We previously proposed a prediction model consisting of 9 CpG sites for forensic age estimation with high practical potentials in Chinese males. Here, we further evaluated the performance of this prediction model in two independent batches of time-series bloodstain samples naturally exposed to room temperature conditions. The first batch consists of 30 Han Chinese males (18-59 years of age) whose peripheral blood was converted into bloodstains on Flinders Technology Association (FTA) cards and naturally exposed to room temperature conditions for different time points up to 3 months. The second batch consists of 99 Han Chinese males (21-66 years of age) whose peripheral blood was divided into 3 replicates, converted into bloodstains on gauze, and naturally exposed to room temperature conditions for 3 months. For each time point and each replicate, the methylation levels at the 9 CpG sites were detected using the EpiTYPER system. Applying the 9-CpG age prediction model to these bloodstain samples resulted in highly accurate age predictions for all time points and replicates (0.81 <R2 < 0.91, 2.94 < MAD < 3.55 years). The updated model combining our previous and current data achieved similarly high prediction results. Therefore, our 9-CpG age prediction model was successfully validated in time-series bloodstain samples converted on both FTA card and gauze under natural room temperature conditions, demonstrating high potentials in future forensic applications to Han Chinese males.
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