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Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay
Published on: September 16, 2012
Evaluation of a dual-color flow cytometry immunophenotyping panel in a multicenter quality assurance program
E L Schenker1, L E Hultin, K D Bauer
1Department of Medicine, UCLA School of Medicine 90024.
Insights
A basic immunophenotyping panel demonstrated consistent results across four laboratories in an AIDS study. This quality assurance program ensured reliable lymphocyte subset measurements for T cells, B cells, NK cells, and CD4+/CD8+ T cells.
Area of Science:
- Immunology
- Clinical Laboratory Science
- Flow Cytometry
Background:
- Accurate lymphocyte subset enumeration is crucial for monitoring immune status, particularly in conditions like HIV/AIDS.
- Multicenter studies require robust quality assurance to ensure inter-laboratory consistency in flow cytometry data.
- Standardized immunophenotyping panels are essential for reliable clinical trial results.
Purpose of the Study:
- To evaluate the inter-laboratory reproducibility of a basic immunophenotyping panel in a multicenter setting.
- To assess the reliability of lymphocyte subset percentage values (T cells, B cells, NK cells, CD4+, CD8+) across participating laboratories.
- To establish a quality assurance program for flow cytometry in an AIDS clinical study.
Main Methods:
- A 6-tube basic immunophenotyping panel using dual-color fluorescein isothiocyanate (FITC) and phycoerythrin (PE) conjugated monoclonal antibodies was employed.
- Four laboratories analyzed 78 peripheral blood specimens over 1.5 years, including HIV-seronegative and HIV-seropositive samples.
- Lymphocyte subset values were corrected using the CD45bright CD14- percentage, and inter-laboratory coefficients of variation (CV) were calculated.
Main Results:
- The panel achieved low inter-laboratory CVs: <3% for total T cells, <5% for CD4+ and CD8+ T cells, ≤17% for B and NK cells, and <8% for CD4T/CD8T ratios.
- The quality assurance program demonstrated high consistency in measuring major lymphocyte subsets.
- A 'lymphosum' quality control check (T+B+NK sum) generally remained within 100 +/- 5% for HIV-seronegative specimens.
Conclusions:
- The basic 6-tube immunophenotyping panel is suitable for comprehensive lymphocyte subset evaluation in clinical studies.
- The panel effectively detects inter-laboratory variations in flow cytometry measurements, serving as a valuable tool for proficiency testing.
- This approach provides robust within-sample quality assurance, ensuring data reliability for multicenter research.
Abstract:
A basic immunophenotyping panel that employed dual-color combinations of fluorescein isothiocyanate (FITC) and phycoerythrin (PE) conjugated monoclonal antibodies (mAb; FITC-CD45/PE-CD14, FITC-IgG1/PE-IgG2, FITC-CD3/PE-CD8, FITC-CD3/PE-CD4, FITC-CD3/PE-CD16 + PE-CD56, and PE-CD19) was utilized in a quality assurance program to determine whether the 4 laboratories participating in a multicenter AIDS study obtained similar lymphocyte subset percentage values for T cells, B cells, NK cells, and CD4+ and CD8+ T cells. Over a 1 1/2 year period, 78 shared peripheral blood specimens were prepared and analyzed in each laboratory. The CD45bright CD14- percentage for each specimen was used to correct that individual's lymphocyte subset values. Interlaboratory coefficients of variation (CV) for the human immunodeficiency virus type I (HIV) seronegative (n = 38) and HIV-seropositive (n = 40) specimens using this panel were < 3% for total T cells; < 5% for CD4+ T cells and CD8+ T cells; < or = 17% for B and NK cells; and < 8% for CD4T/CD8T ratios. The 6-tube basic immunophenotyping panel has several notable features: a) for clinical studies, it permits comprehensive evaluation of an individual's major lymphocyte subsets, i.e., T, B, NK, and CD4+ and CD8+ T cells; b) for interlaboratory proficiency testing programs, it allows the detection of differences among laboratories in measurements of several functionally distinct cell populations; and c) for within-sample quality assurance, it provides several quality control checks, including the lymphosum, i.e., the sum of an individual's corrected T+B+NK values, a sum that was generally 100 +/- 5% on the HIV-seronegative specimens analyzed in this study.
