Ten‐Year Retrospective Study on the Diagnostic Value of Three‐Site Skin Biopsy for Detecting pS129 ‐α‐Synuclein Biomarker: Cervical and Leg Dual‐Site Biopsy as an Effective and Less Invasive Alternative

Background: The in vivo detection of phosphorylated-α-synuclein (pS129-α-syn) via immunofluorescence in cutaneous nerve fibers has emerged as a promising biomarker for diagnosing synucleinopathies, including Parkinson's disease, dementia with Lewy bodies and multiple system atrophy (MSA). However, the variability in biopsy protocols, particularly regarding the choice and number of anatomical sites, limits standardisation and clinical applicability. Objective: To evaluate the diagnostic accuracy of different biopsy site combinations for pS129-α-syn detection in a large cohort of patients with confirmed synucleinopathies, to identify a sampling strategy that ensures high sensitivity while reducing patient burden, promoting methodological standardisation and clinical applicability. Methods: In this 10-year retrospective study, data from 227 patients with Lewy Body Diseases (LBD) (n = 194) or MSA (n = 33), who were identified as positive based on the three anatomical sites (two samples from each site: the cervical region (CE), thigh (TH) and distal leg (LEG)) skin biopsies protocol, were analysed. Diagnostic sensitivity was calculated for each site and combination, stratified by sex and disease duration. Results: The results showed that the CE + LEG combination yielded the highest diagnostic sensitivity in both the LBD (97.68%) and MSA (100%) cohorts, independent of sex and duration. The TH site offered minimal additional diagnostic value when combined with CE and LEG. Conclusions: CE + LEG dual-site skin biopsy provides high diagnostic accuracy for both LBD and MSA, making it a less invasive yet effective alternative to the tri-site protocols (six samples). The distinct deposition patterns observed between disease subtypes warrant further investigation to enhance the biomarker's diagnostic and pathophysiological relevance.