An increased MRP8/14 expression and adhesion, but a decreased migration towards proinflammatory chemokines of type 1 diabetes monocytes.

In the early development of type 1 diabetes macrophages and dendritic cells accumulate around the islets of Langerhans at sites of fibronectin expression. It is thought that these macrophages and dendritic cells are derived from blood monocytes. Previously, we showed an increased serum level of MRP8/14 in type 1 diabetes patients that induced healthy monocytes to adhere more strongly to fibronectin (FN). Here we show that MRP8/14 is expressed and produced at a higher level by type 1 diabetes monocytes, particularly after adhesion to FN, creating a positive feedback mechanism for a high fibronectin-adhesive capacity. Also adhesion to endothelial cells was increased in type 1 diabetes monocytes. Despite this increased adhesion the transendothelial migration of monocytes of type 1 diabetes patients was decreased towards the proinflammatory chemokines CCL2 and CCL3. Because non-obese diabetic (NOD) mouse monocytes show a similar defective proinflammatory migration, we argue that an impaired monocyte migration towards proinflammatory chemokines might be a hallmark of autoimmune diabetes. This hampered monocyte response to proinflammatory chemokines questions whether the early macrophage and dendritic cell accumulation in the diabetic pancreas originates from an inflammatory-driven influx of monocytes. We also show that the migration of type 1 diabetes monocytes towards the lymphoid tissue-related CCL19 was increased and correlated with an increased CCR7 surface expression on the monocytes. Because NOD mice show a high expression of these lymphoid tissue-related chemokines in the early pancreas it is more likely that the early macrophage and dendritic cell accumulation in the diabetic pancreas is related to an aberrant high expression of lymphoid tissue-related chemokines in the pancreas.

Crossover study of three methods for low-level white blood cell counting on two types of flow cytometer.

BACKGROUND: Flow cytometric methods were previously shown to be preferable to microscopic and volumetric methods for counting residual white blood cells (WBCs). In this study, three flow cytometric, low-level WBC counting methods were cross compared using two flow cytometers. METHODS: Double-filtered red cell and platelet concentrates were spiked with different amounts of WBC to obtain panels of unspiked and 0.3, 1.0, 3.3, and 10.0 WBC/microl. The methods of BD Biosciences (BDB), Beckman-Coulter (BC), and an in-house method were performed on flow cytometers from BDB and BC. Samples were measured in ninefold. We required that (a) r(2) be at least 0.98 (linearity), (b) at least 80% of observations fell within 20% of expected values (accuracy), and (c) the coefficients of variation be at least 20% (precision) for samples containing at least 3.3 WBC/microl. RESULTS: For the red cell panel, our requirements were met by the BDB method on both flow cytometers and by the BC and in-house methods on the BDB flow cytometer only. For the platelet panel, our requirements were met on all combinations of methods and flow cytometers, except for the in-house method on the BDB flow cytometer. Intra-assay variation was lowest for the BDB method, irrespective of the type of flow cytometer used. CONCLUSION: Based on accuracy and precision, the BDB method on the BDB flow cytometer produced the best results for counting low-level WBCs.

Comparison of five platforms for enumeration of residual leucocytes in leucoreduced blood components.

The need for quality control of leucoreduction of blood products has led to the development of various methods to count low levels of residual leucocytes. We compared five platforms side-by-side: the Nageotte haemocytometer and four based on fluorescent staining of nuclei: two flowcytometers (Beckman Coulter, BD Biosciences) with methods based on counting beads, a volumetric flow cytometer (Partec) and the microvolumic fluorimeter ImagN2000 (BD Biosciences), all according to their manufacturers' recommended methods. Analysis of double-filtered red cell concentrates (RCCs) and platelet concentrates (PCs), spiked with various numbers of leucocytes, revealed good linearity for all methods over the range of 1.6-32.7 leucocytes/microl, all with r(2) > 0.99. At the rejection level of leucocyte-reduced blood components, i.e. 1 x 10(6) per unit corresponding with approximately 3.3 leucocytes/microl, the Nageotte haemocytometer had low accuracy (0% for RCCs, 56% for PCs), and was relatively imprecise [coefficient of variance (CV) of 34% and 30% respectively]. The Partec flow cytometer gave good results for RCCs (accuracy 67%, CV 22%), but not for PCs (accuracy 0%, CV 25%). The ImagN2000 had an accuracy of 44% for RCCs and 89% for PCs, but the precision was variable (CV 32% for RCCs, 15% for PCs). The best results were obtained with the Beckman Coulter (RCCs: accuracy 86%, CV 13%, PCs: accuracy 67%, CV 16%), and BD Biosciences platforms (RCCs: accuracy 100%, CV 10%; PCs: accuracy 89%, CV 11%). We conclude that, at the rejection level of 1 x 10(6) leucocytes per unit, the widely used Nageotte haemocytometer performs poorly in terms of inaccuracy and imprecision, and that both counting-bead-based, flow cytometric methods performed best.

External quality assessment of flow cytometric HLA-B27 typing.

A biannual external quality assurance (EQA) scheme for flow cytometric typing of the HLA-B27 antigen is operational in The Netherlands and Belgium since 1995. We report here on the results of the first seven send-outs to which 36 to 47 laboratories participated. With the send-out, four specimens from blood bank donors, who had been typed for HLA Class I antigens by complement-dependent cytotoxicity, were distributed. Subtyping of the HLA-B27 allele was performed by PCR-SSP. Ten samples were HLA-B27(pos) (all HLA-B*2705) and 18 were HLA-B27(neg). For flow cytometry, the most widely monoclonal antibody (MoAb) used was FD705, followed by GS145.2 and ABC-m3. The majority of laboratories used more than 1 anti-HLA-B27 MoAb for typing. The HLA-B27(pos) samples were correctly classified as positive by the large majority of participants (median 95%; range 85% to 100% per send out); some participants considered further typing necessary and misclassification as negative was only sporadically seen. The classification of HLA-B27(neg) samples as negative was less straightforward. Ten samples were correctly classified as such by 97% (82% to 100%) of the participants, whereas 64% (range 53% to 70%) of the participants classified the remaining eight samples as HLA-B27(neg). There was no significant prevalence of a particular HLA-B allele among these eight "poor concordancy" samples as compared to the ten "good concordancy" samples. Inspection of the reactivity patterns of the individual MoAb with HLA-B27(neg) samples revealed that ABC-m3 showed very little cross-reactivity apart from its well-known cross-reactivity with HLA-B7, whereas the cross-reactivity patterns of GS145.2 and FD705 were more extensive. The small sample size (n = 18) and the distribution of HLA-B alleles other than HLA-B27 did not allow assignment of specificities to these cross-reactions. Finally, we showed that standardized interpretation of the combined results of two anti-HLA-B27 MoAb reduced the frequency of false-positive conclusions on HLA-B27(neg) samples. In this series, the lowest frequency of false-positive assignments was observed with the combination of the FD705 and ABC-m3 MoAb.

Comparison of single and dual-platform assay formats for CD34+ haematopoietic progenitor cell enumeration.

Most techniques for CD34+ cell enumeration are dual platform assays. That is, they derive absolute numbers of CD34+ cells from either the flow cytometrically assessed per cent (%) CD34+ cells within the nucleated cells and/or the white blood cell count from a haematology cell analyser. Recently, so-called single-platform assays have been developed, in which the absolute number of CD34+ cells is directly derived from a single flow cytometric measurement. The present study aims to compare the variation between eight laboratories in CD34+ cell counts from paired assays of 15 samples using a common single (ProCOUNT) and the local dual-platform method. Six laboratories used the 'SIHON' and two the 'ISHAGE' protocol for CD34+ cell enumeration. Use of the single-platform method reduced the inter-laboratory variation in per cent and absolute numbers of CD34+ cells, as measured by interquartile ranges, by half but did not lead to an appreciable reduction of the inter-laboratory variation in white blood cell counts. Thus, part of the reduced inter-laboratory variation obtained with ProCOUNT may have been a result of the use of standardized procedures and reagents to detect CD34+ cells. In order to eliminate any variation arising from the use of different local protocols for percentage of CD34+ cell assessments, a comparison was made of the ProCOUNT-derived absolute CD34+ cell numbers (i.e. single platform) with the dual-platform absolute CD34+ cell numbers calculated by multiplying ProCOUNT-derived percentage of CD34+ cells and with the corresponding haematology analyser-derived white blood cell count. Regardless, the interquartile ranges of absolute CD34+ cell numbers remained almost a factor of two smaller with the use of the single platform method. Thus, these results suggest that single-platform methodology can reduce the variation in absolute CD34+ cell numbers between laboratories.