UF-100 CHECK液前向散射光强度失控对尿沉渣红细胞光学指标的影响

目的 观察UF-100尿沉渣自动分析仪CHECK液前向散射光强度(FSC)失控对尿液样本红细胞FSC和P70FSC指标的影响.方法 使用UF-100 CHECK液对UF-100进行校准,并于检测前后分别测试70份尿液样本和50份用生理盐水稀释的全血质控物样本并记录校准前37 d和校准后23 d UF-100质量控制结果,使用SPSS11.0对所得数据进行统计学分析.结果 UF-100 FSC失控可在五项定量指标在控情况下出现;校准前后红细胞光学指标FSC、P70FSC和FSC-SDW存在统计学差异;对尿液样本的线性分析可见校准后FSC和P70FSC较校准前分别出现 10.85和 21.27ch漂移.结论 UF-100 CHECK液FSC减低失控提示系统所测定尿液样本红细胞FSC和P70FSC测定值会出现漂移;UF-100质量控制前向散射光强度的减低,势必造成UF-100系统对尿液颗粒的鉴别能力降低;FSC质量控制是UF-100在肾性血尿的鉴别诊断中应用的前提.     

Screening for WBC antibodies by lymphocyte indirect immunofluorescence flow cytometry: superior to cytotoxicity and ELISA?

BACKGROUND: Cytotoxic WBC antibodies are found in patients who have refractoriness to platelet transfusion (RPT) or are experiencing febrile transfusion reactions (FTRs) and in sera giving so called nonspecific hemagglutination by IAT (N/S IAT). Sera from such patients were screened for WBC antibodies regardless of the ability to fix complement using a flow cytometric (FC) lymphocyte indirect immunofluorescence test (LIFT) to compare FC-LIFT with a routine lymphocytotoxicity test (LCT) for WBC antibody detection. STUDY DESIGN AND METHODS: Serum from 104 patients with RPT, 87 with FTR, and 147 with N/S IAT were tested in parallel by using FC-LIFT and LCT. Sera giving discrepant results were re-tested with an HLA class I antibody ELISA to assess whether they were HLA-specific. RESULTS: Of the sera tested, 175 were LIFT positive, and 146 were LCT positive. Fifty-five had antibodies that were detectable only by LIFT; 26 were positive only by LCT. Of these 81 discrepant sera, 30 of 63 were positive in HLA ELISA. CONCLUSION: FC-LIFT detects more WBC antibodies than does LCT or ELISA, and it is a superior screening technique. Because some cytotoxic antibodies are detectable only by LCT, comprehensive WBC antibody screening would require the application of both techniques. However, because FC assessments of cytotoxicity have been described, LCTs may become redundant for WBC antibody screening.     

Analysis of factors affecting quantification of fetomaternal hemorrhage by flow cytometry

BACKGROUND: An analysis was carried out to determine the sources and extent of errors encountered in the quantitation of the volume of fetomaternal hemorrhage (FMH) by flow cytometry. Different assay conditions were compared, to define the simplest, most accurate protocol. STUDY DESIGN AND METHODS: D-, D+, and artificial FMH (mixtures of D+ and D- RBCs) were stained either by a direct method (using FITC-conjugated IgG3 D MoAb [BRAD-3]), with or without dual labeling with PE-conjugated anti-GPA, or by indirect methods (using polyclonal anti-D followed by FITC- or biotin-conjugated anti-IgG reagents). Cells were selected for flow cytometric analysis on the basis of either forward or side scatter (log FSC/log SSC) characteristics or of GPA+ labeling or were unselected. The numbers of events labeled with anti-D were determined from histograms. For some samples, 10 replicates of 500,000 events each were analyzed. RESULTS: Background fluorescent events in 10 directly labeled gated D- samples ranged from 0.007 to 0.023 percent, equivalent to 0.15- to 0.51-mL FMH. Both the use of a gate on log FSC/SSC or the selection of GPA+ events only resulted in a reduction in FMH of 0.3 mL or less. The intra-assay variation in FMH, or sampling error, was found to be approximately 10 percent at low artificial FMH (<10 mL) but greater (< or =50% with a CV of 15%) with D- samples. Direct staining was quicker and produced a lower background than indirect staining. CONCLUSION: The inherent sampling error that is due to the random distribution of rare events throughout the blood sample contributed greatly to the variation in the volume of FMH calculated by flow cytometry. The FMH should not be underestimated. For a routine assay, a simplified protocol and calculation will be sufficiently accurate to determine the dose of prophylactic anti-D that should be given to the patient.     

Flow cytometric assessment of estrogen receptor beta expression in bovine blood neutrophils

The optimisation of a flow cytometric protocol for the determination of the estrogen receptor beta (ERbeta) expression in bovine blood neutrophils is described. The following final incubation conditions were obtained: fixation with 0.25% formaldehyde and 70% methanol, both for 1 h; permeabilisation with 0.05% Triton X-100, overnight labelling at 4 degrees C with the primary antibody diluted at 10 microg/ml and subsequent labelling for 30 min on ice with the fluorescein isothiocyanate-conjugated secondary antibody at 8 microg/ml. Of the three anti-human or anti-rat ERbeta primary antibodies evaluated, only PA1-311 was found to cross-react with bovine cells. Immunoblot analysis supports the obtained results. The flow cytometric technique allows reproducible quantitative determination of the ERbeta protein in neutrophils and may be a valuable tool for future expression studies in these cells of the innate immune system.     

Leukocytes and drug-resistant cancer cells are targets for intracellular delivery by adenoviral dodecahedron

One of the major factors limiting the effectiveness of cancer chemotherapy is inefficient drug delivery. Systems enabling efficient delivery and enhanced intracellular uptake appear particularly promising in this respect. Virus-like particle, adenoviral dodecahedron (Dd), employs receptor-mediated endocytosis for cell penetration and is able to deliver intracellularly dozens of cargo molecules attached to one particle. We focused on studying Dd properties in the context of cancer treatment, showing that intratumoral injection of Dd, assessed in mouse xenograft model, results in vector accumulation in tumor without spreading in off-target organs. Moreover, we demonstrated that Dd is a promising vector targeting leukocytes and drug-resistant cancer cells. Dd uptake by human blood cells analyzed in vitro indicated the preference for leukocytes in comparison to red blood cells and platelets. Furthermore, internalization of Dd-doxorubicin conjugate by drug-resistant cells leads to increased nuclear accumulation of doxorubicin and significant enhancement of cytotoxicity against target cancer cells.     

Antigen-specific responses assessment for the evaluation of Bordetella pertussis T cell immunity in humans

Measurement of antigen-specific T cell responses is an adjunctive parameter to evaluate protection induced by a previous Bordetella pertussis infection or vaccination. The assessment of T cell responses is technically complex and usually performed on fresh peripheral blood mononuclear cells (PBMC). The objective of this study was to identify simplified methods to assess pertussis specific T cell responses and verify if these assays could be performed using frozen/thawed (frozen) PBMC. Three read-outs to measure proliferation were compared: the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) dilution test, the number of blast cells defined by physical parameters, and the incorporation of (3)H-thymidine. The results of pertussis-specific assays performed on fresh PBMC were compared to the results on frozen PBMC from the same donor. High concordance was obtained when the results of CFSE and blast read-outs were compared, an encouraging result since blast analysis allows the identification of proliferating cells and does not require any use of radioactive tracer as well as any staining. The results obtained using fresh and frozen PBMC from the same donor in the different T cell assays, including IFNγ and TNFα cytokine production, did not show significant differences, suggesting that a careful cryopreservation process of PBMC would not significantly influence T cell response evaluation. Adopting blast analysis and frozen PBMC, the possibility to test T cell responses is simplified and might be applied in population studies, providing for new instruments to better define correlates of protection still elusive in pertussis.     

Human CD8(+) T cells and NK cells express and secrete S100B upon stimulation

Previous studies have demonstrated the utility of S100B as a surrogate marker of brain-related pathologies, e.g. neuropsychiatric disorders, and melanoma progression, which have an inflammatory component. This study addresses the relevance of S100B⁺ lymphocytes in mediating such responses. S100B expression was determined in human peripheral blood leukocytes isolated from healthy volunteers using flow cytometry. S100B⁺ lymphocytes were characterised for phenotype, cytokine production and S100B secretion. In addition, we investigated whether S100B activates monocytes and neutrophils.S100B⁺ cells comprised 2-4% of all lymphocytes and the majority displayed a CD3⁺ CD8⁺ phenotype; fewer cells were CD3⁻ CD56⁺ NK lymphocytes. Comparison of S100B⁺ and S100B⁻ CD3⁺ CD8⁺ cells revealed no differences in production of interferon gamma (IFNγ) and interleukin-2 (IL-2). Stimulation of S100B⁺ CD3⁺ CD8⁺ lymphocytes with anti-CD3 or phytohaemagglutinin resulted in release of S100B. High concentrations of recombinant human S100B triggered upregulation of CD11b and membrane shedding of CD62L in granulocytes and monocytes.These findings set the stage for a new field of research addressing a S100B-mediated crosstalk between the innate and adaptive immune systems if close proximity of effector and responder cells accomplishes sufficient local S100B levels. In various physiological and pathological conditions S100B might function as an interface to immunological processes, distinct from known cytokine- and chemokine-mediated pathways.