Discrimination in immunofluorescence between labeled and unlabeled cells by quantitative microfluorometry and computer analysis

Abstract

An automated procedure for discrimination in immunofluorescence between antibody-labeled and unlabeled cells has been developed on the basis of microfluorimetric determination of intensity distributions. After smoothing the raw data for irregularities caused by the scoring statistics optimum fit of the negative distribution to the corresponding positive one was achieved. The procedure was tested in a model system by mixing various known proportions of immunofluorescence-negative and positive plastic beads. In addition, variable mixtures of T-negative CLL cells and normal mononuclear peripheral blood cells were labeled with FITC-conjugated anti-T-antiserum. The expected percentage of T-positive peripheral blood cells agreed satisfactorily with the data measured and computed. Finally, the measured percentage of Ig-positive mononuclear cells from normal peripheral blood was in agreement with the values obtained by other techniques.