温度对手工制备富血小板血浆质量影响的探讨

Effect of temperature on the preparation of platelet-rich plasma

目的探讨温度对手工制备富血小板血浆质量的影响。方法选取南部战区总医院血液中心2020年5月10日~9月10日无偿健康献血员捐献的外周血60mL/人份,共120份。将每人份全血在随机分为3等份,各20mL,共360份,分别分为9(A_(1)、A_(2)、A_(3)、B_(1)、B_(2)、B_(3)、C_(1)、C_(2)、C_(3))组,每组40份,其中A组:PRP制备环境温度设为4±2℃,PRP制备及离心仓内温度分别为A_(1)组:4±2℃,A_(2)组24±2℃,A_(3)组:30±2℃,B组:PRP制备环境温度设为24±2℃,PRP制备及离心仓内温度分别为B_(1)组:4±2℃,B_(2)组:24±2℃,B_(3)组:30±2℃,C组:PRP制备环境温度设为30±2℃,PRP制备及离心仓内温度分别为C_(1)组:4±2℃,C_(2)组:24±2℃,C_(3)30±2℃组。使用富浆法分离制备浓缩血小板,并计算各组血小板回收率,显微镜下血小板形态,应用酶联免疫吸附试验法定量检测血小板源性生长因子(PDGF-BB)、转化生长因子β(TGF-β)、血管内皮生长因子(VEGF)的浓度。结果血小板计数及血小板回收率比较:A_(1)、A_(2)、A_(3)、B_(1)、B_(2)、B_(3)、C_(1)、C_(2)、C_(3),9组中,A_(3) B_(3) C_(3)三组的结果为(489.2±21.47)×10^(9)/L、(57.4±2.3)%,(495.7±23.2)×10^(9)/L、(54.9±1.3)%,(489.4±17.1)×10^(9)/L、(50.7±2.3)%,均出现血小板聚集现象,分别与A_(1)、A_(2),B_(1)、B_(2)和C_(1)、C_(2)比较,差异具有统计学意义(P<0.05)。显微镜下观察:A_(1)、A_(2)、B_(1)、B_(2)、C_(1)、C_(2)6组,收集的PRP中,血小板细胞大小均匀,无聚集。A_(3)、B_(3)、C_(3)3组,收集的PRP中,均有部分血小板聚集成团。生长因子含量检测:未聚集组与聚集组中TGF-β的含量(ng/L)分别为375.0±119.1、183.67±106.2(P<0.05),PDGF-BB的含量(ng/L)分别为933.0±273.0、656±113.0(P<0.05)差异具有统计学意义;VEGF的含量(ng/L)分别为217.5±93.5、155.3±103.4(P>0.05),差异无统计学意义。结论外界环境温度对手工制备PRP制备,离心机机舱的温度保持在24±2℃为佳。

Objective To discuss the effect of temperature on the quality of platelet-rich plasma(PRP)prepared manually.Methods A total of 120 peripheral blood samples(60 mL/person)were collected from healthy voluntary blood donors in the Blood Center of General Hospital of Southern Theater Command from May 10,2010 to September 10,2010.Each whole blood sample(60 mL/person)was randomly divided into 3 aliquots(20 mL each),totaling 360 aliquots,then divided into 9 groups(A_(1),A_(2),A_(3),B_(1),B_(2),B_(3),C_(1),C_(2),and C_(3)),with 40 aliquots in each group.In group A:the ambient temperature for PRP preparation was set to(4±2)℃,and the temperature for PRP preparation and centrifugal bin was(4±2)℃,(24±2)℃and(30±2)℃for group A_(1),A_(2),and A_(3),respectively.In group B:the ambient temperature for PRP preparation was set to(24±2)℃,and the temperature for PRP preparation and centrifugal bin was(4±2)℃,(24±2)℃and(30±2)℃for group B_(1),B_(2) and B_(3) respectively.In group C:the ambient temperature for PRP preparation was set to(30±2)℃,and the temperature for PRP preparation and centrifugal bin was(4±2)℃,(24±2)℃and(30±2)℃for group C_(1),C_(2) and C_(3) respectively.Platelet concentrates were separated and prepared by rich slurry method,and the platelet recovery rate was calculated for each group,the platelet morphology was observed under the microscope,and the concentrations of platelet-derived growth factor(PDGF-BB),transforming growth factorβ(TGF-β)and vascular endothelial growth factor(VEGF)were quantitatively determined using enzyme-linked immunosorbent assay(ELISA).Results The platelet count and platelet recovery rate were compared as follows:among group A_(1),A_(2),A_(3),B_(1),B_(2),B_(3),C_(1),C_(2) and C_(3),the results of A_(3),B_(3) and C_(3)were(489.2±21.47)×10^(9)/L vs(495.7±23.2)×10^(9)/L vs(489.4±17.1)×10^(9)/L and(57.4±2.3)%vs(54.9±1.3)%vs(50.7±2.3)%,respectively,all showed platelet aggregation,and the differences,relative to A_(1) and A_(2),B_(1) and B_(2),and C_(1) and C_(2),were statistically significant(P<0.05).Microscopic observation was as follows:in the PRPs collected from group A_(1),A_(2),B_(1),B_(2),C_(1) and C_(2),the platelet cells were uniform in size and without aggregation;in the PRPs collected from group A_(3),B_(3) and C_(3),some platelets showed aggregation.Determination of growth factor content was as follows:the content of TGF-β(ng/L)and PDGF-BB(ng/L)was 375.0±119.1 vs 183.67±106.2 and 933.0±273.0 vs 656±113.0 in the non-aggregation group and the aggregation group,respectively(P<0.05),while the content of VEGF(ng/L)was 217.5±93.5 vs 155.3±103.4(P>0.05),with no statistically difference.Conclusion The ambient temperature had little effect on the preparation of PRP,and the temperature of the centrifuge bin was better to be maintained at(24±2)℃.