中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (45): 8483-8486.doi: 10.3969/j.issn.1673-8225.2011.45.027

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

PCR-SSO流式荧光微珠法HLA-A*02高分辨分型检测模棱两可结果的统计分析

张容达1，杜  娟2，马晓莉2，张伯伟2   

  1. 1复旦大学物理系生物物理重点实验室，上海市   200433
    2河南省红十字血液中心，河南省郑州市450012
  • 收稿日期:2011-08-01 修回日期:2011-09-05 出版日期:2011-11-05 发布日期:2011-11-05
  • 通讯作者: 张伯伟，硕士，主任技师，教授，河南省红十字血液中心，河南省荥阳市 450012 bowei1919@126.com
  • 作者简介:张容达，男，1990年生，河南省郑州市人，复旦大学物理系2008级学生，主要从事生物物理学实验方法研究。 08300190054@fudan.edu.cn

Statistical analysis for ambiguous results of HLA-A*02 high-resolution genotyping by using PCR-SSO flow fluorescent bead method

Zhang Rong-da1, Du Juan2, Ma Xiao-li2, Zhang Bo-wei2   

  1. 1Key Laboratory of Biophysics, Department of Physics, Fudan University, Shanghai  200433, China
    2Henan Red-Cross Blood Center, Zhengzhou  450012, Henan Province, China
  • Received:2011-08-01 Revised:2011-09-05 Online:2011-11-05 Published:2011-11-05
  • Contact: Zhang Bo-wei, Master, Chief technician, Professor, Henan Red-Cross Blood Center, Zhengzhou 450012, Henan Province, China bowei1919@126.com
  • About author:Zhang Rong-da, Key Laboratory of Biophysics, Department of Physics, Fudan University, Shanghai 200433, China 08300190054@fudan.edu.cn

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摘要：

背景：由于HLA的复杂多态性，实际分型过程中难免会出现一些结果判读模棱两可现象。
目的：统计分析基于PCR-SSO 流式荧光微珠HLA-A*02高分辨分型方法的模棱两可组合结果种类、比例，探讨解决模棱两可解决方法，提高高分辨分型比例。
方法：对河南骨髓库捐献者 1 100人份HLA分型结果进行统计分析，计数法进行分类统计分析软件给出的包含HLA-A*02：01\02：09模棱两可组合代码，计算百分比；与确认为A*02：09的基因多态性分布进行比对，找出与A*02：09关联性较强的等位基因，用补充实验进行复核、确认，建立A*02：01\02：09模棱两可组合的判读方法。
结果与结论：1 039例有效数据中包含A*02：01\02：09模棱两可组合279例(279/1 039，26.853%)，代码种类19种，检出比例较高的有DMDC，DYVK，GMDD，GMDE；34例确认为A*0209的基因多态性统计显示A*0209的出现与A*11：01  (0.147 1)B*07：02(0.264 7)、DRB1*01：01(0.264 7 )、DRB1*03：01(0.117 6)有较强关联，选出高风险标本38例，经检测外显子1~7，并用SBT复核确认有1例结果为A*02：09(代码组合为GMDC)，其余241例未检出A*02：09。提示 A*02：09的出现与B*07：02、DRB1*01：01、DRB1*03：01有较强关联，重点对这些关联标本进行外显子1~7检测，可有效地检出A*02：09。通过统计分析制定合理的判定规则和必要的复核实验可有效地简化操作程序，节省时间，降低费用，提高高分辨比例。

关键词: HLA-A*0201/0209模棱两可, 中华骨髓库, PCR-SSO, HLA分型, 基因

Abstract:

BACKGROUND: Due to the complexity of the HLA typing system, ambiguous situations occur frequently during HLA typing of the donors.
OBJECTIVE: To statistically analyze the ambiguous ratio and combined allele groups of HLA-A*02 high-resolution genotyping by using PCR-SSO flow fluorescent bead method, and to investigate the method to resolve the ambiguous results of HLA-A*02 high-resolution genotyping and improve the ratio of high-resolution genotyping.
METHODS: 1100 pieces of HLA genotyping were collected for statistical analysis. We calculated the percentages of those combinations that contained HLA-A*02:01\02:09 ambiguity, given by the software HLA Fusion 2.0; in the other hand, we searched out those alleles which were highly correlated with A*02:09 by comparing these combination percentages with the A*02:09 genetic polymorphism distribution.
RESULTS AND CONCLUSION: 279 out of 1039 effective samples (279/1039, 26.853%) were found for the results of having A*02:01/02:09 ambiguities that could be categorized into 19 groups in NMDP code. The most frequently groups were DMDC, DYVK, GMDD, GMDE. In another cases, 34 identified as carrying A*02:09 were analyzed appearing that A*02:09 was highly correlated to B*07:02(0.2647), DRB1*01:01(0.2647), DRB1*03:01(0.1176). We selected 38 high risky samples and tested their 4-7 exons, and confirmed by SBT, only 1 sample could be identified as A*0209, while other 241 samples lacked of A*02:09. A reasonable judgment based on statistical analysis and necessary review can effectively simplify the experimental procedures, save time, reduce costs, and increase high-resolution ratio.

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