CD 68

Accurate (EVM11), Biodesign (BL-M68), Caltag Laboratories (BLM68), Dako (KP1, PG-M1, EBM 11), Sanbio/Monosan/Accurate (BLAD8) and Serotec (KiM6).

Apart from EBM 11 which is only applicable to frozen sections, KP1 and PG-M1 monoclonal antibodies are applicable to formalin-fixed paraffin sections, acetone-fixed cryostat sections and fixed-cell smears. Antimacrophage reagents recognizing formalin-resistant epitopes require microwave or enzyme pretreatment with trypsin or pronase before immunostaining to reduce background staining.

The best macrophage reagents produced to date are those recognizing the CD 68 antigen (Knapp, 1989). This 110 kD antigen belongs to a family of acidic, highly glycosylated lysosomal glycoproteins that include the lamp-1 and lamp-2 molecules (Fukuda, 1991). CD 68 is the human homologue of the murine macrosialin antigen (Holness et al, 1993) and is present in the cytoplasmic granules of monocytes, macrophages, neutrophils, basophils and large lymphocytes (Pulford et al, 1990). This antigen is also expressed to some degree in the cytoplasm of some non-hemopoietic tissue. However, the function of the molecule is currently unknown.
The monoclonal antibody KP1 (IgG1, k) was raised against lysosomal granules prepared from lung macrophages (Pulford et al, 1989) and recognizes the 110 kD CD 68 antigen. This antibody labels monocytes and macrophages in a wide range of tissues, e.g. lung macrophages, germinal center macrophages and Kupffer cells. Osteoclasts and myeloid precursors in bone marrow are also strongly labelled. In frozen sections, KP1 stains endothelium and hepatocytes weakly. Strong labeling of blood monocytes (granular/cytoplasmic), neutrophils and basophils is also demonstrated with KP1. KP1 antigen is expressed as an intracytoplasmic molecule, associated with lysosomal granules.
The murine PG-M1 monoclonal antibody (IgG3, K) was raised against spleen cells of Gauchers disease (Falini et al, 1993). Reactivity with cells transfected with a human cDNA encoding for the CD 68 antigen confirms PG-M1 as a member of the CD 68 cluster. In normal tissue, PG-M1 is comparable to KP1; however, in bone marrow paraffin sections, PG-M1 strongly stains macrophages butnot granulocytes and myeloid precursors. PG-M1 also shows immunopositivity with mast cells and synovial cells.

Malignant histiocytosis and true histiocytic lymphoma express the CD 68 macrophage marker (Ralfkiaer et al, 1990). These tumors should be CD 68+, but be unlabelled with antibodies to CD 30, T- and B-cell antigens and cytokeratins. Acute myeloid leukemias (AML) are identified by the presence of CD 68 antigen (Warnke et al, 1989; Thiele et al, 1992). Whilst KP1 recognizes M1-M5 types, PG-M1 immunoreaction is confined to M4 (myelomonocytic) and M5 (monocytic) types of AML. The CD 68 antibodies are also able to distinguish between monocyte/macrophage and lymphoid leukemias. Whilst this is useful in identifying granulocytic sarcoma, some B-cell neoplasms (notably small lymphocytic lymphoma and hairy cell leukemia) show weak cytoplasmic staining in the form of a few scattered granules. Mast cell proliferations and ''plasmacytoid monocytes" are usually stained by both the KP1 and PG-M1 antibodies. The CD 68 antigen is also expressed to varying degrees in Langerhans and interdigitating reticulum cell sarcomas, as well as Langerhans cell histiocytosis (Ruco et al, 1989).
Macrophages may be present either as rare scattered cells or large cellular infiltrates in some T- and B-cell lymphomas, leading to erroneous diagnoses of histiocytic malignancies. Dual immunocytochemical labeling with CD 68 antigen and T/B-cell antigen is useful in delineating the two populations. The identification of macrophages is also crucial in the diagnosis of granulomatous diseases, storage diseases and certain types of lymphadenitis, e.g. Kikuchi's lymphadenitis. In the latter condition, macrophages phagocytosing apoptotic bodies and cells known as "plasmacytoid monocytes" and "crescentic histiocytes" are easily recognized with antibodies against CD 68, avoiding the misdiagnosis of a high-grade lymphoma.

Caution is advised in the immunophenotypic interpretation of histiocytes, since the distinction between "uptake" and "synthetic" patterns should be borne in mind. KP1 would appear to be superior to PG-M1, particularly with respect to the wider recognition of AML. The latter antibody also carries the distinct disadvantage of being demonstrated in about 10% of melanomas. Tissue rich in macrophages is suitable as a positive control.

•Falini B, Flenghi L, Pileri S et al 1993. PG-M1: a new monoclonal antibody directed against a fixative-resistant epitope on the macrophage-restricted form of the CD 68 molecule. American Journal of Pathology 142: 1359-1372.

•Fukuda M 1991. Lysosomal membrane glycoproteins. Structure, biosynthesis, and intracellular trafficking. Journal of Biology and Chemistry 266: 21327-21330.

•Holness CL, Da Silva RP, Fawcett J et al 1993. Macrosialin, a mouse macrophage-restricted glycoprotein, is a member of the lamp/lgp family. Journal of Biology and Chemistry 268:9661-9666.

•Knapp W 1989. Myeloid section report: In: Knapp W et al (eds). Leucocyte typing IV. White cell differentiation antigens. Oxford: Oxford University Press 747-780.

•Pulford KAF, Rigney EM, Micklem KJ et al 1989. KP1: a new monoclonal antibody that detects a monocyte/macrophage associated antigen in routinely processed tissue sections. Journal of Clinical Pathology 42:414-421.

•Pulford KAF, Sipos A, Cordell JL, Stross WP, Mason DY 1990. Distribution of the CD68 macrophage/myeloid associated antigen. Immunology 2: 973-980.

•Ralfkiaer E, Delsol G, O'Connor NTJ et al 1990. Malignant lymphomas of true histiocytic origin. A clinical, histological, immunophenotypic and genotypic study. Journal of Pathology 160:9-17.

•Ruco LP, Pulford KAF, Mason DY et al 1989. Expression of macrophage-associated antigens in tissues involved by Langerhans' cell histiocytosis (histiocytosis X). American Journal of Clinical Pathology 92:273-279.

•Thiele J, Braeckel C, Wagner S et al 1992. Macrophages in normal human bone marrow and in chronic myeloproliferative disorders: an immunohistochemical and morphometric study by a new monoclonal antibody (PG-M1) on trephine biopsies. Virchows Archives A Pathologic Anatomy 421:33-39.

•Warnke RA, Pulford KAF, Pallesen G et al 1989. Diagnosis of myelomonocytic and macrophage neoplasms in routinely processed tissue biopsies with monoclonal antibody KP1. American Journal of Pathology 135:1089-1095. neoplasms in routinely processed tissue biopsies with monoclonal antibody KP1. American Journal of Pathology 135:1089-1095.

Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.