Tubbs Raymond R, Pettay James, Barry Todd S, Swain Eric, Loftus Margaret, Cook James R, Skacel Marek, Paine Gillian, Roche Patrick, Grogan Thomas
Dept of Clinical Pathology, The Cleveland Clinic Foundation and the Cleveland Clinic Lerner College of Medicine, Cleveland, OH 44195, USA.
J Mol Histol. 2007 May;38(2):159-65. doi: 10.1007/s10735-006-9066-1. Epub 2006 Nov 9.
Implementation of interphase fluorescence in situ hybridization (FISH) assays in the clinical laboratory requires validation against established methods. Validation tools in common use include exchange of consecutive sections with another institution that has already established the FISH assay, comparison with conventional banded metaphase cytogenetics, confirmation of specificity using probed normal metaphases, consecutive paraffin sections of a validation set tested by a reference laboratory, and specificity assessment against well characterized cell lines. We have investigated the feasibility of using tissue microarrays (TMA) constructed from murine xenografts as a preliminary specificity-screening tool for validation of interphase FISH assays. Cell lines currently in use for FISH controls are used to generate xenografts in SCID mice which are fixed in formalin and paraffin embedded. A TMA is constructed using duplicate donor cores from the xenograft blocks. Xenografts used represent a wide range of translocations used routinely for formalin fixed paraffin embedded sections evaluated by FISH. Probe cocktails (Abbott-Vysis), for several non-random translocations associated with hematologic neoplasms and soft tissue sarcomas have been used in this manner. On-line deparaffinization, cell conditioning, and prehybridization steps are automated using a staining workstation (Ventana Discovery XT); hybridization and stringency washes are performed manually offline. FISH-probed TMAs are tracked using a Metasystems image scanner and analyzed using classifiers specifically developed for each molecular abnormality. FISH results for each xenograft in the TMA correspond exactly to the genotype previously established for the parent cell line from which the xenograft was prepared. Moderate complexity tissue microarrays constructed from murine xenografts are excellent validation tools for initial assessment of interphase FISH probe specificity.
临床实验室中实施间期荧光原位杂交（FISH）检测需要与既定方法进行验证。常用的验证工具包括与已建立FISH检测的另一机构交换连续切片、与传统带型中期细胞遗传学进行比较、使用探针标记的正常中期细胞确认特异性、由参考实验室对验证集的连续石蜡切片进行检测以及针对特征明确的细胞系进行特异性评估。我们研究了使用由小鼠异种移植构建的组织微阵列（TMA）作为间期FISH检测验证的初步特异性筛选工具的可行性。目前用于FISH对照的细胞系用于在SCID小鼠中生成异种移植，这些小鼠用福尔马林固定并石蜡包埋。使用来自异种移植块的重复供体芯构建TMA。所使用的异种移植代表了通过FISH评估的福尔马林固定石蜡包埋切片常规使用的广泛的易位。以这种方式使用了针对与血液系统肿瘤和软组织肉瘤相关的几种非随机易位的探针混合物（雅培 - 维思）。使用染色工作站（Ventana Discovery XT）自动进行在线脱蜡、细胞预处理和预杂交步骤；杂交和严格洗涤在离线状态下手动进行。使用Metasystems图像扫描仪跟踪FISH探针标记的TMA，并使用针对每种分子异常专门开发的分类器进行分析。TMA中每个异种移植的FISH结果与先前为制备异种移植的亲本细胞系确定的基因型完全对应。由小鼠异种移植构建的中等复杂性组织微阵列是用于间期FISH探针特异性初步评估的优秀验证工具。
