The enumeration of absolute levels of cells and their subsets in clinical samples is of primary importance in human immunodeficiency virus (HIV)؉ individuals (CD4؉ T-lymphocyte enumeration), in patients who are candidates for autotransplantation (CD34؉ hematopoietic progenitor cells), and in evaluating leukoreduced blood products (residual white blood cells). These measurements share a number of technical options, namely, single-or multiple-color cell staining and logical gating strategies. These can be accomplished using single-or dual-platform counting technologies employing cytometric methods. Dual-platform counting technologies couple the percentage of positive cell subsets obtained by cytometry and the absolute cell count obtained by automated hematology analyzers to derive the absolute value of such subsets. Despite having many conceptual and technical limitations, this approach is traditionally considered as the reference method for absolute cell count enumeration. As a result, the development of single-platform technologies has recently attracted attention with several different technical approaches now being readily available. These single-platform approaches have less sources of variability. A number of reports clearly demonstrate that they provide better coefficients of variation (CVs) in multicenter studies and a lower chance to generate aberrant results. These methods are therefore candidates for the new gold standard for absolute cell assessments. The currently available technical options are discussed in this review together with the results of some cross-comparative studies. Each analytical system has its own specific requirements as far as the dispensing precision steps are concerned. The importance of precision reverse pipetting is emphasized. Issues still under development include the establishment of the critical error ranges, which are different in each test setting, and the applicability of simplified low-cost techniques to be used in countries with limited resources. Cytometry (Comm. Clin. Cytometry) 42:327-346, 2000.

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Background: HLA class I peptide tetramers represent powerful diagnostic tools for detection and monitoring of antigen-specific CD8 ؉ T cells. The impetus for the current multicenter study is the critical need to standardize tetramer flow cytometry if it is to be implemented as a routine diagnostic assay. Hence, the European Working Group on Clinical Cell Analysis set out to develop and evaluate a single-platform tetramer-based method that used cytomegalovirus (CMV) as the antigenic model. Methods: Absolute numbers of CMV-specific CD8 ؉ T cells were obtained by combining the percentage of tetramer-binding cells with the absolute CD8؉ T-cell count. Six send-outs of stabilized blood from healthy individuals or CMV-carrying donors with CMV-specific CD8 ؉ T-cell counts of 3 to 10 cells/l were distributed to 7 to 16 clinical sites. These sites were requested to enumerate CD8 ؉ T cells and, in the case of CMV-positive donors, CMV-specific subsets on three separate occasions using the standard method.

Clinical use of flow cytometric (FCM) DNA analysis requires effective quality controls. Thirty‐two laboratories with various degrees of FCM experience participated in the first phase of a quality control program organized by the Association Française de Cytométrie. All received diskettes containing ten list‐mode files and ten histogram files that were derived from FCM analysis of various unfixed tumor specimens. A total of 610 responses on DNA ploidy and cell cycle were obtained with three different DNA analysis softwares: CellFit used by (44% of responses), MultiCycle (44%), and ModFit (12%). After statistical analysis, 31% of the responses were excluded from the final analysis for precise reasons. The groups were too small to carry out a valid analysis of the slight differences in the percentage of cells in the DNA synthesis phase (S%) between CellFit and MultiCycle. To estimate the influence of gating on the final cell‐cycle results, five of the histogram files were derived from corresponding list‐mode files, but the participating laboratories were unaware of this. A good correlation (r = 0.98) was obtained for S% values in the five paired files. The fact that 31% of the responses had to be excluded clearly reflects inadequate training in the use of these analysis softwares and, in some cases, a failure to grasp the biological meaning of the results. In contrast, the laboratories fulfilling consensus recommendations obtained remarkably homogeneous results, showing that standardization is feasible. © 1996 Wiley‐Liss, Inc.

Immunological analysis for cell antigens has been performed by flow cytometry in a qualitative fashion for over thirty years. During that time it has become increasingly apparent that quantitative measurements such as number of antigens per cell provide unique and useful information. This unit on quantitative flow cytometry (QFCM) describes the most commonly used protocols, both direct and indirect, and the major methods of analysis for the number of antibody binding sites on a cell or particle. Practical applications include detection of antigen under‐ or overexpression in hematological malignancies, distinguishing between B cell lymphoproliferative disorders, and precise diagnosis of certain rare diseases.

In 2000, the Belgian Scientific Institute of Public Health introduced a voluntary external quality assessment scheme for lymphocyte immunophenotyping. This paper provides an analysis of the first six surveys. Specimens consisted of fresh EDTA-anticoagulated whole blood and were sent by overnight mail. The 41 participants were surveyed for methodology and were asked to report white blood cell count, percentage of lymphocytes, and percentages and absolute numbers of CD3+, CD4+, CD8+, and CD19+ cells. Median intralaboratory coefficients of variation were 1.0, 1.3, 1.7, and 3.2% for CD3+, CD4+, CD8+, and CD19+ cell percentages, respectively. Interlaboratory variability was consistently lower than 6.5% for CD3+ and CD4+CD3+ cell percentages, and lower than 9.5% for CD8+CD3+ cell percentages. Median coefficients of variation for the absolute values were higher, ranging from 10.1% for CD4+CD3+ cells to 16.5% for CD19+ cells. The percentage of CD4+CD3+ and CD8+ CD3+ cells was in several samples significantly lower than the percentage of total CD4+ and CD8+ cells. The number of laboratories measuring total CD4+ and CD8+ cells decreased by 30% during the programme. Between-laboratory variability remained stable over time. Analysis of individual laboratory performance indicated that some laboratories markedly improved their results.

Clinical use of flow cytometric (FCM) DNA analysis requires effective quality controls. Thirty-two laboratories with various degrees of FCM experience participated in the first phase of a quality control program organized by the Association Française de Cytométrie. All received diskettes containing ten list-mode files and ten histogram files that were derived from FCM analysis of various unfixed tumor specimens. A total of 610 responses on DNA ploidy and cell cycle were obtained with three different DNA analysis softwares: CellFit used by (44% of responses), MultiCycle (44%), and ModFit (12%). After statistical analysis, 31% of the responses were excluded from the final analysis for precise reasons. The groups were too small to carry out a valid analysis of the slight differences in the percentage of cells in the DNA synthesis phase (S%) between CellFit and MultiCycle. To estimate the influence of gating on the final cell-cycle results, five of the histogram files were derived from corresponding list-mode files, but the participating laboratories were unaware of this. A good correlation (r = 0.98) was obtained for S% values in the five paired files. The fact that 31% of the responses had to be excluded clearly reflects inadequate training in the use of these analysis softwares and, in some cases, a failure to grasp the biological meaning of the results. In contrast, the laboratories fulfilling consensus recommendations obtained remarkably homogeneous results, showing that standardization is feasible.