Bagg Adam, Braziel Rita M, Arber Daniel A, Bijwaard Karen E, Chu Albert Y
Department of Pathology and Laboratory Medicine, 7-103 Founders Pavilion, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104-4283, USA.
J Mol Diagn. 2002 May;4(2):81-9. doi: 10.1016/S1525-1578(10)60685-X.
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.
通过评估免疫球蛋白重链（IgH）基因重排来确定单克隆性是一种常用且有用的诊断检测方法。许多进行该检测的实验室通过聚合酶链反应（PCR）来完成。为了评估当前进行IgH基因检测的方法，19个不同的分子病理学协会（AMP）成员实验室分析了29份经过盲法处理的B细胞和T细胞淋巴样肿瘤样本，这些样本包括提取的DNA以及福尔马林固定、石蜡包埋（FFPE）组织，并被要求填写一份技术问卷。从这项研究中可以明显看出，Southern印迹分析仍然是诊断金标准，诊断敏感性和特异性均为100%。然而，在评估相同标本时，IgH PCR检测的性能和结果存在显著异质性，诊断敏感性范围从超过90%到低至20%。许多实验室高估了其IgH PCR检测的诊断敏感性，并且与新鲜/冷冻组织相比，配对FFPE样本检测中的显著下降（从61.3%降至41.8%）未得到充分认识。固定对无法在FFPE样本上进行检测（43.1%）与已经从新鲜或冷冻组织中提取的DNA样本（2.8%）有显著影响。确定了一些影响IgH PCR结果的变量。改善单克隆IgH重排检测的策略包括：在FRIII上游引物中添加FRII（将检测率从57.3%提高到73.6%）以及使用FR3A而非FR3 FRIII引物（将检测率从54.7%提高到69.7%）。尽管评估了众多变量（从DNA提取到PCR产物检测），这使得难以强制要求实验室操作进行改变，但这些发现应该促使诊断分子病理学实验室重新评估其敏感性声明以及方法。如果有足够的样本，病理学家和外科医生都需要确保并非所有提交的材料都已固定。重要的是，需要更大程度的标准化以降低这种关键诊断检测中不可接受的高假阴性率。
