1997 Revised Guidelines for Performing CD4+ T-Cell Determinations in Persons Infected with Human Immunodeficiency Virus (HIV)

Accurate and reliable measures of CD4+ T-lymphocytes (CD4+ T-cells) are essential to the assessment of the immune system of human immunodeficiency virus (HIV)-infected persons (1-3). The pathogenesis of acquired immunodeficiency syndrome (AIDS) is largely attributable to the decrease in T-lymphocytes that bear the CD4 receptor (4-8). Progressive depletion of CD4+ T-lymphocytes is associated with an increased likelihood of clinical complications (9,10). Consequently, the Public Health Service (PHS) has recommended that CD4+ T-lymphocyte levels be monitored every 3-6 months in all HIV-infected persons (11). The measurement of CD4+ T-cell levels has been used to establish decision points for initiating Pneumocystis carinii pneumonia prophylaxis (12) and antiviral therapy (13) and to monitor the efficacy of treatment (14-16). CD4+ T-lymphocyte levels also are used as prognostic indicators in patients who have HIV disease (17, 18) and recently have been included as one of the criteria for initiating prophylaxis for several opportunistic infections that are sequelae of HIV infection (19, 20). Moreover, CD4+ T-lymphocyte levels are a criterion for categorizing HIV-related clinical conditions by CDC's classification system for HIV infection and surveillance case definition for AIDS among adults and adolescents (21).

Most laboratories measure absolute CD4+ T-cell levels in whole blood by a multi-platform, three-stage process. The CD4+ T-cell number is the product of three laboratory techniques: the white blood cell (WBC) count; the percentage of WBCs that are lymphocytes (differential); and the percentage of lymphocytes that are CD4+ T-cells. The last stage in the process of measuring the percentage of CD4+ T-lymphocytes in the whole-blood sample is referred to as "immunophenotyping by flow cytometry" (22-28). Immunophenotyping refers to the detection of antigenic determinants (which are unique to particular cell types) on the surface of WBCs using antigen-specific monoclonal antibodies that have been labeled with a fluorescent dye or fluorochrome (e.g., phycoerythrin [PE] or fluorescein isothiocyanate [FITC]). The fluorochrome-labeled cells are analyzed by using a flow cytometer, which categorizes individual cells according to size, granularity, fluorochrome, and intensity of fluorescence. Size and granularity, detected by light scattering, characterize the types of WBCs (i.e., granulocytes, monocytes, and lymphocytes). Fluorochrome-labeled antibodies distinguish populations and subpopulations of WBCs. Although flow cytometric immunophenotyping is a highly complex technology, methodology for performing CD4+ T-cell determinations has become more standardized between laboratories. The publication of several sets of guidelines addressing aspects of the CD4+ T-lymphocyte testing process (e.g., quality control, quality assurance, and reagents for flow cytometric immunophenotyping of lymphocytes) has contributed to this standardization (29-32).

The CDC guidelines concerning CD4+ T-cell determinations (33) were first published in the MMWR in 1992 to provide laboratorians with the most complete information about how to measure CD4+ T-lymphocytes in blood from HIV-infected persons by using flow cytometry. These guidelines were based on data from scientific literature, information from discussions with technical experts, and experience with related voluntary standards for flow cytometric analyses (29). The 1992 guidelines concluded that more data were needed and that revisions would be published as additional information became available and as important innovations in technology were made. In 1993, a national conference was convened by CDC with sponsorship from the Food and Drug Administration (FDA), National Institutes of Health, and Association of State and Territorial Public Health Laboratory Directors. The objectives of the conference were to review data collected after 1992 and to obtain input about the efficacy of the 1992 guidelines. As a result of the 1993 conference, the revised guidelines for performing CD4+ T-cell determinations in HIV-infected persons were published in 1994 (34).

Since 1994, the field of CD4+ T-cell testing has rapidly expanded. Flow cytometric analyses of T-cell subsets using three- and four-color approaches (in contrast to the two-color approach addressed in previous reports [35,36]), flow cytometric analyses for measuring both the proportion and the absolute numbers of CD4+ T-lymphocytes, and other methods for deriving an absolute CD4+ T-cell count in a blood sample are now commercially available. (Some of these other methods do not depend on the multi-stage process and are collectively referred to in this report as single-platform methods.) Moreover, data evaluating some of the parameters of two-color flow cytometric testing and the routine testing practices of laboratories that provide these testing services have been collected. A second national conference on CD4+ T-lymphocyte immunophenotyping was held in Atlanta, Georgia, on December 12-13, 1995, to discuss these changes. Information shared at the conference and new data collected about laboratory testing practices serve as the basis for the revisions and additions that have been made to the 1994 guidelines. These changes include a) quality assurance (namely, revision of the recommended monoclonal panel to provide a cost-effective solution for laboratories using three-color and four-color approaches), b) the importance of following manufacturers' instructions when using tests and testing devices approved by the FDA, c) recommendations for laboratories performing three- and four-color T-lymphocyte immunophenotyping (TLI), and d) recommendations about the validation and verification procedures that laboratories should conduct before implementing new tests.