Determination of total hemoglobin in forensic blood samples with special reference to carboxyhemoglobin analysis

For the determination of total hemoglobin (Hb) in blood containing elevated carboxyhemoglobin (COHb), a newly developed reagent containing a 100-fold concentration of ferricyanide (20 g/l) and a 2-fold concentration of Sterox SE was compared with a standard reagent (0.2 g/l ferricyanide), the reagent of van Kampen and Zijlstra, using forensic blood samples and experimentally heated blood samples. There were no significant differences between the spectra of hemiglobincyanide (HiCN) solution produced with our reagent and the van Kampen and Zijlstra reagent using experimentally heated blood samples. Although the spectra of HiCN changed gradually with increased heating time and with the passage of time after mixing, the absorbance at 540 nm (A540) did not change until at least 120 min for both the reagents. When forensic blood samples containing elevated COHb were mixed with the van Kampen and Zijlstra reagent, total-Hb concentrations determined 5 min after mixing were 10-20% higher than those determined after 180 min. The overestimates of total Hb determined after 5 min resulted in comparable underestimates of percentage saturation of COHb (COHb%) when COHb% was obtained from the ratio of COHb content, determined by gas chromatogrpahy, to total-Hb concentration in blood. However, there was an extremely good correlation between the values of total Hb in forensic blood samples determined with the van Kampen and Zijlstra reagent after 180 min and those determined with our reagent after 5 min. From the results obtained, our reagent proved to be suitable for the determination of total Hb in forensic science practice.