Quantifying GFAP immunohistochemistry in the brain: introduction of the Reactivity score (R-score) and how it compares to other methodologies

Background: Immunohistochemical upregulation of glial fibrillary acidic protein (GFAP) is commonly used to detect astrogliosis in tissue sections and includes measurement of intensity and/or distribution of staining. There remains a lack of standard objective measures when diagnosing astrogliosis and its severity. New method: Aim was to test a novel semi-quantitative assessment of GFAP which we term reactivity (R)-score, on its reproducibility and sensitivity to measure astrogliosis. The R-score, which is based on the proportion of astrocytes seen at each level of reactivity, was compared to 3 other commonly employed quantification methods in research: (1) thresholding, (2) point-counting, and (3) qualitative grading. Sub-regions of the hippocampus, medulla, and cerebellum were studied in piglet, and 4 human cases with clinically reported astrogliosis. Intra-assay coefficient of variation (CV) and percentage agreement cut-offs of ≤ 20% and ≥ 75% were used respectively to compare amongst the methods, with outcome measures being reproducibility across serial and non-serial sections, resilience to changes in experimental conditions, and inter- and intra-rater concordance. Results: Averaged across 3 brain regions, the intra-assay coefficient of variation (CV) was 5% for R-score, with inter and intra-rater kappa scores being 0.99 and 0.95 respectively. Comparison with existing methods and conclusions: Based on CV values, the R-score was superior to thresholding (CV of 51%) and point-counting (CV of 16%), with the qualitative grade being found to be on par (percentage agreement 95%). Given the ease, reproducibility and selectivity of the R-score, we propose its validity in future research purposes and clinical application.