Optimizing Fixation Protocols to Improve Molecular Analysis from FFPE Tissues

Maraschin, Bruna Jalfim; Silva, Viviane Palmeira da; Rock, Leigha D.; Sun, Hui-Chen; Visioli, Fernanda; Rados, Pantelis Varvaki; Rosin, Miriam P.

doi:10.1590/0103-6440201701211

Cited by 3 publications

(2 citation statements)

References 11 publications

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“…FFPE tissues have several advantages for diagnostic purposes, as they are easy to handle and transport, their processing is low-cost and they can be stored for a long time at room temperature ( 26 ). However, obtaining DNA from this type of sample in sufficient quantity and quality using extraction techniques is a challenge ( 56 ). Several preanalytical factors can influence the analysis of nucleic acids in FFPE samples, such as biospecimen fixation, specimen size, block storage conditions, section thickness, section storage, and others ( 57 ).…”

Section: Discussionmentioning

confidence: 99%

Nested PCR for the Diagnosis of Feline Sporotrichosis From Formalin-Fixed and Paraffin-Embedded Samples Using Different DNA Extraction Protocols

et al. 2022

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Sporotrichosis is a chronic, cosmopolitan granulomatous mycosis that affects humans and animals. The infection is caused by the dimorphic fungi Sporothrix sp. The aims of the present study were to evaluate, standardize and validate a nested PCR technique using two DNA purification kits for the extraction of DNA from formalin fixed and paraffin-embedded tissues (FFPE) for Sporothrix sp. detection. FFPE mycological culture pellet samples of different Sporothrix species (S. chilensis, S. mexicana, S. pallida, S. globosa, S. brasiliensis and S. schenckii) were used as positive controls and clinical FFPE tissue samples of animals positive for Cryptococcus sp., Leishmania infantum and Histoplasma sp. were used as negative controls. Ten clinical FFPE skin samples from cats with sporotrichosis were used to validate the nested PCR. These samples were cut into two distinct paraffin sectioning protocols (5 and 16 μm thick). The paraffin sections were subjected to two different DNA extraction kits (chemical and thermal extractions). A nested PCR was performed on the extracted DNA to identify the genus Sporothrix. The chemical extraction protocol with the 5 μm thick paraffin section was more effective in extracting DNA from Sporothrix sp. from FFPE samples and the nested PCR technique showed the highest sensitivities (100% in the positive controls and of 50% in the skin samples of cats) and specificity (100%). Therefore, the nested PCR using this protocol has great potential to be applied in Sporothrix sp. diagnosis in FFPE samples of cats.

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Section: Discussionmentioning

confidence: 99%

Nested PCR for the Diagnosis of Feline Sporotrichosis From Formalin-Fixed and Paraffin-Embedded Samples Using Different DNA Extraction Protocols

et al. 2022

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“…Fresh frozen samples are regarded to be superior in terms of preservation of nucleic acids, and thus the lack of significance between the tissue types is surprising. However, for the purposes of this study, both FFPE and PFPE samples were optimally preserved, avoiding over-fixation, and both paraffin blocks were kept at −20 °C, which is known to increase nucleic acid quality (Noguchi et al 1997;Maraschin et al 2017;Groelz et al 2018;Schmeller et al 2019). This could account for the improved performance of both types of fixed samples in this study.…”

Section: Discussionmentioning

confidence: 99%

Alternative tissue fixation for combined histopathological and molecular analysis in a clinically representative setting

Meecham

Miranda

Morris

et al. 2021

Histochem Cell Biol

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Formalin is the principal tissue fixative used worldwide for clinical and research purposes. Despite optimal preservation of morphology, its preservation of DNA and RNA is poor. As clinical diagnostics increasingly incorporates molecular-based analysis, the requirement for maintaining nucleic acid quality is of increasing importance. Here we assess an alternative non-formalin-based tissue fixation method, PAXgene Tissue system, with the aim of better preserving nucleic acids, while maintaining the quality of the tissue to be used for vital existing diagnostic techniques. In this study, these criteria are assessed in a clinically representative setting. In total, 203 paired PAXgene Tissue and formalin-fixed samples were obtained. Blind-scored haematoxylin and eosin (H&E) sections showed comparable and acceptable staining. Immunohistochemistry (IHC) staining was suboptimal using existing protocols but improved with minor method adjustment and optimisation. Quality of DNA and RNA was significantly improved by PAXgene tissue fixation [RIN 2.8 versus 3.8 (p < 0.01), DIN 5.68 versus 6.77 (p < 0.001)], which translated into improved performance on qPCR assay. These results demonstrate the potential of PAXgene Tissue to be used routinely in place of formalin, maintaining adequate histological staining and significantly improving the preservation of biological molecules in the genomic era.

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Towards molecular autopsies: Development of a FFPE tissue DNA extraction workflow

et al. 2022

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Optimizing Fixation Protocols to Improve Molecular Analysis from FFPE Tissues

Cited by 3 publications

References 11 publications

Nested PCR for the Diagnosis of Feline Sporotrichosis From Formalin-Fixed and Paraffin-Embedded Samples Using Different DNA Extraction Protocols

Nested PCR for the Diagnosis of Feline Sporotrichosis From Formalin-Fixed and Paraffin-Embedded Samples Using Different DNA Extraction Protocols

Alternative tissue fixation for combined histopathological and molecular analysis in a clinically representative setting

Towards molecular autopsies: Development of a FFPE tissue DNA extraction workflow

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