title: Quantification of cells with specific phenotypes II: determination of CD4 expression level on reconstituted lyophilized human PBMC labelled with anti-CD4 FITC antibody.
abstract: This report focuses on the characterization of CD4 expression level in terms of equivalent number of reference fluorophores (ERF). Twelve different flow cytometer platforms across sixteen laboratories were utilized in this study. As a first step the participants were asked to calibrate the fluorescein isothiocyanate (FITC) channel of each flow cytometer using commercially available calibration standard consisting of five populations of microspheres. Each population had an assigned value of equivalent fluorescein fluorophores (EFF denotes a special case of the generic term ERF with FITC as the reference fluorophore). The EFF values were assigned at the National Institute of Standards and Technology (NIST). A surface-labelled lyophilized cell preparation was provided by the National Institute of Biological Standards and Control (NIBSC), using human peripheral blood mononuclear cells (PBMC) pre-labeled with a FITC conjugated anti-CD4 monoclonal antibody. Three PBMC sample vials, provided to each participant, were used for the CD4 expression analysis. The PBMC are purported to have a fixed number of surface CD4 receptors. On the basis of the microsphere calibration, the EFF value of the PBMC samples was measured to characterize the population average CD4 expression level of the PBMC preparations. Both the results of data analysis performed by each participant and the results of centralized analysis of all participants' raw data are reported. Centralized analysis gave a mean EFF value of 22,300 and an uncertainty of 750, corresponding to 3.3% (level of confidence 68%) of the mean EFF value. The next step will entail the measurement of the ERF values of the lyophilized PBMC stained with labels for other fluorescence channels. The ultimate goal is to show that lyophilized PBMC is a suitable biological reference cell material for multicolor flow cytometry and that it can be used to present multicolor flow cytometry measurements in terms of ABC (antibodies bound per cell) units.
authors: Biosystems and Biomaterials Division, NIST (National Institute of Standards and Technology), Gaithersburg, Maryland, 20899.
authors: L
authors: L
authors: Wang
authors: R
authors: R
authors: Stebbings
authors: A K
authors: AK
authors: Gaigalas
authors: J
authors: J
authors: Sutherland
authors: M
authors: M
authors: Kammel
authors: M
authors: M
authors: John
authors: B
authors: B
authors: Roemer
authors: M
authors: M
authors: Kuhne
authors: R J
authors: RJ
authors: Schneider
authors: M
authors: M
authors: Braun
authors: A
authors: A
authors: Engel
authors: D
authors: D
authors: Dikshit
authors: F
authors: F
authors: Abbasi
authors: G E
authors: GE
authors: Marti
authors: M
authors: M
authors: Sassi
authors: L
authors: L
authors: Revel
authors: S K
authors: SK
authors: Kim
authors: M
authors: M
authors: Baradez
authors: T
authors: T
authors: Lekishvili
authors: D
authors: D
authors: Marshall
authors: L
authors: L
authors: Whitby
authors: W
authors: W
authors: Jing
authors: V
authors: V
authors: Ost
authors: M
authors: M
authors: Vonsky
authors: J
authors: J
authors: Neukammer
mesh_terms: D000906:Antibodies / Q000032:analysis / Q000378:metabolism; D015704:CD4 Antigens / Q000032:analysis / Q000096:biosynthesis*; D018791:CD4 Lymphocyte Count / Q000379:methods / Q000592:standards; D016650:Fluorescein-5-isothiocyanate / Q000032:analysis; D005612:Freeze Drying / Q000379:methods; D005786:Gene Expression Regulation; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000378:metabolism*; D010641:Phenotype
chemical_list: D000906:Antibodies; D015704:CD4 Antigens; D016650:Fluorescein-5-isothiocyanate
keywords: CD4 expression level; FITC; calibration; equivalent fluorescein fluorophore (EFF); measurement uncertainty; quantitative flow cytometry; reference cell material; standard measurement procedure; surface labelled lyophilized PBMC