Fischer C, Knaust K O, Schauer A, Ringert R H
Department of Urology, Georg August University, Goettingen, Germany.
Anticancer Res. 1996 Sep-Oct;16(5A):2575-80.
With the help of the deparaffination technique described by Heldly, it has become possible to conduct large-scale flow-cytometric (FCM) studies, including retrospective DNA analysis of renal cell carcinoma (RCC). The quality of these histograms, however, has to be comparable to the quality of the analyses from fresh tumour tissue. The present study compares the application of FCM on fresh tumour tissue from RCC with paraffin-embedded archive material, thus for the first time, the same combination of stains (ethidium bromide/mithramycin) was used for both materials. From a total of 44 tumours, 125 histograms were obtained from fresh tumour tissue. Aneuploidy was found in 78 (62.4%) of the DNA analyses (coefficient of variation, CV = 5.5%). These 78 aneuploid histograms were compared with 228 sections from the archive material which had been deparaffined and stained. This involved, a deparaffinising period of twelve hours as well the application of pepsin and protease, which was necessary to obtain high-quality histograms (CV = 6.0%). Retrospectively, a total of 58 histograms from 36 cases were available for comparison. Of the archive material, 23/36 tumours were classified as aneuploid; thus new stem cell lines could be detected retrospectively in 63.8% of the cases. The comparison of DNA indices (DI) in the two series produced no significant differences, and the comparison of the mean values for the CV-5.5% for native material histograms and 6.0% for archive material histograms-was not statistically significant. The detritus part in the archive histograms revealed a mean value of 6.3% for native histograms the mean value was 13.4%. These differences were also not significant. These results show that, using the staining combination ethidium bromide/mithramycin, the quality of the histograms obtained with archive material is equal to the quality of the histograms obtained from fresh tumour material. Furthermore, when taking into consideration the clonal heterogeneity of RCC, it is possible to use retrospective analysis to detect aneuploidy with a high degree of certainty and accuracy.
借助赫尔德利描述的脱石蜡技术，开展大规模流式细胞术（FCM）研究，包括对肾细胞癌（RCC）进行回顾性DNA分析已成为可能。然而，这些直方图的质量必须与新鲜肿瘤组织分析的质量相当。本研究将FCM应用于RCC新鲜肿瘤组织与石蜡包埋存档材料的情况进行比较，因此首次对两种材料使用相同的染色组合（溴化乙锭/光神霉素）。从总共44个肿瘤中，从新鲜肿瘤组织获得了125个直方图。在78例（62.4%）DNA分析中发现非整倍体（变异系数，CV = 5.5%）。将这78个非整倍体直方图与已脱石蜡并染色的存档材料的228个切片进行比较。这包括12小时的脱石蜡时间以及胃蛋白酶和蛋白酶的应用，这对于获得高质量直方图（CV = 6.0%）是必要的。回顾性地，总共36例中的58个直方图可用于比较。在存档材料中，23/36个肿瘤被归类为非整倍体；因此在63.8%的病例中可回顾性检测到新的干细胞系。两个系列中DNA指数（DI）的比较未产生显著差异，天然材料直方图CV为5.5%、存档材料直方图CV为6.0%的平均值比较也无统计学意义。存档直方图中的碎屑部分显示天然直方图的平均值为6.3%，该平均值为13.4%。这些差异也不显著。这些结果表明，使用溴化乙锭/光神霉素染色组合，存档材料获得的直方图质量与新鲜肿瘤材料获得的直方图质量相当。此外，考虑到RCC的克隆异质性，使用回顾性分析能够以高度的确定性和准确性检测非整倍体。
