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Severe combined immunodeficiency (SCID) is characterized by a severe deficiency in T cell numbers. We analyzed data collected (n = 307) for PHA-based T cell proliferation from the PIDTC SCID protocol 6901, using either a radioactive or flow cytometry method. In comparing the two groups, a smaller number of the patients tested by flow cytometry had <10% of the lower limit of normal proliferation as compared to the radioactive method (p = 0.02). Further, in patients with CD3+ T cell counts between 51 and 300 cells/μL, there was a higher proliferative response with the PHA flow assay compared to the 3H-T assay (p < 0.0001), suggesting that the method of analysis influences the resolution and interpretation of PHA results. Importantly, we observed many SCID patients with profound T cell lymphopenia having normal T cell proliferation when assessed by flow cytometry. We recommend this test be considered only as supportive in the diagnosis of typical SCID. • PHA proliferation has been traditionally measured in the laboratory by using incorporation of radioactive thymidine (3H-T). • Flow cytometry-based proliferation offers superior analytical resolution, especially in severe T cell lymphopenia. • PHA proliferation is not essential for diagnosis of typical SCID. • The method used to measure PHA proliferationhas an impact on interpretation of results. [ABSTRACT FROM AUTHOR]