Assessment of Interlaboratory Variability of Immunophenotyping

A 3-year interlaboratory proficiency testing for lymphocyte subset phenotyping was initiated as part of the Etalonorme national quality control program. Specimens consisted of fresh whole blood and of lyophilised mononuclear cells (Cytotrol Coulter). The number of participating laboratories was 62 in 1990, 99 in 1991 and 129 in 1992. Statistical analysis indicated that results of phenotyping, expressed as percentages of positive cells, are not related to reagents, instruments or differences in methodology (like, eg time and temperature of incubation). The highest dispersion was observed for total lymphocyte counts and was found to correlate with the type of calibration of the instrument. The coefficients of variation for different lymphocyte subsets were similar if phenotyping was performed on whole blood or lyophilised cells and varied inversely with the percentage of positive cells in each specimen. The consistency of the results indicated that they could serve as a basis for clinical decisions.

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“…The highest CVs were observed for CD19/CD20+ cells, varying from 35 to 39% (fig 2) close to literature data [17,27,33].…”

Section: Whole Bloodsupporting

confidence: 88%

Interlaboratory quality assessment of lymphocyte phenotyping. Etalonorme 1990–1992 surveys

Ducailar

Ouin

1993

Biology of the Cell

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“…The EFF values submitted showed inter-laboratory variation typically ranging from 15,000 to 25,000 with a minimum value of 6,354 and a maximum of 29,197. No outliers were observed for the mean values.…”

Section: Analysis Of Eff Results Submitted By Participantsmentioning

confidence: 99%

“…The inter-lab coefficient variance (CV) of 14% ¼ 3:3% Á ffiffiffiffiffi 18 p (see Table 4) was obtained by centralized analysis of the CD4 expression measurements on identical samples of a single sLL production lot to which all participants only needed to add distilled water. The measurement variance is expected to be much larger when researchers and clinicians select their preferred reagents (antibody clones and fluorophore labels), staining procedures, lysis buffers and fixatives, instrument settings, gating strategies and methods of data analysis (28,29). It is therefore essential for the development of reference cell materials, e.g., sLL, and reference methods (30) to address at least some of these variables to achieve useful level of inter-lab result comparability.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2015

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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. V C 2015 International Society for Advancement of Cytometry Key terms surface labelled lyophilized PBMC; CD4 expression level; FITC; equivalent fluorescein fluorophore (EFF); quantitative flow cytometry; calibration; standard measurement procedure; measurement uncertainty; reference cell material CLUSTER of differentiation 4 (CD4) is a glycoprotein on the surface of T-helper cells, monocytes, macrophages and dendritic cells. As a co-receptor, CD4 amplifies the signal generated by the T cell receptor, which is essential for activation of many molecules involved in the signalling cascade of an activated T cell. The CD4-positive

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“…A third group of publications describes inter-laboratory comparisons and focuses on the reproducibility of DNA flow cytometric results (4,(8)(9)(10)(11)13,18,23,24,26,28,37,41,44,49,50). The laboratories that were compared showed differences in the preparation and staining protocols, the kinds of flow instruments, and the interpretations of the histograms.…”

mentioning

confidence: 99%

Comparison of routine flow cytometric DNA analysis of fresh tissues in two laboratories: Effects of differences in preparation methods and background models of cell cycle calculation

et al. 1998

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Assessment of Interlaboratory Variability of Immunophenotyping

Cited by 32 publications

References 2 publications

Interlaboratory quality assessment of lymphocyte phenotyping. Etalonorme 1990–1992 surveys

Interlaboratory quality assessment of lymphocyte phenotyping. Etalonorme 1990–1992 surveys

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

Comparison of routine flow cytometric DNA analysis of fresh tissues in two laboratories: Effects of differences in preparation methods and background models of cell cycle calculation

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