Immunochromatographic detection of human hemoglobin from deteriorated bloodstains due to methamphetamine contamination, aging, and heating.

Japanese police conduct highly sensitive and quick blood tests to detect human hemoglobin (Hb), because bloodstains left at a crime scene have probative value of circumstantial evidence in a criminal investigation. Although DNA detection from a bloodstain is a useful tool to identify an individual, doing so requires evidence that the bloodstain is of human origin. Stimulant drug abuse and dependence causes major social problems and crimes in Japan, and bloodstains are often found inside syringes seized from drug abusers. In this case, Hb often cannot be detected by conventional testing as high concentrations of stimulants, such as methamphetamine hydrochloride (MA), in blood trigger polymerization of Hb molecules, which become insoluble under non-reducing conditions and can no longer be detected by immunochromatographic detection kits. To overcome this problem, we analyzed methods to detect denatured Hb from bloodstains contaminated with MA. Reduction of polymerized Hb with a strong denaturing agent was required to solubilize polymers into monomers, suggesting that Hb aggregation is caused by aberrant formation of disulfide bonds. Based on these results, we established a pretreatment method, called Fukui's Reduction and Eiken's Dilution (FRED), that enables highly sensitive detection of human Hb from bloodstains mixed with MA by reducing and refolding of denatured Hb. This powerful method can be applied to blood that has been boiled or has otherwise deteriorated for over 20 years.