REFLECTANCE CONFOCAL MICROSCOPY AS A USEFUL DIAGNOSTIC TOOL FOR MONITORING OF SKIN CONTAINING VASCULARIZED COMPOSITE ALLOGRAFT REJECTION: A PRELIMINARY STUDY ON RATS FATIH ZOR, M.D., 1 * HUSEYIN KARAGOZ, M.D., Ph.D., 2,3 ASLI TURGUT ERDEMIR, M.D., 4 YILDIRIM KARSLIOGLU, M.D., 5 CENGIZ HAN ACIKEL, M.D., 6 REZARTA KAPAJ, M.D., 1 SERBULENT GUZEY, M.D., 1 MEHMET SALIH GUREL, M.D., 4 SELCUK ISIK, M.D., 1 and MARIA SIEMIONOW, M.D., Ph.D., D.S.C. 3 Vascularized composite allografts can undergo immune-mediated rejection, and skin biopsies are needed for monitoring of the transplant. However it is an invasive method, and requires processing time and pathological assessment. The purpose of this study is to use a new noninvasive monitoring method of the reflectance confocal microscopy (RCM) to determine severity of the allograft rejection on rats. Five groin flap allotransplantation were performed between 10 male Sprague-Dawley rats. Immunosuppressive therapy with cyclosporine A was given to the recipients during 10 days after surgery and was ended at the 10th postoperative days to allow acute transplant rejection. Following cessation of CsA, concomitant RCM evaluation and skin biopsy was performed every other day from each animal until total rejection of the allograft. Complete rejection of the allograft took nearly about 10 days and 4 or 5 RCM evaluation and skin biopsy was performed from each rat during this period. A total of 17 specimens were evaluated. A scoring system was developed based on the RCM findings. Skin biopsies were evaluated according to the Banff 2007 working classification criteria. RCM evaluation revealed epidermal irregularity and collagen destruction, however mild perivascular inflammation and degeneration of the basal epidermal layer were observed in early and late rejection period respectively with histopathologic evaluation. High correlation was found between the RCM scores and histopathologic grading. The RCM may be the useful tool to reduce the need for skin biopsy for monitoring of the skin contain- ing vascularized composite allograft. V C 2015 Wiley Periodicals, Inc. Microsurgery 36:144–151, 2016. Vascularized composite allotransplantation (VCA) is a new technique used for the reconstruction of large defects that cannot be reconstructed with conventional techniques. Hand and face transplantations are clinical examples of VCA procedures and are performed success- fully although some authors still accept that it’s experi- mental in human. 1,2 To prevent rejection, chronic immunosuppression therapy is given to all recipients. However, acute rejection episodes are almost a routine in these patients, even under immunosuppression therapy. To date, reversible acute rejection episodes have been reported in all clinical VCA patients. Thus, early detec- tion of acute rejection episodes and application of sal- vage procedures are of the utmost importance in these cases. 3,4 Skin is known to be the most antigenic tissue of the body, 5 and skin biopsies have been used for the follow- up of rejection episodes. However, skin biopsy is an invasive, and time-consuming method for both the patient and physician. Reflectance confocal microscopy (RCM) is a new technique that has been used very successfully to image the skin. For 10 years, RCM has been used clinically for the follow-up of pigmented lesions, nevi, and various dermatoses. RCM is a real-time, noninvasive technique, and does not require any processing. The images are evaluated by the physician during the imaging. 6 The aim of this study is to use RCM as a new, non- invasive technique for the follow-up of acute rejection episodes of skin containing VCA, and to define the cor- relation between this new technique and the standard his- topathologic grading of acute rejection on rats. MATERIALS AND METHODS Following approval of the local ethical committee, a total of five groin flap allotransplantation were performed between 10 male Sprague-Dawley rats (five donors and five recipients). All surgical procedures were performed under general anesthesia and sterile conditions. Ketamine (Ketalar V R , Pfizer, United Kingdom) was used for general anesthesia and a surgical microscope (Zeiss OP-MI 6 SD; Carl Zeiss, Goettingen, Germany) was also used. 1 Department of Plastic and Reconstructive Surgery, Gulhane Military Medi- cal Academy, Ankara, Turkey 2 Gulhane Military Medical Academy, Haydarpasa Training Hospital, Depart- ment of Plastic and Reconstructive Surgery, Istanbul, Turkey 3 Department of Orthopaedic Surgery, University of Illinois, College of Medi- cine, Chicago, IL 4 Department of Dermatology, Istanbul Training and Research Hospital, Istanbul, Turkey 5 Department of Pathology, Gulhane Military Medical Academy, Ankara, Turkey 6 Department of Biostatistics and FAVOR Laboratories, Gulhane Military Medical Academy, Ankara, Turkey *Correspondence to: Fatih Zor, M.D., Gulhane Askeri Tip Akademisi, Tip Fakultesi, Plastik ve Rekonstruktif Cerrahi AD, Etlik 06018, Ankara, Turkey. E-mail: fatihzor@yahoo.com Received 5 September 2014; Revision accepted 26 March 2015; Accepted 30 March 2015 Published online 11 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/micr.22419 Ó 2015 Wiley Periodicals, Inc.