Blood Cell Intracellular pH Dynamics and Metabolic Biomarkers Under Simulated Postmortem Interval Conditions: An Exploratory Pilot In Vitro Study

Intracellular pH (pHi) may provide complementary information on postmortem cellular metabolism and homeostasis, but its temporal behavior remains poorly understood. This pilot in vitro study evaluated temporal changes in platelet pHi and pHi in a mixed non-platelet blood-cell population (predominantly erythrocytes and leukocytes), together with plasma glucose, lactate, and ammonia during a simulated postmortem interval (PMI). Venous blood from eight healthy volunteers was maintained at 24 ± 3 °C and analyzed at 0, 3, 6, and 24 h. Intracellular pH was assessed by BCECF-AM flow cytometry, and metabolites were quantified enzymatically. Platelet and non-platelet-cell pHi remained stable during the first 3 h, decreased significantly at 6 h, and increased toward baseline values at 24 h. Glucose concentrations decreased over time, lactate increased progressively, and ammonia increased significantly only at 24 h. Lactate was associated with pHi in both blood-cell populations. Intracellular pH exhibited a biphasic temporal pattern and distinct relationships with metabolic biomarkers during the simulated PMI. An exploratory mixed-effects model incorporating non-platelet-cell pHi, lactate, and glucose explained a substantial proportion of temporal variability (marginal R2 = 0.907) and yielded a leave-one-donor-out cross-validation RMSE of 0.4998 on the log-transformed time scale. These findings support further investigation of multi-marker approaches for PMI estimation.