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Quantification of Cells with Specific Phenotypes I: Determination of CD4+ Cell Count per µL in Reconstituted Lyophilized Human PBMC Pre-labelled with Anti-CD4 FITC Antibody



Richard Stebbings, Lili Wang, Janet Sutherland, Martin Kammel, Adolfas K. Gaigalas, M. John, B. Roemer, M. Kuhne, R. J. Schneider, M. Braun, N. Leclere, D. Dikshit, Fatima Abbasi, Gerald Marti, P. Porcedda, M. Sassi, L. Revel, S. K. Kim, D. Marshall, L. Whitby, Jing Wang, V. Ost, M. Vonski, Joerg Neukammer


A surface-labelled lyophilised lymphocyte (sLL) preparation has been developed using human peripheral blood mononuclear cells (PBMC) pre-labelled with a fluorescein isothiocyanate (FITC) conjugated anti-CD4 monoclonal antibody. The sLL preparation is intended to be used as a reference material for CD4+ cell counting and has been evaluated in the pilot study CCQM-P102. This study was conducted across sixteen laboratories from eight countries to assess the ability of participants to quantify the CD4+ cell count of this reference material and to document cross-laboratory variability plus associated measurement uncertainties. Twelve different flow cytometer platforms were evaluated using a standard protocol that included calibration beads used to obtain quantitative measurements of CD4+ T cell counts. There was good overall cross-platform and counting method agreement with a mean of the laboratory calculated means of 300.4 & 302.9 CD4+ cells per µL for a 1 in 5 and a 1 in 20 dilution, respectively. Greater than 90% of participant data agreed within ±15%. A major contribution to variation of sLL CD4+ cell counts was tube to tube variation of the calibration beads, amounting to an uncertainty of 7.3%. Variation due to preparative steps equated to an uncertainty of 2.6%. There was no reduction in variability when data files were centrally reanalysed. Remaining variation was attributed to instrument specific differences. CD4+ cell counts obtained in CCQM-P102 are in excellent agreement and show the robustness of both the measurements and the data analysis and hence the suitability of sLL for inter-laboratory comparisons and external quality assessment.
Cytometry Part A


CD4+ cell counting, lyophilised cells, flow cytometry, standard measurement procedure, measurement of uncertainty, HIV-1, AIDS, reference material.
Created March 10, 2015, Updated February 19, 2017