oiN:i or i:1noiocx, voi.

183: 116123 (1997)

ANTI GEN RETRI EVAL TECHNI QUES I N
I MMUNOHI STOCHEMI STRY: COMPARI SON OF
DI FFERENT METHODS
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1
Second Serviceof Pathologic Anatomy and Haematopathology Section, Bologna University, I taly
2
I nstituteof Pathologic Anatomy, Siena University, I taly
3
Haematopathology Laboratory, I nstituteof Haematology, Perugia University, I taly
SUMMARY
Routine sections of normal and pathological samples xed in 10 per cent buered formalin or B5, including EDTA-decalcied
bone-marrowbiopsies, weretestedwith61antibodiesfollowingheatinginthreedierent uids: 001M citratebuer (pH 60), 01M
TrisHCl (pH 80), and1mM EDTANaOH solution(pH 80). Thesectionsunderwent either threecyclesof microwavetreatment (5
mineach) or pressurecookingfor 12min. Thealkalinephosphatase/anti-alkalinephosphatase(APAAP) techniquewasusedasthe
standarddetectionmethod;with16antibodiesaslightlymodiedstreptavidinbiotincomplex(SABC)-immunoperoxidasetechniquewas
appliedinparallel. Theresultsobtainedwerecomparedwiththoseobservedwithout any antigenretrieval (AR), or followingsection
digestionwith005per cent proteaseXIV at 37C for 5min. Chess-boardtitrationtestsshowedthat all antibodiesbut oneprotedby
AR. ProteaseXIVdigestionrepresentedthegoldstandardforveantibodies, while55producedoptimal resultsfollowingtheapplication
of heat-basedAR. Bycomparisonwiththeotheruids, EDTAappearedtobesuperiorintermsof bothstainingintensityandthenumber
of markedcells. Theseresults wereindependent of tissueprocessing, immunohistochemical approach, andheatingdevice. Pressure
cookingwasfoundtobemoreconvenient onpractical grounds, asit allowedthesimultaneoushandlingof alargenumber of slidesand
atimesavingof 1min30s, representingtheproper timefor thetreatment. 1997byJ ohnWiley&Sons, Ltd.
J . Pathol. 183: 116123, 1997.
No. of Figures12. No. of Tables4. No. of References29.
KEY WORDSantigenretrieval; immunohistochemistry; xation; standardization
INTRODUCTION
For about 25 years, it was thought that antigen
identicationinroutinesectionswassignicantlylimited
by the masking eect of xatives, which determine
various alterations of tissue structures, such as the
formalin-induced cross-linking of proteins.
13
Conse-
quently, cryostat sectionsrepresented therst choicefor
accurate immunophenotyping, although they often
showed poor cytological detail.
2,3
Meanwhile, proteolytic enzymes (such as trypsin,
pronase, and pepsin) were used to overcome partially
the limitations in antigen accessibility, especially in
formalin-xed samples.
13
They wereapplied to routine
sections under controlled conditions of concentration,
time, and temperature, in order to break some of the
bonds produced by xation and to expose a higher
number of antigenic epitopes.
1
Sincethebeginning of the1990s, new techniques for
antigenretrieval (AR) havebeenproposed, basedonthe
exposure of routine sections to high temperatures in
aqueous salt or protein denaturant solutions for a few
minutes
419
using a Bunsen burner, microwave oven,
pressurecooker, or autoclavefor variabletimes.
4,6,7,15,18
The mechanism by which this approach allows the
detection of most antigensisnot yet completely dened.
Thesenew AR techniques havemainly been applied to
formalin-xed, paran-embedded material, on which
they also signicantly reduce the negative eects of
overxation.
7
This study compares the most popular AR systems,
when applied to heterogeneously treated samples under
dierent methodological conditions.
MATERIALS AND METHODS
Tissuesampleselection
Paran-embedded tissue blocks were randomly
selected fromthe les of the Second Service of Patho-
logic Anatomy/Haematopathology Section of Bologna
University. They had been xed either in 10 per cent
buered formalin (for times ranging from 24h to 1
week) or in B5 (for 22h 30 min).
20
Someof thelatter
blockswerebone-marrowneedlebiopsieswhich, follow-
ing xation, had been decalcied in EDTA for 2h
30 min.
20
All of the specimens had been similarly
processed, as previously described.
20,21
The selected
*Correspondenceto: Professor Stefano A. Pileri, Secondo Servizio
di Anatomia Patologica e Sezione di Ematopatologia, Universit di
Bologna, Policlinico S. Orsola, Via Massarenti 9, 40138 Bologna,
I taly.
Contract grant sponsors: AI RC, Milan; CNR (ACRO 10), Rome;
MURST, Rome; A.B.S.T.E., Bologna.
CCC 00223417/97/09011608 $17.50 Received 27 September 1996
1997 by J ohn Wiley & Sons, Ltd. Accepted 6 March 1997
blocks were representative of a variety of conditions:
normal bone-marrow and placenta, hyperplastic lymph
nodes, malignant lymphomas of all the histotypes of
theRevised EuropeanAmerican LymphomaClassica-
tion,
22
acutenon-lymphoidleukaemias(M1M7accord-
ing to the FAB Classication),
23
breast carcinoma,
colonic carcinoma, gastric carcinoma, prostatic adeno-
carcinoma, endometrial adenocarcinoma, papillary thy-
roid carcinoma, squamous cell carcinoma of thelarynx,
intermediate cell neuroendocrine carcinoma, retino-
blastoma, and rhabdomyosarcoma. Serial sections were
obtained fromeach block, collected on silane-coated or
naturally charged slides (Dako ChemMate Capillary
Gap Slides, BioTek Solutions, U.S.A.), and allowed
to dry overnight (at 37C and 57C, respectively), in
order to ensureoptimal adhesion. Beforeimmunohisto-
chemistry, sections were dewaxed, rehydrated, and
rinsed in running water, as previously described.
20
Antigen retrieval (AR)
Sixty-one specic antibodies (Tables I I V) were
applied to serial sections which had received no pre-
vious treatment or had undergone the following AR
procedures.
Enzymatic digestionRehydrated sections were
immersed in buer solution for 10 min and then trans-
ferred to a 005 per cent proteaseXI V (Sigma, U.S.A.)
solution for 5 min, according to previous reports.
1,3,20
Both the buer and the enzymatic solution had been
pre-warmed in a thermostatic water bath at 37C and
this temperature was maintained throughout the whole
procedure. After theproteolytic treatment, thesections
wereplacedincoldbuer (at 4C) for 10min, inorder to
block theactivity of theenzyme.
Microwave treatment001x citrate buer (Na
citratecitric acid) (pH 60),
7
01x TrisHCl buer (pH
80),
18
and 1mx EDTANaOH solution (pH 80)
15,24
were used as retrieval solutions. Rehydrated sections
wereimmersed in each retrieval solution and processed
in a microwave oven with the power set at 750W for
three cycles (5 min each). The same number of slides
(n=20) was always placed in a rectangular plastic stain-
ing jar (Kartell, U.S.A., codenumber 353), which con-
tained 200ml of retrieval solution and was located in
the centre of the oven, covered by a loose-tting cap.
Thelevel of theuid was constantly maintained during
the procedure by adding distilled water and retrieval
solution alternately followingeach cycle. After thecom-
pletion of thethird cycle, sections wereallowed to cool
at room temperature for 20 min and then rinsed in
distilled water and buer. Microwave ovens with and
without a rotary plate were used (Bauknecht MCI D
1125 Duo, Germany; Philips M742, TheNetherlands).
Pressure cookingTwo litres of each retrieval
solution were placed in a stainless steel 6-litre capacity
pressurecooker with an operating pressureof 103kPa/
15psi (LagostinaNE I rradial Plus, I taly) andbrought to
theboil ona15kW electrichotplatewithout sealingthe
lid. Fifty rehydrated sections wereplaced in metal racks
and lowered into the boiling retrieval solution. The
pressure cooker was then sealed and brought to full
pressure. Theheatingtimebeganonlywhenfull pressure
wasreached. I nthisstudy, heatingtimesof 1min, 1min
30s, and 2min wereemployed. At full time, thecooker
was depressurized and cooled under running water; the
lidwasthenremoved; andthehot buer wasushedout
with cold water froma running tap. Carewas taken to
ensure that the sections did not dry. The cooled slides
werewashed twicein buer solution, prior to immuno-
histochemical staining.
Antibodies and immunohistochemical methods
The61antibodies listed in Tables I I V wereused for
the study and detected by a three-layer-alkaline phos-
phatase anti-alkaline phosphatase (APAAP) tech-
nique,
25
which was carried out either manually or
automatically [on a TechMate 500 immunostainer
(DAKO, Denmark; BioTek, U.S.A.)]. The reaction
was always developed with new fuchsin in steering for
20 min.
25
When a rabbit polyclonal antibody was used
astheprimary reagent, thesection wasincubated with a
mouse anti-rabbit monoclonal antibody diluted 1:30
(DAKO, Denmark, code number M0737) for 30 min,
beforestarting with theAPAAP technique.
Sixteen antibodies were also tested by a slightly
modied streptavidinbiotin complex (SABC) immuno-
peroxidase method, which included overnight incu-
bation in the primary antibody at 4C and was carried
out manually, as previously described.
26
A chess-board titration was performed for each anti-
body, aimingto assessitsoptimal workingdilution with
each AR system.
01x PBS (pH 72) and 01x TBS (pH 76) respect-
ively were used for the APAAP and SABC-based
investigations.
Evaluation of results
The results were independently evaluated by six
observers (CC, BF, LL, SAP, DS, and ES) and were
graded as follows: , completely negative result;
+, weak positivity in a percentage of cells
expected to be positive; ++, weak positivity in all
cellsexpected to bepositive; +++, moderately strong
positivityinall cellsexpectedto bepositive; ++++, very
strongpositivity in all cells expected to bepositive. The
agreement amongfour out of sixobserverswasregarded
as consensus.
RESULTS
Antigen retrieval system
TablesI I V summarizethendingsobtained with the
61 antibodies employed. Theworking dilution for each
reagent was chosen by applying the APAAP technique
and the most ecient AR method, in order to provide
optimal results with both xatives and independently of
117 STANDARDIZATION AND ANTIGEN RETRIEVAL
1997 by J ohn Wiley & Sons, Ltd. oiN:i or i:1noiocx, voi. 183: 116123 (1997)
the length of xation in formalin. Tables I I V also
include the optimal dilutions with the 16 antibodies,
which in parallel were revealed by the immunoperoxi-
dase method. Because of the overnight incubation, the
dilutions with the latter usually appeared higher than
those with the APAAP technique, but the results with
the two methods remained comparable in terms of
sensitivity (Tables I I V).
High temperature vs proteolytic enzymeI n most
instances, high temperatures proved more eective
than protease XI V, by allowing either the emergence
of otherwise undetectable antigens or higher working
dilutions (e.g., KP1/CD68=1:640 vs. 1:100; PG-M1/
CD68=1:20 vs. 1:5; MNF 116/cytokeratins=1:140
vs. 1:70; anti-
1
-fetoprotein=1:16000 vs. 1:2000)
(TablesI I V). Heat-basedantigenretrieval (HBAR) was
TableI Results obtained under dierent methodological conditions with theantibodies most commonly used in thestudy of the
haemolymphopoietic systemand related disorders
Clone Specicity Source Dilution No AgR PT
HBAR+
citrate
HBAR+
TrisHCl
HBAR+
EDTA
O10 CD1a I mmunotech 1:40 + ++ ++++
Poly CD3 DAKO 1:300 ++ + ++ ++++
C8/144B CD8 Dr Mason 1:6 ++ ++ ++++
1:400 + +++ ++++
C3D-1 CD15 DAKO 1:6 + + ++ ++++
1:320 ++ +++ +++ ++++
L26 CD20 DAKO 1:200 + + ++++ ++ ++++
1:3200 + + +++ +++ ++++
I F8 CD21 DAKO 1:10 ++++
MHM6 CD23 DAKO 1:50 + +++ ++++
Ber-H2 CD30 Professor Stein 1:10 ++ +++ ++++
1:320 + +++ ++++
QBEND-10 CD34 BioGenex 1:20 + +++ +++ ++++
1:400 + ++ ++ ++++
BerMACDRC CD35 DAKO 1:5 + ++++ + +
MAB89 CD40 I mmunotech 1:100 ++++
DF-T1 CD43 DAKO 1:200 + +++ +++ ++++
1:1600 ++ +++ ++++ ++++
PD7/26+2B11 CD45 DAKO 1:200 + +++ ++++ ++++
1:4000 + +++ ++++
UCHL-1 CD45R0 DAKO 1:120 + ++ ++ ++++ +++
Ki-B3 CD45R Professor Parwaresch 1:80 ++ + +++ ++++ ++++
1:320 ++ + +++ ++++ ++++
4KB5 CD45RA DAKO 1:20 ++ ++++ +++ +++
CD57 Becton 1:20 ++ ++ +++ +++ ++++
Y2/51 CD61 DAKO 1:5 +++ + + +++
KP1 CD68 DAKO 1:640 + ++ ++++ ++ ++++
PG-M1 CD68 Professor Falini 1:20 + ++ ++ ++ ++++
J CB117 CD79a Dr Mason 1:10 + +++ +++ ++++
Kim-4p Follicular dendritic cells Professor Parwaresch 1:5 ++++ ++ ++ +
DBA.44 Hairy cells Professor Delsol 1:5 ++ ++++ +++ ++++
J C159 GlycophorinA DAKO 1:320 + ++++ +++ +++
NP57 Neutrophilic elastase DAKO 1:10 ++++
M616 FVI I I RAg DAKO 1:6 + ++ ++++ ++ ++++
Poly Lysozyme DAKO 1:800 ++ +++ ++++ ++++ ++++
Poly I gA DAKO 1:2000 + +++ ++++ +++ ++++
Poly I gG DAKO 1:5000 ++ ++++ ++++ ++++ ++++
Poly I gM DAKO 1:5000 ++ ++++ ++++ ++++
Poly I gD DAKO 1:1000 +++ +++ ++++
Poly -I g light chain DAKO 1:10000 ++ +++ ++++ +++ +++
Poly -I g light chain DAKO 1:12000 ++ +++ ++++ +++ +++
Poly Protein S-100 DAKO 1:2000 ++ +++ ++++ +++ ++++
Poly MPO DAKO 1:10000 ++ +++ +++ +++ ++++
CD=cluster of dierentiation; No AgR=no antigen retrieval; PT=proteolytic treatment; HBAR=heat-based antigen retrieval; Poly=polyclonal
antibody; FVI I I RAg=Factor VI I I -related antigen; MPO=myeloperoxidase.
I n bold: overnight incubation of theprimary antibody+SABC technique.
=completely negativeresult; +=weak positivity in a percentageof cells expected to bepositive; ++=weak positivity in all cells
expected to bepositive; +++=moderately strongpositivity in all cellsexpected to bepositive; ++++=very strongpositivity in all cellsexpected
to bepositive.
118 S. A. PILERI ET AL.
1997 by J ohn Wiley & Sons, Ltd. oiN:i or i:1noiocx, voi. 183: 116123 (1997)
particularly suitable for some antibodies, such as
Ber-H2, which in heterogeneously xed material pro-
duces unpredictable results with the enzyme method
(Fig. 1). On the other hand, when switching from
protease XI V to HBAR, the polyclonal antibodies
against I g heavy and light chains did not show a
dramatic increase in their working dilution, but gave a
signicant reduction in background staining. Further-
more, high temperatures gave rise to a remarkable
lowering of dierential staining intensities between the
borders and centre of improperly xed samples, while
they never caused the exposure of non-specic neo-
antigens. The proteolytic enzyme completely abolished
the reactivity of ten antibodies (Tables I I V). On the
other hand, ve reagents denitely beneted from this
approach: I F8/CD21, BerMACDRC/CD35, Kim-4p,
CS 14, and MAB89(TablesI I V and Fig. 2). Thiswas
most evident in formalin-xed material, the staining
being unpredictablein B5-xed samples.
Theantibody NP57(anti-neutrophil elastase) worked
properly only on untreated sections (Table I ). Other
reagents also produced satisfactory staining when
applied without any previous unmasking procedure:
however, their working dilutions in that case appeared
to bemuchlower thanthoseallowedbyappropriateAR
(e.g., DF-T1/CD43=1:50vs. 1:200; Ki-B3/CD45R=1:10
vs. 1:80; 34E11/low molecular weight cytokeratins=
1:25 vs. 1:100; V9/vimentin=1:5 vs. 1:200; HHF35/
human muscleactin=1:25 vs. 1:400).
Comparison of the dierent uids used for HBAR
The best results, in terms of both staining intensity
and thenumber of positivecells, wereachieved by using
1mx EDTA (pH 80) with all but six antibodies (i.e.,
Table I I Results obtained under dierent methodological conditions with a panel of antibodies raised to intermediate lament
proteins
Clone Specicity Source Dilution No AgR PT
HBAR+
citrate
HBAR+
TrisHCl
HBAR+
EDTA
MNF 116 Cytokeratin DAKO 1:140 +++ ++++ ++++ ++++
1:700 +++ ++ +++ ++++
34E12 CytokeratinHMW DAKO 1:100 + +++ ++++ +++
35H11 CytokeratinLMW DAKO 1:100 ++ +++ +++ ++++
V9 Vimentin DAKO 1:200 + +++ +++ ++++
D33 Desmin DAKO 1:200 +++ + ++++ +++ ++++
1:6400 +++ + +++ +++ ++++
2F11 Neurolaments DAKO 1:60 +++ ++ +++ ++++
1:5000 +++ ++ +++ ++++
No AgR=no antigen retrieval; PT=proteolytic treatment; HBAR=heat-based antigen retrieval; HMW=high molecular weight; LMW=low
molecular weight.
I n bold: overnight incubation of theprimary antibody+SABC technique.
=completely negativeresult; +=weak positivity in a percentageof cells expected to bepositive; ++=weak positivity in all cells
expected to bepositive; +++=moderately strongpositivity in all cellsexpected to bepositive; ++++=very strongpositivity in all cellsexpected
to bepositive.
TableI I I Results obtained under dierent methodological conditions with a panel of antibodies raised to cell kinetics, oncogene,
and virus-associated antigens
Clone Specicity Source Dilution No AgR PT
HBAR+
citrate
HBAR+
TrisHCl
HBAR+
EDTA
124 bcl-2 geneproduct DAKO 1:20 +++ ++++ ++++
PG-B6 bcl-6 geneproduct Professor Falini 1:2 ++++
DO-7 p53 DAKO 1:160 +++ +++ ++++
1:900 ++ ++ ++++
c-erbB-2 BioGenex 1:600 + ++ +++ ++++
Rb1 Retinoblastoma geneproduct BioGenex 1:300 + ++++
MI B-1 Ki-67 antigen Professor Gerdes 1:25 + + ++++
CS 14 LMP-1 DAKO 1:7 ++
CCH2+DDG9 CMV DAKO 1:35 +++ +++ ++++ ++++
Poly HP DAKO 1:20 + + +++ ++++ ++++
No AgR=no antigen retrieval; PT=proteolytic treatment; HBAR=heat-based antigen retrieval; LMP-1=latent membrane protein 1;
CMV=cytomegalovirus; HP=Helicobacter pylori.
I n bold: overnight incubation of theprimary antibody+SABC technique.
=completely negativeresult; +=weak positivity in a percentageof cells expected to bepositive; ++=weak positivity in all cells
expected to bepositive; +++=moderately strongpositivity in all cellsexpected to bepositive; ++++=very strongpositivity in all cellsexpected
to bepositive.
119 STANDARDIZATION AND ANTIGEN RETRIEVAL
1997 by J ohn Wiley & Sons, Ltd. oiN:i or i:1noiocx, voi. 183: 116123 (1997)
UCHL1, 4KB5, anti- I g light chain, anti- I g light
chain, 34E12, and HMB45), which proved to bemore
sensitive to the employment of one of the other uids
(Tables I I V). TrisHCl turned out usually to be less
ecient than EDTA, but often superior to citratebuer
(TablesI I V) (Figs38). Theseresultswereindependent
of theheat source, natureof theantibodies (polyclonal
vs. monoclonal), and typeof xation (formalin vs. B5).
Notably, EDTA provided superior results with all the
nuclear antigens tested: Ki-67, bcl-6, p53, and Rb1
(Fig. 9).
Microwave oven irradiation vs. pressure cookingNo
signicant dierences in the immunostaining were
observed using the microwave ovens or the pressure
cooker, AR ecacybeingmainlydueto thetypeof uid
employed. At times, the preservation of cytological
detail was slightly inferior in the sections which under-
went pressure cooking, but this limitation was largely
overcome by shortening the procedure and by the
larger number of slides which could be treated
simultaneously.
Pressure cooking provided the best results, irrespec-
tiveof theuid employed, when thetreatment lasted 1
min 30s.
Tissuexation
Proteolytic enzyme gave occasional advantages in
terms of AR only in formalin-xed material, whilst the
heat-based systems turned out to beequally eectivein
formalin and B5-xed tissuesamples, including EDTA-
decalcied bone-marrowbiopsies(Figs1012). Further-
more, section exposureto high temperatures minimized
Table I VResults obtained under dierent methodological conditions with a panel of antibodies directed against molecules
relevant for thestudy of malignant tumours
Clone Specicity Source Dilution No AgR PT
HBAR+
citrate
HBAR+
TrisHCl
HBAR+
EDTA
HHF35 Actin DAKO 1:400 + ++ +++ +++ ++++
E29 EMA DAKO 1:5 ++ ++ +++ +++ ++++
HMB45 Melanoma-associated antigen DAKO 1:50 + ++++ +++
ER-PR8 PSA DAKO 1:30 + +++ ++++
1:800 + + +++ ++++
Poly -hCG DAKO 1:16000 ++ +++ ++++ +++ ++++
1:50000 ++ +++ +++ +++ ++++
Poly
1
-Fetoprotein DAKO 1:16000 ++ +++ +++ ++++
Thyroglobin DAKO 1:4000 ++ ++ ++++ +++ ++++
Poly Chromogranin A DAKO 1:8000 + + ++ ++++ ++++
BBS/NC/VI -H14 NSE DAKO 1:100 ++ +++ +++ ++++
1D5 Oestrogen receptor DAKO 1:160 ++ ++++
Po Progesteronereceptor DAKO 1:50 ++ ++ ++++
No AgR=no antigen retrieval; PT=proteolytic treatment; HBAR=heat-based antigen retrieval; EMA=epithelial membrane antigen;
PSA=prostatic-specic antigen; -hCG=-human chorionic gonadotropin; NSE=neuro-specic enolase.
I n bold: overnight incubation of theprimary antibody+SABC technique.
=completely negativeresult; +=weak positivity in a percentageof cells expected to bepositive; ++=weak positivity in all cells
expected to bepositive; +++=moderately strongpositivity in all cellsexpected to bepositive; ++++=very strongpositivity in all cellsexpected
to bepositive.
Fig. 1CD30 staining in a lymph nodebiopsy taken froma patient with nodular sclerosing Hodgkins diseaseand xed in formalin for 1 week
(antigen retrieval by EDTA and pressurecooking; APAAP technique)
Fig. 2Detection of follicular dendritic cells in a hyperplastic lymph nodeby theCD21monoclonal antibody (antigen retrieval by proteaseXI V;
APAAP technique)
Figs35Dierent stainingintensitiesof folliclecentrecellsby theJ CB117/CD79amonoclonal antibody when dierent retrieval uidsareapplied
(Fig. 3: Na citrate; Fig. 4: TrisHCl; Fig. 5: EDTA; pressurecooking; APAAP technique)
Figs68Antigenretrieval byNacitrate, TrisHCl, andEDTA givesnegative, moderatelypositive, andstronglypositiveresults, respectively, when
theanti-PSA monoclonal antibody is applied to sections froma prostatic adenocarcinoma (pressurecooking; APAAP technique)
Fig. 9Detection of theproliferation-associated nuclear antigen Ki-67in a hyperplastic lymph nodeby theMI B-1monoclonal antibody (antigen
retrieval by EDTA and pressurecooking; APAAP technique)
Fig. 10Bone-marrowbiopsy: detection of B- (a) and T-cells (b) within a lymphoid reactiveinltrate(antibodies J CB117/CD79a and polyclonal
anti-CD3, respectively; antigen retrieval by EDTA and pressurecooking; APAAP technique)
Fig. 11Bone-marrow biopsy: positivity of neoplastic cells with the KP1/CD68 antibody in a non-lymphoid acute leukaemia of the M5 type
(antigen retrieval by EDTA and pressurecooking; APAAP technique)
Fig. 12Bone-marrowbiopsy: theantibodyagainst myeloperoxidasestainsafewresidual cellsinthesameacutenon-lymphoidleukaemiaasinFig.
11 (antigen retrieval by EDTA and pressurecooking; APAAP technique)
120 S. A. PILERI ET AL.
1997 by J ohn Wiley & Sons, Ltd. oiN:i or i:1noiocx, voi. 183: 116123 (1997)
or abrogated thedierences in stainingamong24h and
1 week formalin-xed samples.
DISCUSSION
I n 1991, two independent groups reported that
exposure of routine sections to high temperatures in
an aqueous medium could improve the detection of
antigens by overcoming the masking eect of formalin
xations.
4,27
Since then, articles dealing with the prob-
lemof AR in routine sections have been ourishing in
theliterature, based on heating or exposureto a strong
alkali or acid. At present, HBAR methodsaredailyused
in histopathology laboratories, as they havebeen found
to be more eective and more easily applicable than
other AR systems. However, theuids and heat sources
employed vary fromsite to site:
16
this does not favour
standardization in immunohistochemistry, which is felt
to be relevant by many researchers and public institu-
tions.
18
Even themechanisms by which thesenewtech-
niques are so eective in antigen unmasking are still
unclear. Cattoretti andSuurmeijer
16
suggestedthat heat
andhydrolysismaybothdenaturateandbreak thetissue
proteins at or near the links made by the formalin
between adjacent amino acids and thought it conceiv-
able that self-assembly of unfolded protein chains with
subsequent restorationof antigenicsitesoccurswhenthe
retrieval solution is allowed to cool. Shi et al.
18
have
stressed that the pH of the retrieval solution is an
important cofactor for some antigens. Morgan et al.
15
have reported that tight complexing of calcium ions
or other divalent metal cations with proteins during
formaldehyde xation can be responsible for masking
certain antigens; thus, the chelation or precipitation of
these ions can represent a critical step in salt-mediated
AR. Thehigh temperatures might beneeded to provide
sucient energy to releasethecalciumionsand divalent
metal cations from the cage-like complexes that they
formwith tissueproteins.
15
The present study gives further support to the
usefulness of HBAR and provides new information in
order to transformthis kitchen approach into a more
standardized tool.
Tissuexation and processing
To thebest of our knowledge, this is therst demon-
stration that HBAR is very eective in re-exposing a
largenumber of relevant antigens both in B5-xed and
in B5-xed, EDTA-decalcied material. This nding
expandsthepreviouslimitedexperienceonthetopicand
answers some pending questions. Ho et al.
28
reported
that microwave AR does not signicantly improve the
detectionof I glight chainsinB5-xedmaterial when4x
urea is applied as the retrieval solution. Cattoretti and
Suurmeijer indicated that the B5-xed tissues can be
stained with a large panel of antibodies after heat
unmasking, basedonour preliminaryresults.
16
Morgan
et al.
15
concluded their paper dealing with EDTA/
EGTA-mediated AR by stressing the need for further
studies to assess whether these uids might be equally
eective in tissues submitted to decalcication. Our
results clearly show that when retrieval solutions other
than4x ureaareemployed, bothB5-xedandB5-xed,
EDTA-decalcied samples are excellent candidates for
HBAR, the quality of immunostaining being improved
with a large variety of polyclonal and monoclonal
antibodies and someantigens becomingreliably accessi-
ble for the rst time (Tables I I V): this statement is
relevant, as B5 xation and EDTA decalcication are
quitecommonly used in haematopathology. Theobser-
vation that HBAR is eective in B5-xed material
further points to the importance of high temperature
as a cofactor in antigen unmasking. This is in keeping
with the nding that even alcohol-xed tissues, which
do not contain cross-linked proteins, benet from the
treatment.
16
Our results with HBAR in formalin-xed samples
parallel those previously reported in the literature.
419
On the whole, HBAR turned out to be superior to
enzymatic treatment in most instances (Tables I I V).
Heat sources
I nour hands, pressurecookingshowedseveral advan-
tagesover microwaveheating. I n particular, asreported
by Norton et al.,
14
it isan eectiveand reliablemethod,
very easy to use, which allows the simultaneous treat-
ment of largebatches of slides; it is not time-consuming
for technologists and it needs only very cheap equip-
ment. Furthermore, pressure cooking avoids the main
drawback of microwaves, namely theoccurrenceof hot
and cold spots, which impairs AR eciency, especially
when heating many slides.
Although heatingof sections is thesametool at work
in both pressurecooking and microwaveheating, some
dierences should exist in the mechanism(s) by which
the two methods produce their positive action. I n fact,
besides the dierent exposure times, the microwave
approach requiresprolonged coolingto achieveoptimal
results, whilepressurecooking does not.
Contrary to what has been thought in the past,
18
overheating does not reduce tissue immunoreactivity.
According to Mason and OLeary
29
who showed that
temperatures from70C to 90C haveno adverseeect
on formalin-xed proteins, it is conceivable that the
bonds produced by xation protect the epitopes from
denaturation to some extent during HBAR. This
hypothesis is supported by theobservation that HBAR
can bedetrimental in samples xed in formalin for very
short times (data not shown): in that case, the high
temperaturesmight denatureproteinsnot yet completely
protected by xation.
AR uids
Three dierent AR uids were employed: 001x
citrate buer (pH 60), 01x TrisHCl (pH 80), and
1mx EDTA (pH 80). The rst is currently the most
widely used medium;
16,18
thesecond representsan inter-
esting proposal from Shi et al.;
18
and the third was
originally described by Morgan et al.
15
for the detec-
tion of the Ki-67 antigen in routine sections and was
122 S. A. PILERI ET AL.
1997 by J ohn Wiley & Sons, Ltd. oiN:i or i:1noiocx, voi. 183: 116123 (1997)
subsequently found to bevery eectivefor theretrieval
of a xation-resistant epitopeof thebcl-6 product.
24
Among these solutions, EDTA turned out to be the
most ecient with the vast majority of the antibodies
tested and independently of the location of the target
molecule(intranuclear, intracytoplasmic, or membrane-
bound). Citratebuer moreoften produced thepoorest
results, bothintermsof immunostainingintensityandin
terms of the number of positive cells. The ecacy of
TrisHCl was intermediate.
I n conclusion, this study provides evidence that the
choice of the retrieval uid is crucial with the new AR
techniques. Two alternatives are possible: either to
chooseamediumwhich guaranteesan excellent average
with most antibodies, or to nd theideal recipefor each
antibody. Theformer approach seems to bemoreprac-
tical if one favours standardization in histopathology:
AR by pressure cooking and EDTA represents a
proposal to that end.
ACKNOWLEDGEMENTS
This paper was supported by grants from AI RC
(Milan), CNR (ACRO 10) (Rome), MURST (Rome),
and A.B.S.T.E. (Bologna).
REFERENCES
1. Pileri S, SerraL, Martinelli G. Theuseof pronaseenhancessensitivityof the
PAP method in the detection of intracytoplasmic immunoglobulins. Bas
Appl Histochem1980; 24: 203207.
2. Taylor CR. Principles of microscopy. I n: Taylor CR, ed. I mmunomicro-
scopy: A Diagnostic Tool for the Surgical Pathologist. Philadelphia: W B
Saunders, 1986; 2022.
3. Falini B, Pileri S, Martelli MF. Histological and I mmunohistological
Analysis of Human Lymphoma. Vol. 9. CRC Press, 1989; 351419.
4. Shi S-R, Key ME, Kalra KL. Antigen retrieval in formalin-xed, paran
embedded tissues: an enhancement method for immunohistochemical stain-
ing based on microwave oven heating of tissue sections. J Histochem
Cytochem1991; 39: 741748.
5. Suurmeijer AJ H. Microwave-stimulated antigen retrieval. A new method
facilitating immunohistochemistry of formalin-xed, paran embedded
tissue. HistochemJ 1992; 24: 597 (Abstract).
6. Cattoretti G, Becker HG, Key G, et al. Monoclonal antibodies against
recombinant parts of the Ki-67 antigen (MI B 1 and MI B 3) detect
proliferating cells in microwave-processed formalin-xed paran sections.
J Pathol 1992; 168: 357363.
7. Cattoretti G, Pileri S, Parravicini C, et al. Antigen unmaskingon formalin-
xed, paran embedded sections. J Pathol 1993; 171: 8398.
8. Gown AM, deWever N, Battifora H. Microwave-based antigenic un-
masking. A revolutionary new technique for routine immunohistochemis-
try. Appl I mmunohistochem1993; 1: 256266.
9. Suurmeijer AJ H, Boon ME. Notes on the application of microwaves for
antigen retrieval in paran and plastic sections. Eur J Morphol 1993; 31:
144150.
10. Van den Berg F, Claessen N, Mulder J , Polak M, Volkers H, Oerhaus
GJ A. Target unmasking uid (TUF) for immunohistochemistry in
(archival) paran embedded tissue. Eur Clin Lab 1993; 20: 24.
11. Leong ASY, Milios J . An assessment of the ecacy of the microwave
antigen-retrieval procedure on a range of tissue antigens. Appl I mmuno-
histochem1993; 1: 267274.
12. MomoseH, Mehta P, Battifora H. Antigen retrieval by microwaveirradia-
tion in lead thiocyanate. Comparison with protease digestion. Appl
I mmunohistochem1993; 1: 7782.
13. Taylor CR, Shi S-R, Chaiwun B, Young L, I mamSA, CoteRJ . Strategies
for improving the immunohistochemical staining of various intranuclear
prognostic markers in formalinparan sections: androgen receptor,
estrogen receptor, progesterone receptor, p53 protein, proliferating cell
nuclear antigen, and Ki-67antigen revealed by antigen retrieval techniques.
HumPathol 1994; 25: 263270.
14. Norton AJ , J ordan S, Yeomans P. Brief, high-temperatureheat denatura-
tion (pressurecooking): a simpleand eectivemethod of antigen retrieval
for routinely processed tissues. J Pathol 1994; 173: 371379.
15. MorganJ M, Navabi H, SchmidKW, J asani B. Possibleroleof tissue-bound
calciumionsincitrate-mediatedhigh-temperatureantigenretrieval. J Pathol
1994; 174: 301307.
16. Cattoretti G, Suurmeijer AJ H. Antigen unmasking on formalin-xed
paran-embedded tissues using microwaves: a review. Adv Anat Pathol
1995; 2: 29.
17. Miller K, Auld J , J essup E, RhodesA, Ashton-Key M. Antigen unmasking
in formalin-xed routinely processed paran wax-embedded sections
by pressure cooking: a comparison with microwave oven heating and
traditional methods. AdvAnat Pathol 1995; 2: 6064.
18. Shi S-R, I mam SA, Young L, Cote RJ , Taylor CR. Antigen retrieval
immunohistochemistry under the inuence of pH using monoclonal anti-
bodies. J HistochemCytochem1995; 43: 193201.
19. Miller RT, EstranC. Heat-inducedepitoperetrieval withapressurecooker.
Suggestions for optimal use. Appl I mmunohistochem1995; 3: 190193.
20. Pileri S. Linfomi maligni non-Hodgkin: dalla tecnica alla diagnostica.
Bologna: Societ EditriceEsculapio, 1985.
21. Pileri S, Falini B, Sabattini E, Bigerna B, Gherlinzoni F, Tazzari PL.
I mmunohistochemistry of malignant lymphomas. Advantages and limita-
tions of the new monoclonal antibodies working in paran sections.
Haematology 1991; 76: 226234.
22. Harris N, J aeE, Stein H, et al. A Revised EuropeanAmerican classica-
tion of lymphoid neoplasms: aproposal fromtheI nternational Lymphoma
Study Group. Blood 1994; 84: 13611392.
23. Bennett J M, Catovsky D, Daniel MT, et al. Proposals for theclassication
of the acute leukemias. A report of the FrenchAmericanBritish (FAB)
co-operativegroup. Br J Haematol 1976; 33: 451458.
24. Flenghi L, BigernaB, Fizzotti M, et al. Monoclonal antibodiesPG-B6aand
PG-B6p recognize, respectively, a highly conserved and a formol-resistant
epitopeon thehuman BCL-6 protein amino-terminal region. AmJ Pathol
1996; 148: 15431553.
25. Cordell J L, Falini B, Erber WN, et al. I mmunoenzymatic labelling of
monoclonal antibodies using immune complexes of alkaline phosphatase
and monoclonal anti-alkaline phosphatase (APAAP complexes). J
HistochemCytochem1984; 32: 219229.
26. Santini D, Ceccarelli C, Mazzoleni G, Pasquinelli G, J asonni VM,
Martinelli GN. Demonstration of cytokeratin intermediate laments in
oocytesof thedevelopingand adult human ovary. Histochemistry 1993; 99:
311319.
27. Shin RW, I waki T, Kitamoto T, Tateishi J . Hydrated autoclave
pretreatment enhances TAU immunoreactivity in formalin-xed normal
and Alzheimers diseasebrain tissue. Lab I nvest 1991; 64: 693702.
28. Ho J , Shintaku I P, Preston M, Said J W. Can microwaveantigen retrieval
replace frozen section immunohistochemistry in the phenotyping of
lymphoidneoplasms?A comparativestudyof kappaandlambdalight chain
staining in frozen sections, B5-xed paran sections, and microwaveurea
antigen retrieval. Appl I mmunohistochem1994; 2: 282286.
29. Mason J T, OLeary TJ . Eects of formaldehyde xation on protein
secondarystructure: acalorimetricandinfraredspectroscopicinvestigation.
J HistochemCytochem1991; 39: 225231.
123 STANDARDIZATION AND ANTIGEN RETRIEVAL
1997 by J ohn Wiley & Sons, Ltd. oiN:i or i:1noiocx, voi. 183: 116123 (1997)