Patients. Peripheral blood samples from patients re­ferred for analysis by the Clinical Cytogenetics Labora­tory of Children’s Hospital of Pittsburgh, PA, USA, were coded prior to being processed for the GPA assay. From a total population of 118 samples, 11 were identified as having no detectable cytogenetic abnormality (and thus were of unknown etiology), as well as being heterozygous for the MN blood group, and therefore informative for the GPA in vivo somatic mutation assay.
GPA Mutation Assay. The “DB6” version of the GPA assay was performed [1]. Briefly, blood samples from MN heterozygotes were double-labeled with monoclonal anti­bodies to the two forms of the GPA protein on the erythro­cyte surface. Five million cells were then analyzed for allele loss phenotypes via flow cytometry.The same laboratory control, a 39-year-old male was analyzed three times in conjunction with the patient sam­ples in this report. Total GPA Mf were 8.8, 11.6 and 11.8 x 10^–6 for these analyses, which are consistent with our experience for this subject, who has been analyzed 69 other times in our laboratory, with an average Mf [ stan­dard deviation (SD)] of 9.7  - 3.9 x 10^–6. Similarly, allele loss mutations occurred at frequencies of 3.6, 4.0 and 7.8 x 10^–6 in these analyses, consistent with our experience for this subject (allele loss Mf = 4.6 x 10^–6); while allele loss and duplication mutants occurred at frequencies of 4.0, 5.2 and 7.6 x 10^–6 , also consistent with other analyses for this individual (Mf = 5.1 x 10^–6 ).
Statistical Analyses. Population comparisons were performed with the t test using Microsoft Excel on ln transformed data.