Optimization of quantitative analysis method for carboxyhemoglobin in severely decomposed spleen tissue influenced by a reducing agent

The concentration of carbon monoxide (CO) in a forensic sample is often determined by directly injecting blood into the oximeter. However, collection of blood sample is not always possible, especially when the subject is a severely decomposed cadaver. In this case, the CO concentration is determined by analyzing spleen tissue using gas chromatography with thermal conductivity detector (GC-TCD). In the previous study, spleen from 125 autopsy cases were analyzed which led to a conclusion that CO intoxication cannot be considered as a definitive cause of death when its concentration is lower than 30 %. The non-CO related cases where the concentration was as high as 30 % were because of high methemoglobin. Methemoglobin (MetHb) is a common product of hemoglobin oxidation caused by decomposition. Since MetHb is unable to bind with oxygen or carbon monoxide, it inhibits the CO saturation of the sample which is a critical step in constructing a calibration curve for CO calculation. Therefore, the importance of incorporating a reducing agent to lower MetHb by reducing it to normal hemoglobin has been proclaimed. To test the viability of reducing agent, where in this case Na₂S₂O₄, as a method to lower MetHb in spleen samples, preliminary tests on blood were conducted by comparing the result of the analysis with and without using the reducing agent. Blood samples with high MetHb were required, so an oxidating agent, sodium nitrite was added to spike the samples, as storing the already collected sample didn’t induce more decomposition and higher MetHb. Using the spiked blood samples, Na₂S₂O₄ was added in various concentrations and analyzed to find the appropriate amount, which was then applied to the spleen samples. The necessity of using the reducing agent was confirmed by comparing the results before and after adding Na₂S₂O₄ to the spleen samples, and the most appropriate concentration was found to be 0.1 mL of 0.574 M Na₂S₂O₄ per 1 mL of blood.