The diagnosis and grading of acute cellular and antibody-mediated rejection (AMR) in lung allograft biopsies is important because rejection can lead to acute graft dysfunction and/or failure and may contribute to chronic graft failure. While acute cellular rejection is well defined histologically, no reproducible specific features of AMR are currently identified. Therefore, a combination of clinical features, serology, histopathology, and immunologic findings is suggested for the diagnosis of AMR.
To describe the perspective of members of the Pulmonary Pathology Society (PPS) on the workup of lung allograft transbronchial biopsy and the diagnosis of acute cellular rejection and AMR in lung transplant.
Reports by the International Society for Heart and Lung Transplantation (ISHLT), experience of members of PPS who routinely review lung allograft biopsies, and search of literature database (PubMed).
Acute cellular rejection should be assessed and graded according to the 2007 working formulation of the ISHLT. As currently no specific features are known for AMR in lung allografts, the triple test (clinical allograft dysfunction, donor-specific antibodies, pathologic findings) should be used for its diagnosis. C4d staining might be performed when morphologic, clinical, and/or serologic features suggestive of AMR are identified.
Allograft rejection is a serious complication in lung transplant because of potential acute graft dysfunction or failure and/or subsequent complications that can lead to chronic graft failure. According to the latest report from the International Society for Heart and Lung Transplantation (ISHLT), 29% of adult patients have at least 1 episode of treated acute rejection between discharge from the hospital and 1-year follow-up after transplant.1 Furthermore, 3.6% and 1.8% of deaths among adult lung transplant recipients within the first 30 days or between 30 days and 1 year post transplant are attributed to “acute rejection,” respectively.1 In addition, the frequency and severity of “acute rejection” episodes are thought to represent the major risk factor for subsequent development of chronic airways rejection/bronchiolitis obliterans syndrome (BOS).2–5
Allograft rejection can be cell mediated or antibody mediated. Cell-mediated rejection is much more common. It is mediated by T lymphocytes that recognize foreign human leukocyte antigens (HLAs) or other antigens.2,6 In contrast, antibody-mediated (or humoral) rejection (AMR) occurs owing to binding of preformed or de novo recipient antibodies directed against antigens that are expressed on the donor organ cells. Acute cell-mediated rejection (ACR) and AMR can occur within days, months, or even years after transplant.
The clinical recognition of ACR and AMR can be difficult as the course might be variable, with some patients being asymptomatic or presenting with symptoms that overlap with other complications and diseases in this patient population. These symptoms might include dyspnea, fever, leukocytosis, and a widened alveolar-arterial oxygen gradient.7 Although ancillary studies together with the clinical presentation of the patient sometimes suggest the presence of ACR or AMR, none of these findings are specific. Therefore, tissue diagnosis is necessary to support a diagnosis of allograft rejection. Transbronchial biopsy to obtain lung tissue is currently the gold standard to assess patients for lung allograft rejection and to distinguish rejection from its clinical mimics such as aspiration, infection, drug toxicity, and recurrent disease. Occasionally, wedge biopsies, explanted lungs (if a patient undergoes retransplant), or autopsy specimens also become available to the pathologist. Recently, the transbronchial cryobiopsy technique became available to obtain lung tissue. Cryobiopsies are reported to yield significantly larger specimens with more alveoli, bronchioles, veins, and venules, and less procedure-related artifact when compared to traditional forceps transbronchial biopsies (Figure 1, A and B).8–10 Concern over higher complication rates in patients undergoing cryobiopsy and limited experience with cryobiopsy in the transplant setting have prevented widespread adoption of the technique in the routine workup of lung allograft recipients. However, a recent study8 did not show a significant difference in complications between cryobiopsies and transbronchial biopsies in the lung allograft setting.
REQUIREMENTS FOR TRANSBRONCHIAL BIOPSIES IN THE EVALUATION OF LUNG ALLOGRAFTS
At least 5 pieces of well-expanded alveolated parenchyma are required for adequate morphologic evaluation of a transbronchial lung allograft biopsy specimen for acute rejection.11 To ensure that these recommendations are fulfilled, the bronchoscopist may need to sample more than 5 pieces. Even more pieces might be necessary to provide biopsy samples of small airways. Specimens should be gently agitated in formalin. Although there are currently no recommendations for the use of cryobiopsies in this setting, in a recent study using cryobiopsies to evaluate rejection in lung allografts, a median of 3 pieces provided twice as many alveoli and small airways than a median of 10 pieces by conventional forceps biopsy.8
A minimum of 3 levels from the paraffin block for hematoxylin-eosin staining for histologic examination are required for examination.11 In addition, “connective tissue stains” such as Trichrome or Verhoeff-Van Gieson stains (or another elastic stain) are recommended to evaluate small airways for the presence of submucosal fibrosis in chronic airways rejection and larger vessels for chronic vascular rejection, respectively. However, given that transbronchial biopsies usually lack larger vessels, these biopsies may be insufficient to assess for chronic vascular rejection. Stains for microorganisms, including Gomori-Grocott methenamine silver stain and a stain for acid-fast bacilli, may be added. While silver staining is routinely performed on lung allograft biopsies in some institutions, they are currently not mandated by the ISHLT because many microbiologic, serologic, and molecular techniques are available and used to identify infections in these patients. Furthermore, the limited sampling implicit in these biopsies may limit the negative predictive value of such stains.11,12 Bronchoalveolar lavage may be performed at the time of biopsy and is useful for the exclusion of infection, but it currently has no clinical role in the diagnosis of acute rejection.
When evaluating a transbronchial biopsy for allograft rejection, all hematoxylin-eosin levels should be reviewed thoroughly, especially since low-grade rejection might only be seen focally, such as in 1 or 2 levels. Furthermore, a low-power view should precede examination under higher power, as most, but not all, rejection and its extent can be initially recognized under low-power microscopy.
DIAGNOSIS OF ACUTE CELLULAR REJECTION IN LUNG ALLOGRAFT BIOPSIES
Acute cellular rejection can affect both vasculature and small airways.11 It is characterized by a mononuclear cell infiltrate around small vessels and capillaries (“acute rejection”) and/or small airways (“small airways inflammation” or “lymphocytic bronchiolitis”). The ISHLT published a revision of the “working formulation for the standardization of nomenclature in the diagnosis of lung rejection” in 2007, which established the diagnostic criteria for ACR.11 This working formulation provides not only the characteristic morphologic features of ACR but also a grading scheme for both acute rejection and small airways inflammation. Grading of ACR is important as treatment and follow-up of the patient are adjusted accordingly. In general, patients with ACR of grade A2 and higher are treated with increased immunosuppression, while treatment of grade A1 rejection is variable and controversial and largely depends on whether the patient is symptomatic or asymptomatic.3,13 However, usually any ACR will prompt closer follow-up biopsy. The treatment of persistent or recurrent ACR is challenging and treatment might include a repeated course of corticosteroids, switch from cyclosporine to tacrolimus, and/or alternative immunosuppressive agents including polyclonal anti–thymocyte globulin, anti–interleukin 2 receptor antagonists, or muromonab-CD3. Evidence also suggests that alemtuzumab, an anti-CD52 monoclonal antibody, might be helpful in refractory ACR. In contrast to ACR, treatment of small airways inflammation/lymphocytic bronchiolitis is not standardized but might include inhaled steroids. Evidence also suggests that azithromycin might be useful in the treatment of small airways inflammation/lymphocytic bronchiolitis.14
Although interobserver and intraobserver variability in grading have been recognized and shown to potentially impact treatment and outcome,8,15–18 this grading scheme is the recommended tool to evaluate posttransplant lung transbronchial biopsies in a standardized fashion. Grading is based on the extent of mononuclear cell infiltrates and the presence or absence of an accompanying acute lung injury; however, clinical findings are not considered in the grading scheme. Interestingly, although cryobiopsies are larger, interobserver reproducibility did not improve with the use of cryobiopsies in a recent study.8
Given the small nature of traditional forceps transbronchial biopsies, sampling bias can occur, as changes in rejection can be quite patchy.
Grading of Acute Cellular Rejection According to the ISHLT Working Formulation
Grading of ACR is described in great detail elsewhere.11 In short, ACR is divided into acute rejection or the “A grade” (Figure 2, A through I) and small airways rejection/lymphocytic bronchiolitis or the “B grade” (Figure 3, A through D) (Table 1). Higher grades of acute rejection are commonly associated with small airways inflammation.
Acute rejection is characterized by a perivascular mononuclear cell infiltrate with or without endothelialitis.11 Most mononuclear cells in acute rejection are T cells, although a few studies have described increased populations of B cells.11,19,20 Overall, the grade of acute rejection increases as the cellular infiltrate becomes more extensive. Beginning in the perivascular stroma, the infiltrate may spread into the adjacent interalveolar septa and, subsequently, into the alveoli. ISHLT grade A0 lacks any morphologic features of acute rejection. In grade A1 rejection, occasional small blood vessels in the alveolated lung parenchyma, particularly venules, are surrounded by a thin ring (2–3 layers) of mononuclear cells. Grade A2 is characterized by more layers of lymphocytes surrounding small vessels. In addition, chronic inflammatory infiltrates are more frequent and might contain occasional eosinophils. Endothelialitis, characterized by subendothelial mononuclear cells, may be noted but is not required for a diagnosis of rejection. In grade A3 rejection, dense perivascular mononuclear cell infiltrates are commonly associated with endothelialitis, eosinophils, and even occasional neutrophils. The inflammatory cell infiltrate extends into the adjacent interalveolar septa and occasionally might extend into adjacent alveoli. Histologic features of acute lung injury may become apparent. Grade A4 is characterized by diffuse perivascular, interstitial, and air space infiltrates of mononuclear cells with prominent alveolar pneumocyte damage and endothelialitis. Paradoxical diminution of perivascular infiltrates can occur as lymphocytes extend into interalveolar septa and alveoli, where they admix with macrophages. High-grade rejection, in general, has morphologic evidence of acute lung injury including organizing pneumonia, fibrin deposition, or hyaline membranes. While grades A1 and A2 are regarded as low-grade rejection, grades A3 and A4 are viewed as high-grade rejection.
While higher-grade acute rejection can usually be readily noted on low-power view, grade A1 rejection might only be detected at higher-power analysis, especially in specimens with procedural artifacts and atelectasis.
Small airways inflammation/lymphocytic bronchiolitis or ISHLT B grade only applies to small airways such as terminal or respiratory bronchioles. Bronchi should be described separately. If no small airways are identified or the biopsy demonstrates overt evidence of infection, the grade “BX” should be used. While ISHLT grade B0 is used if no bronchiolar inflammation is identified, grade B1R (R denotes revision) is defined by lymphocytes in the submucosa of bronchioles. Grade B2R is characterized by a marked infiltrate of both the small airway mucosa and wall. Epithelial damage becomes apparent including necrosis, metaplasia, and/or marked intraepithelial lymphocytic infiltration. Epithelial ulceration, fibrinopurulent exudate, cellular debris, and neutrophilic infiltration might occur.
Mimickers of Acute Cellular Rejection
Histologic features of ACR such as perivascular inflammatory infiltrates can overlap with those of infection.21 In addition, small airways infections usually present with peribronchiolar inflammation. Moreover, ACR and infection can occur together. Therefore, the grading of ACR requires the exclusion of a concurrent infection.11 Specifically in patients with high clinical suspicion for infection, the evaluation of a biopsy for rejection should be done with great caution; in certain situations it might not even be possible to definitely ascribe histologic findings to rejection.
Abundant neutrophils, necrosis, granulomas, and viral cytopathic effect are more commonly seen in infection than in ACR. The presence of histiocytic inflammation and/or mixed chronic and acute inflammation might also favor infection or aspiration over rejection. Predominant neutrophils in the epithelium and submucosa of small airways might favor infection over rejection.22 Evidence suggests that the number of mucosal T cells is higher in small airways rejection/lymphocytic bronchiolitis than in infectious processes.23
Stains for microorganisms (eg, Gomori-Grocott methenamine silver stain, stains for viruses including cytomegalovirus, respiratory syncytial virus, and varicella zoster virus) and correlation with clinical presentation, and culture studies can be helpful and are highly recommended in this regard.
Mimickers of severe acute rejection include conditions that might present with acute lung injury. These conditions include infection, drug toxicity, aspiration, AMR, harvest/reperfusion injury, or recurrence of the primary lung disease. While perivascular chronic inflammation is helpful in the diagnosis of acute rejection, this finding is not entirely specific.
Marked perivascular and/or peribronchiolar or interstitial mononuclear infiltrates might also raise the possibility of posttransplant lymphoproliferative disease (PTLD). In cases that are suggestive of PTLD, an appropriate workup should be performed, including studies for Epstein-Barr virus. Correlation with clinical, radiologic, and culture findings is necessary to rule out or evaluate almost all potential mimics of ACR, especially given the limitations of sample size.
Bronchus-associated lymphatic tissue (BALT) occasionally can mimic ACR. BALT is found in the vicinity of airways, usually contains black anthracotic pigment, and presents as a nodular collection of chronic inflammatory cells that typically does not surround a vessel but might be seen asymmetrically around a vessel (unlike ACR). Deeper sections might be helpful to show these features. Also, epithelial injury, neutrophils, or eosinophils should not be seen in BALT collections.11
DIAGNOSIS OF ANTIBODY-MEDIATED REJECTION IN LUNG ALLOGRAFT BIOPSIES
While diagnostic features of AMR are well established in other organs such as kidney and heart, no specific features of AMR have been established in lung allografts. Nevertheless, AMR likely results in acute and chronic graft dysfunction/failure in a subset of patients.24 Circulating preformed (due to pregnancy, blood transfusions, previous organ transplant) or de novo (occurring after transplant) recipient antibodies are thought to cause AMR. Immune stimulation by prior infections or autoimmunity may also contribute to the development of antibodies in susceptible patients. About 10% to 15% of lung transplant recipients are presensitized to HLA antigens.25 More sensitive and specific assays to detect circulating antibodies suggest that the incidence of preformed anti-HLA antibodies might be higher than previously thought. These preexisting or de novo antibodies can react with antigens that are expressed on donor organ cells, leading to immediate graft loss (hyperacute rejection), accelerated AMR, and/or BOS.26 In fact, studies have consistently demonstrated an increased incidence of acute rejection,27 persistent rejection, BOS,28 or worse overall survival29 in patients with anti-HLA antibodies. Although the optimal treatment of AMR in lung is currently not known owing to difficulties in making the diagnosis and lack of clinical trials, treatment typically includes plasmapheresis, and occasionally, intravenous immunoglobulin or immunomodulatory medications such as rituximab and bortezomib, among others.
Given the potential short- and long-term complications and need for immediate treatment, the diagnosis of AMR is important in patients with lung allografts. However, no morphologic, immunologic, clinical, or radiologic findings specific to lung AMR have been established, making the diagnosis of AMR challenging and likely resulting in its underdiagnosis. The current recommendations of assessment of AMR are summarized in Table 2.
In 2013, the Pathology Council of the ISHLT recommended a multidisciplinary approach to the diagnosis of AMR, using the “triple test” including “presence of clinical allograft dysfunction, circulating donor-specific antibodies [DSAs], and pathologic finding.” 30 Morphologic features and clinical and serologic findings that should prompt immunostaining of a lung allograft biopsy specimen for complement 4d (C4d) either by immunoperoxidase or by immunofluorescence techniques are listed in Table 3, and an example of AMR is shown in Figure 4, A through C. More than 50% of capillary staining by C4d is considered positive. However, a subsequent survey of histopathologists found that cases with these morphologic criteria, specifically, neutrophilic margination, neutrophilic capillaritis and arteritis, and immunophenotypic evidence of AMR, are actually quite uncommon, creating further challenges to identification of cases for AMR workup.31
Deposition of C4d, a complement split product, on the capillary endothelium has been suggested as a surrogate marker for AMR in heart, kidney, liver, and pancreas transplants.32–42 The role of C4d deposition in the diagnosis of AMR in lung allografts, however, is still unclear. Moreover, reproducibility of C4d deposition in allograft lung transbronchial biopsies is problematic, even among pathologists who routinely evaluate C4d in lung allograft biopsies.43 C4d is often difficult to interpret in small lung biopsy specimens because of a relatively high background due to nonspecific binding, such as to elastic fibers and intracapillary serum. In addition, staining is frequently only focal. Moreover, C4d deposition is not specific to AMR, as it can also be seen in infection and harvest/reperfusion injury, or any process that is associated with complement activation.
In 2016 the ISHLT proposed a staging of AMR.31 The proposed staging of clinical AMR (allograft dysfunction, defined as “alterations in pulmonary physiology, gas exchange properties, radiologic features, or deteriorating functional performance,” which may be asymptomatic) is summarized in Table 4. In subclinical AMR (normal allograft function), histologic criteria of AMR are detected on surveillance transbronchial biopsies (with or without C4d and with or without the presence of DSAs) in the absence of allograft dysfunction. When DSAs are identified without other manifestations of AMR (histology, C4d staining, allograft dysfunction), heightened surveillance for allograft dysfunction was recommended. It was noted that ACR and AMR can occur concurrently, but other causes should be excluded. As for ACR, infection needs to be excluded before a diagnosis of AMR should be made.
The ISHLT consensus further recommends that the DSA level and function should not be assessed by using the mean fluorescence intensity of the single antigen bead assay but rather by the antibody titer, as the latter is indicative of antibody load. Immunoglobulin G (IgG) subclasses might also play a role with complement-fixing subclasses, such as IgG1 and IgG3, which may be more damaging. The C1q assay might be suitable to stratify risk in patients with DSAs. However, because of the lack of large data sets and sufficient experience, no recommendations were made in regard to standardization of immunophenotyping and DSA testing.
The 2016 Banff study of the pathology of allograft lungs from patients with circulating DSAs confirmed capillary inflammation, acute lung injury, and endothelialitis as morphologic features in lung allograft biopsies that correlate with the presence of DSAs.44 However, it was emphasized again that none of these histopathologic features were specific to patients with DSAs and that the reproducibility of interpreting these morphologic features is quite problematic even among experienced lung transplant pathologists. Morphologic findings of acute lung injury with diffuse alveolar damage had the highest odds ratio for the presence of DSAs. This study also cautioned the use of C4d immunohistochemical staining for the diagnosis of AMR in lung allografts because of its infrequent diffuse positivity.
Taken together, although a definite diagnosis of AMR cannot be established on morphologic grounds alone at this time, in the correct clinical context, the histopathologic features seen in an allograft transbronchial biopsy may aid in the diagnosis of AMR if other relevant clinical and serologic findings are present.
CONCLUSIONS AND FUTURE DIRECTIONS
Acute cellular rejection should be assessed and graded according to the 2007 working formulation of the ISHLT. While morphologic features of ACR are well defined, there are currently no morphologic, immunophenotypic, clinical, and/or serologic features specific to AMR. At this time, the triple test (clinical allograft dysfunction, DSAa, pathologic findings) is the best approach for the diagnosis of AMR and can guide the clinician to initiate appropriate treatment. C4d by immunofluorescence or immunoperoxidase technique might be performed when morphologic, clinical, and/or serologic features suggestive of AMR are identified. Infection should be excluded before a diagnosis of ACR or AMR is made.
Further research on AMR pathogenesis and diagnosis is needed, as prevention and treatment of AMR appears to be important, based on limited data in lung allografts and experience from other solid organ transplants. Research should be performed in a collaborative effort including pathologists, transplant clinicians, immunologists, and basic science researchers. New diagnostic venues should be explored to gain better understanding and diagnostic accuracy of AMR in lung allografts. For instance, a recent study of kidney allografts used molecular and immune cell functional assays to identify patients with subclinical AMR and ACR.45 In a study by the ISHLT on heart allograft biopsies, natural killer cells and inflammatory genes were assessed.46 This study showed that gene expression sets correlated with endothelial cell injury, DSAs, and AMR. These assays were thought to potentially improve the diagnosis of AMR in heart allografts. Furthermore, gaining experience with transbronchial cryobiopsies and its broader availability might help to make this technique more commonly available for monitoring of lung allograft rejection and its mimickers.
The authors have no relevant financial interest in the products or companies described in this article.