NCCLS document H42-P. cells is an essential factor in the assessment of the immune systems of human immunodeficiency virus (HIV)-infected individuals. The pathogenic process of AIDS is primarily a result of the depletion of CD4+ T cells. Opportunistic infections of every kind and malignant processes develop as CD4 cell levels drop. Case definitions of HIV infection are dependent on CD4 counts as well as on thresholds for the initiation of prophylactic regimens and antiretroviral therapy. The U.S. Public Health Service recommends that CD4+ T-cell levels be monitored every 3 to 6 months in all HIV-infected persons (3, 4). This recommendation means that an accurate and reproducible CD4 count is a fundamental clinical tool for monitoring and treating HIV infection and its complications. The most widely used (and still prevalent) method for CD4 enumeration in the past has been dual- or multiplatform analysis. The total, or absolute, CD4 count is obtained from three clinical measurements, a white blood cell count, a lymphocyte percentage (differential), and CD4+ T-cell measurement using immunophenotyping by flow cytometry. The accuracy and reliability of all three measurements are dependent on the quality assurance procedures in place for the performance of clinical testing, the equipment used, the experience of the technical personnel performing the measurements, and the quality of the samples. In addition, all three measurements have a Z-VEID-FMK predictable range of variation. When all of these variables are considered and the three measurements are multiplied together, any inaccuracies or errors are compounded. Meetings between federal regulatory agencies, clinicians, and people working in the field of flow cytometry have resulted in guidelines that have been established and revised several times in the past 15 years to standardize CD4 testing procedures (8). Revisions have been published in response to new methods of testing and new technologies (2, 5). These steps resulted in widely improved performance of CD4 counts. Over the years, analysis has developed from single-color testing using peripheral blood mononuclear cells to multicolor testing using whole blood. Gating strategies have developed from forward-scatter (FSC) versus side-scatter (SSC) gating on lymphocytes to the use of the CD45 versus SSC gating for clear definition of lymphocyte populations. In 2003, the CDC released the most recent revision specifically to address the need to provide guidelines for the performance of single-platform absolute CD4+ T-cell determinations (5). Z-VEID-FMK In 2000, two multicenter studies were published documenting the superior results obtained for CD4 counts in interlaboratory comparisons (9, 10). These results were superior in terms of their reproducibility, or precision. Z-VEID-FMK There is no true gold standard for the assessment of accuracy in CD4 determinations. It is important to realize that the difference between single- and multiplatform testing is not one of right answers versus wrong answers but of standardized answers. High precision is possible in single-platform testing because the results depend on only one measurement performed on a flow cytometer. There can be biological variations within an individual and variations related to immunosuppressive therapy for individuals involved in long-term studies, EIF4G1 necessitating a need for accuracy and reproducibility within an assay. MATERIALS AND METHODS Immunophenotyping of peripheral blood drawn in EDTA was performed 4 h after blood was drawn from 25 HIV+ patients according to manufacturer’s instructions using a modification of CDC guidelines. The BD Trucount single-platform protocol (BD Trucount tubes; catalog no. 340334; BD Biosciences, San Jose, CA) and a standard, dual-platform, flow cytometry protocol were compared. Samples from all patients were stained by both methods. For the dual-platform protocol, whole-blood samples (100 l per tube) were stained with the recommended 20 l of antibody cocktail (Table ?(Table1)1) according to the manufacturer’s instructions using a modification of the CDC guidelines (2, 11). After staining, cells were lysed with Optilyse C (Beckman Coulter, Hialeah, FL), washed twice, and resuspended in 500 l of phosphate-buffered saline (Quality Biological, Inc.). The samples were analyzed immediately on a BD FACSCanto flow cytometer (Becton Dickinson, CA). TABLE 1. Four-color antibodies by manufacturer test was.