Clin Pathol 1997;50:521-524

A panel of antibodies for the immunostaining ofBouin's fixed bone marrow trephine biopsies

Jean-Luc Gala, Frederique Chenut, Khang Bui Thi Hong, Jean Rodhain,Philippe Camby, Marianne Philippe, Jean-Marie Scheiff

Laboratory of ClinicalMolecular Biology,CliniquesUniversitairesSaint-Luc, UniversiteCatholique deLouvain,Clos-Chapelle-aux-Champs,30-UCLI30.46, B-1200Bruxelles, BelgiumJ-L GalaF ChenutM Philippe

Laboratory ofHaematologicalBiology, CliniquesUniversitairesSaint-Luc, UniversiteCatholique deLouvain, AvenueHippocrate, 30, 1200Bruxelles, BelgiumK B T HongJ RodhainJ-M Scheiff

Laboratory ofPathologyP Camby

Queen Astrid MilitaryHospital, Bruxelles,BelgiumJ-L Gala

Correspondence to:Dr Gala.email: galagsang.ucl.ac.be

Accepted for publication18 February 1997

AbstractAims-To assess a panel of antibodies on

Bouin's fixed bone marrow trephine(BMT) biopsies. These biopsies are widelyused in routine diagnosis of varioushaematological malignancies and may bethe sole material available in many cen-

tres; however, information regarding theimmunostaining of this material is lack-ing.Methods-Biopsies were taken from 72patients presenting with various haemato-logical malignancies (leukaemia, 38;lymphoma, 14; multiple myeloma, 20). Apanel of antibodies was assessed on

Bouin's fixed BMT biopsies by the alkalinephosphatase-antialkaline phosphatasemethod.Results-Three B (MB2, LN-2, Ki-B5)and two T cell lineage antibodies(UCHL-1, CD3-r) reliably identified lym-phoid cells, while MPO-r, Leu-M1I/CD15,and KP-1/CD68 recognised cells from themyeloid or histiocytic/macrophage series.Reed-Sternberg cells were stained byLN-2, Leu-M1, and CD30. Antibodies spe-

cific for plasma cells (VS38) and hairycells (DBA.44) gave a variable pattern ofstaining. Among the proliferation mark-ers, proliferative cell nuclear antigen butnot Ki-67 related antibodies were effec-tive.

Conclusion-This study presents a panelof antibodies with reactivity not restrictedto common fixatives that are also suitablefor Bouin's fixed BMT biopsies.(i Clin Pathol 1997;50:521-524)

Keywords: Bouin's fixative; bone marrow trephinebiopsy; immunostaining

Immunohistochemical evaluation in bone mar-

row trephine (BMT) biopsies appears to be a

useful adjunct to the diagnosis and pathologi-cal staging of haematological neoplasms, andcontributes to a better identification of theinvading malignant cells.'-' The majority ofmonoclonal or polyclonal antibodies (MoAb,PoAb) is useful on unfixed and undecalcifiedsnap frozen material cut in a cryostat, however,such material is not readily available. Fresh or

frozen tissues, which are not suitable for longterm preservation, require complex processingand storage. A large panel of MoAb and PoAbrecognising epitopes that survive histologicalprocessing is available,4 and this allows immu-nodetection of a wide variety of cells in

Table 1 Haematological neoplasms

Neoplasm Number ofsamples

Leukaemia 38Acute lymphoblastic B type 4Acute lymphoblastic T type 2Chronic lymphocytic B type 19Chronic lymphocytic T type 1Hairy cell 3Acute myeloid 9

Lymphomas 14Follicular with t(l 4; 18) 2Mantle zone with t(I 1; 14) 3Burkitt's 5Hodkin's 4

Multiple myeloma 20

Diagnoses were based on typical features found by cytology orhistology, cytogenetics, immunophenotype, biochemistry, andmolecular biology using conventional methods.

routinely fixed, decalcified, paraffin wax em-bedded bone marrow biopsies.'-6 Activity ofthese antibodies is usually assessed on bufferedformalin, B5 or Zenker fixed tissues.3 Very fewdata report the reactivity of the antibodies onBouin's fixed BMT biopsies. Lack of immu-nostaining has been reported on thesebiopsies' 2 that, at least partially, explains whythis fixative is not the first choice for immuno-staining of this material. Nevertheless, Bouin'sfixed BMT biopsies are widely used in routinediagnosis of various haematological malignan-cies, and is the sole material available in manycentres.We report experience of immunostaining

Bouin's fixed BMT biopsies using a panel ofthe newest MoAb and PoAb.4 7 8 Labelling wasassessed on common haematological neo-plasms.

Material and methodsSeventy two samples were obtained frompatients presenting with various haematologi-cal neoplasms (table 1): leukaemia (n = 38),lymphoma (n = 14), and multiple myeloma(n = 20). Bouin's fixed BMT biopsies frompatients with non-malignant haematologicaldisorders (n = 8) were also included. All diag-noses were based on typical features found bycytology or histology, cytogenetics, immuno-phenotype, biochemistry, and molecular biol-ogy using conventional methods.Bone marrow biopsies were all placed in

Bouin's fixative (copper acetate monohydrate25 g, picric acid 40 g, distilled water 1000 ml,formalin 100 ml, acetic acid 15 ml) for nolonger than 24 hours, decalcified for six hoursin 7.5% nitric acid, and embedded in paraffin.Before immunostaining, each Bouin's fixedBMT biopsy was dewaxed and rehydrated. The

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Table 2 Antibodies assessed on Bouin'sfixed bone marrow trephine biopsies

Markers/CD Clone

B cells and plasma cellsKappa-rabbit*Lambda-rabbit*L26/CD204KB5/CD45RAKi-B3/CD45RAKi-B5LN-1/CDw75LN-2/CD74MB2DBA.44VS38C

T cellsUCHL-1/CD45ROCD3-rabbit*

Myeloid/Monocytes/MacrophagesKP1/CD68MPO-rabbit*Leu-MI/CD1 5

Non-lineage restrictedBcl-2Bcl-1 (PRAD 1)DO-7BerH2/CD30PCNAKi-67Ki-67-rabbit*

Source

L264KB5Ki-B3Ki-B5LN-1LN-2MB2DBA.44VS38C

UCHL-1

KP1

Leu-Ml

124DCS-6DO-7BerH2PC10MIB-1

DakoDakoDakoDakoProfessor ParwareschProfessor ParwareschClonabClonabBiotestDakoDako

DakoDako

DakoDakoBecton Dickinson

DakoProfessor BartekNovocastraDakoDakoImmunotechDako

Dako, Dakopatts, Prosan, Belgium; Biotest, Biotest Diagnostics, Dreieich, Germany.Professor Bartek is at the Danish Cancer Society, Copenhagen, Denmark and ProfessorParwaresch is at the Institut fur Hamatopathologie, Kiel, Germany.*Polyclonal antibody.

Table 3 Results of Bouin'sfixed bone marrow trephine immunostaining

Working antibodies(retrieval

MarkerslCD procedure) Dilution Malignancies

B cells and plasma cellsKappa-rabbit + 1/10000 MMLambda-rabbit + 1/10000 MML26/CD20 - (MW) B-LLC, HC, FL, MZ4KB5/CD45RA +/- (MW) 1/25 B-LLC, HC, FL, MZ, CoKi-B3/CD45RA +/- (MW) B-LLC, HC, FL, MZ, CoKi-B5 + 1/1 B-LLC, HC, FL, MZ, CoLN-l/CDw75 - HC, HDLN-2/CD74 + 1/50 B-LLC, HC, HD, FL, MZ, CoMB2 + 1/50 B-LLC, HC, FL, MZ, CoDBA.44 +/- (MW/Enz) 1/50 HCVS38C +/- (MW) 1/50 MM

T cellsUCHL-1/CD45RO + 1/50 T-CLL, T-ALL, CoCD3-rabbit + 1/100 T-CLL, T-ALL, Co

Myeloid/Monocytes/MacrophagesKP1/CD68 + 1/50 AML, MMMPO-rabbit + 1/100 AMLLeu-Ml / CD15 + 1/50 AML, HD

Non-lineage restrictedBcl-2 + 1/80 B-ALL, B-CLL, FL, MMBcl-l (PRAD 1) - (MW) MZDO-7 - (MW) B-CLL*BerH2/CD30 + 1/50 HDPCNA + 1/100 BLKi-67 - (MW) BLKi-67-Rabbit - (MW) BL

B-ALItT-ALL, B cell type/T cell type acute lymphoblastic leukaemia; B-CLIJT-CLL, B celltype/T cell type chronic lymphocytic leukaemia; BL, Burkitt's lymphoma; Co, non-malignantBouin's fixed BMT biopsy; FL, follicular (centrocytic-centroblastic) lymphoma; HD, Hodgkin'sdisease; MM, multiple myeloma; MZ, mantle zone lymphoma; MW, antigen retrieval bymicrowave heating; Enz, antigen retrieval by enzymatic digestion.*B cell CLL patient disclosing a p53 missense mutation.

alkaline phosphatase-antialkaline phosphatasestaining, used to avoid the problem of endog-enous peroxidase, was performed as describedpreviously.6

Antibodies tested and optimal dilutions arelisted in table 2. They were incubated for onehour, except for CD3-r (r = rabbit), Ki-67-r,

lambda, and kappa that were incubatedovernight. Isotype matched monoclonal anti-bodies were included as a control for non-specific binding. Slides were examined under alight microscope and evaluated for stainingintensity, graded from 0 to 5+. Each antibodyunreactive after conventional procedure wasreassessed after microwave heating (five cyclesof three minutes each in citrate buffer pH 5.7)or enzymatic digestion with pronase, accordingto the manufacturer's instructions andliterature.9

ResultsThe results are summarised in table 3.

B CELL AND PLASMA CELLS ASSOCIATEDANTIBODIESKappa and lambda-Both polyclonal antibod-ies positively stained (3+ or 4+) plasma cells inmultiple myelomas, allowing the detection ofmonotypy.L26, 4KB5, Ki-B3, LN-I-We confirmed a lackof staining with L26' 2 on B cell malignancies(chronic lymphocytic, hairy cell) regardless ofmicrowave heating procedure. Unlike previousdata,3 6 neither Reed-Sternberg nor hairy cellleukaemia biopsies reacted with LN-1, and Bcell malignancies were either inconsistentlypositive or very weakly stained (0 or 1+) with4KB5 and Ki-B3.LN-2, MB2, Ki-B5-LN-2, MB2, and themore recently described MoAb Ki-B5' stronglylabelled all chronic lymphocytic B cell andhairy cell leukaemia biopsies (fig 1A), as well asfollicular lymphomas, mantle zone lympho-mas, and normal B lymphocytes (3+ to 5+). Tothe best of our knowledge, the reactivity ofKi-B5 recognising normal and neoplastic Bcells has not been previously reported in BMTbiopsies. LN-2 also identified Reed-Sternbergand Hodgkin's cells in all Hodgkin's diseasebiopsies assessed (3+ or 4+). MB2 reacted asexpected with endothelial cells but stained only50% of multiple myelomas.DBA. 44-Proposed as a hairy cell marker,4 thisMoAb labelled only one of the three hairy cellbiopsies (2+). Noteworthy, staining was mark-edly intensified (4+) by pronase and by micro-wave heating, but only in the single positivecase.VS38-The use of microwave heating wasmandatory with this recently discoveredplasma cell marker.8 Variable immunostainingof normal and malignant plasma was observed(0 to 4+). On most slides, strongly positivemalignant plasma cells, observed on the borderof heavily infiltrated slides (fig 1B), coexistedwith totally negative cells in the centre of thebiopsy, demonstrating the variability of label-ling due to the quality of the fixation (poorer inthe middle of the tissue).

T CELL ASSOCIATED ANTIBODIESUCHL-1, CD3-r-Both T lineage antibodies,which are recommended for the diagnosis ofTcell lymphomas in paraffin wax embeddedsections,10 stained clearly normal and malig-

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Antibodies for the immunostaining ofBouin's fixed bone marrow trephine biopsies

nant T cells. Staining with UCHL-1 wasusually stronger (4+ to 5+) than with CD3-r(2+ to 3+). In concert with others, we foundstaining of myeloid cells with UCHL-1L.

MYELOID/MONOCYTE/MACROPHAGE ASSOCIATEDANTIBODIESKPJ/CD68, MPO, Leu-M1/CD15-KP1 re-acted with marrow monocytes and macro-phages in normal and pathological Bouin'sfixed BMT biopsies (4+ or 5+) (fig 1C), andstrongly labelled myeloid cells in eight of nineacute myeloid leukaemias. Rabbit PoAb(MPO) against myeloperoxidase labelled blastcells in nine of nine myeloid malignancies (4+or 5+) (fig ID). Leu-Ml stained maturegranulocytic cells and monocytes (5+), three ofseven acute myeloid leukaemias (4+ or 5+),

and Reed-Sternberg cells in four of four Hodg-kin's disease biopsies (3+ or 4+) (fig IE).

NON-LINEAGE ASSOCIATED ANTIBODIESBcl-2, Bcl-1, DO-7-Among the antibodies tooncoproteins, bcl-2 but not DO-7 or bcl-l(PRAD D 1/cyclin D1) reacted on Bouin'sfixed BMT biopsies. Anti-bcl-2 MoAb consist-ently stained follicular lymphoma (5+) (fig1F), chronic lymphocytic B cell leukaemia (3+to 5+), acute lymphoblastic leukaemia (4+ or5+), and multiple myeloma (2+ to 5+), but, asexpected, none of the Burkitt's lymphomas."1

CD3O-Like LN-2, CD30 labelled the Reed-Sternberg cells in four of four Hodgkin'sdisease biopsies.PCNA, MiB-1, Ki-67-r-Despite microwaveheating, no staining was observed with either

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Figure 1 (A) Group of hairy cells reacting with Ki-B5; (B) VS38 positive multiple myeloma cells; (C) Cellular contact between KP1 positivemacrophage and plasma cells; (D) Staining of myeloblasts by MPO in a case of acute myeloid leukaemia; (E) Typical Reed-Sternberg cell reacting withLeu-MI; (F) bcl-2 labelling of infiltrating follicular lymphoma cells.

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MoAb or PoAb recognising the Ki-67 antigen.On the contrary, nuclear localisation of prolif-erative cell nuclear antigen (PCNA) was dem-onstrated in more than 80% of the malignantcells in highly proliferative Burkitt's lymphoma(4+ or 5+) without antigen retrieval.

DiscussionAmong the antibodies tested, several demon-strated excellent immunoreactivity on tissuesfrom Bouin's fixed BMT biopsies. The anti-genic specificity on pathological marrows wasin accordance with previously reporteddata.F6 However, the weak and inconsistentimmunolabelling with some MoAb that recog-nise formalin resistant antigen (4KB5, Ki-B3)renders them inappropriate for immunostain-ing of Bouin's fixed BMT biopsies. Lack ofstaining was observed with DBA.44 from twoflorid hairy cell leukaemia biopsies, whereasthey were strongly positive with MB2, suggest-ing that DBA.44 labelling may depend on theexposure to Bouin's fixative. This seems also toapply to VS38 where strong positive or totallynegative labelling of normal and malignantplasma cells was striking on the same slides.Interestingly, this staining variability was notobserved with other antibodies (MB2, LN-2,Ki-B5, UCHL-1, CD3-r, PCNA, CD30, Leu-Ml). Noteworthy, preliminary microwaveheating, recommended by the manufacturer'sinstruction, was not mandatory for immuno-staining of Bouin's fixed BMT with Pab CD3-ror CD30.

In conclusion, reliable immunophenotypingof archival Bouin's fixed BMT biopsies is feasi-ble. While demonstrating the variable degree ofreactivity obtained on Bouin's fixed and decal-cified tissues with some widely used or morerecent antibodies, this study presents a panel ofantibodies suitable for phenotyping the most

common haematological neoplasms on thismaterial.

This study was fully supported by the association SalusSanguinis. We thank Professor Bartek, Danish Cancer Society,Copenhagen, Denmark and Professor Parwaresch, Institut furHamatopathologie, Kiel, Germany for the generous gift of anti-body, Dr M Zandecki, H6pital Calmette, Lille, France for criti-cal reading of the manuscript and useful discussions, andProsan, Dako, Belgium for its contribution.

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