Abstract
Formalin fixation, albeit an outstanding method for morphological and molecular preservation, induces DNA damage and cross-linking, which can hinder nucleic acid screening. This is of particular concern in the detection of low-abundance targets, such as persistent DNA viruses. In the present study, we evaluated the analytical sensitivity of viral detection in lung, liver, and kidney specimens from four deceased individuals. The samples were either frozen or incubated in formalin (±paraffin embedding) for up to 10 days. We tested two DNA extraction protocols for the control of efficient yields and viral detections. We used short-amplicon qPCRs (63–159 nucleotides) to detect 11 DNA viruses, as well as hybridization capture of these plus 27 additional ones, followed by deep sequencing. We observed marginally higher ratios of amplifiable DNA and scantly higher viral genoprevalences in the samples extracted with the FFPE dedicated protocol. Based on the findings in the frozen samples, most viruses were detected regardless of the extended fixation times. False-negative calls, particularly by qPCR, correlated with low levels of viral DNA (<250 copies/million cells) and longer PCR amplicons (>150 base pairs). Our data suggest that low-copy viral DNAs can be satisfactorily investigated from FFPE specimens, and encourages further examination of historical materials.
Highlights
Published: 12 January 2022The preservation of biological specimens through formalin fixation and paraffin embedding (FFPE) provides an invaluable resource for retrospective molecular and histological investigation
The DNAs of frozen and FF samples were eluted in 150 μL of AE buffer, while those of the FFPE samples were in 100 μL of ATE buffer, according to the manufacturer protocol
To compare the yields in each group, following 1 to 10 days of incubation in formalin, we quantified by qPCR the single-copy human RNase P gene
Summary
The preservation of biological specimens through formalin fixation and paraffin embedding (FFPE) provides an invaluable resource for retrospective molecular and histological investigation. This procedure confers stability to the biomolecules of a sample, it induces DNA fragmentation, cytosine deamination, and cross-linking [1], all of which can significantly impair nucleic acid-based testing. In virtue of the minute amounts, polymerase chain reaction (PCR)-based methods have been the gold standard for their high sensitivity and specificity; yet, given their constraints to specific targets and intact template areas, these techniques are affected by the chemical and mechanical transformations induced by the fixation [7]. Albeit valuable, Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations
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