J Clin Pathol 1990;43:719-722

Use of monoclonal antibody KP1 for identifyingnormal and neoplastic human mast cells

H-P Horny, G Schaumburg-Lever, S Bolz, M L Geerts, E Kaiserling

AbstractThe monoclonal antibody KP1 (CD68)was used to stain normal and neoplasticmonocytes and macrophages in rou-

tinely processed, paraffin wax embeddedtissue: mast cells also exhibited strong,consistent cytoplasmic immunoreac-tivity. Light microscopic findings were

corroborated by electron microscopicaland immunocytochemical findings. Thepredominant sites of immunoreactivitywere the specific intracytoplasmic gran-ules of the mast cells. All mast cell sub-types-that is, normal and reactive mastcells, such as those in lymph nodesexhibiting chronic non-specific lymph-adenitis, and malignant or neoplasticmast cells in various types of masto-cytosis-reacted with this antibody. Thisfinding is of diagnostic importance,because mast cell proliferation could bemistaken for histiocyte proliferation. Italso supports the hypothesis that mastcells derive from the bone marrow.

Department of SpecialHistopathology andCytopathology,Institute of Pathology,University ofTubingen, FederalRepublic ofGermanyH-P HornyS BolzE KaiserlingDepartment ofDermatology,University ofTubingen, FederalRepublic ofGermanyG Schaumburg-LeverAkademischZiekenhuis,Ruksuniversiteit,Gent, BelgiumM L GeertsCorrespondence to:Professor E Kaiserling,Pathologisches Institut derUniversitat,Liebermeisterstrasse 8,D-7400 Tubingen, FederalRepublic of Germany.Accepted for publication28 March 1990

The monoclonal antibody KP1 (CD68) is a

reliable reagent for identifying monocytes or

macrophages in routinely processed tissue."13Micklem et al presented evidence that KP1recognises the same glycoprotein (molecularmass of about 110 000) as other widely usedantibodies that detect macrophages (Y2/131,EBMl 1, Ki-M6 and Ki-M7), but can only beused on frozen tissue.4 Because of increasingevidence of a close cytogenetic associationbetween human mast cells and the myelomo-nocytic system, we investigated mast cells invarious reactive and neoplastic disorders(including tumours derived from mast cells)for reactivity with KP1 and other antibodies

detecting macrophages. We wanted to ascer-

tain if the identification of reagents that con-

sistently stain mast cells, in addition to thewell known metachromatic stains (such as

Giemsa and toluidine blue) and the naphtholAS-D chloroacetate esterase reaction,5 couldbe of diagnostic value, even if they were notspecific for these cells. There was strong andreproducible immunohistochemical stainingof normal and reactive and malignant or

neoplastic mast cells with the macrophage-associated monoclonal antibody KPI, butnone with DAKO-MAC 387. KPI immuno-reactivity has also been noted in a case ofsystemic mastocytosis by Wamke et al.2

MethodsThe tissue investigated, which comprised nor-

mal and reactive (hyperplastic) mast cells andmalignant or neoplastic mast cells, and thediagnosis in each case are listed in the table.Specimens were fixed in 5%0 buffered for-malin and embedded in paraffin wax for lightmicroscopical examination. Sections were cutat 5 gm and stained with the Giemsa stain andthe naphthol AS-D chloroacetate esterase re-

action. Immunostaining was performed withthe avidin-biotin complex method describedby Hsu et al.6 The following antibodiesagainst macrophages were used: DAKO-MAC 387 and KP1 (CD68), both monoclonal,and antibodies against a 1-antitrypsin and a 1-

antichymotrypsin, both polyclonal (all pur-

chased from Dakopatts, Hamburg, WestGermany). The specificity of the immuno-reactions was assessed in lymph nodes, wherediffering numbers of macrophages and his-tiocytes were stained by these antibodies.Because one of the two monoclonal anti-macrophage antibodies, KP1, produced

Tissue specimens investigated and corresponding diagnoses

Specimen Mast cell type Diagnosis

Lymph node Normal/reactive Chronic non-specific lymphadenitisLymph node* Normal/reactive Dermatopathic lymphadenitisTumour Reactive/hyperplastic NeurilemmomaTumour Reactive/hyperplastic Epithelioid haemangioma

(angiolymphoid hyperplasia with eosinophilia)Tumour Reactive/hyperplastic Invasive breast carcinomaSkin Neoplastic Urticaria pigmentosaSkin Neoplastic MastocytomaLymph node, bone marrow Neoplastic Systemic mastocytosisBone marrow Neoplastic Systemic mastocytosisBone marrow Neoplastic Systemic mastocytosisLymph node, bone marrow Neoplastic Malignant mastocytosisLymph node Neoplastic Malignant mastocytosisSpleen Neoplastic Malignant mastocytosis

*Also used for electron microscopic studies.Mastocytosis was subdivided into systemic mastocytosis (associated with urticaria pigmentosa-like skin lesions and a relativelygood prognosis) and malignant mastocytosis (without such skin lesions and generally following a rapidly fatal course).'3

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Horny, Schaumburg-Lever, Bolz, Geerts, Kaiserling

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Figure I Cutaneous mastocytosis (urticaria pigmentosa). The corium i

infiltrated by slightly pleomorphic mast cells with metachromatic granulecells exhibit intense cytoplasmic immunostaining by the monoclonal antibc(A Giemsa; B KPl, ABC method).

Figure 2 Chronic non-specific lymphadenitis in a cervical lymph node. Jscattered medium sized macrophages/histiocytes are stained by the monocKPI. The lymphoid cells remain unstained. A fusiform mast cell that exAdiffuse cytoplasmic reactivity with KPI is seen (top centre). (KPl, AB(

Figure 3 Malignant mastocytosis. This lymph node exhibits patchy infilarge, pleomorphic mast cells, all of which are intensely immunostained b3ABC method).

strong, consistent cytoplasmic st

mast cells of all the specimenfurther immunocytochemical sperformed using electron microsc

Electron microscopic investijcarried out on a lymph node drEgenital cellular naevus. The tissuia mixture of 2% paraformaldehyglutaraldehyde in phosphate bu(PBS) (pH 7 4) for four hours. T]were then rinsed in PBS, immerNH4C1 in PBS for one hour, andin PBS.7 The tissue was dehydraialcohols and the temperature

duced to - 35°C. It was then embeddedin Lowicryl at -35°C. Thin sections werecut and mounted on nickel grids coated withFormvar.The immunocytochemical procedure was

carried out as follows: After a short wash inPBS (three times for five minutes) the gridswere floated on drops of normal goat serum(50o in PBS) for 10 minutes. They werethen transferred to drops of the undilutedprimary antibody KP1 (Dako, Hamburg,West Germany) and incubated overnight at4°C. The next day they were rinsed in PBS(three times for five minutes) and thenincubated in a 1 in 5 solution of goat anti-mouse IgG GIO (Janssen, Olen, Belgium)Auro Probe immunogold reagent with 1%Teleostean gelatin (Sigma, Munich, WestGermany) in PBS for 60 minutes in the dark.The grids were washed again in PBS (threetimes for five minutes). The sections weredried, counterstained with 500 uranyl acetatedissolved twice in distilled water (40 minutes)and lead citrate (10 minutes) and examinedwith a Zeiss EMIO electron microscope.

ResultsMast cells were identified in all the tissue

i- specimens by the presence of metachromaticintracytoplasmic granules (shown by theGiemsa stain) and by strong staining fornaphthol AS-D chloroacetate esterase. The

*+ antibody DAKO-MAC 387 did not stain anys of the mast cells, but stained macrophages and

immature myeloid cells. Antibodies to a 1-antitrypsin and a 1 -antichymotrypsin eachstained a large proportion of the mast cells(irrespective of subtype) in every case. KP1produced consistent, strong granular cyto-plasmic staining of all mast cells in all thetissue specimens (figs 1-3).

In the lymph node exhibiting chronic non-specific lymphadenitis, in addition to the

d(eA)nely th loosely scattered mast cells, macrophages,ody KPI (B). sinus histiocytes and plasmacytoid T cells

(plasmacytoid monocytes) were stained byLoosely KP 1, and, as in mast cells, staining in theselonal antibody cells was intracytoplasmic, diffuse, and gran-oibits strong, ular.C method). The lymph node examined by electron

Itration by microscopy exhibited a large number ofyKPJ (KPI, macrophages containing melanosomes.

Macrophage lysosomes were labelled by theantibody KP1. Numerous mast cells were alsopresent: two populations could be distingui-

:aining in the shed by conventional electron microscopy;1S examined, one type was characterised by closely packed;tudies were electron dense granules; the other comprised:opy. degranulated mast cells and was characterisedgations were by low electron density of the granules, whichaining a con- sometimes seemed to merge to form channels.ewas fixed in Mast cells containing granules exhibited,de and 0 2% two principal areas of labelling (fig 4): theiffered saline electron dense granules, with accentuation ofhe specimens labelling in the marginal and most electronsed in 0 5 M dense areas-the gold particles were eitherrinsed again disseminated or clustered in groups of three toted in graded five; and the extra-granular areas. Labellinggradually re- by gold particles here could not always be

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Antibody KP1 staining in human mast cells

related to any particular structures. Small,electron dense granular structures.that lookedlike lysosomes were labelled. No labelling ofthe rough endoplasmic reticulum, which wasplentiful in a few cells, was found. Somecytoplasmic filaments were labelled (fig 4).

Figure 4 Moderately electron dense mast cell granules with scattered and clusteredgoldparticles (short arrows). A few gold particles in intergranular areas are associated withfilamentous structures (long arrows). (KP1).

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Figure 5 Part of an almost completely degranulated mast cell. Some of thedisseminated gold particles are indicated with arrows. (KP1).

Possible labelling of the Golgi apparatus couldnot be assessed as this structure was not seenin any of the mast cells. No granules withscrolls were seen.

In the degranulated mast cells (fig 5) thechannels or granular components, the othermore electron dense areas, were extensivelylabelled. The intergranular cytoplasm waslabelled to a lesser extent.The macrophage phagolysosomes were

much more intensely labelled by KP1 than themast cells and the latter were not labelled atall by the antibody DAKO-MAC 387.

DiscussionWe have shown that there is strong and consis-tent cytoplasmic staining in normal, reactive,and malignant or neoplastic mast cells by themacrophage-associated monoclonal antibodyKPI, first produced and described by Pulfordet al.1 Electron microscopical and immuno-cytochemical investigations showed that thisreactivity was located predominantly in thespecific mast cell granules, even in mast cellsthat were almost completely degranulated. Thereactivity of mast cell granules was consider-ably weaker, however, than that of the phago-lysosomes of macrophages. Our electronmicroscopic investigations also showed thatKPI immunolabelled reticulum cells, epithe-lioid cells, pigmented epidermal cells and cellsthat were probably lymphoid cells, where-asin the macrophages-the lysosomes or phago-lysosomes represented the predominant site ofimmunoreactivity (unpublished observations).So, what are the implications ofthe finding thatmast cells also react with KPl?

Firstly, the reactivity of mast cells with amonoclonal antibody that detects macrophagesis interesting as far as the cytogenesis ofhumanmast cells is concerned. Although mast cells areseen only in small numbers in the bone marrowof healthy adults (their presence at this siteused to be considered abnormal8) and arevirtually absent from the bone marrow ofchildren,9 certain findings indicate a closecytogenetic relation between mast cells and themyelomonocytic system in man:1 Normal and malignant mast cells andmyelomonocytic cells (particularly neutro-phils) share enzyme-cytochemical and im-munocytochemical properties in that both reactfor naphthol AS-D chloroacetate esterase5 andthe leucocyte common antigen,lo and both reactwith the lectin leucoagglutinin" and the mono-clonal antibody MY9.122 There is an extraordinarily high incidence ofmyelodysplastic and myeloproliferative dis-orders in patients with systemic mast cellproliferative disorders, particularly the malig-nant variant.'3 143 Chronic myeloid leukaemia may, in rareinstances, undergo blastic transformation inwhich the blast cells exhibit features ofatypicalmast cells.S""4 Mast cells exhibit reactivity with the mono-clonal antibody KPI (CD68) and with anti-bodies against various other surface membraneantigens (CD9 and CD33) associated with a

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Horny, Schaumburg-Lever, Bolz, Geerts, Kaiserling

late stage ofmonocyte or macrophage differen-tiation."8

If the hypothesis of a bone marrow origin,not only of rodent but also ofhuman mast cellsis accepted,'9 20 the question arises ofhow thesecells pass from the marrow to the connectivetissues where they are normally found, as theyare never encountered in the peripheral blood.One possible explanation is that marrow-derived mast cell progenitors, unlike those ofother blood cells, enter the blood stream. Theseprogenitors differentiate in perivascular sitesinto mature mast cells, which, in contrast totheir progenitor cells, can be detected withstains such as Giemsa or toluidine blue.2" Ofthedifferent types ofleucocytes, the heterogeneouspopulation of monocytes or macrophagesseems the most likely to include the mast cellprogenitors.20 This hypothesis is supported byour finding that normal, reactive (hyper-plastic), and malignant mast cells react with themacrophage-associated monoclonal antibodyKP1.The reactivity of mast cells with KP1 also

presents a more practical problem in that itcould lead to mast cell proliferation being takenfor histiocyte proliferation if this antibodyalone is used to identify macrophages.23We thank Mrs A Mall and Mrs A Adam for their excellenttechnical assistance, Dr U Engst for help with methodologicalproblems, and DrM Ruck for help with the manuscript. We alsothank Dakopatts (Hamburg) and the Vereinigung der Freundeder Universitit Tubingen e.V. for their generous contributionstowards the costs of reproduction of the colour micrographs.

1 Pulford KAF, Rigney EM, Micklem KJ, et al. KP1: a newmonoclonal antibody that detects a monocyte/macrophageassociated antigen in routinely processed tissue sections. JClin Pathol 1989;42:414-21.

2 Warnke RA, Pulford KAF, Pallesen G, et al. Diagnosis ofmyelomonocytic and macrophage neoplasms in routinelyprocessed tissue biopsies with monoclonal antibody KP1.Am JPathol 1989;135:1089-95.

3 Ralfkiaer E, Delsol G, O'Connor NTJ, et al. Malignantlymphomas of true histiocytic origin., A clinical, his-

tofogicaf, inimunopienotypic and genotypic study. JPathol 1990;160:9-17.

4 Micklem K, Rigney E, Cordell J, et al. A human macro-phage-associated antigen (CD68) detected by six differentmonoclonal antibodies. Br J Haematol 1989;73:6-1 1.

5 Leder LD. Uber die selektive fermentcytochemische Dar-stellung von neutrophilen myeloischen Zellen undGewebsmastzellen im Paraffinschnitt. Klin Wochenschr1964;42:553.

6 Hsu SM, Raine L, Fanger H. Use of avidin-biotin-perox-idase complex (ABC) in immunoperoxidase techniques. JHistochem Cytochem 1981;29:577-80.

7 Roth J, Bendayan M, Carlelalm E, Villiger W, Garavito M.Enhancement of structural preservation and immuno-cytochemical staining in low temperature embedded pan-creatic tissue. J Histochem Cytochem 1981;29:663-71.

8 Fadem RS. Tissue mast cells in human bone marrow. Blood1951;6:614-30.

9 WernerW, Krause AR. Die Gewebsmastzellen im menschli-chen Knochenmark. Frankfurt Z Pathol 1965;74:651-8.

10 Horny H-P, Reimann 0, Kaiserling E. Immunoreactivity ofnormal and neoplastic human tissue mast cells. Am J ClinPathol 1988;89:335-40.

11 Schumacher U, Horny H-P, Welsch U. The lectin leuco-agglutinin binds specifically to human granulocytes,monocytes and tissue mast cells: further evidence for acommon 6rigin of the three cell types. Br J Haematol1987;66:405-6.

12 Dalton R, Chan L, Batten E, Eridani S. Mast cell leukaemia:evidence for bone marrow origin ofthe pathological clone.Br J Haematol 1986;64:397-406.

13 Horny H-P, Parwaresch MR, Lennert K. Bone marrowfindings in systemic mastocytosis. Hum Pathol 1985;16:808-14.

14 Travis WD, Li C-Y, Yam LT, Bergstralh EJ, Swee RG.Significance of systemic mast cell disease with associatedhematologic disorders. Cancer 1988;62:965-72.

15 Parkin JL, McKenna RW, Brunning RD. Philadelphiachromosome-positive blastic leukemia: Ultrastructuraland ultracytochemical evidence of basophil and mast celldifferentiation. Br J Haematol 1982;52:663-77.

16 Soler J, O'Brien M, Tavares de Castro J, et al. Blast crisis ofchronic granulocytic leukemia with mast cell and baso-philic precursors. Am J Clin Pathol 1985;83:254-9.

17 Gabriel LC, Escribano LM, Marie J-P, Zittoun R, NavarroJL. Peroxidase activity in circulating mast cells in blastcrisis of chronic granulocytic leukemia. Am J Clin Pathol1986;86:212-9.

18 Valent P, Ashman LK, Hinterberger W, et al. Mast celltyping: demonstration ofa distinct hematopoietic cell typeand evidence for immunophenotypic relationship tomononuclear phagocytes. Blood 1989;73:1778-85.

19 Kitamura Y, Yokoyama M, Matsuda H, Ohno T, Mori KJ.Spleen colony-forming cell as common precursor fortissue mast cells and granulocytes. Nature 1981;291:155-60.

20 Parwaresch MR, Horny H-P, Lennert K. Tissue mast cellsin health and disease. Path Res Pract 1985;179:439-61.

21 Galli SJ. Biology ofdisease. New insights into "The riddle ofthe mast cells": Microenvironmental regulation of mastcell development and phenotypic heterogeneity. LabInvest 1990;62:5-33.

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