title: Quantification of cells with specific phenotypes I: determination of CD4+ cell count per microliter in reconstituted lyophilized human PBMC prelabeled with anti-CD4 FITC antibody.
abstract: A surface-labeled lyophilized lymphocyte (sLL) preparation has been developed using human peripheral blood mononuclear cells prelabeled with a fluorescein isothiocyanate conjugated anti-CD4 monoclonal antibody. The sLL preparation is intended to be used as a reference material for CD4+ cell counting including the development of higher order reference measurement procedures and has been evaluated in the pilot study CCQM-P102. This study was conducted across 16 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 grand mean of the laboratory calculated means of (301.7 ± 4.9) μL(-1) CD4+ cells. Excluding outliers, 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 3.6%. Variation due to preparative steps equated to an uncertainty of 2.6%. There was no reduction in variability when data files were centrally reanalyzed. 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 as a reference material for interlaboratory comparisons and external quality assessment.
authors: Biotherapeutics Group, National Institute for Biological Standards and Control (NIBSC), South Mimms, Potters Bar, Hertfordshire, EN6 3QG, United Kingdom.
authors: Richard
authors: R
authors: Stebbings
authors: Lili
authors: L
authors: Wang
authors: Janet
authors: J
authors: Sutherland
authors: Martin
authors: M
authors: Kammel
authors: Adolfas K
authors: AK
authors: Gaigalas
authors: Manuela
authors: M
authors: John
authors: Bodo
authors: B
authors: Roemer
authors: Maren
authors: M
authors: Kuhne
authors: Rudolf J
authors: RJ
authors: Schneider
authors: Michael
authors: M
authors: Braun
authors: Andrea
authors: A
authors: Engel
authors: Dinesh K
authors: DK
authors: Dikshit
authors: Fatima
authors: F
authors: Abbasi
authors: Gerald E
authors: GE
authors: Marti
authors: Maria Paola
authors: MP
authors: Sassi
authors: Laura
authors: L
authors: Revel
authors: Sook-Kyung
authors: SK
authors: Kim
authors: Marc-Olivier
authors: MO
authors: Baradez
authors: Tamara
authors: T
authors: Lekishvili
authors: Damian
authors: D
authors: Marshall
authors: Liam
authors: L
authors: Whitby
authors: Wang
authors: W
authors: Jing
authors: Volker
authors: V
authors: Ost
authors: Maxim
authors: M
authors: Vonsky
authors: Jörg
authors: J
authors: Neukammer
mesh_terms: D000906:Antibodies / Q000032:analysis; D018791:CD4 Lymphocyte Count / Q000379:methods / Q000592:standards; D015496:CD4-Positive T-Lymphocytes / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate / Q000032:analysis; D005612:Freeze Drying / Q000379:methods; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D010641:Phenotype; D010865:Pilot Projects
chemical_list: D000906:Antibodies; D016650:Fluorescein-5-isothiocyanate
keywords: CD4+ cell counting; acquired immunodeficiency syndrome; flow cytometry; human immunodeficiency virus-1; lyophilized cells; measurement of uncertainty; reference material; relative concentration measurement; standard measurement procedure
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