Immunohistochemical Markers and Post‐Mortem Interval Estimation: A Proof‐Of‐Concept Study on Immunoexpression of Caspase‐8, Annexin A1, Bcl‐2, and NF‐κB in Autoptic Tissues

Estimating the post-mortem interval (PMI) remains a major challenge in forensic practice, as conventional thanatological signs are strongly influenced by biological and environmental variables. Immunohistochemistry is a complementary strategy for identifying tissue biomarkers with time-dependent post-mortem persistence. In this retrospective study, we assessed the immunohistochemical expression of Caspase-8, Annexin A1, B-cell lymphoma 2 (Bcl-2), and Nuclear Factor kappa B (NF-κB) in brain, heart, and liver samples from 32 autopsies with known PMI (1.38-23 days). Cases were stratified into four PMI groups: < 2 days, ≥ 2 to < 5 days, ≥ 5 to ≤ 8 days, and > 8 days. Immunoreactivity was scored semi-quantitatively as weak (1+) or strong (2+). Caspase-8 showed the highest overall positivity and the longest persistence, remaining detectable up to 22 days in heart, 16 days in brain, and 14 days in liver, with complete positivity across tissues up to 8 days. Annexin A1 displayed last positivity at 6.09 days in brain and 7.63 days in heart and liver, being consistently negative beyond 8 days. Bcl-2 expression was restricted to myocardium and detected only in very early PMI cases (< 2 days), whereas NF-κB was consistently negative. These findings support a tissue-specific, multiparametric model for PMI stratification.