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Objectives: To establish a minimally invasive biopsy technique for the analysis of entheseal tissue in patients with psoriatic arthritis (PsA)., Methods: Human cadavers were used for establishing the technique to retrieve tissue from the lateral humeral epicondyle enthesis (cadaveric biopsies). After biopsy, the entire enthesis was surgically resected (cadaveric resections). Biopsies and resections were assessed by label-free second harmonic generation (SHG) microscopy. The same technique was then applied in patients with PsA with definition of entheseal tissue by SHG, staining of CD45+immune cells and RNA extraction., Results: Entheseal biopsies from five cadavers allowed the retrieval of entheseal tissue as validated by the analysis of resection material. Microscopy of biopsy and resection sections allowed differentiation of entheseal, tendon and muscle tissue by SHG and definition of specific intensity thresholds for entheseal tissue. In subsequent entheseal biopsies of 10 PsA patients: the fraction of entheseal tissue was high (65%) and comparable to cadaveric biopsies (68%) as assessed by SHG microscopy. Furthermore, PsA biopsies showed immune cell infiltration and sufficient retrieval of RNA for further molecular analysis., Conclusion: Entheseal biopsy of the lateral epicondyle is feasible in patients with PsA allowing reliable retrieval of entheseal tissue and its identification by SHG microscopy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)