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.     

采用PCR检测淋巴结细胞IgH和TCRγ基因重排对淋巴增殖病的诊断意义

采用PCR体外基因扩增技术研究10例活检淋巴结细胞IgH和TCRγ基因重排,并与组织病理和免疫表型分析进行了对比研究。结果表明:6例病理诊断为非何杰金淋巴瘤(NHL)者中,4例发生相应的IgH或TCR_γ基因克隆性重排,与免疫表型分析一致,另2例同时发生了IgH和TCR_γ基因双重重排。2例病理诊断为坏死性淋巴结炎者,免疫表型分析起源于B细胞,均检测到IgH基因克隆性重排,证明系克隆性恶性B淋巴细胞增殖。表明用PCR检测IgH和TCR_γ基因重排,对恶性淋巴细胞增殖病诊断的敏感性和特异性高于组织病理学和免疫表型分析,具有重要临床应用价值。     

半巢式聚合酶链反应方法检测弥漫性大B细胞淋巴瘤bcl-2/IgH基因重排

目的寻找一种敏感、特异的方法检测弥漫性大B细胞淋巴瘤（DLBCL）bcl-2／IgH基因重排，并通过测定产物的序列，以了解所建方法的可靠性、方法运用专业引物设计软件设计bcl-2／IgH基因重排半巢式PCR引物，对52例经临床病理确诊的DLBCL石蜡包埋组织及10例慢性扁桃体炎患者的新鲜扁桃体组织通过半巢式递降温度梯度PCR（touch down PCR）扩增，检测bcl-2／IgH基因重排，并对其产物进行克隆和序列分析。结果 通过一步法检测到bcl-2／IgH基因重排8例，其中DLBCL6例，新鲜扁桃体组织2例；进行第2次半巢式PCR时仅在DLBCL中发现5例阳性，新鲜扁桃体组织均阴性。将阳性产物在网上序列分析显示：一步法检测到的8例中有3例为假阳性，而半巢式PCR扩增出的5例均为bcl-2／IgH基因重排片段。3例假阳性的片段分别与人类第19号染色体BAC331191，LLNLR-245D11基因片段及1号染色体RP11-498P10基因片段同源。结论常用的检测bcl-2／IgH基因重排引物扩增结果存在一定假阳性，其机制可能是因为人类基因组中存在与常用引物同源性较高的序列。为研究设计的引物与传统的引物结合，进行半巢式PCR可以排除这种假阳性扩增，提高诊断的准确性。     

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.     

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.     

B细胞淋巴瘤bcl-2基因重排与14号染色体微卫星改变的研究

目的 研究bcl-2/IgH基因重排与微卫星DNA改变在B细胞淋巴瘤发生发展中的作用以及bcl-2/IgH基因重排与微卫星不稳定和杂合性缺失之间的关系.方法 应用巢式PCR方法检测bcl-2/IgH基因重排;选取14号染色体上2个微卫星多态性标记,采用聚合酶链反应-单链构象多态性分析法（PCR-SSCP）检测37例B细胞淋巴瘤中微卫星不稳定和杂合性缺失.结果 37例B细胞淋巴瘤中,bcl-2/IgH基因重排频率为24.3％ （9/37）;微卫星不稳定发生率为48.6％ （18/37）,杂合性缺失发生率为40.5％ （15/37）,其中D14S65位点微卫星不稳定和杂合性缺失频率较高,分别为29.7％（11/37）和27％（10/37）.经统计学分析结果显示：D14S65位点bcl-2/IgH基因重排与微卫星不稳定有关（P＜0.05）,与杂合性缺失无关（P＞0.05）;但D14S45位点bcl-2/IgH基因重排与微卫星不稳定和杂合性缺失无关（P＞0.05）.结论 D14S65是B细胞淋巴瘤中敏感的检测位点,bcl-2/IgH基因重排和微卫星不稳定性可能共同导致B细胞淋巴瘤的发生.     

BIOMED-2标准化基因重排检测在非霍奇金淋巴瘤诊断中的意义

目的:利用BIOMED-2标准化免疫球蛋白(Ig)/T细胞受体(TCR)基因重排检测非霍奇金淋巴瘤(NHL)患者骨髓Ig或TCR基因重排,探讨Ig/TCR基因重排在非霍奇金淋巴瘤诊断中的应用意义。方法:从骨髓及石蜡包埋组织(FFPE)标本中提取基因组DNA,采用BIOMED-2系统引物,进行多重PCR扩增并利用PCR片段分析法进行Ig/TCR基因重排的克隆性分析。结果:在235例T细胞非霍奇金淋巴瘤(T-NHL)中,TCRγ和TCRx单克隆重排阳性率分别为57.9%和50.2%,TCRγ和TCRβ的联合检出率为71.9%。在583例B细胞非霍奇金淋巴瘤(BNHL)IgH和IgK单克隆重排阳性率分别为70.7%和69.3%,IgH和IgK的联合检出率为81.6%。套细胞淋巴瘤与滤泡淋巴瘤、弥漫大B细胞淋巴瘤IgH(84.8%对34.0%(P0.001);84.8%对9.2%(P=0.025)和IgK(75.8%vs 50.9%,P0.001;75.8%vs 16.1%(P0.001)重排阳性率差异具有统计学意义。Ig基因重排阳性BNHL患者中,65例(13.7%)患者存在TCR重排阳性。TCR重排阳性T-NHL患者中,未见Ig基因重排阳性。30例弥漫大B细胞淋巴瘤患者FFPE标本有25例(83.3%)检出Ig基因重排,与骨髓标本重排检出率相比较,差异具有统计学意义(P0.001)。结论:利用BIOMED-2标准化Ig/TCR基因重排检测对于淋巴瘤的诊断有辅助性作用,并且利用序列分析法可提高重排检测的敏感性和特异性,对于淋巴瘤的早期诊断有很高的价值。     

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标准化免疫球蛋白基因重排技术在石蜡包埋弥漫大B细胞淋巴瘤组织中的应用

目的探讨石蜡包埋组织标本应用BIOMED-2标准化免疫球蛋白(IG)基因重排技术检测弥漫大B细胞淋巴瘤(DLBCL)的可行性。方法从45例石蜡包埋组织标本中提取DNA,采用BIOMED-2系统引物进行多重聚合酶链反应(PCR)扩增,并应用PCR片段分析法进行IG基因重排的克隆性分析。结果将DNA由原浓度100~500 ng/μL稀释至50~100 ng/μL后,DNA扩增最大片段(300~400 bp)含量由10.0%提高到90.0%;45例DLBCL石蜡切片标本提取DNA进行免疫球蛋白重链基因(IGH)和免疫球蛋白kappa基因(IGK)克隆性分析,IGH+IGK阳性率达到84.4%;15例反应性淋巴组织增生标本进行IG/T淋巴细胞抗原受体(TCR)克隆性分析,未见克隆性重排。结论稀释DNA是唯一可以既提高DNA扩增最大片段又能提高克隆性检出率的方法。在DLBCL的诊断和淋巴组织增生性疾病的鉴别诊断中,BIOMED-2标准化体系检测IGH基因克隆性重排是一种快速、可靠的方法,具有重要的临床应用价值。     

非霍奇金淋巴瘤患者骨髓单个核细胞BCL-2/IgH、IgH基因重排的检测

本研究检测非霍奇金淋巴瘤(NHL)患者BCL-2/IgH基因主要断裂区重排、IgH基因重排,并探讨其对疾病的早期诊断、疗效评价等方面的意义。分别提取70例NHL(60例B-NHL、10例T-NHL)、7例淋巴结炎性肿大患者、20名正常人的骨髓单个核细胞DNA,通过PCR方法检测BCL-2/IgH、IgH基因重排,以凝胶电泳出现相应条带者为阳性,并与患者病理组织学检查进行比较,探讨此两种基因重排发生的相关因素,比较化疗后的动态变化。结果表明,①30例弥漫大B细胞淋巴瘤患者(DLBCL)中,10例骨髓BCL-2/IgH重排阳性(33.3%),与30例其它B-NHL(除外滤泡淋巴瘤FL及DLBCL)阳性率(6.7%)、20名正常人阳性率(5%)比较均有显著性差异(p均<0.05)。所有T-NHL及7例淋巴结炎性肿大患者BCL-2/IgH重排均为阴性;②对8例BCL-2/IgH基因重排阳性患者进行动态监测,经2个疗程R-CHOP治疗后BCL-2/IgH基因重排明显减少,PCR半定量结果均值由初治0.59降至0.16(p<0.05),6个疗程R-CHOP治疗后PCR半定量均值为0,BCL-2/IgH基因重排完全转阴;③BCL-2/IgH基因重排阳性患者中LDH水平升高占81.8%,在重排阴性患者中占28.6%,两组间有统计学差异(p<0.05)。然而,BCL-2/IgH基因重排与淋巴瘤分期、是否伴有全身症状、β2-MG水平、骨髓侵犯、肝脏脾脏侵犯均无显著相关性;④20例DLBCL(均为初治)骨髓单个核细胞DNA检测显示,9例IgH基因重排阳性(45%);30例其它B-NHL(均为初治或复发患者,DLBCL除外)中14例IgH基因重排阳性(46.7%),其两组间无统计学差异性(p>0.05),但对照组20名正常人、10例T细胞淋巴瘤患者及7例淋巴结炎性肿大患者均为阴性;⑤对7例IgH基因重排阳性患者进行动态监测表明,1个疗程的R-CHOP治疗就能显著减少IgH基因重排,PCR半定量结果均值由初治0.42降至0.13(p<0.05),2个疗程后PCR半定量均值为0,重排完全消除;⑥IgH基因重排阳性患者中LDH水平升高占90%,在重排阴性患者中占30%,两组间有统计学差异(p<0.05);IgH基因重排与淋巴瘤分期、是否伴有全身症状、β2-MG水平、骨髓侵犯、肝脏脾脏侵犯均无显著相关性。结论:BCL-2/IgH、IgH基因重排均可作为B-NHL早期诊断及评价疗效的特异性指标,这两种重排均与LDH水平相关;BCL-2/IgH基因重排对DLBCL特异性较高。     

Application of BIOMED-2 primers in fixed and decalcified bone marrow biopsies: analysis of immunoglobulin H receptor rearrangements in B-cell non-Hodgkin's lymphomas

The detection of clonality in lymphomas was recently improved by the BIOMED-2 approach, but analysis of fixed tissues is limited. Here, we adapted the BIOMED-2 protocol for examining immunoglobulin H (IgH) receptor rearrangements in fixed, decalcified bone marrow biopsies (BMBs) for clonality analysis in B-cell non-Hodgkin's lymphomas (B-NHL). The study included 111 decalcified BMBs (12 formalin fixed and 99 glutardialdehyde fixed), with B-NHL (n = 85), T-NHL (n = 8), or reactive infiltrates (n = 18). Initially, IgH FRIII polymerase chain reaction (PCR) analysis of crude DNA extracts from 75 glutardialdehyde-fixed BMBs (B-NHLs) using a standard seminested PCR resulted in clonal peaks in 46 of 75 (61.3%) BMBs compared with 19 of 70 (27.1%) for the original BIOMED-2 protocol. Modifications to both DNA extraction and PCR reaction improved the detection rate to 26 of 36 (72.2%) for BIOMED-2 primers, including 10 of 15 (66.7%) cases not detected by our standard IgH analysis. Moreover, introducing the same modifications for analysis of the FRII region by BIOMED-2 primers revealed clonal peaks in 19 of 36 (52.8%) B-NHLs compared with 5 of 70 (7.1%) for the original BIOMED-2 protocol. Together, analysis of FRII and FRIII regions by the modified BIOMED-2 protocol increased the detection rate to 31 of 36 (86.1%), particularly for BMBs with histological evidence of follicular lymphoma (FRIII, 70%; FRII, 90%). In summary, this study provides an improved protocol for detection of clonality by IgH-specific BIOMED-2 primers in fixed, decalcified bone marrow biopsies.     

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.     

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.     

原发中枢神经系统淋巴瘤的脑脊液细胞学诊断

  目的  研究脑脊液细胞学方法对原发中枢神经系统淋巴瘤(primary central nervous system lymphoma, PCNSL)的诊断意义。  方法  回顾性分析21例脑脊液细胞学阳性的PCNSL患者的临床资料, PCNSL的诊断综合应用脑脊液细胞学、脑脊液免疫细胞化学、脑脊液淋巴细胞基因分析和流式细胞分析等方法。  结果  21例患者临床-神经影像学分型包括脑脊膜型13例, 实质型4例, 室管膜(下)型3例, 眼型1例。脑脊液细胞学检查21例均见淋巴瘤细胞或异型淋巴细胞。脑脊液免疫细胞化学检查17例呈B淋巴细胞优势, 符合B淋巴细胞来源淋巴瘤, 10例Ki67阳性细胞比例在40%~60%。7例流式细胞分析中5例以B细胞为主, B细胞比例66%~87%;1例以NK/T细胞为主。脑脊液淋巴细胞基因重排检测3例为IgH单克隆, 1例为T细胞受体单克隆。最终6例行脑活检并确诊PCNSL。综合上述结果, 21例确诊PCNSL, 其中B细胞型19例, T细胞型2例。  结论  综合应用脑脊液细胞学、免疫细胞化学、细胞基因重排检测和流式细胞分析的脑脊液学方法, 是诊断PCNSL的重要途径。     

T cell receptor gamma-chain gene polymerase chain reaction to diagnose central nervous system involvement by cutaneous T cell lymphoma

The authors describe a patient who was suspected of having cutaneous T cell lymphoma involvement of the brain despite repeatedly negative cerebrospinal fluid (CSF) cytology, inconclusive flow cytometry, and no discrete lesion for brain biopsy. The diagnosis was made by polymerase chain reaction (PCR) analysis that showed a monoclonal T cell receptor γ-chain gene rearrangement in the CSF, identically sized to that present in a skin biopsy specimen. Thus, PCR could be used early and routinely to diagnose central nervous system spread of T cell lymphomas, because of its potentially superior sensitivity and specificity to CSF cytology.     

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.     

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.     

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.