Comparison of the staining of peripheral blood T lymphocytes by various anti-CD8 and HLA-DR monoclonal antibodies.
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.
Kootte
A M
AM
Department of Medicine, University Hospital, Leiden, The Netherlands.
Langelaar
R A
RA
Tanke
H J
HJ
Van der Voort
E A
EA
Paul
L C
LC
D000328:Adult; D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis*; D016827:CD8 Antigens; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006681:HLA-D Antigens / Q000032:analysis*; D006684:HLA-DR Antigens / Q000032:analysis*; D006801:Humans; D007202:Indicators and Reagents; D016030:Kidney Transplantation; D007958:Leukocyte Count; D008297:Male; D010641:Phenotype; D010799:Phycoerythrin; D013194:Staining and Labeling / Q000379:methods*; D013601:T-Lymphocytes / Q000032:analysis / Q000145:classification*; D013861:Thiocyanates
D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D016827:CD8 Antigens; D005452:Fluoresceins; D006681:HLA-D Antigens; D006684:HLA-DR Antigens; D007202:Indicators and Reagents; D013861:Thiocyanates; D010799:Phycoerythrin; D016650:Fluorescein-5-isothiocyanate