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4

Histochemistry: A Case of Unappreciated Beauty ? RenC J. Buesa Fern Forest Road, Cooper City, FL

Why Talk About Beauty? This article stresses the nature of histology as a work of It all started during the second half of the 17th century

art of intrinsic beauty and the fact that 95% of all the pro- with the discovery of protozoans by Leewenhauk, the ob- ~edures presently used were developed between 1841 and servation of blood cells inside the capillaries in the wing 1949. The article also includes information about the time membranes of bats by Malpighi, and the feathers, fish required to complete the most common histochemistry Pro- scales, and the porous appearance of slivers of cork by tocols, with an overall average of 39 minutes for each. Sala- ~ ~ ~ k ~ . ries ranges and the cost Per slide are also presented. The All early microscopists were amazed by what they saw costs vary from $ 0.78 Per slide for ~rotocols to detect with their primitive microscopes, and they all wrote about organisms in tissue using laboratory prepared solutions and the beauty of their subjects of observation. They also real- performed manually in batches of 10 slides, to $ 10.11 per ized that big organisms had to be teased or squashed to slide for immunohistochemical tests completed by hand in show their microscopic features, so maceration, teasing, and batches of 40 slides using commercially prepared reagents. compressing, which ultimately lead to specimen cutting or The advantages and limitations of autostainers for histo- microtomy, became the starting point of our trade: histo- chemistry and immunohistochemical procedures also are discussed. Photomicrographs are examples of the beauty of ~ ~ ~ l ~ i ~ ~ from those early days when the microscopist our finished work that, because of their perception as mere both and studied the objects, the 19th century wit- diagnostic tools, usually receive less than 1 minute of nessed the rapid development of instruments and techniques attention in the pragmatism-ruled pathology environ- that has continued to our days (Table 1) (I), and with them ment. (The J Histotechnol 26:91, 2003) a new type of line of work also developed, the histology

technician who since the beginning provided the finished Submitted: April 16, 2003; Accepted April 29, 2003 slides used in all types of studies, from botany and zoology

to human research and medicine. Key words: histology as beautiful art, salaries in histology, In perspective, the advances have been enormous, but time to complete histology procedures, costs per slide, despite all the new pieces of equipment now available and autostainers all the modern and sophisticated methods, there is some-

Table 1. Historical Development of Histology Procedures (1)

Period

Before 1900 Between 1901 From 1950 to

Fixation, processing, sealing-mounting, and general preservation and display of specimens

Histochemical procedures Whole mount, injections, electron and phase microscopy,

and other general procedures Total of "traditional" histology proceduresa Immuno and biomolecular proceduresb "The bulk (95%) of all the "traditional" histology procedures were developed between the year of the first published recipe (1841) and 1949. 'Aside from h~stoenzymology (1937) and immunofluorescence (1942), the rest of the present array of advanced and esoteric histology procedures were developed dur~ng the second half of the 20th century.

The Journal of Histotechnology I Vol. 26, No. 2 1 June 2003

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Table 2. Time Required to Cbmplete Histochemical (HC) and Immunohistochemical (IHC) Procedures

HC Proceduresa Minutes required to cotnplete No. procediires % of all C~imcilated %

<I5 minutes 10 27 27 16 to 30 minutes 10 27 54 31 to 45 minutes 6 16 70 46 to 60 minutes 3 8 7 8

1 to 2 hours 4 11 89 2 to 3 hours 0 0 89 3 to 4 hours 1 3 92 Overnight 3 8 100

IHC Procedures with Different Detectioiz ~ ~ s t e t n s ~ , -

Slides per ruiz ABC system LSAB+ systenz Envisioiz systenz

1 to 12 2.0 to 2.4 hours 1.5 to 1.6 hours 1.7 to 1.8 hours 13 to 24 2.25 to 2.75 hours 1.5 to 1.8 hours 1.8 to 2.0 hours 25 to 36 2.5 to 4.1 hours 2.0 to 2.5 hours 2.2 to 2.9 hours 37 to 48 3.5 to 4.8 hours 2.5 to 3.5 hours 3.0 to 5.0 hours

Average time per slide (minutes/slides) accordiizg witlz the number of slides/ri~iz~

Slides/run Minutes/slide

7 20.0 12 12.0 19 8.0 24 6.5 3 1 6.0 3 6 5.0 43 4.8 48 4.5

"Thirty-seven most frequent procedures performed manually, some with microwave oven boosts. the DAKO autostainer.

'Average timetslide for the three detection systems and the DAKO autostainer

thing that all the present technology has been unable to the staining procedure, the human element, the pride and substitute: the loving hand of the histology technician, the the feeling of accomplishment that any of us experience care that he or she puts in the art of producing micro- when, at the end of the whole procedure, we look at the scopic slides, the "touch," the appreciation for beauty in slide and feel the intimate satisfaction of a beautiful job the contrast of colors, for the differentiation "end point" of done!

Table 3. Average National Salaries for Histotechnicians (HT), Histotechnologists (HTLs), and Histology Supervisors (HT-Sup)

Category (dollars/ho~ir)

Aspect HT HTL HT-Sip

Average national salary in 1992 12.00 13.60 17.60 Average national salary in 2000 15.30 18.00 21.00 Hourly salary increase between 1992 and 2000

(dollars/hour) +3.30 +4.40 +3.40 Hourly salary increase between 1992 and 2000

(as a percentage in 8 years) +27.5% +32.4% +19.3% Salary increase as an average annual

percentage = (above percentage) + 8 +3.44% +4.05% +2.41% (Average annual percentage) x 2 [for the

two years between 2000 and 20021 +6.9% +8.1% +4.8% Factor to calculate mean 2002 salary 1.069 1.08 1 1.048 Average salary at the end of 2002 = average

salary in 2000 x above factor $16.36 $19.46 $22.01

92 Histochemistry: A Case of Unappreciated Beauty? 1 Buesa

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-4 43mm5m kits 263 1-30 Cbt31mms-g 1.30 s o h r t i o n s m i d *- a17 0.24

bbor MSES* 138 0.9 T d c o g t p e r s ~

With ~ m n g - 4 kits 391 1.84 W i wlulim 2.60 1.44

T&iie-dorrrwoEk,lmown-theb ~~e b d h w d d y (Z3) intheseme tbat beautyisthehabwkofwu~l%isbeautybnotonly widenthtbe-c-hof f a r ~ ~ a y d ~ n d r t d n g t b E s e ~ d t h e ~ - e # M i ~ ~ a ~ m ~ M i ~ S o E i e t g Q f L o n - dm ~gtbeV~~age,bntdso, forexample , in the atmugeat of&ozeo c o l d sesame seeds in om days (4). Euerywbmbkinwrd;l i lyworir,beitaperf&%- tion. a crisp rolltine staining. a perfedy oriented shave skin

biopsy, an o p h d l y differentiated s i k stain or any slide dme with cam. we find beauty of which to be proud!

Materials and Medhads This article does dot only discuss ttie h a l y of our tmk

but also prides. some pradd iasight about aur work h apcts te-g the t h e required and the c a t i avdvd in rompleting it. To ~cc0mpEisb thew fmo ~~bjedves, f have usrd the results of a national d a q survey 15) a d h e summary of dl the pmdwiviry studies Z have perfamed during my career that extended from 1952 to my &mnt last year and W ~1~ pdim from lab assistant to sitpenisor. manager, and consultant in histology. The tis- sues usea tu prepare tbe * t o m i w h e d slides were processed wiih mineral oil (63; dl ibe &ohtiom for the his- ~~ m s were prepad in the Istbomtoq from pure chemicals and dry dyes a d dl the protocols were perform4 manually. All the p h d m - taken with & r e m apcbmmatk abjeaives using an automatic film m n e m s r a t e ~ o ~ ~ t h ~ f ~ e l I ~ t b e c o s t and the time to complete pmmml. The WU- s i o n d ~ ~ ~ t h a t ~ w i i b q d

t d times can show d i f f ~ o c e s in the ~~ddide.

F@r@l.A, & u k ~ & E ( a u & c ~ ~ a 1 4 0 : l . t ' ~ s t s i n a d by lab aides a! an average ~ o s t ol W.WdIide B. Msmds t6dwotW at 4Q:I (Wrnin at SZ87I~lideE

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EiewLwgDowrXtTakeToCreateAR~Bsa~? Completing a bistubgy slide inch& many steps, &h

wirb its own time fktur. There is involved in gmsshg d cassetting th specimeo; the autmmic tissue processQrs require the manual wka of maimmmce, as well ss those ~ ~ t o g e t t i q g t h e s p e c i m e n s i n d o u t ~ g a n d atling are ~ n W y manual e o n s even w b using m i a t o m s with a m m t i e blmk &anm for trirmning arrd even sedmbg, Some s d l laba&es M t have aute ~ a r m d t s e w h o l e ~ e o f d s t a i P i q g ~ i s mud. Wdtiag c a s m s a d slid& is d y a manual task arsd when wmlipphg is atnomated the cuversQqxm have tobe laded andda&dmandIy.as w d l a s m y

Wtht lbwe steps stress the fan that himlogy is m a i n l y a d ~ v i t y a n d t h i s v t x y f z c t ~ p t e t o t h e treautydtkslidesandtothe*thate;lehhbmd d a i v e s f m m ~ a c t i v i t p ~ i n w m e ~ 9 n d s r e p s , i s qite time mmming.g. Sel- th 37 maat hquwtly re- FM ' try(HC)pocedmesTdtodo,itwas *=thanw-bemdiakgs* W an hour (Table 2) with d d m mge h m 5 m i n n c e s ( ~ r e d d A ~ O ] t 6 ~ e r n i g h t ~ ~ - y l ~ ~ I ) e G a l a a t t r a , d t b e c l a s s k P T ~ ~ r e - ~ g i n a n o v e t a l l a v a g e o f 3 9 ~ n b e 6 T b e ~ f a r t i s s u e c ~ w ~ 4 3 min~teseaAandfortbe

detection of p&6@m io tissue tbe average is 18 minutes per ptwol. NOR inclnded in these ihmkbns is the the wed for dewaxing to hydmtioa at the starl aad dehyctrating t r r ~ g a r t f i e e n d o f a I i y t I m h Q i f t b a t a ~ a b w t 3 0 minuwto thetataldtrration ofany J3c w n s p e c i d s ~

With regards to i r m n u n ~ t o c ~ IIHC) pmtocoIs, the duration varieg with the dewtion system rmd the nmn- ber of slides mil antigem tu detm per run, and one m~ Mlude about 2 honrs # coritpletc dewaxing to hydrating smd the beat-indnced epitoge retrieval steps, regadas of wbewphe run is fiaished m u a l l y or with an>amo&m W h the M C promo1 is totally mwd. it will last about 7.5 bun versus 3 horn of manual *ps wben completed automaticrrlly. The salary e o m p m t attached ta this time d i f f ~ , d o n g w i t h t b e w ~ inthedeli~eny and wmmptbn I# rmgea&, justifqr tbe initial cast or the re- agents pumhse commitments aSgociated with some au@ stabm systems.

w h a t r F + t h e ~ ~ o f A l l T h k ~ Them are d x y diBemw between hkmechicians

(H'rs). hist-lom m 1 , d ~~ E a p T h s (HT-Sup] and Meen m&ns of d~ oonnhy (3. Supmi- sors em more but their aanual salary average inrxease was the lowest amongst dn'm gmups (Table 3). with tbe bi- a V w ~WESp0Idhg b tbe

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acmrding with the survey, W e t coast salaries are tkhighestinahemtim. ForeachgrmrpW,IFfl,and H T - S ~ ) t h e ~ v e m g e ~ g a l a r y ~ n t ~ 1992 and20ZK)wascalcdafedandusedto~thesalaryat tbe emj of 2W32 for each (Table 3).

It was alsg considered tlpu HC procedur%s are usrvllly completed by HTs and HTLs, and therefore their salaries were averaged to calcnlh the cost of d~~~pdilre~. The

@aria of HTL and HT-Sup w m averaged ta dcnrlate &e cost of IHC p r m e d m s k m w his is tbe usual distrihtion of tfiese tasks in the majority of 1aWat:ories. In the cost per -dare. tbe reagents used -and their amounts and if the -gem were bought as kikief, as c o m m m d l y p a e p d s e Iulions. or if a11 the salutims we= made in the Iabmtmy h m pure chemicals and dry dyes alse were included,

The average time per prooedure (Table 21 times the ev- erage d a t y per techid cakgory (Table 3) served to 4- caJate thesalary mqxmntof thecmt, to which thema- *rids corn were added to get to t l ~ total cost per typ: of prdm (Table 4). Finally. a l l h e HC protmls were ass& as if- by hand tn batches of 10 slides, d the M C in batches of 41) slides when done m d y . and in M e s af48 slidmif run wi& an autm-. It is a fad that the ma per slide inversely depends on the numhf of slides per batch or run and this hsts to be taken into consideration when scheduling the task in the hhatwy.

For both I32 and KHC procedum, it is widen1 fhat it is much c b q m to use soMions prepared in the hhrabry. T h o s e ~ g ~ ~ h a r g u e o n p n d s ~ m s s - sumed p a r e r wnsistency of cclmercid mlotims, but L jnstdbge with this mtiw -use, on tbe o m haad, it is not that d i € h d t ta prepare a solution and, on tbe &r, none of these KC p n d m s ax expeeted ra provide quan- titative results and therefore allow for some leeway in the intensity (quality) of tbe reaction.

E i s a l s o e ~ t h s t i t i s ( ~ 1 ~ d e m b l y to^ th aab rqplk for lk w-iadh epi* retrieval of MCaswel I&su&gananta&wr .~TBeofa I l~ - mrcially prepmi rageats followed by a manual pme- h ~ m t l s s p m e l a l m m t o r y m s l d e ~ n t s d a n ~ o . stainer is of tbe order of almost 2 1 (Tabfe 4). The pse of autosminrs for HC pmedum is chqioned

mwadaysbymanydI&gteewitb~snatiotkwitb some - . . l m m a t m s . U i ~ ~ l e ~ a m o s t a i a e r s m s p cial2y Sui~fwmcmbecaurre~results gwiyde- pend arr the ammcy and consistency of all the steps. Also, youwil lwtknowtheontcomeofttreMC:~lmti l you add the chromogen, d if the gmtoool was c o m p w manually you win not know if yon missed a wh& step or just did not tmf same slides; ttme dways wiil Ix m e dwbt factm w b cmfnmkd with a "mg&ive'* outcome. W i t h m ~ i n g d ~ c o n d i ~ h a r e n o s d problems; bides, is no .step (except for tbe counterstaining) that will require m y "chromatic" or manual medon. With regards to m t o s ~ far HC prmdu~% I ltbld a

different cphim: they are m l d l e fa ~ - ~ e l a b ~wbere~veraInmsof thearmprotoco l travea ,be c o m p E e E d d g i t y ~ ~ t b a t d a n Q ? ~ ~ ~ plex diffefentMon q s . like tfie romine bematoxylin md msin (H&E) a d some HC ptwok that are stmightfar- ward, like the periodic acid Schiff, Mabry's and Masson's trkhmes, crystal violet, muciamh, van Gieson, d c h blue, and Frussian bhe. Any atber HC s& q n k h g w e micmsEoprcally ~~ step is not ideally suited £or au- ~andthes l ide snsoa l l y~ tno luute i thcro t l er -or undermixid kcawe the compuw-dri'ven pmcmol of the autassakr cannot change to aqmpemak for d S e m e s in the nature and characteristics of either the &sue or the seetion (at I& this is my pin t of viewI).

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The Faas of Ibaty! It daes not bave to be a camplea @uff to hnd b e a q

in our slides even thp "le" rmrtine HBtE (the everyday H& even when done with an autnstakr) can coin* with tliE mom ~ O U S W n ' s wichrome ia rev* tfie oucfwr inner Wty (Figure 11.

T b e c h r o ~ ~ ~ o f o w w ~ g o e % f r o m b l a e tored anb in-ktween mvealing tissue compwents mgmz 2) as well as pathogens (Figure 3) with an enwmms array of prmcduces where gold and silver b m to m a m m and gray and even ~ pitch bIack wirh different hues of w o w to brown ~ ~ j p 4 ) . F ~ 5 A i s a o exampleof the fact th nature pmvides tbe wt2iae, the sketch, and we litl-inthecolor~ T h i s i s a n u a s t a i w d d ~ 1 o f s k e I d muscle photogqhd with p b e conmt d the fallowing ~ p h o t o s a r e ~ t e s o f t k ~ m d e d u s i n g a s m a a y ~ ~ ~ i n ~ ~ ~ ~ o f i h e s a m e s p e c i m e a As it has been said e l s e w k {2), "... the pthlogkt kwws the dtslinaian and the ~hnologis i is the master of the trail."

S o f i u T h a v e ~ t o t b e l w a t r t y ~ o f H C p m e - ~ b u t w h a t ~ ~ C s l i d e s t t l a t i a s o m e ~ o r i e s ~ ~ t h e ' k w t i n e s p e ~ i a I ~ ~ a r ) f C s l i d e s ? A r e tbey not beautiful atso? l k devehpmi afMC teEhtliques cansh td a b r d t h q h of unqnestiodle nqaitude

and importaam, s real diagwic quanhi~ kap W the chromatic pDint of view, IHC slides are a barn and meXchhg twwtme moWtDny! It does no4 matter e c h a n t i g e n i s d e t e e t e d , ~ y g r e d v i e w d o f ~ ~ c o l w aadt f ie~~~asional htyfwndins~meofthest idaEde- pen& on tbe c ~ ~ c s of the tissue itself, but not on fhepmEu&lm*

W b e n ~ ~ 0 U t o f m e r e ~ f s w n e t i m e s " ~ w i t f i ~ m e

frwmion ) i s e v d d c m s I bave timed the patholo- gists wide reviewing o9r speFial padmes. h their quest for speed, it is e m cmmm paaim fm them to remove d t e s l i d e ~ ~ t h e n M x h a a i c a l s c a g e , d ~ s l i d e s are moved udm the objectives by quick and smnerimes ~ ~ ~ j e d c s , likeapmtidigitatar. Itis* e a s ~ ~ m a r y ta nse dbjdves of the lowest m a g d i d o n available (usually 2:l or ewp 1:l) that. &ho@ provide the b ~ g e d ~ ~ o f t h e d m t o s e S e c t t h e a r - a s o f ~ c ~ ~ t t r a t w i l l b e v i e , w e d i n ~ l l o t e dmil using high-powlet objdm, pw the possibility o f ~ a d t g t t r e ~ d e t a i I s h t l l e ~ g ( s o ~ alemomlxztumd).

Wd v W ~ bEtweea WOW, well m n d - i n g o n t b n a a r r e o f h ~ ' -ft,tbeaveragetimerequired

~ A A , B Z e l & o w s k y ' s s i ~ ~ g o a r i c m a r lCk1 {WnpmreduA at Wh91/sli&). 1.PerMk acid merheaamim s ihrat 25-1 170rain- et slide). C, Gamori's &dm pmcedute mr ml (45-min pwdm at S1.5Usiide). D. hhatory-prrpared s~lver +ate prw&m (8) ar 4&1 (mnrin procedm at 82.WsEde).

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m view a slide is arr#md 30 s e d and seldwn more than Imiarde ,a lobwghsrrmes l idesare~ iWkfmthe find dhgmis is reached. Tbe average 39 miu- r t qkd m finish a mutiae HC slide wilt eojoyjnst arolmd "1 minute. of fame" before it is relegated to the &Iivion of the slides ~ t a b i n e t f a t ~ t a ~ o m n n t i l i t i s h a l l y d i . .'d!

Isitnotfairtosaytbathk&misUygetstoka~oF unappreciated beanty? ThE an" concept -6 from two Weremi viewpoints: for us histotech, although we of ow wok for patient care, the finished slick is our immediate gad, but for oar direct "cli- ents" (the pathologists), it is just a diagnostic tool, a means, hut never a goal.

Because oftbe above, we can appreciate the quality of the finiskd slide and *juice in its beauty, but the pahhpists are usually impnious ro this aspect Beauty and ztrt aw our distinction; let's always work in a way that we can keep bth, even when our Final product can -me a case of ''unappreciated beauty"!

ltlmmENw 1.3wsa RJ: W o g y L Z w k . Science & Business Irr3titrcr-e.

Ncsth Miami l k v & pp 141, 1M 2. L h p - C d E: I& beauty makers. or the pl- of his-

tology. I HktoteEhnol25:71-73, ZUX! 3. M c k SB: 3cim-m imihtes art. 3 H ~ e ~ I 26:7 . 2 a 4. ~ r s S R : T b a t t d ~ ~ f o r f m a e n ~ o n . P a r t

I: A systm far pmckh face dowu uyaembeddisg of tissue using free* temperatme+mbddhg we%. J Histotechno1 26: 1 1-19.203

5. We&PA: ASCPUWX]wageddaqvacancysurvey lab M d 32,2Q01 and @ h t t p : / ~ . a s c p . o r g h ~ W s u ~ 1M

6. Btlwa RJ: M h e d oil: the bwt xylene substitute for t i w e m g yet? J Hist&chnoi 23143-149.

7. Busa W: PhoQpbtmptic acid e l i m i n w m y 1 nibate as a sens ibr kfote h silver i m p p a h n of Helicobacter py- lori 3. Hktuwdtwi 24:113-116.2001

8. Buesa Rk Silver proteinate imprepatbn In under two how. J HistorechnoI24:259-263, #W] I