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.
Laboratory for Clinical and Tumor Immunology, Department of Internal Oncology, Erasmus Medical Center-Daniel den Hoed Cancer Center, Groene Hilledijk 301, 3075 EA Rotterdam, The Netherlands. gratama@immh.azr.nl
Jan W
JW
Gratama
Jaco
J
Kraan
Mike
M
Keeney
Viv
V
Granger
David
D
Barnett
D017252:CD3 Complex / Q000097:blood; D015704:CD4 Antigens / Q000097:blood; D016827:CD8 Antigens / Q000097:blood; D002624:Chemistry, Clinical / Q000379:methods; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods*; D006801:Humans; D017493:Leukocyte Common Antigens / Q000097:blood*; D010865:Pilot Projects; D015203:Reproducibility of Results; D016176:T-Lymphocyte Subsets / Q000166:cytology*
D017252:CD3 Complex; D015704:CD4 Antigens; D016827:CD8 Antigens; D017493:Leukocyte Common Antigens