Background/Purpose: Enthesitis as hallmark feature of psoriatic arthritis (PsA) has been mostly addressed through clinical assessment and imaging evaluation, such as MRI or musculoskeletal ultrasound. Data regarding a more specific molecular analysis of enthsesis are however lacking, due to the difficulties in harvesting entheseal tissue. Up to date, retrieval of entheseal tissue constitutes a technical challenge, due to the unclear definition of which entheseal structure would qualify for a feasible biopsy. Here, we propose a minimally invasive biopsy technique of human entheses, for the analysis of entheseal tissue in patients with PsA.

Methods: We obtained elbows from 5 cadavers from the Institute of Anatomy and recruited 10 patients with active elbow enthesitis PsA who fulfilled the classification criteria for PsA (CASPAR). Biopsies were done with Blakeslay forceps after identifying the lateral epicondyle, the olecranon and the radial head by ultrasound. In cadavers, the whole enthesis (=cadaver resection material) including adjacent tendon, muscle and bone was surgically resected after the biopsy for further analysis. All materials (cadaver biopsies, PsA biopsies, cadaver resection material) were fixed and embedded in paraffin for sections. The material was stained by hematoxylin/eosin, safranin O and trichrome or analyzed without staining using multi-photon microscopy (Zeiss LSM 880 NLO Intravital microscope, Jena, Germany) for capturing secondary harmonic generation (SHG). All images had a 16-bit depth and were obtained at 2.05 µs/pixel. We obtained pro sample 3 measurements of SHG signal intensity (SHG-I) from entheses, tendon, muscle and bone and used the average of these measurements as final intensity. Optimal out-of-sample SHG-I thresholds to discriminate tissue types and their 95% confidence intervals were estimated by repeated ROC curve analyses. Immunofluorescence and RNA isolation were performed for protein and RNA evaluation of the biopsies.

Results: The anatomical specimens from cadaver were adopted for establishing the entheseal biopsy procedure. Here, the retrieval of entheseal tissue was validated by analysis of the resection material. Whereas, the difference between entheseal, tendonal and muscular components within the small entheseal biopsies was not achieved by standard histochemistry, SHG microscopy of both biopsy and cadaveric resection material allowed differentiation of the different tissues as well as the definition of specific intensity thresholds for entheseal tissue. The same method for entheseal biopsy was then successfully applied to 10 PsA patients. Here, the fraction of entheseal tissue was high (65%) and comparable to the fraction retrieved in cadaveric biospies (68%) as assessed by SHG microscopy. Moreover, further bimolecular analysis showed immune cells infiltration in the entheseal tissue of PsA patients and confirmed good quality RNA retrieval.

Conclusion: Entheseal biopsy of the tendon plate of the lateral epicondyle is feasible in PsA patients allowing reliable retrieval of entheseal tissue and its identification by SHG microscopy, for further bimolecular analysis and a better treatment strategy for enthesitis in the future.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/entheseal-biopsy-approach-an-ultrasound-guided-method-for-retrieval-of-entheseal-tissue-from-psoriatic-arthritis-patients/