Human blood PIG‐A mutation and micronucleated reticulocyte flow cytometric assays: Method optimization and evaluation of intra‐ and inter‐subject variation

We previously described flow cytometry‐based methods for scoring the incidence of micronucleated reticulocytes (MN‐RET) and PIG‐A mutant phenotype reticulocytes (MUT RET) in rodent and human blood samples. The current report describes important methodological improvements for human blood analyses, including immunomagnetic enrichment of CD71‐positive reticulocytes prior to MN‐RET scoring, and procedures for storing frozen blood for later PIG‐A analysis. Technical replicate variability in MN‐RET and MUT RET frequencies based on blood specimens from 14 subjects, intra‐subject variability based on serial blood draws from 6 subjects, and inter‐subject variation based on up to 344 subjects age 0 to 73 years were quantified. Inter‐subject variation explained most of the variability observed for both endpoints (≥77%), with much lower intra‐subject and technical replicate variability. The relatively large degree of inter‐subject variation is apparent from mean and standard deviation values for MN‐RET (0.15 ± 0.10%) and MUT RET (4.7 ± 5.0 per million, after omission of two extreme outliers). The influences of age and sex on inter‐subject variation were investigated, and neither factor affected MN‐RET whereas both influenced MUT RET frequency. The lowest MUT RET values were observed for subjects