IMMUNOLOGICAL DIFFERENTIATION OF ABSOLUTE LYMPHOCYTE COUNT WITH AN INTEGRATED FLOW CYTOMETRIC SYSTEM - A NEW CONCEPT FOR ABSOLUTE T-CELL SUBSET DETERMINATIONS

We describe a method to obtain results for immune status monitoring th at uses a three-test panel, comprised of isotype control and 2 specifi c Mab tests (CD4/CD8/CD3 and CD16/CD19/CD3), in conjunction with a flo w cytometer that directly measures absolute counts. Automated software is used for lineage-specific gating of three-color immunofluorescence to determine lymphocyte and lymphocyte subset counts, The autogating function of this software is shown to yield equivalent results to manu al analysis by an expert user, and to be effective when as few as 25 t arget cells are present. The software is also shown to perform automat ic quality control checks of the sample preparation, reagent, and auto mated analysis. We demonstrate that the sum of T (CD3+), B (CD19+), an d natural killer (NK, CD16+CD3-) cells, as a determination of all lymp hocytes, correlates well with lymphocytes measured using a light scatt er differential. Moreover, T+B+NK lymphocyte count is shown to be less error-prone than lymphocyte count from light scatter differential, an d to minimize errors that arise from between-technician variation in s ample preparation, Our data suggest that the new approach that we desc ribe could offer an alternative to the traditional two-stage methods f or measuring absolute counts of lymphocyte subsets for immune status m onitoring, As such this method could reduce, through objective automat ed analysis, testing cost and complexity, without sacrificing the qual ity of results. (C) 1995 Wiley-Liss, Inc.