Consistency of routine measurements of CD4+, CD8+ peripheral blood lymphocytes

Aboulker, J; Autran, Brigitte; Beldjord, Kheïra; Touraine, F.; Debré, Patrice

doi:10.1016/0022-1759(92)90187-x

Cited by 26 publications

(22 citation statements)

References 11 publications

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“…Both singleplatform assay methods decreased between-laboratory %CV by nearly half compared with the predicate method. The within-laboratory and between-laboratory precision that we observed using a conventional multiple-platform method was nearly identical to the precision observed in other multisite studies that analyzed similar specimens (1,11,14).…”

Section: Discussionsupporting

confidence: 85%

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Multisite Comparison of CD4 and CD8 T-Lymphocyte Counting by Single- versus Multiple-Platform Methodologies: Evaluation of Beckman Coulter Flow-Count Fluorospheres and the tetraONE System

Reimann

O’Gorman

Spritzler

et al. 2000

Clin Diagn Lab Immunol

115

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New analytic methods that permit absolute CD4 and CD8 T-cell determinations to be performed entirely on the flow cytometer have the potential for improving assay precision and accuracy. In a multisite trial, we compared two different single-platform assay methods with a predicate two-color assay in which the absolute lymphocyte count was derived by conventional hematology. A two-color method employing lymphocyte light scatter gating and Beckman Coulter Flow-Count fluorospheres for absolute counting produced within-laboratory precision equivalent to that of the two-color predicate method, as measured by coefficient of variation of replicate measurements. The fully automated Beckman Coulter tetraONE System four-color assay employing CD45 lymphocyte gating, automated analysis, and absolute counting by fluorospheres resulted in a small but significant improvement in the within-laboratory precision of CD4 and CD8 cell counts and percentages suggesting that the CD45 lymphocyte gating and automated analysis might have contributed to the improved performance. Both the two-color method employing Flow-Count fluorospheres and the four-color tetraONE System provided significant and substantial improvements in between-laboratory precision of absolute counts. In some laboratories, absolute counts obtained by the single-platform methods showed small but consistent differences relative to the predicate method. Comparison of each laboratory's absolute counts with the five-laboratory median value suggested that these differences resulted from a bias in the absolute lymphocyte count obtained from the hematology instrument in some laboratories. These results demonstrate the potential for single-platform assay methods to improve within-laboratory and between-laboratory precision of CD4 and CD8 T-cell determinations compared with conventional assay methods.

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Section: Discussionsupporting

confidence: 85%

“…The accurate quantitation of these cells in blood is crucial for providing clinical care to HIV-1-infected patients and for the systematic evaluation of new therapeutic modalities. However, previous studies have identified substantial variability in results between laboratories performing these assays (1,5).…”

mentioning

confidence: 99%

Multisite Comparison of CD4 and CD8 T-Lymphocyte Counting by Single- versus Multiple-Platform Methodologies: Evaluation of Beckman Coulter Flow-Count Fluorospheres and the tetraONE System

Reimann

O’Gorman

Spritzler

et al. 2000

Clin Diagn Lab Immunol

115

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“…First, do all instruments discriminate lymphocytes from other blood components equally? Reports that lymphocyte counts differ by up to 40% between laboratories due to the use of different hematology instruments (1,4,8,20) suggest that not all instruments measure lymphocytes equally. Furthermore, Cohen et al (5) reported that the Coulter STKS was less accurate than the Coulter S + IV in determining lymphocyte and granulocyte counts in HIV patients and that the discrepancy increased with increasing red blood cell mean corpuscular volume.…”

Section: Discussionmentioning

confidence: 99%

“…One tube from each donor was retained at Ortho Diagnostic Systems, Inc., stored overnight at room temperature, then processed and run at approximately the same time as the other study sites. Throughout this paper, study sites are referred to numerically, as sites [1][2][3][4]. Fifty samples were shipped, and a complete set of all requested analyses was received for 41 samples.…”

Section: Sam P I E Smentioning

confidence: 99%

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Standardization of absolute CD4⁺ lymphocyte counts across laboratories: An evaluation of the ortho CytoronAbsolute flow cytometry system on normal donors

et al. 1995

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The Ortho CytoronAbsolute is a flow cytometer designed to provide direct absolute counts of lymphocytes and their subsets from a single instrument. This study was designed to determine the performance of four geographically separated CytoronAbsolute instruments using 24-h-old, shipped, whole blood samples and to compare the results obtained on the CytoronAbsolute to those obtained using combinations of hematology instruments and other flow cytometers. The absolute count feature of the CytoronAbsolutes located at the four sites were cross calibrated and gave across-site coefficients of variation (CVs) of <4.0% for absolute count and 8.2% for absolute lymphocyte count. The calibration was stable for at least 2 months. Absolute lymphocyte counts and lymphocyte percentage immunophenotypes were determined on blood from 50 healthy human immunodeficiency virus (HIVI-seronegative donors. There were no significant site-to-site differences (each P > .05) in CD3+/CD4+ absolute lymphocyte counts determined on the CytoronAbsolute. In contrast, there was a significant site-to-site difference ( P < .001) between sites 2 and 3 and sites 3 and 4 in the absolute CD3+/CD4+ lymphocyte counts determined via the conventional method of combining a flow cytometry-derived percentage with a hematology instrument-derived lymphocyte count. There was no significant difference (P = .388) in CD3+/CD4+ lymphocyte percent determinations between the CytoronAbsolute and the FACScan or Profile II flow cytometers used in this study. These results demonstrate that different operators can cross calibrate CytoronAbsolutes for absolute CD3+/CD4+ lymphocyte subset determinations, even over large geographic distances. o 1995 Wiley-Liss, Inc.

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Analysis of variation in results of flow cytometric lymphocyte immunophenotyping in a multicenter study

et al. 1997

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Fifty‐five laboratories participated in a send‐out study of four peripheral blood samples comparing a standard protocol vs. local protocols for flow cytometric lymphocyte immunophenotyping. The standard protocol included centrally provided reagents, instrument setup using triple‐fluorescent microbeads and a three‐color, whole‐blood immunostaining technique based on fluorescein isothiocyanate and phycoerythrin‐labeled monoclonal antibodies, erythrocyte lysis, washing, fixation, and identification of nucleated cells by the DNA/RNA stain LDS‐751. Data analysis guidelines included lymphocyte selection using CD45,CD14‐assisted “backgating” on forward (FSC) and sideward (SSC) light scatter and placement of fluorescence (FL) markers on the basis of the isotype control staining. Most (i.e., 77%) of the variation in results of percentage lymphocyte subset assessments using the standard protocol was explained by laboratory, sample, background FL, and the interaction between laboratory and sample. Purity and completeness of the FSC,SSC lymphogate, background FL, flow cytometer type, and flow cytometer setup (which were either partly or entirely determined by laboratory) contributed significantly to the variation. The effect of the leukocyte differential count on the variation in absolute numbers of lymphocyte subsets was particularly large in lymphopenic samples. The use of this standard protocol vs. local protocols did not reduce the interlaboratory variation. Instrument incompatibility with the standard protocol (e.g., incompatible filter combinations for LDS‐751 detection) and lack of experience of many participants with three‐color flow cytometry (in particular with the use of LDS‐751) may have contributed to that result. We suggest that training and experience in a universally applicable standard protocol are critical for minimization of interlaboratory variation in flow cytometric immunophenotyping. Cytometry 30:166–177, 1997. © 1997 Wiley‐Liss, Inc.

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Consistency of routine measurements of CD4+, CD8+ peripheral blood lymphocytes

Cited by 26 publications

References 11 publications

Multisite Comparison of CD4 and CD8 T-Lymphocyte Counting by Single- versus Multiple-Platform Methodologies: Evaluation of Beckman Coulter Flow-Count Fluorospheres and the tetraONE System

Multisite Comparison of CD4 and CD8 T-Lymphocyte Counting by Single- versus Multiple-Platform Methodologies: Evaluation of Beckman Coulter Flow-Count Fluorospheres and the tetraONE System

Standardization of absolute CD4⁺ lymphocyte counts across laboratories: An evaluation of the ortho CytoronAbsolute flow cytometry system on normal donors

Analysis of variation in results of flow cytometric lymphocyte immunophenotyping in a multicenter study

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Consistency of routine measurements of CD4+, CD8+ peripheral blood lymphocytes

Cited by 26 publications

References 11 publications

Multisite Comparison of CD4 and CD8 T-Lymphocyte Counting by Single- versus Multiple-Platform Methodologies: Evaluation of Beckman Coulter Flow-Count Fluorospheres and the tetraONE System

Multisite Comparison of CD4 and CD8 T-Lymphocyte Counting by Single- versus Multiple-Platform Methodologies: Evaluation of Beckman Coulter Flow-Count Fluorospheres and the tetraONE System

Standardization of absolute CD4+ lymphocyte counts across laboratories: An evaluation of the ortho CytoronAbsolute flow cytometry system on normal donors

Analysis of variation in results of flow cytometric lymphocyte immunophenotyping in a multicenter study

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Standardization of absolute CD4⁺ lymphocyte counts across laboratories: An evaluation of the ortho CytoronAbsolute flow cytometry system on normal donors