Inoue T, Nabeshima K, Kataoka H, Koono M
Department of Pathology, Miyazaki Medical College, Japan.
Pathol Int. 1996 Dec;46(12):997-1004. doi: 10.1111/j.1440-1827.1996.tb03580.x.
Although several factors affecting the sensitivity of polymerase chain reaction (PCR) amplification from formalin-fixed tissues have been investigated mostly by experiments, the feasibility of archival formalin-fixed, paraffin-embedded tissue samples stored in pathology departments for PCR amplification has rarely been examined directly. Thus, the feasibility of 74 archival unbuffered 10% formalin-fixed, paraffin-embedded tissues for PCR amplification with primers producing a 190 b.p. DNA segment of p53 exon 5 was investigated. Fixation time was the critical factor influencing the sensitivity of PCR amplification. All (6/6) of the samples fixed for only 1 day, 44% (7/16) of the samples fixed for 2-3 days and 14% (4/28) of the samples fixed for 4-6 days showed successful amplification, while no amplification was obtained for the samples fixed for 7 days or more. The peak size of DNA extracted from the archival tissues decreased as the fixation time became longer. Experiments using xenografted tumor tissues fixed for various times showed longer permissible fixation time; up to 9 days of fixation, decreasing amounts of PCR products were obtained while no amplification was obtained for the samples fixed for 12 days or more. The time in paraffin seemed to be a minor factor for PCR amplification since all of the 1 day fixation samples, including those that had been embedded for up to 5 years, resulted in efficient amplification. The size of the amplified DNA segments, however, could be another factor influencing the sensitivity of amplification because even the 1 day fixation samples showed less amplification of 345 b.p. DNA compared with those of 167 and 262 b.p. DNA. Additionally, a point mutation was detected in the amplified p53 products from archival tissues using a non-isotopic method, temperature gradient gel electrophoresis. In conclusion, archival tissue samples that had been fixed immediately for only up to 1 day were constantly available for PCR amplification of approximately 200 b.p. DNA segments, suggesting that surgical specimens should be subjected to cutting and paraffin embedding just after 1 day or less fixation for subsequent use in PCR amplification.
尽管影响从福尔马林固定组织中进行聚合酶链反应（PCR）扩增敏感性的几个因素大多已通过实验进行了研究，但病理科保存的存档福尔马林固定、石蜡包埋组织样本用于PCR扩增的可行性很少被直接检测。因此，研究了74份存档的未缓冲10%福尔马林固定、石蜡包埋组织样本，用引物扩增p53基因第5外显子的190个碱基对DNA片段的PCR可行性。固定时间是影响PCR扩增敏感性的关键因素。仅固定1天的所有样本（6/6）、固定2 - 3天的样本中有44%（7/16）以及固定4 - 6天的样本中有14%（4/28）显示成功扩增，而固定7天或更长时间的样本未获得扩增。从存档组织中提取的DNA的峰值大小随着固定时间延长而减小。使用不同固定时间的异种移植肿瘤组织进行的实验显示允许的固定时间更长；固定长达9天时，获得的PCR产物量减少，而固定12天或更长时间的样本未获得扩增。石蜡包埋时间似乎对PCR扩增是一个次要因素，因为所有固定1天的样本，包括那些已包埋长达5年的样本，都实现了高效扩增。然而，扩增DNA片段的大小可能是影响扩增敏感性的另一个因素，因为即使是固定1天的样本，与167和262个碱基对的DNA相比，345个碱基对DNA的扩增也较少。此外，使用非同位素方法温度梯度凝胶电泳在存档组织扩增的p53产物中检测到一个点突变。总之，立即固定仅1天的存档组织样本可一直用于约200个碱基对DNA片段的PCR扩增，这表明手术标本应在固定1天或更短时间后立即进行切割和石蜡包埋，以供后续PCR扩增使用。
