Human DNA decays faster with time than viral dsDNA: an analysis on HPV16 using pathology archive samples spanning 85\xa0years

Sara Nicolás-Párraga,Laia Alemany,Francesc Xavier Bosch,Ana Félix,Montserrat Torres,Ignacio G Bravo,Eugenia Cruz,Silvia De Sanjosé

doi:10.1186/s12985-021-01529-9

Abstract

BackgroundQuality of the nucleic acids extracted from Formalin Fixed Paraffin Embedded (FFPE) samples largely depends on pre-analytic, fixation and storage conditions. We assessed the differential sensitivity of viral and human double stranded DNA (dsDNA) to degradation with storage time.MethodsWe randomly selected forty-four HPV16-positive invasive cervical cancer (ICC) FFPE samples collected between 1930 and 1935 and between 2000 and 2004. We evaluated through qPCR the amplification within the same sample of two targets of the HPV16 L1 gene (69 bp, 134 bp) compared with two targets of the human tubulin-β gene (65 bp, 149 bp).ResultsBoth viral and human, short and long targets were amplified from all samples stored for 15 years. In samples archived for 85 years, we observed a significant decrease in the ability to amplify longer targets and this difference was larger in human than in viral DNA: longer fragments were nine times (CI 95% 2.6–35.2) less likely to be recovered from human DNA compared with 1.6 times (CI 95% 1.1–2.2) for viral DNA.ConclusionsWe conclude that human and viral DNA show a differential decay kinetics in FFPE samples. The faster degradation of human DNA should be considered when assessing viral DNA prevalence in long stored samples, as HPV DNA detection remains a key biomarker of viral-associated transformation.

Highlights

Quality of the nucleic acids extracted from Formalin Fixed Paraffin Embedded (FFPE) samples largely depends on pre-analytic, fixation and storage conditions
Differential quality of viral and human DNA retrieved from FFPE samples with storage time
In samples stored for 85 years, overall congruence between the amplification results obtained for human and for HPV16 DNA (Table 2) was low (21% concordance)

Summary

Introduction

Quality of the nucleic acids extracted from Formalin Fixed Paraffin Embedded (FFPE) samples largely depends on pre-analytic, fixation and storage conditions. In pathology routine most biopsies and surgical samples are formalin fixed paraffin embedded (FFPE) to preserve tissue structure and cellular morphology. The information targeted is usually nucleic acid sequencing, but tissue preservation in paraffin blocks is variable and depends on a number of factors in the different steps of the process that affect quantity and quality of the extracted nucleic acids [4, 5]. Overfixation or the use of a fixative molecule with multiple active groups, e.g. glutaraldehyde, hardens the tissue by cross-linking multiple molecules and can induce physical fragmentation of the nucleic acids during extraction [7, 8]. DNA damage induced by fixation conditions can hinder downstream applications based on amplification techniques [10, 11]

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Conclusion