Inter-laboratory evaluation of the VISAGE enhanced tool and models for age estimation from blood and buccal cells

Over the past decade, numerous assays for forensic age estimation based on the analysis of DNA methylation markers have been developed, demonstrating significant potential for use in criminal investigations. Despite these advancements, only few comprehensive evaluation studies were published. In this study, we present findings of an extensive inter-laboratory evaluation of the VISAGE Enhanced Tool and its associated statistical models for epigenetic age estimation in blood and buccal swabs. Six laboratories conducted reproducibility, concordance, and sensitivity assessments using DNA methylation controls alongside blood and saliva samples to evaluate the tool's technical performance. Results demonstrated consistent and reliable DNA methylation quantification across all participating laboratories, with the tool maintaining sensitivity even with a DNA input of 5 ng for bisulfite conversion. To evaluate the age estimation models, 160 blood and 100 buccal swab samples were analysed in three laboratories. The models achieved mean absolute errors (MAEs) of 3.95 years for blood and 4.41 years buccal swabs, which represents an increase of ∼0.7 years for both tissues to the results from the original VISAGE testing set. When comparing results of each laboratory with the original VISAGE testing set, significant differences were found only for age estimation results from blood of one laboratory with an underestimation of chronological age observed within the entire range tested at that laboratory. When excluding this laboratory, the MAE decreased to 3.1 years (N = 89). No significant differences among laboratories were found for buccal swabs. Overall, this study confirms that the VISAGE Enhanced Tool performs robust DNA methylation quantification and reliable age prediction, however protocol and model validation within each laboratory is required upon implementation.