Are pre-analytical factors fully considered in forensic FFPE molecular analyses? A systematic review reveals the need for standardised procedures.

The need for molecular analyses has become increasingly common in the forensic sciences, particularly in forensic pathology, to better shape the causes of death. This approach is called the "molecular autopsy," where conventional medico-legal findings are often enhanced with specific molecular tests to provide reliable clinical and forensic diagnoses. In this context, FFPE (Formalin-Fixed Paraffin-Embedded) tissue samples collected during forensic autopsies are the only available specimens in retrospective studies for molecular DNA and/or RNA analyses. It is well known that pre-analytical parameters such as the agonal time, the PMI (Post-Mortem Interval), the fixation procedures, and the FFPE ageing and storage conditions can deeply impact the quality and quantity of the recovered nucleic acids, thus influencing the reliability of the downstream molecular tests. In the present study, we reviewed the recent forensic literature to establish whether these parameters are reported. Our survey showed that up to 34.9% and 40.5% of the 50 selected studies on DNA and RNA, respectively, reported the pre-analytical parameters mentioned above. Many publications did not report the length of agony (if any), which is an important parameter in RNA-based studies and estimations of the PMI; in addition, even relevant information on formalin tissue fixation procedures was often missing, thus impairing any critical evaluation of the PCR-based results. To address these issues, we propose the use of a simple form we set up to be filled out by Forensic Pathologists, where each pre-analytical step concerning the tissue samples collected during autopsy is accurately described and reported. In our opinion, this standardization will help the forensic community compare and evaluate the results of different molecular tests, thus increasing the reliability of the molecular results in forensics.