Hair root staining with Hematoxylin: Increasing the rate of obtaining DNA profiles in forensic casework

Abstract

Hair evidence collected during a forensic investigation has the potential to provide valuable sourcing information through DNA analysis of its root. Over time, hair examiners at the North Carolina State Crime Laboratory observed hair roots being sent for DNA analysis were not yielding profiles as expected. Recent advancements in the Forensic Biology Section’s detection limits prompted research into whether changes to the current root removal protocol could increase the likelihood of developing a DNA profile from a hair root. An internal validation was completed for the method of Hematoxylin staining to screen telogen roots for DNA analysis. Over 900 head hairs from approximately 15 living donors were examined for telogen roots. Those roots were stained using Hematoxylin and examined for nuclei. The roots were separated into groups based on nuclei present: Group I (1–10 nuclei), Group II (11–20 nuclei), Group III (21–30 nuclei), Group IV (31–40 nuclei), and Group V (41 or greater nuclei). A set of 64 roots, including a Negative Control (0 nuclei) and a Positive Control (anagen or catagen roots), were sent for quantitative DNA analysis. The data showed a clear separation between Groups I and II, where 36% of Group I verses 80% of Group II passed the quantification cutoff. All samples in the Negative Control, Group I, and the Positive Control were then amplified along with a representative sample in Groups II through V. After amplification, the delineation between Groups I and II maintained with 27% of Group I verses 89% of Group II obtaining DNA profiles. As a result, the nuclei required to obtain a potential DNA profile at our laboratory is eleven or more. Prior to Hematoxylin, anagen, catagen, or telogen roots with follicular tissue were sent for DNA analysis, resulting in 32% of roots yielding DNA results. Following implementation of Hematoxylin into casework in March 2019, the success rate has more than doubled to 69%, illustrating the utility of Hematoxylin root staining in improving casework efficiency. Through this methodology, only roots with the best potential to develop a DNA profile are sent for testing, thereby decreasing DNA caseload, cost, and time of analysis. In fact, Hematoxylin staining has resulted in a 14% reduction in the number of hair roots forwarded for DNA analysis, meaning hairs not meeting nuclear threshold are preserved for future examinations.