HISTOLOGIC®

Vol. XXXVIII, No. 1 May 2005

silver (GMS) stain is probably themost widely used fungal stain,however, the somewhat capriciousnature of silver staining maychallenge even the mostexperienced of histologypractitioners. Insufficient silverimpregnation, overstaining thatresults in high background staining,or undesirable precipitate may allcontribute to difficulties in findingstained fungal organisms. Properdetermination of staining endpointwith a microscope, in manyinstances, is essential to asuccessful outcome.

As a result, some pathologists willroutinely order a periodic acid-Schiff (PAS) stain in the hope ofattaining a rapid and more fool-proof stain. This method is amongthe most frequently employedtechniques in histology labora-tories. It is relatively simple toperform and reliable, even ininexperienced hands. The PASstain will reliably demonstratethe polysaccharide-laden wall ofmost fungal organisms, with theexception of Histoplasmacapsulatum.2

A Common MistakeWhen Staining for Fungi

Vinnie Della Speranza, MS, HTL(ASCP)Rena Fail, HT(ASCP)

Medical University of South CarolinaCharleston, SC

dellav@musc.edu

Fungal stains remain an important tool in the histology laboratory’sdiagnostic arsenal for identifying infectious microorganisms.Dermatomycoses, or superficial fungal infections involving the skin, arerarely a serious health problem in man; however, systemic mycoses withsuch organisms as Cryptococcus neoformans and Candida albicans cancause life-threatening infections, especially among those who are immunesuppressed. Therefore, fungal stains are often ordered with some urgencyby the surgical pathologist when hints of fungal infection are observedwith the H&E stain.

Most fungi are fairly large and their walls are rich in polysaccharides(1,2-glycol groups), which can be demonstrated with Schiff’s reagent orhexamine (methenamine) silver solutions.1 Both techniques rely on theuse of an oxidizer (periodic acid or chromic acid) to create aldehydebinding sites for the Schiff or silver ions. The Grocott’s methenamine

Managing Editor, Gilles LefebvreScientific Editor, Vinnie Della Speranza,

MS, HTL(ASCP) HT, MT

IN THIS ISSUE

A Common Mistake When Stainingfor Fungi…………………………………… 1

Fatty Tissue Fixation Using MicrowaveTechnology………………………………… 3

Methods for Assessment of Proliferation in an in vitro Tissue Model of Oral Epithelia…………………………… 6

Rapid Detetion of Lipid in Livers for Transplantation…………………………11

To B-5 or Not To B-5 ………………………15Letter From the Editor………………………18

Fig. 1. Positive fungus control. PAS stain, 400X Fig. 2. Positive fungus control. Chromic acid-Schiff stain,400X

1

2

One drawback to employing thePAS for fungal staining, however,is that it will stain polysaccharideswherever they appear in tissues.This method will stain many intra-cellular components in varioustypes of normal cells, from a widevariety of animal species. Some ofthese intracellular components arelisted below3:

• glycogen

• neutral mucosubstances

• some epithethial sulfomucinsand sialomucins

• colloid of the thyroid

• basement membranes

• reticular fibers

• paneth cell granules

• adrenal chromaffin cells

• neuronal glycolipids

• intranuclear inclusionsin vas deferens

• afibrillar cells of arteriolesof kidney

• granules withinreticuloendothelial cells

• salivary glands

• gastric and Brunner’s glands

• mucin from respiratory tract

• acinar and endocrine glands,including the prostate in man

Staining of these elements insome tissues may be distractingwhen trying to find PAS-stainedfungal organisms. As a result,pathologists who utilize the PASfor fungal staining will oftenrequest the stain with diastasedigestion in an attempt tominimize staining structuresof little or no interest.

Interestingly, few histologypractitioners question the use ofthe oxidizer periodic acid in thePAS stain, yet we use the oxidizerchromic acid in the GMS stain for

fungi. In fact, if we were to substituteperiodic acid for chromic acid in theGMS stain, essentially we will haveperformed the Jones’ method, whichspecifically stains basementmembranes and reticular fibers.

Yes, the Jones’ technique candemonstrate fungi; however, it willalso stain many other structures,making the discovery of fungalorganisms much more difficult.Whether we are using Schiff orsilver stain, the weaker periodic acidoxidizer creates aldehyde bindingsites in many more tissue structures.In this example, we can see how theselection of an oxidizing agent cansignificantly determine whichstructures will be stained. Thischoice will maximize the probabilityof achieving the desired results.

These observations were actuallyquite well known a generation ortwo ago. However, this older, yetstill important information cansometimes be overlooked. Modernhistology practitioners can use thisinformation today to guide surgicalpathologists in selecting the mostappropriate staining methodavailable.

Bauer (1933) is credited with theearliest report that the oxidationof sugars (1,2-glycols) with chromicacid creates aldehydes that willbind the Schiff molecule.4 Manyothers, including Hotchkiss,McManus (1960), Spicer, Pearse,and Lillie continued to study thealdehyde-Schiff reaction usingvarious oxidizers. It became wellunderstood that the strength of theoxidizer, the duration of exposure,and the density of sugars in thetissue structures of interestdetermine the location where theSchiff molecule will bind.

Both chromic acid and potassiumpermanganate, which are strongeroxidizers than periodic acid, alsoproduce aldehydes inpolysaccharides, but they furtherattack and destroy those aldehydesif given sufficient time. “Because of

the further oxidation of aldehydeby chromic acid, less density ofaldehyde reaction is achieved andstructures with fewer glycol groupsare not demonstrated, such asbasement membranes andcollagenous and reticular fibers.”5

This statement by Lillie tells uswhy chromic acid is the betteroxidizer when staining fungi, sincethese organisms have a greaterdensity of sugar (polysaccharides)in their cell wall.

While chromic acid continuesfurther oxidation of allpolysaccharides, completeconversion in the areas of heaviestconcentration takes longer,allowing these to remain reactivewith Schiff (or silver).2 Since thewalls of fungi are particularly richin 1,2-glycol groups, they stainmore intensely than most otherreactive components in the tissuesection. The contrast between fungiand background is often moredistinct than what is seen induplicate sections stained by thePAS reaction3 (see Figs. 1 and 2).This discussion suggests that iffungi are present in your tissuesection, they are less likely to bemissed when a stronger oxidizerlike chromic acid is used.

Work in our laboratory hasconvinced us that using chromicacid as oxidizer, followed by Schiff,yields results superior to PAS forfungal staining, as backgroundstaining is greatly decreased. Thisapproach is as simple as doing atypical PAS, yet avoids the need toperform a diastase digestion, whichadds unnecessary work andexpense to fungal staining. Themethod reported by Casella3

utilizing potassium permanganateas oxidizer is another suitablealternative to periodic acid andyields similar results to chromicacid when staining fungi.

Interestingly, chromic acid-Schiff(CAS) is remarkably similar to themethod by Gridley6 that can be

found in many histology textsexcept that Gridley’s techniquealso requires the use of aldehydefuchsin following Schiff. This isdone to achieve more intensestaining.7 The aldehyde fuchsin actsas an aldehyde and occupiesuninvolved linkages of the Schiffreagent, thus reinforcing the depthof the stain.2 While we haveconfirmed that aldehyde fuchsindoes in fact intensify the stainedorganisms, we speculate that theGridley method is not widely usedtoday because the aldehyde fuchsinstain requires the use ofparaldehyde, a controlled substancethat may be problematic to obtainin some laboratories.

In conclusion, the next time yourpathologist requests a PAS forfungus, we recommend that yousuggest the CAS as a much betteralternative. It makes little sense toutilize an oxidizer that will createaldehydes in undesirable locationsonly to have to use an enzyme totry to eliminate them later. Betterto avoid creating them in the firstplace. A simple demonstration withparallel stained sections, onestained with PAS and the otherwith CAS, will convince even themost skeptical observer.

References1. Bancroft JD, Gamble M. Theory and Practice

of Histological Techniques. 5th ed. New York, NY:Churchill Livingstone; 2002:335-338.

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

3. Thompson SW. Selected Histochemical andHistopathological Methods. Springfield, Ill:Charles C. Thomas; 1966:480-481, 496-499.

4. Bauer H. Mikroskopisch-chemischer Nachweis vonGlykogen un einigen anderen Polysacchariden,Zeitschrift fur Mikroskopisch-AnatomischeForschung. 1933;33:143-160.

5. Lillie RD. Histopathologic Technique and PracticalHistochemistry. 3rd ed. New York, NY: McGraw Hill;1965:195.

6. Gridley MF. A stain for fungi in tissue sections.Am J Clin Pathol. 1953;23:303-307.

7. Humason GL. Animal Tissue Techniques. SanFrancisco, Calif: W.H. Freeman and Co; 1972:396-397.

Fatty Tissue FixationUsing Microwave

Technology

Mary Faith Abbuhl, MSHTL(ASCP)QIHC

Andrea Williams, HT(ASCP) University Hospitals of Cleveland

Cleveland, OHMary.Abbuhl@uhhs.com

AbstractThe microwave oven has been inuse in the histology laboratory formany years. In recent years,laboratory-grade microwaveinstruments have become morewidely adopted as standardequipment in many laboratories.1These microwave units may beused to accelerate a number oflaboratory procedures, includingfixation, processing, decalcification,special staining, and immuno-histochemical (IHC) staining.

Our histology laboratory has beenusing microwave technology forspecial stain procedures andantigen retrieval techniques inimmunostaining procedures. Mostrecently, we investigated the use ofthe microwave to aid in fixation offatty tissue specimens. These tissuespecimens often do not process

well during routine tissueprocessing.2 Tissue thickness andfat content are two main factorscausing the problem of under-processed tissue.

IntroductionDiagnostically, the most critical fattytissues are breast and lymph nodespecimens.Any delay in processingthese specimens may affect patientcare.We are continually faced withinadequate processing of fattytissues, most often due to both thethickness of the sections whengrossed in and their fat content.The use of alcoholic formalin inpreprocessing and processing, oftenrecommended as a solution to thisproblem, is not an option for someimmunohistochemical procedures,especially those typically used forbreast samples.We have found thatformalin fixation done in amicrowave oven offers a suitablealternative when preparing fattysamples.

Materials and MethodsFor this study we collected alimited number of samples (n=20)of breast tissue from mastectomiesand breast reductions. Parallelsections of these tissues were cut inat the grossing table at threethicknesses—approximately 3 mm,5 mm, and 6 mm—and placed intoplastic tissue cassettes.

3

Fig. 1. Routinely fixed and processed breast tissue stained with H&E. 200X

4

*Background staining observed. †Crisp nuclear detail. MW = microwave fixed tissue.

The cassettes were then placed andheld in 10% formalin (Val TechDiagnostics, Brackenridge, PA).

One group of cassettes wasprocessed routinely using aTissue-Tek® VIP 3000 processor(Sakura Finetek, Inc., Torrance,CA), while parallel blocks werefixed in a MicroMED T/T™microwave oven (HackerInstruments, Winnsboro, SC)followed by routine processingin the VIP 3000 processor. Thecassettes to be microwaved wereplaced into the MicroMED T/Tcassette holder in a large (400 ml)glass container that was filled with

10% formalin. A program forfixation was created that allowed

for a 5 minute ramp-up oftemperature of the formalin to60°C, and this temperature wasmaintained with microwavestimulation for 55 minutes.The microwaved tissues were thentransferred to the routineprocessor.

Tissue blocks were routinelyembedded in paraffin andsectioned.3 Each sample was stainedwith H&E (Richard AllanScientific, Kalamazoo, MI) andMasson’s trichrome (Poly ScientificR&D Corp., Bayshore, NY).In addition, IHC was performedfor estrogen receptors (ER),progesterone receptors (PR),and HER-2/neu (Hercep Test,DakoCytomation, Carpinteria, CA).

ResultsA preliminary study of 20 patientsamples was conducted. Thesespecimens were all extremely fattytissues. Tissues of 3 mm thicknessor less consistently processed wellafter microwave fixation, requiringno additional processing, whileroutinely fixed 3 mm sectionsdid not consistently process wellin the routine processor. Manyrequired reprocessing. The tissuesof greatest thickness, approxi-mately 6 mm, did not process well,even after microwave fixation.The tissues of intermediatethickness, approximately 5 mm,were inconsistent in processing,irrespective of the methodemployed for fixation.

Fig. 2: Microwave fixed breast tissue stained with H&E. Nuclear detail is improved. 200X

Fig. 3. Routinely fixed and processed breast tissue stained with Masson’s trichrome. 200X

Table 1

Sample H&E Trichrome ER PR HER-2/neu

#1 Very Good Excellent Excellent* Excellent* Negative*

#1 MW Excellent Very Good† Excellent Excellent Negative

#2 Very Good Very Good Very Good Rare Negative

#2 MW Excellent Very Good† Very Good Negative Negative

#3 Excellent Very Good Excellent Excellent* Negative*

#3 MW Excellent† Very Good† Excellent Excellent Negative

5

We chose 3 samples to evaluatestaining consistency whencomparing tissues fixed in themicrowave to those fixed routinely.H&E and trichrome stains, as wellas three immunostains, werecompared for each (Table 1).With respect to nuclear detail, themicrowave fixed tissue showedsuperior staining with H&E(Figs. 1 and 2). In many cases it wasnecessary to cut the routinely fixedtissue thicker in order to obtain abetter section, due to the tissue’sfat content. Overall, microwavefixed tissue could be sectionedthinner, thereby yielding a higherquality sample to viewmicroscopically. The trichromestain displayed similar results,however, the nuclear stainingappeared slightly paler but crisperin the microwave fixed tissue(Figs. 3 and 4).

Immunostaining analysis of ERand PR receptors and HER-2/neushowed comparable staining(Figs. 5-8). Nonspecific backgroundstaining seen in the routinelyfixed tissue was completely absentin the microwave fixed tissue.Only one sample showed very rarePR staining in the routinely fixedtissue but not in the microwavefixed tissue. This discrepancy maybe attributable to sampling butwarrants further study. None ofthe tissues studied was found to bepositive for HER-2/neu, preventingus from evaluating the quality ofimmunostaining with this marker.Although the tissues were negativewith this antibody, nonspecificbackground staining commonlyseen in the routinely fixed tissueswas virtually absent in themicrowave fixed tissues.

ConclusionHistorically, we have found thatroutine fixation and processingof even the thinnest of fatty tissuesections frequently requiresreprocessing in our laboratory.Even though the number of

Fig. 4. Microwave fixed breast tissue stained with Masson’s trichrome. Note the crisp nuclear detail. 200X

Fig. 5. Routinely fixed and processed breast tissue stained with IHC for estrogen receptor protein. Some backgroundstaining is noted in intercellular areas and ductal lumens. 400X

Fig. 6. Microwave fixed breast tissue stained with IHC for estrogen receptor protein. Tissue shows minimal backgroundstaining. 400X

specimens observed in this study isvery small, we believe that formalinfixation of fatty tissue specimensprocessed using microwavetechnology, especially with tissuessectioned at a reasonable thickness,helps to prevent the need forreprocessing of these tissues.

Microwave fixation of fatty tissuesdoes not appear to create anyundesirable artifacts with H&E,trichrome, or immunohistochemicalstaining. On the contrary, it appearsto help decrease nonspecific IHCbackground staining seen in some

routinely prepared sections andimprove the nuclear detail of thecells overall.

References1. Buesa RJ. Haven’t you calibrated your microwave

oven yet? J Histotechnol. 2000;25(1):39-41.2. Johnson ML. A technique for correcting poorly

processed paraffin blocks. HistoLogic. 2003;36(1):21-22.

3. Carson FL. Histotechnology: A Self-Instuctional Text.2nd ed. Chicago, Ill: American Society of ClinicalPathology; 1997:25-42.

Methods forAssessment of

Proliferation in anin vitro Tissue Model

of Oral EpitheliaGail L. Ackermann, DDS, M Dent

Elizabeth Chlipala, BS, HTL(ASCP) QIHC

Premier Laboratory, LLCUniversity of Colorado

Boulder, COliz@premierlab.com

Introduction EpiOral™ and EpiGingival™ culturesare organotypic in vitro models oforal and gingival epitheliumdeveloped by MatTek Corporation(Ashland, MA). Normal human oralkeratinocytes are differentiated intotissues with a noncornified, buccalphenotype, or a cornified, gingivalphenotype (Fig. 1). The same cellsare used to propagate both tissues.The epithelium is cultured on amicroporous membrane, MilliporeMillicell® CM cell culture inserts(Millipore Corp., Billerica, MA)on a plastic disk utilizing serum-freemedia. The model is used to assessthe response of the epithelium tovarious stimuli including prolifer-ative agents.1 We report here onmethods used to assess proliferationin this model, including Ki-67immunohistochemical staining,number of cells in the basal celllayer, thickness of the basal celllayer, and thickness of theepithelium.

Materials and Methods Histology MethodsEpiOral and EpiGingival cultureswere fixed in 4% paraformaldehydefor 24 hours and then transferredto phosphate buffered saline(PBS). The cultures were removedfrom the cell culture wells with an8 mm biopsy punch, processedbetween biopsy pads, and bisectedinto two approximately equalhalves prior to paraffin embedding.Two 4-micron sections were cut;one was immunohistochemicallystained with the cell proliferation

6

Fig. 7. Routinely fixed and processed breast tissue stained with IHC for progesterone receptor protein. 400X

Fig. 8. Microwave fixed breast tissue stained with IHC for progesterone receptor protein. 400X

marker Ki-67. Ki-67 is a nuclearprotein that is expressed in allactive phases of the cell cycle(G1, S, G2, and M-phases) but isabsent in resting cells (G0-phase).2The second section was stainedby standard methods withhematoxylin and eosin (H&E).

Proliferating cells were immuno-histochemically stained with amonoclonal mouse anti-human

Ki-67, clone MIB-1(DakoCytomation, Carpinteria,CA). A pretreatment of heat-induced epitope retrieval in10 mmol/L citrate buffer, pH 6.0was utilized prior to the primaryantibody incubation. The primaryantibody was incubated at 1:150for 30 minutes at room temperaturethen visualized using EnVision+mouse (DakoCytomation,Carpinteria, CA) followed by

DAKO Liquid DAB+(DakoCytomation, Carpinteria,CA). The sections were counter-stained with hematoxylin andcoverslipped prior to assessment.

Analysis MethodsFour different parameters wereused to determine the proliferativeeffects of various treatments: overallthickness of the epithelium, basalcell layer thickness, number of basalcell nuclei, and number of Ki-67positive nuclei. The H&E stainedslides were used to measure theoverall thickness of the specimensand the thickness of the basal layer.The outer edges of the specimenwere not evaluated since these werenot representative of the entirespecimen. Specifically, we found theedges to be much thicker than thecenter of the specimens and theconcentration of Ki-67 positive cellswas much higher at the peripherythan in the center of the specimen.We believe that this was primarilydue to culture conditions. The thick-ness of the specimen was measuredat 20X magnification on a ZeissAxioskop 2 Plus microscope(Fig. 2A). Measurements were takenat three equidistant points along thelength of each of the two specimensusing an ocular micrometer (10 mmscale with 100 divisions, KR-207Klarmann rulings), totaling sixmeasurements per specimen(Fig. 3). The raw results of thesemeasurements were then convertedto microns by taking into accountthe magnification factor using

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Fig. 1. Specimen A is an image of an EpiOral construct, which is a noncornified buccal mucosa consisting of basal and suprabasal layers. Specimen B is the EpiGingival construct,a cornified gingival mucosa consisting of basal, suprabasal, and cornified layers. H&E, 400X

Fig. 2A. Measurements for specimen thickness were taken with the 20X objective utilizing an ocular micrometer,as demonstrated by the black double arrow, while measurements of the basal cell layer were taken with the 40Xobjective, as demonstrated by the green double arrow (EpiGingival construct). H&E, 400X

Fig. 2B. Artificial separation of the cornified layers was taken into account and not measured when overall specimenthickness measurements were taken in the EpiGingival constructs (EpiGingival construct). H&E, 400X

the following equation: ocularmicrometer measurement/objectiveused x 1000 = microns.

In a portion of the EpiGingivalspecimens there was an artificialseparation of the keratinous layerthat was due to processing. Theseparation was excluded from themeasurement of the thickness(Fig. 2B). The thickness of thebasal cell layer was measured at40X magnification (Fig. 2A).

The number of Ki-67 positive nucleiand basal cell nuclei were countedat 20X magnification using theKi-67 stained section. The entirespecimen, excluding the outer edges,was used to count the Ki-67 positivenuclei. All cells that had any nuclearstaining were counted as positive forKi-67 (Fig. 4). This figure demon-strates the variable intensity ofstaining with this antibody. In somecells there is focal staining of thenucleus, whereas in other cells theentire nucleus is stained. This con-forms to previous observations andreflects the short half-life of theprotein.2 Each bisected portion wascounted, so a total of two numberswere generated per specimen. Thetotal number of nuclei in the basallayer was estimated by countingthree representative lengths of1 mm that were evenly spacedalong the entire construct, totaling6 counts per specimen (Fig. 3).

The data generated from the indi-vidual measurements and cell countswere entered into a Microsoft Excelspreadsheet to tabulate individualand group means (Fig. 5). Graphicalrepresentation of the data is shownin Fig. 6.

Results and Discussion The appearance of EpiOral andEpiGingival cultures is shown inFig. 1. The most notable differencebetween them is that one isnonkeratinizing and the other iskeratinizing. In Fig. 2B, theseparation of the keratinized layerfrom the underlying epithelium canbe seen. This separation wascreated during processing. TheEpiGingival cultures were highlysusceptible to this artifact. Greatcare must be taken in the handlingof these specimens from grossingto embedding, to decrease thepotential for this artifact. Figs. 7Band 7D show the increasednumbers of cells that are positivefor Ki-67 following application ofan agent that induces proliferation,and Figs. 7A and 7C demonstrateepithelium that is relativelyinactive. The cells that are positivefor Ki-67 tend to be in the basalcell layer, as would be expected.There is an increased number of

cells in the basal cell layer andincreased thickness of the basal celllayer associated with the increasednumber of Ki-67 positive cells. Inaddition, the overall thickness ofthe epithelium was increased.

The results generated from theanalysis of several groups ofcultures under different conditionsindicate that the parameters mea-sured are capable of elucidatingdifferences in the cultures causedby the agents being tested. In somecases, however, there was con-siderable variability betweenindividual specimens (perhaps dueto varying culture conditions) thatmade statistical analysis difficult.We recommend using groups ofmore than 5 specimens per groupto avoid this potential problem.

Conclusion Measurement of epithelialthickness, basal cell layer thickness,number of cells in the basal celllayer, and number of Ki-67 positivecells provides a robust method forassessment of proliferation inEpiOral and EpiGingival cultures.

References1. EpiOral Model (ORL-200) and EpiGingival Model

(GIN-100) Technical Specifications. Available at:http://www.mattek.com.

2. Scholzen T, Gerdes J. The Ki-67 protein: from theknown and the unknown. J Cell Physiol.2000;182(3):311-322.

Fig. 4. All cells that contained any brown staining were counted as positive for Ki-67. Note that in some cells onlya portion of the nucleus stains, while in others the staining is more diffuse (EpiGingival construct). Ki-67, 400X

Fig. 3. Representative measurements of total thickness of epithelium and thickness of basal cell layer were taken atpoints A, B, and C. The numbers of basal cell nuclei were counted along three areas on each section at D, E, and F.The double arrow denotes the area in which the basal cell nuclei were counted (EpiOral construct). H&E, 40X

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Fig. 7. Specimens A and C are normal, untreated, mature EpiOral and EpiGingival constructs. Specimens B and D are treated constructs, which demonstrate increases in Ki-67positive cells, basal cell nuclei, basal cell layer thickness, and epithelial thickness.

Fig. 5. Example of a portion of the Excel spreadsheet.

Fig. 6. Graphical representation of the data generated.

9

Epithelial Thickness

Basal Cell Layer Thickness

Kl-67 Positive Nuclei

Basal Cell Nuclei

Group Section A - Total Thickness (µm) Section B - Total Thickness (µm)

Specimen Number 1 2 3 Average 1 2 3 Average Average

Group 1

No Treatment

A 105 115 120 113.33 110 110 115 111.67 112.50

B 130 125 105 120.00 115 110 115 113.33 116.67

C 105 115 120 113.33 110 120 105 111.67 112.50

MEAN 113.33 118.33 115.00 115.56 111.67 113.33 111.67 112.22 113.89

SE 8.33 3.33 5.00 2.22 1.67 3.33 3.33 0.56 1.39

Mea

n +

SE C

ell C

ount

s

Mea

n +

SET

hick

ness

Mea

sure

men

ts (

µm)

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Rapid Detection ofLipid in Livers for

TransplantationRena Fail, HT(ASCP) QHIC

With the Technical Assistance ofJennifer German, BSMedical Universityof South Carolina

Charleston, SCfailm@musc.edu

AbstractThere is a substantial increase inthe demand for organs available fortransplantation due to the increaseduse of transplantation as a methodto treat end-stage liver disease.Increasing the donor pool hasresulted in the inclusion of botholder donors and those donors whohave some high-risk conditions.Retransplantation or death mayresult from transplanting a liverwith 30% or greater fat content. Asa result, donor livers with greaterthan 30% fat content are deemedunsuitable. Time is of the essence intransplanting harvested organs—aliver is only viable for 12-16 hoursafter removal from the donor.Therefore, a rapid method for thedetection of lipid is necessary todetermine the viability of donorlivers.1

IntroductionA core biopsy from a donor liverwas brought to the frozen sectionroom, sections cut, and slides givento the technician in the specialstains lab. We had recently changedthe oil red O procedure from oneusing isopropanol to another usingpropylene glycol. Even though theisopropanol method was quick(15 minutes), the results were oftendirty. Droplets of the dye solutionwould be deposited on and aroundthe tissue, making interpretationdifficult. The lack of adifferentiation step in the methodprobably accounted for this.Switching to the propylene glycolmethod allowed for some controlof differentiation, producing acleaner, crisper stain. However, the

lengthy fixation of the tissue in10% neutral buffered formalinspecified in the method was adrawback for rapid diagnosis ofliver steatosis. Formalin fixation isdesired in order to fix the tissueproteins, which aids in the retentionof lipids. It does not fix the lipid,but traps it while maintaining thearchitecture of the tissue.2 The totaltime for this staining procedurewas 90 minutes. With a transplantsurgeon and his transplant teampacing in the hallways, and animpatient pathologist and aresident waiting for this slide, it wasobvious that adjustments had to be

made. Some steps were shortenedbefore the slides were given to thepathologist. The stain lookedgood—clean and crisp. Fig. 1 is theresult of the stain performed on theliver allograft. As the photo depicts,the fat content is greater than 30%.

Though commonly referred to as adye, oil red O is actually a pigmentthat functions as an oil-solublecolorant. It is less soluble in hydro-alcoholic solvents than in lipoidsubstances.The solvent used must beable to extract the excess dye with-out dissolving the lipid in the tissueto be stained.Three fundamental

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Fig 1. Donor liver biopsy demonstrates fat content that is more than 30%. Oil red O stain was performed as describedin this article. 400X.

Fig. 2. Donor liver biopsy, with no dye droplets to interfere with interpretation. Oil red O stain was performed usingthe propylene glycol method described below. 600X

requirements for the solvent usedin fat staining were suggested byChiffelle and Putt (1951):

1. The solvent should not extractany lipids from the tissue section.

2. The solvent should dissolvesufficient amounts of pigmentto yield brilliant staining results.

3. The stain should be easy, rapid,and economical to prepare, andstable in solution.

Of the two common solvents usedfor oil red O, propylene glycol ispreferred because it removes nolipid, whereas minimal amounts oflipid are removed with isopropanol.Introduced by French in 1926, oilred O (Sudan red B, solvent red 27)is classified as one of the Sudandyes, which have been in generaluse since the late 1800s. The depthof its color makes it the preferredchoice for staining lipids.3,4

MethodPurpose: Oil red O stain candemonstrate neutral lipids in frozentissue sections, determining theviability of donor livers, as well asfind fat that appears in abnormalplaces, such as in the kidneys or

brain. It is also useful in verifyingfat embolism as a cause of death,as well as demonstrating fat embolithat may develop and enter thecirculatory system after a bonefracture or a crushing injury ina fatty body area. Degeneratingmaterial containing fat, such as cellmembranes or myelin, may coalesceinto fat droplets that can be seenwith fat-detecting stains, differ-entiating between fine lipid dropletsand fine glycogen droplets. Tumorswith unusual morphology, such asliposarcomas, can be differentiatedfrom other types of tumors(rhabdomyosarcomas) using theoil red O stain.5

Principle: The basis for staining lipidswith an oil-soluble dye is its greatersolubility in lipoid substances than inthe usual hydroalcoholic dye solvents.This is a physical method of staining.The dye must not be water soluble,must be strongly colored, and mustact with tissue constituents only insolution.The solvent choice is critical.

Fixation: Air dry 2 frozen sectionslides. Place one in 10% neutral buf-fered formalin (NBF), in case there isan adhesion problem resulting in thetissue washing off the slide. Proceedwith staining the second slide.

Specimen: To ensure thepreservation of all lipids, freezingthe tissue is preferred. Routineprocessing removes all but a fewprotein-bound lipids. Frozensections are cut at 5 microns.

Quality Control: Previously cutfrozen sections of fatty liver or skinare used as control slides.

Precautions:• Wear suitable protective clothing.• Lab coats, gloves, and safety

glasses are required.• Use propylene glycol under

a fume hood.• Gloves are necessary to avoid

absorption of dyes into skin.• Dispose of hazardous waste

in appropriate receptacles.

Solutions:Mayer’s Hematoxylin

0.5% Oil Red OOil red O powder ……………0.5 gPropylene glycol …………100.0 ml

Add a small amount of propyleneglycol to the oil red O and mix well.Gradually add the remainder of thepropylene glycol, stirringperiodically. Heat gently until thesolution reaches 95°C. (DO NOTALLOW THE SOLUTION TOGO OVER 100°C.) Stir whileheating. Filter through coarse filterpaper while still warm. Allow tostand overnight at room tempera-ture. Filter again before use.

85% Propylene GlycolPropylene glycol……………85.0 mlDistilled water………………15.0 ml

NOTE: Store the oil red O solutionand 2 coplin jars of 85% propyleneglycol in a 60°C oven.

Staining Procedure:1. Dip briefly in distilled water.2. Dip slides in undiluted propylene

glycol.3. Stain in oil red O solution

for 1 minute at 60°C.4. Differentiate by dipping twice

in each of 2 changes of 85%propylene glycol at 60°C.

5. Rinse in 2 changes of distilledwater.

12

Fig. 3. Donor liver biopsy showing dye droplets that make interpretation difficult. Oil red O stain used with propanol.400X

6. Counterstain in Mayer’shematoxylin for 30 seconds.Be sure to filter hematoxylinprior to use if not filtered daily.

7. Rinse in distilled water.Do not use acid alcohol.Do not dehydrate throughalcohols.

8. Mount in an aqueous mountingmedium.

Results:Fat ………………………Intense redNuclei …………………………Blue

Procedure Notes:1. Synthetic resinous media contain

organic solvents that will dissolvelipids; therefore, the use of anaqueous mounting medium isvital. Permanent aqueousmounting media are NOT suitable.

2. Do not exert any pressure on thecoverglass, as this may displace thefat. Do not try to press air bubblesout if they are present in thesection, as the fat in the section isliquid and mobile. If necessary,remove the coverglass by soakingthe slide in water.

3. Oil red O is more soluble in lipidthan in propylene glycol; the 60°Coven keeps the solution warmenough to liquefy lipids that aresolid at room temperature,thereby allowing the dye to enterthe lipid more readily.

Procedure Limitations:Simple lipids may be removed byany fixative or solution containingalcohol or organic solvents. As aresult, sections that are paraffinembedded cannot be used. Onlyfrozen sections may be used forthis procedure.

References1. Kim SH, Lee JM, Kim JH, et al. Appropriateness of

a donor liver with respect to macrosteatosis: appli-cation of artificial neural networks to US images—initial experience. Radiology. 2005;234:793-803.Available at: http://radiology.rsnajnls.org. AccessedMarch 31, 2005.

2. Bancroft JD, Stevens A, eds. Theory and Practiceof Histological Techniques. 3rd ed. New York, NY:Churchill Livingstone; 1990:216-217.

3. Troyer H. Principles and Techniques ofHistochemistry. 1st ed. Boston, Mass: Little, Brownand Co; 1980:126-137.

4. Lillie RD. HJ Conn’s Biological Stains. 8th ed.Baltimore, Md: Williams & Wilkins; 1969:104-105.

5. Sheehan DC, Hrapchak BB. Theory and Practice ofHistotechnology. 2nd ed. St. Louis, Mo: Battelle Press;1987:202-205.

13

Sometimes once is not enough.

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The HistoLogic® Archives enables users to accessarticles from past HistoLogic® issues dating back to1971. Just type in a keyword in our archive searchengine or look up an article by subject category.It’s that simple.

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15

To B-5or Not To B-5

Deb Croegaert, AS, HT(ASCP)University of Iowa Hospitals

and Clinics Iowa City, IA

debra-croegaert@uiowa.edu

AbstractDespite years of controversy due tohazards associated with its use, B-5fixative has found wide acceptanceas a fixative for hematopoietic(bone marrow) and lymphoreticular(lymph node) tissues because tissuefixed in this solution will demon-strate beautiful nuclear detail.1

The purpose of this study was toexplore whether a safer alternativefixative with similar advantagescould be used to replace B-5 in ourlaboratory. Using lymphoid tissue,sections were split and fixed in B-5,neutral buffered formalin (NBF),and several products marketed asB-5 substitutes. The specimenswere routinely processed and thenstained with hematoxylin and eosin(H&E). Immunohistochemical(IHC) staining with the markersCD30, BCl-2, and Cyclin D1 wasalso assessed. The slides wereevaluated under blind studyconditions by several pathologistsfor comparison.

MaterialsAZF™ Fixative: Newcomer Supply,Middleton, WI

B-5 Stock Solution2:Mercuric chloride……………133.4 gSodium acetate ……………… 27.8 gDistilled water ………………1400 ml

Dissolve mercuric chloride indistilled water while heating. Afterit is dissolved, add sodium acetateand mix. Filter the solution.

B-5 Working Solution: Add 3 mlof full-strength formalin to 30 mlof stock solution before use.

B-Plus™: BBC Biochemical,Stanwood, WA

Presto-fixe II™: SPECTRA-TINTAvailable at: http://www.spectra-tint.com/index.html10% Neutral Buffered Formalin(NBF): Fisher Diagnostics,Springfield, NJ Z-5™: Anatech Ltd., Battlecreek, MIZinc Formalin, buffered: Richard-Allan Scientific, Kalamazoo, MI

MethodsFresh tonsil tissue was sectioned2-3 mm thick and placed in5 different fixatives followingmanufacturer’s protocols, in parallelwith sections fixed in NBF and B-5prepared in our laboratory. All ofthe sections were fixed for 4 hoursat room temperature.AZF: AZF is a working solution.Recommended fixation is at least3-4 hours.B-5: Make working solution fromthe stock solution just before use.Recommended fixation time is4-8 hours.B-Plus: B-Plus is a workingsolution. Recommended fixationtime is 3-4 hours.Presto-fixe II: Presto-fixe II is aworking solution. Recommendedfixation time is 4-8 hours.NBF: NBF is a working solution.Recommended fixation time is4-8 hours.

Z-5 is purchased as a stock solutionand is diluted with 95% reagent-grade alcohol when opened for use.The bottle is good for 1 year.

Recommended fixation time is4 hours for small surgical tissue.The specimen is washed in NBFbefore routine processing.Zinc Formalin, buffered: Zincformalin is a working solution.Recommended fixation time is4-8 hours.

All specimens were routinelyprocessed on a closed system tissueprocessor through 10% NBF,graded alcohols, xylene, andparaffin. The sections for H&E werecut at 2 µm, and those sectioned forIHC were cut at 4 µm, using arotary microtome.

Mercury pigment resulting fromfixation in mercury-based fixativeswas removed from the B-5 fixedsections prior to staining using thefollowing protocol2:1. Place in Weigert’s iodine

(1 part iodine and 3 parts water)for 5 minutes.

2. Wash in distilled water.3. Place in 5% sodium thiosulfate

for 5 minutes.4. Wash in tap water.

The slides were labeled A-G andgiven to several pathologists, whowere given a ballot to rate the slideson a scale of 1-5 (1 being the highestdegree of resemblence to thosefixed in B-5 and 5 being the lowest).IHC was then performed comparingthe B-5 fixed tissue with those fixedin the two top-scoring replacementfixatives, which were zinc formalinand B Plus, along with B-5.

B-5 fixative Buffered zinc formalin B-Plus

Fig. 1. Tonsil tissue, H&E. 400X. All comparable with good nuclear detail, clarity, and contrast.

16

Slides for IHC were stored overnightin a 37°C incubator. Prior tostaining, the slides were placed in

a 60°C oven for 30 minutes. Heatinduced epitope retrieval (HIER)pretreatment was done using

citrate buffer (pH 6.0) in amicrowave pressure cooker asper our usual protocol for thesemarkers (CD30, BCl-2, andCyclin D1).

DiscussionThe first written records noting thehazards of mercury date back tothe first century AD. In the 1500s,the vapor hazards associated withmercury distillation preventedworkers from working more than1 month per year.3 The phrase “madas a hatter,” linked to the madcaphatmaker in Lewis Carroll’s classicAlice in Wonderland, relates to thedisease found in the hatmakingindustry of the 19th century.A mercury solution used in theprocess of turning fur into felt tomake hats produced toxic fumes,which the workers inhaled; thiseventually led to accumulationof mercury in the workers’ bodies.The symptoms included trembling,loss of coordination, slurred speech,loosening of teeth, memory loss,depression, irritability, and anxiety.The mad hatter syndrome is stillused today to describe the effectsof mercury poisoning.4 We nowknow that mercury poses a seriousenvironmental hazard, whichwill persist in a bioactive form thatcan contaminate the food chain.Fish in many of the nation’swaterways have been found to becontaminated with mercury.5

Today, there is considerable pres-sure from regulatory agencies toremove mercury from use inlaboratories. This is to reduce thewaste stream that could lead toenvironmental contamination, aswell as to eliminate the hazards forlaboratory staff. As a result, severalmanufacturers now offer substitutefixatives that are touted to providethe benefits of mercury fixationwithout the hazards associatedwith exposure to this heavy metal.However, resistance to eliminatingmercury-based fixatives is due tothe failure of many of theseproducts to achieve theperformance and results expectedfrom fixatives such as B-5.

B-5 fixative Buffered zinc formalin B-Plus

Fig. 3. Tonsil tissue, BCl-2. 400XA. Negative staining. B. Strong cytoplasmic staining. C. Strong cytoplasmic staining.

B-5 fixative Buffered zinc formalin B-Plus

Fig. 2. Tonsil tissue, CD30. 400XA. Faint cytoplasmic staining. B. Good membranous staining. C. Strong membranous staining

with golgi apparent.

B-5 fixative Buffered zinc formalin B-Plus

Fig. 4. Tonsil tissue, Cyclin D1. 400XA. Negative staining. B. Moderate-strong nuclear

staining.C. Strong nuclear staining,

with nonspecific staining.

But now the American HospitalAssociation (AHA) is working withthe Environmental ProtectionAgency (EPA) to get all hospitalsto voluntarily agree to eliminate allmercury by 2005.5 The College ofAmerican Pathologists (CAP) hasincluded a new standard in itsaccreditation checklist (LaboratoryGeneral Checklist) asking, “Doesthe laboratory have a written planto reduce or eliminate mercury?”6

Interestingly enough, the title ofthe article parodies the famous line

from Shakespeare’s Hamlet, wherethe main character manages tofeign, or not to feign, his ownmadness. Can we, after reviewingall the facts and knowing whatmandates are in our future, make asane decision to use B-5 or not touse B-5?

References1. Carson FL. Histotechnology: A Self-Instuctional Text.

2nd ed. Chicago, Ill: American Society of ClinicalPathology; 1997:19.

2. Sheehan DC, Hrapchak BB. Theory and Practice ofHistotechnology. 2nd ed. St. Louis, Mo: Battelle Press;1987:42-47.

3. Sax NI. Dangerous Properties of Industrial Materials.5th ed. New York, NY: Van Nostrand Reinhold Co;1979:1-10.

4. Complementary Medical Association (CMA).Mad Hatter Syndrome. Available at:http://www.the-cma.org.uk/HTML/hatter.htm.Accessed April 11, 2005.

5. Lead and Mercury Alliance for a Healthy Tomorrow.Available at:http://www.cleanproduction.org/Risk/us_safe_chem.htm.Accessed April 7, 2005.

6. College of American Pathologists. Laboratory generalchecklist changes. Available at:http://www.cap.org/apps/cap.portal?_nfpb=true&_pageLabel=lab_accred_book.Accessed April 7, 2005.

7. Agency for Toxic Substances and Disease Registry(ATSDR). Available at:http//:atsdr1.atsdr.cdc.gov:8080/hazdat.html.

17

Table 1. A Comparison of B-5 With the Substitute Fixatives Studied

B-5 Fixative B-5 Substitute

Preparation Made from stock solution prior to use Of those studied, most were supplied asworking solutions. One required dilution

Washing None needed Of those studied, two required washing prior toplacing tissue into formalin following fixation in thesubstitute, to prevent precipitation of buffer salts

Corrosivity Corrosive, avoid contact with metals None of the studied substitutes was corrosive

Post-fixation Morphology No artifacts when postfixed Some affect morphology adversely when usedas a postfixative

Artifact Pigment Mercury pigment must be None of the substitutes caused the formation removed prior to staining of pigment artifact

Microtomy B-5 fixation can make tissues brittle No effect on microtomyand more difficult to section,especially if tissues are fixed for long periods

Environmental Concerns Mercuric chloride easily reaches the waste All contain zinc and formaldehyde.water system during mercury pigment Formaldehyde will break down in the environment.removal. Mercury leaches out of tissues Zinc is not considered an environmental hazardduring processing. Distillation is not an option. Transformed to methyl mercury in the environment, which accumulates in fish that may be consumed by humans7

Health Hazards Mercuric chloride is highly toxic Formaldehyde is toxic to humans and is a regulatedand excessive exposure levels can carcinogen. Zinc ingestion is not toxic in small permanently damage the brain quantities3

and kidneys. May be fatal if inhaled,swallowed, or absorbed through the skin;extremely destructive to tissue andmucus membranes3

Disposal Must be collected and removed by an Must be disposed of as formaldehyde waste.EPA-licensed chemical waste hauler.3 Some waste water authorities may permit neutralized Cost is approx. $500 per 55 gallon drum formaldehyde to be expelled into sewer3

1818

On behalf of HistoLogic,I would like to expressmy appreciation to allof you who havesupported this publicationthrough the submission ofmanuscripts over the past34 years. HistoLogic wascreated in 1971 in anattempt to keep histology

practitioners around the globe connected to oneanother through the sharing of relevant,practical scientific and technical information.Since that time, it has been read worldwide bymore than 1,000,000 individuals. It is my sincerehope that the information that has appearedwithin these pages during my editorship hasbeen of benefit to you in your work.

We understand that many of our colleaguesmay not have the opportunity to further theirprofessional development through attendanceat symposia and workshops. For this reason,the appearance of sound scientific informationin your mailbox becomes all the more importantto aid you in keeping your theoreticalknowledge complete and up to date. As a result,we at HistoLogic remain committed to providingyou with the highest caliber publication possible.

Few things contribute more to the advancementof science and technology than the sharing ofknowledge and ideas with our colleagues.Indeed, this communication is the very foundationupon which new ideas are stimulated and newdevelopments and discoveries are built. I believethat as scientists we have a responsibility to sharewhat we know. It matters not whether youpublish your work in HistoLogic, the Journal ofHistotechnology, or any other scientific publication,only that you do, in fact, share your knowledge.Knowledge shared in print will always remainone of the most effective tools for reaching largenumbers (often tens of thousands) of individuals.

This is where you come in. If you have neverconsidered submitting your work for publication,I urge you to consider the opportunity. I know thatsome may be daunted by the perception that it isvery difficult to get one’s work published.HistoLogic is interested in any manuscript that isscientifically valid. I’ve conversed with some

colleagues who believe that they don’t writewell enough to successfully publish an article.However, as the scientific editor, I am availableto assist you in any way possible. I am happy tohelp you construct your article, so please do notallow this concern to discourage you fromcoming forward with your ideas.

For those who are interested in submitting amanuscript to HistoLogic, I urge you to contactme directly at dellav@musc.edu, or you maycorrespond with me via the mailing address includedon the back of this issue. I prefer that manuscriptsbe prepared using word processing software suchas Microsoft® Word or Corel® WordPerfect.®

Articles that appear in HistoLogic are requiredto have relevant scientific or technical referenceswhich should be listed in the order in which youcite them in your article. References must besufficiently detailed as to allow the reader tolocate the information you used whenconstructing your manuscript. We prefer thatreferences are formatted according to theAMA Manual of Style, and I can assist you withgetting your references into that format, ifnecessary. We encourage you to include colorimages that serve to illustrate key points in yourarticle for the reader. While we prefer these inelectronic format, including jpeg and tiff, we canalso work with high-quality color prints and slides.All slides should have a caption that includes suchrelevant information as tissue type, stain used,magnification, and any remarkable features to bebrought to the attention of the reader.

Most important of all, please be sure to includethe most expedient way for me to communicatewith you. E-mail is preferred over postal mailing,but if e-mail is not available, please include atelephone number where you can be reached.

I hope that you will seriously consider makinga contribution to the histology community bysubmitting a manuscript to HistoLogic. I lookforward to hearing from you.

Vinnie Della Speranza, Scientific Editor

Letter From the EditorLetter From the EditorSubmitting Articles to HistoLogic

Microsoft Word and Corel WordPerfect are registered trademarks of their respective owners.

D i s c o v e r l o v ea t f i r s t s l i d e .

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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.

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