T、B细胞受体基因重排克隆性分析的应用要略

T、B细胞的受体基因重排克隆性分析可协助诊断淋巴组织的疑难病例，为此综述受体基因重排克隆性分析的理论与实践要领，包括基本原理、假阴性、假阳性、交叉性、敏感性、阳性率、分析方法的选择和具体运用及结果判定。     

淋巴瘤TCRγ及TCRβ克隆性基因重排分析

T淋巴细胞免疫球蛋白受体包括αβ和76两组,共涉及到相应的4条基因重排并编码合成。检测淋巴组织中这4处基因重排为克隆性还是非克隆性,可以鉴别组织中淋巴细胞是克隆性增生的瘤细胞还是非克隆性反应性增生的淋巴组织,对淋巴瘤的诊断有重要意义。临床中常用的检测为TCRγ或TCR8两种基因,结果发现,仅检测其中的一项,并非所有的T细胞淋巴瘤均可出现克隆性基因重排,其中不能排除假阴性的可能。所以,本研究将同一组T细胞淋巴瘤标本采用两种基因重排检测,观察是否可以提高阳性率。     

抗原受体基因重排克隆性检测在淋巴瘤诊断中的应用

本研究旨在探讨抗原受体基因重排克隆性检测在淋巴瘤诊断中的意义。收集分析了31例淋巴瘤组织,石蜡包埋切片,HE染色后观察形态,免疫组织化学分析免疫表型。采用BIOMED-2标准化试剂盒检测抗原受体基因重排克隆性。结果表明,31例病例中形态学及免疫组织化学分析疑似T细胞淋巴瘤12例,T细胞反应性增生1例,B细胞淋巴瘤16例,B细胞反应性增生2例。基因重排克隆性检测免疫球蛋白(Ig)阳性率94.44%(17/18),T细胞抗原受体(TCR)阳性率92.31%(12/13),2例阴性。最终12例确诊为T细胞淋巴瘤,B细胞淋巴瘤17例,反应性增生2例,阳性检出率为93%。结论:抗原受体重排基因克隆性分析是诊断淋巴瘤的一种有效辅助手段。     

BIOMED-2方法检测恶性淋巴瘤骨髓侵犯的基因重排研究

本研究探讨BIOMED-2方法检测非霍奇金淋巴瘤(NHL)患者骨髓中免疫球蛋白(IG)和T细胞受体(TCR)基因克隆性重排的可行性,并初步评价其临床价值。采用BIOMED-2系统检测73例NHL(B-NHL 55例,T-NHL 18例)患者骨髓中IGH、IGK、IGL基因和TCRβ、TCRγ、TCRδ基因的克隆性重排,与骨髓穿刺细胞形态学进行比较,评价其与病理特征、临床分期等的相关性。结果表明:73例NHL中31例检测出IG或TCR基因重排,阳性率42.5%,高于骨髓穿刺细胞形态学阳性率24.7%(18/73),差异具有统计学意义(p<0.05);其中B-NHL阳性率为40.0%(22/55),T-NHL阳性率为50.0%(9/18),两者差异无统计学意义(p>0.05)。PCR检测阳性率和AnnArbor分期相关,Ⅲ/Ⅳ期患者阳性率高于Ⅰ/Ⅱ期,差异具有统计学意义(p<0.05)。结论:BIOMED-2检测IG和TCR基因重排是判断淋巴瘤骨髓浸润的有效方法,比骨髓细胞形态学更为敏感,有助于临床分期、预后判断和治疗选择。PCR检测阳性率和Ann Arbor分期相关,与淋巴瘤恶性程度、年龄、治疗状态、有无B组症状及是否累及脾脏无关。     

免疫球蛋白基因重排分析在淋巴瘤诊断中的初步应用研究

用免疫球蛋白重链Ｊ区（ＪＨ），Ｋ轻链及λ轻链Ｃ区（ＣＫ，Ｃλ２）基因探针及Ｓｏｕｔｈｅｒｎ印迹杂交技术，对２２例非何杰金氏淋巴瘤进行分析。结果显示１３例尿Ｂ细胞淋巴瘤均出现重链基因重排，其中１０例有Ｋ或λ轻链基因重排；３例Ｔ细胞淋巴瘤中，１例出现重链，１例出现Ｋ轻链基因重排。１例分类未明淋巴瘤出现重链基因重排。５例未作免疫表型分析的淋巴瘤中有４例出一现重链，３例出现Ｋ轻链，１例出现λ轻链基因重排。     

胃肠道淋巴瘤TCRγ基因重排分析及其意义

胃肠道淋巴瘤是最常见的结外淋巴瘤之一 ,其诊断较为复杂 ,误诊率高。诊断通常要根据临床症状、形态学、免疫组织化学和分子生物学等综合判断。由于T细胞受体γ(TCRγ)基因重排发生在T细胞分化的早期 ,并且在两种不同表型的T细胞 (αβ和γδ)中都存在TCRγ基因重排 ,因此 ,TCRγ基因重排成为检测淋巴瘤T细胞单克隆基因重排常用的靶基因。我们选用TCRγ通用引物 ,采用PCR、琼脂糖凝胶电泳及单链构象多态性分析 (SSCP) ,检测并分析了 4 0例胃肠道淋巴瘤基因重排的情况 ,探索这些方法在胃肠道淋巴瘤诊断和研究中的意义。材料和方法1…     

克隆性基因重排检测在NHL诊断中的意义

目的探明PCR克隆性基因重排检测（基因诊断）在NHL诊断中的意义。方法应用病理组织学、免疫组化、基因诊断方法三结合方案分析178例与会诊31例NHL诊断的作用。结果分析结果显示：以病理组织学为主,辅以免疫组化确诊NHL114例。疑难、早期、微量标本共64例与会诊31例均作了基因诊断。总的阳性率为61．4％（58／95）。其中IgH阳性13例（22．4％）,TCRβ芝邱阳性41例（70．6％）,双克隆性4例（7％）。本室基因诊断送检率为35．8％（64／178）,总确诊率为93．7％（60／64）。结论本文分析证明基因诊断是NHL三结合诊断中确诊疑难病例及分型的最有效的方法。     

Ig/TCR基因重排与基因突变检测在淋巴瘤诊断中的临床价值

目的 探讨Ig/TCR基因重排和基因突变在淋巴瘤诊断中的应用价值.方法 从227例患者石蜡包埋组织中提取基因组DNA,使用BIOMED-2系统引物和聚丙烯酰胺凝胶电泳法对标本基因组DNA中IGH、IGK、IGL、TCRG、TCRB、TCRD的重排进行分析,并对其中6例疑难病例进行相关基因突变二代测序检测.结果 178例...     

非霍奇金淋巴瘤基因重排研究

本文阐述了在非霍奇金淋巴瘤中作为肿瘤克隆性标志的抗原受体基因重排和某些组织学亚型淋巴瘤中由染色体易位引起的特异性癌基因重排，并探讨这些分子标记在淋巴瘤病因学中的作用及其临床应用。     

免疫球蛋白重链基因重排在B淋巴细胞系统肿瘤中的…

单克隆性免疫球蛋白重链基因重排作为Ｂ细胞系肿瘤的分子标志，已广泛应用于肿瘤的早期诊断及鉴别诊断，特别是对微小残留病的检测，对指导治疗和判断预后具有重要意义。本文对其在临床中的研究应用作一综述。     

BIOMED-2 multiplex immunoglobulin/T-cell receptor polymerase chain reaction protocols can reliably replace Southern blot analysis in routine clonality diagnostics

To establish the most sensitive and efficient strategy of clonality diagnostics via immunoglobulin and T-cell receptor gene rearrangement studies in suspected lymphoproliferative disorders, we evaluated 300 samples (from 218 patients) submitted consecutively for routine diagnostics. All samples were studied using the BIOMED-2 multiplex polymerase chain reaction (PCR) protocol. In 176 samples, Southern blot (SB) data were also available, and the two types of molecular results were compared. Results of PCR and SB analysis of both T-cell receptor and immunoglobulin loci were concordant in 85% of samples. For discordant results, PCR results were more consistent with the final diagnosis in 73% of samples. No false-negative results were obtained by PCR analysis. In contrast, SB analysis failed to detect clonality in a relatively high number of samples, mainly in cases of low tumor burden. We conclude that the novel BIOMED-2 multiplex PCR strategy is of great value in diagnosing patients with suspected B- and T-cell proliferations. Because of its higher speed, efficiency, and sensitivity, it can reliably replace SB analysis in clonality diagnostics in a routine laboratory setting. Just as with SB results, PCR results should always be interpreted in the context of clinical, immunophenotypical, and histopathological data.     

运用BIOMED-2 PCR检测脑脊液中的恶性B淋巴细胞

本研究旨在建立一种检测弥漫性大B细胞淋巴瘤（DLB CL）中枢神经系统（CNS）累及患者脑脊液（CSF）中恶性B淋巴细胞的敏感的方法.对9例考虑有CNS累及风险的DLBCL患者采集其CSF,离心获取细胞沉淀,在直接裂解后运用BIOMED-2 PCR检测免疫球蛋白重链（IgH）基因重排（恶性B淋巴细胞特征性改变）,并将此方法的敏感性与细胞学检测及流式细胞术进行比较.此外,通过一系列数量/浓度的肿瘤细胞,分析两种样品处理方式（细胞直接裂解法和传统DNA提取法）导致的敏感度差异,并评估直接裂解法联合BIOMED-2 PCR方案的敏感度.结果显示,BIOMED-2 PCR检测到5例DLBCL患者的CSF中存在克隆性IgH基因重排（恶性B淋巴细胞“阳性”）,而细胞学检测和流式细胞术均只能明确检测2例为“阳性”,表明BIOMED-2 PCR敏感性高于细胞学检测和流式细胞术.另外,经过改进的样品处理方式——细胞直接裂解法比传统的DNA提取能获得更高的BIOMED-2PCR敏感度,前者可以检测到浓度低至1％、细胞数低至20个的肿瘤细胞.结论：细胞直接裂解联合BIOMED-2PCR是一种敏感的、非常适用于CSF（细胞数少）的检测方法,可辅助诊断DLBCL病例的CNS累及.     

Ig基因重排检测在B细胞性淋巴瘤诊断中的应用

目的探讨Ig基因重排检测在B细胞性淋巴瘤中的诊断价值。方法选取B细胞性淋巴瘤30例、淋巴组织反应性增生30例,提取DNA,应用BIOMED-2引物系统中的47条引物进行PCR扩增,核酸分子异源双链凝胶电泳分析Ig基因重排结果。结果 30例B细胞性淋巴瘤中检测出Ig克隆性重排,包括IGH(A+B)克隆性重排23例,IGK克隆性重排23例,IGL克隆性重排5例,IGH(A+B)+IGK克隆性重排30例,IGHA+IGK克隆性重排29例;在30例淋巴组织反应性增生中未检测到Ig克隆性重排。结论 Ig基因重排是诊断B细胞性淋巴瘤的有用工具。     

非霍奇金淋巴瘤患者免疫球蛋白及T细胞受体基因重排研究

目的 研究非霍奇金淋巴瘤(NHL)患者骨髓或外周血免疫球蛋白(Ig)/T细胞受体(TCR)基因单克隆重排特点及其临床意义.方法 以BIOMED-2引物系统及多重PCR方法对139例NHL患者骨髓或外周血标本进行Ig及TCR基因重排检测.结果 在B系NHL(B-NHL)中,82例慢性淋巴细胞白血病(CLL)患者中有70例(85.4％)检出Ig基因单克隆重排,其中IgH、IgK、IgL单克隆重排阳性率分别为46.3％(38例)、62.2％(51例)和1.2％(1例).其他类型的B-NHL患者有39.4％(33例中有13例)检出Ig基因单克隆重排,其中IgH和IgK单克隆重排阳性率分别为33.3％(11例)和39.4％(13例),未检测到IgL基因单克隆重排.T系NHL(T-NHL)患者中有50.0％(24例中有12例)检出TCR基因单克隆重排,其中TCRB和TCRG单克隆重排阳性率分别为8.3％(2例)和45.8％(11例),TCRB和TCRG双重基因重排阳性率为4.2％(1例),未检测到TCRD单克隆重排.11例早期(Ann Arbor Ⅰ、Ⅱ期)与46例晚期(Ⅲ、Ⅳ期)患者的Ig/TCR基因单克隆重排阳性率分别为36.4％和45.6％;10例惰性患者和47例侵袭性患者的Ig/TCR基因单克隆重排阳性率分别为40.0％和44.7％,差异均无统计学意义(P值均＞0.05).除外CLL的57例NHL患者骨髓涂片检测骨髓侵犯阳性率为12.3％,明显低于骨髓基因重排检测阳性率(43.9％),两者差异有统计学意义(P＜0.05).敏感性试验表明多重PCR检测恶性克隆的敏感性为3.12％～6.25％.结论 NHL的临床分期和恶性程度不同,其Ig/TCR基因单克隆重排阳性率有差异,但未能证实其相关性.联合应用BIOMED-2多重引物可提高PCR检测骨髓和(或)外周血Ig/TCR基因单克隆重排的敏感性.多重PCR对NHL患者骨髓侵犯检测的敏感性优于骨髓涂片检查,有助于早期发现骨髓侵犯及预防复发.     

Application of microfluidic technology to the BIOMED-2 protocol for detection of B-cell clonality

The BIOMED-2 protocol is widely used for detecting clonality in lymphoproliferative disorders. The protocol requires multiple PCR reactions, which are analyzed by either capillary electrophoresis (GeneScan analysis) or heteroduplex PAGE analysis. We tested a microfluidic chip-based electrophoresis device (Agilent 2100 Bioanalyzer) for the analysis of B-cell clonality using PCR for the three framework subregions (FR) of the Ig heavy chain gene (IGH) and PCR for two rearrangements occurring in the Ig κ chain gene (IGK-VJ and IGK-DE). We analyzed 62 B-cell lymphomas (33 follicular and 29 nonfollicular) and 16 reactive lymph nodes. Chip-based electrophoresis was conclusive for monoclonality in 59/62 samples; for 20 samples, it was compared with GeneScan analysis. Concordant results were obtained in 45/55 IGH (FR1, FR2, and FR3) gene rearrangements, and in 34/37 IGK gene rearrangements. However, when the chip device was used to analyze selected IGK gene rearrangements (biallelic IGK rearrangements or IGK rearrangements in a polyclonal background), its performance was not completely accurate. We conclude, therefore, that this microfluidic chip-based electrophoresis device is reliable for testing cases with dominant PCR products but is less sensitive than GeneScan in detecting clonal peaks in a polyclonal background for IGH PCR, or with complex IGK rearrangement patterns.     

T cell receptor gene rearrangement lineage analysis reveals clues for the origin of highly restricted antigen-specific repertoires

Due to ordered, stage-specific T cell receptor (TCR)-beta and -alpha locus gene rearrangements and cell division during T cell development, a given, ancestral TCR-beta locus VDJ rearrangement might be selected into the mature T cell repertoire as a small cohort of "half-sibling" progeny expressing identical TCR-beta chains paired with different TCR-alpha chains. The low frequency of such a cohort relative to the total alphabeta TCR repertoire precludes their direct identification and characterization in normal mice. We considered it possible that positive selection constraints might limit the diversity of TCR-alpha chains selected to pair with beta chains encoded by an ancestral VDJ-beta rearrangement. If so, half-sibling T cells expressing structurally similar, but different TCR-alpha chains might recognize the same foreign antigen. By single cell polymerase chain reaction analysis of antigen-specific TCRs selected during a model anti-tumor response, we were able to identify clusters of T cells sharing identical VDJ-beta rearrangements but expressing different TCR-alpha chains. The amplification of residual DJ-beta rearrangements as clonal markers allowed us to track T cells expressing different TCR-alpha chains back to a common ancestral VDJ-beta rearrangement. Thus, the diversity of TCR-alpha's selected as partners for a given VDJ-beta rearrangement into the mature TCR repertoire may indeed be very limited.