Reprocessing of hair roots using conventional forensic DNA analysis following RapidHIT® ID processing

Hair traces are frequently recovered at crime scenes and represent a valuable source of forensic DNA, although the cellular content of hair roots varies substantially depending on growth stage, with shed hairs typically containing limited DNA. Rapid DNA platforms such as the RapidHIT® ID (RHID) system enable expedited STR profiling; however, the impact of RHID processing on downstream laboratory-based analyses remains an important consideration. In this study, the ability to reprocess hair roots following RHID analysis was evaluated, and Diamond™ Nucleic Acid Dye (DD) was employed to assess cellular retention and degradation associated with the RHID workflow. Hair roots representing a range of nuclei counts were initially assessed using DD staining and fluorescence microscopy, processed using the RHID system, and subsequently recovered from the cartridge for conventional forensic DNA analysis. Post‑RHID assessment using DD enabled visualisation of remaining cellular material and provided an indication of substrate integrity following rapid DNA processing. Recovered hair roots were subjected to standard lysis, extraction, and STR amplification protocols to determine the feasibility of downstream profiling. Results demonstrated that hair roots, particularly those with higher initial nuclei counts, could be successfully reprocessed following RHID analysis, yielding DNA profiles suitable for comparison and further forensic applications. DD staining indicated that cellular material was retained post‑RHID, supporting the suitability of recovered hair roots for additional testing. In contrast, shed hairs were less likely to yield usable profiles after reprocessing, reflecting their inherently limited cellular content rather than RHID‑induced degradation. These findings demonstrate that RHID processing does not preclude subsequent laboratory analysis of hair roots and highlight the value of hair root visualisation prior to processing, as demonstrated here using Diamond™ Dye (DD). Collectively, this approach supports the use of rapid DNA workflows while preserving evidentiary material for confirmatory and extended forensic analyses.