were subdivided into fourgroups according to erythropoietin dosage. Testresults of all groups were redetermined in fourweeks, after erythropoietin treatment. In the sub-group of hemodialysis patients with anemia thetreatment with recombinant human erythropoi-etin resulted in an increase in hemoglobin levels,

PosteriPosters


Saèci 4. hrvatskog kongresa medicinskih biokemi~ara POSTERI

eritrocita, došlo je do porasta koncentracije he-moglobina i poboljšanje kvalitete ìvota.E-mail: [email protected]

decrease in RBC transfusion required, and im-proved quality of life.E-mail: [email protected]

P1-2

ODRE\IVANJE TRANSFERINSKIHRECEPTORA, TRANSFERINA I @ELJEZA

U REFERENTNOJ SKUPINI @ENA

Mladina B., Sapunar A., Dujmov I., Ivelja N.

Klini~ka bolnica Split, Odjel za medicinsku laboratorijskudijagnostiku, Split

Receptori transferina (sTfR) su transmem-branski proteini prisutni na površini ve}ine sta-nica. Oni veù transferin na površinu stanice iprenose ga u stanicu. Uloga receptora transfe-rina je opskrba stanica èljezom. Koncentracijatransferinskih receptora u plazmi je pribli`noproporcionalna koli~ini ukupnog transferina. Ciljrada bio je ispitati povezanost sTfR, transferina ièljeza u referentnoj skupini èna. Receptoritransferina i transferin odre|ivani su nefelome-trijski na BN Pro Spec (Dade Behring). @eljezo jeodre|ivano na analizatoru Hitachi 917. Obra|e-na su 34 uzorka (ène, 18-24 godine). Izme|usTfR i transferina na|ena je statisti~ki zna~ajnapovezanost (F=5,4; p=0,026). Koeficijent korela-cije iznosio je 0,38, a koeficijent determinacije0,145. Izme|u sTfR i èljeza na|ena je statisti~kizna~ajna negativna povezanost (F=6,8; p=0,014).Koeficijent korelacije iznosio je -0,42, a koefi-cijent determinacije 0,175. STfR je neovisan pa-rametar statusa èljeza, koji raste kod nedostat-ka èljeza.E-mail: [email protected]

P1-2

DETERMINATION OF SOLUBLETRANSFERRIN RECEPTOR,

TRANSFERRIN AND IRON IN AREFERENCE GROUP OF ADULT FEMALES

B. Mladina, A. Sapunar, I. Dujmov, N. Ivelja

Department of Medical Laboratory Diagnosis,Split Univerity Hospital, Split

Transferrin receptors are transmembrane pro-teins present on the surface of most cells. It bindsiron-loaded transferrin to the cellular surfaceand transports it into the cell. The role of solubletransferrin receptor (sTfR) is cell supply withiron. The concentration of sTfR in plasma is ap-proximately proportional to the quantity of totalbody transferrin. The aim of the study was tocompare sTfR, transferrin and iron in a referencegroup of adult females. sTfR and transferrin weremeasured by immunonephelometry on a BN ProSpec (Dade Behring). Iron was determined on aHitachi 917. Our clinical study included 34 fe-males aged 18-48 years. There was a significantcorrelation between sTfR and transferrin (F=5.4;p=0.027). Coefficient correlation was 0.38 and de-termination coefficient 0.145. A significant nega-tive correlation was observed between sTfR andiron (F=6.8; p=0.014). Correlation coefficient was-0.42, and determination coefficient 0,175. sTfR isan independent parameter of iron status and isincreased in iron deficiency.E-mail: [email protected]

P1-3

TOPLJIVI SERUMSKI TRANSFERINSKIRECEPTOR (sTfR) I INDEKS sTfR/log

FERITIN U SIDEROPENI^NOJ ANEMIJI IANEMIJI KRONI^NE BOLESTI

Margeti} S., Topi} E., Kvaternik M., Ruì}-Ferenc D.,Bubenik V.

Klini~ka bolnica “Sestre milosrdnice”, Klini~ki zavod zakemiju, Odsjek za laboratorijsku hematologiju, Zagreb

Cilj rada bio je ispitati vrijednosti koncentra-cije topljivog serumskog transferinskog receptora(sTfR) i indeksa sTfR/log feritin (sTfR/logF) u si-

P1-3

SOLUBLE SERUM TRANSFERRINRECEPTOR (sTfR) AND sTfR/log FERRITININDEX IN IRON DEFICIENCY ANEMIA AND

ANEMIA IN CHRONIC DISEASE

S. Margeti}, E. Topi}, M. Kvaternik, D. Ruì}-Ferenc,V. Bubenik

Laboratory of Hematology, Clinical Institute of Chemistry,Sestre milosrdnice University Hospital, Zagreb

The aim of the study was to assess the concen-trations of soluble serum transferrin receptor(sTfR) and sTfR/log ferritin index (sTfR/logF)



deropeni~noj anemiji i anemiji u reumatoidnomartritisu (anemija kroni~ne bolesti). U istraì-vanje je bio uklju~en 61 kontrolni ispitanik i 96anemi~nih bolesnika. U zdravih ispitanika izmje-rene vrijednosti sTfR i indeksa sTfR/logF nisu sestatisti~ki zna~ajno razlikovale ovisno o spolu iìvotnoj dobi. Rezultati su pokazali da su u bo-lesnika sa sideropeni~nom anemijom izmjerenevrijednosti sTfR i sTfR/logF statisti~ki zna~ajnove}e u odnosu na kontrolnu skupinu ispitanika iskupinu ispitanika s anemijom u reumatoidnomartritisu. Na temelju dobivenih rezultata zaklju-~ujemo da je odre|ivanje sTfR uz izra~un indeksasTfR/logF pouzdan laboratorijski test u dijagno-stici sideropeni~ne anemije. Ispitani parametrisu pokazali zadovoljavaju}u klini~ku primjenji-vost i u diferencijalnoj dijagnostici sideropeni~neanemije i anemije kroni~ne bolesti. ROC anali-zom je indeks TfR/logF pokazao bolji dijagno-sti~ki potencijal u odnosu na parametar sTfR. Ubolesnika s anemijom kroni~ne bolesti vrijednostiispitanih parametara, sTfR i sTfR/logF, nisu sestatisti~ki zna~ajno razlikovale ovisno o koncen-traciji C-reaktivnog proteina, što upu}uje na toda na ispitane parametre ne utje~e akutni ilikroni~ni upalni proces.E-mail: [email protected]

values in iron deficiency anemia and anemia inrheumatoid arthritis (anemia of chronic disease).The study included 61 apparently healthy volun-teers as a control group and a total of 96 anemicpatients. In healthy subjects there were no signifi-cant sex and age differences in sTfR and sTfR/logF values. The results showed that sTfR andsTfR/logF levels were significantly higher in pa-tients with iron deficiency anemia than in controlsubjects and those with anemia in rheumatoidarthritis. The results showed sTfR and sTfR/logF to be reliable parameters in the diagnosis ofiron deficiency anemia. On differential diagnosisof iron deficiency and anemia of chronic disease,the tested parameters of sTfR and sTfR/logF alsoshowed acceptable clinical utility. ROC analysisshowed a higher discriminating power of sTfR/logF versus sTfR in the diagnosis of iron defi-ciency anemia as well as on differential diagnosisbetween iron deficiency anemia and anemia ofchronic disease. In patients with anemia ofchronic disease, the tested parameters of sTfRand sTfR/logF showed no significant differencesaccording to C-reactive protein concentration.These results suggest the tested parameters not tobe affected by acute or chronic inflammatory dis-ease.E-mail: [email protected]

P1-4

IMUNOFENOTIPIZACIJA URAZLIKOVANJU TRIHOLEUKEMIJE ISPLENI^NOG LIMFOMA S VILOZNIM

LIMFOCITIMA

Kardum-Paro M.M.1, Šiftar Z.1, Nazor A.1, Kušec R.1,2,Kardum-Skelin I.3, Šušter~i} D.3, Jakši} B.2

1 Zavod za klini~ku kemiju, 2 Klinika za unutarnje bolesti,3 Laboratorij za citologiju Klinike za unutarnje bolesti, KB

“Merkur”, Zagreb

Triholeukemija (TL) je poseban oblik kroni~nelimfoproliferativne bolesti pra}en pancitopeni-jom, splenomegalijom i infiltracijom koštane srìmononuklearnim stanicama (trihocitima) znako-vitog fenotipa (CD103+CD25+CD11c+CD19+CD22+). Temeljem znakovite morfološke slike iimunofenotipskog profila B-limfocita diferenci-jalno dijagnosti~ka potvrda TL zna~i isklju~enjesli~ne limfoproliferativne bolesti (LPB), kao što jespleni~ni limfom s viloznim limfocitima (SLVL)(CD103-CD25-CD11c-/+CD19+CD22+). Cilj radabio je usporedbom rezultata citomorfološke ana-lize i imunofenotipizacije utvrditi razinu slaganja

P1-4

FLOW CYTOMETRY METHOD INDISTINCTION BETWEEN HAIRY CELLLEUKEMIA AND SPLENIC LYMPHOMA

WITH VILLOUS LYMPHOCYTES

M.M. Kardum-Paro1, Z. Šiftar1, A. Nazor1, R. Kušec1,2,I. Kardum-Skelin3, D. Šušter~i}3, B. Jakši}2

1 Institute of Clinical Chemistry, 2 University Department ofMedicine, 3 Laboratory of Cytology, University Department

of Medicine, Merkur University Hospital, Zagreb

Hairy cell leukemia (HCL) is a rare form ofclonal B-cell malignancy characterized by pancy-topenia, splenomegaly and bone marrow infiltra-tion with malignant B-lymphocytes. On differen-tial diagnosis of HCL, another B-cell lymphopro-liferative disorder such as splenic lymphomawith villous lymphocytes (SLVL) has to be ex-cluded based on the cell morphology and theirimmunophenotypic profile. The aim of the studywas to compare cell morphology and cell immu-nophenotyping to assess their diagnostic impactin differentiating HCL from SLVL. During threeyears, 9 diagnoses of HCL, 4 diagnoses of SLVL



dviju razli~itih metoda i korisnost imunofeno-tipizacije u razlikovanju ovih dvaju entiteta.Citomorfološkom analizom koštane srì (14 uzo-raka) tijekom tri godine dijagnosticirano je 9 slu-~ajeva TL (tartarat-rezistentna kisela fosfataza(TRAP) pozitivni), 4 slu~aja SLVL (od toga 2 mo-gu}a LPB) (TRAP negativni, anti-TL monoklon-sko protutijelo DBA44 pozitivni) i 1 slu~aj B-prolimfocitne leukemije (B-PLL), te je u~injenaimunofenotipizacija metodom proto~ne citome-trije. Unutar skupine TL (9), imunofenotipiza-cijom je u 6 slu~ajeva (6/9; 66,7%) opisan zna-kovit fenotip trihocita (CD103+CD25+CD11c+CD19+CD22+ uza sna`nu koekspresiju biljegaCD103/ CD22); u dva slu~aja ekspresije biljegaCD103 i CD25 nisu uo~ene (CD103-CD25-CD11c+CD19+CD22+; bez koekspresije biljegaCD103/CD22) (?varijanta TL), dok je u jednomslu~aju TL ekspresija biljega CD103, CD25 iCD11c u koštanoj srì bila negativna, a naknad-nom imunofenotipizacijom stanica u punktatuslezene je uo~ena samo jasna ekspresija biljegaCD11c na B-limfocitima (?SLVL; ?atipi~na TL).Unutar skupine SLVL (4) imunofenotipizacija jeopisala 1 SLVL (1/4; 25%) znakovitog fenotipa(CD103-CD25-CD11c-/+CD19+CD22+) i tri LPB(CD103-CD25+CD11c+CD19+CD22+FMC7+) (?1atipi~an B-CLL; ?2 B-NHL) (mogu}a pozitivnostanti-TL monoklonskog protutijela DBA44 u LPBdo 5%), dok je B-PLL temeljem fenotipa opisankao SLVL (CD103-CD25-CD11c+CD19+CD22+FMC7-), a ponovljena analiza ukazala je na istifenotip, 1c+CD19+CD22+. Sna`na i jasna ekspre-sija antigena CD25 i CD103 1c+CD19+CD22+ jenajspecifi~nija i najosjetljivija odlika trihocita, ali1c+CD19+CD22+ ne i B-limfocita u SLVL, te je udiferencijalnoj 1c+CD19+CD22+ dijagnostici ovihdvaju entiteta imunofenotipizacija i 1c+CD19+CD22+ odre|ivanje ekspresije ovih dvaju anti-gena neophodno. 1c+CD19+CD22+ Poradi sli~no-sti SLVL i ostalih LPB (varijante TL,CLL, PLL)1c+CD19+CD22+ dvojbene slu~ajeve je uputnoistraìti i citogenetski.E-mail: [email protected]

(two LPD possible) (TRAP negative, anti-HCLmonoclonal DBA 44 antibody positive), and 1 B-PLL were made on the basis of cell morphologyand tartarate-resistant acid phosphatase (TRAP)positivity. Within the group of HCL diagnoses (9),a characteristic immunophenotypic profile wasdescribed in 6 cases (6/9; 66.7% consistency)(CD103+CD25+CD11c+ CD19+CD22+; coexpress-ion CD103/CD22 obtained); because of negativityof CD103 and CD25 (CD103-CD25-CD11c+CD19+CD22+; without coexpression CD103/CD22) two cases were considered as a HCL vari-ant. Only in one case there was no expression ofeither CD103 or CD25, or CD11c, however, obvi-ous positivity of CD11c on B-cells was found inthe spleen (?SLVL; ?atypical HCL). Within thegroup of SLVL (4) only 1 SLVL with a character-istic immunophenotypic profile was described (1/4; 25.0% consistency) (CD103-CD25-CD11c-/+CD19+CD22+), 3 were considered for LPD(CD103-D25+CD11c+CD19+CD22+FMC7+) (?1atypical B-CLL; ? 2B-NHL) (anti-HCL mono-clonal DBA 44 antibody positivity possible up to5%). One B-PLL was considered as SLVL(CD103-CD25-D11c+CD19+CD22+FMC7-) andreanalysis indicated the same profile. Accord-ingly, strong and obvious positivity of CD25 andCD103 on B-cells as the most specific and sensi-tive markers for HCL makes the FCM useful inthe differential diagnosis of these two entities.However, when SLVL or other LPDs are suspected(HCL variants, CLL, PLL), cytogenetics should beemployed.E-mail: [email protected]



P1-5

KARAKTERIZACIJA AKUTNEMIJELOI^NE LEUKEMIJE PROTO^NOM

CITOMETRIJOM

Šiftar Z.1, Kardum-Paro M.M.1, Kušec R.2, Nazor A.1,Kardum-Skelin I.3, Šušter~i} D.3, Jakši} B.H.2, Minigo H.2

1 Zavod za klini~ku kemiju, 2 Hematolo{ki odjel Interneklinike, 3 Citolo{ki odjel Interne klinike, KB “Merkur”,

Zagreb

Imunofenotipizacija proto~nom citometrijomje korisna metoda za analizu blastnih stanica,odre|ivanje linijske pripadnosti i stadija zrelostiu akutnim leukemijama. Cilj studije bio je utvr-diti korisnost standardnog panela protutijela kojise rabi u dijagnostici akutne mijeloi~ne leuke-mije (AML), te pokušati prikazati u~inkovitostdodatnih ne-mijeloi~nih biljega CD2, CD19 iCD56 u pre-poznavanju AML t(8;21), AMLt (15;17) i AMLinv (16) podtipova. U studiju je uklju-~en 61 bolesnik s AML koji su dijagnosticirani urazdoblju od 2000. do 2003. u našoj ustanovi.AML su dalje klasificirane kao AML0, AML1,AML2, AML2 t(8,21), AML3 t(15;17), AML4,AML4Eo inv(16), AML5, AML6, AML s multili-nijskom displazijom i bifenotipska leukemija, s 5,4, 20, 3, 3, 11, 2, 1, 3, 8 i 1 slu~ajem. Najkorisnijibiljezi za postavljanje dijagnoze AML osim AML6su HLAD/DR, CD33, CD34, CD13, MPO i CD117u frekvenciji od 95, 92, 80, 80, 75 i 56%. Nadalje,na|eno je da je MPO zna~ajno niì (p<0,05) uAML0 nego u zrelijim tipovima, što je potvr|enoMann Whitneyom neparametrijskom analizom,za razliku od CD34 koji je baš suprotno viši. Eri-troleukemija je dijagnosticirana na osnovi pozi-tivnosti glikoforina A, a za AML3 je znakovitnegativan HLAD/DR, sa zna~ajno višim vrijed-nostima CD33 nego u drugim tipovima. Dodatninapor je u~injen da se dijagnosticiraju entiteti spovratnim citogenetskim translokacijama testi-ranjem na izraàj CD2, CD19 i CD56 na blastnimstanicama. CD2 je bio pozitivan u oba slu~ajaAML4Eo inv(16), a CD19 u 1/3 i CD56 u 1/1slu~aju AM2 t(8;21). Preporu~ena kombinacija bi-ljega je korisna u dijagnozi AML. Odre|ivanje iz-raàja CD2, CD19 i CD56 na blastima je obe}a-vaju}e i potrebno ga je potvrditi u daljnjem radu.E-mail: [email protected]

P1-5

FLOW CYTOMETRICCHARACTERIZATION OF ACUTE

MYELOID LEUKEMIA - ARETROSPECTIVE STUDY

Z. Šiftar1, M.M. Kardum-Paro1, R. Kušec2, A. Nazor1, I.Kardum-Skelin3, D. Šušter~i}3, B.H. Jakši}2, H. Minigo2

1 Institute of Clinical Chemistry; 2 Department ofHematology, 3 Department of Cytology, University

Department of Medicine, Merkur University Hospital,Zagreb

Immunophenotyping by flow cytometry is auseful method for assesement of blast cells, theirlineage determination and maturity stage inacute leukemias. The study was designed toevaluate the usefulness of the standard panel ofantibodies used in flow cytometric determinationfor the diagnosis of acute myeloid leukemia(AML), and to try to display the efficiency of addi-tional non-myeloid markers CD2, CD19 andCD56 to recognize AML t(8;21), AML t(15;17) andAMLEo inv(16) subtypes. Sixty-one patients withAML included in the study were diagnosed at ourhospital between 2000 and 2003. AMLs were fur-ther classified as: AML0, AML1, AML2, AML2t(8;21), AML3 t(15;17), AML4, AML4Eo inv(16),AML5, AML6, AML with multilineage dysplasiaand biphenotypic leukemia, with 5, 4, 20, 3, 3, 11,2, 1, 3, 8 and 1 case, respectively. The most usefulmarkers contributing to the diagnosis of AML, ex-cept for AML6, were HLAD/DR, CD33, CD34,CD13, MPO and CD117 at a frequency of 95, 92,80, 80, 75 and 56%, respectively. Moreover, MPOwas found to be significantly lower (p<0.05) inAML0 than in more mature types, as confirmedby Mann Whitney nonparametric analysis. Onthe contrary, CD34 was higher. The diagnosis oferythroleukemia was based on glycophorin Apositivity, and AML3 was characterized byHLAD/DR negativity, with a significantly higherlevel of CD33 than in the other types of AML. Anadditional effort was made to diagnose entitieswith reccurent cytogenetic translocation by test-ing the expression of CD2, CD19 and CD56 onblast cells. CD2 was positive in both cases ofAML4Eo inv(16), CD19 in 1/3 and CD56 in 1/1case of AML2 t(8;21). The proposed combinationof markers proved to be useful in the diagnosis ofAML. Determination of CD2, CD19 and CD56 ex-pression on blasts seems promising, however, itshould be confirmed in future studies.E-mail: [email protected]



P1-6

UÊSTALOST MOLEKULARNIHIZOFORMA BCR/ABL p210 I NJIHOVO

KLINI^KO ZNAÊNJE U BOLESNIKA SKRONI^NOM MIJELOI^NOM

LEUKEMIJOM

Vrani} T.1, Maruši}-Vrsalovi} M.1, Manduši} T.1, Šiftar Z.1,Kardum-Skelin I.2, Minigo H.3, Jakši} B.R.3, Ku{ec R.1

1 Zavod za klini~ku kemiju, Odjel za molekularnudijagnostiku, 2 Citolo{ki odjel, 3 Hematolo{ki odjel Klinike

za unutarnje bolesti, KB “Merkur”, Zagreb

Fuzijski onkogen BCR/ABL translokacijet(9;22)(q34;q11) dovodi do spajanja eksona 3 ili 2BCR gena s eksonom 2 ABL gena. Transkriptib3a2 ili b2a2 kodiraju protein 210kD (p210). Rje-|i transkripti su e1a2 koji kodira manji protein(p190) te e19a2 koji kodira ve}i protein (p230).Cilj rada bio je istraìti u~estalost p210 BCR/ABLizoforma b3a2 i b2a2, javljanje p190 BCR/ABLmolekularnog oblika, te usporediti molekularneBCR/ABL izotipove s klini~kim, hematološkim ibiokemijskim parametrima. U istraìvanje suuklju~ena 24 bolesnika s kroni~nom mijeloi~nomleukemijom (KML) u razdoblju od 1986. do 2003.godine. BCR/ABL preuredba dokazana je citoge-netski i RT-PCR-om (metoda BIOMED-1) kodsvih bolesnika. Svi su imali fuzijski transkriptp210 BCR/ABL i to b3a2 (75%) ili b2a2 (25%).Jedan bolesnik je iskazao koekspresiju b3a2 ib2a2 transkripta. Kod dvoje bolesnika (8,3%) na-|en je p190 BCR/ABL subklon. Kaplan-Meierovaanaliza pokazala je bolje (ali ne zna~ajno) pre-ìvljenje kod tipa b2a2 u odnosu na b3a2. Wil-coxonov test za korelaciju tipova transkripataprema broju leukocita, trombocita, bazofila, mije-loidnih blasta u perifernoj krvi i koštanoj srì,LDH, veli~ini slezene, dobi i spolu nije pokazaozna~ajnu razliku. Ipak, koncentracija hemoglo-bina bila je zna~ajno viša (p=0,036) kod b3a2 tipaprema b2a2 [130g/L(108-146) vs. 103g/L(76-167)].Nije bilo korelacije izme|u pojave fibroze u ko-štanoj srì ili pove}ane eozinofilne mijelopoeze stipovima molekularnih transkripata. Dvoje odšestoro bolesnika s b2a2 tipom iskazala su slo-ènu kromosomsku preuredbu t(9;6;22) i + der(22) t(9;22). Zamijetili smo nešto niù pojavnostb2a2 molekularne izoforme p210 BCR/ABL (25%)u odnosu na literaturne podatke (40%). Moleku-larni podtip b2a2 fuzijskog transkripta nemabolju prognosti~ku zna~ajnost, iako korelira s vi-šim koncentracijama hemoglobina. Potrebno jenastaviti istraìvanje s ve}im brojem bolesnika.E-mail: [email protected]

P1-6

FREQUENCY OF BCR/ABL p210MOLECULAR ISOFORMS AND THEIR

CLINICAL SIGNIFICANCE IN PATIENTSWITH CHRONIC MYELOGENOUS

LEUKEMIA

T. Vrani}1, M. Maruši}-Vrsalovi}1, T. Manduši}1, Z. Šiftar1,I. Kardum-Skelin2, H. Minigo3, B.R. Jakši}3, R. Ku{ec1

1 Institute of Clinical Chemistry, Department of MolecularDiagnosis, 2 Department of Cytology, 3 Department of

Hematology, Merkur University Hospital, Zagreb

BCR/ABL fusion oncogene caused by translo-cation t(9;22)(q34;q11) fuses exon 3 or 2 of BCRgene to exon 2 of ABL gene. Transcripts b3a2 orb2a2 encode p210 kD protein (p210). Less fre-quent are transcript e1a2 translated into smallerprotein (p190) or larger (e19a2) fusion with p230.This study was designed to determine the fre-quency of p210 isoforms b3a2 and b2a2, inci-dence of p190 molecular variant, and to correlatemolecular isotypes with clinical, hematologic andbiochemical parameters. Twenty-four chronic my-elogenous leukemia (CML) patients diagnosedfrom 1986 till 2003 were included in the study.BCR/ABL rearrangement was confirmed by cy-togenetics and RT-PCR (BIOMED-1 method) inall patients. All patients had p210 fusion tran-script of either b3a2 type (75%) or b2a2 type(25%). One (4.2%) patient showed coexpression ofb3a2 and b2a2 type of fusion transcript. Two(8.3%) patients had the p190BCR/ABL subclone.Kaplan-Meier analysis showed apparent (not sig-nificant) survival advantage for b2a2 over b3a2type. Wilcoxon test for correlation of transcripttypes to white blood cell counts, platelets,basophils, myeloid blasts in PB and BM, LDH,spleen size, sex or age showed no significant dif-ference. However, hemoglobin level was signifi-cantly higher (p=0.036) for b3a2 transcript typecompared to b2a2 [130g/L (108-146) vs. 103 g/L(76-167), respectively]. There was no correlationfor the existence of BM fibrosis or increased eosi-nophilic myelopoiesis with molecular transcripttypes. Of interest, two of six b2a2 type patientshad complex chromosomal rearrangements, i.e.t(9;6;22) and + der (22) t(9;22). We observed asomewhat lower incidence of b2a2 molecularisoform of p210BCR/ABL (25%) than expected(40% according to literature). Certain prognosticsignificance favoring b2a2 type was found. Addi-tional studies in a greater number of patients arewarranted.E-mail: [email protected]



P1-7

PRA]ENJE MINIMALNE OSTATNEBOLESTI U AKUTNIM MIJELOI^NIM

LEUKEMIJAMA SEMI-KVANTITATIVNOMMETODOM RT-PCR

Šiftar Z.1, Bobeti}-Vrani} T.1, Maruši}-Vrsalovi} M.1,Kardum-Skelin I.2, Jakši} B.3, Minigo H.3, Kušec R.1

1 Zavod za klini~ku kemiju, 2 Citolo{ki odjel, 3 Hematolo{kiodjel Interne klinike, KB “Merkur”, Zagreb

Novom klasifikacijom Svjetske zdravstveneorganizacije malignih bolesti hematopoetskog ilimfoidnog tkiva izdvojene su akutne mijeloi~neleukemije (AML) s povratnim citogenetskim pro-mjenama: AML s t(8;21)(q22,q22), AML1/ETO;AML s t(15;17)(q11-12) i varijante, PML/RARalfa; i AML s inv(16)(p13;q22) ilit(16;16)(p13;q11), CBFbeta/MYH11X kao zasebnientiteti. Molekularna dijagnostika je korisna ivrlo osjetljiva metoda koja se rabi za postavljanjedijagnoze AML i pra}enje minimalne ostatne bo-lesti (MOB) nakon postupaka intenzivne kemo-terapije. Studija je tako osmišljena da pokaèkako testiranje na molekularne biljege pomaè uprocjeni uspješnosti terapije, razotkriva MOB i~ak predvi|a recidiv bolesti. Desetoro bolesnika sAML dijagnosticirani su u posljednje dvije godineu našoj ustanovi kao pozitivni na AML1/ETO(n=4), PML/RARalfa (n=4) ili CBFbeta/MYH11X(n=2) fuzijske gene. Molekularni biljezi su potvr-|eni standardnom semi-kvantitativnom meto-dom RT-PCR prema preporukama BIOMED-1.Izraàj transkripata se pratio tijekom terapije inakon nje, prema utvr|enim terapijskim progra-mima. MOB je na|ena nakon šest mjeseci iliduljeg razdoblja u 6 bolesnika. Dvoje bolesnikabilo je još uvijek pozitivno na CBFbeta/MYH11X,a troje na AML1/ETO molekularni transkript,bez klini~kih znakova bolesti. Citomorfološkimpregledom koštane srì nisu na|eni blasti, osim uslu~aju s pozitivnim AML1/ETO, kada ih je na-|en minimalan broj. Bolesnik koji je bio nepre-kidno dulje od 16 mjeseci pozitivan na PML/RARalfa ušao je u recidiv. Istodobno su u koš-tanoj srì na|eni blasti jedino kada je marker biopovišen preko dva loga. Pra}enje molekularnihbiljega pomaè u utvr|ivanju MOB i njezine pro-gresije, predvi|aju}i recidiv bolesti i prije klini~-ke pojave.E-mail: [email protected]

P1-7

MONITORING OF MINIMAL RESIDUALDISEASE IN ACUTE MYELOID LEUKEMIA

BY SEMI-QUANTITATIVE RT-PCR

Z. Šiftar1, T. Bobeti}-Vrani}1, M. Maruši}-Vrsalovi}1, I.Kardum-Skelin2, B. Jakši}3, H. Minigo3, R. Kušec1

1 Institute of Clinical Chemistry, 2 Department of Cytology,3 Department of Hematology, University Department of

Medicine, Merkur University Hospital, Zagreb

The new World Health Organization classifi-cation of neoplastic disease of hematopoietic andlymphoid tissues recognizes acute myeloid leuke-mias (AML) with recurrent cytogenetic translo-cations: AML with t(8;21)(q22,q22), AML1/ETO;AML with t(15;17)(q11-12) and variants, PML/RARalfa; and AML with inv(16)(p13;q22) ort(16;16)(p13;q11), CBFbeta/MYH11X as distinctentities. Molecular diagnosis is a useful and verysensitive method used for the diagnosis of AMLand monitoring of minimal residual disease(MRD) after intensive chemotherapeutic proce-dures. The study was designed to show that mo-lecular marker testing helps demonstrate thera-peutic efficacy, to disclose MRD, and even to pre-dict the disease relapse. Ten AML patients in-cluded in the study were diagnosed at our institu-tion as positive for AML1/ETO (n=4), PML/RARalfa (n=4) or CBFbeta/MYH11X (n=2) fu-sion genes during the past two years. Molecularmarkers were confirmed by the standard semi-quantitative RT-PCR method according toBIOMED-1 protocol. Expression of transcriptswas monitored during and after the course oftherapy, according to therapeutic schedules. Aftersix months or later, MRD was observed in six pa-tients. Two patients were still positive forCBFbeta/MYH11X and three for AML1/ETOmolecular transcripts, without clinical signs ofthe disease. These observations were not con-firmed by cytomorphological examinations ofbone marrow, except in case with AML1/ETOpositivity, when a minimal number of blasts wasfound. Relapse occurred in the patient continu-ously positive for PML/RARalfa for 16 months.At the same time, blasts in the bone marrow wereonly found when marker positivity increased overtwo logs. Accordingly, molecular marker monitor-ing helps in diagnosing MRD and its progression,and in predicting the disease relapse even beforeits clinical manifestation.E-mail: [email protected]



P1-8

OTKRIVANJE AML1/ETO PRIJEPISA UBOLESNIKA S T(8;21) PRI DIJAGNOZI I

TIJEKOM TERAPIJE POMO]UKVANTITATIVNE POLIMERAZNE

LANÂNE REAKCIJE

Zadro R.1, Kastelic R.2, Bulum J.3, Raji} Lj.4,Stavljeni}-Rukavina A.1, Labar B.3

1 Klini~ki zavod za laboratorijsku dijagnostiku KBC“Zagreb”, 2 Abbott Diagnostics, Predstavni{tvo u RH,

3 Interna klinika, 4 Klinika za pedijatriju, KBC “Zagreb”,Zagreb

Translokacija t(8;21) jedna je od naj~eš}ih pre-uredba gena u akutnim mijeloi~nim leukemijama(AML) s u~estaloš}u od oko 20% odraslih i 40%dje~jih AML-M2. Posljedica ove translokacije jespajanje gena AML1 na kromosomu 21q i genaETO na kromosomu 8q u zdruèni gen AML1/ETO. Prijepis aml1/eto moè se otkriti kvalita-tivnom i kvantitativnom lan~anom reakcijom po-limerazom (PCR) kod bolesnika pri dijagnozi i uklini~koj remisiji. Cilj rada bio je usporediti re-zultate kvalitativnog i kvantitativnog PCR u ot-krivanju prijepisa aml1/eto te izmjeriti koli~inuprijepisa aml1/eto u uzorcima koštane srì bole-snika u razli~itim vremenskim to~kama od tre-nutka postavljanja dijagnoze do završetka tera-pije. Uporabom tehnologije Light Cycler izve-dena je analiza 50 uzoraka RNK izolirane stan-dardnim metodama iz koštane srì 13 bolesnikakod kojih je pri dijagnozi dokazan prijepis aml1/eto kvalitativnom PCR. Usporedba dobivenih re-zultata pokazala je potpunu podudarnost ishodatesta kvalitativno i kvantitativno PCR. Odre-|ena je grani~na vrijednost od 0,27 za omjeraml1/eto:G-6-PDH iznad koje su rezultati pro-glašeni pozitivnima. Taj raspon pri dijagnozi jeiznosio 0,31-2,35. Šestoro bolesnika s t(8;21) kodkojih je postignuta potpuna remisija bolesti pra-}eni su u razli~itim vremenskim razmacima na-kon postavljene dijagnoze i uvodne terapije.Uo~en je postupan pad razine prijepisa aml1/etood trenutka postavljanja dijagnoze (raspon 1,0-2,35) na manje od 0,3 jedan do tri mjeseca nakonpo~etka terapije. U zaklju~ku, kvantifikacija os-tatne bolesti tehnologijom Light Cycler pouzdanaje i osjetljiva metoda pra}enja dinamike iš~eza-vanja malignog klona stanica.E-mail: [email protected]

P1-8

MOLECULAR MONITORING OF AML1/ETOTRANSCRIPT IN AML PATIENTS AT

DIAGNOSIS AND FOLLOW-UP BYQUANTITATIVE PCR

R. Zadro1, R. Kastelic2, J. Bulum3, Lj. Raji}4, A.Stavljeni}-Rukavina1, B. Labar3

1 Clinical Institute of Laboratory Diagnosis, ZagrebUniversity Hospital Center, 2 Abbott Diagnostics,

Representative Office in Croatia, 3 University Department ofMedicine, 4 University Department of Pediatrics, Zagreb

University Hospital Center, Zagreb

The t(8;21) is one of the most commontranslocations in acute myeloid leukemia (AML)occurring in approximately 20% of adult and 40%of pediatric AML-M2 patients. The result of thistranslocation is the fusion gene AML1/ETOwhose transcripts can be detected by qualitativeand quantitative PCR in patients at diagnosis aswell as in clinical remission. Using Light Cyclertechnology, we analyzed 50 samples of 13 patientswho were positive for AML1/ETO transcript atdiagnosis by use of qualitative RT-PCR. In allstudy patients, positive results by qualitative RT-PCR were in agreement with the results obtainedby Light Cycler technology. By comparison of thetwo methods we established the cut-off value foraml1/eto transcript:G-6-PDH ratio of 0.27, whilethe same ratio ranged from 0.30-2.35 at diagno-sis. Additionally, six patients with t(8;21) AMLwere studied by quantitative RT-PCR at differenttime intervals (1-16 months) after therapy initia-tion. We observed a constant decrease in aml1/etotranscript:G-6-PDH ratio from the time of diag-nosis (range 1.0-2.35) to below 0.30 at 1 to 3months of therapy initiation. We conclude thatquantification of residual disease with Real TimeRT-PCR is a reliable and sensitive method tomonitor the dynamics of the malignant clone dis-appearance.E-mail: [email protected]



P1-9

ZNAÎ LI NIZAK BROJ TROMBOCITAUVIJEK I TROMBOCITOPENIJU?

Bernt T., Kralik S., Mayer V., Suchanek E.

Klinika za ènske bolesti i porode, KBC “Zagreb”, Zavod zaklini~ku biokemiju i biologiju reprodukcije, Zagreb

U~estalost trombocitopenije u trudno}i kre}ese od 4% do 8% i predstavlja zna~ajan problem iza trudnicu i za fetus. Terapiju treba zapo~eti štoranije, ali je prije potrebno isklju~iti pseudotrom-bocitopeniju (PTCP) kao laboratorijski artefakt.PTCP je naj~eš}e posljedica agregacije Tr podutjecajem antikoagulacijske otopine EDTA, aagregacija se stajanjem poja~ava. U radu je pri-kazana 27-godišnja tre}erotkinja (2 zdravo ro|e-ne djece) koja je primljena u Kliniku u 34. tjednutrudno}e zbog izrazito niskog broja Tr, neupad-ljive anamneze i bez klini~kih znakova krvare-nja. Neposredno nakon va|enja krvi (37 oC) do-bivene su vrijednosti Tr 19x109/L (Cell Dyn 3200,Abbott, K3 EDTA 7,5% BD). Odre|ivanjem brojaTr u istom uzorku krvi 1 sat nakon va|enja (sob-na temperatura) uo~en je skok vrijednosti Tr(115x 109/L), a 2 sata nakon va|enja 134x 109/L.Sli~ne vrijednosti dobivene su i s Na-citratomkao antikoagulansom. Mikroskopskom analizomrazmaza bojenih MGG odmah nakon va|enja, te1 i 2 sata nakon va|enja uo~ena je prisutnostve}eg broja agregata Tr u prvom uzorku. Uhistogramima raspodjele veli~ine Tr ustanovljenesu zna~ajne razlike izme|u I. brojanja te II. i III.brojanja. Promjene histograma I. brojanja ukazu-ju na velik broj agregata. U nama dostupnojliteraturi nismo za opisani slu~aj našli sli~nepodatke. Broj i veli~ina agregata se stajanjemsmanjuju. Proto~nom citometrijom u uzorku sEDTA i Na-citratom na|ena je povišena koli~inaprotutijela IgG i IgM vezanih za trombocite. Me-|utim, testom antitrombocitnih protutijela nemoè se razlu~iti radi li se o pseudotromboci-topeniji ili mogu}e o pravim autoprotutijelima.Zanimljivo je da je u ove trudnice uo~en nizakbroj Tr u uzorcima krvi brojanim odmah nakonva|enja (37 oC), koji se je stajanjem pove}avao. Udostupnoj literaturi (CC i Medline) nismo našlisli~ne podatke. Iako su optimalne temperaturena kojima se doga|a agregacija Tr pod utjecajemEDTA ispod 37 oC i poja~ava se stajanjem, u ovetrudnice je situacija bila upravo obrnuta. U ta-kvim slu~ajevima preporu~a se pa`ljiva analizakrvnih razmaza i histograma Tr, kao antikoa-gulans treba rabiti amonijev oksalat, a Tr brojitiu Burkerovoj komorici.

P1-9

DOES A LOW PLATELET COUNT ALWAYSIMPLY THROMBOCYTOPENIA?

T. Bernt, S. Kralik, V. Mayer, E. Suchanek

Department of Clinical Chemistry and ReproductionBiology, University Department of Gynecology and

Obstetrics, Zagreb University Hospital Center, Zagreb

The prevalence of thrombocytopenia in preg-nancy is between 4% and 8%, and is a seriousproblem for both the pregnant woman and the fe-tus. Treatment should be initiated as early as pos-sible. To avoid misdiagnosis, it is necessary torecognize pseudothrombocytopenia (PTCP) as apossible laboratory artifact prior to therapy ini-tiation. The most common reason for this artifactis in vitro platelet clumping in blood samples col-lected with EDTA as anticoagulant. The clump-ing activity increases several hours from bloodcollection. A 27-year-old pregnant woman in the34th week of third pregnancy (two healthy chil-dren) with a very low platelet count, and withoutany disorder or signs of bleeding is presented. Im-mediately upon blood collection (37 oC), the plate-let count was 19x109/L (Cell Dyn 3200, Abbott, K3EDTA 7.5% BD). In the blood sample, the plate-let count was 115x109/L at one hour and134x109/L at two hours of blood collection (roomtemperature). Similar values were observed in theblood sample collected with citrate as anticoagu-lant. Numerous platelet clumps were verified bymicroscopic analysis of MGG stained bloodsmears prepared from EDTA and citrate-antico-agulated blood in the sample analyzed immedi-ately upon blood collection. The occurrence of acharacteristic histogram pattern indicated thepresence of massive platelet clumping in theblood sample analyzed immediately upon bloodcollection. The histogram pattern of the bloodsample at two hours of collection showed noplatelet clumps. No similar data were found inthe available literature. In EDTA-blood specimenand citrate-anticoagulated blood specimen, thepresence of IgG and IgM antiplatelet antibodieswas detected by flow cytometry. It is not possibleto determine whether there is a PTCP or someother type of thrombocytopenia by testingantiplatelet antibodies. In this pregnant woman,platelet count was very low in both EDTA- andcitrate-anticoagulant blood immediately uponblood sampling. Blood smear prepared from thisspecimen demonstrated clumping. However,platelet count increased progressively over time inboth specimens, and there was no clumping. Al-though the clumping activity is greater at a tem-



perature below 37 oC, and is also stronger a fewhours of blood collection, in this case it was justopposite. We found no such data in the availableliterature. To avoid severe misinterpretation insuch patients, blood smears as well as platelethistograms should be analyzed. Blood must becollected in ammonium oxalate and plateletscounted in Burker chamber.

P1-10

SADR@AJ HEMOGLOBINA URETIKULOCITIMA - CHr: PARAMETAR ZA

PROCJENU NEDOSTATKA @ELJEZA UBOLESNIKA LIJEÊNIH

HEMODIJALIZOM

Perovi} E.1, Sambunjak J.1, Klari} D.2, Val~i} A.1

1 Odjel za laboratorijsku dijagnostiku, 2 Odjel za unutarnjebolesti; Op}a bolnica Zadar, Zadar

Bolesnici lije~eni hemodijalizom su osobitopodlo`ni nedostatku èljeza. Prvotni razlog tomuje gubitak krvi tijekom dijalize, nadalje u~estalasu okultna krvarenja iz gastrointestinalnog trak-ta (GIT), a ometena je i apsorpcija èljeza u GIT-u. Uza sideropeniju uporabom rekombinantnoghumanog eritropoetina (rHu-EPO) javlja se fun-kcionalni nedostatak èljeza. Uobi~ajene pretra-ge za odre|ivanje statusa èljeza su: serumskiferitin, % zasi}enja transferina, a u novije vri-jeme se odre|uje koncentracija topljivog recep-tora transferina (sTfr). Nedostatak odre|ivanjaferitina jest to što je on reaktant akutne fazeupale, pa je ~esto la`no povišen (dijagnosti~kaosjetljivost 88%, a specifi~nost samo 63%). Posto-tak zasi}enja transferina ima dijagnosti~ku osjet-ljivost 81%, a specifi~nost 63%. sTfr je bolji po-kazatelj statusa èljeza, ali odre|ivanje nije do-voljno standardizirano. Odre|ivanje sadràja he-moglobina u retikulocitima (CHr) je idealno zaprocjenu nedostatka èljeza u bolesnika lije~enihhemodijalizom, jer izravno mjeri èljezo na raziniprekursora eritrocita. Kako su oni prisutni ucirkulaciji samo oko 24 sata pretraga predstavljatrenutni prikaz stausa èljeza. Jedino pretragakoja se odnosi na retikulocite moè biti visokoosjetljiva i specifi~na za dijagnozu nedostatkaèljeza. U 115 bolesnika lije~enih hemodijalizomodre|eni su: %Rtc (χ=1,78; SD=0,76) i CHr(χ=32,70; SD=2,45) na hematološkom analizatoruAdvia 120 (Bayer Diagnostic, Tarrytown, NY,SAD) . CHr <30 pg imalo je 23 (20%) bolesnika,što vrijednost CHr <30 pg ~ini to~nim, vrlo osjet-ljivim i specifi~nim pokazateljem nedostatka è-

P1-10

RETICULOCYTE HEMOGLOBIN CONTENT- CHr: A PARAMETER IN THE

EVALUATION OF IRON DEFICIENCY INHEMODIALYSIS PATIENTS

E. Perovi}1, J. Sambunjak1, D. Klari}2, A. Val~i}1

1 Department of Laboratory Diagnosis, 2 Department ofMedicine, Zadar General Hospital, Zadar

Hemodialysis patients are particularly proneto iron deficiency. The primary reason for this isthe loss of blood during dialysis treatment alongwith the loss through occult gastrointestinalbleeding, and even dietary iron absorption is re-duced. Besides iron deficiency, functional iron de-ficiency is often present because of the treatmentwith recombinant human erythropoietin (rHu-EPO). The commonly used tests for iron statusassessment are: serum ferritin, % of transferrinsaturation, and recently soluble transferrinreceptor (sTfr). The shortcoming of ferritin deter-mination refers to its potent activity as an acutephase reactant, thus being often falsely elevated(diagnostic sensitivity and specificity is 88% and63%, respectively). Transferrin saturation has adiagnostic sensitivity and specificity of 81% and63%, respectively. sTfr is a better marker of ironstatus, however, yet not adequately standardized.Reticulocyte hemoglobin content (CHr) is an idealtest for iron deficiency in hemodialysis patientsbecause it is a direct measure of iron at the levelof erythrocyte precursors. They exist in the circu-lation for approximately only one day, thus pro-viding a ’snapshot’ of iron status. Only a test thatevaluates reticulocytes can be highly sensitiveand specific for the diagnosis of iron deficiency. %Rtc (χ=1.78; SD=0.76) and CHr (χ=32.70;SD=2.45) were determined in 115 hemodialysispatients on an Advia 120 hematology analyzer(Bayer Diagnostic, Tarrytown, NY, USA). CHr<30 pg is an accurate, very sensitive and specificmeasure of iron deficiency. When assessment ofCHr is included in clinical protocols for the treat-ment of anemia in chronic renal failure patients,



P2Bolesti jetre

Liver diseases

P2-1

DIJAGNOSTI^KA VRIJEDNOSTODRE\IVANJA TROPONINA I U

BOLESNIKA S AKUTNIMKOLECISTITISOM

Jagi} V.1, Babi} @.2, Lazi} J.1, Rumenjak V.1, Maruši} M.2

1 Zavod za medicinsko laboratorijsku dijagnostiku 2 Klinikaza unutarnje bolesti, Op}a bolnica “Sveti Duh”, Zagreb

Dijagnosti~ka vrijednost pove}ane koncentra-cije sr~anog troponina I. (cTnI) potvr|ena je i do-kazana u dijagnostici akutnog infarkta miokar-da, u otkrivanju ošte}enja miokarda u nestabil-noj angini pektoris te u perioperacijskom ošte-}enju miokarda, kao i u kontuziji srca. Me|utim,odre|ivanje koncentracije cTnI pokazalo se kori-snim dijagnosti~kim pokazateljem i u nekim dru-gim klini~kim situacijama. Cilj ovoga rada bio jedijagnosti~ka procjena korisnosti odre|ivanjakoncentracije cTnI kod bolesnika s akutnim ko-lecistitisom. Ispitivanje je obuhvatilo 15 bolesni-ka (6 muškaraca i 9 `ena) starosti 28- 80 godina.Kod svih bolesnika je na osnovi klini~kih, labo-ratorijskih i ultrazvu~nih nalaza postavljena di-jagnoza akutnog kolecistitisa uzrokovanog kole-litijazom. Akutno koronarno zbivanje isklju~enoje na osnovi anamnesti~kih podataka, nalazaEKG i sr~anih biljega. Na osnovi klini~kih i la-boratorijskih parametara isklju~ena je i dijag-noza akutnog pankreatitisa. Koncentracija cTnIodre|ivana je imunofluorometrijskom metodom(Dimension, Dade Behring). Referentna vrijed-nost za cTnI bila je do 0,05 µg/L, a vrijednosti 0,6-1,5 µg/L smatrane su kao sumnja na akutniinfarkt miokarda. U skupini bolesnika (n=5) kodkojih je izmjerena debljina stijenke >5 mm vri-jednosti cTnI bile su 0,15-0,25 µg/L (srednjavrijednost 0,21 µg/L, SD 0,04 µg/L). Ostalibolesnici (n=10) imali su vrijednosti cTnI do 0,05

ljeza. Kada se odre|ivanje CHr uklju~i u klini~kesmjernice lije~enja anemije u bolesnika s kroni~-nim zatajenjem bubrega, dobiti }e se bolji uvid ustatus `eljeza te }e se znatno smanjiti ukupnitroškovi lije~enja ovih bolesnika.E-mail: [email protected]

it will provide a better insight into the iron statusand will reduce the overall cost of patient care.E-mail: [email protected]

P2-1

DIAGNOSTIC VALUE OF TROPONIN-IIN PATIENTS WITH ACUTE

CHOLECYSTITIS

V. Jagi}1, @. Babi}2, J. Lazi}1, V. Rumenjak1, M. Maruši}2

1 Institute of Medical Laboratory Diagnosis, 2 UniversityDepartment of Medicine, Sveti Duh General Hospital,

Zagreb

Diagnostic value of the increased concentra-tion of cardiac troponin I (cTnI) has been provedin the diagnosis of acute myocardial infarction,in detection of myocardial lesion in unstable an-gina pectoris, in perioperative myocardial lesionas well as in myocardial contusion. However, de-termination of cTnI concentration has proved tobe a useful diagnostic marker also in some otherclinical situations. The aim of the study was toassess the diagnostic value of cTnI concentrationdetermination in patients with acute cholecysti-tis. The study included 15 patients (6 men and 9women) aged 28-80 yrs. In all patients, the diag-nosis of acute cholecystitis due to cholelithiasiswas based on clinical, laboratory and ultrasoundfindings. Acute coronary disease was excluded onthe basis of history data, ECG findings and car-diac markers. The diagnosis of acute pancreatitiswas also excluded on the basis of clinical andlaboratory parameters. The concentration of cTnIwas determined by the immunofluorometricmethod (Dimension, Dade Boehring). Referencevalue of cTnI was set at 0.05 µg/L, and values of0.6-1.5 µg/L were considered suspect of acutemyocardial infarction. In the group of patients(n=5) with gallbladder wall thickness of >5 mm,cTnI values were 0.15-0.25 µg/L (mean 0.21 µg/L, SD 0.04 µg/L). In other patients (n=10) cTnIvalues were up to 0.05 µg/L and myocardial wallthickness <5 mm. Only one patient had cTnI val-



µg/L i debljinu stijenke <5 mm. Samo jedan odovih bolesnika imao je vrijednosti cTnI 0,1 µg/L.Pove}ane vrijednosti cTnI >0,05 µg/L kod bole-snika s akutnim kolecistitisom mogu ukazivatina zadebljanje stijenke ù~nog mjehura, ali i bitiznakom ošte}enja miokarda zbog èstine akutnogoblika kolecistitisa, hemodinamskih promjena ihipoksije miokarda.E-mail: [email protected]

ues of 0.1 µg/L. The increased cTnI values >0.05µg/L in patients with acute cholecystitis couldpoint to gallbladder wall thickening, but couldalso be a sign of myocardial lesion due to the ag-gressive acute form of cholecystitis, hemodynamicchanges and myocardial hypoxia.E-mail: [email protected]

P2-2

ODRE\IVANJE MAGNEZIJA UKRONI^NIH ALKOHOLIÂRA

Ruljan~i} N.1, Rumenjak V.2

1 Psihijatrijska bolnica Jankomir, Medicinsko-biokemijskilaboratorij; 2 Zavod za medicinsko-laboratorijskudijagnostiku, Op}a bolnica “Sveti Duh”, Zagreb

Magnezij je drugi po zastupljenosti kation ustanici. Kofaktor je u više od 325 stani~nih enzi-ma. Malnutricija, nedostatak fosfata i vitaminaD, acidoza/alkaloza, gastrointestinalni gubitak ipoja~ano izlu~ivanje magnezija putem bubregasu alkoholom izazvani patofiziološki procesi kojiutje~u na koncentraciju magnezija. Cilj rada bioje odrediti koncentraciju ukupnog i fiziološki ak-tivnog ioniziranog magnezija u plazmi bolesnikas kroni~nim alkoholizmom. U hepariniziranojplazmi 33 ispitanika s dijagnosticiranim kroni~-nim alkoholizmom koncentracija ukupnog mag-nezija je odre|ivana kolorimetrijski (metoda kal-magit, referentni raspon 0,60-1,11 mmol/l), akoncentracija ioniziranog magnezija potencio-metrijski ion-selektivnim elektrodama (NovaBiomedical Ultra State Profile C, referentniraspon 0,53-0,67 mmol/l ). Kod svih ispitanika suse izmjerene koncentracije ukupnog magnezijakretale od 0,59 do 1,11 mmol/l (prosje~na vri-jednost 0,85 mmol/l), a koncentracije ioniziranogmagnezija od 0,40 do 0,63 mmol/l (prosje~navrijednost 0,50 mmol/l). Sniène vrijednostiukupnog magnezija (<0,60 mmol/l) su zabiljeènekod 6,06% ispitanika, dok ih je 60,60% imalo sni-ène vrijednosti ioniziranog magnezija (<0,53mmol/l). Prema dobivenim rezultatima zapaènesu sniène vrijednosti u koncentraciji ioniziranogmagnezija u kroni~nih alkoholi~ara, ali su zaodre|ivanje statusa magnezija potrebna daljnjaispitivanja.E-mail: [email protected]

P2-2

DETERMINATION OF MAGNESIUM INCHRONIC ALCOHOLICS

N. Ruljan~i}1, V. Rumenjak2

1 Laboratory of Medical Biochemistry, Jankomir PsychiatricHospital; 2 Institute of Medical Laboratory Diagnosis, Sveti

Duh General Hospital, Zagreb

Magnesium (Mg) is the second most abundantcation in the cells. It is a cofactor in more than325 cell enzymes. Malnutrition, phosphate andvitamin D deficiencies, acidosios/alkalosis,gastrointestinal loss and urinary Mg wastage areall alcohol induced pathophysiological processeswhich have some effect on the magnesium concen-tration. The aim of the study was to determine theconcentration of total blood magnesium andphysiologically active form, ionized magnesiumin the plasma of patients with chronic alcohol-ism. In heparinized plasma of 33 patients withdiagnosed chronic alcoholism, the concentrationof total blood magnesium was determined byspectroscopy (calmagit method, reference range0.60-1.11 mmol/l) and of ionized magnesium byuse of ion-selective electrodes (Nova BiomedicalUltra State Profile C, reference range 0.53-0.67mmol/l). In all patients, the concentration of to-tal blood magnesium ranged from 0.58 to 1.11mmol/l (mean 0.85 mmol/l ) and of ionized mag-nesium from 0.40 to 0.63 mmol/l (mean 0.50mmol/l ). Lower blood levels of total blood mag-nesium (<0.60 mmol/l) were recorded in 6.06% ofpatients; in 60.60% of patients the levels of ion-ized magnesium were lower than 0.53 mmol/l.Thus, the measured levels showed lower concen-tration of ionized magnesium in chronic alcohol-ics, however, additional studies are needed to de-termine the magnesium status in this patientpopulation.E-mail: [email protected]



P2-3

REZULTATI TESTIRANJA TRUDNICA INOVIH DAVATELJA KRVI NA BILJEGE

HEPATITISA B I SIFILISA

Mihaljevi} I., Lovri} M., Strahija-Tomi} K., Balija M.,Juki} I.

Hrvatski zavod za transfuzijsku medicinu, Zagreb

Prenatalna skrb uklju~uje ispitivanje krvitrudnica na biljege spolno prenosivih bolesti, i tosifilisa i hepatitisa B, a sukladno nalazima imu-noprofilaksu novoro|ene djece. Na podru~ju gra-da Zagreba sustavno ispitivanje trudnica naHBsAg provodi se posljednje tri godine. Svrharada bila je ispitati proširenost spolno prenosivihbolesti hepatitisa B i sifilisa u trudnica gradaZagreba i okolice i usporediti ih s rezultatimaistih ispitivanja u novih davatelja krvi muška-raca u dobi od 18-20 godina i èna s podru~jaRepublike Hrvatske kao primjera spolno najak-tivnije populacije. Skupina A: trudnice testiranena HBsAg i anti Treponema (T.) pallidum od 1.sije~nja 2000. do 31. prosinca 2002. u Hrvatskomzavodu za transfuzijsku medicinu (HZTM); sku-pina B: novi davatelji krvi, mladi muškarci kojisu krv prvi puta dali u istom razdoblju; skupinaC: ène koje su dale krv prvi puta u istom raz-doblju. Svi uzorci krvi ispitani su HBsAg EIAtestom osjetljivosti >0,2 ng/mL krvi, Ortho TestSystem 3 i Murex HBsAg V3 GE36 i EIA testomza T. pallidum protutijela Trepanostika -TP, Bio-merieux (Organon) ili ICE Syphilis Abbott (Mu-rex). Reaktivnost je potvr|ivana testom drugogproizvo|a~a, ostalim HBV biljezima, a u sifilistestu RPR i TPHA testom. Tijekom tri godineispitano je 16.280 trudnica, 20.315 novih dava-telja muškaraca izme|u 18 i 25 godina starosti i5.461 èna novih davatelja krvi. U~estalostHBsAg pozitivnih po skupinama bila je 0,54%,0,43% i 0,36%. Tako je 95% HBsAg pozitivnih uskupini B i 100% u skupinama A i C pokazivaloanti HBe reaktivnost uz izostanak biljega akutneinfekcije anti HBcIgM. U~estalost protutijela naT. pallidum bila je niska, u skupini trudnica0,024%, u muških novih davatelja krvi 0,004%, au ènskih 0,018%. U~estalost HBV u svim trimaskupinama ispitanika kretala se oko 0,4% i nijepokazala tendenciju pada, što bi moglo predstav-ljati minimalnu u~estalost HBV postignutu prijeu~inka nedavno uvedenog obveznog cijepljenjamla|ih adolescenata. Nositeljstvo obiljeàva antiHBe reaktivnost, što znatno smanjuje mogu}nostprijenosa s majke na dijete. Sifilis mjeren reak-tivnoš}u EIA anti TP testa nije proširen me|u

P2-3

SYPHILIS AND HEPATITIS B MARKERSCREENING OF PREGNANT WOMEN AND

FIRST TIME BLOOD DONORS IN THEZAGREB AREA

I. Mihaljevi}, M. Lovri}, K. Strahija-Tomi}, M. Balija,I. Juki}

Croatian Institute of Transfusion Medicine, Zagreb

Prenatal care includes prepartal maternalblood screening for markers of some sexuallytransmitted diseases, i.e. syphilis and hepatitis B,and subsequent neonatal immunoprophylaxis ac-cording to the findings thus obtained. In theZagreb area, prepartal maternal screening forHBsAg has been performed for the last threeyears. The aim of the study was to determine therate of hepatitis B and syphilis in pregnantwomen from the City of Zagreb and its surround-ings, and to compare it with the results of the re-spective nation-wide testing performed in first-time male and female blood donors aged 18-20 asa population of highest sexual activity. Group A:pregnant women tested at Croatian Institute ofTransfusion Medicine (CITM) for HBsAg andanti Treponema (T.) pallidum from January 1,2000 till December 31, 2002; group B: male first-time blood donors aged 18-25 presenting duringthe same period; and group C: female first-timeblood donors presenting during the same period.Blood samples were tested by HbsAg EIA, sensi-tivity >0.2 ng/Ml blood, Ortho Test System 3,Murex HBsAg V3 GE36, and EIA for T. pallidumTrepanostika-TP Biomerieux (Organon) or ICESyphilis Abbott (Murex). Reactivity was con-firmed by a test from a different manufacturer,other HBV markers, and by RPR and TPHA testfor syphilis testing. During the study period, 16280 pregnant women, 20 315 male first-timeblood donors aged 18-25 and 5 461 female first-time blood donors were tested. The overall HBsAgprevalence was 0.54% in group A, 0.43% in groupB, and 0.36% in group C. So, 95% of HBsAg posi-tive subjects in group B and 100% in groups Aand C had anti Hbe positive HBV profile withnegative anti HBcIgM. The prevalence of anti T.pallidum (anti TP) was low in all three studygroups (0.024%, 0.004 % and 0.018%, respec-tively). The prevalence of HBV in all three groupswas ∼ 0.4%, showing no declining tendency, prob-ably representing the minimal HBV prevalencereached before the possible effect of recently intro-duced compulsory vaccination of young adoles-cents. The carrier state was found to be character-



spolno aktivnom populacijom i ima u~estalost od0,014%.E-mail: [email protected]

P2-4

RAZINE ENDOTELINA (1-21) URAZLIÎTIM STUPNJEVIMA ALKOHOLNE

CIROZE JETRE

Antoljak N.1, Peran N.2, Vrki} N.1, Begonja A.1, Topi}E.1, Duvnjak M.3

1 Klini~ki zavod za kemiju, KB “Sestre milosrdnice”, Zagreb,2 Op}a bolnica [ibenik, [ibenik, 3 Klinika za

gastroenterologiju i hepatologiju, KB “Sestre milosrdnice”,Zagreb

Endotelin (ET) je protein sastavljen od 21aminokiseline, a ima snaàn vazokonstrikcijskiu~inak. Nastaje i otpušta se iz endotelijskih sta-nica u obliku preproendotelina, pri~em nastajeme|uproizvod, veliki endotelin. Ciroza jetre jebolest kod koje nastaju ascites i portalna hiper-tenzija pra}ena promjenom sistemne i regionalnehemodinamike. Povišene razine ET na|ene su uplazmi bolesnika s alkoholnom cirozom jetre. Ciljrada bio je usporediti razine ET(1-21) u bolesnikas razli~itim stupnjevima alkoholne ciroze jetre.ET(1-21) je mjeren u plazmi bolesnika pomo}uenzimskog imunotesta (Biomedica, Graz, Aus-trija) s pragom mjerljivosti od 0,05 pmol/L. Ustudiju je bilo uklju~eno 60 bolesnika s doka-zanom alkoholnom cirozom jetre i 86 zdravihispitanika odgovaraju}e dobi i spola. Statisti~kaanaliza napravljena je pomo}u kompjutorskogprograma Sigmastat i Excel. Razine ET(1-21) bi-le su zna~ajno više u bolesnika (0,55, raspon 0-6,19 pmol/L) nego u zdravih ispitanika (0,12, ra-spon 0-1,67 pmol/L, p<0,001). Bolesnici s ascite-som i/ili varikozitetima jednjaka su imali viševrijednosti nego bolesnici bez tih promjena. Samou podskupini bolesnika s teìm stupnjem bolestina|ene su više vrijednosti endotelina u odnosuna zdrave ispitanike ili ostale bolesnike. Nega-tivna korelacija na|ena je izme|u ET(1-21) i se-rumskih albumina, protrombinskog vremena inatrija (r=-0,36, r=-0,37, r=-0,33), a pozitivna sgama-globulinima (r=0,44, p<0,05). Klini~ka va-ljanost ET(1-21) mjerena površinom ispod krivu-lje ROC bila je visoka (A=0,72, p<0,05). Endotelinmoè biti koristan biljeg nastalih vaskularnih pro-mjena u bolesnika s alkoholnom cirozom jetre.E-mail: [email protected]

ized by anti HBe reactivity, thus greatly reducingthe possibility of maternal transmission to thechild. As assessed by EIA anti TP test, syphiliswas not found to be widely spread among thesexually active population, showing a prevalenceof 0.014%.E-mail: [email protected]

P2-4

ENDOTHELIN (1-21) LEVELS IN VARIOUSDEGREES OF ALCOHOLIC LIVER

CIRRHOSIS

N. Antoljak1, N. Peran2, N. Vrki}1, A. Begonja1, E.Topi}1, M. Duvnjak3

1 Clinical Institute of Chemistry, Sestre milosrdniceUniversity Hospital, Zagreb, 2 [ibenik General Hospital,

[ibenik, 3 University Department of Gastroenterelogy andHepatology, Sestre milosrdnice University Hospital, Zagreb

Endothelin (ET) is a potent vasoconstrictorpeptide, made up of 21 amino acids. It is pro-duced and released from endothelial cells in theform of preproendothelin whereby an intermedi-ate big-endothelin is formed. Liver cirrhosis is ac-companied by the onset of ascites, portal hyper-tension and changes in systemic and regionalhemodynamics. Increased ET levels related to im-paired systemic circulation have been reported inliver cirrhosis patients. The aim of the study wasto compare ET(1-21) levels in patients with vari-ous degrees of alcoholic liver cirrhosis. ET(1-21)was measured in plasma by ELISA test(Biomedica, Graz, Austria) with a detectablelimit of 0.05 pmol/L. Sixty proven alcoholic livercirrhosis patients and 86 healthy volunteersmatched for sex and age were included in thestudy. Statistical analysis was done by Sigmastatand Excel computer program. ET(1-21) levelswere significantly higher in patients (0.55, range0-6.19) than in healthy control group (0.12, range0-1.67 pmol/L, p<0.001). Patients with ascitesand esophageal varices had higher values thanthose without them. Only patients with the mostsevere grade of liver disease had higher ET val-ues as compared to other subgroups and healthyvolunteers. There were negative correlations be-tween ET(1-21) and serum albumins, pro-thrombin time and sodium (r=-0.36, r=-0.37, r=-0.33, respectively), and positive correlation withgama-globulins (r=0.44, p<0.05). Clinical accu-racy of ET(1-21) is high (P=0.72, p<0.05), so itmight be a useful marker of vascular changes inthese patients.E-mail: [email protected]



P2-5

SADR@AJ ZASI]ENIH I NEZASI]ENIHMASNIH KISELINA U TKIVU

REGENERIRAJU]E JETRE MIŠEVANAKON DJELOMI^NE HEPATEKTOMIJE

Giacometti J.1, Tota M.1, ]uk M.2, Radosevic-Stasic B.2

1 Zavod za kemiju i biokemiju, 2 Zavod za fiziologiju iimunologiju, Medicinski fakultet, Rijeka, Hrvatska

Ošte}enje jetre ili djelomi~na hepatektomija(pHx) poti~e brz rast preostalog jetrenog tkiva,koji uklju~uje promjene genske ekspresije i izlu-~ivanja odre|enih ~imbenika rasta, kao i tipi~nestrukturne promjene. Posebnu ulogu u tim proce-sima imaju zasi}ene (SFA) i nezasi}ene (UFA)masne kiseline koje kontroliraju procese prolife-racije i apoptoze, kao i funkciju limfati~nih sta-nica odgovornih za lu~enje brojnih citokina i cje-lokupni imuno nadzor. îni se da pritom mono-nezasi}ene kiseline (MUFA) odràvanjem nor-malne fluidnosti stani~nih membrana umanjujuštetan u~inak polinezasi}enih masnih kiselina(PUFA) koje poti~u lipidnu peroksidaciju. No,dok neke od n-6 PUFA stimuliraju lu~enje pro-upalnih citokina TNFalfa, IL-6 i reaktivnih kisi-kovih radikala, neke od n-3 PUFA mogu imati isuprotan u~inak. Usto, neki kemijski peroksi-zomski induktori mogu potaknuti i proliferacijuhepatocita aktiviranjem NF-kappaB u Kupffero-vim stanicama i stimulacijom stvaranja TNFalfa,ali regulacijski mehanizmi su još uvijek nedo-voljno poznati. Nastoje}i istraìti neke aspekteovih zbivanja, primjenom plinske kromatografijeispitivali smo tkivne sadràje SFA, MUFA iPUFA (n-3 i n-6) u regeneriraju}oj jetri miševa od2. do 21. dana nakon pHx. Rezultati su pokazalida se sastav FA u tkivu jetre izrazito mijenjanakon pHx. Postotak SFA se zna~ajno smanjio(C18:0 nakon 24 h, a C16:0 nakon 48 h). Isto-dobno, nakon 24 h zna~ajno je porastao postotakC18:1n-9 (oleinska kiselina, OA) i C18:3n-3 (alfa-linolenska kiselina), a smanjio se postotakC20:4n-6 (arahidonska kiselina, AA) i C22:6(dokozaheksaenska kiselina, DHA), ukazuju}i nato da u regeneraju}oj jetri po~etno opadaju ak-tivnosti desaturaza delta6 i delta5. Sadràj AA sedo sedmog p.o. dana normalizirao, no više vri-jednosti OA i niè vrijednosti DHA su trajale i 7.p.o. dana, naglašavaju}i da bi i promjena me|u-sobnih odnosa MUFA i n-3 i n-6 PUFA moglaimati zna~ajnu ulogu u aktiviranju i zaustav-ljanju brzog rasta jetre nakon pHx.E-mail: [email protected]

P2-5

GROWTH-ASSOCIATED CHANGES INFATTY ACID COMPOSITION IN MURINEREGENERATING LIVER AFTER PARTIAL

HEPATECTOMY

J. Giacometti1, M. Tota1, M. ]uk2, B. Radosevic-Stasic2

1 Department of Chemistry and Biochemistry, 2 Departmentof Physiology and Immunology, School of Medicine,

University of Rijeka, Rijeka, Croatia

Injury or removal of liver tissue leads to fastliver growth, involving changes in gene expres-sion, growth factor production and morphologi-cal structure. Unsaturated fatty acids, which arecontrolling proliferation and apoptosis as well asthe lymphatic functions responsible for cytokineproduction and maintenance of immune surveil-lance, might be important biological mediators inthese events. Monounsaturated fatty acids(MUFA) providing an adequate fluidity to the bio-logical membranes seem to diminish the hazardof lipid peroxidation which affects polyunsatu-rated fatty acids (PUFA). Some n-6 compoundscan increase formation of the proinflammatorycytokines TNFalfa and interleukin-6, and of reac-tive oxygen species, while some n-3 PUFA opposethese effects. It seems that chemical peroxisomeproliferators also increase hepatocyte prolifera-tion by the mechanisms involving activation ofnuclear factor-kappaB and production ofTNFalfa by Kupffer cells, however, the mecha-nisms are still being investigated. To elucidatethese events, we estimated by gas chromatogra-phy tissue levels of saturated fatty acids (SFA),MUFA and PUFA (n-3 and n-6) in the regenerat-ing mice liver from second until 21st day afterpartial hepatectomy (pHx). The data showed thathepatic FA composition was markedly affected bypHx. The proportion of SFA significantly de-creased (C18:0 at 24 h and C16:0 at 48 h). Simul-taneously, at 24 h, the proportion of C18:1n-9(oleic acid, OA) and C18:3n-3 (alfa-linolenic acid)markedly increased, while the proportion ofC20:4n-6 (arachidonic acid, AA) and C22:6(docosahexaenoic acid, DHA) decreased, suggest-ing the activity of delta6 and delta5 desaturasesto be initially impaired in the regenerating liver.The AA content recovered on day 7 p.o., while thehigher level of OA and low levels of DHA per-sisted until day 7 p. o., emphasizing the role ofthe interrelationship between MUFA, n-3 and n-6polyunsaturated fatty acids for the initiation andinhibition of liver regeneration.E-mail: [email protected]



P3-1

PRA]ENJE UPALNOG ODGOVORANAKON AORTOKORONARNOG

PREMOŠTAVANJA OVISNO O PRIMJENIIZVANTJELESNOG KRVOTOKA

Brki} K.1, Sutli} @.1, Bio~ina B.1, Rude` I.1, Uni} D.1,Bari} D.1, Šprajc N.2

1 Odjel za kardijalnu kirurgiju, 2 Zavod za laboratorijskudijagnostiku, Klini~ka bolnica “Dubrava”, Zagreb

Primjena izvantjelesnog krvotoka (cardiopul-monary bypass, CPB) kod kardiokirurških opera-cija poti~e razvoj sustavnog upalnog odgovoraorganizma. Pritom dolazi do aktiviranja razli~i-tih proteinskih sustava plazme i razli~itih stani-ca. U svrhu pra}enja toga odgovora do danas suse rabili razli~iti upalni biljezi: citokini, kemoki-ni, komplement, proteini akutne faze, prokalci-tonin i neopterin, ali još uvijek ne postoji dovolj-no osjetljiv i specifi~an biljeg. Cilj studije bio jepratiti kinetiku izlu~ivanja upalnih biljega kojitvore jezgru upalnog odgovora (interleukin-6,komplement C3 i C4, neopterin i CRP) tijekomsedmodnevnog poslijeoperacijskog razdoblja. Is-pitanici su bili podijeljeni u dvije skupine: sku-pina CABG (n=35) i skupina OPCAB (n=45) ovis-no o tome jesu li bili podvrgnuti operaciji revas-kularizacije miokarda uz uporabu CPB ili bez.Uzorci seruma sakupljani su prema slijede}emrasporedu: ujutro dan prije operacije (T1), 6 (T2),12 (T3) i 24 (T4) sata nakon završetka operacije,te tre}eg (T5), petog (T6) i sedmog dana (T7)nakon operacije. IL-6 i neopterin odre|eni sumetodom ELISA, a C3, C4 i CRP turbidime-trijskom metodom na analizatoru OlympusAU600. Skupina CABG pokazala je zna~ajno viševrijednosti neopterina u to~kama T2 (p<0.001),T3 (p<0.001), T4 (p=0.001) i T6 (p=0.003), te IL-6u to~ki T7 (p=0.015), dok je skupina OPCABpokazala zna~ajno povišenje u to~ki T3 za C3(p=0.008) i C4 (p). Kinetika CRP nije se razliko-vala izme|u skupina. Kinetika neopterina upra-vo ukazuje na razliku utjecaja primjene CPB naorganizam, dok se kinetika C3, C4 i IL-6 razli-kuje samo u nekim to~kama pra}enja. Ova sepojava mo`e objasniti interindividualnim razli-kama u odgovoru kako na samu operaciju (sku-pina OPCAB) tako i na utjecaj CPB (skupinaCABG). Stoga je potrebno ispitati interindividu-

P3Bolesti kardiovaskularnog sustava i dijagnostika hitnih stanja

Cardiovascular diseases and emergency diagnosis

P3-1

MONITORING INFLAMMATORYRESPONSE AFTER AORTOCORONARYBYPASS DEPENDING ON THE USE OF

CARDIOPULMONARY BYPASS

K. Brki}1, @. Sutli}1, B. Bio~ina1, I. Rude`1, D. Uni}1,D. Bari}1, N. Šprajc2

1 Department of Cardiac Surgery, 2 Institute of LaboratoryDiagnosis, Dubrava University Hospital, Zagreb

The use of cardiopulmonary bypass (CPB) incardiac surgery induces a state of systemic in-flammatory response during which differentplasma proteins and blood cells are activated.For the purpose of monitoring this response, vari-ous inflammatory markers have been used todate, e.g., cytokines, chemokines, complement,acute phase proteins, procalcitonin andneopterin, however, none of them sensitive andspecific enough. The aim of the study was tomonitor the kinetics of inflammatory markerswhich are the core of inflammatory response(interleukin-6, complement C3 and C4, neopterinand CRP) during a seven-day postoperative pe-riod. Two groups of subjects were included: CABG(n=35) and OPCAB (n=45), depending on the useof CPB during arterial revascularization. Serumsamples were collected as follows: in the morningon the day before the surgery (T1), at 6 (T2), 12(T3) and 24 (T4) hours postoperatively, and onday 3 (T5), 5 (T6) and 7 (T7) postoperatively. IL-6and neopterin were measured by ELISA method,and C3, C4 and CRP by turbidimetric method onan Olympus AU600 autoanalyzer. CABG groupshowed significantly higher neopterin levels at T2(p<0.001), T3 (p<0.001), T4 (p=0.001) and T6(p=0.003), and IL-6 levels at T7 (p=0.015).OPCAB group showed significantly higher levelsof C3 and C4 at T3 (p<0.001 and p=0.008, respec-tively). CRP kinetics did not differ between thegroups. Neopterin kinetics directly pointed to dif-ferent CPB effects on the body, whereas C3, C4and IL-6 kinetics differed only at some timepoints. This could be explained byinterindividual differences in the response to bothsurgical trauma (OPCAB group) and CPB(CABG group). Therefore, the effects ofinterindividual differences on the release of in-flammatory markers should be examined to better



alne razlike u mehanizmima otpuštanja upalnihbiljega kako bi se razumjeli procesi koji se od-vijaju u organizmu tijekom kardiokirurške ope-racije.E-mail: [email protected]

understand the process going on duringcardiothoracic surgery.E-mail: [email protected]

P3-2

BILJEZI PROTEINURIJE KODKARDIOLOŠKIH BOLESNIKA S

PREMOSNICAMA ARTERIJSKIHGRAFTOVA

Matiši} D.1, Bili} K.1, Rogi} J.1, ^voriš}ec D.1,Gašparovi} H.2, Jeli} I.2

1 Klini~ki zavod za laboratorijsku dijagnostiku i 2 Klinika zakardiokirurgiju, Klini~ki bolni~ki centar “Zagreb”, Zagreb

Za vrijeme presa|ivanja premosnica arterij-skim graftovima bolesnici su priklju~eni na eks-trakorporalnu cirkulaciju. Tijekom takvog za-hvata rad bubrega je oslabljen, a za o~ekivati su iošte}enja pojedinih bubre`nih struktura. Boles-nici su nakon zahvata podvrgnuti terapiji vera-pamilom, koji se redovito rabi u kardijalnoj ki-rurgiji u svrhu lije~enja supraventrikularnih arit-mija te kao mo}no sredstvo za prevenciju spazmapremosnica arterijskim graftovima. Bolesnici spostoje}om bubre`nom disfunkcijom te bolesnicialergi~ni na verapamil nisu uzimani u obradu. Usvrhu pra}enja bubre`ne funkcije, kod 38 bole-snika kojima su ugra|ene premosnice arterij-skim graftovima odre|ivani su u drugoj jutarnjojmokra}i albumin kao pokazatelj ošte}enja glome-rularne bazalne membrane, te alfa-1-mikro-globulin kao pokazatelj ošte}enja proksimalnihtubula. Istodobno je odre|en i kreatinin u mo-kra}i. Uzorci su uzimani prijeoperacijski te po-tom prvog, tre}eg i petog dana poslije operacije.Kako su u obradu uzimani bolesnici bez postoje}ebubre`ne disfunkcije, u prijeoperacijskim uzor-cima su u ve}ine bolesnika na|ene vrijednostialbumina i alfa-1-mikroglobulina u referentnomrasponu. Prvoga dana poslije operacije kod ve-}ine bolesnika na|ene su zna~ajne promjene uizlu~ivanju albumina i/ili alfa-1-mikroglobulina,dok se tre}eg i petog dana nakon operacija vri-jednosti albumina i alfa-1-mikroglobulina vra-}aju u referentni raspon. Rezultati pokazuju dasu albumin i alfa-1-mikroglobulin biljezi kojimase mo`e pratiti rano ošte}enje bubrega kod bo-lesnika s premosnicama arterijskih graftova.E-mail: [email protected]

P3-2

MARKERS OF PROTEINURIA INCARDIOLOGIC PATIENTS WITH

ARTERIAL GRAFT BYPASS

D. Matiši}1, K. Bili}1, J. Rogi}1, D. ^voriš}ec1, H.Gašparovi}2, I. Jeli}2

1 Institute of Clinical Laboratory Diagnosis, and2 University Department of Cardiac Surgery, Zagreb

University Hospital Center, Zagreb

During arterial bypass grafting, patients areconnected to extracorporeal circulation. Renal ac-tivity is diminished during such a surgical proce-dure, and damages to some renal structures maybe expected. After the procedure, patients are sub-jected to therapy with verapamil which is regu-larly used in cardiac surgery for treatment of su-praventricular arrhytmias and as a potent medi-cation to prevent bypass spasms. Patients withexistent renal dysfunction and patients allergic toverapamil were not included in the study. In or-der to monitor renal function in 38 patients witharterial bypass grafting, albumin was deter-mined in the second morning urine sample as anindicator of glomerular basal membrane damage,and alpha-1-microglobulin as an indicator ofproximal tubular damage. Also, creatinine inurine was determined. The samples were takenpreoperatively, and on days 1, 3, and 5 after thesurgical procedure. As only patients without pre-existent renal dysfunction were monitored, albu-min and alpha-1-microglobulin values were inmost patients within the reference range. On day1 following the surgery, significant changes werefound in albumin and/or alpha-1-microglobulinsecretion in most patients, while albumin and al-pha-1-microglobulin regained reference rangevalues on days 3 and 5. Our results demonstratedthat albumin and alpha-1-microglobulin aremarkers that could be used to monitor early renallesions in patients undergoing arterial bypassgrafting.E-mail: [email protected]



P3-3

KONCENTRACIJE ENDOTELINA-1 KODBOLESNIKA S INFARKTOM MIOKARDATIJEKOM FIBRINOLITI^KE TERAPIJE

Rumenjak V.1, Jug M.2, Popovi} M.4, Goldner V.4,Brklja~i} V.3

1 Zavod za medicinsko laboratorijsku dijagnostiku, Op}abolnica “Sveti duh”; 2 Klinika za kardiovaskularne bolesti;

3 Klini~ki zavod za laboratorijsku dijagnostiku, Klini~kibolni~ki centar “Zagreb”, 4 Medicinski fakultet, Zagreb

Endotelin-1 jedan je od najsna`nijih dosad po-znatih vazokonstriktora. Danas su poznata triizomerna oblika: endotelin-1, endotelin-2 i endo-telin-3, od kojih endotelin-1 ima najsna`niji va-zokonstrikcijski u~inak. U raznim studijama po-kazalo se da su vrijednosti endotelina-1 povišeneu akutnom infarktu miokarda. Cilj rada bio jeispitati promjene u vrijednosti endotelina-1 kodbolesnika s infarktom miokarda koji su pod-vrgnuti fibrinoliti~koj terapiji. Ispitivanje je obu-hvatilo 78 bolesnika (30 `ena, 48 muškaraca,srednja dob 62 godine) s klini~ki potvr|enim in-farktom miokarda. Fibrinoliti~ka terapija primije-njena je kod 62 bolesnika, a kod drugih su po-stojale kontraindikacije. Kontrolnu skupinu ~i-nili su bolesnici na programu kroni~ne dijalize ibolesnici s kardiovaskularnim bolestima, ali bezinfarkta miokarda. Endotelin-1 odre|ivan je en-zim imuno analizom na ~vrstom nosa~u (R&DSystems, SAD), a CKMB maseni i mioglobin en-zim imuno analizom (Dade-Behring, Austrija).Koncentracije endotelina pra}ene su tijekom 72sata od prijma u bolnicu. Uspješnost fibrinoliti~-ke terapije procjenjivana je na temelju norma-lizacije ST spojnice u EKG-u, vidljivog isplav-ljivanja enzima i na temelju klini~ke slike. Naosnovi ovih kriterija fibrinoliti~ka terapija bila jeuspješna u 39, a neuspješna u 23 bolesnika.Koncentracije endotelina tijekom fibrinoliti~keterapije pokazale su sli~no kretanje kao i tradi-cionalni biljezi (CKMB maseni i mioglobin). Bo-lesnici kod kojih su izmjerene vrijednosti endo-telina >2,5 SD od srednje vrijednosti za cijeluskupinu imali su ve}u u~inkovitost fibrinoliti~keterapije. Rezultati ovoga ispitivanja pokazali suda pra}enje koncentracije endotelina-1 tijekomfibrinoliti~ke terapije kod bolesnika s akutniminfarktom miokarda mo`e biti dodatni biljeg uprocjeni veli~ine ošte}enja miokarda, kao i u pro-cjeni uspješnosti terapije.E-mail: [email protected]

P3-3

ENDOTHELIN-1 LEVELS DURINGFIBRINOLYTIC THERAPY IN PATIENTS

WITH MYOCARDIAL INFARCTION

V. Rumenjak1, M. Jug2, M. Popovi}4, V. Goldner4, V.Brklja~i}3

1 Institute of Medical Laboratory Diagnosis, Sveti Duh GeneralHospital; 2 University Department of Cardiovascular Diseases;3 Clinical Institute of Laboratory Diagnosis, Zagreb University

Hospital Center, 4 School of Medicine, Zagreb

Endothelin-1 is one of the most potentvasoconstrictors currently known. Today, threeisomeric types are known: endothelin-1,endothelin-2 and endothelin-3, of whichendothelin-1 has the strongest vasoconstrictive ef-fect. Various studies showed elevated endothelin-1levels in acute myocardial infarction. The aim ofthe study was to investigate changes inendothelin-1 levels in patients with myocardialinfarction subjected to fibrinolytic therapy. Thestudy included 78 patients (30 females and 48males, mean age 62 years) with clinically provenmyocardial infarction. Fibrinolytic therapy wasadministered to 62 patients, while in otherscontraindications were present. Control groupconsisted of patients on chronic dialysis andthose with cardiovascular diseases but withoutmyocardial infarction. Endothelin-1 was deter-mined by enzyme immunoassay on solid carrier(R&D Systems, USA), and mass CKMB and my-oglobin by enzyme immunoassay (Dade-Behring,Austria). Endothelin levels were monitored dur-ing 72 hours of hospital admission. The success offibrinolytic therapy was estimated on the basis ofof ST segment normalization in ECG, observedenzyme washout, and clinical presentation. Onthe basis of these criteria, fibrinolytic therapywas successful in 39 and unsuccessful in 23 pa-tients. Endothelin levels during fibrinolytictherapy showed similar trends to those of conven-tional markers (mass CKMB and myoglobin). Inpatients with endothelin levels >2.5 SD of meanvalue for the whole group, the efficacy of fibrino-lytic therapy was higher. The results of this studyshowed that monitoring of endothelin-1 levelsduring fibrinolytic therapy in patients with acutemyocardial infarction could be an additionalmarker in the assessment of the extent of myocar-dial damage, and in the evaluation of therapysuccess.E-mail: [email protected]



P3-4

ENDOTELIN I ^INITELJ RASTA JETREU BOLESNIKA S KORONARNOM BOLESTI

SRCA

Antoljak N.1, Vrki} N.1, Begonja A.1, Nikoli}-HeitzlerV.2, Bulj N.2

1 Klini~ki zavod za kemiju, 2 Odjel za kardiologiju, KB“Sestre milordnice”, Zagreb

^initelj rasta jetre (HGF) je citokin uklju~en uregeneraciju tkiva, angiogenezu i nastanak kola-terala. Otkriven je kao protein koji poti~e rasthepatocita, no ima u~inak i na druga tkiva. Endo-telin (ET) je protein sa~injen od 21 aminokiselines va`nim fiziološkim i patofiziološkim djelova-njem u nastanku zatajenja srca. Najva`nija jeperiferna vazokonstrikcija i poticanje miocita ifibroblasta. Cilj rada je bio ispitati mogu li vri-jednosti HGF i ET u plazmi bolesnika biti od ko-risti u dijagnozi akutnog infarkta miokarda. ET iHGF su mjereni u plazmi 42 zdrava ispitanika i31 bolesnika s akutnim infarktom miokarda pri-likom hitnog prijma u bolnicu. Uklju~eni su ustudiju prema standardnim bikemijskim i klini~-kim kriterijima. Razine ET i HGF mjerene suenzimimunološkim testom (Biomedica, Austria,odnosno R&D, SAD). Statisti~ka analiza u~injenaje kompjutorskim programom Sigmastat. Zna-~ajnost razlike izme|u skupina ispitanika testi-rana je Mann-Whitneyevim testom, a klini~kavaljanost analizom ROC. Razine HGF bile su višeu gotovo svih bolesnika nego u zdravih ispita-nika: 12,446 (1,007-12,858) ng/mL u odnosu na0,854 (0,517-1,347) ng/mL, p<0,001. Razine ETbile su tako|er više u bolesnika: 0,72 (0,05-8,5)pmol/L u odnosu na 0,37 (0,05-1,6) pmol/L,p<0,001. Prema analizi ROC, HGF je odli~an bi-ljeg akutnog infarkta miokarda, površina ispodkrivulje A=0,94, p<0,001. Najbolja prijelomnavrijednost bila je 1,47 ng/mL. Klini~ka valjanostendotelina bila je nešto slabija, A=0,7, p<0,01, aprijelomna vrijednost 0,6 pmol/L. Zaklju~ujemoda su vrijednosti HGF bolji pokazatelj akutnoginfarkta miokarda nego vrijednosti endotelina.E-mail: [email protected]

P3-4

ENDOTHELIN AND HEPATOCYTEGROWTH FACTOR IN PATIENTS WITH

CORONARY ARTERY DISEASE

N. Antoljak1, N. Vrki}1, A. Begonja1, V. Nikoli}-Heitzler2,N. Bulj2

1 Clinical Institute of Chemistry, 2 Department ofCardiology, Sestre milosrdnice University Hospital, Zagreb

Hepatocyte growth factor (HGF) is a cytokineinvolved in tissue regeneration, angiogenesis andlateral vessel growth. It was firstly recognized asa molecule that stimulates hepatocyte prolifera-tion, but it is now known to be a cytokine withnumerous functions. Endothelin (ET) is a 21-amino acid peptide that has potent physiologicand pathophysiologic effects involved in the de-velopment of heart failure. These include effectson arterial smooth muscle cells that cause intenseperipheral vasoconstriction and stimulation ofcardiac myocytes and fibroblasts. The purpose ofthe study was to explore whether HGF and ETvalues could be used in the diagnosis of myocar-dial infarction. ET and HGF were measured inplasma of 42 healthy volunteers and 31 acutemyocardial infarction patients immediately uponadmission to our hospital. They were included inthe study according to clinical and standard bio-chemical criteria. HGF and ET1-21 levels weremeasured by ELISA test (R&D, USA, andBiomedica, Austria, respectively). Statisticalanalysis was made by Sigmastat computer pro-gram. Differences between groups were tested byMann-Whitney test and clinical accuracy by ROCanalysis. The HGF levels were elevated in almostall patients compared to healthy group: 12.446(1.007-12.858) ng/mL vs. 0.854 (0.517-1.347) ng/mL, p<0.001. Endothelin levels were also higherin the patient group: 0.72 (0.05-8.5) pmol/L vs.0.37 (0.05-1.6 pmol/L, p<0.001. ROC analysisshowed HGF to be an excellent marker of myocar-dial infarction with area under curve A=0.94,p<0.001. The best cut-off value was 1.47 ng/mL.The clinical accuracy for endothelin was A=0.7,p<0.01 and best cut-off value 0.6 pmol/L. So, weconcluded that HGF values could prove more use-ful than ET values in the detection of acute myo-cardial infarction patients.E-mail: [email protected]



P3-5

SR^ANI NATRIJURETSKI PEPTIDI(ANP, BNP) KAO INDIKATORI FUNKCIJE

DESNE I LIJEVE KLIJETKE

@igman M.1, Lukinac Lj.1, Balenovi} A.1, Planinc D.2,Kusi} Z.1

1 Klinika za onkologiju i nuklearnu medicinu, 2 Klinika zaunutarnje bolesti, Klini~ka bolnica “Sestre milosrdnice”,

Zagreb

Cilj rada bio je usporediti koncentracije cirku-liraju}ih ANP i BNP u skupinama zdravih ispi-tanika i kardioloških bolesnika. Unutar skupinekardioloških bolesnika analizirali smo vrijednostiANP i BNP s obzirom na stupanj poreme}ajafunkcije kako lijeve tako i desne klijetke. RazinaANP i BNP odre|ivana je metodama IRMAtvrtke CIS bio International (SHIONORIA ANP iSHIONORIA BNP, Francuska). Sr~ana bolest di-jagnosticirana je klini~kom kardiološkom obra-dom, a hemodinamski pokazatelji funkcije klijet-ka odre|ivani su scintigrafski radionuklidnomventrikulografijom u mirovanju pomo}u 99mTc-pirofosfatom obilje`enih eritrocita. Uspore|enesu vrijednosti ANP i BNP u serumu 64 zdravaispitanika (skupina A) i 186 kardioloških bole-snika koji su bili podijeljeni u tri skupine (B,C,D)s obzirom na stupanj ošte}enja lijeve i/ili desneklijetke: skupina B (n=75) globalna istisna frak-cija (GEF) desno i lijevo >40%; skupina C (n=70)GEF lijeve klijetke <30%, a GEF desne >40%; iskupina D (n=41) GEF <30% desno i lijevo. Me-dijan vrijednosti ANP (pg/mL) i BNP (pg/mL)prema skupinama: skupina A* 6,7 i 3,1; skupinaB** 98,5 i 71; skupina C** 390 i 194,2; skupinaD** 1250 i 680. *p<0,01 A vs. (B, C, D); **p<0,01B vs. C, C vs. D, B vs. D. Zna~ajno više vrijed-nosti natrijuretskih peptida na|ene su u sr~anihbolesnika u odnosu na zdravu populaciju, što jeposljedica remodeliranja sr~anih klijetka i slab-ljenja funkcije srca. Dobiveni rezultati upu}ujuna mogu}nost neinvazivnog ispitivanja funkcijelijeve i desne klijetke na osnovi serumskogodre|ivanja razine ANP i BNP, što bi bilo osobitova`no za sr~ane bolesnike kod kojih se planiratransplantacija srca.E-mail: [email protected]

P3-5

ATRIAL NATRIURETIC PEPTIDE (ANP)AND BRAIN NATRIURETIC PEPTIDE(BNP) AS INDICATORS OF LEFT AND

RIGHT VENTRICULAR FUNCTION

M. @igman1, Lj. Lukinac1, A. Balenovi}1, D. Planinc2,Z. Kusi}1

1 University Department of Oncology and Nuclear Medicine,2 University Department of Medicine, Sestre milosrdnice

University Hospital, Zagreb

The aim of the study was to compare the levelsof ANP and BNP determined in healthy popula-tion and patients with cardiovascular disease,and to assess the difference in ANP and BNP con-centrations between the patients with left andright ventricular disorders. The concentrations ofANP and BNP were determined by IRMA kitsSHIONORIA ANP and SHIONORIA BNP, CISbio International, France (as part of the jointproject). Heart failure was diagnosed by clinicalexamination and hemodynamic functions wereevaluated by radionuclide ventriculography atrest with 99mTc-pyrophosphate labeled erythro-cytes. Serum values of ANP and BNP in the groupof 64 healthy subjects (group A) were comparedwith the values of 186 cardiologic patients di-vided into three groups (B, C and D) according tothe grade of heart failure (left and/or right ven-tricle): group B (n=75) had global ejection frac-tion (GEF) left and right >40%; group C (n=70)with GEF left <30% and right 40%; and group D(n=41) with GEF left and right <30%. Medianvalues of ANP (pg/mL) and BNP (pg/mL) ac-cording to groups: group A* 6.7 and 3.1; groupB** 98.5 and 71; group C** 390 and 194.2; groupD** 1250 and 680. *p<0.01 A vs. (B, C, D);**p<0.01 B vs. C, C vs. D, B vs. D. ANP and BNPwere significantly higher in cardiologic patientsthan in healthy subjects. The results suggestedthe measurement of basal ANP and BNP to be anoninvasive parameter for evaluation of the leftand right ventricular function in patients withvarious forms of heart failure, especially in thosescheduled for heart transplantation.E-mail: [email protected]



P3-6

ODRE\IVANJE TROPONINA I. NAKONOPERACIJSKE UGRADNJE SRÂNIH

PREMOSNICA

Fressl G., Rogi} D., Belina D., Fu~ek M.

Zavod za laboratorijsku dijagnostiku, Klini~ki bolni~kicentar “Zagreb”, Zagreb

Imunokemijsko odre|ivanje troponina I. (TnI)nakon operacijske ugradnje sr~anih premosnica(tzv. bypass operacije) u posljednje se vrijemeautomatski uklju~uje u poslijeoperacijski panelpretraga tijekom boravka bolesnika u jedinici in-tenzivne skrbi. S obzirom na to da razina TnI userumu izravno korelira s opsegom sr~anog ošte-}enja, odre|ivanje ovoga parametra u navede-nom kontekstu ima potencijalno zna~ajnu pre-diktivnu vrijednost. Pojedina~ne studije ukazujuna ~injenicu da dinamika poslijeoperacijskog po-rasta (vrijeme postizanja vršne koncentracijeTnI), kao i vrijeme potrebno za povrat do nor-malne razine predstavljaju zna~ajne prediktoredugoro~nog ishoda. U svrhu racionalizacije pri-mjene ove pretrage cilj ovoga rada bio je utvrditioptimalne vremenske razmake poslijeoperacij-skog mjerenja TnI. U tu svrhu je TnI mjeren kod24 bolesnika podvgnutih bypass operaciji u ra-zli~itim vremenskim to~kama, i to izme|u 15 i120 sati nakon operacije (fluoroimunokemijskametoda, instrument i reagensi tvrtke Dade). U87% bolesnika vršna koncentracija TnI doseg-nuta je u razdoblju izme|u 15 i 24 sata poslije-operacijski. U 75% došlo je do zna~ajnog sma-njenja koncentracije TnI nakon 30 do 72 sata.Poslijeoperacijski tijek u svih bolesnika s nave-denom dinamikom bio je uredan. Sukladno ovimrezultatima, a s obzirom na vrijeme poluìvotaTnI u serumu, preporu~ujemo poslijeoperacijskouzimanje krvi za ovu pretragu u dvjema vre-menskim to~kama, i to 24 te 72 sata nakonoperacijskog zahvata. Ako je koncentracija udrugoj to~ki zna~ajno nià, a klini~ki tijek ure-dan, nije potrebno daljnje odre|ivanje TnI.E-mail: [email protected]

P3-6

CARDIAC TROPONIN I DETERMINATIONAFTER CORONARY ARTERY BYPASS

SURGERY

G. Fressl, D. Rogi}, D. Belina, M. Fu~ek


Immunochemical determination of cardiactroponin I (cTnI) after coronary artery bypasssurgery has lately been included among the testsregularly performed postoperatively during thepatient’s stay in intensive care unit. As cTnI con-centration is in direct correlation with the extentof cardiac damage, the determination of this pa-rameter in these conditions has a potentially sig-nificant predictive value. Individual studies havedemonstrated that the dynamics of postoperativecTnI elevation (the period needed to reach peakcTnI concentration) and the time needed to regainnormal concentration are important predictors oflongterm outcome. To rationalize the use of thistest, the aim of this study was to determine opti-mal intervals for postoperative measurement ofcTnI. cTnI was therefore determined in 24 pa-tients undergoing bypass surgery at various timepoints, i.e. 15 and 120 hours after the surgery(fluoroimmunochemical method, instrument andreagents by the Dade Company). The peak cTnIconcentration was reached postoperatively in 87%patients during the period of 15-24 hours. In 75%of patients, cTnI concentration was significantlydecreased after 30-72 hours. Postoperative coursewas normal in all patients with theabovementioned dynamics. According to these re-sults and considering the half-life of serum cTnI,we recommend postoperative blood sampling forthis test at two time points, i.e. at 24 and 72 hourspostoperatively. If the concentration is signifi-cantly lower at the 2nd time point and the clini-cal course is normal, further TnI determination isnot necessary.E-mail: [email protected]



P3-7

POLIMORFIZMI GENA ZA CETP UBOLESNIKA S KORONARNOM BOLEŠ]U

SRCA (ZAGREBA^KA KOHORTA)

Feren~ak G.1, Grškovi} B.1, Cheng S.2, Skodlar J.3,Šesto M.3, Stavljeni}-Rukavina A.1

1 Klini~ki bolni~ki centar “Zagreb” i Medicinski fakultet,Zagreb, 2 Roche Molecular Systems, Inc., 3 Specijalna

bolnica za vaskularnu kirurgiju i kardiologiju “Magdalena”,Krapinske Toplice

Koncentracija HDL-kolesterola negativno ko-relira s rizikom od koronarne bolesti srca (KBS).Kolesterol-ester transfer protein (CETP) ima sre-dišnju ulogu u metabolizmu ovog lipoproteina istoga bi mogao utjecati na pove}anu sklonost ate-rosklerozi. Cilj istraìvanja bio je odrediti utje~uli C(-629)A, I405V ili D442G varijante CETP ge-na na rizik od KBS. Na temelju rezultata angio-grafije ispitanici su podijeljeni u KBS (n=479) ikontrolnu skupinu (n=200). Genotipizacija je pro-vedena multipleks PCR-om nakon kojeg je slije-dilo otkrivanje umnoènih alela pomo}u linearnopoloènih imobiliziranih sljedno-specifi~nih son-da na najlonskim trakama. Svi su genotipovi biliu Hardy-Weinbergovoj ravnoteì. Relativni riziciza pojedine genotipove izra~unati su logisti~komregresijom nakon korekcije po dobi, spolu, loga-ritmiranim vrijednostima triglicerida, ukupnom iHDL-kolesterolu, lipoproteinu (a), fibrinogenu, teprisutnosti dijabetesa, hipertenzije i pušenja. VVgenotip CETP gena bio je povezan s pove}animrizikom obolijevanja od KBS (OR 2,32, 95% CI1,07-5,04, p=0,035). CETP C(-629)A varijanta ni-je povezana s KBS. Samo je jedan ispitanik izKBS skupine bio nositelj varijante D442G. Multi-varijatna analiza uz dob, spol, dijabetes, hiper-tenziju i pušenje kao kovarijate nije pokazalazna~ajan utjecaj varijante I405V na serumskekoncentracije lipida. Me|utim, AA genotip u pro-motoru povezan je s višim koncentracijama HDL-kolesterola, njegovih subfrakcija i apolipoprote-ina A I (p<0,001). Zaklju~eno je da nosioci VVgenotipa imaju zna~ajno pove}an rizik obolije-vanja od KBS u hrvatskoj populaciji, a AA ge-notip utje~e na koncentracije HDL-kolesterola iapolipoproteina A I.E-mail: [email protected]

P3-7

POLYMORPHISMS OF CETP GENE INPATIENTS WITH CORONARY ARTERY

DISEASE (ZAGREB COHORT)

G. Feren~ak1, B. Grškovi}1, S. Cheng2, J. Skodlar3, M.Šesto3, A. Stavljeni}-Rukavina1

1 Zagreb University Hospital Center and School of Medicine,Zagreb, 2 Roche Molecular Systems, Inc.,

3 Magdalena Special Hospital for Cardiovascular Surgeryand Cardiology, Krapinske Toplice

HDL-cholesterol concentration is inversely re-lated to the risk of coronary artery disease (CAD).Cholesteryl ester transfer protein (CETP) has acentral role in the metabolism of this lipoproteinand might alter the susceptibility to atherosclero-sis. The aim of our study was to determinewhether C(-629)A, I405V or D442G variants ofthe CETP gene have an impact on the risk of de-veloping CAD. Based on angiography results, thepatients were divided into CAD (n=479) and con-trol (n=200) groups. Genotyping was performedby multi-target PCR followed by detection of am-plified alleles with linear arrays of immobilizedsequence-specific probes on nylon membranestrips. All the genotypes were in Hardy-Weinbergequilibrium. Odds ratios (OR) for the genotypeswere calculated by logistic regression after ad-justing for age, sex, log (triglyceride), total andHDL-cholesterol, lipoprotein (a), fibrinogen, dia-betes, hypertension and smoking. The VV geno-type of the CETP gene was associated with an in-creased risk of developing CAD (OR 2.32, 95% CI1.07-5.04, p=0.035). CETP C(-629)A variant wasnot associated with CAD. Only one patient withD442G variant was found in the CAD group.Multivariate analysis with age, sex, diabetes, hy-pertension and smoking as covariates did notshow any significant effect of I405V variant onserum concentrations of lipids. However, AA geno-type in the promotor was associated with higherconcentrations of HDL-cholesterol, its subfrac-tions and apolipoprotein A I (p<0.001). We con-clude that the VV genotype carriers are at a sig-nificant risk of developing CAD in the Croatianpopulation, and AA genotype is a determinant ofHDL-cholesterol and apolipoprotein A I levels.E-mail: [email protected]



P3-9

POLIMORFIZMI GENA PON1 I PON2 UBOLESNIKA S KORONARNOM BOLEŠ]U

SRCA (ZAGREBA^KA KOHORTA)

Feren~ak G.1, Grškovi} B.1, Cheng S.2, Skodlar J.3,Šesto M.3, Stavljeni}-Rukavina A.1

1 Klini~ki bolni~ki centar “Zagreb” i Medicinski fakultet,Zagreb, 2 Roche Molecular Systems Inc., 3 Specijalna bolnicaza kardiovaskularnu kirurgiju i kardiologiju “Magdalena”,

Krapinske Toplice

Paraoksonaza (PON) je enzim vezan za HDL,a svoj antioksidantni u~inak ostvaruje uklanja-njem proizvoda lipidne peroksidacije. Otkrivenoje nekoliko polimorfnih lokusa u genima PON1

P3-8

UTJECAJ POLIMORFIZAMA U GENU ZALIPOPROTEIN-LIPAZU NA KORONARNUBOLEST SRCA I PLAZMATSKE LIPIDE U

ZAGREBA^KOJ KOHORTI

Pašali} D.1, Feren~ak G.2, Grškovi} B.2, Cheng S.3, FijalB.3, Šesto M.4, Skodlar J.A.4, Stavljeni}-Rukavina A.2

1 Zavod za kemiju i biokemiju, Medicinski fakultetSveu~ili{ta u Zagrebu; 2 Klini~ki bolni~ki centar “Zagreb”,

Zagreb; 3 Roche Molecular Systems, Inc.; 4 Specijalnabolnica za kardiovaskularnu kirurgiju i kardiokirurgiju

“Magdalena”, Krapinske Toplice

Ispitali smo povezanost izme|u polimorfiza-ma u genu za lipoprotein-lipazu (LPL) (-93T/G,D)N, N291S i S447X), koronarne bolesti srca(KBS) i koncentracije lipida. Uklju~eni su hrvat-ski ispitanici s (n=479) i bez (n=200) angiogra-fijom potvr|ene KBS. Polimorfizam N291S bio jezna~ajno povezan s KBS (OR: 0,36; p: 0,048).HDL2-kolesterol i vrijednosti apolipoproteina A Ibile su zna~ajno više u nenositelja -93T/G I D9Npolimorfizama u skupini KBS (p: 0.017 i 0.028).Genska varijanta N291S nije zna~ajno utjecalana vrijednosti lipida ni u jednoj od skupina. Ni`itrigliceridi i viši HDL2-kolesterol u kontrolnojskupini bili su povezani s mutacijom S447X (p:0.043 i 0.056). Zaklju~ujemo da bi promjena ugenu za lipoprotein-lipazu mogla utjecati na vri-jednosti lipida i doprinositi riziku obolijevanja odKBS.E-mail: [email protected]

P3-8

EFFECT OF LIPOPROTEIN LIPASE GENEPOLYMORPHISMS ON CORONARY

ARTERY DISEASE AND PLASMA LIPIDSIN ZAGREB COHORT

D. Pašali}1, G. Feren~ak2, B. Grškovi}2, S. Cheng3, B.Fijal3, M. Šesto4, J.A. Skodlar4, A. Stavljeni}-Rukavina2

1 Department of Chemistry and Biochemistry, ZagrebUniversity School of Medicine, Zagreb

2 Zagreb University Hospital Center, Zagreb3 Roche Molecular Systems Inc.

4 Magadalena Special Hospital for Cardiovascular Surgeryand Cardiology, Krapinske Toplice

We investigated the relationship between lipo-protein lipase (LPL) gene polymorphisms (-93 T/G, D9N, N291S, and S447X), coronary artery dis-ease (CAD) and lipid traits. We examinedCroatian patients with (n=497) and without(n=200) angiographically confirmed CAD. TheN291S polymorphism was significantly associ-ated with CAD (OR: 0.36; p: 0.048). HLD2-choles-terol and apolipoprotein A I levels were signifi-cantly higher in non-carriers of the -93T/G andD9N polymorphisms in the CAD group (p: 0.017and 0.028, respectively). The N291S genetic vari-ant did not show any significant difference be-tween carriers and non-carriers in either studygroup for any of the lipids. Lower triglyceride andhigher HDL2-cholesterol levels in the controlgroup were associated with carriers of the S447Xmutation (p: 0.043 and 0.056, respectively). Weconclude that modification in the lipoprotein li-pase gene might influence lipid levels and conferthe risk of CAD.E-mail: [email protected]

P3-9

POLYMORPHISMS OF PON1 AND PON2GENES IN PATIENTS WITH CORONARYARTERY DISEASE (ZAGREB COHORT)

G. Feren~ak1, B. Grškovi}1, S. Cheng2, J. Skodlar3, M.Šesto3, A. Stavljeni}-Rukavina1

1 Zagreb University Hospital Center and School of Medicine,Zagreb, 2 Roche Molecular Systems Inc., 3 Magdalena

Special Hospital for Cardiovascular Surgery andCardiology, Krapinske Toplice

Paraoxonase (PON) is a high density lipopro-tein-linked enzyme which exerts its antioxidanteffect by removing lipid peroxidation products.Several polymorphic loci have been identified in



(M55L i R192Q) i PON2 (S311C). Cilj istraìvanjabio je utvrditi imaju li navedene genske varijanteutjecaja na rizik od koronarne bolesti srca (KBS).Na temelju rezultata angiografije ispitanici supodijeljeni u skupinu KBS (n=479) i kontrolnuskupinu (n=200). Genotipizacija je provedenamultipleks PCR-om, nakon koje je slijedilo otkri-vanje umnoènih alela pomo}u linearno polo-ènih imobiliziranih sljedno-specifi~nih sonda nanajlonskim trakama. Svi su genotipovi bili uHardy-Weinbergovoj ravnoteì. Relativni riziciza pojedine genotipove izra~unati su logisti~komregresijom nakon korekcije prema dobi, spolu,logaritmiranim vrijednostima triglicerida, ukup-nom i HDL-kolesterolu, lipoproteinu (a), fibri-nogenu, te prisutnosti dijabetesa, hipertenzije ipušenja. Genotip LL gena PON1 povezan je spove}anim rizikom od KBS (OR 1,92, 95% CI1,12-3,33, p=0,017), dok preostala dva polimorfnamjesta nisu povezana s KBS. Multivarijatna ana-liza uz dob, spol, dijabetes, hipertenziju i pušenjekao kovarijate nije pokazala zna~ajan utjecajanaliziranih lokusa na serumske koncentracijeukupnog, HDL-, LDL-kolesterola, triglicerida, li-poproteina (a), apolipoproteina A I, B i E. Za-klju~eno je kako genotip LL zna~ajno doprinosiriziku od KBS u hrvatskoj populaciji.E-mail: [email protected]

PON1 (M55L and R192Q) and PON2 (S311C)genes. The aim of our case-control study was todetermine whether these genetic variants had animpact on the risk of developing coronary arterydisease (CAD). Based on angiography results, thepatients were divided into CAD (n=479) and con-trol (n=200) groups. Genotyping was performedby multi-target PCR followed by detection of am-plified alleles with linear arrays of immobilizedsequence-specific probes on nylon membranestrips. All the genotypes were in Hardy-Weinbergequilibrium. Odds ratios (OR) for the genotypeswere calculated by logistic regression after ad-justing for age, sex, log (triglyceride), total andHDL-cholesterol, lipoprotein (a), fibrinogen, dia-betes, hypertension and smoking. The LL geno-type of the PON1 gene was associated with an in-creased risk of developing CAD (OR 1.92, 95% CI1.12-3.33, p=0.017), whereas the other two poly-morphic sites did not show significant associa-tion with CAD. Multivariate analysis with age,sex, diabetes, hypertension and smoking ascovariates did not show significant effect of any ofthe three polymorphic loci tested on serum con-centrations of total, HDL-, LDL-cholesterol,triglycerides, lipoprotein (a), apolipoproteins AI,B and E. In conclusion, the LL genotype confers asignificant risk of developing CAD in theCroatian population.E-mail: [email protected]

P3-10

POVEZANOST POLIMORFIZAMA UGENIMA KOJI KODIRAJU

KOAGULACIJSKE I ÎMBENIKEFIBRINOLIZE S KORONARNOM

BOLEŠ]U SRCA

Coen D.1, Feren~ak G.1, Grškovi} B.1, Cheng S.2, ŠestoM.3, Skodlar J.3, Stavljeni}-Rukavina A.1

1 KBC “Zagreb”, Zagreb, 2 Roche Molecular Systems Inc.,3 Specijalna bolnica za kardiovaskularnu kirurgiju i

kardiologiju “Magdalena”, Krapinske Toplice

Koronarna bolest srca (KBS) je multifaktor-ska bolest uvjetovana genskim i ~imbenicimaokoline. Budu}i da aktivacija koagulacije igraklju~nu ulogu u stvaranju tromba, genske mu-tacije proteina uklju~enih u koagulaciju i fibri-nolizu mogu imati ulogu u patogenezi KBS. Ciljje istraìvanja bio utvrditi postoji li povezanostizme|u genskih ~imbenika za koje se pretpo-stavlja da su povezani s pove}anom sklonoš}utrombozi s rizikom od KBS: polimorfizmi 4G/5G iG11053T u PAI-1 genu, G-455A u genu za fibrino-

P3-10

ASSOCIATION OF COAGULATION ANDFIBRINOLYSIS GENE POLYMORPHISMS

WITH CORONARY ARTERY DISEASE

D. Coen1, G. Feren~ak1, B. Grškovi}1, S. Cheng2, M.Šesto3, J. Skodlar3, A. Stavljeni}-Rukavina1

1 Zagreb University Hospital Center, Zagreb, 2 RocheMolecular Systems Inc., 3 Magdalena Special Hospital for

Cardiovascular Surgery and Cardiology, Krapinske Toplice

Coronary artery disease (CAD) is a multifacto-rial disease influenced by both genetic and envi-ronmental determinants. Since coagulation acti-vation plays a key role in thrombus formation,genetic mutations of the proteins involved in co-agulation and fibrinolysis may have a role in thepathogenesis of CAD. The aim of the study was toinvestigate the association between the risk ofCAD and genetic factors thought to be associatedwith an increased tendency to thrombosis:polymorphisms 4G/5G and G11053T of PAI-1gene, G-455A of fibrinogen gene, G10976A and -323 0/10-bp of factor VII gene, G1691A of factor



gen, G10976A i -323 0/10-bp u genu za faktor VII,G1691A u genu za faktor V i G20210A u genu zaprotrombin. Analizirane su DNK 479 bolesnika sangiografski dokazanom KBS (>50% stenoze ubar jednoj koronarnoj arteriji) i 200 kontrolnihosoba bez KBS (<10% stenoze). Polimorfizmi sugenotipizirani multilokusnim linearnim blotomza genske kandidate za kardiovaskularne bolesti(Roche Molecular Systems Inc, SAD). Nakon ko-rekcije s obzirom na ostale rizi~ne ~imbenike do-biveni su slijede}i relativni rizici i 95%-tni inter-vali pouzdanosti (dominantni i recesivni modeli):PAI-1 4G/5G i 11053 TT (OR 1,24 i 0,84, 95% CI0,84 do 1,83 i 0,56 do 1,25), fibrinogen -455AA(OR 1,05, 95% CI 0,75 do 1,50), faktor VII10976AA i -32310/10-bp (OR 0,58 i 0,59, 95% CI0,11 do 2,97 i 0,15 do 2,25). Zaklju~eno je kakoistra`ivani polimorfizmi nisu povezani s rizikomod KBS u prou~avanoj populaciji.E-mail: [email protected]

V gene, and G20210A of prothrombin gene. Weperformed a case-control study evaluating 479patients with angiographically documented CAD(>50% stenosis of at least one coronary artery)and 200 controls without CAD (<10% stenosis).The polymorpisms were genotyped using amultilocus genotyping assay for candidate mark-ers of cardiovascular disease risk (Roche Molecu-lar Systems Inc, USA). After adjustment for otherrisk factors, the following odds ratios (OR) andconfidence intervals (95% CI) were obtained(dominant and recessive models): PAI-1 4G/5Gand 11053 TT (OR 1.24 and 0.84, 95% CI 0.84 to1.83 and 0.56 to 1.25, respectively), fibrinogen -455AA (OR 1.05, 95% CI 0.75 to 1.50), factor VII10976AA and -32310/10-bp (OR 0.58 and 0.59,95% CI 0.11 to 2.97 and 0.15 to 2.25, respectively).In conclusion, the studied polymorphisms werenot associated with CAD in this cohort.E-mail: [email protected]

P3-11

BILJEZI FIBRINOLIZE ZA VRIJEMEOPERACIJE REVASKULARIZACIJE

MIOKARDA

@ivkovi} M.1, Brki} K.2, Niki} N.3, Hašperger D.1,Romi} @.1

1 Zavod za laboratorijsku dijagnostiku, 2 Odjel za kardijalnukirurgiju, 3 Odjel za anesteziju, reanimaciju i intenzivnu

skrb, Klini~ka bolnica “Dubrava”, Zagreb

Revaskularizacija miokarda naj~eš}a je kar-diokirurška operacija izvo|ena danas u svijetu.Tradicionalno se izvodi uz primjenu izvantjeles-nog krvotoka. Prolaz krvi preko površina nepo-krivenih endotelom izaziva poreme}aj koagu-lacije, komplementa i fibrinoliti~kog sustava, tzv.sustavni upalni odgovor. Suvremene kiruršketehnike omogu}uju izvo|enje revaskularizacijemiokarda i bez primjene izvantjelesnog krvotoka,a o pogodnosti bolesnika za taj na~in izvo|enjaodlu~uje operater temeljem obilje`ja same bole-sti. Cilj studije bio je pra}enjem biljega fibrino-lize PAI-1, plazminogena i D-dimera utvrditi raz-liku u aktivnosti fibrinolize za vrijeme izvo|enjarevaskularizacije miokarda s obzirom na na~inizvo|enja. Provedeno je prospektivno ispitivanjeu 28 bolesnika koji su programski operirani zbogkoronarne bolesti srca u KB “Dubrava” tijekomo`ujka i travnja 2003. godine. Od toga je 14 bo-lesnika operirano uz primjenu izvantjelesnogkrvotoka (CABG), a drugih 14 bez njega (OPCAB).Svi su primili istu vrstu anestezije, iste vazoaktiv-ne lijekove, te je primijenjen isti hemodinamski

P3-11

FIBRINOLYTIC MARKERS DURINGARTERIAL REVASCULARIZATION

M. @ivkovi}1, K. Brki}2, N. Niki}3, D. Hašperger1, @.Romi}1

1 Institute of Laboratory Diagnosis, 2 Department of CardiacSurgery, 3 Department of Anesthesiology, Resuscitation and

Intensive Care, Dubrava University Hospital, Zagreb

Arterial revascularization is the most commoncardiothoracic surgery performed in the world.Traditionally, it is performed using extracorpor-eal circulation. The blood passage acrossnonphysiological surfaces causes disorders of co-agulation, complement and fibrinolytic system,known as whole body inflammatory response.Modern surgical techniques can provide myocar-dial revascularization also without using extra-corporeal circulation. The objective of the studywas to determine the difference in fibrinolytic ac-tivity by monitoring the fibrinolytic markers PAI-1, plasminogen and D-dimer during myocardialrevascularization depending on the way it is per-formed. The prospective study of 28 patients whounderwent cardiac surgery for coronary heartdisease was performed during March and April2003. Fourteen patients underwent surgery withextracorporeal circulation (CABG), and the other14 without it (OPCAB). They all received thesame type of anesthesia, the same vasoactivedrugs, and the same hemodynamic supervisionwas used. Antifibrinolytics were not used. Plasmasamples were collected in four phases of the proce-



nadzor. Antifibrinolitici nisu primijenjeni. Plaz-ma je uzorkovana u ~etiri faze operacije, te slije-de}eg jutra. Aktivnost PAI-1 i koncentracija plaz-minogena odre|ene su kromogenom metodom naanalizatoru BCT (Behring Coagulation Timer), akoncentracija D-dimera aglutinacijskom meto-dom na analizatoru STA Compact (Roche/Boeh-ringer Mannheim). Aktivnost PAI-1, koncentra-cije plazminogena i D-dimera pokazale su znatnove}e promjene u skupini CABG. Aktivnost PAI-1i koncentracija D-dimera naglo su rasle za vri-jeme primjene izvantjelesnog krvotoka, a potroš-nja plazminogena bila je ubrzana. Slijede}ega ju-tra vrijednosti parametara izjedna~ene su u objeskupine. Dakle, aktivnost fibrinolize ve}a je uskupini CABG. Sustavni upalni odgovor uzrok jeja~oj fibrinolizi u skupini CABG, dok je fibrino-liza u skupini OPCAB normalan fiziološki odgo-vor organizma na sam operacijski zahvat.E-mail: [email protected]

dure and on the next morning. PAI-1 activity andplasminogen concentration were determined us-ing chromogenic method on a BCT analyzer, andD-dimer concentration was determined using ag-glutination method on an STA Compact analyzer.PAI-1 activity, plasminogen and D-dimer concen-trations showed significantly greater changes inthe CABG group. PAI-1 activity and D-dimer con-centration were increased during the use of extra-corporeal circulation, while plasminogen con-sumption was accelerated. On the next morning,the values of fibrinolytic markers were leveled inthe two groups. Accordingly, fibrinolytic activitywas more pronounced in the CABG group. Thewhole body inflammatory response was the causeof stronger fibrinolysis in the CABG group, whilefibrinolysis in the OPCAB group was the normalphysiological response to the operative procedureper se.E-mail: [email protected]

P3-12

PROFIL LIPIDA I FENOTIPOVI APO H UISPITANIKA S DISLIPIDEMIJAMA KOJIISKAZUJU RAZLIÎTU DISTRIBUCIJU

VELIÎNA LDL ÊSTICA

Trbojevi}-êpe M., Vogrinc @., Zrinski-Topi} R.,Stavljeni}-Rukavina A.

Klini~ki zavod za laboratorijsku dijagnostiku Medicinskogfakulteta Sveu~ilišta u Zagrebu, Klini~ki bolni~ki centar


Pove}ana koncentracija ukupnog i LDL kole-sterola nije znakovit nalaz u ve}ine osoba s ko-ronarnim bolestima. Ukazano je na izrazito ate-rogeniji u~inak malih, gustih LDL ~estica. Meha-nizam kojim se one stvaraju još nije razjašnjen.Oko 40% beta2-glukoproteina I u plazmi je veza-no za lipoproteine, što je dovelo do njegova ozna-~avanja kao apolipoproteina H. Uloga Apo H umetabolizmu lipida je zasad nejasna, premda ondjeluje na uklanjanje triglicerida iz krvi štakora iaktivira lipoprotein-lipazu. Studije s izoelektri~-nim fokusiranjem otkrile su da je to heterogenamolekula s pet glavnih izoforma. ApoH2 je naj-~eš}i alel u svim do danas ispitanim rasama, aslijede ApoH3 i ApoH1. Cilj ovoga rada bio jeispitati mogu}nost da na promjenu veli~ine LDL~estice tako|er utje~e i strukturni polimorfizamApoH. Stoga smo ispitali u~estalost pojedinih ale-la u osoba koje su imale razli~itu distribucijuveli~ina LDL ~estica: s preteìto velikim LDL ~es-ticama (’profil’ A), srednjim (’profil’ I) i malim

P3-12

LIPID PROFILE AND APO HPHENOTYPES IN DISLIPIDEMIC

SUBJECTS SHOWING DIFFERENT LDLPARTICLE SIZE DISTRIBUTION

M. Trbojevi}-êpe, @. Vogrinc, R. Zrinski-Topi}, A.Stavljeni}-Rukavina


Elevated total and LDL cholesterol is notcharacteristic of most individuals who sufferfrom coronary artery disease. It has already beenshown that the presence of small, dense LDL par-ticles is particularly atherogenic. The mecha-nism(s) responsible for their production remainunclear. Approximately 40% of the plasma beta2-glycoprotein I is associated with lipoproteins,which has led to its designation as apolipoproteinH. The role of apo H in lipid metabolism is stilluncertain, although it affects triglyceride clear-ance in the rat and is reported to be the activatorof lipoprotein lipase. Isoelectric focusing studieshave revealed apoH to be a heterogeneous mol-ecule with five major isoforms. The ApoH2 is themost common allele, followed by ApoH3 andApoH1, in all racial groups examined to date.The aim of this study was to evaluate the possibil-ity that the alterations in LDL particle size arealso influenced by structural polymorphism ofApo H. Thus, we determined the frequency distri-butions of the three apo H alleles in subjects char-



(’profil’ B). Skupina od 138 dobrovoljnih davalacakrvi s normalnom koncentracijom lipida u krvi inormalnom distribucijom veli~ina LDL ~estica(’profil’ A) izabrani su kao kontrolna skupina zaispitivanje polimorfizma ApoH u našoj populaciji.U~estalost ApoH alela je ispitana kod 386 osoba srazli~itim dislipidemijama koje su kasnije klasi-ficirane i po preteìtom ’profilu’ veli~ina LDL ~e-stica: A (n=127), I (n=111) i B (n=148). Distri-bucija LDL ~estica odre|ena je metodom gradi-jentne elektroforeze u poliakrilamidnom gelu,koja je razvijena u našem laboratoriju. Feno-tipovi ApoH su odre|eni metodom izoelektri~nogfokusiranja i imunoblota. îmbenici koji su po-vezani sa smanjenjem veli~ine LDL ~estica supove}ana koncentracija triglicerida u krvi i sma-njena koncentracija HDL kolesterola. Pove}anakoncentracija kolesterola u krvi udruèna s nis-kom koncentracijom triglicerida dovodi do stva-ranja velikih ~estica LDL. Nije na|ena statisti~kizna~ajna razlika u u~estalosti pojedinih alelaApoH izme|u kontrolne skupine i osoba s dislipi-demijama koje iskazuju razli~itu distribuciju ve-li~ina LDL ~estica.E-mail: [email protected]

acterized by different LDL particle size distribu-tions: with predominant large LDL subclasses(pattern A), intermediate (I), and small denseLDL particles (pattern B). A group of 138 healthyblood donors with normal lipid profile and nor-mal distribution of LDL particle size (LDL pat-tern A) were selected as a control group for theevaluation of apo H polymorphism in our popula-tion. The frequency distributions of the three ApoH alleles were demonstrated in 386 subjects withvarious dyslipidemias that were later classifiedalso according to the predominant LDL pattern:A (n=127), I (n=111) and B (n=148). LDL sizephenotyping was performed by non-denaturinggradient polyacrylamide gel electrophoresis de-veloped in our laboratory. ApoH phenotypes weredetermined by isoelectric focusing followed byimmunoblotting. The factors associated with thedecreased LDL size were elevated triglyceridesand decreased HDL cholesterol. Increased plas-ma cholesterol accompanied by the low level oftriglycerides produce large LDL particles. We didnot find a significant difference between the dis-tribution of apo H phenotypes in control individu-als and dyslipidemic subjects showing differentdistribution of LDL particle size.E-mail: [email protected]

P3-13

RAZINA HOMOCISTEINA PLAZME UBOLESNIKA NA HEMODIJALIZI

Škovraga S., Garilovi} J., V~elik M.

Hematolo{ko-biokemijski laboratorij, Op}a ùpanijskabolnica, Poèga

Kardiovaskularne bolesti su glavni uzrok smr-ti u bolesnika na hemodijalizi. Hiperhomocistei-nemija koja je vjerojatno uzrokovana poreme}enimbubre`nim metabolizmom predstavlja jedan od ~im-benika rizika za kardiovaskularne bolesti kod di-jaliziranih bolesnika u zadnjem stadiju bubre`ne bo-lesti. Cilj rada bio je odrediti i usporediti razinuhomocisteina prije i nakon hemodijalize u bole-snika u zadnjem stadiju bubre`ne bolesti. Ana-liziran je 21 uzorak EDTA-plazme bolesnika uzadnjem stadiju bubre`ne bolesti (10 m, 11 `) pri-je i nakon hemodijalize. Razina homocisteina jeodre|ivana na analizatoru IMX (Abbott) meto-dom fluorescentne imunopolarizacije (FPIA). Re-ferentne vrijednosti homocisteina su od 5 do 15µmol/l. Usporedno su odre|ene koncentracije ure-je, kreatinina, alkalne fosfataze i magnezija u se-

P3-13

TOTAL PLASMA HOMOCYSTEINE LEVELSIN PATIENTS WITH END-STAGE RENAL

DISEASE

S. Škovraga, J. Garilovi}, M. V~elik

Laboratory of Hematology and Biochemistry, CountyGeneral Hospital, Poèga

Cardiovascular disease is the leading cause ofmorbidity and mortality in patients with endstage renal disease. Elevated total plasma homo-cysteine, which is probably due to impaired renalmetabolism, is among the most common cardio-vascular risk factors in patients with chronic re-nal failure. The aim of the study was to determineand compare total homocysteine levels in patientswith end stage renal disease before and afterhemodialysis. EDTA samples were collected from21 patients (10 M, 11 F) before and after hemodi-alysis. Plasma homocysteine levels were meas-ured by automated fluorescence polarization im-munoassay (FPIA) on an IMX analyzer (Abbott).The reference range for homocysteine is 5-15µmol/l. The levels of urea, creatinine, alkaline



rumu svih bolesnika prije i nakon hemodijalize,kao i koncentracije kolesterola, HDL-kolesterola,LDL-kolesterola i triglicerida u serumu prije he-modijalize. Razina homocisteina prije hemodija-lize kretala se od 16,09 do 38,74 µmol/l (srednjavrijednost 30,22 µmol/l), a nakon hemodijalize od10,46 do 25,68 µmol/l (srednja vrijednost 17,94µmol/l). Dakle, nakon hemodijalize nastupa zna-~ajan, prosje~no 41%-tni pad razine homocisteinau plazmi bolesnika u odnosu na razinu homo-cisteina prije hemodijalize.E-mail: [email protected]

phosphatase and magnesium were measured insera of all patients before and after hemodialysis,and those of serum total cholesterol, HDL choles-terol, LDL cholesterol and triglycerides beforehemodialysis. The mean measured plasma homo-cysteine level was 30.22 (range 16.09-38.74) µmol/lbefore, and 17.94 (range 10.46-25.68) µmol/l af-ter hemodialysis. Thus, elevated plasma homo-cysteine levels were observed in all patients withend stage renal disease before hemodialysis. Asignificant 41% mean reduction in plasma homo-cysteine levels was observed after as compared tothe predialysis homocysteine levels.E-mail: [email protected]

P3-14

PRIKAZ SRÂNIH PARAMETARA URANOJ DIJAGNOZI AKUTNOG INFARKTA

MIOKARDA

Hrabri} S., ûka-Husnjak S., Dvornik Š.

Zavod za laboratorijsku dijagnostiku, Klini~ki bolni~kicentar “Rijeka”, Rijeka

Sr~ani biokemijski parametri su vrlo zna~ajniza rano dijagnosticiranje akutnog infarkta mio-karda (AIM), o ~emu naj~eš}e ovisi tijek i ishodbolesti. Odre|ivanje sr~anih parametara trebabiti osjetljivije i specifi~nije kako bi se u štokra}em razdoblju potvrdio AIM. Cilj ove studijeje bio prikazati kompatibilnost izme|u 3 raspo-loìva troponinska testa, kao i ostalih sr~anih para-metara (CK, CKMB mass i MYO) u ranoj dija-gnozi AIM-a. U studiju je bilo uklju~eno 37 bole-snika (13 ` i 24 m), od kojih je 6 primljeno prekohitne slu`be, a 31 ispitanik je bio smješten uKoronarnu jedinicu KBC “Rijeka”. Koncentracijatroponina T (cTnT) u serumu ispitanika mjerenaje na analizatoru Elecsys 1010 (kemiluminis-centna metoda), koncentracija troponina I (cTnI)je mjerena na analizatorima Axsym-Abbott(MEIA) i Dade Behring Dimension RxL (imuno-reakcija s monoklonskim protutijelima). Serum-ska koncentracija CK, CKMB mass i MYO je bilaodre|ena na analizatoru Dimension RxL. Rezul-tati su pokazali da su vrijednosti sr~anih para-metara u bolesnika bili povišeni: CK (65%),CKMB mass (59%), cTnI (65%), MYO (49%) icTnT (57%). Usporedbom troponina na razli~itimanalizatorima dobivena je visoka korelacija i sta-tisti~ka zna~ajnost (r2=0,9704, p<0,05) kod odre-|ivanja na Axsymu, nià korelacija (r2=0,8905)odre|ivanjem na Elecsysu 1010 u odnosu na tro-

P3-14

CARDIAC MARKERS IN THE EARLYDIAGNOSIS OF ACUTE MYOCARDIAL

INFARCTION

S. Hrabri}, S. ûka-Husnjak, [. Dvornik

Department of Laboratory Diagnosis, Rijeka UniversityHospital Center, Rijeka

Cardiac markers are very important in theearly diagnosis of acute myocardial infarction(AMI) as well as in the prediction of the diseaseoutcome. The measurement of cardiac markershas to be highly sensitive and specific in order toconfirm AMI as early as possible. The aim of thisstudy was to compare three commercially avail-able troponin tests and other cardiac markers(CK, CKMB mass and MYO) in the early diagno-sis of AMI. Thirty-seven patients were included inthe study (13 F and 24 M), 6 of them from theEmergency Service, and 31 from the CoronaryUnit. Elecsys 1010 analyzer (chemiluminescencemethod) was used to test troponin T (cTnT) serumlevels, while troponin I (cTnI) levels were meas-ured on Axsym-Abbott (MEIA) and Dade BehringDimension RxL (immunoassay with specificmonoclonal antibody) analyzers. CK, CKMBmass and MYO were measured on a DimensionRxL analyzer. Results showed increased levels ofcardiac markers in AMI patients: CK (65%),CKMB mass (59%), cTnI (65%), MYO (49%) andcTnT (57%). Comparison of the cTnI test per-formed on Dimension RxL with that on Axsymyielded a high and statistically significant corre-lation (r2=0.9704, p<0.05), which was lower oncomparison to Elecsys 1010 (r2=0.8905). Therewas a good correlation between CK and cTnI(r2=0.8094, p<0.05) on the Dimension RxL



ponine mjerene na analizatoru Dimension RxL.Dobra korelacija na|ena je izme|u CK i cTnI(r2=0,8094, p<0,05) na analizatoru Dimension.Rezultati ove studije pokazali su visoku osjet-ljivost odre|ivanja cTnI (65%) na analizatoru Di-mension RxL u odnosu na ostale sr~ane parame-tre kod postavljanja rane dijagnoze AIM. cTnI jenajbolji pokazatelj AIM i najdu`e ostaje povišenu serumu, za razliku od mioglobina koji se zakratko vrijeme vra}a na normalnu vrijednost.Rezultati su pokazali dobru korelaciju troponinakoji su odre|eni na trima razli~itim analizato-rima.E-mail: [email protected]

analyzer. These results indicated a higher sensi-tivity of cTnI (65%) on the Dimension RxLanalyzer relative to other cardiac markers in theearly diagnosis of AMI. Thus, cTnI is a better in-dicator of AMI that remains elevated in the se-rum for a longer period of time, while myoglobinreturns to normal values very fast. All commer-cially available troponin tests showed good corre-lation.E-mail: [email protected]

P3-15

KONCENTRACIJA C-REAKTIVNOGPROTEINA U LIKVORU TIJEKOM

AKUTNOG INFEKTIVNOG MENINGITISA

Suljevi} E., Jadri} R.

Institut za klini~ku kemiju i biokemiju, Medicinski fakultet,Sarajevo, BiH

Niska osjetljivost i specifi~nost tradicionalnihlaboratorijskih pretraga postale su nedostatne zato~no dijagnosticiranje i po~etak odgovaraju}eglije~enja u bolesnika s bakterijskim meningi-tisom. C-reaktivni protein (CRP) mogao bi bitikorisna dodatna pretraga za brzu dijagnozu bak-terijskog meningitisa. Ispitali smo u~inkovitostodre|ivanja CRP u likvoru tijekom akutnog bak-terijskog meningitisa. CRP smo analizirali osjet-ljivim imunoturbidimetrijskim testom na anali-zatoru Dimension RXL (Dade Behring). Koncen-tracije CRP u likvoru i diferencijalna bijela krvnaslika (BKS) mjerene su u 20 bolesnika (dob 1-50godina) s akutnim bakterijskim meningitisom, teu kontrolnoj skupini od 25 osoba bez infekcije iupale. Povišene razine CRP na|ene su u 94%bolesnika s bakterijskim meningitisom. Srednjavrijednost razine CRP bila je 0,25 (raspon 0,10-0,55) mg/L u kontrolnoj skupini i 21,4 (raspon10,2-100) mg/L u skupini bolesnika s bakterij-skim meningitisom. Manja no raznolika povi-šenja zapa`ena su u bolesnika s akutnim virus-nim meningitisom. Osjetljiv test za CRP u lik-voru bio bi vrijedna dodatna pretraga konven-cionalnim pretragama kod akutnog bakterijskogmeningitisa.E-mail: [email protected]

P3-15

C-REACTIVE PROTEIN CONCENTRATIONIN CEREBROSPINAL FLUID DURING

ACUTE BACTERIAL MENINGITIS

E. Suljevi}, R. Jadri}

Institute of Clinical Chemistry and Biochemistry, School ofMedicine, University of Sarajevo, Sarajevo, Bosnia &

Herzegovina

The low sensitivity and specificity of tradi-tional laboratory tests have become insufficientfor the accurate diagnosis and initiation of ap-propriate treatment in patients with bacterialmeningitis. C-reactive protein (CRP) may be auseful supplement for the rapid diagnosis of bac-terial meningitis. We investigated the effective-ness of CRP determination in cerebrospinal fluid(CSF) during acute bacterial meningitis. CRPwas analyzed by a sensitive immunoturbidimet-ric assay on a Dimension RXL analyzer (DadeBehring). CSF concentrations of CRP and differ-ential white blood cell count (WCC) were meas-ured in 20 patients (age range, 1-50 years) pre-senting with acute bacterial meningitis, and in acontrol group of 25 individuals free from infec-tion and inflammation. Elevated levels of CRPwere found in 94% of patients with bacterial men-ingitis. The mean CRP level was 0.25 (range 0.10-0.55) mg/L in the control group and 21.4 (range10.2-100) mg/L in the group of patients with bac-terial meningitis. Smaller but variable elevationswere observed in patients with acute viral menin-gitis. A sensitive assay for CRP in CSF would bea useful adjunct to conventional studies in acutebacterial meningitis.E-mail: [email protected]



P3-16

LIPIDNI STATUS SKUPINE RADNOSPOSOBNIH ISPITANIKA

Mirjani}-Azari} B.1, \eri} M.2, Popovi} M.1, Sani~aninZ.3

1 JZU Dom zdravlja, Banja Luka, BiH; 2 Klini~ki centar,Novi Sad, Srbija i Crna Gora; 3 Medicinski fakultet, Banja

Luka, BiH

Hiperlipoproteinemije predstavljaju jedan odnajzna~ajnijih rizi~nih ~imbenika za nastanakkoronarne bolesti srca. Cilj istraìvanja bio jeprocijeniti zastupljenost hiperlipoproteinemijakako bi se uvidjelo je li potrebno pristupiti pre-venciji hiperlipoproteinemija kod nas. Ispitano je250 radnika na redovnom sistematskom pregle-du. Koncentracija ukupnog kolesterola i trigli-cerida odre|ivana je enzimskom metodom(CHOD-PAP,GPO-PAP) na biokemijskom anali-zatoru Hitachi 902, a HDL-kolesterol u super-natantu poslije precipitacije drugih lipoproteinafosfo-volframat-magnezij reagensom. LDL-kole-sterol odre|ivan je ra~unskim putem po formuliFriedvalda i suradnika. Utvr|eno je da je ukupnikolesterol grani~no povišen kod 44,04% muška-raca i 44,68% èna, a visoko rizi~ne vrijednostiimalo je 43,12% muškaraca i 39,01% èna. Tri-gliceridi su bili grani~no povišeni kod 22,02%muškaraca i 19,15% èna, a visokorizi~ne vrijed-nosti imalo je 37,61% muškaraca i 9,93% èna.HDL-kolesterol je bio grani~no snièn kod55,96% muškaraca i 41,84% èna, dok je 5,50%muškaraca i 1,42% èna imalo visokorizi~ne vri-jednosti. LDL-kolesterol je bio grani~no povišenkod 31,96% muškaraca i 21,43% èna, dok je vi-sokorizi~ne vrijednosti imalo 43,30% muškaraca i42,14% èna. Hiperlipoproteinemija je zabilje-èna kod 62,8% ispitanika. Zabrinjavaju}e visokau~estalost poreme}aja metabolizma lipida i lipo-proteina ukazuje na potrebu uklju~ivanja analizaprve etape stepenaste laboratorijske dijagnostikeu rutinske pretrage u okviru sistematskih pre-gleda radnika. Neophodna je i edukacija cjelo-kupnog stanovništva u primjeni mjera za spre~a-vanje hiperlipoproteinemija.E-mail: [email protected]

P3-16

LIPID STATUS IN A GROUP OFSUBJECTS CAPABLE OF WORK

B. Mirjani}-Azari}1, M. \eri}2, M. Popovi}1, Z.Sani~anin3

1 JZU Health Center, Banja Luka, Bosnia & Herzegovina;2 Clinical Center, Novi Sad, Serbia & Montenegro; 3 School

of Medicine, Banja Luka, Bosnia & Herzegovina

Hyperlipoproteinemias are one of the majorrisk factors for coronary heart disease. The aim ofthe study was to determine the rate of hyper-lipoproteinemias in order to estimate the need oftaking preventive measures for hyperlipoprote-inemias in our country. A group of 250 workerswere examined at regular systematic check-up.Concentrations of total cholesterol andtriglycerides were determined by the enzymaticcolorimetric method (CHOD-PAP,GPO-PAP) on aHitachi 902 analyzer, and HDL-cholesterol wasdetermined in the supernatant upon precipitationof other lipoproteins, with the addition of phos-photungstic acid and MgCl2. LDL-cholesterolwas calculated according to the formula ofFriedvald and coworkers. Borderline elevation oftotal cholesterol was found in 44.04% of male and44.68% of female, and high risk values were re-corded in 43.12% of male and 39.01% of femalesubjects. Borderline elevation of triglycerides wasrecorded in 22.02% of male and 19.15% of femalesubjects, whereas high risk values were recordedin 37.61% of male and 9.93% of female subjects.Borderline decrease in HDL-cholesterol was ob-served in 55.96% of male and 41.84% of femalesubjects, whereas 5.50% of male and 1.42% of fe-male subjects showed high risk values. Border-line elevation of LDL-cholesterol was recorded in31.96% of male and 21.43% of female subjects,whereas high risk values were found in 43.30% ofmale and 42.14% of female subjects. Hyper-lipoproteinemia was recorded in 62.8% of studysubjects. The prevalence of lipid metabolism dis-orders was alarmingly high, pointing to the needof adding these tests in the first-step procedures ofgraduated laboratory diagnosis as routine testson regular systematic check-ups. Proper educa-tion of the population at large in the measures ofhyperlipoproteinemia prevention is warranted.E-mail: [email protected]



P3-17

UTJECAJ VALSARTANA NA AKTIVNOSTNa+/K+-ATPaze U KORI BUBREGA

ŠTAKORA

Kneèvi} A., Samarìja I.

Zavod za farmakologiju, Farmaceutsko-biokemijski fakultetSveu~ilišta u Zagrebu, Zagreb

U skupini antihipertenzivnih lijekova koji dje-luju na renin-angiotenzinski sustav nalaze se in-hibitori konvertaze angiotenzina (ACE-inhibito-ri) i antagonisti angiotenzinskih receptora (AT

1-

blokatori). AT1-receptori za angiotenzin II. smje-

šteni su u bubregu unutar tubularnog i vasku-larnog sustava, i to u proksimalnim tubulima(luminalna i bazolateralna membrana), distal-nim tubulima i sabirnim kanali}ima te aferent-nim i eferentnim arteriolama glomerula. Prisut-na raspodjela AT

1-receptora ukazuje na fiziološko

zna~enje angiotenzina II. u regulaciji ravnoteèteku}ine i elektrolita, naro~ito Na+ i bikarbo-nata, zatim glomerularne filtracije te kortikalne imedularne mikrocirkulacije (bubre`ne hemo-dinamike). Cilj ovoga rada bio je utvrditi utjecajvalsartana (AT

1-blokatora) na aktivnost Na+/K+-

ATPaze, membranskog enzima zna~ajnog za odr-àvanje membranskog potencijala i stani~nog vo-lumena. Aktivnost enzima se odre|ivala u izoli-ranim membranskim vezikulama kore bubregaštakora u kojoj su najviše zastupljeni proksimal-ni i distalni tubuli. Mjeru enzimatske aktivnostije predstavljao oslobo|eni anorganski fosfor kojise odre|ivao spektrofotometrijski. Postupci izo-lacije membrana su uslijedili 1 sat nakon i.p.primjene lijeka odnosno fiziološke otopine. Podkontrolnim uvjetima aktivnost enzima u kori jeiznosila 28,5±7,6 µmol P

i/mg proteina/h (n=6).

Pod utjecajem valsartana (160 mg/100g tjelesnemase) aktivnost enzima se u prosjeku pove}alaza 29% i iznosila je 37,8±6,1 µmol P

i/mg proteina/

h. Iz navedenoga se moè zaklju~iti da valsartannakon jednokratne primjene pove}ava aktivnostNa+/K+-ATPaze. Taj porast aktivnosti enzima jevjerojatno nastao kao posljedica neposredne inhi-bicije AT1-receptora preko kojih se provodi regu-lacija Na+/H+ izmjene u luminalnoj membrani iNa+/HCO

3- kotransporta u bazolateralnoj mem-

brani proksimalnih tubula. Stoga pove}anje ak-tivnosti Na+/K+-ATPaze moè biti kompenzacij-ska reakcija u svrhu odràvanja ravnoteè elek-trolita u organizmu. Za o~ekivati je da bi dugo-trajna primjena AT

1-blokatora mogla izazvati slo-

ènije odgovore na razini membranskih trans-portnih sustava i posljedi~no aktivnost pumpe.

P3-17

EFFECT OF VALSARTAN ON Na+/K+-ATPase ACTIVITY IN THE RAT RENAL

CORTEX

A. Kneèvi}, I. Samarìja

Department of Pharmacology, Zagreb University School ofPharmacy and Biochemistry, Zagreb

The angiotensin-converting enzyme inhibitors(ACE-inhibitors) and angiotensin II receptor an-tagonists (AT1-antagonists) belong to the antihy-pertensive class of drugs which act on the renin-angiotensin system. The intrarenal AT1-receptorsare mostly located within the tubular and vascu-lar systems which include proximal tubules (lu-minal and basolateral membrane), distal tubuleand collecting ducts as well as glomerular affer-ent and efferent arterioles. The present distribu-tion of AT1-receptors stresses the physiologic im-portance of angiotensin II in the regulation ofglomerular filtration, cortical and medullary mi-crocirculation in addition to maintaining balanceof body fluids and electrolytes, especially sodiumand bicarbonate. The aim of this study was to in-vestigate the effect of valsartan (AT1-antagonist)on Na+/K+-ATPase activity. Na+/K+-ATPase is amembrane bound enzyme which regulates cell po-tential and volume. The enzyme activity wasmeasured on isolated rat cortical membrane vesi-cles which were enriched in proximal and distaltubular cells. The released inorganic phosphorusrepresented the measure of Na+/K+-ATPase activ-ity. The preparation of membrane vesicles pro-ceeded for 1 hour after i.p. application ofvalsartan (160 mg/100 g body mass) or saline.The enzyme control activity was 28.5±7.6 µmolPi/mg protein/h (n=6). Under the influence ofvalsartan the enzyme activity increased by 29%,to a value of 37.8±6.1 µmol Pi/mg protein/h. Inorder to maintain the electrolyte balance, the in-crease of enzyme activity might have been a com-pensatory reaction to the reduced Na+ reabsorp-tion due to the inhibition of AT1-receptors at theluminal membrane (regulation of Na+/H+ ex-change) and basolateral membrane (regulation ofNa+/HCO3

- cotransport). It appears thatlongterm use of AT1-antagonists could elicit morecomplex responses of the membrane transportsystems and consequently the pump activity.



P4-1

PROMJENA BAZNIH VRIJEDNOSTIBIOKEMIJSKIH BILJEGA KOŠTANE

PREGRADNJE OSTEOKALCINAI BETA-CROSSLAPSA

Rako I.1, Bulatovi} G.1, Baši}-Markovi} N.2

1 Medicinsko-biokemijski laboratorij, Poliklinika “Medico”,Rijeka, 2 Ordinacija op}e medicine, Rijeka

Više bazne vrijednosti osteokalcina i beta-crosslapsa zna~ajno su povezane s brìm gubit-kom koštane mase u menopauzi. @ene s visokimvrijednostima ovih biljega gube tijekom ~etirigodine 2 do 6 puta više koštane mase nego one snormalnim vrijednostima. Prednost odre|ivanjakoštanih biljega je u tome što nas obavještavaju ometaboli~noj aktivnosti koštanoga tkiva, dokdenzitometrijski nalaz daje uvid u trenutnu koš-tanu strukturu i masu. Cilj ovoga rada bio jedokazati da su bazne vrijednosti biljega koštanepregradnje, kao i njihov porast nakon osam mje-seci, u prosjeku viši kod èna u menopauzi. Pra-}ena je promjena koncentracije PTH prilikompromjene koncentracije koštanih biljega, te ko-relacija osteokalcina i beta-crosslapsa. Koncen-tracija osteokalcina i beta-crosslapsa odre|ivalase imunokemijskom metodom elektrokemilumi-nijscencije u serumu 33 ène u premenopauzi i 19èna u menopauzi bez terapije. Prosje~ne baznevrijednosti osteokalcina i beta-crosslapsa kod è-na u premenopauzi bile su niè od vrijednostiistih kod èna u menopauzi. Prosje~na koncen-tracija PTH tako|er je bila nià kod èna u pre-menopauzi, a rasla je s porastom koncentracijekoštanih biljega pregradnje. Nije dokazan zna-~ajniji porast koncentracije koštanih biljega uskupini èna u menopauzi u razdoblju od osammjeseci. Koeficijenti korelacije osteokalcina ibeta-crosslapsa u objema skupinama (r1=0,621;r2=0,837) pokazali su zna~ajnu i vrlo visoku me-|usobnu povezanost ovih dvaju biokemijskih bi-ljega koštane pregradnje. Više bazne vrijednostiosteokalcina i beta-crosslapsa kod èna u meno-pauzi, kao i njihov porast nakon osam mjesecikod velikog broja èna (naro~ito beta-crosslapsau 71,4%), ukazuju na va`nost njihovog odre|iva-nja u menopauzi, jer nas obavještavaju o poja-~anoj koštanoj pregradnji u toj ìvotnoj dobi.Dokazani porast koncentracije PTH u menopauzi

P4Bolesti koštanog i vezivnog sustavaBone and connective tissue diseases

P4-1

CHANGES IN THE BASELINE LEVELS OFOSTEOCALCIN AND BETA-CROSSLAPS,

BIOCHEMICAL MARKERS OF BONETURNOVER

I. Rako1, G. Bulatovi}1, N. Baši}-Markovi}2

1 Laboratory of Medical Biochemistry, Medico Polyclinic,Rijeka, 2 Ordinacija op}e medicine, Rijeka

Higher baseline levels of osteocalcin and beta-crosslaps are significantly associated with ahigher rate of bone loss in the menopause. Over a4-year period, women with higher levels of thesemarkers have a rate of bone loss 2- to 6-fold thatin women with normal levels. Determination ofthese bone turnover markers is highly useful forproviding information on the bone metabolic ac-tivity, whereas densitometry just provides an in-sight into the bone mass status at the time ofmeasurement. The aim of the study was to dem-onstrate that baseline levels of bone turnovermarkers and their increase after eight monthswere on an average higher in menopausalwomen. The concentration of PTH was monitoredin parallel with changes in the bone turnovermarkers, as well as correlation betweenosteocalcin and beta-crosslaps. Serum concentra-tions of osteocalcin and beta-crosslaps were deter-mined in 33 premenopausal and 19 menopausalwomen (no therapy) by electrochemiluminescenceimmunoassay. The mean baseline values ofosteocalcin and beta-crosslaps in premenopausalwomen were lower than the respective values inmenopausal women. The mean concentration ofPTH was also lower in premenopausal womenand increased with the rising concentration ofbone turnover markers. There was no significantrise in the concentration of bone turnover markersin menopausal women after eight months. Theparametric correlation tests revealed significantpositive correlations between osteocalcin andbeta-crosslaps in both groups of women. Higherbaseline levels of osteocalcin and beta-crosslapsin menopausal women, and their increase aftereight months in the majority of women (especiallyof beta-crosslaps in 71.4%), suggest the impor-tance of their determination in menopause be-cause they provide information on the increasingbone turnover in this age. The demonstrated in-



moè se povezati s postupnim opadanjem kon-centracije kalcija u serumu, što u kombinaciji svisokim vrijednostima beta-crosslapsa kod ènau menopauzi moè zna~iti opasnost od osteo-poroze ili njen po~etak. U takvim slu~ajevimapreporu~a se napraviti denzitometriju kako bi sepotvrdio brì gubitak koštane mase što ga pred-vi|aju koštani biljezi pregradnje.E-mail: [email protected]

crease in PTH concentration in menopause maybe associated with gradual decrease of serum cal-cium concentration which, in combination withelevated levels of beta-crosslaps in menopausalwomen, could imply the risk of osteoporosis oreven onset of the process. In these cases, densitom-etry is recommended to confirm precipitated boneloss predicted by bone turnover markers.E-mail: [email protected]

P4-2

VRIJEDNOSTI KOŠTANIH BILJEGAOSTEOKALCINA I BETA-CROSSLAPSA UPLAZMI KOD @ENA OD 35-55 GODINA NA

PODRU^JU SPLITA

Zlodre S.

Poliklinika za urologiju i citologiju sa specijalisti~kimmedicinsko-biokemijskim laboratorijem “Analiza”, Split

Osteoporoza je heterogeni poreme}aj defini-ran kao stanje smanjene koštane mase po jedinicivolumena s normalnim odnosom minerala i ma-triksa. To je bolest koja danas u svijetu ima epi-demijske razmjere. Biokemijske pretrage se rabekod dijagnosticiranja bolesti kao dopuna denzi-tometriji, pomo} pri odlu~ivanju o terapijskompostupku, predvi|anju rizika budu}ih prijeloma ipra}enju terapije. S obzirom na to da je kost re-zultat procesa neprestanog izgra|ivanja i razgra-|ivanja u kojem sudjeluju brojni ~imbenici, jednaskupina biokemijskih pretraga ukazuje na stu-panj izgradnje, a druga razgradnje kosti. Osteo-kalcin je protein koji sintetiziraju osteoblasti, pase njegove koncentracije u serumu smatraju bi-ljegom stvaranja kosti. Naziv beta-CrossLaps ilibeta CTx ozna~ava popre~no vezano mjesto okta-peptida C-terminalnog telopeptida kolagena tipaI koji sadrì isoaspartil (beta-aspartil) peptidnuvezu u njenom L-entantiomernom obliku. Raz-gradnjom kolagena nastaju razgradni proizvodikoji dospijevaju u cirkulaciju. Neki od njih sadrèizomeriziranu skupinu beta-CrossLaps i pokaza-telj su stupnja razgradnje kosti. Oba parametrapokazuju visoku dijagnosti~ku specifi~nost i os-jetljivost kao biljezi koštane pregradnje. Cilj stu-dije bio je utvrditi mogu}nost upotrebe ovih bi-ljega kao neinvazivne metode za ispitanika uprocjeni rizika nastanka osteoporoze. Analiziranoje 40 plazma pripremljenih s K2EDTA kao anti-koagulansom, nasumce odabranih ènskih osobau dobi od 35-55 godina na podru~ju Splita, kojenisu imale nikakve osteoporotske komplikacije.

P4-2

PLASMA CONCENTRATIONS OF BONEMARKERS OSTEOCALCIN AND BETA-CROSSLAPS IN WOMEN AGED 35-55

FROM THE SPLIT AREA

S. Zlodre

Laboratory of Medical Biochemistry, Analiza Polyclinic ofUrology and Cytology, Split

Osteoporosis is a heterogeneous dysfunctiondefined as a state of decreased bone mass per vol-ume unit with normal mineral and matrix ratio.Today, the disease has acquired epidemic propor-tions worldwide. Biochemical tests are used inaddition to densitometry to diagnose the disease,to help in making therapeutic decisions, to pre-dict the risk of future fractures, and in therapymonitoring. Since bone is the result of a constantprocess of formation and resorption called boneremodeling, which includes numerous differentfactors, a group of tests reflect the degree of boneformation and others the degree of boneresorption. Osteocalcin is a protein synthesized byosteoblasts, so its concentrations are considered amarker of bone formation. Beta-CrossLaps orbeta-CTx is the name of the crosslinked octapep-tide of C-terminal telopeptide collagen type I,which contains isoaspartyl (beta-aspartyl) pep-tide bond in its L-enantiomeric form. During thecollagen type I breakdown, degradation productsreach the circulation. Some of them contain theisomerized beta-CrossLaps bond and indicate thedegree of bone degradation. Both parametersshow high diagnostic specificity and sensitivityas markers of bone remodeling. The aim of thestudy was to evaluate the use of these markers asa noninvasive method to assess the risk of oste-oporosis. Forty plasma specimens prepared withK2EDTA as anticoagulant, obtained from ran-domly selected women free from osteoporosis dis-comforts, aged 35-55, from the Split area weretested. Osteocalcin and beta-CrossLaps concen-



Koncentraciju osteokalcina i beta-CrossLapsa od-re|ivali smo testovima tvrtke Roche Diagnostics:Osteokalcin kat. broj 2 149 133; beta-CrossLapskat. broj 1972308 na aparatu Elecsys 1010, send-vi~ imunokemijskom metodom elektrokemilumi-niscencije. Koncentracija osteokalcina u refe-rentnom rasponu (11-43 ng/mL) na|ena je u92,5%, a takva koncentracija beta-CrossLapsa(<0,573 pg/mL) u 95,0% uzoraka. Pove}ane kon-centracije osteokalcina na|ene su u 3 (7.5%)uzorka, a sniène ni u jednom uzorku. Pove}anekoncentracije beta-CrossLapsa na|ene su u 5(12.5%) uzoraka. Dobiveni rezultati ukazuju napotrebu odre|ivanja navedenih koštanih biljegauz denzitometriju. Preporuka bi bila pra}enjevrijednosti parametara tijekom odre|enog raz-doblja.E-mail: [email protected]

trations were determined by use of Roche Diag-nostics tests, Osteocalcin cat. number 2149133and Beta-CrossLaps, cat. number 1972308. Ana-lyses were performed on an Elecsys 1010 instru-ment by the sandwich immunoassay electro-chemiluminescent technique. Osteocalcin concen-trations within the reference range (11-43 ng/mL)were found in 92.5%, and beta-CrossLaps withinthe reference range (<0.573 ng/mL) in 87.5% ofthe samples. Osteocalcin concentrations aboveand below the reference range were found in 3(7.5%) and none of the samples, respectively.Beta-CrossLaps concentrations above the refer-ence range were found in 5 (12.5%) samples.Study results pointed to the neccessity of deter-mining specific bone markers in addition to den-sitometry. It is recommended to follow up the val-ues of these parameters over a longer period oftime.E-mail: [email protected]

P4-3

PROTUTIJELA NA OKSIDIRANI LDLU SISTEMSKOM LUPUSU I

REUMATOIDNOM ARTRITISU

Beljan B.1, Matica J.1, Bili}-Zulle L.1,2, Fiši} E.1, Dvornik Š.1

1 KBC Rijeka, 2 Medicinski fakultet Sveu~ilišta u Rijeci, Rijeka

Oksidirani LDL stvara se uslijed djelovanjaslobodnih radikala na LDL, pri~em nastaju imu-nogeni epitopi koji mogu uzrokovati stvaranjeprotutijela na oksidirani LDL (oLAB). Povišenevrijednosti oLAB nalaze se kod bolesnika s kar-diovaskularnim bolestima, dijabetesom, sistem-skim eritematoznim lupusom (SLE), preeklamp-sijom i hipertireozom. Sniène vrijednosti nalazese kod akutnog infarkta miokarda, ishemijskogmo`danog udara i akutne septikemije. Cilj radabio je odrediti koncentraciju oLAB u serumuispitanika sa SLE i reumatoidnim artritisom(RA), te ih usporediti s koncentracijama u kon-trolnoj skupini. Koncentracija oLAB odre|ena jeu serumu 29 ispitanika sa SLE (4 muškarca, 25èna), 26 ispitanika s RA (3 muškarca, 23 ène) i28 ispitanika iz kontrolne skupine (3 muškarca,26 èna) heterogenom enzimimunokemijskommetodom s reagencijama tvrtke BiomedicaGmbH (Biomedica Gruppe, Be~, Austrija). Pro-cjena izmjerenih vrijednosti u~injena je premapreporu~enim referentnim vrijednostima proiz-vo|a~a: 94-315 mU/ml. Izmjerene vrijednostioLAB izme|u skupina uspore|ene su Kruskal-Wallisovim testom. Zaklju~ivanja su izvedena za

P4-3

ANTIBODIES AGAINST OXIDIZED LDL INSYSTEMIC LUPUS ERYTHEMATOSUS

AND RHEUMATOID ARTHRITIS

B. Beljan1, J. Matica1, L. Bili}-Zulle1,2, E. Fiši}1, S. Dvornik1

1 Rijeka University Hospital Center, Rijeka, 2 Rijeka UniversitySchool of Medicine, Rijeka

Oxidized LDL is generated by the action of freeradicals and results in the formation of modifiedepitopes which are immunogenic. In consequence,antibodies against oxidized LDL (oLAB) are gen-erated. Elevated oLAB concentrations are foundin sera of patients with cardiovascular diseases,diabetes, systemic lupus erythematosus (SLE),preeclampsia and hyperthyroidism. ReducedoLAB concentrations are found in acute myocar-dial infarction, ischemic stroke and acute septi-cemia. The aim of study was to evaluate serumoLAB levels in patients with SLE and rheuma-toid arthritis (RA), and to compare them with thelevels in healthy subjects. oLAB values weremeasured in sera of 29 subjects with SLE (4 maleand 25 female), 26 subjects with RA (3 male and23 female), and 28 control subjects (3 male and26 female). Quantitative determination of oLABwas performed using the Biomedica GmbH(Biomedica Gruppe, Vienna, Austria) enzyme im-munoassay kit. The reference range of 94-315mU/ml recommended by the manufacturer wasused in the study. The values determined in thethree groups were compared using Kruskal-



razinu zna~ajnosti p<0,05. U skupini ispitanikasa SLE medijan i raspon izmjerenih vrijednostibio je 409 mU/ml (16-3149); s RA 327 mU/ml (62-3967) i u kontrolnoj skupini 295 mU/ml (8-2767).Kod 14% ispitanika sa SLE, 11% s RA i 18% izkontrolne skupine izmjerene su sni`ene vrijed-nosti oLAB; kod 24% ispitanika sa SLE, 39% sRA i 36% iz kontrolne skupine izmjerene su nor-malne vrijednosti, a kod 62% ispitanika sa SLE,50% s RA i 46% iz kontrolne skupine izmjerenesu povišene vrijednosti oLAB. Kruskal-Walliso-vim testom nije prona|ena statisti~ki zna~ajnarazlika me|u skupinama ispitanika (p=0,451). Iz-mjereni rezultati ne ukazuju na postojanje zna-~enja odre|ivanja protutijela na oksidirani LDLu dijagnostici SLE i RA. Za potvrdu dijagnosti~kevrijednosti oLAB potrebna su daljnja istra`i-vanja.E-mail: [email protected]

Wallis test. Statistical significance was set atp<0.05. The median and range of oLAB were 409(16-3149) mU/ml in SLE, 327 (62-3967) mU/mlin RA, and 295 (8-2767) mU/ml in control sub-jects. Relative to the reference range, decreasedoLAB concentration was recorded in 14% of SLE,11% of RA and 18% of control subjects; normaloLAB concentration was recorded in 24% of SLE,39% of RA and 36% of control subjects; and el-evated oLAB concentration was found in 62% ofSLE, 50% of RA and 46% of control subjects.Kruskal-Wallis test yielded no statistically sig-nificant difference in oLAB concentrations amongthe three groups of subjects (p=0.451). Our resultsshowed serum oLAB values to be of no diagnosticvalue in the assessment of patients with SLE andRA as compared with healthy subjects. Addi-tional studies are recommended.E-mail: [email protected]

P4-4

IMUNOLOŠKI STATUS KOD BOLESNIKASA SISTEMSKIM ERITEMATOZNIM

LUPUSOM

Peran N., Meniga D., Reškov @., Pamukovi}-Jaram D.

Biokemijsko-hematološki laboratorij, Op}a bolnica Šibenik,[ibenik

Sistemski eritemski lupus (SLE) je kroni~naupalna autoimuna bolest nepoznate etiologijekod koje tjelesni imunološki sustav koji normalnoštiti tijelo od bolesti po~inje napadati vlastita tki-va. Cilj ovoga rada je bio odrediti neke imuno-loške parametre kod bolesnika sa SLE: protutijelapotiv jezgre (ANA), protutijela protiv dvolan~aneDNA (anti-dsDNA), lupus antikoagulant (LAC)protutijela protiv negativno nabijenih fosfolipidaili kompleksa fosfolipida s proteinima, cirkulira-ju}e imunokomplekse (CIC), komponente kom-plementa C3 i C4. Ispitano je 22 bolesnika saSLE i kontrolna skupina od 20 zdravih ispita-nika. ANA su odre|ena metodom indirektne imu-nofluorescencije na stanicama Hep-2 (BioSys-tems). Anti-dsDNA su odre|ena metodom indi-rektne imunofluorescencije na hemoflagelatu Cri-thidia luciliae (Biosystems). LAC je odre|en vre-menom dRVV (BCT, Dade Behring). CIC je odre-|en nefelometrijski, a C3 i C4 turbidimetrijski(Dade Behring). Pozitivan ANA test, titar ve}i od1:40 imalo je 95% bolesnika sa SLE, ali je i 15%zdravih ispitanika imalo pozitivan ovaj test, titar1:40; 75% bolesnika sa SLE imalo je anti-dsDNA,

P4-4

IMMUNE STATUS IN PATIENTS WITHSYSTEMIC LUPUS ERYTHEMATOSUS

N. Peran, D. Meniga, @. Reškov, D. Pamukovi}-Jaram

Laboratory of Biochemistry and Hematology, ŠibenikGeneral Hospital, Šibenik

Systemic lupus erythematosus (SLE ) is achronic inflammatory autoimmune disease of un-known etiology, in which the body’s immune sys-tem, which normally protects the body from dis-ease, starts to attack its own tissues. The aim ofthis study was to determine some immunologicparameters in patients with SLE: antibodiesagainst cell nucleus (ANA), antibodies againstdouble-stranded DNA (anti-dsDNA), lupus anti-coagulant (LAC) antibodies against negativelycharged phospholipids or phospholipid com-plexes with proteins, circulating immune com-plexes (CIC), and complements C3 and C4. Thestudy included 22 SLE patients and a controlgroup of 20 healthy subjects. ANA were deter-mined by immunofluorescence assay on Hep-2cells (BioSystems), anti-dsDNA by immunofluo-rescence assay using Crithidia luciliae hemofla-gellate (Biosystems), LAC by dRVV time (BCT,Dade Behring), CIC by nephelometric technique,and C3 and C4 by turbidimetric assay (DadeBehring). Positive ANA test, titer >1:40 was re-corded in 95% of patients with SLE, and in 15%of healthy subjects with a titer 1:40; 75% of SLEpatients and none of control subjects had positive



dok niti jedan zdravi ispitanik nije imao ovaprotutijela; 40% bolesnika sa SLE imalo je LAC,dok je u 23% bolesnika bila prisutna venska iliarterijska tromboza. Niti jedan zdravi ispitaniknije imao ova protutijela; CIC su bili prisutni kod90% bolesnika sa SLE, ali i kod 10% zdravihispitanika; C3 i C4 su bili snièni kod 90% bole-snika sa SLE. ANA test ima veliku osjetljivost,ali ne i specifi~nost za dijagnozu SLE. Anti-dsDNA test je visoko specifi~an za SLE. Prisut-nost LAC pove}ava rizik od tromboembolijskihbolesti. CIC su prisutni u serumu ve}ine bole-snika sa SLE, ali nisu specifi~ni za SLE. Potroš-nja komplementa je zna~ajno pove}ana u SLE,što izaziva sniènje serumskog komplementa.E-mail: [email protected]

anti-dsDNA; 40% of SLE patients had positiveLAC; 23% of SLE patients and none of controlsubjects had venous or arterial thrombosis; CICwere present in 90% of SLE patients, but also in10% of healthy individuals. C3 and C4 were lowin 90% of SLE patients. ANA test was found to beof high sensitivity but inadequate specificity forthe diagnosis of SLE. Anti-dsDNA test is highlyspecific for SLE. The presence of LAC increasesthe risk of thromboembolism. CIC are present inserum of most SLE patients but are not specificfor SLE. Serum complement is decreased due tothe significantly increased complement consump-tion in SLE.E-mail: [email protected]

P4-5

IZOENZIMI ALKALNE FOSFATAZE UBENIGNOJ TRANZITORNOJ

HIPERFOSFATAZEMIJI

Milanovi}-Stipkovi} B., Jur~i} Z., Topi} E., @aja-Franulovi} O.


Tranzitorna hiperfosfatazemija je rijedak po-reme}aj izrazitog povišenja ukupne aktivnosti al-kalne fosfataze. Javlja se naj~eš}e u dje~joj dobi.Nema specifi~nih simptoma i otkriva se slu~ajno.U razdoblju od travnja 2001. do studenoga 2002.godine otkriveno je sedmoro djece s izrazito viso-kom aktivnoš}u alkalne fostataze u serumu. Upetero djece prona|eno je i pridruèno pove}anjeaktivnosti 5-nukleotidaze. Prosje~na dob djece jebila 12,9 (raspon 7-21) mjeseci. Razlog hospi-talizacije bio je razli~it: infekcija respiracijskogtrakta, enteropatija, anemija i prijeoperacijskepretrage. Na osnovi laboratorijskih nalaza i kli-ni~kog pregleda isklju~ena je bolest jetre ili me-taboli~ka bolest kostiju. Alkalna fosfataza odre-|ivana je metodom IFCC-a reagencijama tvrtkeOlympus na automatskom analizatoru AU 600Olympus. Izoenzimi AP odre|ivani su na agaroz-nom gelu Isopal Plus - Beckman. Radi boljeg raz-dvajanja koštanog i jetrenog izoenzima primije-njena je temperaturna denaturacija na 56 0C/10minuta i obrada neuraminidazom. Ukupne aktiv-nosti alkalne fosfataze u ispitivane djece bile suprosje~no 3665 (raspon 1628-5469) IU/L. Razdva-janjem izoenzima dokazano je da porastu ukupneaktivnosti uglavnom doprinosi koštani izoenzim.

P4-5

ALKALINE PHOSPHATASE ISOENZYMESIN BENIGN TRANSITORYHYPERPHOSPHATASEMIA

B. Milanovi}-Stipkovi}, Z. Jur~i}, E. Topi}, O. @aja-Franulovi}


Transitory hyperphosphatasemia is a rare dis-order characterized by a marked increase in totalalkaline phosphatase (AP) activity. It usually oc-curs in childhood, has no specific symptoms, andis detected by accident. From April 2001 till No-vember 2002, seven children (mean age 12.9,range 7-21 months) with extremely high serumAP activity were seen. An associated increase inthe 5-nucleotidase activity was detected in fivechildren. They were hospitalized for various rea-sons like respiratory infection, enteropathy,anemia and preoperative screening. Based onlaboratory findings, radiologic and clinical ex-amination, hepatic disease and metabolic bonedisease were excluded. AP was determined by theIFCC method using Olympus reagents on an AU600 Olympus autoanalyzer. AP isoenzymes weredetermined on Beckman Isopal Plus agarose gel.Temperature denaturation at 56 0C/10 min andneuraminidase treatment were used for betterseparation of the bone and hepatic isoenzymes.The pooled mean AP activity in the study chil-dren was 3665 (range 1628-5469) IU/L.Isoenzyme separation demonstrated the boneisoenzyme to predominantly contribute to the to-tal AP activity increase. Five children also



Petero djece imalo je tako|er i lagani porast je-trenog izoenzima. Do normalizacije aktivnosti al-kalne fosfataze došlo je spontano u prosjeku za5,1 (raspon 1,5-9) mjeseci. Pravodobnim prepo-znavanjem ovoga biokemijskog fenomena mogu-}e je izbje}i zamke pogrešne dijagnoze i nepo-trebnih pretraga.E-mail: [email protected]

showed a mild hepatic isoenzyme elevation. TheAP activity normalized spontaneously in a meanof 5.1 (range 1.5-9) months. Timely recognition ofthis biochemical phenomenon can help avoid thetraps of false diagnosis and unnecessary testing.E-mail: [email protected]

P5-1

METODA ODRE\IVANJA KATEKOLAMINAU MOKRA]I POMO]U TEKU]INSKE

KROMATOGRAFIJE VISOKOGRAZLU^IVANJA UZ FLUORESCENTNI

DETEKTOR

Petek-Tarnik I., Petek M.

Endokrinološki laboratorij, Klini~ka bolnica “Sestremilosrdnice”, Zagreb

Katekolamini (adrenalin, noradrenalin i dopa-min) imaju va`nu ulogu u `iv~anom sustavu i uregulaciji krvnog tlaka. Njihovo je odre|ivanjevrlo va`no u dijagnosticiranju i pra}enju simpa-toadrenalnih tumora, primjerice, feokromocito-ma i neuroblastoma. Cilj rada je bio poboljšatimetodu odre|ivanja katekolamina u mokra}i.Opisana je metoda kojom se u kratkom vremenuistodobno odre|uju adrenalin, noradrenalin i do-pamin pomo}u teku}inske kromatografije viso-kog razlu~ivanja uz fluorescentni detektor. Dabismo uzorak pripremili za nanošenje na kolonurabili smo metodu Antona i Sayrea, koju smoprilagodili koli~ini uzorka. Odvajanje smo pro-vodili na koloni Sephasil peptide C18 (250x4.6x5mm), uz mobilnu fazu koju su ~inili fosfatni pufers dodatkom oktansulfonske kiseline i acetonitrilu omjeru 93 : 7 v/v, pri pH 3,05. Rezultate dobi-vene novom metodom usporedili smo s rezul-tatima metode teku}inske kromatografije uzelektrokemijski detektor, koja je ve} prije po-stavljena u našem laboratoriju. Ispitali smo uzor-ke 200 mokra}a, a rezultati su se vrlo dobroslagali, što je potvr|eno koeficijentom korelacijeod 0,9879. Normalne vrijednosti adrenalina(10,9-65,5 nmol/dU), noradrenalina (32,5-271,9nmol/dU) i dopamina (<2500 nmol/dU) nisu serazlikovale usporedbom s dosadašnjom metodom.

P5Endokrinološke bolesti

Endocrine diseases

P5-1

DETERMINATION OF URINARYCATECHOLAMINES BY HIGH

PERFORMANCE LIQUIDCHROMATOGRAPHY WITH

FLUORESCENCE DETECTION

I. Petek-Tarnik, M. Petek

Laboratory of Endocrinology, Sestre milosrdnice UniversityHospital, Zagreb

Catecholamines (epinephrine, norepinephrineand dopamine) play an important role in the cen-tral nervous system and in the regulation of bloodpressure. The determination of urinary cate-cholamines is essential for the diagnosis andmonitoring of sympathoadrenal tumors such aspheochromocytomas and neuroblastomas. Theaim of the study was to improve the method usedfor determination of urinary catecholamines. TheHPLC method for fast simultaneous determina-tion of epinephrine, norepinephrine and dopa-mine in 24-h urine with fluorescence detection isdescribed. A modified micro version of Anton andSayre was used for sample preparation. The sepa-ration was performed on a Sephasil peptide C18column (250x4.6x5 mm) using mobile phase con-taining phosphate buffer with octanesulfonic acidand acetonitrile (93:7 v/v, pH 3.05). The newmethod was compared to the HPLC-EC methodpreviously established at our laboratory. A totalof 200 urine samples were examined. The resultswere in good agreement. Correlation coefficientwas 0.9879. Normal values of epinephrine (10.9-65.5 nmol/dU), norepinephrine (32.5-271.9nmol/dU) and dopamine (<2500 nmol/dU) didnot differ from those obtained by the comparativemethod. Sample preparation is simple and quick,requiring no expensive reagents or equipment,



Uvo|enjem nove metode zaklju~ili smo da je pri-prema uzorka brza, a sam sustav viskotla~neteku}inske kromatografije ne traì skupe oto-pine, što metodu ~ini pogodnom za laboratorijskurutinu.E-mail: [email protected]

which makes the method suitable for laboratoryroutine.E-mail: [email protected]

P5-2

EVALUACIJA METODA PRETRA@IVANJAMIKROALBUMINURIJE

Vu~i}-Lovren~i} M., Boì~evi} S., Ljubi} S., Boèk T.,Mesi} R., Ro~i} B., Metelko @.

Sveu~ilišna klinika “Vuk Vrhovac”, Zagreb

Mikroalbuminurija (izlu~ivanje albumina >30mg/24 h) u oboljelih od še}erne bolesti prethodidijabeti~noj nefropatiji i ~imbenik je rizika zarazvitak kardiovaskularnih bolesti. U ovom is-traìvanju procjenjivane su dvije metode pretra-ìvanja mikroalbuminurije u drugoj jutarnjojmokra}i: kvantitativno odre|ivanje albuminsko-kreatininskog omjera (A/K; imunokemijska me-toda, Bayer Diagnostics, Tarrytown, SAD, alkal-ni pikrat, Trace Scientific, Melbourne, Australija;autoanalizator Olympus AU600, Tokio, Japan) isemikvantitativno odre|ivanje albuminurije po-mo}u reagens-trake (Micral II, Roche Diagnos-tics, Mannheim, Njema~ka). U istraìvanje subili uklju~eni bolesnici (n=128, M/@=59/69, dob37-74 god.) s tipom 2 še}erne bolesti, koji su došlina redovnu godišnju dijabetološku kontrolu. Danprije dolaska na kontrolu bolesnici su premastrukturiranim uputama skupili 24-satnu mo-kra}u za kvantitativno odre|ivanje albuminurije(imunokemijska metoda, Bayer Diagnostics, Tar-rytown, SAD; autoanalizator Olympus AU600,Tokio, Japan), što je ujedno bila metoda uspo-redbe (’zlatni standard’) za obje testirane metodepretraìvanja. Izra~unata je dijagnosti~ka speci-fi~nost, osjetljivost te pozitivna i negativna pre-diktivna vrijednost za obje metode pretraìvanjamikroalbuminurije. Specifi~nost, osjetljivost, po-zitivna prediktivna vrijednost, negativna predik-tivna vrijednost: A/K 87%, 93%, 95%, 83%; MicralII 72%, 93%, 88%, 83%. Rezultati upu}uju na do-bru dijagnosti~ku pouzdanost metoda za pretra-ìvanje mikroalbuminurije, pri~em kvantitativnoizmjereni albuminsko/kreatininski omjer ima vi-šu specifi~nost u odnosu na reagens-traku (87%vs. 72%), dok je osjetljivost metoda jednaka(93%). Obje metode podjednako su korisne upretraìvanju mikroalbuminurije i mogu pouzda-no identificirati bolesnike kod kojih je potrebno

P5-2

EVALUATION OF SCREENING METHODSFOR MICROALBUMINURIA

M. Vu~i}-Lovren~i}, S. Boì~evi}, S. Ljubi}, T. Boèk,R. Mesi}, B. Ro~i}, @. Metelko

Vuk Vrhovac University Clinic for Diabetes, Endocrinologyand Metabolic Diseases, Zagreb

Microalbuminuria (albumin excretion >30mg/24 h) in diabetic patients precedes diabeticnephropathy and represents a risk factor for car-diovascular disease. In this study we evaluatedtwo screening methods for microalbuminuria us-ing second morning urine samples: quantitativedetermination of albumin/creatinine ratio (ACR;immunochemical method, Bayer Diagnostics,Tarrytown, USA, and alkaline picrate method,Trace Scientific, Melbourne, Australia, respec-tively; Olympus AU600 autoanalyzer, OlympusOptical Co., Tokyo, Japan), and semiquantitativedetermination of albuminuria using reagent-strip(Micral II, Roche Diagnostics, Mannheim, Ger-many). The study included type 2 diabetic pa-tients (n=128, M/F=59/69, age 37-74 years) whoattended the University Clinic for their regularyearly assessment. On the day before assessment,the patients collected 24-hour urine according tostructured instructions for quantitative determi-nation of albuminuria (immunochemical method,Bayer Diagnostics, Tarrytown, USA; OlympusAU600 autoanalyzer, Olympus Optical Co., To-kyo, Japan), which was used as a ’gold standard’,i.e. comparison reference method for both screen-ing methods studied. Diagnostic specificity, sensi-tivity, and positive and negative predictive valueswere calculated for both screening methods formicroalbuminuria. Specificity, sensitivity, posi-tive predictive value and negative predictivevalue: ACR 87%, 93%, 95% and 83%; and MicralII 72%, 93%, 88% and 83%, respectively. The re-sults indicated a fair diagnostic reliability of thetwo microalbuminuria screening methods, withhigher specificity of the quantitatively determinedalbumin/creatinine ratio when compared to test-strip (87% vs. 72%), whereas sensitivity of themethods was the same. Both methods proved to be



u~initi kvantitativnu analizu izlu~ivanja albu-mina.E-mail: [email protected]

equally useful in the screening for microalbu-minuria, and could reliably identify the patientsin need of quantitative assessment of the albuminexcretion rate.E-mail: [email protected]

P5-3

UÎNAK PROTEINA IZ URTICAE FOLIUM- LISTA KOPRIVE NA KONCENTRACIJU

GLUKOZE U KRVI NOD MIŠEVA

Petlevski R.1, Hadìja M.2, Slijep~evi} M.2, Jureti} D.1

1 Zavod za medicinsku biokemiju i hematologiju,Farmaceutsko-biokemijski fakultet Sveu~ili{ta u Zagrebu,

2 Zavod za molekularnu medicinu, Institut “Ru|erBo{kovi}”, Zagreb

Za više od 1000 biljnih vrsta opisan je hipo-glikemijski u~inak, ali za manji broj njih je iznanstveno potvr|en. Svjetska zdravstvena or-ganizacija preporu~ila je daljnja istraìvanja utom podru~ju. Cilj ovoga istraìvanja bio je ispi-tati hipoglikemijski u~inak proteina male mo-lekularne mase (3-15 kDa) izoliranih iz lista ko-prive na ìvotinjskom modelu še}erne bolesti.Proteini su izolirani iz liofiliziranog maceratadroge metodom gel filtracije na koloni SephadexG-25. Eluirane frakcije s kolone koje su pokazalemaksimum apsorbancije na valnoj duìni od 280nm (frakcije broj 9, 10 i 11) su pulirane, dijali-zirane preko no}i, liofilizirane i ~uvane na -20 oCdo analize. Še}erna bolest je u NOD miševa in-ducirana i.v. injekcijom aloksan monohidrata (75mg/kg tj.t.). Dijabeti~ni NOD miševi zatim supodijeljeni u dvije skupine. Jedna skupina NODmiševa tretirana je i.v. sterilnom otopinom PBS-a(0,33 mL/mišu) (n=6), a druga izoliranim pro-teinima iz Urticae folium - lista koprive u kon-centraciji od 1,6 mg /mišu u 0,33 mL sterilnogPBS-a (n=3). Koncentracija glukoze u krvi u ovimdvjema skupinama ìvotinja odre|ena je meto-dom glukoza oksidaza-peroksidaza (GOD-PAP)nakon 10, 30, 60 i 240 min od po~etka tretmana.Proteini male molekularne mase (3-15 kDa)izolirani iz Urticae folium - lista koprive pokazalisu zna~ajan hipoglikemijski u~inak tijekom cije-log trajanja tretmana. U usporedbi s dijabeti~-nim NOD miševima tretiranim samo sterilnomotopinom PBS-a (kontrolna skupina) ispitani hi-poglikemijski u~inak iznosio je 32,3% u 10. mi-nuti; 34,1% u 30. minuti; 30,6% u 60. minuti, te30,9% u 240. minuti od trenutka primjene uodnosu na kontrolnu krivulju. Dokazani hipo-glikemijski u~inak vaàn je za smanjenje post-

P5-3

EFFECT OF URTICAE FOLIUM - NETTLELEAF DERIVED PROTEIN ON BLOOD

GLUCOSE IN NOD MICE

R. Petlevski1, M. Hadìja2, M. Slijep~evi}2, D. Jureti}1

1 Department of Medical Biochemistry and Hematology,Zagreb University School of Pharmacy and Biochemistry,

2 Department of Molecular Medicine, Rudjer Bo{kovi}Institute, Zagreb

The aim of the study was to investigate thehypoglycemic effect of low molecular mass (3-15kDa) protein isolated from the drug Urticae fo-lium - nettle leaf (Urtica dioica L. - Urticaceae)on an animal model of diabetes mellitus. Morethan 1000 plant species have been reported to ex-ert hypoglycemic effects, but few of them havebeen investigated. The World Health Organiza-tion has recommended additional studies in thefield. Protein was isolated from the lyophilizedmacerate of the drug by the method of gel filtra-tion on a Sephadex G-25 column. The eluted frac-tions from the column showing maximal absorb-ance at a wavelength of 280 nm were pooled (Nos.9,10 and 11), dialyzed overnight, lyophilized andstored at -20 oC until analysis. Diabetes was in-duced in NOD mice by i.v. injection of alloxanmonohydrate (75 mg/kg body mass). DiabeticNOD mice were divided into two groups of micetreated i.v. with sterile PBS solution (0.33 mL/mouse) (n=6) and with nettle leaf-derived proteinat a concentration of 1.6 mg protein/mouse in0.33 mL sterile PBS (n=3). The concentration ofblood glucose in the two groups of animals wasdetermined by the method of glucose oxidase-per-oxidase (GOD-PAP) at 10, 30, 60 and 240 minfrom the treatment. The low molecular mass pro-tein isolated from nettle leaf showed a significanthypoglycemic effect throughout the treatment pe-riod. In comparison with diabetic NOD micetreated with sterile PBS solution alone, thehypoglycemic effect recorded in the other group ofdiabetic NOD mice was 32.3% in 10th minute;34.1% in 30th minute; 30.6% in 60th minute and30.9% in 240th minute from the beginning oftreatment. This hypoglycemic effect is importantfor the reduction of postprandial hyperglycemia,



prandijalne hiperglikemije koja ima va`nu uloguu nastanku makrovaskularnih komplikacija u še-}ernoj bolesti.E-mail: [email protected]

which plays a major role in the development ofmacrovascular complications of diabetes melli-tus.E-mail: [email protected]

P5-4

AKTIVNOST GLUTATION PEROKSIDAZE IKONCENTRACIJA SELENIJA UHIPERTIREOZNIH BOLESNICA

Mayer Lj.1, Romi} @.1, Škreb F.1, Ba~i}-Vrca V.1,êpelak I.2, @ani}-Grubiši} T.2

1 Zavod za laboratorijsku dijagnostiku, Klini~ka bolnica“Dubrava”, 2 Farmaceutsko-biokemijski fakultet, Zagreb

Hipertireoza je kao hipermetaboli~no stanjepra}ena pove}anim iskorištenjem kisika, pove-}anim stvaranjem reaktivnih spojeva kisika i po-sljedi~no promjenama u aktivnosti antioksida-tivnih enzima. Glutation peroksidaza (GPX) svojantioksidativni u~inak temelji na kataliziranjuhidrolize vodikovog i organskih hidroperoksida.U strukturi aktivnog centra svake od ~etiripodjedinice ovoga enzima nalazi se atom selenijauklopljen u selenocistein. Cilj ovoga istraìvanjabio je odrediti ovisnost aktivnosti GPX i kon-centracije selenija o primjeni terapije, odnosnoduljini trajanja bolesti, te ispitivanje povezanostinjihove aktivnosti, odnosno koncentracije u razli-~itim vrstama uzoraka. U istraìvanje je uklju-~eno 70 èna: 14 s novodijagnosticiranom Grave-sovom boleš}u (skupina A), 28 s hipertireozom naterapiji (skupina B) i 28 zdravih èna (kontrolnaskupina). Skupinu B dodatno smo raš~lanili nadvije podskupine s obzirom na aktivnost TSH.Aktivnost GPX mjerena je u uzorku pune krvi, aselenij u serumu pomo}u AAS. Aktivnost GPX jeu svim ispitivanim skupinama bila statisti~kizna~ajno smanjena u odnosu na vrijednostiizmjerene u kontrolnoj skupini. Smanjenje je bilonajizraènije u skupini A (pribli`no 21%), dok jeu skupini B bilo manje (11%). Izmjerena kon-centracija selenija nije se statisti~ki zna~ajnorazlikovala ni u jednoj skupini bolesnica u odno-su na kontrolnu. Smanjenje aktivnosti GPX uhipertireozi još je jedna potvrda o postojanju ok-sidacijskoga stresa u hipertireozi. Njegov inten-zitet varira, ovisan je o duljini trajanja bolesti iprimjeni terapije. Selenij, iako kofaktor GPX, neprati dinamiku enzima. S obzirom na razli~ituraspodjelu selenija u serumu i stanicama oprav-dano bi bilo u slijede}im istraìvanjima kon-centraciju selenija mjeriti u stanicama (eritro-

P5-4

ACTIVITY OF GLUTATHIONEPEROXIDASE AND SELENIUM

CONCENTRATION IN HYPERTHYROIDWOMEN

Lj. Mayer1, @. Romi}1, F. Škreb1, V. Ba~i}-Vrca1, I.êpelak2, T. @ani}-Grubiši}2

1 Institute of Laboratory Diagnosis, Dubrava UniversityHospital, 2 School of Pharmacy and Biochemistry, Zagreb

Hyperthyroidism as a hypermetabolic state isaccompanied by an increased oxygen utilization,increased production of reactive oxygen species,and consequently by changes in the activities ofantioxidant enzymes. The antioxidative effect ofglutathione peroxidase (GPX) is based oncatalyzing hydrolysis of hydrogen and organicperoxides into less active forms. In the structureof the active site of GPX selenium is incorporatedin the form of selenocysteine. The aim of thisstudy was to determine dependence of GPX activ-ity and selenium concentration on the use oftherapy and duration of the disease. Seventywomen were included in the study: 14 with newlydiagnosed Graves’ disease without therapy (groupA); 28 hyperthyroid women on therapy (group B);and 28 healthy women (control group). Group Bwas additionally divided into two subgroups ac-cording to TSH activity. The activity of GPX wasmeasured in whole blood, and selenium in serum.The activity of GPX was statistically significantlylower in all patient groups as compared with con-trol group. The decrease was most pronounced ingroup A (approximately 21%), and less pro-nounced in group B (approximately 11%). Theconcentration of selenium showed no statisticallysignificant differences in any patient group ascompared with control group. The decrease inGPX activity in hyperthyroid patients is anotherconfirmation of the existence of oxidative stress inthis disease. The intensity of oxidative stress var-ies depending on the disease duration and use oftherapy. Selenium, although a cofactor of GPX,does not follow the dynamics of the enzyme. Con-sidering variable serum and cellular seleniumdistribution, additional studies appear justifiedto measure selenium concentration in cells that



citima, leukocitima, ~ak i trombocitima) koje vje-rodostojnije opisuju status selenija u organizmu,manje su osjetljive na dnevne varijacije i ~akkoreliraju s aktivnoš}u selenoenzima.E-mail: [email protected]

more accurately reflect the status of selenium(less sensitive to daily variations) and even corre-late with the activity of selenoenzymes.E-mail: [email protected]

P6-1

POLIMORFIZAM GLUTATIONS-TRANSFERAZE P1 I RIZIK OD

ALZHEIMEROVE BOLESTI

@untar I.1, Kalanj-Bognar S.2, Topi} E.3, Štefanovi}M.3, Petlevski R.1, Demarin V.4, Maleš @.1

1 Farmaceutsko-biokemijski fakultet, 2 Medicinski fakultetSveu~ili{ta u Zagrebu, 3 Klini~ki zavod za kemiju i 4 Klinika

za neurologiju, KB “Sestre milosrdnice”, Zagreb

Alzheimerova bolest (AD, engl. Alzheimer’sdisease) je neurodegenerativna bolest nepozna-tog uzroka. Središnji ìv~ani sustav izrazito jeosjetljiv na ošte}enja izazvana razli~itim kemij-skim tvarima (neurotoksinima), pri~em razinaošte}enja uvelike ovisi o genetskoj osnovi u~in-kovitosti enzimskog sustava detoksifikacije. Glu-tation transferaze (GSTe) su višestruka genetskaobitelj II. faze enzimske detoksifikacije i štite sta-ni~ne makromolekule od citotoksina i karcino-gena. Klasa pi GST (GSTP1) je najrasprostra-njeniji nehepati~ni enzim. Cilj istraìvanja bio jeodrediti genetsku raspodjelu polimorfizmaA313G petog eksona gena GSTP1 u zdravih is-pitanika iz Hrvatske i ispitivanje mogu}e pove-zanosti polimorfizma s AD. Genotip A313G genaGSTP1 odre|en je metodom PCR-RFLP u kon-trolnoj skupini zdravih ispitanika (n=180) i sku-pini ispitanika s AD (n=40). Klini~ka dijagnozaAD potvr|ena je nalazima neuropsiholoških, la-boratorijskih i radioloških (kompjutorizirana to-mografija) pretraga. U~estalost polimorfizma ge-na GSTP1 u zdravih hrvatskih ispitanika sli~naje literaturnim podacima za druge populacije.Analiza je pokazala prisutnost utvr|eno homozi-gotnog, mutiranog (GG) genotipa u 20% ispita-nika s AD i 8% zdravih ispitanika. Heterozigotni(AG) genotip zabiljeèn je u 25% AD i 46% zdra-vih ispitanika. Zdravi (AA) genotip bio je utvr|enkod 55% AD i 46% zdravih ispitanika. Razlika uraspodjeli polimorfizma petog eksona gena

P6Neurodegenerativne bolestiNeurodegenerative diseases

P6-1

GLUTATHIONE S-TRANSFERASE P1POLYMORPHISM AND RISK OF

ALZHEIMER’S DISEASE

I. @untar1, S. Kalanj-Bognar2, E. Topi}3, M.Štefanovi}3, R. Petlevski1, V. Demarin4, @. Maleš1

1 School of Pharmacy and Biochemistry, 2 School ofMedicine, University of Zagreb, 3 Clinical Institute of

Chemistry and 4 University Department of Neurology, Sestremilosrdnice University Hospital, Zagreb

Alzheimer’s disease (AD) is a neurodegene-rative disorder of unknown etiology. Many chemi-cals can affect the nervous system and some ofthem require metabolic activation to induce theirtoxic effects, so that genetic polymorphisms thatencode for defective forms of the detoxifying en-zymes can further increase the risk of effects fromexposure to neurotoxicants. Glutathione transfe-rases (GSTs) are a multiple gene family of phaseII enzymes that catalyze detoxifying endogenousreactions with glutathione and protect cellularmacromolecules from damage caused by cytotoxicand carcinogenic agents. The pi class of GST(GSTP1) is known as the most ubiquitous andprevalent of the GST isoenzymes in nonhepatictissues. The aim of the study was to determine thegenotype distribution for the GSTP1 A313G exon5 polymorphism in Croatian healthy subjects andto investigate the association between GSTP1polymorphism and AD. The A313G GSTP1 geno-types were determined by PCR-RFLP method insamples of healthy controls (n=180) and AD pa-tients (n=40). Clinical diagnosis of AD was con-firmed through a battery of neuropsychologicaland laboratory tests and radiology (computerizedtomography) studies. The frequency of GSTP1polymorphism in the Croatian healthy subjectswas in the range reported for other populations.Analysis showed 20% of homozygous mutant(GG) genotype in AD patients and 8% in controls,



GSTP1 izme|u skupine ispitanika s AD i kontrol-ne skupine bila je statisti~ki zna~ajna (p=0,019).Tako|er je na|eno statisti~ki zna~ajano pove}a-nje rizika od AD u ispitanika s homozigotnim,mutiranim (GG) genotipom (OD=2,964, CI=1,149-7,646). Naši preliminarni rezultati upu}ujuna mogu}u povezanost polimorfizma A313Gpetog eksona gena GSTP1 s AD.E-mail: [email protected]

25% of heterozygous (AG) genotype in AD patientsand 46% in controls, while wild type (AA) geno-type was detected in 55% of AD patients and 46%of controls. There was a statistically significantdifference between AD patients and controls inthe distribution of the GSTP1 exon 5 polymor-phism (p=0.019). Additionally, there was a statis-tically significant increase in AD (OD=2.964,CI=1.149-7.646) for homozygous mutant (GG)genotype. Our preliminary results demonstratethat the GSTP1 A313G exon 5 polymorphism isassociated with the risk of AD.E-mail: [email protected]

P6-2

HITOTRIOZIDAZA U PRA]ENJUENZIMSKE ZAMJENSKE TERAPIJE

GAUCHEROVE BOLESTI U HRVATSKOJ

Maradin M.1, Fumi} K.1, Mrsi} M.2

1 Klini~ki zavod za laboratorijsku dijagnostiku, 2 Klinika zahematologiju, Klini~ki bolni~ki centar “Zagreb”, Zagreb

Gaucherova bolest je naju~estalija lizosomskabolest nakupljanja uzrokovana smanjenom kata-liti~nom koncentracijom beta-glukozidaze, zbog~ega dolazi do nakupljanja glukocerebrozida ulizosomima makrofaga. Kod simptomatskih bole-snika s Gaucherovom boleš}u zamije}en je izrazitporast kataliti~ne aktivnosti serumske hitotrio-zidaze (100-5.000 puta), ljudskog analoga hitina-ze, podrijetlom iz aktiviranih makrofaga. Manjezna~ajno pove}anje aktivnosti na|eno je kod jošnekih drugih lizosomskih te razli~itih granulo-matoznih imunoloških ili zaraznih bolesti. Stogase povišena aktivnost hitotriozidaze u serumusmatra specifi~nom za Gaucherovu bolest i slu`iu pra}enju u~inkovitosti lije~enja. U ovom suradu izneseni prvi podaci o promjenama aktiv-nosti hitotriozidaze tijekom enzimske zamjensketerapije bolesnika s Gaucherovom boleš}u uHrvatskoj. Do danas je dijagnosticirano ukupno 7bolesnika u dobi od 24-32 godine. Petoro bole-snika prima i.v. Ceradase (1200 IU svaki drugitjedan) u razdoblju od 6-24 mjeseca. Aktivnosthitotriozidaze u serumu mjerena je uza sintetskisupstrat 4-MUF-beta-D-N,N’,N’’-triacetil hitotri-ozid. U prvih 12 mjeseci lije~enja zamije}en jepad kataliti~ne aktivnosti hitotriozidaze za 14%-66% od po~etne vrijednosti. Aktivnost hitotriozi-daze se mo`e smatrati najboljim biokemijskimparametrom koji korelira s op}im stanjem bole-snika.E-mail: [email protected]

P6-2

CHITOTRIOSIDASE IN MONITORINGENZYME REPLACEMENT THERAPY FOR

GAUCHER’S DISEASE IN CROATIA

M. Maradin1, K. Fumi}1, M. Mrsi}2

1 Clinical Institute of Laboratory Diagnosis, 2 UniversityDepartment of Hematology, Zagreb University Hospital

Center, Zagreb

Gaucher’s disease is the most common lyso-somal storage disease caused by decreased cata-lytic concentration of β-glucosidase that results inglucocerebroside accumulation in macrophagelysosomes. A pronounced increase (100- to 5000-fold) in the catalytic activity of serum chito-triosidase, a human analog of chitinase originat-ing from activated macrophages, was observed insymptomatic patients with Gaucher’s disease. Aless significant increase in this activity has beenrecorded in some other lysosomal and variousgranulomatous immunologic or infectious dis-eases. Elevated chitotriosidase activity is there-fore considered specific for Gaucher’s disease andis used to monitor therapy efficacy. We presentinitial data on changes in chitotriosidase activityduring enzyme replacement therapy in Croatianpatients with Gaucher’s disease. A total of 7 pa-tients aged 24-32 years were diagnosed. Five pa-tients were administered i.v. Ceradase (1200 IUevery other week) for a period of 6-24 months. Se-rum chitotriosidase activity was determined us-ing a synthetic substrate 4-MUF-beta-D-N,N’,N’’-triacetyl chitotriosidase. A decrease in the cata-lytic activity of chitotriosidase by 14%-66% of itsinitial value was observed during the first 12months of therapy. Chitotriosidase activity can beconsidered the best biochemical parameter that isin correlation with the general status of the pa-tient.E-mail: [email protected]



P7-1

POUZDANOST ODRE\IVANJAPROTROMBINSKOG VREMENA I

AKTIVIRANOG PARCIJALNOGTROMBOPLASTINSKOG VREMENA NA

COAGUCHEK PRO DM

Coen D., Miloš M., Zadro R.

Klini~ki bolni~ki centar “Zagreb”, Klini~ki zavod zalaboratorijsku dijagnostiku, Zagreb

CoaguChek Pro DM je prijenosni koagulacij-ski monitor koji omogu}ava odre|ivanje protrom-binskog vremena (PV), aktiviranog trombopla-stinskog vremena (APTV) i aktiviranog vremenazgrušavanja u punoj kapilarnoj ili venskoj krvi.Cilj studije bio je utvrditi pouzdanost rezultataPV-a i APTV-a dobivenih na Coaguchek Pro DMusporedbom s rezultatima standardnih labora-torijskih metoda. Ukupno je obra|eno 54 ispita-nika (19 na oralnoj antikoagulacijskoj terapiji, 5na kontinuiranoj infuziji visokomolekularnim he-parinom, 5 s cirozom jetre te 25 zdravih dobro-voljaca). Kod svih ispitanika istodobno su uzetiuzorci pune kapilarne krvi za odre|ivanje naCoaguChek Pro DM (Roche Diagnostics, Njema~-ka) te venske krvi s citratom kao antikoagulan-som za odre|ivanje u koagulacijskom labora-toriju. Za odre|ivanje PV-a i APTV-a u punoj krvirabljene su odgovaraju}e jednokratne CoaguChek Pro APTT i PTN (ze~ji tromboplastin,ISI=2,0) test-kazete. Mjerenje PV-a i APTV-a ukoagulacijskom laboratoriju izvršeno je na ana-lizatoru BCS (Dade Behring, Njema~ka) uz upo-rabu Innovina (ISI=0,92) odnosno Actina FS. Uzdravih ispitanika dobiveni referentni raspon zaAPTV (23,7-43,9 s) na CoaguChek-u podudaraose s rasponom deklariranim od proizvo|a~a(20,2-40,8 s), dok je referentni raspon standardnelaboratorijske metode iznosio 24,0-33,0 s. Unato~dobroj korelaciji (r=0,885) rezultata APTV-a iz-me|u metoda, vrijednosti izmjerene na CoaguChek-u su u pravilu bile više, a razlike su bileve}e kod viših APTV-a. Srednja razlika izme|uINR-a dobivenih na CoaguChek-u i standardnomlaboratorijskom metodom iznosila je 0,22 (ras-pon: -0,51-1,13). U 16/19 (80,9%) slu~ajeva vrijed-nosti INR-a nisu se razlikovale više od 0,5. Iakoje na|ena dobra korelacija (r=0,935), vrijednostiINR-a su se podudarale s objema metodama sa-mo u podru~ju INR=1,0-2,0. Kod ispitanika s

P7Procjena analiti~kih tehnika i metoda

Evaluation of analytical techniques and methods

P7-1

RELIABILITY OF PROTHROMBIN ANDACTIVATED PARTIAL THROMBOPLASTIN

TIME ON COAGUCHEK PRO DM

D. Coen, M. Miloš, R. Zadro


The CoaguChek Pro DM (Roche Diagnostics,Germany) is a bedside coagulation monitor forthe measurement of prothrombin time (PT), acti-vated partial thromboplastin time (aPTT) andactivated clotting time in fresh capillary or ve-nous whole blood. The aim of the study was toevaluate the reliability of PT and aPTT resultsobtained on CoaguChek by comparing them withstandard laboratory methods. A total of 54 pa-tients were included in the study (19 receivingoral anticoagulant therapy, 5 on unfractionatedheparin therapy, 5 with liver cirrhosis, and 25healthy volunteers). Capillary whole blood sam-ples were obtained by fingerprick simultaneouslyto citrated venous blood collection. Whole bloodPT and aPTT were determined by using appro-priate CoaguChek Pro APTT and PTN (rabbitbrain thromboplastin, ISI=2.0) test cartridges.Laboratory PT (Innovin, ISI=0.92) and aPTT(Actin FS) were performed on a BCS (DadeBehring, Germany) coagulation analyzer. The ob-tained reference interval for CoaguChek aPTT inhealthy subjects (23.7-43.9 s) was close to thatstated by the manufacturer (20.2-40.8 s), as com-pared to the laboratory reference interval (24.0-33.0 s). Despite good correlation (r=0.885) foraPTT, there was an important dispersion of val-ues and a trend towards longer clotting timeswith CoaguChek, with increasing difference withmore prolonged aPTTs. The mean difference be-tween CoaguChek and laboratory INR was 0.22(range: -0.51-1.13). In 16/19 (80.9%) cases INRvalues differed by less than 0.5. Although goodcorrelation (r=0.935) was obtained between INRresults with the two methods, comparable valueswere observed only in the INR range 1.0-2.0. Inthe patients who achieved the targeted level of an-ticoagulation as determined by standard labora-tory method, higher INR values were always ob-tained with CoaguChek; they were in 2 cases out-side the therapeutic range and thus may have er-roneously indicated the need of anticoagulant



vrijednostima INR-a u terapijskom podru~ju(INR=2,0-3,5) odre|enima standardnom labora-torijskom metodom dobiveni su viši rezultati naCoaguChek-u, koji su u 2 slu~aja bili iznad te-rapijskog podru~ja, što bi moglo uputiti na po-grešnu izmjenu doze lijeka. Zbog zna~ajnih raz-lika u rezultatima dobivenima objema meto-dama potrebno je ustanoviti razloge neslaganjaprije uporabe CoguChek Pro DM u razli~itimklini~kim slu~ajevima.E-mail: [email protected]

dose change. As substantial differences were ob-tained between CoaguChek and standard labora-tory methods, the reason for the discrepancyshould be further investigated before the use ofCoaguChek in various clinical situations.E-mail: [email protected]

P7-2

NEUSKLA\ENOSTI REZULTATAKEMIJSKE ANALIZE MOKRA]E TEST

TRAKOM U VANJSKOJ PROCJENI RADAMEDICINSKO-BIOKEMIJSKIH

LABORATORIJA

Sikirica M.1, Jureti} D.2, Flegar-Meštri} Z.1

1 Zavod za klini~ku kemiju, Klini~ka bolnica “Merkur”,2 Farmaceutsko-biokemijski fakultet, Zagreb

Kemijska analiza mokra}e test trakama se od1997. godine provodi kao dio analize mokra}e unacionalnom programu vanjske procjene kakvo}erada medicinsko-biokemijskih laboratorija u Hr-vatskoj. Svrha programa, uz procjenu kakvo}emjerenja, je i trajna edukacija kako bi se do-prinijelo što ve}oj primjeni standardizirane, pre-poru~ene metode rutinske analize mokra}e. Pro-gram se temelji na kemijskoj analizi sintetskidobivene mokra}e test trakama o~itavane vizu-alno ili na ~ita~u mokra}nih test traka. Dvade-setpet (15%) medicinsko-biokemijskih laborato-rija o~itavanje izvodi na ~ita~u mokra}nih testtraka, pa se rezultati dobiveni uporabom 10 vrstatest traka razli~itih proizvo|a~a izra`avaju poje-dina~no po parametru, semikvantitativno u SIjedinicama (mmol/L) ili kvalitativno u arbitrar-nim jedinicama (0/neg; 1/+; 2/++; 3/+++; 4/++++).Prema evaluaciji test traka Urine Reagent Strips,MDA Evaluation Report, 98, 65, dobiveni rezul-tati su objedinjeni u koncentracijske raspone(najni`a i najviša koncentracija) za svaki ispi-tivani parametar svih test traka. Dobiveni rezul-tati pokazuju preklapanja gdje iste pozitivne ka-tegorije razli~itih test traka korespondiraju srazli~itim koncentracijama mjerenog parametra.Ova preklapanja rezultata su posljedica razli~itihkoncentracija reaktanata (za iste parametre),zbog ~ega je i podjela rastu}ih koncentracija (or-dinalna skala) u klase razli~ita. Radi unapre-

P7-2

INCONSISTENCY OF THE RESULTS OFURINALYSIS USING MULTIPROPERTYSTRIPS IN THE EXTERNAL QUALITY

ASSESSMENT

M. Sikirica1, D. Jureti}2, Z. Flegar-Meštri}1

1 Institute of Clinical Chemistry, Merkur UniversityHospital, 2 School of Pharmacy and Biochemistry, Zagreb

Urinalysis using multiproperty strips hasbeen performed as part of urinalysis within thenational program of External Quality Assessmentfor medical biochemistry laboratories in Croatiasince 1997. Apart from the measurement qualityassessment, the aim of the program is continuouseducation to contribute to the use of standardized,recommended method of routine urinalysis to thelargest possible extent. The program is based onchemical analysis of synthetically produced urinewith dipsticks read visually or on a reading de-vice. In 25 (15%) medical biochemistry laborato-ries the reading is done on reading devices, thusthe results obtained by using 10 types ofmultiproperty strips of various manufacturersare expressed individually for each parameter,semiquantitatively in SI units (mmol/L) or quali-tatively in arbitrary units (0/neg; 1/+; 2/++, 3/+++, 4/++++). According to the Urine ReagentStrips, MDA Evaluation Report, 98, 65, the ob-tained results are united in concentration ranges(lowest and highest concentration values) for eachparameter investigated in all strips. The obtainedresults show overlapping where the same positivecategories of various strips correspond with vari-ous concentrations of the parameter measured.This overlapping of the results is due to variousreactant concentrations (for the same param-eters), thus rendering the classification of ascend-ing concentrations (ordinal scale) variable aswell. Aiming at promoting good laboratory prac-



|ivanja dobre laboratorijske prakse, ove neuskla-|enosti rezultata ukazuju na potrebu standar-dizacije koncentracija reaktanata za iste anali-ti~ke postupke.E-mail: [email protected]

tice, this inconsistency in the results indicates theneed of standardization of reactant concentra-tions for the same analytical procedures.E-mail: [email protected]

P7-3

UTJECAJ AUTOMATIZIRANOGPOSTUPKA ODRE\IVANJA NA

REZULTATE PROTROMBINSKOGVREMENA PV-INR U PROGRAMU

VANJSKE PROCJENE RADAMEDICINSKO-BIOKEMIJSKIH

LABORATORIJA

Povjerenstvo za vanjsku procjenu kakvo}e radamedicinsko-biokemijskih laboratorija Hrvatskog

dru{tva medicinskih biokemi~ara:Parag G.1, Jureti} D.2, Flegar-Meštri} Z.1

1 Klini~ka bolnica “Merkur”, Zavod za klini~ku kemiju,Zagreb, 2 Farmaceutsko-biokemijski fakultet, Zagreb

Uspješnost medicinsko-biokemijskih laborato-rija u odre|ivanju protrombinskog vremena PV-INR iz rezultata vanjske procjene kakvo}e radaprocijenjena je za svaki pojedini laboratorij u raz-doblju prije i nakon uvo|enja automatiziranogpostupka na koagulometru. Primjenom odgova-raju}e statisti~ke obrade rezultati su izra`eni kaopostotak odstupanja od ciljne vrijednosti i kaoindeks standardne devijacije, prema kojemu jemedicinsko-biokemijski laboratorij klasificirankao odli~an, vrlo dobar, dobar, zadovoljavaju}i iloš. U prvom pregledu vanjske procjene kakvo}erada iz koagulacije koja je zapo~ela 1995. godinesudjelovala su 103 laboratorija, od kojih su 53(52%) rabila ru~nu tehniku za izvo|enje testa.Rezultati PV-INR su pokazali visok interla-boratorijski koeficijent varijacije (KV=17,9%) inizak postotak zadovoljavaju}ih rezultata (33%).Nezadovoljavaju}i ishod rezultata djelomice jebio uzrokovan i uporabom niskoosjetljivih re-agensa (ISI >1,5). Automatizirani postupak odre-|ivanja na koagulometru uveden je do 2002.g. u89% medicinsko-biokemijskih laboratorija. Pre-gled rezultata kroz vanjsku procjenu kakvo}e ra-da nakon uvo|enja nove tehnologije uz primjenuvisokoosjetljivih reagensa pokazuje sni`enje KV(7,2%) i tendenciju porasta zadovoljavaju}ih re-zultata (80%). Me|utim, i primjena ru~nogpostupka odre|ivanja uz uporabu visokoosjetlji-vih reagensa rezultira kontinuiranom kvalitetomrezultata (15% medicinsko-biokemijskih labora-torija). Uvo|enje automatizacije uz uporabu os-

P7-3

EFFECT OF AUTOMATEDCOAGULOMETER DETERMINATION ONPROTHROMBIN TIME PT-INR RESULTS

IN MEDICAL BIOCHEMISTRYLABORATORY EXTERNAL QUALITY

ASSESSMENT PROGRAM

Commission on External Quality Assessment ofMedical Biochemistry Laboratories of the Croatian

Society of Medical Biochemists:G. Parag1, D. Jureti}2, Z. Flegar-Meštri}1

1 Institute of Clinical Chemistry, Merkur UniversityHospital, Zagreb, 2 School of Pharmacy and Biochemistry,

Zagreb

The performance of medical biochemistrylaboratories was assessed by PT-INR determina-tion during a period before and after the intro-duction of automated coagulometers. Accordingto the standard deviation index, registered labo-ratories are classified as excellent, very good,good, sufficient and poor. A total of 103 laborato-ries participated in the first survey in 1994, 52%of them using manual technique. The interlabo-ratory coefficient of variation was quite high(CV=17.9) and only 33% showed satisfactory per-formance. At that time, a great number of labora-tories used reagents with low sensitivity (ISI>1.5). In 2002, 89% of laboratories used auto-matic coagulometers. The introduction of auto-matic coagulometers and use of high specific rea-gents showed 80% satisfactory performance andCV 7.2. Although 15% of participating laborato-ries still used manual technique along with theuse of high-sensitive reagents, their results werevery good. The introduction of automatic coagu-lometers and use of high-sensitive reagents im-proved standardization in PT-INR determina-tion, and resulted in satisfactory performanceand very successful laboratory classification.E-mail: [email protected]



jetljivijih reagensa doprinjelo je provo|enju stan-dardizacije u odre|ivanju protrombinskog vre-mena, poboljšanju kakvo}e ishoda testa i us-pješnosti medicinsko-biokemijskog laboratorija.E-mail: [email protected]

P7-4

ANALITI^KA PROCJENA ANALIZATORAZA ODRE\IVANJE KONCENTRACIJE Na,

K, Cl, Ca2+ I pH U SERUMU I PUNOJKRVI

Milardovi} S.1, Rumenjak V.2

1 Fakultet kemijskog inènjerstva i tehnologije; 2 Zavod zamedicinsko laboratorijsku dijagnostiku, Op}a bolnica “Sveti

Duh”, Zagreb

Mjerenje koncentracije elektrolita jedno je odnaj~eš}ih odre|ivanja kako u medicinsko bioke-mijskim laboratorijima, tako i u odre|ivanjimauz bolesnika. Razvijeni su brojni analizatori naosnovi ionsko selektivnih elektroda. U ovom raduprikazan je analizator za odre|ivanje natrija, ka-lija, klorida, ioniziranog kalcija i pH u punoj krvii serumu, razvijen u suradnji Zavoda za op}u ianorgansku kemiju Fakulteta kemijskog inè-njerstva i tehnologije i Zavoda za medicinskolaboratorijsku dijagnostiku Op}e bolnice “SvetiDuh” kao tehnološki projekt Ministarstva zna-nosti i tehnologije (TP-01-0125-03). Analizator jekonstruiran kao samostalan ure|aj s automat-skom kalibracijom, a mjerna }elija konstruiranaje kao višestruki senzor za istodobno mjerenjekoncentracije (aktivnosti) svih pet iona. Kon-strukcija ovoga višestrukog senzora je izvornirad i pod zaštitom je patenta. Višestruki senzorne zahtijeva odràvanje i u cijelosti se zamjenjujenakon 1000 uzoraka (serum ili puna krv). Ana-liti~ka procjena analizatora u~injena je premapropisima ECCLS (1986.) i pokazala je dobrerezultate. Postignuti rezultati pokazali su da jeovaj analizator prikladan kao zamjena za pla-meni fotometar, tim više što se njime moèmjeriti i koncentracija Ca2+.E-mail: [email protected]

P7-4

ANALYTICAL ASSESSMENT OF THEANALYZER FOR DETERMINATION OF

SODIUM, POTASSIUM, CHLORIDE, Ca2+CONCENTRATIONS AND pH IN SERUM

AND WHOLE BLOOD

S. Milardovi}1, V. Rumenjak2

1 Zagreb University School of Chemical Engineering andTechnology; 2 Institute of Medical Laboratory Diagnosis,

Sveti Duh General Hospital, Zagreb

Measurement of electrolyte concentrations is be-coming one of the fundamental analyses in medicalbiochemistry laboratories as well as in point-of-caretesting. There are many analyzers based on ion se-lective electrodes. We describe an analyzer for themeasurement of the concentration (activity) of so-dium, potassium, chloride, Ca2+ and pH in serumand whole blood. The analyzer has been developedin collaboration between Department of Generaland Inorganic Chemistry, School of Chemical Engi-neering and Technology, University of Zagreb, andDepartment of Medical Laboratory Diagnosis, SvetiDuh General Hospital, as part of the Croatian Min-istry of Science and Technology project (TP-01-0125-03). The analyzer is made as an automatic de-vice with self-calibration, the measuring cell beinga multisensor for simultaneous measurement of theconcentration (activity) of five ions. The measure-ment cell is under patent pending. The multisensorrequires no maintenance but is replaced after 1000samples (serum or whole blood). Analytical assess-ment of the analyzer was done according to ECCLS(1986) rules, and it showed good results for all pa-rameters. It is concluded that this analyzer couldreplace flame photometers in medical biochemistrylaboratories.E-mail: [email protected]



P7-5

IZRADA NACIONALNOG STANDARDA ZAHEPATITIS B POVRŠINSKI ANTIGEN

(HBsAg)

Glavaš K.1, Mihaljevi} I.2

1 Pliva d.d., Istra`ivanje; 2 Hrvatski zavod za transfuzijskumedicinu, Zagreb

Širenje hepatitisa B predstavlja sve ve}izdravstveni problem kod nas i u svijetu. Testi-ranje krvi davatelja smatra se jednom od boljihmjera prevencije širenja ove bolesti. Iz tog raz-loga smo pokušali napraviti naš nacionalni stan-dard za HBsAg (0.5 IU/l) po uzoru na britanski,te ispitati njegovu stabilnost kroz ~etiri mjeseca iponovljivost. Pritom smo rabili Me|unarodnistandard za HBsAg (SZO) i serume dobivene izplazma dobrovoljnih davatelja krvi pozitivnih naHBsAg. Nakon spajanja seruma u jedan jedin-stveni HBsAg smo otkrivali enzimsko-imuno-loškim testovima: HBsAg ELISA test System 3,Ortho Clin. Diagnostic i HBsAg Version 3 GE34/36, Murex. Usporedba ba`darnih krivulja Me|u-narodnog standarda i našega nacionalnog stan-darda pokazala je da se niske koncentracijeHBsAg (0-1 IU/l) mogu uspore|ivati, ali bi treba-lo napraviti ve}a razrje|enja. Rezultati dobiveniusporedbom ba`darne krivulje našega standardas krivuljama Virusne kontrole kvalitete (viralquality control, VQC) pokazali su da je naš stan-dard dovoljno osjetljiv da bi bio primjenjiv u kon-troli testiranja na HBsAg (sample/cut off, S/CO,vrijednost 0,5 IU/l u Ortho testu bila je izme|u1:32 i 1:64 VQC razrje|enja, a osjetljivost Orthotesta je bila 1:128). Srednja vrijednost S/CO mje-rena tijekom ~etiri mjeseca testom Murex bila je9,68±4,7, što je dokazalo stabilnost našega stan-darda uz dodatak konzervansa Na-azida. Ponov-ljivost testa Murex mjerena uporabom ovogastandarda bila je zadovoljavaju}a s obzirom na toda su se vrijednosti 46 od ukupno 48 uzorakanalazile unutar raspona S/CO od 4,9-6,1.E-mail: [email protected]

P7-5

DEVELOPMENT OF NATIONALHEPATITIS B SURFACE ANTIGEN (HBsAg)

STANDARD

K. Glavaš1, I. Mihaljevi}2

1 Pliva Co.; 2 Croatian Institute of Transfusion Medicine,Zagreb

The spread of hepatitis B presents a hugehealth problem in Croatia and worldwide. Donorblood testing is considered to be a good measurein the spread prevention. Therefore, we tried todevelop a national HBsAg standard (0.5 IU/l)based on the British National HBsAg Standard.We also tried to assess its four-month stabilityand reproducibility. For this purpose, we usedWHO International HBsAg Standard and seraconverted from HBsAg positive donor plasma.Upon pooling of the sera, HBsAg were detected bythe HBsAg ELISA test System 3, Ortho Clin. Di-agnostic and HBsAg Version 3 GE34/36, MurexEnzyme Immunoassays. Comparison of the WHOstandard curve with our pool standard curve re-vealed that low HBsAg levels (0-1 IU/l) werecomparable, however, we had to make higher di-lutions. Results obtained by comparison of ourstandard curve with the viral quality control(VQC) curves indicated our standard to be sensi-tive enough to be used as a HBsAg testing control(sample/cut off (S/CO) value of 0.5 IU/l inOrtho test was between 1:32 and 1:64 VQC dilu-tions, and Ortho test sensitivity was 1:128). Themean S/CO value measured by Murex test dur-ing the four-month period was 9.68±4.7, provid-ing evidence for our standard stability with Na-azide as a preservative. The reproducibility ofMurex test using this standard was satisfactory,as 46 of 48 samples fell within the S/CO range of4.9-6.1.E-mail: [email protected]

P7-6

USPOREDBA TRIJU RAZLI^ITIHTESTOVA ZA CRP U SERUMU

Marevi} S., Sokoli} B., Neumann I., Petres B.

Klinika za infektivne bolesti “Dr. Fran Mihaljevi}’’,Zagreb

C-reaktivni protein (CRP) je protein akutnefaze koji se sintetizira u jetri pod kontrolom in-terleukina-6. CRP je danas nezaobilazna pretra-

P7-6

COMPARISON OF THREE SERUM CRPASSAYS

S. Marevi}, B. Sokoli}, I. Neumann, B. Petres

Dr. Fran Mihaljevi} University Hospital for InfectiousDiseases, Zagreb

C-reactive protein (CRP) is an acute phaseprotein synthetized in the liver under the controlof interleukin-6 (IL-6). Today, CRP is an impor-



ga u dijagnostici, terapiji i pra}enju akutnih ikroni~nih bakterijskih infekcija te autoimunih imalignih bolesti. Na tr`ištu su danas dostupnirazli~iti imunokemijski testovi za kvantitativnoodre|ivanje koncentracije CRP-a. Cilj ove studijebio je usporediti vrijednost triju testova za odre-|ivanje CRP-a u serumu od tri razli~ita proizvo-|a~a: Olympus, Randox i Orion Diagnostica.Koncentracije CRP-a odredili smo u serumu na-sumice odabranih bolesnika (N=69) Klinike zainfektivne bolesti “Dr. Fran Mihaljevi}’’. Sva tritesta temelje se na imunoturbidimetrijskom od-re|ivanju CRP-a, a ispitivanje je provedeno nabiokemijskom analizatoru Olympus AU400. Do-biveni rezultati obra|eni su linearnom regresi-jom, izra~unati su koeficijenti korelacije, a sta-tisti~ka zna~ajnost testirana je sparenim Wil-coxon testom na razini zna~ajnosti p<0,05. Koefi-cijenti korelacije i jednad`be regresije iznosili suza: Olympus vs. Orion r=0,9879, (y=0,9363x+1,5164); Orion vs. Randox r=0,9934 (y=1,2413x-6,8303) i Randox vs. Olympus r=0,9850 (y=0,747x+7,1306). Nije na|ena statisti~ki zna~ajnarazlika izme|u testova Olympus i Orion (p=0,0543) te Orion i Randox (p=0,0752), ali je posto-jala statisti~ki zna~ajna razlika izme|u testovaOlympus i Randox (p=0,0308). Nepreciznost zasva tri testa bila je ispod CV 10%. Linearnomregresijom i korelacijom na|ena je visoka pove-zanost izme|u svih triju testova za CRP (r>0,98).Statisti~ki zna~ajna razlika postojala je samo iz-me|u testova Olympus i Randox, ali se ne smatraklini~ki zna~ajnom. Sva tri testa pokazala suujedna~enu kvalitetu reagensa.E-mail: [email protected]

tant analyte for the diagnosis, therapy and fol-low up of acute and chronic bacterial infectionsas well as of autoimmune and malignant dis-eases. Currently there are many commerciallyavailable immunochemistry assays for quantita-tive determination of CRP. The aim of the studywas to compare the value of three assays for CRPdetermination in serum, manufactured by Olym-pus, Randox and Orion Diagnostica. CRP con-centrations were measured in serum of randomlyselected patients (N=69) from Dr. Fran Mihaljevi}University Hospital for Infectious Diseases. Allthree assays are based on immunoturbidimetry,and were performed on an Olympus AU400 bio-chemistry analyzer. Results were statisticallyanalyzed by linear regression analysis, correla-tion coefficients were calculated, and statisticalsignificance was tested by paired Wilcoxon test(p<0.05). The correlation coefficients and linearregression lines were: Olympus vs. Orionr=0.9879 (y=0.9363x+1.5164); Orion vs. Randoxr=0.9934 (y=1.2413x-6.8303); and Randox vs. Ol-ympus r=0.9850 (y=0.747x+7.1306). There was nostatistically significant difference between Olym-pus and Orion (p=0.0543), and Orion andRandox (p=0.0752), however, it was found be-tween Olympus and Randox (p=0.0308). The im-precision for all three assays was below CV 10%.Accordingly, linear regression analysis and corre-lation coefficients showed a high correlation be-tween all three CRP assays (r>0.98). There was astatistically significant difference between Olym-pus and Randox, however, the difference was notclinically relevant. All three assays showed prac-tically the same quality of reagents.E-mail: [email protected]

P7-7

UTJECAJ FIBRINOGENA I LUPUSANTIKOAGULANATA NA PROCJENUAKTIVNOSTI ANTIKOAGULANTNOG

PUTA PROTEINA C DADE BEHRINGOVIMPROC GLOBAL TESTOM

Paukovi}-Sekuli} B., Salamuni} I., Galetovi} A.,Bilopavlovi} N.

Odjel za medicinsko laboratorijsku dijagnostiku, Klini~kabolnica “Split”, Split

Sustav proteina C (PC) je va`an mehanizam uregulaciji koagulantne aktivnosti. Poreme}aji po-ve}avaju rizik za vensku trombozu. ProC Globaltest omogu}uje procjenu globalne antikoagu-lantne aktivnosti PC i poreme}aja u nasljednim

P7-7

EFFECT OF FIBRINOGEN AND LUPUSANTICOAGULANTS ON EVALUATION OF

THE ACTIVITY OF PROTEIN CANTICOAGULANT PATHWAY BY DADE

BEHRING PROC GLOBAL TEST

B. Paukovi}-Sekuli}, I. Salamuni}, A. Galetovi}, N.Bilopavlovi}

Department of Medical Laboratory Diagnosis, SplitUniversity Hospital, Split

Protein C (PC) system is an important mecha-nism in the regulation of coagulant activity. Therelated disorders increase the risk of venousthrombosis. ProC Global test enables evaluationof the global anticoagulant activity of PC and dis-



(APC rezistencija, nedostatak PC i PS) i ste~enim(visoka koncentracija fibrinogena, visoka vrijed-nost FV, FVIII, lupus koagulanta (LA-a)) koagu-lacijskim poreme}ajima. Cilj ispitivanja je bio od-rediti utjecaj povišenih koncentracija fibrinogena>3,5 g/l i lupus antikoagulanta (LA) >1,3 na nor-malizirani omjer (NR) ProC Global testa kod 101bolesnika (59 @ i 42 M) s idiopatskom trombozom.NR, NR FV Leiden (FVL), fibrinogen i LA odre-|eni su koagulacijskom metodom testovima tvrt-ke Dade Behring na instrumentu Behring Co-agulation Timer (BCT). Za NR kao cut off uzetaje vrijednost 0,8, a za NR FVL 0,9. NR<0,8 (ra-spon 0,61- 0,93) dobiven je kod 67,6% bolesnika sfibrinogenom >3,5 g/l i 46,4% bolesnika (raspon0,64-0.96 ) s LA >1,3. NR FVL <0,9 imao je jedanbolesnik s normalnom koncentracijom fibrinoge-na i normalnim LA, jedan bolesnik s koncentra-cijom fibrinogena >3,5 g/l i dvoje bolesnika s LA>1,3. Rezultati pokazuju da povišene vrijednostifibrinogena i LA-a smanjuju NR i treba ih uzeti uobzir kod procjene rezultata ProC Global testa.Poreme}ene vrijednosti su posljedica hiperak-tivnosti prokoagulantnog sustava kod visokihkoncentracija fibrinogena i interferencije hetero-gene skupine protutijela koja ~ine LA s aktiv-nostima PC ovisnim o fosfolipidima.E-mail: [email protected]

orders in hereditary (APC resistance, deficiencyof PC and PS) and acquired (high concentrationof fibrinogen, high value of FV, FVIII, LA) coagu-lation disorders. The aim of the study was to in-vestigate the effect of increased concentrations offibrinogen >3.5 g/l and lupus anticoagulant (LA)>1.3 on normalized ratio (NR) of ProC Global testin 101 patients (59 F and 42 M) with idiopathicthrombosis. NR, NR FV Leiden (FVL), fibrinogenand lupus anticoagulant were determined by thecoagulation method using Dade Behring tests ona Behring Coagulation Timer (BCT). A NR valueof 0.8 and NR FVL value of 0.9 were taken as cutoff values. NR <0.8 (range 0.61-0.93) was re-corded in 67.6% of patients with fibrinogen >3.5g/l and 46.4% of patients (range 0.64-0.96) withLA >1.3. Only one patient with normal fibrinogenconcentration and normal LA, one patient withfibrinogen concentration >3.5 g/l and two pa-tients with LA >1.3 had NR <0.9. Study resultsshowed the increased values of fibrinogen and LAto decrease NR, and that it should be taken in ac-count when evaluating the results of ProC Globaltest. The abnormal values are the consequence ofhyperactivity of the procoagulant system at highfibrinogen concentrations and interference of theheterogeneous group of LA constituting antibod-ies with the phospholipid dependent PC activi-ties.E-mail: [email protected]

P7-8

PROCJENA REAGENSA TVRTKE PLIVAZA ODRE\IVANJE KALCIJA, FOSFORA I

MAGNEZIJA

@upani} D., Vlaši}-Tanaskovi} J.

Medicinsko biokemijski laboratorij, Op}a bolnica Pula,Pula

Hormonska regulacija metabolizma kalcija,fosfora i magnezija je me|usobno povezana. Kal-cij se u krvnom serumu javlja kao difuzibilni inedifuzibilni, fosfor kao anorganski i organski, a3/4 magnezija vezano je u obliku apatita, dok seostatak nalazi u ioniziranom obliku. Odgovornisu za mnogobrojne vitalne funkcije organizma, testoga zauzimaju vrlo va`no mjesto u laborato-rijskoj dijagnostici. Cilj studije bio je procijenitiPlivine reagense za odre|ivanje kalcija, fosfora imagnezija, njihovu usporedivost s reagensimakoji se ve} rabe u našem laboratoriju, te pri-mjenjivost za rad na automatskim analizatorimaserije Hitachi 717. Metode za odre|ivanje kalcija,

P7-8

EVALUATION OF PLIVA REAGENTS FORDETERMINATION OF CALCIUM,

PHOSPHORUS AND MAGNESIUM

D. @upani}, J. Vlaši}-Tanaskovi}

Medical Biochemistry Laboratory, Pula General Hospital,Pula

Hormone regulation of the calcium (Ca), phos-phorus (P) and magnesium (Mg) metabolism isinter-related. Ca is present in serum in diffusibleand undiffusible forms, P as organic and inor-ganic, and 3/4 of Mg are bound to hydroxyapa-tite whereas the rest is found in ionized form.They are responsible for many vital functions andthus have an important role in laboratory diag-nosis. The aim of the study was to evaluate Plivareagents for Ca, P and Mg determination, and toassess their comparability with the reagents usedat our laboratory and their applicability for workon Hitachi 717 autoanalyzers. Methods for deter-mining Ca, P and Mg were applied to the



fosfora i magnezija postavljene su prema propisuproizvo|a~a reagensa i prilago|ene radu na auto-matskom analizatoru Hitachi 717. Procjena re-agensa obuhvatila je odre|ivanje nepreciznostiunutar dana, iz dana u dan, neto~nosti, te uspo-redbu s reagensima tvrtka Big Blue i Roche. Ne-preciznost unutar dana odre|ena je višekratnimmjerenjem (30x) odabranih parametara u uzor-cima komercijalnog referentnog seruma, a iz da-na u dan u uzorcima kontrolnih seruma s dekla-riranim vrijednostima u normalnom i patološ-kom podru~ju. Ispitivanje neto~nosti provedenoje na 30 uzoraka seruma bolesnika. Svi rezultatimjerenja obra|eni su u programu za statistikuExcel. Dobiveni rezultati pokazali su niske koefi-cijente varijacije za nepreciznost unutar dana(KVCa = 4,04%, KVP = 4,96%, KVMg = 1,65%), teiz dana u dan (KVCaNOR = 3,08%, KVCaPAT =3,25%, KVPNOR = 7,33%, KVPPAT = 3,99%,KVMgNOR = 5,95%, KVMgPAT = 3,54%). Podu-darnost rezultata dobivenih uporabom reagensatvrtka Pliva, Big Blue, Roche i vlastitih reagensaza AAS izra`ena je koeficijentom korelacije (rCa= 0,991, rP = 0,992 i rM g= 0,962). Visoka po-novljivost rezultata, stupanj to~nosti i podudar-nost s drugim regensima ~ine Plivine reagense zamjerenje kalcija, fosfora i magnezija prihvatlji-vim za svakodnevno laboratorijsko odre|ivanje.E-mail: [email protected]

analyzer using manufacturer’s instructions andslightly adjusted for use on a Hitachi 717autanalyzer. Reagent evaluation included deter-mination of within-day and between-day impreci-sion, inaccuracy, and comparison with Big Blueand Roche reagents. Within-day imprecision wasdetermined by multiple measurements (30x) ofchosen parameters in commercial reference sera,and between-day imprecision in control serumsamples with declared values of normal andpathologic range. Studies of inaccuracy were per-formed by use of 30 patient samples. All resultswere statistically tested. Study results showedlow variation coefficient for within-day impreci-sion (CVCa=4.04%, CVP=4.96%, CVMg=1.65%)and between-day imprecision (CVCaNOR = 3.08%,CVCaPAT = 3.25%, CVPNOR = 7.33%, CVPPAT =3.99%, CVMgNOR = 5.95%, CVMgPAT = 3.54%).Comparison of the results obtained by use of BigBlue, Roche and own (for AAS) reagents was ex-pressed by the coefficient of correlation (rCa=0.991, rP = 0.992 and rMg=0.962). It is concludedthat the use of Pliva reagents for Ca, P and Mgdetermination yielded highly reproducible resultsof high accuracy and compliance with other rea-gents, making them acceptable for daily use inclinical laboratories.E-mail: [email protected]

P7-9

PROCJENA LASERSKOGHEMATOLOŠKOG ANALIZATORA CELL

DYN 3700 SL

Suljevi} E., Fazli} M., ]ori} J.

Klini~ki centar Univerziteta u Sarajevu, Institut za klini~kuhemiju i biohemiju, Sarajevo, BiH

Istra`ivanje parametara krvne slike je u pro-gramu svih zdravstvenih laboratorija. Hemato-loške pretrage danas se isklju~ivo rade na hema-tološkim analizatorima. Automatizacija hemato-loških laboratorija je posljedica velikog broja za-htjeva za pretragama, pravodobnog izdavanjanalaza i mogu}nosti primjene suvremenih teh-nologija. U ovom radu izvedena je procjena he-matološkog analizatora Cell Dyn 3700 SL(Abbott). Ispitana je pouzdanost rezultata u 219uzoraka krvi s K3EDTA pomo}u parametarapreciznosti, to~nosti, osjetljivosti i specifi~nosti,utjecaj prenošenja uzoraka i korelacija s anali-zatorom MAXM Retti (Coulter). Ispitani su he-matološki parametri: leukociti (WBC), neutro-

P7-9

EVALUATION OF THE CELL DYN 3700 SLHEMATOLOGY ANALYZER

E. Suljevi}, M. Fazli}, J. ]ori}

Institute of Clinical Chemistry and Biochemistry, SarajevoUniversity Hospital Center, Sarajevo, Bosnia and Herzegovina

A study of blood count parameters is includedin the program of all medical laboratories. Today,all hematology tests are exclusively performed onhematology analyzers. Automation of hematologylaboratories is the result of the great number ofrequests for these tests, timely reporting of find-ings, and possibility of using sophisticated tech-niques. In this study, evaluation of the Cell Dyn3700 SL laser hematology analyzer (Abbott) wasperformed. Reliability of the results obtained in219 K3EDTA blood samples was assessed by useof the following parameters: precision, accuracy,sensitivity and specificity, effect of sample trans-fer, and correlation with MAXM Retti, Coulter.The following parameters were tested: whiteblood count (WBC), neutrophils (NEU), lympho-



filni leukociti (NEU), limfociti (LYM), monociti(MONO), eozinofilni leukociti (EOS), bazofilnileukociti (BASO), eritrociti (RBC), hemoglobin(HGB), hematokrit (HCT), srednji volumen eri-trocita (MCV), srednji sadràj hemoglobina u eri-trocitima (MCH), srednja koncentracija hemo-globina u eritrocitima (MCHC) i trombociti(PLT). Rezultati su pokazali da preciznost anali-zatora zadovoljava reproducibilnost za parame-tre WBC, RBC, HGB, HCT, MCV, MCH, MCHC iPLT (KV=0,13 za MCV do 3,62 za trombocite).Vrijednosti koeficijenata koleracije (r) usporedbedvaju analizatora su bili zadovoljavaju}i osimkod MCHC (r=0,64), što je u skladu s literatur-nim podacima. To~nost diferencijalne krvne slikeispitana je na hematološkom analizatoru i mikro-skopskom metodom. Rezultati koeficijenata ko-relacije granulocita i limfocita bili su zadovo-ljavaju}i (r=0,97), što se ne odnosi na monocite(r=0,86). Podaci postotaka prenošenja uzoraka sumanji od 1%. Parametri osjetljivosti i specifi~-nosti zadovoljavaju analiti~ke kriterije. Potvr-|eno je da je laserski hematološki analizator CellDyn 3700 SL pouzdan hematološki analizator zaodre|ivanje krvne slike. Analizator se sa svojimprogramom diferencijalne krvne slike moè upo-trijebiti za istraìvanje i razdvajanje normalnihpa i patoloških krvnih slika, uz dopunu mikro-skopske metode kojom se potvr|uju distribu-cijske ili morfološke promjene leukocita.E-mail: [email protected]

cytes (LYM), monocytes (MONO), eosinophils(EOS), basophils (BASO), red blood cells (RBC),hemoglobin (HGB), hematocrit (HCT), mean cor-puscular volume (MCV), mean corpuscularhemoglobin (MCHC) and platelets (PLT). The re-sults confirmed the analyzer precision to fulfillthe reproducibility for the following parameters:WBC, RBC, HGB, MCV, MCH, MCHC and PLT(CV=0.13 for MCV through 3.62 for PLT). Corre-lation coefficient values (r) obtained throughoutthe comparison of the two analyzers were satis-factory except for MCHC (r=0.64), which is in ac-cordance with literature data. The accuracy ofleukocyte differentiation was tested on hemato-logy analyzer and by the method of microscopicdifferentiation. Correlation coefficients for granu-locytes and lymphocytes were satisfactory (r=0.97), which did not hold for monocytes (r=0.86).Percentage data on sample transfer were below1%. The sensitivity and specificity parametersmet the analytical criteria. Accordingly, reliabil-ity of the Cell Dyn 3700 SL hematology analyzerfor blood count determination was confirmed.The analyzer and its program for differentialwhite blood count can be used for research andseparation of normal and pathologic blood countswith the addition of microscopic methods con-firming the distribution or morphologic changesof leukocytes.E-mail: [email protected]

P7-10

ANALITI^KA PROCJENA ANALIZATORAOLYMPUS AU2700

Juri~ek J.1, Brki} K.2, Šprajc N.1, Romi} @.1

1 Zavod za laboratorijsku dijagnostiku, 2 Odjel za kardijalnukirurgiju, Klini~ka bolnica “Dubrava”, Zagreb

Cilj je prikazati analiti~ku procjenu biokemij-skog analizatora Olympus AU2700, koja je prove-dena prema preporukama Europskog odbora zaklini~ko-laboratorijske standarde (ECCLS). Ispi-tivani su glukoza, ureja, kreatinin, ukupni prote-ini, albumini, ukupni kalcij, urati, kolesterol, tri-gliceridi, LDH, CK, GGT, K, Na, Cl, C3, C4, feri-tin i transferin. Procjena je obuhvatila nepreciz-nost iz dana u dan, nepreciznost unutar serije,neto~nost i usporedna odre|ivanja OlympusomAU600. Nepreciznost iz dana u dan odre|ivana jeu triplikatu u kontrolnim serumima Level 1,Level 2 i skupnom serumu. Nepreciznost unutarserije odre|ivana je u 30 uzastopnih mjerenja u

P7-10

ANALYTICAL EVALUATION OF OLYMPUSAU2700 ANALYZER

J. Juri~ek1, K. Brki}2, N. Šprajc1, @. Romi}1

1 Institute of Laboratory Diagnosis, 2 Department of CardiacSurgery, Dubrava University Hospital, Zagreb

The objective is to present analytical evalua-tion of the Olympus AU2700 clinical chemistryanalyzer, performed according to the EuropeanCommittee for Clinical Laboratory Standards(ECCLS) guidelines. The following analytes weretested: glucose, urea, creatinine, total proteins, al-bumins, calcium, uric acid, cholesterol, triglyce-rides, LDH, CK, GGT, K, Na, Cl, C3, C4, ferritinand transferrin. The evaluation consisted of de-termination of between-day imprecision, within-run imprecision, inaccuracy and correlation withOlympus AU600. Between-day imprecision wasdetermined in control sera Level 1, Level 2 andpool sera. Within-run imprecision was deter-



kontrolnim serumima Level 1, Level 2, te skup-nom serumu. Neto~nost mjerenja prikazana jekao postotak odstupanja (R%) srednje izmjerenihvrijednosti od srednje deklariranih vrijednostikontrolnih seruma. Usporedba analizatora pro-vedena je pomo}u 50 uzoraka u razli~itim kon-centracijskim podru~jima. Analiti~kom procje-nom analizatora Olympus AU2700 dobivena jeniska nepreciznost iz dana u dan i unutar serije(KV zadovoljavaju}i), prihvatljiva neto~nost zasve analite (R zadovoljavaju}i) i visok stupanjkorelacije s Olympusom AU600 (zadovoljavaju}ikoeficijenti korelacije od 0,9630 do 0,9996). Pro-vedena analiti~ka procjena pokazala je da je Ol-ympus AU2700 pouzdan biokemijski analizatorkoji osigurava brz i siguran rad uz uporabu mini-malnih koli~ina uzoraka i reagensa, što mu osi-gurava mjesto u svakom medicinsko-biokemij-skom laboratoriju.E-mail: [email protected]

mined in 30 consecutive measurements in controlsera Level 1, Level 2 and pool sera. Inaccuracywas shown as a percentage of deviation from con-trol serum mean values. Comparison of theanalyzers was performed using 50 samples invarious concentration ranges. Low between-dayand within-run imprecision (satisfactory CV%),and acceptable inaccuracy for all analytes (satis-factory R%) as well as a high rate of correlationwith Olympus AU600 were found for OlympusAU2700 (satisfactory coefficient of correlation).Analytical evaluation showed the OlympusAU2700 to be a reliable biochemistry analyzer,which ensures rapid and safe work using mini-mal quantities of samples and reagents, thus be-ing suitable for routine medical biochemistrylaboratory.E-mail: [email protected]

P7-11

USPOREDBA BRZINE SEDIMENTACIJEERITROCITA ODRE\ENE POMO]U

AUTOMATIZIRANOG SUSTAVASEDITAINER I BD VACUTAINERA

Matica J., Bili}-Zulle L., Beljan B., Fiši} E., Dvornik Š.

Klini~ki bolni~ki centar “Rijeka”, Rijeka

Automatizirani sustav Seditainer (BectonDickinson) rabi varijaciju standardne Wester-grenove tehnike za odre|ivanje brzine sedimen-tacije eritrocita. Isti je antikoagulans (3,8% oto-pina natrijevog citrata), kao i omjer krvi i anti-koagulansa. Glavne razlike su u visini stupcakrvi (Seditainer 82 mm, Westergren 200 mm) teu polo`aju epruvete tijekom o~itavanja (Seditai-ner nagib od 20o prema okomici, Westergren oko-mit polo`aj). Sustav Seditainer o~itavanje provo-di pomo}u video kamere u razmacima od 5 mi-nuta te izra~unava udaljenost izme|u vrha stup-ca krvi i dodirne površine izme|u plazme i sta-nica. Odnos izme|u brzine sedimentacije dobi-vene pomo}u sustava Seditainer nije linearan uusporedbi s Westergrenovom metodom, stoga na-kon višestrukog mjerenja tijekom 20 minuta su-stav matemati~ki prilago|ava vrijednosti premaWestergrenovoj metodi. Cilj rada bio je uspore-diti brzinu sedimentacije eritrocita odre|enepomo}u automatiziranog sustava Seditainer sbrzinom sedimentacije eritrocita u BD vakutai-nerima za odre|ivanje brzine sedimentacije eri-

P7-11

COMPARISON OF ERYTHROCYTESEDIMENTATION RATE USING

SEDITAINER AUTOMATED SYSTEM ANDBD VACUTAINERS

J. Matica, L. Bili}-Zulle, B. Beljan, E. Fiši}, [. Dvornik

Rijeka University Hospital Center, Rijeka

Seditainer automated system (Becton Dickin-son) uses a variation of the standard Westergrentechnique for determination of erythrocyte sedi-mentation rate (ESR). The anticoagulant (3.8%sodium citrate) as well as the blood to anticoagu-lant ratio are the same. The major differences arein the blood column height (Seditainer 82 mm,Westergren 200 mm) and tube positioning duringthe reading (Seditainer inclination at 20o fromthe vertical, and Westergren vertical). The Sedi-tainer automated system uses a video camerathat scans the racks in the instrument at 5-minintervals. The system calculates the distance be-tween the blood zero level and plasma/cell inter-face. The ESR recorded in Seditainer tube wasnot linear when compared with Westergrenmethod. After multiple determinations during 20-min reading period, the system mathematicallyadjusted the values according to Westergrenmethod. The aim of the study was to compare theESR obtained by Seditainer automated systemwith that obtained by BD vacutainers. ESR wasdetermined in 179 subjects by use of Seditainer



trocita. Brzina sedimentacije eritrocita odre|enaje kod 179 ispitanika pomo}u automatiziranogsustava Seditainer (1,8 ml) i modificiranomWestergrenovom metodom u BD vakutainerima(Becton Dickinson, 1,6 ml). Me|usobni odnosizmjerenih vrijednosti ispitan je korelacijom, anjihova povezanost izraèna je jednad`bom prav-ca Passing i Bablok regresije. Passing i Bablokregresijom dobiven je visok koeficijent korelacijeizme|u ovih dviju metoda (r=0,96). Odsje~akpravca regresije na osi y iznosi -1,57, a nagibpravca 0,91 za vrijednosti automatiziranog susta-va Seditainer kao zavisne varijable. Prednostautomatiziranog sustava Seditainer je u brziniizvo|enja analize (20 minuta), a visoka podudar-nost sa standardnom metodom ~ini ga pogodnimza uporabu u rutinskom laboratorijskom radu.E-mail: [email protected]

system (1.8 ml) and BD vacutainers (BectonDickinson, 1.6 ml). The relationship between thetwo methods was calculated using correlationand Passing and Bablok regression. Correlationcoefficient between the two methods was r=0.96.According to Passing and Bablok regressionequation: intercept=-1.57 and slope=0.90 forSeditainer automated system as a dependentvariable. High correlation with the standardmethod and short time of analysis (20 minutes)make the Seditainer automated system suitablefor use in laboratory routine.E-mail: [email protected]

P7-12

ODRE\IVANJE AKTIVNOSTI LAKTATDEHIDROGENAZE - NAŠA ISKUSTVA I

ODLUKA O METODI IZBORA

Leni~ek-Krleà J., ]uri} D., Tadi} I., Brki} K., @iì}V., Naki} M.

Klinika za dje~je bolesti, Zagreb

Za odre|ivanje aktivnosti laktat dehidroge-naze (LDH) danas se gotovo isklju~ivo rabi me-toda kontinuiranog mjerenja pod optimiranimuvjetima. Razli~itost dobivenih rezultata poslje-dica je uporabe razli~itih supstrata i pufera. Na-ša su iskustva pokazala da razli~iti proizvo|a~itest reagencija koje primjenjuju razli~ite tipovereakcija nemaju dobro obra|en i prilago|en ra-spon referentnih vrijednosti, uz istodobno nepri-hvatljivo odstupanje u vanjskoj i gotovo idealno uunutarnjoj procjeni kontrole kvalitete. Naš je ciljbio ispitati i uvesti onu metodu koja }e zadovoljitisva tri kriterija: u praksi primjenjiv i dobro raz-ra|en referentni raspon, te dobru unutarnju ivanjsku kontrolu kvalitete. U analizu je uklju-~eno 125 slu~ajno odabranih seruma neovisno odobi i 50 uzoraka komercijalnog kontrolnog seru-ma prilago|enog proizvo|a~u reagensa. Tipovireakcija koje smo ispitivali rabile su: piruvat kaosupstrat i TRIS pufer (A), laktat kao supstrat iAMP pufer (B), te piruvat kao supstrat i fosfatnipufer (C). Sva odre|ivanja su u~injena na istomanalizatoru uz propisane uvjete komercijalnihtest reagencija razli~itih proizvo|a~a. Rezultatiispitivanja pokazali su slijede}e: aktivnosti LDH

P7-12

DETERMINATION OF LACTATEDEHYDROGENASE ACTIVITY - OUR

EXPERIENCE AND DECISION ON THEMETHOD OF CHOICE

J. Leni~ek-Krleà, D. ]uri}, I. Tadi}, K. Brki}, V.@iì}, M. Naki}

Zagreb Children’s Hospital, Zagreb

The method of continuous measurement underoptimal conditions is now almost exclusively usedfor determination of lactate dehydrogenase(LDH) activity. Variability in the results obtainedis due to the use of different substrates and buffer.Our experience shows that various manufacturersof test reagents using various types of reactionlack well developed and adjusted range of refer-ence values, along with unacceptable deviation onexternal quality control assessment and nearlyideal agreement on internal quality control as-sessment. Therefore, the aim of the study was toidentify and introduce the method that wouldmeet the following criteria: well defined referencerange applicable in practice, and good internaland external quality control. A total of 125 ran-domly selected sera irrespective of age and 50commercial control serum samples adjusted tothe reagent manufacturer’s requirements wereanalyzed. The reaction types tested utilized: pyru-vate as substrate and TRIS buffer (A), lactate assubstrate and AMP buffer (B), and pyruvate assubstrate and phosphate buffer (C). All determi-nations were performed on the same analyzer



istoga uzorka odre|ene tipom reakcije B bile sunajniè, a tip reakcije A imao je dvostruko viševrijednosti. Vrijednosti mjerene tipom reakcije Cnalazile su se izme|u ovih dviju vrijednosti. Po-nu|eni rasponi referentnih vrijednosti od proiz-vo|a~a za svaki pojedini tip reakcije ne zadr-àvaju taj odnos. Korelacijom ovih triju tipovareakcije dobili smo slijede}e koeficijente kore-lacije: 0,976 za reakcije B i C; 0,986 za reakcije Ai C; i 0,957 za reakcije A i B. Kontrolni serumišto ih preporu~uje proizvo|a~ rabljeni su za svakitip reakcije i nalazili su se uz minimalna od-stupanja u sredini dozvoljenog raspona. Vanj-skom procjenom kvalitete kroz godinu danaaktivnosti LDH nalazile su se izvan dozvoljenogodstupanja mjerene tipom reakcije B. Naše is-kustvo pokazalo je da su u praksi najprihvat-ljivije vrijednosti dobivene tipom reakcije C uzkorekciju i utvr|ivanje vlastitih referentnih vri-jednosti za pojedine dobne skupine, a ispravnostnaše odluke pokazati }e slijede}a vanjska kon-trola kvalitete.E-mail: [email protected]

with due care of the required conditions for com-mercial test reagents from different manufactur-ers. Study results showed the LDH activity in thesame sample to be lowest when determined bytype B reaction and two-fold that when deter-mined by type A reaction, whereas the values ob-tained by type C reaction were in-between thesetwo. The ranges of reference values provided bythe manufacturers for each individual type of re-action did not maintain this relationship. Corre-lation of the three types of reaction yielded the fol-lowing correlation coefficients: 0.976 for reactiontypes B and C; 0.986 for reaction types A and C;and 0.957 for reaction types A and B. Controlsera recommended by the manufacturer wereused for each type of reaction and were in themiddle of the allowed range, with only minimaldeviations. External quality assessment over aone-year period showed the values of LDH activ-ity to be beyond the allowed deviation when deter-mined by type B reaction. Our experience sug-gested the values obtained by use of type C reac-tion to be most acceptable in practice, with duecorrection and setting of own reference values forparticular age groups. The next external qualitycontrol will determine the validity of our decision.E-mail: [email protected]

P7-13

UTJECAJ RAZLIÎTIH METODAPROCJENE REAKCIJSKE KRIVULJE NA

REZULTATE AKTIVIRANOGPARCIJALNOG TROMBOPLASTINSKOG

VREMENA

Miloš M., Coen D., Zadro R.


Suvremeni opti~ki koagulometri omogu}ujupra}enje cjelokupnog tijeka procesa stvaranjaugruška mjerenjem promjena u apsorpciji ilitransmisiji, te biljeènjem tih promjena u oblikureakcijske krivulje. Postoji više metoda evalu-acije reakcijske krivulje, koje u razli~itim to~-kama krivulje biljeè vrijeme zgrušavanja. Zbogtoga se u istom uzorku na istom koagulometrurazli~itim metodama evaluacije mogu dobiti raz-li~iti rezultati. Cilj rada bio je usporediti rezul-tate aktiviranog parcijalnog tromboplastinskogvremena (APTV-a) dobivene trima razli~itim me-todama procjene reakcijske krivulje. U 104 uzor-ka plazme odre|en je APTV (Actin FS, DadeBehring) pomo}u triju metoda procjene reakcij-

P7-13

IMPACT OF DIFFERENT EVALUATIONMODES FOR THE DETECTION OF

CLOTTING TIMES ON aPTT RESULTS

M. Miloš, D. Coen, R. Zadro


Modern photo-optical coagulometers collectoptical data throughout the course of clot forma-tion by monitoring changes in light absorbance ortransmittance. By using different evaluationmodes, they offer the possibility to measure clot-ting time at different points of clot formation. Ac-cordingly, different results for the same assaymay be obtained even on the same analyzer. Theaim of the study was to compare activated partialthromboplastin time (aPTT) results obtainedwith 3 different evaluation modes. aPTT wasmeasured (Actin FS, Dade Behring) in 104plasma samples using the fixed absorbance, drift-ing baseline and point of inflexion evaluationmodes on 2 Dade Behring coagulation analyzers(BCS and BCT). Discrepant aPTT results wererecorded in all study samples, being always pro-



ske krivulje: zadane promjene apsorbancije (fixedabsorbance), pomaka osnovne linije (driftingbaseline) i to~ke infleksije (point of inflexion) na 2koagulacijska analizatora (BCS i BCT, DadeBehring). U svim uzorcima dobiveni su viši re-zultati metodom evaluacije krivulje pomo}u me-tode to~ke infleksije (srednja vrijednost: 39,4 s;raspon: 20,5-99,1 s) u odnosu na rezultate dobi-vene metodama pomaka osnovne linije (srednjavrijednost: 33,3 s; raspon: 20,2-78,5 s) i zadanepromjene apsorbancije (srednja vrijednost: 31,2s; raspon: 19,0-80,8 s). Tijekom istraìvanja u 2razli~ite situacije zabiljeèni su neto~ni rezultatikoji se mogu uo~iti samo uvidom u reakcijskukrivulju. Prvo, kod 4 normalna uzorka dobivenesu izrazito kratke vrijednosti APTV-a metodomzadane promjene apsorbancije. Drugo, u 5 uzora-ka uop}e nije zabiljeèn ugrušak metodom po-maka osnovne linije usprkos normalnim reak-cijskim krivuljama. U svim ovim slu~ajevimadruge dvije metode evaluacije dale su ispravanrezultat. Zaklju~eno je kako razlike u dobivenimrezultatima ukazuju na potrebu uspostavljanjareferentnih vrijednosti specifi~nih i za metoduprocjene reakcijske krivulje. Da bi se dobio to~anrezultat, predlaèmo pregled reakcijske krivuljesvih sumnjivih rezultata i, ako je potrebno, po-navljanje mjerenja drugom metodom procjene.E-mail: [email protected]

longed with the point of inflexion evaluationmode (mean 39.4 s, range 20.5-99.1 s) as com-pared with the results obtained by the driftingbaseline (mean 33.3 s, range 20.0-78.5 s) or fixedabsorbance (mean 31.2 s, range 19.0-80.8 s)evaluation mode. During the study, inaccurate re-sults which were identified by visual inspection ofthe reaction curves were observed on two occa-sions. Firstly, extremely short aPTT results wereobtained in 4 normal samples by fixed absorb-ance. Secondly, despite normal reaction curves in5 samples, no clot result was measured by thedrifting evaluation mode. In all these cases, theremaining two evaluation modes yielded correctresults. It is concluded that differences betweenthe results obtained by different evaluation modesimplicate the need to establish not only reagent-coagulometer specific but also evaluation mode-specific reference intervals. According to our expe-rience, only visual analysis of the reaction curveprovides complete information on the entire clot-ting process. In order to obtain correct results, wesuggest confirmation of all suspected values byinspection of the reaction curve and, if needed, re-peated testing with other evaluation modes.E-mail: [email protected]

P7-14

PROCJENA UÎNKOVITOSTIHEMATOLOŠKIH BROJAÂ BAYER ADVIA120 I ABBOTT CELL-DYN (MODELI 3200 I

4000) U ODRE\IVANJU PETODJELNEDIFERENCIJALNE KRVNE SLIKE

Juri~evi} M., Alpeza I., Zadro R.

Klini~ki zavod za laboratorijsku dijagnostiku Medicinskogfakulteta Sveu~ilišta u Zagrebu i KBC “Zagreb”, Zagreb

Uz to~no brojanje krvnih stanica, svrha suvre-menog hematološkog broja~a je i smanjenje po-trebe ru~nog diferenciranja leukocita mikrosko-piranjem obojenih krvnih razmaza. Cilj rada bioje procijeniti u~inkovitost triju razli~itih hema-toloških broja~a i usporediti dobivene rezultate srezultatima za diferencijalnu krvnu sliku dobi-venim mikroskopiranjem. Analiza diferencijalnekrvne slike izvedena je na 150 slu~ajno oda-branih uzoraka krvi bolni~kih ispitanika isto-dobno na tri hematološka analizatora: BayerAdvia 120, Abbott Cell-Dyn (modeli 3200 i 4000) iru~nom metodom diferenciranja pod mikrosko-

P7-14

EFFICACY ASSESSMENT OFDIFFERENTIAL BLOOD COUNT ON

BAYER ADVIA 120 AND ABBOTT CELL-DYN HEMATOLOGY ANALYZERS

(MODELS 3200 AND 4000)

M. Juri~evi}, I. Alpeza, R. Zadro


Besides precise blood cell count, a modernhematology instrument should eliminate the needof technical review and microscopy on differentialblood analysis. The aim of the study was to evalu-ate the differential performance of three hema-tology instruments and compare it with manualdifferential count. Analysis of differential bloodcount was performed on 150 inpatient rando-mized blood samples on three blood counters:Bayer Advia 120, and Abbott Cell Dyn 4000 and3200, along with manual differentiation. Forcomparison with the method of microscopy, truetable analysis was carried out for each instru-



pom. Prema kriterijima što ih je predloìoNCCLS (dokument H20-A) za procjenjivanjeu~inkovitosti odre|ivanja petodjelne diferenci-jalne krvne slike (nesegmentirani >7%, nezreligranulociti >3%, blasti >3%, varijabilni limfociti>7%) izra|ena je tablica to~nosti za svaki poje-dini instrument u usporedbi s mikroskopskommetodom. Dobiveni su slijede}i rezultati za Advia120, Cell-Dyn 4000 i 3200: osjetljivost (%) 96,9;88,0 i 89,4; specifi~nost (%) 67,0; 58,3 i 83,3;u~inkovitost (%) 60,1; 61,4 i 84,9. Uklju~ivanjempregleda grafi~kog prikaza kao dodatnog kriteri-ja u procjeni diferencijalne krvne slike dobivenaje jednako dobra osjetljivost (%): 98,4; 98,5 i 96,7,ali zna~ajno niè vrijednosti za specifi~nost (%):18,2; 29,4 i 38,0 i u~inkovitost (%): 51,6; 67,3 i61,4. U zaklju~ku, dobivena je visoka osjetljivostprocjene petodjelne diferencijalne krvne slikezbog tehnološke usavršenosti svih triju instru-menata, a razli~ite vrijednosti specifi~nosti iu~inkovitosti su rezultat postavljenih kriterijapojave signala u svakom pojedinom instrumentu.Utjecaj takve procjene dobivanja petodjelne dife-rencijalne krvne slike daje zadovoljavaju}u u~in-kovitost uporabe instrumenata u rutinskom ra-du. Nasuprot tome, visoka osjetljivost grafi~kogprikaza diferencijalne krvne slike rezultat je vi-sokog postotka la`no pozitivnih procjena, što imaza posljedicu potrebu za provjerom rezultata podmikroskopom, a to zna~i smanjenje ru~nog di-ferenciranja na 30%.E-mail: [email protected]

ment according to the criteria proposed byNCCLS. The results obtained on Advia 120, CellDyn 4000 and Cell Dyn 3200 were as follows (%):sensitivity: 96.9, 88.0 and 89.4; specificity: 67.0,58.3 and 83.3; and efficacy: 60.1, 61.4 and 84.9,respectively. Inclusion of an overview of scatter-plot patterns as an additional criterion in differ-ential blood count assessment yielded equally ac-ceptable sensitivity values: 98.4, 98.5 and 96.7,but significantly lower values for specificity: 18.2,29.4 and 38.0, and efficacy: 51.6, 67.3 and 61.4,respectively. In conclusion, high sensitivity of dif-ferential blood count assessment was obtaineddue to technologic sophistication of the instru-ments, whereas different values of specificity andefficacy were the result of different criteria deter-mining the occurrence of signal in each instru-ment. The consequence of high sensitivity and lowspecificity of the scatterplot pattern is the need ofmicroscopy verification of the results thus ob-tained.E-mail: [email protected]

P7-15

SEDIMENTACIJA ERITROCITA:USPOREDBA SUSTAVA ESR

(ERYTHROCYTE SEDIMENTATION RATE)S REFERENTNOM WESTERGRENOVOM

METODOM

Kunovi} B., Nimac A.

Medicinsko-biokemijski laboratoij, Zagreb

Mjerenje brzine sedimentacije eritrocita u kr-vi je jednostavna rutinska metoda koja se je do-sad izvodila Westergrenovom metodom kao refe-rentnom, no uvo|enjem automatiziranih anali-zatora ova je pretraga postala brà i jednostav-nija. Prikazati }emo naše rezultate koje smodobili na analizatoru VES-MATIC 20 u usporedbis našom dotada rutinskom, Westergrenovom me-todom jednokratnim sustavima Greiner. Naanalizatoru VES MATIC 20 postupak je potpuno

P7-15

ERYTHROCYTE SEDIMENTATION:COMPARISON OF ERYTHROCYTE

SEDIMENTATION RATE (ESR) SYSTEMAND WESTERGREN REFERENCE

METHOD

B. Kunovi}, A. Nimac

Laboratory of Medical Biochemistry, Zagreb

The measurement of the erythrocyte sedimen-tation rate (ESR) in blood is a simple routinemethod to date performed by use of Westergrenreference method, however, the introduction ofautoanalyzers has made the test more rapid andsimpler. Results obtained on the VES-MATIC 20analyzer were compared with the formerly rou-tine Westergren method using disposable Greinersystem. Using the VES MATIC 20 analyzer, theprocedure is completely standardized and auto-



standardiziran i automatiziran, a istodobno jeskra}eno i vrijeme pretrage. Za 25 minuta dobijuse rezultati za 20 uzoraka, što odgovara mjerenjuod jednog sata Westergrenovom metodom. Uz to,smanjena je i koli~ina krvi potrebna za pretragu,što je povoljno za bolesnika. Temeljem dobivenihrezultata i njihovom statisti~kom obradom za-klju~ujemo da je mjerenje brzine sedimentacijepomo}u analizatora VES-MATIC 20 standardi-zirano, to~nije, brè i s manjim volumenom krvi,a time povoljnije za bolesnika.E-mail: [email protected]

mated, and the test time is shortened. Results for20 samples are obtained in 25 minutes, corre-sponding to 1-hour measurement by Westergrenmethod. Also, a smaller amount of blood is re-quired, which is favorable for the patient. The re-sults obtained in the study and their statisticalprocessing indicated the ESR measurement onVES-MATIC 20 analyzer to be a standardized,more accurate and faster procedure requiringlesser blood volume, thus being preferable for thepatient.E-mail: [email protected]

P7-16

USPOREDBA KONCENTRACIJA LAKTATAIZME\U UZORAKA KAPILARNE KRVI,

VENSKE PUNE KRVI I VENSKE PLAZMEUZETIH U MIROVANJU

êpi} K.1, Šupe-Domi} D.1, Dujmov I.1, Tocilj J.2

1 Odjel za medicinsko laboratorijsku dijagnostiku,2 Odjel za plu}ne bolesti, Klini~ka bolnica “Split”, Split

Laktat predstavlja vezu izme|u aerobnog ianaerobnog metabolizma. Koncentracija laktatau krvi ovisna je o sintezi u miši}nim stanicama ieritrocitima te o metabolizmu u jetri. Koncen-tracija laktata se obi~no odre|uje iz venske pla-zme. Cilj ovoga istraìvanja bio je vidjeti postojeli razlike izme|u koncentracije laktata u kapi-larnoj krvi, venskoj punoj krvi i venskoj plazmi.Koncentracija laktata u uzorku je odre|ivanaenzimskom metodom. Uzorci su uzeti od 40 zdra-vih èna (starosti 18-34 godine) za vrijeme miro-vanja. Razlike izme|u koncentracije laktata ukapilarnoj krvi (srednja vrijednost 1,83±0,37nmol/l) i venskoj plazmi (srednja vrijednost1,56±0,43 nmol/l) te razlike izme|u koncentracijelaktata u venskoj punoj krvi (srednja vrijednost1,86±0,28 nmol/l) i venskoj plazmi (srednja vri-jednost 1,56±0,43 nmol/l) bile su statisti~ki zna-~ajne (p<0,01). Korelacija izme|u koncentracijelaktata u venskoj punoj krvi i venskoj plazmi bilaje linearna i pozitivna (0,62). Statisti~ka analizarezultata pokazala je da razlika izme|u kon-centracije laktata u kapilarnoj krvi i venskojpunoj krvi nije statisti~ki zna~ajna. Ovi rezultatiupu}uju na to da se pra}enje koncentracije lak-tata treba uvijek provoditi iz uzorka uzetog naisti na~in. Ova je studija dala zanimljive rezul-tate koje bi trebalo potvrditi na ve}em broju ispi-tanika.E-mail: [email protected]

P7-16

DIFFERENCES IN LACTATECONCENTRATION AMONG CAPILLARYBLOOD, VENOUS WHOLE BLOOD ANDVENOUS PLASMA SAMPLED AT REST

K. êpi}1, D. Šupe-Domi}1, I. Dujmov1, J. Tocilj2

1 Department of Medical Laboratory Diagnosis,2 Department of Pulmonology, Split University Hospital, Split

Lactate makes the link between aerobic andanaerobic metabolism. The concentration of lac-tate in the blood is dependent on the rate of itsproduction in muscle cells and erythrocytes andthe rate of metabolism in the liver. Venous plasmais the most suitable sample for routine lactate de-termination. Differences among capillary blood,venous whole blood and venous plasma lactateconcentrations (LC) were compared. Enzymaticmethod was used for the measurement of LC insamples obtained from 40 healthy women (age18-34) at rest. The differences between capillaryblood and venous plasma LC were statisticallysignificant (p<0.01), an so were those between ve-nous whole blood and venous plasma LC (p<0.01;linear positive correlation 0.62). However, therewas no significant difference between capillaryblood and venous whole blood LC. These resultsindicate that blood lactate measurements andmonitoring should always be performed in thesame sample (capillary blood or venous wholeblood or venous plasma). This study suggests theneed of additional studies in a larger sample.E-mail: [email protected]



P7-17

PROVJERA METODE ZA ODRE\IVANJEKONCENTRACIJE PROOKSIDATIVNIH

TVARI

Sokoli} I., Rumenjak V., Dravinski @., Milardovi} S.

Op}a bolnica “Sveti Duh”, Zavod za medicinsko-laboratorijsku dijagnostiku, Zagreb

Oksidativni status odraàva ravnoteù izme-|u antioksidativne obrane organizma i oksidansau organizmu. Poreme}aj ove ravnoteè (nedo-voljan unos antioksidansa ili poja~ano stvaranjeoksidansa) dovodi do stanja organizma nazvanogoksidativni stres koji se usko povezuje s degene-rativnim i patološkim procesima u organizmu,bilo da se o njemu govori kao o uzroku ili po-sljedici patološkog stanja. Klju~ni preduvjet pro-cjene oksidativnog statusa organizma je mogu}-nost pouzdanog mjerenja koncentracije prooksi-dativnih tvari koje su ishodište kaskadne reak-cije kojom nastaju ošte}enja proteina, lipida,DNK i ostalih stani~nih elemenata. Cilj ovogarada bila je procjena metode za mjerenje koncen-tracije prooksidativnih tvari te odre|ivanje kon-centracije biljega oksidativnog statusa kod zdra-vih osoba. Procjenjivana je metoda odre|ivanjakoncentracije prooksidativnih tvari u serumu te-meljena na Fentonovoj reakciji. U reakciji poFentonu Fe2+ u prisutnosti hidroperoksida i lipo-peroksida stvara hidroksil radikal koji mijenjaboju kromogena, u ovom slu~aju N,N-dimetil-p-fenilen-diamina (DMPD). Intenzitet obojenjaproporcionalan je koncentraciji peroksida u uzor-ku i mjeri se fotometrijski. Kalibracija je izvede-na pomo}u terc-butil-peroksida, a rezultat izra-èn u µmol/l. Metoda je ispitana na uzorku se-ruma 98 zdravih muškaraca u dobi od 25-55 go-dina starosti. Ponovljivost mjerena iz dana u dandaje SD 0,031 µmol/l i CV 8%. Procjena osjet-ljivosti metode dala je granicu od 30 µmol/l, ametoda se pokazala linearnom do 700 µmol/l.To~nost metode ispitana testom iskori{tenja (re-covery) iznosila je 122%. Prooksidativne tvari userumu ispitanika kretale su se u rasponu kon-centracija od 43-474 µmol/l. Ispitana metodaodre|ivanja koncentracije prooksidativnih tvaripokazala je dobre rezultate, što ~ini temelj zadaljnja istraìvanja oksidativnog statusa bole-snika.E-mail: [email protected]

P7-17

EVALUATION OF THE METHOD FORDETERMINATION OF PROOXIDATIVE

SPECIES CONCENTRATIONS

I. Sokoli}, V. Rumenjak, @. Dravinski, S. Milardovi}

Institute of Medical Laboratory Diagnosis, Sveti DuhGeneral Hospital, Zagreb

Oxidative status is a balance between the con-centration of oxidants and antioxidative defensemechanisams in the body. Disturbance of thisbalance may be caused by excessive oxidant pro-duction or by a decrease in the antioxidant capac-ity, which results in a condition known asoxidative stress. Oxidative stress is closely relatedto almost every pathologic and degenerative proc-ess, either as the cause or direct consequence. Akey to accurate assessment of prooxidative statusis reliable measurement of prooxidative speciesconcentrations. The aim of the study was toevaluate the method of prooxidative species deter-mination based on Fenton reaction. In the pres-ence of lipoperoxides and hydrogen peroxide,Fe2+ produces hydroperoxyl radical, which turnsN,N-dimethyl-p-phenylenediamine (DMPD) intofairly long lived colored radical cation. The inten-sity of color is proporrtional to the prooxidativespecies concentration in the sample, and is meas-ured photometrically. Calibration was performedwith tert-butyl-peroxide, and results were ex-pressed in µmol/l. The measurement was per-formed in plasma samples obtained from 98healthy men aged 25-55. Day-to-day reproducibil-ity yielded SD 0.031 µmol/l and CV 8%. Thelimit of sensitivity was 30 µmol/l. The method islinear to up to 700 µmol/l. Recovery test yieldedsensitivity of 122%. The prooxidative species con-centration in serum was 43-474 µmol/l. Themethod for determination of prooxidative speciesconcentration showed good results and warrantsadditional investigations of oxidative status inboth healthy population and pathologic cases.E-mail: [email protected]



P8-1

LABORATORIJSKI INFORMACIJSKISUSTAV U IZGRADNJI

Plasaj T.1, Lukinac Lj.2, Nöthig-Hus D.2, Kusi} Z.2

1 Odjel za informati~ku obradu podataka, 2 Klinika zaonkologiju i nuklearnu medicinu, KB “Sestre milosrdnice”,

Zagreb

Laboratorijski informacijski sustav (LIS) uve-den je u Odsjeku za dijagnostiku bolesti štitnja~eKlinike za onkologiju i nuklearnu medicinu KB“Sestre milosrdnice” još 1989. godine. Postoje}iLIS napisan je u DOS-u, ali je prilago|en za radu okruènju MS Windows. Sastoji se iz dva os-novna dijela koji se nadopunjuju i koji su jošuvijek u fazi izgradnje. Prvi dio (PROGRAM 1.)omogu}uje upis osnovnih podataka bolesnika,odabir terapije (lijek, doza) i pretraga koje trebaizvršiti, upis vrijednosti rezultata izvršenih pre-traga, a omogu}uje i upit o svim upisanim ispi-tanicima (ispis svih posjeta s rezultatima izvrše-nih pretraga), pisanje radnih lista i naljepnica teizdavanje nalaza. Unutar PROGRAMA 1. postojipodprogram POSEBNI PARAMETRI kojim jemogu}e dopuniti podatke o odre|enom ispitaniku(lipemi~ni serum, steroidi, hepatitis C, trudno}a/tromjese~je). Drugi dio (PROGRAM 2.) daje razli-~ite vrste izvještaja o napravljenim pretragama,npr. rezultate odabranih skupina ispitanika (abe-cednim redom ili po vremenskom redoslijedu)prema napomeni (ordinarius, dijaliza), vrstamapretraga te izboru, npr. samo povišenih vrijed-nosti tiroksina (H T4), ispis rezultata svih posjetaza odre|enog bolesnika, kao i statisti~ku obradu(referentne vrijednosti, godišnji broj bolesnika shipertireozom - ispis po mjesecima). Poseban dioodnosi se na dijagnosti~ki modul (Neuronskamreà/Assistant algoritam) koji na temelju labo-ratorijskog algoritma predlaè hipotetsku kli-ni~ku dijagnozu. Privremenu datoteku izabranihpodataka mogu}e je obraditi u Excelu (MS Of-fice), što omogu}uje daljnju obradu u nekomdrugom statisti~kom programu, npr. Med Calcu.Baza podataka sadrì ukupno 328 883 zapisa orezultatima 28 pretraga za 63 140 ispitanika (123658 posjeta). Ovaj LIS, putem kojega je rad ulaboratoriju znatno unaprije|en, i dalje je otvo-ren za dopunu novim parametrima.E-mail: [email protected]

P8-1

LABORATORY INFORMATION SYSTEMUNDER DEVELOPMENT

T. Plasaj1, Lj. Lukinac2, D. Nöthig-Hus2, Z. Kusi}2

1 Department of Data Processing, 2 University Department ofOncology and Nuclear Medicine, Sestre milosrdnice

University Hospital, Zagreb

At the University Department Oncology andNuclear Medicine, Sestre milosrdnice UniversityHospital in Zagreb the first Laboratory Informa-tion System (LIS) was installed in 1989 and sincethen it has undergone constant improvement. Ba-sically, it was designed for the DOS environmentbut it was also adapted for use in MS Windows.This LIS consists of two interconnected parts,PROGRAM 1 and PROGRAM 2. PROGRAM 1allows the following: input of patient data,therapy data, test results, patient information(date of visits, results, therapy, special com-ments), printing of worksheets, labels and patientreports. A special subprogram (SPECIFIC PA-RAMETERS) additionally describes the types ofserum (lipemia, hepatitis C), taking of the ster-oids or presence of pregnancy. PROGRAM 2 givesoutput data, e.g., selected reports by names ordates of specific test results, selection of patientsby certain comments (pregnancy/trimesters,hyperthyroid patients or patients diagnosed asthyroid cancer patients), or selection of patientsaccording to type of therapy, diagnosis or in-creased/decreased test results. In addition, sta-tistical analysis of data can be done (referenceranges, total number of patients with increasedTSH level per month). A special part is a diagnos-tic module (Neuron Net/Assistant algorithm),which can provide a hypothetical clinical diagno-sis, e.g., hypothyroidism, on the basis of test re-sults. The temporarily selected database could bealso processed by Excel (MS Office) and other sta-tistical programs such as Med Calc. The databasecontains a total of 328 883 data (63 140 patients,123 658 visits) for 28 different tests. This LIS isstill open for further graduation with other usefulparameters.E-mail: [email protected]

P8Laboratorijski informati~ki sustavi

Laboratory information systems



P8-2

DOSTAVA NALAZA ELEKTRONI^KOMPOŠTOM VANJSKIM KORISNICIMA

KLINI^KOG LABORATORIJA

Brki} K.1, Vitunjski-Englert B.2, Lovre~ek J.2,Petrove~ki M.2,3

1 Odjel za kardijalnu kirurgiju, 2 Zavod za laboratorijskudijagnostiku, KB “Dubrava”, Zagreb; 3 Katedra za

informatiku, Medicinski fakultet Sveu~ili{ta u Rijeci, Rijeka

Uporaba elektroni~ke pošte jednostavan je,brz i rasprostranjen na~in priop}avanja danas,pa je stoga logi~na ~injenica da je elektroni~kapošta pronašla primjenu i u medicini. Tehnolo-giju elektroni~ke pošte mogu}e je primijeniti ukomunikaciji izme|u lije~nika i bolesnika, lije~-nika i laboratorija, te izme|u laboratorija i nji-hovih korisnika. Cilj studije bio je procijenitiglavne postavke za brzu i pouzdanu dostavu la-boratorijskih nalaza elektroni~kom poštom vanj-skim korisnicima laboratorija, te uvesti ovu us-lugu u svakodnevni rutinski rad Zavoda za labo-ratorijsku dijagnostiku Klini~ke bolnice “Dubra-va”. Istraìvanje je provedeno od travnja 2002. dooùjka 2003. godine. Obuhva}ene su dvije skupi-ne ispitanika: skupina A (n=161) kojima su rezul-tati laboratorijskih pretraga poslani pomo}uelektroni~ke pošte i skupina B (n=568) koji surabili usluge laboratorija tijekom jednog nasum-ce odabranog mjeseca, kako bi se ispitale zna-~ajke (dob, spol, vrst osiguranja) prosje~ne popu-lacije koja dolazi u laboratorij. Za oblikovanjeelektroni~ke ina~ice laboratorijskog nalaza u za-šti}eni pdf-oblik primijenjena je programska pot-pora Acrobat Reader tvrtke AdobeTM. Tijekomjedne godine usluge laboratorija rabilo je 8786vanjskih korisnika, a ukupno je elektroni~kompoštom poslan 161 (1.8%) laboratorijski nalaz.Skupina A bila je statisti~ki zna~ajno mla|a odskupine B (medijani 38 i 59 godina; p<0,001).Skupina A imala je 74% radno osiguranih osoba,za razliku od skupine B gdje je 56% ispitanikaspadalo u ovu kategoriju osiguranja (p<0,001).Zaklju~eno je kako je razra|eni sustav dostaverezultata laboratorijskih pretraga pomo}u elek-troni~ke pošte jednostavan i pouzdan, a omo-gu}uje brù i ekonomski povoljniju uslugu ko-risnicima laboratorija. Svaki laboratorij moèvrlo jednostavno ovu dopunsku uslugu uklopiti uizbor svojih usluga, ~ak i pomo}u tek jednogra~unala koje ima pristup na Internet.E-mail: [email protected]

P8-2

DELIVERING CLINICAL LABORATORYRESULTS BY E-MAIL TO OUTPATIENT

USERS

K. Brki}1, B. Vitunjski-Englert2, J. Lovre~ek2, M.Petrove~ki2,3

1 Department of Cardiac Surgery, 2 Institute of LaboratoryDiagnosis, Dubrava University Hospital, Zagreb;

3 Computerization Department, Rijeka University School ofMedicine, Rijeka

Since using e-mail communication is a simple,fast and frequently accessible feature, it is under-standable that it has now found application alsoin health industry. E-mail technology can be ap-plied to communication between physicians andpatients, physicians and laboratory, and labora-tory and their consumers. The aim of the studywas to evaluate the main propositions for fastand reliable delivery of laboratory results by e-mail to all outpatient users and to apply it indaily practice at the Department of LaboratoryDiagnosis, Dubrava University Hospital. Thestudy was carried out from April 2002 till March2003. There were two groups of subjects: group A(n=161) to whom laboratory results were sent bye-mail, and group B (n=161) who used laboratoryservices through one randomly selected month,with the purpose to describe the average popula-tion (age, sex and health insurance) usually at-tending the laboratory. Acrobat Reader AdobeTMwas used to prepare a safe electronic version oflaboratory report. During the one-year period,there were 8786 outpatient users and 161 (1.8%)laboratory results were sent using e-mail. GroupA was significantly younger than group B (me-dian 38 and 59 years, respectively) (p<0.001). Ingroup A, 74% of subjects were employed, in con-trast to 56% in group B (p<0.001). The elaboratedsystem for laboratory result delivery by e-mail issimple and reliable, offering faster and less ex-pensive service to laboratory consumers. Anyclinical laboratory could incorporate this addi-tional service into their package of services, evenwith only one computer with Internet access.E-mail: [email protected]



P8-3

STRUKTURA I PRETPOSTAVKEELEKTRONI^KOG POSLOVANJA U

KLINI^KOM LABORATORIJU

Šrenger V., Rogi} D., Stavljeni}-Rukavina A.

Klini~ki zavod za laboratorijsku dijagnostiku Medicinskogfakulteta Sveu~ilišta u Zagrebu i Klini~kog bolni~kog centra


Uporaba Interneta i elektroni~kog poslovanjaklini~kom laboratoriju donose kudikamo ve}uinteraktivnost, povezanost i brzinu u usporedbi stradicionalnim, ali zna~e i radikalnu promjenu.Elektroni~ko poslovanje prije svega je organi-zacijsko, a tek potom tehnološko pitanje. Organi-zacija poslovanja mora biti utemeljena na infor-macijskoj tehnologiji (IT). Me|utim, djelokrugIT-a u klini~kom laboratoriju nije samo automa-tizacija postoje}ih procesa rada i prikupljanje in-formacija, nego rješavanje problema, pruànjeusluga te integracija svih procesa rada. Klini~kilaboratorij primjenom interaktivnog elektroni~-kog poslovanja postaje virtualna organizacija(mre`na organizacija). Da bi se to postiglo, trebaprovesti slijede}e: informatizaciju svih internihprocesa, reinènjering poslovanja, poslovanje us-mjereno prema krajnjim korisnicima usluga, tepotpunu integraciju elektroni~kog poslovanjaprema bolnici u cjelini. Elektroni~ko se poslova-nje dugo vremena temeljilo na konceptu EDI(Electronic Data Interchange), no danas se rabiInternet (Web i e-pošta) kao komunikacijska in-frastruktura poslovanja i povezivanja svih sudio-nika u procesu rada. Tradicionalni programi suviše usmjereni ka poboljšanju unutarnje u~inko-vitosti i internih poslovnih procesa, dok rješenjakoja rabi Internet nude jeftinije i brè poveziva-nje s ve}im brojem sudionika poslovnoga procesa.Razmjena va`nih informacija doga|a se u real-nom vremenu. Danas se mogu rabiti i ve} gotoviposlovni operativni sustavi za planiranje resursapoduze}a (ERP Systems - Enterprise ResourcePlanning Systems). Jedan od njih je i SAP (Sys-tem, Applications and Products in Data Process-ing), najrašireniji svjetski sustav ERP. SAP jeprogram koji sadrì integrirani skup informacij-skih rješenja za stvaranje integriranog poslovnoginformacijskog sustava, a mogao bi se primijenitii kao osnova laboratorijskog informacijskog su-stava.E-mail: [email protected]

P8-3

ARRANGEMENT AND CONDITIONS OFELECTRONIC BUSINESS IN CLINICAL

LABORATORY

V. Šrenger, D. Rogi}, A. Stavljeni}-Rukavina


The use of Internet and electronic business inthe clinical laboratory brings along immenselyincreased interactivity, interconnection and ra-pidity compared to traditional operations, butalso implicates radical change. Electronic busi-ness is primarily organizational, and only then atechnologic issue. Organization of business opera-tions must be based on information technology(IT). However, the scope of IT in clinical labora-tory involves not only the automation of currentlyapplied work processes and gathering of informa-tion but also solving problems, provision of serv-ices, and integration of all work processes. By theapplication of interactive electronic business,clinical laboratory becomes a virtual organiza-tion (network organization). To achieve thismodel, the following tasks should be imple-mented: computerization of all internal processes,re-engineering of operations, operations orientedtowards service end-users, and complete integra-tion of electronic operations directed towards thehospital as a whole. Electronic business used tobe based for a long period on the EDI (ElectronicData Interchange) concept that has currentlybeen superseded by Internet (Web and e-mail) as acommunication infrastructure in operations andintegration of all participants in the work proc-ess. Traditional software is being increasinglyoriented towards improving internal efficacy andinternal processes, while solutions that utilizeInternet offer inexpensive and rapid connection toa large number of participants in the businessprocess. Exchange of important information takesplace in real time. Presently, completed businessoperational systems can be used to plan companyresources (ERP Systems - Enterprise ResourcePlanning Systems). SAP (System, Applicationsand Products in Data Processing), which is one ofthem, is the most widespread ERP system world-wide. SAP is a software containing an integratedset of informatic solutions for setting up of inte-grated business information system that couldalso be applied as a basis for laboratory informa-tion system.E-mail: [email protected]



P8-4

LABORATORIJSKI INFORMATI^KISUSTAV

Štefanovi} M.1, Topi} E.1, Tešija A.1, Vukeli} N.1,Polenus S.2, Markovi} I.2, Šimi} G.A.2, Polenus A.2

1 Klini~ki zavod za kemiju, KB “Sestre milosrdnice”,2 Samson informatika d.o.o., Zagreb

Suradnjom stru~njaka Zavoda i tvrtke Sam-son informatika razvijen je informati~ki pro-gramski paket koji je omogu}io informatizacijulaboratorija, uspostavu mre`nog sustava unutarlaboratorija, elektroni~ku vezu izme|u admini-strativnog i analiti~kog rada laboratorija, pove-zivanje laboratorijskih i klini~kih odjela za elek-troni~ki prijenos podataka te prijenos podataka uobra~unsku slu`bu bolnice. Ovaj laboratorijskiinformati~ki sustav (LIS) je višestruko osiguranpri kontroli upisa, rada analizatora i ispisa na-laza te pohrani podataka. Biološki uzorak bole-snika nakon ulaska u laboratorij dobiva naljep-nicu s bar kodom, tj. magnetskim zapisom kojinosi u sebi sve relevantne podatke o bolesniku,kao i o zahtjevu za laboratorijskom obradom.Uzorak s bar kodom distribuira se na radilištalaboratorija i uklju~uje u analiti~ke sustave kojiautomatski izra|uju analize zapisane u bar kodu.Završen nalaz se nakon autorizacije od stranelaboratorijskog stru~njaka elektroni~ki šalje naklini~ke odjele. Veza izme|u LIS i klini~kih od-jela ostvarena je putem opti~kog mre`nog su-stava na razini bolnice. Na klini~kim odjelimabolnice dodatni informati~ki program nazvanPreglednik nalaza omogu}ava uvid u nalaz bole-snika koji je upravo završen, kao i pregled pri-jašnjih nalaza istoga bolesnika. Na tom Pre-gledniku lako se moè u}i u Katalog pretraga kojisadrì saèt pregled svih laboratorijskih pretragakoje se mogu izraditi u Zavodu tijekom redovitogradnog vremena, odnosno za potrebe hitnih bo-lesnika tijekom 24 sata, te pruìti lije~niku po-datak o klini~koj zna~ajnosti kao i o cijeni pre-trage. Program tako|er daje uvid u poslovanje iprocjenu kvalitete rada Zavoda, a zapis u dato-teci ostaje trajan tijekom vremena predvi|enogzakonom. LIS je tako pripremljen da se jedno-stavno moè uklopiti u bolni~ki informati~ki su-stav. Uz LIS unutar bolnice, Zavod putem elek-troni~ke pošte ([email protected]) ostva-ruje izravnu komunikaciju izme|u stru~njakaZavoda i pu~anstva odgovaraju}i na sve pojedi-na~ne upite.

P8-4

LABORATORY INFORMATION SYSTEMS

M. Štefanovi}1, E. Topi}1, A. Tešija1, N. Vukeli}1, S.Polenus2, I. Markovi}2, G.A. Šimi}2, A. Polenus2

1 Clinical Institute of Chemistry, Sestre milosrdniceUniversity Hospital, 2 Samson informatika Co., Zagreb

A computer program package has been devel-oped by collaboration of Institute professionalsand Samson informatika Co., which has enabledlaboratory computerization, establishment of thenetwork system within the laboratory, electronicconnection between the administrative and ana-lytical laboratory activities, connection of labora-tory and clinical departments to the electronicdata transfer, and data transfer to the Hospitalaccounting department. This Laboratory Infor-mation System (LIS) has been provided with mul-tiple protection through control of data entry,analyzer performance, report printing, and datastorage. On entering the laboratory, the patient’sbiological sample is allocated a bar code, i.e.magnetic record containing all relevant data onthe patient and test requests. The sample pro-vided with the bar code is distributed to the labo-ratory work sites and included in analytical sys-tems for the tests stated in the bar code to be auto-matically performed. Upon authorization by theresponsible laboratory professional, the completefinding is electronically forwarded to clinical de-partments. The connection between LIS and clini-cal departments is realized through the fiberopticnetwork system at the Hospital level. At clinicaldepartments of the Hospital, an additional soft-ware entitled Laboratory Finding Browser allowsfor an insight into the current patient’s findingsas well as in his/her previous findings. TheBrowser allows for access to the Test Catalog,which provides a brief survey of all laboratorytests available at the Institute during office hoursor 24 hours for emergency cases, also informingthe physician on the clinical relevance and cost ofa particular test. The program also offers infor-mation on the Institute business issues and per-formance quality assessment, the records beingstored for a time period warranted by legal provi-sions. LIS has been so designed as to be easily in-corporated in the Hospital computer system. Inaddition to in-house LIS, the Institute has devel-oped direct communication between Institute pro-fessionals and the population at large, answeringall individual queries by e-mail ([email protected]).



P9-1

POLIMORFIZAMMETILENTETRAHIDROFOLAT

REDUKTAZE KAO ÎMBENIK RIZIKA ZARAK GLAVE I VRATA

Begonja A., Topi} E., Štefanovi} M., Šimundi} A.M.


Metilentetrahidrofolat reduktaza (MTHFR)katalizira konverziju 5, 10-metilentetrahidrofo-lata, koji se rabi u sintezi purina i timidilata, u 5-metiltetrahidrofolat, koji je neophodan za sintezumetionina. U~estali genetski polimorfizamC677T MTHFR-a (uzrokuje promjenu alanina uvalin u 225. kodonu) smanjuje aktivnost enzimaMTHFR, ~ime se povisuje razina 5, 10-metilen-tetrahidrofolata. Ovo moè štititi stanice od po-grešne inkorporacije uridilata, te ošte}enja DNKzbog pove}ane raspoloìvosti timidilata. Cilj ovo-ga rada bio je ispitati polimorfizam MTHFR kaogenetski ~imbenik rizika za rak glave i vrata.Mogu}i utjecaj polimorfizma MTHFR C677T ispi-tivan je u studiji parova u koju je bilo uklju~eno120 zdravih ispitanika, te 82 bolesnika s rakomglave i vrata. Polimorfizam MTHFR utvr|en jepomo}u PCR-RFLP. U skupini bolesnika muškogspola na|ena je zna~ajno manja frekvencija Talela (27,4% u skupini bolesnika naprama 34,6%u kontrolnoj skupini, p=0,007) s dva puta manjimrizikom za rak glave i vrata (OR = 0,52, 95%CI=0,32-0,80). Kod heterozigotnih nositelja geno-tipa 677CT (28,6% me|u bolesnicima naprama59,3% me|u kontrolama) utvr|en je 3,5 puta ma-nji rizik za rak glave i vrata (OR = 0,28, 95%CI=0,14-0,54, p<0,001) u usporedbi s genotipo-vima CC i TT. Frekvencije genotipova zadovolja-vale su Hardy-Weinbergovu ravnoteù u objemaskupinama. Daljnja ispitivanja na ve}em brojuispitanika neophodna su za procjenu polimor-fizma MTHFR kao ~imbenika rizika za nastanakraka glave i vrata.E-mail: [email protected]

P9-1

METHYLENETETRAHYDROFOLATEREDUCTASE POLYMORPHISM AND RISK

OF HEAD AND NECK CANCER

A. Begonja, E. Topi}, M. Štefanovi}, A.M. Šimundi}


Methylenetetrahydrofolate reductase (MTHFR)catalyzes the conversion of 5, 10-methylenetetra-hydrofolate, utilized for purine and thymidylatesynthesis, to 5-methyltetrahydrofolate, necessaryfor methionine synthesis. A common C677T poly-morphism in MTHFR gene (coverts an alanine toa valine in codone 225) reduces the activity of theMTHFR enzyme and increases the levels of 5, 10-methylenetetrahydrofolate. This may protect cellsfrom DNA damage due to uridylate misincorpo-ration because of the availability of thymidylate.The aim of this study was to investigate MTHFRpolymorphism as a genetic risk factor for headand neck cancer. The possible influence of theMTHFR C677T polymorphism was studied in acase-control study of 120 healthy subjects and 82patients with head and neck cancer. MTHFRgenotypes were determined by PCR-RFLP. A sig-nificant decrease of T allele was observed in themale subgroup (27.4% in cases vs. 34.6% in con-trols, p=0.007) with a 2-fold reduction in the riskof head and neck cancer (OR=0.52, 95% CI=0.32-0.80). Men with heterozygous 677CT genotype(28.6% in cases vs. 59.3% in controls) had a 3.5-fold decreased risk of head and neck cancer(OR=0.28, 95% CI = 0.14-0.54, p<0.001) com-pared to CC and TT genotypes. Genotype frequen-cies were in Hardy-Weinberg equilibrium in bothgroups. Further studies in a larger number ofsubjects are needed to investigate the role ofMTHFR C677T polymorphism as a cancer riskfactor.E-mail: [email protected]

P9Razno

Miscellanea



P9-2

LABORATORIJSKI POKAZATELJI UINFEKCIJI NEMATODOM TRICHINELLA

SPIRALIS

Šimek S., Pirija M., Honovi} L.

Medicinsko biokemijski laboratorij, Op}a bolnica Pula, Pula

Trihineloza je bolest uzrokovana nematodomTrichinella spiralis. Najva`niju etiološku uloguza razvoj trihineloze u ~ovjeka ima trihineloznosvinjsko meso (osobito šunka i kobasice) koje jenedovoljno sušeno, smrznuto ili pe~eno. Bolest seobi~no javlja sporadi~no ili u manjim epidemi-jama. Jedna takva epidemija javila se u dijelustanovništva srednje i sjeverne Istre (okolica Po-re~a, Pazina i Buzeta) u listopadu 2002. godine.Velika raznolikost simptoma u po~etku bolesti(gastointestinalni simptomi, bol u miši}ima, ede-mi u licu, klonulost...) bolest ~ine lako zamje-njivom, te je prije svega nu`na to~na anamnezauz hematološke, biokemijske i serološke pretra-ge. U razdoblju od 15. listopada do 5. studenoga2002. godine na Odjel za infektivne bolesti OBPula zaprimljeno je 54 bolesnika, od kojih za 46(19 @ i 27 M) postoje potpuni klini~ki podaci ozarazi trihinelom. Bolesnici su bili prosje~ne dobiod 40 godina, a u bolnici su zadr`ani u prosjeku15 dana. Nakon prijma u bolnicu svim bolesni-cima je odre|ena KKS (hematološki broja~ CellDyn 3500) i neki od biokemijskih parametara(CK, LDH, AST, ALT, GGT, ureja, KREAT-bioke-mijski analizator Hitachi 717). Nakon prijma ubolnicu 26 (56,52%) bolesnika je imalo leuko-citozu (L sv=15,4x109/L). Vrijednosti eozinofilakod svih su bolesnika bile višestruko povišene(Eoz sv=27,07%). Povišene vrijednosti pokazalesu i slijede}i biokemijski parametri: CK sv=454U/l, AST sv=45 U/l, ALT sv=47 U/l te LDH sv=646U/l. Primjenom terapije (Vermox) i poboljšavanjaop}eg zdravstvenog stanja vrijednosti leukocita ieozinofila su se snizile (L sv=9,59x109/L, Eozsv=16,67%). Dobiveni laboratorijski nalazi poka-zali su dobru korelaciju vrijednosti eozinofila ikreatin kinaze (r=0,903). Zaklju~eno je kako to~-ni anamnesti~ki podaci uz DKS u kojoj se osobitoisti~e eozinofilija, te povišene vrijednosti CKpredstavljaju pouzdane laboratorijske pokazate-lje infekcije nematodom Trichinella spiralis, oso-bito u drugoj, tkivnoj fazi bolesti.E-mail: [email protected]

P9-2

LABORATORY PARAMETERS INTRICHINELLA SPIRALIS INFECTION

S. Šimek, M. Pirija, L. Honovi}

Laboratory of Medical Biochemistry, Pula General Hospital,Pula

Trichinosis is a disease caused by the nema-tode Trichinella spiralis. The most importantetiologic factor for the development of trichinosisis infected and inadequately prepared pork (espe-cially ham and sausages). The disease usually oc-curs sporadically or in smaller epidemics. Suchan epidemic struck a part of the population incentral and north Istria (Pore~, Pazin and Buzet)in October 2002. At the beginning of the diseasevariable symptoms (gastrointestinal, musclepain, facial edema, collapse) make trichinosiseasily mistaken for some other diseases. For thesereasons it is necessary to take thorough historyand perform accurate hematologic, biochemicaland serologic testing. During the period from Oc-tober 15 till November 15, 2002, 54 patients wereadmitted to the Department of Infectious Dis-eases, Pula General Hospital, 46 (19F and 27 M)of them with complete clinical data indicatingtrichinella infection. The mean age of patientswas 40 years, and their mean hospital stay 15days. On admission to the hospital, completeblood count (CBC; Cell Dyn 3500) and some bio-chemical tests (Hitachi 717) were performed in allpatients. Initial tests showed 26 patients to haveleukocytosis (Lav=15.4x109/L). The values ofeosinophils were significantly increased in all pa-tients (Eos av=27.07%). Other biochemical pa-rameters were also increased (CKav=454 U/l,ASTav=45 U/l, ALTav=47 U/l and LDHav=464U/l). Upon therapy initiation (Vermox) and im-provement of the patient general health condition,the values of leukocytes and eosinophils de-creased (Lav=9.59x109/L and Eosav=16.67%).Laboratory results showed good correlation be-tween the values of CK and eosinophils (r=0.903).Accordingly, thorough history data together withleukocyte differentials, particularly eosinophiliaand increased values of CK, present reliable toolsfor diagnosing infection with Trichinella spiralis.E-mail: [email protected]



P9-3

KLORIRANI UGLJIKOVODICI UMAJÎNOM MLIJEKU NA PODRU^JUPRIMORSKO-GORANSKE @UPANIJE

Keki} M., @ivkovi} A., Me|ugorac B.

Zavod za javno zdravstvo Primorsko-goranske ùpanije,Rijeka

Maj~ino mlijeko je zbog svog jedinstvenog sa-stava optimalan izbor prehrane za novoro|en~e.Naàlost, maj~ino mlijeko sadrì i mnoge one~iš-}iva~e, me|u kojima su organoklorirani pesticidi,poliklorirani bifenili (PCB) i njihovi kongeneri. Naj-va`niji razlog za njihovu analizu je lipofilna naravovih spojeva, zbog koje su oni prisutni u tkivima iteku}inama bogatim mastima. Uzorci maj~inogmlijeka sakupljeni su u KBC Rijeka, Klinika zaginekologiju i porodiljstvo, tijekom 2001. godine.Uzorci maj~inog mlijeka sakupljeni su u sterilneposudice u koli~ini od 40-50 ml. Za odre|ivanjekloriranih pesticida, polikloriranih bifenila i nji-hovih kongenera primijenjena je metoda plinskekromatografije (AOAC 970.52). Radni uvjeti: Tk185 oC, 1 min; 5 oC/min - 230 oC, 15 min; Ti 250 oCi Td 350 oC; plin nositelj bio je dušik, 3,5 ml/min;“make-up” plin dušik, 24 ml/min, splitless. Ko-li~ina uzorka uba~ena u kromatograf bila je 1 µl.Za kromatografiranje je upotrebljena kolonaRTxR-35, 30 m, 0,32 mm ID, 0,25 µm df Restek.Za identifikaciju i kvantifikaciju rabljeni su iz-vorni standardi: Standard Pesticide MIX AB-Res-tek PCB congener standard #1 i kit Aroclors,Supelco. DDT i njegovi metaboliti, kao i ve}inadrugih organokloriranih pesticida na|eni su u75% analiziranih uzoraka (p,p’-DDE prosje~no78,7 ng/g mlije~ne masti i p,p’-DDT prosje~no26,7 ng/g mlije~ne masti). PCB su identificirani usvim analiziranim uzorcima (prosje~no 369 ng/gmlije~ne masti). Zna~ajna korelacija je na|ena iz-me|u maj~ine dobi i koli~ine DDT-a i PCB-a.Temeljem eksperimentalnih rezultata zaklju~uje-mo da postoji trend opadanja koncentracije or-ganokloriranih pesticida, dok razine PCB-a osta-ju nepromijenjene.

P9-3

HUMAN MILK AND ORGANOCHLORINEHYDROCARBONS IN THE PRIMORSKO-

GORANSKA COUNTY

M. Keki}, A. @ivkovi}, B. Me|ugorac

Public Health Institute of the Primorsko-Goranska County,Rijeka

Human milk is the best food for a newborn forits unique composition. Unfortunately, humanmilk contains many harmful compounds such asorganochlorine pesticides, polychlorinated biphe-nyls (PCB) and their congeners. The most impor-tant reason for their analysis in human milk isthe lipophilic character of these compounds thatmakes them present in highest concentrations intissues and liquids rich in fat. Human milk sam-ples were manually collected into sterilized bot-tles at a quantity of 40-50 ml. The AOAC 970.52method was used to determine organochlorine hy-drocarbons on a Varian 3400 CX gas chromato-graph equipped with 63Ni ECD. Operating con-ditions were: Tc 185 oC for 1 min, 5 oC - 230 oC, 15min, Ti 250 oC and Td 350 oC. The carrier gaswas nitrogen, 3.5 ml/min, and make-up gas ni-trogen 24 ml/min. The splitless injection modewas used. The injected volume was 1 µl and thecolumn was RTxR-35, 30 m 0.32 mm ID, 0.25 µmdf, Restek. The standards Pesticide MIX AB-Restek, PCB congener standard #1 and KitAroclor from Supelco were used. DDT isomerswere detected as major chlorine pesticides in 75%of all samples (p,p’-DDE average 78.7 ng/g milkfat and p,p’-DDT average 26.7 ng/g milk fat).PCBs were detected in all samples (average 369ng/g milk fat) as well as PCB congener 138 (aver-age 34.6 ng/g milk fat). Significant correlationswere found between maternal age and DDT andPCBs. It is concluded that there is a decreasingtrend in the content of organochlorine pesticides,while PCBs remain unchanged.

P9-4

UÎNAK AKTIVNOSTI LIPOPROTEINLIPAZE U SERUMU NA HDL I LDL

Qujeq D., Fariver A.

Babol University of Medical Sciences, Babol, Iran

Lipoprotein lipaza je enzim s aktivnoš}u gli-ceril ester hidrolaze, koji se nalazi na endotelnoj

P9-4

EFFECTS OF SERUM LIPOPROTEINLIPASE ACTIVITY ON HDL AND LDL

D. Qujeq, A. Fariver

Babol University of Medical Sciences, Babol, Iran

Lipoprotein lipase is an enzyme with glycerylester hydrolase activity, which is located on the



površini izvanjetrenih kapilara. Ovaj se enzimpomo}u heparina otpušta u krvotok, gdje se moèmjeriti njegova aktivnost. Njegova fiziološkafunkcija je hidroliza hilomikrona i VLDL trigli-cerida, koji se prenose u tkivo kao slobodnemasne kiseline. Smatra se kako je niska razinakolesterola lipoproteina visoke gusto}e snaànneovisni rizi~ni ~imbenik u razvoju koronarne bo-lesti srca. Lipoprotein lipaza utje~e na metabo-lizam lipoproteina niske gusto}e i lipoproteina vi-soke gusto}e. Samo je nekoliko izvješ}a o va`-nosti lipoprotein lipaze u serumu. U ovom smoradu ispitivali odnos izme|u aktivnosti lipopro-tein lipaze u serumu i koncentracija lipoproteina.Mu`jaci štakora Sprague-Dawley (280-330 g) dr-àni su u prostoriji s ograni~enim pristupom ukontroliranim uvjetima temperature (23±5 oC) iciklusa svjetla/tame (12:12 h). @ivotinje su imalepristup hrani za glodavce i vodi ad libitum.Uzorci venske periferne krvi (2 ml) uzimani suizme|u 8 i 13 sati, ostavljeni 30 min da se zgru-šaju na sobnoj temperaturi, odmah su centri-fugirani na 2500 o/min kroz 5 min, te je serumodvojen i pohranjen bez odlaganja na -20 oC doanalize lipoproteina u serumu. Lipoprotein lipa-za i koncentracija lipoproteina u serumu odre-|ene su enzimatski homogenom metodom. Re-zultati su pokazali srednju vrijednost aktivnostilipoprotein lipaze u serumu od 36,58±7,29 µmol/ml/min. Tako|er je aktivnost lipoprotein lipaze userumu pokazala pozitivnu korelaciju s lipo-proteinima visoke gusto}e (r=0,62) te negativnukorelaciju s lipoproteinima niske gusto}e (r=-0,39). Naši rezultati ukazuju na to da bi aktiv-nost lipoprotein lipaze mogla biti ~imbenikompromjena u metabolizmu lipoproteina.E-mail: [email protected]

endothelial surface of extrahepatic capillaries.The enzyme is released into the circulation byheparin, where its activity can be determined. Itsphysiological function is the hydrolysis ofchylomicrons and VLDL triglycerides, which aretransported into the tissue as free fatty acids. Alow level of serum high-density lipoprotein cho-lesterol is considered to be a potent independentrisk factor in the development of coronary heartdisease. Lipoprotein lipase affects low-density li-poprotein and high-density lipoprotein metabo-lism. There are only few reports on the impor-tance of lipoprotein lipase in serum. In this workwe investigated the relationship between serumlipoprotein lipase activity and lipoprotein concen-trations. Male Sprague-Dawley rats (280-330 g)were housed in a restricted-access room with con-trolled temperature (23±5 oC) and light-dark cy-cles (12:12 h). Standard rodent food and tap wa-ter were provided ad libitum. Venous peripheralblood samples (2 ml) were obtained from ani-mals. The samples were obtained between 8 a.m.and 1 p.m., and allowed to clot at room tempera-ture for 30 min, were immediately centrifuged at2500 xg for 5 min, and the serum was removedand stored without delay at -20 oC until analysisof serum lipoproteins. Serum lipoprotein lipaseand lipoprotein concentrations were determinedenzymatically by the homogeneous method. Theresults showed the mean serum lipoprotein lipaseactivity to be 36.58±7.29 µmol/ml/min. Also, se-rum lipoprotein lipase activity correlated posi-tively with high-density lipoprotein (r=0.62) andinversely with low-density lipoprotein (r=-0.39).Our results suggest that lipoprotein lipase activ-ity may be a factor in the modulation of lipopro-tein metabolism.E-mail: [email protected]

P9-5

ANTIKARDIOLIPINSKA PROTUTIJELAKAO ÎMBENIK RIZIKA U TRUDNO]I

Posavec Lj., Košec V., Stan~i} V., Ga}ina P., Kopr~inaM., Rai} B., Petek M.

Klini~ka bolnica “Sestre milosrdnice”, Endokrinološkilaboratorij Interne klinike, Zagreb

Cilj rada bio je na temelju ishoda trudno}a iuz primijenjenu terapiju ispitati utjecaj antikar-diolipinskih protutijela (ACL) kao ~imbenika ri-zika u trudno}i. Skupini od 35 trudnica koje suprethodno imale dva spontana poba~aja izmje-rena je koncentracija antikardiolipinskih protuti-

P9-5

ANTICARDIOLIPIN ANTIBODY AS A RISKFACTOR IN PREGNANCY

Lj. Posavec, V. Košec, V. Stan~i}, P. Ga}ina, M.Kopr~ina, B. Rai}, M. Petek

Laboratory of Endocrinology, University Department ofMedicine, Sestre milosrdnice University Hospital, Zagreb

The aim of the study was to analyze pregnancyoutcome in a sample of women with elevated lev-els of anticardiolipin antibodies (ACL) and to as-sess therapeutic efficacy. The concentration ofACL (IgG- and IgM-ACL) was determined byELISA in a group of 35 pregnant women with two



jela (IgG i IgM) enzimimunološkom metodom.Osam (22,8%) trudnica imalo je pozitivne objefrakcije imunoglobulina, deset (28,6%) ih je imalopozitivna samo IgG-ACL, a 22 (62,8%) samo IgM-ACL. Sve trudnice s pozitivnim ACL (n=23) pod-vrgnute su antiagregacijskoj terapiji s niskim do-zama (oko 80 mg Andola na dan). Dvjema trud-nicama dijagnosticiran je antifosfolipidni sin-drom i trudno}e su završile spontanim poba~a-jem. Prijevremeni porod uslijedio je kod dvijetrudnice, dok su ostale trudno}e završile sretnoterminskim porodom. Iz dobivenih rezultata mo-è se zaklju~iti da postojanje ACL pove}ava rizikod spontanog poba~aja ako se pojavljuju u prvomtrimestru trudno}e i u ve}oj koncentraciji. Zbogtoga bi ènama s višestrukim spontanim poba-~ajem trebalo ACL odre|ivati kao test probira.Optimalnim terapijskim protokolom koji uklju-~uje antiagregacijsku terapiju u niskim dozamaili u kombinaciji sa supkutanim heparinom zna-~ajno se poboljšava uspješnost završetka tru-dno}e.E-mail: [email protected]

consecutive spontaneous abortions. Eight (22.8%)women were positive for both Ig fractions, 10(28.6%) were only IgG-ACL positive, and 22(62.8%) were only IgM-ACL positive. All womenwith positive ACL (n=23) were allocated totherapy with low dose aspirin (80 mg daily). Theantiphospholipid syndrome was diagnosed in twocases and these pregnancies ended in spontane-ous abortion. Two women had preterm delivery,and other pregnancies had normal outcome.Thus, ACL appears to increase the risk of abor-tion when present in the first trimester and athigh concentrations. Optimal therapeutic proto-col with low dose of aspirin alone or with subcu-taneous heparin significantly improves thechance of successful pregnancy outcome in pa-tients with ACL.E-mail: [email protected]

P9-6

ANTIOKSIDACIJSKI KAPACITET SERUMAU ODNOSU NA BIOKEMIJSKE

PARAMETRE

Salamuni} I., Katalini} V., Miloš M.

Klini~ka bolnica “Split”, Odjel za klini~ku laboratorijskudijagnostiku, Split

Aerobni organizmi su tijekom evolucije razvilisloèn sustav zaštite kao protuteù mogu}emštetnom djelovanju oksidacijskih procesa. Slo-bodni radikali reagiraju i s makromolekulama(lipidi), a razli~ite kemijske promjene mogu bitiuzrokom funkcijskog ošte}enja stanica, tkiva iorgana. Cilj rada bio je odrediti antioksidacijskikapacitet seruma (FRAP-ferric reducing ability/antioxidant capacity); koli~inu produkata lipidneoksidacije (TBARS-thiobarbituric acid reactivespecies); koli~inu hidroperoksida (FOX-2-ferrousoxidation in xylenol orange) kod “naizgled” zdra-vih muškaraca u odnosu na biokemijske parame-tre aspartat aminotransferazu (AST), alaninaminotransferazu (ALT), gama glutamil transfe-razu (GGT), bilirubin, glukozu, kolesterol itrigliceride. Od 166 uzoraka seruma “naizgled”zdravih muškaraca (skupina A) njih 25% (n=42)su imali sve biokemijske parametre unutar re-ferentnog raspona (skupina B). U skupini A do-bili smo zna~ajnu korelaciju za FRAP (779,4-

P9-6

PARALLEL INVESTIGATION OF SERUMANTIOXIDANT POWER AND

BIOCHEMICAL PARAMETERS

I. Salamuni}, V. Katalini}, M. Miloš

Department of Clinical Laboratory Diagnosis, SplitUniversity Hospital, Split

The potentially harmful reactive oxygen spe-cies are being constantly produced during normalaerobic metabolism and are safely removed by avariety of biological antioxidants. The antioxi-dant power of serum and amount of lipid oxida-tion products were determined in sera of 166 ap-parently healthy males. Serum antioxidant ca-pacity was determined as ferric reducing ability/antioxidant capacity (FRAP assay). The forma-tion of lipid oxidation products was evaluated asthiobarbituric acid reactive species serum test(TBARS). The ferrous oxidation in xylenol orange(FOX-2) assay was used for the measurement oftotal serum hydroperoxides. The following bio-chemical parameters were determined: AST, ALT,GGT, bilirubin, glucose, cholesterol, triglyceridesand hemoglobin. Group A included 166 sera,whereas group B was a subgroup of group A andincluded only sera with biochemical parameterswithin the reference limits (n=42). There was nosignificant correlation between TBARS and bio-



1409,9 µmol/L) i bilirubin (1,7-48,9 µmol/L), p=0,024; FRAP i hemoglobin (119-172 g/L), p=0.0318; FRAP i glukozu (4,3-8,5 mmol/L), p=0,0241; FRAP i trigliceride (0,35-13,59 mmol/L),p=0,0091. U skupini B dobili smo zna~ajnu ko-relaciju za FRAP (779-1313 µmol/L) i bilirubin,p=0,013. Za TBARS u skupini A (1,178±0,32µmol/L) i skupini B (1,26±0,287 µmol/L) nismodobili zna~ajnu korelaciju s biokemijskim para-metrima. U skupinama A i B za razinu hidro-peroksida (FOX-2 1,90-6,94 µmol/L) nismo dobilizna~ajnu korelaciju s biokemijskim parametri-ma. Rezultati ukazuju na povezanost antioksi-dacijskog kapaciteta i biokemijskih parametarakoji djeluju kao antioksidansi u serumu.E-mail: [email protected]

chemical parameters in either of the studygroups. In group B, there was a significant corre-lation between FRAP and bilirubin. Study resultspointed to the association of serum antioxidantcapacity and biochemical parameters acting asserum antioxidants.E-mail: [email protected]

P9-7

PROCJENA ZRELOSTI PLU]A FETUSAODRE\IVANJEM BROJA LAMELARNIH

ÊSTICA U AMNIJSKOJ TEKU]INI

Kralik S., Mayer V., Bernt T., Suchanek E.

Zavod za klini~ku biokemiju i biologiju reprodukcije,Klinika za ènske bolesti i porode, KBC “Zagreb”, Zagreb

Lamelarne ~estice proizvodi tip II. alveolarnihstanica u pove}anim koli~inama s napredova-njem trudno}e. Sastoje se gotovo isklju~ivo odfosfolipida i predstavljaju skladišta surfaktanta.Odre|ivanje broja lamelarnih ~estica daje objek-tivnu procjenu koli~ine surfaktanta u amnijskojteku}ini. Kako je promjer lamelarnih ~estica iz-me|u 1 i 5 µm, njihov se broj moè odrediti po-mo}u trombocitnog kanala u gotovo svakom he-matološkom broja~u stanica. Cilj rada bio jeusporediti rezultate dobivene brojanjem lamelar-nih ~estica s drugim biokemijskim pokazateljimazrelosti fetalnih plu}a u amnijskoj teku}ini kojise rutinski odre|uju u našem Zavodu: omjeromlecitin/sfingomijelin (L/S) i fosfatidil glicerol(PG). U 378 amnijskih teku}ina dobivenih am-niocentezom u trudnica od 18. do 42. tjedna tru-dno}e odre|eni su L/S, PG i broj lamelarnih ~e-stica. L/S i PG su odre|ivani metodom tanko-slojne kromatografije fosfolipida. Broj lamelarnih~estica odre|en je brojanjem trombocita iz 50 µlsvjeè, necentrifugirane amnijske teku}ine po-mo}u hematološkog broja~a “ABX-Micros”. Bio-kemijsku zrelost plu}a fetusa definirali smo kaozreli L/S ili PG, a nezrelost kao nezreli i L/S i PG.Broj lamelarnih ~estica manji od 15000 bio je100% specifi~an za biokemijsku nezrelost fetal-nih plu}a (osjetljivost 89%, pozitivna prediktivna

P9-7

ASSESSMENT OF FETAL LUNGMATURITY BY AMNIOTIC FLUID

LAMELLAR BODY COUNTS

S. Kralik, V. Mayer, T. Bernt, E. Suchanek

Department of Clinical Biochemistry and ReproductionBiology, University Department of Gynecology and

Obstetrics, Zagreb University Hospital Center, Zagreb

Lamellar bodies are produced by type II alveo-lar cells in increasing quantities as gestation ad-vances. They are composed almost entirely ofphospholipids and represent a storage form ofsurfactant. Quantification of lamellar bodies pro-duces an objective estimate of the quantity of sur-factant in amniotic fluid (AF). Because lamellarbody diameter is between 1 and 5 µm, lamellarbody count can be determined using plateletchannel of a commercial cell counter. The aim ofthe study was to compare lamellar body countwith the lecithin/sphingomyelin ratio (L/S) andphosphatidylglycerol (PG) analysis in testing fe-tal lung maturity. Lamellar body count, L/S andPG were assessed in 378 amniotic fluid samplesobtained by amniocentesis from pregnant womenfrom 18th to 42nd week of pregnancy. L/S andPG were determined by thin layer chromatogra-phy of phospholipids. An ABX-Micros hematologyanalyzer was used to determine lamellar bodycounts. A lamellar body count of less than 15000was 100% specific for biochemical lung immatu-rity (sensitivity 89%, positive predictive value(PPV) 100%, negative predictive value (NPV)99%). A lamellar body count of greater than50000 was 100% specific for biochemical lungmaturity with a sensitivity of 96%, PPV of 100%



vrijednost (PPV) 100%, negativna prediktivnavrijednost (NPV) 99%). Broj lamelarnih ~esticave}i od 50000 bio je 100% specifi~an za bioke-mijsku zrelost fetalnih plu}a (osjetljivost 96%,PPV 100%, NPV 60%). Metoda odre|ivanja brojalamelarnih ~estica u svrhu procjene zrelosti fe-talnih plu}a pokazala se pouzdanom, brzom i jef-tinom, pa se moè preporu~iti kao test probiranakon kojega bi se radila tankoslojna kroma-tografija fosfolipida samo ako je broj ovih ~esticave}i od 15000, a manji od 50000.E-mail: [email protected]

and NPV of 60%. Lamellar body count was foundto be a rapid, simple and universally availablemethod with excellent specificity and good sensi-tivity. It could serve as an extremely cost-effectivescreening test for fetal lung maturityE-mail: [email protected]

P9-8

PROBIR NA NOVU AMINOKISELINSKUDEHIDROGENAZU IZ IRANSKOGA TLA

KAO IZVORA MIKROORGANIZAMA

Omidinia E.1, Khathami S.1, Omumi A.R.2,Mirzahosieni H.1, Thaherkhani H.3, Samadi A.4

1 Biochem. Dept. Pasteur Institute of Iran, 2 Biotech. Dept.Pasteur Institute of Iran, 3 Parasitology Dept., Medical

Faculty, Hamadan University and Medical Sciences,Hamadan, Iran, 4 Kirksville College of Osteopathic

Medicine, Kirksville, USA

Provedeno je ispitivanje radi pronalaènja no-ve aminokiselinske dehidrogenaze iz iranskogatla kao izvora mikroorganizama. Primijenjene surazli~ite podloge uz dodatak L-metionina, L-fe-nilalanina, L-lizina ili L-triptofana. Ukupno je684 sojeva izolirano iz 400 uzoraka tla; 37 od ovihproizvo|a~a imalo je aktivnost o NAD ovisneaminokiselinske dehidrogenaze. Izolirani su pro-izvo|a~i fenilalanina i glutamat dehidrogenaze teidentificirani kao Bacillus sp. odnosno Delfeciaacidovorance. Nedavno je izolirana bakterija uzprimjenu neutralne podloge koja je sadràvala L-metionin. Razina proizvodnje enzima kod opisa-nih sojeva kretala se od 40 do 60 U/L. Tako|er jeispitivana uloga specijalne (baffled) posude, kojase pokazala vrlo djelotvornom u proizvodnji en-zima.E-mail: [email protected]

P9-8

SCREENING FOR NEW AMINO ACIDDEHYDROGENASE FROM IRANIAN SOIL

AS A SOURCE OF MICROORGANISMS

E. Omidinia1, S. Khathami1, A.R. Omumi2, H.Mirzahosieni1, H. Thaherkhani3, A. Samadi4

1 Biochem. Dept. Pasteur Institute of Iran, 2 Biotech. Dept.Pasteur Institute of Iran, 3 Parasitology Dept., Medical

Faculty, Hamadan University and Medical Sciences,Hamadan, Iran, 4 Kirksville College of Osteopathic

Medicine, Kirksville, USA

A study was done to find a new amino aciddehydrogenase from Iranian soil as a source ofmicroorganisms. Various media were used with asupplement of either L-methionine, L-phenyla-lanine, L-lysine, or L-tryptophan. A total of 684strains were isolated from 400 soil samples; 37 ofthese producers were found to have an activity ofNAD-dependent amino acid dehydrogenase. Theproducers of phenylalanine and glutamate dehy-drogenase were isolated and identified as Bacil-lus sp. and Delfecia acidovorance, respectively.Recently, a bacterium was isolated using a neu-tral medium containing L-methionine. The levelof enzyme production by the reported strainsranged from 40 to 60 U/L. Also, the role of baf-fled flask was studied, and we found it very effec-tive in enzyme production.E-mail: [email protected]



P9-9

ODRE\IVANJE KONCENTRACIJETROMBOPOETINA I IL-6 U DJECE SREAKTIVNOM TROMBOCITOZOM U

TIJEKU PNEUMONIJE

Dodig S.1, Raos M.1, Kova~ K.1, Nogalo B.1, Benko B.2,Glojnari} I.3, êpelak I.4

1 Specijalna bolnica za bolesti dišnog sustava djece imladeì, 2 Imunolo{ki zavod, 3 Pliva d.d., 4 Farmaceutsko-

biokemijski fakultet, Zagreb

Trombocitoza moè biti primarna - klonalnaili sekundarna - reaktivna kao posljedica brojnihupalnih bolesti, nekih imunoloških, hematološ-kih i zlo}udnih bolesti te kao posljedica primjenenekih lijekova. Osnovna proizvodnja trombocitaovisna je o koncentraciji trombopoetina (TPO).Ovim smo radom èljeli ispitati zašto se samo uneke djece u tijeku pneumonije pojavljuje trom-bocitoza. Ispitano je 68 djece s pneumonijom: 17bolesnika s normalnim brojem trombocita i 51bolesnik s pove}anim brojem trombocita. Labo-ratorijska obrada uklju~ivala je odre|ivanje se-dimentacije eritrocita, broja leukocita i trom-bocita, koncentracije hemoglobina, C-reaktivnogproteina (CRP), interleukina 6 (IL-6) i TPO. Bo-lesnici s normalnim brojem trombocita i normal-nom koncentracijom TPO imali su statisti~kizna~ajno ubrzanu sedimentaciju eritrocita i pove-}anu koncentraciju CRP-a te umjereno, ali nestatisti~ki zna~ajno, pove}anu koncentraciju IL-6. Djeca s trombocitozom imala su statisti~ki zna-~ajno pove}an broj leukocita, pove}anu koncen-traciju TPO, dok je koncentracija CRP-a i IL-6bila pove}ana u nekih bolesnika. U djece s reak-tivnom trombocitozom postojala je anemija. Re-aktivna trombocitoza o~itovala se u tijeku pne-umonije uglavnom u djece s anemijom.E-mail: [email protected]

P9-9

THROMBOPOIETIN AND IL-6 INCHILDREN WITH THROMBOCYTOSIS

DURING PNEUMONIA

S. Dodig1, M. Raos1, K. Kova~1, B. Nogalo1, B. Benko2,I. Glojnari}3, I. êpelak4

1 Special Hospital for Respiratory Diseases in Children andAdolescents, 2 Institute of Immunology, 3 Pliva Co., 4 School

of Pharmacy and Biochemistry, Zagreb

Thrombocytosis can occur as clonal thrombo-cytosis or reactive thrombocytosis during chronicinflammatory or infectious disorder, iron-defi-ciency anemia, acute blood loss, hemolytic stateand due to some drug administration. The base-line platelet production is dependent onthrombopoietin (TPO). The aim of the study wasto find out why thrombocytosis occurs only insome children with pneumonia. Sixty-eight chil-dren with pneumonia were included in the study.Blood samples of 17 patients with normal plateletcount and 51 from patients with thrombocytosiswere used for TPO, interleukin (IL-6), C-reactiveprotein (CRP), erythrocyte sedimentation rate(ESR) and complete blood count determination.CRP and ESR were increased in patients withnormal platelet count. TPO and leukocyte countwere increased, and hemoglobin was decreased inpatients with thrombocytosis. Reactive thrombo-cytosis during pneumonia was found to mainlyoccur in children with anemia.E-mail: [email protected]

P9-10

PRA]ENJE RAZINE CIKLOSPORINA (2SATA NAKON PRIMLJENE DOZE) U

BOLESNIKA S TRANSPLANTIRANIMBUBREGOM

Hrabri} S., ûka Husnjak S., Dvornik Š., Orli} P.

Klini~ki bolni~ki centar “Rijeka”, Rijeka

Neoral je mikroemulzijski pripravak ciklo-sporina (CsA) koji se rabi za odràvanje imu-nosupresije nakon transplantacije bubrega. Pra-}enje koncentracije CsA 2 sata nakon primljene

P9-10

MONITORING RENAL TRANSPLANTPATIENTS BY MEASUREMENT OF 2-HOUR POST DOSE CYCLOSPORINE

LEVELS

S. Hrabri}, S. ûka Husnjak, [. Dvornik, P. Orli}

Rijeka University Hospital Center, Rijeka

Neoral is a cyclosporine (CsA) microemulsionwhich has been used to maintain immunosup-pression after renal transplantation. Monitoringcyclosporine (CsA) concentrations 2 hour after



doze (C2) je nova metoda za procjenu apsorpcije imetabolizma CsA u pojedinog bolesnika. Niskarazina CsA moè rezultirati akutnim odbaciva-njem transplantata, a visoka razina nefrotok-si~noš}u. Cilj studije je bio ustanoviti je li pra-}enje razine C2 bolji pokazatelj u odràvanjutransplantata od dosadašnjeg na~ina pra}enjempo~etne razine C0. Razina CsA mjerena je u pu-noj krvi (EDTA) 17 bolesnika s transplantiranimbubregom (88 uzoraka) u razdoblju od prosinca2001. do travnja 2003. godine. Koncentracija CsAodre|ena je na analizatoru Dimension RxL (DadeBehring) imunokemijskom tehnikom s mono-klonskim protutijelima specifi~nim za CsA bezprethodne obrade uzorka. Rezultati su podijeljeniu tri skupine prema proteklom vremenu od tran-splantacije: prvi mjesec, drugi mjesec, tre}i i višemjeseci nakon transplantacije. U prvoj skupinisrednja vrijednost ±SD C0 je bila 292±148 ng/ml,u drugoj 244±144 ng/ml i u tre}oj 206±64 ng/ml.Srednja vrijednost ±SD C2 kod bolesnika u prvommjesecu nakon transplantacije je bila 1364±547(min 466, max 2563) ng/ml; u drugom mjesecu C2je bila 1191±661 (min 272, max 2954) ng/ml; inakon više od tri mjeseca C2 je bila 1228±417(min 681, max 1997) ng/ml. Srednja vrijednost±SD kreatinina u prvoj skupini je bila 135±45µmol/L, u drugoj skupini 162±82 µmol/L i u tre}oj110±36 µmol/L. Zabiljeèna je epizoda odbaciva-nja u dvoje bolesnika (12%) devetoga i dvadeset-osmoga dana nakon transplantacije. Pra}enjerazine C2 u krvi bolesnika s transplantiranimbubregom je bolji pokazatelj odràvanja tran-splantata od pra}enja razine C0, a istodobno seprati terapijski u~inak kako ne bi nastupilotoksi~no djelovanje lijeka zbog previsoke doze.E-mail: [email protected]

drug administration (C2) is a novel method forevaluation of CsA absorption and metabolism inindividual patients. The aim of the study was toassess whether 2-hour post-dose monitoring (C2)is superior to pre-dosage monitoring (C0) of CsAin renal transplant recipients (RTRs). The level ofcyclosporine was determined in EDTA samples(whole blood) in 17 RTRs (88 samples) from De-cember 2001 till April 2003. CsA concentrationwas determined on an RxL Dimension analyzer(Dade Behring) using the immunoassay methodwith specific monoclonal antibody withoutmanual sample pretreatment. Results were di-vided into three groups according to the timeelapsed from transplantation: one month, twomonths, and three or more months post-trans-plantation. The mean ±SD C0 was 292±148,244±144 and 206±64 ng/ml in the one-month, 2-month and ≥3-month post-transplantation group,respectively. The respective mean ±SD C2 was1364±547 (min 466, max 2563), 1191±661 (min272, max 2954) and 1228±417 (min 681, max1997) ng/ml. The mean ±SD creatinine was135±45, 162±82 and 110±36 µmol/L in the one-month, 2-month and ≥3-month post-transplanta-tion group, respectively. Two (12%) patients expe-rienced rejection crisis on day 9 and 28 from kid-ney transplantation. Monitoring of C2 level inRTRs is a better indicator of transplant mainte-nance than monitoring of C0 level, whereas CsAconcentration is useful in assessing drug toxicitydue to high dosage.E-mail: [email protected]

P9-11

BIOKEMIJSKI I HEMATOLO[KIPARAMETRI U BOLESNIKA S

HEMORAGIJSKOM VRU]ICOM SBUBRE@NIM SINDROMOM

Skorvaga S., Garilovi} J., V~elik M.

O@B Poèga, Hematolo{ko-biokemijski laboratorij, Poèga

Obra|eni su nalazi 31 bolesnika (21 muška-rac, 10 èna) u dobi od 18 do 60 godina, koji sulije~eni na Infektivnom odjelu O@B Poèga urazdoblju od 25. oùjka do 10. prosinca 2002.godine. Svi su bolesnici imali pozitivne serološkenalaze (Puumala virus 27 i Dobrava virus 4).

P9-11

BIOCHEMISTRY AND HEMATOLOGYPARAMETERS IN PATIENTS WITH

HEMORRHAGIC FEVER WITH RENALSYNDROME

S. Skorvaga, J. Garilovi}, M. V~elik

Laboratory of Hematology and Biochemistry, CountyGeneral Hospital, Poèga

Laboratory findings from 31 patients (21 Mand 10 F) aged 18-60 years, treated at the Depart-ment of Infectious Diseases, County General Hos-pital in Poèga from March 25 till December 10,2002, were analyzed. All patients had positive se-



Obra|eni bolesnici imali su blag (n=9), srednjeteàk (n=17) i teàk (n=5) klini~ki oblik bolesti.Bolesnici su bili hospitalizirani 5-19 dana (pro-sjek 11 dana), a u bolnicu su se javili izme|u 1. i10. dana bolesti. Analize su ra|ene na hemato-loškom analizatoru Cell Dyn 3500 (Abbott), tebiokemijskom analizatoru Alcyon 300 (Abbott),Vitros 250 (Ortho Clinical Diagnostics). Premavrijednostima trombocita, ureje i kreatinina bo-lest smo podijelili u 4 stadija. U prvom stadijubolesti koji traje 1-2 dana svi laboratorijski na-lazi bili su u okviru referentnih vrijednosti. Udrugom stadiju bolesti koji traje 3-7 dana po~elesu padati vrijednosti trombocita i kretale su se od38 do 136x109/L (r.v. 140-440x109/L). Vrijednostileukocita kretale su se od 2,00 do 14,1x109/L (r.v.5,00-10,00x109/L) uz izraènu neutrofiliju. Vri-jednosti ureje i kreatinina bile su u okviru re-ferentnih vrijednosti. Kod svih bolesnika bila jeprisutna proteinurija. U tre}em stadiju bolestikoji traje 4-9 dana vrijednosti trombocita vratilesu se na normalu, a porasle su vrijednosti urejeod 2,01 do 15,06 mmol/L (r.v. 2,5-6,3 mmol/L) ikreatinina od 61,9 do 558 µmol/L (r.v. @: 44,0-97,0µmol/L, M: 53,0-106 µmol/L). Proteinurija je jošuvijek bila prisutna. U ~etvrtom stadiju bolestilaboratorijski su se nalazi vra}ali na normalu.Vrijednosti SE 1 h kretale su se od 4 do 86 (r.v. @:do 12, M: do 5). Vrijednosti CRP-a kretale su seod 9,3 do 284 (r.v. do 10,00 mg/L). Dakle, odstu-panje vrijednosti trombocita, ureje i kreatininaod referentnih vrijednosti korelira sa stadijembolesti.E-mail: [email protected]

rologic test (Puumala virus in 27 and Dobravavirus in 4 patients). According to symptoms, thepatients were divided into 3 groups: benign (n=9),moderate (n=17) and severe (n=5) form of the dis-ease. The length of hospitalization was 5-19(mean 11) days. Measurements were made in pa-tient sera on a Cell Dyn 3500 (Abbott) counter,Alcyon 300 (Abbott) and Vitros 250 (Ortho Clini-cal Diagnostics) clinical chemistry analyzer. Ac-cording to serum levels of platelets (PLT), bloodurea nitrogen (BUN) and creatinine, the diseasewas classified into four stages. In the first stage,which lasted for 1-2 days, all parameters werewithin the reference range. In the second stage,which lasted for 3-7 days, the values of PLT de-creased to 38-136x109/L (reference range 140-440x109/L), the values of white blood cells (WBC)were 2.00-14.1x109/L (r.r. 5.00-10.00x109/L), andthere was a marked increase in granulocytes. Se-rum values of BUN and creatinine were withinthe reference range, while proteinuria persisted inall patients. In the third stage of the disease,which lasted for 4-9 days, serum values of PLTreturned to the reference range, while the valuesof BUN and creatinine increased, ranging from2.01 to 15.06 mmol/L (r.r. 2.5-6.3 mmol/L) andfrom 61.9 to 558 µmol/L (r.r. F: 44.0-97.0 µmol/L,M: 53.0-106 µmol/L). Patients still had proteinu-ria. In the fourth stage, all parameters returnedto the reference range. The values 1-h ESR were4-86 (r.r. F: <12, M: <5), and those of CRP rangedfrom 9.3 to 284 (r.r. <10.00 mg/L). It is concludedthat deviations in the values of PLT, BUN andcreatinine from reference ranges follow the dis-ease stages.E-mail: [email protected]

P9-12

UTVR\IVANJE SPOLA DJETETA IZ KRVIMAJKE U RANOJ TRUDNO]I

Wagner J.1, Habek D.2, Lauc G.1

1 Medicinski fakultet Osijek, DNK laboratorij, 2 Klini~kabolnica Osijek, Odjel za ginekologiju i porodiljstvo, Osijek

Današnja prenatalna dijagnostika zasniva sena metodama poput amniocenteze i biopsije ko-rionskih resica. Obje metode su zbog svoje in-vazivnosti povezane s rizikom pojave razli~itihkomplikacija kako kod fetusa, tako i kod majke.Zbog toga se u medicinskoj genetici ve} duljevrijeme nastoje razviti sigurnije metode pre-natalne dijagnostike. Svrha ove studije bilo jedokazivanje fetalne DNK te utvr|ivanje spola fe-

P9-12

ANALYSIS OF FETAL GENDER FROMMATERNAL PLASMA IN EARLY

PREGNANCY

J. Wagner1, D. Habek2, G. Lauc1

1 DNA Laboratory, School of Medicine, 2 Department ofGynecology and Obstetrics, Osijek University Hospital, Osijek

At present, prenatal diagnosis requires sam-pling of fetal cells by invasive procedures such asamniocentesis and chorionic villus sampling.These invasive procedures, however, present lowbut definite risks for both the fetus and themother. To avoid this potential risk, it is a long-sought goal of medical genetics to develop safermethods for prenatal diagnosis. The purpose of



tusa iz krvne plazme majke. Metoda kojom bi seprenatalno utvr|ivao spol djeteta mogla bi se pri-mjenjivati kao preliminarni test koji bi prethodioinvazivnim metodama poput amniocenteze i bi-opsije korionskih resica. Test bi osobito bio ko-ristan kod fetusa za koje postoji vjerojatnost X-vezanog recesivnog naslje|ivanja. Slobodna fe-talna DNK i DNK majke izolirana je iz krvneplazme majke primjenom QIAamp DNA Mini ki-ta. O~eva DNK izolirana je iz FTA papiri}a chelexekstrakcijom. Amplifikacija mikrosatelitnih sek-venca (kratka uzastopna ponavljanja) izvršena jemultipleks lan~anom reakcijom polimeraze, afragmenti su razdvojeni kapilarnom elektrofo-rezom. Dokazana je prisutnost fetalne DNK ukrvnoj plazmi majke. Spol djeteta utvr|en jeanalizom amelogeninskog lokusa, preostali am-plificirani fetalni aleli sluìli su kao kontrolauspješnosti umnaànja fetalne DNK. Zaklju~enoje kako se neinvazivno utvr|ivanje spola fetusamoè primijeniti u svrhu izbjegavanja invazivnihprenatalnih dijagnosti~kih metoda (poput amnio-centeze i biopsije korionskih resica) u slu~aje-vima X-vezanog recesivnog naslje|ivanja. Ako sena ovaj na~in utvrdi da je fetus ènskog spola,tada primjena invazivnih prenatalnih dijagno-sti~kih metoda nije potrebna.E-mail: [email protected]

this study was to detect circulating fetal DNA inmaternal plasma and to determine fetal gender.The method for diagnosing fetal gender can beused as a pre-test to determine whether invasiveprenatal diagnosis such as amniocentesis andchorionic villus sampling should be performed ona fetus having a risk of X-linked recessive inherit-ance. Cell-free DNA was isolated from maternalplasma using the QIAamp DNA Mini Kit. Weused multiplex fluorescent PCR amplification ofshort tandem repeats to detect maternal and fetalalleles in the maternal plasma samples. PaternalDNA was isolated from FTA cards using theChelex extraction and amplified by multiplexfluorescent PCR in order to determine fetus-spe-cific alleles in the maternal plasma samples. Us-ing this method we were able to determine fetusspecific alleles in the maternal plasma samples.Fetal gender was determined by analysis of theamelogenin locus, while other amplified lociserved as a control of successful amplification offetal DNA. Accordingly, the noninvasive prenataldiagnosis of fetal gender can be used instead ofinvasive prenatal diagnostic methods such asamniocentesis and chorionic villus sampling incases of X-linked recessive inheritance. If the fetushas a risk of X-linked recessive inheritance and isfound to be female, the use of invasive prenataldiagnosis and associated risks can be avoided.E-mail: [email protected]

P9-13

DIJAGNOSTI^KO ZNAÊNJEODRE\IVANJA MORFOLOGIJE

ERITROCITA U MOKRA]I FAZNOKONTRASTNIM MIKROSKOPOM

Kronja-Negro J.1, Naki} D.2, Val~i} A.1

1 Odjel za laboratorijsku dijagnostiku, 2 Odsjek zanefrologiju i hemodijalizu Odjela za interne bolesti Op}e

bolnice Zadar, Zadar

Odre|ivanje morfologije eritrocita u mokra}ivrijedan je dijagnosti~ki postupak pri klasifika-ciji podrijetla hematurije, te za rano pra}enjebolesnika s izoliranom mikroskopskom hema-turijom nepoznate etiologije. Prema današnjimkriterijima smatra se da je hematurija ’ne-glo-merularnog’ podrijetla ako prevladavaju (>80%)izomorfni eritrociti; ’glomerularnog’ podrijetla uprisutnosti >80% dismorfnih eritrocita ili najma-nje 5% akantocita (G1 stanica); te ’miješana’ akosu oba tipa eritrocita prisutna u pribli`no istomomjeru. Cilj studije bio je odrediti dijagnosti~kozna~enje ove pretrage u diferencijalnoj dijagno-

P9-13

DIAGNOSTIC ROLE OF URINARYERYTHROCYTE MORPHOLOGY

DETERMINATION BY PHASE-CONTRASTMICROSCOPY

J. Kronja-Negro1, D. Naki}2, A. Val~i}1

1 Department of Laboratory Diagnosis, 2 Division ofNephrology and Hemodialysis, Department of Medicine,

Zadar General Hospital, Zadar

The analysis of urinary erythrocyte morphol-ogy is a useful and reliable method for the earlywork-up and follow-up of patients with isolatedmicroscopic hematuria of unknown origin. It isalso a valuable diagnostic procedure for determi-nation of the origin of hematuria. Today,hematuria is classified as ’glomerular’ if there aremore than 80% of dysmorphic erythrocytes, andas ’non-glomerular’ if more than 80% of isomor-phic erythrocytes or at least 5% of all erythrocytesare acanthocytes (G1 cells). Hematuria is definedas ’mixed’ when the two types of cells are presentin a roughly equal percentage. Acanthocytes are a



stici glomerularnih bolesti. U razdoblju od 4.listopada 2002. do 4. lipnja 2003. godine obra-|ene su 92 mokra}e ambulantnih i odjelnih ne-froloških bolesnika. Kao uzorak isklju~ivo se ra-bio svje`, srednji mlaz prve jutarnje mokra}e.Morfologija eritrocita ocjenjuje se fazno kon-trastnim mikroskopom u dvije glavne kategorije:izomorfnu i dismorfnu. Preporuka je da se prvotraè akantociti kao podtip dismorfnih eritrocitaspecifi~nog prstenastog oblika s jednom ili viševezikula, prisutnost kojih strogo indicira hema-turiju glomerularnog podrijetla. Va`no je uo~iti iostale elemente koji mogu biti dijagnosti~ki in-dikativni. Prema prethodno navedenim kriteri-jima glomerularna hematurija prona|ena je u45% ispitanika, dok se kod ostalih mogla dija-gnosticirati miješana hematurija s prevlaš}u dis-morfnih eritrocita. Kod samo jednog ispitanika spotvr|enom dijagnozom hemoliti~ko-uremijskogsindroma utvr|ena je postglomerularna hema-turija. Ispitanici kod kojih je prona|ena glome-rularna hematurija procijenjeni su ili }e se pro-cjenjivati drugim dijagnosti~kim postupcima naprisutnost glomerularne bolesti. Zlatni standardza utvr|ivanje glomerularne bolesti je nalaz bi-opsije bubrega, koji je ujedno jedini relevantanpokazatelj dijagnosti~kog zna~enja odre|ivanjamorfologije mokra}nih eritrocita. Biopsijom bu-brega koja je dosad napravljena kod 33% bole-snika s laboratorijskim nalazima glomerularnehematurije potvr|ena je glomerularna bolest ipostavljena dijagnoza. Odre|ivanje morfologijemokra}nih eritrocita fazno kontrastnim mikro-skopom je neinvazivna, jeftina, jednostavna i po-uzdana metoda u dijagnostici glomerularnih bo-lesti.E-mail: [email protected]

subtype of dysmorphic erythrocytes. The aim ofthe study was to assess diagnostic significance ofthe method in the differential diagnosis ofglomerular diseases. From October 4, 2002 tillJune 4, 2003, 92 urine samples of nephrologic pa-tients were analyzed. The first morning mid-stream urine was examined within 1 h from col-lection. Erythrocyte morphology was evaluated byphase-contrast microscopy. Using the above crite-ria, hematuria of glomerular origin was found in45% of study patients, whereas others had mixedhematuria with a predominance of dysmorphicerythrocytes. Only one patient with a confirmeddiagnosis of hemolytic-uremic syndrome hadhematuria of a nonglomerular origin. The pa-tients with hematuria of glomerular origin havealready been or will be evaluated by other diag-nostic procedures for the presence of glomerulardisease. Kidney biopsy is the gold standard toverify the presence of glomerular disease, and theonly relevant indicator for the diagnostic signifi-cance of urinary erythrocyte morphology determi-nation. To date, kidney biopsy was done in 33% ofpatients with laboratory findings of hematuria ofglomerular origin. The presence of glomerulardisease was confirmed and the diagnosis estab-lished. Determination of urinary erythrocyte mor-phology by phase-contrast microscopy is a simple,noninvasive, inexpensive and reliable method inthe diagnosis of glomerular disease.E-mail: [email protected]

P9-14

KLIRENSI KREATININA (IZRAÛNATI IPROCIJENJENI) - USPOREDBA

REZULTATA

Predovan G., Baljak N., Kronja-Negro J., Val~i} A.

Op}a bolnica Zadar, Odjel za laboratorijsku dijagnostiku,Zadar

Odre|ivanje klirensa kreatinina jedna je odrutinskih laboratorijskih pretraga pri obradi os-teoporoze. U posljednje vrijeme u našem se la-boratoriju znatno pove}ao broj zahtjeva za tupretragu. Kako se radi o preteìto starijoj ènskojpopulaciji, sakupljanje 24-h mokra}e je ponekadvelik problem, pa je naša zamisao bila usporeditirezultate izra~unatih klirensa s rezultatima pro-

P9-14

CREATININE CLEARANCES(CALCULATED AND ESTIMATED) -

RESULT COMPARISON

G. Predovan, N. Baljak, J. Kronja-Negro, Val~i} A.

Department of Laboratory Diagnosis, Zadar GeneralHospital, Zadar

Determination of creatinine clearance is one ofthe routine laboratory tests in osteoporosis. Re-cently, the number of requests for this test in ourlaboratory has considerably increased. Since ourcatchment population are mainly elderly females,the 24-hour urine collection may present a greatproblem. Therefore, our idea was to compare theresults of calculated clearances with the results of



cijenjenih klirensa uz koje smo ra~unali i indekstjelesne mase. Pretpostavili smo da bi se u slu-~aju normalnih vrijednosti kreatinina u serumu inormalne diureze ovi rezultati mogli podudarati,pa bismo mogli predloìti da se u slu~ajevima ka-da se klirens traì samo radi obrade za osteo-porozu ne skuplja 24-h mokra}a. Drugi je cilj bionapraviti obradu naših rezultata za klirens kre-atinina prema godinama i spolu, te usporeditidobivene rezultate s vrijednostima po Thomasu.U obradu smo uzele potencijalno zdrave osobe:121 ènu u dobi od 20-79 godina i 60 muškaracau dobi od 16-80 godina. Klirens kreatinina smoodre|ivali na ure|ajima tvrtke Olympus i s nji-hovim izvornim reagensima. Primijenili smo me-todu Jaffe kinetike (kolor test). Naš referentniraspon bio je do 106 µmol/l. Rezultati u skupinièna: jako dobro podudaranje u donjim granica-ma izme|u izra~unatog i procijenjenog klirensa,dok su gornje vrijednosti bile više kod izra~u-natih klirensa. Naši rezultati izra~unatih kliren-sa kreatinina slaù se s rezultatima po Thomasuprema donjoj vrijednosti, dok su gornje vrijed-nosti bile izrazito više. Rezultati u skupini mu-škaraca: tako|er dobro podudaranje u donjimvrijednostima, osim za raspon od 30-50 godina(procijenjeni klirensi ve}i). Gornje vrijednosti bi-le su više kod izra~unatih klirensa. Slabije sla-ganje s rezultatima po Thomasu; donje vrijed-nosti bile su više, osim za raspon od 50-59 godina.Gornje vrijednosti bile su visoke. Stoga sma-tramo da kod potencijalno zdravih osoba pro-cijenjeni klirens moè zamijeniti izra~unati kli-rens kreatinina. Iako je pomo}u indeksa tjelesnemase potvr|eno da naši ispitanici imaju preko-mjernu tjelesnu teìnu, podudarnost rezultatanije bila upitna. Na osnovi dobivenih rezultataodrediti }emo vlastite referentne vrijednosti zaklirens kreatinina.E-mail: [email protected]

estimated clearances, coupled with body mass in-dex (BMI) calculation. We assumed that, in caseof normal creatinine values in serum and normaldiuresis, those results might match. This wouldthen allow us to suggest that, in cases when theclearance is only requested for osteoporosis analy-sis, the 24-hour urine collection may not be neces-sary at all. The second aim was to analyze thementioned creatinine clearances according to ageand sex, and to compare the results obtained withThomas values. About 180 potentially healthysubjects were included in the study: 121 womenaged 20-79 and 60 men aged 16-80. Creatinineclearance was analyzed on Olympus analyzersusing original Olympus reagents. The Jaffe ki-netic color test was used, reference range up to106 µmol/l. Women: very good matching at lowerlimits between the calculated and estimatedclearance, whereas calculated clearances showedhigher upper limits than estimated clearances.Our results of calculated clearance matched Tho-mas’ lower values, while upper values were sig-nificantly higher. Men: good matching also ob-served for lower values, except for the 30-50 agerange (estimated clearances were higher). Uppervalues were higher in calculated clearances.There was poorer matching with Thomas’ results,lower values were slightly higher, except for the50-59 age range. Upper values were high. We sug-gest that estimated clearance could replace calcu-lated creatinine clearance in potentially healthysubjects. Although our patients were overweight,the match of results was unquestionable. Basedon the results obtained, we will determine ourown reference values for creatinine clearance.E-mail: [email protected]

P9-15

POREME]AJ METABOLIZMA GLUKOZEKOD TERAPIJE ATIPI^NIM

ANTIPSIHOTICIMA

Dundovi} S.1, Fija~ko M.2, Pavela J.2, Šeri} V.2, Cetina N.2

1 Medicinsko biokemijski laboratorij, O@B Našice, Na{ice2 Odjel za medicinsku biokemiju, KB Osijek, Osijek

Atipi~ni antipsihotici olanzapin i risperdon ra-be se u lije~enju shizofrenije kao nova generacijaantipsihotika. Unato~ njihovom dobrom terapij-skom u~inku kod duè primjene ovih lijekova

P9-15

IMPAIRED GLUCOSE METABOLISMASSOCIATED WITH ATYPICAL

ANTIPSYCHOTIC DRUGS

S. Dundovi}1, M. Fija~ko2, J. Pavela2, V. Šeri}2, N. Cetina2

1 Laboratory of Medical Biochemistry, County General Hospital,Našice, 2 Department of Medical Biochemistry, Osijek University

Hospital, Osijek

Olanzapine and risperdone are atypical antip-sychotic medications used in the treatment ofschizophrenia. Treatment with antipsychotic



moè do}i do poreme}aja u metabolizmu glukoze.Ispitivanje je provedeno u skupini bolesnika kojisu primali olanzapin (n=17) i risperdon (n=18) tekontrolnoj skupini (n=15). Proveden je OGT test(prema preporuci SZO), te su odre|ene koncen-tracije glukoze (enzimsko odre|ivanje s heksoki-nazom, Olympus); inzulina (ELISA, Mercadone);C-peptida (ELISA, Mercadone) u odre|enim vre-menskim razmacima (0, 60, 120, 180 min). U 54%bolesnika utvr|ena je ošte}ena tolerancija glu-koze (koncentracija glukoze bila je natašte <6,6mmol/L, a nakon 120 minuta od 7,7 do 11,0mmol/L). Ošte}ena tolerancija glukoze utvr|enaje u 59% bolesnika koji su primali olanzapin i u50% bolesnika koji su primali risperdon. U 97%bolesnika koncentracija inzulina i C-peptida po-rasla je 4-6 puta nakon optere}enja glukozom.Rezultati ukazuju na to da se kod primjene olan-zapina i risperdona u lije~enju shizofrenije kaokomplikacija moè razviti hiperglikemija kojanadalje moè dovesti do dijabeti~ne ketoacidoze idijabetes melitusa. Da bi sprije~ili ove kompli-kacije preporu~a se raditi probir na dijabetesmelitus u odre|enim vremenskim razmacima.E-mail: [email protected]

drugs can be associated with impaired glucosemetabolism. Schizophrenic patients treated witholanzapine (n=17) or risperdone (n=19) and 15healthy control subjects were included in thestudy. We evaluated OGTT (according to WHOrecommendations); glucose concentration wasquantified by the hexokinase method (enzymaticUV test, Olympus); insulin and C-peptide concen-trations were quantified by ELISA (Mercadone)at specific time intervals (0, 60, 120 and 180min). Impaired glucose metabolism was con-firmed in 54% of patients (fasting plasma glucose<6.6 mmol/L, 2-h OGTT plasma glucose 7.7-11.0mmol/L). Impaired glucose tolerance was veri-fied in 59% of patients treated with olanzapineand 50% of patients treated with risperdone. In97% of patients treated with atypical antipsi-chotics, the concentrations of insulin and C-pep-tide increased 4- to 6-fold after OGTT. Atypicalantipsychotics can produce a number of side ef-fects such as hyperglycemia and glucose intoler-ance that may potentially lead to the developmentof diabetic ketoacidosis and diabetes mellitus. Wepropose that patients taking atypical antipsy-chotic drugs be screened for diabetes mellitusevery few months.E-mail: [email protected]

P9-16

KVALITATIVNA ANALIZAAMINOKISELINA NEKIH SVOJTA RODA

HYPERICUM TANKOSLOJNOMKROMATOGRAFIJOM

Maleš @.1, Biluši}-Vunda} V.1, Plazibat M.2,Hazler-Pilepi} K.1, Kos N.1

1 Zavod za farmaceutsku botaniku Farmaceutsko-biokemijskog fakulteta, 2 Botani~ki zavod Prirodoslovno-matemati~kog fakulteta Sveu~ilišta u Zagrebu, Zagreb

Hypericum (H.) L. - pljuskavica, biljni je rod izporodice Hypericaceae zastupljen u hrvatskojflori s manje od dvadeset svojta od kojih su uovom radu istraìvane slijede}e: H. hirsutum L.,H. montanum L. i H. perforatum L. H. hirsutum -~upavodlakava pljuskavica je trajnica do 100 cmvisoka, cijela gusto obrasla kratkim dlakama. UHrvatskoj raste na bazi~nim, svjeìm tlima odbrdskih do pretplaninskih poloàja. H. monta-num - gorska pljuskavica je rijetko dlakava traj-nica, nešto nià od prethodne, a nalazimo ju nasli~nim staništima. H. perforatum - rupi~astapljuskavica je trajnica s podankom koja narastedo 90 cm visine. Na širokom prostoru areala vr-ste opisane su razne podvrste, varijacije i forme

P9-16

QUALITATIVE ANALYSIS OF AMINOACIDS IN SOME TAXA OF THE GENUS

HYPERICUM BY THIN-LAYERCHROMATOGRAPHY

@. Maleš1, V. Biluši}-Vunda}1, M. Plazibat2, K. Hazler-Pilepi}1, N. Kos1

1 Department of Pharmaceutical Botany, School ofPharmacy and Biochemistry, 2 Institute of Botany, School of

Science, University of Zagreb, Zagreb

Hypericum L. - St. John’s wort, is a genus be-longing to the family Hypericaceae, which com-prises less than twenty taxa in the Croatian flora,of which the following were investigated: H.hirsutum L., H. montanum L. and H. perforatumL. H. hirsutum - hairy St. John’s wort is an erect,pubescent perennial up to 100 cm high. InCroatia it grows on basic, fresh soils from themountainous to the subalpine zone. H.montanum - mountain St. John’s wort is an erect,slightly pubescent perennial, somewhat shorterthan the previous one, which occurs on similarhabitats. H. perforatum - common or perforate St.John’s wort is an erect, glabrous rhizomatousperennial up to 90 cm high. Throughout the large



od kojih u Hrvatskoj rastu: H. perforatum subsp.angustifolium (DC) Gaudin (uskolisna rupi~astapljuskavica), H. perforatum L. subsp. perforatum(širokolisna rupi~asta pljuskavica) i H. perfora-tum subsp. veronense (Schrank) Fröhlich (sitno-lisna rupi~asta pljuskavica). Rupi~asta pljuska-vica dolazi na razli~itim staništima, od morskerazine do pretplaninskog podru~ja, s tim da jetipi~na podvrsta ~eš}a u kontinentalnim kraje-vima, a ostale dvije u primorskom dijelu Hrvat-ske. Nadzemni dijelovi širokolisne rupi~astepljuskavice sadrè ~itav niz biološki aktivnihsastavnica: flavonoide, procijanidine, hipericine,eteri~no ulje i trijeslovine. Ljekovitosti vrsta rodaHypericum, a poglavito vrste H. perforatum, pri-daje se veliko zna~enje, osobito kao biljnom anti-depresivu. Cilj ovoga rada bio je utvrditi prisut-nost aminokiselina u nadzemnim dijelovima ~u-pavodlakave (H. hirsutum), gorske (H. monta-num) i triju podvrsta rupi~aste pljuskavice (H.perforatum) skupljenim u razli~itim podru~jimaHrvatske. Tankoslojna kromatografija istraìva-nih uzoraka provedena je na celulozi F, u dvjemasmjesama otapala: n-butanol-aceton-ledena oc-tena kiselina-voda (35:35:10:20 V/V/V/V) i n-bu-tanol-ledena octena kiselina-voda (40:10:10 V/V/V). Otkrivanje odijeljenih aminokiselina prove-deno je nakon prskanja kromatograma reagen-som ninhidrin. Nadzemni dijelovi istraìvanihsvojta sadràvali su slijede}e aminokiseline: le-ucin, izoleucin, fenilalanin, valin, triptofan, me-tionin, gama-aminomasla~nu kiselinu, tirozin,prolin, alanin, treonin, glicin, serin, arginin,histidin i ornitin. Aminokiselinski sastav je ovisioo biljnoj svojti i nalazištu.E-mail: [email protected]

geographical range of distribution a number ofsubspecies, varieties and forms have been recog-nized, the following occurring in Croatia: H.perforatum subsp. angustifolium (DC) Gaudin,H. perforatum L. subsp. perforatum and H.perforatum subsp. veronense (Schrank) Fröhlich.Perforate St. John’s wort grows across differenthabitats from the sea level to the subalpine belt,but in Croatia the typical subspecies is more fre-quent in the inland, and the others in the littoralareas of the country. Chemical studies of theaerial parts of H. perforatum subsp. perforatumindicated a number of biologically active sub-stances: flavonoids, procyanidins, hypericins, es-sential oil and tannins. This plant is widely usedin Europe for its antidepressive effects as an al-ternative to medical synthetic drugs. This studyinvestigated the presence of amino acids in theaerial parts of H. hirsutum, H. montanum andthree subspecies of H. perforatum collected at dif-ferent localities in Croatia. Thin-layer chroma-tography of the investigated samples was per-formed on cellulose F using two solvent systems:n-butanol-acetone-glacial acetic acid-water(35:35:10:20 V/V/V/V) and n-butanol-glacialacetic acid-water (40:10:10 V/V/V). Detection ofseparated amino acids was carried out by theninhydrin reagent. The aerial parts of the investi-gated species contained the following amino ac-ids: leucine, isoleucine, phenylalanine, valine,tryptophan, methionine, gamma-aminobutyricacid, tyrosine, proline, alanine, threonine, gly-cine, serine, arginine, histidine and ornithine.The composition of amino acids was dependenton the investigated taxa and locality.E-mail: [email protected]

P9-17

CMVpp65 ANTIGENEMIJA U ODNOSU NASEROLOŠKI CMV STATUS PRIMATELJA

TRANSPLANTATA BUBREGA

Aralica M.1, Orli} P.2, Dvornik Š.1

1 Zavod za laboratorijsku dijagnostiku, 2 Interna klinika,Klini~ki bolni~ki centar “Rijeka”, Rijeka

Infekcija CMV (citomegalovirusom) naj~eš}aje infekcija koja se javlja u prvim mjesecima na-kon transplantacije organa. CMV status prima-telja ima zna~ajnu ulogu u njenoj pojavnosti.Naša retrospektivna studija imala je za cilj utvr-diti povezanost izme|u serološkog CMV statusaprimatelja i pozitivnog testa antigenemijeCMVpp65 u prva tri tjedna nakon transplantaci-

P9-17

CMVpp65 ANTIGENEMIA ASSAY AND CMVSEROLOGY IN KIDNEY TRANSPLANT

RECIPIENTS

M. Aralica1, P. Orli}2, [. Dvornik1

1 Institute of Laboratory Diagnosis, 2 University Departmentof Medicine, Rijeka University Hospital Center, Rijeka

Cytomegalovirus (CMV) infection is the mostcommon infection in the first months after organtransplantation. CMV status of the donor and re-cipient is an important factor in the incidence ofinfection. This retrospective study was under-taken to determine the relationship between CMVrecipient serology and positive result of CMVpp65antigenemia assay (CMVpp65) in the first three



je bubrega. Prijeoperacijski CMV status primateljautvr|en je odre|ivanjem CMV IgG i IgM MEIA iimunoturbidimetrijskom metodom. Imunocito-kemijski test antigenemija CMVpp65 (CMVpp65)izvodili smo jedanput na tjedan poslijeoperacijskikroz prva tri tjedna. CMVpp65 je pozitivan akose u sedimentu leukocita izoliranih iz krvi (2,0 G/L) na|e jedna pozitivna stanica. Od ukupno 11boesnika 9 ih je bilo CMV IgG+/IgM-, a samo 2 subila CMV IgG+/IgM+. U skupini IgG+/IgM- bole-snika 15 rezultata na CMVpp65 je bilo pozitivno,a 12 negativno. U prvom tjednu pozitivna su bila4 bolesnika (jedan je imao >100 pozitivnih stani-ca na 2,0 G/L), u drugom tako|er, s najviše 4pozitivne stanice u pripravku, dok je u zadnjemtjednu bilo 6 pozitivnih bolesnika s najviše 2 po-zitivne stanice na 2,0 G/L. Zaklju~eno je kako jetest antigenemije CMVpp65 koristan u ranomotkrivanju infekcije CMV kod bolesnika s trans-plantiranim bubregom. Treba ga rabiti i za pra-}enje tijeka CMV infekcije u prvim tjednima na-kon transplantacije neovisno o serološkom sta-tusu bolesnika.E-mail: [email protected]

weeks after kidney transplantation. CMV serol-ogy of recipients was preoperatively performed forIgG and IgM by MEIA and immunoturbidimetricassay. We performed immunocytochemicalCMVpp65 once a week in the first three weekspostoperatively. Even one antigen positive leuko-cyte in a cell spin preparation (CSP) with 2.0 G/Lleukocytes confirms positive test result. Elevenpatients, mean age 46.6±11.2 (M:F=5:6) were fol-lowed up. Immunosuppression included antilym-phocyte antibody as induction therapy andmycophenolate mofetil, cyclosporine and pred-nisolone. Nine of the 11 patients had IgG+/IgM-CMV serology and only 2 patients had IgG+/IgM+. In IgG+/IgM- combination CMVpp65 werepositive in 15 and negative in 12 cases. In the firstweek of testing we observed 4 positive cases (oneof them had >100 antigen positive leukocytes inCSP), the same were found in the second week oftesting but this time the maximal number of posi-tive leukocytes was 4. In the last week of testing 6cases were positive but the maximal number ofpositive leukocyte was 2. In the IgG+/IgM+ com-bination, one of two patients had 75 antigen posi-tive leukocytes in CSP in the first week of testing,the number of positive cells was 10 in the secondweek, to finally become negative at the end of thestudy period. Accordingly, CMVpp65 was foundto be useful in the early detection of CMV infec-tion. It should be used for monitoring of CMV in-fection in the first weeks after transplantation, ir-respective of the recipient’s CMV serology.E-mail: [email protected]

P9-18

RASPODJELA PREOSJETLJIVOSTI NAARTROPODE KOD STANOVNIŠTVA

ZAGREBA^KE @UPANIJE

Cigula-Kurajica V., Popovi}-Grle S., Zovko V., BunetaL.

Medicinsko-biokemijski laboratorij, Klinika za plu}nebolesti “Jordanovac”, Zagreb,

Glavni alergenski sastojci ku}ne prašine na-stali su od artropoda. Najva`niji izvor alergena uku}noj prašini su ku}ne grinje, poput Dermato-phagoides pteronyssinus i Dermatophagoides fa-rinae. Drugi va`an inhalacijski alergen u ku}nojprašini, iako manje raširen, jest `ohar, Blatellagermanica. Alergijski rinitis najuobi~ajeniji jeoblik preosjetljivosti na inhalacijske alergene ilokaliziran je u mukozi nosa i konjunktivi, dok jealergijska astma lokalizirana u bronhima. U raz-

P9-18

DISTRIBUTION OF HYPERSENSITIVITYTO ARTHROPODS IN THE ZAGREB

COUNTY POPULATION

V. Cigula-Kurajica, S. Popovi}-Grle, V. Zovko, L.Buneta

Laboratory of Medical Biochemistry, Jordanovac UniversityHospital for Lung Diseases, Zagreb

The principal allergenic components of housedust are of arthropod origin. The most importanthouse dust allergenic source are house dust mites,such as Dermatophagoides pteronyssinus andDermatophagoides farinae. The other importantaeroallergen in house dust, although less spread,is cockroach, Blatella germanica. Allergic rhinitisis the most common form of hypersensitivity toinhalatory allergens. It is localized at nasal mu-cosa and conjunctiva, whereas allergic asthma is



doblju od 1. velja~e 2002. do 1. lipnja 2003. pro-u~avana je senzibilizacija bolesnika s respira-cijskim alergijama na inhalacijske alergene za-tvorenih prostora: Blatella germanica, Dermato-phagoides pteronyssinus i Dermatophagoides fa-rinae. Analizirani su podaci 55 bolesnika ob-ra|enih u ambulanti ili na 2. bolni~kom odjelu.Postavljene dijagnoze bile su rinitis, astma, terinitis i astma, dok je manji broj bolesnika imaodruge dijagnoze. Specifi~na IgE protutijela od-re|ivana su u serumu dobivenom iz venske krvi.Mjerenja su provedena po metodi FEIA naure|aju UniCAP 100E (Pharmacia, Švedska).Cilj studije bilo je istraìvanje raspodjele pre-osjetljivosti na artropode: Blatella germanica,Dermatophagoides pteronyssinus i Dermatopha-goides farinae kod stanovništva s respiracijskimalergijskim poreme}ajima na podru~ju Zagre-ba~ke ùpanije. U skupini od 55 testiranih osobas dijagnozama rinitisa (11%), astme (36%), rini-tisa i astme (36%), odnosno ukupno s astmom(72%) samo je 7/55 bolesnika imalo reakciju naalergen Blatella germanica (klasa 1-3). Istodobnosu 22 ispitanika pokazala preosjetljivost na Der-matophagoides pteronyssinus, a 23 na Derma-tophagoides farinae. Kod svih 7 ispitanika s uo~e-nom senzibilizacijom na Blatella germanica (vri-jednosti izme|u 0,36 i 17,0 kUA/l - klasa 1-3) bilaje povišena i vrijednost specifi~nih IgE protuti-jela na Dermatophagoides pteronyssinus i Der-matophygoides farinae (vrijednosti izme|u 0,36 i100 kUA/l - klasa 1-6) uz izrazito povišen ukupniIgE. Rezultati istraìvanja pokazuju da u popu-laciji bolesnika s alergijama dišnoga sustava po-stoji podjednaka preosjetljivost na Dermatopha-goides pteronyssius i Dermatophagoides farinae.Preosjetljivost na Blatella germanica manje jeizraèna, ~emu je vjerojatan razlog manja izloè-nost ovom alergenu.E-mail: [email protected]

an allergy manifestation localized in the bronchi.During the period from February 1, 2002 tillJune 1, 2003, the sensitization of patients withrespiratory allergies to house aeroallergens Bla-tella germanica, Dermatophagoides pteronyssi-nus and Dermatophagoides farinae was studied.The data collected from 55 patients treated asoutpatients or inpatients were analyzed. The pa-tients had the diagnoses of rhinitis, asthma orboth, whereas other diagnoses were recorded in afew cases. Specific IgE antibodies were deter-mined in serum obtained from venous blood. Themeasurements were performed by the FEIAmethod on an UniCAP 100E device (Pharmacia,Sweden). The aim of this study was to assess thedistribution of hypersensitivity to the arthropodsBlatella germanica, Dermatophagoides pterony-ssinus and Dermatophagoides farinae in patientswith respiratory allergic disorders living in theZagreb county. In the group of 55 study patientswith the diagnoses of rhinitis (11%), asthma(36%), rhinitis and asthma (36%), i.e. totalasthma (72%), only 7/55 patients showed reac-tion to the Blatella germanica allergen (class 1 to3). At the same time, 22 patients were hypersensi-tive to Dermatophagoides pteronyssinus and 23patients to Dermatophagoides farinae. All 7 pa-tients with hypersensitivity to Blatella germanica(range 0.36-17.0 kUA/l - class 1-3) showed in-creased values of specific IgE to Dermato-phagoides pteronyssinus and Dermatophagoidesfarinae (range 0.36-100 kUA/l - class 1-6) with amarked increase in total IgE. Study resultsshowed the patients with respiratory allergies tobe comparably sensitive to Dermatophagoidespteronyssinus and Dermatophagoides farinae.The hypersensitivity to Blatella germanica ap-pears to be less pronounced, probably due tolower exposure.E-mail: [email protected]

P9-19

ODRE\IVANJE IgE U SERUMU DJECE SASTMOM I POLENOZOM

Dodig S., Brajkovi} M., @iv~i} J., Raos M., Kova~ K.,Nogalo B.

Specijalna bolnica za bolesti dišnog sustava djece i mladeì,Zagreb

U dijagnostici alergijskih bolesti se uz in vitropostupke primjenjuje i odre|ivanje serumskekoncentracije ukupnih i specifi~nih IgE. Cilj stu-dije bio je odrediti koncentraciju ukupnih i speci-

P9-19

SERUM IgE IN CHILDREN WITH ASTHMAAND POLLINOSIS

S. Dodig, M. Brajkovi}, J. @iv~i}, M. Raos, K. Kova~,B. Nogalo

Special Hospital for Respiratory Diseases in Children andAdolescents, Zagreb

Besides in vitro tests, determination of totaland allergen-specific IgE in serum concentrationsis used in the diagnosis of allergic diseases. Theaim of the study was to determine the possible



fi~nih IgE u serumu djece s astmom i polenozom,te utvrditi postoji li razlika u koncentraciji IgE userumu djece s monosenzibilizacijom odnosnopolisenzibilizacijom na inhalacijske alergene. Uastmati~ara s polisenzibilizacijom koncentracijaukupnoga IgE bila je ve}a nego u djece s mono-senzibilizacijom. Pokreta~ astme je alergen gri-nje iz ku}ne prašine, Dermatophagoides ptero-nyssinus (Der p). Koncentracija specifi~nih IgEve}a je na alergen grinje, Der p, od koncentracijespecifi~nih IgE na alergene peluda. U djece s po-lenozom sezona peludacije utje~e na koncen-traciju ukupnih i specifi~nih IgE. Preosjetljivostna višestruke alergene podrazumijeva i ve}e kon-centracije IgE u serumu. U djece s polenozomtreba imati u vidu vrijeme uzimanja krvi, budu}ida je u vrijeme peludacije ve}a koncentracijaalergena u zraku, pa time i ve}i alergenski poti-caj organizma na sintezu specifi~nih IgE.E-mail: [email protected]

difference between total and specific IgE in serumconcentration in children with monosensitizationor polysensitization to inhalant allergens. Theconcentration of total serum IgE was greater inasthmatic children with polysensitization. Thetrigger for asthma was house dust mite, Derma-tophagoides pteronyssinus (Der p). The concen-tration of specific IgE to Der p was greater thanthe concentration of specific IgE to pollen aller-gens. Pollination season was found to influencetotal and specific IgE concentration in childrenwith pollinosis. Thus, children with hypersensi-tivity to multiple allergens will have higher se-rum concentration of IgE than children withmonosensitization, as pollination (more allergensin the air) implies greater stimulation of the bodyfor specific IgE synthesis.E-mail: [email protected]

P9-20

UÎNAK DEKSAMETAZONA,AZITROMICINA I KLARITROMICINA NA

KONCENTRACIJU SERUMSKOG AMILOIDA PROTEINA U EKSPERIMENTALNOM

MODELU UPALE NA BALB/C MIŠEVIMA

Glojnari} I., Erakovi} V., Parnham M.J.

Pliva, Istraìva~ki institut, Zagreb

Serumski amiloid A (SAA) je protein akutnefaze koji se sintetizira u jetri pod utjecajemcitokina. Cilj studije bio je ispitati u~inak makro-lidnih antibiotika azitromicina i klaritromicinana koncentraciju SAA tijekom akutne upale, tega usporediti s u~inkom deksametazona. U ènkimiševa soja BALB/c izazvana je upala supku-tanim injiciranjem 2% otopine srebrnog nitrata.Deksametazon je injiciran intraperitonealno udozama od 5, 10, 20 i 30 mg/kg, 1 sat nakon srebr-nog nitrata. Makrolidi (20, 40 i 80 mg/kg) suaplicirani oralno u 0,5% karboksimetil celulozisvakih 12 sati (klaritromicin) ili jednom na dantijekom 3 dana (azitromicin), po~evši 1 sat ili 24sata prije injiciranja srebrnog nitrata. Koncen-tracije SAA (ELISA, Tridelta, Irska) u serumuodre|ivane su prije po~etka pokusa, te 6, 12, 24,48 i 72 sata nakon injekcije srebrnog nitrata.Srednja vrijednost koncentracija SAA (maksi-mum 24 h nakon srebrnog nitrata) bila je sta-tisti~ki zna~ajno nià (p<0,05) nakon primjenedeksametazona (kod svih doza u usporedbi s ota-

P9-20

INHIBITION OF SERUM AMYLOID APROTEIN RELEASE BY

DEXAMETHASONE, AZITHROMYCIN ANDCLARITHROMYCIN DURING

EXPERIMENTAL INFLAMMATION INBALB/c MICE

I. Glojnari}, V. Erakovi}, M.J. Parnham

Pliva, Research Division, Zagreb

SAA, an acute phase protein, is produced pri-marily by the liver in response to cytokines. Wetested the macrolide antibiotics azithromycin andclarithromycin versus dexamethasone for theircapacity to inhibit SAA release in response to aninflammatory agent. A sterile inflammation wasinduced in female BALB/c mice with 2% silvernitrate, s.c. Dexamethasone, 5, 10, 20 or 30 mg/kg, was given i.p. 1 h later. Macrolides (20, 40 or80 mg/kg) were given orally in 0.5% carboxy-methylcellulose, every 12 h (clarithromycin) oronce daily for 3 days (azithromycin), starting 1 or24 h before silver nitrate injection. SAA levels(ELISA, Tridelta, Ireland) in blood were assayedbefore, 6, 12, 24, 48 and 72 h after silver nitrateinjection (baseline). Mean SAA levels (max. 24 h)were significantly inhibited (p<0.05) by dexam-ethasone at 24 h (all doses vs. vehicle) and 48 h (5mg/kg) after baseline; by clarithromycin at 24 h(80 mg/kg vs. vehicle) and 48 h (40 and 80 mg/kg) after baseline; and by azithromycin at 12 h



palom) te nakon 48 sati u dozi od 5 mg/kg.Primjenom klaritromicina dobivene su zna~ajnoniè srednje vrijednosti SAA od vrijednosti pri-mjenom otapala i to 24 sata (doza od 80 mg/kg) i48 sati (doze od 40 i 80 mg/kg) nakon injekcijesrebrnog nitrata. Primjenom azitromicina izmje-rene su zna~ajno niè srednje koncentracije SAAu odnosu na otapalo i to 12 sati nakon injekcijesrebrnog nitrata za doze 20 i 40 mg/kg. Za-klju~ujemo da je SAA pogodan biokemijski biljegza in vivo testiranje spojeva s potencijalnom pro-tuupalnom aktivnoš}u.E-mail: [email protected]

(20 and 40 mg/kg vs. vehicle) after baseline. Inconclusion, SAA seems to be a valuable markerfor the in vivo testing of compounds with poten-tial anti-inflammatory activity.E-mail: [email protected]

P9-21

ANALIZA AKTIVNOSTI LEKTINA KOJIVE@E MANOZU U HUMANOM SERUMU

Šupraha-Goreta S., Flögel M., Lauc G.

Farmaceutsko-biokemijski fakultet, Zavod za biokemiju imolekularnu biologiju, Zagreb

S ciljem poboljšanja metoda za odre|ivanjelektinske aktivnosti u kompleksnom biološkomuzorku, posljednjih nekoliko godina u našem la-boratoriju razvijamo novu tehniku nazvanu gli-koprobama (Glycobiology 10:357, 2000). Gliko-probe se sastoje od oligosaharidnog liganda, foto-reaktivnog umreìva~a i digoksinskog kraja kojiomogu}uje direktnu analizu lektinske aktivnostiu kompleksnim biološkim uzorcima. Lektin kojiveè manozu (MBL) je lektin tipa C s tipi~nomtopljivom kolektinskom strukturom. Djeluje kaomolekula koja prepoznaje i moè vezati razli~iteše}erne strukture na površinama mikroorgani-zama. Njegova sposobnost aktiviranja komple-menta ~ini ga klju~nom komponentom u prvojcrti obrane, kao i u nekim patološkim procesima.Kako bi se mogla analizirati aktivnost MBL uhumanom serumu sintetizirali smo specifi~nuglikoprobu koja sadrì Manoza9-N-Acetilgalak-tozamin2 (Man9) glikan kao ligand. Rabe}iMan9-glikoprobu izmjerili smo aktivnost MBL u30 uzoraka seruma osoba oboljelih od reuma-toidnog artritisa i 25 uzoraka seruma zdravihkontrola. Va`no je napomenuti da je ovo prvi pri-mjer otkrivanja lektinske aktivnosti, a ne samoukupne koli~ine MBL lektina. Prona|ena je vrlovelika biološka varijabilnost MBL aktivnosti uobje prou~avane skupine, no nije prona|ena zna-~ajnija statisti~ka razlika izme|u skupina. Ovajrezultat ne podupire hipotezu da vezanje MBLna degalaktozirane oligosaharide na IgG dopri-nosi patologiji reumatoidnog artritisa.E-mail: [email protected]

P9-21

ANALYSIS OF MANNOSE BINDINGLECTIN ACTIVITY IN HUMAN SERA

S. Šupraha-Goreta, M. Flögel, G. Lauc

Department of Biochemistry and Molecular Biology, Schoolof Pharmacy and Biochemistry, Zagreb

Aiming to improve the tools for lectin analysis,we have recently developed a new class of complexneoglycoconjugates named Glycoprobes (Glycobi-ology 10:357, 2000). Glycoprobes consist of an oli-gosaccharide ligand, photoreactive cross-linkerand a digoxin tag, and enable direct analysis oflectin activity in complex biological samples.Mannose-binding lectin (MBL) is a C-type lectinwith a typical soluble collectin structure. It func-tions as a pattern recognition molecule that bindsrepeating sugar arrays on many microbial sur-faces. Its ability to activate complement responsemakes it an important player in the first line ofdefense, as well as in some pathological proc-esses. To be able to analyze MBL activity in hu-man serum we synthesized a specific glycoprobecontaining mannose9-N-acetylgalactosamine2(Man9) glycan as a ligand. Using Man9-glyco-probe we assayed MBL activity in 30 sera of pa-tients with rheumatoid arthritis and 25 sera ofmatching healthy controls. It is important to notethat this was the first example where activity, andnot the simple presence of MBL protein was as-sayed. Very large biological variability of MBLactivity was found to exist in both study groups,while there appeared to be no significant differ-ence between the groups. This result does not sup-port the hypothesis that binding of MBL todegalactosylated oligosaccharides on IgG contrib-utes to the pathology of rheumatoid arthritis.E-mail: [email protected]



P9-22

PRA]ENJE LIJEÊNJA AFEKTIVNIHPOREME]AJA LITIJEM

Samoš}anec K.1, Papi}-Futa~ D.1, Topi} E.1, PotkonjakJ.2, Thaller V.2

1 Klini~ki zavod za kemiju, 2 Klinika za psihijatriju iKlini~ka bolnica “Sestre milosrdnice”, Zagreb

Litij poznat kao ’stabilizator raspoloènja’ po-kazao se lijekom izbora u prevenciji i lije~enju bo-lesnika s bipolarnim poreme}ajem. Kontinuira-na kontrola serumske razine litija je neophodnazbog njegove uske terapijske širine (0,5-1,2mmol/l) i zna~ajnih nuspojava u terapijskom po-dru~ju. Na Klinici za psihijatriju KB “Sestremilosrdnice” u dvogodišnjem razdoblju lije~eno jelitijem 190 bolesnika (110 èna i 80 muškaraca) ito dozama od 300, 600 i 900 mg na dan. Kon-centracija litija mjerena je natašte kolorimetrij-skom metodom koja se temelji na vezanju litija izuzorka s azo bojom 6-dodecil-6-(2'-hidro-ksi-5'-(2'-4'-dinitrofenilazo) benzil-13,13-dimetil-1,4,6,11-tetraoksaciklotetradekanom (Vitros Li slides,Ortho-Clinical Diagnostics, a Johnson-Johnsoncompany, analizator Kodak Ektachem 250). Uèna koje su primale 300 mg litija (n=13) srednjaserumska razina bila je 0,28 (0,20-0,59) mmol/l,uz dozu od 600 mg (n=45) bila je 0,58 (0,25-1,17)mmol/l, a uz dozu od 900 mg (n=52) 0,68 (0,24-1,17) mmol/l. U muškaraca niti jedan bolesniknije primao dozu od 300 mg, dok je uz dozu od600 mg (n=32) srednja serumska razina lijekaiznosila 0,51 (0,25-0,99) mmol/l, a uz dozu od 900mg (n=46) bila je 0,64 (0,2-1,48) mmol/l. Dvojebolesnika nije uzimalo terapiju. Kao što se vidi,vrlo je velika raspršenost serumskih koncentra-cija, što potvr|uje preporuku kontinuiranog pra-}enja koliko zbog zna~ajnih nuspojava u terapij-skom rasponu, toliko radi kontrole redovitog uzi-manja terapije.E-mail: [email protected]

P9-22

THERAPEUTIC MONITORING OFLITHIUM IN THE TREATMENT OF

AFFECTIVE DISORDERS

K. Samoš}anec1, D. Papi}-Futa~1, E. Topi}1, J.Potkonjak2, V. Thaller2

1 Clinical Institute of Chemistry, 2 University Department ofPsychiatry and Sestre milosrdnice University Hospital,

Zagreb

Lithium known as a mood stabilizer hasshown to be therapy of choice in the preventionand treatment of patients with bipolar disorder.Continuous monitoring of lithium level in serumis needed for its narrow therapeutic range (0.5-1.2 mmol/l) and serious side effects associatedwith therapeutic range. During a two-year period,190 patients (110 F and 80 M) were treated at theUniversity Department of Psychiatry, Sestremilosrdnice University Hospital, with daily lithi-um doses of 300, 600 or 900 mg. Fasting lithiumconcentration was measured by the colorimetricmethod based on sample lithium binding to theazo dye 6-dodecyl-6-(2'-hydroxy-5'-(2'-4'-dinitro-phenylazo) benzyl-13.13-dimethyl-1,4,6,11-tetra-oxacyclotetradecane (Vitros Li slides, Ortho-Clinica Diagnostics, a Johnson-Johnson com-pany, Kodak Ektachem 250 analyzer). In femalepatients receiving 300 mg of lithium (n=13), themean serum level of lithium was 0.28 (0.20-0.59)mmol/l, in those on 600 mg (n=45) it was 0.58(0.25-1.17) mmol/l, and in those on 900 mg(n=52) it was 0.68 (0.24-1.17) mmol/l. In thegroup of male patients, none of the patients re-ceived the dose of 300 mg of lithium, whereas inthose receiving 600 mg (n=32) the mean serumlevel of lithium was 0.51 (0.25-0.99) mmol/l, andin those on 900 mg (n=46) it was 0.64 (0.2-1.48)mmol/l. Two patients did not take their therapyat all. Accordingly, serum concentrations oflithium show great dispersion, thus supportingthe recommendation for continuous therapeuticmonitoring for both major side effects associatedwith lithium therapeutic range and as a controlof patient’s compliance with the therapeutic regi-men prescribed.E-mail: [email protected]



P9-23

ANIONSKI PROCIJEP IHIPOALBUMINEMIJA U BOLESNIKA

JEDINICE INTENZIVNOG LIJEÊNJA

Pavi} M., Milevoj L., Hreljac M., Broni} A.

Klinika za traumatologiju, Zagreb

Anionski procijep (anion gap, AG) definira sekao razlika koncentracija neizmjerenih aniona ineizmjerenih kationa u serumu. AG je izra~unatpomo}u slijede}eg izraza: [Na+K] - [Cl +HCO3].Kod fiziološkog pH svi su serumski proteini pri-sutni u anionskom obliku, te uravnoteùju udionatrijevog pozitivnog naboja. Albumin je serum-ski protein prisutan u najve}oj koncentraciji,sadrì relativno veliku gusto}u naboja te se po-kazalo da mu je udio u fiziološkom AG oko 75%.Cilj ispitivanja bio je procijeniti odnos hipoal-buminemije i vrijednosti AG, te istraìti kore-laciju izme|u razine hipoalbuminemije i AG ubolesnika jedinice intenzivnog lije~enja. Odre-|ivali smo koncentracije elektrolita i albumina userumu u skupini od 58 bolesnika jedinice in-tenzivnog lije~enja i 21 zdravog davatelja (ukup-no 555 mjerenja). U uzorcima smo odre|ivalinatrij, kalij i kloride indirektnom ISE tehnikomna analizatoru Dimension ES. Koncentracije al-bumina odre|ivali smo metodom bromkrezol ze-lenila na sustavu Kodak Ektachem DT II. Uzorcikapilarne krvi analizirani su pomo}u acidobaz-nog analizatora IL1306. AG smo izra~unali natemelju gore navedenog matemati~kog izraza. Uskupini zdravih davatelja srednja vrijednost AGbila je 16 mmol/L, a srednja vrijednost koncentra-cije albumina 43 g/L. Izrazita hipoalbuminemija(srednja vrijednost 26 g/L) bila je znakovita zaskupinu bolesnika i oni su pokazali statisti~kizna~ajno niì AG (srednja vrijednost 8 mmol/L) uodnosu na kontrolnu skupinu ispitanika. Naši surezultati potvrdili kako bolesnici s hipoalbumi-nemijom imaju statisti~ki zna~ajno niè vrijed-nosti AG u odnosu na kontrolnu skupinu, iakonije na|ena korelacija izme|u pojedina~nih kon-centracija albumina i sniènog AG u bolesnikajedinice intenzivnog lije~enja (r=0,26, p<0,05).E-mail: [email protected]

P9-23

ANION GAP AND HYPOALBUMINEMIA INCRITICALLY ILL PATIENTS

M. Pavi}, L. Milevoj, M. Hreljac, A. Broni}

University Hospital of Traumatology, Zagreb

Anion gap (AG) is defined as the difference be-tween the concentration of unmeasured anionsand unmeasured cations in serum. AG was calcu-lated as [Na+K] - [Cl +HCO3]. At normal pH allserum proteins are anions and thereby counter-balance the portion of sodium positive charge. Al-bumin, the most abundant serum protein, con-tains a relatively high charge density and conse-quently has been shown to be responsible for ap-proximately 75% of the normal AG. The aim ofthe study was to assess the relationship betweenhypoalbuminemia and AG, and to correlate thedegree of hypoalbuminemia with the value of AGin critically ill patients. Serum electrolyte and al-bumin concentrations were determined in a groupof 58 critically ill patients and 21 healthy volun-teers (a total of 555 measurements). Samples wereanalyzed for sodium, potassium and chloride bythe indirect ISE method on a Dimension ES. Al-bumin concentration was estimated by thebromcresol green method on a Kodak EktachemDT II System. Capillary blood samples wereanalyzed on an IL1306 blood gas analyzer. AGwas calculated using the above equation. Studyresults showed the mean value of AG to be 16mmol/L and mean albumin 43 g/L in the controlgroup of healthy donors. Marked hypoalbumi-nemia (mean 26 g/L) was common in critically illpatients and they had a significantly lower AG(mean 8 mmol/L) compared to the control group.Study results confirmed the group of patientswith hypoalbuminemia to have a lower AG thancontrol subjects, although there was no signifi-cant correlation between individual albumin con-centrations and decreased AG in the critically illpatients (r=0.26, p<0.05).E-mail: [email protected]



P9-24

DIJAGNOSTI^KA VRIJEDNOSTMIJELOPEROKSIDAZAANTINEUTROFILNIH

CITOPLAZMATSKIH PROTUTIJELA UDIJAGNOZI BRZO PROGRESIVNOG

GLOMERULONEFRITISA

Fija~ko M., Pavela J., Cetina N.

Klini~ka bolnica Osijek, Osijek

Mijeloperoksidaza antineutrofilna citoplaz-matska protutijela (MPO-ANCA) vaàn su doda-tak serološkom probiru bolesnika s brzo pro-gresivnim glomerulonefritisom (BPGN). Mada seMPO-ANCA nalazi marginalno dodaju dijagnozipauci-imuno bubre`nog vaskulitisa, koja se te-melji na biopsiji bubrega, oni mogu navesti naranu dijagnozu. MPO-ANCA nalazi sugeriraju di-jagnozu u ve}ine bolesnika s idiopatskim (pauci-immune) BPGN i bubre`nim vaskulitisom, i po-dupiru dijagnozu u bolesnika s visokim vrijed-nostima MPO-ANCA. Ovaj je podatak vaàn sto-ga što u ovih bolesnika naglo propada funkcijabubrega, a reagiraju na ranu terapiju. Prikazu-jemo troje bolesnika koji su zaprimljeni s kli-ni~kim i laboratorijskim nalazima BPGN.E-mail: [email protected]

P9-24

DIAGNOSTIC VALUE OFMYELOPEROXIDASE ANTINEUTROPHILCYTOPLASMIC ANTIBODY TESTING IN

THE DIAGNOSIS OF RAPIDLYPROGRESSIVE GLOMERULONEPHRITIS

M. Fija~ko, J. Pavela, N. Cetina

Osijek University Hospital, Osijek

Myeloperoxidase antineutrophil cytoplasmicantibody (MPO-ANCA) tests are an importantadjunct to serologic screening of patients withrapidly progressive glomerulonephritis (RPGN).Although MPO-ANCA results add marginally tothe diagnosis of pauci-immune renal vasculitismade on the basis of information from renal bi-opsy, they may lead to an early diagnosis. MPO-ANCA results may suggest the diagnosis in mostpatients with idiopathic (pauci-immune) RPGNand renal vasculitis, and strongly support the di-agnosis in patients with high MPO-ANCA levels.This information is important because these pa-tients experience rapid deterioration of renalfunction but will respond to early therapy. We re-port on three patients who presented with theclinical and laboratory findings of RPGN.E-mail: [email protected]

P9-25

UTJECAJ SPOLA, DOBI I TERAPIJEORALNIM ANTIKOAGULANSIMA NA

RAZINU ANTIGENA SLOBODNOGPROTEINA S

Radi} M.1, Coen D.2, Leni~ek-Krleà J.3, Zadro R.2

1 Farmaceutsko-biokemijski fakultet, 2 Klini~ki zavod zalaboratorijsku dijagnostiku, 3 Klinika za dje~je bolesti, KBC


Protein S (PS) je o vitaminu K ovisan gli-koprotein koji se nalazi u plazmi u dva oblika:40% je slobodni protein S, a 60% je vezano naC4b-vezivni protein. Samo slobodni protein S jefunkcionalno aktivan i sudjeluje kao kofaktoraktiviranog proteina C u reakciji inaktiviranjaaktiviranih faktora zgrušavanja V. i VIII. Odre-|ivanje koncentracije antigena slobodnog PS(PS:Ag) klju~no je za dijagnozu nasljednog nedo-statka PS, koji je rizi~ni ~imbenik za nastanakvenske tromboze. Uz to, na razinu slobodnogPS:Ag utje~u mnogobrojni fiziološki i patološki~imbenici, izme|u ostalog i terapija oralnim anti-koagulansima (OAT). Cilj studije bio je utvrditi

P9-25

IMPACT OF SEX, AGE AND ORALANTICOAGULANT THERAPY ON THE

FREE PROTEIN S LEVEL

M. Radi}1, D. Coen2, J. Leni~ek-Krleà3, R. Zadro2

1 School of Pharmacy and Biochemistry, 2 Clinical Instituteof Laboratory Diagnosis, 3 University Department of

Pediatrics, Zagreb University Hospital Center, Zagreb

Protein S (PS) is a vitamin K-dependent glyco-protein that circulates in plasma in two forms:40% in a free form and 60% conjugated to theC4b-binding protein. Only free PS is functionallyactive as a cofactor of activated protein C in theinactivation of coagulation factors Va and VIIIa.Quantitative determination of free PS antigen(PS:Ag) is essential for the diagnosis of PS defi-ciency that is a well-established risk factor for ve-nous thrombosis. However, free PS:Ag levels areaffected by various physiologic and pathologicfactors including oral anticoagulant therapy(OAT). The aim of the study was to determine theeffects of sex, age and OAT on the level of freePS:Ag. Free PS:Ag was measured by using in-



postoji li utjecaj spola, dobi i stupnja OAT narazinu slobodnog PS:Ag. Koncentracija slobodnogPS:Ag odre|ena je vlastitom enzimimunoke-mijskom metodom uporabom specifi~nih protu-tijela na humani PS (Dako, Danska) nakon pret-hodnog taloènja citratne plazme s polietilen-glikolom 6000. Za odre|ivanje referentnog ra-spona analizirane su plazme 46 zdravih odraslihdobrovoljaca (18 èna i 28 muškaraca) i 33 djece(dob: 3-17 godina). Nadalje je koncentracija slo-bodnog PS:Ag odre|ena i u plazmama 92 bole-snika na OAT kojima je prethodno odre|eno pro-trombinsko vrijeme. Srednja vrijednost koncen-tracije slobodnog PS:Ag u djece je iznosila 97,2%,a referentni raspon 68,2-136,2%, s tim da nijebilo statisti~ki zna~ajne razlike u vrijednostimaizme|u djevoj~ica i dje~aka. Za razliku od toga,dobivena je zna~ajna razlika (p<0,05) u koncen-tracijama slobodnog PS:Ag u muškaraca u od-nosu na ène (srednja vrijednost: 116,8% odnosno92,8%), pa su stoga utvr|eni zasebni referentnirasponi za muškarce (77,3-158,0%) i za ène(64,0-111,2%). U skupini bolesnika na OAT(INR=0,81-8,91) koncentracije slobodnog PS:Agiznosile su od 12,8% do 115,4% (srednja vrijed-nost: 53,9%). Za terapijsko podru~je OAT (INR=2,0-3,5) srednja vrijednost koncentracije slobod-nog PS:Ag iznosila je 49,0% (raspon: 27,2-73,3%).Zaklju~eno je kako spol ima zna~ajan utjecaj nakoncentracije slobodnog PS:Ag u zdravoj odraslojpopulaciji. Vrijednosti u muškaraca su više zapribli`no 26% u odnosu na one u èna.E-mail: [email protected]

house ELISA with specific antibodies to humanPS (Dako, Denmark) in the supernatant ofplasma after precipitation with PEG 6000.Plasma samples from 46 healthy adult volunteers(18 women, 28 men) and 33 healthy children, age3-17 years, were tested to establish referenceranges. Additionally, free PS:Ag was measured inplasma samples obtained from 92 patients receiv-ing OAT. The mean level of free PS:Ag in childrenwas 97.2% with a reference range of 68.2-136.2%and no significant difference between boys andgirls. Since a significant difference (p<0.05) wasobtained in free PS:Ag levels between men andwomen (mean 116.8% and 92.8%, respectively),sex specific reference ranges were determined(men: 77.3-158%, women: 64.0-111.2%). In thegroup of patients on OAT with INR values from0.81 to 8.91, the levels of free PS:Ag ranged from12.8% to 115.4% (mean 53.9%). For the therapeu-tic range (INR=2.0-3.5), the obtained mean levelof free PS:Ag was 49.0% (range: 27.2-73.3%). Ac-cordingly, sex has an important impact on freePS:Ag level only in adult population, resulting inapproximately 26% higher values in men than inwomen.E-mail: [email protected]

P9-26

DETECTION OF FETAL RHESUS-STATUSAND SEX BY USING CELL FREE FETAL

DNA IN MATERNAL PLASMA

J. Atamaniuk, A. Karimi, M.M. Müller

Kaiser Franz Josef Hospital,Institute for Laboratory Diagnosis, Vienna, Austria

The risk of severe hemolytic disease of the fetusand newborn due to Rhesus D incompatibility oc-curs in approximately 15% of pregnancies. In ad-dition, pregnancies with male Rh D positive fe-tuses are more severely affected by hemolytic dis-ease of the newborn. They have a three-fold mor-tality recorded in female Rh D positive fetuses.The aim of this study was to evaluate anoninvasive prenatal method to identify fetal RhD status and sex in Rh D negative pregnantwomen. Cell free plasma DNA was isolated from20 maternal plasma samples, 600 µl plasma, and

P9-26

OTKRIVANJE FETALNOG RHESUSSTATUSA I SPOLA POMO]U

BEZSTANI^NE FETALNE DNK UMAJÎNOJ PLAZMI

Atamaniuk J., Karimi A., Müller M.M.

Kaiser Franz Josef Hospital, Institute for LaboratoryDiagnosis, Be~, Austrija

Rizik od teške hemoliti~ne bolesti fetusa i no-voro|en~eta zbog nepodudarnosti Rhesus D jav-lja se u otprilike 15% trudno}a. Uz to, trudno}e smuškim Rh D pozitivnim fetusima zahva}ene suteòm hemoliti~nom boleš}u novoro|en~eta ismrtnost je tri puta viša nego kod ènskih Rh Dpozitivnih fetusa. Cilj ove studije bio je proci-jeniti neinvazivnu prenatalnu metodu identifici-ranja fetalnog Rh D statusa i spola u Rh D ne-gativnih trudnica. Bezstani~na plazmatska DNKizolirana je u 20 uzoraka maj~ine plazme, 600 µl



plazme, 2 µl je uporabljeno kao templat za re-akciju PCR. PCR je primijenjena za dokazivanjeRh D, SRY i beta globina kao referentnog gena.Gestacijska dob bila je od 10. do 38. tjedna. Ispi-tana su tri uzorka iz prvog trimestra, 8 uzorakaiz drugog trimestra, te 9 uzoraka iz tre}eg tri-mestra. Usporedba rezultata dobivenih pomo}uPCR i serološkom dijagnostikom nakon poro|ajapokazala je da su Rh D zna~ajke sukladno utvr-|ene u 19 od 20 uzoraka. La`no negativan rezul-tat zabilje`en je za jedan uzorak iz prvog trime-stra, mo`da zbog niske razine DNK fetalne plaz-me. Šesnaestoro novoro|en~adi bilo je Rh D po-zitivno, a ~etvoro Rh D negativno. Spol fetusato~no je odre|en u svim uzorcima. Naši podacipokazuju kako je naša metoda PCR uz primjenumaj~ine krvi prikladna za odre|ivanje spola i RhD fetusa po~evši od drugog trimestra.E-mail: [email protected]

2 µl were used as template for PCR reaction. PCRwas used to detect Rh D, SRY and beta globin asa reference gene. Gestational age was from 10th to38th week. Three samples from the first, 8 sam-ples from the second and 9 samples from the thirdtrimester were tested. Comparison of the resultsobtained by PCR with serologic diagnosis afterdelivery showed that Rh D characteristics weredetermined concordantly in 19 of 20 samples.One sample from the first trimester tested falsenegative, possibly due to low levels of fetalplasma DNA. Sixteen newborns were Rh D posi-tive and 4 were Rh D negative. The gender of thefetuses was correctly determined in all samples.Our data indicate that our PCR method usingmaternal blood is appropriate to determine thegender as well as Rh D of fetuses beginning withsecond trimester.E-mail: [email protected]


ZNANSTVENE RADIONICE Saèci 4. hrvatskog kongresa medicinskih biokemi~ara

ZR1FarmakogenetikaPharmacogenetics

Znanstvene radioniceScientific workshops

ZR1-1

FARMAKOGENETIKA: DIJAGNOSTI^KOSREDSTVO U LABORATORIJSKOJ

MEDICINI

Topi} E.

Klini~ki zavod za kemiju, Klini~ka bolnica “Sestre

milosrdnice”, Zagreb

Razlike u metabolizmu lijeka i odgovora nalijek izme|u dviju osoba s istom dozom i istomtjelesnom teìnom mogu biti izazvane prolaznimuzrocima, kao što su inhibicija i indukcija enzi-ma, ili trajnim uzrocima, kao što su genetskamutacija, delecija ili amplifikacija gena. Frek-vencija genetske promjene koja je ve}a od 1% upopulaciji izraàva se kao genetski polimorfizam.Farmakogenetski polimorfizmi se mogu o~itovatina farmakokinetskoj i farmakodinamskoj razini.Farmakokinetska razina bavi se polimorfizmimagena zbog kojih dolazi do promjena koncentracijalijeka i njegovih metabolita na mjestima njihovamolekularnog djelovanja (polimorfizmi enzimametabolizma lijekova, transporteri lijekova), dokse farmakodinamska razina bavi polimorfizmimagena povezanih s u~inkom i mehanizmom djelo-vanja lijeka, a nevezani su s njegovom koncen-tracijom (receptori, ionski kanali). Vezu izme|ugenetske predispozicije i u~inkovitosti lijeka pro-u~ava farmakogenetika na temelju kojih ispiti-vanja se utvr|uje specifi~ni fenotip. Na temeljusposobnosti metaboliziranja lijekova genetski sepolimorfizam povezuje s tri vrste fenotipova. Fe-notip ekstenzivnog metabolizma lijeka znakovitje za normalnu populaciju; fenotip slabog meta-bolizma udruèn je s nakupljanjem specifi~nihsupstrata lijeka i tipi~no je autosomno recesivnosvojstvo nastalo mutacijom i/ili delecijom obaju

ZR1-1

PHARMACOGENETICS: A DIAGNOSTICTOOL IN LABORATORY MEDICINE

E. Topi}


The variation in drug metabolism and drugresponse between two individuals of the samebody weight and on the same drug dosage can bedue to transient causes such as enzyme inhibitionand induction, or to permanent causes such asgenetic mutation, gene deletion or amplification.A genetic mutation occurring at a frequency of>1% in a population is designated as genetic poly-morphism. Pharmacogenetic polymorphisms canmanifest at the pharmacokinetic and pharmaco-dynamic levels. The pharmacokinetic level refersto gene polymorphisms that modify the concen-tration of a drug and its metabolites at the site oftheir molecular action (polymorphisms of drug-metabolizing enzymes, drug transporters),whereas the pharmacodynamic level refers topolymorphisms of the genes associated with thedrug effect and mechanism of action unrelated toits concentration (receptors, ion channels). Phar-macogenetics investigates the relationship be-tween genetic predisposition and drug efficacy, onthe basis of which studies the specific phenotypeis being defined. According to drug metabolizingability, genetic polymorphism is associated withthree phenotypes: extensive metabolizer pheno-type characteristic of normal population; poormetabolizer phenotype associated with accumula-tion of specific drug substrates, a typically auto-somal recessive trait generated by mutation and/or deletion of both alleles responsible for


Saèci 4. hrvatskog kongresa medicinskih biokemi~ara ZNANSTVENE RADIONICE

ZR1-2

UTJECAJ GENOTIPA CITOKROMA P450CYP2C9 NA OSJETLJIVOST PREMA

TERAPIJI VARFARINOM

Grdi} M.1, Coen D.2, Zadro R.2

1 Farmaceutsko-biokemijski fakultet,2 Klini~ki zavod za lab. dijagn. Medicinskog fakulteta

Sveu~ili{ta u Zagrebu i KBC “Zagreb”, Zagreb

Varfarin je oralni antikoagulans koji se naj-~eš}e rabi u klini~koj praksi za prevenciju i lije-~enje u bolesnika s tromboembolijskim poreme-}ajima. Doza lijeka koja je potrebna za postizanjeoptimalnog terapijskog u~inka razlikuje se u po-jedinih bolesnika i do 120 puta. Jedan od mnogo-brojnih ~imbenika koji utje~u na intraindividu-alnu varijabilnost u odgovoru na terapiju varfa-rinom je i genetski polimorfizam osnovnog en-zima koji metabolizira varfarin, citokroma P450CYP2C9 s varijantama CYP2C9*2 i CYP2C9*3sa sniènom enzimskom aktivnoš}u u odnosu nadivlji tip (CYP2C9*1). Utjecaj genotipa CYP2C9na osjetljivost prema terapiji varfarinom je ispi-tan u 63 bolesnika. Za svakog je bolesnika izra-~unata tjedna doza (TD) varfarina i odre|enoprotrombinsko vrijeme. Genotipizacija polimor-fizama CYP2C9 je izvedena lan~anom reakcijompolimeraze. Vrijednosti INR u terapijskom pod-ru~ju (2,0-3,5) dobivene su u 30 bolesnika, a u

ZR1-2

EFFECT OF CYTOCHROMEP450 CYP2C9 GENOTYPE ON WARFARIN

SENSITIVITY

M. Grdi}1, D. Coen2, R. Zadro2

1 School of Pharmacy and Biochemisty, University ofZagreb, and 2 Clinical Institute of Laboratory Diagnosis,

Zagreb University Hospital Center, Zagreb

Warfarin is an oral anticoagulant that is mostcommonly used for prevention and treatment inpatients with thromboembolic disorders. The dos-age required to achieve optimal therapeutic effectvaries up to 120-fold between individual patients.Interindi-vidual variability in response to warfa-rin therapy is attributed to a multitude of factors,including genetic polymorphism of the principalenzyme involved in warfarin metabolism, cyto-chrome P450 CYP2C9 with variant allelesCYP2C9*2 and CYP2C9*3. The effect of CYP2C9genotype on warfarin sensitivity was studied in63 warfarin-treated patients. In each patient theweekly dose of warfarin was recorded and pro-thrombin time was determined. Genotyping forCYP2C9*2 and CYP2C9*3 was performed byPCR RFLP. Therapeutic INR values (2.0-3.5)were achieved in 30 patients, whereas subthera-peutic INR values (1.5-2.0) were obtained in 19patients. In 10 patients, the INR values were

alela odgovornih za fenotipsku ekspresiju; feno-tip ultraekstenzivnog metabolizma rezultira po-ve}anim metabolizmom lijeka i to je autosomnodominantno svojstvo koje proizlazi iz amplifika-cije gena. Genetski polimorfizam transportera li-jeka i receptora stvara pak osnovu za fenotip spolaganom i brzom apsorpcijom lijeka, odnosnološu ili u~inkovitu interakciju s receptorima. Pri-stup farmakogenetskog probira u predvi|anjuodre|enog fenotipa zasnovan je na identifikacijialela koji pokazuju osjetljivost ve}u od 95%, atemelji se na PCR amplifikaciji i digestiji restrik-cijskim endonukleazama pojedina~nog polimorf-nog alela (single nucleotide polymorphism, SNP).Kao krajnji cilj istraìvanja farmakogenskih poli-morfizama postavlja se izrada farmakogenetskeosobne iskaznice za ostvarenje koje je neophodnobolje poznavanje mehanizama genskih predispo-zicija prema neèljenim reakcijama, razvoj teh-nologija za mapiranje SNPova, razvoj jeftinijihmetoda genotipizacije, te razrada eti~kih krite-rija koji trebaju osigurati gensku ravnopravnost.E-mail: [email protected]

phenotypic expression; and ultraextensive meta-bolizer phenotype associated with enhanced drugmetabolism, occurring as an autosomal domi-nant trait resulting from gene amplification. Ge-netic polymorphism of drug transporters andreceptors underlies a phenotype characterized byslow and fast drug absorption, respectively, i.e.poor or efficient interaction with receptors. Theconcept of pharmacogenetic screening in predict-ing particular phenotypes is based on identifica-tion of alleles showing >95% sensitivity by use ofPCR amplification and restriction endonucleasedigestion of single nucleotide polymorphism(SNP). The endpoint of the research in pharmaco-gene polymorphisms is development of personalpharmacogenetic card, which requires better un-derstanding of the mechanisms of gene predispo-sition to undesired responses, development oftechnologies for SNP mapping, development ofinexpensive genotyping techniques, and develop-ment of ethical criteria to ensure gene equity.E-mail: [email protected]



subterapijskom podru~ju (1,5-2,0) u 19 bolesnika.U 10 bolesnika vrijednosti INR bile su <1,5, a>3,5 u 4 bolesnika. U~estalost genotipovaCYP2C9 je iznosila: 66,7% za *1/*1, 20,6% za *1/*2, 11,1% za *1/*3 i 1,6% za *2/*3, a frekvencijealela *1, *2 i *3 bile su 0,83, 0,11 i 0,06. Zapostizanje terapijskog u~inka potrebne TD varfa-rina iznosile su 9,0-105,0 (medijan 33,4) mg, stim da su se zna~ajno razlikovale (p<0,05) kodbolesnika s genotipom *1/*1 (medijan 40,5; ra-spon: 23,2-105,0 mg) u odnosu na bolesnike s ge-notipom *1/*2 (medijan 31,5; raspon 10,5-46,5mg) i *1/*3 (medijan 23,6; raspon 9,0-63,0 mg).Za subterapijske vrijednosti INR medijani TD ubolesnika s genotipovima *1/*1, *1/*2 i *1/*3iznosili su 40,5, 30,7 i 16,1 mg. U 9/10 bolesnika svrijednostima INR <1,5 utvr|en je genotip *1/*1,a medijan TD varfarina iznosio je 26,2 mg. Ana-liza genotipa CYP2C9 moè se upotrijebiti zapredvi|anje osjetljivosti na terapiju varfarinom sobzirom na to da je kod heterozigota s polimor-fnim alelima potrebna zna~ajno manja doza zapostizanje jednakog antikoagulacijskog u~inka uodnosu na homozigote za divlji tip.E-mail: [email protected]

<1.5, while in 4 patients they were >3.5. The dis-tribution of CYP2C9 genotypes was as follows:66.7% for *1/*1, 20.6% for *1/*2, 11.1% for *1/*3 and 1.6% for *2/*3. Allele frequencies for *1,*2, and *3 were 0.83, 0.11 and 0.06, respectively.The weekly warfarin doses required to achievetherapeutic INR range (2.0-3.5) ranged between9.0-105.0 mg (median:33.4 mg), and were signifi-cantly different (p<0.05) in patients with wildtype *1/*1 (median dose 40.5, range 23.2-105.0mg) genotype as compared to patients with *1/*2(median dose 31.5, range 10.5-46.5 mg) and *1/*3 genotype (median dose 23.6, range 9.0-63.0mg). In patients with subtherapeutic INR values,the median weekly dose was 40.5 mg in patientswith *1/*1 genotype, 30.7 mg in patients with*1/*2 genotype and 16.1 mg in patients with *1/*3 genotype. Nine of 10 patients with INR values<1.5 were homozygous for CYP2C9*1 allele, andthe median weekly dose was 26.2 mg. Asheterozygotes with polymorphic alleles requiresignificantly lower doses to achieve the same anti-coagulant effect as wild type homozygotes, theanalysis of CYP2C9 genotype could predict war-farin sensitivity.E-mail: [email protected]

ZR1-3

FARMAKOGENETIKA U TERAPIJIEPILEPSIJE: VA@NOST POLIMORFIZAMA

CYP2C9, CYP2C19 I GENA MDR1 ZAINDIVIDUALIZACIJU TERAPIJE

FENITOINOM

Boìna N., Grani} P., Lali} Z., Hajnšek S., TramišakI., Stavljeni}-Rukavina A.


Farmakokinetski parametri antiepileptika fe-nitoina znatno variraju me|u bolesnicima. Feni-toin je supstrat citokroma P-450 enzimaCYP2C9, CYP2C19 i transportnog P-glikoprote-ina kodiranog genom MDR1. Cilj rada bio je ana-lizirati genske polimorfizme CYP2C9, CYP2C19 iMDR1 i odrediti njihov utjecaj na biološku raspo-loìvost fenitoina u 64 osobe; ustanoviti korelirali koncentracija fenitoina i metaboli~ki odnos fe-nitoina (FMR) prema glavnom metabolitu u plaz-mi 5-(4-hidroksifenil)-5-fenilhidantoin (p-HPPH)s polimorfizmom CYP2C9, CYP2C19 i genaMDR1. Metode PCR-RFLP su primijenjene za ot-krivanje naj~eš}ih alela: CYP2C9*1,*2,*3,CYP2C19*1,*2,*3; te za otkrivanje mutacije

ZR1-3

PHARMACOGENETICS IN THERAPY OFEPILEPSY: ROLE OF CYP2C9, CYP2C19AND MDR1 GENE POLYMORPHISMS IN

PHENYTOIN THERAPYINDIVIDUALIZATION

N. Boìna, P. Grani}, Z. Lali}, S. Hajnšek, I.Tramišak, A. Stavljeni}-Rukavina


The anticonvulsant drug phenytoin (PHT) ex-hibits nonlinear pharmacokinetics with largeinterindividual variation. Phenytoin is a sub-strate of the cytochrome P-450 enzymes CYP2C9and CYP2C19, and P-glycoprotein transporter,encoded by the MDR1 gene. The aim of the studywas to examine the genetic polymorphisms ofCYP2C9, CYP2C19 and MDR1, and their effectson the phenytoin bioavailability in 64 individu-als, and to determine whether phenytoin concen-trations and phenytoin metabolic ratios (PMR) tothe major phenytoin metabolite (HPPH) correlatewith these polymorphisms . The methods of PCR-RFLP were used for the most frequent alleles:CYP2C9*1,*2,*3, CYP2C19*1*2, *3, and C3435T



C3435T u genu MDR1. Vrijednosti koncentracijafenitoina i metabolita u plazmi 12 h nakon uzi-manja 300 mg lijeka per os sluìle su za fenoti-pizaciju. Analiza fenitoina (FT) i metabolita (p-HPPH) provedena je metodom HPLC. U~estalostalela CYP2C9*1,*2,*3 bila je 0,76, 0,145 i 0,095,za CYP2C19*1,*2 iznosila je 0,85 i 0,15. U~esta-lost genotipova MDR1bila je 0,281 za CC, 0,468za CT i 0,25 za TT. Najviše koncentracije fenito-ina zabiljeène su u osoba s genotipom *1/*1, anajniè (statisti~ki zna~ajno razli~ite, p=0,046) uosoba s genotipom *2/*2 i *3/*3. Metaboli~ki od-nosi u genotipu *1/*1 bili su statisti~ki zna~ajnorazli~iti (p=0,002) prema *1/*2 ili *1/*3 genotipu i(p=0,042) prema *2/*2 ili *3/*3 genotipu. Meta-boli~ki odnosi u genotipu CC-MDR1 bili su sta-tisti~ki zna~ajno razli~iti od genotipa TT (p=0,043). Polimorfizmi CYP2C9, CYP2C19 i MDR1gena imaju utjecaj na bioraspoloìvost fenitoinate stoga farmakogenetska analiza moè biti kori-sna u individualizaciji terapije.E-mail: [email protected]

mutations of MDR1 gene. The 12 h plasma con-centrations after 300 mg oral dose of phenytoinwere used for phenotyping. PHT and p-HPPHwere analyzed by the HPLC method. The frequen-cies of CYP2C9*1,*2,*3 were 0.760, 0.145, and0.095; and of CYP2C19*1,*2 they were 0.85 and0.15, respectively. The frequencies of MDR1 geno-types were 0.281 for CC, 0.468 for CT, and 0.25for TT. The highest phenytoin concentrationswere recorded in individuals with *1/*1 geno-type, and lowest (p=0.046) in individuals with*2/*2 i *3/*3 genotypes. PMR in *1/*1 genotypewere different (p=0.002) to *1/*2 ili *1/*3 geno-types; (p=0.042) to *2/*2 or *3/*3 genotypes.PMR in *1/*1 genotypes were different (p=0.002)to *1/*2 or *1/*3 genotypes; (p=0.004) to *2/*2or *3/*3 genotypes. PMR in CC-MDR1 genotypewere different (p=0.043) to TT genotype. TheCYP2C9, CYP2C19 and MDR1 gene polymor-phisms were found to influence phenytoin bio-availability, thus pharmacogenetic studies couldprove useful in therapy individualization.E-mail: [email protected]

ZR1-4

GENSKI POLIMORFIZAM CYP2D6 ULIJEÊNJU NEUROPSIHIJATRIJSKIH

BOLESNIKA

Štefanovi} M.


CYP2D6 je enzim koji metabolizira neke umozgu prisutne endogene spojeve, a tako|er ivelik broj lijekova me|u kojima su antipsihotici iantiparkinsonici koji ~esto izazivaju štetne nus-pojave. Oslabljeni metaboli~ki kapacitet enzimamogao bi biti povezan s povišenim rizikom premaduševnim i neurološkim bolestima te rizikomnastanka štetnih nuspojava pri njihovu lije~enju.Prvi cilj istraìvanja bio je metodom multipleksalel specifi~ne lan~ane reakcije polimeraze (poly-merase chain reaction, PCR) utvrditi moè li seodre|ivanjem nefunkcionalnih alela CYP2D6*3,*4, *6, *7, i *8 u bolesnika predvidjeti odgovor nalije~enje i time utjecati na bolji i sigurniji odabirlijeka. Drugi cilj je bio usporediti 145 zdravih is-pitanika s duševnim (shizofrenija, n=73; depre-sija, n=36) i neurološkim (Parkinsonova, PB;n=41; Alzheimerova bolest, AB; n=35) bolesni-cima te ustanoviti postoji li u bolesnika s nefunk-cionalnim enzimom povišena sklonost prema timbolestima. Rezultati usporedbe duševnih bolesni-

ZR1-4

CYP2D6 GENETIC POLYMORPHISM INTREATMENT OF NEUROPSYCHIATRIC

PATIENTS

M. Štefanovi}


CYP2D6 enzyme metabolizes some brain cir-culating endogenous compounds as well as manydrugs, antipsychotics and antiparkinsonics. Pa-tients using these drugs often suffer from severeside effects. Decreased metabolic capacity couldalso be associated with an increased risk of psy-choses and neurologic diseases, and also with arisk of serious side effects during treatment. Thefirst aim of our study was to determine (by multi-plex allele specific PCR) if testing for nonfunc-tional CYP2D6 alleles *3, *4, *6, *7 and *8 couldpredict patient’s response to treatment, and thuslead to a more appropriate and safer choice oftreatment. The second aim was to compare 145healthy subjects with patients and to determine ifthose with nonfunctional enzyme were at an in-creased risk of disease development. We investi-gated psychotic (schizophrenia, n=73; depression,n=36) and neurologic (Parkinson’s, PD; n=41;Alzheimer’s disease, AD; n=35) patient groups.Comparison of psychotic patients with distinctive



ka s izraènim nuspojavama na lijekove kojeuzimaju (n=38) potvrdili su zna~ajno povišenuu~estalost *4 (relativni rizik, RR=2,6) i *6(RR=5,3) nefunkcionalnih alela CYP2D6 u uspo-redbi s bolesnicima bez nuspojava (n=97). U sku-pini s nuspojavama na|eno je i znatno više *4/*4genotipa i sporometaboliziraju}eg fenotipa (RR=7,1). U bolesnika s PB razlika je za podskupinu snuspojavama (n=28) prema podskupini bez nus-pojava (n=28) bila manja i odnosila se samo naCYP2D6*4 alel (RR=4,4). Druga razina istraì-vanja potvrdila je za alel CYP2D6*4 povišen rizikprema shizofreniji (RR=2,5), a za potpuno neak-tivni enzim (oba alela nefunkcionalna) relativnije rizik iznosio 5,0. U skupini depresija (n=36)nismo utvrdili povezanost s polimorfizmomCYP2D6. U podskupinama PB podijeljenim pre-ma teìni bolesti (ljestvice Hoehn i Yahr) utvrdilismo da su nosioci alela *4 skloniji razvitku teìhoblika bolesti 2,7 puta. Homozigoti za neaktivnealele skloniji su pogoršanju bolesti ~ak 7,9 puta.Prema rezultatima za AB, RR za alel *4 iznosio je3,7, dok homozigoti nisu pokazali razliku premazdravim kontrolama. Ispitivanjem polimorfizmaCYP2D6 mogu}e je predvidjeti odgovor bolesnikana lije~enje. CYP2D6 se tako|er moè promatratii kao jedan od ~imbenika rizika za nastanakshizofrenije i PB, dok je njegova zna~ajnost unastanku AD manja.E-mail: [email protected]

side effects (n=38) and those without them (n=97)confirmed a significantly higher frequency ofCYP2D6*4 nonfunctional allele (relative risk,RR=2.6) and *6 (RR=5.3). The side effect groupalso had a significantly higher frequency of *4/*4genotype as well as poor metabolizer phenotype(RR=7.1). The difference between the side effectsubgroup (n=28) of PD patients and non-side ef-fect subgroup (n=28) was lower and was relatedjust to CYP2D6*4 allele (RR=4.4). The secondstudy level confirmed an increased risk for per-sons with CYP2D6*4 allele to develop schizophre-nia (RR=2.5). Persons with completely inactiveenzyme were at an even greater risk (RR=5.0). Inthe group with depressions (n=36) we found noassociation with CYP2D6 polymorphism. In PDsubgroups ranked according to the disease sever-ity (Hoehn and Yahr staging), carriers of *4 allelewere found to have a 2.7-fold risk of developingmore severe forms of the disease. Homozygotes forinactive alleles were at a 7.9-fold risk to develop amore severe form of the disease. The results forAD showed RR for *4 allele to be 3.7, whilehomozygotes showed no difference from controls.It is possible to predict the patient’s therapeuticresponse by CYP2D6 genotyping. CYP2D6 poly-morphism may also be considered as one of themajor risk factors in the development of schizo-phrenia and PD, whereas its role in AD develop-ment is less significant.E-mail: [email protected]

ZR1-5

HOMOZIGOT CYP2C9*3 ALELA - PRIKAZSLUÂJA BOLESNIKA NA TERAPIJI

VARFARINOM

Samardìja M.1, Topi} E.2, Štefanovi} M.2

1 KB Osijek, Zavod za transfuzijsku medicinu, Osijek;2 KB “Sestre milosrdnice”, Klini~ki zavod za kemiju, Zagreb

Uloga genske varijabilnosti u interindividu-alnom odgovoru na lijekove predstavlja zna~ajnopodru~je znanstvenog interesa s mogu}im va`-nim utjecajem na terapijski odgovor. CYP2C9 jeodgovoran za metabolizam S-varfarina. UzCYP2C9*1 alel (divlji tip) naj~eš}e zastupljenialeli u bijelaca su CYP2C9*2 i CYP2C9*3 koji uhomozigotnom obliku dovode do stvaranja enzi-ma niske aktivnosti (12%, odnosno manje od 5%)u odnosu na divlji tip. Cilj studije bio je ispitatiutjecaj polimorfizma CYP2C9 na varijabilnostdoze varfarina. U ispitivanje je bilo uklju~eno358 ispitanika: 181 bolesnik na terapiji varfari-

ZR1-5

A HOMOZYGOTE FOR CYP2C9*3 ALLELICVARIANT - CASE REPORT OF A PATIENT

ON WARFARIN THERAPY

M. Samardìja1, E. Topi}2, M. Štefanovi}2

1 Department of Transfusion Medicine, Osijek UniversityHospital, Osijek; 2 Clinical Institute of Chemistry, Sestre

milosrdnice University Hospital, Zagreb

The role of genetic variability in interindi-vidual responsiveness to drugs is an emergingarea of interest with potentially important thera-peutic consequences. CYP2C9 is responsible forthe metabolism of S-warfarin. Besides its wildtype allele CYP2C9*1, two common allelic vari-ants are known in Caucasians, CYP2C9*2 andCYP2C9*3, which in homozygous state expressenzymes of very low activity (only 12% and 5% oftotal wild type activity, respectively). The aim ofthe study was to evaluate the impact of CYP2C9polymorphism on the variability of warfarin dose



ZR2-1

LABORATORIJSKI STANDARDI UDIJAGNOSTICI I PRA]ENJU TERAPIJE

AUTOIMUNIH BOLESTI

Malenica B.

Zavod za imunologiju, Klini~ki zavod za laboratorijskudijagnostiku, KBC “Zagreb” i Referentni centar za klini~ko-

laboratorijsku imunodijagnostiku hematoloških iimunoloških bolesti Ministarstva zdrastva Republike

Hrvatske, Zagreb

Laboratorijska dijagnostika autoimunih bole-sti uklju~uje odre|ivanje parametara op}e labo-ratorijske dijagnostike i specifi~ne imunodija-gnosti~ke metode. Parametri osnovne laborato-

ZR2-1

LABORATORY STANDARDS IN THEDIAGNOSIS AND THERAPY MONITORING

OF AUTOIMMUNE DISEASES

B. Malenica

Laboratory of Immunology, Clinical Institute of LaboratoryDiagnosis, Zagreb University Hospital Center and Reference

Center for Clinical Laboratory Immunodiagnosis ofHematologic and Immune Diseases of the Ministry of

Health, Republic of Croatia, Zagreb

Laboratory diagnosis of autoimmune diseasesincludes parameters of both standard laboratoryexaminations and specific immunodiagnosticprocedures. Standard laboratory examinations

ZR2Autoimune bolesti

Autoimmune diseases

nom (Marivarin) dulje od mjesec dana (102 ène i79 muškaraca, prosje~na dob 60 godina, raspon19-80 godina) i 177 dobrovoljnih davatelja krvi(61 èna i 116 muškaraca, prosje~na dob 34,5godina, raspon 18-65 godina) kao kontrolna sku-pina. Genotipizacija CYP2C9*1, *2 i *3 alela ra-|ena je PCR-RFLP metodom primjenom restrik-cijske endonukleaze Ava II, Nsi I i Kpn I. U~e-stalost alela CYP2C9*2 i CYP2C9*3 u kontrolnojskupini iznosila je 12,4% odnosno 3,7%, a u sku-pini bolesnika 17,4% i 6,6% (nema statisti~kizna~ajne razlike). U skupini bolesnika otkrivenje jedan genotip CYP2C9*3/*3. Radilo se o 56-go-dišnjem muškarcu koji je hospitaliziran pod dg.Insultus cerebrovascularis, Fibrillatio atriorum,Hypertensio arterialis. Po~etna doza Marivarinaiznosila je 4,0 mg (2,8 INR), optimalna doza 1,5mg (1,83 INR), duìna uzimanja lijeka 8 mj.,vrijeme postizanja terapijskog optimuma 60dana. Dakle, bolesnik s rijetkim genotipomCYP2C9*3/*3 zahtijevao je vrlo nisku dozu varfa-rina i dugo vrijeme za postizanje terapijskogoptimuma. Krvarenje je izbjegnuto pa`ljivim i~estim pra}enjem PV-a, stoga bi odre|ivanje ge-notipa CYP2C9 prije uvo|enja terapije zasigurnoimalo i ekonomsku opravdanost.E-mail: [email protected]

requirements. A total of 358 subjects were in-cluded in the study. Patient group included 181patients on warfarin (Marivarin) therapy (102 fe-male and 79 male, mean age 60 years, range 19-80 years). All patients had been treated with war-farin therapy for more than a month. Controlgroup included 177 unrelated healthy volunteersfrom the Croatian general population (61 femaleand 116 male, mean age 34.5 years, range 18-65years). PCR-RFLP genotyping for CYP2C9*1, *2and *3 alleles was performed by using Ava II, NsiI and Kpn I restriction endonucleases. The fre-quency of CYP2C9*2 and CYP2C9*3 alleles inthe control group was 12.4% and 3.7%, and in thepatient group 17.4% and 6.6%, respectively (nostatistically significant difference). In the patientgroup, however, one homozygous CYP2C9*3/*3genotype was detected. A 56-year-old man washospitalized under the diagnosis of Insultuscerebrovascularis, Fibrillatio atriorum, Hyper-tensio arterialis. Therapy was initiated with a4.0 mg dose of Marivarin (2.8 INR), whereas opti-mal dose was 1.5 mg. (1.83 INR), duration oftherapy 8 months, time of optimization therapy60 days. The patient with rare CYP2C9*3/*3genotype required a very low dose of warfarinand very long time for therapy optimization.There was no bleeding because of careful and of-ten PT monitoring, so CYP2C9 genotyping priorto therapy initiation also appears to be cost effec-tive.E-mail: [email protected]



rijske dijagnostike (SE, CRP, proupalni citokini)omogu}uju razlikovanje upalnih od neupalnih,odnosno autoimunih od degenerativnih i para-reumatskih bolesti. Specifi~na imunodijagnosti-ka uklju~uje dokazivanje specifi~nih autoanti-tijela, imunih T-limfocita (citokina), poreme}ajasustava komplementa, te odre|ivanje haplotipaHLA. Unato~ navedenim mogu}nostima labora-torijski imunodijagnosti~ki standard u dijagno-stici stanovite autoimune bolesti je dokaz spe-cifi~nih serumskih autoantitijela i njihovih cilj-nih autoantigena. Prikazati }emo algoritme do-kazivanja antinuklearnih antitijela (ANA), anti-neutrofilnih citoplazmatskih antitijela (ANCA),te skupine autoantitijela koja obilje`avaju auto-imune bolesti jetre i crijeva. Istaknuti }emo zna-kovita autoantitijela za pojedinu autoimunu bo-lest, povezanost razine (titar, koli~ina) stanovitogautoantitijela s aktivnoš}u i raširenoš}u bolesti,te vrijednost stanovitog autoantitijela u klini~kojkontroli bolesnika i procjeni djelotvornosti tera-pije. Nasuprot tome, odre|ivanje citokina vrlo seselektivno rabi u rutinskoj laboratorijskoj imu-nodijagnostici autoimunih bolesti. Oni su va`ni urazumijevanju imunopatogeneze i pouzdan te-melj za primjenu i kontrolu djelotvornosti speci-fi~ne imunoterapije stanovite autoimune bolesti.Analiza sustava komplementa doprinosi dijagno-sti~kom postupku i klini~kom pra}enju bolesnikas autoimunim bolestima uzrokovanim imuno-kompleksima.E-mail: [email protected]

(SE, CRP, proinflammatory cytokines) make dis-tinctions between degenerative or pararheumaticand systemic autoimmune diseases. Specificimmunodiagnostic procedures include detectionof specific autoantibodies, immune T-lymphocytes(cytokines), complement function and HLA haplo-type determination. Besides all these possibilities,laboratory standard in the immunodiagnosis ofautoimmune diseases is the determination of spe-cific serum autoantibodies and their targetautoantigens. Detection algorithm of antinuclearantibodies (ANA), antineutrophil cytoplasmicautoantibodies (ANCA) and the group od auto-antibodies which characterize liver and bowelautoimmune diseases is presented. Furthermore,autoantibodies which characterize each of thesystemic rheumatic diseases, systemic vascu-litides, and each of the liver and bowelautoimmune diseases are presented, and theircorrelation with clinical disease activity andtreatment efficacy is discussed. On the otherhand, laboratory determination of cytokines isvery selective in routine immunodiagnosis.Firstly, their detection is obvious in planning andclinical monitoring of therapeutic efficacy of spe-cific immunotherapy. Complement analysis helpsus in the diagnostic procedure and clinical moni-toring of patients with “immune complex”autoimmune diseases.E-mail: [email protected]

ZR2-2

NOVI BIOMARKERI U DIJAGNOSTICI IPRA]ENJU BOLESNIKA S

REUMATOIDNIM ARTRITISOM

Te{ija A.

Klini~ki zavod za kemiju, Klini~ka bolnica“Sestre milosrdnice”, Zagreb

Klini~ke manifestacije koje prate reumatoidniartritis (RA) rezultat su destruktivnih procesa uzglobnoj hrskavici, sinovijalnom i koštanom tki-vu. U svrhu procjene te`ine bolesti u upotrebi subrojne standardizirane metode stupnjevanja naosnovi intenziteta boli i razine pokretljivosti. La-boratorijsko pra}enje bolesnika s RA ograni~enoje uglavnom samo na pokazatelje upalnog proce-sa, kao što su CRP ili SE, koji nisu specifi~nisamo za procese u zglobu i slabo koreliraju sošte}enjem hrskavice. Radiografska metoda još

ZR2-2

NEW BIOMARKERS IN THE DIAGNOSISAND MONITORING OF PATIENTS WITH

RHEUMATOID ARTHRITIS

A. Te{ija


Clinical manifestations of rheumatoid arthri-tis (RA) are the results of destructive processes injoint cartilage, synovial and bone tissues. To as-sess the severity of the disease, a number of stand-ardized rating systems according to pain and mo-bility indices are in use. Laboratory monitoring ofRA patients is still limited mainly to inflamma-tion parameters such as CRP and ESR, which arenot joint specific and correlate poorly with carti-lage damage. Radiography is still a gold stand-ard to assess joint damage in RA, although it al-



uvijek predstavlja zlatni standard za procjenustupnja ošte}enja zglobova u RA, iako ne omo-gu}uje rano otkrivanje ošte}enja niti je prikladnaza pra}enje uspješnosti terapije. Specifi~ni i os-jetljivi biokemijski markeri koji odraàvaju pore-me}aje u metabolizmu hrskavi~nog, sinovijalnogili koštanog tkiva mogli bi imati presudnu uloguu otkrivanju ranog ošte}enja, procjeni rizika zarazvoj izrazito destruktivnog oblika RA, a time iu odabiru terapije. Potencijalno vrijedni bioke-mijski markeri odnose se na molekule svih trijutkiva koja zahva}a destruktivni proces: hrska-vice, sinovijuma i subhondralne kosti. Osnovnekomponente izvanstani~nog matriksa zglobnehrskavice su kolagen tipa II. i proteoglikan agre-kan, pa fragmenti ovih dvaju proteina pred-stavljaju potencijalne biokemijske biljege sinteze(hondroitin sulfat, propeptidi prokolagena tipaII.), odnosno razgradnje hrskavice (fragmentiproteinskog dijela agrekana, keratan sulfat, piri-dinolin, alfa lanci i telopeptidi kolagena tipa II.).Uz fragmente agrekana i kolagena, biljeg sintezehrskavi~nog tkiva je i protein YKL-40, odnosnoCOMP (cartilage oligomeric matrix protein), ma-triksne metaloproteinaze (MMP) i njihovi inhi-bitori (TIMP) kao biljezi razgradnje. Potencijalnibiljezi aktivnosti sinovijalnog tkiva su fragmentikolagena tipa I. i III., te nekolagenski proteiniCOMP, hijaluronat, MMP i TIMP. Kona~no, po-kazatelji koštane pregradnje su N i C propeptidiprokolagena tipa I., koštana ALP i osteokalcin zasintezu, odnosno deoksipiridinolin, piridinolin,N- i C- telopeptidi i BSP (bone sialoprotein) zarazgradnju. Upotreba navedenih biljega još jeuvijek ograni~ena nedovoljnim poznavanjem me-tabolizma i klirensa ovih molekula, kao i dopri-nosa ostalih tkiva njihovoj koncentraciji u sino-vijalnoj teku}ini, serumu i mokra}i.E-mail: [email protected]

lows neither early detection of joint tissue damagenor efficient monitoring of treatment. Specificand sensitive biochemical markers reflecting ab-normalities in the metabolism of cartilage, syno-vial and bone tissues could have a major role inthe detection of early damage and in identifyingpatients at a high risk of rapid destructive type ofRA. Potentially valuable biomarkers include mol-ecules of each of the three tissues affected by de-structive processes: cartilage, synovium andsubchondral bone. The main components of theextracellular matrix of articular cartilage aretype II collagen and aggrecan, so that the frag-ments of these two proteins represent potentialbiomarkers of synthesis (chondroitin sulfate, typeII procollagen propeptides) and degradation (coreprotein fragments, keratan sulfate, pyridinoline,alpha chains and telopeptides of type II collagen).Protein YKL-40 is also a marker of cartilage syn-thesis, whereas COMP (cartilage oligomeric ma-trix protein), matrix metalloproteinases (MMPs)and their inhibitors (TIMPs) are markers of carti-lage degradation. Candidate markers of synovialtissue activity are fragments of type I and III col-lagen molecules and noncollagen proteins COMP,hyaluronate, MMPs and TIMPs. Finally, markersof bone turnover are type I procollagen N- and C-propeptides, bone ALP and osteocalcin for synthe-sis, and deoxypyridinoline, pyridinoline, N- andC-telopeptides and BSP (bone sialoprotein) fordegradation. Use of these biomarkers is still lim-ited by insufficient knowledge of the metabolismand clearance of these molecules as well as of thecontribution of nonjoint tissues to their synovialfluid, serum and urine concentrations.E-mail: [email protected]

ZR2-3

AUTOIMUNI ASPEKTI CELIJAKIJE ICROHNOVE BOLESTI U DJECE

@iì} V.1, Brumen V.2, Radalj @.3, Naki} M.1

1 Klinika za dje~je bolesti Zagreb, Medicinski fakultetSveu~ili{ta u Zagrebu 2 [kola narodnog zdravlja “Andrija[tampar”; 3 Institut za medicinska istraìvanja i medicinu

rada, Zagreb

Celijakija je kroni~na imuno posredovana bo-lest tankog crijeva, klini~ki obiljeèna malap-sorpcijom zbog trajnog nepodnošenja glutena.Crohnova bolest je kroni~na upalna bolest još

ZR2-3

AUTOIMMUNE ASPECTS OF CELIAC ANDCROHN’S DISEASE IN CHILDREN

V. @iì}1, V. Brumen2, @. Radalj3, M. Naki}1

1 University Department of Pediatrics, Zagreb UniversityHospital Center; 2 Andrija Štampar School of Public Health,3 Institute of Medical Research and Occupational Medicine,

Zagreb

Celiac disease is a chronic, immune-mediateddisease, presented as malabsorption due to per-manent gluten intolerance. Crohn’s disease is achronic inflammatory disease of as yet unknown



uvijek nerazjašnjene etiologije, koja naj~eš}ezahva}a krajnji dio tankog crijeva, ali se moèproširiti i na cijeli probavni sustav. Cilj rada jeusporedno prikazati doseg seroloških testova udijagnosticiranju i klini~kom pra}enju tijeka ce-lijakije odnosno Crohnove bolesti. Preventivni iprediktivni dosezi testa na antiglijadinska pro-tutijela razreda IGA [AGA-IgA] odnosno razredaIgG [AGA-IgG], testa na endomizijalna protuti-jela razreda IgA [EMA-IgA] i testa na protutijelarazreda IgA na tkivnu transglutaminazu [tTG-IgA], koji se rabe u serodijagnostici celijakije,prikazani su pokazateljima osjetljivosti (O), spe-cifi~nosti (S) te pozitivne prediktivne vrijednosti(PPV). Usporedno se prikazuju pokazatelji tes-tova koji se rabe u serodijagnostici Crohnove bo-lesti (test na antineutrofilna citoplazmatska pro-tutijela [ANCA] i test na protutijela na Saccharo-myces cerevisiae [ASCA]. Test AGA-IgA pokazujevrlo zadovoljavaju}e zna~ajke: O=91%, S=92%,PPV=95%. Test AGA-IgG je izrazito osjetljiv(O=97%), no niskospecifi~an test (S=74%), ~imemu je umanjena i PPV (87%). Osobito su zado-voljavaju}e zna~ajke testa EMA-IgA (O=93%;S=96%; PPV=98%). Me|utim, daleko najboljimpokazao se test tTG-IGA (O=96%, S=99%,PPV=99%). U oboljelih od Crohnove bolesti ovi sutestovi mahom negativni (s iznimkom ina~e ni-skospecifi~nog testa AGA-IgG kao op}enitog po-kazatelja upalnog crijevnog procesa). Stoga serabi kombinacija testova ANCA/ASCA relativnoniske osjetljivosti (O=68%), ali zato visoke spe-cifi~nosti (S=92%). Dakle, zna~ajke seroloških te-stova koji se rabe u dijagnosticiranju celijakijeomogu}uju njihovu primjenu u ranoj dijagnosticibolesti, pra}enju njezina tijeka u razdoblju bez-glutenske dijete, odre|ivanju najpogodnijeg ~asaza biopsiju, otkrivanju asimptomatskih oblikabolesti u klini~ki zdravih srodnika i osoba u kojihje celijakija pridruèna a ne osnovna bolest, te umasovnim preventivnim pregledima op}e popu-lacije. Nedostatnost i nespecifi~nost serodija-gnostike Crohnove bolesti upu}uje klini~are na toda uporište za dijagnozu ove bolesti traè u kli-ni~kom o~itovanju i kolonoskopskim nalazimabolesnika.E-mail: [email protected]

etiology, most commonly affecting terminal ileum,however, possibly spreading throughout the diges-tive system. The aim is to present in parallel thescope of serologic tests used in the diagnosis andclinical follow-up of celiac disease and Crohn’s dis-ease. The scope of tests for antigliadin class IgAand class IgG antibodies (AGA-IgA and AGA-Ig),antiendomysial class IgA antibodies (EMA-IgA)and tissue transglutaminase class IgA antibodies(tTG-IgA) used in the prevention and prediction ofchildhood celiac disease is presented by their sensi-tivity (S), specificity (Sp) and positive predictivevalue (PPV). The same parameters concerning se-rodiagnosis of Crohn’s disease (antineutrophil cy-toplasmic antibody (ANCA) and anti-Saccharomy-ces cerevisiae antibody test (ASCA) are given inparallel. The characteristics of AGA-IgA testshowed it to be very satisfactory: S=91%, Sp=92%,PPV=95%. AGA-IgG test was found to be highlysensitive (S=97%), but of a low specificity(Sp=74%), the latter diminishing its PPV (87%).Especially satisfactory were the characteristics ofEMA-IgA test (S=93%, Sp=96%, PPV=98%). How-ever, tTG- IgA test turned out to be superior to all(S=96%, Sp=99%, PPV=99%). In patients sufferingfrom Crohn’s disease, the aforementioned testswere mostly negative (with the exception of other-wise low-specific AGA-IgG test, which generally in-dicates bowel inflammation). Therefore, a combi-nation of ANCA/ASCA tests is used, which has arelatively low sensitivity (68%) but high specificity(92%). Accordingly, the characteristics of serologictests used in the diagnosis of celiac disease allowfor their use in the early diagnosis, follow-up dur-ing the period of gluten-free diet, assessment ofoptimal biopsy timing, identification of silentforms of the disease in clinically healthy relativesand in patients mainly suffering from some otherdisease, and in mass screening of the general popu-lation. As for Crohn’s disease, due to the insuffi-ciency and nonspecificity of the serodiagnosis, cli-nicians are forced to rely upon clinical presenta-tion and colonoscopic findings.E-mail: [email protected]



ZR2-4

DISTRIBUCIJA O APO H OVISNIHAUTOPROTUTIJELA I APO H FENOTIPOVA

UNUTAR ODABRANE SKUPINEISPITANIKA KOJI ISKAZUJU LUPUS

ANTIKOAGULANS

Vogrinc @., Trbojevi}-êpe M., Coen D.,Stavljeni}-Rukavina A.

Klini~ki zavod za laboratorijsku dijagnostiku, Klini~kibolni~ki centar ”Zagreb”, Zagreb

Na|eno je da ApoH (beta 2-glukoprotein I)izgra|uju pet ’suchi’ domena (I-V) te da postojinjegov genetski polimorfizam. Naj~eš}a su trialela (ApoH1, ApoH2, ApoH3) koji odre|uju petfenotipova. ApoH je tako|er ciljni antigen zaodre|enu skupinu ’antifosfolipidnih’ autoprotu-tijela. Neka o Apo H-ovisna protutijela tako|eriskazuju i aktivnost lupus antikoagulansa (LAK).Studije ukazuju na to da je njihova prisutnostpovezana s ve}im rizikom za razvoj tromboze,poba~aja i trombocitopenije. Cilj rada bio je ispi-tati mogu}nost da su kod manje u~estalih alelaApoH prisutne strukturne ili konformacijskepromjene u ’o LAK-ovisnim’ domenama, što moèpotaknuti stvaranje ovoga tipa o ApoH-ovisnog/LAK+ autoimunog odgovora. Izabrali smo ispita-nike s produljenim APTT vremenom, potvr|e-nom LAK aktivnoš}u i bez dokazanih ili s u vrloniskim titrovima prisutnim protutijelima na ’~i-sti’ kardiolipin. Mnogi su ispitanici imali nekeklini~ke i serološke zna~ajke sistemskog erite-matoznog lupusa i visoku u~estalost klini~kihsimptoma antifosfolipidnog sindroma. Protutijelaovisna o ApoH dokazana su komercijalnim kom-pletom pretraga antikardiolipin/apoH ELISA.Fenotipovi ApoH dokazani su metodom izoelek-tri~nog fokusiranja i imunoblota. Naši su rezul-tati pokazali da je 63,5% (47/74) naših izabranihispitanika (LAK+) tako|er imalo i protutijela naApoH. Dokazana je visoka pozitivna korelacijaizme|u LAK aktivnosti i titrova protutijela naapoH. Me|utim, ispitanici pozitivni na ApoH is-kazali su razli~it koagulacijski profil; na|ena jeskupina s visokim aktivnostima LAK u odnosuna anti ApoH titrove (nelinearna korelacija,r=0,874) i skupina s niskim aktivnostima LAK(linearna korelacija, r=0,929). Raspravlja se oklini~kom zna~enju stvaranja takvih razli~itih ti-pova protutijela ApoH/LAC+. U ovom radu nijena|ena statisti~ki zna~ajna razlika u u~estalostipojedinih fenotipova ApoH izme|u kontrolneskupine i skupine osoba s o ApoH-ovisnim/LAK+autoprotutijelima.E-mail: [email protected]

ZR2-4

DISTRIBUTION OF APO H DEPENDENTAUTOANTIBODIES AND APO H PHENO-TYPES AMONG SELECTED SUBJECTSSHOWING LUPUS ANTICOAGULANT

ACTIVITY

@. Vogrinc, M. Trbojevi}-êpe, D. Coen,A. Stavljeni}-Rukavina


It has been found that apo H (beta-2-glycopro-tein I) is composed of five suchi domains (I-V)and its genetic polymorphism has been reported.Three common alleles (ApoH1, ApoH2, ApoH3)that determine five different phenotypes have alsobeen reported. Apo H is also the target antigen forbinding of certain ’antiphospholipid’ antibodies.It has been reported that antibodies directedagainst domain III and domain IV also exert lu-pus anticoagulant activity (LAC). Studies indi-cate that autoantibodies with LAC activity arestronger risk factors for thrombosis, fetal loss andthrombocytopenia. The aim of our study was toevaluate the possibility that the presence of lessfrequent apoH phenotypes, i.e. alleles, may inducestructural or conformational changes in these’LAC-dependent’ regions that may initiate apo H-dependent/LAC+ type of autoimmune response.We selected patients showing prolonged APTT,confirmed LAC activity and none or low titers ofantibodies to ’pure’ cardiolipin (n=74). Many ofthem had some clinical or serologic features ofsystemic lupus erythematosus and a high preva-lence of clinical manifestations of antiphospholi-pid syndrome. Antibodies to apoH were deter-mined by commercially available anticardio-lipin/apoH ELISA kit. ApoH phenotypes weredemonstrated by isoelectric focusing followed byimmunoblotting. Our results showed 63.5% (47/74) of our selected patients (LAC+) to also haveelevated apoH-dependent antibody titers. Astrong positive correlation was observed. How-ever, anti ApoH positive patients showed distinctcoagulation profiles: a group with high LA po-tency according to anti ApoH titers (nonlinearcorrelation, r=0.874) and a group with low LApotency (linear correlation, r=0.929). Clinical sig-nificance of such distinct types of ApoH/LAC+autoantibodies is discussed. We found no signifi-cant differences in the distribution of apoH phe-notypes between control subjects (n=166) and pa-tients with apoH-dependent/LAC+ autoantibod-ies.E-mail: [email protected]


INDUSTRIJSKE RADIONICE Saèci 4. hrvatskog kongresa medicinskih biokemi~ara

Industrijske radioniceIndustry workshops

IRProcjena analiti~kih tehnika i metoda

Evaluation of analytical techniques and methods

IR-2

VENOSAFE - SIGURNOST BEZKOMPROMISA

De Meyer I.

Terumo Europe

Posljednjega desetlje}a svjedoci smo sve ve}ihzahtjeva za sigurnijim vakuumskim epruvetamaza uzimanje krvi koje se ne mogu razbiti. To jedovelo do usavršavanja i pojave na trìštu plas-ti~nih vakuumskih epruveta. U tome je jedan odvode}ih proizvo|a~a bio Terumo Europe, koji jevakuumske epruvete od polietilenskog terefta-lata (PET) uveo još prije desetak godina. Kako biodgovorio na sve ve}e zahtjeve za standardiza-cijom u laboratorijskoj sredini te slijedom ustraj-ne potrage za rješenjima potpune sigurnosti, Te-rumo Europe je 2002. godine proizveo Venosafe,tre}u generaciju vakuumskih epruveta za uzima-nje krvi. Ova nova epruveta PET nudi višestrukeprednosti: ne propušta plin i jam~i nazna~eni vo-lumen uzete krvi (omjer) kroz cijeli vijek traja-nja; ne}e se razbiti ako padne na tvrdu površinu;otporna je na centrifugiranje pri velikim brzina-ma; teìna joj je za oko 30% manja od teìne istetakve staklene epruvete; potpuno }e izgorjeti uuvjetima spaljivanja medicinskog otpada. Nada-lje, primjena novog sigurnosnog zatvara~a omo-gu}ava lagano ru~no i automatsko otvaranje idobro se uklapa u automatizirani laboratorij. Ia-ko se u po~etku plastiku smatralo manje inert-nom od stakla, brojne su studije dokazale da suplasti~ne vakuumske epruvete prikladne kao pri-bor za prikupljanje krvi: nisu zapaène nikakveinterferencije kod uobi~ajenih klini~ko kemijskihodre|ivanja, hormonskih analiza, terapijskogpra}enja lijekova, odre|ivanja krvne grupe i ko-agulacije. Vrijednost epruveta Venosafe ilustri-

IR-2

CLINICAL PERFORMANCE OF A NEWVACUUM BLOOD SAMPLING TUBE:

VENOSAFE - SAFETY WITHOUTCOMPROMISE

I. De Meyer

Terumo Europe

In the last decade, the demand for safer, virtu-ally non-breakable evacuated tubes for blood col-lection has steadily increased. This trend resultedin the development and commercial marketing ofplastic evacuated tubes. One of the leading com-panies was Terumo Europe introducing polyethy-lene terephthalate (PET) evacuated tubes as earlyas more than 10 years ago. In order to serve theincreasing demand for standardization in labo-ratory setting and based on persistent search forultimate safety solutions, in 2002 Terumo Europeintroduced Venosafe, third generation of vacuumblood collection tubes. This new PET tube offersthe following advantages: they are gas-tight andguarantee the indicated draw-volume (ratio)throughout their lifetime; do not break whendropped on a hard surface; resist centrifugationat high speed; weigh about 30% less than theirglass counterparts; and are almost completelycombustible under incineration conditions com-monly used for medical waste. Furthermore, theuse of a newly developed safety closure allowseasy manual and automatic opening and fits verywell into the automated laboratory. Althoughplastic was initially perceived as less inert thanglass, a number of studies have proved that plas-tic evacuated tubes are suitable for use as bloodcollection devices: no interference was noticed forcommon clinical chemistry determinations, hor-mone analysis, therapeutic drug monitoring,


Sa`eci 4. hrvatskog kongresa medicinskih biokemi~ara INDUSTRIJSKE RADIONICE

rati }emo novijim ispitivanjima ovih epruveta sgelom, aktivatorom koagulacije, heparinom i ci-tratnim otopinama.E-mail: [email protected]

blood group determination and coagulation. Thevalidation of Venosafe tubes will be illustrated byrecent studies on Venosafe tubes with gel, coagu-lation activator, heparin and citrate solutions.E-mail: [email protected]


INDEKS Saèci 4. hrvatskog kongresa medicinskih biokemi~ara

Indeks autoraAuthor index

Albert WH. 15Alpeza I. 103Antoljak N. 62, 67Aralica M. 125Atamaniuk J. 133Babi} @. 59Ba~i}-Vrca V. 88Ba}a-Vrakela I. 33Balenovi} A. 68Balija M. 61Baljak N. 122Bari} D. 64Bari{i} I. 27Bari{i} K. 11Ba{i}-Markovi} N. 80Begonja A. 62, 67, 111Belina D. 69Beljan B. 82, 100Benko B. 118Bernt T. 57, 116Bili} K. 65Bili}-Zulle L. 82, 100Bilopavlovi} N. 96Bilu{i} Vunda} V. 124Bio~ina B. 64Bobeti}-Vrani} T. 55Böcher O. 15Bo{njak N. 49Boèk T. 86Boì~evi} S. 86Boìna N. 137Brajkovi} M. 127Brki} K. 64, 73, 99, 101, 108Brklja~i} V. 46, 66Broni} A. 131Brumen V. 142Bubenik V. 50Bulatovi} G. 80Bulum J. 56Bulj N. 67Buneta L. 126Cetina N. 123, 132Cheng S. 70, 71, 72Cigula-Kurajica V. 126Coen D. 72, 91, 102, 132, 136,

144Cremonesi L. 28

êpelak I. 14, 88, 118êpi} K. 105ûka-Husnjak S. 76, 118ûkovi}-âvka S. 21^vori{}ec D. 65]ori} J. 98]uk M. 63]uri} D. 101Davidovi}-Mrsi} S. 17De Meyer I. 146Demarin V. 89Dodig S. 118, 127Dravinski @. 106Dujmov I. 49, 50, 105Dumi} J. 31Dundovi} S. 123Duvnjak M. 62Dvornik [. 76, 82, 100, 118, 125\eri} M. 78Erakovi} V. 128Fariver A. 113Fazli} M. 98Feren~ak G. 70, 71, 72Ferrari F. 28Ferrari M. 28Fertek D. 30Fija~ko M. 123,132Fijal B. 71Fi{i} E. 82, 100Flegar-Me{tri} Z. 42, 92, 93Flögel M. 26, 31, 129Foglieni B. 28Franceschi M. 18Fressl G. 69Fu~ek M. 69Fumi} K. 24, 90Gabela O. 26Ga}ina P. 114Galetovi} A. 96Garilovi} J. 75, 119Ga{parovi} H. 65Giacometti J. 63Glava{ K. 95Glojnari} I. 118, 128Goldner V. 66Grani} P. 137Grdi} M. 136

Griesmacher A. 40Gr{kovi} B. 25, 70, 71, 72Gutierrez B. 15Habek D. 120Hadìja M. 87Hajn{ek S. 137Ha{perger D. 73Hazler-Pilepi} K. 124Herak-Kramberger CM. 10Honovi} L. 112Horvath AR. 38Hrabri} S. 76, 118Hreljac M. 131Hruba P. 30I{~i}-Be{ J. 33Ivankovi} G. 15Ivelja N. 50Jadri} R. 77Jagi} V. 59Jak{i} B. 51, 53, 54, 55Jeli} I. 65Jug M. 66Juki} I. 61Juki} T. 18Jur~i} Z. 84Jureti} D. 87, 92, 93Juri~ek J. 99Juri~evi} M. 103Juri~i} Lj. 46Kalanj-Bognar S. 89Kardum-Paro M.M. 51, 53Kardum-Skelin I. 51, 53, 54, 55Karimi A. 133Kastelic 56Katalini} V. 56, 115Keki} M. 113Khathami S. 117Klari} D. 58Kneèvi} A. 79Kopr~ina M. 114Kos N. 124Ko{ec V. 114Kova~ K. 118, 127Kozi} S. 33Kralik S. 57, 116Kricka L. 6Kronja-Negro J. 121, 122


Saèci 4. hrvatskog kongresa medicinskih biokemi~ara INDEKS

Kunovi} B. 104Kusi} Z. 18, 68, 107Ku{ec R. 51, 53, 54, 55Kutela N. 33Kvaternik M. 50Labar B. 56Lali} Z. 137Lauc G. 120, 129Lazi} J. 59Leni~ek-Krleà J. 101, 132Lovre~ek J. 108Lovri} M. 61Luka~-Bajalo J. 9Lukinac Lj. 18, 68, 107Ljubi} S. 86Malenica B. 140Male{ @. 89, 124Mandu{i} T. 54Maradin M. 90Marevi} S. 95Margeti} S. 50Markovi} I. 110Maru{i} M. 59Maru{i}-Vrsalovi} M. 54, 55Mastilica M. 37Mate{a N. 18Matica J. 82, 100Mati{i} D. 65Mayer Lj. 88Mayer V. 57, 116Me|ugorac B. 113Meniga D. 83Mesi} R. 86Metelko @. 86Mihaljevi} I. 61, 95Milanovi}-Stipkovi} B. 84Milardovi} S. 94, 106Milevoj L. 131Milo{ M. 91, 102, 115Minigo H. 53, 54, 55Mirjani}-Azari} B. 78Mirzahosieni H. 117Mladina B. 50Mrsi} M. 90Müller MM. 7, 25, 133Naki} D. 121Naki} M. 101, 142Nazor A. 51, 53Nekulova M. 19Neumaier M. 23Neumann I. 95Niki} N. 73Nikoli}-Heitzler V. 67Nimac A. 104Nogalo B. 118, 127Nöthig-Hus D. 107Omidinia E. 117Omumi AR. 117Ore{kovi} S. 34

Orli} P. 118, 125Palicka V. 30Pamukovi}-Jaram D. 83Papi}-Futa~ D. 130Parag G. 93Parnham MJ. 128Pa{ali} D. 71Paukovi}-Sekuli} B. 96Pavela J. 123, 132Paveli} K. 5Pavi} M. 131Pecen L. 19Peran N. 62, 83Perkov S. 42Perovi} E. 58Petek M. 85, 114Petek-Tarnik I. 85Petkovi} I. 27Petlevski R. 87, 89Petres B. 95Petrik J. 14Petrove~ki M. 108Pirija M. 112Planinc D. 68Plasaj T. 107Plazibat M. 124Polenus A. 110Polenus S. 110Popovi} M. 66, 78Popovi}-Grle S. 126Posavec Lj. 114Potkonjak J. 130Predovan G. 122Qujeq D. 25, 113Radalj @. 142Radi} M. 132Radosevic-Stasic B. 63Rai} B. 45, 114Raji} Lj. 56Rako I. 80Raos M. 118, 127Re{kov @. 83Ro~i} B. 86Rogi} D. 36, 46, 69, 109Rogi} J. 65Romi} @. 73, 88, 99Rude` I. 64Ruljan~i} N. 60Rumenjak V. 41, 59, 60, 66, 94,

106Rumora L. 12Ruì~ka K. 25Ruì}-Ferenec D. 50Saboli} I. 10Salamuni} I. 96, 115Samadi A. 117Samardìja M. 139Samarìja I. 13, 79Sambunjak J. 58

Samo{}anec K. 130Sandberg S. 38Sani~anin Z. 78Sansovi} I. 27Sapunar A. 49, 50Serti} J. 20, 26Sikirica M. 92Simeon-Rudolf V. 43Simickova M. 19Simonsson P. 39Sinha P. 29Skodlar J. 70, 71, 72Skorvaga S. 119Slijep~evi} M. 87Sokoli} B. 95Sokoli} I. 106Stan~i} V. 114Stavljeni}-Rukavina A. 3, 26, 56,

70, 71, 72, 74, 109, 137, 144Stenirri S. 28Stieber P. 16Strahija-Tomi} K. 61Suchanek E. 57, 116Suljevi} E. 77, 98Sutli} @. 64[eri} V. 123[esto M. 70, 71, 72[iftar Z. 51, 53, 54, 55[imek S. 112[imi} G. 110[imundi} AM. 32, 111[korvaga S. 75[kreb F. 88[prajc N. 64, 99[renger V. 109[tefanovi} M. 89, 110, 111, 138,

139[upe-Domi} D. 105[upraha-Goreta S. 129[urina B. 43[u{ter~i} D. 51, 53Tadi} I. 101Te{ija A. 110, 141Thaherkhani H. 117Thaller V. 130Tocilj J. 105Topi} E. 50, 62, 84, 89, 110, 111,

130, 135, 139Tota M. 63Trami{ak I. 137Trbojevi}-êpe M. 74, 144Uni} D. 64Val~i} A. 58, 121, 122Valik D. 19V~elik M. 75, 119Vince A. 33Vitunjski-Englert B. 108Vla{i}-Tanaskovi} J. 97Vogrinc @. 74, 144


INDEKS Sa`eci 4. hrvatskog kongresa medicinskih biokemi~ara

Vrani} T. 54Vrki} N. 62, 67Vuceli} B. 21Vu~i}-Lovren~i} M. 86Vukasovi} I. 44Vukeli} N. 110Wagner J. 120

Zadro R. 22, 56, 91, 102, 103,132, 136

Zivna H. 30Zivny P. 30Zlodre S. 81Zovko V. 126Zrinski-Topi} R. 26, 74@aja-Franulovi} O. 84

@ani}-Grubi{i} T. 12, 88@igman M. 68@iv~i} J. 127@ivkovi} A. 73@ivkovi} M. 113@i`i} V. 101, 142@untar I. 89@upani} D. 97

sa@eci 4. hrvatskog kongresa medicinskih ...biochemia medica god. 13, prilog broju 1–2, 2003. 3...

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