Analysis of variation in results of flow cytometric lymphocyte immunophenotyping in a multicenter study

Fifty-five laboratories participated in a send-out study of four peripheral blood samples comparing a standard protocol vs. local protocols for flow cytometric lymphocyte immunophenotyping. The standard protocol included centrally provided reagents, instrument setup using triple-fluorescent microbeads and a three-color, whole-blood immunostaining technique based on fluorescein isothiocyanate and phycoerythrin-labeled monoclonal antibodies, erythrocyte lysis, washing, fixation, and identification of nucleated cells by the DNA/RNA stain LDS-751. Data analysis guidelines included lymphocyte selection using CD45,CD14-assisted "backgating" on forward (FSC) and sideward (SSC) light scatter and placement of fluorescence (FL) markers on the basis of the isotype control staining. Most (i.e., 77%) of the variation in results of percentage lymphocyte subset assessments using the standard protocol was explained by laboratory, sample, background FL, and the interaction between laboratory and sample. Purity and completeness of the FSC,SSC lymphogate, background FL, flow cytometer type, and flow cytometer setup (which were either partly or entirely determined by laboratory) contributed significantly to the variation. The effect of the leukocyte differential count on the variation in absolute numbers of lymphocyte subsets was particularly large in lymphopenic samples. The use of this standard protocol vs. local protocols did not reduce the interlaboratory variation. Instrument incompatibility with the standard protocol (e.g., incompatible filter combinations for LDS-751 detection) and lack of experience of many participants with three-color flow cytometry (in particular with the use of LDS-751) may have contributed to that result. We suggest that training and experience in a universally applicable standard protocol are critical for minimization of interlaboratory variation in flow cytometric immunophenotyping.