We evaluate the consequences of processing alcohol-fixed tissue in a processor previously used for formalin-fixed tissue. Biospecimens fixed in PAXgene Tissue Fixative were cut into three pieces then processed in a flushed tissue processor previously used for formalin-fixed, paraffin-embedded (FFPE) blocks (neutral buffered formalin [NBF]^+ve), a formalin-free system (NBF^−ve), or left unprocessed. Histomorphology and immunohistochemistry were compared using hematoxylin/eosin staining and antibodies for MLH-1, Ki-67, and CK-7. Nucleic acid was extracted using the PAXgene Tissue RNA/DNA kits and an FFPE RNA extraction kit. RNA integrity was assessed using RNA integrity number (RIN), reverse transcription polymerase chain reaction (RT-PCR) (four amplicons), and quantitative RT-PCR (three genes). For DNA, multiplex PCR, quantitative PCR, DNA integrity number, and gel electrophoresis were used. Compared with NBF^−ve, RNA from NBF^+ve blocks had 88% lower yield and poorer purity; average RIN reduced from 5.0 to 3.8, amplicon length was 408 base pairs shorter, and Cq numbers were 1.9–2.4 higher. Using the FFPE extraction kit rescued yield and purity, but RIN further declined by 1.1 units. Differences between NBF^+ve and NBF^−ve in respect of DNA, histomorphology, and immunohistochemistry were either non-existent or small in magnitude. Formalin contamination of a tissue processor and its reagents therefore critically reduce RNA yield and integrity. We discuss the available options users can adopt to ameliorate this problem:

我们评估在以前用于福尔马林固定组织的处理器中处理酒精固定组织的后果。在 PAXgene Tissue Fixative 中固定的生物样本被切成三块，然后在冲洗过的组织处理器中进行处理，该处理器之前用于福尔马林固定、石蜡包埋 (FFPE) 块（中性缓冲福尔马林 [NBF] ^+ve）、无福尔马林系统 (NBF ^-ve)，或未处理。使用苏木精/伊红染色和 MLH-1、Ki-67 和 CK-7 抗体比较组织形态学和免疫组织化学。使用 PAXgene Tissue RNA/DNA 试剂盒和 FFPE RNA 提取试剂盒提取核酸。使用 RNA 完整性数 (RIN)、逆转录聚合酶链式反应 (RT-PCR)（四个扩增子）和定量 RT-PCR（三个基因）评估 RNA 完整性。对于 DNA，使用多重 PCR、定量 PCR、DNA 完整性数和凝胶电泳。与 NBF ^{-ve相比，来自 NBF +ve}的 RNA块的收率降低 88%，纯度较差；平均 RIN 从 5.0 降低到 3.8，扩增子长度缩短了 408 个碱基对，Cq 数提高了 1.9-2.4。使用 FFPE 提取试剂盒挽救了产率和纯度，但 RIN 进一步下降了 1.1 个单位。NBF ^+ve和 NBF ^-ve在 DNA、组织形态学和免疫组织化学方面的差异要么不存在，要么幅度很小。因此，组织处理器及其试剂的福尔马林污染会严重降低 RNA 产量和完整性。我们讨论了用户可以采用来改善这个问题的可用选项：