J Clin Pathol 1989;42:414-421 Laboratory techniques KP1: a new monoclonal antibody that detects a monocyte/macrophage associated antigen in routinely processed tissue sections K A F PULFORD, E M RIGNEY, K J MICKLEM, M JONES, W P STROSS, K C GATTER, D Y MASON The Nuffield Department of Pathology, John Radcliffe Hospital, Oxford SUMMARY A new monoclonal antibody, KP1, raised against a lysosomal fraction of human lung macrophages, recognises a fixation-resistant epitope in a wide variety of tissue macrophages (such as Kupffer cells germinal centre, splenic, and lamina propria macrophages), and in granulocyte precursors. Its broad reactivity with cells of the mononuclear phagocytic lineage was established by testing on routinely processed samples of normal and reactive lymphoid tissues. Interdigitating reticulum cells were unstained or showed limited cytoplasmic staining while Langerhans' cells and follicular dendritic reticulum cells were unreactive. KPI recognises a molecule of about 110 kilodaltons in macrophage-rich human tissue when tested by either immunoprecipitation or Western blotting (although the latter procedure also shows two additional components with molecular weights of 70 and 40 kilodaltons). KP1 should be of considerable value for studying disorders of the monocyte/macrophage system, including both reactive and neoplastic states (such as true histiocytic proliferations). Monocytes, macrophages, and other elements of the mononuclear phagocytic system have several vital roles in healthy subjects, including antigen processing, secretion of a wide range of molecules, and phagocytosis.' Despite their obvious importance progress in defining antigenic molecules specific for these cells which can be used to trace their distribution in normal and pathological human tissue has been limited. This emerged from the analysis of anti- myeloid antibodies performed in the Third Inter- national Workshop on Leucocyte Differentiation Antigens2 which defined several new molecules on mononuclear phagocytes according to the CD system (CD3 1, CD32, CD35, CD36). Many of these markers, however, are also expressed on other tissues such as CD26 on platelets and CD31 on endothelium, or are absent from, or present at only low levels on, tissue macrophages. None of these new antigens, nor any of the CD antigens reactive with monocytes identified at earlier workshops (CDll, CD13, CD14, CD16 or CD 18) could be categorised as "pan-macrophage" and the nearest approach to the definition of such markers in the Third Workshop lay in the identifica- Accepted for publication 6 October 1988 tion, by staining tissue sections, of a group of five antibodies which showed closely similar reactions, and which all stained selectively most tissue macrophages (antibodies 24, Y2/131, Ki-M6, Y1/82A, EBM1 1). The molecular targets for these antibodies remain poorly defined, however, and all of these reagents suffer from the practical limitation, in the context of diagnostic pathology, of not reacting with routinely processed paraffin wax embedded tissue. In this paper we describe a new monoclonal antibody (KPI) which recognises most human macrophages in routinely processed tissue, and for which the target molecule can be identified by immunocytochemical techniques. Material and metbods PREPARATION OF ANTIGEN Samples of fresh normal human lung, removed during surgery for lung carcinoma, were obtained from this hospital. Cells were extracted by irrigating the lung tissue with HEPES buffered RPMI 1640 medium containing 10% fetal calf serum (FCS, Gibco Biocult, Long Island Co, Ltd). The resulting washings were spun down and separated on a Lymphoprep 414 on March 19, 2020 by guest. Protected by copyright. http://jcp.bmj.com/ J Clin Pathol: first published as 10.1136/jcp.42.4.414 on 1 April 1989. Downloaded from
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JClin Pathol 1989;42:414-421
Laboratory techniquesKP1: a new monoclonal antibody that detects amonocyte/macrophage associated antigen in routinelyprocessed tissue sections
K A F PULFORD, E M RIGNEY, K J MICKLEM, M JONES, W P STROSS,K C GATTER, D Y MASON The Nuffield Department ofPathology, John Radcliffe Hospital, Oxford
SUMMARY A new monoclonal antibody, KP1, raised against a lysosomal fraction of human lungmacrophages, recognises a fixation-resistant epitope in a wide variety of tissue macrophages (such asKupffer cells germinal centre, splenic, and lamina propria macrophages), and in granulocyteprecursors. Its broad reactivity with cells of the mononuclear phagocytic lineage was established bytesting on routinely processed samples of normal and reactive lymphoid tissues. Interdigitatingreticulum cells were unstained or showed limited cytoplasmic staining while Langerhans' cells andfollicular dendritic reticulum cells were unreactive. KPI recognises a molecule of about 110kilodaltons in macrophage-rich human tissue when tested by either immunoprecipitation or Westernblotting (although the latter procedure also shows two additional components with molecularweights of 70 and 40 kilodaltons).KP1 should be of considerable value for studying disorders of the monocyte/macrophage system,
including both reactive and neoplastic states (such as true histiocytic proliferations).
Monocytes, macrophages, and other elements of themononuclear phagocytic system have several vitalroles in healthy subjects, including antigen processing,secretion of a wide range of molecules, andphagocytosis.' Despite their obvious importanceprogress in defining antigenic molecules specific forthese cells which can be used to trace their distributionin normal and pathological human tissue has beenlimited. This emerged from the analysis of anti-myeloid antibodies performed in the Third Inter-national Workshop on Leucocyte DifferentiationAntigens2 which defined several new molecules onmononuclear phagocytes according to the CD system(CD3 1, CD32, CD35, CD36). Many of these markers,however, are also expressed on other tissues such asCD26 on platelets and CD31 on endothelium, or areabsent from, or present at only low levels on, tissuemacrophages. None of these new antigens, nor any ofthe CD antigens reactive with monocytes identified atearlier workshops (CDll, CD13, CD14, CD16 orCD 18) could be categorised as "pan-macrophage"and the nearest approach to the definition of suchmarkers in the Third Workshop lay in the identifica-
Accepted for publication 6 October 1988
tion, by staining tissue sections, of a group of fiveantibodies which showed closely similar reactions, andwhich all stained selectively most tissue macrophages(antibodies 24, Y2/131, Ki-M6, Y1/82A, EBM1 1).The molecular targets for these antibodies remain
poorly defined, however, and all of these reagentssuffer from the practical limitation, in the context ofdiagnostic pathology, of not reacting with routinelyprocessed paraffin wax embedded tissue. In this paperwe describe a new monoclonal antibody (KPI) whichrecognises most human macrophages inroutinely processed tissue, and for which the targetmolecule can be identified by immunocytochemicaltechniques.
Material and metbods
PREPARATION OF ANTIGENSamples offresh normal human lung, removed duringsurgery for lung carcinoma, were obtained from thishospital. Cells were extracted by irrigating the lungtissue with HEPES buffered RPMI 1640 mediumcontaining 10% fetal calf serum (FCS, Gibco Biocult,Long Island Co, Ltd). The resulting washings werespun down and separated on a Lymphoprep
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Use ofKPI to detect monocyte/macrophage associated antigen
(Nygaard) density gradient. Macrophages accountedfor about 80% of the total white cell count. These cellswere washed and a total of 4 x 10' resuspended in1[5 ml 0 34M sucrose containing 1 mM iodoaceta-mide, 0 2 mM phenylmethyl sulphonyl fluoride, 0 5%sodium azide, 10 mM leupeptin and Pepstatin A(Sigma Chemical Co.). The suspension was sonicatedand the nuclei pelleted by centrifugation at 300 g forfive minutes. The supernatant was then spun at 8000 gfor 15 minutes and the resulting supernatant stored at- 70°C. This material constituted the antigen used forimmunisation.
IMMUNISATION SCHEDULE AND FUSIONBalb/c mice were immunised three times intraperiton-eally at eight day intervals with 50 ig antigen emul-sified in Freund's complete adjuvant. A fusion wascarried out three days after the last injection accordingto a previously described technique3 and supernatantswere screened initially on cryostat sections of humanlung and tonsil. Further testing was carried out onparaffin wax sections of lung tissue fixed in formolsaline.
CELLS AND CELL LINESRoutine bone marrow smears taken for diagnosticpurposes and smears of human peripheral blood wereobtained from the haematology department. Slideswere air-dried overnight and stored at - 20°C. Theywere fixed before use in a variety of different fixatives,including buffered formol acetone and acetonemethanol, as detailed elsewhere.4
TISSUE SECTIONSNormal tonsil was obtained from the Ear, Nose andThroat Department of the Radcliffe Infirmary,Oxford.Fixed tissue: Tissues obtained fresh were fixed inunbuffered formol saline before paraffin wax embed-ding. Routinely processed paraffin wax embeddedtissue biopsy specimens were obtained from the his-topathology department. All tissues had been fixed informol saline, with the exception of bone marrowtrephine biopsy specimens which were fixed in formolsaline containing 1% glacial acetic acid. Routinesections from several other hospitals which had beenfixed in B5 fixative were also analysed.Fresh tissues: Tonsil samples were snap frozen inliquid nitrogen. Cryostat sections of 6 ,um thicknesswere cut, dried, fixed and stored as previously des-cribed.5
IMMUNOENZYMATIC LABELLINGTissue sections and cell smear preparations werestained using the alkaline phosphatase:anti-alkalinephosphatase (APAAP) method.56 Before immuno-
staining, paraffin wax sections of formalin fixed tissuewere dewaxed, hydrated, and then incubated for 20-30minutes in 0 1% trypsin solution in 0 1% calciumchloride solution (pH 7 8).
SODIUM DODECYL SULPHATE-POLYACRYLAMIDEGEL ELECTROPHORESIS (SDS-PAGE) ANDWESTERN BLOTTINGFresh spleen tissue in which there was a heavymacrophage infiltration was obtained after splenec-tomy from a case of B cell lymphoma. The tissue wasstored frozen at - 70°C until use. Samples werehomogenised in a hypotonic 20 mM Tris (pH 7-5)buffer, the resulting suspension centrifuged at 70 000 gfor 20 minutes, and the supernatant removed. Thepellet was rehomogenised in the Tris buffer, this timecontaining 2%/o Tween 40 (Sigma Chemical Co.) andspun at 4000 g to remove nuclei before a high speed70 000 g spin. The 70 000 g supernatant was retainedand the pellet was washed with 20 mM Tris buffer. Thewashed pellet was rehomogenised in the Tris buffercontaining 1% Nonidet P40 (NP40), spun at 70 000 g,and the supernatant retained. A pellet of 2 x 108 U937cells was lysed in 1% NP40. After a 70 000 g spin thesupernatant was collected.
All procedures were performed at 4°C in thepresence of the metabolic inhibitors described above.The three different spleen lysates, the U937 lysate,
and the antigen used for immunisation were run on a7 5% SDS polyacrylamide gel slab.7 Proteins weretransferred electrophoretically overnight by Westernblotting8 on to nitrocellulose paper using a TRANS-BLOT apparatus (Biorad). After blocking free proteinbinding sites with bovine serum albumin (BSA) in TBS(0 5M Tris-HCI, pH 7-6, diluted in 1:10 in 1 SM saline)the nitrocellulose paper was incubated for 30 minutesin KPI antibody or in an irrelevant monoclonalantibody. After washing, the APAAP stainingprocedure was then completed.6 Molecular weightstandards transferred on to nitrocellulose paper werestained with 0 1% Fast Green9 to calibrate the blots.
IMMUNOPRECIPITATIONA crude membrane preparation from a spleen rich inmacrophages was solubilised in 2% NP40 and thepreparation enriched for glycoproteins by lentil lectinaffinity chromatography. The I M x-methyl man-noside eluate from the lectin column was dialysed anda sample (100 pg) was labelled with lmCi['2'51] iodide(Amersham) in the presence of 20 ,ig Iodogen (Pierce).
Labelled material was gel filtered to removeunbound iodine and precleared twice with affinitypurified rabbit anti-mouse Ig antibody bound toStaphylococcus aureus. Monoclonal antibodies (in theform of undiluted hybridoma culture supernatants)were mixed with the pre-cleared extract, and after one
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416hour rabbit anti-mouse Ig bound to S aureus wasadded. After a further hour the immunoabsorbent waswashed three times in buffer containing 1% NP40 and500 mM sodium chloride. Immunoprecipitates wereanalysed by SDS-PAGE followed by autoradiographyof the dried gel using an enhancing screen.
FACS ANALYSISWhite cells were obtained either by centrifugation onTrisil-Ficoll or by red cell lysis of whole blood. In thelatter procedure samples of normal peripheral bloodwere treated for 12 minutes with red cell lysingsolution consisting of ammonium chloride (8-29 g/l),sodium edetic acid (0-37 mg/ml), and potassiumbicarbonate ([-0 g/l). The resulting white cells werewashed four times in Dulbecco "A" medium (Oxoidcode BR 14a) containing 2-5% normal human serumand then incubated for 30 minutes at 40C with KPIantibody (undiluted hybridoma culture supernatant).After washing twice, the cells were incubated for afurther 30 minutes with fluorescein isothiocyanate(FITC)-conjugated goat anti-mouse Ig (Sigma code F-2012) diluted 1/20. The cells were washed twice in1 5% formaldehyde in PBS and analysed in a FAC-Scan (Becton Dickinson) flow cytometer using anargon ion laser of 15mW at 488 nm. Lymphocyte,granulocyte, and monocyte populations were analysedseparately by gating on forward and side scattermeasurements.
Results
On preliminary screening of the hybridomas obtainedusing spleen cells from a mouse immunised with thehuman macrophage antigen, 18 supernatants stainedmacrophages selectively in cryostat sections of humanlung and tonsil. Only one supernatant, however,stained most macrophages in these tissues. This super-natant also stained macrophages in paraffin waxsections of lung tissue fixed in formol saline. The cellsproducing this supernatant were cloned to produce thecell line KPl.
BIOCHEMICAL CHARACTERISATION OF THE KPIANTIGENImmunoprecipitation of a radiolabelled homogenateof macrophage-rich human spleen showed a proteinband with a molecular weight ofabout 110 kilodaltons(fig 1).The results of Western blotting are shown in fig 2.
Under reducing conditions KPI stained three bandswith molecular weights of 110, 70, and 40 kilodaltonsin the lung antigen preparation and in NP40 lysates ofU937 cells and spleen. The bands consistently detectedin several different experiments were relatively diffuse.No staining of material at these positions was seen in
Fig 1 Immunoprecipitation ofa "251 labelled lysate ofhumanmacrophage-rich tissue. Track A: prominent band about 10kilodaltons is precipitated by antibody KPJ. Track B:antibody MHM23 precipitates LFA-1/Mac-J (CD11/18)complex but no material with the same molecular weight asthe KPJ antigen. Track C: control immunoprecipitation inwhich no antibody was used.
Fig 2 Western blotting of the KPJ antigen. Lanes A and Bshow 110, 70, and 40 kilodalton bands detected by KPJ underreducing conditions in extracts oflung (lane A) and U937cells (lane B). An additional band (220 kilodaltons) isdetected in the spleen lysate under non-reducing conditions(lane C), and also a decrease of the 70 kilodalton band andabsence of the 40 kilodalton band.
control blots stained with an unrelated monoclonalantibody. Under non-reducing conditions KPI reac-ted with an additional band of 220 kilodaltons in theNP40 spleen lysate (fig 2), and in these preparationsthe 70 and 40 kilodalton bands were less intense. Thetwo spleen lysates prepared in the absence of detergentor in the presence ofTween 40 gave identical results onWestern blotting with those obtained using the NP40lysate.
REACTIONS OF KPI ON TISSUE SECTIONSThe results of immunocytochemical studies with KPlIantibody on routinely processed sections of normaltissues are summarised in the table. Trypsinisation oftissue sections fixed in formol saline before stainingconsiderably enhanced the intensity of the labelling
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*Staining in all cells was diffusely present in the cell cytoplasm.
obtained with KPI. Trypsination was not requiredwhen staining tissues fixed in B5 fixative.
Normal tissueThe antibody reacted with tissue macrophages in awide range of tissues, including lung macrophages (fig3), germinal centre macrophages (fig 4), Kupffer cellsin the liver (fig 5) and bone marrow macrophages (fig6). In the latter tissue the antibody also reacted withmyeloid precursors (fig 6), and this reaction wasconfirmed by staining smears of human bone marrowin which myelocytes and also many mature granu-locytes were strongly labelled. Megakaryocytes andcells of erythroid lineage were not labelled by KPI insmears or sections of bone marrow.
In the spleen KPl antibody labelled macrophages ingerminal centres and in red pulp. No staining ofmarginal zone macrophages was seen. In the thymusKPI stained both medullary and cortical macro-phages. Langerhans' cells, interdigitating reticulumcells, and follicular dendritic cells were all unstained,with the exception (as detailed below) of a few of theinterdigitating reticulum cells seen in dermatopathiclymphadenopathy. Tissue polymorphs were usuallynot stained by KPI or showed only weak labelling. Nostaining was seen of any non-haemopoietic cell types(such as epithelium, muscle, etc.).
Reactive lymphoid tissuesA variety of reactive lymph nodes (comprising at leasttwo examples of follicular hyperplasia, sinus histio-cytosis, sarcoidosis, tuberculosis, cat scratch disease,dermatopathic lymphadenopathy and toxoplasmosis)were studied and broad reactivity against cells ofmacrophage lineage was observed, epithelioid granu-
lomas and multinucleated cells being particularlystrongly labelled (figs 7 and 8). Most of the abnormalinterdigitating reticulum cells characteristic of der-matopathic lymphadenopathy were negative (fig 9),although a minority of these cells showed weakpositivity, usually localised to a small region close tothe nucleus.
REACTIONS OF KPI ON BLOOD CELLSKPI stained all monocytes and most granulocytes insmears of peripheral blood. The staining ofmonocyteswas granular in appearance and suggested anintracytoplasmic location of the antigen, possiblyassociated with lysosomal granules. Analysis by flowcytometry showed strong reactivity of all monocyteswith KPl when white cells were prepared by the wholeblood lysis method, but not when white cells separatedon lymphoprep were analysed. Granulocytes gave aweak or negative reaction while lymphocytes andplatelets were unstained.
Discussion
As the antigen recognised by KPl is resistant toconventional fixatives such as formalin and B5, thisantibody may be of practical importance for diagnos-tic pathologists. KP1 seems to recognise an antigenwhich persists throughout cell maturation from themonocyte to the mature macrophage stage. Indeed,the amount of KPl antigen present in macrophagesmay increase when these cells are activated andmature, as indicated by the greater intensity of stainingof tissue macrophages compared with that seen inperipheral blood monocytes. This finding is in contrastto that obtained for myeloid cells, in which the KPIantigen is weaker in mature granulocytes than in thebone marrow precursors of these cells.The only KPI positive cells in lymphoid tissue
sections other than classic macrophages were theintermediate sized rounded cells seen in T cell areas.These were shown in subsequent studies to correspondto the cell type known as "plasmacytoid T cells", andtheir reaction with antibody KPI provides additionalevidence that they are related to monocytes/macro-phages.'°
If the reactions of antibody KPl are compared withthose ofpreviously published monoclonal anti-macro-phage antibodies, the greatest similarity is withantibodies Y 1/82A", EBMI 112, Ki-M6'3 and Ki-M7'4.The first three of these antibodies were clustered into apossibly homogeneous group in the Third Inter-national Workshop on Leucocyte DifferentiationAntigens2 on the basis of their "pan-macrophage"pattern of reactivity on tissue sections. There are,however, minor differences between the KPI and thereactions of those other monoclonal antibodies, whichsuggest that they may recognise different antigens.
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Fig 3 Labelling ofalveolar macrophages by antibody KPI.Immunostaining in thisfigure and in figs 4-9 was performedby the APAAP technique on trypsinisedparaffin waxsections.
Fig 4 Labelling of tingible body macrophages in reactivelvmphoid tissue with antibody KPI.
4
r;-,s,.0
A.,t.:,
VVt
XIf I.,-XFig 5 Staining of Kupffer cells in the liver by antibody KPI.
Antibodies Ki-M6 and Ki-M7, for example, werereported as reacting with antigens which differ inmolecular weight (60 and 29 kilodaltons, respectively)from the molecules detected by KPI.' '4 EBM 11antibody shows some similarity to KPI in that it hasbeen reported as reacting by Western blotting withmolecules of 120, 70, 64 and 22 kilodaltons.' EBM 1Iantibody is reported to react in tissue sections withinterdigitating reticulum cells and Langerhans' cells,however, whereas KPI does not seem to react withthese cell types.'2'2On the basis of published data it seems that KPI is
different in terms of the molecule(s) which it recognisesand in its immunocytochemical reactions, frompreviously reported anti-macrophage monoclonalantibodies.'67 It should be noted, however, that themolecular weights of the target antigens reported forthe monoclonal antibodies which it resembles mostclosely in its staining reactions that is, Y I /82A,Ki-M6, Ki-M7 and EBM II have not been indepen-
Fig 6 Strong labelling ofmacrophages (arrowed) in a bonemarrow trephine biopsy specimen by antibody KPI andweaker staining ofman) mYeloidprecursors.
dently validated by other laboratories; and indeed, aresult has been reported in immunoprecipitationexperiments for Ki-M6 (90 kilodaltons) which differsfrom the values obtained by Western blotting.2 Fur-thermore, we have not attempted a direct comparisonbetween the immunocytochemical reactions of theseother antibodies and those of antibody KP1, and thepublished reactions of these antibodies were obtainedprimarily using cryostat sections (in which antigensare usually better preserved than in paraffin waxsections), whereas KPI was evaluated on formalin*fixed tissue. Further analysis of the antigens recog-nised by all of these antibodies is therefore required iftheir association is to be established with certainty.
Interestingly, the material detected by KPlantibody by immunoprecipitation shows a diffusepattern on gel electrophoresis, suggesting that it maybe extensively glycosylated. The finding that antigenicmaterial of the same molecular weight was detected byKPI in both detergent-free spleen lysates and in those
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Use of KPI to detect monocyte/macrophage associated antigen 419
- *A
,' "'.'S fX 0;f..''"",,;j* ''4',w-1 ht
Fig 7 Cat scratch disease biopsy specimen stained with antibody KPI. A cluster ofmacrophages is arrowed in (a) and shownat higher power in (b).
II fa
'4~~~~~~~~~~~~~~~~~~~~~~~~~~~4
Fig 8 Sarcoidosis in human spleen. (a) KPI positive granulomata around a lzYmphoid area: (b) granuloma at highermagnification, itith a multinucleate giant cell (arrow1ed)
.- > s ^s n x 5 x * tS containing Tween 40 or NP40 indicates that the;96t. WS **i < t i ~ ^ , 5 -; antigen is probably present in the cytosol, microsomal,~~ z" t* tt t % 4 k ~~~andsurfacemembrane fractionsofmacrophages.This+'*t;*** 9+ > ri * 2 s4 s. raises the possibility that, as found for other mon-
a 1'~~~~~~~~~~~~~~~~~~~~~~~~~1
magnifican, wh a m e g. ocyte/macrophage antigens, the KPI antigen isI'.. antigen isprobablypresentinlysosomes,thecontentsofwhichmovesto>,t s3 >* ; *9the cell surface and to the extracellular environment
*4 % -1-.q-r- A. during exocytosis. The fact that the separationCv rs.*a - -;^4 procedure used for isolating human blood white cells8 |13 4 ° ew ^ *; . < influenced whether KPI antigen could be detected on
A.'9?t #4,W the cell surface by flow cytometry also supports thispt *
C4ss ^ * xtw ' \
> iyv ~ view.'9 Further work is needed to elucidate the bio-W ,*8w.tt*.4 * ->0> E: - * ^ g .~ t*- chemical nature of the KP1 antigen and to define in
detail its subcellular localisation.Fig 9 Dermatopathic lymphadenopathr showing dot-like Functional studies have indicated that the mono-reactivity with antibody KPI in some atypical interdigitating cyte/macrophage family can be divided into tworeticulum cells (arrowed). groups. The first group, comprising follicular den-
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420 Pulford, Rigney, Micklem, Jones, Stross, Gatter, Masondritic cells, interdigitating reticulum cells, and Langer-hans' cells, have an accessory role in the immuneresponse,21 22 particularly in terms of presentingantigen to lymphoid cells. In contrast, cells in thesecond group, comprising tissue macrophages such asKupffer cells and lung alveolar macrophages, performa microbicidal and phagocytic role.23 Recent studieswith monoclonal antibodies have shown that thesetwo groups can also be distinguished on the basis oftheir antigenic differences. Some antibodies, such asanti-CDl lc (pI50,95) EBM 1IIs and Ki-M 124, labelLangerhans' cells, interdigitating reticulum cells anddendritic reticulum cells, in addition to phagocytictissue macrophages, while other antibodies label onlyaccessory cells25 or tissue macrophages.'3 1426 KPItherefore seems to be another anti-macrophage anti-body which reacts with phagocytic tissue macrophagesbut not with accessory cells. The only exception lies inits reaction with interdigitating reticulum cells in casesof dermatopathic lymphadenopathy (fig 9). This re-action (which was not seen in non-dermatopathicnodes), however, consisted of a small dot-like area ofreactivity confined to a minority of the cells. Thereactions of KP1 therefore provide support for theview that the monocyte/macrophage family can bedivided into two groups which share a common stemcell but which show considerable functional andbiochemical divergence.2728Most tissue samples available to diagnostic patho-
logists, even with the recent growth in the use ofcryostat sections for immunostaining, have beenembedded in paraffin wax after fixation in formalin (orless commonly with Bouin's or with mercury basedfixatives). The availability of an antibody which candetect macrophages in this type ofmaterial is thereforeof considerable potential in the context of routinehistopathology. We are aware of only two othermonoclonal antibodies recognising macrophageassociated antigens in conventionally processedparaffin wax embedded tissue, and each of these is lesssuitable for routine use than KPI antibody. Ki-M7antibody is described as staining paraffin wax sectionsbut this reactivity is said to be inconsistent, macro-phages sometimes being weakly stained or unlabel-led.'4 The other antibody, MAC387, is restricted in itsreactions to a subpopulation of tissue macrophages,being unreactive with germinal centre macrophages orKupffer cells, and it also labels squamous epitheliumand granulocytes strongly.2930
It may be added that the analysis of the pattern ofimmunocytochemical reactivity of KPI in this studyhas been performed almost entirely on routinelyprocessed tissue. It is likely that in this tissue there issome reduction in antigenic reactivity, and cells withlow levels of antigen expression may therefore appearartefactually negative. This is suggested by the fact
that KPl stains granulocytes strongly in marrow andblood smears (which have been only briefly exposed tofixative) whereas in routine tissue sections they wereweak or unreactive. A full description of the spectrumof KPI reactivity will therefore require systematicanalysis of cryostat tissue sections and cultured celllines of haemopoietic and non-haemopoietic origin.Nevertheless, even if the antigen were present at lowlevels in some cells of non-myeloid/mononuclearphagocyte origin, the results of the present studyindicate clearly that in the context of routine patho-logy it is highly selective for the myeloid and mononu-clear phagocyte lineage.One area of obvious use for antibody KPI is in
assessing the number and distribution of tissue macro-phages in pathological conditions (such as inflamma-tion and neoplasia). It may also be of value in thediagnosis of malignant histiocytic tumours. The detec-tion of these tumours is difficult on morphologicalgrounds alone, and no fully satisfactory antigenicmarkers detectable in routinely processed tissues areavailable. Use of KPI may thus help to resolve thelong standing problem of how to distinguish betweentrue histiocytic malignancies and high grade neo-plasms of lymphoid origin (such as anaplastic largecell lymphoma/"Ki-1 lymphoma").
This work was supported by grants from theLeukaemia Research Fund and the Cancer ResearchCampaign. KCG is a Wellcome senior research fellowin clinical science.
Addendum
Experiments performed in the authors' laboratorysince the submission of this paper have shown that themonoclonal anti-macrophage antibodies Y I /82A,"EBMI 1,12 and Ki-M6'3 all recognise the 110 000kilodalton glycoprotein detected by antibody KPI(Micklem et al, unpublished observations).
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Requests for reprints and for antibody samples to: Dr D YMason, Haematology Department, John Radcliffe Hospital,Oxford OX3 9DU, England.
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athol: first published as 10.1136/jcp.42.4.414 on 1 April 1989. D
