FISH 实验室质量控制

这些指南最初是为 ProbeChek™ 对照玻片产品编写的,但对于使用 CEP® 或 LSI® 探针进行计数来建立高质量实验室程序而言,是一个极好的起点。

CEP 或 LSI 探针杂交程序应按照探针产品随附的试剂盒说明书中的说明进行。强烈建议使用探针产品试剂盒说明书中所示的最佳性能荧光显微镜设备、滤光片和程序,以便使用 ProbeChek™ 对照品对 CEP 和 LSI 探针信号进行最佳杂交、准确评估和判读。杂交前应使用相差显微镜对检测标本进行评估,以确定是否适合 FISH。用于 FISH 的玻片的建议指南如下:

  • 细胞应均匀分布在玻片上。出现细胞聚集区域表示玻片制备或样本制备不佳。放大 400 倍(总放大倍数)视野下,至少应有八个细胞。更少的细胞表明细胞浓度对于最佳计数来说过低。 
  • 细胞核和中期染色体不应有表现为“相亮”。“相亮”细胞表明玻片制备条件并非最佳。在制备玻片前,始终使用优质淋巴细胞制备物来优化玻片制备条件。
  • 细胞核和中期细胞周围不应有可见的细胞质。细胞质会阻碍杂交,导致杂交效率低和结果不准确。

应对照实验室确定的质量参数来评价每张杂交玻片。使用适当的显微镜、照明和滤光片组,应评估杂交的特异性、探针信号强度和信噪比(背景噪声),确定杂交是否适合分析。靶标区域中至少 85% 的细胞核应易于计数。应有最小的背景或核荧光“噪声”。使用最低 400 倍的放大倍数时,应当容易观察到探针信号。玻片制备物上的大部分细胞核应与周围细胞核分离,不应被细胞质覆盖。存在许多核聚集(超过 3 个核)区域可以排除足够且均匀的核分布以精确计数。不符合这些标准的制备物不应用于信号计数。评估时,应分别对每个探针进行信号计数,但同时计数 CEP X 和 CEP Y 探针是合理的。

 

下载 PDF 格式玻片制备文档

玻片制备和原位杂交质量控制的建议文档格式。

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