The human immunodeficiency virus (HIV) global epidemic has necessitated the routine enumeration of T-lymphocyte subsets, which has created a need for external quality assurance (EQA). The United Kingdom National External Quality Assessment Scheme (UK NEQAS) for Immune Monitoring provides EQA for 296 laboratories in 40 countries. In 1993, UK NEQAS developed and incorporated into its program stabilized whole blood that enables the accurate monitoring of laboratory performance. Overall, the mean interlaboratory coefficient of variation (CV) for percentage CD4 ؉ T-lymphocyte subset enumeration has fallen from 15% to less than 5%, as a direct result of the increased use of CD45/ side scatter (SSC) gating. Laboratories using alternative gating strategies (i.e., CD45/CD14 or forward scatter [FSC]/SSC) were about 7.4 times more likely to fail an EQA exercise. Furthermore, the adoption of single-platform technology resulted in a reduction of the overall mean interlaboratory CV for absolute CD4 ؉ T lymphocytes from 56% (prior to the widespread use of single-platform technology) to 9.7%. Individual laboratory deficiencies were also identified using a performance monitoring system and, through re-education by collaboration with the coordinating center, satisfactorily resolved. In conclusion, during the last 9 years, the UK NEQAS for Immune Monitoring program has highlighted the significant technological advances made by laboratories worldwide that undertake lymphocyte subset enumeration. Cytometry (Clin. Cytometry) 50:102-110, 2002.

BackgroundThe phenotypic diagnosis of von Willebrand disease (VWD) is a multistep process with classification dependent on the quantification of von Willebrand factor (VWF) multimeric structure. VWF multimer analysis is a technically challenging, lengthy and non‐standardised assay, usually performed in specialist laboratories. Recently, a new semi‐automated multimer assay, the Hydragel 5 von Willebrand multimers (H5VWM) has become available.ObjectivesThis study, performed in two European centres, compared existing in‐house multimer assays to the H5VWM in individuals with and without VWD.ResultsOverall agreement of 91.1% was observed in 74 individuals with normal VWF levels, 57 patients grouped as type 1 VWD, 33 type 2A, 16 type 2B, 28 type 2M, 11 type 2N. Patients tested following Desmopressin or VWF concentrate, with thrombotic thrombocytopenic purpura and acquired von Willebrand syndrome were also evaluated. Many of the discrepancies between methods were in patients with genetic mutations linked to more than one type of VWD including p.R1374C/H and p.R1315C. Quantifiable multimer results were available within one working day. Densitometry improved the interpretation of the multimers with slight structural variations that were not apparent by visual inspection of the in‐house method.Conclusions5VWM was a rapid, sensitive, standardised assay which used existing technology and could be included as an initial screen of VWF multimers in a VWD diagnostic algorithm in conjunction with traditional multimer analysis.

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

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 costeffective 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

The pathogenic mechanisms underlying the development of microvascular complications in insulin-dependent diabetes mellitus (IDDM) remain obscure despite the fact that retinopathy develops in the majority [1][2][3], and nephropathy in around 30 % [3,4]. Although duration of diabetes and poor glycaemic control are established risk factors, they appear to account for only a proportion of an individual's risk [5]. We have recently demonstrated that, in subjects with retinopathy but not those without, mean urinary albumin excretion rate increases steeply when the mean diastolic blood pressure rises above 75 mmHg. It thus appears that members of a subgroup of diabetic subjects may have abnormal renal vulnerability Diabetologia (1997) Summary The interrelationships between fibrinogen, von Willebrand factor, a marker of vascular endothelial cell damage, and serum lipids were explored in well-characterised subjects with insulin-dependent diabetes mellitus. The 2091 subjects were enrolled into a cross-sectional, clinic-based study of complications, from 16 European countries: the EURODIAB IDDM Complications study. The anticipated significant relationships between both plasma fibrinogen and plasma von Willebrand factor concentrations and age and glycaemic control, and between fibrinogen and body mass index, were noted. Fibrinogen, adjusted for age and glycated haemoglobin concentration, was also related to smoking habits and was higher in the quartiles with highest systolic and diastolic blood pressures. There was a clustering of vascular risk factors, with a positive relationship between plasma fibrinogen and serum triglyceride concentrations in both genders and between fibrinogen and total cholesterol in males. An inverse relationship between fibrinogen and high density lipoprotein cholesterol was also apparent in males. A prominent feature was a positive relationship between both fibrinogen and von Willebrand factor and albumin excretion rate (p < 0.001 and p < 0.003 respectively) in those with retinopathy but not in those without this complication. In view of previous observations on blood pressure and albuminuria in these subjects the findings are consistent with the hypothesis that microalbuminuria and increased plasma von Willebrand factor are due to endothelial cell perturbation in response to mildly raised blood pressure in subjects with retinopathy. Fibrinogen may also contribute to microvascular disease and its relationships to lipid vascular risk factors suggest a possible pathogenic role in arterial disease in diabetes. [Diabetologia (1997) to mildly raised blood pressure and that retinopathy is a close correlate of this risk [6].von Willebrand factor (vWF) is synthesised by vascular endothelial cells and megakaryocytes and an increased concentration in plasma can serve as a marker of endothelial cell damage [7][8][9]. Such an increase is a feature of diabetic nephropathy [10]. Recent studies in small numbers of subjects with IDDM suggest that vWF is a sensitive marker of incipient nephropathy and that a r...