Ai Xiaofei, Fu Qianqian, Wang Jun, Zheng Yingchun, Han Cong, Li Qinghua, Sun Qi, Ru Kun
Department of Pathology, Institute of Hematology and Blood Diseases Hospital, CAMS & PUMC, Tianjin 300020, China.
Zhonghua Xue Ye Xue Za Zhi. 2014 Jun;35(6):495-8. doi: 10.3760/cma.j.issn.0253-2727.2014.06.004.
To explore the feasibility of detecting lymphoma with the application of BIOMED-2 standardized immunoglobulin/T cell receptor (IG/TCR) gene rearrangement system in formalin fixed paraffin-embedded (FFPE) tissue samples, and to discuss the relationship between the longest amplification fragment of extracted DNA and positive detection rate of different IGH V-J primers.
DNA was extracted from 50 cases of FFPE tissue samples. Multiplex-PCR amplifications were performed and then the IG/TCR gene rearrangements were analyzed using BIOMED-2 standardized clonality analysis system.
(1)When the DNA concentration was diluted to 50-100 ng/μl from 100-500 ng/μl, the proportion of the longest amplification fragment (300-400 bp) of DNA was improved from 10.0% to 90.0% in 30 cases of diffuse large B cell lymphoma (DLBCL) wax roll samples (P<0.01). The positive rate of IGH+IGK was increased from 46.7% to 83.3%, the difference was statistically significant (P=0.006). The lengths of the longest amplification fragments of DNA were all longer than 300 bp in the paraffin section samples of DLBCL. The positive rate of IGH+IGK of these samples was 96.7%. The difference of the positive rate of IGH+IGK between the wax roll samples and the paraffin section samples had no statistical significance (P=0.195). (2)When the concentration of DNA was high, most of the longest amplification fragments of extracted DNA were 100 bp or 200 bp, and the detection rate of short fragment IGH FR3 was more stable than that of long fragment IGH FR1. (3)The clonality analysis of TCRG+TCRB in all 13 cases of peripheral T cell lymphoma samples showed positive results, while no positive IG/TCR clones were found in 7 cases of reactive lymphoid tissue hyperplasia in control group.
Dilution of DNA is the only method to improve not only the proportion of longest fragment amplification but also the detection rate of clonality. The detection rate of IGH FR3 would not be affected by the concentration of DNA. The application of BIOMED-2 standardized IG/TCR gene rearrangement system in FFPE tissue samples plays an important role in the lymphoma diagnosis.
探讨应用BIOMED-2标准化免疫球蛋白/T细胞受体（IG/TCR）基因重排系统检测福尔马林固定石蜡包埋（FFPE）组织样本中淋巴瘤的可行性，并探讨提取DNA的最长扩增片段与不同IGH V-J引物阳性检出率之间的关系。
从50例FFPE组织样本中提取DNA。进行多重聚合酶链反应（Multiplex-PCR）扩增，然后使用BIOMED-2标准化克隆性分析系统分析IG/TCR基因重排。
（1）在30例弥漫性大B细胞淋巴瘤（DLBCL）蜡块样本中，当DNA浓度从100 - 500 ng/μl稀释至50 - 100 ng/μl时，DNA最长扩增片段（300 - 400 bp）的比例从10.0%提高至90.0%（P<0.01）。IGH+IGK阳性率从46.7%提高至83.3%，差异有统计学意义（P = 0.006）。DLBCL石蜡切片样本中DNA最长扩增片段长度均大于300 bp，这些样本的IGH+IGK阳性率为96.7%。蜡块样本与石蜡切片样本的IGH+IGK阳性率差异无统计学意义（P = 0.195）。（2）当DNA浓度较高时，提取的DNA最长扩增片段大多为100 bp或200 bp，短片段IGH FR3的检出率比长片段IGH FR1更稳定。（3）13例外周T细胞淋巴瘤样本的TCRG+TCRB克隆性分析均显示阳性结果，而对照组7例反应性淋巴组织增生中未发现阳性IG/TCR克隆。
DNA稀释是提高最长片段扩增比例及克隆性检出率的唯一方法。IGH FR3的检出率不受DNA浓度影响。BIOMED-2标准化IG/TCR基因重排系统在FFPE组织样本中的应用对淋巴瘤诊断具有重要作用。
