Quantitative Fluorescence Measures for Determination of Intracellular Perforin Content

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Abstract
We present methodologic details and operating characteristics of a procedure with whole blood for the quantitative assessment of intracellular perforin within distinct lymphocyte subsets. Using this method, we analyzed 20 healthy controls and 2 individuals with an inherited deficiency of perforin. The mean ± standard deviation perforin contents of natural killer (NK) cells and cytotoxic T cells of healthy controls were 3,561 ± 1,157 and 500 ± 779 relative number of molecules (rMol) of antiperforin antibody bound per cell, respectively. The NK cell perforin contents of individuals with heterozygous and homozygous perforin deficiency (familial hemophagocytic lymphohistiocytosis) were 2,260 and 212 rMol of antiperforin antibodies per NK cell. While the homozygous deficiency was found to be associated with negligible antiperforin binding, the heterozygous condition was associated with a level of perforin binding that was below the 15th percentile for healthy individuals. Because 83% of this subject's NK cells were shown to bind to antiperforin antibodies by conventional flow cytometry (relative to the normal range of 81% ± 25%), quantitative cytometry may be more sensitive than conventional cytometric methods in identifying cytolytic defects.