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Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disorder in which correct diagnosis is essential for effective patient management. Demonstration of deficiency of glycosylphosphatidylinositol (GPI)-linked antigens from red cells and/or granulocytes by flow cytometry represents a highly specific diagnostic test for PNH. Currently, no external quality assessment (EQA) programme or reference material is available for whole-blood PNH testing (red cells and leucocytes) by flow cytometry., Methods: In order to address this issue, we report the development of a stabilized whole-blood PNH sample. We present the results of a detailed time course study by flow cytometry that demonstrates the stability of GPI-linked antigen expression on granulocytes and red cells in a stabilized PNH peripheral blood sample, using a previously described method., Results: The PNH cells, as well as the coexisting normal red cell and granulocyte populations, remained stable for up to 120 days, both in terms of immunophenotypic and light scatter characteristics. Subsequent samples were used for a PNH EQA programme and issued to 92 laboratories worldwide., Conclusions: This study has highlighted that PNH testing by flow cytometry has significant problems with regard to false-positive and -negative results. In addition, the variation in GPI-linked antigen detection methods has highlighted the urgent need for standardized protocols., (Copyright © 2008 Clinical Cytometry Society.)

Background: Long-term stabilized blood samples are potentially useful as positive or negative procedure controls for flow cytometric HLA-B27 screening, and could serve as test samples in an external quality assessment (EQA) scheme. We evaluated long-term stabilized whole blood specimens as prepared for the UK NEQAS for Leucocyte Immunophenotyping EQA scheme (Sheffield, UK)., Methods: Peripheral blood samples were obtained from nine blood bank donors with known HLA-B typing. Short-term stabilization with Trans-FIXtrade mark was performed before shipment to Sheffield. Thereafter, long-term stabilization was performed. Commercially available HLA-B27 mAb were tested periodically between 1 week and 12 months on (i) fresh, (ii) short-term stabilized, and (iii) long-term stabilized blood samples using a stain, lyse, and wash technique. We compared the forward scatter (FSC), sideward scatter (SSC), and fluorescence signals of lymphocytes as a function of time. Furthermore, a pilot send-out with stabilized blood samples of four blood bank donors was distributed among the participants to the Benelux EQA scheme for HLA-B27 screening, and results were compared with historical EQA data obtained using nonstabilized blood samples from the same donors., Results: There were no major effects on FSC and SSC characteristics of lymphocytes. Background fluorescence of stabilized samples increased and specific fluorescence of stabilized HLA-B27 positive samples decreased as compared with fresh samples. However, discrimination between the investigated HLA-B27 positive and HLA-B27 negative samples remained feasible poststabilization. In the pilot send-out, the results obtained with stabilized samples were less concordant than with the corresponding fresh samples due to variable quality of the stabilized samples., Conclusion: Long-term stabilized whole blood samples are potentially useful as true HLA-B27 positive and true HLA-B27 negative control cells for daily and longitudinal quality control of flow cytometric HLA-B27 screening. In the same way, long-term stabilized samples may be used for EQA purposes. However, these samples are currently not feasible for reagent validation purposes. Extensive quality control of long-term stabilized samples is necessary before distribution in multicenter surveys., ((c) 2008 Clinical Cytometry Society)

Background: We have previously reported a flow rate calibration method for the determination of absolute CD4(+) T-lymphocyte counts that removes the need for the addition of latex beads to each sample. However, a limitation with this approach is that a calibration factor (CF) needs to be applied to adjust for differences in viscosity between latex bead suspensions and biological specimens. We have also demonstrated the value of using stabilized whole blood samples in external quality assessment (EQA) studies; such samples have a stable absolute lymphocyte count for over 1 year, at 4 degrees C. It was successfully demonstrated that this material can be used as a flow rate biocalibration (FRB) material for use as a flow cytometric control to provide a sample with a known CD4(+) T-lymphocyte count. Such material has advantages over latex bead technology as it can act as a full process control as well as having the same matrix and viscosity characteristics as the test material, thus removing the need for a CF., Methods: In this study, we have analyzed 268 consecutive normal, abnormal, and HIV(+) samples using FRB, incorporating the PanLeucoGating approach and compared this to the MultiSet method, defined as the predicate., Results: Percentage similarity statistics revealed the following: 0-3,000 CD4(+) cells/mul mean percentage difference (MPD; bias) 1.2%, 95% CI of 5.6-8%; 0-200 CD4(+) cells/microl MPD of 1.25%, 95% CI of 11.63-14.13%; 201-500 CD4(+) cells/microl MPD of 1%, 95% CI of 4.6-6.6%., Discussion: This study demonstrates that stabilized whole blood can be used for FRB. It has the advantage of being a full process control, in addition to costing less than latex beads with highly comparable results. As bench top flow cytometers are extremely stable, this is a low cost and robust alternative to bead based methods for generating absolute CD4 counts., (Copyright 2006 International Society for Analytical Cytology.)

Background: Reports suggest that flow rate (FR) is constant on bench top flow cytometers. Therefore, if FR is constant, the volume acquired in a fixed time period will also be constant, enabling absolute leucocyte counting using flow rate calibration (FRC)., Methods: FR stability was ascertained on a standard FACSCalibur by counting TruCount beads suspended in phosphate buffered saline over 120 s. Studies using two lysing solutions (FACS lysing solution and PharM Lyse) and corresponding sample lysates established a lysing solution calibration factor (CF). Absolute CD4(+) T-lymphocyte counts on 10 peripheral blood samples determined using FRC were compared with the predicate method TruCount/MultiTEST, incorporating MultiSET software. Linearity studies were also performed at three different flow rates., Results: A high degree of linearity over a wide range of counts (50 to >1,600 CD4(+) T lymphocytes/microl) at all three pressures was observed. Importantly, there was no significant difference from the predicate method when appropriate lysing solution CF was used., Conclusions: Using a simple calibration procedure and incorporation of an appropriate lysing solution CF, we show that FRC can easily be performed. The technical details that underpin this novel approach for absolute leucocyte enumeration are provided., (Copyright 2003 Wiley-Liss, Inc.)

Background: CD4(+) T-lymphocyte enumeration is vital for monitoring disease progression in individuals positive for the human immunodeficiency virus (HIV), and as a result, there is a need to develop cost-effective protocols that provide accuracy, precision, and affordability. Recently, PanLeucoGating has been shown to fulfill these requirements; however, although comparable to state-of-the-art single-platform protocols (SP), there is still a requirement for an accurate total white cell count. To overcome this limitation, we recently developed a flow-rate based calibration method that enables the PanLeucoGating protocol to be used as a SP approach, and in this study show that this approach can be used for CD4(+) T-lymphocyte enumeration., Methods: A total of 113 HIV samples were analyzed using three protocols: (a) state-of-the art SP bead-based method (MultiSet; predicate protocol), (b) PanLeucoGating protocol used as a dual-platform (DP) approach, and (c) the newly developed flow rate-based SP approach. We demonstrate that flow rate calibration can be achieved easily and that the method is highly comparable to the state-of-the-art SP method., Results: A high correlation was observed between the predicate protocol and the SP PanLeucoGating approach over the whole range of CD4 counts tested (r(2) = 0.9928; bias 8 cells/microl), including the clinically relevant range (e.g., 0-200 CD4 cells/microl; bias 0 cells/microl). For batched samples, the cost of providing a CD4(+) T-lymphocyte count was reduced to approximately US $1., Conclusions: The SP PanLeucoGating is a cost-effective approach to CD4(+) T-lymphocyte enumeration that maintains accuracy and precision., (Copyright 2003 Wiley-Liss, Inc.)

Background: Flow cytometric enumeration of CD34+ hematopoietic sterm and progenitor cells (HPC) is the reference point for undertaking apheresis and evaluation of adequacy for PBSC engraftment. An external quality assurance (EQA) scheme for CD34+ HPC enumeration has been operational in Belgium, Netherlands and Luxemburg (Benelux) since 1995. Within this group, a multicenter survey was held to validate the state-of-the-art methodology, i.e., multiparametric definition of HPC based on light scatter, expression of CD34 and CD45, and counting beads (i.e., 'single platform ISHAGE' method)., Methods: 'Real-time' EQA was used to monitor the application of the single-platform ISHAGE method by 36 participants. Three send-outs of stabilized blood with CD34+ cell counts 35-60 cells/microl were distributed to 36 participants, who were required to assay the samples on three occasions using the standard assay and their local techniques. These results were compared with thosed obtained by 111-116 UK NEQAS participants testing the same specimens., Results: Using the single platform ISHAGE methods, between-laboratory coefficients of variations (CVs) as low as 10% were achieved. Intra-laboratory CVs were < 5% for approximately 50% of the participants. Local single-platform techniques yielded between-laboratory CVs as low as 9% in both Benelux and UK NEQAS cohorts. In contrast, the lowest between-laboratory CVs using dual-platform techniques were 17% (Benelux) and 21% (UK NEQAS), respectively., Conclusion: The single-platform ISHAGE method for CD34+ cell enumeration has been validated by an international group of 36 laboratories. The observed varation between laboratories allows a meaningful comparison of CD34+ cell enumeration.

Background: Major causes of interlaboratory variation in low-level WBC counting are the gating strategies and staining methods employed. To overcome these limitations, a stable low-level WBC control preparation (termed daily run control [DRC]) was developed that when coupled with a new gating strategy will enable international standardization., Study Design and Methods: Both a whole blood preparation (stability of more than 12 months; target WBC count of 20 cells/microL with defined fluorescence values) and a new gating strategy were developed and used with a staining kit (LeucoCOUNT [Becton Dickinson BioSciences] providing the basis for standardization). These were then combined and used to crosscalibrate seven different flow cytometers. After standardization with the DRC, comparative studies were undertaken with fresh samples with a WBC range of <1 to 60 cells per microL., Results: The developed gating strategy enabled the DRC WBCs to be positioned to within four channels of the expected target fluorescence 1 value (2.3% variation) and within three channels of the target fluorescence 2 value (0.7% variation) on all evaluated instruments. Subsequent analysis of any sample meant that the WBCs always occupied the same "sample space," irrespective of flow cytometer platform and without the need for repositioning of the analysis region and/or gate., Conclusion: The cross calibration and standardization of flow cytometers used for low-level WBC counting (irrespective of platform) are attainable with this United Kingdom National External Quality Assessment Scheme strategy. Its adoption should reduce interlaboratory CVs and provide a practical approach for the rapid identification of operator and machine problems.

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., (Copyright 2002 Wiley-Liss, Inc.)

We tested the feasibility and precision of affordable CD4+ T cell counting in resource-poor settings using a recently standardised fixative, TransFix in whole blood (WB) by flow cytometry (FCM). The precision of the assays was established under optimal conditions for single-platform FCM such as the volumetric CytoronAbsolute and the bead-based FACSCan. Fresh WB samples from HIV-seropositive and seronegative patients were tested in Tanzania and South Africa, fixed and sent to the UK for reanalysis 7 days later. Correlation, bias and limits of agreements were analysed by linear regression and the Bland-Altman test. Absolute CD4+ T cell counts remained stable for at least 10 days when TransFix was added to WB in 1:10 dilution at 20-25 degrees C, and for 7 days when added in 1:10 or 1:5 dilution to samples stored to mimic 'tropical' conditions at 37 degrees C. Higher temperatures such as 42 degrees C were tolerated for only short periods since the recovery had decreased to 63% by day 3. The reproducibility of lymphocyte subset analysis remained unchanged by TransFix with coefficient of variations <6% for all T cell subsets. Absolute CD4+ T cell counts and CD4+ T cell % values on fixed samples in the UK showed a high correlation with the results using fresh samples in Tanzania (r=0.993 and 0.969, respectively) and with the samples handled in Johannesburg (r=0.991 and 0.981) with minimal bias. Primary CD4 gating using only a single CD4 antibody also remained accurate in TransFixed samples (r=0.999). Thus, TransFix permits optimal fixation and transport of WB samples in the developing world for FCM to local regional laboratories and for quality assurance in international centres. When used together with inexpensive primary CD4 gating, TransFix will allow reliable and affordable CD4+ T cell counting by FCM in resource-poor settings.

As quantitative flow cytometry is being increasingly used to characterize non-malignant and malignant disorders, interlaboratory standardization becomes an important issue. However, the lack of standardized methods and process controls with predefined antibody binding capacity values, limits direct interlaboratory comparison. The present study has addressed these issues using a stable whole blood product and a standardized antigen quantification protocol. It was demonstrated that: (i) a standard technical protocol can result in a high degree of interlaboratory concordance; (ii) interlaboratory variation of less than 12% can be achieved for CD4 antibody binding capacity values; and (iii) stable whole blood can be used as a process control with predefined antibody binding capacity values. Furthermore, using such an approach, a normal range was established for CD3, CD4 CD8 and CD19. These antigens appear to be expressed in a hierarchical manner, a factor that could be used as a procedural quality control measure.

The European Working Group on Clinical Cell Analysis (EWGCCA) has, in preparation for a multicentre peripheral blood stem cell clinical trial, developed a single-platform flow cytometric protocol for the enumeration of CD34+ stem cells. Using this protocol, stabilized blood and targeted training, the EWGCCA have attempted to standardize CD34+ stem cell enumeration across 24 clinical sites. Results were directly compared with participants in the UK National External Quality Assessment Scheme (NEQAS) for CD34+ Stem Cell Quantification that analysed the same specimens using non-standardized methods. Two bead-counting systems, Flow-Count and TruCount, were also evaluated by the EWGCCA participants during trials 2 and 3. Using Flow-Count, the intralaboratory coefficient of variation (CV) was

To determine the potential advantage of single-platform technology in the enumeration of CD4+ T lymphocyte and CD34+ stem cells, data has been analysed from the UK NEQAS for Leucocyte Immunophenotyping schemes. The inter-laboratory CVs for CD4+ T lymphocyte counts were consistently lower for single-platform (mean 13.7%, range 10-18.3%) compared to dual-platform methodology (mean 23.4%, range 14.5-43.7%). Subgroup analysis of single-platform users demonstrated mean overall inter-laboratory CVs of 17.2%, 13% and 7.1% for the FlowCount, TruCount and volumetric approach respectively. The lowest inter-laboratory CVs obtained for a single sample by each single platform approach were 4% (TruCount), 4.4% (volumetric), 4.6% (FACSCount) and 12.7% (FlowCount). Similarly, the mean inter-laboratory CV for CD34+ stem cell enumeration using non-standardized single-platform approaches was 18.6% (range 3.1-36.9%) compared to 28.6% (range 19-44.2%) for the dual-platform technology. Our results suggest absolute cell subset enumeration should be performed by single-platform technology and that such an approach should improve the quality control of multi-centre clinical trial data for CD4+ T lymphocyte and CD34+ stem cells.

Summary. The European Working Group on Clinical Cell Analysis (EWGCCA) has, in preparation for a multicentre peripheral blood stem cell clinical trial, developed a single-platform flow cytometric protocol for the enumeration of CD34[sup +] stem cells. Using this protocol, stabilized blood and targeted training, the EWGCCA have attempted to standardize CD34[sup +] stem cell enumeration across 24 clinical sites. Results were directly compared with participants in the UK National External Quality Assessment Scheme (NEQAS) for CD34[sup +] Stem Cell Quantification that analysed the same specimens using non-standardized methods. Two bead-counting systems, Flow-Count and TruCount, were also evaluated by the EWGCCA participants during trials 2 and 3. Using Flow-Count, the intralaboratory coefficient of variation (CV) was less than or equal to 5% in 39% of the laboratories (trial 1), increasing to 65% by trial 3. Interlaboratory variation was reduced from 23.3% (trial 1) to 10.8% in trial 3. In trial 2, 70% of laboratories achieved an intralaboratory CV less than or equal to 5% using TruCount, increasing to 74% for trial 3; the interlaboratory CV was reduced from 23.4% to 9.5%. Comparative analysis of the EWGCCA and the UK NEQAS cohorts revealed that EWGCCA laboratories, using the standardized approach, had lower interlaboratory variation. Thus, the use of a common standardized protocol and targeted training significantly reduced intra-and interlaboratory CD34[sup +] cell count variation. [ABSTRACT FROM AUTHOR]

Abstract: In this report, we have evaluated the effects of a TransFix®-based stabilisation technique on leukocyte scatter characteristics, immunophenotyping, membrane permeability, absolute cell counting and morphology to extend previously reported flow cytometric data focused on the lymphocyte population. We show that scatter characteristics, immunophenotyping and absolute cell counting are well preserved, particularly in the lymphocyte population. Nevertheless, a general increase in membrane permeability, evaluated by propidium iodide (PI) uptake, was observed in TransFix®-treated leukocyte subsets. Ultrastructural observations show selective morphological preservation (up to 10 days of storage) of lymphocytes and, to a lesser extent, of monocytes. In contrast, granulocytes have necrosis-like features, although the plasma membrane seems well preserved. Therefore, electron microscopy observations reflect modifications induced in different cell populations as evidenced by flow cytometry (FC). The data indicate that this short-term stabilisation method is particularly suitable for the analysis of human lymphocytes and it is a good procedure for quality control programmes for inter- and intra-laboratory performance evaluation; good results are obtained with respect to antigen definition and absolute cell counting procedures. Any apoptotic pathways in leukocyte subsets are blocked for at least 10 days. [Copyright &y& Elsevier]

Leucocyte counts of < 5 × 106 per blood transfusion product are currently recommended in the UK in order to reduce transfusion-related infections and febrile reactions. Routine leucocyte depletion, however, requires the development of reliable internal and external quality assurance (EQA) programmes. We report preliminary findings from the UK NEQAS for Low-Level Leucocyte Counting from 18 UK Transfusion Centres over a four month period. Data analysis showed that the IMAGN 2000 had the lowest CVs (range 7.5–36%, mean 16.7) for samples with counts of 5–30 cells/μl when compared to the flow cytometric (range 13.8–88%, mean 29.5) and Nageotte methods (range 20.6–117%, mean 61.8). In addition, laboratories using commercial nuclear stains (LeucoCOUNTTM) had consistently lower CVs than those using ‘in-house’ propidium iodide staining methods. Important differences in flow cytometric gating strategies were also identified. This study highlights the current variability in low level leucocyte counting, especially within the critical range of 5–30 cells/μl (equating to < 5 × 106/l). The acceptance of consensus protocols, including gating strategies and nuclear staining techniques, is required to reduce the observed interlaboratory variation. Finally, we demonstrate that stabilized blood preparations can be successfully used to provide a national/international low-level leucocyte EQA scheme. [ABSTRACT FROM AUTHOR]