A simultaneous three-color T cell subsets analysis with single laser flow cytometers using T cell gating protocol comparison with conventional two-color immunophenotyping method

Abstract

We describe a method for simultaneous analysis of CD3, CD4, and CD8 positive cells from whole blood utilizing single laser flow cytometers. All three T cell values are attained from a single test tube. CD4 and CD8 positive cells are identified only if they are CD3 positive. Thus the values obtained by this method for T helper/inducer and T cytotoxic/suppressor cells can be reported directly as a percentage of T lymphocytes. Analysis for CD4 and CD8 positive cells is accomplished, by first gating on CD3 positive T lymphocytes, hence the approach is referred to as a T gating method. As the third dye, conjugated to anti-CD3 monoclonal antibodies (MAbs), we utilized peridinin chlorophyll protein (PerCP), a new red fluorochrome. The proposed method may prove to be practical for monitoring disease progression in AIDS, where longitudinal T helper/inducer and T cytotoxic/suppressor cell enumeration must be performed unambiguously by a simple, reproducible, and fast method.