Reduction of variation in T-cell subset enumeration among 55 laboratories using single-platform,three or four-color flow cytometry based on CD45 and SSC-based gating of lymphocytes

BACKGROUND: Enumeration of CD4(+) and CD8(+) T-cell subsets provides relevant information for diagnosis and monitoring of patients with cellular immunodeficiencies. As a result, an external quality assurance scheme was implemented in Belgium, The Netherlands, and Luxembourg in 1995. A workshop was held to train the participants in state-of-the art technology for assessment of absolute T-cell subset counts (i.e., a three or four-color, single-platform assay with lymphocyte gating based on CD45 and sideward light scatter) with the aim to achieve between-site coefficients of variation (CVs) <10% and within-site CVs <5% for > or =75% of the participants. METHODS: Three send-outs of stabilized blood from a healthy donor were distributed to 55 laboratories, each with the request to perform the standard assay on three occasions. For comparison, each laboratory performed its local technique in parallel. RESULTS: With the standard technique, between-site CVs of approximately 8% (CD3+ T cells), approximately 9% (CD4+ T cells), and approximately 10% (CD8+ T cells) were achieved. Within-site CVs were <5% for 82% (CD3+ T cells) and approximately 70% (CD4+ and CD8+ subsets) of the participants. Local techniques yielded between-site CVs of 13%-17% for CD3+, CD4+, and CD8+ T cells. CONCLUSIONS: The state-of-the-art technology for T-cell subset enumeration was implemented successfully among 55 Belgian-Dutch laboratories and resulted in significant reductions of between-site variation of absolute CD3+, CD4+, and CD8+ T-cell counts.     

Selection of lymphocyte gating protocol has an impact on the level of reliability of T-cell subsets in aging specimens

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.     

Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells in leukapheresis product and bone marrow for clinical transplantation: a comparison of three methods

Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells (HSCs) is widely used for evaluation of graft adequacy of peripheral blood and bone marrow stem cell grafts. In the present study, we review and compare the major counting techniques of stem and progenitor cells. The methods are: the Milan/Mullhouse protocol, two-platform ISHAGE (International Society of Hematotherapy and Graft Engineering) and single-platform ISHAGE analysis system. According to the Milan/Mulhouse protocol, HSCs are identified by CD34 antibody staining and easy gating strategy. The ISHAGE guidelines for detection of CD34+ cells are based on a four-parameter flow cytometry method (CD34PE/CD45PerCP staining, side and forward angle light scatter) thus employing multiparameter gating strategy. With two-platform ISHAGE protocol, an absolute CD34+ count is generated by incorporating the leukocyte count from an automated hematology analyser. The single-platform ISHAGE method to determine the absolute CD34+ count directly from a flow cytometer includes the use of Trucount tubes (Becton Dickinson) with a known number of fluorescent beads. CD34+ cells were quantified in mobilized peripheral blood, collected by leukapheresis, and bone marrow from 42 samples from patients with hematological malignancies. The differences against the means display low disagreement between the Milan/Mulhouse and ISHAGE protocols, with discrepancies of up to 2.5% (two-platform ISHAGE)--2.6% (single-platform ISHAGE) in enumeration of CD34+ cells in leukapheresis product and 4.8% (two-platform ISHAGE)--4.9% (single-platform ISHAGE) in bone marrow. Our results show high correlation among all three methods. Since the three protocols are compatible, choosing the most convenient in terms of costs, simplicity and compliance with clinical results appears to be a logical consequence.     

T-cell subset counting and the fight against AIDS: reflections over a 20-year struggle

The story of T-lymphocyte subset immunophenotyping technology is reviewed on the occasion of the 20th anniversary of CD4 T-cell enumeration. Over time, immunophenotyping has evolved into precise, reliable, but complicated and expensive technology requiring fresh blood samples. The gating technologies that were universally adapted for clinical flow cytometry for the past decade relied on rapidly deteriorating morphological scatter characteristics of leukocytes. This special issue dedicated to CD4 T-cell enumeration features most of the available new options that will have a significant impact on how this technology will be implemented within the first decade of the 21st century. In a series of original publications, including the new NIH guideline for T-cell subset enumeration, contemporary gating protocols that use immunologically logical parameters are presented as part of the more reliable and affordable immunophenotyping alternative. Some of the improvements addressed here include the costs of the assays and the capacity to monitor interlaboratory and intralaboratory performances. It is clear that an effective attack on the human immunodeficiency virus (HIV) epidemic has to embrace resource-poor regions. Reducing the cost of the assay while improving reliability and durability is a move in the right direction.     

Enumeration of CD34+ cells in cord blood: a variation on a single-platform flow cytometric method based on the ISHAGE gating strategy

Single-platform flow cytometric absolute cell counting protocols provide increased robustness for CD34+ cell enumeration by limiting potential sources of imprecision. However, samples with any cellular fragmentation or debris, such as cord blood samples, provide challenges for these assays. We describe a simple, robust absolute CD34+ cell counting protocol, suitable for cord blood, using TRUCOUNT absolute count tubes (BD Biosciences, San Jose, CA) and a modified ISHAGE (International Society for Hematotherapy and Graft Engineering) gating strategy. An advantage of TRUCOUNT tubes is that each tube is supplied with a known number of lyophilized fluorescent beads. The method includes no-wash fixative-free ammonium chloride red blood cell lysis and the viability dye, 7-amino actinomycin D, to exclude dead cells. The threshold was set on CD45 expression in the FL1 channel and an exclusion gate in the forward scatter channel reduced debris. No manual adjustment of the gating regions was required, even for samples in less than optimal condition. Comparison of the TRUCOUNT-ISHAGE protocol with the original dual-platform ISHAGE assay (n = 30) and the single-platform ISHAGE protocol using Flow-Count Fluorospheres (Beckman Coulter, Fullerton, CA; n = 22) showed high correlation (R(2) = 0.949 and 0.989, respectively) and no significant difference or bias for samples ranging from 22 to 600 CD34+ cells per microliter. Results are presented that demonstrate the detrimental effect of a fixative-containing lysis reagent when used in a lyse-and-wash procedure. The TRUCOUNT-ISHAGE protocol combines the attributes of TRUCOUNT tubes and the ISHAGE gating strategy to provide a single-platform protocol capable of achieving readily standardization of CD34+ cell enumeration.     

Evaluation of a universal template for single-platform absolute T-lymphocyte subset enumeration

The single-platform absolute T-lymphocyte subset analysis was evaluated utilizing a universal protocol in a Canadian multicenter study with the collaboration of the members of the Canadian HIV Trials Network (CTN). Participants used flow cytometers and reagents of their choice for labeling and lysing whole blood. Over a 2-year period, CTN laboratories performed single-platform absolute T-lymphocyte subset enumerations on fresh and commercial stabilized blood products using commercially available microfluorospheres TruCount and Flow-Count. This multicenter evaluation demonstrated that the application of a universal template for single-platform analysis provides a generic approach that embraces a wide array of immunophenotyping settings available in clinical laboratory.     

Analyses of quality assessment studies using CD45 for gating lymphocytes for CD3(+)4(+)%

BACKGROUND: The purpose of this study was to assess whether laboratories which do not use CD45 for gating lymphocytes with three- (or four-) color flow cytometry (non-CD45 laboratories) for CD3(+)4(+)% and CD3(+)8(+)% do worse on quality assessment (QA) studies than laboratories which do use CD45 (CD45 laboratories). METHODS: Data came from blood specimens donated by 62 donors (50 HIV-positive) assayed over 2 years (November, 1996-October, 1998) by 35 laboratories in the NIAID DAIDS Flow Cytometry QA Program. RESULTS: Non-CD45 laboratories were significantly more likely to be classified as having unacceptable inter-laboratory results (far from the group median) than CD45 laboratories (5.6% vs 1.5%, P = 0.005 for CD3(+)4(+)%; 10.4% vs 5.0%, P = 0.007 for CD3(+)8(+)%). The intra-laboratory range of results on blinded replicates was significantly more likely to be deemed unacceptable (range >4%) in non-CD45 laboratories than in CD45 laboratories for CD3(+)8(+)% (14. 5% vs 3.5%, P = 0.002) but not for CD3(+)4(+)% (2.6% vs 1.5%, P = 0. 62). These differences in favor of CD45 gating were observed even though the non-CD45 laboratories had been doing three-color flow cytometry in the QA program significantly longer (P = 0.05) than the CD45 laboratories, and so would be expected to have fewer problems with the assay. CONCLUSIONS: Laboratories which choose to use a single CD3/CD4/CD8 tube for immunophenotyping may be sacrificing both accuracy and reproducibility.     

A new single-platform method for the enumeration of CD34+ cells

Guidelines for flow cytometric enumeration of CD34⁺ hematopoietic stem cells (HSC) recommend the use of a single-platform assay. The SCE kit has recently been commercialized by BD Biosciences. Results obtained with this newly available kit were compared with CD34⁺ cell enumerations obtained in parallel with already commercialized diagnostic kits; fresh peripheral blood, apheresis, cord blood (CB) and bone marrow (BM) samples, as well as thawed apheresis and CB samples, were assayed. The SCE kit produced data for CD34⁺ enumeration that correlate well with data produced with the older assays (r²≥0.9). Practical advantages were the ability to enumerate viable CD34 cells in all kinds of HSC products, the absence of bead pipetting (which decreases results precision) and a gating strategy complying with international recommendations. A major disadvantage was the absence of specific software for data analyses and presentation of results.     

Precise CD4 T-cell counting using red diode laser excitation: for richer,for poorer

BACKGROUND: Measuring CD4 T-cell counts at low cost is relevant in dealing with the human immunodeficiency virus (HIV) epidemic throughout the developing world. The recently introduced novel concepts in gating strategies and sample stabilization facilitate affordable immunophenotyping by flow cytometry. However, the impact of these developments is still limited by the high cost of currently available flow cytometers. METHODS: Diode lasers emitting 10-15 mW at 635 nm are one-tenth the size and cost and require one thousandth the power of an equivalent 488-nm argon ion laser. We used the available 635-nm diode-based flow cytometers, including PA-II, Luminex 100, SuperMot, and FACSCalibur, to investigate whether these instruments can generate reliable CD4 counts when used with allophycocyanin (APC) and cyanin-5 (Cy5)-labeled CD4 antibodies. RESULTS: We document the feasibility of obtaining leucocyte differential counts using orthogonal side scatter (SSC) without the need for forward scatter (FSC). Accurate CD4% values among lymphocytes and leucocytes can be obtained by primary CD4 gating using a single CD4 monoclonal antibody conjugated to APC or Cy5. Double immunofluorescence (IF) staining with CD4-APC (FL1) and CD45-APC-Cy7 (FL2) introduces pan-leucogating for a convenient assessment of absolute CD4 counts on double platforms. We demonstrate that small flow cytometers with laser diodes are capable of delivering absolute CD4 T-cell counts with a precision similar to the performance of the current state-of-the-art single-platform instruments (e.g., the CytoronAbsolute; R(2) = 0.961). In this respect, they appear to be superior to the nonflow CD4 counting techniques. CONCLUSIONS: Accurate CD4 counts can be generated at minimal cost on red diode laser-operated flow cytometers, retaining the potential for high throughput capacity without compromising precision. With further improvements in volumetric technology and clinical software, these cytometers may develop into a new generation of inexpensive battery-operated laboratory hardware that combines cellular phenotyping with bead-based multiplexing immunoassays for (HIV) serology.     

Compatibility of Stabilized Whole Blood Products with CD4 Technologies and Their Suitability for Quality Assessment Programs

Background

CD4 T cell enumeration is the most widely used prognostic marker for management of HIV disease. Internal quality control and external quality assessment (EQA) programs are critical to ensure reliability of clinical measurements. The utility of stabilized whole blood products (SWBP) as a test reagent for EQA programs such as Quality Assessment and Standardization for Immunological measures relevant to HIV/AIDS (QASI) program have been demonstrated previously. Since then, several new commercial SWBPs and alternative CD4 enumeration technologies have become available. Seven SWBPs were evaluated on seven different enumeration platforms to determine which product(s) are most suitable for EQA programs that support multiple analytical technologies.

Method

Assessment of SWBPs was based on two criteria: (1) accuracy of CD4 T cell measurements and; (2) stability under sub optimal storage conditions.

Results

Three SWBPs (Multi-Check, StatusFlow and CD4 Count) showed accurate CD4 T-cell absolute count and percentage values across six of the enumeration platforms. All products retain stability up to 18 days at 21–23°C with the exception of Multi-Check-high on FacsCount and Multi-Check-Low and StatusFlow-Low on Pima. One of the products (CD4 Count) retained stability for three days on all platforms tested when stored at 37°C.

Conclusion

This study demonstrated that the characteristics of commercially available SWBPs vary across multiple CD4 platforms. The compatibility of testing panels for EQA programs with multiple analytical platforms needs to be carefully considered, especially in large multiplatform CD4 EQA programs. The selection of a suitable cross-platform SWBP is an increasing challenge as more reagents and platforms are introduced for CD4 T-cell enumeration.     

Enumeration of CD4(+) T-cells in the peripheral blood of HIV-infected patients: an interlaboratory study of the FACSCount system.

The aim of the present study was to assess the interlaboratory reproducibility of the FACSCount system for the enumeration of peripheral blood (PB) CD4(+) T-cells. In each of the seven participating centers, both previously stained and unstained PB samples (n = 49) were received and either analyzed or stained and then analyzed. Interlaboratory reproducibility was checked in two different groups of centers (n = 3 and n = 4) where the study was performed in parallel. In addition, both the intralaboratory precision and accuracy of this system were analyzed in comparison with results obtained with conventional flow cytometry. Accordingly, upon comparing both methods, a high degree of correlation was observed in the total number of CD3(+) T-cells (coefficient of correlation of 0.9750 +/- 0.0184, slope of the best linear fit: 0. 9214 +/- 0.0311, y-intercept of 12 +/- 47) as well as in the number of CD3(+)/CD4(+) (coefficient of correlation of 0.9794 +/- 0.1457, slope of the best linear fit: 0.9463 +/- 0.0753, y-intercept of -11 +/- 36) and CD3(+)/CD8(+) (coefficient of correlation of 0.9728 +/- 0.0192, slope of the best linear fit: 0.9682 +/- 0.0735, y-intercept of 7 +/- 95) major subsets. In addition, low coefficients of variation (CV) were obtained for replicates, indicating the method's high degree of accuracy. The present study shows that with respect to the interlaboratory reproducibility reported for most techniques used for the enumeration of PB CD4(+) T-cells, the FACSCount system results in data with much lower coefficients of variance (CVs) (mean CV of less than 10%). Upon measuring the impact on results of different variables associated with either sample preparation or data acquisition and analysis, our study clearly shows that data acquisition and analysis does not influence the results by increasing variability since the coefficients of variation obtained for samples prepared in the same laboratory under the same conditions and read in different laboratories with different instruments were identical to those obtained for the replicates of the same samples read in each individual center. In contrast, interlaboratory variability, although low, significantly increased when sample preparation was carried out in different laboratories, suggesting that pipetting still represents the major source of variability in the FACSCount system.     

Quality assurance of human papillomavirus testing

The External Quality Assurance (EQA) in medical microbiology in the Czech Republic is well organized. It is coordinated by the Accreditation Department of the Centre of Epidemiology and Microbiology (AD-CEM) of the National Institute of Public Health in Prague. Since 1993 when the first samples were sent out the number of programmes and participating laboratories has been rapidly increasing. EQA for Human papillomavirus (HPV) has been available since 2000. As has been shown for other programmes, the EQA for HPV has proved to be useful, helping to improve the accuracy of analyses and contributing to the standardization of methods of HPV DNA testing. EQA for HPV has been well received by routine laboratories, demonstrated by a high number of these institutions voluntarily participating in EQA.     

External Quality Assessment for Tuberculosis Diagnosis and Drug Resistance in the European Union: A Five Year Multicentre Implementation Study

BackgroundExternal quality assurance (EQA) systems are essential to ensure accurate diagnosis of TB and drug-resistant TB. The implementation of EQA through organising regular EQA rounds and identification of training needs is one of the key activities of the European TB reference laboratory network (ERLTB-Net). The aim of this study was to analyse the results of the EQA rounds in a systematic manner and to identify potential benefits as well as common problems encountered by the participants.MethodsThe ERLTB-Net developed seven EQA modules to test laboratories’ proficiency for TB detection and drug susceptibility testing using both conventional and rapid molecular tools. All National TB Reference laboratories in the European Union and European Economic Area (EU/EEA) Member States were invited to participate in the EQA scheme.ResultsA total of 32 National TB Reference laboratories participated in six EQA rounds conducted in 2010–2014. The participation rate ranged from 52.9% - 94.1% over different modules and rounds. Overall, laboratories demonstrated very good proficiency proving their ability to diagnose TB and drug-resistant TB with high accuracy in a timely manner. A small number of laboratories encountered problems with identification of specific Non-tuberculous Mycobacteria (NTMs) (N = 5) and drug susceptibility testing to Pyrazinamide, Amikacin, Capreomycin, and Ethambutol (N = 4).ConclusionsThe European TB Reference laboratories showed a steady and high level of performance in the six EQA rounds. A network such as ERLTB-Net can be instrumental in developing and implementing EQA and in establishing collaboration between laboratories to improve the diagnosis of TB in the EU/EEA.     

Results of the National External Quality Assessment for Toxoplasmosis Serological Testing in China

Background

Toxoplasmosis is typically diagnosed by serologic testing. External quality assessment (EQA) of clinical laboratories could ensure the accuracy and reliability of serological tests. We assessed the quality of toxoplasma serological assays in Chinese clinical laboratories by an EQA performed between 2004 and 2013 by the National Center for Clinical Laboratories.

Methodology and Findings

EQA panels were prepared and shipped at room temperature to participating laboratories that employed toxoplasma IgG and IgM serological detection. By 2013, 5,384 EQA test reports for toxoplasma-specific IgM and 2,666 reports for toxoplasma-specific IgG were collected. Enzyme-linked immunosorbent (ELISA) and chemical immunofluorescent assays were the most commonly used detection methods. The overall coincidence rates of negative samples were better than those of positive samples. The overall EQA score for toxoplasma-specific IgM detection ranged between 84.3% and 99.6%. The ratio of laboratories that achieved correct IgG detection ranged from 61.1% to 99.3%. However, the inter- and intra-assay variabilities were found to be considerable. The most common problem was failure to detect low titers of antibody.

Conclusion

The EQA scheme showed an improvement in toxoplasma serological testing in China. However, further optimization of assay sensitivity to detect challenging samples remains a future challenge.     

CD45-assisted PanLeucogating for accurate,cost-effective dual-platform CD4+ T-cell enumeration

BACKGROUND: North American and European guidelines for dual-platform (DP) flow cytometry recommend absolute CD4 T-cell counts to be calculated from two parameters: the absolute lymphocyte counts obtained on a hematology analyzer and the percentages of CD4+ cells among lymphocytes (CD4%/lympho) obtained by flow cytometry. Nevertheless, the identification of lymphocytes is error-prone: a poor match between these common denominators in the two systems is the main source of inaccuracy. In contrast, total leucocyte counts (white cell counts [WCC]) and CD4% among the gated CD45+ leucocytes (CD4%/leuco) can be determined with greater accuracy. METHODS: We introduced "PanLeucogating," i.e., we used total leucocytes as the common denominator for improving the precision of DP absolute CD4 counting. Correlations and Bland-Altman tests were used for statistical analysis. RESULTS: First, 22 stabilized blood product samples were provided by U.K. National External Quality Assessment Scheme (NEQAS) and a higher accuracy and precision of CD4 counts were documented using PanLeucogating compared with lymphocyte gating. Next, 183 fresh and 112 fixed (TransFix) whole blood samples were used to compare DP methods and single-platform (SP) methodology, including both volumetric and bead-based techniques. A particularly high correlation and comparable precision of absolute CD4 counts were observed between the SP volumetric method and DP PanLeucogating (R(2) = 0.990; bias 6 +/- SD 17%). The SP volumetric method showed lower levels of agreement with the DP lymphocyte gating (R(2) = 0.758; bias 14 +/- SD 51%) and with the SP bead-based method (R(2) = 0.923; bias 4 +/-SD 31%). CONCLUSIONS: These observations show that DP leucocyte counts (WCC) should replace lymphocyte counts as the "common denominator" although CD4%/lympho values can, as an extra step, be also provided readily if requested. When coupled with quality control for WCC on hematology analyzers, the DP method with CD45 PanLeucogating represents a robust CD4 T-cell assay that is as accurate as the SP volumetric technique. This DP method uses only two, CD45 and CD4, antibody reagents and can be run on any pair of hematological analyzer plus flow cytometer.     

Impact of standardization on clinical cell analysis by flow cytometry

The evolution of flow cytometry from a research tool to a pivotal technology for clinical diagnostic purposes has required significant efforts to standardize methods. The great advantage of flow cytometry is that it's applications are highly amenable to standardization. Here, we review the efforts that have been made for flow cytometric applications in four major fields of clinical cell analysis: CD4+ T-cell enumeration, CD34+ hematopoietic stem and progenitor cell enumeration, screening for the HLA-B27 antigen and leukemia/lymphoma immunophenotyping. These standardization efforts have been parallelled by the establishment of external quality assessment (EQA) schemes in many countries worldwide. The goal of these EQA exercises has been primarily educa-tional, but their results will increasingly serve as a basis for laboratory accreditation. This important development requires that the EQA schemes, in particular the quality of the distributed samples and the procedures for evaluating the results, meet the highest standards.     

Monitoring standards for molecular genetic testing in the United Kingdom, the Netherlands, and Ireland

Molecular genetic techniques have entered many areas of clinical practice. Public expectations from this technology are understandably high. To maintain confidence in this technology, laboratories must implement the highest standards of quality assurance (QA). External quality assessment (EQA) is recognized as an essential component of QA. The United Kingdom National External Quality Assessment Service (UKNEQAS) for Molecular Genetics, first set up in 1991, is currently the longest provider of EQA to molecular genetic testing laboratories in the UK, The Netherlands, and Ireland. Errors in the scheme are sporadic events. However, evidence from this and other EQA schemes suggests that a residual error rate persists, which should be taken into account in clinical practice. This EQA scheme has evolved from the respective scientific bodies of the constituent countries and retains a strong emphasis on collective peer review. It is essential that the steps taken to ensure quality in this rapidly expanding field are clear and transparent to participants and public alike. We describe the procedures developed and the governance imposed to monitor and improve analytical and reporting standards in participant laboratories and we compare our experiences with those of equivalent EQA services in the United States.     

International External Quality Assessment Study for Molecular Detection of Lassa Virus

Lassa virus (LASV) is a causative agent of hemorrhagic fever in West Africa. In recent years, it has been imported several times to Europe and North America. The method of choice for early detection of LASV in blood is RT-PCR. Therefore, the European Network for Diagnostics of ‘Imported’ Viral Diseases (ENIVD) performed an external quality assessment (EQA) study for molecular detection of LASV. A proficiency panel of 13 samples containing various concentrations of inactivated LASV strains Josiah, Lib-1580/121, CSF, or AV was prepared. Samples containing the LASV-related lymphocytic choriomeningitis virus (LCMV) and negative sera were included as specificity controls. Twenty-four laboratories from 17 countries (13 European, one African, one Asian, two American countries) participated in the study. Thirteen laboratories (54%) reported correct results, 4 (17%) laboratories reported 1 to 2 false-negative results, and 7 (29%) laboratories reported 3 to 5 false-negative results. This EQA study indicates that most participating laboratories have a good or acceptable performance in molecular detection of LASV. However, several laboratories need to review and improve their diagnostic procedures.     

Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. European Working Group on Clinical Cell Analysis

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.