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。将固定在PAXgene组织固定剂中的生物样本切成三段，然后在冲洗过的组织处理器中进行处理，该处理器先前用于福尔马林固定的石蜡包埋（FFPE）块（中性缓冲福尔马林[NBF] ^{+ ve}），无福尔马林的系统（NBF ^-ve），或者不做任何处理。使用苏木精/曙红染色和MLH-1，Ki-67和CK-7抗体比较组织形态学和免疫组化。使用PAXgene组织RNA / DNA试剂盒和FFPE RNA提取试剂盒提取核酸。使用RNA完整性编号（RIN），逆转录聚合酶链反应（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，使用了多重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块的产率降低了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数增加了1.9-2.4。使用FFPE提取试剂盒可挽救产量和纯度，但RIN进一步下降了1.1个单位。在DNA，组织形态学和免疫组织化学方面，不存在NBF ^{+ ve}和NBF ^-ve之间的差异，或者差异很小。因此，组织处理器及其试剂的福尔马林污染严重降低了RNA产量和完整性。我们讨论了用户可以用来缓解此问题的可用选项：