a brief review of alzheimer’s disease by using conventional silver staining … · 2007-09-16 ·...

Vol. XXXVII, No. 2 December 2004

IntroductionAlzheimer’s disease is a slowlyprogressive cerebral degenerationcharacterized by dementia.Increasing age and the presence ofthe beta-amyloid protein in neuriticplaques are risk factors associatedwith AD.1 Extensive research isongoing in an attempt to betterunderstand AD. The followingtheories have been proposed for itsetiology: invasion by slow viruses,acetylcholinesterase deficiency,aluminum excess, autoimmuneresponses, genetic predisposition,amyloid protein deposition, andvascular changes. In patients withAD, neurons and neuritic processesare lost. The gyri narrow, the sulciwiden, and cortical atrophybecomes apparent (Fig. 2B).1

A common finding in the brains ofAlzheimer’s disease patients is aloss of approximately 200 g in aspan of 3 to 8 years. The atrophy isbilateral and symmetric and targetsthe frontal and hippocampal cortex(Fig. 2). A definitive Alzheimer’sdiagnosis is based on large

A Brief Review of Alzheimer’s Diseaseby Using Conventional Silver Staining

ProceduresDr. Alicia A. Zuniga, CLS, HTL (ASCP); Patricia Gonzalez, BS, HT;

Antonio Fernandez, MDSchool of Natural and Health Sciences

Barry University, Miami Shores, [email protected]

AbstractAlzheimer’s Disease (AD) is the leading cause of dementia. As many as10% of all people 65 years of age and older have AD, and as many as50% of those 85 and older have the disease. The high morbidity andmortality associated with this disorder and its increasing prevalence inaging populations require the histology laboratory to have in its arsenalreproducible staining methods to render this diagnosis in biopsy sections.We evaluated different silver stains to identify which is most useful formaking the diagnosis of AD in histological sections. We found that theBielschowsky silver stain is the best method to demonstrate ADpathology in our laboratory. The senile or neuritic plaques were the mostconspicuous histologic lesion observed in sections stained with theBielschowsky technique.

Managing Editor, Gilles LefebvreScientific Editor, Vinnie Della Speranza,

MS, HTL (ASCP) HT, MT

IN THIS ISSUE

A Brief Review of Alzheimer’s Diseaseby Using Conventional Silver StainingProcedures …………………………………21

Combined UCHL-1/PASHStaining in the Diagnosis ofRenal Transplant Rejection ………………25

Cryomicrotomy of Murine Tissuesfor Research Immunohistochemistryand Routine Hematoxylin and EosinStaining. Part II ……………………………29

Alcian Blue-H&E-Metanil YellowStain for Diagnosing Barrett’s Esophagus …………………………………35

Double Embedding:Double the Trouble?………………………39

Mark Your Calendar!Educational Opportunities in 2005 ………41

Fig. 1. Single photon emission computerized tomography. A) Altered blood circulation in an AD brain.B) Normal brain.

A B

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numbers of microscopicneurofibrillary tangles, neuropilthreads, neuritic plaques, andgranulovacuolar degenerationpresent in several tissue samples ofcerebral cortex. Symptoms includeloss of memory, judgment andreasoning, difficulty with day-to-day function, and changes in moodand behavior. The utilization ofimaging studies in the diagnosis ofAD is done mainly to excludestructural lesions such as subduralhemorrhage and brain tumors.Figure 1 shows a single photonemission computerized tomographyindicating how blood is circulatingin normal (B) and AD brain (A).

We evaluated the Bielschowskysilver stain, Bodian silver stain, andmodifications of the methenaminesilver stain for the utility indemonstrating microscopic changesin brains from patients withAlzheimer’s disease.

Materials and MethodsBrain samples from three patientswho died from cardiorespiratoryfailure and had a history of progres-sive cognitive decline were fixed in10% buffered formalin for 14 days.Several tissue blocks from each casewere cut on a rotary microtome.Sections of 8-10 µ were floated ona water bath at 41°-43°C in distilled

water, collected onto precleaned,charged slides, and dried overnightin a 39°C oven, deparaffinized, andrehydrated to distilled water priorto staining. Staining techniquesincluded the Bielschowsky silverstain, Bodian silver stain,hematoxylin & eosin, and amodification of the methenaminesilver stain, using both microwaveand conventional procedures.

Slides were observed under a lightmicroscope and cortical areas wereselected at 40X, 100X, and 400Xmagnifications using the DP12Olympus® Microscope DigitalCamera System (OlympusAmerica, Melville, NY).The pictures recorded in theSmartMedia™ installed in thecamera system were viewed withthe software provided with theDP12-BSW. Saved pictures wereprinted using a Hewlett PackardDeskjet 660 color printer.

ResultsThe preliminary results of thisoverview indicate that theBielschowsky silver stain2 is thebest staining method used in ourlaboratory to demonstrate ADpathology. The conventionalprocedure was compared to themicrowave modification.3 The twotechniques differed in the amountof time employed to do theprocedures but results were

Fig. 3A. Section of cerebral cortex revealing the presence of neuritic plaques.H&E, 40X

Fig. 3B. At higher magnification, the uniform size and spherical shape of the neuriticplaques are observed. Silver stain, 100X

Fig. 2A. Normal brain. Fig. 2B. The brain of a patient with AD showscortical atrophy, characterized by slender gyriand prominent sulci.

A B

A B

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conclusive in both. The majormicroscopic features observed inthe sections of cortex from patientswith Alzheimer’s disease wereneurofibrillary tangles and neuriticplaques (Figs. 3, 4). Theneurofibrillary tangles found withinthe cytoplasm of abnormal neuronsconsist of fibrous proteins that arewound around each other as pairsof helical filaments. The maincomponent of tangles1 (Fig. 4A, 4B)is an abnormal phosphorylatedform of a normally occurringmicrotubule-associated protein(MAP), termed tau, which isnecessary to stabilize neuronalmicrotubules for proper axonaltransport. The abnormallyphosphorylated tau is less ableto bind microtubules, probablycausing microtubuledepolymerization, disrupted axonaltransport, compromised synapticneurotransmission, and finally,neuronal death. These tangles areresistant to chemical and enzymaticbreakdown, and they persist inbrain tissue long after the neuronin which they arose has died anddisappeared. The observedneurofibrillary tangles arecomposed of argentaphilic fibersarranged in irregular bundles,knots, and curves.

The senile or neuritic plaques werethe most conspicuous histologiclesions observed in sections stainedwith the Bielschowsky technique.

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Fig. 4A. The cytoplasm of a neuron is distended by neurofibrillary tangles.H&E, 100X

Fig. 4B. A Bielschowsky silver stain demonstrates the fibrillary characterof the cytoplasmic inclusions. 100X

Fig. 5. Section of cortex from a patient with AD, demonstrating senile plaques. Bielschowsky stain,microwave method. 40X

A B

Fig. 6. Section of cortex. Numerous senile plaques are observed. Bielschowsky stain, microwave method. 100X


The neuritic plaques (Figs. 3, 5, 8,9, 10) are patches or flat areascomposed of clusters ofdegenerating nerve terminalsarranged around a central core ofß-amyloid peptide (BAP). Neuriticplaques are argentaphilic andcontain abundant glial processes aswell as deposits that stain positivelyfor amyloid.4 These plaques werefound in areas of the cerebralcortex that are linked tointellectual function. BAP is afragment of a much larger

membrane-spanning amyloidprecursor protein (APP). Thefunction of APP is unclear, but itappears to be associated with thecytoskeleton of nerve fibers.4

Normally, the degradation of APPinvolves cleavage in the middleof the BAP portion of themolecule, with both fragmentsbeing lost in the extracellular fluid.In Alzheimer’s disease, theAPP molecule is cut at both endsof the BAP segment, therebyreleasing an intact BAP molecule

that accumulates in neuriticplaques as amyloid fibrils5 (Fig. 9).The core of these plaques containsa distinct form of BAP, which is40 amino acids in length. BAP isderived from proteolysis of a muchlarger (695 amino acids)membrane-spanning amyloidprotein.5 Under the microscope,different developmental stages ofneuritic plaques were observed. InFigs. 3A, 5, 6, 7, diffuse plaques areobserved; primitive plaqueswithout a ß-amyloid core areobserved in Figs. 3A, 6, 7. Classicneuritic plaques are observed atlow magnification in Fig. 3B (100X)and at higher magnification inFig. 10 (400X). In our laboratory,the quantitative studies of differentplaques and comparison of stainingmethods in serial sections areunderway, as well as the electronmicroscopy of such cases.

DiscussionThe incidence of the progressiveneurological disorder known asAlzheimer’s disease ischaracterized by loss of memory,carelessness about personalappearance, emotional disturbancesthat progress to completedisorientation, severe deteriorationin speech, incontinence, andstereotypical repetitive movements.Pathologic changes include corticaldegeneration that is more markedin frontal, temporal, and parietallobes. Characteristic degenerationincludes a decrease in neurons inregions of the brain that areresponsible for cognition, memory,and other thought processes. Thecause of AD has not been fullyelucidated, but there have beensignificant advances in ourunderstanding of the origin of bothAD-associated amyloid in theneuritic plaques and theneurofibrillary tangles in thecytoplasm of neurons.

In our AD cases, paraffin-processed tissue sections of cortexstained with Bielschowsky silverstain have given consistent results.In the future, we plan to do more

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Fig. 7. Three different types of neuritic plaques are observed. Diffuse plaques, primitive plaqueswithout a ß-amyloid core, and classic plaques are evident. 100X

Fig. 8. Senile plaques. Bielschowsky stain, microwave method. 400X


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quantitative and morphologicalanalysis. Application of themicrowave modification to varioussections may translate intosignificant savings in time andresources. The application of thisstaining technique to ADpathology offers many possibilitiesfor the study and quantification ofneuritic plaques, neurofibrillarytangles, and granulocorticaldegeneration using both lightmicroscopy and transmissionelectron microscopy.

References1. Reusche E. Silver staining of senile plaques and

neurofibrillary tangles in paraffin sections: a simpleand effective method. Pathol Res Pract.1991;187:1045-1049.

2. Bancroft JD, Stevens A, eds. Theory and Practiceof Histological Techniques. 4th ed. New York, NY:Churchill Livingstone; 1996.

3. Carson FL. Histotechnology: A Self-Instructional Text.2nd ed. Chicago, Ill: American Society of ClinicalPathology; 1997.

4. Navarro A, Tolivia J, del Valle E. Congo red methodfor demonstrating amyloid in paraffin sections.J Histotechnol. 1999;22(4):1-5.

5. Navarro A, Astudillo A, del Valle E, Gonzalez C,Tolivia J. Immunohistochemical presence ofapolipoprotein D in senile plaques. J Histotechnol.2001;24(1):45-48.

CombinedUCHL-1/PASHStaining in the

Diagnosis of RenalTransplant Rejection

Ranjitha Veerappan, MD;Lou Ellen Miller, HT (ASCP);

Piyush Joshi, MD;George A. Youngberg, MD

Pathology DepartmentEast Tennessee State University

Johnson City, [email protected]

IntroductionDetermining if the cause of renalallograft dysfunction in the earlyposttransplant period is due torejection or other reasons is adaunting task for the clinician.Clinical signs and symptoms aloneare not helpful in clearlydistinguishing the numerous causesof renal dysfunction. Variousnoninvasive tests including imagingstudies have also been unsuccessfulin distinguishing rejection fromother causes.1 Renal allograft biopsyhas, therefore, emerged as a keyelement in aiding in the accuratediagnosis of transplant rejection.

An allograft biopsy in the context ofacute rejection not only helps toconfirm the diagnosis, but it alsoreveals if the rejection is a result ofhumoral (antibody-mediated) orcellular immunologic mechanisms.This distinction determines thetherapeutic course and the success-ful reversal of acute rejection.Cellular rejection is the morecommon type and is typically mani-fested by a T-lymphocyte attackdirected against renal tubules (andoccasionally against blood vessels).Microscopically, the early stages arecharacterized by edema and focalinfiltration of the interstitium andperitubular capillaries by lympho-cytes. In later stages, plasma cells,monocytes, and macrophagesappear along with a more diffuselymphocytic infiltrate. Invasion ofthe tubular epithelium by

Fig. 9. Senile plaque. Bielschowsky stain, microwave method. 400X

Fig. 10. Senile plaque stained with Bielschowsky (microwave method) at 400X. The senile plaque presents anabnormal spherical structure that consists of an amyloid core surrounded by dystrophic neurites, astrocytes,and microglial cells.

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lymphocytes is a characteristicfinding of tubulointerstitialrejection.2 To standardize diagnosesand avoid false positives, a formalsystem for interpreting renalallograft biopsies was developed.3

In this system, the diagnosis ofacute cellular tubulointerstitialrejection is based on the findingof tubulitis. The criterion fordiagnosing tubulitis is theidentification of intraepitheliallymphocytes in the tubules.

The interpretation of tubulitis hastraditionally been done usingperiodic acid-Schiff hematoxylin(PASH) staining, which clearlyidentifies the tubular basementmembranes and, therefore, aids inthe accurate assessment of thelocation of the lymphocytes (insideversus adjacent to the tubule).However, a significant drawbackrelated to the PASH stain is thatintraepithelial lymphocytes cannotalways be reliably distinguishedfrom tubular epithelial cell nuclei.Additional stains such as leukocytecommon antigen (LCA) and aT-cell stain (UCHL-1) thatspecifically identifies lymphocyteshave been used to address thisissue. However, these stains againraise the issue of the location of thelymphocytes (inside versusadjacent to the tubule), since thebasement membrane cannot bevisualized by these methods. Theideal stain, therefore, would be theone that combines the ability of thePASH stain to visualize thebasement membrane and alsoidentify the lymphocytes with aspecific marker such as LCA orUCHL-1. This method wouldgreatly facilitate and improve theaccuracy of diagnosing acutecellular tubulointerstitial rejection.Such a stain has recently beendeveloped4 and can be producedusing the Ventana Benchmarkautomated immunohistochemistrystaining system and the NexES™special stains instrument.

Case ReportA 32-year-old man with a liveunrelated donor renal allograft was

noted to have rising creatinine. Hisimmunosuppressive regimen con-sisted of rapamycin and steroids,with no mycophenolate mofetil orcalcineurin inhibitors. The elevatedcreatinine did not respond to pulsemethylprednisolone, so a percu-taneous renal biopsy was obtained.

MethodsRoutine light microscopic renalbiopsy stains, including PASH andhematoxylin and eosin (H&E),were made using standard methods.

A UCHL-1/PASH stain wasprepared according to the methodof Resch et al,4 using a Ventana(Ventana, Tucson, AZ) combinedautomated immunohistochemical(BenchmarkTM) and histochemical(NexES) stainer. Briefly, the renalcore biopsy was received inCarson’s fixative, processedovernight, and paraffin embedded.The sections were cut at 3 micronsand stained on the Benchmarkusing the iViewTM DAB paraffindetection kit (Ventana, Tucson, AZ)

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Fig. 1. Tubulitis in the renal biopsy. Lymphocyte cytoplasm stains brown. Tubular borders are clearly delineated.UCHL-1/PASH, 400X

Fig. 2. Distinguishing lymphocytes from tubular cells can be difficult on a standard PASH stain. PASH, 400X


with the T-cell (CD45RO)antibody. After the antibody step,the slides were removed fromthe Benchmark, rinsed in DIwater/Dawn® dishwashing liquid,and placed on the NexES forroutine PASH stain. The slideswere then dehydrated throughgraded alcohols, cleared in xylene,and mounted with a permanentmounting medium.

ResultsThe biopsy demonstrated moderateinterstitial inflammation involvinggreater than 25% of the corticalarea. The inflammatory infiltrateconsisted predominantly oflymphocytes, with some admixtureof plasma cells, eosinophils, andneutrophils. Several tubularprofiles showed more than fourmononuclear cells per tubularcross-section. This was most clearlydemonstrated on the PASH/T-cellstain (Fig. 1). No tubular cross-sections contained more than tenmononuclear cells. The PASH/T-cellstain confirmed that the majorityof lymphocytes present wereT-cells. A diagnosis of acute cellularrejection, tubulointerstitial typewas made (Banff 1997 acuterejection, type IA).

DiscussionThe key element in making adiagnosis of acute cellularrejection, tubulointerstitial type,is the identification of tubulitis.This can be challenging becauselymphocytes in foci of presumptivetubulitis can be difficult todistinguish from tubular nuclei onstandard stains like H&E or PASH(Fig. 2). Immunohistochemicalstains for LCA or T-cell markerscan reliably identify thelymphocytes, but it may be difficultfor these stains to distinguishwhether the lymphocytes are trulyinside the tubular basementmembrane or merely adjacent tothe tubule (Fig. 3). Combining abasement membrane stain (PAS)with a T-cell stain (UCHL-1)elegantly solves the problemof both specifically identifying

T-lymphocytes and preciselydetermining their location (Fig. 4).Thus, the presence of tubulitiscan quickly and accurately beconfirmed or excluded. Thiscombination of UCHL-1/PASHgreatly improves the accuracyof diagnosing acute cellulartubulointerstitial rejection andhelps determine the therapeuticcourse and prognosis.

References1. Brown ED, Chen MY, Wolfman NT, Ott DJ,

Watson NE Jr. Complications of renaltransplantation: evaluation with US and radionuclideimaging. Radiographics. 2000;20(3):607-622.

2. Rosai J. Rosai and Ackerman’s Surgical Pathology.8th ed. Elsevier Science; 1996:1115-1116.

3. Racusen LC, Solez K, Colvin RB, et al. The Banff 97working classification of renal allograft pathology.Kidney Int. 1999;55(2):713-723.

4. Resch L, Yu W, Fraser RB, et al. T-cell/periodicacid-Schiff stain: a useful tool in the evaluationof tubulointerstitial infiltrates as a componentof renal allograft rejection. Ann Diagn Pathol.2002;6(2):122-124.

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Fig. 3. It is difficult to identify borders of tubules on a standard lymphocyte stain; determining the locationof the lymphocytes (outside versus inside the tubular basement membranes) can be problematic. LCA, 400X

Fig. 4. Lymphocytes are clearly identified, and their precise location in relation to tubular basement membranesis easy to determine on the PASH/T-cell stain. UCHL-1/PASH, 400X


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IntroductionCryomicrotomy Part II is acontinuation of “Preparation andSnap Freezing Part I” thatappeared in the May 2004 issueof HistoLogic.1 The terms cryo-microtomy and cryotomy aresynonyms and will be used inter-changeably in this article. Althoughthe techniques presented can beapplied to clinical situations, thisdiscussion is directed primarilytoward research laboratoriesworking with rodents and otheranimal tissues. A major complaintby researchers is that frozensections (FS) have very poormorphology when compared toeither paraformaldehyde (PFA)or neutral buffered formalin (NBF)fixed paraffin tissues. However, ourlaboratory encountered majorproblems using these aldehydefixatives for murine lymphocyte(CD) marker immuno-histochemistry (IHC) andimmunofluorescent (IFA) staining.Unfortunately, and as others havediscovered, some murine CDantigens are so compromised byaldehyde cross-linkage thatattempts at heat induced epitoperetrieval (HIER) or enzymedigestion fail to recover anti-genicity, and yield no IHC or IFAstaining. Fluorescence and confocallaser scanning microscopy (CLSM)are also plagued with increasedautofluorescence as a result ofaldehyde fixation.2 Consequently,cryotomy and organic solventfixation became our major optionsto avoid aldehyde fixatives forsuccessful IHC and IFA. Wenoticed our tissue morphologywas very good afteracetone/alcohol fixation andsomewhat less so after acetone

fixation. With organic solventfixatives, we have excellent IHCand IFA staining and never haveto use antigen recovery methods.

Frozen sections may be obtainedfrom either fresh frozen tissues ortissues previously fixed in PFA orparaformaldehyde-lysine-periodate(PLP).3 Fixed tissues must becryoprotected in 30% sucroseovernight at 4°C. Tissue collectionand snap freezing methods werediscussed at length previously.1 As areminder, our research tissues arenever frozen inside a cryostat usinga Peltier cooling device or heatextractor. Cryostat freezing temper-atures are warmer than the snapfreezing temperatures discussed inPart I. Consequently, cryostat tissuefreezing is slower and creates large,damaging ice crystal formations,known as freezing artifact, in tissuespaces (Fig. 1). Hematoxylin and

eosin (H&E) and other specialstains (oil red O for lipids) can bedone with FS, in addition toIHC/IFA methods. This article willdiscuss general cryostat setup andusage, ergonomics, cryostat chambertemperature differentials, optimalsectioning temperatures fordifferent tissues, microtome blades,hints on specimen block orientation,cryosectioning, biohazards, as wellas block and FS handling andstorage.

CryostatA primary consideration is whichcryostat is best suited for researchcryomicrotomy. With so manychoices, any cryostat availableshould work well. Cryostats comewith either manual or motorizedoperation, and the latter is becomingmore popular because it helps inpreventing repetitive motioninjuries. Our cryostats need littlespace, are easy to defrost and clean,are ergonomically comfortable touse, and have excellent, uncrowdedchamber space to work in and tohold multiple blocks. A tall,comfortable chair with a pneumaticlever permits seat height adjustmentto accommodate tall and shorttechnicians. During cryostatoperation, technicians should sitrather than stand to help preventstress on back, arms, and neck. Jarsof stain or fixative should not be

Cryomicrotomy of Murine Tissuesfor Research Immunohistochemistry andRoutine Hematoxylin and Eosin Staining

Part IIGayle M. Callis, MT, HT, HTL(ASCP)

Veterinary Molecular BiologyMontana State University, Bozeman, MT

[email protected]

Fig. 1. Freezing artifact in muscle shows severely damaged morphology by water ice crystal formation.Muscle is no longer recognizable. NBF-fixed frozen section, H&E. 40X

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placed on top of cryostat or insidethe chamber in order to preventspills in a chamber or on electroniccomponents of the instrument.A counter or cart placed next to acryostat provides an extra workarea within easy reach of theoperator. Our laboratory has onecryostat designated for BiosafetyLevel 2 prion tissues, and it is keptin a separate room.

Cryostat Chamber andTemperature Differentials Cryostats have warm and cold areaswithin the chamber, and these areascan be used to make cryosectioningless difficult. Our cryostats havemetal bars with holes to hold metaldisks (or tissue chucks) and a Peltiercooling plate. These bars keepblocks 10°C colder than the pro-grammed temperature of theinstrument cabinet. Mountingblocks using cold bars or a Peltierdevice can cool blocks in excess of-30°C to -40°C. These colder tem-peratures can make some tissuestoo cold for immediate sectioning.After using a thermometer to findour cryostat chamber’s warm/coldspots, we learned that areas imme-diately adjacent to the microtomewere the same temperature as thechamber temperature at the micro-tome block and knife holders.Newly mounted, colder blocks areplaced next to the microtome toequilibrate to a desired sectioningtemperature for 20 minutes beforecryotomy. We can store up to20 mounted blocks in a handyplastic microcentrifuge tube racknext to the microtome to keep theblocks ready for sectioning at thedesired temperature. Immediatesectioning of -30°C to -40°C blocksresulted in shattered, ugly liver,brain, and spleen frozen sections.One must be aware of the cryostat’scooling capabilities, temperaturedifferentials, and how theseconditions can be optimizedto get a good frozen section.

Mounting BlocksOnto Metal ChucksTissue-Tek® O.C.T. Compound(Sakura Finetek, Torrance, CA)

embedded blocks removed from a-80°C freezer must be placed in thecryostat chamber to allow them toequilibrate to sectioning tempera-ture. This takes approximately 20to 30 minutes. To speed up our tem-perature equilibration, we nowmount -80°C blocks immediatelyafter removal from the freezer. Thekey is to wait for the tissue to be atthe required sectioning tempera-ture. Large blocks are mounted onlarge block holders to ensure goodsupport under the whole block. Wepurchased additional mushroom-style chucks with waffle-weave topsand shortened the stems so the backof the disk fits flat against themicrotome. A generous drop ofOCT is applied under the block toprovide firm support and avoidoverhanging edges. Bubbles trappedunder the block lead to chatter,“chopped” blocks with lost tissue, orblock separation during sectioning.Bubbles can be avoided by pushingthe block firmly into the OCT usinganother cold disk. Mineralized boneblocks are mounted on waffle-weave chucks using 2% methyl-cellulose, water soluble (AldrichChemical Co., Milwaukee, WI).Frozen methylcellulose is extremelyhard for extra holding power and

vibration-free frozen bonesectioning.

Cryosectioning TemperaturesTechnicians should testcryosectioning temperatures forany given tissue using their chosencryostat. Because tissues havedifferent sectioning qualities,densities, and homogeneity (liver,spleen, brain), we select asectioning temperature best suitedto cut a good FS from the tissue.Cryosectioning brain, liver, spleen,fatty tissue, and bone can be verydifficult if the tissue and bladetemperatures are not optimal andidentical. After trial and error, wefound temperature settings andguidelines that worked best for us(Fig. 2). One must be willing tochange the temperature in order toobtain a good frozen section.Mineralized bone is routinelycryosectioned using CryoJane®

Tape-Transfer System(Instrumedics, Hackensack, NJ).Acid decalcified bone must bethoroughly rinsed in running tapwater followed by 30% sucrosecryoprotection. Residual acid inunwashed decalcified bonetrimmings could corrode metalcryostat parts. EDTA-decalcified

Suggested Cryosectioning Temperatures for Tissues

Cryosectioning Temperature Tissue of Blade and Tissue

Normal or inflamed tissues; -20°Clymph nodes, small intestine,Peyer’s patches, kidney, and stomach

Spleen, liver, brain, spinal cord -17°C to -16°CBrain, prefixed/sucrose cryoprotected -19°C or warmer for thick 50 µm sections

Skin folded into “V” shape -20°C with hair oriented inside the “V”

Mineralized bone -28°C to -32°C with CryoJane Extremely fatty tissue, oily mink skin

Lung—fresh, unfixed, OCT-filled -20°CLung—fresh, not OCT-filled -26°C with CryoJane Lung and mouse nasal turbinates—PFA- -26°C prevents sucrose or PLP-fixed with 30% sucrose oozing out of frozen tissue blockcryoprotection

Fig. 2. Temperature settings used in our lab. Cryosectioning temperature settings may vary depending on the cryostat.

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and sucrose-cryoprotected mousenasal turbinates are easilysectioned using high-profiledisposable blades.

Microtome BladesOur laboratory prefers Accu-Edge®

(Sakura Finetek, Torrance, CA)high-profile (over low-profile)disposable blades. We found nodifference in sharpness betweenhigh- or low-profile blades, butwider, thicker high-profile bladesproved more stable duringcryotomy. However, techniciansshould choose the optimal blade fortheir needs. The blade is movedfrequently to ensure the sharpestedge; care is taken to avoiddamaging the edge with a brush orgauze. The blade holder must becleaned, lubricated, and properlyadjusted, with no loose screws orlevers. Angle settings can bechanged by an accidental looseningof the adjusting levers orunfamiliarity with knife holderoperation. A desired blade anglesetting can be marked using aSharpie® marker on top and bottomof the knife holder for a quickrealignment of a “lost” angle setting.A used blade should not be returnedto the package slot but disposed ofin a biohazard sharps container.Mineralized bone can be sectionedwith either a D or C profile tungstencarbide (TC) tipped, steel backcryostat knife (Delaware DiamondKnives, Wilmington, DE). Tungstencarbide blades are expensive andmust be kept extremely sharp and inprime condition for bone cryotomy.Steel blades, C profile, work well forall tissues except bone and must befree of nicks or rust, and sharpenedfrequently with a good knifesharpener or reconditioningservice. A dull blade of any designwill be useless for cutting goodfrozen sections.

CryomicrotomyBlock OrientationThe block and/or tissue is orientedin the microtome holder to providea path of least resistance duringsectioning. Rather than place ablock with the long edge of tissue

parallel to the blade edge, we turnthe block so it looks like a diamondwith one corner pointed downtoward the blade edge (Figs. 3, 4).This allows the block and tissue tocontact the edge en pointe with lessresistance as the tissue begins tosection. One can actually feel thisresistance during a sectioningstroke as block and tissue meetthe blade. Initial tissue orientationat embedding is also importantto enable cryotomy. 2

CryosectioningProper safety precautions must betaken for known or potentiallybiohazardous agents. A dedicatedcryostat or special biosafetycontainment area may be necessarywhen working with infectiousbacteria, viruses, and prions intissue. Gloves have not hinderedcryosectioning techniques ormanipulation requiring finedexterity. Reusable silk glove liners(www.wintersilks.com) may be

Fig. 3. Block with mouse spleen. Block face oriented to blade edge for resistance-free cryotomy and uncompressedspleen FS. Blue dotted line indicates where OCT is trimmed away using room temperature, Teflon®-coated razorblade. This shapes the block and removes excess OCT, if needed.

Fig. 4. Mouse spleen in block oriented as a (pink) diamond for “path of least resistance” cryotomy. Styrofoamfinger rest is seen on left side of blade holder. Anti-roll plate is removed for brush technique sectioning.Trimmings are pushed behind blade with large blue cleaning brush.

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worn inside disposable gloves tokeep hands warm and dry duringlong cutting sessions.

Cryotomy is very easy to learn withpatience and practice. How othersperform this task varies greatly; thisled to a lively demonstration anddiscussion at a hands-on workshopat the 2003 NSH.4 We prefer toremove or not use the anti-rollplate in favor of the cold “brushtechnique.” As a 5 µm FS beginsto pass over the blade edge, a finecamel’s hair brush is used tocapture the curl, and the section isgently guided but not brushed ontothe blade holder (Fig. 5). The brushtechnique is very fast, does notprevent serial sectioning, andavoids the need for repeatedcleaning and adjustment of theanti-roll plate. A styrofoam squaretaped onto the blade holder allowsthe technician to rest the littlefinger for a relaxed hold on thebrush; it also aids in frostbiteprevention (Fig. 4). The anti-rollplate is reinstalled to keep thick,50 µm brain sections from curlingover, as thick section manipulationis harder to control with a brush.

The cryostat flywheel should beturned slowly and continuously sothe blade passes through the tissuesmoothly. Hesitation during flywheelrotation creates a jerky motion andthe tissue section will have uneventhick/thin areas. A light grip of theflywheel handle works best. A FScan be picked up two ways from theblade holder. With the first method,a room temperature slide is loweredwith a quick, featherlight motiontoward, and then away from, the FSto attract (also known as flash dry)the section onto the slide. Thesection must never be pushedagainst the holder by a slide or theFS will melt and refreeze onto theholder. The other method requiresthat slides be cooled inside thecryostat. After a FS is cut, it ismanipulated, using the brush, fromthe holder onto the top of a coldslide. The section will adhere to theslide by warming the back of theslide with your hand or finger.

Block Storage After sectioning, blocks are sealedwith a thin OCT layer smearedacross the block face and allowedto freeze. Sealed blocks helpprevent tissue from drying outduring freezer storage. Large dropsof OCT on top of tissue areavoided to prevent melting andrefreezing of tissue. One should beable to recut a sealed block and notlose valuable tissue to excessiveretrimming. Never store frozenblocks in a cryostat chamber or self-defrosting freezer. Defrost cyclescan cause freeze/thaw damage toantigens and create freezingartifact in tissue. Sealed blocks canbe wrapped with aluminum foil,marked with a Sharpie marker, putinto ziplock bags or 50 ml screwtop centrifuge tubes, and stored ina -80°C freezer for a period ofyears, or a -27°C freezer for aperiod of months. We have beenable to resection tissue after 6years of storage at -80°C whileretaining excellent morphology,antigenicity, and sectioning quality.

Cryostat decontamination remainscontroversial as some productshave a high water content(Clorox®), which causes ice buildup,some are corrosive to cryostatparts, and others allow for toxic

fume exposure (formalin).Some cryostat manufacturers toutdecontamination features, but thesemay not work for infectious agentsresistant to heat, formalin, or lowClorox concentration.

Frozen Section Handlingand StorageFrozen sections on slides destinedfor IHC/IFA should not be storedin a cryostat immediately aftersectioning. When a FS is movedfrom the cryostat to room tem-perature, water condensation formson top of the FS. This condensationcan cause damage to tissuemorphology. An exception is FSfor laser capture microdissection(LCM) and in situ hybridization(ISH) work.4 After slide mounting,these FS are kept on dry ice(approximately -90°C) or in a blackslide box surrounded by dry iceinside the cryostat to minimizeRNA loss.

How a FS should be storeddepends on what ultimately will bedone to the section. For an H&Estain, a fresh, unfixed FS can beimmersed immediately into roomtemperature NBF or PFA, fixed for10 minutes or more, rinsed withdistilled water, and stained with aroutine H&E method. We found

Fig. 5. Brush technique used to guide section onto blade holder before pickup onto a glass slide, while block staysdown and behind the blade. Note side of finger resting on styrofoam for relaxed brush control.

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that these H&E stained FS hadgood morphology preservationcomparable to paraffin-embeddedtissues (Fig. 6).

Frozen Sections for IHC/IFAProceduresIn our lab, antigen stability dictatesFS storage conditions, e.g., temper-ature and time. Our perfused fixedtissue (PFA or PLP) FS are air driedat RT for 30 minutes (minimum)and placed into either a black slidebox (25 slots) or 5-slide mailer(s)inside a ziplock bag. Nylon bags filledwith 16 mesh silica gel were placedinto storage containers to maintaindry conditions. Freezer storage canbe at -80°C (for several weeks) orsometimes at -27°C (for up to aweek), with -80°C the preference.Before IHC staining, the slide boxis removed from the freezer andnot opened for 30 minutes to allowthe sections to equilibrate to roomtemperature. Boxes are never to beopened to take out just a few slidesand then reclosed and returned tothe freezer. Freeze/thawing ofsections and water condensationmust be avoided to prevent loss ofantigenicity. These freezer storageconditions and the temperature

equilibration are done with all ourunfixed and fixed frozen sections.

Fresh unfixed frozen sections arehandled in 4 ways. It is recom-mended that optimal fixationparameters (length of air drying, typeof fixative, fixation time, and temper-ature) be determined for your labor-atory’s IHC/IFA staining success.

1. Fresh tissue FS are air dried for30 minutes (minimum) andstored unfixed using previouslydescribed freezer storageprocedures. On the day before weperform IHC, stored sections areremoved from -80°C and allowedto air dry at RT overnight insidethe closed box. These FS arefixed just before beginning theIHC protocols.

2. Fresh tissue FS are air dried30 minutes to several hours, thenfixed in 4°C acetone, air dried todisperse acetone fumes, thenstored as described. Acetone-fixed sections are removed fromthe freezer on the day of stainingusing described temperatureequilibration of sections in theclosed slide box.

3. Fresh tissue FS are air driedovernight at RT. Dry FS are fixedin either RT 75% acetone inabsolute ethanol (5 min) andbuffer rinsed, or 4°C acetone(10 min), air dried and bufferrinsed, followed by IHC staining.These are the preferred fixationmethods in our lab.

4. In one day, fresh tissue FS arecut, air dried for 30 minutes(minimum), fixed with a 4°Cacetone method, and IHC stained.

SummaryFrozen sections, although notalways popular, are often whatresearchers must accept in orderto obtain good immunostainingresults. Our laboratory nowperforms more cryotomy thanparaffin microtomy, and we enjoyexcellent tissue morphology andIHC staining results. On a personalnote, I actually prefer doingcryotomy to paraffin work. Thecryosectioning technique is easy tolearn and teach to others. Bycleverly using a cryostat chamber’swarm and cold spots and choosinga sectioning temperature suitablefor a tissue, cryotomy can beefficient and trouble free. Withoutexception, a blade edge must besharp and free of nicks. Withproper snap freezing, carefulcryosectioning, block and sectionhandling, fixation, and staining, thesometimes dreaded FS can behighly acceptable. For those whohave done paraffin sectionsexclusively and suddenly have todo cryomicrotomy, be patient, payattention to fine detail, relax, andenjoy the challenge of getting theperfect frozen section.

References 1. Callis G. Preparation and snap freezing of murine

tissues for research immunohistochemistry androutine hematoxylin and eosin staining. HistoLogic.2004;37(1):4-7.

2. Billinton N, Knight AW. Seeing the wood through thetrees: a review of techniques for distinguishing greenfluorescent protein from endogenousautofluorescence. Anal Biochem. 2001;291:175-197.

3. McLean IW, Nakane PK. Periodate-lysine-paraformaldehyde fixative: a new fixation forimmunoelectron microscopy. J Histochem Cytochem.1974;22:1077-1083.

4. Callis G, Sterchi DL. Cryomicrotomy for researchmurine immunohistochemistry and laser capturemicrodissection. National Society for HistotechnologyS/C. Louisville, Ky; 2003. Workshop #3.

Fig. 6. Fresh bovine skin, 5 µm FS, NBF fixed, then stained with H&E. 20X

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35

Alcian Blue-H&E-Metanil Yellow Stain

for DiagnosingBarrett’s EsophagusWendi Edgett, BS, HTL(ASCP)

Genesys Regional Medical CenterHealth Park Campus

Grand Blanc, [email protected]

IntroductionBarrett’s esophagus is a conditionthat can lead to adenocarcinoma.Proper diagnosis and follow-uptreatment is very important forpatients diagnosed with this disorder.Sometimes it can be difficult todiagnose.The Alcian Blue-H&E-Metanil Yellow (AB-H&E-MY)stain is very effective indistinguishing between Barrett’s andother gastrointestinal disorders. It issimple to perform, and the resultscan be easily duplicated from onestaining batch to another.This stainhelps save time and money bycombining multiple stains todemonstrate many structures.

DiscussionThe gastrointestinal (GI) tract iscomposed of four distinct layers:the mucosa, submucosa, muscularispropia, and adventitia. Theepithelium of the GI tract variesdistinctly as you transition fromone area of the tract to the next,from the esophagus through thestomach and the intestine. Theepithelium is very specific to theproperties and purpose of eacharea of the tract. The esophagus iscomposed of stratified squamousepithelium that provides protectionfrom contact with the food weswallow (Fig. 1). The point in whichthe esophagus and the stomachmeet is called the gastro-esophageal (GE) junction, wherethere is a distinct change from thestratified squamous epithelium ofthe esophagus to the absorptivetubular epithelium of the stomach(Fig. 2). The epithelium of thestomach has mucus-secretingglands that contain neutral mucins.

The intestine is composed oftubular epithelium that containsmucin-secreting goblet cells.1

Barrett’s esophagus is a conditionthat occurs when the normalsquamous epithelium of theesophagus is replaced by intestinalcolumnar epithelium. This arisesfrom the chronic irritation causedby gastroesophageal reflux disease(GERD). Acid from the stomachcauses the cells of the esophagusto be damaged. When they areregenerated, they may be replacedwith the columnar epitheliumsimilar to that found in the

intestine. This is called metaplasia.An individual with the presenceof intestinal metaplasia will bemonitored with repeat biopsiesapproximately every 2 years if nodysplasia is found and every6 months if dysplasia is present.2-5

Endoscopic biopsies comprise alarge percentage of the specimenswe process in the lab, and amongthese biopsies, a large number areesophageal biopsies. These biopsiesare generally very small fragmentsof tissue that routinely are cut withtwo or three levels per block. Bypicking up a section from each level

Fig. 1A. GI tract layers. H&E; 10X Fig. 1B. Esophageal mucosa. H&E; 40X

Fig. 2. Gastroesophageal (GE) junction. H&E; 40X

Mucosa

Submucosa

Muscularis propia

Muscularis mucosa

Epithelium

Lamina

Esophagus

Stomach


for the special stain at the sametime that the sections for hema-toxylin and eosin (H&E) stainingare cut, it gives the pathologist theopportunity to evaluate both slidesat the same time.

H&E, the most frequently usedstain in histology labs, stains a vastarray of tissue structures, providinga simple and comprehensivestarting point for the pathologist tomake a diagnosis. The hematoxylinis used to stain the nuclearchromatin, which renders the nucleiblue. The eosin is used to stain theconnective tissue, whosecomponents appear in three shadesof pink—red blood cells stain thedarkest, muscle stains slightlylighter, and collagen stains thelightest shade of pink.

The alcian blue stain is primarilyused to stain for acidmucopolysaccharides. At a pHof 2.5 it will stain both sulfated(sulphomucins) and carboxylated(sialomucins) mucopolysaccharides,which are found in the goblet cellsin the intestine. The alcian blue dyeis a very large, positively chargedmolecule in solution that will bindto the negatively charged lowdensity mucin found in the gobletcells, staining them blue.6 Thealcian blue can be modified todistinguish between the acidmucins by modifying the pH ofthe staining solution. In theAB-H&E-MY stain, alcian blueis used at a pH of 2.5. The gastricmucosa of the stomach iscomposed of mucus-secreting cells.These cells contain neutralmucopolysaccharides and typicallydo not stain with alcian blue;however, in some circumstances,there can be some variable, weaklypositive staining seen with alcianblue, yielding a very pale blue.6

Metanil yellow is a counterstainthat stains collagen yellow. Someprocedures call for the use ofsaffron (a rather expensive spice);however, for economical purposes,metanil yellow has been substitutedfor saffron in this report. When themetanil yellow is combined with

the alcian blue, the mucin inBarrett’s esophagus stains abrilliant turquoise color. In somesituations, it can become difficult todetermine if the glands present inan esophageal biopsy are gastricmucosa or intestinal metaplasia, asseen in Barrett’s esophagus. Byusing the AB-H&E-MY stain, thecells of Barrett’s esophagus aremore easily demonstrated.

Case StudiesThe following are three casestudies that represent the clinicalsignificance of the stain. In all threecases, the pathologist was presentedwith an H&E at three levels and an

AB-H&E-MY of consecutivesections of the same three levels.

1. A 77-year-old Caucasian malesuffers from chronic reflux andhas a history of Barrett’sesophagus. He also has a smallhiatal hernia. The patient wasscheduled for a follow-up endo-scopic biopsy to determine ifdysplasia was present. The biopsywas obtained from the longsegment of Barrett’s esophagus,28-40 cm from the incisors. TheH&E revealed glandular mucosawith infiltration of plasma cellsand lymphocytes, and the biopsywas negative for dysplasia.

Fig. 3A, 3B. Biopsy from the long segment of Barrett’s esophagus, 28-40 cm from the incisors, demonstratingthe esophageal mucosa. H&E. A) 40X; B) 400X

Fig. 3C, 3D. Biopsy from the long segment of Barrett’s esophagus, 28-40 cm from the incisors, demonstratingthe presence of specialized intestinal metaplasia. AB-H&E-MY. C) 100X; D) 200X

A

C D

B

36


Fragments showed ulceration anderosion with the presence ofgranulation tissue. The presenceof specialized intestinal meta-plasia was demonstrated by theAB-H&E-MY (Fig. 3C, 3D). Thepatient was diagnosed withulceration with erosion andgranulation tissue and chronicallyinflamed, nondysplastic Barrett’sesophagus. The patient is recom-mended to undergo a repeatbiopsy in 2 years.

2. A 56-year-old Caucasian male hasa history of Barrett’s esophagus.His last endoscopic evaluation was2 years previous, indicatingBarrett’s esophagus and showingno dysplasia.The patient wasscheduled for a follow-upendoscopic biopsy to determine ifdysplasia is present.The biopsy

was obtained from the longsegment of Barrett’s esophagus,25-40 cm from the incisors.TheH&E revealed chronicallyinflamed glandular tissue, withnumerous plasma cells andlymphocytes in the lamina propria,and the biopsy was negative fordysplasia.The AB-H&E-MYdemonstrated a large number ofgoblet cells present (Fig. 4C, 4D).The patient was diagnosed withchronic active inflammation ofnondysplastic Barrett’s esophagus.The patient is recommended toundergo a repeat biopsy in 2 years.

3. A 65-year-old Caucasian femalecame to the emergency roompresenting with symptoms ofdiarrhea, bloody stool, andtenderness in the lowerabdominal area; the symptoms

have been present for 10 days andworsen after she eats. She has ahistory of dysphagia (difficultyswallowing), esophagitis(inflammation of the esophagus),and GERD. The patient wasscheduled for an endoscopic biopsyto rule out Barrett’s esophagus, andthe biopsy was taken from theregion of the GE junction. TheH&E revealed esophageal mucosaof squamous epithelium that wasnegative for dysplasia. The gastricmucosa has a large amount oflymphatic infiltrate in the laminapropria. The AB-H&E-MYconfirms the presence of focalizedintestinal metaplasia (Fig. 5C, 5D).The patient was diagnosed withchronically inflamed, nondysplasticBarrett’s esophagus.

Materials and MethodsTissue Preparation and Sectioning Hollandes fixative is the preferredfixative for gastrointestinal biopsies.Hollandes is a picric acid-basedfixative that is very effective inpreserving the mucin in the tissue.6However, since mucin does notbreak down as quickly as othercarbohydrates, neutral bufferedformalin can be used. Routinelyprocessed paraffin sections are cutat 4 µm, and sections for the routineH&E and the AB-H&E-MY are cutat the same time.

Solutions3% Acetic Acid

Glacial acetic acid…………3.0 mlDistilled water……………97.0 mlStir together. Stable at roomtemperature for months.

Alcian Blue, pH 2.5Alcian blue …………………1.0 g3% acetic acid …………100.0 mlThymol crystalsDissolve alcian blue in acetic acid.Check pH; adjust the pH to 2.5,using acetic acid as needed. Add afew crystals of thymol to preventmold growth. Solution is stable atroom temperature for monthsand may be reused until weak.

Mayer HematoxylinCommercially made

Fig. 4A, 4B. Biopsy from the long segment of Barrett’s esophagus, 25-40 cm from the incisors, demonstratingthe mucosa. H&E. A) 40X; B) 400X

Fig. 4C, 4D. Biopsy from the long segment of Barrett’s esophagus, 25-40 cm from the incisors, showing a large numberof goblet cells, indicative of intestinal metaplasia in the esophageal mucosa. AB-H&E-MY. C) 100X; D) 400X

A

C D

B

37


0.25% Hydrochloric AcidHydrochloric acid, conc. …2.5 mlDistilled water …………997.5 mlCarefully add hydrochloric acid tothe distilled water slowly. Stableat room temperature for months.

0.25% Ammonia WaterAmmonium hydroxide, conc.1.0 mlDistilled water …………399.0 mlSlowly add ammoniumhydroxide to distilled water. Usefor 1 day only.

EosinCommercially made

0.25% Metanil YellowMetanil yellow ……………0.25 gDistilled water …………100.0 mlGlacial acetic acid ………0.25 ml Mix together well. Stable at roomtemperature for up to 1 year.

Method1. Deparaffinize and bring

sections to water2. Stain with alcian blue, pH 2.5

solution, 15 minutes3. Wash well with water4. Stain in Mayer hematoxylin,*

4 minutes5. Rinse in running water,

several changes6. Differentiate in 0.25%

hydrochloric acid, 2-3 seconds7. Rinse in running water, several

changes8. Blue in 0.25% ammonia water,

2-3 seconds9. Rinse well in running water,

several changes10. Place in 70% ethanol, 1 minute11. Stain with eosin solution,

1 minute12. Dehydrate in 95% ethanol,

30-60 seconds

13. Dehydrate in 100% ethanol,two changes, 30 seconds each

14. Place in metanil yellowsolution,† 1 minute

15. Rinse with ethanol, 2 changes,10 dips each

16. Clear with xylene, 3 changes,2 minutes each

17. Mount in a resinous medium

*For automated stainers, run a program on the stainerthat takes the slides from water, through your routineH&E, and stops at the second change of absoluteethanol.

†Timing of the metanil yellow is critical. If stained fortoo long, increased background staining will occur.

ResultsNuclei—blue Cytoplasm—pink-red Mucin—turquoise forBarrett’s esophagus goblet cells(some gastric mucin will stain afaint blue)Collagen—yellow Smooth muscle—salmon

ConclusionWhen the three stains used in theAB-H&E-MY are combined, theyyield dark blue nuclei, pinkish-redcytoplasm, turquoise mucin inBarrett’s esophagus vs. eithernonstaining or pale blue mucin ofherniated tissue, yellow collagen,and salmon smooth muscle. Thisstain gives the pathologists acolorful demonstration of thevarious components of thegastrointestinal tract with which tomake a diagnosis. It is very usefulin diagnosing Barrett’s esophagusand distinguishing it from other GIconditions.

References1. Burkitt HG, Young B, Heath JW. Wheater’s Functional

Histology: A Text and Colour Atlas. 3rd ed. ChurchillLivingstone; 1993.

2. Blount PL, Cowan DS, Reid BJ. Fred Hutchinson CancerResearch Center and the University of Washington. An in-depth information source about Barrett’s esophagus.Available at: http://barrettsinfo.com/index.htm.

3. Levine DS. Barrett’s esophagus. Sci Am. 1994;1:16-25.4. Johns Hopkins Pathology Web site. Barrett’s esophagus:

understanding Barrett’s. Available at:http://pathology2.jhu.edu/beweb/menu_understanding.cfm.

5. Reid BJ, Blount PL, Rubin CE, Levine DS, Haggitt RC,Rabinovitch PS. Flow cytometric and histologic progressionto malignancy in Barrett’s esophagus: prospectiveendoscopic surveillance of a cohort. Gastroenterology.1992;102:1212-1219.

6. Bancroft JD, Gamble M. Theory and Practice ofHistological Techniques. 5th ed. New York, NY: ChurchillLivingstone; 2002:163-169, 178-183.

Fig. 5A, 5B. Biopsy from the region of the GE junction, demonstrating esophageal and gastric mucosa.H&E. A) 40X; B) 400X

Fig. 5C, 5D. Biopsy from the region of the GE junction, demonstrating the presence of focalized intestinal metaplasia.AB-H&E-MY. C) 100X; D) 400X

A

C D

B

38


39

Double Embedding:Double the Trouble?M. Reid, MLT; C. Goodwin, MLT

Department of Pathology& Laboratory Medicine

Mount Sinai Hospital University of Toronto, Canada

[email protected]

AbstractTraditionally, double embedding1 isused to process large and densebone blocks. While the quality of thesections is excellent, there are safetyissues to be considered, such asexposure of technologists tochemical fumes. Ether, celloidin,and chloroform can be toxic to therespiratory system. In addition, thesechemicals are a fire hazard for thelaboratory. Automation of doubleembedding has been precluded bythe use of these hazardous solutionsduring processing. This lack ofautomation results in a totallymanual procedure, which takes15 days to complete, increasing thetechnologist’s hands-on time anddelaying the time to diagnosis. Inthe interest of reducing turnaroundtimes and exposure to volatilechemicals, we have developed anautomated processing schedule toembed large and dense bone blockson a standard processor. By usingprolonged times in graduatedalcohols, xylenes, and paraffin, wehave been able to reduce thespecimen processing time to 3 days.Blocks embedded using thisprocessing technique demonstrateno difference in cutting, and thehistological features of the tissue arepreserved. Decreasing turnaroundtimes without sacrificing qualityhas had a positive impact on thecost of processing and quality ofpatient care.

IntroductionCutting large bone blocks presentsone of the greatest challenges forthe histotechnologist. Every stageof processing must be adequatelyperformed to ensure good qualitysections. Double embedding usingcelloidin has been the method ofchoice because of its excellent

results. There is little shrinkage ofthe tissue since no heat is usedduring processing. Tough tissueis not made any harder; soft tissueis completely impregnated andsupported; and the relationship ofdifferent tissues is maintained.

There are also some disadvantagesto double embedding. The ether,celloidin, and chloroform used inprocessing pose a respiratory dangerfor the technologist. The chemicalsmust be used in an adequatelyventilated area, preferably a fumehood, which means that this methodcannot be automated. Performingeach step manually increases thetechnologist’s hands-on time. It takes15 days to complete the processingfrom dehydration to waximpregnation. This greatly

delays the turnaround time forpatient diagnosis. In order toreduce both the turnaround timeand exposure of the technologistto hazardous chemicals used indouble embedding, we set out todevelop an automated processingmethod that maintains thehistological appearance butshortens the processing time.

Materials and MethodsRepresentative 5 mm thick sectionswere sampled from human femoralhead, tibial plateau, and femur(cross-section). The tissues varied insize from 2x2 to 5x5 cm in cross-section. The tissues were fixed in10% buffered formalin (0.1Mphosphate buffer, pH 7.4) (VWR,Ontario, Canada) for 48 hours.All blocks were decalcified in

*Paraplast is manufactured by Tyco Healthcare/Kendall, Mansfield, MA.

Table 1 DOUBLE EMBEDDING SCHEDULE

70% alcohol 72 hours 21ºC 100% alcohol 24 hours 21ºC 100% alcohol 24 hours 21ºC 100% alcohol 24 hours 21ºC 50% ether/alcohol 24 hours 21ºC 1%-2% celloidin in ether alcohol 72 hours 21ºC Chloroform 24 hours 21ºC 100% xylene 24 hours 21ºC 100% xylene 24 hours 21ºC Paraplast®* X-tra wax 24 hours 21ºC Paraplast X-tra wax 24 hours 21ºC Embed in Paraplast X-tra wax

Table 2 AUTOMATED PROCESSING SCHEDULE

70% alcohol 18 hours 35°C No vacuum Agitation on 90% alcohol 3 hours 35°C No vacuum Agitation on 100% alcohol 3 hours 35°C No vacuum Agitation on 100% alcohol 3 hours 35°C No vacuum Agitation on 100% alcohol 3 hours 35°C No vacuum Agitation on100% alcohol 3 hours 35°C No vacuum Agitation on 100% xylene 4 hours 35°C No vacuum Agitation on 100% xylene 5 hours 35°C No vacuum Agitation on Paraplast wax 4 hours 60°C No vacuum Agitation on Paraplast wax 3 hours 60°C No vacuum Agitation on Paraplast wax 3 hours 60°C No vacuum Agitation on Paraplast X-tra wax 6 hours 60°C No vacuum Agitation on Embed in Paraplast X-tra wax


40

8% formic acid (pH 2.0) (FisherScientific Co., Ltd., Toronto, Canada)until decalcified. This wasdetermined by gross examination.One set of blocks was processedusing the double embedding method(Table 1). The adjacent sections wereprocessed using an automatedmethod on a Tissue-Tek® VIP™

Processor (Sakura Finetek, Torrance,CA) (Table 2). The methods aredetailed in Tables 1 and 2.

ResultsAfter processing, the integrity of thebone samples was maintained. Therewas no difference noted in the easeof sectioning the blocks, and allblocks were able to produce ribbons.There was no distortion orsignificant shrinkage observed in thetissue or the sections obtained fromblocks that were processed using theautomated cycle, compared to thesections from the double embeddedblocks. Light microscopyexamination demonstrated that thehistological appearance of tissuesections from blocks processed usingthe automated method (Fig. 1C, 1D)were preserved and appearedidentical to those seen in tissuesections from blocks processed bythe double embedding method(Fig. 1A, 1B).

The processing time was reducedfrom 15 days using the doubleembedding schedule to 3 daysusing the automated processingschedule. The technologist’s hands-on time was decreased approx-imately tenfold using theautomated schedule. Eliminatingthe use of chloroform, ether, andcelloidin also reduced the tech-nologist’s exposure to dangerousfumes, with no obvious effects onhistology.

Conclusions1) A long automated processing

cycle can replace the doubleembedding method for largeand dense bone blocks.

2) This method decreasedprocessing turnaround timefivefold and should impactfavorably on the length of timeto diagnosis.

3) There was reduced chemicalexposure for technologists, aspotentially harmful chemicalswere eliminated.

Reference1. Bancroft JD, Gamble M. Theory and Practice of

Histological Techniques. 5th ed. New York, NY:Churchill Livingstone; 2002:281.

Fig. 1. Histological sections of decalcified bone that have been processed either by the double embedding method(A, B) or the long automated schedule (C, D). There is no difference in the histological appearance of the bone.Hematoxylin and eosin: A and C, 40X; B and D, 250X

Sometimes once is not enough.

That’s why Sakura features theHistoLogic® Archives on itsweb site at www.sakuraus.com.Whether you want to reviewrecent advances or decades-oldinnovations in histology, youcan find ample material inour archives.

The HistoLogic® Archivesenables users to access articlesfrom past HistoLogic® issuesdating back to 1971. Just typein a keyword in our archivesearch engine or look up anarticle by subject category.It’s that simple.

The HistoLogic® Archives.Another resource thatdemonstrates Sakuradedication to histology.

AccessHistoLogic®

Archives

A B

C D

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41

January 19 National Society for HistotechnologyTeleconference 1:00 pm Eastern Time (301) 262-6221Title: “Mohs Histographic Surgery

for Histologists”Speaker: Clifford Chapman, HTL(ASCP)QIHC

January 21 University of Texas Health Sciences Ctr/ San AntonioTeleconference 12:00 pm Central Time (800) 982-8868Title: “Basic Principles of Fixation”Speaker: Barry Rittman, PhD

February 16 National Society for HistotechnologyTeleconference 1:00 pm Eastern Time(301) 262-6221Title: “QC, QA and QI: Qualifiers,

Quirks and Questions”Speaker: Sandra Dolar, BA, CT(ASCP)

February 18 University of Texas Health Sciences Ctr/ San AntonioTeleconference 12:00 pm Central Time (800) 982-8868Title: “Packaging Regulations for Diagnostic

Specimens Have Changed Again”Speaker: Linda Durbin, President

EXAKT Technologies, Inc.Oklahoma City, OK

March 3-5 Indiana Society for HistotechnologySite: Downtown Hilton, Indianapolis, IN Contact: LaDonna Elpers (812) 985-5900

ext 128 Email: [email protected]

March 16 National Society for HistotechnologyTeleconference 1:00 pm Eastern Time (301) 262-6221Title: “Grossing Procedures for the Histotech”Speaker: Michael LaFriniere, PA, HT(ASCP)

March 18 University of Texas Health Sciences Ctr/ San AntonioTeleconference 12:00 pm Central Time (800) 982-8868Title: “Dermatopathology: Histology

and Surgical Grossing”Speaker: Clifford M. Chapman,

MS, HTL(ASCP),QIHC

March 18-19 Kentucky Society for HistotechnologySite: Louisville, KYContact: Renee Matherly (502) 852-5587Email: [email protected]

March 26 Massachusetts Society of HistotechnologySite: Fenway Park, Boston, MAContact: Jason Burrill (978) 658-6000 x1229Email: [email protected]

April 15 University of Texas Health Sciences Ctr/San AntonioTeleconference 12:00 pm Central Time (800) 982-8868Title: “Immunohistochemical Staining

Techniques to Localize Treponemapallidum Spirochetes”

Speaker: Hazel V. Dalton, BA, HT, QIHC (ASCP)

April 15-16 Region I SymposiumSite: Doubletree Hotel, Windsor Locks, CTContact: Jason Burrill (978) 658-6000 x1229Email: [email protected]

April 20 National Society for HistotechnologyTeleconference 1:00 pm Eastern Time(301) 262-6221Title: “Prions: Working With Infectious

Proteins”Speakers:Lisa Manning, BS, RT

and Stefanie Czub, DVM

April 21-24 Texas Society for Histotechnology Site: Marriott Dallas/Plano

at Legacy Town Center Contact: Donna Willis (817) 878-5644 Email: [email protected]

April 22-23 Region VII & Colorado Societyof HistotechnologySite: Beaver Run Resort, Breckenridge, COContact: John McGinley (970) 491-3041Email: [email protected]: www.coloradohisto.org

April 22-23 North Carolina Society for HistopathologyTechnologistsSite: Greensboro Airport MarriottContact: Delorise Williams, President

(919) 558-1200Email: [email protected]

April 27-29 Wisconsin, Minnesota, Iowa (Tri-State Symposium) Site: Radisson, Rochester, MN Contact: Sue Ubl (608) 262-5432 Email: [email protected]

April 29-30 Region I Symposium Site: Quality Inn, Vernon, CT Contact: Denise Woodward (860) 429-5163 Email: [email protected]

April 30 - Washington State Histology SymposiumMay 1 Site: Washington Athletic Club, Seattle, WA

Contact: Linda Cherepow (206) 667-1378Email: [email protected]

May 6-7 New York State Histological SocietySite: Holiday Inn, Binghamton, NYContact: Judy LaDuc (518) 897-2247 Email: [email protected]

Mark Your Calendar! Educational Opportunities in 2005


42

May 12-13 Illinois Society for Histotechnologists State MeetingSite: Peoria, ILContact: Dana Spears (309) 344-2451Email: [email protected]

May 18 National Society for HistotechnologyTeleconference 1:00 pm Eastern Time (301) 262-6221Title: “Mastering the Trichrome Stain—

From Troubleshooting to Diagnosis”Speaker: M. Lamar Jones, BS, HT(ASCP)

May 20 University of Texas Health Sciences Ctr/San AntonioTeleconference 12:00 pm Central Time (800) 982-8868Title: “Specimen Preparation for Laser

Microdissection (LCM)”Speaker: Diane L. Sterchi, MS, HT/HTL

(ASCP), EM (MSA)

May 20-21 Michigan SocietySite: Traverse City Park Place Hotel

Traverse City, MI Contact: Paula Bober (313) 729-1891 Email: [email protected]

June 3-4 Louisiana Society of HistotechnologySite: Wyndham Metairie-New Orleans

Metairie, LA Contact: Jane Goodman (504) 897-8830Email: [email protected]

June 15 National Society for HistotechnologyTeleconference 1:00 pm Eastern Time (301) 262-6221Title: “Tissue Microarrays”Speakers:Wanda Jones, HT(ASCP)

and Paul Billings

June 17 University of Texas Health Sciences Ctr/San AntonioTeleconference 12:00 pm Central Time (800) 982-8868Title: “Decalcified Bone Sections

in Animal Models of Osteoarthritis”Speaker: Elizabeth A. Chlipala, BS,

HTL(ASCP)QIHC

July 15 University of Texas Health Sciences Ctr/San AntonioTeleconference 12:00 pm Central Time (800) 982-8868Title: “Quick Fix: Safe Handling

of Histological Fixatives”Speaker: Maureen Doran, BA, HTL(ASCP)

July 20 National Society for HistotechnologyTeleconference 1:00 pm Eastern Time (301) 262-6221Title: “Immunohistochemical Panels

in Dermatopathology”Speaker: David Tacha, PhD, HT(ASCP)

August 19 University of Texas Health Sciences Ctr/San AntonioTeleconference 12:00 pm Central Time (800) 982-8868Title: “What Every Histotech

Should Know About Water”Speaker: Ethel Macrea, AA, HT(ASCP), QIHC

September 10-14 NATIONAL SOCIETYFOR HISTOTECHNOLOGYSYMPOSIUM/CONVENTIONSite: Fort Lauderdale, FLContact: NSH Office

4201 Northview Drive, Suite 502Bowie, Maryland 20716-2604(301) 262-6221

Fax: (301) 262-9188Email: [email protected]

September 16 University of Texas Health Sciences Ctr/San AntonioTeleconference 12:00 pm Central Time (800) 982-8868Title: “Microwave Usage in Today’s

Histology Lab”Speaker: Donna Willis, HT/HTL(ASCP)

October 21 University of Texas Health Sciences Ctr/San AntonioTeleconference 12:00 pm Central Time (800) 982-8868Title: “The Biology of Cancer”Speaker: Jerry Santiago, BS, HTL

(ASCP)QIHC

November 5 Connecticut Society of Histotechnologists TBA Contact: Denise Long Woodward (860) 429-5163 Email: [email protected]

November 16 National Society for HistotechnologyTeleconference 1:00 pm Eastern Time (301) 262-6221Title: “Ionizing Radiation Safety

in the Histology Laboratory”Speaker: Cheryl Culver-Schultz, MS

November 18 University of Texas Health Sciences Ctr/San AntonioTeleconference 12:00 pm Central Time (800) 982-8868Title: “Handling Bone Specimens”Speakers:Gillian Rittman, ONC

and Barry Rittman, PhD


J o i n t h e R e v o l u t i o n .

Welcome to the latest Tissue-Tek®

innovations in clinical pathology.Evolving histology

into a new dimension of total automation,

streamlined productivity,and enhanced workflow.

Specimen preparation is now possiblein just 2 hours for same-day results.

For more information,visit our web site at www.sakuraus.com

©2004 Sakura Finetek U.S.A., Inc.


Torrance, CA 90501 U.S.A.Phone: (800) 725-8723


To receive your own copy of HistoLogic,® or to have someone added to themailing list, submit home address to: Sakura Finetek U.S.A., Inc.,1750 West 214th Street,Torrance, CA 90501.

The editor wishes to solicit information, questions, and articles relating tohistotechnology. Submit these to: Vinnie Della Speranza, HistoLogic® Editor,165 Ashley Avenue,Suite 309,Charleston,SC 29425.Articles,photographs,etc,will not be returned unless requested in writing when they are submitted.

PRESORTEDSTANDARD

U.S. POSTAGEPAID

CAROL STREAM, ILPERMIT #1Sakura Finetek U.S.A., Inc.

1750 West 214th StreetTorrance, CA 90501

44

The foundat ion of spec imen standardizat ion

Accu-Edge® Grossing Tools

Precision grossing tools bringstandardization to the otherwise variableprocess of grossing.The new Tissue-Tek®

Accu-Edge® set of grossing tools provides accurate cutting for exact specimen size and thickness.

• Unique grossing board features2 adjustable wells

• Grossing forks in 3 sizes make it easier to gross soft specimens

• Trimming and scalpel blades deliveruniform results for consistent tissueprocessing with the Tissue-Tek® Xpress™

Rapid Tissue Processor

Visit our web site at www.sakuraus.com

©2004 Sakura Finetek U.S.A., Inc.


Torrance, CA 90501 U.S.A.Phone: (800) 725-8723

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