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A technic using fluorescent immunospheres was developed to identify simultaneously two surface antigens on individual lymphocytes while preserving Wright's stained cell morphology. Small numbers (10,000-20,000) of cells were studied from peripheral blood or bone marrow samples from normal control subjects and from patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). Samples were studied for surface antigens using OKT-11, OK-Ia-1, B1, and J5. Comparison was made with results obtained from the same patients by indirect immunofluorescence. Correlation between results obtained with immunospheres and indirect immunofluorescence was excellent (r = 0.97). In addition, 35 cerebrospinal fluid samples from children with ALL were tested using the immunosphere method alone. Results obtained with spinal fluid lymphocytes agreed with previously reported results using similar methodology. It is concluded that the use of fluorescent microspheres provides a method for the combined evaluation of cell morphology and surface antigens on small, heterogeneous cell populations.

Ficoll-Hypaque separated normal human peripheral blood lymphocytes undergo appreciable blastogenesis in RPMI 1640-pooled human serum or - autologous plasma. During the 1st week, granulated blasts with peroixdase activity and ultrastructural properties of granulocytes precursors appear. These cells, which appear to be relateed to CFC (colony-forming cells), contribute to the mitotic population in these unstimulated "control" cultures prior to 6 days.

The identification of small numbers of leukemic cells in the cerebrospinal fluid (CSF) presents a diagnostic problem in the treatment of children with acute lymphoblastic leukemia (ALL). We adapted a latex sphere rosetting technique to allow us to identify simultaneously cell surface markers and cell morphology in 199 CSF samples from 34 patients and 14 control subjects. In patients without leukemic meningitis, the majority of CSF lymphocytes (69%) were found to be mature T cells positive for OKT11. A much smaller number of cells (8%) were found to be B cells positive for la. In these children, only 3% of CSF lymphoid cells expressed the common acute lymphoblastic leukemia antigen (CALLA). Similar results were found in the control subjects. By contrast, 28 CSF samples from nine children with varying numbers of CSF lymphoblasts had much greater proportions of CALLA- and la-positive CSF cells (24% to 96%). Leukemic meningitis was present in one of these patients and later developed in four others. However, three patients with small numbers of lymphoblasts present but with low proportions of CALLA-positive CSF cells (less than 5%) subsequently had normal CSF examinations. We found the use of this rosetting technique valuable in providing information complementary to that obtained from cell morphology alone about the possible malignant nature of small numbers of lymphoblast-like CSF cells seen on cytocentrifuge preparations in children with ALL.