Characteristics of Monoclonal Antibody Measurements in Human Peripheral Blood

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Abstract
Three areas of monoclonal antibody measurements using flow cytometry have been presented. These include a description of a dual immunofluorescent method for measuring two antibodies simultaneously, the effects of blood storage on enumeration of helper (H) and suppressor (S) cells, and the relationship between absolute lymphocyte count and H/S ratio in both control and AIDS patients. These studies reveal that a dual immunofluorescent labeling method is useful for enumerating lymphocytes from peripheral blood which bear the helper, suppressor and/or thymus-derived (T) cell receptors. Fluorescein (FL)-conjugated Leu-3a + 3b antibodies were used to enumerate helper T-lymphocytes, while the B-phycoerythrin (B-PE)-conjugated Leu-2a antibodies were utilized for enumerating suppressor T-lymphocytes. Dual immunofluorescently stained lymphocytes, prepared from whole blood, were analyzed by flow cytometry. Two light scatter parameters, (forward and 90 degree scatter) were used to define the lysed erythrocyte, lymphocyte, monocyte, and granulocyte populations. Only the lymphocytes were analyzed for dual immunofluorescence activity. The helper and suppressor distributions from 167 control patients were as follows: The average percentage +/- SD of the helper and suppressor cells were 42.8 +/- 7.5 and 21.6 +/- 6.4, respectively. The H/S ratio was 2.17 +/- .75. These studies show that the H/S ratio can be determined in a single preparative sample and analyzed by dual immunofluorescence in a single flow cytometric analysis even though the H/S ratio may vary from normal during a disease condition. The dual immunofluorescent assay enables one to correlate the activities of two antibodies against cell surface receptors and allows the measurement of a large number of samples in a minimal time. This study also compared the effects of anticoagulant, storage time, and temperature on the phenotypic determination of the percentages of helper and suppressor T-lymphocytes in human peripheral blood. Blood was drawn in ACD, heparin, and EDTA and stored for up to 4 days at room temperature or 4 degrees C. Phenotypic determination of helper/suppressor lymphocytes was most stable for ACD or heparinized blood at room temperature. Marked changes were observed in the percentages of helper cells at 4 degrees C, whereas the percentages of suppressor cells did not change appreciably regardless of the anticoagulant storage time or temperature. Finally, the relationship between ALC and the H/S ratio in control and AIDS patients was determined. The ALC varied considerably in both control and patient populations as a function of time. Conversely, the H/S ratio remained constant.(ABSTRACT TRUNCATED AT 400 WORDS)