Ultra-early post-mortem interval (PMI) estimation based on longitudinal plasma protein dynamics in humans: A pilot study

Accurate estimation of the post-mortem interval (PMI) during the earliest phase after death remains one of the most problematic tasks in forensic pathology. Traditional tanatological methods show limited reliability within the first hours after death due to marked inter-individual variability and strong dependence on environmental and physiological factors. Increasing attention has therefore been directed toward molecular approaches capable of capturing the biological processes occurring immediately after death. In this context, blood represents a promising but still insufficiently explored matrix for ultra-early PMI estimation. A prospective longitudinal pilot study was conducted on ten adult patients who died from natural causes at Intensive Care Unit of Magna Graecia University of Catanzaro under controlled conditions. Peripheral venous blood samples were collected from upper limb veins (primarily antecubital veins) using standard sterile techniques at precisely defined post-mortem time points ranging from death certification to 120 min after death. Plasma concentrations of twelve candidate proteins involved in apoptosis, mitochondrial metabolism, cellular stress response, cytoskeletal integrity and tissue injury were quantified using sandwich ELISA assays. Longitudinal data were analyzed using linear mixed-effects models, complemented by non-parametric correlation analyses and exploratory multivariate approaches. Several biomarkers exhibited reproducible and statistically significant time-dependent plasma concentration changes within the first two hours after death. Caspase-9 and pyruvate dehydrogenase E1 showed early peaks followed by progressive declines, consistent with rapid apoptotic activation and metabolic collapse. Structural proteins such as β-actin and myosin indicated concordant kinetic patterns, while Hsp70 displayed an early decrease. Cardiac injury markers showed more complex temporal dynamics, whereas LDH and Bcl-2 did not show consistent associations with post-mortem time. Combined proteomic profiles allowed exploratory discrimination between ultra-early and early PMI intervals. These findings provide preliminary original human evidence supporting the forensic potential of plasma-based multiparametric biomarker approaches for ultra-early PMI estimation and suggest that molecular data may meaningfully complement traditional methods during the most challenging post-mortem phase, although further large-scale studies are required to confirm these preliminary results.