Blood grouping of mixed bloodstains using immunocytochemical methods

Immunocytochemical methods to determine the ABO blood group of each blood of mixed bloodstains have been developed. Mixed bloodstains were made on surgical blades and a cedar board. The blades were dipped into blood and then dipped into blood of a different group at intervals of 30, 20, 15, 10 and 5 s. Two drops of blood were dropped on a cedar board and then two drops of blood of a different group were dropped there at the same intervals. The bloodstains were dried for a week. The blood samples were removed from the blades or the cedar board and processed according with a routine histological method. Three serial thin sections were obtained. After deparaffinization, the sections were treated in papain solution for 2 h at 36 degrees C, to unmask antigenic sites on red cell membranes. The labeled streptavidin-biotin (LSAB) and peroxidase-anti-peroxidase (PAP) methods were used to detect A and B antigens, and an indirect immunocytochemical method for H antigen. These immunocytochemical methods showed specific immunologic reactions and allowed determination of the blood group of each blood of mixed bloodstains. Further, these methods indicated a possibility to determine who was stabbed first, in cases where two or more victims were stabbed with a single knife.