流式细胞术检测血小板-单核细胞聚集的影响因素

Efficient measurement of platelet-monocyte aggregates in whole blood by flow cytometry

目的探讨流式细胞术检测全血血小板单核细胞聚集(PMAs)的影响因素。方法分别采用枸橼酸钠和K2EDTA作为抗凝剂,多聚甲醛为固定剂,静脉采集9名志愿者全血,室温和4℃条件下放置不同时间。采用抗CD14PE单克隆抗体标记单核细胞,抗FITCCD42a单克隆抗体标记血小板,流式细胞仪测定各研究条件下PMAs占各自单核细胞总数的百分比。结果在室温和4℃条件下,6h内5mmol/LK2EDTA和0.5%的多聚甲醛不会增加体外PMAs的形成,但它们均能使已形成的PMAs减少。枸橼酸钠抗凝条件下,体外PMAs的形成随时间延长而显著增加,抽血后室温放置30min和抽血后即刻(10min)PMAs测定值之差<3%,分别为6.53%±1.75%和8.80%±2.33%(P<0.05)。结论体外全血PMAs测定易受多种因素影响,抗凝剂枸橼酸钠不影响PMAs的形成及其稳定性,采用枸橼酸钠抗凝需在抽血后短时间内(<30min)测定PMAs。

Objective To study the influence of the measurement of the platelet-monocyte aggregates （PMAs） by using of flow cytometry （FCM）. Methods Anticoagulated peripheral venous bloods from nine healthy donors were incubated with a PE-CD14 MAb （ monocyte marker） and a FITC-CD42a MAb （ platelet marker） for 20 min and the formations of PMAs were measured by use of FCM. The factors such as fixative, anticoagulant, storage time and temperature were analyzed. Results The PMAs of citrated whole blood increased with the time elapsed in 6 h after blood drawing when they were stored in the room temperature. The PMAs of each time point showed significant difference （ P 〈 0. 05 ）. Furthermore, the citrated samples stored in 4℃ caused more PMAs formation. The formation of PMAs did not increase with the time and temperature changed when the whole blood were anticoagulated with K2 EDTA or citrate sodium added with 0. 5% Paraformaldehyde （PFA）. However, both of them can cause the PMAs disaggregate, which may affect the measurement of the PMAs formed in vivo. Conclusions The citrate sodium may not influence the formation and stability of the PMAs. Measuring the citrated whole blood quickly after drawing the blood appears to reflect closely the PMAs in vivo.