Flow cytometric dot plot analysis for the calculation of f and μ. BLCLs were analyzed after staining sequentially with a mixture of unconjugated anti–human CD48, CD55, and CD59, PE-conjugated rabbit anti–mouse immunoglobulin antibody, and anti-HLA-DR–FITC. The number of events is shown in each quadrant. The mutant frequency (f) is calculated as the number of GPI– cells divided by the number of GPI+ cells. (A) Normal GPI+ cells mixed with GPI– cells from a patient with PNH. (B) BLCLs from healthy donor 1, where the frequency of mutants is 22 × 10–6 (C) BLCLs from a patient with ataxia-telangiectasia, with increased GPI– cells (f = 245 × 10–6). (D-F) To calculate the mutation rate, preexisting mutants are eliminated from the population by collecting the upper 50th percentile of the distribution curve after staining with anti-CD59. The collected GPI+ cells are then returned to culture and expanded. The mutation rate is determined by the formula μ = f ÷ d, where d represents the number of cell divisions occurring in vitro after sorting.24 (D) Analysis of a BLCL from healthy donor 2 after expansion after flow sorting. f = 6.4 × 10–6, d = 8 cell divisions, and μ = 8 × 10–7 mutations per cell division. (E-F) Analyses of 2 representative cell lines from PNH patients. (E) Analysis of a BLCL from PNH patient 1 after expansion after flow sorting. f = 4.73 × 10–6, d = 4.24 cell divisions, and μ = 11.2 × 10–7 mutations per cell division. (F) Analysis of a BLCL from PNH patient 2 after expansion after flow sorting. f = 5.05 × 10–6, d = 10.6 cell divisions, and μ = 4.76 × 10–7 mutations per cell division.
