The goals of this study were to optimize processing methods of cryopreserved peripheral blood mononuclear cells (PBMC) for immunological assays, identify acceptance parameters for the use of cryopreserved PBMC for functional and phenotypic assays, and to define limitations of the information obtainable with cryopreserved PBMC. Blood samples from 104 volunteers (49 human immunodeficiency virus-infected and 55 uninfected) were used to assess lymphocyte proliferation in response to tetanus, candida, and pokeweed-mitogen stimulation and to enumerate CD4 ؉ and CD8 ؉ T cells and T-cell subpopulations by flow cytometry. We determined that slowly diluting the thawed PBMC significantly improved viable cell recovery, whereas the use of benzonase improved cell recovery only sometimes. Cell storage in liquid nitrogen for up to 15 months did not affect cell viability, recovery, or the results of lymphocyte proliferation assays (LPA) and flow cytometry assays. Storage at ؊70°C for <3 weeks versus storage in liquid nitrogen before shipment on dry ice did not affect cell viability, recovery, or flow cytometric results. Storage at ؊70°C was associated with slightly higher LPA results with pokeweed-mitogen but not with microbial antigens. Cell viability of 75% was the acceptance parameter for LPA. No other acceptance parameters were found for LPA or flow cytometry assay results for cryopreserved PBMC. Under optimized conditions, LPA and flow cytometry assay results for cryopreserved and fresh PBMC were highly correlated, with the exception of phenotypic assays that used CD45RO or CD62L markers, which seemed labile to freezing and thawing.
New analytic methods that permit absolute CD4 and CD8 T-cell determinations to be performed entirely on the flow cytometer have the potential for improving assay precision and accuracy. In a multisite trial, we compared two different single-platform assay methods with a predicate two-color assay in which the absolute lymphocyte count was derived by conventional hematology. A two-color method employing lymphocyte light scatter gating and Beckman Coulter Flow-Count fluorospheres for absolute counting produced within-laboratory precision equivalent to that of the two-color predicate method, as measured by coefficient of variation of replicate measurements. The fully automated Beckman Coulter tetraONE System four-color assay employing CD45 lymphocyte gating, automated analysis, and absolute counting by fluorospheres resulted in a small but significant improvement in the within-laboratory precision of CD4 and CD8 cell counts and percentages suggesting that the CD45 lymphocyte gating and automated analysis might have contributed to the improved performance. Both the two-color method employing Flow-Count fluorospheres and the four-color tetraONE System provided significant and substantial improvements in between-laboratory precision of absolute counts. In some laboratories, absolute counts obtained by the single-platform methods showed small but consistent differences relative to the predicate method. Comparison of each laboratory's absolute counts with the five-laboratory median value suggested that these differences resulted from a bias in the absolute lymphocyte count obtained from the hematology instrument in some laboratories. These results demonstrate the potential for single-platform assay methods to improve within-laboratory and between-laboratory precision of CD4 and CD8 T-cell determinations compared with conventional assay methods.
Human immunodeficiency virus type 1 (HIV-1) infection results in impaired immune function that can be measured by changes in immunophenotypically defined lymphocyte subsets and other in vitro functional assays. These in vitro assays may also serve as early indicators of efficacy when new therapeutic strategies for HIV-1 infection are being evaluated. However, the use of in vitro assays of immune function in multicenter clinical trials has been hindered by their need to be performed on fresh specimens. We assessed the feasibility of using cryopreserved peripheral blood mononuclear cells (PBMC) for lymphocyte immunophenotyping and for lymphocyte proliferation at nine laboratories. In HIV-1-infected patients with moderate CD4؉ lymphocyte loss, the procedures of density gradient isolation, cryopreservation, and thawing of PBMC resulted in significant loss of CD19 ؉ B cells but no measurable loss of total T cells or CD4 ؉ or CD8 ؉ T cells. No significant changes were seen in CD28 ؊ CD95 ؉ lymphocytes after cell isolation and cryopreservation. However, small decreases in HLA-DR ؉ CD38 ؉ lymphocytes and of CD45RA ؉ CD62L ؉ were observed within both the CD4 ؉ and CD8 ؉ subsets. Fewer than 10% of those specimens that showed positive PBMC proliferative responses to mitogens or microbial antigens lost their responsiveness after cryopreservation. These results support the feasibility of cryopreserving PBMC for immunophenotyping and functional testing in multicenter AIDS clinical trials. However, small changes in selected lymphocyte subsets that may occur after PBMC isolation and cryopreservation will need to be assessed and considered in the design of each clinical trial.Therapeutic approaches that reduce the rate of human immunodeficiency virus type 1 (HIV-1) replication result in fewer clinical events and prolong the survival of infected individuals (10,22). In addition to an increase in CD4 T cells, immune function as measured by in vitro assays also improves significantly in response to antiretroviral treatments. Following initiation of highly active antiretroviral therapy, functionally relevant lymphocyte subsets in blood begin to return to normal levels. In addition, in vitro proliferation and cytokine secretion by peripheral blood mononuclear cells (PBMC) also show improvement (4, 14, 16, 24). As a result, in vitro measurements of immune function are being explored as a substitute for clinical endpoints in therapeutic trials (21). Furthermore, quantitating immune function through the use of these assays will provide a better definition of the immune reconstitution that occurs during antiretroviral therapy and may advance our understanding of AIDS pathogenesis.The in vitro assays currently used to measure immune function are technically complex, prone to variability, and usually performed in real time on fresh specimens. The problems of performing these assays efficiently and reproducibly are compounded in multicenter clinical trials. In these clinical trials, specimens are collected over many months and at multiple l...
The Ortho CytoronAbsolute is a flow cytometer designed to provide direct absolute counts of lymphocytes and their subsets from a single instrument. This study was designed to determine the performance of four geographically separated CytoronAbsolute instruments using 24-h-old, shipped, whole blood samples and to compare the results obtained on the CytoronAbsolute to those obtained using combinations of hematology instruments and other flow cytometers. The absolute count feature of the CytoronAbsolutes located at the four sites were cross calibrated and gave across-site coefficients of variation (CVs) of <4.0% for absolute count and 8.2% for absolute lymphocyte count. The calibration was stable for at least 2 months. Absolute lymphocyte counts and lymphocyte percentage immunophenotypes were determined on blood from 50 healthy human immunodeficiency virus (HIVI-seronegative donors. There were no significant site-to-site differences (each P > .05) in CD3+/CD4+ absolute lymphocyte counts determined on the CytoronAbsolute. In contrast, there was a significant site-to-site difference ( P < .001) between sites 2 and 3 and sites 3 and 4 in the absolute CD3+/CD4+ lymphocyte counts determined via the conventional method of combining a flow cytometry-derived percentage with a hematology instrument-derived lymphocyte count. There was no significant difference (P = .388) in CD3+/CD4+ lymphocyte percent determinations between the CytoronAbsolute and the FACScan or Profile II flow cytometers used in this study. These results demonstrate that different operators can cross calibrate CytoronAbsolutes for absolute CD3+/CD4+ lymphocyte subset determinations, even over large geographic distances. o 1995 Wiley-Liss, Inc.
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-
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