A comparative study of the quality of DNA obtained from fresh frozen and formalin-fixed decalcified paraffin-embedded bone marrow trephine biopsy specimens using two different methods
A comparative study of the quality of DNA obtained from fresh frozen and formalin-fixed decalcified paraffin-embedded bone marrow trephine biopsy specimens using two different methods
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DOI：
10.1136/jcp.2006.045294
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发表时间：
2008-01-01
影响因子：
3.4
通讯作者：
Dahlstrom, J. E.
中科院分区：
文献类型：
--
作者：
Talaulikar, D.;Gray, J. X.;Dahlstrom, J. E.
背景资料：考虑到其预后价值，使用免疫球蛋白重链和轻链对非霍奇金淋巴瘤（NHL）进行分子分期重新引起了人们的兴趣。目的：比较使用两种方法从新鲜冷冻和福尔马林固定的脱钙石蜡包埋（FFDPE）骨髓环钻扩增DNA用于分子分期的效率。方法：在从13份FFDPE和14份新鲜冷冻环钻活检标本中手动提取DNA后，使用两种方法测试扩增能力：使用In Vivo Scribe免疫球蛋白重链（IgH）克隆性试剂盒中提供的扩增对照主混合物，其产生96-600碱基对（bp）之间的5个扩增子;结果：使用第一种方法，与新鲜冷冻样品相比，FFDPE环钻产生的扩增子的平均最大长度在300 bp处在统计学上较低，所有新鲜冷冻样品产生的扩增子的大小高达600 bp（p < 0.001）。b-珠蛋白基因的实时扩增显示，新鲜冷冻样品的平均交叉阈值显著低于FFDPE样品（23.48（95% CI 22.47至24.48）vs 33.64（95% CI 32.15至35.12）; p < 0.001）。尽管可从新鲜冷冻和FFDPE环钻样品中提取可扩增的DNA用于IgH/IgL分析，新鲜冷冻的标本作为模板DNA的来源是上级的，特别是对于较高碱基对的PCR产物。
Background: Given its prognostic value, there is renewed interest in molecular staging in non-Hodgkin's lymphoma (NHL) using immunoglobulin heavy and light chain (IgH, IgL) gene rearrangements.Aims: To compare the efficiency of DNA amplification from fresh frozen and formalin-fixed decalcified paraffin-embedded (FFDPE) bone marrow trephines for use in molecular staging using two methods.Methods: After manually extracting DNA from 13 FFDPE and 14 fresh frozen trephine biopsy specimens, two methods were used to test for amplifiability: use of the amplification control master mix supplied in the In Vivo Scribe immunoglobulin heavy chain (IgH) clonality kit, which creates 5 amplicons between 96-600 base pairs (bp); and real-time amplification of the beta-globin gene.Results: Using the first method, the mean maximum length of amplicons generated from FFDPE trephines was statistically lower at 300 bp compared to fresh frozen samples, all of which generated amplicons up to 600 bp in size (p < 0.001). Real-time amplification of the b-globin gene showed that the mean crossing threshold of fresh frozen samples was statistically lower than that of FFDPE samples (23.48 (95% Cl 22.47 to 24.48) vs 33.64 (95% Cl 32.15 to 35.12); p < 0.001).Conclusions: Although amplifiable DNA can be extracted from both fresh-frozen and FFDPE trephine samples for IgH/IgL analysis, freshly frozen specimens are superior as a source of template DNA, especially for higher base pair PCR products.