The pathologist assessing each of these samples assigned, in a blinded fashion, a grade of histologic rejection to the sample, based on the Banff grading system with attention to the degree of inflammation present. To score inflammation, the number of dermal lymphocytic perivascular cuffs was averaged over at least three 20× fields. Perivascular cuffs were defined as circumferential inflammatory cells immediately surrounding a blood vessel. If perivascular cuffs were present in the sample, the cuff thickness was estimated based on the number of lymphocytes from the blood vessel to the outer edge, for which the number was also averaged over at least three 20× fields. As perivascular cuffs are often not completely symmetrical in nature, the thickest portion of the cuff was used to define the degree of inflammation present (Table 5). Samples were then given an overall inflammation score, based on the following criteria: “none” = no perivascular cuffs; “minimal” = <5 cuffs, no more than 2 cells thick; “mild” = <5 cuffs, 3 cells thick or more; “moderate” = 5 to 15 cuffs, any thickness; “severe” = >15 cuffs, any thickness (Figure 2). Swine-specific histologic findings were correlated with the level of clinical rejection in a revised histological grading system.
Of the graft skin sections evaluated (n = 126), 15 were given Grade 0, 59 given Grade 1, 28 given Grade 2, 14 given Grade 3, and 10 given Grade 4 (Table 4). Along with inflammation, epidermal inflammatory cell infiltration and keratinocyte necrosis were recorded for each sample. With review of the samples, it was noted that not all specimens fit into the grading system outlined by the Banff criteria. Specifically, there were samples with significant inflammation but without epidermal infiltrates, and conversely, there were samples without significant inflammation but that did have epidermal infiltration. These characteristics were considered and stratified into subcategories within Grade 2 and Grade 3 of the proposed criteria. After full analysis of all of the samples, swine-specific trends and particular cellular characteristics were compiled to construct a new grading system for the skin component in swine VCA (Table 2).
Rejection Grade 0 consists of normal dermal and epidermal skin without evidence of inflammation. In swine (as well as human) skin, there are always a small amount of perivascular lymphocytic infiltrates present in normal skin biopsies, which must be accounted for in giving rejection grades to allografts.21 However, in Grade 0 rejection (Figure 3A), no epidermal changes are seen. Grade 1 (Figure 3B) also does not have epidermal changes; however, there is a mild perivascular lymphocytic infiltrate present, increased from the sparse lymphocytic infiltrate seen in normal porcine skin histology.
As previously mentioned, we have stratified Grade 2 rejection into two subcategories: Grade 2A (Figure 3C) and 2B (Figure 3D). This subdividing accounts for specimens that contain paucicellular perivascular inflammation but do have some epidermal infiltration without keratinocyte necrosis. Grade 2A is defined as moderate perivascular infiltrate based on cuff characteristics (Table 5) without epidermal involvement. The defining characteristic of Grade 2B rejection is the presence of epidermal inflammation; although there is often perivascular dermal lymphocytic inflammation, it can range from very few lymphocytes to moderate lymphocytic cuffing and accounts for up to but not necessarily moderate perivascular inflammatory cell presence with the aforementioned epidermal infiltration of inflammatory cells. Similarly, Grade 3 has been partitioned into 3A (Figure 3E) and 3B (Figure 3F). Rejection Grade 3A is characterized by moderate or severe inflammation with multifocal single cell epidermal necrosis. Grade 3B is characterized by variable dermal inflammation (up to severe) with multifocal, full-thickness, epidermal necrosis. Although both 3A and 3B feature epidermal necrosis, the key difference between the grades is that 3A has only single cell keratinocyte necrosis that does not affect the entire thickness of the epidermis (Figure 3E), while 3B has larger, multifocal areas of necrosis that involves the entire thickness of the epidermis, resulting in large areas of ulceration. However, in Grade 3B there are still areas of intact epidermis, while in Grade 4 the rejection is defined by diffuse, full-thickness, epidermal necrosis affecting the entire site.
For internal validation, all samples were also scored by a trained second independent, blinded party (M.G.), using the proposed porcine VCA skin rejection grades. A statistical analysis was performed to evaluate the discordance between the histological and clinical assessments of each sample. Given the subjectivity and lack of accepted standardization in grading of clinical rejection, association was evaluated in a dichotomous fashion using low-grade rejection, defined as Grades 0, 1, and 2, and high-grade rejection, defined as Grade 3 or Grade 4. A McNemar test was used to evaluate relationship between the low- and high-grade histologic and clinical rejection scores for each sample. Because the paired nominal data had few discordant pairs, a mid-p McNemar test was used.22 The analysis resulted in a P value of 0.3, showing no evidence of discordance between the histologic and clinical grading systems.
Through the review of all specimens, graft capillary thrombosis was not appreciated. However, occasional occurrences of graft arteriopathy was noted, which were retrospectively found to be more frequent in those grafts that had been allowed multiple episodes of rejection (Figure S1, SDC, http://links.lww.com/TP/B713).
Experimental studies using swine models have been a staple in the preclinical study of VCA, due in part to the similarities between swine and human skin as well as the ease in operating on and assessing progress in this particular large animal model.9 The Banff 2007 Working Classification for Vascularized Composite Tissue Allografts provided the first unified criteria for the grading of skin rejection in VCA in humans.7 This classification greatly improved our ability as a field to compare and learn from other patients in this relatively rare procedure as well as to provide an objective measure to follow individual graft progression, assisting in both graft monitoring and titration of immunosuppressive treatment. However, while these criteria were also considered to be fairly applicable to the experimental swine models, as the skin is largely similar, there has not been an in-depth analysis of grading criteria as they pertain to histologic findings in swine skin. Given the importance of an accurate comparative model, we created these swine-specific grading criteria for skin rejection.
By retrospectively studying rejection in a large number of VCA transplants in MGH minipigs, we have proposed new, more refined rejection criteria specific to the MGH minipig, based on the original Banff criteria. Although we have highlighted many aspects of the striking similarity of pig skin anatomy and healing compared with that of human skin,10-18 pigs are different than humans both in their behavior and in some aspects of their inflammatory response. Pigs are more likely to traumatize skin posttransplantation, so small superficial pustules are not uncommon incidental findings. Anecdotally, pigs also have a more heavily eosinophilic component to their granulocytic inflammatory response compared to humans. However, the features of skin rejection, namely lymphocytic perivascular dermal inflammation and epidermal inflammation and necrosis, are strikingly similar.
The Banff 2007 Classification of skin rejection in VCA stratify the rejection grades by amount of inflammatory infiltrate present. Specifically, Grades 0 to 4 histologic rejection are defined in part by no or rare inflammation, mild inflammation, moderate inflammation, severe inflammation, and necrosis, respectively.7 In our proposed new grading criteria, we have subdivided rejection Grades 2 and 3 into 2A/2B and 3A/3B. Within Grade 2 rejection, we have distinguished between rejection characterized by moderate dermal inflammation without epidermal involvement and rejection characterized by variable dermal inflammation but inflammatory cell infiltration of the epidermis. This delineation is important, as in our experience, epidermal cellular involvement tends to correlate better with worse clinical rejection when compared to strictly dermal perivascular inflammation despite paucicellular inflammation that may not correlate with the moderately cellular inflammation assigned to Grade 2. For Grade 3, we have distinguished between epidermal necrosis that is single cell (3A) versus numerous (3B), accounting for the possibility of multifocal epidermal necrosis with different levels of inflammatory infiltration. Where the Banff 2007 Criteria defines Grade 3 histologic rejection by dense inflammation with epidermal involvement, we have noted in swine rejection that fairly severe epidermal necrosis may be associated with relatively few inflammatory cells in the dermis, and yet still quickly progress to Grade 4 rejection. This necessitated a subdivision of Grade 3 rejection that included more severe epidermal necrosis with or without large perivascular lymphocytic cuffs in the dermis. With this new definition of Grade 2 and 3 rejection in VCA skin in a swine model, we can accurately place histopathological grades by biopsy including the details that might otherwise have assigned other grades to these specimens.
Using our revised, swine-specific rejection criteria, we have drawn several conclusions from acutely rejecting animals both in general pattern and in specific details. Although granulocytes (neutrophils and eosinophils) as well as macrophages were present in rejecting skin samples, the vast majority of infiltrating inflammatory cells were lymphocytes. Of these, most were T cells, with fewer B cells (Figure S2, SDC, http://links.lww.com/TP/B713), consistent with previous findings.5,23,24 While overall inflammation is a major component of our modified grading criteria, the most clinically relevant factor seems to be the extent of inflammatory infiltration into the epidermis. Similar to human VCA rejection, our group found that swine grafts could be rescued up to but not including Grade 4 histopathologic rejection, which is characterized by diffuse epidermal necrosis.7 Notably, even those that had histologic Grade 3B rejection—with multifocal epidermal necrosis—were able to be rescued using standard immunosuppressive treatment (steroid bolus treatment and calcineurin inhibitor) due to the ability of the graft to reepithelialize. We also found that dermal inflammation could be quite significant, but if the epidermis was not involved, the clinical appearance was much less severe with a relatively low clinical rejection score (Grade 2 or lower) (Figures 1 and 3). We also did not include inflammation in the subcutis in the rejection scoring system, as subcuticular inflammation does not reflect the appearance or behavior of the graft; clinically important inflammation is restricted to the dermis and epidermis. Neutrophilic inflammation was significantly correlated with Grade 4 rejection (Figures 2 and 3). However, neutrophilic dermatitis is not considered specific to the pathogenesis of rejection; rather, neutrophils are a generic response to tissue damage (in this case, epidermal necrosis).25,26
Through this extensive review of pathologic specimens, it became increasingly evident that the accurate assessment of rejection and grading relies not only on a good grading system but also on the technical aspects of obtaining, preserving, and staining the biopsy as well. When evaluating graft rejection, it is important to interpret the histologic appearance in the context of the gross appearance. Ideally, multiple biopsies should be obtained from multiple sites. Significant differences in histologic appearance can occur within the same graft even millimeters apart. The clinical rejection of an experimental graft should not necessarily be predicted based on one punch biopsy taken from a focal area of epidermal necrosis, as reepithelialization of the necrotic area may occur if the rest of the graft survives and the necrotic area is small. Specimen preparation is also of importance, as the maintenance of tissue architecture is relevant to enable slide staining and get high quality, consistent specimens to evaluate pathologically. Though we did have some excluded samples for which the biopsy and/or fixing or embedding provided slides with insufficient tissue to adequately assess, the rest of our samples were uniform enough that they could be adequately compared. However, in our experience, 24 hours of formalin fixation followed by placing the sample in ethanol before paraffin embedding provided the optimal preparation.
As mentioned previously, it can be difficult to ensure that these grafts remain without scratching or traumatic injury, as this can cause inflammation unrelated to rejection that can confound histologic appearance. The grafts are insensate, so preventing the animal from injuring the graft requires diligence and attention. For this purpose, our included animals were all maintained in single-animal runs after transplantation to avoid graft damage from another pig. The cohort was also housed in specialized runs with protective polyethylene paneling that provides smooth walls to the enclosure. The animals are seen at least once a day to assess graft condition. This prevents the majority of animal scratching of the graft in our studies and largely mitigates the concern for inflammation unrelated to rejection. Furthermore, we do not currently have complete knowledge on the effect of the experimental treatment regimens on the skin and histological outcomes. Most of the animals received tacrolimus therapy either for a set time period or in pulsed dosing, though a few had costimulation blockade or cellular therapy. None of the regimens correlated with any particular rejection grade, but as our study evaluated skin samples only in the context of whether or not they were rejecting and independent of the individual treatment regimens, we cannot exclude confounding of the different experimental treatments on the skin rejection grade.
The importance of this proposed grading system lies in its implications for future studies. As the histologic grading system shows correlation to the clinical grading system, it can be used in the setting of an acute rejection episode to delineate the severity of the episode, often not homogenous throughout the graft. However, with accurate grading of acute rejection episodes, we can also evaluate the relationship between acute rejection clinical appearance, the grade of acute rejection, and the development of chronic rejection changes and other long-term outcomes. Graft arteriopathy was noted in several specimens, particularly in those that experienced multiple episodes of acute rejection (Figure S1, SDC, http://links.lww.com/TP/B713), possibly representing chronic changes. It has been shown that increased number of acute rejection episodes is associated with increased risk of chronic rejection27,28; while this has not been studied in depth in translational models, an accepted and reproducible grading system for the acute episodes will prove important in a thorough investigation into this topic.
In the current era of rapid medical and surgical advancements, adequate preclinical models are crucial to continued medical research and patient safety. Because of the limited patient population in the relatively young field of VCA, preclinical models are even more critical to our understanding of the relevant immunomodulatory processes and our discovery of less toxic and more effective treatment regimens. These new criteria defined here for histologic grading of skin rejection in swine—with the grading criteria paralleling those of the Banff Classification—provide a uniformity in histopathological assessment and contribute to the ability to analyze findings in swine preclinical models in the evaluation of VCA rejection.
We acknowledge the contributions and assistance in the large animal operations of the entire Johns Hopkins VCA Laboratory team as well as our veterinarian colleagues—specifically Dr. Jessica Izzi and Dr. Caroline Garrett—and the veterinary technicians Melanie Adams, Kristy Koenig, and Gabriella Dancourt. We acknowledge the support of the Army, Navy NIH, Air Force, VA and Health Affairs regarding the AFIRM II effort under award CTA05: W81XWH-13-2. The U.S. Army Medical Research Acquisition Activity, 820 Chandler Street, Fort Detrick MD 21702–5014 is the awarding and administering acquisition office. Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the Department of Defense. In addition, we acknowledge support of the FY12 Restorative Transplantation Research (RTR) program under award W81XWH-13-2-0060 MR120034P10.
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