Springer Nora L, Smith Eliza, Brooks Marjory B, Stokol Tracy
Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.
Am J Vet Res. 2014 Oct;75(10):879-85. doi: 10.2460/ajvr.75.10.879.
To develop a flow cytometric assay to quantify platelet-derived microparticles (PMPs) in equine whole blood and plasma.
Citrate-anticoagulated whole blood from 30 healthy adult horses.
Platelet-poor plasma (PPP) was prepared from fresh whole blood by sequential low-speed centrifugation (twice at 2,500 × g). Samples of fresh whole blood and PPP were removed and stored at 4° and 24°C for 24 hours. Platelet-derived microparticles were characterized in fresh and stored samples on the basis of the forward scatter threshold (log forward scatter < 10(1)) and labeling with annexin V (indicating externalized phosphatidylserine) and CD61 (a constitutive platelet receptor). A fluorescent bead-calibrated flow cytometric assay was used to determine microparticle counts. Platelet counts, prothrombin time, and activated partial thromboplastin time were measured in fresh samples.
Significantly more PMPs were detected in fresh whole blood (median, 3,062 PMPs/μL; range, 954 to 13,531 PMPs/μL) than in fresh PPP (median, 247 PMPs/μL; range, 104 to 918 PMPs/μL). Storage at either temperature had no significant effect on PMP counts for whole blood or PPP. No significant correlation was observed between PMP counts and platelet counts in fresh whole blood or PPP or between PMP counts and clotting times in fresh PPP.
Results indicated that the described PMP protocol can be readily used to quantify PMPs in equine blood and plasma via flow cytometry. Quantification can be performed in fresh PPP or whole blood or samples stored refrigerated or at room temperature for 24 hours.
开发一种流式细胞术检测方法，用于定量马全血和血浆中血小板衍生微粒（PMPs）。
来自30匹健康成年马的枸橼酸盐抗凝全血。
通过连续低速离心（2500×g，两次）从新鲜全血中制备乏血小板血浆（PPP）。采集新鲜全血和PPP样本，分别在4℃和24℃保存24小时。根据前向散射阈值（对数前向散射<10(1)）以及用膜联蛋白V（表明磷脂酰丝氨酸外化）和CD61（一种血小板组成性受体）标记，对新鲜和保存样本中的血小板衍生微粒进行表征。使用荧光微球校准的流式细胞术检测方法确定微粒计数。在新鲜样本中测量血小板计数、凝血酶原时间和活化部分凝血活酶时间。
新鲜全血中检测到的PMPs显著多于新鲜PPP（中位数，3062个PMPs/μL；范围，954至13531个PMPs/μL）（中位数，247个PMPs/μL；范围，104至918个PMPs/μL）。在任何一个温度下保存对全血或PPP中的PMP计数均无显著影响。在新鲜全血或PPP中，PMP计数与血小板计数之间，以及在新鲜PPP中PMP计数与凝血时间之间均未观察到显著相关性。
结果表明，所描述的PMP检测方案可通过流式细胞术轻松用于定量马血液和血浆中的PMPs。定量可在新鲜PPP、全血或冷藏或室温保存24小时的样本中进行。
