Abstract:
The ability to determine the age of a human dermal injury is important in forensic work. It helps answer questions about the timing of the injury and incident, the relationship of the injury to the incident, the order of infliction of the injuries and the survival time after the injury, which are critical to the reconstruction of a crime. However, there is currently no method for the reliable estimation of human dermal injury age. In this research, the mRNA expression of seventeen markers, involved in injury healing, were screened in intravital human dermal injuries, using duplex PCR assays, to determine their suitability for use in the development of a multiplex PCR assay to study mRNA expression for human dermal injury age estimation. The seventeen mRNA markers included: dual specificity phosphate 1 (DUSP1), interleukin 7 (IL7), P-selectin (SELP), vascular cell adhesion molecule 1 (VCAM1), tenascin C (TNC), cluster of differentiation 14 (CD14), E-selectin (SELE), interleukin 6 (IL6), tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL1β), chymase 1 (CMA1), collagen type III alpha I (COL3A1), laminin 5 (LAMA5), interleukin 2 (IL2), collagen type I alpha I (COL1A1), collagen type I alpha II (COL1A2), and vascular endothelial growth factor A (VEGFA). The screening identified DUSP1, IL7, IL6, TNFα, IL1β, CMA1, COL3A1, IL2 and VEGFA as suitable mRNA markers and these were incorporated into a traditional end point PCR based multiplex PCR assay. 18S ribosomal RNA (18S rRNA) was also included in the assay as an endogenous control and for the normalisation when evaluating mRNA expression levels. The multiplex PCR assay was used to detect, analyse and evaluate the mRNA expression of the markers in intravital human dermal injuries during the injury healing process. Based on the multiplex PCR assay detections, successful mRNA expression evaluations were only possible for DUSP1, IL7, TNFα, IL1β, CMA1 and VEGFA. DUSP1, IL7, TNFα, and VEGFA showed an initial decrease in mRNA expression during the early phase of injury healing, followed by an increase in mRNA expression towards the middle and late phases; IL1β and CMA1 mRNA expression was limited to the early phase. Using regression modelling, the mRNA expressions of IL7, TNFα and IL1β were identified as statistically significant predictors of injury age and were used in the development of a regression model for human dermal injury age estimation. The model was able to distinguish between injuries of different ages (hours), but requires improvement before forensic application. Additionally, a comparison of the detection and evaluation using endpoint PCR and real-time PCR for the mRNA markers DUSP1, IL6, CMA1, COL3A1 and VEGFA was conducted, and it was found that the two methods gave similar results.