more than just a mandatory exercise - sakura-
TRANSCRIPT
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. XXXVI, No. 1, May 2003
Human Resources at CollaborativeGroup, Ltd., a manufacturer ofpersonal care products. Previouslyhe served as Assistant Director forHospital Safety at the StateUniversity of New York at StonyBrook. He has published in TheNew England Journal of Medicine,contributed to book chapters, and isan adjunct instructor at Stony BrookDepartment of Health, Technologyand Management. He has over25 years of experience in the safetyand chemical hygiene business.When John was in charge of safetyat my former place of employment,he and his crew became notoriousfor things like wandering into alab and looking in the refrigeratorfor flammables, and calling inthe county bomb squad to removepicric acid.
Although it will never be noted onthat sign-out sheet passed around atthe end of the safety class, myexposure to safety dogma is actuallyfar more intensive than the annualmandatory session. You see,
More Than Just a Mandatory ExerciseRobert A. Skinner, HTL(ASCP)
Dana Gaddy, PhD, Associate ProfessorCenter for Orthopaedic Research, Dept. of Orthopaedic Surgery
University of Arkansas for Medical SciencesLittle Rock, AR
“Mandatory Safety Training Sessions.” Mandatory. Makes it sound likeyou have no choice but to attend. Well, I figured out that I actually hadchoices. I actually did not HAVE to go to a session. Of course, then Iwould be subject to suspension, thus not getting paid and potentiallyhaving to find other employment. Those were three potential options Ichose not to initiate, so I went. No session = no paycheck? Now there’san incentive. It’s amazing how the reading of that “deficiency memo”prompts you into finding that hour to attend a session.
I just attended my 26th mandatory annual safety session. So I’ve beenthrough more exaltations of “Don’t let the picric acid dry out or it couldexplode”; “Make sure the glassware is heat rated or it could explode”;“Don’t put chemicals like acetone in the refrigerator or they couldexplode”, than some of the session presenters. Since my anniversary dateis in 2 months I guess I’ll be attending two sessions in less than 4 monthsso statistically that should make me safer than most. John Marchese is asafety expert. He has a Master’s degree in Mechanical/EnvironmentalEngineering and currently serves as the executive director for Safety and
Managing Editor, Gilles LefebvreScientific Editor, Vinnie Della Speranza,
MS, HTL (ASCP) HT, MT
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IN THIS ISSUE
More Than Just a Mandatory Exercise……… 1
Remembrances of Opportunities Lost …… 4
Beware—Some Things You Don’t Want to Share…………………………… 6
A Method to Repair Freeze Artifactin Skeletal Muscle Biopsies……………… 10
Mercury’s Rising…………………………… 12
Enhanced Detection of Microorganisms in Tissue………………… 12
Tools to Facilitate and Standardize Grossing …………………… 17
A Technique for Correcting Poorly Processed Paraffin Blocks……… 21
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Mr. Marchese and I have beenfishing partners for nearly as longas we have been in our respectiveprofessions, and some of the safety-related things you learn while out onthe boat or answering an emergencypage are not readily available in asafety class. The one thing I’veappreciated from John over the yearsis his frustration in trying to reach anaudience that is highly skilled andhighly intelligent but often notterribly interested in the message.
When a wiseguy like me asks“where’s the refrigerator?” on theboat so I can “get even” for theacetone search, this prank sparksthe type of dialogue not normallyheard in a typical safety trainingsession. When I postulate in John’spresence that exploding picric acidmight be urban legend since nobodywe know has actually experienced itin the cumulative centuries ofexperience between us, his responsetypically ends with, “you know,people like you make my job moredifficult . . . .”
Despite the playful banter, I’vecome to realize that I’ve learned alot from him—a guy who does notview safety simply as a profession,but as a way of life. He’s bouncedlecture concepts off of me as asounding board before they were
delivered to an audience. I’vecritiqued the in-house videos thatwere produced to drive home thedangers of sharps handling. I’veheard the stories and statisticssupporting the dangers ofhousehold coffee makers in theworkplace to the point where thelast thing I do when I leave thehouse is make sure the coffeemaker is not only turned off butunplugged. Believe me, you canlearn a lot during a 30-minute runback to the dock.
So when the clock radio alarmsounded to the news on September5, 2002, informing the listeners ofCentral Arkansas that there hadbeen an overnight fire in one of theresearch buildings where I work,my first words to my wife were, “Ibet someone left a coffee maker onovernight and caused a fire.” I knewthe majority of the research staffworking on that floor. I knew theywere well trained. I knew they allwere supposed to have attended theannual mandatory safety session. Sothinking back to a conversationI once had with John who said,“coffee makers should be includedin every annual safety session,” mymoney was on this as the mostlikely cause of the fire at ourfacility. Little did I know whatreally caused it.
This photo depicts the aftermath ofwhat happens when approximately50 ml of isopentane is placed ina loosely capped 1-L bottle andthe bottle is placed in a freezer thatis not explosion-proof. Eventuallythe freezer thermostat will kick onwhen the isopentane vaporscontained in the freezer aresufficient to support ignition. It’sthe same basic thing that happensto gasoline vapors in a car enginecylinder ignited by the spark plug.Ignition: Blast Off.
It might suffice at this point tosimply say, “See what happens?Don’t do this,” but the forensicsbehind the event are interesting.Isopentane (2-methyl butane) iscommonly used to flash freezebiologic specimens. I have usedthe reagent in this manner for overa decade to snap freeze musclebiopsies for enzyme histochemistryand immuno workups in severalwork settings without incident.
The placement of the looselycapped bottle of remnantisopentane into that particularfreezer was at the behest of anexperienced PhD-level researcher,who had stored isopentane infreezers in a similar mannernumerous times over 15 yearsin other settings without incident.This was not the act of anuninformed novice technician orsome volunteer student helper. Infact, some thought actually wentinto the process. Putting theisopentane in the freezer wouldsave the time that would otherwisebe needed to cool it from roomtemperature all the way downto the requisite temperature(approximately -160°C). Also, byleaving the lid ajar, vapor pressurebuildup sufficient to break the capor the bottle could be avoided. Andsince this was done successfullynumerous times before, what reasonwould there be to think that thisparticular time would lead to suchan explosive outcome?
It was subsequently learned thatthe investigator had worked in adifferent facility with brand-new
equipment. The freezer in the priorfacility was an explosion-proof unit.The freezer in our facility was not.A number of important featuresdistinguish explosion-proof freezersfrom common household units. Inexplosion-proof units, the controlsand electrical components thatcould potentially cause a spark(such as motors and condensers) arelocated outside the cold box or areinsulated from the interioratmosphere of the storagechamber.1 Some units havecompressors and other electricalcomponents mounted on top toavoid heavier-than-air vapors thatcan accumulate along the floor.Price is another distinguishingcharacteristic. One can purchase ahousehold 20-cu-ft. upright freezerfor under $500.00, while anexplosion-proof unit of the samesize will cost over $2000.00. In thisinstance, the investigator did nottake into consideration theconstruction of the freezer despitethe label above the door handledesignating the unit as “NotSuitable For Flammable Liquids.”2
The Material Safety Data Sheetfor isopentane specifies that itsflash point is -51°C,3-5 which is bydefinition “the minimumtemperature at which a liquid givesoff vapor in a test vessel sufficientto form an ignitable mixture withair near the surface of the liquid.”One might anticipate that as thetemperature continues to climbpast the -51°C mark, continuedvaporization might cause sufficientpressure buildup to warrant leavingthe bottle cap ajar. But a far moresignificant factor can also begleaned from the MSDS: “Vapor isheavier than air and may travelalong ground; distant ignition ispossible.”3,4
The compressor and motor onthis freezer were located underthe storage box, however, thethermostat was located insidethe storage box. This compoundsthe possibilities, but the fact thatthe freezer door detached andjettisoned some 12 feet from the
freezer leads one to believe thatignition was probably caused bythe interior thermostat after therequisite accumulation of vapors.It was a big bang for such a smallvolume of liquid.
The cost accounting, includingreplacement of equipment, books,and reagents, is approximately$165,000.00. This does not take intoaccount the facility cleanup andreconstruction, water damage to thefloors below, loss of productivity,loss of data, and potential loss offuture funding due to misseddeadlines. It could be a year beforethe actual complete loss total isrealized and the final numbers willfar exceed the quarter milliondollar mark.
In summary, an experienced PhDresearcher, in an effort to shorten aprocedure time by about 2 minutesand to reuse approximately 100 mlof reagent (value approximately$2.00), places a volume offlammable liquid into a looselycapped bottle and stores it in aregular household freezer. Theliquid gases off into the confines of the freezer and at 3:50 AM when thethermostat clicks on to start thecompressor motor, a spark from thethermostat ignites the collected
vapors and blows the door offthe freezer. The blast severs acompressed air line which in effectcreates a “flamethrower across thelab,”according to the firefighters atthe scene, ultimately costing theinstitution a huge amount ofmoney. But, if you think about it,it could have been a lot worse.Suppose the thermostat had clickedon at 3:50 in the afternoon whilepeople were working at thebenches across from the freezer.Even worse, a spark from thefreezer’s interior light could haveignited the vapors when the freezerdoor was opened by lab personnel.The potential for loss of life wassubstantial.
Needless to say, this incidentprompted a campuswidemobilization to check for potentiallyflammable materials located in non-rated refrigeration units and forgood reason. Chilled acetone iscommonly used for a variety oflaboratory procedures. We all needto maintain vigilance in dealing withthe storage of flammables. Far toooften we hear the warningsregarding these reagents once a yearat the mandatory safety trainingsession and then file them in ourmental recesses because we don’tthink we’ll have immediate use for
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the information. It’s only after anevent like ours that people areprompted to check the following:
Are your refrigeration units(refrigerator and freezer) explosion-proof or rated for the storage offlammables?
If your units are not rated for thisapplication, is there anything inthem that shouldn’t be there?
Are your non-rated refrigerationunits clearly labeled as notapproved for the storage offlammable liquids? Are youmaintaining the necessary vigilanceto heed the warnings?
Have you familiarized yourself withthe MSDS for the materials you areworking with? Do you have theinformation necessary to properlyand confidently work with thesetypes of materials?
AcknowledgmentsThe authors wish to extend their appreciation to Mr.John Marchese, whose knowledge and relentless pursuitof safety in the workplace provided the inspiration forthis report.The authors wish to thank Larry J. Suva, PhD, andRichard W. Nicholas, MD, for their photographiccontributions.
References1. The purchase and use of laboratory refrigerators &
freezers with flammable liquids, University ofCalifornia at San Francisco. Available at:http://www.ehs.ucsf.edu.
2. Safe storage of laboratory chemicals,Texas A&M University. Available at:http://safety.science.tamu.edu/chemstorage.html.
3. Isopentane. Available at: http://www.inchem.org.4. 2-methyl butane, MSDS. Available at:
http://physchem.ox.ac.uk/MSDS/ME/2-methylbutane.html.
5. Isopentane, MSDS. Available at:http://msds.ehs.cornell.edu/msds/msdsdod/a195/m97379.htm.
Additional Sources of Information1. Occupational exposure to hazardous chemicals in
laboratories, Occupational Safety & HealthAdministration. U.S. Department of Labor.29 CFR 1910.1450. Available at:http://www.osha.gov/pls/oshaweb/owadisp.showdocument?p table=STANDARDS&p id=10106&p textversion=FALSE.
2. National Research Council RecommendationsConcerning Chemical Hygiene in Laboratories (Non-Mandatory) — 1910.1450 App A. Available at:http://www.osha.gov/pls/oshaweb/owadisp.showdocument?p table=STANDARDS&p id=10107&p textversion=FALSE.
Remembrances ofOpportunities Lost
Jules M. Elias, PhDProfessor Emeritus, HSC
Stony Brook, SUNYVisiting Professor, College of
Naturopathic MedicinePortland, OR
In the early 1960s, after completingmy Master’s in Hematology, I wentto work at the Brookhaven NationalLaboratories in Upton, New York.I spent over 6 hours a day cuttingserial sections of Bouin’s-fixedmouse spleens that displayedmacroscopic nodules derived fromstem cells of normal mouse bonemarrow. The goal of the seniorscientists was to reconstruct thespleen at the microscopic level todetermine its hemodynamics (openor closed system). To keep mysanity, I offered to do cytogenetickaryotyping of bone marrowaspirates obtained from patients inthe leukemia outpatient clinic. Thetechnique, developed by Nowell andHungerford in the 1940s, was a wayto induce peripheral bloodlymphocytes to dedifferentiate andenter the cell cycle. After a 3-daygrowth in culture, the cells weretreated with colcemid to arrest themin mitosis. The cells weresubsequently harvested, andconventional air-dried preparationswere stained with a standardGiemsa solution obtained from thehematology laboratory.Photomicrographs taken of cellscaught in metaphase were enlargedsufficiently to allow cutting out ofhomologous chromosomes toprepare a karyotype of a patient’sblood. The goal was to find thePhiladelphia chromosome, whichwas thought to be chromosome 21with a deletion of its long arm,pathognomonic for chronic myeloidleukemia.
After many months of karyotypingI discovered a problem with theGiemsa staining. Instead of
obtaining the uniform deep blue-black staining that facilitatedphotomicrography, I was gettingdark and light ribbon-like staining.When I checked the pH of theGiemsa, I determined that theoriginal batch of stain obtainedfrom the hematology lab hadbecome alkaline. I promptlydiscarded that batch, obtained freshGiemsa, and restained thepreparations to obtain the expectedresult. All future batches of Giemsawere routinely pH checked to avoidthis aberrant result.
Approximately 6 months later, anarticle appeared in the prestigiousresearch journal Nature thatdescribed the use of an “alkalineGiemsa” method to produce bandstaining of metaphasechromosomes. These investigatorsdetermined that homologouschromosomes display identicalbanding patterns, which greatlyenhanced the ability to discerntranslocations and deletions ofportions of chromosomes. In fact,later researchers using the alkalineGiemsa technique determined thatthe Philadelphia chromosome wasnot chromosome 21, as wasoriginally thought, but chromosome22 that had lost portions of the longarm! I was annoyed with myself fornot having the insight to take whatlooked like a lab error and explorewhat a fortuitous change in pHcould do to improve chromosomeanalysis.
A decade later while employed atStony Brook Health SciencesCenter, I routinely scored paraffinsections of breast tissuesimmunostained forestrogen/progesterone receptors(ER/PgR-ICA). Since Long Islandis considered to be one of fourregions where the incidence ofbreast cancer exceeds the nationalaverage, the opportunity to observethe rare cases displayingcytoplasmic receptor staininginstead of the expected nuclearstaining presented itself. Becausethis pattern of staining had not beendescribed as being biologically
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significant and did not meet thecriteria for being scored as apositive result, these cases werescored as negative. Moreover, caseswith cytoplasmic staining were notfollowed to determine if thisvariation had any clinical value.Parenthetically, a major criticism ofthe ER/PgR-ICA was that it did notmeasure biochemical functionalityof the receptor and, therefore, didnot unequivocally select the patientswho would benefit from some formof endocrine therapy. In essence, theER/PgR by IHC was a static assay.To overcome this limitation, theimmunohistochemicaldemonstration of pS2, a proteinproduct that was intimatelyassociated with steroid receptorfunction, was performed. Wesubsequently published a reportthat validated the value of pS2 withregard to ER/PgR functionality;those patients with positivecytoplasmic staining were morelikely to score a high positive forboth ER and PgR.
Decades later a report entitled,“Cytoplasmic localization of wild-type p53 in glioblastomas
correlates with expression ofvimentin and glial fibrillary acidicprotein” (Neuro-Oncology, July2002), recalled my experience withcases that were scored ER-ICAnegative because of cytoplasmicstaining. These investigatorsdetermined that the cytoplasmicaccumulation of wild-type p53 intumor cells indicates that the tumorsuppressor gene is inactive withregard to growth-repressivefunctions. Their study suggestedthat vimentin expression is a strongindicator for the cytoplasmicexpression of p53 in tumors thatalso express GFAP. Again, mybiological frustration level wastweaked as there were isolatedreports that vimentin expression inbreast tumors was indicative of apoor prognosis.
These experiences are a reminderof how often we choose to ignorethose laboratory results that do notmeet the established standard andthus, missing potentially importantobservations. James Watson andFrancis Crick, who discovered theduplex structure of DNA, personifythe ultimate seekers of phenomena
that others chose to ignore,consciously or otherwise. Theyvisited the laboratories of severalkey DNA researchers and analyzedtheir data. From this heterogeneousdatabase, Watson and Crick wereable to synthesize a model of DNAstructure that still stands. By beingreceptive to the potential in thework of other equally brilliantscientists who did not see the bigpicture and consequently missedthe opportunity to make asignificant scientific contribution,Watson and Crick have gained apermanent place in science history.
The scientific literature is repletewith experiments that did notproduce expected results, and thosesensitive enough not to discard datathat do not produce the expectedoutcome seem to make the leapsthat contribute to the advancementof science and medicine. SherlockHolmes knew this in his earlycareer as a research chemist whenhe remarked that a fellow scientistwould never succeed in hisexperiments because he saw onlywhat he expected to see. Progresscomes from those who see beyondwhat we expect to see.
Having said all that, if I were stillactive in the field, I would take theinteresting opportunity todetermine if cytoplasmic ER-ICAstaining is indeed indicative of anonfunctioning estrogen receptor.A call for all ER-ICA casesdisplaying this unexpected patternwould provide enough material fora retrospective study ofimmunostaining for pS2. Defyingthe great Sherlock, I predict thatthe majority of cases will lack pS2or have only weak cytoplasmicstaining. Thus, we could deduce thefunctional status of the estrogenreceptor without having to performmolecular analysis. I can’t do it, so Ioffer the suggestion to some futureresearcher looking to make asignificant contribution to theadvancement of knowledge.
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Fig. 1. Estrogen receptor staining in human breast by immunohistochemistry most commonly results in a nuclearstaining pattern. 200X. Photo courtesy of John Metcalf, MD, Medical University of South Carolina.
Beware—SomeThings You Don’t
Want to ShareJane Chladny, HT (ASCP)
University of IllinoisCollege of Veterinary Medicine
Urbana, [email protected]
Maureen Doran, BS, HTL (ASCP)School of Medicine
Southern Illinois UniversityCarbondale, IL
Laboratory personnel working inhuman or veterinary medicine areat risk of exposure to a variety ofinfectious agents. Workers mustprotect themselves from age-oldinfectious diseases like tuberculosis,emerging diseases such as West Nilevirus, and agents used forbioterrorism. Safe handling ofinfectious agents requires early riskassessment. Knowledge of thenumber of laboratory infections,route of transmission, infectiousdose, incubation time prior to theonset of symptoms, and severity ofthe disease provides the informationneeded to develop appropriatetechniques and equipmentsafeguards.
In 1949, Sulkin and Pike publishedthe first in a series of surveys aboutlaboratory-associated infectionssummarizing 222 viral infections,21 of which were fatal.1 In at leastone-third of the cases, the source ofinfection was considered to beassociated with the handling ofinfected animals and tissues.Accidents were recorded in only12% of the reported cases.2 By 1976,data indicated that, of the nearly4000 cases of laboratory-associatedinfections, fewer than 20% wereassociated with a known accident.3
Tuberculosis (TB), one of the mostwidespread infectious diseases, maybe encountered when working withhuman and animal samples. Itremains the most common cause of
death among adults worldwide,killing 2 to 3 million people eachyear. All known mycobacterialpathogens of animals,Mycobacterium bovis being themost common, are transmissibleto humans. Laboratory-acquiredmycobacterial infection has beendocumented and there is directcorrelation between the frequencyof infection in laboratory workersand the number of positive sampleshandled. Medical personnel are atrisk of acquiring TB in the courseof their work through the inhalationof contaminated droplet nuclei.Infected tissue specimens orcultures have the potential to createaerosols in the laboratory and atautopsy or necropsy. Persons inenvironments in which theinhalation of infected aerosol ispossible should wear appropriaterespiratory protective devices. In1978, two pathologists in Californiaand a histotechnologist fromanother hospital seroconvertedpositive for TB after being presentwhen frozen sections were takenfrom a lung infected with TB. Thetissue block was cooled in thecryostat with a compressed gascoolant, which created a heavyaerosol.4,5
As stated in the December 2002issue of HistoLogic, there havebeen reports that Mycobacteriumtuberculosis has been culturedfrom surgical and autopsy tissuesafter weeks of formalin fixation.6
Until there are firm data thatunequivocally prove that theorganism cannot retain viabilityin tissues after formalin fixation,added precautions are warrantedwhen working with tissuescontaining this agent.
Zoonotic diseases and infections arenaturally transmissible fromvertebrates to humans. Up to 200diseases have been identified asbeing shared between man andanimals. These diseases or infectionsmay be asymptomatic and trivial inanimals but have serious or deadlyresults in humans. The death ofElizabeth Griffin, a 22-year-old
animal caretaker at EmoryUniversity, is a distressing exampleof the potential danger of a zoonoticdisease. Ms. Griffin developedocular inflammation 2 weeks aftera splash of an unknown substanceto her eye while working withmacaques. She sought treatmentat a university facility and wasdischarged. Four weeks later shesuccumbed to the B-virus infection.7
OSHA cited the primate researchcenter with a willful, seriousviolation and proposed penaltiesof over $100,000 for the death ofan employee.8 Herpesvirus simiae(B-virus) is a member of the herpesgroup of viruses that is common inold-world monkeys of the macaquegenus. The disease, which is similarto cold sores in humans, causes mildor inapparent infection in monkeys;however, infections in humans canbe fatal. B-virus disease in humansis characterized by a variety ofsymptoms that include vesicularskin lesions at or near the site ofexposure, localized neurologicalsymptoms, and encephalitis.Laboratory personnel having directcontact with macaques are at thegreatest risk of infection.Transmission to humans usuallyoccurs via exposure tocontaminated monkey salivathrough bites, scratches, andsplashing.
Between 1950 and 1960, two-thirdsof occupational infections with B-virus were related to polio vaccinetesting on macaques. Recent casesoften coincide with use of macaquesin retroviral research. One case ofB-virus infection occurred followinga technician’s exposure tocontaminated cell cultures of simianorigin.9 Laboratory workers whohave the potential for exposure tomacaques and macaque tissuesshould be properly trained and wearpersonal protective equipment(PPE) as recommended. Ms.Griffin’s death would have beenprevented if she had simply beenwearing goggles.
Emerging diseases pose aparticular risk because of the
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unknown course of the newdisease. West Nile virus (WNV) isa disease relatively new to theUnited States. The first reportedoutbreak occurred in New York inlate 1999. By 2002, only 3 yearslater, West Nile virus was reportedin 44 states. In Illinois alone, it wasreported that 778 people becameill and 52 died from the disease.There were 4007 laboratory-positive human cases reportednationwide to the CDC/Arbonetas of January 29, 2003.10
Most West Nile virus infections inhumans are subclinical. Overtdisease is estimated to occur inapproximately 1 in every 100infections. Mild cases of West Nilevirus infections may cause a slightfever or headache. Those withmore severe infections may exhibita rapid onset of a high fever withhead and body aches, disorien-tation, tremors, convulsions, andpossibly paralysis and death.Symptoms occur 3 to 14 days aftera bite from an infected mosquito.Persons 50 years of age or olderare at greatest risk for developingcomplications from naturalinfection. In animals, the greatestnumber of deaths has occurred inbirds and horses. There is apreventative vaccine now availablefor horses.
There is documentation that WNVwas passed from one person toanother through blood transfusionand organ donation.11 The youngestperson infected with WNV was ababy reportedly infected bydrinking breast milk from themother who became ill and testedpositive for WNV after receivinga blood transfusion. Althoughthe baby has remained healthy,a blood sample from the infantdemonstrated IgM antibodies tothe WNV, an indication that theinfant had been infected.12
The degree of risk to postmortemworkers from ill or dead animalsand humans is undetermined at thistime. West Nile virus is groupedwith most of the arboviruses known
to have caused lab-acquiredinfections. There are twodocumented cases of laboratory-acquired WNV from a scalpel cutand a contaminated needlepuncture and one case of a personworking with WNV who developedneutralizing antibodies withoutclinical symptoms.13 The CDCconsiders WNV a Biosafety Level(BSL) 3 agent. However, becausemany labs are not equipped forBSL 3 practices, BSL 2 proceduresare acceptable if modifications areapproved by the laboratory director.Modifications are as follows:exhaust air is discharged outdoors,access is restricted when work is inprogress, ventilation is balanced toprovide directional airflow into theroom, and recommended standardmicrobiological practices and safetyequipment regulations for BSL 3are rigorously followed.14
Just as emerging diseases are ofconcern to healthcare andlaboratory workers, so are diseasescaused by agents that have noknown effective treatment andare resistant to conventionalinactivation procedures.Transmissible SpongiformEncephalopathies (TSEs) arecaused by prions, proteinaceousparticles smaller than the smallestvirus. TSEs are fatal, degenerativediseases affecting the centralnervous system. Prion diseases, orTSEs, include Creutzfeldt-JakobDisease (CJD) and variant CJD,BSE, scrapie, transmissible minkencephalopathy, feline spongiformencephalopathy, chronic wastingdisease (CWD) in deer and elk,kuru, Gerstmann-Straussler-Scheinker syndrome (GSS), andfatal familial insomnia (FFI).15
Animal prion diseases, except BSE,are not regarded as a public healththreat, but all TSEs should behandled with care.
The highest concentration of prionsis in CNS tissues and theircoverings. High concentrations arealso suspected in the spleen, thymus,lymph nodes, and lung.15 Formalinfixation inactivates most disease
agents, however with prions, tissuesthat are formalin fixed and paraffinembedded remain infectious.
The efficiency of disinfectants ininactivating prions is unclear. Someexperts recommend that equipmentand surfaces may be decontaminatedwith 2N sodium hydroxide over a1-hour period.15 The followingprocedure substantially reduced theinfectivity levels in brain tissue ofrodents infected with CJD, BSE, andscrapie agents. Tissues, well-fixed informalin, were placed in 98% formicacid for 1 hour then rinsed well inH2O and returned to formalinbefore processing.16
Prosectors should wear cut-resistantgloves to avoid puncture of the skin.If accidental contamination of skinoccurs, swab the area with 1Nsodium hydroxide for 5 minutes,then wash with copious amounts ofwater.15 Gloves should be wornwhen handling and sectioningtissues. Dispose gloves, paraffinshavings, and wipers used to cleanthe surface of the water bath into abiohazard bag for incineration. Allresidues, liquid and solid, fromprocessing, embedding, and stainingin the laboratory should bediscarded into a leak-proofbiohazard bag for incineration.17-19
Liquid waste may be collected in a4-L waste bottle containing 600 mlof 6N sodium hydroxide.15
It is believed that ingestion is themost common route of exposure forprion disease, but CJD has beenspread by way of surgicalprocedures (grafts) and even byusing a surgical suite that was notproperly decontaminated after aprocedure on a CJD patient.20, 21
Until recently, laboratory workershave given little thought to agentsused in bioterrorism. After theanthrax scare of 2001, there isgreater chance that laboratoriesmay encounter the organism. ATexas laboratory worker who wastesting specimens from the 2001attack contracted the cutaneousform of the disease, and last year
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a worker in the anthrax research labat the United States Army MedicalResearch Institute of InfectiousDisease (USAMRID) testedpositive for anthrax exposure.The CDC states that these casesdemonstrate the importance ofsafety procedures and vaccinationfor lab workers who routinelyhandle anthrax.22, 23
There are three forms of thedisease: cutaneous, gastrointestinal,and inhalation. If untreated, all canlead to septicemia and death. Of thethree, the inhalation route is themost severe. The organism is mostlikely to reach the bloodstream anddisseminate toxin throughout thebody. If untreated, the fatality rateby the inhalation route is90%-100%, as opposed to thecutaneous route, which is 5%-20%.Antibiotic therapy (penicillin) isoften effective if administered earlyor used as prophylaxis.24, 25
Naturally occurring anthraxendospores remain viable in soil fordecades. In 1996 a cow in Ontariodied from anthrax. The ownerreported that cows had died ofanthrax on the same farm 65 yearspreviously. Recent excavations inthat area unearthed the viableorganism. Similarly, anthraxoccurred in cattle in South Dakotaand New Mexico following oilcompany drilling and earth work in1997 and 1998.26
Postmortem dissection should notbe performed on animals withsuspected anthrax. Exposure to aircauses the vegetative form in thecarcass to form stable endospores.If a veterinarian suspects anthrax,he should submit the tip of an earto minimize environmentalcontamination and exposure to thepublic and laboratory personnel.Blood should be collected with aslittle contact as possible. Allinstruments and materials shouldbe autoclaved, incinerated, orchemically disinfected aftercontamination with anthrax.27,28
Workers who are motivated toachieve a safe working environment
and who are provided with accurateinformation can establish adequatebiosafety controls. Diligence in theapplication of safe standardoperational procedures andpreventative measures is required todecrease the incidence and severityof laboratory-acquired infections.The hurried, sloppy, absent-minded,unconcerned technician will createthe maximum hazard. The cautious,skilled, conscientious, and attentivetechnician will create minimalhazard. The knowledge, thetechniques, and the safetyequipment necessary to preventmost laboratory-acquired infectionsare available. It is the responsibilityof the laboratory professional toaccept the importance of a safeworking environment.
References1. Sulkin SE, Pike RM. Survey of laboratory-acquired
infections. Am J Public Health. 1951;41(7):769-781.2. Pike RM. Laboratory-associated infections: summary
and analysis of 3921 cases. Health Lab Sci.1976;13(2):105-114.
3. Pike RM. Past and present hazards of working withinfectious agents. Arch Pathol Lab Med.1978;102:333-336.
4. Tuberculosis infection associated with tissueprocessing—California. MMWR. 1981;30(6):73-74.
5. Tuberculosis infection from preparation of frozensections. N Engl J Med. 1981;305(3):167.
6. Della Speranza V, Richardson M. Does formaldehydekill Mycobacterium tuberculosis, HistoLogic.2002;35(2):37-38.
7. Fatal Cercopithecine herpesvirus 1 (B virus) infectionfollowing a mucocutaneous exposure and interimrecommendations for worker protection. MMWR.1998;47(49):1073-1076, 1083.
8. Lamborn C. OSHA and Yerkes reach settlement overemployee death. Lab Animal. 1999;28(2):11.
9. Wells DL, Lipper SL, Hilliard JK, et al. Herpesvirussimiae contamination of primary Rhesus monkeykidney cell cultures. Centers for Disease Controlrecommendations to minimize risks to laboratorypersonnel. Diagn Microbiol Infect Dis. 1989;12:333-335.
10. CDC Office of Communication, West Nile virusupdate. Available at:http://www.cdc.gov/od/oc/media/index.htm. Accessed2003.
11. Garmendia AE, Van Kruiningen HJ, French RA. TheWest Nile virus: its recent emergence in NorthAmerica. Microbes Infect. 2001;3(3):223-229.
12. CDC. West Nile virus activity—United States:investigations of the West Nile virus infections inrecipients of blood transfusion and organtransplantation. MMWR. 2002;51:884, 895.
13. The Subcommittee on Arbovirus Laboratory Safetyof the American Committee on Arthropod-BorneViruses. Scherer WF, Head. Laboratory safety forarboviruses and certain other viruses of vertebrates.Am J Trop Med Hyg. 1980;29(6):1359-1381.
14. Epidemic/Epizootic West Nile Virus in theUnited States: Revised Guidelines for Surveillance,Prevention, and Control. CDC workshop.Fort Collins, CO; April 2001.
15. CDC Biosafety in Microbiological and BiomedicalLabs. 4th ed. US Department of Health and HumanServices; May 1999:135-143.
16. Brown P, Wolff A, Gajdusek DC. A simple andeffective method for inactivating virus infectivity informalin-fixed tissue samples from patients withCreutzfeldt-Jakob Disease. Neurology. 1990;40:887-890.
17. Advisory Committee on Dangerous Pathogens.Precautions for work with human and animaltransmissible spongiform encephalopathies. GreatBritain: HMSO;1994.
18. Infection Control Guidelines for TransmissibleSpongiform Encephalopathies. United Kingdom:World Health Organization; March 20, 1996.
19. Budka H, Aguzzi A, Brown P, et al. Tissue handling insuspected Creutzfeldt-Jakob Disease (CJD) and otherhuman spongiform encephalopathies (prion diseases).Brain Pathol. 1995;5:319-322.
20. Thadani V, Penar PL, Partington J, et al. Creutzfeldt-Jakob disease probably acquired from cadaveric duramater graft. J Neurosurg. 1988; 69:766-769.
21. Bernoulli C, Siegfried J, Baumgartner G, et al. Dangerof accidental person to person transmission ofCreutzfeldt-Jakob disease by surgery. Lancet.1977;1(8009):478-479.
22. Update: Investigation of anthrax associated withintentional exposure and interim public healthguidelines. MMWR. 2001;50(41):889-893.
23. Anthrax escapes lab room at USAMRID; workerexposed. CIDRAP. April 23, 2002.
24. Collier J, Young J. Attacking anthrax. Sci Am.2002;286(3):48-50, 54-59.
25. Dixon TC, Meselson M, Guillemin J, Hanna PC.Anthrax. N Engl J Med. 1999;341(11):815-826.
26. Human anthrax associated with an epizootic amonglivestock—North Dakota, 2000. MMWR.2001;50(32):677-680.
27. CDC protocol for anthrax cases. CAP Today.2001;86-87.
28. Turnbull PCB. Guidelines for the Surveillance andControl of Anthrax in Human and Animals. 3rd ed.World Health Organization, Deptartment ofCommunicable Diseases Surveillance and Response.1998.
Additional Sources Acha PN, Szyfres B. Zoonoses and CommunicableDiseases Common to Man and Animals. 2nd ed.Washington, DC: Pan American Health Organization.Pan American Sanitary Bureau, Regional Office of theWorld Health Organization; 1987.
Miller CD, Songer JR, Sullivan JF. A twenty-five yearreview of laboratory-acquired human infections at theNational Animal Disease Center. Am Ind Hyg Assoc J.1987;48(3):271-275.
Pike RM. Laboratory-associated infections: incidence,fatalities, causes, and prevention. Annu Rev Microbiol.1979;33:41-66.
Prescott LM, Harely JH, Klein DA. Microbiology.Dubuque, Iowa: Wm. C. Brown Publishers; 1990.
Web SitesBiological MSDS. Available at:http://www.hc-sc.gc.ca/pphb-dgspsp/msds-ftss/index.html.
Centers for Disease Control and Prevention (CDC).Available at:http://www.cdc.gov/health/, andhttp://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm.
Chronic Wasting Disease. Available at:http://www.dfg.ca.gov/hunting/wasting.html.
New Zoonoses Homepage (ed: PHLS ZoonosesAdvisory Group). Available at:http://www.cdsc.wales.nhs.uk/zoo.htm.
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A Method to RepairFreeze Artifact inSkeletal Muscle
BiopsiesRena Fail, HT (ASCP)Vinnie Della Speranza
MS, HTL(ASCP)Medical University of South Carolina
Charleston, [email protected]
AbstractEnzyme histochemistry is anessential component of thediagnostic evaluation of skeletalmuscle biopsies.1 Muscle enzymesare demonstrated in fresh frozenmuscle that is ideally free of artifactto facilitate microscopic evaluation.Muscle specimens should bereceived in the laboratory freshfor rapid freezing, but far toofrequently we receive specimensfrom outside hospitals that aresubmitted incorrectly, eitherfloating in saline or having alreadybeen frozen in OCTTM Compoundat the point of origin prior toshipment. In these instances weoften find ourselves working withsamples that are obscured withfreeze artifact. We exploredtechniques to improve samples thatwould otherwise be unreadable.
IntroductionMany small hospitals are ill-equipped for snap freezing whichrequires access to liquid nitrogenor dry ice. For this reason, weinstruct our outside clients to sendtwo samples of muscle, one thatis clamped in a rayport biopsyclamp and immersed in4% paraformaldehyde for lightand electron microscopy studies.The second piece is requestedfresh, to be placed onto a saline-dampened piece of gauze and putin a biohazard bag that is shippedon ice either the same day orovernight. Inevitably, some clientsfail to follow our instructions,leaving us with a compromisedsample (see Fig. 1).
Slow freezing, which may includefreezing samples inside of acryostat or other low-temperaturefreezer will typically yield a samplethat is riddled with freeze artifact,causing gross distortion of thetissue.
Rapid freezing at ultracoldtemperature is imperative to yieldan optimal sample for diagnosis.2
Underlying disease processes can
either be masked by ice crystalartifact or the artifact itself canmimic a disease process, confusingthe diagnosis, e.g., lipid storagemyopathies. Freezing artifact alsomakes it very difficult for thepathologist to assess fiber types.If a muscle is full of vacuoles, thepathologist may order stains forlipid and glycogen in order todetermine if the vacuoles are a partof the pathology or artifact. Some
Fig. 1. Skeletal muscle sample riddled with ice crystal artifact resulting from inadequate freezing technique.H&E stain. 100X
Fig. 2. Skeletal muscle sample thawed and refrozen to correct for prior ice crystal artifact. H&E stain. 100X
10
instances of freeze artifact may beso severe that a definitive diagnosiscannot be made from the availablematerial. For those cases it is bestto thaw the tissue in cold normalsaline and refreeze. Often this willlead to a vast improvement inmorphology and little if any loss ofenzyme reactivity (see Fig. 2).
MethodWe prefer to mount the musclespecimen protruding out of a gumtragacanth-supporting mediumwhich provides a firmer hold thanOCTTM and will effectively hold themuscle in the correct orientationduring freezing. To prepare themuscle for this technique it isnecessary to remove as much of thegum tragacanth as possible fromthe initially frozen block by cuttingit away from the specimen with asharp blade. Once this is achieved,place the muscle in cold (5-7°C)normal saline for a period of30 minutes. We believe that thesalt content of the saline aids indrawing away the excess watermolecules from the muscle fibersthat formed the ice crystals duringthe initial freezing of the sample. Itis important to blot the specimenthoroughly after removing it fromthe saline. You can use anyabsorbent pad, or several layers ofpaper towels. This will effectivelyeliminate the excess moisture onthe exterior of the sample which isnow ready to be refrozen.
Method for Snap Freezing TissuesYou will need the followingmaterials and equipment:
• 10% gum tragacanth• 2-methyl butane (isopentane)• Liquid nitrogen• Cork square approximately 2x2 cm• 300 ml beaker• Small styrofoam container• Long forceps• -70°C freezer• Ultralow-temperature
thermometer
1. Using a tongue blade or flatspatula, place a mound of gumtragacanth approximately 1 cmhigh on the cork.
2. With a small spatula, open a smallhole in the center of the moundabout 3 mm deep.
3. Carefully place the musclespecimen on top of the gumtragacanth, orienting the tissueso that a cross section of musclefibers can be obtained. Avoidgetting the gum material onto thesurfaces that will be sectioned asit will not cut. Very gently pushthe base of the specimen into thegum tragacanth about 1-2 mm.
4. A marking pen may be used towrite the specimen number on asmall strip cut from a file card.Slip one end of the strip into thegum tragacanth against the cork,taking care to leave the numberexposed from the gum.
5. Immerse a beaker containing 2-methyl butane into an insulatedcontainer of liquid nitrogen. Caremust be exercised to establish thecorrect amount of liquid nitrogen,taking into consideration that thebeaker of 2-methyl butane willdisplace the liquid nitrogen.Ideally, the nitrogen will risesufficiently to surround thebeaker which will accelerate
cooling. However, do not allowany liquid nitrogen to overflowinto the beaker and contaminatethe 2-methyl butane. If thisoccurs, you need to get a freshbeaker of 2-methyl butane andstart again. Cool the 2-methylbutane to at least -150°C,verifying with a low-temperaturethermometer. If liquid nitrogenis not available, dry ice may besubstituted to cool the 2-methylbutane if a thermometer is usedto monitor the temperature.
6. Pick up the block with longforceps by the edge of the cork;turn block upside down andplunge it into the beaker of2-methyl butane for 30 seconds(longer if the specimen is thick),swirling the block while freezing.Transfer the block to a -70°Cfreezer until you are ready tosection.
7. Affix the cork base to a cryostatchuck with OCTTM for sectioning.
8. Cut enough sections to performthe required stains and enzymes.
ConclusionThis simple method has beenreliable in our hands and is useful toremove or reduce ice crystal artifactthat has rendered a musclespecimen uninterpretable. It isreproducible and relatively easy toperform if sufficient care isexercised to conduct refreezing atultralow temperature.
AcknowledgmentThe authors wish to thank Robert A. Skinner, BS,HTL(ASCP), University of Arkansas for MedicalSciences, for his advice and insights into the use ofthis technique.
ReferencesBancroft S, Stevens S. Theory and Practice of HistologicalTechniques. 3rd ed. Churchill Livingstone; 1990:84.Dubowitz V. Muscle Biopsy: A Practical Approach.2nd ed. Bailliere Tindall; 1985:623.
11
SAFETYPRECAUTIONS:
2-methyl butane is highlyflammable and harmfulif swallowed or inhaled.It affects the centralnervous system, causesirritation to skin, eyes,and respiratory tract.Contact with liquid nitrogencan cause frostbite. Avoidinhalation, wear cold-insulating gloves, face shieldor eye protection.
12
Mercury’s RisingVinnie Della Speranza, MS,
HTL(ASCP)Medical University of South Carolina
Charleston, [email protected]
The shipping of pathology cases,notably slides and paraffin blocksvia the postal service, is a commontask for many of us. Even in anage when computer electronicsmake the rapid transmittal ofmicroscopic images over theinternet simple and convenient,most consulting pathologistsprefer to receive the slidesthemselves, wishing not to belimited to only those microscopicfields offered electronically by thereferring physician. We alsoencourage referring facilities toinclude the paraffin blocks withany cases they send forconsultation to give us maximumflexibility to render the diagnosisin the shortest time possible. Theneed for additional or repeatstaining of outside cases that havebeen fixed or processed at theirpoint of origin with protocolsunfamiliar to us cannot be
anticipated in advance, and attimes, are critical to thecompletion of the case.
Those of us who are charged withthe responsibility of packagingpatient materials for shipmentprobably exercise greater care withthe glass slides which we know to befragile. This is understandable asexperience has taught us that evenan intact parcel on arrival is noguarantee that the contents haven’tbeen reduced to multiple shards ofcolored glass.
I will admit to you that even I havegiven less thought to the packagingof paraffin blocks leaving myfacility. After all, they are, for themost part, shock resistant; and untilrecently I would have expectedthem to resist any of the rigors oftransportation that I could imagine.
As you can see in Fig. 1, the sendertook extra care to wrap this block inbubble wrap packaging materialwhich we know protects even themost fragile of shipments. It is clearthat this block was subjected toelevated temperatures, and given theinsulating properties of the air-filled
plastic wrap, the temperature of theshipping environment must havebeen extreme.
In the past I’ve not consideredsending cold packs along withparaffin blocks, but this experiencesuggests that it would serve us wellto consider the climates we ship toand take appropriate precautions toprevent this unfortunate outcome.Despite our best efforts in thisparticular instance, we were unableto recover the specimen from thepackaging material.
Enhanced Detectionof Microorganisms
in TissueHerbert Skip Brown, BA,
HT(ASCP)Lab Management Consultants
St. Louis, [email protected]
AbstractIn the field of histopathology, twokey challenges present themselvesas pivotal in achieving diagnosis:for the pathologist, the abilityto microscopically identify anddistinguish individual pathogensand/or cellular changes withina tissue host; for thehistotechnologist, the ability toreliably target suspected agentsand/or cellular disruptions, andhighlight them in such a way asto enhance the pathologist’s abilityto selectively view them apart fromall other tissue elements.
The term microorganism bydefinition denotes the extrememinuteness or microscopic/ultramicroscopic size and nature ofan organism. Our ability to observethem oftentimes relies on the fact
Fig. 1. The bubble wrap melted into the paraffin block, contributing to the loss of the specimen.
that they generally exist inaggregate communities asinterdependent elements thatfunction as a unit. Given theirability to grow and reproducewithin a host independent of otherlike organisms, pathologists mustbe able to identify even a singleorganism. In a survey ofpathologists who were asked tocomment on the factors mostaffecting their ability to discern thepresence of infectious particles intissues, the overwhelming responseleaned toward three reasons:1) minimal populace of organismsin tissue; 2) inadequate dyeintensity of stain; and 3) highdegree of background noise(counterstain) masking out theorganisms. While the availability ofhigh-powered microscopes haslargely facilitated the detection ofscant organisms, the greatestchallenges remain with thehistotechnologist to demonstratethese pathogens when present,in a clear and distinct manner.If organisms are not able to beviewed clearly enough todistinguish their morphology,they may go undetected or bemisidentified. In some instancesstain artifact may be mistaken forthe presence of microorganisms,when in fact none are present.
While a number of microorganismsare pathogenic to humans, thisarticle will focus on acid-fastbacteria to illustrate three methodsof enhancing the detection of theseorganisms. The first two are verysimilar in nature, both using similarreagents and heat inducement. Thethird method utilizes a non-antibody fluorescent dye markerthat provides optimal contrastresolution even for single non-aggregated organisms.
Contrast SensitivityOptimal organism detectionrequires a higher sensitivity andaffinity of a stain toward the targetagent and an optimal balance ofbackground color that willdelicately contrast and thereforehighlight the organism. Contrastsensitivity may be defined as the
13
Fig. 1. Mycobacterium tuberculosis is illustrated with three varying degrees of contrast. A. Overstaining of methyleneblue counterstain masking the bacilli. B. Optimal counterstain (m. blue) with distinguishable red organisms.C. Aggregate and individual organisms using Acri-Fluor fluorescent stain.
A
B
C
ability of the visual system todistinguish between an object and itsbackground color.Various cells of thehuman eye participate inthe visualization of an object,distinguishing such characteristics ascolor, size, shape, contrast, etc.Thesecells process the information intoneural codes and transmit them tothe brain, which in turn decodes andformats all of the information intovisual perception.1 Most personshave normal contrast sensitivity andcan readily perceive an object in acolor environment. If, however, thatenvironment has a low contrast, suchas a pink object with a redbackground, or a brown object with adark tan background, the sensitivitycan be sufficiently diminished as torender an object difficult todistinguish morphologically, if nottotally impossible to see.This conceptis illustrated by the images in Fig. 1.Histotechnologists must be attentiveto a stain procedure that will presenta sharp visual image to theirpathologist, and make it easy to seeoutline and detail of the organismthrough contrast.
Principle/Primary StainBy nature, acid-fast bacilli possessa semipermeable membranesurrounded by a lipid layer. Oncethe primary stain is applied(commonly carbol fuchsin), thislipid layer enables the bacillus toresist the decolorizing effects in thesteps that follow. When using thisstain, all cells, including acid-fastbacilli, are stained a deepcharacteristic red with carbolfuchsin, then the tissue is
decolorized with a mild acid-alcohol to differentiate the bacteriafrom other tissue elements. Non-acid-fast bacteria will not retain thestain after decolorizing. The tissueis counterstained with a softlycontrasting stain of light blue oraqua, usually with methylene blue.2
The most commonly used primarydye solution in acid-fast staining iscarbol fuchsin, which contains aphenylmethane dye (new fuchsin,basic fuchsin, or pararosaniline) inan alcoholic-phenol solution. Thephenol in the solution helpsenhance the basic fuchsin dye andpromotes a stronger binding of dyeto the organism. Two commonformulas of carbol fuchsin arefound in the Ziehl-Neelsen andKinyoun techniques. Both stainsimpart a rich red color to acid-fastbacilli. Kinyoun’s, because of itshigher concentration of dye, isoften preferable for smaller, morefilamentous bacteria, or forprocedures requiring a rapidstaining time. Heating of theprimary stain with a microwave orconvection oven has been shown topromote enhanced staining andalso decrease staining time.
In a previous study wedemonstrated that there is a directrelationship between staining timein carbol fuchsin and depth of colorachieved in the bacteria. Slidesections of tissue containingtuberculosis in the form of lesionsof aggregate colonies were stainedwith carbol fuchsin. Microwaveirradiation was used with the slidesleft to incubate in the warmedsolution for increments of1 minute, 5 minutes, 10 minutes,and 15 minutes. The slides stainedfor 1 minute expressed only a fainttinge of pink color and had a verylow contrast resolution.
Progressively we could see amarked increase in intensity instaining from 5 minutes through15 minutes. A distinct stainingpattern associated with theduration of staining time becameapparent. With the staining times of
1, 5, and 10 minutes, the differencein color was more noticeable in thecenter of the tubercular lesion thanon the outer periphery. Bacilli thatwere in the more central portion ofthe lesion showed a deeper stainthan those on the outer areas.When stained for 1 minute,organisms on the outer edge werealmost translucent, demonstratingvery little stain retention, while thecentral area of the lesion was aslightly more intense pink. Thebacteria at the periphery of thelesion stained very well whenincubated in carbol fuchsin for10 minutes but remained slightlyweaker than those in the centralarea. Since we know that all tissueelements will generally stain withcarbol fuchsin, it was apparent thatthe older bacilli (those on theperiphery of the lesion) appearedto lose their ability to retain thedye. One theory is that with theaging of bacilli there is aprogressive loss of lipid content inthe surrounding capsule layer, thusmaking it more susceptible to acid-alcohol rinses.
PROCEDURE A(Microwave Technique)This method utilized a modificationof the Ziehl-Neelsen techniquewith microwave heating of thecarbol fuchsin solution. An 800-wattlaboratory microwave instrument(Fig. 2) was used (H2100, SigmaDiagnostics).3 Sections of infectedlung were taken at 4 microns,mounted, and dried on slides. Theslides were deparaffinized, hydratedto water, and placed in 40 ml ofcarbol fuchsin solution containedin a plastic coplin jar. With a ventedlid placed on top, the slides wereirradiated at 50% power for30 seconds, then gently agitatedwith a plastic pipette (3-4 draws),and incubated for 10 minutes.Slides were then rinsed in distilledwater to remove excess stain. Afterrinsing, the slides were placed inmalachite green solution (Sigma)for 1 minute at room temperature.Malachite green solution serves asa differentiator and counterstain.4
It simultaneously removes excesscarbol fuchsin while leaving
14
Fig. 2. Sigma H2100 microwave oven.
a contrasting turquoise-greenbackground. This step added tothe quality control by consistentlyapplying the same level ofoptimized differentiation andcounterstain to the procedure,thus eliminating variability fromslide to slide for those done inthe same special stain run. Whilethe turquoise-green backgroundappeared to offer a higher contrastsensitivity to the bacilli thanmethylene blue, both wereexcellent counterstains when usedproperly (see Fig. 3). Slides wererinsed in tap water aftercounterstaining, air dried, thendipped in xylene and mounted witha permanent mounting medium.Results demonstrated bacteria in adeep red with a green to turquoisebackground.
PROCEDURE B(Convection Transfer Heating)The second procedure is aproprietary variation of the Fitemethod for acid-fast bacilli whereslides are sectioned, dried, thenplaced in a xylene-peanut oilmixture for deparaffinization,blotted, and stained. The Fitemethod was developed primarilyto demonstrate more thinlyfilamentous types of acid-fast
bacteria such as Nocardia and leprabacilli. Morphologically,Mycobacterium leprae are thinnerorganisms than M tuberculosis witha reduced lipid layer content. Thisdifference accounts for less dyeuptake by the organisms due to adecreased diameter and diminished resistance to alcohol and aciddecolorization. The xylene-peanutoil mixture is believed to enhancethe lipid layer and the alcoholstypically used in the slidedeparaffinization scheme areeliminated from this stain method.Heat was applied through the useof the StainQuickTM System (Cel-Tek, Inc.) in which six staining jarsare housed in a stain rack witheach container holding itsrespective dye or reagent for theprocedure (see Fig. 4). Thecontainer reserved for the carbolfuchsin has an outer metal sleeve,which heats the solution to 53°C.Four-micron paraffin sections weredried and placed in ParaclearTM.The slides were transferred intowarmed Enhancing FluidTM (Cel-Tek, Inc.), which is a xylene-peanutoil mixture, for 1 minute. Slideswere washed in warm runningwater for 3 minutes and placed inthe prewarmed jar of carbol
fuchsin for 1 minute. After primarystaining they were washed again inwarm running water for 1-2 minutesthen decolorized in 2 changes ofdecolorizer (5% sulfuric acid in25% ethanol) for 1 minute each.Slides were washed in warmrunning tap water for 1 minute,counterstained in methylene bluefor 30 seconds, and rinsed again inwarm water. They were thenblotted, allowed to air dry, dippedin ParaclearTM, and coverslipped.The organized rack system makesthe procedure flow very quicklyand with ease. Resultsdemonstrated acid-fast bacilli in adeep red color with a light bluecontrasting background.
PROCEDURE C (Fluorochrome)The last method of acid-fastdetection discussed here providesthe highest degree of visualcontrast and gives the microscopistthe best opportunity to identifyindividual organisms within a tissuematrix. Acri-Fluor FluorescentStainTM (Scientific DeviceLaboratory, Inc.)6 utilizes a non-antibody fluorochrome dye thatcan be visualized under themicroscope after excitation withultraviolet light (see Fig. 5). Theelectrons of the fluorochrome dye,in their excited state, emit energyin the form of visible light (yellow,orange, red), thus causing theorganism to visibly fluoresceagainst a dark green to blackbackground.
Four-micron paraffin sectionswere placed on a slide, dried,deparaffinized, and hydrated towater. Care must be taken to avoidhaving the tissue dry out duringstaining. Slides were laid outhorizontally on a staining rackor countertop for staining. Dropsof the Acri-Fluor dye were placedon the slide just liberally enoughto cover the tissue; slides werestained for 15 minutes at roomtemperature, then rinsed indeionized water and allowedto drain. Drops of DestainerSolutionTM (Scientific DeviceLaboratory, Inc.) were placed ontissue sections and allowed to stand
15
Fig. 3. Mycobacterium tuberculosis stained with heat-induced Ziehl-Neelsen technique for acid-fast bacteria.(Oil immersion)
for 2 minutes. As in procedure A,the Destainer Solution acts as asimultaneous decolorizer andcounterstain. Slides were rinsed indeionized water, drained, air dried,and coverslipped with an aqueousmounting medium. After staining,the slides were viewed with afluorescence microscope. Acid-fast bacteria appeared bright yellowto orange against a dark backgroundof dark green to black (see Fig. 6).This procedure is often used as thefinal confirmation to rule out thepresence of acid-fast bacteria whenother attempts have failedto produce the suspected organisms,as contrast sensitivity is enhanced.When optimally performed, it allowsone to more easily discern themorphology of individual bacilli.
SummaryEach of the procedures presentedabove offers an enhanced targetingability of the primary dye throughits specific chemistry and/or theinducement of heat. With eachstain, bacteria were presented asrichly colored, well-defined entities,whether they were in aggregatecolonies or as single organisms.Additionally, optimal backgroundcolor provided the ability to furtherenhance identification of organismsthrough improved contrast.
ConclusionIn histological staining, success inthe detection of microorganisms isas much a part of contrastsensitivity as it is primary staineffectiveness. Histotechnologistsmust find ways of optimizing theirability to target and visualize thesemicroscopic structures. Contrastsensitivity of the eye must beconsidered with attempts made tooptimize the crispness of perceivedstructures, making it easier for theeye to locate and identify. Whilethe pathologist and histotechnologistmay rarely interact whileperforming their respective duties,their roles are intimately linkedand must be in alignment to ensureoptimal diagnostic success. Thepathologist must be able to trustthat all steps were taken tomaintain the integrity of methodsFig. 6. Enhanced visualization of acid-fast bacteria is shown using fluorescent stain technique.
Mycobacterium tuberculosis bacilli are illustrated in bright yellow.
Fig. 5. Acri-Fluor Fluorescent Stain KitTM (Scientific Device Laboratory, Inc.).
Fig. 4. StainQuickTM System (Cel-Tek, Inc.).
16
and procedures, and that thepicture being presented to them isan accurate and clear representationof tissue elements within. Thehistotechnologist must strive notonly to perform analyses, but alsoto create a perfect picture,demonstrating and highlighting,or eliminating, structures thatmight be misidentified aspathogenic organisms.
References1. Vision Sciences Research Corporation.
ContrastSensitivity.net. Available at:http://www.contrastsensitivity.net. Accessed June 2002.
2. Sheehan DC, Hrapchak BB. Theory and Practiceof Histotechnology. 2nd ed. Columbus, Ohio:Battelle Press; 1980.
3. Sigma Diagnostics. Ease of Use and ApplicationsManual—Accumate H2100 Microwave. St. Louis, Mo:Sigma Diagnostics; 1996.
4. Horobin RW, Bancroft JD. Troubleshooting HistologyStains. New York, NY: Churchill Livingstone; 1998.
5. Cel-Tek, Inc. StainQUICK System for Acid FastStaining. Product No. 1955 (Insert). Glenview, Ill; 1997.
6. Scientific Device Laboratory, Inc. Acri-FluorFluorescent Stain. Product No. 384 (Insert).Des Plaines, Ill; 1997.
Tools to Facilitate and Standardize
GrossingRita Romaguera, MD
Mehdi Nassiri, MDAzorides R. Morales, MDDepartment of Pathology
University of Miami/Jackson Memorial Hospital
Miami, FL
Grossing, a term that refers toexamination and dissection ofsurgical specimens, along withpreparation of sections from thosetissues requiring processing, is theinitial step in the practice ofsurgical pathology. While textbooksand manuals of surgical pathologyteach about the sampling ofspecimens, they are silent regardingthe dimensions of individual tissueslices, other than to suggest athickness of 2 to 4 mm.Unfortunately, it is not anuncommon practice to place asmuch tissue as will fit into aspecific size cassette. Althoughthis liberal approach to grossing
17
Fig. 1. These photos show slicing of tissue that is held in place by either A) index finger, or B) plastic holder.
B
A
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may have been permitted byconventional processing methodslasting 12 hours or longer, it failswith rapid tissue processing nowdone by microwave-based methodsand by shortened conventionalassays. The requirement to adjustthe length of the processing cycleaccording to the thickness of tissuesections is discussed in publicationsof microwave-based methods suchas Visinoni et al,1 and Willis andHinshew.2 Conversely, Moralesand associates advanced the notionof standardizing tissue sectionsto accommodate the processingcycle in microwave methods.3
Appropriate tools are required toobtain proper slices of tissue forprocessing and are essential tostandardization of tissue sections,but grossing tools createdspecifically for pathology are rare.A review of the literature andMEDLINE searches for “grossingtools” or “pathology tools” failed toyield a single reference. There are,however, several publications aboutthe birth and evolution of surgicalinstruments that have evolved withthe advances of surgical ingenuityand its demands. It has beencustomary to use the sameinstruments developed for thepractice of surgery for grossing.It is a tradition, passed on from onegeneration to the next, with poorresults and no standardizationin grossing the tissue.
During the course of developingand implementing an automatedmicrowave-based rapid tissueprocessing system at the Universityof Miami/Jackson MemorialHospital, the need to standardizethe dimensions, and particularlythe thickness of tissue sections,became readily apparent. To thatend, we created two tools thatgreatly facilitate grossing, not onlyfor microwave-based methods,but also in conventional tissueprocessing. These tools areillustrated in Figs. 1-5.
Fig. 2. Grossing board with slotted metal plate and knife-guiding assembly.
Fig. 3. Tissue sample obtained as illustrated in Fig. 1.
20
As previously reported3 and alsodescribed in detail elsewhere,4 thegrossing board permits preparationof uniform tissue slices of thedesired thickness. It consists ofa board with a slotted metal plateand a track for the knife or surgicalblade (Figs. 1, 2). The slots serve aswells for the placement of thetissue and the knife-guidingassembly keeps the cutting blade inplace. As the bottom surface of theslots are parallel to the cuttingsurface, sliding the cutting blade inthe track along the surface of thecutting board yields tissue pieces ofuniform thickness, which facilitatesprocessing, as well as microtomy.Because the depths of the slots areeasily adjusted, sections of desiredthickness are obtained. Moreover,the slots can be built large enoughto facilitate serial slicing of organs,such as prostate and breast.
While the grossing board greatlyfacilitates sampling of solid organsand tumors, flat structures such asskin and small tubular organs likethe appendix, fallopian tubes, andumbilical cord, are not amenablefor its use. For those, we developedanother tool that holds the tissuein place to permit sampling ofuniform desired thickness. This toolis particularly helpful in grossingorgans composed of layers ofdifferent structures that slide overeach other during sectioning, suchas the bowel and gallbladder.Additionally, tissues that are softand slippery, such as adipose tissue,or fatty tissues, such as breast, areeasily handled with this tool. Thetool is composed of a handle and ahead supporting a double array offour tines (Fig. 5). The gap betweenthe double array of tines ispredetermined to accommodatethe desired thickness of tissuesections (Fig. 5 inset). As illustratedin Fig. 4, the tissue is held in placewhile sliding the blade against theouter surfaces of the tool, thusyielding a section of the desiredthickness in the gap providedbetween the double array of tines.
Fig. 4. These photos show slicing of bowel with the assistance of the grossing tool: A) sliding the blade against theouter surface of the tines; B) slice of tissue occupying gap between tines.
A
B
This contrasts with the commonpractice of holding such tissueswith forceps during sectioning,a procedure which producesirregular slices of tissue, varyingconsiderably in dimensionsand thickness.
In summary, we describe two toolsthat allow standardization andgreatly facilitate grossing byproviding tissue sectionsconsistently uniform in thickness.
References1. Visinoni F, Milios J, Leong AS-Y, et al. Ultra-rapid
microwave/variable pressure induced histoprocessing:description of a new tissue processor. J Histotechnol.1998;21:219-224.
2. Willis D, Hinshew J. Microwave technology in thehistology laboratory. HistoLogic. 2002;35:1-5.
3. Morales AR, Essenfeld H, Essenfeld E, et al.Continuous-specimen-flow, high-throughput, 1-hourtissue processing. Arch Pathol Lab Med. 2002;126:583-590.
4. Morales AR, Essenfeld E, Essenfeld H, inventors.University of Miami, Miami, FL, assignee. Pathologygrossing board. US patent 6,513,803, B2. Feb. 2003.
A Techniquefor Correcting
Poorly ProcessedParaffin BlocksMichael L. Johnson, BS,
HTL, HT(ASCP)Spokane, WA
Every histotechnologist isfamiliar with the frustrationsof trying to cut a block that wasunderprocessed and “mushy”because it was grossed in toothick. In some cases, adjusting themicrotome to a thicker setting(6 or 8, or even 10 microns),re-embedding the specimen aftersqueezing the tissue to removeresidual xylene, or freezing withfreeze spray, may make it possibleto obtain a section. Some of theseblocks are so poorly processed,however, that they cannot be cutat all no matter what tricks weattempt in order to get that onesection the pathologist needs.When nothing works to obtain asatisfactory section, the tissuemust be reprocessed. This istypically a time-consuming,hands-on process.
Histotechs are always looking fora way to automate the process inorder to save time and energy.One of the most commontechniques is to first melt theblock down to remove theparaffin. The tissue is then putback in the cassette, placed in atissue processor, and run througha purge or cleaning cycle. Thetissue emerges with the paraffinand xylene removed and wet with100% alcohol. Then the tissue ismanually run through 95% andthen 70% alcohol before it isplaced back in formalin andreprocessed with that night’ssurgical tissues. This works
satisfactorily in most cases.However, it does subject theunprotected tissue to hot xyleneand hot alcohol which is quiteharsh and can cause distortion ofthe cellular morphology.
Several years ago, thehistotechnologists at Sacred HeartMedical Center (Spokane, WA)returned from an NSH symposiumwith a novel technique forreprocessing tissues that involved far less technical time, and it sparedthe tissue from the harsh treatmentof a purge cycle (see Fig. 1). Thisprocedure involves melting theblock down and blotting off theexcess paraffin before putting thetissue back in the cassette andplacing the cassette directly intoformalin for reprocessing with theregular run of tissues for that night.The results are remarkably goodwith less handling time.
The rationale for reprocessingblocks this way is simple. Theportions of the block that wereadequately processed initially arespared further dehydration as theremaining paraffin insulates thetissue from the effects of alcohol,which in excess can make sometissues very hard and brittle.The paraffin remaining in thispreviously processed block iseventually removed in xylene onthe processor and the entiresample is then reinfiltrated withnew paraffin. The portion of theblock that was previouslyunderprocessed is reexposed tothe effects of fixative, dehydrant,clearing agent, and then infiltratedwith paraffin. When complete, allparts of the block are properlyprocessed and infiltrated, and theblock cuts very well. Undoubtedly,part of the reason the tissueprocesses better the second timearound is that it is thinner becausesome of it was cut away duringinitial attempts to cut the block.
21
Fig. 5. Grossing tool consisting of handle, tine-supportinghead, and double array of tines (inset).
Although the originator of thismethod is unknown to me, Ithought it would be a usefultechnique to share with thehistotechnology community.
Perhaps someone among usknows who first reported thismethod so that he or she canreceive credit for such a noveland useful technique.
22
Fig. 1. Above are photos of tissue blocks reprocessed using A) the new technique described, in which the melted-down block is placed back in formalin and reprocessed, and B) the purge technique. Nuclear detail is quite good inboth cases, but nuclei seem to show somewhat more distortion using the purge technique.
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