北京大学学报(医学版) ›› 2014, Vol. 46 ›› Issue (1): 111-114.

• 论著 • 上一篇    下一篇

自然沉降法分离抗凝全血建立生物样本库的特点

陈智滨1 ,林琴 2 , 马长华2, 刘凯宁1 ,孟焕新1△   

  1. (北京大学口腔医学院·口腔医院 1.牙周科,2.检验科,北京100081)
  • 出版日期:2014-02-18 发布日期:2014-02-18

Characteristic of sample banks isolated from EDTA-blood by sedimentation method

CHEN Zhi-bin , LIN Qin , MA Chang-hua , LIU Kai-ning, MENG Huan-xin   

  1. (1.Department of Periodontology, 2. Department of Clinical Laboratory, Peking University School and Hospital of Stomatology, Beijing 100081, China)
  • Online:2014-02-18 Published:2014-02-18

摘要: 目的:评估用自然沉降法分离EDTA抗凝外周静脉全血(抗凝全血)建立血浆、白细胞、基因组DNA生物样本库的特点。方法: 采集符 合纳入标准的29名志愿者抗凝全血,进行血常规检测作为对照组;然后将抗凝全血分装到4支EP管中,每支1 mL,分为0.5、1、2、3 h  4个试 验组,于室温静置自然沉降0.5、1、2、3 h后吸取血浆层,测量其体积,检测血浆层中白细胞总数及中性粒细胞、淋巴细胞的百分比;将含有 白细胞的血浆层离心后,分装为无细胞血浆管和白细胞沉淀管,用部分白细胞提取DNA,建立血浆、白细胞、DNA 3种生物样本库。结果: 0.5 h 组血浆体积(241.72±101.52)μL显著低于1 h组(317.24±97.50)μL、2 h组(371.03±91.66)μL和3 h组(408.97±97.43)μL, P<0.05。1 h组血浆体积显著低于2 h组和3 h组,P<0.05;2 h组显著低于3 h组,P<0.05。0.5 h组白细胞总数[(2.50×106)±(1.48×106 )]显著高于2 h组[(1.47×106)±(7.19×105) ]和3 h组[(1.21×106)±(7.41×105)]的白细胞总数,P<0.05,与1 h组[(2.29×106)± (1.17×106)]比较差异无统计学意义,P>0.05;1 h组显著高于2 h和3 h组,P<0.05;2 h组与3 h组差异无统计学意义,P>0.05。4个试验组白 细胞总数均显著低于对照组。中性粒细胞百分比:0.5 h组(54.14%±11.65%)显著高于1、2、3 h组(46.66%±12.70%,39.17%±12.33%, 43.25%±14.54%),P<0.05,显著低于对照组(60.53%±8.46%),P<0.05;1 h组显著高于2 h组,P>0.05; 1 h组与3 h组比较差异无统计学意义 ,P>0.05;3 h组与1 h、2 h组比较差异无统计学意义,P>0.05。淋巴细胞百分比:0.5 h组(35.09%±10.84%)显著低于1、2、3 h组(41.48% ±12.20%、47.96%±12.27%、45.50%±13.71%),显著高于对照组(30.98%±7.33%), P<0.05;1 h组显著高于对照组、0.5 h组,显著低于2 h、3 h组,P<0.05;2 h组显著高于对照组、0.5 h、1 h组,P<0.05,与3 h组之间差异无统计学意义,P>0.05。结论:  抗凝全血自然沉降法 是一种无化学试剂干扰、低物理因素影响、低消耗、操作简单的分离白细胞血浆层的方法;0.5~1 h是抗凝全血自然沉降获取含白细胞血浆层的 最佳时间段;利用一份抗凝全血可以分离出血浆、白细胞和DNA 3种生物样本,建立3种生物样本库。

关键词: 血沉, 生物标本库, 血浆, 白细胞计数, DNA

Abstract: Objective:To assess the characteristics of establishing the different sample banks of plasma, leukocytes and DNA by sedimentation method of isolating from ethylene diamine tetraacetic acid(EDTA)-blood and to clarify the sedimentation method of leukocyte isolation and plasma volume by comparative data and recommended procedures for applicability. Methods:In the study, 29 EDTA-bloods were obtained, the total amounts of leukocytes and the percentage of neutrophile granulocytes, and lymphocytes in the EDTA-blood detected as a control group and then assigned equally into 4 EP tubes with 1 mL EDTA-blood per tube as 4 test groups, then the 4 tubes were placed with the EDTA-blood at room temperature and the plasma layers were isolated at 0.5, 1, 2 and 3 h, receptively. The total amount of leukocytes and the percentage of neutrophile granulocytes, and lymphocytes were detected by automated hematology analyzer at the clinical laboratory. The volume of the plasma was also measured at the same time. Results:The plasma volume at 0.5 h [(241.72±101.52)μL] was substantially lower than those at 1 h[(317.24±97.50)μL], at 2 h[(371.03±91.66)μL], and at 3 h [(408.97±97.43)μL] , P<0.05. The plasma volume at 1 h was substantially lower than those at 2 h and 3 h (P<0.05). The total amount of leukocytes in the plasma layer at 0.5 h (2.50 ×106±1.48×106) group was substantially higher than the amount of 2 or 3 h groups respectively (1.47×106±7.19×105 ,1.21 ×106±7.41×105), P<0.05. Significant difference was not found between 0.5 h group and 1 h group (2.29×106±1.17×106) , P>0.05. The total amount of leukocytes in the plasma layer in1h group was substantially higher than that in 2 h and 3 h groups (P<0.05). There was no significant difference between 3 h group and 2 h group (P>0.05). The total amount of leukocytes in the plasma layer of the 4 test groups was substantially lower than that in the control group (P<0.05). The percentage of neutrophile granulocytes (54.14%±11.65%) in the plasma layer in 0.5 h group was substantially higher than those in 1 h, 2 h and 3 h groups (46.66%±12.70%,39.17%±12.33%,43.25%±14.54%), P<0.05, respectively, which was the substantially lower than that in the control group (60.53%±8.46%), P<0.05. The average value of the percentage of neutrophile granulocytes in the plasma layer in 1 h group was substantially higher than that in 2 h group (P<0.05). There was no significant different between 3 h group and both 1 h, 2 h groups (P>0.05). The mean percentage of lymphocytes in the plasma layer in 0.5 h group (35.09%±10.84%) was substantially lower than those in the plasma layer in 1 h, 2 h and 3 h groups, respectively ( 41.48%± 12.20%, 47.96%±12.27%, 45.50%±13.71%), which was significant higher than that in the control group(30.98%±7.33%), P<0.05. The average value of the percentage of lymphocytes in the plasma layer in 1 h group was substantially higher than those in the control group and 0.5 h group, but was substantially lower than those in 2 h and 3 h groups (P<0.05). The average value of percentage of lymphocytes in the plasma layer in 2 h group was substantially higher than those in the control group, 0.5 h and 1 h groups (P<0.05). There was no significant difference between 2 h and 3 h groups (P>0.05). Conclusion:The best period of time in obtaining leukocytes is 0.5-1 h sedimentation of EDTA-blood. Both the plasma layer and leukocytes can be separated and obtained at the same time from the same sample by the sedimentation method of EDTA-blood. The sedimentation of EDTA-blood has the least interference of both chemical and physical factors, as well as a ready operation, which can establish the plasma, leukocytes and DNA sample banks for various aspects of research.

Key words: Blood sedimentation, Biological specimen banks, Plasma, Leukocyte count, DNA

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