1

Technical Bulletin for Histotechnology • Managing Editor, Gilles Lefebvre • Scientific Editor, Vinnie Della Speranza, MS, HTL (ASCP) HT, MTVol. XXX, No. 1, April 1999

Vinnie Della Speranza Named New Scientific Editor of Histo-Logic

The invitation to serve as Scientific Editor of Histo-Logic was an unexpected surprise to Vinnie DellaSperanza, MS, HTL(ASCP)HT, MT. Although he hasserved a number of professional organizations invarious capacities during his career, he’d never reallyimagined himself in this new role. Despite an alreadyfull schedule, he didn’t hesitate to accept the challenge.

“When the invitation to serve Histo-Logic wasextended to me, I found myself reflecting on the richhistory this publication has enjoyed over the years. Irecall with special affection the Histo-Logics ofyesterday and the valuable information theycontained that I, for one, found so especially useful

in my work at the bench.The opportunity to continuethe rich legacy of those esteemed colleagues whopreceded me as scientific editor is a treasured honor.”

Vinnie is a familiar face to many at the NSHSymposium/Convention each year where he haspresented workshops and participated in manycommittee, membership, or board meetings. Althoughhe has long been active in histotechnology, he beganhis career as a medical technologist after earning hisbaccalaureate of science degree with a double majorin medical technology and biology from the StateUniversity of New York at Stony Brook. It didn’t takehim long to realize that histology was his first love.

“Jules Elias was one of my professors in college. Ivividly remember how his enthusiasm and passionfor the discipline were so contagious for me. In fact,Jules introduced me at the time to a local pathologist

Vinnie Della Speranza Named New Scientific Editor of Histo-Logic……………………………… 1Something Old, Something New...…………………………… 2A Comparison of Staining Methodsfor Helicobacter pylori ……………………………………… 3Welcome Tom Lieb …………………………………………… 8Breaking News... New Weapon in the Fight Against Breast Cancer………………………………………… 8All in a Day’s Work! ………………………………………… 11A Closer Look at Xylene Substitutesto Reduce the Hazardous Waste Stream ……………………15Histotechs to Invade the Smallest Statein the Union ………………………………………………… 18Meet Dennis Keast ………………………………………… 19High School Eligibility Route forHT(ASCP) May End by 2005 ………………………………20Get to Know Ross Lindmeyer ………………………………20

No reader should utilize or undertake procedures in Histo-Logic articles unless thereader, by reason of education, training, and experience, has a completeunderstanding of the chemical and physical properties of all materials to be utilizedand the function of each material by which any procedure is accomplished. Theprocedures discussed in these articles represent the opinions and experiences of theindividual authors. Sakura Finetek U.S.A., Inc. assumes no responsibility or liabilityin connection with the use of any procedure discussed herein.

© 1999 Sakura Finetek U.S.A., Inc.

IN THIS ISSUE

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who was looking for someone with my training to helpbring immunohistochemical techniques into his lab ata nearby community hospital. Although I had otherjob offers at the time, I found myself drawn to theopportunity to become more involved with histology.”

A year and a half later, Della Speranza was promotedto Histology Supervisor where he remained until late1979, when he was invited to serve as TechnicalDirector for the Anatomic Pathology Laboratoriesunder Jules for the new, soon to open, UniversityHospital at Stony Brook. There he earned his masterof science degree with an emphasis in immunologyand pathology. Along the way, he also earned his HTand HTL credentials with the ASCP.

It was Jules Elias who encouraged him to begin hispublic speaking activities, first at local campus work-shops and eventually at the NSH symposium/convention. Today, Della Speranza receives invita-tions to present workshops at national, regional, andlocal conferences. This year he is scheduled to givepresentations to the Louisiana and New Yorksocieties, a reference laboratory in Florida, and theNSH in Providence, R.I. Della Speranza has authoreda number of articles throughout his career. In the pastyear he wrote an invited chapter entitled “SafetyIssues for the Clinical Laboratory” for the textbookSaunders Manual of Clinical Laboratory Science; aneditorial for the Journal of Histotechnology entitled“Are We the Victims of Our Own Success?”; and anASCP Tech Sample entitled “The H&E Stain,Concepts and Pitfalls.”

Della Speranza is presently serving his third term asRegion 1 Director for the National Society forHistotechnology. Despite a heavy regional andnational agenda, he remains active with the NewYork State Histotechnological Society where he hasserved two terms as president and over 15 years onthe board of directors. In 1998, he was the firstrecipient of the New York State HistotechnologicalSociety’s President’s award for outstandingcontributions and distinguished service.

It is as much Della Speranza’s activities outside ofwork that make him an excellent choice to serveHisto-Logic. Two evenings a week he makes time ina hectic schedule to provide instruction to adults,some from disadvantaged backgrounds, who struggleto begin a career in the health field. He currentlyteaches phlebotomy and medical terminology.

“The opportunity to assist and encourage indi-viduals looking for their start in a health career isincredibly rewarding for me. I am reminded of themany, like Jules, who freely extended a helpinghand to me throughout my career. I feel compelledto do the same for the next generation of healthcare practitioners. I believe that we have a respon-sibility to pass on what we know. To do anythingless would be a missed opportunity to help othersand to shape the future of the profession.”

His energy and an attitude like this leave littledoubt that Della Speranza brings the right stuff toHisto-Logic.

Something Old, Something New…Vinnie Della Speranza, Scientific Editor

Histo-Logic began as the vision of Lee Luna who, in1969, imagined the value of having a vehicle by whichhistotechs could communicate as a discipline. Pre-dating even the Journal of Histotechnology, the first

“I believe that we have theresponsibility to pass on what weknow.To do anything less wouldbe a missed opportunity to help

shape the future of the profession.”

Countdown to the 21st CenturyNoteworthy Milestones Leading to the Present Day

Compiled by Vinnie Della Speranza

1816 Rene Laenner inventsthe stethoscope

1798 Edward Jennerdevelops the concept of

vaccination to fight small pox

1828 William Harveydescribes the

circulation of blood

1833 First descriptionof the use of hardening agents

in microtechnique

1839 Chevalierfirst coins the

term microtome

1842 The frozen sectionis accidentally discovered

by Stilling

1843 First clearing agent,turpentine, is used

by Varby

1860 Rudolph Virchowexplains the cellular basis

for disease

1860 Firstsilver staining

techniques appear

1800s

1865 Bohmerfirst to use

alum hematoxylin

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1869 Paraffinembedding is first used

1875 Hematoxylin and eosin firstused in combination

1876 AlexanderGraham Bell develops

the telephone

1878 Thomas Edisonforms the Edison Electric Co.,

predecessor of GE

1879 Edison’s first light bulb,a carbon filament lamp

1880 Rotary microtomefirst appears

1892 Trillat first describesthe preservative effects

of formaldehyde

1892 Rudolph Dieselpatents the internalcombustion engine

1895 Marconi pioneersthe wireless

telegraph (radio)

1893 Karl Benz andHenry Ford build thefirst four wheel cars

issue of Histo-Logic appeared in July, 1971, sponsoredby the Lab-Tek Division of Miles Laboratories.

Over the years, many of us have squirreled awaythose old issues of Histo-Logic, replete with stainmodifications, processing tips, tricks, and trouble-shooting strategies. They saved my life more thanonce, always seeming to contain information that Ineeded to know in my daily work.

Today, the rich legacy of Histo-Logic continues withthe guidance and commitment of Sakura Finetek.Together we are working hard to provide you, ourreaders, with a document that I hope you will con-tinue to keep for years to come. If your collection isincomplete, by the way, you can view old issues ofHisto-Logic at Sakura’s web site at www.sakuraus.com.

Beginning with this issue, you will see some changesthat I am very excited about. Be sure to read thesection All in a Day’s Work! where we will regularlyfeature a different workplace where you, ourreaders and colleagues, are often unsung heroesfacing some rather interesting challenges. Whoknows, maybe we’ll see your group featured!

I know that you will enjoy our Countdown to the 21st

Century, an interesting collection of facts and trivialooking back at the noteworthy events that haveshaped society and our discipline as the centurydraws to a close.

To quote Lee Luna, “Reading Histo-Logic should befun as well as educational.” I couldn’t agree more.

Won’t you consider sharing your ideas, tricks, tips,shortcuts and yes, even your opinions with the rest ofus? Histo-Logic is the forum that brings us all a bitcloser. Histotechs too often believe, in error, that ifthey know something, everyone else must know it too.Someone will always benefit through our collectivesharing. So if you have an idea or brainstorm buthaven’t written before, don’t let that stop you. Simplycontact me and I’ll be happy to help you get it intoprint.

I hope that you will take a moment occasionally totell us how we’re doing. I’d like you to think of Histo-Logic as your publication, one that will chase awaythose workday blues and maybe even bring a fewsmiles along the way.

A Comparison of Staining Methodsfor Helicobacter pyloriKim Rhatigan-Drexler MA, HTL(ASCP)Histology SupervisorUniversity Hospital & Medical CenterStony Brook, N.Y.

AbstractHelicobacter pylori is a spiral-shaped bacterium thathas been found to be an important pathogen livingin the stomach lining of patients suffering fromchronic gastritis. It is a nonsporing, S-shaped, gram-negative rod measuring approximately 3.5 by 0.5microns and is present in a high percentage ofpatients with chronic gastritis affecting the gastricantrum and corpus. H. pylori infection of the gastricmucosa is present in 90% to 100% of patients withduodenal ulcer and 70% of those with gastric ulcer.1

It has also been associated with many of the mostimportant diseases involving gastroduodenal tissue.2

Visualization of H. pylori may be seen with thestandard Hematoxylin and Eosin stain in paraffinsections, however, the H & E can be unreliablebecause the organisms are often hard to distinguishfrom the mucosa since contrast between theorganism and the intraluminal mucous layer is notdistinct. A greater number of organisms can beidentified through the use of the many special stainsthat are found to color this pathogen. This articlecompares 6 different methods for the visualization ofthis organism in gastric biopsies which are evaluatedfor effectiveness, ease of use, and reproducibility.

IntroductionH. pylori has been determined to be an importantpathogen in man. It produces a number of productswhich damage the lining of the stomach. It wasdiscovered in Perth, Australia by Robin Warren andBarry Marshall in 1982. The organism lives in thestomachs of most people throughout the world,although in countries like Australia, US, UK andSweden less than 50% are infected and mostchildren are Helicobacter free.1 Once the infection ispresent, it may persist for many years, if not for life.Inflammation disappears if the infection issuccessfully treated with a regimen of tetracycline,

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metronidazole, and Pepto-BismolTM(bismuth).H. pylori is now accepted as a cause of:

• inflammation of the lining of the stomach(gastritis)

• ulcers in the first part of the small bowel(duodenum)

• stomach (gastric) ulcers• carcinoma of the stomach• Mucosal Associated Lymphoid Tissue (MALT)

lymphoma of the stomach

H. pylori has a unique way of adapting to the harshenvironment of the stomach. The inside of thestomach is bathed in gastric juice which is rich indigestive enzymes and concentrated hydrochloricacid. The stomach is protected from its own gastricjuice by a thick layer of mucus that covers thestomach lining. H. pylori takes advantage of thisprotection by living in the mucus lining. H. pylorihas adapted to life in this harsh environment byusing an enzyme it possesses called urease. Ureaseconverts urea, of which there is an abundant supplyin the stomach (from saliva and gastric juices), intobicarbonate and ammonia, which are strong bases.This creates a cloud of acid neutralizing chemicalsaround the organism, protecting it from the acid inthe stomach.1 This reaction offers an important testfor the diagnosis of H. pylori called the breath test.

H. pylori has another advantage allowing it to residein the mucus lining of the stomach. The immunesystem normally will respond by sending neutrophils,killer T lymphocytes, and other infection fightingagents to the site of a H. pylori infection. However,these potential H. pylori eradicators cannot reach theinfection because they cannot easily get throughstomach lining. They do not go away either, though,and the immune response continues to grow andgrow. Polymorphs die and spill their destructivecompounds (superoxide radicals) on stomach liningcells. Over time, gastritis and perhaps eventually apeptic ulcer results. It may not be the organism thatcauses peptic ulcer as much as the persistentinflammation of the patient’s stomach lining inresponse to the organism’s presence.1

Most duodenal ulcers are also caused by H. pylori.The pathogen can move into the duodenum andcause inflammation there as well. This may develop

Sayeed method

Gimenez method

Giemsa stain

1896 William Roentgendiscovers x-rays

1895 German physicianLudwig Rehn is first to sew up

a heart wound successfully

1897 Coplin stainingjar is introduced

1898 First photographstaken with

artificial light

1903 Wright brothersfirst airplane flight

1903 The EKGis developed

1905 Einsteinformulates his

theory of relativity

1908 Bakelite, thefirst synthetic plastic,

is developed

1909 First automatictissue processor

1912 The Titanicsinks

1912 Swiss chemistdevelops cellophane

1900s

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into an ulcer and in some cases severe bleeding orperforation into the abdomen can result.

H. pylori is believed to be transmitted orallythrough the ingestion of waste-tainted food orwater. In addition, it is possible that H. pylori couldbe transmitted from the stomach to the mouththrough gastroesophageal reflux (in which a smallamount of the stomach’s contents is involuntarilyforced up the esophagus) or belching, commonsymptoms of gastritis. The bacterium then could betransmitted person-to-person through oral contact.1

Despite the availability of simple screening tests,gastric biopsy is the most definitive method ofdetecting H. pylori infection. The histologist is oftencalled upon to perform methods that candemonstrate the presence of this organism.

MethodsSix special stain methods were used on the samegastric biopsy specimen to stain for H. pylori. Thebiopsy was fixed in 10% neutral buffered formalin atroom temperature and processed on a Sakura VIPtissue processor through ethanol and xylene intoParaplast paraffin wax. Sections were cut at3 microns, placed onto clean glass slides and dried at60°C for one hour prior to staining. The results ofeach method were assessed microscopically by threeindependent observers.

1. Cresyl Echt Violet (CEV)3

This method utilizes a metachromatic dye, cresylecht violet (also called cresyl violet acetate) to stainthe bacteria.3 The bacteria stand out well becausethe mucous layer does not stain heavily with theCEV and provides a nice contrast for the organism.It is a very easy and quick stain utilizing a 0.1%aqueous working solution of CEV. The slides aredeparaffinized, hydrated to water, and stained inthe CEV for 3 minutes. They are then rinsed indistilled water, dehydrated, cleared, and mounted.

2. May-Grünwald Giemsa4

This stain uses solutions of both Jenner’s andGiemsa to acquire the results. Slides aredeparaffinized, hydrated to water, and treated withmethanol. They are incubated in Jenner’s workingsolution and then in Giemsa working solution for 10minutes each. Slides are differentiated in 0.1% acetic

Cresyl echt violet stain

Rapid method

Dieterle stain

1915 First transcontinentaltelephone call, from NYC

to San Francisco

1922 The American Societyof Clinical Pathologists

(ASCP) is founded

1922 Insulin is isolatedand used to save a dying baby

1924 A Ford touringcar costs $290

1924 MGM and Columbia Pictures

open

1926 ASCP begins plansfor a technician

registration program

1926 B.F. Goodrichpioneers synthetic rubber

1927 Charles Lindberghflies non-stop from

New York City to Paris

1927 Babe Ruth hits60 home runs, a record

that will stand for 33 years

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acid and then dehydrated through tertiary butanol toxylene. This stain is relatively fast and easy, howevercare must be taken not to over-differentiate the slides.

3. Dieterle-Microwave Modification4

This silver method utilizes many solutions to stain thebacteria black. Slides are deparaffinized, hydrated towater, and put into uranyl nitrate for the first step for2 minutes. The slides are then incubated in silvernitrate solution for 5 minutes. Exposure to a devel-oper solution containing hydroquinone, sodium sul-phite, acetone, formaldehyde, and pyridine turns thebacteria black.The background is yellowish-brown.

4. Sayeed Staining Method2

This kit, purchased from Poly Scientific, Bay Shore,New York (cat# K090), uses periodic acid as anoxidizer in the initial step. Slides are deparaffinized,hydrated to water, and oxidized in 0.5% periodic acidfor 1 minute. They are then treated with Coleman’sFeulgen solution for 2 minutes. From here they arestained with Mayer’s Hematoxylin, rinsed in water toblue, and stained with 10.0% Methylene Blue workingsolution. Bacteria are stained blue and the mucin isstained magenta.

5. Rapid Staining Method5

This Poly Scientific kit (cat# K086) requires slides tobe oxidized in 1.0% periodic acid for 10 minutesfollowed by treatment with 5.0% sodiummetabisulfite for 5 minutes. After a rinse in water,slides are stained in 1.0% Alcian Yellow for 5 minutes.After another rinse in water, the slides are placed in1.0% Toluidine Blue working solution for 3 minutes.The Helicobacter pylori are stained blue, the mucinyellow, and the background is pale blue.

FIXATION: 10% buffered formalinSECTIONS: paraffin, 3-5 microns

SOLUTIONS:1. Periodic acid 1% aqueous2. Sodium metabisulfite 5% aqueous3. Alcian Yellow(C.I. 12840) 1% (filter before use)4. Toluidine Blue (C.I. 52040) stock (1% aqueous)5. Sodium hydroxide 3%6. Toluidine Blue working solution: PREPARE

FRESH 0.5 ml of Toluidine Blue stock—50.0 mL distilled water2 drops 3% sodium hydroxide

STAIN PROCEDURE:1. Deparaffinize and hydrate slides to deionized

water2. Oxidize in 1% periodic acid aq for 10 minutes at

room temperature3. Rinse well in tap water4. Sodium metabisulfite 5% for 5 minutes5. Tap water rinse for 2 minutes6. Stain in Alcian Yellow 1% for 5 minutes7. Rinse well in tap water8. Stain in freshly prepared Toluidine Blue working

solution for 3 minutes9. Rinse well in tap water

10. Blot dry, dehydrate quickly, clear and mount

RESULTS:Helicobacter pylori blueMucin yellowBackground pale blue

6. Gimenez Technique(Poly Scientific kit# K072) This method uses ZiehlNeelsen Carbol Fuchsin to stain the bacteria andmalachite green as a counterstain. Slides are deparaf-finized, hydrated to water, and stained in the workingcarbol fuchsin for 5 minutes. After a rinse in water,slides are counterstained in 0.8% malachite greenand then air dried prior to mounting.

Discussion of ResultsOut of the six methods we tried, three independentobservers favored the staining achieved with theRapid method. This method provided clear, crispbacteria as well as good morphology and distinctive,yellow staining of mucin. The organisms stood outvery well against the background and were easy tolocate. The morphology of the biopsy was very clearand distinct. This method offers the technologist aneasy technique that is quick and reproducible.

The CEV (cresyl echt violet) method was deemedsecond best in our trials. The bacteria were veryeasy to locate and the background was notdistracting. It is a very simple and fast stain and it isinexpensive enough that it could be used routinelyon all gastric and duodenal biopsies.

The Giemsa method provided adequate staining ofthe bacteria. However the bacteria were not crispand appeared muddy in some microscopic fields. The

1928 Amelia Earhart is thefirst woman to fly across the

Atlantic ocean

1928 Alexander Flemingdiscovers penicillin

1928 The ASCP Boardof Registry is formed

1929 Stock Marketcrashes

1929 Blue Cross is formedby Texas teachers and

Baylor University in Dallas

1930 ASCP BORawards its

first certification

1930 The US Food and Drug Administrationis established as a consumer protection agency

1930 Karl Landsteinerdiscovers the

major blood types

1935 Scottish physicistSir Robert Watson Watt

builds radar to detect aircraft

1935 The USSocial Security Act

is enacted

1928 Rice Krispiesbegin to snap,crackle, pop

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Giemsa stain does not provide good tissuemorphology which is a drawback to this method. Thebackground was not as sharp as that achieved withthe Rapid method. The technique requires differ-entiation, a potential pitfall for the inexperiencedtechnologist and a potential source of inconsistencyfrom run to run, especially when different individualsperform the stain from day to day.

The Gimenez method provided a nice color contrastof the bacteria and the background. It is a simple stainbut we experienced a precipitate of the carbol fuchsinthat could distract from the organisms. An advantageof the Gimenez method is that other acid-fastorganisms such as Rickettsiae may be demonstrated.Methods offering a better staining result make work-ing with the hazards of carbol fuchsin unnecessary.

The Sayeed method, like the Rapid technique,provided for the staining of the bacteria as well asthe mucin. However, it was our experience that thecolor contrast is not distinct, making organismidentification slow, tedious, and uncertain. We foundthe mucin staining to be distracting and felt the

organisms were obscured with this method. We alsoexperienced inconsistent results with this methodthat were not explored further.

The Dieterle is the least recommended stain forH. pylori for a number of reasons.The precipitate thatis common among silver techniques in general maydistract from, or be mistaken for the black staining H.pylori. There is not enough contrast between thebacteria and the background to locate the bacteriaeasily. It is also a labor intensive and time consumingtechnique as compared to the other methods. Thereagents in the Dieterle stain are also quite toxic andcostly. Silver stains can be capricious, with stainingrepeats more likely to be necessary with this method.

ConclusionThe Rapid method reported by Leung et al is asimple, reproducible method that provides greatcontrast between the bacteria and the backgroundand was determined to be the method of choice atour facility. The H. pylori organisms may be locatedquickly and easily and the method also providesgood tissue morphology and distinct mucin staining.

1937 First jet engineis built

1936 Fluorescentlight is invented

1938 Teflon makesits debut

1939 Television is demonstrated atthe New York World’s Fair

1940 RCA demonstratesthe first

electron microscope

1941 First aerosolspray cans

1941 George Papanicolau develops the Pap smearto screen for cervical cancer, dramatically reducing

the 26,000 annual deaths from this disease

1945 The first Tupperware product,the polyethylene cup

1948 Firstmicrowave oven

1948 Velcromakes its debut

Table 1. Comparison of Staining Methods for Detection of H. pylori

Cresyl Echt Giemsa Dieterle Sayeed Rapid Gimenez Violet (May- (Microwave Stain Kit Stain Kit Kit (CEV) Grünwald) Modification) (#K090)* (#K086)* (#K072)*

Time to 15 minutes 28 minutes 45 minutes 19 minutes 30 minutes 18 minutescomplete(approx.)

Stain Results:

Color of Purple Blue Black Blue Blue RedH. pylori

Background Light Purple Light Blue Yellowish Magenta Yellow GreenColor (Mucin)/ (Mucin)

Blue

Remarks: Good results— Bacteria are Background Bacteria are Excellent Toxic reagent; Bacteria are visible but is distracting— hard to results—great carbol fuchsineasy to identify not crisp; silver method distinguish color contrast precipitate

tissue can be in the mucin— between may bemorphology capricious; variable bacteria and problematicless than toxic reagents results from background—optimal run to run bacteria are

crisp

* stain kits purchased from Poly Scientific R&D Corp., Bay Shore, NY

The interpretation of the slide is less timeconsuming than the other methods. Our secondchoice, the cresyl echt violet method, is a simple,economical method that can easily be performedwith good reproducibility in most laboratorieswhere cost is the first concern.

The importance of detecting H. pylori and the easewith which it can be identified is of growing impor-tance to pathologists and gastroenterologists todayas the eradication of H. pylori is essential to thetreatment of ulcer disease.

AcknowledgmentsThe author thanks Joel Israel and Linda Chen fortechnical assistance; Bernard Lane, MD forparticipation in stain evaluation, and Kathy DaSilvafor the photography.

References:1. Dunn, et al. Helicobacter pylori. Clin Microbiol Rev, Am Soc Microbiol. Oct.

1997:721.2. Cohen L, Sayeeduddin, M, et al. A new staining method for identification of

Helicobacter pylori and simultaneous visualization of gastric morphologicfeatures. Mod Pathol. 1997;10(11):1160-1161.

3. Gomes C. Rapid cresyl echt violet staining method for identifying Helicobacterpylori. On Stage, NY State Histotechnological Society. 1993;16(2).

4. Carson F. Histotechnology: A Self-Instructional Text. 1980:109.5. Leung JK, Gibbon KJ, Vartanian RK. Rapid staining method for Helicobacter

pylori in gastric biopsies. J Histotech. 1996;19(2):131-132.

WelcomeTom LiebMeet Tom Lieb, new AreaManager for Washington,Northern Oregon, Idaho,Montana, and Alaska.

Tom was born in Oklahoma,and attended the Universityof Oklahoma where hereceived a B.S. in Zoology.

He has held two positions in laboratory-related com-panies, Scientific Products and Fisher/IL as a DiagnosticSpecialist. His interests include scuba diving, skiing, andwhitewater rafting.

Breaking News…New Weapon in the Fight AgainstBreast CancerVinnie Della Speranza, Scientific Editor

HERCEPTIN (Transtuzumab), the latest weapon inthe fight against metastatic breast cancer, is ahumanized monoclonal antibody that targets theHER2/neu protein in metastatic breast cancerpatients. In approximately 30% of patients with breastcancer, the HER2 protein is over-expressed as part ofthe process of malignant transformation and tumorprogression. Over-expression of the HER2 protein onthe surface of breast cancer cells suggests that it couldbe a target for this therapeutic antibody(HERCEPTIN) which is a humanized monoclonalantibody that binds with high affinity to the HER2protein and has been shown to inhibit theproliferation of human tumor cells that over-expressHER2 protein in vitro and in vivo.

The HercepTest (available through Dako Corp.) isthe first Class III PMA FDA-approved IHC test thataids in the assessment of patients for whomHERCEPTIN treatment is being considered.HercepTest is an IHC method for demonstratingHuman Epidermal growth factor Receptor 2(HER2). HER2 is a protein component of normalepithelial cells and is pathologically abundant in avariety of cancer cells, in particular, some forms ofbreast cancer. The discovery of HER2 (also calledp185HER2) was prompted by studies showing thatsmall quantities of the gene encoding for it, known as

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1951 UNIVAC, the firstelectronic computer, is usedby the US Census Bureau

1952 The British Cometis the first

jet airliner service

1952 F. John Lewis performs the firstsuccessful open heart surgery

1952 Jonas E. Salkdevelops a successful

polio vaccine

1953 Sony’s pocket transistor radiomakes its debut

1953 IBM’s first computer,the 701, is introduced

1954 Fluoroscein isothiocyanate (FITC)becomes the first antibody-labeling agent

1956 Fortran, the first computerprogramming language, is developed

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Proven ReliabilitySakura Finetek U.S.A., Inc.

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With the Cyto-Tek® MonoPrep™ System,the high technology lies in the patenteddesign, not in the use, making the process easyand affordable, and the results consistent. Forfine needle aspirates, urines, bronchial

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HER2 proto-oncogene, c-erbB-2 or HER2/neu, couldtransform normal cells into cancerous lines. Theprotein HER2 is thought to mediate these trans-formations by transmitting growth signals from thecell membrane to the nucleus, increasing cell division.

“The proto-oncogene c-erbB-2 (HER2/neu) is ampli-fied in about 30% of human breast carcinomas. Thetransmembrane product of this gene has become auseful target for antibody localization and/or therapy.Detection of c-erbB-2 amplification in tissue there-fore, is now an important part of overall breast cancerpatient management.”1 Protein over-expression hasalso been demonstrated in the absence of gene ampli-fication so that in target-directed therapies, detectionof the protein product may be more relevant thananalysis of gene copy number.2 HercepTest directlyvisualizes the presence of the protein targeted byHERCEPTIN therapy, not its genetic determinants.

HercepTest is performed on two tissue sections (onefor primary antibody and one for negative reagentcontrol). The method uses an enhanced polymersystem that avoids the use of avidin-biotin techniques.The polymer is labeled with multiple HRP enzymesand secondary anti-rabbit antibodies, thus enhancingsensitivity without the background staining that canoccur from endogenous biotin. The detectionantibody in the HercepTest is a polyclonal rabbitprimary antibody that has a high affinity for theHER2 antigen in formalin-fixed, paraffin-embeddedtissues. Binding to non-HER2 protein is claimed to beminimal by the manufacturer. The specificity of theHercepTest primary antibody for HER2 protein hasbeen demonstrated by Western blot analysis usingestablished cell lines and stable transfectants. The

procedure takes about 3 hours to perform and isinterpreted by a pathologist.

Stained sections are evaluated using a graded scale,which is interpreted as positive or negative.Strongly positive (+3 staining intensity) and weaklypositive (+2 staining intensity) scores correlatewith response to therapy, while patients withnegative scores (+1 and 0 staining intensity) are nottreated with HERCEPTIN. These positive ornegative results, in combination with the patient’sclinical history and other diagnostic tests, aid inclassification of abnormal cells/tissues and providea basis for HERCEPTIN treatment selection.

Anti-HER2 therapy has been described as a truebreakthrough. Given the high prevalence of breastcancer, it is valuable to have a diagnostic test that canaccurately identify those patients most likely tobenefit from HERCEPTIN therapy. By reliablyidentifying HER2 positivity, the HercepTest enableswomen with metastatic breast cancer to be con-sidered for a new therapeutic option that may signifi-cantly extend their lives and decrease morbidity.

Editor’s note:Those interested in learning more about the HercepTestshould contact their Dako representative or visit theHercepTest Information Center at www.dakousa.com

References:1. Nassiri M, Planas L, Meng L, Assefi L, Morales AR. Mod Pathol. Annual

Meeting Abstracts, March 1997.2. Slamon DJ, Godolphin W, Jones LA, et al. Studies of the HER2/neu proto-

oncogene in human breast and ovarian cancer. Science. 1989;244:707-712.3. HercepTest product literature, Dako Corp. 1998.

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1962 Tissue-Tek EmbeddingRings become available to

histotechs in the lab

1962 John Glennorbits the earth

1957 The first satellites,Sputnik I & II, are launched

by the Soviet Union

1957 Erythromycin isdeveloped for penicillin-

allergic patients

1960 Dr. Albert Starr andengineer J.L. Edwards design

the artificial heart valve

1960 Frank Colton developsthe first oral contraceptive

1960 Roger Maris tiesBabe Ruth’s home run

record (60)

1961 Watson and Crickdecode the DNA molecule

1961 Soviet cosmonautYuri Gagarin is first person

to orbit the earth

1961 Alan Shepard makesthe first U.S. space flight

11

Alfred Ngbokoli must prepare several hundred perfect slides each week.

Each work day begins with a morning strategy session. (Left to right, AlfredNgbokoli, Dr. Tracey McNamara, and Dr. John Trupkiewicz)

1963 Cassette tapemakes its debut

1963 Dr. Michael DeBakeyis first to use an artificial

heart during surgery

1963 Hepatitis B isdiscovered and isolated

1963 Patent is issuedfor the first

computer mouse

1965 First space walks byAmerican and Russian

astronauts

1965 Medicare andMedicaid are enacted

1967 Dr. Christian Barnardperforms the first human heart transplant

1967 IBM builds thefirst floppy disk

1967 The Clinical Laboratory ImprovementAct (CLIA’67) establishes minimum quality

standards for clinical laboratories

All in a Day’s Work!By Vinnie Della Speranza, Scientific Editor

All in a Day’s Work! is a salute to the efforts ofhistotechs everywhere, those unsung heroes who oftengo unrecognized for their contributions to the advance-ment of health and science. Please contact the editor ifyou would like to see your work featured in thiscolumn.

“She was a big-eyed baby with a look that wasdescribed as an impish grin. Many were heartbrokenwhen she died suddenly at the age of sixteen monthsafter a brief and mysterious illness. Her name wasKumari. A short while later doctors realized thatanother baby, this time an eighteen-month-old malenamed Astor, had died years earlier of the same viralillness at another facility. Testing performed on storedfixed tissues revealed that the fatal virus belonged tothe Herpes family of viruses known to cause diseasein people and animals.”

These cases appeared in a recent report by DeniseGrady in the New York Times. In some ways, theyresemble countless examples that each of us mightshare of lost heroic attempts to conquer disease atany one of our facilities. What keeps us all going inthe face of defeat are our success stories, but fewwould disagree that we learn as much, and perhapsmore, from our failures as we do from those caseswith happy endings. What makes this story differentis that Kumari and Astor were baby elephants.

I had an opportunity recently to visit a very uniquepathology service located just north of New YorkCity at the Bronx Zoo. There, my gracious hosts,Alfred Ngbokoli, histology supervisor, and Dr.Tracey McNamara, chief of the pathology service forthe Wildlife Conservation Society (WCS), took meon a tour that left me feeling that each of us needs todo more. They didn’t give me a sales pitch. Frankly,they didn’t need to. They are scientists with a passionthat is contagious. They simply gave me the facts,which I share with you here. The facts speak forthemselves.

What makes this facility so unique is that while thereare 184 accredited zoos in North America, only fourhave pathology departments, and only three areequipped with histology labs! WCS services fourNYC zoos, the New York Aquarium and the WildlifeSurvival Center in Georgia, a refuge for endangeredspecies, with a total animal population of approx.18,000, representing almost 1500 different species.

Until my fateful visit to the WCS, I simply regardedzoos as a place for amusement, a fun place to take thekids. It turns out that modern zoos are so much moreand that the public never gets to see the mostimpressive part of any zoo, the dedicatedprofessionals working behind the scenes on behalf ofconservation. “The global human population, now atalmost six billion, is increasing by 85 million peopleeach year and as they multiply, humans appropriatewildlife habitats. Calculations suggest that if tropicalrain forests continue to be felled at the current rate,one-quarter of all wild species on earth will be lost in50 years” according to the 1998 annual report of the

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WCS. While zoo collections provide the only contactmost of us will ever have with wild animals, theirconservation efforts to save endangered speciesplace a much greater importance upon their workthan our simple entertainment.

At first glance, the histology laboratory looks muchlike yours or mine. A clean, well-equipped facility.Cubes of formalin, bottles of stains and reagents, andthe expected mountains of well-organized paraffinblocks and H&E stained slides, as well as therequisite jars of various sizes containing formalin-fixed organs of past studied cases are all within view.But as I look more closely, I begin to notice smalldetails that remind me that I am in no ordinaryhistology lab. A window looks out into a meticulouslyclean necropsy area, much larger than any autopsyfacility I have seen. The stainless steel cooler runsalmost to the twenty-five-foot high ceiling, strongevidence that hints at the clientele that are servedhere. Its second inner door opens out onto a loadingdock where trucks may pull up to deliver their cargo.A large electric hoist is suspended from the ceiling. Iam told however that it is not uncommon for necrop-sies to be performed on the floor when the patient istoo large to do otherwise. Those too large to bring tothe facility are necropsied in the field.

I glance closer at the jars of stored organs, eachlabeled with the appropriate case number as yours ormine would be. My mouth drops as I look moreclosely and the labels reveal that I am peering atorgans of siberian tiger, panda, crocodile heart, harborseal kidney, elephant, giraffe, cottonmouth snake,egyptian tortoise, penguin, and surinam toad, to namejust a few. I laugh out loud as I come across a jarcontaining a stuffed toy monkey wearing sunglasses, ataste of Alfred’s humor poking fun at us mere mortalswho only work with human specimens, I suspect.

A typical workday begins with a morning strategysession that first identifies any new cases that mayhave come in. Here the day’s priorities are estab-lished. Large animal necropsies require the participa-tion of many. Barring this, Alfred’s day will likely befilled with the sectioning and staining of a highvolume of paraffin sections from formalin fixedtissues, all to be stained with at least an H&E, but arather large volume of special stains, including PASand silvers for fungi, Kinyoun’s AFB, mucicarminefor cryptococcosis and silvers for spirochetes, as wellas iron stains by the hundreds may be needed.Working alongside the medical staff, the slides thatAlfred prepares each day can lead to discoveries thatare important to animal health and be of benefit towildlife worldwide.

13

1969 Neil Armstrongwalks onthe moon

1969 Videocassette tapeis developed

1969 Boeing747 Jumbo Jetenters service

1969 Scanning electronmicroscope is built

1970 The compact disk (CD)is introduced

1970 Ludwig Sternberger develops theperoxidase-anti-peroxidase (PAP) method,

revolutionizing immunohistochemistry

1970 The Tissue-Tek II Cassettebecomes available (in clear only!)

1971 Intel introduces the first

microprocessor chip

1971 Histo-Logicis created

by Lee Luna

1972 MedicalCAT scanning

developed

Large animals may be necropsied on the floor.

Animals too large to transport are necropsied in the field.

It is not unusual to find zoo veterinarians usingadvanced technologies like magnetic resonanceimaging, computerized tomography, ultrasonography,endoscopy, and mammography on zoo patients,according to Dr. McNamara. But the lack of rapiddiagnostic services at the vast majority of zoos istroubling. “The death of an animal in a collection isregrettable, but not to learn from it when there arestill so many unknowns in the field of comparativemedicine is truly wasteful,” she said.“The best hope ofavoiding losses may rest in rapid detection, necropsyand histopathology, of an initial or ‘index’ case of whatcould easily develop into an epidemic involving manyanimals.”

In truth, there continues to remain large gaps in theknowledge base of the health problems of non-domestic species. Whenever there is an unexplaineddie-off of animals in the wild, such as the death ofseals in the North Sea discovered to be due to a pre-viously unknown virus, or the death of large numbersof lions in the Serengeti caused by, of all things,canine distemper, scientists like McNamara arereminded of just how little is known about exoticspecies.

This brings us back to Kumari and Astor. In modernzoos it has become common practice to create mixedspecies exhibits where the risk of interspecies diseasetransmission is becoming a serious concern. Thedeaths of Kumari and Astor, the first Asian elephantsborn at the National Zoo in Washington D.C., and

the Bronx Zoo, respectively, led to the discovery thatAfrican elephants commonly carry a herpes strainthat is relatively harmless in them but is deadly intheir Asian counterparts. It is this very virus that isbelieved to be the major cause of death worldwideamong young Asian elephants in captivity. Thepractice followed by modern zoos of keeping rare orendangered species in flocks or herds creates a largeelement of risk since illness in one may rapidlyspread through an entire group. Indigenous wildlifeincluding rodents, raccoons, and migratory waterfowlpose another threat of disease introduction into zooanimals. The tools that veterinarians employ in petsand farm animals, such as antibiotics and vaccines,are simply not available for the treatment of exoticspecies simply because their effects in thesepopulations have not been sufficiently studied andthey could actually do more harm than good.

McNamara is working hard to change this. She isactively engaged in collecting diagnostic and necropsyinformation and images that are shared electronicallyvia telepathology to veterinarians in remote locationswithout the facilities found at WCS. Each month,Alfred is asked to prepare several hundred ‘perfect’slides that are shared with zoos around the world orincorporated into teaching sets. This past year,

14

1973 MRI medical imagingintroduced

1973 The Tissue-Tek IIopaque Cassette appears

(in white only!)

1975 The first personal computer

debuts

1975 Microsoft is founded byBill Gates & Paul Allen

1976 Concorde supersonic airlinemakes first passenger flight

1976 Steve Wozniak and Steve Jobsbuild computer circuit board and

form Apple Computer Co.

1978 Tissue-Tek II Cassettesfirst appear with colors

(yellow, blue, green, white)

1977 First test tube baby born in England

1978 Tissue-Tek III Uni-Cassetteappears (in white only!)

Dr. McNamara standing near the oversized refrigerator and electric hoist neededfor large animals.

Alfred preparing a leopard for necropsy.

McNamara obtained a large collection of koda-chromes and glass slides from the AFIP which con-tains a wealth of information that she intends to sharewith her colleagues. I learned that the AFIP’s interestin animal health and disease traces back to the civilwar when the study of pathology in horses was seen tohave strategic importance to government soldiers.

McNamara feels that “a strong argument can bemade for the need for diagnostic pathology servicesfor all zoological collections. Perhaps even more sothan their domestic counterparts, zoo animals are inurgent need of accurate and rapid pathology support.Information gained from review and interpretation ofsurgical biopsy, cytologic, and necropsy material by apathologist empowers zoo staff to take managementsteps to halt or prevent disease problems. Only thenwill it be possible to ensure that these high riskanimals do not succumb to a preventable disease forsimple want of a diagnosis.”

(editor’s note: readers may find additional informationabout issues confronting the world’s wildlife atwww.wcs.org , homepage of the Wildlife ConservationSociety)

References:1. McNamara T. Emerging infections in captive wildlife. In: Nelson AM, Horsburgh

CR, eds. Pathology of Emerging Infections 2, Washington, DC: ASM Press; 1998:chap 18.

2. McNamara T. The role of pathology in conservation. In: Fowler ME, Miller RE,eds. Zoo and Wild Animal Medicine: Current Therapy, Philadelphia, Pa: WBSaunders Co; 1999: chap 2.

3. Wildlife Conservation Society annual report, 1998.

A Closer Look at XyleneSubstitutes to Reduce theHazardous Waste Stream Deborah Maini, MS, CT(ASCP)HTLCytotechnologistNorth Shore University HospitalManhasset, NY

AbstractXylene has been known for many years to be ahazardous chemical used in large quantities inhistology laboratories. This solvent has a toxic effecton human health and the environment that is well

documented. However, its excellent reputation as aclearing agent has made it difficult for alternatives tobe considered in the laboratory. One key feature ofxylene that has made research into alternatives morepopular is the high expense associated with itsdisposal. Reducing the xylene waste stream may beaccomplished through recycling or the use ofsubstitute clearing agents. Recycling systems requirea significant capital outlay but are considered costeffective and payback of capital expenditure isquick–approximately one year, making recycling anattractive proposition to hospital administrators.More importantly, it reduces the laboratorygenerated waste stream for this hazardous material.Replacing xylene with substitute clearing agents mayalso be cost effective, but only if disposal costs can beeliminated. Substitutes however, may provide a saferwork environment for staff. Some substitutes,specifically the short chain aliphatic hydrocarbons,may also be recycled. In this study, the performanceof popular xylene substitutes available today iscompared to xylene.

IntroductionXylene continues to be the solvent of choice forcarrying out the clearing and deparaffinizing of tissuesduring processing and staining in histology labs.4 It isalso used in the staining schemes employed in bothhistology and cytology laboratories. In 1971, the totalxylene production in the U.S. was 612,325,000 gallons,up 14% from the previous year.5 It has been estimatedthat today histology laboratories alone consume morethan 1.5 million gallons of xylene each year.4 Thisoccurs despite the known hazards of this materialwhich have been well documented. In 1975, NIOSH(National Institute for Occupational Safety & Health)issued its criteria recommending a standard foroccupational exposure to xylene. This was done todevelop a program to reduce the occupationaldiseases arising from chronic exposure to thiscompound.At that time, NIOSH found xylene toxicityto have a narcotic effect on workers causing muscularweakness, reduced muscle coordination, and mentalconfusion, as well as irritation to skin and mucousmembranes including the conjunctiva and respiratorytract. OSHA has stated that xylene may have a targetorgan effect on fetuses. Repeated exposure canproduce neurotoxic effects leading to irreversibleCNS damage, as well as damage to the liver, kidneys,

15

1980 PortableVCR-camera debuts

1981 First flight of areusable space craft,

the space shuttle Columbia

1981 First IBMpersonal computer

1981 Earliest cases of immunodeficiency that will eventuallybe called AIDS are noted in New York and California

1983 The cardiac pacemaker wasdeveloped by Wilson Greatbatch

1983 AT&T operates the world’slargest fiberoptic communication system

connecting New York to Washington

1982 First implanted artificial heartdesigned by Robert Jarvik

1979 The first Tissue-TekVacuum Infiltration Processor

(V.I.P.) appears

and gastrointestinal tract.4 NIOSH recommended thatworker exposure levels not be permitted to exceed100 ppm time weighted average over a ten hourperiod, which continues to be the OSHA enforcedstandard today. The problems associated with workingwith xylene are compounded by the fact that it maytake 72 hours or more to clear from the body.4 This isespecially problematic for technologists who may berequired to work more than a five day work weekwhich will effectively prevent the solvent from beingfully cleared from the body in such individuals.

Its toxicity and flammability characteristics makexylene a hazardous chemical waste whose disposal isregulated by the US Environmental ProtectionAgency. In addition, accredited medical laboratoriesare required by the CAP to have a hazardous wasteminimization plan in place. Although relativelyinexpensive to purchase, the disposal costs of xylenecan be high since it is not permissible to dischargexylene into the environment via wastewater (draindisposal). The EPA requires that hazardous waste becollected by the generator of the waste and disposedof by a waste hauler licensed by that agency. Federallegislation (RCRA) holds the waste generator (thelaboratory) responsible from ‘cradle to grave’ for anyharm that comes to the environment caused by afacility’s hazardous chemical waste.1 Disposal costs arerapidly consuming a tremendous chunk of alaboratory’s budget which is especially burdensome inthis era of managed care.

The best waste minimization strategy is to replacehighly hazardous chemicals with less hazardous ornon-hazardous products.2 A number of xylenesubstitute products have been offered to laboratoriesby chemical suppliers to provide a safer alternative forlaboratory professionals. These may be categorized aslimonene-based clearants, aliphatic hydrocarbons andhydrocarbon blends.The blends are hazardous in theirown right and so are not considered further here.Once thought to be quite safe, limonene has beenfound to be a sensitizer, predisposing laboratoryprofessionals to chronic allergic reactions afterprolonged exposure, posing a serious health risk.

Substitutes classified as aliphatic hydrocarbons havereceived the greatest attention.They appear to be lowin reactivity and toxicity. Manufacturers claim thatthese clearing agents are non-irritating and non-

sensitizing. Their PEL of 300 ppm per 8 hours, whichis well above that of xylene, would be difficult toachieve in a well ventilated room without heating thecompound.1 Substitutes falling into this group may befurther divided into long-chain and short-chainaliphatic hydrocarbons. Short-chain aliphatics aremore volatile and have a flash point below 110°F(those with a flash point below 100°F are classified as‘flammable’ and have special storage requirements),while the long-chain varieties have flashpoints above140°F. The short-chain substitutes are lighter inweight and will penetrate tissues faster than theirheavier cousins.1 They may also remove lipids moreeffectively but are relatively intolerant of watercontamination, requiring the laboratory professionalto be vigilant in adhering to a strict solution rotationschedule. Long-chain aliphatics approach theconsistency of a light oil.

Materials and MethodsTwo short-chain aliphatic hydrocarbon and two long-chain aliphatic hydrocarbon xylene substitutes wereevaluated in this study. The long-chain aliphatichydrocarbons selected were Slide-Brite Clearant (S &S Company, Albany, GA) and Micro-Clear (Micron,Fairfax, VA). Both of these clearants are considerednon-toxic and non-flammable, which simplifies storagerequirements. They also offer the possibility ofrecycling or drain disposal with permission from localwaste water authorities. The short-chain aliphatichydrocarbons selected were Pro-Par Clearant(Anatech Ltd., Battle Creek, MI) and ShandonXylene Substitute (Shandon-Lipshaw, Pittsburgh, PA).These also are non-toxic but their low flash pointsrequire special storage as flammable liquids whichmakes permission for drain disposal unlikely.

Five parallel pieces of human tissue were cut fromeight surgical specimens. Representative sections ofskin, colon, lung, placenta, prostate, spleen, and twocases of uterus were selected. Tissues submitted forprocessing were approximately 3 mm thick. Tissueswere fixed in 10% neutral buffered formalin (FisherScientific) and processed overnight on a SakuraVacuum Infiltration Processor (VIP) tissue processorwith a fourteen hour processing cycle (Table 1)utilizing 10% NBF, graded ethanols and cleared inxylene or one of the substitute clearants studied.Tissues were infiltrated and embedded in Tissue-Prepembedding medium (Fisher Scientific).

16

1984 Macintoshcomputer goes

on sale

1984 Genetic (DNA)fingerprinting becomes

available

1985 Microsoft and IBM createthe operating system for

the IBM PC

1985 The first gene amplificationtechnology, Polymerase Chain

Reaction, is discovered

1986 CDCuniversal precautions

are developed

1989 The National Institutes for Health begin the

human genome mapping project

1990 The HubbleSpace Telescope

is deployed

1991 OSHA’s BloodbornePathogens Standards

are established

1991 Genetically alteredmicroorganisms are sold

for commercial use

Four-micron sections were cut from each of theblocks, dried at 60°C for one hour and stainedtogether H&E on a Leica Autostainer. Respectiveslides were deparaffinized and cleared on the slidestainer with xylene or the appropriate xylenesubstitute before being mounted. The stained slideswere evaluated blindly by eight pathologists whosescores were averaged. Sections were assessed fornuclear detail, cytoplasmic membrane, cytoplasmicclarity, hematoxylin staining, eosin staining, andoverall appearance of the section for each clearingagent and graded with a score ranging from 1(unacceptable) to 5 (best) (results summarized intable 2). Scores from one pathologist who found allslides to be unacceptable were deleted from the study.

ResultsOverall, the performance of the xylene substitutesstudied was found to be inferior to xylene by theevaluators. The performance of each substitute is sum-marized in table 2. Numerical scores represent theaverage score among the observers for each criteriastudied. Bias arising from the familiarity of thereviewers with tissues processed and stained withxylene as the clearant cannot be ruled out.Two, Pro-Par(Anatech, Ltd. Battle Creek, MI) and Slide Brite (S&S,Georgia) were found to more closely approximate themicroscopic appearance of tissues prepared with xyleneand warrant further study to see if results can beimproved.They were also among the least expensive.

DiscussionThis study suggests that xylene substitutes may offera viable alternative to the hazards associated withxylene for laboratory staff. It is important to notethat substitutes rarely perform identically to thematerial being replaced. The user must be preparedto adjust procedures to accommodate suchdifferences. Substitutes may only offer economicadvantage if disposal costs can be eliminated, eitherthrough recycling, burning as a fuel supplement, ordrain disposal. Local water authorities must becontacted for approval to discharge a substitute intothe waste water, despite any manufacturer’s claimsthat this is acceptable for their product. Due to thelimited nature of this study, the frequency withwhich substitutes may have to be removed from theprocessor or staining instruments relative to xylenecould not be assessed. The economics of xylenesubstitute use require a closer look since it is welldocumented that the aliphatic hydrocarbons are lesstolerant of water contamination than xylene, posingpotential difficulties especially in more humidclimates.

Table 1Tissue Processing Scheme—Sakura VIP

• note: all stations utilized pressure/vacuumStation Solution Time Temp1 10% neutral formalin 90 min 42°C2 10% neutral formalin 90 min 42°C3 60% ethanol 30 min 42°C4 95% ethanol 60 min 42°C5 95% ethanol 60 min 42°C6 100% ethanol 60 min 42°C7 100% ethanol 60 min 42°C8 100% ethanol 60 min 42°C9 clearant 60 min 42°C

10 clearant 60 min 42°C11 paraffin 40 min 60°C12 paraffin 40 min 60°C13 paraffin 40 min 60°C14 paraffin 40 min 60°C

17

Table 2Averaged Scores of Clearant Performance

Assessed Blindly by Seven Observers UtilizingA Scale of 1-5 (1 = poor, 5 = excellent)

ShandonMicro- Slide- Xylene

Category Clear Xylene Brite Sub. Pro-Par

Nuclear 3.71 4.0 3.86 3.29 4.00Detail

Cytoplasmic 3.71 4.0 3.86 3.43 3.29Detail

Cytoplasmic 3.43 4.14 3.71 3.43 3.57Clarity

Hematoxylin 3.57 4.14 3.71 3.71 3.43Staining

Eosin 3.43 4.0 3.57 3.43 3.57Staining

Overall 3.60 4.2 3.83 3.42 3.80appearance

Aver. Score 3.6 4.1 3.8 3.45 3.6

1992 The Internetbecomes available

to the public

1992 The finalCLIA’88 regulations

are published

1993 Intelintroduces the Pentium

microprocessor chip

1994 The Hubbletelescope discovers the

Black Hole

1995 Microsoft releasesWindows ’95

operating system

1995 DVD isintroduced

1997 The firstseptupletsare born

1998 Mark McGwirehits 70 home runs

1998 Viagra ismarketed to correct

male impotence

1996 World Chess champion Gary Kasparovloses first game to IBM computer program

called Deep Blue

1997 Dolly the sheep is cloned

by Scottish scientists

References:1. Dapson JC, Dapson R. Hazardous Materials in the Histopathology Laboratory,

Regulations, Risks, Handling and Disposal. 3rd ed. 1995 Anatech LTD; 1995: 127-138.

2. Ornelas PE, Hogrefe RF, Dapson R, Dapson JC. The role of recycling andchemical substitution in pollution prevention programs. Lab Med. 1998, 29(6):356-359.

3. Ornelas PE, Hogrefe RF, Dapson R, Dapson JC. The laboratory’s role inreducing hazardous waste. Lab Med. 1998; 29(5) 287-290.

4. Algeo LA. Xylene, Controversial mainstay in histology laboratories. Adv MedLab Professionals. 1994; 6(8):8-9.

5. NIOSH Criteria Documents, Criteria for a Recommended Standard:Occupational Exposure to Xylene, DHHS Publication no. 75-168, 1975.Available at: http://www.cdc.gov.niosh/75-168.html.

6. Wynnchuk M. Evaluation of xylene substitutes for paraffin tissue processing.J Histotech. 1994; 17(2): 143-149.

7. Jones R, Morton A, and Moghissi A. Comparison of deparaffinization agents foran automated immunostainer. J Histotech. 16(4):367-368.

Histotechs to Invade the SmallestState in the UnionBy Vinnie Della Speranza

This fall, histotechs from all over the nation andsome from distant points around the globe, willdescend on Providence, Rhode Island as theNational Society for Histotechnology hosts its annualSymposium/Convention, October 16–21. Those whomay be visiting the state of Rhode Island andProvidence Plantations (the full name for the state)for the very first time will find that they are in for areal treat. Only 50 miles long by 40 miles wide, (somesay an hour long and a half hour wide), every part ofthe state is accessible without big city delays. Onlyone hour from Boston and three hours from NewYork City, techs will be able to learn by day and playby night.

Providence is one of the last undiscovered great citiesin the United States. Newsweek Magazine has namedProvidence one of the “Hottest Cities” in America.And Parade Magazine listed Providence as the “safestcity in the continental U.S.” Why all of the accolades?With a wealth of art galleries and loft studios, a TonyAward-winning theater company, a series ofBroadway shows at the Performing Arts Center, astate orchestra, two ballets, fine museums, collegetheatre, choral groups and a nationally-esteemedschool of design, Providence packs an artistic punch.

The state’s capital, Providence has a long and richhistory. Founded in 1636 as a bastion of religious

freedom by a preacher from Salem, Massachusettswho fell into disfavor with the morally conservativepuritans, Roger Williams thanked God forhis “merciful providence.” Located at the confluenceof three rivers at the head of Naragansett Bay,Providence has a long history of sea trade with allparts of the world, including the near and far east. Thesea made Providence wealthy in the 18th century aswitnessed by the number of grand Victorian homesflanking the city’s river banks. One family in partic-ular, the Browns, ascended quickly to the highestranks of Providence business and society, endowingBrown University and bringing back lovely treasuresand artwork from China (on exhibit at the JohnBrown House).The Industrial Revolution of the 1800sshifted the focus of the city’s economy to manu-facturing, counting textiles, jewelry and silverwaremanufacturing among its largest industries.Providence was once deemed one of the “FiveIndustrial Wonders of the World”.

One of Providence’s charms is that you can seevirtually the entire city on foot.A brief stroll from theConvention Center will allow visitors to take in mostof the historic and architectural sites, including thenew Providence Riverwalk’s beautiful promenadethat stretches from the capitol area to the head of theBay. A short hike up College Hill brings you toBrown University and the Rhode Island School ofDesign, as well as several interesting museums andlibraries. One can stroll up Smith Hill to the historicState House, cruise the shops, art galleries, andrestaurants of Downcity or walk to Federal Hill, oneof the city’s great ethnic neighborhoods. Unlike bigcities, Providence has the charm of a place wherenatives rarely walk more than just a few blocksbefore meeting someone they know.

Do you enjoy good food? You’ll be delighted to findthat Providence is a first class restaurant city withbargain prices. According to the Boston Globe, whenBoston’s finest chefs dine out they go to Providence.Frequent appearances on the Top Ten lists of suchmagazines as Gourmet, Food & Wine, and BonAppetit keep the city’s dining experience in the publiceye. After departing from a local eatery, Julia Childwas heard to remark, “I wouldn’t change a thing.”Seafood is a specialty in Providence but some of thefinest Italian food in the world is also found here, aswell as French, Indian, Thai, Japanese, Greek, and

18

Sources for Countdown to the 21st Century:Twenty-five years of laboratory medicine. Lab Med. 1995;26(1).Jones TE. History of the light microscope. 1995. Available at: http://www.utmem.edu/personal/thjones/hist/hist_mic.htm.History of western biomedicine Karolinska Institute. Available at http://www.mic.ki.se:80/west.html#west 4.Milestones in cardiology. The Heart: An Online Exploration. The Franklin Institute Science Museum. 1996. Available at http://sln2.fi.edu/biosci/history/firsts.html.The national inventors hall of fame. Available at http://www.invent.org/book/index.html.The history of medicine. Galaxy 1993-1999. Available at http://orion.einet.net/galaxy/Medicine/History.html.Milestones of technology. The Mercury Center. 1997. Available at http://www.4.mercurycenter.com/special/techmuseum/milestones/

Portuguese. A major international culinary institute(Johnson & Wales University) keeps the kitchens ofthe city supplied with its talented chefs. If casual ismore your style, try the bevy of cafes andcoffeehouses around the city that welcome you for acup of java, the daily newspaper, and plenty ofpeople-watching.

And after a full day of meetings, the city is full ofnightlife. There are dozens of clubs, theaterperformances, and concerts every weekend in theDowncity area lasting well into the early hours of themorning. For anyone interested in venturing furtherout, Newport, located on the state’s 32 miles of coast,is a 40 minute drive. Autumn is a wonderful time toconsider an excursion to the orchards of westernRhode Island or to see the fall foliage. You mightconsider bringing the family along to enjoy all thatthe area has to offer.

Just before press time, Kerry Crabb, NSH ConventionChair told Histo-Logic that this year’s conventionprogram will include 98 workshops, almost half ofwhich will be presented for the first time inProvidence. In keeping with the meeting’s new format,workshops and seminar presentations will be offeredeach day. On Saturday morning, a special plenarysession will be sponsored by the Society for AppliedImmunohistochemistry entitled “Selected Topics inImmunohistochemistry.” A change from pastmeetings, the poster exhibits this year will be locatedin the center of the vendor exhibit hall to make themmore accessible to the attendees. Those traveling toProvidence by auto may be interested to learn thatsome of the convention hotels will offer free parking,a substantial savings for those on a budget. Be sure tocheck the conference program for details.

It isn’t too early to plan to join the NSH inProvidence, October 16-21. Convention week iscertain to be educational and memorable. Programswill be mailed in late Spring to all NSH members. Ifyou aren’t a member, you will surely want to takeadvantage of the member rates for registration andworkshop fees. Visit the NSH website at www.nsh.orgfor membership information or call (301)262-6221.

Sources:Greater Providence Visitors and Convention Bureau,http://www.providencecvb.com/

Guest Guide to Greater Providence & Warwick, from the editors of Rhode IslandMonthly, Anchor Publishing, 1997.

Providence Meeting and Destination Guide, Greater Providence Visitors andConvention Bureau, 1994.

MeetDennis KeastSakura Finetek U.S.A.,Inc., is proud to announcethe appointment ofDennis Keast as the newArea Manager for Ohioand Michigan.

Dennis attended OaklandUniversity in Rochester,Michigan where he earneda B.A. degree in Psy-chology. He has been insales for five years.

Dennis’s interests include football, hockey, chess, andreading.

19

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The editor wishes to solicit information, questions, and articles relating tohistotechnology. Submit these to: Vinnie Della Speranza , Histo-LogicEditor, L2, 766 University Hospital, Stony Brook, NY 11794-7025. Articles,photographs, etc, will not be returned unless requested in writing when theyare submitted.

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High School Eligibility Route forHT(ASCP) May End by 2005On February 18, 1999 the NSH board of directorsapproved a motion to formally request the ASCPBoard of Registry to discontinue the Route IIIeligibility option for histotechnician (HT) certifica-tion by January 1, 2005. Route III eligibility presentlyallows individuals with a high school diploma to sitfor the H.T. exam after receiving on-the-job trainingand two years of work experience. The NSH boardhas taken this position believing that the complexityof new technologies available now and on thehorizon will require the histotechnician of the futureto possess a greater academic foundation in order toperform successfully on the job.

This request to the ASCP Board of Registry willculminate a 10-year effort by NSH to increase theeducational standards for histology technicians. Ifapproved, it will put the histology technician on thesame level as the medical laboratory technician(MLT), with the same educational standards.

NSH president Sumiko Sumida said, “Histologytechnicians today are expected to do more than justproduce microscopic slides for the pathologists. Manylaboratories are expecting histology technicians to“gross-in” small specimens. CLIA requires an AA

degree or 60 semester hours of college for anyonewho starts performing this task after April 25, 1995.NSH would like to ensure that all future HT(ASCP)certified technicians can meet the CLIA regulationsto perform gross description.”

Technicians who have not yet taken the HT certifica-tion examination and who do not possess academictraining beyond high school are urged to make plansto do so now. Their eligibility to take the exam afterJanuary 1, 2005 may be lost.

Get to KnowRoss LindmeyerMeet Ross Lindmeyer, newArea Manager for Iowa,Upper Peninsula Michigan,Minnesota, Nebraska, theDakotas, and Wisconsin.

Ross comes to us witha sales career of over15 years with Cardinal

Health, Inc. as a Hospital/ Managed Care consultant inpharmaceutical distribution.

His interests include golf, gardening, fishing, and otheroutdoor activities.