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Microscopic Quality Control of Hematoxylin and Eosin – Know your Histology Chapter 16 ixation and issue rocessing
Jamie M. Nowacek B H C CM QHC M
Reise an uate y Jo
n . iernan MB C
B
c
Breast References
Breast tissue contains blood vessels, collagenous stroa, lleelln Bd “nuclear staining it alu eato lin”
Biotechnic and Histochemistry iation te cells and tissues to ic te are alied, fi atives also cause
oeitelial cells and acinar eitelial cells te latter cells are luna l “te ualit control dilea in histotecnolog sical canges to cellular and e tracellular coonents
aected b te enstrual ccle, or e ale, oeitelial cells a ossible anser” Histologic te structure o a tissue is deterined b te saes and si§es o
ave clear ctolas and sall dense nuclei during te ollicular sternberg ss ¡ “histolog or patologists” liincott acroolecules in and around cells te rincial acroolecules ¦iable cells ave surace ebranes tat are iereable to large,
ase and suc canges need to be considered en assessing aven, piladelia sa, ¡, , , , , ¡, inside a cell are roteins and nucleic acids in vertebrate anials, droilic olecules
i ation, eseciall in organic liuids tat
, , , ¡¡, ¡¡¡, ¡, ¡, te acroolecules on te outside suraces o cells and in te
h&e ualit control ¢oung B, heat £¤ “¤eater’s
unctional histolog” t ed dissolve or disrut te liids o te cell ebrane, allos relativel
curcill livingstone, edinburg, , ¡, e tracellular saces are glcoroteins and roteoglcans, in large olecules to enetrate and escae
urterore, te ctolas
H&E Quality Control Material erosceno ¦p “di
iore’s atlas o histolog” t edition, ic uc carbodrate aterial is covalentl ¨oined to rotein becoes ereable to acroolecules, oring a roteinaceous
liincott ¤illias & ¤ilins, piladeliasa, ¡, , olecules carbodrates are droilic© te old uc ater in
or te dail c o te h&e stain i ould suggest te olloing netor suicientl orous to allo urter enetration o large
te e tracellular sace, b drogen bonding tere is also, o course, olecules in tis conte t, “large olecules” include tose o arain
colon – dierentiation o uscle and collagen, inaroriate uc ater inside cells© ater accounts or about ¡ª o te eigt a , tose o antibodies used or iunostaining, and larger de
staining o ucin, clear staining o eitelial vesicular nuclei o te uan bod uton, artur c ¡ Textbook of Medical olecules dierent fi atives result in dierent degrees o orosit
assessing lasa cell staining is uite elul in tat a clear cloc Physiology t ed piladelia ¤B saunders in bones coagulants suc as ercuric cloride, icric acid, or §inc sulate
ace arrangeent o nuclear croatin ill indicate an adeuate and teet dro aatite, a crstalline ineral containing calciu result in a larger ore si§e tan do noncoagulant fi atives, suc as
dierentiation o te eato lin and osate ions, doinates te e tracellular doain, togeter
it collagen, a fibrous rotein oraldede, glo al or glutaraldede soe fi atives are bot
sin – deonstration o blue eratoaline granules, dierentiation coagulant and noncoagulant
or e ale, acetic acid coagulates
o eratin ro collagen and nerves, definition o te reticular an essential art o all istological and ctological tecniues nuclear croatin but not roteins tus, coagulant fi atives are
aillar border o te deris is reservation o cells and tissues as te naturall occur to generall beneficial or subseuent arain sections in addition to
idne – identification o baseent ebranes and bot ro ial accolis tis, tissue blocs, sections or sears are usuall acilitating ereation o sections b antibodies, coagulant fi atives
and eerent tubules te idne contains a ide diversit o cells iersed in a fi ative fluid, altoug in te case o sears, erel can also increase te e osure o antigenic sites, a conseuence
ro tose e ibiting dense croatin gloerular tuts to tose dring te rearation acts as a or o reservation te fi atives o te deoration o acroolecular saes tat constitutes
aving a dusting o croatin aterial cuboidal cells o collecting eloed revent autolsis b inactivating lsosoal en§es, and coagulation crosslining iedes enetration o arain but
tubules luna te stabili§e te fine structure, bot inside and beteen cells, b increases te sical strengt o te tissue, eseciall i ro§en
aing acroolecules resistant to dissolution b ater and oter sections are needed ceical addition o fi ative olecules, it
Conclusion liuids
i atives also inibit te grot o bacteria and olds tat or itout crosslining, can odi antigenic sites and suress
it is aarent tat ualit h&e sections are iortant in obtaining a give rise to utreactive canges iunostaining tis is a ell non eect o oraldede, oten
confident, accurate diagnosis te above revie is an attet to list in eroring teir rotective role, fi atives denature roteins b reversible b one o an antigen retrieval rocedures
tose icroscoic eatures tat istotecnologists sould assess in coagulation, b oring additive coounds, or b a cobination o
order to obtain a ualit stain nortunatel, fi ation and rocessing in attets to cobine te best roerties o dierent coounds
coagulation and additive rocesses a coound tat adds ceicall used or fi ation, an i tures ave been develoed ost contain
la a uge role in section ualit and it is diicult to assess staining to acroolecules stabili§es structure ost eectivel i it is able to
ualit in isolation bot coagulant and noncoagulant ingredients ¬ter substances
cobine it arts o to dierent acroolecules, an eect non in fi ative solutions ave actions suc as controlling te osotic
an o te above recoendations are based on ersonal as crosslining te result is conorational canges in te structure ressure, ad¨usting te h, and counteracting srinage or selling
e erience and as suc sould be oen or debate critical discussion o roteins and subseuent inactivation o ost en§es soe caused b anoter ingredient srinage or selling tat occurs
is areciated and encouraged it is also aarent tat noledge liids, roteins, carbodrates and inerals are e tracted b fi ative evenl troug a secien usuall does not atter, but severe
o routine istolog and reerence to classic istolog te ts suc as liuids in ic te are soluble
i ation canges bot ceical and distortion results ro canges in te si§es o soe but not all arts
tose listed in te eerences is iortant antigenic rofiles o roteins te dilea o fi ation as alas been o a tissue, as en tubular structures srin itin teir surrounding
tat it introduces soe artiact in order to ave a rotective eect connective tissue, or en artificial saces are ored around cells
B definition, fi atives cange te original ceical and sical
coositions o tissues in addition to altering te ceical nature o
140 special stains and h & e special stains and h & e 141
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ixation and issue rocessing ixation and issue rocessing
iation efore araffin Emein nortunatel, desite raid enetration o tissues, te ceical
ost sales used or staining noral and atological tissues reactions o oraldede it tissue roteins, eseciall te
are ebedded in arain, and a nuber o fi atives ave been oration o etlene bridges, occur slol a coonl ade
orulated it tis in ind te ost coonl used fi atives are istae it oralincontaining fi atives is to underfi te tissue
discussed ere tere is an abundance o secialt fi atives tat sall ×× ieces fi ed in nB
or ours ill generall
ill not be covered ere but a be ound in te reerences given in so good ctolasic reservation and nuclear detail addition
te bibliogra o oraldede is largel colete in ours, but crosslining
reactions continue or at least to ees large, sot seciens
ormale
ye suc as ole uan brains reuire ¡ ees in nB
to becoe
fir enoug to cut into slices ro ic sales can be taen or
oralin is an article o coerce containing ª v ® ª istolog ¦ariations in tie and conditions o fi ation cause te
oraldede ic is a gas in ater ost o te oraldede a¨orit o robles in istoceistr
is resent as soluble olers, ic deoleri§e on dilution
oralin also contains about ª etanol, ic is added
i ation rooundl aects istological and iunoistoceical
b te anuacturer to retard te oration o iger olers, staining, tecnicians, atologists and researc orers ust
ic eventuall all out o solution as araoraldede ¬ld tereore decide on te ost aroriate etod asects to consider
bottles o oralin, eseciall ater storage in a cold lace, oten are teerature, si§e o te storage container, volue ratio, salt
contain deosits o tis ite oder
oraldede in solution concentration, h and incubation tie
oraldede fi ation is
also deteriorates b reacting it itsel canni§§aro reaction and ticall erored at roo teerature sing a lo and ide
canging into etanol and oric acid onoeric oraldede secien container to allo or adeuate enetration and ease o
e ists alost entirel as etlene drate, an addition coound retrieval b a tecnician is te best coice or adeuate volue ratio
ored b a reversible reaction it ater
oraldede itsel is in addition, volue ratio o fluid to tissue and – secien
te coound tat reacts it roteins© it is resent at an e treel ticness are recoended or good enetration to revent selling
lo concentration in a “ª oraldede” solution, but is roduced or srining o te cells, an isotonic solution buered to h – is
instantl ro etlene drate olloing reoval ro solution in recoended, to aintain ultrastructure and inii§e cell distortion
te reactions o fi ation tese ceical roerties o oraldede te sorter te tie elased beteen reoving te sale ro te
are suari§ed in
igure bod and iersing it in fi ative, te better te duration o e osure
te ost coonl used fi ative or istoatolog is a ª aueous to te fi ative ust be otii§ed or eac secien te
or e ale,
solution o oraldede, oten called ª oralin because it is it as been noted in a cap surve tat glcogen reservation in
ade b tenold dilution o oralin
or about ears tis fi ative liver can be sub¨ect to fi ation arteacts as nB
enetrates slol Figure 1. Some reactions of formaldehyde with water, with itself, and with proteins.
as also included inorganic salts to aintain a near neutral h and troug te eatocte ebrane, te glcogen associated it te
an osotic ressure siilar to tat o aalian e tracellular fluid ctolasic rotein atri is dislaced to one side o te cell tis
te solution is called neutral buered oralin, or nB
it fi es not observation is reerred to as olari§ation and is considered art o
b coagulation, but b adding to te sidecains o basic aino te orolog en discerning te stain result
igure glcogen
acids, eseciall lsine, and to te aide nitrogen atos o etide olari§ation tis is a great e ale o o seciens tat ave
linages crosslining etlene bridges are ored ere to rolonged e osure to fi atives can deonstrate e tree orolog
oraldede binding sites are close togeter see
igure tis canges lcogen olari§ation is less ared ater fi ation in a
results in loered ereabilit to acroolecules but te structures nonaueous liuid, in ic tis olsaccaride is not soluble
o rotein olecules are not greatl altered te sall si§es o te
etlene glcol and oraldede olecules allo raid enetration
conseuentl tis fi ative is suitable or large or sall seciens
14 special stains and h & e special stains and h & e 14
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ixation and issue rocessing ixation and issue rocessing
Figure 2. A paraffin section of Mercuric C
lorie lco
olic iaties
formaldehyde-fixed liver stained by the eac fi ative reserves orolog dierentl, tus tere are ultile nB
is te ost idel used fi ative, desite not being te best
periodic acid-Schiff method, illustrating otions
or soe cells and tissues nB
does not rovide adeuate
polarization of glycogen (magenta within one or ever urose tis is because te sortcoings o nB
are
the hepatocytes. uclei are counter- fi ation e ales are connective tissues, soe structures containing not ver serious, rovided tat te seciens are fi ed or suicient
stained with hemalum (blue. ucins, and bone arro ercuric cloride hgcl adds to roteins tie oreover, te aearances o oraldedefi ed tissues
and brings about raid coagulation it resulting granularit tat is are ailiar to atologists, o ave trained teir visual sstes
too fine to be visible it ligt icrosco lillie’s B fi ative is ª to recogni§e noral and abnoral eatures in sections cut ro
aueous oraldede it hgcl and acetic acid tis routinel fi ed aterials
i ture enances nuclear detail, ic is iortant or identiing
noral and abnoral cell tes in bone arro eatooietic arguents ave been ade or canging te routine fi ative to one
tissue seciens te coagulation o nuclear croatin is an eect o several nonaueous i tures, and strong cases can be ade
o te acetic acid te ercuric cloride ensures raid structural or clare’s fluid etanol and acetic acid, b volue, carno’s
stabili§ation and also acilitates brigt staining b an o te des fluid etanol, cloroor and acetic acid, ¡ and puctler’s
used in icrotecniue te reasons or tis eect on stainabilit etacarn carno it etanol instead o etanol tese
lyoal Bouin’s lui are not understood liuids, ic fi b coagulation o roteins and croatin, contain
lo al is te silest dialdede, it te orula ¬hc—ch¬ lie pol andré Bouin ¡ invented several fi ative i tures in te no ater te alost coletel dedrate te tissue, so tat te
oraldede, it readil ors drates and olers decoosition a dirt looing bron crstalline reciitate, robabl ercurous tie or rocessing into arain a is sorter tan ater nB
or
ears © te one ost oten associated it is nae, first cloride hg cl ors in all arts o tissues fi ed in i tures
b a o te canni§§aro reaction occurs raidl in neutral or alaline ublised in , contains ª oraldede ª oralin, oter aueous fi atives tere are also an i tures containing an
containing hgcl © it is called ercur igent and ust be reoved, alcool usuall etanol, oralin, acetic acid and ª to ª ater,
solutions aueous solutions ust be buered to about h to be acetic acid and icric acid, in ater picric acid enetrates
stable, and te ust also contain a sall roortion o etanol, b seuential treatents it iodine and sodiu tiosulate solutions, oten called a
a or
aa or naed or teir inventors, o include
tissues rater slol, coagulating roteins and causing soe beore staining Because it contains ercur, B is sub¨ect to to ic
ic catal§es te reaction o glo al it roteins addition to and srinage it also des te tissue ello te acetic acid coagulates tellesnic§ around , Bodian s and davidson s in
crosslining o roteins is uicer tan te euivalent reactions o aste disosal regulations, ic al to te fi ative solution and an a
a i ture te ceical reactions o oraldede it roteins
nuclear croatin and ooses te srinage caused b icric acid ever solution tereater tat as been containated it ercur
oraldede adeuate fi ation o sall seciens b glo al is te reactions o oraldede ave alread been discussed© at te are not retarded b buering to a nearneutral h all tese alcoolic
acieved in one our ecords ust be et o all B volues used in te laborator fi atives contain acetic acid, ic roduces caracteristic atterns
h o Bouin’s fluid tese occur ore raidl tan in nB
te coleentar eects o te tree ingredients o Bouin’s solution o coagulated nuclear croatin, acilitating te recognition o cell
te glo albased fi atives available to laboratories are sold as inc salts tes nuclei tat ave been in nB
or a ee or ore e ibit less
reade solutions it trade naes and undisclosed coositions or ell togeter to aintain orolog seciens are usuall
fi ed in Bouin’s or ours prolonged storage in tis acidic i ture te to icit o ercur coounds as discouraged teir use in ronounced atterns o croatin
evidentl tere are no ublised recies or fi atives o roven
eicac tat contain glo al as te rincial ingredient prorietar causes drolsis and loss o stainable dna and na toroug laboratories ¬ te oter eleents in rou iib o te eriodic alcool etanol or etanol alone instantl coagulates roteins
glo al fi atives are reorted to be useul or general istolog and asing ater fi ation is necessar table ²n, cd, hg, §inc is te least to ic ²inc sulate as been but causes considerable distortion o te icroanato in ieces
iunoistoceistr te dierences ro oraldede nB
used or ore tan a centur in astringent lotions and ee dros, o anial tissue tese unanted canges are oosed b dilution
lie ercuric cloride, icric acid enances subseuent staining, ic reduce te selling o inflaed suraces b coagulating
fi ation are eseciall it anionic “acid” des parain sections o oraldede o te alcool it cloroor iiscible it ater, ater, and
e travasated lasa roteins te first §incoralin fi ative as or acetic acid ic coagulates croatin and ooses srinage,
a suression o staining o arginineric roteins b anionic des fi ed tissues are usuall iersed or a e ours in a icric acid robabl te one ublised b pa
is in , onl tree ears ater
solution Bouin’s fluid is coonl used beore staining b te being iscible it ater, alcool and drocarbons alcool
tis atters i ou need to stain panet cells, eosinoil leuoctes te discover tat oraldede reserved tissue arcitecture ver alone etanol is usuall reerred is suitable or fi ing tin laer
or te tails o serato§oa, and tricroe etods tricroe stains use cobinations o anionic des ell in recent decades several intelligentl orulated fi atives ave
it osotungstic or osoolbdic acid to iart contrasting rearations suc as blood fils or cell cultures solid seciens
b antigen retrieval is less reuentl reuired tan ater fi ation in nB
³
been develoed a e are entirel coagulant, it ²n as te onl taen ro atients it gout are usuall fi ed in ª etanol or
colors to ctolas, collagen fibers and oter coonents o tissues ingredient liel to iobili§e roteins ost §inccontaining fi atives
te trade secrec associated it glo alcontaining fi atives a subseuent istoceical detection o sodiu urate crstals, ic
reclude teir use in researc and in diagnostic alications ere also contain oraldede, aing te coarable to B and oter can be dissolved out o te tissue b ater
te atologist a be interrogated about is coetence and ercurcontaining solutions
noledge o aterials used in te laborator
144 special stains and h & e special stains and h & e 14
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ixation and issue rocessing ixation and issue rocessing
ecalcification na and reove te urine and riidine bases ro dna it is also
ierative to as te acid out o te tissue i reservation o nuclear Stain echniue hicness of Sections ecommended ixative ixatives to Avoid
olloing fi ation, tere are tecniues available to te istotecnician
to irove icroto and staining ualit eac tecniue ust be dna is iortant, or i istoceical etods or nucleic acids or 3–4 μm NBF
en§e activities are intended, a celating agent is reerred to an Gram smear 95%
evaluated or eac assa to reserve orolog and rovide te best
stain ossible table te first etod, going in cronological order acid suall te disodiu salt o edta is used, it te h ad¨usted Warthin-Starry 3–4 μm NBF (critical)
o istolog, is decalcification o seciens tat a be diicult to cut to a level beteen and decalcification b edta uc longer tan
on a icrotoe because o calciu carbonate or osate deosits decalcification b acids – ees rater tan das Helicoacter 3–4 μm NBF
decalcification can be acieved eiter b acids or b celating agents asson’s richrome 3–4 μm NBF Boin’s
irst, ae sure te tissue as been adeuatel fi ed and rinsed
ell to revent an undesired reaction it te decalciing agent olloial ron 3–4 μm NBF chromate
dilute ineral acids drocloric or nitric or oric acid can be used oer 3–4 μm NBF B5 oay Boin’s ecal
eectivel i te end oint o decalcification is onitored careull hoanine
nuclear and ctolasic detail are coroised i seciens are
e osed or too long to acidic decalciing agents, ic can e tract
smear citrateacetone
San Blac 3–4 μm NBF Boin’s
Table 1. Stain optimization. roen μm
eer 3–4 μm NBF B5 ecal
Stain echniue hicness of Sections ecommended ixative ixatives to Avoid ast ell smear
F
3–4 μm FF NBF - Florie smear alcoholic ormalin
FB smear 95%
lcian Ble 3–4 μm NBF
Bielschosy 3–4 μm FF NBF cicarmine 3–4 m NBF
roen Felen 3–4 μm NBF Boin’s
Boian 3–4 μm NBF
Gomori’s richrome 3–4 μm NBF
ono e − μm FF NBF
roen lastic an Gieson 3–4 μm NBF
3–4 μm FF il e 3–4 μm NBF
S roen μm NBF
Fontana asson 3–4 μm NBF
ones μm FF NBF alcim 3–4 μm NBF alcohol
GS 3–4 μm FF NBF Bile 3–4 μm NBF
smear 95%
H 3–4 μm B5
rate rystals 3–4 μm alcohol aeos NBF
allory’s
ron 3–4 μm NBF
smear air ry ol Fast Ble 3–4 μm NBF
eticlin 3–4 μm NBF
´ca
® citrate ³ acetone ³ oralin
Snoo’s
14 special stains and h & e special stains and h & e 14
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