不同抗凝剂种类、操作和保存方法对血浆微粒检测影响

Influences of different anticoagulants,preparation and preservation methods on detection of circulating microparticles

目的探讨不同抗凝剂种类、标本处理方式、保存温度及时间对血浆微粒(MPs)检测效果的影响。方法采集2013年8月—2014年5月在苏州大学附属第二医院住院治疗的40例系统性红斑狼疮患者、41例类风湿性关节炎患者和52名健康对照者血浆标本;采用流式细胞术(FCM)计数不同抗凝剂[枸橼酸盐、乙二胺四乙酸二钾(EDTA-K2)、肝素]、标本处理方式[洗涤贫血小板血浆(PPP)和直接PPP]、保存温度(-4、-20、-80℃)及时间(1、3、7、10、14 d)血浆中MPs的含量。结果采用枸橼酸盐、EDTA-K2、肝素抗凝剂和无抗凝剂血浆中MPs含量分别为(1 527.0±620.4)、(981.4±247.9)、(877.7±176.2)和(480.8±112.2)μL,差异有统计学意义(F=9.11,P<0.01);系统性红斑狼疮患者、类风湿性关节炎患者和健康对照者直接PPP MPs含量分别为(1 972.4±2 850.7)、(3 347.8±3431.5)和(2 157.7±1 901.1)μL,均高于洗涤PPP的(1 406.4±2 205.7)、(2 375.6±2 500.2)和(1 502.8±1 337.1)μL(均P<0.01);与新鲜血浆标本MPs含量比较,-4℃、-20℃下血浆标本保存1、3、7、10 d和-80℃下血浆标本保存3、7、10 d MPs含量均下降,差异均有统计学意义(P<0.05);而在3个温度下保存14 d MPs含量开始升高,与新鲜血浆标本MPs含量比较,差异均无统计学意义(P>0.05);分别对3个温度下保存1、3、7、10、14 d血浆标本MPs含量比较,差异均无统计学意义(P>0.05)。结论检测血浆MPs含量时宜选用新鲜EDTA-K2抗凝标本且无需多次离心洗涤,短期内标本保存在-4℃、-20℃、-80℃下均可。

Objective To investigate effects of anticoagulant,sample preparation,storage temperature and time on detection of circulating microparticles（ MPs）. Methods The blood samples of 40 systemic lupus erythematosus and 41 rheumatoid arthritis inpatients from the Second Affiliated Hospital of SoochowUniversity,and 52 healthy donors were collected. MPs of the samples treated with different anticoagulants（ sodium citrate,ethylenediaminetetraacetic acid-dipotassium[EDTA-K2],heparin）,preparation methods（ washed platelet-poor plasma[PPP],and directed PPP）,storage temperatures（- 4 ℃,- 20 ℃,- 80 ℃）,and times（ 1,3,7,10,and 14 days） were detected with flowcytometer and compared. Results The content of MPs for the samples treated with sodium citrate plasma,EDTA-K2,heparin,and without anticoagulant were 1 527. 0 ± 620. 4,981. 4 ± 247. 9,877. 7 ± 176. 2,and 480. 8 ± 112. 2 μL,respectively,with significant differences（ F = 9. 11,P〈0. 01）. The MPs content of directed PPP samples from systemic lupus erythematosus patients,rheumatoid arthritis patients,and healthy donors were 1 972. 4 ± 2 850. 7,3 347. 8 ± 3 431. 5,and 2 157. 7 ± 1 901. 1 μL,respectively,and were all higher than those of washed PPP samples（ 1 406. 4 ± 2 205. 7,2 375. 6 ± 2 500. 2,and 1 502. 8± 1 337. 1μL）（ all P〈0. 01）. Compared with those of the fresh plasma samples,the MPs content decreased significantly for the samples stored at the temperature of- 4 ℃,- 20 ℃,and- 80 ℃ and stored for 1,3,7,and 10 days（ all P〈0. 05）,but the content showed no significant difference when stored for 14 days at the temperature of- 4 ℃,- 20 ℃,and- 80 ℃（ all P〉0. 05）; there were also no significant differences in MPs content among the samples with various storage duration（ 1- 14 days） at the three different temperatures（ all P〉0. 05）. Conclusion MPs content should be detected for fresh samples treated with EDTA-K2 and without of repeated washing and the storage temperature of- 4 ℃,- 20 ℃,and- 80 ℃ could selected for short time preservation of plasma sample.