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Molecular analysis of H&E- and Papanicolau-stained samples-systematic review.
Histochemistry and Cell Biology ( IF 2.3 ) Pub Date : 2020-05-05 , DOI: 10.1007/s00418-020-01882-w
Alexandra Pote 1 , Otília Boghenco 1 , Ana Marques-Ramos 1, 2
Affiliation  

Molecular pathology allows the identification of causative agents in infectious diseases and detection of biomarkers important for prediction of disease susceptibility, diagnosis and personalized therapy. Accordingly, nucleic acid-based methods have gained a special role in clinical laboratories particularly to evaluate solid and hematological tumors. Extraction of nucleic acids is commonly performed in microdissected formalin-fixed paraffin-embedded (FFPE) or cytological samples that had been previously evaluated through the use of hematoxylin and eosin (H&E) or Papanicolau (Pap) stains, respectively. Although the effect of both stains on nucleic acids integrity has been explored by several authors, the results are not consistent and require further examination. Accordingly, the goal of this review was to assess the influence of H&E and Pap stains on DNA and RNA integrity and to address the mechanism by which each staining compromises molecular based-analysis. The analyzed studies demonstrate that H&E- and Pap-staining result in low DNA recovery and some degree of DNA fragmentation. Additionally, it is concluded that hemalum inhibits PCR by interfering with DNA extraction, preventing DNA polymerase attachment and possibly by rescuing divalent cations. Accordingly, proper sample purification and adjustment of PCR conditions are of key importance to achieve satisfactory results by PCR in H&E- and Pap-stained samples. Furthermore, although H&E results in RNA fragmentation, it is possible to perform expression analysis in H&E-stained frozen sections, using RNase-free conditions, low amounts of hematoxylin and a rapid protocol from sample collection to RNA analysis. It The effect of Pap-staining on RNA integrity remains to be determined.

中文翻译:

H&E和Papanicolau染色样品的分子分析-系统评价。

分子病理学可以识别传染病中的病原体,并检测对预测疾病易感性,诊断和个性化治疗很重要的生物标志物。因此,基于核酸的方法已在临床实验室中发挥了特殊作用,特别是在评估实体和血液肿瘤方面。核酸的提取通常在显微解剖的福尔马林固定石蜡包埋(FFPE)或先前分别通过使用苏木精和曙红(H&E)或帕潘尼古劳(Pap)染色剂评估的细胞学样品中进行。尽管几位作者已经探讨了两种染色剂对核酸完整性的影响,但结果不一致,需要进一步检查。因此,本次审查的目的是评估H&E和Pap对DNA和RNA的完整性进行染色,以解决每种染色都会损害基于分子的分析的机理。分析的研究表明,H&E和巴氏染色会导致DNA回收率低和一定程度的DNA片段化。另外,得出的结论是,血红素通过干扰DNA提取,防止DNA聚合酶附着并可能通过拯救二价阳离子来抑制PCR。因此,适当的样品纯化和PCR条件的调整对于通过PCR在H&E和巴氏染色的样品中获得令人满意的结果至关重要。此外,尽管H&E导致RNA片段化,但仍可以使用无RNase的条件在H&E染色的冷冻切片中进行表达分析,苏木精含量低,从样品收集到RNA分析的操作流程迅速。巴氏染色对RNA完整性的影响尚待确定。
更新日期:2020-05-05
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