Red cell microparticle enumeration: validation of a flow cytometric approach.
Red cell microparticle enumeration: validation of a flow cytometric approach.
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DOI：
10.1111/j.1423-0410.2011.01577.x
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发表时间：
2012-07
期刊：
影响因子：
2.7
通讯作者：
Lee JS
中科院分区：
文献类型：
--
作者：
Xiong Z;Oriss TB;Cavaretta JP;Rosengart MR;Lee JS
红细胞（RBC）微粒（MP）计数的临床应用越来越受到关注，因为它们被认为是输血后和镰状细胞病中凝血和炎症的效应物。在MP计数方面不存在统一的方法，一个关键的限制是缺乏内部验证过程。我们提出并验证了流式细胞术的方法，其中利用内标。使用从RBC单位或从志愿者获得的血浆样品中分离的MP计数血型糖蛋白A+膜联蛋白V+事件。将绝对计数（7 μm）和校准珠（0 μ m 5、0 μ m 9和3 μm）以固定比例的混合物加入每份样品中。最初基于荧光阈值选择RBC MP，并且0.5-μ m和0.9-μm珠定义MP的上和下光散射分布。使用7 μ m 6：3 μm微珠事件的比例作为内标，以验证样品间MP计数的精密度（变异系数= 2.5 - 7.2%），并在富血小板血浆（PRP）和无血小板血浆（PFP）中保持恒定。从用离子载体和增加的钙浓度刺激的全血中获得的PRP和PFP中的RBC MP计数增加，在研究的每个浓度下，PRP显示出比PFP更高的MP计数。该方法是检测生物样本中RBC MP计数的有用策略，前提是流式细胞仪可以可靠地区分校准微珠的大小。
There is growing interest in the clinical application of red blood cell (RBC) microparticle (MP) enumeration as they have been postulated to be effectors of coagulation and inflammation following transfusion and in sickle cell disease. No uniform approach in MP enumeration exists and a key limitation is the lack of an internal validation process. We present and validate a flow cytometric approach where an internal standard is utilized. Glycophorin A+ Annexin V+ events were enumerated using MPs isolated from RBC units or plasma samples obtained from volunteers. A mixture of absolute counting (7Æ6 μm) and calibration beads (0Æ5, 0Æ9 and 3 μm) at a fixed ratio was added to each sample. RBC MPs were initially selected based upon a fluorescence threshold, and the 0Æ5- and 0Æ9-μm beads defined the upper and lower light scatter distribution of MPs. The ratio of 7Æ6:3-μm bead events was used as an internal standard to validate the precision of MP enumeration across samples (coefficient of variation = 2·5–7·2%) and remained constant in both platelet-rich plasma (PRP) and platelet-free plasma (PFP). RBC MP counts increased in both PRP and PFP obtained from whole blood stimulated with ionophore and increasing calcium concentrations, with PRP showing higher MP counts than PFP at every concentration studied. This method is a useful strategy to detect RBC MP counts across bio-samples provided that the flow cytometer can reliably discriminate the size of the calibration beads.