免疫磁珠分离淋巴细胞在流式交叉配型试验中的应用及研究  

A new approach of rapidly isolating lymphocytes from whole blood with immune magnetic beads for organ transplant flow cytometry cross-matching

作　　者：郭靖[1,2] 罗奇志[1] 田芳[1] 郭旭丽[1] 罗伟光[1] 邹义洲[1] Guo Jing Luo Qizhi Tian Fang Guo Xuli Luo Weiguang Zou Yizhou(Department of Immunology, School of Basic Medical Science, Xiangya School of Medicine, Central South University, Changsha 410008, China Department of Immunology, School of Medicine, Ji Shou University, Jishou 416000, Chin)

机构地区：[1]中南大学湘雅医学院免疫学系,长沙410008 [2]湖南省吉首大学医学院微生物与免疫学教研室,吉首416000

出　　处：《中华细胞与干细胞杂志（电子版）》2017年第3期146-151,共6页Chinese Journal of Cell and Stem Cell（Electronic Edition）

基　　金：国家自然科学基金面上项目(81571562)

摘　　要：目的探讨免疫磁珠快速分离法及密度梯度离心(Ficoll)分离法分离外周血单个核细胞(PBMC)在流式细胞交叉配型中的应用比较。方法取10例交叉配型供体外周抗凝血各10 ml,受体非抗凝血5 ml,分别采用密度梯度离心与免疫磁珠阴性选择试剂盒从相同体积的抗凝血中分离PBMC。用血细胞计数比较两种方法分离的WBC、PLT、RBC粒度分布和PBMC中淋巴细胞的纯度;用细胞流式和淋巴细胞表面标记荧光抗体检测两种方法分离得到的PBMC中T、B和NK细胞的数量质量;将免疫磁珠阴性分离和密度梯度离心法分离的供者PBMC与受者血清共孵育,加入羊抗人IgG-FITC,洗涤后加入抗人CD3-PE、抗人CD19-APC单克隆抗体,再用流式细胞仪检测细胞表面荧光强度。采用方差分析和t检验进行统计学分析。结果免疫磁珠阴性分离的PBMC数量是Ficoll分离PBMC的0.42倍,但淋巴细胞的比例[(99.2±0.08)﹪]高于PBMC分离的淋巴细胞比例[(82.5±5.27)﹪(t=9.91,P<0.01)],且两种方法分离的PBMC中RBC分别为(0.001±0.001)×10~6/μl和(0.02±0.009)×10~6/μl(t=6.64,P<0.001);血小板的数量分别为(1.00±0.05)×10~3/μl和(196.00±4.21)×10~3/μl差异均有统计学意义(t=146.46,P<0.01)。流式细胞检测免疫磁珠分离的PBMC中CD3^+T细胞为(81.33±4.60)﹪,CD19^+B细胞为(8.41±0.87)﹪,CD3-CD56^+NK细胞为(9.35±0.67)﹪和CD3^+CD56^+NKT细胞为(2.47±0.07)﹪。而Ficoll分离的PBMC中CD3^+T细胞为(37.36±3.27)﹪,CD19^+B细胞为(5.79±0.94)﹪,CD56^+NK细胞为(6.60±0.91)﹪,且差异均有统计学意义(t=24.64、6.470、7.70、51.31,P均<0.01)。T和B淋巴细胞流式交叉配型试验中,设门定量读取T和B淋巴细胞与受者血清中抗体结合情况,密度梯度离心获得的淋巴细胞中混有血小板等,使流式检测结果中会混有假阴性,而免疫磁珠分离法没有出现假阴性结果。证明免疫磁珠分离的PBMC可应用于临床交叉配型试验。结论免疫磁珠阴性选择分�Objective To compare immunomagnetic bead and density gradient centrifugation （Ficoll） for the separation of peripheral blood mononuclear cells （PBMC） in flow crossmatching. Method Hemocytometer was used to count and compare the numbers of WBC, PLT, RBC isolated. For quality and quantity analysis of isolated T, B and NK cells in PBMC, flow cytometry was used to detect lymphocyte surface markers by specific fluorescent antibodies. The donor PBMC was used to detect antibodies in the recipient serum. Anti-human CD3-PE and antihuman CD19-APC monoclonal antibody were used for identification of T and B cells. Statistical analysis was performed using variance analysis and t test. Result The number of PBMC Macs separation is 0.42 times the Ficoll separation of PBMC, but the proportion of lymphocytes （99.2±0.08 ） % higher than that of PBMC isolated lymphocyte ratio （ 82.5 ±5.27） % （t = 9.91, P 〈 0.01 ）, and two methods of separating PBMC RBC respectively （0.001± 0.001 ）× 10^6/[.tl and （0.02 ± 0.009）× 10^6/μl （t = 6.64, P 〈 0.001 ）; the number of platelets were （ 1.00 ± 0.05 ）× 10^3/μl and （ 196.00± 4.21 ）× 10^3/μl had statistical significance difference （t = 146.46, P 〈 0.01 ）. Flow cytometry showed that CD3＋ T cells in PBMCs isolated from immunomagnetic beads were （8.41 ± 0.87） %, CD3-CD56＋NK cell （9.35 ± 0.67） % and （2.47 ± 0.07） % of CD3＋CD56＋NKT cells. Ficoll isolated PBMCs contain （37.36 ± 3.27） % CD3＋ T cells, （5.79 ± 0.94） % CD19＋ B cells and （6.60 ±0.91） % CD56＋ NK cells, and the differences were statistically significant （t = 24.64, 6.470, 7.70, 51.31, P 〈 0.01 ）. In the cross matching test ofT and B lymphocytes, quantitative read T and B lymphocytes and the serum antibody binding were tested. As density gradient centrifugation in lymphocyte mixed with platelets, so the flow cytometry results will be mixed with false negative, but the immune magnetic beads separation does not appear false negative resul

关 键 词：外周血 单核细胞 离心法 梯密度 免疫磁珠分离 组织相容性试验 

分 类 号：R392-33[医药卫生—免疫学]