Tbakhi A, Totos G, Pettay J D, Myles J, Tubbs R R
Department of Anatomic Pathology, Cleveland Clinic Foundation, Ohio 44195, USA.
Mod Pathol. 1999 Mar;12(3):272-8.
It has been suggested that neutral buffered formalin (NBF)-fixed, paraffin-embedded, or fresh specimens might provide satisfactory DNA templates for polymerase chain reaction (PCR) assays used in establishing the clonality and presumptive B-cell lineage of lymphoma. The suitability of other fixatives used by hematopathologists, such as B5, is still undetermined. Thirty cases were identified from the files of the Cleveland Clinic Foundation, Cleveland Ohio, that showed abnormal immunoglobulin heavy chain (IgH) rearrangement by Southern blot analysis (SBA). Corresponding paraffin-embedded tissue samples fixed in NBF (21 cases), B5 (18 cases), Hollande's fixative (17 cases), zinc formalin (ZF) (5 cases), and Bouin's fixative (3 cases) were studied. With use of consensus primers against the framework 3 (FR3) and FR2 regions of the VH gene, paired against JH primer(s), PCR analysis was performed. bcl-2/IgH translocation was also studied. Ten reactive lymphoid samples were used as controls, and 40 cases were evaluated. Successful amplification of a clonal proliferation was manifested as one or two discrete narrow bands in the appropriate size range. The sensitivity of detecting clonality was 95, 94, 67, 80, and 0% for NBF, Hollande's fixative, B5, ZF, and Bouin's fixative, respectively. Although NBF and Hollande's fixative were 100% specific, consistent false-positive results were a major problem with B5-fixed tissue. Paraffin-embedded tissue, fixed in NBF, Hollande's fixative, and ZF solutions, may be used for DNA extraction and PCR assays for establishing B-cell clonality. The precipitating fixative B5 and Bouin's solution should not be used for this purpose until the issue of false-positive results is resolved.
有人提出，中性缓冲福尔马林（NBF）固定、石蜡包埋或新鲜的标本可能为用于确定淋巴瘤克隆性和推定B细胞谱系的聚合酶链反应（PCR）检测提供令人满意的DNA模板。血液病理学家使用的其他固定剂，如B5，其适用性仍未确定。从俄亥俄州克利夫兰诊所基金会的档案中确定了30例经Southern印迹分析（SBA）显示免疫球蛋白重链（IgH）重排异常的病例。研究了相应的用NBF固定（21例）、B5固定（18例）、霍兰德固定剂固定（17例）、锌福尔马林（ZF）固定（5例）和Bouin固定剂固定（3例）的石蜡包埋组织样本。使用针对VH基因框架3（FR3）和FR2区域的共有引物，并与JH引物配对，进行PCR分析。还研究了bcl-2/IgH易位。使用10个反应性淋巴样样本作为对照，并对40例病例进行了评估。克隆增殖的成功扩增表现为在适当大小范围内出现一条或两条离散的窄带。NBF、霍兰德固定剂、B5、ZF和Bouin固定剂检测克隆性的灵敏度分别为95%、94%、67%、80%和0%。尽管NBF和霍兰德固定剂的特异性为100%，但B5固定组织的主要问题是一致的假阳性结果。用NBF、霍兰德固定剂和ZF溶液固定的石蜡包埋组织可用于DNA提取和PCR检测以确定B细胞克隆性。在假阳性结果问题解决之前，不应将沉淀固定剂B5和Bouin溶液用于此目的。
