Comparison of the staining of peripheral blood T lymphocytes by various anti-CD8 and HLA-DR monoclonal antibodies

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T cell subset analysis with monoclonal antibodies is performed in variety of clinical situations, including the monitoring of transplant recipients. We compared the percentages CD8+ cells in the peripheral blood of ten normal volunteers and 26 renal transplant patients using fluorescein isothiocyanate (FITC)- or phycoerytrin (PE)-conjugated Leu2a and Leu2b monoclonal antibodies (mAb). Furthermore, the percentage of HLA-DR+ cells within this population was determined using two anti-HLA-DR antibodies of differing isotype (designated 203 and 243) and either an FITC or a PE label. For both controls and transplant recipients, the PE-conjugated Leu2a and Leu2b mAbs gave significantly higher percentages of positive cells than the FITC-conjugated antibodies (P less than 0.01). Furthermore, the percentage of Leu2b+ cells was higher than the percentage of Leu2a+ cells, irrespective of the fluorochrome used (P less than 0.01); this was particularly true for samples with less than or equal to 10% Leu2a+ cells. Cell sorting experiments indicated that up to 30% of the Leu2a- population were able to bind OKT8 or Leu2b mAb in blood samples with a low percentage of Leu2a+ cells. The percentage of Leu2+ cells that were stained with anti-HLA-DR mAb 203 or 243 varied considerably, depending on the fluorochrome and the isotype of the antibodies. We conclude that the analysis of peripheral blood T cells with mAbs that apparently have the same specificity may give significantly different results, depending on the patient population, the fluorochrome and the isotype of the antibodies.

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