免疫磁珠法分选骨髓CD34^＋细胞：纯度影响因素的分析

背景：免疫磁珠分选技术已成为分选CD34^＋细胞的主要方法之一,在长期实验中发现诸多因素影响其分离纯度,包括磁珠与细胞孵育时干冰与碎冰的使用、磁珠与细胞孵育及分离时摇床的摇摆方向、红细胞裂解液裂解单个核细胞的使用等。目的：在免疫磁珠分选CD34^＋细胞过程中分别对上述影响因素进行分析改进,以提高分选CD34^＋细胞的纯度。设计、时间及地点：细胞学体外对照观察,于2008-07/2009-03在太原市中心医院皮肤科实验室完成。材料：骨髓象筛选正常的人骨髓29份,由太原市中心医院血液科提供,取材均经患者同意。CD34^＋免疫磁珠为Dynal公司产品。方法：采用淋巴细胞分离液密度梯度离心法分离培养人骨髓单个核细胞,用免疫磁珠法分选骨髓CD34^＋细胞,对其分选过程中的主要环节如冰块与碎冰、摇床的种类以及红细胞裂解液的使用进行方法改进。方法1：按试剂盒提供方法进行磁珠常规分选CD34^＋细胞。方法2：在方法1基础上,用碎冰替代冰块进行磁珠与单个核细胞孵育。方法3：在方法2基础上,用万向摇床替代水平摇床。方法4：在方法3基础上,向单个核细胞中预先加入红细胞裂解液裂解残留红细胞。主要观察指标：通过流式细胞仪检测不同方法分选出的CD34^＋细胞纯度。结果：随着不同环节的逐渐改进,CD34^＋细胞纯度由（32.7±6.6）%升至（84.5±5.2）%,方法1,2,3,4所分选出的CD34^＋细胞纯度逐渐升高,组间方差分析差异显著（F=76.209,P〈0.01）,Bonferroni法两两比较差异亦有非常显著性意义（P〈0.01）。结论：碎冰、万向摇床及红细胞裂解液的使用能有效提高免疫磁珠法分选人骨髓CD34^＋细胞的纯度。

Immunomagnetic beads method for separating bone marrow CD34~＋ cells：An analysis of purity influencing factors

BACKGROUND：Immunomagnetic beads method has been a main method of sorting CD34^＋ cells. Numerous studies have shown that many factors affected its isolation purity,including usage of dry ice and brash ice when incubating magnetic bead and cells,wagging direction of the shaker when incubating and isolating magnetic bead and cells,and usage of splitting mononuclear cells using erythrocyte lysis buffer. OBJECTIVE：To improve the purity of CD34^＋ cells by changing the influence factors of immunomagnetic bead separating bone marrow CD34^＋ cells. DESIGN,TIME AND SETTING：The in vitro control observation experiment was performed at the Laboratory of Department of Dermatology,Taiyuan Central Hospital from July 2008 to March 2009. MATERIALS：Human bone marrow（29 samples） was obtained from the Department of Hematology,Taiyuan Central Hospital after the agreement of patients. CD34^＋ cells immunomagnetic beads were supplied by Dynal Company. METHODS：Human bone marrow mononuclear cells were separated by lymphocyte separating medium and density gradient centrifugation. Bone marrow CD34^＋ cells were sorted by the immunomagnetic beads method. The main processes were improved,such as usage of dry ice and brash ice,type of the shaker and usage of erythrocyte lysis buffer. Method 1：separating of CD34^＋ cells conventionally according to the kit;Method 2：the ice was replaced by the crushed ice based on conventional separating method;Method 3：based on method 2,the horizontal shaker was instead by the universal shaker;Method 4：on the basis of the method 3,the erythrocyte lysis buffer was mixed into mononuclear cells to crack residual erythrocytes. MAIN OUTCOME MEASURES：The purity of CD34^＋ cells was measured by flow cytometry. RESULTS：The purity of CD34^＋ cells was gradually increased after improving different steps,from（32.7±6.6） % to（84.5±5.2） %,the difference was significant（F=76.209,P 0.01） . There were significant differences among each method using Bonferroni pairwise comparison（P 0.01） . CONCLUSION：The use of crushed ice,universal shakers and erythrocyte lysis buffer can effectively improve the purity of bone marrow CD34^＋ cells.