Reduction of variation in T‐cell subset enumeration among 55 laboratories using single‐platform, three or four‐color flow cytometry based on CD45 and SSC‐based gating of lymphocytes

BACKGROUND: Enumeration of CD4(+) and CD8(+) T-cell subsets provides relevant information for diagnosis and monitoring of patients with cellular immunodeficiencies. As a result, an external quality assurance scheme was implemented in Belgium, The Netherlands, and Luxembourg in 1995. A workshop was held to train the participants in state-of-the art technology for assessment of absolute T-cell subset counts (i.e., a three or four-color, single-platform assay with lymphocyte gating based on CD45 and sideward light scatter) with the aim to achieve between-site coefficients of variation (CVs) <10% and within-site CVs <5% for > or =75% of the participants. METHODS: Three send-outs of stabilized blood from a healthy donor were distributed to 55 laboratories, each with the request to perform the standard assay on three occasions. For comparison, each laboratory performed its local technique in parallel. RESULTS: With the standard technique, between-site CVs of approximately 8% (CD3+ T cells), approximately 9% (CD4+ T cells), and approximately 10% (CD8+ T cells) were achieved. Within-site CVs were <5% for 82% (CD3+ T cells) and approximately 70% (CD4+ and CD8+ subsets) of the participants. Local techniques yielded between-site CVs of 13%-17% for CD3+, CD4+, and CD8+ T cells. CONCLUSIONS: The state-of-the-art technology for T-cell subset enumeration was implemented successfully among 55 Belgian-Dutch laboratories and resulted in significant reductions of between-site variation of absolute CD3+, CD4+, and CD8+ T-cell counts.