Minimum information to report about a flow cytometry experiment on extracellular vesicles: Communication from the ISTH SSC subcommittee on vascular biology

The Extracellular Vesicle Flow Cytometry Working Group ( http://www.evflowcytometry.org ) is formed by members of the International Society for Extracellular Vesicles (ISEV), the International Society for Advancement of Cytometry (ISAC), and the International Society on Thrombosis and Haemostasis (ISTH). This working group of flow cytometry experts develops guidelines for best practices regarding flow cytometry detection of extracellular vesicles. To improve rigor and standardization, this working group published a framework outlining the minimal information to report about a flow cytometry experiment on extracellular vesicles (MIFlowCyt-EV) in the Journal of Extracellular Vesicles, the ISEV journal, in 2020. In parallel, an article explaining MIFlowCyt-EV was published in Cytometry Part A, one of the ISAC journals, and now will be introduced to the ISTH as an SSC Communication in the Journal of Thrombosis and Haemostasis. The goal of this SSC Communication is to explain why flow cytometry is becoming the instrument of choice to characterize single extracellular vesicles, the obstacles that have been identified and (mostly) overcome by developing procedures to calibrate flow cytometers, and the relevance of reporting minimal information to improve reliability and reproducibility of experiments in which flow cytometers are used for characterization of extracellular vesicles.     

非阻塞性冠状动脉粥样硬化患者血管内皮功能与左室舒张功能的关系

目的 探讨非阻塞性冠状动脉粥样硬化患者血管内皮功能与左室舒张功能的关系。方法 选取2013.6～2015.6宣武医院心脏内科收治的因胸痛疑诊冠心病、经冠脉造影检查证实为非阻塞性冠状动脉粥样硬化症（冠状动脉狭窄＜50%）患者119例，其中男性55例，女性64例，年龄31～85岁，平均年龄60岁。专业培训人员采用标准化问卷调查记录患者临床资料，行超声心动图和肱动脉血流介导内皮依赖性血管舒张功能（FMD）检测，分为FMD正常组（FMD＞10%）和减低组（FMD≤10%）。结果 两组人群基本临床资料无显著性差异（P值均＞0.05）。左室结构指标室间隔厚度、左室后壁厚度、左室舒张末内径、左室质量指数两组间比较无显著性差异（P值均＞0.05）。左室收缩功能指标每搏输出量、心输出量、左室短轴缩短率及射血分数组间比较也无显著性差异（P值均＞0.05）。FMD降低组较FMD正常组E/A比值显著减低（P＜0.001）、E/e’比值显著升高（P=0.006），控制年龄等影响因素，偏相关分析显示FMD与E/A比值存在显著正相关（r=0.261，P=0.005），FMD与E/e’呈显著负相关（r=-0.203，P=0.033）。结论 在非阻塞性冠状动脉粥样硬化人群中，内皮功能与左室舒张功能密切相关，内皮功能不全患者左室舒张功能明显减退。     

清热解毒、散风除翳法对细菌性角膜炎的作用机制研究

目的:观察清热解毒、散风除翳法对细菌性角膜炎模型鼠的疗效及其作用机制。方法:将60只Wistar大鼠随机分为5组,即模型组、小剂量组、大剂量组、对照组以及空白组，每组12只大鼠。采用细菌性角膜炎动物模型症状评定观察清热解毒、散风除翳法的疗效,通过细菌清除率、角膜细菌菌落计数,光学显微镜观察大鼠角膜的病理组织学改变,并采用流式细胞仪检测CD4+,CD8+及CD4+/CD8+的水平讨论清热解毒、散风除翳法的作用机制。结果:1)清热解毒、散风除翳法可降低细菌性角膜炎性反应状评分,提高细菌清除率,改善角膜基质层的病理结构;2)清热解毒、散风除翳法可提高细菌性角膜炎模型鼠外周血血CD4+、CD8+的表达水平,具有剂量依赖性(P<0.05),CD4+/CD8+比值相对稳定,各组间差异无统计学意义(P>0.05)。结论:清热解毒,散风除翳法能有效改善细菌性角膜炎,其作用机制可能与上调大鼠外周血中CD4+,CD8+含量有关。     

Quantitative application of flow cytometry for the analysis of circulating human T cells: A preclinical pharmacokinetic study

As the application of flow cytometry to a quantitative pharmacokinetic study with adoptive T cell therapy is new, we aimed to investigate the quantitativity of flow cytometry-based analysis for the pharmacokinetic assessment of circulating human T cells in a preclinical study.We evaluated the selectivity, linearity, accuracy, precision, and sensitivity of flow cytometry-based analysis for human CD8⁺ T cells in immunodeficient mouse blood. The CD3/8/45-positive cell population was successfully distinguished from the negative population. Linear regression analysis for the calibration curve showed good linearity and recovery was approximately 100%. Acceptable inter- and intra-day precision and accuracy were observed and the lower limit of quantification (30 cells/50 μL) was validated with acceptable precision and accuracy. Blood concentrations of human CD8⁺ T cells in immunodeficient mice were then evaluated after administration using this method and the time-concentration profile of human T cells in mice was successfully assessed.The present study is the first to clarify the quantitativity of flow cytometry-based analysis for circulating human T cells in animals. The concept of the present study would be applicable to quantitative pharmacokinetics/efficacy or safety analysis of adoptive T cell therapy.     

Variable HLA expression on deceased donor lymphocytes: Not all crossmatches are created equal

Flow cytometric crossmatch tests are used to detect donor-specific antibody and determine eligibility for transplantation. Crossmatch sensitivity is dependent on lymphocyte quality, to include HLA expression on the cell surface. The impact of HLA expression variability on crossmatch reactivity was examined using lymphocytes isolated from different donor types: deceased donor (DD) versus living donor (LD) and tissue sources (blood, spleen, or lymph nodes). HLA class I expression was similar on B cells isolated from LD blood, DD spleen, and DD lymph nodes, but significantly lower on B cells isolated from DD blood (p = 0.0004). In contrast, class II expression on B cells and class I on T cells were significantly higher in LD blood than all DD tissues. Within DD tissues, spleen provided the highest expression of class II on B cells and class I on T cells. HLA expression on B cells, but not T cells, was impacted by memory (CD27+) versus non-memory status. Importantly, HLA expression differences on lymphocytes isolated from the same donor but different tissues impacted crossmatch outcomes. HLA expression is impacted by multiple factors and should be routinely monitored to ensure crossmatch sensitivity and to reconcile crossmatch strength with solid phase HLA antibody analyses.     

Agreement between blood draw techniques for assessing platelet activation by flow cytometry

It is widely believed that assays of platelet activation are susceptible to preanalytical variables related to blood draw technique. We assessed platelet activation by whole blood flow cytometry and investigated the effects of: (1) drawing blood into vacuum tubes or manually aspirated syringes, and (2) discarding the first drawn blood sample (discard tube). Platelet P-selectin expression and platelet-monocyte complexes were measured by flow cytometry under both basal conditions and following stimulation with 0.1, 1, or 10 µM ADP. Bland-Altman plots demonstrated agreement between results for vacuum tube and syringe-aspirated samples with an a priori-defined clinically relevant agreement limit of 5%. Agreement of results was also observed between discard tube and second draw samples for both vacuum-driven and manually aspirated blood. We conclude that a vacuum tube or a manually-aspirated syringe can be used when assessing platelet activation by flow cytometry and that there is no need for a discard tube.