Two different staining methods for staining, visualizing, and counting nuclei in evidentiary hair roots, 4′, 6-diamidino-2-phenylindole (DAPI) and hematoxylin, were compared based on a review of relevant literature. Ease of use, safety, stability, and efficacy were evaluated. Overall, both staining methods were found to be successful, despite the individual challenges observed with each method. DAPI staining requires a fluorescence microscope for visualization. Hematoxylin staining is less effective on dark pigmented hair roots, can overstain light-colored hairs, requires a rinse step, and may cause polymerase chain reaction (PCR) inhibition in some cases. Based on these findings, DAPI staining is recommended when a fluorescence microscope is available. Studies show hematoxylin is an effective nuclear stain for hair roots under a simple compound microscope; however, a fluorescence microscope may still be required. Either stain can be added to a forensic hair examination workflow to increase the efficiency and efficacy of nuclear DNA analysis from hair roots.
Get full access to this article
View all access options for this article.
References
1.
PfeifferH, HuhneJ, OrtmannC, et al.Mitochondrial DNA typing from human axillary, pubic and head hair shafts - success rates and sequence comparisons. Int J Legal Med, 1999; 112(5):287–290; doi: 10.1007/s004140050251
2.
AdmireL, CarsonM, CrawfordK, et al.Hair root staining with Hematoxylin: Increasing the rate of obtaining DNA profiles in forensic casework. Forensic Sci Int, 2023; 343:111544; doi: 10.1016/j.forsciint.2022.111544
3.
BoonenT, VitsK, HosteB, HubrechtF. The visualization and quantification of cell nuclei in telogen hair roots by fluorescence microscopy, as a pre-DNA analysis assessment. Forensic Sci Int Genet Suppl Ser, 2008; 1(1):16–18; doi: 10.1016/j.fsigss.2007.10.098
4.
BourguignonL, HosteB, BoonenT, et al.A fluorescent microscopy-screening test for efficient STR-typing of telogen hair roots. Forensic Sci Int Genet, 2008; 3(1):27–31; doi: 10.1016/j.fsigen.2008.08.006
5.
BrooksEM, CullenM, SztydnaT, et al.Nuclear staining of telogen hair roots contributes to successful forensic nDNA analysis. Aust J Forensic Sci, 2010; 42(2):115–122; doi: 10.1080/00450610903258136
6.
EdsonJ, BrooksEM, McLarenC, et al.A quantitative assessment of a reliable screening technique for the STR analysis of telogen hair roots. Forensic Sci Int Genet, 2013; 7(1):180–188; doi: 10.1016/j.fsigen.2012.10.001
7.
LawasM, OtterstatterLM, ForgerLV, et al.A quantitative method for selecting a hair for nuclear DNA analysis. Forensic Sci Int Genet, 2020; 48:102354; doi: 10.1016/j.fsigen.2020.102354
8.
LeeSY, HaEJ, WooSK, et al.A rapid nuclear staining test using cationic dyes contributes to efficient STR analysis of telogen hair roots. Electrophoresis, 2017; 38(13–14):1771–1779; doi: 10.1002/elps.201700024
9.
LepezT, VandewoestyneM, Van HoofstatD, et al.Fast nuclear staining of head hair roots as a screening method for successful STR analysis in forensics. Forensic Sci Int Genet, 2014; 13:191–194; doi: 10.1016/j.fsigen.2014.08.003
10.
OtterstatterL, Espinoza QuirogaIA, BettsSM, et al.Revisiting the hair root visible nuclei count threshold for nuclear DNA typing. Forensic Genom, 2023; 3(4):138–149; doi: 10.1089/forensic.2023.0017
11.
TarnowskiBI, SpinaleFG, NicholsonJH. DAPI as a useful stain for nuclear quantitation. Biotech Histochem, 1991; 66(6):296–302; doi: 10.3109/10520299109109990
AvwioroG. Histochemical uses of hematoxylin: A review. J Phys Conf Ser, 2011; 1:24–34.
14.
CooperJA, MintzBR, PalumboSR, et al.Assays for determining cell differentiation in biomaterials. In: Characterization of Biomaterials.Woodhead Publishing; 2013:101–137; doi: 10.1533/9780857093684.101
Leica Biosystems. Harris Hematoxylin Safety Data Sheet. Model Work Health and Safety Regulations. Version 1.1. Nussloch, Germany; 2018. Available from: file:///C:/Users/82bur/Downloads/Harris_Haematoxylin_AU.pdf [Last accessed: April6, 2025].
23.
Newcomer Supply. Hematoxylin Stain, Harris Modified. Version 1.4. Waunakee, WI; 2017. Available from: https://www.newcomersupply.com/sds/ [Last accessed: April6, 2025].
SzaboS, JaegerK, FischerH, et al.In situ labeling of DNA reveals interindividual variation in nuclear DNA breakdown in hair and may be useful to predict success of forensic genotyping of hair. Int J Legal Med, 2012; 126(1):63–70; doi: 10.1007/s00414-011-0566-5
