POS0842 NON-INVASIVE COHERENT RAMAN IMAGING OF INVOLVED FOREARM SKIN REVEALS ALIGNED COLLAGEN IN DERMIS OF SYSTEMIC SCLEROSIS PATIENTS

Abstract

BackgroundSystemic sclerosis (SSc) is an immune-mediated connective tissue disease with clinical hallmark of inflammation, vasculopathy and fibrosis resulting in abnormal collagen and intercellular matrix formation in the dermis and internal organ. Skin severity correlates with systemic complications and mortality in SSc.Many skin assessment tools studied do not fulfilled all standards set by OMERACT which include criterion validity, construct validity, discrimination, responsiveness, reliability and feasibility.Modified Rodnan skin score (mRSS) is commonly used as outcome measure in clinical trials of SSc. It is however, limited by inter-rater variability. Studies also showed that skin biopsy samples from body part of SSc patients with clinically normal mRSS had demonstrable pathological deposition of collagen. While repeat skin biopsy to track longitudinally, may be unacceptable to patients. Hence, ongoing research for non-invasive tools are encouraged.ObjectivesWe follow through our previous work studying commercially available non-invasive imaging tools in SSc skin assessment by using Coherent-Raman scattering microscopy (CRS).MethodsSkin biopsies of 4mm were obtained from SSc (n=3) and HC (n=1). Skin samples were collected at anterior surface of forearm from newly diagnosed diffuse SSc which were treatment naïve. Samples were frozen at -80c prior to analysis under CRS at skin depth of 150-200um. The laser setting was 3% pump (802nm) and 6% stokes (1045nm) to avoid tissue burn. 3 contrast methods used are Coherent anti-Stokes Raman scattering (CARS) for imaging of lipids, Second Harmonic Generation (SHG) for collagen and Two-Photon Fluorescence (TPF) for elastin fibers. We analysed collagen and elastin fibers in the skin samples which is disease relevant end products of fibroblast activation in SSc. Descriptive study of the skin CRS characteristics is reported.ResultsThe SHG analysis revealed denser collagen fibers in dermal layer of diseased skin. It also appears that the collagen deposition occurs in more superficial layers of the skin. Peak of collagen curve were at depth of 110-130um HC vs 75-90um in SSc. Collagen fibres were more aligned in the SSc (Figure 1).Figure 1.Microscopy analysis of SSc (B) vs HC (A) skin. I: 3D image of biopsy samples with three contrasts. Red is CARS for lipid, blue is SHG for collagen and green is TPF for elastin fibers. II. SHG. Imaging revealed denser and more aligned collagen fibers in SSc.Furthermore, TPF revealed larger number of disordered elastin fibres in the dermal layer of SSc than HC.ConclusionNailfold capillaroscopy is used to define and stage micro-vasculopathy in SSc. Inflammatory pro-fibrotic processes on the other hand cause abnormal collagen and intercellular matrix formation in the dermis and internal organ.With the demand of better tools in diagnosis of early SSc and therapeutic research, our work with CRS prove better in objective evaluation of skin changes at the molecular level. Here, we demonstrate that the SHG is altered in the early diffuse SSc skin with increased and aligned collagen in the SSc dermis compatible with mRSS score. Others have observed this alignment of the collagen, and we have published that SSc fibroblasts migrate/invade along aligned collagen and modify the underlying extracellular matrix, adding collagens I and III, cross-linking enzymes and other factors including TSP-1.Our future work include:1.Generating a computer module in defining pathological collagen level2.Analysis of metabolites and pathological pathways in SSc3.In-vivo trials with novel therapeutic peptides.4.Lastly, manufacturing of non-invasive handheld device that is capable of diagnosis subclinical SSc and for outcome measure in clinical-therapeutic researchReferences[1]Abignano G, et al. Virtual skin biopsy by optical coherence tomography: the first quantitative imaging biomarker for scleroderma. Ann Rheum Dis. 2013[2]Ahmed Abdi B, et.al. Use of Patterned Collagen Coated Slides to Study Normal and Scleroderma Lung Fibroblast Migration. Sci Rep. 2017AcknowledgementsWe want to acknowledge our colleagues and patients in Royal Free Hospital, to make this project a success.Disclosure of InterestsNone declared