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Composite Tissue Allotransplantation: Classification of Clinical Acute Skin Rejection

Cendales, Linda C.1,6; Kirk, Allan D.2; Moresi, J Margaret3; Ruiz, Phillip4; Kleiner, David E.5

doi: 10.1097/
Original Articles: Clinical Transplantation

Background. Composite tissue allotransplantation (CTA) is a recently introduced option for limb replacement and reconstruction of other nonreconstructible tissue defects. As with recipients of other allotransplants, CTA recipients can experience rejection episodes that are presumed to be mediated by immune mechanisms similar to those affecting solid organ grafts. However, a systematic examination of this process has not been performed, and there are no standardized criteria for the description of severity or type of rejection

Methods. We collected biopsies from human limb allografts and abdominal walls in various stages of rejection for histological and immunohistochemical analysis to formulate a CTA rejection scheme. Biopsies were ranked by severity and reproducibility of the system was tested using a second set of biopsies. Tissue slides were examined blindly by three pathologists and the nonparametric Kendall coefficient of concordance (W) was used to assess the amount of agreement among the pathologists in their classification grades.

Results. Rejection initially appeared as a perivascular infiltrate progressing to involve the dermis. Arteritis was observed only in the medium to large size arteries of the subcutis. Myositis was seen occasionally. Perineural involvement without frank neuritis was present in advanced rejection. The infiltrate was predominantly CD4+ in milder cases and CD8+ in advanced cases. HLA-DR was minimally expressed in keratinocytes even in severe rejection. Kendall's W was 0.9375 (p≤0.0001).

Conclusions. Based on this survey, we proposed an initial classification system for acute rejection in the skin of a CTA and demonstrated that this system is easily reproduced by independent pathologists.

1 Orthopedic Section, Office of the Clinical Director, Intramural Research Program, National Institute of Arthritis and Musculoskeletal and Skin Disease, National Institutes of Health, Department of Health and Human Resources, Bethesda, MD.

2 Transplantation Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Resources, Bethesda, MD.

3 Department of Dermatology, Johns Hopkins Medical Institutions, Baltimore, MD.

4 Department of Pathology, University of Miami, Miami, FL.

5 Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Department of Health and Human Resources, Bethesda, MD.

This study was funded by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases and the Division of Intramural Research of the National Institute of Diabetes, Digestive and Kidney Diseases, United States National Institutes of Health.

6 Address correspondence to: Linda C. Cendales, M.D., Center Drive 10, MSC 1102, CRC Room 1-5140, Bethesda, MD 20892-1102. E-mail:

Received 27 December 2004. Revision requested 19 March 2005.

Accepted 8 July 2005.

Composite tissue allotransplantation (CTA) is a relatively new discipline introduced for the treatment for functionally significant tissue or limb defects. It differs from other vascularized allografts in the diversity of tissues comprising the graft. In the past 5 years 24 hand transplants and 8 abdominal walls have been reported (1–2). Most CTA recipients have experienced reversible episodes of acute rejection (2–5), but given the infrequent application of CTA, no single group has accumulated sufficient experience to determine histological patterns of rejection. As such, a grading scheme for ranking the pathological severity of rejection, specifically one based on the most accessible component of the graft, the skin, has not been established. Moreover, although CTA rejection has been presumed to be mediated by mechanisms similar to solid organ transplantation, it has not been determined whether CTA tissues differ in their propensity to attract an allospecific infiltrate, or if some elements are spared rejection.

We therefore collected samples from international centers in which clinical hand or abdominal wall CTA has been performed. We evaluated the distribution, intensity and phenotype of the inflammatory infiltrate of the skin at different stages of clinical rejection, documented which tissues were involved, and when possible, made retrospective clinical correlations of the histological findings. Based on this initial survey we proposed a classification system for grading acute rejection based on the skin of composite tissue allograft, and demonstrated that this system was reproducible by independent pathologists. We suggest that this system serve as a foundation for subsequent standardized reporting of rejection in the field, and recommend that a repository be established to facilitate the systematic analysis of CTA rejection and the response of rejection to immunosuppressive therapies.

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We collected 29 specimens from transplanted human limbs and abdominal walls previously procured at various time points after transplantation (1 month-3 years), and at various stages of clinical rejection (mild to severe, requiring amputation). The contributing physicians obtained the specimens per their institutional protocols and also contributed a description of the gross clinical signs of rejection. Immunosuppression varied widely based on institutional protocols and was not correlated with the time of biopsy or the rejection scores from this assessment. For the human limb specimens, we received nine formalin fixed full thickness skin biopsies containing subcutaneous tissue, one specimen in slides after hematoxylin-eosin (H&E) staining, and one as a block including skin, nerve, tendon, bone and muscle. Formalin-fixed specimens were used for H&E staining and immunohistochemical studies performed using antibodies directed against CD3, CD8, CD20, CD68, HLA-DR, CD21 (Dako, Carpenteria, CA), and CD4 (Novocastra). The specimens from abdominal walls were 18 full thickness skin biopsies from seven patients obtained at various times posttransplant that were fixed in formalin, paraffin-embedded and stained with H&E. To generate criteria for rejections, 11 biopsies were ranked by severity based on the intensity of the overall infiltration, and the degree of involvement of adnexa and the epidermis. Muscle and nerve were available in two specimens and were examined, but were not used in the severity ranking. Histological ranking was retrospectively correlated with the reported severity of the rejection episode. Reproducibility of the system was tested using a second set of 18 biopsies. Tissue slides were examined blindly by three pathologists: two surgical pathologists with experience in transplantation and one dermopathologist. The pathologists categorized the specimens according to the newly defined system. The nonparametric Kendall coefficient of concordance (W) was used to assess the amount of agreement among the three pathologists in their classification grades (6).

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Rejections in the limb transplant cases were characterized by the contributing clinical investigators as patchy or generalized erythematous rash localized exclusively to the allograft that ranged from a mild rash to, in one case, erythematous-scaly papules, superficial erosion and necrosis requiring revision amputation of the limb due to rejection (5). Abdominal wall cases had clinical findings that ranged from unremarkable findings to erythema limited to the allograft. In one case the allograft was lost due to thrombosis. All specimens demonstrated at least some degree of perivascular infiltrate suggesting that this is one of the earliest findings in rejection. This infiltrate continued with involvement of the dermis and epidermis in apparent proportion to the clinical degree of rejection as assessed by contributing clinicians. Adnexal structures, particularly the epithelial sheathes of hair follicles, were involved with increasing severity through the series. Confluent necrosis of the epidermis or other structures was not seen except in the most severe case. Based on the blinded ranking of 11 specimens from limb transplants and the apparent progression of involvement of dermal vessels, adnexal structures and epidermis, we defined a simple classification scheme (Figs. 1 and 2).





Although the majority of specimens examined were punch biopsies of skin, a small number of specimens were obtained that showed the deeper structures. Two muscle samples were evaluated and myositis was seen in both. However, since muscle was not obtained in all cases, it was not clear how the severity or the tempo of myositis related to the dermal changes or clinical appearance of rejection. Arteritis of medium and large arteries of the subcutis was observed only in the most severe lesions, and was associated with vascular thrombosis (Fig. 3). Because the majority of cases examined did not sample arteries of the subcutis, it is not possible to completely exclude arteritis from lesser degrees of rejection. However, the consequences of arteritis, namely infarction, were not seen except in the most severe case. Although perineural inflammation was seen, possibly due to involvement of the perineural vascular plexus, frank neuritis was not seen even in the most severe cases of rejection. Thus, most of the tissue elements other than nerve appeared susceptible to a cellular immune response.



Immunohistochemical staining showed that the infiltrate in histologically mild cases was predominantly CD4+ T-cells, and was extensive in some areas (Fig. 2). CD8+ cells appeared in greater numbers as the severity of the infiltrate worsened. CD20 stains were only available on the most severe case of rejection. In this case, large aggregates of B cells were found in the dermis and subcutaneum and scattered individually within the band of subepidermal inflammation. The lymphoid aggregates of this case also contained dendritic cells as demonstrated by CD21 staining. Essentially no dendritic cells were seen in biopsies from lesser degrees of rejection. Macrophages were scattered within the inflammatory infiltrates at all degrees of inflammation. Interestingly, HLA-DR was only minimally expressed in keratinocytes even in severe rejection. This is specifically unlike T-cell mediated graft versus host disease which induces vigorous up-regulation of HLA-DR in the epidermis (7). Eosinophils were few in number but were noted in most of the biopsies. Neutrophils were only seen in the most severe case, and then only associated with areas of epidermal necrosis. Thus, CTA rejection appeared as a perivascular and predominantly CD4+ T-cell infiltrate coincident with the appearance of a visible erythematous rash. As rejection progressed clinically, the intensity and distribution of the infiltrate progressed and became increasingly characterized by CD8+ cells. All elements of the graft, with the possible exception of neurons, were involved in the infiltrate.

To test the reproducibility of the system, three pathologists from different institutions classified 18 cases from abdominal wall transplants not used in the original definition of the system. Absolute agreement varied between 13 of 18 cases to 17 of 18 cases. To evaluate the degree of agreement, we used the nonparametric Kendall's coefficient of concordance (W), which is a very robust measure of agreement that can take on values between 0 and 1 (i.e. no agreement to complete agreement). Kendall's W can both be used to statistically test whether there is significant agreement and to provide an easily interpreted nonparametric Spearman correlation among pairs of pathologists. For these data, the computed value of Kendall's W (corrected for ties) was 0.9375 and the P value for testing whether this represents and amount of agreement greater than expected by chance was highly significant (P≤0.0001). The corresponding average Spearman correlation among pairs of raters was 0.906. Since this is on the standard scale for correlation, the observed value shows very high agreement among the three independent pathologists.

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Composite tissue allografts are complex tissue units composed of skin, connective tissues, nerves, bone marrow, and vascular tissue serving a single function. This embryological germ layer diversity (endoderm, mesoderm and ectoderm) distinguishes CTAs from other vascularized allografts. Rejection of human limbs and abdominal walls often presents as generalized swelling and rash (2–3) and/or asymptomatic, erythematous scaly papules (5) limited to the allograft. Treatment involves systemic or topical steroids, increased baseline immunosuppression, or T-cell depletion (4, 5). Terminal rejection with necrosis mandates graft removal (5). Although CTA rejection is presumed to be mediated by mechanisms similar to solid organ transplantation, the pathologic changes in biopsy tissue have not been systematically described. In this manuscript we have demonstrated the salient histological and immunohistochemical features and progressive nature of CTA rejection from two different clinical graft sources (hand and abdominal wall). We have established a system of histopathological description and have shown this to be useful for communicating the status of a graft between independent pathologists.

Allografts that contain skin are unique in that rejection can be appreciated visually. Indeed we have shown that significant perivascular infiltration appears coincident with a skin rash. However, given the heterogeneity of composite tissue allografts, and the potential that repeated inflammation could manifest itself as chronic dysfunction, it is important to begin cataloging the histology of CTA rejection in terms more objective than visual inspection. Although we agree that visualization of skin changes can be used as a clinical indicator of rejection, the sensitivity and specificity of rash as a marker of rejection remains to be established, as does evidence of histological response to therapy.

To allow objective comparisons of clinical experience between centers, a grading system is necessary to classify histopathological rejection in the allografted skin (the most accessible and likely to be biopsied component). Based on our initial observations, we propose the following preliminary classification system to standardize reporting of CTA rejection (Fig. 2): Grade 0: nonspecific changes. No or only mild lymphocytic infiltration without involvement of the superficial dermal structures or epidermis. Grade 1: mild rejection. Superficial perivascular inflammation with involvement of superficial vessels and without involvement of overlying epidermis. Grade 2: moderate rejection. Features of Grade 1 with involvement of the epithelium of adnexal structures. Grade 3: severe rejection. Bandlike superficial dermal infiltrate with more continuous involvement of the epidermis and middle and deep perivascular infiltrate. Grade 4: necrotizing rejection (not shown). Includes features of grade 3 along with frank necrosis of the epidermis or other tissues.

Scoring systems have been designed in animals and humans (8, 9). Observations in the former include the appearance of visual skin and histologic signs of rejection appearing simultaneously in 3 of 10 treated swine. In two animals, visual changes preceded histologic diagnosis and in five animals, a histologic picture appeared before noticeable changes in the skin surface. Therapy was based on cyclosporine, mycophenolate mofetil, and prednisone. In contrast with humans, drug doses in this study were not adjusted based on the clinical progress of the animals, despite the occurrence of rejection.

A scoring system has been put forward in patients undergoing full-thickness cadaver skin transplant for large abdominal defects (9). These transplants included one or both rectus abdominus muscles with the investing fascia, the overlying subcutaneous tissue and skin in patients in which failure to close the abdomen was encountered at the time of visceral transplants. In contrast with limb amputees who are generally in good health and undergo hand transplantation, the patients who received abdominal wall transplants suffered intestinal failure from a life-threatening disease that adds additional complexity to the clinical picture. The scale ranges from no rejection to severe rejection with too few cases in each category studied to determine a statistical rate of concordance among the pathologists evaluating the specimens. In two occasions, the grade of the skin rejection was indeterminate. Eighteen specimens from this case series were included in this study. In our study, we have expanded upon the immunohistochemical characterization of these patients and determined which features are common between abdominal wall grafts in critically ill individuals, and limb grafts in otherwise healthy recipients. Observations from a case report of graft rejection in a human hand allograft at month 29 posttransplant suggest clinicopathologic features similar to those seen in chronic lichenoid cutaneous graft-versus-host disease (5). Larger numbers of patients will be required to determine the frequency of this long term complication of human skin transplantation.

Many questions remain with respect to classifying rejection in CTA. These include whether the involvement of different structures (adnexae, epidermis, vessels, etc.) connotate differences regarding outcome, or whether sampling induces diagnostic bias. Similarly, the impact of sampling on diagnostic bias remains undefined; it is also unclear whether the score should be based on the worst, or the predominant, lesion in the seen.

Certainly, bias will also be unavoidable in the choice of lesions biopsied, thus clinical guidelines for biopsy will need to be established in the near future. Finally, chronic changes will also need to be classified and correlated with functional impairment. Many of these questions will be better answered through animal experimentation. To that end, we have established a nonhuman primate model of CTA that includes all CTA elements (10).

Systematic and prospective examination of larger cohorts of patients must be evaluated to address these questions. As it is clear that in the foreseeable future no single center will be able to answer these vital questions, we recommend that a repository for clinico-histopathological correlation be established. The author can be contacted to receive tissues and initiate dialogue regarding the cataloguing of biopsy tissue. Reporting to international organizations with an interest in CTA will be facilitated greatly by the ability to compare results objectively, and this in turn, will advance the field in keeping with its deserved place with other accepted forms of allotransplantation.

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The authors gratefully acknowledge the selfless contributions to this study through the generous gifts of allografted tissue received from Darla Granger, Warren Breidenbach, Carolyn Burns, Carrie Marcell, Jean-Michel Dubernard, Nadey Hakim, Raimund Margreiter, Deborah Weppler, Andreas Tzakis, Frederic Schund, and Carlo Van Holder. The authors would like to thank Bob Wesley for his expert statistical support.

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Composite tissue allotransplantation; Acute rejection; Scoring system

© 2006 Lippincott Williams & Wilkins, Inc.