Direct and indirect flow cytomyetric enumeration of 6‐thioguanine‐resistant human peripheral blood lymphocytes

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Abstract
Methods based on flow cytometry and sorting, auto radiography, and cloning were used to evaluate the potential for the enumeration of 6‐thioguanine‐resistant human peripheral blood lymphocytes assumed to be deficient with respect to the enzyme hypoxanthineguanine‐phosphoribosyl‐transferase. Flow cytometric sorting of proliferating cells in the late S‐and the G2‐stages by means of DNA content, as measured by propidium iodide fluorescence, enabled an enrichment of variant cells to about 99%. The main source of false events was contaminating doublets of G0/G1 cells appearing in the sorting region. Doublet discrimination measured as the difference between pulse height and area (Ortho‐50) accomplished no further improvement. A combination of propidium iodide fluorescence and bromodeoxyuridine incorporation, measured by fluorescent anti‐bromodeoxyuridine‐DNA antibodies, allowed flow cytometric enrichment to about 99.99% of variant cells. By sorting of 3H‐thymidine‐labeled cell nuclei from the late S‐and the G2‐phases and subsequent autoradiographic evaluation, partly resistant variants could be discriminated; variant frequencies of the same magnitude as for the cell cloning methods were obtained.

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