HISTOLOGIC ®

T e c h n i c a l B u l l e t i n f o r H i s t o t e c h n o l o g y

®

Vol. XXXII, No. 1, May 2000

IntroductionStaining for amyloid is commonlyrequested of clinical histology labor-atories. The Congo Red method isconsidered to be the gold standard,especially when stained preparationsare viewed with polarized light. Mostreferences caution, however, thatthick (8-10 microns) paraffin sectionsare required if one is to achieve theapple-green birefringence that is saidto be characteristic of Congo Red-

stained amyloid deposits. Failure toutilize thick paraffin sections mayrender small amyloid depositsindistinguishable from nonspecificbackground typically associatedwith binding of the dye to collagenand elastin fibers. Our laboratoryutilizes a novel approach to achievehigh contrast visualization ofamyloid in thin paraffin sections ofkidney when viewed with afluorescence microscope.

MethodsAlkaline Congo Red Technique(Putchtler, Sweat and Levine, 1962) 5

The use of a staining solution fullysaturated with sodium chlorideincreases the specificity of the results.

Fixation: 10% neutral bufferedformalin

Sections: 2-micron paraffin sections

Solutions:1. Mayer’s Hematoxylin (Poly

Scientific R&D Corp., Bayshore,NY)

2. Alcoholic Sodium Chloride(saturated)80% alcohol … … … … … 500.0 mlSodium chloride … … … … 5.0 gm

3. 1% Sodium HydroxideDistilled water … … … … 100.0 mlSodium hydroxide … … … 1.0 gm

4. Alkaline Alcoholic SodiumChloride (working) Alcoholic sodium chloride … … … … … … … 50.0 ml1% sodium hydroxide … … 0.5 ml

IN THIS ISSUE

A Novel Approach for the Demonstration of Amyloid in Thin (2 micron) Sections of Kidney … … … … … … 1

Artifact in Tissues Held in 70% Ethanol… … … … … … … … … … … … … … 3

A Breath of Fresh Air– Using Respirators in the Workplace … … … … … … … … … … … … 4

Histotechnology: The Next 100 Years … … … 6

All in a Day’s Work … … … … … … … … … … 11

Elastic Tissue Staining in Human Skin … … … … … … … … … … … … 12

The History and Use ofHematoxylin … … … … … … … … … … … … … 14

Memoirs of a Self-Made Histotech(Part One) … … … … … … … … … … … … … … 17

A Novel Approach for theDemonstration of Amyloid in

Thin (2 micron) Sections of KidneyRena Fail, HT (ASCP) and Sally Self, MD

Medical University of South CarolinaCharleston, SC 29425

Managing Editor, Gilles LefebvreScientific Editor, Vinnie Della Speranza,

MS, HTL (ASCP) HT, MT

Fig. 1A. Congo Red stained 2-micron section of kidney viewed with fluorescence microscopy. 400�

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5. Stock Congo Red Solution(saturated)Alcoholic sodium chloride … … … … … … … 250.0 mlCongo Red … … … … … … 0.5 gm

6. Working Congo Red Solution(prepare fresh) Stock Congo Red solution … … … … … … … 50.0 ml1% sodium hydroxide … … 0.5 mlFilter: Use within 15 minutes

Stain Procedure:1. Deparaffinize and hydrate to

distilled water.

2. Stain nuclei with Mayer’shematoxylin for 10 minutes.

3. Blue in running water for5 minutes.

4. Rinse in 3 changes of distilledwater.

5. Treat with Alkaline alcoholicsodium chloride working solutionfor 20 minutes.

6. Stain in Congo Red workingsolution for 30 minutes for thinsections.

7. Dehydrate rapidly in 3 changesof 100% alcohol, clear in3 changes of xylene, and mountin permount.

ResultsAmyloid:

Light microscopy: deep pink tored

With polarization: apple-greenbirefringence

With fluorescence: bright pink tored whenviewed with546 nm (green)illumination

Nuclei: blue

DiscussionAmyloid is a homogeneous, highlyrefractile substance staining readilywith Congo Red. Congo Red-stained amyloid appears pink tored by light microscopy, and apple-green with polarizing microscopy.This reaction is shared by all formsof amyloid and is believed to be

due to the cross ß-pleatedconfiguration of amyloid fibrils.This ß-pleated sheet configurationgives amyloid its polarscopicappearance when stained withCongo Red dye. Fine rigid non-branching fibrils, 7.5-10 nm indiameter of indeterminate lengthare seen in thin sections byelectron microscopy. In kidneys, thefibrils are found within themesangium and subendothelium,and occasionally within thesubepithelial space. The amyloidfibrils may extend into thebasement membrane, resulting inthickening and splitting of thestructure. The adjacent epithelialfoot processes are obliterated.1

The demonstration of amyloidosis inbiopsies is important because someforms, notably those secondary toinflammatory processes, may beslowed or controlled with theadministration of anti-inflammatorymedications. Appropriate specialstains are needed to demonstrateamyloid, particularly in end stagekidneys. Amyloidosis is difficult todistinguish from other late chronicglomerular diseases. Those mostcommonly used differentiating stainsare the Congo Red, Vassar andCulling’s Thioflavine T, andHighman’s Methyl Violet.2

Nephrologists at our hospital beganusing the smaller 18- and 20-gaugeneedles to obtain kidney biopsies.Though less invasive and lesspainful for the patient, they posed aproblem for our laboratory. Ourtechnologists are required to obtain20-23 consecutive slides with twoserial sections of the kidney biopsyon each slide. These sections areroutinely cut at 2 microns. Twelve toeighteen of these slides are stainedwith H&E, Masson’s Trichrome,PAS, and Jones’ Periodic Acid-Methenamine Silver (PAMS). Fiveextra unstained slides are retainedfor future studies to eliminate theneed to recut the block. Whenamyloid staining becomesnecessary, the need to obtainthicker recuts can often yieldsections bearing little resemblanceto the original sections.

Congo Red (polarized),Thioflavine T, and the meta-chromatic stains such as MethylViolet are methods we have usedpreviously for staining amyloid.Metachromatic methods, by theirvery nature, are nonspecific. Tissuecomponents other than amyloid,including fibrinoid, arteriolarhyaline, keratin, intestinalmuciphages, Paneth’s cells, zymogengranules, and jutaglomerularapparatus all stain withThioflavine T.3 As a result, CongoRed is the method of choice.

Fig. 1C. Congo Red stained 2-micron section of kidneyviewed with polarizing microscopy. 400�

Fig. 2A. Congo Red stained 10-micron section of liverviewed with fluorescence microscopy. 400�

Fig. 1B. Congo Red stained 2-micron section of kidneyviewed with brightfield microscopy. 400�

A few years ago, we had a slide thatwas stained with Thioflavine T whichproved to be ambiguous. Congo Redwas requested for comparison. Inorder to view a comparable section,it was necessary to perform the stainon a 2-micron section. Congo Red-stained amyloid appears red whenviewed by light microscopy, and paleapple-green when polarized. On a2-micron section, Congo Red viewedby either light microscopy orpolarization is very pale, but doesexhibit a pale green hue underpolarization. In searching theliterature we discovered thatamyloid exhibits a faint blue-greenautofluorescence. Following stainingwith Congo Red, amyloid exhibits apink-red fluorescence whenilluminated with 546 nm (green)wavelength light. This is the samechannel (BP 546/12 excitation filter)on our fluorescent scope that we useto examine Auramine-Rhodaminestains. We further learned that thicksections can sometimes producefalse positives. To avoid this, we usea fully saturated NaCl Congo Red

solution which suppresses false posi-tive staining of collagen and elastin.4

Fluorescence of the 2-micron sectionappeared to be very sensitive andvery specific. Amyloid fluorescedbright pink to red even on 2-micronsections (see Figs. 1A and 2A).

Unlike Vassar and Culling’sThioflavine T or Highman’s MethylViolet, Congo Red-stained sectionsare permanent. They do not fadeover time. Congo Red staining canstill be seen years later with bothlight and fluorescent microscopy.Using 2-micron sections stained withCongo Red and viewed on therhodamine channel is the easiest andmost definitive way to diagnoseamyloid. Fluorescence microscopy ofCongo Red-stained sections candetect even minute deposits ofamyloid.

References1. Spicer SS. Histochemistry in Pathologic Diagnosis.

1st ed. New York, NY: Marcel Dekker, Inc.; 1987: 510-515.

2. Burkholder PM. Atlas of Human Glomerular Pathlogy.Hagerstown, Md: Harper and Row;1933: 346-349.

3. Bancroft JD, Stevens A. Theory and Practice ofHistological Techniques. 3rd ed. New York, NY:Churchill Livingstone; 1990:155-175.

4. Elghetantany MT, Saleem A, Barr, Kayer. The Congored stain revisited. Annals of Clinical and LaboratoryScience.1989; 19(3):190-195.

5. Humason GI. Animal Tissue Techniques, 3rd ed.San Francisco, Calif: W.H. Freeman & Company;1972:3342-344.

Artifact in TissuesHeld in 70% Ethanol

M. Jane Chladny, HT(ASCP)Laboratories of Veterinary

Diagnostic MedicineCollege of Veterinary Medicine

University of Illinois2001 S. Lincoln Ave.

Urbana, IL 61802

AbstractArtifactual vacuole formation in thewhite matter of rat central nervoussystem tissues exposed to70% ethanol may resemblepathologic changes. Vacuolization inthe fiber tracts of rat central nervoussystem tissue has been documented.An experiment was conducted todetermine if similar vacuolizationoccurs in the white matter of otherspecies. Samples of mouse, rat, cat,

dog, sheep, cow, horse, and humanbrain, previously well fixed in 10%neutral buffered formalin, were heldin 70% ethanol for 24 and 48 hours.A similar piece of brain from eachspecies held in 10% formalin wasused as a control. After routineprocessing, vacuolization artifact inthe fiber tracts was observed in thetissues held in 70% ethanol in allspecies examined.

IntroductionIn a research facility or histologylaboratory where all tissues arewell fixed prior to processing, anadditional fixation step on thetissue processor is unnecessary.As practiced in some laboratories,a decision was made to substitute70% ethanol for 10% neutralbuffered formalin as the initialsolution on the tissue processor.The removal of formalin from theprocessor was instituted not onlyfor waste minimization, but also forthe health and safety of laboratorypersonnel. This modification of theprocedure produced well-processedtissues. For weekend processing,tissues were loaded into a processoron Friday afternoon, and held in70% ethanol on delay mode untilprocessing on Sunday evening.

Following implementation of themodified processing schedule, apathologist observed microscopicvacuolization in the white matter ofrat brain sections used in atoxicology study. Because ofinconsistencies in the appearance ofvacuoles in different dose groups, itwas questioned whether thevacuoles were lesions produced by

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Fig. 2C. Congo Red stained 10-micron section of liverviewed with polarizing microscopy. 400�

Fig. 1. Horse brain, formalin-fixed overnight processing.100�

Fig. 2B. Congo Red stained 10-micron section of liverviewed with brightfield microscopy. 400�

the test material or artifact. Whenthe accompanying histologypaperwork was examined, it wasnoted that the vacuoles appearedonly in animals whose tissues wereembedded on Mondays.

McLarrin reports that vacuolizationmay occur in the white matter ofrat central nervous system tissuesheld in 70% ethanol longer than48 hours.1 Furthermore, Luna statesthat changes may be observed in ratcentral nervous system tissues in aslittle as 20 hours of exposure.2 Theobserved vacuoles were processingartifacts, not lesions as firstsuspected. Formalin was returned tothe first station on the processor toprevent the artifact in centralnervous system (CNS) tissues.

The appearance of vacuoles in rattissue prompted us to investigatethe effect of exposure to 70%ethanol on brain tissue of otherspecies.

Materials and MethodsSamples of brain were collectedfrom a mouse, rat, cat, dog, sheep,cow, horse, and human. Threepieces from the same area of theformalin-fixed brains were trimmedfor evaluation. One piece was leftin 10% neutral buffered formalinto serve as a control. Another piecewas placed in 70% ethanol for24 hours. The third piece wasplaced in 70% ethanol for 48 hours.Subsequent to exposure, all wereprocessed using the followingroutine overnight processingschedule:

10% neutral buffered formalin 1 hr10% neutral buffered formalin 1 hr60% ethanol 1 hr80% ethanol 1 hr95% ethanol 1 hr95% ethanol 1 hr100% ethanol 1 hr100% ethanol 1 hrxylene substitute 1 hrxylene substitute 1 hrparaffin 15 minparaffin 15 minparaffin 15 minparaffin 15 min

Processed tissues were embedded inparaffin, sectioned at 3 microns, andstained with hematoxylin and eosin.

ResultsMicroscopic examination showedsome vacuolization in the fibertracts of all species after 24 hoursof exposure to 70% ethanol,although a marked increase invacuolization and a spongyappearance was observed at48 hours exposure. There was littlechange in the appearance ofperivascular clearing in both thewhite and gray matter of the threetreatment groups. Perivascularclearing is considered a commonartifact, secondary to immersionfixation.

DiscussionThe use of 70% alcohol is endorsedby many as an acceptable holdingmedium for fixed tissues.Vacuolization artifact can becomeproblematic if 70% ethanol isused for CNS tissues awaitingprocessing. Although it is necessaryto remove tissues from 10%formalin for immunohistochemistryto prevent masking of antigenicity,changes will occur in the whitematter of CNS tissues held in 70%alcohol for more than 20 hours.This artifact was present not onlyin rat CNS fiber tracts, aspreviously reported, but it was alsoobserved in the seven other speciestested.

AcknowledgmentsThe author thanks Dr. E. J. Ehrhart for microscopicinterpretation and photomicrography, and Rena Fail,Medical University of South Carolina, for human sampleexperimentation.

References1. McLarrin G. Vacuoles in the fiber tracts of rat CNS

tissues. J Histotech. 1982; 5(4): 171.2. Luna L. Histopathologic Methods and Color Atlas of

Special Stains and Tissue Artifacts. Gaithersburg, Md:American Histolabs, Inc., Publications Division; 1992:591.

A Breath ofFresh Air – Using

Respiratorsin the Workplace

Maureen Doran HTL(ASCP)Southern Illinois University

School of MedicineHistology Center, 2055Carbondale, IL 62901

Engineering controls are anemployee’s best form of protectionfrom airborne contaminants. Inenvironments where engineeringcontrols are not feasible, respiratorsare used to protect workers. Whererespirators are used, theOccupational Safety and HealthAdministration (OSHA) standardfor respiratory protection must befollowed. OSHA’s recently revisedrespiratory protection standard,29 CFR 1910.134, was published onJanuary 8, 1998 and becameeffective April 8, 1998. The U.S.Department of Health and HumanServices/NIOSH publication #99-143recommends that a respiratoryprotection program contain theitems described below.

Written Standard OperationalProcedures (SOPs) describing theselection and use of respiratorsmust be developed. Informationand guidance needed for theproper selection, use, and care ofthese devices must be included.One person must administer theprogram and be responsible forimplementing all aspects of theprogram. The program’sadministrator should have a

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Fig. 2. Horse brain, formalin fixed, followed by 48 hrs in70% alcohol. 100�

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background that provides sufficientknowledge to develop and overseea respiratory protection program.

Respirator selection is based on thehazard to which the worker isexposed. A hazard assessment needsto be conducted to determine thetype of respirator protection that isneeded. The assessment shouldinclude a surveilance of work areaconditions including exposuremeasurements/estimates. NIOSHhas developed a new set of regula-tions for testing and certifyingnonpowered, air-purifying,particulate-filter respirators. Thiscertification provides for nine classesof particulate filters with three levelsof efficiency. All of these newparticulate respirators meet theCDC’s performance criteria forprotection against tuberculosis. TheHEPA filters which were certifiedunder 30 CFR11 are still acceptablefor use in TB environments. Moreprotective respirators may beneeded for certain high riskprocedures. OSHA does not allowthe use of chemical cartridge respir-ators against substances with poor orunknown warning properties. Insituations where the identity of thecontaminant is unknown, or whenthe concentration of thecontaminant is very high, a supplied-air respirator should be used.

The respirator user must be trainedin the correct use of the respiratorand be aware of its limitations. Thistraining must include instructions forwearing, adjusting, and checking theseal of the respirator. A user sealcheck determines whether arespirator has been put on andadjusted to fit properly. This is doneevery time a respirator is worn.

All tight-fitting respirators must befit tested before an employee isrequired to use respiratory protec-tion. The standard specifies fouraccepted qualitative fit testprotocols. Qualitative fit testing isacceptable as long as the con-taminant concentration does notexceed 10 times the contaminant’spermissible exposure limit. Fit tests

are used to select the respiratorthat provides an adequate andcomfortable fit. Fit testing isconducted at regular intervals, andalso if there has been a change inthe work environment or theemployee’s health.

Respirators must be cleaned anddisinfected on a regular basis. If areplaceable-filter respirator is wornby more than one person, it mustbe cleaned and disinfected aftereach use. Respirators should beinspected during cleaning, anddamaged or deteriorated partsmust be replaced. Disposable-particulate respirators can bereused, but cannot be worn bymore than one person. Theserespirators must be discarded ifthey are soiled, physically damaged,or the user experiences increasedbreathing resistance.

Respirators must be stored in aconvenient, clean, sanitary location.They should be protected fromdust, harmful chemicals, sunlight,moisture, and excessive heat orcold. Often replaceable-filterrespirators are stored in resealableplastic bags, but this is notrecommended for particulate filter-type respirators. It is important toinsure that respirators are drybefore storage.

Not all workers are capable ofwearing a respirator. Certain facialfeatures or injuries can prevent aproper face-to-respirator seal.Individuals with breathing orpulmonary difficulties can developCOPD from chronic laboredbreathing associated with theirdisease. Pulmonary functiontesting can determine anemployee’s ability to toleraterespirator use without sufferingfrom restricted air flow. Thestandard requires that anemployer obtain a writtenrecommendation regarding theemployee’s ability to wear therespirator. Depending on eachstate’s licensing agency, either aphysician or other licensed healthcare professional is allowed to

perform the medical evaluationrequired by the written respiratoryprogram. The medical evaluationand/or exam is used to determinethat a worker is physically able todo the work safely while wearingthe respirator.

Wearing a respirator is a majorinconvenience to most workers.Even the best respiratory programwill fail if workers do not complywith the program. OSHAdetermined that respirators usedwithout proper training and fittesting can increase the employee’spotential for exposure. Theemployee must feel susceptible tothe disease or the condition relatedto the hazard, according to Becker’sHealth Belief Model [1974]. Theworker must believe that thebenefits of wearing the respiratoroutweigh the inconvenience.

Sources of Additional InformationAmerican National Standards Institute. Practices for

Respiratory Protection, Z88.2, 1992.American Industrial Hygiene Association Respiratory

Protection Manual, AIHA 1993.NCCLS. Clinical Laboratory Safety; Approved

Guideline. NCCLS document GP17-A (ISBN 156238-300-0)1996.

NIOSH Guide to the Selection and Use of ParticulateRespirators Certified Under 42 CFR. Cincinnati,Ohio: DHHS, CDC, NIOSH; DHHS (NIOSH)Publication No. 96-101, 1996.

The OSHA Respiratory Protection Standard(29 CFR.134).

U.S. Department of Health and Human Services. NIOSHRespiratory Protection Program in Health CareFacilities. DHHS(NIOSH) Publication No.99-143,1999.

Histotechnology:The Next100 Years

M. Lamar Jones, BS, HT(ASCP)

IntroductionAs we move into the nextmillennium it is tempting toimagine where the discipline mightbe going. Before we can lookahead, it is only appropriate thatwe reflect over the last 100 yearsof histotechnology to appreciatehow the discipline has changed inthat time. About one-third into the

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20th century we saw several trendsthat helped shape the histo-pathology laboratory, one of whichwas automated tissue processing.The late 1960s and early 1970sbrought about some uniquechanges that impacted the workforce up to the 1990s. The mostdramatic advances took placewithin the last decade, setting thestage for histotechnology to moveforward into the new millennium.Some of these advances include:

• the development of techniques toamplify and hybridize DNA andRNA probes to microbial andhuman genomes

• digital imaging of tissues and theadvent of confocal microscopy

• the advent of the discipline ofinformation technology

• the utilization of microchips toenhance the capability ofautomation in histology

• the wider availability ofstreamlined reagent systems thatbrought sophisticatedtechnologies within reach ofthose with minimal training

Few would argue that thehistopathology laboratory isprobably the most manual andlabor-intensive area of pathology,and as such, is an area ripe forautomation. Only a few areas ofhistotechnology have had any typeof automation thus far; notablyprocessing, staining, and morerecently, coverslipping. In recentyears the skilled workforce hasdwindled, driving the need foradvances in instrumentation and

technology. This reality combinedwith the exciting developments incomputer technology have openedup the possibilities for furtherexciting changes. Because the fieldis rapidly changing, we need to becreative and flexible in order tokeep pace and change with it.

What types of instrumentation willwe see in the next several years? Inthe next 50 years? How will we copewith the changes brought about bytechnology? What will education forhistotechnologists be like? How willwe prepare for the changes that willreshape our histopathologylaboratories? This article will offer aglimpse of how future generations ofhistotechnologists may practice theircraft.

AutomationThe histopathology laboratorycontinues today to be a labor-intensive area of diagnosticpathology. With budget restraints,personnel shortages, and qualityissues, automation can offer solu-tions to many problems. Why willthe emphasis on automationincrease in the decades ahead?

In contrast to human labor,automation offers:

• consistent volume and qualityof output

• rapid turnaround

• high speed and volume ofoutput

• fewer possible errors

• constant availability

• consolidation of tests

• predictable cost

One type of automation consists ofhigh end systems that move thespecimen from the receiving area ofthe lab through the analyzingprocess, and then to be discarded orstored. Such systems are controlledby information systems that help toimprove test ordering, as well asverification. Another type of auto-mation is the modular system. Thissystem will allow equipment to be

operated either independently ortogether as work stations. It is idealfor small labs, can stand alone orform work stations, as other piecesof equipment can be added ifneeded. The mission for labs is toreduce manual processes, increaseproductivity, and achieve rapidturnaround times for procedures.And laboratory personnel can becross-trained, which will increaseflexibility and mobility. Today,consolidated work stations, robotics,and modular equipment areincorporated into the histopathologylaboratory.

GrossingThe Gross Lab will be an integralpart of both the surgical suite andthe histopathology laboratory.In many hospitals, the histology labis now located relatively close to thesurgical suite. The histotechnologistmay work very closely with thesurgical team in taking andpreparing the tissue specimenimmediately after removal fromthe patient. The histotechnologist’straining will include a greateremphasis on anatomy and surgicalprocedure. As enrollments inpathology residency programsdecline, it may become increasinglynecessary for histotechs to becapable of working alone.

The specimen will be placed ontothe gross preparation area,described, and automaticallydigitally photographed. Magneticresonance or x-ray technologies willalso be utilized for appropriatespecimens with all resulting imagesbecoming part of the patient’spermanent record. Imaging willoccur by voice commands to thecomputer that controls the tracksystem. A tissue processing cassettewill be “ stamped” out with a barcode that identifies that patient andspecimen. The patient name, barcode and surgical number will berecorded digitally onto a tinymicrochip that will travel along with,and become a permanent part of thespecimen. Data on this processorchip will be permanent and can beaccessed at any time with a specialpen capable of being carried in a lab

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coat pocket, which will decipher thecode on the chip and then be playedback in a human voice.

“ Stamped” cassettes will move on atrack system in front of thegrossing area and the specimenswill be placed in the cassettes.A divider will be placed roboticallybetween specimens. Once thespecimens are placed into thecassettes, they will be sealedrobotically and taken to theprocessing chamber by the tracksystem. The cassettes will be placedinto the proper processing chamber,according to the size and type oftissue that is in the processingcassette. This will allow for theproper processing of each size oftissue. Care must be taken to placethe specimen into the processingcassette exactly the way that it willbe embedded, since this will be thelast opportunity for humanintervention before the sections areprepared and analyzed automatically.

ProcessingTissue processing will also have adifferent approach. The tissueprocessor unit will be built into thelaboratory. In this manner, thereagents will be piped into thelaboratory’s processor. The reagentswill be purchased and shipped in andconnected to transport lines to theprocessor. The processor will bevoice activated so there are nobuttons to press, or any need tocalculate the start, stop, and delaytimes. There will be a viewing screento allow any specimen to be seen asmagnified in the processing cycle inany reagent. This will also allowlipids, water, and other impurities tobe seen as they are removed andreplaced in the processing cycle.During processing, the density andspecific gravity of the tissues andreagents will be recorded. Thereagents will be pumped into theprocessing chamber and whencompleted, the used reagent will bepumped out directly to a disposalarea. Technologists will no longerhandle hazardous reagents. And thetissues may be embedded in somemedium other than paraffin. Thismedium will be pumped into the

processing chamber as the finalreagent and solidified around thespecimen. In a closed system such asthis, processing will be completed inas little as an hour for routinesurgicals, and biopsies in fifteenminutes or less.

MicrotomyTissue specimens may no longer besectioned with a microtome. Oncethe tissue specimens are processedand solidified, they will be tracked toan instrument called a tissueanalyzer. Here the tissues will besectioned with a laser or highpressure vibrating system. Once thesection is obtained, it will bemounted onto a grid that will beplaced into an imaging system afterstaining. Unlike paraffin, thesynthetic embedding medium willpermit any required staining methodto occur without prior removal.

StainingUp until the last several years,staining microscope slides in thehistopathology laboratory has beenprimarily performed manually.Today it is not at all uncommon tohave an automated H & E slidestainer even in the smallesthistopathology laboratory. And forhigh-volume labs there areautomated special stain andimmunohistochemistry stainersavailable and in use. Staining tissuesections may likely be utilized formany more years and will remain avital part of histotechnology. TheH & E may still be utilized, but thereal art and science of histo-technology will center aroundgenetic code “ staining.” More of theimmunohistochemistry and in situhybridization will become thestandard. The nice, artistically andscientifically prepared microscopeslides may be replaced bycomputerized charts, graphs, andplates generated by the computer-driven image analyzer. Perhaps onlyinfectious disease cases will actuallyhave a stained glass slide preparedfor documentation. Staining will beperformed by robotics to includestaining and coverslipping in oneprocess all on a microchip-embedded microslide!

MicroscopesThe microscope for the pathologistmay very well be attached to a“ lazy boy” or some type of com-mand center pilot seat. Sectionswill be robotically transferred tothe imaging system, which will notonly provide visual images but willmeasure the density of stainmolecules in the section.This willallow the computer to recreatethree-dimensional images ofmicroscopic structures, providingthe pathologist with a capability forvisualization of structural abnor-malities never before imagined.Images will be projected onto acomputer screen that covers oneentire wall, providing a level ofresolution and detail previouslyunheard of. Image quality will farexceed anything seen today as aresult of enhancements tocomputer and electronic chips. Thiswill be called a “ video-scope.”

The video-scope will either beremote controlled or voice activated.The system will automaticallyanalyze and interpret the sectionimages, offering the pathologist, bythe sounds of a human voice, a run-down of the measured parametersand possible diagnoses. And shouldthe pathologist need to see the“ gross,” or the “ block,” then therequest would be made by simplevoice command. After the block issectioned by the tissue analyzer, itwill be filed robotically. Uponrequest, the cassette can be retrievedrobotically and viewed on the screenby the pathologist. For QC purposes,the bar code would be scanned toreveal the proper patient andsurgical number.

Bar codesBar codes are already used in manyfacets of the clinical laboratory. Barcodes permit the tracking andverification of data such as thatcontained on:• specimen labels• reagent labels• accession labels• request labels• microslide labels

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Within the next decade, we will seetissue cassettes embossed with barcodes.

There are tremendous advantages tousing bar codes in the laboratory.They offer an efficient and error-freemeans of tracking that is inexpen-sive, and they provide a high degreeof security. Some present-dayimmunostainers utilize bar codes totrack patient slides, the stain that isneeded for those slides, and thereagents needed to perform thatstain.

Bar codes will revolutionize thehistopathology laboratory, so muchso that all patient and specimentracking, as well as stain perfor-mance, will be bar code-driven.

ImmunohistochemistryMost immunohistochemicalprocedures are performed in thehistopathology or specialprocedures laboratories. Whilemany laboratories once relied uponmanual methods to complete thesestains, more recently instrumen-tation has been developed topermit automated staining to bedone reliably and reproducibly.I envision that a device will bedeveloped which will radicallychange the way immuno-histochemistry is performed. It mayconsist of a small electronic chipthat may be read by a computer.A piece of fresh or fixed tissue willbe placed directly onto the chipand viewed on a computer screen.Relevant data will be entered intothe system and an antibodyapplied, making it possible for theresult to be read directly. This maybe the immuno stain and/or the

image analysis of the future, calledthe “ immuno chip.”

Molecular PathologyIt has been only in the last fewyears that the word “ molecular”has entered into the vocabulary ofthe histotechnologist. Already manyof these tests are being performedin the histopathology laboratory.And we will see more of these typesof tests performed by histo-technologists, perhaps in the next10-15 years. Molecular techniqueswill become the gold standard. Thepractice of molecular pathology willchange the medical field greatly, andit is off to a good start even now. Wenow see molecular pathology as atool for diagnosing patients withinfectious diseases and geneticallytransmitted diseases. Many histo-technologists are already per-forming DNA/RNA hybridizationand apoptosis assays. For many ofthe infectious diseases andorganisms, the histopathologylaboratory may be the microbiologylab of tomorrow. Microslidesproperly prepared can be retrievedfrom the slide file for many yearsupon request, whereas micro cultureplates only last a few days. Probesand antibodies will demonstratemicroorganisms with a high degreeof sensitivity and specificity.

Information TechnologyThe world of computerization hasonly entered the histopathologylaboratory in the last several years.In most cases, histopathology wasprobably the last part of thelaboratory to become computerized.But today, the computer is the“ way of life.” In the very nearfuture, even as we speak, the com-plete testing process in pathologywill include:

• electronic reporting

• electronic signature/verification

• full auto fax support

• auto-encoded reports containingCPT 4, snowmed, and ICD 9codes

• code compliance with regulatoryagencies

• image processing

• intranet and web pages fortelepathology and consults

• voice recognition features

• direct third-party payer ormanaged care billing

The histopathology laboratory ofthe future will have morecomputerization than everimagined. Histotechnologists willbecome quite skilled andknowledgeable with computers andinformation technology.

EducationFormal education forhistotechnologists will be morenecessary than ever before. As aresult of the impact thathistotechnologists will have on thehealth care industry, training willbecome more sophisticated.A bachelor’s degree plus at least a2-year concentrated histopathologyresidency program will be required.Upon graduation, a Master ofScience in Histopathology will beawarded. Licensure, as well ascertification, will become thenational standard for all tech-nologists. The residency programwill involve such training as actualcase studies, and tissue and organmanagement. Histopathology willreplace histotechnology, due to theincreased sophistication of thetechnology that practitioners of thediscipline will be required toemploy. The technological changesof the discipline that I have alludedto will bring about a long desiredelevation of professional statureand compensation.

It is unclear when changes of thesort that I have imagined mayarrive. What is certain is thatchange will come. Technologicalinnovations will bring opportunityto those who are ready to meetthe challenge. Are we as histo-technologists going to be ready?Will we take the challenge? If not,we will be left behind, for tech-nological change waits for no one.There is no question that thehistotechnology discipline of the

future will require a level ofknowledge, skill, and sophisticationbeyond anything previouslyimagined. Nevertheless, the properhandling of tissue specimens willstill be done best in the hands of awell-trained, professionalhistotechnologist!

M. Lamar Jones is consultant atHistological Consultants, Inc.,Alabaster, AL.

Sources of Additional InformationDadoun R, Krempel G. Progressive automation taking

labs into 21st century. Advance for MedicalLaboratory Professionals. 1999;11(25):8-10.

Blick K. Facing up to facts: labs now in the informationbusiness. Advance for Medical LaboratoryProfessionals. 1999;11(25):11-13.

Kaul K. Mind-boggling developments forecast formolecular pathology. Advance for MedicalLaboratory Professionals. 1999;11(25):14-15.

Miller M. Bar coding for the next millennium - usingstandards. Advance for Medical LaboratoryProfessionals. 1999;11(16):12-15.

Narayanan S. Technology and laboratoryinstrumentation in the next decade. MedicalLaboratory Observer. 2000;32(1):25-31.

All in a Day’s Work !M. Jane Chladny, HT(ASCP)

Laboratories of VeterinaryDiagnostic Medicine

College of Veterinary Medicine University of Illinois2001 S. Lincoln Ave.

Urbana, IL 61802

All in a Day’s Work! is a salute tothe efforts of histotechs everywhere,those unsung heroes who often gounrecognized for their contributionsto the advancement of health andscience. Please contact the editor ifyou would like to see your workfeatured in this column.

The Laboratories of DiagnosticMedicine, part of the University ofIllinois College of VeterinaryMedicine, offer a variety of serviceswhich provide histotechnologistswith daily challenges and continuallearning. The primary responsibilityof the histopathology laboratory isto provide diagnostic support toclinicians at the university’s largeand small animal clinics,veterinarians within the state ofIllinois, and even some prac-titioners out of state. Specimens canrange from a small lesion taken

from a domestic animal, tonecropsy tissues from a herd animalwith a suspected infectious disease.It is sometimes necessary forowners to sacrifice one sick animalfor disease diagnosis to prevent awidespread outbreak in the herd.Along with routine H&E slides, thelaboratory performs up to twentydifferent special stains per day froma routine offering of about fiftystains. Additionally, immuno-histochemical procedures areutilized for the diagnosis of tumorsand infectious disease. Immuno-histochemistry can be challenging inan animal lab. Because species crossreactivity is usually not known,experimentation with variousantigen unmasking procedures,antibody dilutions, incubation times,and detection systems must be done.Those antibodies that do cross reactare titered for optimum staining ineach species requiring theimmunohistochemical procedure.Currently, the laboratory offersnineteen infectious disease markersand thirty antibodies for tumoridentification or differentiation.

Besides services for domestic andfood animals, the universitysupports a zoo pathology program,which enables veterinarypathology residents to specialize indiseases of zoo animals.

Submissions from the Lincoln Parkand Brookfield Zoos and SheddAquarium in Chicago, supplydiversity in the species seen by thelaboratory. The type of animalsubmitted for necropsy can causethe diagnostic workload to fluctuate

a great deal. Tissues from a finchcan be contained in a couple ofblocks, or if a tiny pipe fish dies, theentire specimen is embedded in oneblock. However, if a beluga whale,or a horse exhibiting neurologicsigns, is necropsied, nearly sixtycassettes may be submitted.Because pathologists and residentshave come to expect only thehighest quality slides, some tissuespresent an extraordinary challengefor the microtomist. Marinemammals and birds are unusuallydifficult to section. Many large,dense bones and keratinizedspecimens, such as hooves and nails,require pretreatment and specialhandling. Oversized specimens, suchas large animal eyes, are embeddedusing lead Ls and placed on 3x2"slides at sectioning.

Slide preparation and staining forresearch projects is anotherimportant function of the histologylaboratory at the College ofVeterinary Medicine. In manycases, histotechnologists must assistan investigator, who may beunfamiliar with histologicprocedures, to determine themethods most appropriate tocomplete his project. Thelaboratory has been involved inmany interesting and unusualprojects. As one might expect, someresearch done at the College ofVeterinary Medicine is conductedfor the betterment of animals.Annual trips to Africa supply the labwith tissues from wild animals founddead. Those tissues are examined inan effort to learn more about the

Fig. 1. One of the many step sectionsthrough the dorsal plane of an entirefrog.

Fig. 2. Section of large bone including joint on3x2'' slide.

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animals in their natural habitat,thereby aiding animals held incaptivity in zoos. Also, theinformation obtained by theexamination of these tissues is sentback to Africa to help wildlifemanagement with health problemsof their free range populations.One study examined samples ofAlaskan seals and whales forpossible metals toxicity.

Aside from research to benefitanimals, many of the projectsinvolving the histology laboratoryare for the advancement ofknowledge to benefit humans.Studies have been done oncryptosporidium and rotovirus inan attempt to block effects of theagent which produces diarrheaand is fatal for many children inthird world countries andunderdeveloped areas of the UnitedStates. Animal models have beenused to study amyotrophic lateralsclerosis (ALS) and multiplesclerosis (MS). In the MS project,investigators are working to identifythe gene responsible for the disease.Once that information is obtained,further research may produce amethod to block or alter the geneproduct.Another project is underwayto target cells responsible for a braintumor which affects children.

In addition to research to studydiseases, animal models have beenused in projects concerning humanhealth and safety. Appetite andnutrition have been evaluatedthrough varying zinc intake. Thenutritional benefits of eating certainfoods, such as broccoli, on diseaseprevention have been researched. Anongoing project studies the effects of

ultrasound on animal and humanhealth. Adverse changes in the lungshave been observed in severalspecies. The results of this researchproject will be used to establish thesafety of ultrasound use on humans.

Tasks are not limited to traditionalslide preparations. Much like ahospital lab, the daily routineconsists of H&Es, special stains, andimmunohistochemical procedures;however, on occasion, glycol-methacrylate has been utilized,making embedding, sectioning, andstaining a deviation from the norm.Plant tissues have been processedand special stains applied to sectionsof soybean roots for identificationof fungus. The laboratory has evenprocessed and cut industrial sludge.Another function of the lab includespreparation of slide sets used tosupport teaching of veterinary andveterinary pathology students. Inmost cases, 100 slides from a singleblock are prepared for theveterinary student teaching set.

Histotechnologists at the Universityof Illinois find the best of bothworlds: a diagnostic lab, as in ahospital, and a lab which supportsresearch. Four of the fivehistotechnologists were previouslyemployed by hospital laboratories.The diversity of tasks, species, andtissues found at the College ofVeterinary Medicine provides astimulating and challengingenvironment. The busy laboratory isstaffed with excellent technologistswho meet those challenges whilegaining expertise in a wide range ofhistologic techniques.

Elastic Tissue Stainingin Human Skin

Yufeng Yu, MDClifford M. Chapman, MS, HTL

(ASCP), IQCPathology Services, Inc.

640 Memorial DriveCambridge, MA 02139

AbstractThere are several methods used inhistology to visualize elastic tissue.Different methods stain thickelastic, and thinner elaunin andoxytalan fibers to varying degrees.We report on the effectiveness ofthese methods in staining elasticfibers in specimens of human skin.

IntroductionElastic fibers are an integral andimportant component of skin. Thesefibers are composed of two majorparts: a central core of elastin that issurrounded by microfibrils.1,2 Thickelastic fibers present in the reticulardermis contain both parts. Thesethick elastic fibers connect tothinner elaunin fibers in thepapillary dermis which, in turn, forma plexus with very thin oxytalanfibers that project perpendicularlytoward the dermal-epidermaljunction. These two types of thinnerelastic fibers are composed mostly ofmicrofibrils with little or no elastin.3

There are many different methodsused in histology to stain elastictissue. Different methods result inthe various elastic fibers stainingto different degrees. We report onthe effectiveness of some of thesemethods in staining elastic fibersin specimens of human skin.

MethodsSpecimen PreparationHuman skin specimens were fixed in10% neutral buffered formalin for24 hours, and then processedthrough a graded series of ethanol,cleared in xylene, and infiltrated inparaffin on a Sakura VIP tissueprocessor over a total processingtime of 8 hours. Paraffin sectionswere cut on a rotary microtome at a

12

Fig. 3. Embedding oversized specimen using lead Ls.

Fig. 4. Histology laboratory staff (left to right): MelanieCox, Dana Browning, Gigi Simon, Jane Chladny, andKaren Gebbink.

thickness of 5 microns, picked up onlysine-coated slides, and heated in anoven at 65oC for 45 minutes. Aftercooling, slides were deparaffinized inthree changes of xylene for5 minutes each, and hydrated todistilled water before staining.

Staining ProceduresFour different methods for stainingelastic tissue were utilized: VerhoefVan Gieson,4 Gomori’s aldehydefuchsin,5 Weigert’s resorcin,6 andMiller stain.7 Serial sections offormalin-fixed, paraffin-embeddedhuman skin were prepared andnumbered. Hematoxylin and eosinstaining was used in addition to theabove elastic tissue stains onadjacent sections from the sameparaffin blocks.

Procedure: Miller stain for elastictissue

0.5% Potassium PermanganatePotassium Permanganate … 0.5 gDistilled water … … … … 100 ml

1.0% Oxalic AcidOxalic acid … … … … … … … 1.0 gDistilled water … … … … 100 ml

Miller Elastic StainVictoria Blue 4R(C.1 42563) 1 gNew fuchsin(C.1 42520) … … 1 gCrystal violet(C.1 42555)… … 1 g

Dissolve in 200 ml of hot distilledwater, then add in following order:

Resorcin … … … … … … … … 4 gDextrin … … … … … … … … … 1 g30% ferric chloride (fresh)50 ml

Boil for 5 min then filter while hot.Transfer precipitate plus filterpaper to original beaker and re-dissolve in 200 ml of 95% ethanol.Boil on a hot plate, or in a waterbath for 15 to 20 min. Filter andmake up to 200 ml with 95%ethanol. Finally, add 2 ml ofconcentrated hydrochloric acid.

Purchase Miller stain:Rowley Biochemical (Danvers,MA) catalog # SO-709

Van Gieson stainAcid fuchsin, 1% aqueous 2.5 mlPicric acid,saturated aqueous… … … 97.5 ml

Purchase Van Gieson stain:Rowley Biochemical (Danvers,MA) catalog # SO-463

Method:1. Deparaffinize and hydrate

slides to distilled water.

2. Place into 0.5% potassiumpermanganate for 5 min.

3. Wash 7x with tap water.

4. Place into 1.0% oxalic acid for3 min.

5. Wash 7x with tap water.

6. Rinse in 95% ethanol for1 min.

7. Place into Miller Elastic Stainfor 1-2 hours at roomtemperature.

8. Rinse 7x in tap water.

9. Rinse in 95% ethanol: 2 times5 seconds each.

10. Rinse in water.

11. Place into Van Gieson’s stainfor 2 min.

12. Dehydrate rapidly through95% ethanol – two changes5 seconds each.

13. 100% ethanol – 2 times2 min each.

14. Xylene – 3 times 3 min each.

15. Coverslip with Permount.

RESULTS:elastic fibers … … … … blue/blackmast cells… … … … … … … … blackred blood cells … … … … … greenconnective tissue … … … … … red

Procedure: Weigert’s resorcinfuchsin elastic tissue stain.

Weigert’s resorcin fuchsinSD Alcohol, 3A … … … 70% v/vHydrochloric acid … … … 1% v/vResorcin-fuchsin … … 0.2% w/vDistilled water … … … … 29% v/v

Purchase Weigert’s resorcin stain:Rowley Biochemical (Danvers,MA) catalog # F-370-1

Method:1. Deparaffinize and hydrate to

distilled water.

2. Immerse slides in the Weigert’sresorcin fuchsin, and leave6 hours to overnight at roomtemperature.

3. Remove slides from stain andwash in running tap water untilall color comes out.

4. Dehydrate through alcohols andxylene.

5. Mount with Permount.

RESULTS:elastic fibers … … … light to dark

purple

ResultsWhile hematoxylin and eosinstaining shows collagen in variousshades of pink, specific elastictissue fibers cannot be identified(Fig. 1). In contrast, the VerhoeffVan Gieson method shows thickblack-stained elastic fibers in thedermis. However, the thinnerelaunin fibers that run up andunder the epidermis cannot bediscerned (Fig. 2).

The Gomori aldehyde fuchsinmethod stains both the thickelastic fibers in the dermis, and thethinner elaunin fibers thatapproach the epidermis, a darkpurple, which contrasts well withthe light green counterstain.However, the thinnest oxytalanfibers are still difficult to identify(Fig. 3). Weigert’s resorcin methodresults in dark purple-blackstaining of the finest oxytalanfibers that extend into the dermis.Additionally, thick elastic fibersand elaunin fibers are stained.While this staining is precise anddelicate, there is virtually nocounterstain (Fig. 4).

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Fig.1. Hematoxylin and eosin. 780�

Miller’s method stains all elasticfibers. Thick fibers in the dermis,thinner elaunin fibers leading fromthe dermis, and the thinnestoxytalan fibers extending into theepidermis are stained dark black.This staining contrasts well with thenuclear and connective tissuecounterstains (Fig. 5).

DiscussionThe major function of skin is toprotect the body against exteriorforces resulting from substances,chemicals, and microbes. While theepidermis is composed of tightlypacked cells which secrete keratinproteins, the underlying dermis ismade up of ground substance,collagen, and elastic fibers. Theelastic fibers form the basis for theintegrity and elasticity of the skin.8

Thick elastic fibers in the reticulardermis are composed of a core ofelastin surrounded by microfibrils,and stain readily with all methodsemployed herein. However, thethinner elaunin and oxytalan fibersare not stained with the VerhoeffVan Gieson method. In addition,sections must be oxidized withpotassium permanganate in boththe Miller and Gomori methods inorder for both elaunin andoxytalan fibers to be stained. Thisoxidation results in disulfide groupsbeing formed, which cause thefibers to become basophilic andable to be stained by thesemethods.3

The finest oxytalan fibers arevisualized by the Weigert’s resorcinmethod. Under high powermagnification these fibers can beseen extending to the basementmembrane of the dermal-epidermaljunction.

In the clinical histology laboratory,it is not surprising that the primarymethod of staining elastic tissue isthe Verhoeff Van Gieson. Increasedelastic tissue can manifest itselfclinically as elastofibroma orpseudoxanoma elasticum.Conversely, decreased elastic tissuein skin may appear as macularatrophy or cutis laxa, which is adegenerative change in the elasticfibers.8 Thus, the visualization ofthick elastic fibers in the reticulardermis using the Verhoeff VanGieson method is appropriate.

However, in a research setting, it isimportant to know exactly whichfibers are being investigated. Using

an inappropriate method couldresult in false negative results.Visualization of the thinner elauninand oxytalan fibers requires the useof methods other than the VerhoeffVan Gieson. These methods must beevaluated for each use to determineif they will stain the elastic fibertype under investigation.

AcknowledgmentsThe authors wish to thank Ms. Karen Carlson andPathology Services, Inc. for their assistance and supportduring the preparation of this manuscript.

References1. Vollenweider RS, et al. Elastic fibers in scar tissue.

J Cutan Pathol. 1996; 23:37-42.2. Cotta-Pereira G, et al. Oxytalan, elaunin and elastic

fibers in the human skin. J Invest Derm. 1976;66:143-148.

3. Bancroft JD, Stevens A. Theory and Practice ofHistological Techniques. 4th ed. New York, NY:Churchill Livingstone; 1998: chap 7.

4. Verhoeff FH. Some new staining methods of wideapplicability including a rapid differential stain forelastic tissue. JAMA. 1908; 50:876.

5. Gomori G. Aldehyde-fuchsin: a stain for elastic tissue.Am J Clin Pathol. 1950; 20:665-666.

6. Weigert C. Ueber eine Methode zur Farbung elastischeFasern. Zentrab Allg Pathol. 1898; 9:289-292.

7. Miller PJ. An elastin stain. Medical Lab Technol. 1971;28:148-149.

8. Barnhill RL. Textbook of Dermatopathology.McGraw-Hill; 1998: chap 17.

The History and Useof Hematoxylin

Maria G. SinghalThe Methodist Hospital

School of HistotechnologyHouston, Texas

Origin of HematoxylinHematoxylin is a compoundextracted from Haematoxyloncampechianum, which is a derivativeof the third largest family of plants –the legumes. The Aztecs used thiscompound to dye the fabric for theirrobes. The word “ Haematoxylon”means “ blood wood” and“ campechianum” stands for the factthat both the trees and MexicanIndians who discovered the dye arenative to the Bay of Campecheregion. It was Hernando Cortez whofirst found that Indians used thethorny plants to make the violet andblack dye. Spaniards introduced thisdye to the European markets whereit competed well with indigo, theoriginal violet dye from India. TheEnglish also produced a similar dye

14

Fig. 5. Miller stain. 780�

Fig. 3. Gomori aldehyde fuchsin. 780�

Fig. 4. Weigert’s resorcin. 780�

Fig. 2. Verhoeff Van Gieson. 780�

and called it “ logwood,” since it wasshipped as unextracted logs.Haematoxylin was first produced byCherrul in 1810, and Erdmannworked out the formula in 1842. Itwas discovered later that the actualstaining occurred as a result of theoxidation product of Haematoxylin,called hematein.

PropertiesIn its pure form, Hematoxylin is acolorless powdery substance whichhas a tendency to crystallize intoeither tetragonal prisms containingthree molecules of water, orrhombic crystals with one moleculeof water. It is a very weak acidic dyewith an isoeletric point aroundpH 6.5. It attains staining propertiesupon conversion to hematein and inthe presence of various electrolytes.Oxidation of Hematoxylin occurswhen hydrogen atoms are removed,forming hematein (see Fig. 1).

Several oxidizing agents can be usedto achieve oxidation of Hema-toxylin, such as sodium iodate,potassium permanganate, ormercuric oxide. First exposure toatmospheric oxygen in the presenceof ultraviolet light will causeformation of hematein, the latterbeing responsible for the color-related properties attributed toHematoxylin. Pure Hematoxylin issoluble in water, ethanol, methylcellosolve, and ethylene glycol.

ContaminantsThe pure dye is difficult to obtainon a commercial scale. Commercialsamples may be contaminated withwood from other parts of the tree,such as bark. These are insoluble andcan be filtered out, but they decrease

the effectiveness of the stain. Crystalcontaminants may be due to soilfrom roots of the tree, or due to theattempts to stabilize the powderusing sodium sulfite or antioxidants.

Uses in HistologyThe first biologist who tried to useHematoxylin was Waldeyer in 1863.However, he was not verysuccessful in staining the axiscylinders of nervous tissue and,therefore, he gave up usingHematoxylin in favor of anotherdye. Two years later, Bohmersuccessfully employed Hematoxylinin combination with alum todevelop his staining formula. In1872, Merkel used Hematoxylin tostain muscles of animals inpolarization studies. A great dealwas accomplished in the field ofHematoxylin staining during thelate 1800s. In 1879 (not fullyconfirmed), Kleinenberg proposeda formula for Hematoxylincontaining alum and calciumchloride dissolved in 70% alcohol.Around this time, Delafield’sHematoxylin was also proposed ina formula which was furnished byPrudden in 1885, in response to aninquiry. Renaut and Ehrlich gavediffering formulas for glycerinatedalum-Hematoxylin in 1881 and1886, respectively. However, themost important development inalum-Hematoxylin was made byMayer in 1891. He emphasized theuse of aluminum chloride asopposed to alum. During the 1880s,Bohmer, Heidenhain, Weigert,Apathy, and later, Platnerdeveloped methods introducing theuse of chromium compounds withHematoxylin. Later, Flesh and Palproposed modifications toWeigert’s formula. During the late1880s and 1890s, researchersemployed iron, molybdenum,vanadium, and salts of othermetals. Benda, Heidenhain, andWeigert developed iron-Hematoxylin, differing in the use ofiron salts and the method ofapplication. Mallory worked withmolybdenum, and Wolters workedwith vanadium. These solutionsproduce different staining pictures

due to the electrolytes and themordanting action of the metalsalts.

Common uses of HematoxylinHematoxylin is one of the mostvaluable natural dyes used innuclear staining today. Its primaryuse consists of staining histologictissue samples for microscopicanalysis. Two of the most importantuses are: (1) staining in thePapanicolaou procedure for thediagnosis of cytopathologyspecimens, and (2) in combinationwith the contrast stain eosin,making the popular H&E stain fortissues in routine pathology. Itsindustrial use has declined over theyears but it is still used to dyetextiles, furs, and to prepare inks.It is used as a reagent for thedetection and determination ofvarious metals in fluids, extracts,and tissue.

Tissues that can be stained withHematoxylinHematoxylin is extensively used tostain nucleic acids. Therefore, itdemonstrates both nucleic DNA, aswell as nucleic and cytoplasmicRNA. Various modifications havebeen used to stain a variety ofsubstances, including mucins,mitochondria, muscle striations,myelin sheaths, and dentinaltubules.

Staining ProcedurePreparation of Harris’sHematoxylin:

Hematoxylin… … … … … … … 5 gAbsolute ethyl alcohol… … 50 mlAmmonium aluminum sulfate… … … … … … … … … 100 gDistilled water … … … … 1000 mlMercuric oxide … … … … … 2.5 g

Dissolve Hematoxylin in alcohol.Completely dissolve theammonium aluminum sulfate inwater with heat. When bothsolutions are dissolved, mix the twosolutions, and bring to a rapid boil.Remove from heat. Collect 50 mlof this solution. Label “ withoutoxidizer,” and put aside forempirical testing. To this solution,

15

O

HO O

OH

OH

HO

CC

CH2

CH2

O

HO OH

OH

OH

HO

CHC

CH2

CH2

Hematein Hematoxylin

Fig. 1. Chemical Structures

16

Fig. 2. Empirical test.

add mercuric oxide slowly. Plungeflask into ice. Cool rapidly. Thestain is ready for use as soon as ametallic sheen develops on thesurface of the solution. Filterbefore use.

Empirical Testing1. Odor:

A good Hematoxylin stainshould have a wine smell.

2. Color:Color should be a deep purple-red.

3. Tap Water Test:A few drops of good stain in tapwater should turn bluish-black.

4. Filter Paper Test:A good stain, when placed on apiece of filter paper, shoulddiffuse and produce a patternwith a maroon color ending in adark purple.

Progressive vs regressive staining Progressive stains demonstrate par-ticular elements and are selectivelycontrolled by time and solutionconcentration. Staining continueswith periodic viewing under themicroscope until the desired resultsare observed. Regressive stainsutilize the principle of rapidstaining, only the tissue componentsare stained. The entire tissue is over-stained and the excess solution isremoved by differentiation until thedesired stains are visible.

DifferentiationDifferentiation is a process whereexcess Hematoxylin is graduallyremoved using a dilute acid rinse.Other reagents such as basic media,mordant, buffers, and oxidizershave been used as differentiating

agents (bluing). Different alkalinesolutions have been used toachieve bluing results: tap water,ammonia solutions, Scott’ssolutions, and lithium carbonatesolutions. Sections must bereplaced in an alkaline solutionfollowing the differentiation step.

Common causes of artifacts instaining due to the nature ofHematoxylinWeakly stained section:

– Hematoxylin under-oxidized;Hematoxylin over-oxidized.

– Staining time too short.– Over- or under-differentiation.

Nuclei not clear blue:– Insufficient bluing.

Section stained too strongly:– Insufficient differentiation.

Common alternatives toHematoxylinBrazilinCelestine blueMethylene blueToluidine blueThionineAzure

Pros and cons of Hematoxylin usePros:

– The most widely used nuclearstain.

– Rapid, reproducible results in awide range of tissues.

– Staining can be easily modifiedto suit individual needs.

Cons:– Expensive.– Results can vary if quality

control is not maintained.– Hematoxylin solutions easily

oxidize.

Fig. 3. Gastric mucosa, foveolar epithelium. Stained withHematoxylin-eosin (H/E). 250� (oil immersion)

Fig. 4. Mucinousadenocarcinoma; signet ring cells. Stainedwith Hematoxylin-eosin (H/E). 250� (oil immersion)

Fig. 5. Chronic inflammation; plasma cells.Stained with Hematoxylin-eosin (H/E).250� (oil immersion)

Fig. 6. Ganglioneurofibromatosis; ganglion cells.Stained with Hematoxylin-eosin (H/E).250� (oil immersion)

Fig. 7. Colon; normal crypt in cross-section.Stained with Hematoxylin-eosin (H/E).250� (oil immersion)

17

Fig. 8. Neurofibroma; nerve bundles. Stained withHematoxylin-eosin (H/E). 250� (oil immersion)

Fig. 9. Lymphoid follicle; lymphocytes. Stained withHematoxylin-eosin (H/E). 250� (oil immersion)

AcknowledgmentsJuan Lechago, M.D., Ph.D. The Methodist

Hospital/Baylor College of Medicine.Jo Ellen Atkins, BS, HT. The Methodist Hospital.Barry Rittman, Ph.D. UT-Houston Dental Branch,

personal reference notes.Subhendu Chakraborty, MS. Baylor College of Medicine,

visual assistance.

References1. Clark G. History of Staining. 3rd ed. Baltimore, Md:

Williams & Wilkins; 1983.2. Conn HJ. The History of Staining. Geneva, NY: Book

Service of the Biological Stains Commission; 1933.3. Conn HJ. Biological Stains. 8th ed. Baltimore, Md:

Waverly Press, Inc; 1969.4. Sheehan D, Hrapchak B. Theory and Practice of

Histotechnology. 2nd ed. St. Louis, Mo: C.V. Mosby Co;1980.

Memoirs of a Self-Made Histotech

(Part One)

Jeffrey S. SilvermanHT, HTL(ASCP) QIHCSouthampton Hospital

Southampton, NY

I’ve been privileged to havesomehow found myself engaged inthe honorable field ofhistotechnology, helping todiagnose disease in my fellowhumans and their companion

animal pets for the last quarter ofthe century. As a self-taughthistotech who learned all he didthrough passion for histology, andluck in meeting lots of friendlyemployers and mentors, I’ve beenasked to reflect back on thechanges I’ve witnessed in myvaried career.

I was the quintessential sciencenerd in school, dissecting frogs andpigs in grade school science fairs.Prior to that, I always took thingsapart, watches and clocks, old TVsand radios from friends andneighbors. One favorite neighborhad an auto repair business and heused to bring me carburetors,generators and such to dissect aswell. My mother used to drag meto antique stores a lot when I wasfourteen. On one such trip I foundan antique copy of AnimalMicrology by Michael F. Guyer(1906-1946 editions). I bought awell microtome from EdmundScientific and practiced sectioningpotatoes and cork. At gradeschool, I used to collect thescience supply catalogs from theteachers and pore over thecontents. My earliest memories ofmicroscope slides were in juniorhigh school, where there was a boxof someone’s ancient kidney andskin sections, probably preparedby some science teacher during hiscollege histo course. After lookingat them, I immediately set aboutdissolving off the coverslips andplaying with whatever other stainswere available in the school, usingmethods that I gleaned from mybooks.

Lo and behold, when I got to highschool (class of 1971), I found thatsomeone had purchased a rotarymicrotome that I was told no onecould ever remember using. It wasby an unknown Japanese manu-facturer and even had a conveyorbelt for the ribbons. It had a steelknife and a whet stone and strop!I made a paraffin processing set-up. We found an old asbestos-insulated incubator, put a 60 wattlight bulb in it and made a paraffin

oven. Somehow, the school, circa1969-71, had a budget for this stuff,and in high school I pored overFisher, Turtox, and CarolinaBiological catalogs orderingparaffin, whatever fixings forZenkers and Bouin’s that weren’tin the chemistry labs, an aluminumBoeckel slide drying plate, slides,coverslips, a few Peel-Away plastictissue capsules, and dry stains likeorange G, and aniline blue toconcoct Masson Trichrome andHeidenhein’s Azan stains (fromthe old books).

I managed to obtain some newerbooks that told of one-stepdehydrant clearants like dioxane,so we bought some and that’s whatI used for processing. Toxic as hell,and I played with it with no PPEsand no hood during my highschool years. I used xylene andcellosolve (ethylene glycolmonoethyl ether) to deceratesections and stain. Next, at school,we euthanized a rat and later aguinea pig with ether in a bigmayonnaise jar from the kitchen,dissected them, and used all thedifferent fixatives on variousorgans. The processing and stainingset-up was in my home in thebasement because there was noroom at the school. So I carried thetissues home on the bus in fixative,transferred the tissues fromfixative by hand through dioxane,and infiltrated them in the bulb-heated paraffin oven. Embeddingwas from a pitcher in the oven intoa unique Unimold from Lipshaw, atwo-piece mold that made a blockwith a paraffin “ stem” for theobject clamp.

I did the microtomy at school in aprep room between two class-rooms. One day I slit open myfinger and was too embarrassed tointerrupt the class, so I appliedpressure until the bell rang. Icarried ribbons of sections home inmy mother’s nylon stocking boxesand mounted them later ontoslides smeared with homemadeMayer’s albumen after floatingthem on water placed onto slides I

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had sitting on the slide warmer!With Sly and the Family Stone andthe latest Beatles 45 blasting onthe Hi-Fi upstairs in the den, Iprepared trichromes of guinea pigepididymis, kidney, and everythingelse, in my parent’s basement.Damn near burned out thelaundry sink with all thechemicals. The slides are still inuse at my high school thirty yearslater.

Early in my senior year, thepremed club visited thelaboratories of our local countyhospital, the Nassau CountyMedical Center. I told them of myexperience and asked them aboutemployment. I ended upvolunteering every day afterschool and all that summer. Istarted out preparing five-galloncarboys of phosphate bufferedformalin and decal solution for thegross rooms, cleaning cassettes,filing slides and blocks and othersuch scutwork. I embeddedautopsies in checkerboard grids ofaluminum squares. The surgicalswent into Tissue-Tek embeddingrings from rewashed steelcassettes, but I didn’t yet dosurgicals. I also dehydrated scoresof vials of vaginal cell suspensionswith amyl acetate for examinationunder the newly invented scanningelectron microscope, to elucidatechanges in the hormonal cycle anddisease.

My mentor in histology, DickSchroeder, knowing of myexperience with things tinctorial,gave me the method for the MovatPentachrome stain, told me thatthey wanted to use it for certainlesions and asked if I couldshorten it from 18 hours to one ortwo. I did, by substituting 1 hourBouin’s mordant instead of alonger time in Zenkers’s, and byusing an acid Orcein Verhoeff tostain nuclei and elastica in one-half hour, replacing both thepersnickety resorcin fuchsin elasticand Weigert hematoxylin steps ofthe original method. Heating somesolutions also sped up the process.

I published the result inHisto-Logic in 1972 under the lateLee Luna, who took me to taskuntil I could argue that mymodifications were either originalor referenced. I was 19 years old.During my senior year I loved thelab so much, I paid classmates todrive me to the hospital duringclasses – I played hooky. Thatsummer, I worked on largersurgicals, cleaned up the backlog of“ posts,” and learned special stainslike PAS, retic, etc. It was a sad daywhen I had to leave to attendColumbia College 20 miles away inNYC. But I soon got a part-timejob sectioning pig skin for theradiation research lab at NCMC.Man, a real job! There was also aten-bed hospital on the east endthat generated maybe 20 blocks aweek, and I did those also forthirty bucks or so on the weekends.It wasn’t long before my studiessuffered and I was on academicprobation for a year. I got a jobthrough an employment agency for$150 a week at Saint BarnabasHospital in the Bronx, commutingwith my next door neighbor in themorning who dropped me off atthe subway. I took the LIRR backhome, all for $150 a week. But itwas here that I really started tolearn pathology.

St. Barnabas was a small, leisurelypaced private hospital on a hill inthe Bronx, formerly known as theHome for Incurables, surroundedby tenements on three sides andLittle Italy (Arthur Avenue) onthe other. Boy, the lunches we usedto have on Arthur Avenue. For asmall place, we saw some amazingcases. This was the firstopportunity for me to regularly sitin on grossing, and it was for metantamount to beginning apathology residency that wouldlast for twenty years. I seem tohave a photographic memory forpathology and it wasn’t longbefore I became proficient inidentifying lesions, often resolvingfrozen section problems for thedocs after having just read about“ peritoneal gliomatosis secondary

to ovarian teratoma.” I voraciouslyread all of the journals and all ofthe textbooks in the office andbegan a slide collection.

I attended meetings of the NYCPathologists Club slide seminars asa guest of my pathologist. In 1973,after attaining the necessary twoyears of training and experiencenecessary to sit for the HT exam ofthe ASCP, I took and passed theexam. Collecting slides reallymultiplied my accumulation ofknowledge since I used to take ourrare cases back to NCMC to sharewith Dr. Vincent Palladino and hisresidents and attendings. His activetraining program had numerousconferences and received countlessdonations of wet tissue, rangingfrom classics to rarities, from all ofhis former residents. I used toprepare, for free, sections of all ofthese donations and specialconference cases, as well as hisconsults, to the relief of theoverworked histolab upstairs,where I had first trained. I wastheir “ pet histologist” and I didn’teven work there. I used to racethrough traffic from the Bronx toEast Meadow, Long Island to getthere before the chief left for theday.

In this way, I amassed an amazingcollection and saw many timeswhat most practicing pathologistsdo during their service rotation,because this went on everyday.Through Dr. Palladino, I servedvariously as seminar histo-technologist, preparing 3000 slidesof lymphomas one year, andprojectionist for slide seminars ofthe Nassau/Suffolk PathologySocieties, working with suchluminaries as Drs. LaurenAckerman and Robert Scully.I gave informal slide seminars forthe residents in the sign-out roomof all the materials I brought orprepared for Dr. Palladino.

Look for Part 2 of JeffreySilverman’s memoir in your nextissue of Histo-Logic.

19

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