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. (C) 2000 Wiley-Liss, Inc.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/1097-0320%2820001215%2942%3A6%3C327%3A%3AAID-CYTO1000%3E3.0.CO%3B2-F

Cytofluorometric methods for assessing absolute numbers of cell subsets in blood

Volumetric capillary cytometry (VCC) is a new technology that involves the detection and enumeration of dually fluorochrome-labeled cells in a precise volume. We compared the accuracy and precision of VCC with the accuracy and precision of flow cytometry and hematology (F&H) for the measurement of the absolute numbers of CD4 and CD8 T cells in the whole blood of patients infected with human immunodeficiency virus. Five laboratories, each with a different F&H system and certified by the National institute of Allergy and Infectious Diseases flow cytometry proficiency testing program, were shipped aliquots of the same samples from a central site in addition to procuring samples locally. In general, the VCC technology generated CD4 and CD8 T-cell counts which were lower than those obtained with F&H. Intralaboratory variability of replicate CD4 T-cell determinations was similar for both technologies except in the local samples with CD4 counts less than 200/microliters, where the VCC variability was higher than the F&H variability. Interlaboratory variability on replicate CD4 T-cell counts made by VCC was significantly less than that when counts were made by F&H. The VCC instrument has automated CD4 and CD8 T-cell enumeration in whole blood and has consolidated the process to a single platform. Its performance in this evaluation indicates that it may represent a viable alternative to F&H for obtaining absolute T-cell subset counts.

Inter- and intrainstitutional evaluation of automated volumetric capillary cytometry for the quantitation of CD4- and CD8-positive T lymphocytes in the peripheral blood of persons infected with human immunodeficiency virus. Site Investigators and the NIAID New CD4 Technologies Focus Group.

Mathematical model for the effect of reverse transcriptase inhibitor on the dynamics of HIV-1 infection model with three delays is proposed and analyzed. We begin this model with proving the positivity and boundedness of the solution. We analyze the stability of the viral free steady state and the infected steady state of the system. Also we have carried out bifurcation analysis along with an estimated length of delay to preserve the stability behaviour. By developing a few Lyapunov functionals, we obtain conditions ensuring global stability of steady states. Numerical simulations are carried out to explain the mathematical conclusions.

Global stability analysis of HIV-1 infection model with three time delays

Volumetric capillary cytometry (VCC) is a new technology that involves the detection and enumeration of duallyfluorochrome-labeled cells in a precise volume. We compared the accuracy and precision of VCC with the accuracy and precision of flow cytometry and hematology (F&H) for the measurement of the absolute numbers of CD4 and CD8 T cells in the whole blood of patients infected with human immunodeficiency virus. Five laboratories, each with a different F&H system and certified by the National Institute of Allergy and Infectious Diseases flow cytometry proficiency testing program, were shipped aliquots of the same samples fromacentralsiteinadditiontoprocuringsampleslocally.Ingeneral,theVCCtechnologygeneratedCD4and CD8T-cellcountswhichwerelowerthanthoseobtainedwithF&H.IntralaboratoryvariabilityofreplicateCD4 T-cell determinations was similar for both technologies except in the local samples with CD4 counts less than 200/ml, where the VCC variability was higher than the F&H variability. Interlaboratory variability on replicate CD4 T-cell counts made by VCC was significantly less than that when counts were made by F&H. The VCC instrument has automated CD4 and CD8 T-cell enumeration in whole blood and has consolidated the process to a single platform. Its performance in this evaluation indicates that it may represent a viable alternative to F&H for obtaining absolute T-cell subset counts.

Inter- and Intrainstitutional Evaluation of Automated Volumetric Capillary Cytometry for the Quantitation of CD4- and CD8-Positive T Lymphocytes in the Peripheral Blood of Persons Infected with Human Immunodeficiency Virus

A CD4 monoclonal antibody which reacts with CD4+ lymphocytes but does not react significantly with monocytes was generated and used to develop a simple method for CD4 count. In a comparison with standard flow cytometry, high correlation was obtained. The developed method can be an alternative to flow cytometry for resource-limited countries.

A Simple Manual Rosetting Method for Absolute CD4+ Lymphocyte Counting in Resource-Limited Countries

The purpose of this study was to evaluate a new alternative fluorescence immunoassay method (Zymmune CD4/CD8 Cell Monitoring Kit; Zynaxis, Inc., Malvern, Pa.) for determining the absolute CD4+ and CD8+ T-lymphocyte concentrations in whole blood. The investigation was performed as a two-site comparison of the reference whole blood flow cytometric method with the Zymmune method. In this investigation, a total of 166 patient samples were evaluated of which approximately 20% were from human immunodeficiency virus-positive individuals. The mean value for samples performed by the Zymmune CD4 assay was 1,094 (range, 74 to 2,586) cells per microliters, while the reference method yielded a mean of 890 (range, 35 to 2,033) cells per microliter. The correlation coefficient for regression analysis was 0.940. The mean value for samples performed by the Zymmune CD8 assay was 700 (range, 212 to 1,813) cells per microliter, while the reference method yielded a mean of 546 (range, 82 to 2,158) cells per microliter. The correlation coefficient for regression analysis was 0.921. No site-specific differences or trends in CD4 or CD8 values were seen when the data were analyzed by site of collection. The average precision of the CD4 assay varied from 6 to 14%, corresponding to the high and low concentration ranges. For CD8, the average precision varied from 8.3 to 16% over the respective high to low concentration ranges. We conclude that the Zymmune CD4/CD8 Cell Monitoring Kit method provides absolute CD4+ and CD8+ T-lymphocyte concentrations which are equivalent to those given by the reference flow cytometric method.

Determination of CD4 and CD8 lymphocyte subsets by a new alternative fluorescence immunoassay.

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Determination of CD4 and CD8 lymphocyte subsets by a new alternative fluorescence immunoassay.