Abstract 3763: Development of a plucked hair immunohistochemisty biomarker platform
Authors: Jackson Ben R. , Somerville Tim , Booth Catherine 
Published: February 2011

Abstract
Background
Plucked hair is a valuable surrogate tissue to monitor pharmacodynamic (PD) responses to treatment. Hair collection is minimally invasive, simple and permits frequent sampling. We have previously demonstrated the feasibility, and utility of monitoring mRNA expression in individual plucked human scalp hairs to correlate targeted gene expression changes in response to a range of oncology treatments. RNA analysis permits the examination of downstream events resulting from changes in target activation. To directly monitor target response to treatment, immunohistochemistry (IHC) protocols have now been developed.
Methods
Scalp hairs were plucked from healthy, normal volunteers (aged 18-42 yrs) and fixed and processed for IHC. IHC protocols were developed for whole mount and sectioned hairs (using fluorescence and DAB detection methods) for a range of markers of drug action, including markers of cell proliferation and growth factor receptor phosphorylation. Levels of inter- and intra-donor variation were also assessed by labeling for Ki67 and analyzing 7 anagen hairs from each donor. Once labeled, hairs were imaged and the label quantified by a range of methods, including manual counting of labeled nuclei and semi-automated analysis of number of labeled nuclei by Image-Pro Plus® software.
Results
Results showed that for a variety of antigens in plucked hair, the IHC method developed was a reproducible technique for both whole mounts and sectioned hair. One standard protocol could be used for each antibody, with adjustments made to primary antibody concentrations and the region of the hair analyzed. From each set of 7 scalp hairs approximately 5 were suitable for analysis of IHC labeling. The number of cells labeled for Ki67 was quantified manually from 10 volunteers (5 hairs per donor) in a 0.4mm length of the outer root sheath and this gave a percentage variation of 38% and 23% for all volunteers and male only volunteers, respectively. This inter-donor variation was higher than the intra-donor variation which was 27% and 20% for all volunteers and male only volunteers, respectively. This analysis was performed in parallel using Image-Pro Plus® which has the advantage of increased speed of analysis and removal of potential error introduced by using different human scorers. Values were comparative with manual counting with inter-donor variation of 37% (all donors), 19% (only male donors), and intra variation of 29% (all donors) and 23% (only male donors). Hairs processed 6 weeks after fixation retained 60-80% of original antigenicity dependant on the antigen analyzed.
Conclusion
The results presented demonstrate that IHC labeling and quantification protocols can be developed for plucked human hairs. The range of inter- and intra-donor variation was established. This provides a simple, acceptable technique for collecting and analyzing surrogate epithelial tissue to monitor direct cellular response to drug treatment.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3763.