In the early 1980’s, the principal discovery of the pathogenesis of human immunodeficiency virus (HIV) infection was the recognition of an alteration in peripheral blood CD4 T-cell levels. Since that time, the enumeration of the absolute number of CD4+ T-cells by flow cytometry, and the measurement of HIV RNA levels by molecular techniques has proven critical for the diagnosis and prognostication of HIV infection and the management of patients receiving anti-viral therapy.
Presently, most laboratories utilize three- or four-color immunophenotypic analysis for lymphocyte subset enumeration, with a CD45-side scatter gate to identify the lymphocyte population and to eliminate dead cells, debris and degranulated granulocytes from analysis. Three-color analysis is usually performed with two labeled specimens (i.e., CD45-CD3-CD4 and CD45-CD3-CD8) while four-color analysis is performed with a single labeled specimen (i.e., CD45-CD3-CD4-CD8).
In the past, the absolute lymphocyte count was separately determined on the hematology analyzer and then used in conjunction with flow cytometric data to calculate absolute CD4+ and CD8+ counts. The accuracy of this analysis has been recently improved by introduction of methods that allow for direct measurement of absolute cell numbers with the flow cytometer only. Studies of HIV-infected patients in the research laboratory have led to a number of discoveries awaiting widespread clinical utilization. At present, the most important is quantitative measurement of immune activation by measurement of CD38 expression on CD8+ T-cells. Some studies indicate CD38 expression superior to measurement of the viral load by HIV RNA for predicting disease progression and survival in HIV-infected patients. Other flow cytometric assays under study for the management of HIV-infected patients include measurement of the frequency of antigen-specific immune responses, functional assays for antigen-specific cytokine responses, measurement of cell turnover, programmed cell death, and HIV viral burden.
Flow cytometric analysis was instrumental in the discovery of some primary (congenital) immunodeficiency diseases, a heterogeneous group of diseases of the host defense systems which commonly present in childhood as chronic or recurrent infection, failure to thrive, unusual infections, allergic disorders and leukocyte adhesion disorders, and is commonly used for the diagnosis and management of these diseases.(15.)
