july 2013 2 innovation & information - immundiagnostik … · hemoglobin/haptoglobin reliable...

Innovation & Information

July 2013

Immundiagnostik AG • Stubenwald-Allee 8a • 64625 Bensheim • Germany • Tel.: +49 (0) 62 51/7 01 90-0 • Fax: +49 (0) 62 51/84 94 30 • [email protected] • www.immundiagnostik.com

Novel venues in diagnostics and therapy monitoring of breast cancer patients• 5-FU chemotherapy: PCR analysis of DPYD gene mutations helps to avoid side effects (page 9)• Tamoxifen treatment: PCR diagnostics of the CYP2D6 gene for an individualized therapy (page 9)

Diagnostics of infectious diseases in gastroenterology• Comprehensive ELISA and PCR portfolio for a specific detection of pathogens in clinical routine (page 10)

Lab-Tip for a successful participation in interlaboratory trials (page 11)

Hemoglobin/HaptoglobinReliable detection and prevention of colon cancer (page 3)

Alpha 1-AntitrypsinDiscover protein loss in time (page 5)

NEW ON THE MARKET

i2Calprotectin (PhiCal® ELISA) and TNFa Blocker Monitoring Personalized diagnostics and therapy control of inflammatory bowel diseases (page 2)

Zonulin Tight junction marker reveals: Probiotics influence the intestinal barrier (pages 4-5)

Meetings & Events (page 11)

sIgA and ß-DefensinMeaningful status marker for the gut-associated immune system (page 4)

Focus on Gastroenterology

EDN, DAO, TransglutaminaseComprehensive test panel for allergies, histamine intolerance, and gluten sensitivity (pages 6-8)

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i2 Innovation & Information

1

Therapy of infl ammatory bowel diseases

• TNFa-Blocker-Monitoring (Infl iximab drug level, e.g. REMICADE®)• TNFa-Blocker-Monitoring (Adalimumab drug level, e.g. HUMIRA®)• TNFa-Blocker-ADA (total antibodies against Infl iximab, e.g. REMICADE®)• TNFa-Blocker-ADA (antibodies against Infl iximab e.g. REMICADE®)• TNFa-Blocker-ADA (antibodies against Adalimumab e.g. HUMIRA®)• TNFa-Blocker-ADA (antibodies against Etanercept e.g. ENBREL®)• TNFa (Tumor Necrose Factor a)

Diagnostics of infl ammatory bowel diseases

• PhiCal® Calprotectin (MRP 8/14)• Lysozyme• Myeloperoxidase (MPO)• PMN-Elastase

Intestinal barrier malfunction

• Alpha 1-Antitrypsin • Zonulin

Status of the gut-associated immune system

• secretory Immunoglobulin A (sIgA)• sIgA-1• sIgA-2• ß-Defensin 2

Food intolerances

• anti-human-tissue-transglutaminase IgG• anti-human-tissue-transglutaminase IgA• anti-human-epidermal-transglutaminase IgA• anti-human-tissue-transglutaminase sIgA• Diaminooxidase (DAO)• Histamine• EDN (EPX)

Colon cancer: Prevention & early detection

• Hemoglobin, human (Hb)• Hemoglobin-Haptoglobin-complex

(Hb-Hp-complex)

Diagnostics of infectious diseases

• Helicobacter pylori antigen• Norovirus• Enterovirus• Salmonella sp.

One-Stop-Shop for Gastroenterology Comprehensive test portfolio for stool and serum samples

ID-PRODUCTS ON THE MARKET

Monitoring of inflammatory activity, drug level, and immune reaction enables an individualized therapy

Tumor necrose factor alpha (TNFα) is a pro-inflammatory cy-tokine, that is released by cells of the immune system and which sustains inflammatory reactions. In the therapy of chro-nic inflammatory pathologies, such as Crohn‘s disease, ulcera-tive colitis and rheumatoid arthritis, TNFα blocker (anti-TNFα antibodies) are used to treat the underlying inflammation.

The clinical efficacy of TNFα blocker correlates with the indivi-dual drug level of a patient. The drug level on the other hand is influenced by bioavailability, pharmacokinetics, and immu-nogenicity of the TNFα blocker.

Immundiagnostik offers several test systems for a personalized TNFα blocker therapy: The TNFα blocker monitoring ELISAs enable a quantitative determination of the drugs Infliximab, Adalimumab, or Etanercept in serum and plasma. Other ELISA tests detect anti-drug antibodies (ADA) against Infliximab or Adalimumab and serve as a tool to monitor the reaction of the immune system to this substances, since ADA may cause allergic reactions to infusions and may lead to therapy failure.

Until now, most tests have solely determined free ADA which were not bound to the drug. However, ADA-drug complexes have frequently been the cause of non-interpretable test re-sults. Immundiagnostik now presents a novel test that mea-sures free as well as drug-bound ADA. This test is especially useful for monitoring patients with a detectable drug level, e.g. shortly after a TNFα blocker application.

Calprotectin is released in large quantities by granulocytes during inflammatory processes and faecal calprotectin is an established, reliable marker of intestinal inflammation. The determination of calprotectin in stool is therefore ideal for the differentiation of inflammatory and functional gastrointesti-nal diseases. Monitoring faecal calprotectin concentrations in patients suffering from inflammatory bowel syndrome (IBD) is a good instrument to control treatment efficacy (e.g. of a TNFα blocker therapy).

Elevated levels are an early and reliable indicator for a relapse in IBD patients. Our well published PhiCal® Calprotectin ELISA has been validated in several clinical studies and features high specificity, a broad linear measuring range as well as short in-cubation times – the ideal test for clinical routine.

Regular external interlaboratory controls attest a consistently high quality of the PhiCal® Calprotectin ELISA.

The PhiCal® Calprotectin test together with the ELISAs for TNFα blocker monitoring and ADA monitoring are a powerful toolbox for diagnostics and therapy control of IBD patients, truly optimizing personalized treatment.

Calprotectin & TNFa blocker

Specific: Monoclonal test system Precise: Cut-off at 50 µg/g Fast: Only 1 hour total incubation time Reliable: With stabilization buffer Effective: Best linearity up to 2100 µg/g Practical: Also available as 1-point calibration

PhiCal® Calprotectin ELISA

Flexible: For IBD therapy with Infliximab (e.g. Remicade®) & Adalimumab (e.g. Humira®)

Precise: Sensitive & quantitative determination Practical: drug level monitoring in 2 hours Service: Contract analysis of all determinations

TNFa blocker ELISA portfolio

Innovation & Information i2

2

Nanda KS, Cheifetz AS, Moss AC (2013)Impact of Antibodies to Infliximab on Clinical Outcomes and Serum Infli-ximab Levels in Patients with Inflammatory Bowel Disease (IBD): A Meta-Analysis. Am J Gastroenterol 108(1): 40-47

Kopylov U, Mazor Y, Yavzori M, Fudim E, Katz L, Coscas D, Picard O, Chowers Y, Eliakim R, Ben-Horin S (2012) Clinical utility of anti-human lambda chain-based enzyme-linked immu-nosorbent assay (ELISA) versus double antigen ELISA for the detection of anti-infliximab antibodies. Inflamm Bowel Dis 18(9): 1628-1633

Molander P, Af Bjorkesten CG, Mustonen H, Haapamaki J, Vauhkonen M, Kolho KL, Farkkila M, Sipponen T (2012) Fecal calprotectin concentration predicts outcome in inflammatory bowel disease after induction therapy with TNFalpha blocking agents. Inflamm Bowel Dis 18(11): 2011-2017

.................................................................................PhiCal® Calprotectin ELISA K 6927PhiCal® Calprotectin (1-point calibration) ELISA K 6967

TNFa blocker monitoring ELISAs:• Drug level of Infl iximab (e.g. Remicade®) K 9655• Drug level of Adalimumab (e.g. Humira®) K 9657

TNFa blocker ADA ELISAs:• Antibodies against Infl iximab (e.g. Remicade®) K 9650• Antibodies against Adalimumab (e.g. Humira®) K 9652• Antibodies against Etanercept (e.g. Enbrel®) K 9653• Total antibodies against Infl iximab (e.g. Remicade®) K 9654.................................................................................

Diagnostics and therapy control of infl ammatory bowel diseases

Stool sample preparation made easy

Stool sample tube facilitates the hygienic preparation of a defined faecal solution

Immundiagnostik‘s ELISAs enable the quantitative determi-nation of many stool parameters in gastroenterology. These assays require exactly defined stool solutions. In conventional lab routine, faecal samples have to be weighed, mixed with buffer and centrifuged. During this procedure, lab personnel is confronted with direct stool contact and the difficulty of ac-curate weighing of specimens with variable consistencies.

The stool sample application system (SAS) is a tube that has been specifically designed for a simple, hygienic handling of stool samples. The tube enables the preparation of a defined faecal sample solution with minimal stool contact and wi-thout multiple steps that require special equipment.The innovative tube contains a dipstick with notches that hold exactly 15 mg of stool and thus facilitates clean and ex-

act sampling. After the stick has been placed back into the tube (thereby stripping off excess materi-al), a simple shaking step is suffi cient to dissolve the faecal material in the buff er. The suspension is now ready-to-use for ELISA-analysis and can be processed according to the respective manual.



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K 6998SAS = Cat. No. for an empty SAS-tube

The catalogue number of a fi lled SAS-tube is derived from the ELISA catalogue number of the respective parameter:

ELISAcat.no.SASP1 = SAS-tube with buff er (dilution 1:50)ELISAcat.no.SASP2 = SAS-tube with buff er (dilution 1:100)

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Established ELISAs deliver reliable results

Colorectal cancer almost always develops over the course of many years without causing any symptoms. During this time period, pre-cancerous lesions or initial tumor stages can be detected and removed with high chances of healing – the best prevention of colon cancer.

Early detection of colon cancer is based on the search for oc-cult blood in stool. Many studies in recent years have confir-med that the immunological analysis of human hemoglobin with an ELISA is more sensitive and specific than the com-monly used guaiac-based test. Even small traces of blood can be reliably detected in faecal samples without negative inter-ference of food or drug components.

Free hemoglobin is generally bound by haptoglobin, a se-rum protein. The hemoglobin/haptoglobin (Hb/Hp) complex is more stable than free hemoglobin in faecal samples and is therefore better traceable after a long bowel passage. While hemoglobin assays detect bleedings in the lower intestinal tract, the determination of the hemoglobin/haptoglobin complex provides additional information on blood from lar-ger polyps (see adjacent figure from Lüthgens et al.) or adeno-mas in the upper intestinal tract.

The combination of both ELISAs yields highest sensitivity in the early detection of colorectal cancer.

Lüthgens K, Maier A, Kampert I, Sieg A, Schmidt-Gayk H (1998) Hemoglobin-Haptoglobin-Complex: A Highly Sensitive Assay for the Detection of Fecal Occult Blood. Clin Lab 44: 543-551

Sieg A, Thoms C, Lüthgens K, John MR, Schmidt-Gayk H (1999) Detection of colorectal neoplasms by the highly sensitive hemoglobin-haptoglobin complex in feces. Int J Colorectal Dis 14(6): 267-271

Reliable early detection of colon cancer by measuring hemoglobin and hemoglobin/haptoglobin complex in stool

Hemoglobin ELISA K 7816DHemoglobin (1-point calibration) ELISA K 7836DHb-Hp-Complex ELISA K 7817DHb-Hp-Complex (1-point calibration) ELISA K 7837D

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Normal mucosa Polyps Polyps Polyps Carcinoma < 5 mm 6-19 mm > 20 mm

µg H

b-H

p/ g

Sto

ol

MeanMedia

Median and mean values of the Hb-Hp concentration in stool samples from patients with normal gut mucosa, polyps of different sizes or carcinoma (Lüthgens et al., 1998, Fig. 9)



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Status of the gut-associated immune system

sIgA and ß-Defensin2 are meaningful markers

In humans, the gut is the largest interface with the environment. The intestinal mucosa represents a protective and highly selective bar-rier that permits the absorption of nutrients but effectively prevents the invasion of pathogens, such as viruses, bacteria or parasites.The intestinal barrier consists of a mucous layer, the gut epithelium and the intestinal immune system.The mucosa represents a first acel-lular barrier that minimizes the contact of the gut epithelium with luminal microorganisms. The cells of the intestinal epi-thelium release numerous anti-microbial peptides to ward off pathogens and to regulate the number of commensal gut bacteria. The Defensin family of small cationic peptides is an important part of this anti-microbial immune system. The peptides prevent the invasion of microorganisms and stimu-late the immune system. The expression of ß-Defensin2 is trig-gered by contact with bacteria or by inflammation signalling molecules. Low ß-Defensin2 levels in stool indicate a weak gut-associated immune system that corresponds with a reduced colonisation resistance and an elevated mucosal permeability. High ß-Defensin2 concentrations, on the other hand, indicate a local inflammation process.

Another component of the gut-associated immune system are specific immune globulins of the subclass A (IgA), which are produced in the Lamina propria independent of serum IgA synthesis and which are transported through the epithelial la-

ß-Defensin2

yer into the intestinal lumen. Here, they are present as cova-lently bound IgA-dimers, called secretory IgA (sIgA).In the bowel, sIgA neutralizes bacteria and toxins, thereby assuming a key role in the maintenance of intestinal barrier function. There are two IgA-subclasses, IgA1 and IgA2, that vary in their amino acid sequence, their glycosylation, and their distribution. IgA1 is synthesized primarily in the mucosa of the air passages, the upper intestinal tract and the small in-testine. IgA2 dominates in the colon mucosa. Low sIgA concentrations in stool samples are an indicator for in-creased activity of microbial proteases, an unhealthy „modern“ life style or a specific immune suppression. High sIgA levels are a marker for an over-reaction of the immune system to micro-organisms, food components or food additives. The determi-nation of sIgA1 or sIgA2 provides information on the affected intestinal region.

The analysis of ß-Defensin2 and sIgA is an ideal test combinati-on for the status analysis of the gut-associated immune system.

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sIgA ELISA K 8870sIgA (1-point calibration) ELISA K 8880sIgA-1 ELISA K 6863sIgA-2 ELISA K 6864

ß-Defensin2 ELISA K 6500

Kapel N, Benahmed N, Morali A, Svahn J, Canioni D, Goulet O, Ruemmele FM (2009) Fecal beta-defensin-2 in children with inflammatory bowel diseases. J Pediatr Gastroenterol Nutr 48(1): 117-120

Pabst O (2012) New concepts in the generation and functions of IgA. Nat Rev Immunol 12(12): 821-832

Tight junction marker Zonulin reveals: Probiotics influence the intestinal barrier

Zonulin ELISA for research on intestinal barrier function

Zonulin regulates the intercellular contacts in the intestinal epithelial layer. The protein binds to a specific receptor on the surface of epithelial cells in the intestinal layer, thereby acti-vating a biochemical signalling cascade. This process causes the tight junctions between the epithelial cells to open so that substances from the intestinal lumen can pass through. A lack of tight junction control associated with an unregulated passage of molecules through the intestinal epithelium may induce inflammation and autoimmune diseases. Accordingly, elevated Zonulin levels are present in patients with celiac di-sease or Type-I Diabetes and their relatives.

Athletes, such as long distance runners and triathletes often suffer from gastrointestinal problems due to excessive strain that leads to an intestinal barrier malfunction with resulting inflammatory reactions. Symptoms include nausea, stomach and bowel cramps, vomitting and diarrhea. In addition, the dis-rupted barrier also promotes endotoxemia, the infiltration of pathogens and toxins into the bloodstream, thereby increasing the susceptibility for infections and autoimmune diseases.

Lamprecht et al. tested on a collective of 23 trained endurance athletes whether the daily intake of probiotics improves the function of the intestinal barrier. The investigators chose Zo-nulin as a marker for gut barrier integrity. They determined

Determination of inflammatory activity in bowel diseases

Intestinal protein loss is a serious consequence of various sy-stemic or local gastrointestinal diseases (e.g. allergies, chronic inflammation, malignancies). These pathologies damage the mucosal integrity and/or cause lymphostasis, thereby leading to an increased transfer of plasma proteins into the bowel lumen. Subsequently, hypoproteinemia accompanied with edema may develop. This condition is diagnosed by exclusion of other sources of protein loss and by proof of an elevated alpha-1 Antitrypsin (a1-AT) concentration in stool.

In serum, a1-AT represents the majority of serine protease in-hibitors and protects tissues from protease damages during inflammation. The protein is synthesized primarily in the liver but also to a small extent in intestinal macrophages, mono-cytes, and intestinal epithelial cells. Since a1-AT is relatively resistent against enzymatic digestion, the secreted amount in stool reflects the internal concentration of the protein.

An elevated a1-AT stool concentration is therefore a widely recognized marker for intestinal protein loss and for an increa-sed mucosal permeability.

The analytical quality of Immundiagnostik‘s alpha 1-Antitryp-sin ELISA surpasses by far the conventional radial immunodif-fusion (RID) technique in the determination of serum, stool and tissue culture supernatants. In direct comparison, the concentrations measured with the ELISA were approximately 30% above the corresponding RID levels.

Cell culture supernatants of an intestinal cell line as well as fa-ecal samples of lymphostasis patients yielded negative results


5


unremarkable and within normal range before exer-cise (< 11.3 pg . mL-1), but we observed a significantincrease from pre to post exercise above normal inboth groups (P = 0.001, Figure 5) at baseline and after14 weeks of treatment.

DiscussionAthletes exposed to high intense exercise show increasedoccurence of GI symptoms like cramps, diarrhea, bloating,nausea, and bleeding [31,32]. These symptoms have beenassociated with alterations in intestinal permeability and

Figure 3 Plasma concentrations of carbonyl groups bounded on protein in trained men before and after 14 weeks of treatment, andpre/post a triple step test cycle ergometry. Pro with probiotics supplemented group, Plac placebo group, Ex exercise, Tx treatment, wk week;n = 11 (probiotic supplementation), n = 12 (placebo). Values are means ± SD. There was a significant differences from pre to post exercise (except“Pro wk14”): PEx < 0.05; and a tendential difference between groups after 14 wk of treatment: PTx < 0.1 (ANOVA).

Figure 2 Stool concentrations of zonulin in trained men before and after 14 weeks of treatment. Pro with probiotics supplementedgroup, Plac placebo group, Tx treatment, wk week; n = 11 (probiotic supplementation), n = 12 (placebo). Values are means ± SD. There was asignficant difference between groups after 14 wk of treatment: PTx < 0.05 (ANOVA).

Lamprecht et al. Journal of the International Society of Sports Nutrition 2012, 9:45 Page 8 of 13http://www.jissn.com/content/9/1/45

Decrease of Zonulin concentration in stool samples in correlation with probiotics supplementation (according to Lamprecht et al., 2012, Fig. 2)

Probioticsweek 0

Probioticsweek 14

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Zonulin ELISA (determination in stool) (K 5600)Zonulin ELISA (determination in serum) (K 5601)

Lamprecht M, Bogner S, Schippinger G, Steinbauer K, Fankhauser F, Hallstroem S, Schuetz B, Greilberger JF (2012). Probiotic supplementation affects markers of intestinal barrier, oxidation, and inflammation in trained men; a randomized, double-blinded, place-bo-controlled trial. J Int Soc Sports Nutr 9(1): 45.

Alpha 1-Antitrypsin indications Suspected enteral protein loss Crohn‘s disease Necrotising enterocolitis Chronic mesenterial ischemia Viral, bacterial, allergic, autoimmune inflammations

Faust D, Spirchez Z, Armbruster FP, Stein J (2001) Determination of alpha1-proteinase inhibitor by a new enzyme linked immunosorbant assay in feces, serum and an enterocyte-like cell line. Z Gastroenterol 39(9): 769-774

.................................................................................a1-Antitrypsin ELISA K 6750a1-Antitrypsin (1-point calibration) ELISA K 6760a1-Antitrypsin (Clearance) ELISA K 6752.................................................................................

Zonulin at the beginning of the trial and after 14 weeks of probiotics- or placebo-treatment. At the start, all athletes dis-played elevated Zonulin concentrations (reference value <30 ng/ml), indicating a mild increase in gut permeability. After 14 weeks, only the Zonulin levels of the probiotic-treated group were normal, not those of the placebo group. The authors conclude that the supplementation of probiotics appears to improve the intestinal barrier in athletes. The practical value of this finding is to reduce the susceptibility of this group to endotoxemia, inflammation, infections, and allergies.

with RID. Our ELISA could detect a1-AT in all of these samples, in some of them even in very high concentrations.These results clearly prove that the a1-AT ELISA is far more sensitive than the conventional method and that it recognizes not only hepatic but also enteral a1-AT. The discrepancy of both methods and hence the superiority of the ELISA to RID is especially striking in the analysis of extremely high enteral protein losses. The combination of two specific antibodies in our a1-AT ELISA widely excludes the possibility of false negative results there-by enabling a reliable diagnostics of enteral protein loss.

Alpha 1-Antitrypsin in faeces is a marker for enteral protein loss

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Comprehensive test portfolio reveals underlying causes

Food intolerances summarize a number of symptoms that ap-pear particularly after the consumption of certain foods and are due to various underlying pathologies.

Food allergies (e.g. cow‘s milk allergy) are caused by a specific reaction of the immune system to a certain food component. Typically, IgE-antibodies are involved in this immune defense but there are also IgE-independent food allergies (e.g. gluten sensitivity).

However, food intolerances without specific involvement of the immune system are far more often than true food aller-gies. Among underlying causes for intolerances are defect en-zymes or transport proteins that prevent the degradation or absorption of a particular food as well as an immediate phar-macological effect of food ingredients on the metabolism (e.g. in histamine intolerance).

EDN: Distinction of allergy and intolerance

EDN (eosinophil-derived neurotoxin) is one of several, stron-gly positively charged, basic proteins that are released by ac-tivated eosinophils. The physiological task of EDN is to exert a cytotoxic effect to defend parasites. In response to a chronical-ly alert immune system (e.g. in allergies and asthma) however, EDN can cause tissue damage. Elevated EDN levels in stool are a reliable marker for an intestinal food allergy. In serum, EDN concentrations are indicative for asthma activity in children.

The determination of intestinal permeability and faecal EDN are the best diagnostic tools for non-IgE-mediated cow‘s milk allergy in toddlers. Cow‘s milk allergy can be IgE-mediated, non-IgE-mediated or both, which complicates diagnosis. Clas-sical immunological blood tests using specific IgE antibodies are often inconclusive and an elimination diet with subse-quent provocation is elaborate and not always indicated.

Prof. Kapel and colleagues examined 25 toddlers with su-spected cow‘s milk allergy and tested the diagnostic quality of several parameter in comparison to conventional allergy diagnostic methods (determination of specific antibodies and skin prick tests). An elimination diet with subsequent cow‘s

milk provocation confirmed an allergy in 14 toddlers. The determination of faecal EDN in this patient group was the best diagnostic parameter for cow‘s milk allergy next to the analysis of the intestinal permeability.

The measurement of faecal EDN is therefore a promising, non-invasive tool for the diagnosis of cow‘s milk allergy, especially in children with non-IgE-mediated allergy.

DAO analysis sheds light on histamine intolerance

Histamine is a powerful, tightly controlled physiological mes-senger protein, produced by special cells and stored intracel-lularly until release. Excessive physiological histamine release that is not counteracted by enzymatic degradation as well as consumption of histamine-rich food can lead to a rise of hista-mine levels in the circulation. These small elevations in hista-mine concentration above the normal range may already cau-se histamine intolerance (HIT). Typical HIT-symptoms include gastrointestinal problems, nasal obstruction or runny nose, headache, dysmenorrhea, hypotension, arrhythmia, urticaria, itchiness, flushing, and asthma attacks.

Diamine oxidase (DAO) is responsible for histamine degrada-tion in the extracellular space. The enzyme is synthesized pri-marily in the intestinal mucosa. A small portion continuously transfers into circulation, DAO-amount and -activity is therefo-re detectable in serum.

Kalach N, Kapel N, Waligora-Dupriet AJ, Castelain MC, Cousin MO, Sauvage C, Ba F, Nicolis I, Campeotto F, Butel MJ, Dupont C (2013)Intestinal permeability and fecal eosinophil-derived neurotoxin are the best diagnosis tools for digestive non-IgE-mediated cow‘s milk allergy in toddlers. Clin Chem Lab Med 51(2): 351-361

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EDN ELISA (K 6811)

EDN ELISA (1-point calibration) (K 6821)

Diagnostics of food intolerances



Frequent headaches or migraine Running or congested nose after consumption

of histamine-rich food Edema Swollen eye lids Erythema Sleeping disorders Gastrointestinal problems, diarrhea Monitoring of a histamine-free diet

Indications for DAO determination

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Sessa A, Perin A (1994) Diamine oxidase in relation to diamine and polyamine metabolism. Agents Actions 43(1-2): 69-77

Zopf Y, Baenkler HW, Silbermann A, Hahn EG, Raithel M (2009) The differential diagnosis of food intolerance. Dtsch Arztebl Int 106(21): 359-369

Maintz L, Yu CF, Rodriguez E, Baurecht H, Bieber T, Illig T, Weidinger S, Novak N (2011)Association of single nucleotide polymorphisms in the diamine oxidase gene with diamine oxidase serum activities Allergy, DOI: 10.1111/j.1398-9995.2011.02548.x.

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DAO REA (Serum) (K 8220)

DAO ELISA (Serum) (K 8500)

Histamine ELISA (Stool) (K 8213)

Histamine ELISA (EDTA, Plasma, Urine) (K 8212)

Gluten-related disorders: Comprehensive diagnostics of complex pathologies

Up to this date, there is a controversial debate about the existence of gluten sensitivity in addition to celiac disease and wheat allergy.

The discussion reflects the strain of patients with gluten-de-pendent symptoms, who fall through the cracks of conventi-onal diagnostic examinations because they don‘t match the inherent laboratory diagnostics criteria.

The clinical course of gluten sensitivity is often unspecific and frequently lacks text book-like symptoms such as villious atro-phy in the bowel, vomitting, or diarrhea. Instead, many forms are clinically silent and only much later lead to a pronounced form of celiac disease (= gluten-sensitive enteropathy, GSE).

Hence, a suspected gluten sensitivity should at any rate be analysed in depth. Especially children could otherwise suff er from irreversible growth retardations. Avoiding gluten-contai-ning foods also lowers the cancer-risk of patients with gluten sensitivity.

Immundiagnostik offers a broad panel of laboratory dia-gnostics that is even able to diagnose gluten-sensitive pati-ents with untypical clincial symptoms.

Sapone A, Bai J, Ciacci C, Dolinsek J, Green P, Hadjivassiliou M, Kaukinen K, Rostami K, Sanders D, Schumann M, Ullrich R, Villalta D, Volta U, Catassi C, Fasano A (2012) Spectrum of gluten-related disorders: consensus on new nomenclature and classification. BMC Medicine 10(1): 13

Di Sabatino A, Giuffrida P, Corazza GR (2013) Still waiting for a definition of nonceliac gluten sensitivity. J Clin Gastroenterol Feb 18. [Epub ahead of print]

Recurrent intestinal problems Growth retardation in children Iron deficiency anemia Neurological disorders (e.g. depression) Symptoms of rheumatoid arthritis Increased number of miscarriages Infertility Dermatitis herpetiformis Duhring

Indications for the determination of gluten sensitivity

DAO production may be decreased by enterocyte damage during the course of a gastrointestinal disease. In addition, its activity is competitively inhibited by biogenic amines, alcohol, or certain drugs. A shortage of co-factors, such as vitamin C, vitamin B6, copper or manganese ions can also reduce DAO activity.In patients with HIT symptoms, DAO concentration and activi-ty should therefore be determined in the circulation.

Immundiagnostik offers two complementing test systems for this purpose: The worldwide first DAO ELISA quantifies the amount of DAO, the DAO REA (3H) on the other hand analyses the activity of the enzyme. The combination of both assays enables the reliable determination of DAO insufficiency and DAO activity loss.


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Our proposal to a stepwise laboratory diagnostics of gluten sensitivity:

anti-tTG IgA in serum

(Antibodies against tissue transglutaminase)

positive

negativ (possibly false negative

because of IgA deficiency)

IgA deficiency ?(Total IgA) IgA deficiency

IgA deficiency not existent

anti-tTG IgG / anti-Gliadin IgG

in serum

positive

negative

anti-tTG sIgA / anti-Gliadin sIgA

in feces

positive

negative

anti-eTG IgA(antibodies against epidermal

transglutaminase)

anti-Gliadin IgA

Gluten sensitivityis existing

in all likelihood

positive

negative

Gene analysisHLA DQ 2/8

If serology and analysis of fecal parameter do not come to a clear result, HLA-DQ2 and HLA-DQ8 determination

is useful in exclusion of celiac disease.

Additional diagnostics

negative

positive

Our product portfolio for the diagnostics of gluten sensitivity

Serum diagnostics:• Anti-tissue transglutaminase IgA (Anti-tTG-IgA) ELISA K 9399• Anti-tissue transglutaminase IgG (Anti-tTG-IgA) ELISA K 9398• Anti-epidermal transglutaminase IgA (Anti-eTG-IgA) ELISA K 9396• Anti-gliadin IgA ELISA K 9310• Anti-gliadin IgG ELISA K 9300

Stool diagnostics:• Anti-tissue transglutaminase sIgA (Anti-tTG-sIgA) K 9393• Anti-gliadin sIgA ELISA K 9311

HLA DQ-Typisation• MutaGEL® HLA-DQ PCR KE09020

MutaCHIP® 5-FU PCR and MutaCHIP® TAMOX PCR support the personalized therapy

Individual therapy monitoring as part of a personalized medici-ne has gained importance in recent years. Immundiagnostik‘s new products for the treatment of tumor patients cover the areas of diagnosis, therapy choice, and therapy monitoring.

The PCR-analysis for Tamoxifen treatment and the genetic risk profile analysis for 5-Fluorouracil chemotherapy are useful tools to choose and adjust individual treatments.

MutaCHIP ® 5-FU PCR5-Fluorouracil (5-FU) is a cytostatic agent that is part of a stan-dard tumor therapy. However, patients with lacking or signi-ficantly reduced activity of the enzyme dihydropyrimidine dehydrogenase (DPD) carry an elevated risk to suffer from severe and even fatal adverse side effects. This risk can be re-duced or avoided with a genotypic analysis of the DPYD gene which codes for the DPD enzyme. One alteration in the DPYD gene is the DPD*2A mutation, which – together with 13 other possible gene variations – is responsible for the loss of normal enzymatic activity.

The PCR analysis with the MutaCHIP® 5-FU kit can detect all of these mutations before the start of a chemotherapy. The MutaCHIP® 5-FU genotyping thereby enables the individual optimization of a 5-FU therapy without harmful side effects.

NEW PRODUCTS


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MutaCHIP® 5-FU KF390006MutaCHIP® TAMOX KF390005

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Survival rate of breast cancer patients under Tamoxifen therapy in correlation to different CYP2D6 enzyme activity (according to Brauch and Schroth et al. 2009, Fig. A1 B)

Appendix

Recurrences (%)

CYP2D6Genotyping

EM hetEM/IM PM

14.5 18.6 14.8

11.9

*4 only

4.423.71pihCilpmA

A

C

0

Rela

pse-

Free

Sur

viva

l (p

ropo

rtion

)

Time (years)

1.0

0.8

0.6

0.4

0.2

2 4 6 8 10

Log rank (EM:PM) P = .15

EMhetEM/IMPM

B

0

Rela

pse-

Free

Sur

viva

l (p

ropo

rtion

)

Time (years)

1.0

0.8

0.6

0.4

0.2

2 4 6 8 10

Log rank (EM:PM) P = .003

EMhetEM/IMPM

Fig A1. Kaplan-Meier distribution and recurrence rates for time-to-breast cancer recurrence in 492 patients with breast cancer treated with tamoxifen. (A) Outcomestratification between CYP2D6 phenotypes extensive metabolizer (EM), heterozygous EM/intermediate metabolizer (hetEM/IM), and poor metabolizer (PM) by *4 only.(B) Outcome stratification between CYP2D6 phenotypes by comprehensive allele genotyping (AmpliChip; Roche Diagnostics, Pleasanton, CA). (C) Comparison ofrecurrence rates between CYP2D6 metabolizer subgroups indicating a shift to higher recurrence rates in PM on the basis of comprehensive genotyping for correctphenotype assessment.

Brauch et al

6 © 2012 by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY

Downloaded from jco.ascopubs.org on November 23, 2012. For personal use only. No other uses without permission.Copyright © 2012 American Society of Clinical Oncology. All rights reserved.

Survival rate of patients with strong CYP2D6 enzyme activity

Survival rate of patients with weak CYP2D6 enzyme activity

MutaCHIP® TAMOX PCRTamoxifen is a selective estrogen receptor modulator which is routinely applied to treat estrogen-receptor-positive (ER+) breast cancer. The drug is converted by the enzyme cyto-chrome P450 2D6 (CYP2D6) into its active metabolites 4-hy-droxytamoxifen and endoxifen. The enzymatic activity of CYP2D6 hence has a direct influence on the success rate of Tamoxifen treatment. 50% of the caucasian population carry a variant of the CYP2D6 gene and are therefore not able to metabolize Tamoxifen correctly.In a recent publication, Brauch, Schroth, and colleagues de-monstrate the correlation of a reduced CYP2D6 activity and a poor response to Tamoxifen therapy (s. Figure below).The CYP2D6 genotyping can identify patients with reduced enzyme activity before the start of a Tamoxifen therapy and recommend them for an early alternative treatment with arom-atase inhibitors to reduce the risk of recurrent tumor formation.

With the MutaCHIP® system for genotyping of the CYP2D6 en-zyme Immundiagnostik now delivers the diagnostic tool for a personalized adjustment of the Tamoxifen therapy.

Brauch H and Schroth W et al. (2012)Tamoxifen use in postmenopausal breast cancer: CYP2D6 matters. Journal of Clinical Oncology, Vol 31(2) :176-180

Novel venues in diagnostics and therapy monitoring of cancer patients


10

NEW PRODUCTS

Comprehensive ELISA and PCR portfolio for a specifi c detection of pathogens in clinical routine

Immundiagnostik offers a multitude of test systems for the de-tection of infectious agents in a variety of matrices.

Our real time PCR-Kits identify genetic variants of pathogens with unsurpassed specificity and sensitivity for a fast, easy, and efficient diagnosis. The kits enable the detection of DNA

or RNA of all common bacterial and viral agents in biological fluids (see table below) – ideal for daily routine in clinical la-boratories.

The ELISA panel offers the flexibility of pathogen detection in stool, serum, or plasma samples. In addition, special versions for automates or CLIA are available on demand.

Diagnostics of infectious diseases in gastroenterology

Pathogen detection

Cat. No. Parameter

KBECVGK01 Cytomegalovirus IgG

KBECVMK02 Cytomegalovirus IgM µ capt

KBEDIGK22 Dengue Fever IgG

KBEDIMK23 Dengue Fever IgM

KBEEVAK-27 EBV VCA IgA

KBEEVGK03 EBV VCA IgG

KBEEVMK04 EBV VCA IgM

KBEHLAK09 Helicobacter pylori IgA

KBEHLMK26 Helicobacter pylori IgM

KBEHLGK08 Helicobacter pylori IgG

KBEHSGK05 Herpes simplex virus 1 IgG

KBEHGGK24 Herpes simplex virus 1&2 IgG

KBEHGMK25 Herpes simplex virus 1&2 IgM µ capture

KBEHHGK06 Herpes simplex virus 2 IgG

KBEHHMK34 Herpes simplex virus 2 IgM

KBEMVGK10 Measles IgG

KBEMVMK11 Measles IgM

KBEMCGK42 Mycoplasma IgG ELISA

KBEMCMK43 Mycoplasma IgM ELISA

KBEMUGK12 Mumps IgG

KBEMUMK13 Mumps IgM

KBERVGK14 Rubella IgG

KBERVMK15 Rubella IgM µ capture

KBESPGK16 Syphilis IgG

KBESPMK17 Syphilis IgM

KBETXGK18 Toxoplasma IgG

KBETXMK19 Toxoplasma IgM µ capture

KBETBGK38 Tuberculosis IgG

KBETBMK39 Tuberculosis IgM

KBEVIGK20 Varicella zoster IgG

KBEVIMK21 Varicella zoster IgM

ELISA panel for serum/plasma analysis

Cat. No. Parameter

Enterovirus

KP2900296 MutaREAL® real time RT-PCR

KG290296 MutaREX® real time RT-PCR (+ extr. ctrl.)

KP1900296 MutaPLATE® real time RT-PCR (open sys.)

KG190296 MutaPLEX® real time RT-PCR (open systems) (+ extraction control)

Norovirus

KP2934196 MutaREAL® real time RT-PCR


KP1934196 MutaPLATE® real time RT-PCR (open sys.)


Verotoxin (stx 1/2 genes)

KP190096 MutaFAST® VTEC stx 1/2 real time PCR (for open systems)

Salmonella spec.



Clostridium diffi cile (Toxin B)

KE19011 MutaPLATE® real time RT-PCR (open sys.)

PCR detection in all biological matrices

Cat. No. Parameter

VIRHW/E-017 Serazym Adenovirus

VIRHW/E-045 Serazym Astrovirus

VIRHW/E-020 Serazym Rotavirus

VIRHW/E-018 Serazym Entamoeba histolytica

VIRHW/E-040 Serazym Clostridium diffi cile(Toxin A/B)

VIRHW/E-030 Serazym Verotoxin 1+2

VIRHW/E-093 Serazym Campylobacter

Helicobacter pylori Antigen

K 6921 Polyclonal ELISA

K 6922 Monoclonal ELISA

ELISA panel for stool samples*

*Kit versions for automates also available

LAB-TIP

Practical tips for a successful participation in interlaboratory trials

A correct step-by-step execution of the ELISA protocol greatly infl uences the quality of results and is the basis for a successful partici-pation in interlaboratory trials. We have collected a few practical tips for the optimal handling of stool samples that we would like to share with our customers, esp. with those who participate in interlaboratory trials:

• Reconstitute lyophilized sample material as indicated by the trial organisation. Adhere to the gi-ven incubation time to achieve a homogenous solution. Otherwise, non-dissolved specimen residues (due to a shorter incubation period) will cause false low results.

• All notches of the sample tube dipstick need to be fi lled with specimen, even if the provided volume is low. An insuffi cient amount of specimen will inevitably lead to false low data.

• Do not store sample material in the refrigerator (4°C). We recommend to store the sample at -20°C for repeated use on several days.

In addition, we recommend to always use the latest kit version. On a continuous basis we advance and optimize our laboratory diagnostics according to customer needs and clinical requirements.

The new test versions are customer-friendly, state-of-the-art, and users benefi t from faciliated protocols. Especially accredited labora-tories with external validation can rely on best results with the latest kit versions – a warrant for successful participation in interlabo-ratory trials.

If you have questions regarding stool sample preparation, application of a new kit version, or kit handling on automates, please con-tact our laboratory team:

[email protected]

Immundiagnostik AGStubenwald-Allee 8a64625 Bensheim, GermanyTel.: +49 (0) 62 51-70 19 00Fax: +49 (0) 62 51-84 94 [email protected]

i2 Innovation & Information / July 2013

IMPRINT

Find out more

www.immundiagnostik.com

• AACC 28. July - 01. August, Houston / USA Booth No. 2726

• ESPEN 31. August - 03. September, Leipzig Booth No. 10

• BIHE 17. - 19. September, Baku / Azerbaijan

• ASBMR 04. - 07. October, Baltimore / USA Booth No. 406

• UEGW 12. - 16. October, Berlin Hall 15 / Booth No. 17

• ASN 05. - 10. November, Atlanta / USA Booth No. 1724

MEETINGS & EVENTS


11

Stool sample

Dipstick

july 2013 2 innovation & information - immundiagnostik … · hemoglobin/haptoglobin reliable...

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