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From Clinical to Pathological: The Evolution of Diagnostics in Antibody Mediated Rejection

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Jennifer Conway, MD
Stollery Children's Hospital
Edmonton, AB, Canada

Antibody mediated rejection (AMR) has mystified the transplant community for many years and continues to generate discussion and controversy. AMR occurs when an antibody targets donor endothelium, which sets the stage for an immunological assault by activation of complement and other mechanisms. While this entity was first described in the 1980s, the transplant community continues to refine and develop a universal definition of AMR. Much of this work has been done through the International Society of Heart and Lung Transplant (ISHLT), with the first cardiac working group formulating a definition in 1990 with further development and expansion in 2004. The 2004 definition focused on pathological findings and graft dysfunction in the presence of a donor specific antibody (DSA). Over the subsequent years the definition and focus has moved away from clinical sequelae towards a purely pathological diagnosis where graft dysfunction and DSA are no longer part of the definition. This last modification published in 2013 combines both the histological and immunopathological (IP) features seen on biopsy to define pathological AMR (pAMR). This approach to AMR diagnosis is reflective of the criteria set forth for cellular rejection with no requirement for clinical symptoms or additional diagnostics. The important histological features include: capillary injury with swollen endothelial cells, intravascular capillary macrophages, interstitial edema and hemorrhage. The (IP) features rely on either immunohistochemistry staining on paraffin embedded samples or immunofluorosence staining of frozen samples to identify markers of complement deposition and the presence of intravascular macrophages. Complement deposition is predominantly detected by C4d staining with a positive stain defined as >50% of the capillaries staining positive. Macrophages located within the capillary beds are identified by CD68 staining and a positive stain occurs when >10% of the capillaries are involved.

A number of pitfalls may occur during the interpretation of the biopsy samples making the diagnosis of AMR difficult. Early in the post-transplant period some of the histological and immunological findings may be present secondary to ischemic damage existing at the time of transplant or secondary to post-operative management. In addition, while C4d staining may be considered the hallmark of AMR, false positive results can occur after treatment with intravenous immunoglobulins, rituximab or reflecting a non-specific inflammatory reaction and therefore the biopsy may be uninterpretable. These examples highlight the importance of a strong pathology team, good internal positive and negative controls and excellent communication between the clinical and pathological team in order to guide the diagnosis of pAMR.

Since the new guidelines for AMR diagnosis there are now five different categories that a biopsy sample can be labeled with including pAMR 0 (negative for histological and IP studies), pAMR1(h) (histological findings present/IP negative), pAMR1 (I) (IP positive-either C4d or CD68 and histology negative), pAMR2 (histology and IP positive) and pAMR3 (severe pAMR with advanced histological features and positive IP). In pediatrics it is unclear what the relationship is between these various pathological categories and clinical outcomes. To date, and association between pAMR 3 and worse cardiovascular outcomes in children has been identified but unlike in adults the significance of subclinical AMR remains unclear.

These categories, while helpful in further categorizing the pathological features of AMR, leave a number of questions for those approaching a patient with a new biopsy finding of pAMR. What are the future implications of asymptomatic AMR? Which categories of pAMR require treatment? Which categories of pAMR affect long-term outcomes? What treatment is indicated for which pAMR category? Do the findings in the adult literature parallel what we would expect in the pediatric patients? All of is further complicated by the evolving field of antibody detection with its own complexities including the use of various thresholds to define significant donor specific antibodies, various detection systems, the unclear role of non-HLA antibodies and reporting of newer HLA antibodies, such as DQ. How to integrate the biopsy findings with the antibody results has become somewhat of an art with room for further scientific guidance.

While there have been a number of adult papers that have started to address some of these questions there is little literature in pediatrics to provide direction. However, this new diagnostic standard for AMR provides a starting point for collaborative efforts to tackle these important questions in pediatrics and shed light on this complex topic. ■

Disclosure Statement: The author has no conflicts of interest to disclose.

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