Quantification of cells with specific phenotypes II: determination of CD4 expression level on reconstituted lyophilized human PBMC labelled with anti-CD4 FITC antibody.

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.

Flow cytometric differentiation of erythrocytes and leukocytes in dilute whole blood by light scattering.

We have determined differential scattering cross sections of sphered erythrocytes integrated over the solid angle accepted by our detectors at wavelengths varying between 379.5-632.8 nm, employing Ar+-, Kr+-, and HeNe-laser radiation. Integrated differential cross sections for forward and orthogonal light scatter exhibit a pronounced minimum at about lambda = 413 nm, caused by the strong absorption of light by oxyhemoglobin. Experimental data are in good agreement with values calculated by the Mie theory. Simultaneous measurements of forward and orthogonal light scatter using Kr+-laser radiation (lambda = 413.1 nm) allow flow cytometric differentiation of (native) erythrocytes, thrombocytes, and leukocytes as well as subpopulations of leukocytes, i.e., granulocytes, lymphocytes, and monocytes in dilute whole-blood samples. Lysis of red blood cells or staining of white blood cells is not required to obtain absolute leukocyte counts or differential white blood cell counts.