Dual immunofluorescent analysis of human peripheral blood lymphocytes

These studies reveal that a dual immunofluorescent labeling method is useful for enumerating cells from human peripheral blood that bear the helper, suppressor, and/or T-cell receptors. Fluorescein (FL)-conjugated Leu-3a + 3b antibodies were used to enumerate Helper (H) T-lymphocytes, while the B-phycoerythrin (B-PE)-conjugated Leu-2a antibodies were utilized for quantitating suppressor (S) T-lymphocytes. FL-conjugated Leu-4 antibodies were used to measure the T-lymphocyte activity. Dual immunofluorescent stained lymphocytes, prepared either from whole blood or by Ficoll-Hypaque, gradient cell separation, were analyzed by flow cytometry. Two light scatter parameters, forward and 90 degree, were used to define the lysed erythrocyte, lymphocyte, monocyte, and granulocyte populations. Only the lymphocytes were analyzed for dual immunofluorescence activity. The helper, suppressor, and T-lymphocyte distributions from 100 controls were as follows: The average percentages +/- SD of the helper and suppressor cells were 41.2 +/- 7.2 and 23.0 +/- 7.2, respectively. The H/S ratio was 1.99 +/- 0.77, while the T-cell distribution on 28 patients was 71.4 +/- 7.7. The Ficoll-Hypaque purified lymphocytes and lysed whole blood lymphocytes compared favorably in their H/S ratios. A comparison was made between the percentages of helper and suppressor cells enumerated by fluorescent microscopy and flow cytometry in which correlation coefficients of 0.80 and 0.86 were determined, respectively. These studies show that helper and suppressor T-lymphocytes can be quantitated simultaneously by flow cytometry, which enables one to correlate the phenotypic activities of two antibodies against cell surface receptors and permits the measurement of a large number of samples in a minimal time.