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.     

Immunoglobulin heavy chain gene analysis in lymphomas: a multi-center study demonstrating the heterogeneity of performance of polymerase chain reaction assays

Determination of monoclonality through an evaluation of immunoglobulin heavy chain (IgH) gene rearrangements is a commonly performed and useful diagnostic assay. Many laboratories that perform this assay do so by the polymerase chain reaction (PCR). To evaluate current methods for performing IgH gene testing, 19 different Association of Molecular Pathology (AMP) member laboratories analyzed 29 blinded B cell and T cell lymphoid neoplasm samples of extracted DNA and formalin-fixed, paraffin-embedded (FFPE) tissue and were asked to complete a technical questionnaire. From this study, it is clear that Southern blot analysis remains the diagnostic gold standard, with a 100% diagnostic sensitivity and specificity. There was, however, remarkable heterogeneity in the performance of, and results obtained from, IgH PCR assays with diagnostic sensitivity ranging from over 90% to as low as 20%, when evaluating the same specimens. Many laboratories overestimate the diagnostic sensitivity of their IgH PCR assay, and there was a significant, and under appreciated, drop-off (from 61.3% to 41.8%) in detection in paired FFPE as compared with fresh/frozen tissues. Fixation has a dramatic impact on the inability to perform the test on FFPE (43.1%) versus DNA already extracted from fresh or frozen tissue (2.8%). A number of variables that affected the outcome of IgH PCR were identified. Strategies that improved the detection of monoclonal IgH rearrangements include: the addition of FRII to the FRIII upstream primer (increasing detection from 57.3% to 73.6%) and the use of the FR3A rather than the FR3 FRIII primer (increasing detection from 54.7% to 69.7%). Although numerous variables (from DNA extraction to PCR product detection) were evaluated, making it difficult to mandate alterations in laboratory practice, these findings ought to prompt diagnostic molecular pathology laboratories to reevaluate their claims of sensitivity, as well as their methodologies. Both pathologists and surgeons need to ensure that not all submitted material is fixed, if there is adequate sample. Importantly, there is a need for greater standardization to reduce the unacceptably high false negative rate of this crucial diagnostic assay.     

IgH基因克隆性重排检测在B细胞淋巴瘤诊断中的应用

目的建立B-NHL临床诊断中IgH基因克隆性重排检测基本方法.方法应用PCR方法,采用IgH FR3A引物,检测了7例B-NHL及4例反应性增生的淋巴结石蜡样本中IgH基因重排情况.结果黏膜相关型边缘区B细胞淋巴瘤（MALT）3例中有2例（2/3）,弥漫大B细胞淋巴瘤2例中2例（2/2）,滤泡淋巴瘤1例中0例（0/1）,套细胞淋巴瘤1例中1例（1/1）检测到IgH基因的克隆性重排.总检测率为5/7（71%）,4例淋巴结反应性增生病例病例均为IgH基因的多克隆性重排.结论PCR-FR3A在鉴别淋巴组织肿瘤性或反应性增生和最终确诊B-NHL上是一项有效的辅助方法.     

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.     

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。     

Improvement of clonality detection rate in multiple myeloma using fluorescent IgH PCR with different sets of primers

The IgH rearrangement provides a useful marker of clonality in B-cell malignancies and amplification of this rearrangement is the method of choice to monitor the residual tumor cells in multiple myeloma (MM). The critical point of this analysis was the false-negative rate observed at diagnosis in patients presenting tumor cells well above the limit of detection. The aim of this study was therefore to increase the clonality detection rate by IgH polymerase chain reaction (PCR). Bone marrow DNA from 37 MM patients were analyzed at diagnosis. IgH PCR with agarose gel detection was performed between framework regions FR3 and FR1, both in combination with 5 different primers in FR4. Fluorescent IgH PCR with highly resolutive capillary electrophoresis was used to improve the detection and to size clonal PCR products. Sixty-two percent of the clonal rearrangements were initially detected with JHD primer specific to the JH segments 1,2,4,5. The use of JH3 and JH6 homologous primers increased the detection rate to 78%, whereas a consensus JH primer only reached 67% of positivity. The lowest detection rates were obtained with JHExt and JH3 with a detection of respectively 43 and 14%. However, three rearrangements were exclusively amplified by JHExt and two additional cases were detected by JH3. The combined use of primers yielded the best score with 89% of positivity. With Genescan analysis, two additional cases showed a monoclonal rearrangement improving the detection rate to 95%. The use of multiple sets of primers along with a highly sensitive genescan analysis makes possible the follow-up of minimal residual disease for most MM patients.     

非霍奇金淋巴瘤抗原受体基因重排检测中双重重排现象的初步研究

目的　检测非霍奇金淋巴瘤 (NHL)抗原受体基因双重重排的发生率及初步探讨其病理意义和可能机制。方法　采用PCR方法 ,联合使用IgHFR3A及FR2A、TCRγ、TCRβ引物 ,检测 12 5例NHL患者IgH基因及TCR基因克隆性重排 ;检测B NHL和T NHL中抗原受体基因双重重排的发生率 ,并对其中的 117例NHL患者采用免疫组织化学EnVision两步法检测Ki6 7蛋白表达并计算增殖指数 ,分析NHL不同重排类型与增殖指数间的关系。结果　联合使用IgHFR3A及FR2A、TCRγ、TCRβ引物 ,96例B NHL患者中有 8例 (8% )检测到发生双重重排 ,2 9例T NHL患者中有 5例 (17% )检测到发生双重重排。B NHL中 6 5例检测到IgH基因克隆性重排 ,8例发生双重重排 ,15例为多克隆重排 ;T NHL中有 15例检测到TCR基因克隆性重排 ,5例发生双重重排 ,9例为多克隆重排。上述病例同时平行检测了Ki6 7,并计算增殖指数 ,经One way检验 ,在B NHL和T NHL中 ,克隆性重排、双重重排及多克隆重排的淋巴瘤增殖指数差异无显著性 (P >0 .0 5 )。结论　淋巴瘤抗原受体基因的双重重排在成熟NHL中并非罕见 ;增殖指数与NHL基因双重重排无相关性     

IgH基因重排PCR分析在淋巴组织细针穿刺中的辅助诊断价值

目的 探讨Ign基因重排检测在细针穿刺诊断淋巴组织病变中的价值.方法 收集淋巴组织细针穿刺病例159例,应用半巢式PCR检测IgH基因重排.将基因重排检测结果与细胞学诊断及组织学随访结果进行比较.结果 穿刺标本中,IgH基因重排检测B-NHL阳性率为72.22%,RH阳性率仅为3.92%.本法敏感性为72.22%,特异性达到96.08%.结论 IgH基因重排检测对辅助淋巴组织细针穿刺标本的良、恶性判断具有一定价值,并且单克隆性结果更有意义.但在淋巴瘤和转移性非淋巴细胞恶性肿瘤的鉴别上不建议应用.     

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.     

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.     

BIOMED-2多重聚合酶链反应检测抗原受体基因重排在淋巴增殖性疾病诊断中的应用

目的 建立敏感而有效的检测免疫球蛋白(Ig)和T细胞受体(TCR)基因重排的方法,并探讨在淋巴增殖性疾病诊断和鉴别中的作用.方法 采用BIOMED-2多重聚合酶链反应,检测来自54例淋巴增殖性疾病患者的58份淋巴组织标本,分析抗原受体基凶重排状况及其克隆来源.结果 在88.0%(25份标本中22份)B细胞淋巴瘤/白血病和53.3%(15份标本中8份)T细胞淋巴瘤中检出Ig/TCR呈单克隆重排;在17例淋巴组织非恶性增殖患者的病理活检标本中,14例(82.4%)呈多克隆重排;在合格的57份标本中有44份(77.2%)的结果与最终诊断相符.联合检测Igλ和TCRδ并未提高Ig/TCR单克隆重排的检出率,但可能有助于Igλ和TCRδ+淋巴瘤的诊断.结论 对于分析淋巴增殖性疾病,尤其是不典型病例的克隆来源状况,BIOMED-2多重聚合酶链反应检测抗原受体基凶重排是迅速、敏感而可靠的方法.     

Detection of T-regulatory cells has a potential role in the diagnosis of classical Hodgkin lymphoma

Previous studies have demonstrated an increase in T-regulatory cells in the involved lymph nodes and peripheral blood of patients with Hodgkin lymphoma. Our study examined whether the detection of T-regulatory cells by flow cytometry could distinguish classical Hodgkin lymphoma (CHL) from benign cases and B-cell non-Hodgkin lymphomas (B-NHL). We measured CD4, CD25, and CD152 in 14 CHLs, 2 nodular lymphocyte-predominant Hodgkin lymphomas, 31 B-NHLs, and 54 benign cases. All T-regulatory cell parameters, including percent lymphocytes CD4+/CD152+ and CD4+/CD25+/CD152+, and mean and median CD152 expression in CD4+/CD25+ lymphocytes, were higher in CHL than in B-NHL and benign. Mean CD152 in CD4+/CD25+ lymphocytes distinguished CHL from benign with 79% sensitivity and 100% specificity, and from B-NHL with 71% sensitivity and 90% specificity. Overall, our results show that T-regulatory cells are increased in CHL and their detection may be a useful tool in differentiating CHL from other entities.     

B-cell clonality determination using an immunoglobulin kappa light chain polymerase chain reaction method

To augment the detection of clonality in B-cell malignancies, we designed a consensus primer kappa light chain gene (Igkappa) polymerase chain reaction (PCR) assay in combination with a consensus primer immunoglobulin heavy chain gene (IgH) PCR assay. Its efficacy was then evaluated in a series of 86 paraffin tissue samples comprising neoplastic and reactive lymphoproliferations. Analysis after PCR was accomplished by 10% native polyacrylamide gel electrophoresis after heteroduplex pretreatment of PCR products and by a post-PCR chip-based capillary electrophoresis analytic method. Overall, 49 of 68 (72%) of mature B-cell neoplasms yielded discrete Igkappa gel bands within the predicted size range with no clonotypic Igkappa products observed among reactive lymphoid or T-cell proliferations. The application of Igkappa PCR improved overall sensitivity from 81% with IgH PCR alone to 90% with combined Igkappa/IgH PCR, with this effect being most notable in germinal center-related lymphomas. Sequencing of positive Igkappa rearrangements revealed that most rearrangements involved members of the Vkappa1 (40%) and Vkappa2 (34%) gene families along with Jkappa1 (26%), Jkappa2 (23%), and Jkappa4 (51%) gene segments. Involvement of Vkappa pseudogenes was identified in 24% of cases with Vkappa-KDE rearrangements. Our results demonstrate the efficacy of Igkappa PCR in improving the detection rate of clonality in B-cell neoplasms and further introduce a novel post-PCR chip-based capillary electrophoresis analytic method for rapid PCR fragment size evaluation.     

PCR analysis of the immunoglobulin heavy chain gene in polyclonal processes can yield pseudoclonal bands as an artifact of low B cell number

Polymerase chain reaction (PCR)-based analysis for detecting immunoglobulin heavy chain gene (IgH) rearrangements in lymphoproliferative disorders is well established. The presence of one or two discrete bands is interpreted as a monoclonal proliferation, whereas a smear pattern represents a polyclonal population. Prompted by our observation of discrete bands in histologically reactive processes with a relative paucity of B cells, we sought to determine whether low numbers of B cells in biopsy specimens could artifactually produce pseudomonoclonal bands. We performed IgH PCR analysis on serially diluted DNA samples from 5 B cell non-Hodgkin’s lymphomas (B-NHLs), 5 reactive lymph nodes, 5 reactive tonsils and 10 microdissected germinal centers from a lymph node with follicular hyperplasia. We also assessed multiple aliquots of DNA samples from small biopsy specimens of reactive lymphocytic processes from the stomach (5 cases). PCR products were evaluated using high resolution agarose or polyacrylamide gels, and DNA sequencing was performed on IgH PCR products from two reactive germinal centers, which yielded monoclonal bands of identical size. All 5 B-NHLs harboring monoclonal B cell populations yielded single discrete bands, which were maintained in all dilutions. By contrast, all of the reactive lesions with polyclonal patterns at 50 ng/μl starting template concentration showed strong pseudomonoclonal bands at dilutions of 1:1000 to 1:1500 in placental DNA. Two of the microdissected reactive germinal centers that showed bands of identical size on duplicate reactions were proven to have different IgH sequences by sequencing. We conclude that specimens containing low numbers of polyclonal B cells may produce pseudomonoclonal bands on IgH PCR analysis. IgH PCR analysis should be performed on multiple aliquots of each DNA sample, and only samples that yield reproducible bands of identical size can be reliably interpreted as monoclonal.     

Enhanced sensitivity with a novel TCRgamma PCR assay for clonality studies in 569 formalin-fixed, paraffin-embedded (FFPE) cases.

BACKGROUND: Clonal rearrangement of genes encoding the immunoglobulins (Ig) and T-cell antigen receptors (TCR) are considered to be useful markers for the diagnosis of lymphoma and for determining the clonal origins of B- and T-cell populations in lymphoid neoplasms. METHODS AND RESULTS: Polymerase chain reaction-based clonality assays for TCRgamma, TCRbeta, and immunoglobulin (Ig) heavy chain (IgH) gene rearrangements were evaluated for diagnostic sensitivity and specificity in 569 formalin-fixed, paraffin-embedded (FFPE) tissues. Combined TCRbeta and TCRgamma assays enhanced the routine detection of TCR clonality to 90% of all peripheral T-cell lymphoma (PTCL) cases. IgH clonality was detected in 59% of 241 peripheral B-cell lymphoma (BCL) cases and 6% of 169 PTCL cases. Of 452 lymphomas, 5% could not be classified phenotypically as B or T lineage after immunohistochemical and clonality studies. Of all BCL cases analyzed, 24% had detectable TCRbeta and/or TCRgamma clonality. Of these BCL with biclonal results, 47% were extranodal lymphomas from skin and various tissues. CONCLUSIONS: Clonality assays were useful for distinguishing reactive or benign lymph nodes from neoplastic lymphoid infiltrates in most cases. The inclusion of TCRb and TCRg assays in the assessment of lymphomas results in a significant increase in the sensitivity of clonality detection, but is of limited utility in assessing the T- or B-cell phenotype of the tumor.     

多重PCR法检测初诊成人急性淋巴细胞白血病患者克隆性免疫球蛋白和T细胞受体基因重排

目的 应用多莺PCR方法检测初诊成人急性淋巴细胞白血病(ALL)患者的克隆性免疫球蛋白(Ig)和T细胞受体(TCR)基因重排,为实时定量RT-PCR(RQ-PCR)法监测ALL患者体内的微量残留病(MRD)奠定基础.方法 参照BIOMED-2协作组制定的Ig和(或)TCR检测方法,设计96条不同的PCR引物,分成14个混合管,通过多重PCR,分别检测患者骨髓单个核细胞的IgH、IgK、TCRB、TCRG、TCRD克隆性基因重排.结果 在22例成人B系ALL患者中,Ig克隆性重排检出率为96%,其中IgH为86%,IgK为14%.在18例成人T系ALL患者中,TCR克隆性重排检出率为100%,其中TCRB为83%,TCRG为78%,TCRD为39%.两个及两个以上克隆性标志物的检出率在B和T系ALL中分别为91%(22例中20例)和89%(18例中16例).结论 BIOMED-2协作组设计的14管多重PCR引物和方法,几乎可检测到淋巴细胞白血病患者体内所有占优势的克隆性T、B细胞增殖群体,方法简便、可靠、覆盖面广,适用于成人ALL患者基因重排检测和MRD监测.     

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。     

多重PCR检测基因抗原受体重排在儿童急性淋巴细胞白血病诊断中的应用

目的 建立多重PCR方法检测免疫球蛋白（Ig）和T细胞受体（TCR）基因重排,探讨在儿童急性淋巴细胞白血病诊断和鉴别中的作用.方法 参照BIOMED-2协作组制定的Ig和（或）TCR检测方法,对儿童ALL患儿114 例,分别检测患者骨髓单个核细胞的IgH、IgK、TCRG和TCRB基因重排.结果 在105例B系儿童ALL患者中,克隆重排的检出率分别为IgH 73%、IgK34%、TCRG 44%和TCRB 35%,所有B-ALL患者Ig基因重排检出率可达85%;在11例T系ALL患者中,克隆重排的检出率分别为TCRB 82%和TCRG 73%,T-ALL 患者TCR基因重排检出率可达100%.结论 BIOMED-2引物系统可以检测出绝大多数儿童ALL患者的Ig/TCR基因重排,是一种有效的检测工具,值得在临床中推广使用.     

PCR检测B-ALL IgH基因重排

为了探讨急性淋巴细胞白血病(ALL)患儿免疫球蛋白重链(IgH)基因重排的临床价值,应用多聚酶链反应(PCR)技术检测25例B-ALL患儿IgH基因。结果表明,25例中19例在初诊或复发时检出克隆性重排,并对此19例随访观察48次,平均随访时间为11.2月(2～25月)。IgH-PCR预测ALL复发率为58.8％,占复发病例的80％。阳性克隆检出时间先于形态学复发的平均时间为10.3周(1～28.8周)。本方法敏感性0.5％。该研究提示,IgH-PCR对监测 B-ALL的微量残留病(MRI)具有一定实用价值。但IgH-PCR阴性并不能排除即将复发,阳性可作为预测复发的信号。在随访的病例中,阳性者先后均复发。     

Detection of T-cell receptor gene rearrangement in children with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis using the BIOMED-2 multiplex polymerase chain reaction combined with GeneScan analysis

BackgroundHemophagocytic lymphohistiocytosis (HLH) is a serious immune disorder. Epstein–Barr virus (EBV) is the most common trigger of secondary HLH. T-cell receptor (TCR) gene rearrangement is detectable in half of patients with EBV-associated HLH using Southern blotting and conventional polymerase chain reaction (PCR) analyses. However, its clinical significance is unclear. The impact of TCR gene clonality on the outcome of patients with EBV-HLH should be evaluated using more sensitive methods. In this study, we used BIOMED-2 multiplex PCR and GeneScan analysis to evaluate TCR gene rearrangement in childhood EBV-HLH.MethodsSix children with EBV-HLH were enrolled in this study. EBV load in blood was quantified by real-time PCR. TCR gene rearrangement was analyzed by BIOMED-2 multiplex PCR.ResultsAll 6 patients showed monoclonal peaks in TCRβ and/or TCRγ at diagnosis. Serial monitoring of one patient disclosed a change in the rearrangement pattern of the TCR genes in response to immunochemotherapy.ConclusionThese findings suggest that BIOMED-2 multiplex PCR, a highly sensitive method for detecting T-cell clonality, is useful for predicting the therapeutic response in childhood EBV-HLH.