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
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
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DOI：
10.1016/s1525-1578(10)60591-0
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发表时间：
2005-11-01
影响因子：
4.1
通讯作者：
Fisch, P
中科院分区：
文献类型：
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作者：
Lassmann, S;Gerlach, UV;Fisch, P
淋巴瘤中克隆性的检测最近通过BIOMED-2方法得到了改进，但固定组织的分析受到限制。在这里，我们采用BIOMED-2协议检查免疫球蛋白H（IgH）受体重排固定，脱钙骨髓活检（BMB）的克隆性分析B细胞非霍奇金淋巴瘤（BNHL）。该研究包括111例脱钙BMB（12例福尔马林固定，99例戊二醛固定），B-NHL（n = 85），T-NHL（n = 8）或反应性浸润（n = 18）。最初，IgH FRIII聚合酶链反应（PCR）分析的粗DNA提取物从75戊二醛固定的BMB（B-NHL）使用标准的套式PCR导致克隆峰在46 75（61.3%）BMB相比，19 70（27.1%）的原始BIOMED-2协议。DNA提取和PCR反应的改进提高了BIOMED-2引物的检出率为26/36（72.2%），包括10/15（66.7%）例未检出我们的标准IgH分析。此外，通过BIOMED-2引物引入相同的修饰用于FRII区域的分析，与原始BIOMED-2方案的70个中的5个（7.1%）相比，在36个（52.8%）B-NHL中的19个中发现了克隆峰。总之，通过修改的BIOMED-2方案分析FRII和FRIII区域将检测率提高到36例中的31例（86.1%），特别是对于具有滤泡性淋巴瘤组织学证据的BMB（FRIII，70%; FRII，90%）。总之，本研究提供了一种改进的方案，用于在固定的脱钙骨髓活检中通过IgH特异性BIOMED-2引物检测克隆性。
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 (BNHL). 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.