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Background Automation in HIV clinical flow cytometry when appropriately applied brings considerable standardisation benefits. The Canadian Immunology Quality Assessment Program (CIQAP) detected situations where operators did not manually override automated software in the event of improper output on the Epics XL and FC500 CD4 immunophenotyping platforms. The automated gating algorithm identifies lymphocytes using a double gate strategy based on CD45 × side scatter (SS) gating and a light scatter FS × SS gate known to fail with sub optimal specimens. Method To generate correct interpretation and results CIQAP introduced a simple protocol modification, bypassing the light scatter gate to include all cells characterized by the CD45 gate. Seventeen problem cases were reanalysed for both absolute and relative T-cell subsets accuracy and compared to the CIQAP group mean values. Results were found to be associated with the percentage of lymphocytes excluded by the automated light scatter gate. Results The modified manual protocol resolved poor performance in 14 instances out of 17 problem cases. It was found to improve accuracy when the light scatter gate excluded greater than 5% of the cells. The remaining three cases had a lymphocyte recovery of greater than 94.6% in the original automated analysis. Conclusion There is a risk in relying solely on automated gating procedures when using the Epics XL and FC500 CD4 immunophenotyping platforms. Laboratory managers have the responsibility to intervene when required. EQA providers are equally responsible to alert the clinical laboratories of the need to update operator training to deal with stressed specimens. © 2016 International Clinical Cytometry Society.

Background The global HIV/AIDS pandemic and guidelines for initiating anti-retroviral therapy (ART) and opportunistic infection prophylaxis demand affordable, reliable, and accurate CD4 testing. A simple innovative approach applicable to existing technology that has been successfully applied in resource-challenged settings, PanLeukogated CD4 (PLG), could offer solutions for cost saving and improved precision. Methods Day-old whole blood from 99 HIV+ donors was simultaneously studied in five North-American laboratories to compare the performance of their predicate methods with the dual-platform PLG method. The predicate technology included varying 4-color CD45/CD3/CD4/CD8 protocols on different flow cytometers. Each laboratory also assayed eight replicate specimens of day-old blood from 10 to 14 local donors. Bias and precision of predicate and PLG methods was studied between- and within-participating laboratories. Results Significantly (P < 0.0001) improved between-laboratory precision/coefficient of variation (CV%) was noted using the PLG method (overall median 9.3% vs. predicate median CV 13.1%). Within-laboratory precision was also significantly (P < 0.0001) better overall using PLG (median 4.6% vs. predicate median CV 6.2%) and in 3 of the 5 laboratories. PLG counts tended to be 11% smaller than predicate methods (P < 0.0001) for shipped (median of predicate—PLG = 31) and local specimens (median of predicate—PLG = 23), both overall and in 4 of 5 laboratories (median decreases of 4, 16, 20, and 21% in shipped specimens); the other laboratory had a median increase of 5%. Conclusion Laboratories using predicate CD4 methods similar to those in this study could improve their between-laboratory and their within-laboratory precision, and reduce costs, by switching to the PLG method after adequate training, if a change (usually, a decrease) in CD4 counts is acceptable to their health systems. © 2008 Clinical Cytometry Society

Background The global struggle with human immunodeficiency virus (HIV) and the battle to develop affordable CD4 T-cell counting technology are both unfulfilled goals in 2008. The need for such instrumentation is more critical now as implementation of antiretroviral therapy (ART) is in progress in many resource limited regions. Major scaling-up efforts in rural situations are difficult to implement without laboratory infrastructure. CD4 T-cell counting is especially critical when trying to reach individuals with HIV to have them enrolled in ART as soon as they qualify for treatment based on CD4 count. Method This review covers both the chronological evolution and the scientific milestones of technological development of affordable immunophenotyping. It is more focused on flow cytometry but does consider the potential contribution by digital image cytometry. Results Thus far flow cytometry offered only modest progress toward affordable immunophenotyping. A list with desirable features is offered for side by side comparison. Digital image cytometry has yet to show its enormous affordable market potential. Conclusions It is possible to develop truly affordable, portable flow cytometry but it is not here yet. There are some hopeful signs as there are innovative and practical technical components appearing at regular intervals. However, so far the technical breakthroughs have been fragmented efforts without any attempts to consider intercorporate collaboration to optimize critical mass and synergy. The smaller players in the industry have made some progress toward meeting the monumental needs in Africa and Asia. Digital image cytometry may well be the ultimate winner in the affordable technology race. © 2008 Clinical Cytometry Society

Background: A new generation of bench-top flow cytometers with digital signal processing to perform suspension array technology (SAT) based bead array assays as well as leukocyte immunophenotyping is now available. These hybrid instruments provide an opportunity for the development of a more cost effective multitasking platform to support infectious disease treatment in resource limited countries. Methods: We report the development and testing of two modules compatible with the hybrid flow cytometers. The first module is an eleven HIV-1 protein bead array (PBA) for the detection of circulating antibodies and the second is a cell based T-cell enumeration assay. Results: The HIV-1 PBA was tested in parallel with two enzyme immunoassays (EIAs) for the detection of plasma antibodies from 4 HIV-1 seroconversion panels and a low antibody titer panel. The PBA as well as the two EIAs performed equally for the detection of antibody positive samples from all seroconversion panels. One antibody positive sample from the low antibody titer panel was missed by the PBA together with one of the two EIAs tested. A parallel analysis of the HIV-1 PBA with Western blot (a confirmatory test for HIV infection) using plasma from nine HIV-1+ individuals showed that the HIV-1 PBA detected more of the gp41 and gp120 antibody positive samples. Preliminary CD4 T-cell immunophenotyping results from 14 HIV+ and 10 HIV− whole blood specimens with the hybrid flow cytometer platform compared well to conventional flow cytometry data. Conclusion: The successful combination of bead and cell based assays on a single hybrid instrument demonstrated the potential utility of a multitasking platform. The results presented are providing groundwork for future development of more cost effective modular architecture for a flexible flow cytometry based platform. © 2006 International Society for Analytical Cytology

Background: Exceptionally robust cell preparations are needed for quality assessment programs (QAPs) such as the International Program for Quality Assessment and Standardization for Immunological Measures (QASI) relevant to HIV/AIDS. A suitable product must withstand environmental stress related to transportation for a minimum of 6 days. The two objectives of this study are (1) to evaluate the performance of various commercial preparations with multicenter participation and (2) to evaluate the robustness of stabilized blood cell products. Methods: Phase 1: The performance of stabilized blood cell products was evaluated in a multicenter QAP utilizing various staining procedures and flow cytometers. Absolute cell enumeration was achieved using single-platform T-cell subset methodology. Phase 2: The robustness of stabilized blood cell products was evaluated by monitoring T-cell subset values from samples stored at 4°C, 22°C, and 37°C for up to 10 days. Results: The largest interlaboratory variation in both absolute and relative T-cell values was 16% in samples with CD4 levels >400 cells per microliter and 21% in samples with CD4 levels

Measurements of CD4 T-cell levels are essential for the assessment of human immunodeficiency virus (HIV) disease course, clinical staging, epidemiological studies, and decisions regarding prophylactic therapies against opportunistic infection. Until now, only in the industrialized countries was T-cell subset monitoring considered a practical option to assess disease progression. The Quality Assessment and Standardization for Immunological Measures Relevant to HIV/AIDS (QASI) program was established in 1997 to meet performance assessment for immunophenotyping laboratories in countries where such service is not available. The QASI program is provided at no cost to any laboratory in a resource-poor setting that wishes to participate. This report describes the beneficial impact of participation in the QASI program. Carefully selected commercial stabilized whole blood preparations were sent regularly to participating laboratories. Participants reported the T-cell subset values they obtained by flow cytometry. Once the aggregate mean values for the T-cell subsets were established for the shipment, a comprehensive and confidential report was sent to each laboratory. The results from five consecutive shipments were analyzed. The coefficient of variation decreased from 7.2% to 4.7% and from 14.2% to 8.8% for percent and absolute CD4 T-cell counts, respectively. With the implementation of the QASI program using commercial stabilized whole blood specimens, it is possible to reduce interlaboratory error. This study illustrates that a quality assessment program can improve the overall performance of laboratories. Reducing interlaboratory variation can enhance significantly the effectiveness of multicenter HIV vaccine or drug trial evaluation.

BACKGROUND In the past decade, human immunodeficiency virus (HIV) lymphocyte immunophenotyping has evolved significantly. New fluorochromes, new multicolor reagents, enhanced instruments, and the capacity to provide absolute cell counts using the single-platform technique have all contributed to the reliability of T-cell subset measurements. In this study, four gating protocols were evaluated to select the most robust method for T-cell subset enumeration. Methods: Peripheral blood specimens from 21 HIV+ and 20 HIV− individuals were monitored up to 96 h. Aliquots of specimens were stored at room temperature and analyzed at 6 (baseline), 48, 72, and 96 h. Aliquots were stained with CD45-fluorescein isothiocyanate (FITC)/CD3PC5/CD4RD1/CD8ECD. Data analysis was performed with all four gating protocols. Results: Only with fresh blood did all protocols provide similar results. From samples that were 48 h old, the choice of gating strategy had a dramatic impact on immunophenotyping results. The largest deviations from baseline values occurred at 96 h and gating protocols that included dual light scatter gates provided the greatest shift of T-cell subset values over time. The gating protocols that were based exclusively on cell lineage-specific gates gave the most robust T-cell values up to 96 h. Conclusion: By selecting the appropriate gating protocol, the temporal integrity of specimens can be extended up to 4 days. Cytometry (Clin. Cytometry) 50:53–61, 2002. © 2002 Wiley-Liss, Inc.

This brief review of the development of gating strategies for CD4 1 T-cell enumeration is really the story of contemporary clinical flow cytometry. It is the chronicle of its birth, and its slow invasion into the clinical immunology laboratory over the past 15 years. The driving force behind the technological evolution of leukocyte immunophenotyping was, and still is, the impact of the HIV/AIDS pandemic on the discipline of immunology. While, at times, this review of analytical flow cytometry makes metaphorical excursions into the literary world, the objective is not to trivialize technology. Rather, it attempts to illustrate how, in his drive to solve the practical problems of modern medicine, man’s essentially eclectic nature excels in an environment of diverse knowledge. This review will examine how technological advancements, such as the introduction of clinical instruments with multi-color capabilities, and the evolution of leukocyte gating strategies, has influenced the evolution of leukocyte immunophenotyping. Cytometry 30:157‐165, 1997. r 1997 Wiley-Liss, Inc. Key terms: immunology; metaphor; multi-color capabilities In his metaphorical essay, ‘‘Godel, Escher, Bach: An Eternal Golden Braid’’ (1), Pulitzer Prize winner Douglas Hofstadter exemplified the impact of great trilogies on humanity by weaving the contributions of three universal giants into a unified concept. The impact of Hofstadter’s trilogy far outweighs the impact generated by the emergence of leukocyte immunophenotyping, but it does serve as a compelling example of how disciplines which are conceptually far apart can still fuse and consolidate knowledge. Contemporary clinical flow cytometry also required the unifying force of several powerful trilogies. The events that would culminate in the birth of the current state-of-the-art flow cytometry were initiated in three different countries and were based on three fundamentals derived from as many different scientific disciplines. The first essential step in the development of present-day analytical cytology dates back to nineteenth century Germany, where Paul Ehrlich first described the fundamental extrinsic properties of leukocytes (2). The second important step took place in Stockholm during the 1930’s, where Caspersson and his colleagues unraveled some of the crucial elements of modern analytical cytology. In the course of making some precise microspecrophotometric absorption measurements of unstained cells, they defined some of the essential intrinsic properties of leukocytes (3). In Boston, during the early 1940’s, Coons, Creech, and Jones developed a cell labeling technology for microscopy which enabled researchers to detect fluorescent antibodies bound to cell surface epitopes (4). Their qualitative measurement of extrinsic properties of cells was the third essential step leading to modern immunophenotyping of leukocytes (5).

Background: A significant worldwide mobilization effort to treat people with HIV disease began in 2003. Most guidelines for initiating antiretroviral therapy require reliable and reproducible CD4 T-cell counting. Therefore, any effort that improves global availability of quality managed assessment schemes for CD4 T-cell enumeration is a positive achievement for the clinical management of AIDS on a worldwide scale. Methods: The Canadian QASI-Quality Management System (QMS) has been in operation for over a decade. More recently, QMS has fine-tuned its strategy to optimize its global impact in the fight against the HIV/AIDS pandemic. Three modifications were implemented: (1) introduction of skills and knowledge transfer workshops pertaining to the initiation of national quality management programs for CD4 counting, (2) introduction of a road map to establish domestic EQAP for countries that are ready, and (3) introduction of a statistical analysis package which permits continuous monitoring of global impact of the QASI-QMS. Results: Based on QASI-QMS distribution of specimens over four consecutive participation cycles, there was decreased interlaboratory variation for both low and medium CD4 T-cell levels. After three cycles of consecutive participation, there is an average of 38 and 26% error reduction reported for the mid and low CD4 levels, respectively. Conclusion: The program improvements mentioned earlier appear to have had a profound effect with regard to enhancing the performance of laboratories participating in the QASI-QMS. Specifically, there is a significant reduction in interlaboratory variability of CD4 T-cell counts resulting from continuous participation in the QASI-QMS. © 2009 Clinical Cytometry Society