Lymphocyte Subset Enumeration for Immunodeficiency Disease
In the early 1980’s, the principal discovery of the pathogenesis of hu- man 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 cy- tometry, and the measurement of HIV RNA levels by molecular tech- niques 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 immunophe- notypic analysis for lymphocyte subset enumeration, with a CD45- side scatter gate to identify the lymphocyte population and to elimi- nate 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 deter- mined 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 introduc- tion 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 measure- ment of the frequency of antigen-specific immune responses, func- tional assays for antigen-specific cytokine responses, measurement of cell turnover, programmed cell death, and HIV viral burden.22
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 pres- ent in childhood as chronic or recurrent infection, failure to thrive, unusual infections, allergic disorders23, 24 and leukocyte adhesion dis- orders, and is commonly used for the diagnosis and management of these diseases.25