Estimation of bloodstain deposition time within a 24-h day-night cycle with rhythmic mRNA based on a machine learning algorithm

Estimating the time that bloodstains are left at a crime scene can provide invaluable evidence for law enforcement investigations, including determining the time of the crime, linking the perpetrator to the crime scene, narrowing the pool of possible suspects, and verifying witness statements. There have been some attempts to estimate the time since deposition of bloodstains, i.e., how much time has passed since the bloodstain was left at a crime scene. However, most studies focus on the time interval of days. As far as we know, previous study have been conducted to estimate the deposition time of blood within a 24-h day-night cycle. To date, there is a lack of studies on whether rhythmic mRNA of blood is suitable for bloodstain samples. In this study, we estimated the bloodstain deposition time within a 24-h day-night cycle based on the expression of messenger RNAs (mRNAs) by real-time quantitative polymerase chain reaction. Bloodstain samples were prepared from eight individuals at eight time points under real and uncontrolled conditions. Four mRNAs expressed rhythmically and were used to construct a regression model using the k-nearest neighbor (KNN) algorithm, resulting in a mean absolute error of 3.92 h. Overall, using the rhythmic mRNAs, a machine learning model was developed which has allowed us to predict the deposition time of bloodstains within the 24-h day-night cycle in East Asian populations. This study demonstrates that mRNA biomarkers can be used to estimate the bloodstain deposition time within a 24-h period. Furthermore, rhythmic mRNA biomarkers provide a potential method and perspective for estimating the deposition time of forensic traces in forensic investigation. Case samples in forensic analysis are usually limited or degraded, so the stability and sensitivity of rhythmic biomarkers need to be further investigated.