Ascorbic acid and vitamin C-containing beverages delay the leucomalachite green reaction to detect latent bloodstains

The leucomalachite green (LMG) test is one of catalytic tests for the detection of latent bloodstains and generally used in forensic field because of convenience and cost/time-effectiveness. However, contamination of latent bloodstains at crime scenes can interfere with the LMG reaction, resulting in false-negative or false-positive decisions. Herein, we examined if ascorbic acid and vitamin C (l-ascorbic acid or ascorbate)-containing beverages affect the LMG reaction. Ascorbic acid showed the inhibitory activities on the LMG reaction in a dose-dependent manner. Similarly, vitamin C-containing beverages also inhibited the LMG reaction and the inhibitory effects were proportional to the concentrations of vitamin C in beverages. It was also identified that as incubation time after adding LMG reagent to the mixtures of blood and ascorbic acid or beverages was increased, the inhibitory effects of ascorbic acid vitamin C-containing beverages on LMG test were disappeared. These results suggest that the LMG reaction is delayed but not stopped by ascorbic acid and vitamin C-containing beverages. Neither incubation at room temperature around 20-25°C nor the addition of acetic acid affects the inhibitory activity of ascorbic acid on LMG reaction. We also showed that ascorbic acid does not affect DNA stability, allowing us to obtain full short tandem repeat (STR) profiles through amplification of DNA using commercial STR kits. In conclusion, ascorbic acid and vitamin C-containing beverages delayed the LMG reaction, suggesting that it should be considered that negative results of LMG test could be false negative due to contamination of bloodstains with inhibitory factors on LMG test.