Controversies in defining cardiac antibody-mediated rejection: Need for updated criteria

What specimens should be used to assess cardiac AMR? 

Early studies of AMR in cardiac allografts used frozen tissue samples obtained at the time of routine endomyocardial biopsy. Such samples were considered highly desirable for this purpose in the original ISHLT grading schema because they obviate the artifacts associated with routine formalin-fixed paraffin-embedded (FFPE) tissue processing, which includes tissue fixation, dehydration and lipid extraction associated with xylene infiltration prior to paraffinization.15 Furthermore, FFPE tissue processing for immunoperoxidase staining has been shown to be less desirable for detection of complement components on lipid-containing membranes, which are the hallmark of AMR.16 More recent experience suggests that these processing issues can be surmounted by using other fixatives and different immunohistochemical methods,17, 18 but recent comparative studies have highlighted the diminished sensitivity and differences in pattern found on FFPE tissue samples (Figure 2A).19, 20, 21 Inter-rater disparity in immunohistochemical studies has also recently been reported. The inter-rater concordance rate was much better for immunofluorescence staining than for staining on FFPE tissues (kappa 0.9 vs 0.3). Current efforts defined at the recent Tenth Banff Conference on Allograft Pathology focused on establishing acceptable processing requirements for cardiac biopsies, although both sample types will continue to be used until an obvious new standard emerges. It is significant that the Banff Conference on Renal Allograft Pathology continued the emphasis on use of frozen tissue for the detection of AMR in renal biopsy specimens.22

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Figure 2. (A) False negative C4d by immunohistochemistry. Note the pale and interrupted pattern of the brown reaction product in capillaries affected by AMR (arrows). Controls for C4d by immunohistochemistry were positive. Immunofluorescent staining for C4d was strongly and diffusely positive on the same case (inset). (B) False positive C4d by immunofluorescence. In this slide, it is impossible to determine whether the pattern represents capillary and/or sarcolemmal membrane staining. The significance of the sarcolemmal staining pattern is unknown. Drying artifact involving the frozen tissue (arrow) may have contributed to this pattern. (C) Fibrin by immunofluorescence in mild AMR. Fibrin is usually not seen in acute AMR, except when it is severe or has been present for several weeks. This slide shows the fibrin pattern of rare perivascular foci in a case of mild acute AMR present for several weeks. The patient's previous AMR episode was negative for fibrin. The HLA-DR capillary staining in this case was bright and diffuse, as shown in (E). (D) Fibrin by immunofluorescence in severe AMR. Fibrin is often seen in patients with severe, long-standing AMR or in those who are not compliant with maintenance immunosuppression. The slide shows large aggregates of fibrin in areas of capillary damage. HLA-DR staining of these capillaries was paradoxically weak (F), consistent with capillary damage. (E) HLA-DR by immunofluorescence in mild AMR. Slide from a patient with mild acute AMR, positively staining for both C3d and C4d. This slide is stained with HLA-DR and highlights the bright uniform staining of upregulated capillary endothelium when AMR is present. (F) HLA-DR by immunofluorescence in severe AMR. This slide, taken from the same patient as in (D), shows the pattern of HLA-DR staining found in patients with long-standing or severe AMR. The pattern of HLA-DR staining is weak and not linear due to damaged vessel walls. Many capillaries do not show staining (arrows).

What analytes should be measured? 

Original definitions of AMR involved finding the colocalization of immunoglobulin (IgG or IgM) and complement components (C3d, C1q and then C4d). From the early studies, it became obvious that interstitial staining with immunoglobulin reagents complicated this definition. Many investigators have rejected the value of staining for immunoglobulin as part of this definition. However, some reports, particularly where accommodation is being investigated, retain immunoglobulin staining to clarify alternative potential reasons for complement staining.23, 24 Immunoglobulin detection may also be helpful in situations of AMR where complement is not bound by antibodies involved.25

Recently, it has been reported that C4d staining alone is sufficient evidence of AMR if it is detected in a generalized and smooth distribution in all capillaries.26, 27 Others have disputed this claim and urge evaluation of both C3d and C4d to assure that the staining is related to AMR.28, 29, 30 At the recent Banff meeting, it was determined that the optimal staining by immunofluorescence should at least include both C3d and C4d. Non-immunologic causes of C4d expression have been reported, including viral infections, reperfusion injury and treatment with monoclonal antibodies and anti-thymocyte preparations (Figure 2B).31, 32, 33, 34, 35

The value of other analytes is still controversial. Based on experimental evidence in animal allografts, we and others have also urged evaluation of HLA-DR, which helps to define the location of capillaries by immunofluorescence and serves as further evidence of endothelial activation and/or capillary damage.8, 36, 37 The role of fibrin detection has also been disputed, although there have been numerous reports of the adverse impact of fibrin accumulation in endomyocardial biopsies, indicating the hypercoagulability of the vascular endothelium in such situations.38, 39 There are also recent reports detailing the interaction of platelets and endothelial cells in AMR in response to HLA alloantibody.40, 41 In the future, it may well be that other analytes, including regulator proteins and chemokines, will also need to be assessed to fully characterize this process in all its forms.30, 41, 42

The distribution and intensity of staining of these reactants have not been standardized in a grading scheme. There is a precedent in the renal transplant literature to consider only diffuse staining as significant,20, 21 but a recent report has challenged this assumption in a careful study using outcome as an endpoint.43

Donor-specific antibodies: Diagnostic criterion or risk factor? 

Pre-sensitization to HLA Class I and/or Class II antibodies from various causes predisposes heart transplant recipients to higher rates of AMR and worse outcomes.44 New advances in solid-phase assays have allowed a more accurate and discriminate appraisal of preformed antibodies. As a result, sensitized patients awaiting suitable heart donors can now be better risk-stratified and screened by virtual cross-match. Similarly, the appearance of post-transplantation de novo antibodies, especially when donor-specific, increases the risk of AMR, allograft dysfunction and coronary allograft vasculopathy.9, 10, 11, 12 Yet, cardiac AMR has been diagnosed in the absence of anti-HLA antibodies, albeit much less commonly. Some of the clinically relevant non-HLA antibodies that have been incriminated in allograft rejection and/or bad outcomes comprise autoantibodies directed against cardiac proteins and vimentin or against antigens expressed on donor endothelial cells.45, 46 It is therefore still controversial whether to consider the detection of donor-specific antibodies in recipients as an early indicator of an immune response heralding AMR, or simply as a risk factor among others.

The progress in methodology in the field also brings to light some new unresolved issues in need of further exploration and consideration when formulating future diagnostic guidelines for cardiac AMR. These include: deciding which antibodies from a growing list are clinically pertinent for monitoring; understanding how their specificity and titer may influence whether they are protective or detrimental to the cardiac allograft; and elucidating mechanisms behind the apparent resistance of some allografts (accommodation) to humoral injury.47

What is the incidence/prevalence of cardiac AMR? 

The true incidence of cardiac AMR is poorly characterized for a number of reasons. Screening for AMR in asymptomatic patients is not standard practice, especially since most transplant programs only define it when clinically manifest. It is therefore likely that cardiac AMR is underreported, that is if we accept the premise that AMR can be subclinical. If AMR exists as a subclinical entity as has been reported, it can present as acute self-limited or repetitive episodes or as chronic low-grade, ultimately leading to cardiac allograft vasculopathy or dysfunction and heart failure. Last, the incidence of AMR is not constant and varies decrementally over time after transplantation.

What has been reported to date in much of the literature is perhaps best described as prevalence of symptomatic AMR. The prevalence of lone symptomatic AMR ranges between 10% and 15%, whereas the prevalence of AMR when routinely being surveilled (symptomatic and asymptomatic) or when diagnosed concurrently with acute cellular rejection (mixed rejection) can surpass 40%.48, 49, 50, 51 This wide range is also the result of the lack of prior standardized diagnostic criteria as well as the diversity of the populations studied in their predisposition to AMR.

Are histologic criteria alone adequate to screen for cardiac AMR? 

Screening criteria for AMR currently include the histologic findings of capillary endothelial swelling and macrophage activation and the presence of allograft dysfunction. If identified, confirming the diagnosis is carried on by immunofluorescence or immunoperoxidase staining. Therefore, our ability to diagnose or exclude cardiac AMR effectively rests on the assumption that histologic parameters alone have adequate specificity and sensitivity. In a large study of biopsy specimens with confirmed AMR by immunofluorescence, histologic changes were found to have acceptable specificity to rule out AMR. On the other hand, the sensitivities of capillary endothelial swelling (63%) and macrophage vascular adherence (30%) were disappointingly low.52 In further analyzing the data, a receiver operating characteristic (ROC) curve was plotted combining various semi-quantitative scores of both histologic parameters. Even then, the area under the curve was 0.759 indicating that up to roughly 25% of cases of cardiac AMR may be missed under the present recommended screening approach. It is generally accepted that cardiac AMR becomes manifest mostly early after transplantation.3, 4, 48, 49, 51 Heart transplant recipients without AMR during that period are usually less likely to develop it later on.53 Whether to include immunohistochemical testing on all endomyocardial biopsy specimens, at least in the first few weeks after transplantation, deserves serious consideration in future AMR diagnostic guidelines.

Issues concerning the requisite allograft dysfunction to diagnose cardiac AMR are debated in what follows.

Should asymptomatic cardiac AMR be disregarded? 

The existing ISHLT guidelines questionably assume that cardiac AMR is always symptomatic or that it should not be diagnosed in the absence of allograft dysfunction. This definition is counterintuitive and conflicts with the proposed stages of AMR progression in solid-organ transplants,54, 55, 56 as well as with recently described experience relating asymptomatic AMR with worse cardiovascular mortality and higher likelihood of cardiac allograft vasculopathy.57, 58, 59 Cardiac AMR should be perceived as a pathologic continuum with potential ensuing clinical involvement. This paradigm change in our approach to cardiac AMR can be accomplished by eliminating the allograft dysfunction requisite from any future AMR diagnostic scheme. Alternatively, cardiac dysfunction or other clinical manifestations may be part of a constellation of pathologic, laboratory and clinical criteria, none of which alone are diagnostically definitive of AMR. Reconciling these diverse diagnostic elements of cardiac AMR is complex enough that it may lend itself nicely to a set of major and minor diagnostic criteria, similar to what has been done in other immunologic diseases.

Should the severity of cardiac AMR be graded? 

The most up-to-date review of the grading system for cardiac biopsies designates acute AMR as either absent (AMR 0) or present (AMR 1), without any provision of grading its severity. Yet, more agree that the extent of histologic and immunopathologic features of AMR varies between biopsies.3, 4, 51, 52 With more severe AMR, histologic changes may progress from low-grade endothelial cell activation and macrophage vascular adherence to vascular permeability and, eventually, capillary damage with ensuing interstitial pleomorphic inflammation and myocyte necrosis. Immunofluorescent immunoglobulin and complement deposits may become more or less prominent and diffuse and can be seen in the interstitium along with fibrin as vessels get injured with more severe AMR (Figure 2C and D). The integrity of capillaries is important to assess, since, with severe injury and fibrin accumulation, they may be unable to express complement components diagnostic of AMR. Staining capillaries routinely with HLA-DR, which is upregulated during rejection, would be useful in this regard.42 HLA-DR staining in severe rejection is often diminished or expressed in patches because of capillary injury (Figure 2E and F). These diverse immunohistologic features of cardiac AMR based on severity were additionally found to be clinically relevant as they were linked to an incremental risk of cardiovascular mortality in one large series.60 Even in the absence of a clinical correlate, it would make sense to devise a consensus severity scale for cardiac AMR similar to what has been done for cellular rejection.

AMR of the cardiac allograft is a complex immunologic entity with pathologic and clinical characteristics that are difficult to standardize. Yet, it was encouraging to see pathologists and clinicians gather in the presence of a delegate from the Board of Directors of the ISHLT at the recent Banff Conference to lay the basis for what should hopefully develop into much-needed updated diagnostic guidelines for cardiac AMR.