Highly enriched preparations of monocytes, B and T lymphocytes, and granulocytes from 18 normal donors were serotyped in parallel in a complement-dependent cytotoxicity assay using allogeneic and heterologous antisera defining three independent tissue antigen systems. HLA and B-lymphocyte tissue antigens were detected on human monocytes although granulocyte antigens were absent. By cytotoxicity testing the presence of Ia-like antigens on monocytes was significantly diminished compared to the autologous B-lymphocyte population and has important implications in B-lymphocyte serology. The study indentified a number of human antisera obtained from multitransfused subjects and pre- and post-transplant organ recipients that were non-HLA and appeared to define monocyte-associated antigens. The serological implications of surface antigen expression on human monocytes compared with other peripheral blood cells are discussed.

Using a microcytotoxicity assay, the serological reactivity of human granulocytes, namely neutrophils and eosinophils, and chronic myeloid leukemia (CML) cells and cultured CML cell lines (K562, NALM-1) were examined. Mature granulocyte forms and cord granulocytes are readily lysed by specific granulocyte cytotoxins that do not react with random T and B lymphocytes, monocytes, red blood cells, or platelets. Furthermore, certain antisera were preferentially cytotoxic for eosinophil-enriched populations. Granulocytotoxin detected antigens on one of three CML blast cell populations tested and K562, but failed to react with NALM-1. By cytotoxicity, mature granulocytes were poor targets for B2-microglobulin and the appropriate HLA antisera although both sera types are absorbed with granulocytes. Furthermore, granulocytes did not possess B-lymphocytes (Ia-like) or blood group A, B, and Rh (D) antigens. Except for K562, both HLA and heterologous B-lymphocyte antisera were cytotoxic for the CML blast cell populations tested.

Some aspects influencing the serologic outcome of complement-dependent granulocyte cytotoxicity are presented. Fieoll-Hypnque (density 1.060) gradient centrifugation with hypotonic lysis of red blood cells yielded populations of granulmytes with greater than 90 per cent purity. Granulocytes exposed to papain (0.01%) at 24 C for 12 minutes showed enhanced serologic reactivity compared with untreated cells. In addition, enhanced cytotoxicity was promoted by 5 C (cold) as opposed to 22 C (warm) incubation of granulocytes and antibody in the first stage of the microcytotoxicity assay and emphasizes the temperature dependence of the seroiogic reactions. Neutrophil-specific leukoagglutinins were not cytotoxic under optimal in vifm conditions. Conversely, a dgnbkant proportiam of antibodies seh?cted for cytotoxicity failed to agglutinate granulocytes. Cold reactive granulocytotoxins are complement dependent, IgM in nature, and 2-mercaptaethanol sensitive and do not appear to be Immune complexes. By optimizing reaction conditions, granulocyte microcytotoxicity is a valuable addition to in vitro assays detecting immune sensitization against granulocyte surface antigens. THE NUMEROUS methodologies that describe the detection of granulocyte cell surface antigens testify as to the difficulties experienced with the present in vitro techniques. Such methods as immunofluorescence, antiglobulin consumption and polymorphonuclear function studies ,5*6*19~24*32 have definite limitations and are not suited to routine screening of antisera. To date, leukoagglutination assays18,20.26 have met with the most success in the definition of neutrophilspecific antigen groupsz1 although spontaneous aggregation of granulocytes and the need for functionally intact normal cells hamper this technique. Complement-dependent cytotoxic assays have utilized