Human T-lymphotropic virus type III (HTLV-III) or lymphadenopathy-associated virus (LAV) is tropic for human T cells with the helper-inducer phenotype, as defined by reactivity with monoclonal antibodies specific for the T4 molecule. Treatment of T4+ T cells with monoclonal antibodies to T4 antigen blocks HTLV-III/LAV binding, syncytia formation, and infectivity. Thus, it has been inferred that the T4 molecule itself is a virus receptor. In the present studies, the surfaces of T4+ T cells were labeled radioactively, and then the cells were exposed to virus. After the cells were lysed, HTLV-III/LAV antibodies were found to precipitate a surface protein with a molecular weight of 58,000 (58K). By blocking and absorption experiments, this 58K protein was identified as the T4 molecule. No cell-surface structures other than the T4 molecule were involved in the antibody-antigen complex formation. Two monoclonal antibodies, each reactive with a separate epitope of the T4 molecule, were tested for their binding capacities in the presence of HTLV-III/LAV. When HTLV-III/LAV was bound to T4+ T cells, the virus blocked the binding of one of the monoclonal antibodies, T4A (OKT4A), but not of the other, T4 (OKT4). When HTLV-III/LAV was internally radiolabeled and bound to T4+ T cells which were then lysed, a viral glycoprotein of 110K (gp110) coprecipitated with the T4 molecule. The binding of gp110 to the T4 molecule may thus be a major factor in HTLV-III/LAV tropism and may prove useful in developing therapeutic or preventive measures for the acquired immune deficiency syndrome.

A single-platform technology that uses an internal bead standard and three-color flow cytometry to determine CD4 and CD8 absolute counts was evaluated for reproducibility and agreement. Values obtained using TruCount absolute-count tubes were compared to those obtained using a two-color predicate methodology. Sixty specimens from human immunodeficiency virus type 1-infected donors were shipped to five laboratories. Each site also analyzed replicates of 14 human immunodeficiency virus type 1-infected local specimens at 6 h and again at 24 h. The interlaboratory variability was significantly less with TruCount (median difference in percent coefficient of variation [%CV] between the two methods was ؊8% and ؊3% for CD4 and CD8, respectively) than with the predicate method. Intralaboratory variability was smaller, with a median difference in %CV of ؊1% for both CD4 and CD8 with 6-h samples and ؊2% and ؊3% for CD4 and CD8, respectively, with 24-h samples. Use of TruCount for shipped samples resulted in a median CD4 count change of 7 cells (50th estimated percentile) when all laboratories and CD4 strata were combined. For on-site samples, the median CD4 count change was 10 CD4 cells for 6-h samples and 2 CD4 cells for 24-h samples. Individual site biases occurred in both directions and cancelled each other when the data were combined for all laboratories. Thus, the combined data showed a smaller change in median CD4 count than what may have occurred at an individual site. In summary, the use of TruCount decreased both the inter-and intralaboratory variability in determining absolute CD4 and CD8 counts.

Since the publication of the "Three-color supplement to the NIAID/DAIDS Guideline for Flow Cytometric Immunophenotyping" in 1996 (1), significant scientific and technological advances in the development and production of reagents, instrumentation, and software have increased the use of multicolor flow cytometry in both research and clinical laboratories. With the increased adoption of three and four-color flow cytometry as the preferred methodology in determining patients' CD4 and CD8 T-cell counts, it has become apparent that a gating strategy that integrates the bright CD45 cells reduces interlaboratory and intralaboratory variability. Traditionally, a lymphocyte gate for immunophenotyping is derived from a bivariate frequency distribution histogram that includes 90°side scatter (SSC) and forward light scatter (FSC) frequency patterns. This type of histogram configuration is called a homogenous gating protocol (2). The advantage of the combination of bright CD45 fluorescence and light scatter, a heterogeneous gating protocol, was first reported in 1993 (3). Over the past few years, it has been determined that this alternative approach provides a more reproducible and accurate lymphocyte gate (4 -6). This heterogeneous method will be referred to as the CD45 gating method.The purpose of this article is to update the 1996 NIAID/ DAIDS Guideline and include the use of the CD45 gating method to minimize measurement variability with multicolor flow cytometry for the enumeration of T-cell subsets. Some information is provided about the advantages of four-color flow cytometry and the use of single-platform bead-based technology for determining absolute and percentage of lymphocytes. The addition of integrated fluorosphere counting provides a single-platform protocol that facilitates the simultaneous determination of both absolute and percentage of lymphocyte subsets. The specifications and recommendations were developed for use in laboratories supporting clinical trials and epidemiolog-

Health and Welfare Canada, Ottawa, Ontario (F.M.); and Division of AIDS, NIAID, NIH, Bethesda, Maryland (D.L., J.K.)'Section nurnberingsystern isdesigned to maintain continuity with the 1993 "NIAID DAIDS Guidelinesfor Flow Cytornetric Immunotyping" (1).

Idiopathic CD4+ T-lymphocytopenia differs from HIV infection in its immunologic characteristics and in its apparent lack of progression over time. Nothing about the immunologic or viral-culture studies performed in these patients or about their family members or blood donors suggests that a transmissible agent causes this condition.