Advancing forensic accuracy: mitigating methemoglobin interference in postmortem carbon monoxide analysis using...

Analyzing carbon monoxide concentration within an individual is crucial. The analysis of CO content in a tissue sample is performed using gas chromatography. The concentration is calculated based on a linear equation derived from the calibration curve created with the CO-fortified sample. However, when methemoglobin (MetHb) is formed from putrefaction, it inhibits CO binding to the sample and may lead to inaccurate results. MetHb results from the iron oxidation of normal heme hemoglobin (HHb), and by treating the sample with a reducing agent, it can be converted back to HHb. To investigate the effect of the reducing agent on spleen CO analysis, each sample was divided into two parts. One was treated with a 0.574 M sodium dithionite solution (Na₂S₂O₄), a reduced sample, and the other was treated with a rinse solution, serving as the control sample, with both undergoing the same preparation process and analyzed using Gas Chromatography with a Thermal Conductivity Detector (GC-TCD). Spleen samples from 60 autopsy cases were analyzed. The results indicated that 48 cases showed lower CO levels when the sample was reduced compared to the control sample, where the difference of the control and reduced samples ranged from 2.21 to 93.24%, with a median value of 13.83%. 12 cases exhibited no difference, where the difference between control and reduced sample ranged from 0.05 to 1.57%, with a median value of 0.67%. Our findings demonstrate that MetHb formed during decomposition can significantly inhibit CO binding in spleen tissue, leading to overestimation of CO levels when no reducing agent is used. Therefore, incorporating sodium dithionite treatment into GC-TCD methods improves the accuracy of postmortem CO quantification, particularly in putrefied samples.