2576: Optimization of a differential cytokine profile-based non-invasive diagnostic and predictive tool for reliable diagnosis of acute rejection in VCA

Background

Current methods of detection of early acute transplant rejection relies on invasive tissue biopsies and time-consuming histological analysis We propose an alternative method using adhesive discs to analyze molecular changes in cells sampled from the epidermis of a vascular composite allotransplant (VCA), to detect markers of acute rejection We aim to validate efficiency of skin stripping as a non-invasive predictor and sensitive diagnostic test for acute rejection in VCA.

Methods

Using an established VCA rat model, we transplanted composite flaps from donor Brown-Norway rats to age-matched recipient Lewis rats Following cyclosporine for 5 days, we inspected daily for clinical signs of rejection and sampled transplanted skin with adhesive CuDerm-discs at each time point up to rejection We performed QRT-PCR on sampled cells for cytokines associated with early rejection We sampled flaps for biopsies and histology to corroborate the disc data.

Results

CuDerm-disc-sourced PCR revealed that expressions of MCP1, MIP1α, MIP1β and CXCL10 increased progressively with mild and advanced rejection, compared to the immunosuppressed stage (p < 005) MIP3α and CXCL9 showed significant upregulation at mild rejection (19-fold, 70-fold, respectively), but a downregulation during advanced rejection (4-fold,20-fold, respectively, p < 005) Comparison of mild and advanced rejection showed highly significant differential cytokine expression (p < 001) We verified the sensitivity and validity of the CuDerm-disc method by comparison of mRNA expression in VCA biopsies and found cytokine detection comparable between both methods The mild and advanced rejection cytokine profiles from discs also corresponded with the Banff classification of cellular allograft rejection of the respective flap histologies.

Conclusions

Skin stripping, when compared to traditional tissue biopsy, is a comparable and reliable analytical tool The cytokine profiles gathered from skin stripping are distinct and capable of detecting the earliest stages of acute rejection, as well as distinguishing from advanced rejection, stages which are difficult to analyze definitively using traditional histology Our results clearly demonstrate the promise of skin stripping as a non-invasive tool in predicting and diagnosing early rejection, prior to onset of advanced rejection.