Toward methodological standardization in forensic immunohistochemistry: a critical appraisal of 144 post-mortem studies and a proposed evaluative framework

Immunohistochemistry (IHC) is widely applied in post-mortem forensic investigations. Its evidentiary value depends critically on methodological standardization. Following PRISMA guidelines and PROSPERO registration (CRD420251063965), we systematically searched PubMed, Scopus, and Web of Science for original IHC studies on human autopsy material. A total of 144 studies were included and evaluated against six predefined methodological criteria (M1–M6) addressing slide interpretation metrics, multi-reader assessment, observer blinding, concordance reporting, quality control, and predefined positivity thresholds. IHC was applied across diverse forensic domains, most frequently in traumatic brain injury (16%), sudden cardiac death (14%), COVID-19-related death (13%), and wound vitality/wound dating (11%). Quantitative or semi-quantitative slide reading (M1) was adopted in 75.7% of studies, and quality control measures (M5) in 52.8%. By contrast, blinded reading (M3) was implemented in only 40% of applicable studies, inter-observer concordance (M4) in 10%, and predefined positivity thresholds (M6) in only 4.2% of studies. Only 38.9% of studies met three or more methodological criteria, and a single study satisfied all six criteria. Forensic IHC demonstrates broad application and considerable inferential ambition, but its methodological foundations remain inconsistently consolidated. We propose that future forensic IHC studies place greater emphasis on analytical validation, standardized interpretation criteria, assessment of observer agreement, and systematic reporting of relevant pre-analytical variables in order to improve reproducibility and comparability.