Hyperspectral Imaging for Objective Assessment of Skin Prick Test Reactions: A Single-Subject Proof-of-Concept Study of Interobserver Reliability and Spectral Index Separation

Background/Objectives: Hyperspectral imaging (HSI) enables non-contact assessment of tissue oxygenation and perfusion-related properties and may provide objective information beyond conventional visual assessment of skin prick test (SPT) reactions. This proof-of-concept study evaluated the interobserver reliability and within-subject spectral separation of HSI-derived indices during SPT. Methods: One healthy adult volunteer underwent standardised SPT on two separate days with histamine as the positive control, NaCl as the negative control, normal skin, and a non-reactive allergen site. HSI was acquired at 0, 5, 10, 15, and 45 min using a Tivita® Tissue camera. Three raters independently assessed four predefined regions of interest. Five indices were analysed: near-infrared reflectance (NIR), tissue oxygen saturation (StO2), tissue haemoglobin index (THI), tissue water index (TWI), and tissue lipid index (TLI). Interobserver reliability was assessed using ICC(3,1), and within-subject separation was described using baseline-corrected trapezoidal area under the curve (AUCbc) and delta-AUCbc values. Results: StO2 showed the most consistent interobserver reliability, with good-to-excellent ICCs across all four ROIs at both visits. THI showed excellent reliability at the histamine and NaCl sites, whereas NIR showed excellent reliability at the histamine site and good reliability at the NaCl site. Reliability at the less clearly defined control sites was more variable across indices and visits. StO2 provided the largest cumulative separation of the histamine response from all controls (mean delta-AUCbc: +1204 vs. NaCl, +1300 vs. normal skin, and +1238 vs. non-reactive allergen; index-units × min). THI showed secondary separation, NIR moderate separation, TWI marginal separation, and TLI an inverse response. Conclusions: HSI, particularly StO2, with complementary information from THI and NIR, showed reproducible within-subject spectral changes during SPT. These exploratory findings support prospective validation in a larger multi-subject, multi-allergen cohort.