4℃保存全血对血小板形态及功能的影响

Effects of venous blood preservation at 4 ℃ on platelet morphology and function

目的探讨全血保存温度对血小板(PLT)形态及功能的影响。方法选择30名健康志愿者为研究对象,采集空腹肘静脉血于含乙二胺四乙酸二钾抗凝剂的真空管,充分抗凝,采血即刻(对照组)及4℃(4℃组)、室温保存(室温组)4、8、24h制备血涂片,进行瑞氏-姬姆萨复合染色液染色,显微镜下观察血小板的形态、结构;并使用血细胞分析仪检测对照组及4℃组、室温组4、8、24h的PLT数量、大血小板比例(P-LCR)、平均血小板体积(MPV)和血小板分布宽度(PDW);流式细胞术检测对照组及4℃组、室温组4、24hPLT表面活化标志物CD62P的表达;全波长扫描式多功能读数仪检测对照组及4℃组、室温组4、24hPLT内Ca^2+浓度。结果随着保存时间的延长,4℃组和室温组PLT体积增大,且室温组PLT体积增大更加明显,并出现空泡现象,PLT结构较疏松。与对照组比较,室温组和4℃组保存4、8、24h的P-LCR、MPV、PDW均显著增加(P<0.05)。室温组保存4、8、24h的P-LCR、MPV和PDW两两比较差异均无统计学意义(P>0.05)。4℃组保存8、24h的P-LCR和MPV与4h比较显著增大(P<0.05);4℃组保存24h的P-LCR和MPV与8h时比较差异无统计学意义(P>0.05)。4℃组保存4、8h的PDW比较差异无统计学意义(P>0.05),保存24h的PDW与4、8h时比较显著增大(P<0.05)。与室温组相同时间点比较,4℃组保存4、8h的P-LCR、MPV和PDW显著降低(P<0.05)。4℃组与室温组保存24h的P-LCR、MPV和PDW比较差异无统计学意义(P>0.05)。对照组、室温保存4、8、24h组和4℃保存4、8、24h组各组之间的PLT计数两两比较差异均无统计学意义(P>0.05)。与对照组比较,室温组保存4、24h及4℃组保存4、24h的血小板表面CD62P表达水平均显著升高(P<0.05)。与室温组同时间点比较,4℃组保存4、24h的血小板表面CD62P表达水平显著降低(P<0.05)。室温组保存24h时的血小板表面CD62P表达水平与4h时比较差异无统计学意义(P>0.05)。4℃组保存24h时的血小板表面CD62P表达水平与4h时比较显著升高(P<0.05)。与对照组比较,室温组保存4、24hPLT内Ca^2+水平显著升高(P<0.05)。对照组及4℃组4、24h的PLT内Ca^2+水平两两比较均无显著差异(P>0.05)。与室温组同时间点比较,4℃组保存4、24h的PLT内Ca^2+水平显著降低(P<0.05)。室温组保存24h时的PLT内Ca^2+水平与4h时比较显著升高(P<0.05)。结论全血标本4℃保存并在4h内完成检测,可以最大限度维持PLT原始状态,提高PLT检测相关指标的准确性。

Objective To investigate the effect of whole blood preservation temperature on platelet morphology and function. Methods Thirty healthy volunteers were selected as subjects. Fasting elbow venous blood was collected and kept in vacuum tubes containing ethylenediaminetetraacetic acid dipotassium anticoagulant to fully anticoagulation. Blood smear was prepared immediately after blood collection(control group),at 4 ℃(4 ℃ group) and room temperature(room temperature group) for 4,8 and 24 hours,and then stained with Wright-Giemsa complex staining solution. The morphology and structure of platelets were observed under the microscope. The number of platelets,platelet large cell ratio(P-LCR),the mean platelet volume(MPV) and the platelet volume distribution width(PDW) of the control group,the 4 ℃ group and the roomtemperature group preserved for 4,8 and 24 h were detected by hematological analyzer. Flow cytometry was used to detect the expression of platelet activation marker CD62 P in the control group,4 ℃ group and room temperature group preserved for 4,24 hours. The all-wavelength scanning multifunctional reading instrument was used to check the Ca2 +concentration of platelets in the control group,4 ℃ group and room temperature group preserved for 4,24 hours. Results With the prolongation of preservation time,the platelet volume of the room temperature group and 4 ℃ group became larger and larger. And the platelet volume in the room temperature group increased more significantly,with cavitation phenomenon,and platelet structure was loose. Compared with the control group,the P-LCR,MPV and PDW in the 4 ℃ group and room temperature group stored for 4,8 and 24 hours was significantly increased(P < 0. 05). There was no significant difference in the P-LCR,MPV and PDW by paired-comparisons among the preserved time of 4,8 and 24 hours in the room temperature group(P > 0. 05). Compared with the 4 ℃ group preserved for 4 hours,the P-LCR and MPV was higher than that in the 4 ℃ group preserved for 8 hours and 24 hours(P < 0. 05). There was no significant difference in the P-LCR and MPV between the preserved time of 8 hours and 24 hours in the 4 ℃ group(P > 0. 05). There was no significant difference in the PDW between the preserved time of 4 hours and8 hours in the 4 ℃ group(P > 0. 05). The PDW in the 4 ℃ group preserved for 4 hours and 8 hours was higher than that preserved for 24 hours(P < 0. 05). Compared with the same time point in room temperature group,the P-LCR,MPV and PDW in the 4 ℃ group preserved for 4,8 hours were significantly decreased(P < 0. 05). There was no significant difference in the P-LCR,MPV and PDW between the 4 ℃ group and room temperature group preserved for 24 hours(P > 0. 05). There was no significant difference in the the number of platelet among the control group,4 ℃ group and room temperature group preserved for 4,8 and 24 hours(P > 0. 05). Compared with the control group,the level of CD62 P in the room temperature group and4 ℃ group preserved for 4 and 24 hours was higher(P < 0. 05). Compared with the same time point in room temperature group,the level of CD62 P in the 4 ℃ group preserved for 4 and 24 hours was lower(P < 0. 05). There was no significant difference in the level of CD62 P between the preserved time of 4 hours and 24 hours in the room temperature group(P >0. 05). The level of CD62 P in the 4 ℃ group preserved for 24 hours was higher than that in the 4 ℃ group preserved for 4 hours(P < 0. 05). Compared with the control group,the level of Ca2 +concentration of platelets in the room temperature group preserved for 4,24 hours was higher(P < 0. 05). There was no significant difference in the level of Ca2 +concentration of platelets by paired-comparisons between the control group and the 4 ℃ group preserved for 4 hours and 24 hours(P > 0. 05).Compared with the same time point in room temperature group,the level of Ca2 +concentration of platelets in the 4 ℃ group preserved for 4 hours and 24 hours was lower(P < 0. 05). The level of Ca2 +concentration of platelets in the room temperature group preserved for 24 hours was higher than that in the room temperature group preserved for 4 hours(P < 0. 05).Conclusion The whole blood samples were stored at 4 ℃ and tested within 4 hours,which can maintain the original state of platelets as much as possible and improve the accuracy of relevant indicators of platelets.