Inhibition of acid phosphatase detection in mixed semen-blood training samples by anti-coagulants present in blood collection tubes

Abstract Acid phosphatase detection using fast blue or fast black reagent is routinely performed at the RCMP National Forensic Laboratory Services as a presumptive test for semen. In training exercises, samples consisting of semen mixed with blood were reported to yield negative results with the fast black reagent. To determine the cause of the inhibition, mixed semen-blood stains were prepared using different blood sources, anti-coagulants, and layering techniques, before being tested with the fast blue or the fast black reagent. The anti-coagulant sodium fluoride (NaF) was shown to cause false negative results with the fast black reagent and to impair detection when using the fast blue reagent while no inhibition was observed when dipotassium EDTA (K2 EDTA) was used as anti-coagulant. Four different toothpastes containing fluoride were also tested for potential inhibition of semen detection. Inhibition was observed when semen was mixed with three of the undiluted toothpastes but not with diluted toothpastes (1 in 20 in water). These findings highlight the importance of using appropriate blood sources when preparing mixed semen-blood stains for the purpose of training and proficiency testing in a forensic laboratory. However, forensic casework samples are not expected to contain NaF at the inhibiting levels.