Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Development Of A Non-invasive Diagnostic And Predictive Tool For Acute Rejection In Vca Using Differential Cytokine Profiling
Nakul Rao, MD, Piul Rabbani, PhD; Daniel Ceradini, MD; Rohini Kadle, BS; Anna Zhou, BA; Nicholas Brownstone, BA.
NYU, New York, NY, USA.

PURPOSE:
Detection of early acute transplant rejection relies heavily on invasive organ or tissue biopsies and histological analysis for proper diagnosis and survival of the transplant. We propose an alternative detection method using skin stripping of epidermis and molecular analysis, to detect known markers of acute rejection. We hypothesize that skin stripping can be used as a non-invasive predictor and as a sensitive early diagnostic test for early acute rejection in transplantation.
METHODS:
Using an established rat model for vascular composite allograft (VCA), we transplanted composite flaps from donor Brown-Norway rats to age-matched recipient Lewis rats. We treated recipient rats with cyclosporine for five days and inspected daily for clinical signs of rejection. We sampled transplanted flap skin with 15 adhesive CuDerm sampling discs at each of the progressive time points up to rejection: day 5 of immunosuppression, first detection of clinical mild rejection and advanced rejection. Using skin cells from the discs, we performed quantitative RT-PCR to detect cytokine genes, CXCL9, CXCL10, MCP1, MIP1α, MIP1β and MIP3α, indicative of early rejection in skin. We also harvested corresponding 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 statistical significance during each of the two clinical stages of rejection, relative to the immunosuppressed stage: MCP1expression - 30x and 65x, MIP1α- 10x and 500x, MIP1β- 10x and 200x, CXCL10 expression- 10x and 95x, at mild and advanced rejection, respectively, p<0.05. MIP3α and CXCL9 showed significant upregulation at mild rejection with 19 fold and 70 fold changes in expression, respectively, and a downregulation at advanced rejection with 4 fold and 20 fold changes, respectively, p<0.05. All differential cytokine expression was highly significant between mild and advanced rejection as well with p<0.01. We verified the sensitivity of the CuDerm method by comparison with QRT-PCR of flap 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.
CONCLUSION:
Skin stripping, when compared to traditional tissue biopsy, is a comparable and reliable analytical tool. The cytokine profiles for the progression of rejection are distinct and capable of detecting the earliest stages of acute rejection 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.


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