Determination of optimal replicate number for validation of imprecision using fluorescence cell‐based assays: Proposed practical method

Background Assay validation includes determination of inherent imprecision across the reportable range. However, specific practical guidelines for determinations of precision for cell‐based fluorescence assays performed on flow cytometers are currently lacking. Methods Replicates of 10 or 20 measurements were obtained for flow cytometric assays developed for clinical in vitro diagnostic use, including neutrophil CD64 expression for infection/sepsis detection, fetal red cell enumeration for fetomaternal hemorrhage detection, human equilibrative nucleoside transporter 1 quantitation in leukocytes for possible correlation with drug responsiveness, and CD34+ hematopoietic stem cell enumeration of apheresis products, using up to three different instrument platforms for each assay. For each assay, the mean, 95% confidence intervals (95% CIs) of the mean, standard deviation, and coefficient of variation (CV) of sequential replicates were determined. Results For all assays and most instrument platforms,