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

✍ Scribed by Jan W. Gratama; Jaco Kraan; René Van den Beemd; Berend Hooibrink; Dirk R. Van Bockstaele; Herbert Hooijkaas

• Publisher: John Wiley and Sons
• Year: 1997
• Tongue: English
• Weight: 209 KB
• Volume: 30
• Category: Article
• ISSN: 0196-4763
• DOI: 10.1002/(sici)1097-0320(19970815)30:4<166::aid-cyto2>3.0.co;2-i

✦ Synopsis

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 phycoerythrinlabeled 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,CD14assisted ''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

This study was performed under the auspices of the Foundation for Immunophenotyping in Hemato-Oncology (SIHON), the Foundation for Quality Control in Medical Immunology (SKMI), the Foundation for Quality Control of Hospital Laboratories (SKZL), and the Belgian Association for Cytometry (BVC/ABC), with the participation of (in alphabetical order): M.