A stepwise strategy to distinguish menstrual blood from peripheral blood by Fisher’s discriminant function

Blood samples are the most common and important biological samples found at crime scenes, and distinguishing peripheral blood and menstrual blood samples is crucial for solving criminal cases. MicroRNAs (miRNAs) are important molecules with strong tissue specificity that can be used in forensic fields to identify the tissue properties of body fluid samples. In this study, the relative expression levels of four different miRNAs (miR-451, miR-205, miR-214 and miR-203) were analysed by real-time PCR, with 200 samples from 5 different body fluids, including two kinds of blood samples (peripheral blood and menstrual blood) and three kinds of non-blood samples (saliva, semen and vaginal secretion). Then, a strategy for identifying menstrual and peripheral blood based on Fisher’s discriminant function and the relative expression of multiple miRNAs was established. Two sets of functions were used: Z1 and Z2 were used to distinguish blood samples from non-blood samples, and Y1 and Y2 were used to distinguish peripheral blood from menstrual blood. A 100% accuracy rate was achieved when 50 test samples were used. Ten samples were used to test the sensitivity of the method, and 10 ng or more of total RNA from peripheral blood samples and 10 pg or more of total RNA from menstrual blood samples were sufficient for this method. The results provide a scientific reference to address the difficult forensic problem of distinguishing menstrual blood from peripheral blood.