Pathology of Lung Rejection: Cellular and Humoral Mediated
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Acute rejection is an important risk factor for bronchiolitis obliterans syndrome, the clinical manifestation of chronic airway rejection in lung allograft recipients. Patients with acute rejection might be asymptomatic or present with symptoms that are not specific and can be also seen in other conditions. Clinical tests such as pulmonary function tests and imaging studies among others usually are abnormal; however, their results are also not specific for acute rejection. Histopathologic features of acute rejection in adequate samples of transbronchial lung biopsy of the lung allograft are currently the gold standard to assess for acute rejection in lung transplant recipients. Acute alloreactive injury can affect both the vasculature and the airways. Currently, the guidelines of the 2007 International Society of Heart and Lung Transplantation consensus conference are recommended for the histopathologic assessment of rejection. There are no specific morphologic features recognized to diagnose antibody-mediated rejection (AMR) in lung allografts. Therefore, the diagnosis of AMR currently requires a “triple test” including clinical features, serologic evidence of donor-specific antibodies, and pathologic findings supportive of AMR. Complement 4d deposition is used to support a diagnosis of AMR in many solid organ transplants; however, its significance for the diagnosis of AMR in lung allografts is not entirely clear. This chapter discusses the currently recommended guidelines for the assessment of cellular rejection of lung allografts and summarizes our knowledge about morphologic features and immunophenotypic tests that might help in the diagnosis of AMR.
KeywordsAntibody-mediated rejection Humoral rejection Cellular rejection Lung Lymphocytic bronchiolitis
Acute rejection is the host’s response to the recognition of the graft as foreign. It can occur days, months, or even years after transplantation. Rejection can be divided into cellular and humoral forms. Acute cellular rejection is the predominant type of acute rejection of lung allografts . It is mediated by T lymphocytes that recognize foreign human leukocyte antigens (HLA) or other antigens [1, 2]. Humoral rejection is mediated by preformed or de novo recipient antibodies (therefore, also referred to as antibody-mediated rejection [AMR]) against antigens of the donor organ cells.
Acute rejection is an important complication in patients with lung allografts. Twenty-nine percent of adult patients have at least one episode of treated acute rejection between discharge from the hospital and 1 year after transplantation . Moreover, 3.6% and 1.8% of all deaths that occur within the first 30 days or between 30 days and 1 year following lung transplantation are due to acute rejection, respectively . In addition, the frequency and severity of acute rejections are thought to represent the major risk factor for the subsequent development of bronchiolitis obliterans syndrome (BOS) [1, 4, 5, 6].
HLA mismatch, genetic and recipient factors, type of immunosuppression, vitamin D deficiency, and infection are risk factors of acute rejection. For instance, the recipient alloimmune response is thought to be related to the recognition of differences to donor antigens leading to acute lung allograft rejection. Indeed a higher degree of HLA mismatch has been shown to increase the risk of acute rejection although this effect is not consistent across all HLA loci or studies [4, 7, 8, 9, 10]. Mismatches at the HLA-DR, HLA-B , and HLA-A  loci, as well as a combination of all three loci , appear specifically important. For instance, acute rejection within 2 months after transplantation has been shown to be associated with HLA-DR mismatch, while acute rejection at 4 years has been found to be associated with HLA-B mismatch .
Several host genetic characteristics have been studied that may modulate acute lung rejection. For instance, a genotype leading to increased IL1- production may protect against acute rejection , while a multidrug-resistant genotype (MDR1 C3435T) appears to predispose to persistent acute rejection that is resistant to immunosuppressive treatment .
The incidence of acute rejection appears to be age-dependent, with the lowest incidence of acute rejection in infants (< age 2) . However, children have a higher risk for acute rejection than adults . Furthermore, the registry of the International Society of Heart and Lung Transplantation (ISHLT) showed that the incidence of acute rejection between discharge and 1-year follow-up was slightly higher in younger adult lung allograft recipients (age 18–34 years) (36%)  when compared to the entire adult population in which 29% had at least one acute rejection episode . The incidence of acute rejection does not seem to change in older lung transplant recipients (age 65 and higher) .
Regimens of immunosuppression might also play a role in acute rejection. For instance, the rate of acute rejection in the first year after transplantation was highest among recipients who were on cyclosporine-based regimens and lowest among those on tacrolimus-based regimens .
Vitamin D deficiency might also play a role in acute rejection. A study found that 80% of lung recipients were 25(OH)D deficient around the time of transplantation and that vitamin D-deficient recipients had more episodes of acute cellular rejection and infection . A similar association between vitamin D deficiency and acute rejection has been described in other solid organ recipients including the liver, kidney, and heart. Although the exact mechanism for this phenomenon is not entirely clear, it is speculated that (1) vitamin D might slow down the maturation of antigen-presenting cells as in vitro studies have shown, (2) vitamin D might induce dendritic cells to acquire tolerance, and/or (3) a synergistic effect between vitamin D analogs and immunosuppressants occurs .
Viral infections have also been thought to modulate the immune system and to increase alloreactivity. Indeed, a high incidence of acute rejection has been found in lung transplant recipients after community-acquired respiratory tract infections with human influenza virus, respiratory syncytial virus, rhinovirus, coronavirus, and parainfluenza virus [20, 21, 22]. Chlamydia pneumoniae infection has also been linked to the development of acute rejection in one study . The significance of CMV infections and the impact of CMV prophylaxis strategies on acute rejection frequency are not clear at this time .
The clinical course of acute rejection can be variable. Acute rejection is often identified on surveillance transbronchial biopsy in an asymptomatic patient. If symptoms occur, they might be non-specific and overlap with those seen in other complications and diseases in this patient population. These symptoms might include dyspnea, fever, leukocytosis, and a widened alveolar-arterial oxygen gradient. Higher-grade rejection appears to cause more severe symptoms and can lead to acute respiratory distress . In patients with rejection, pulmonary function testing may show a decrease in forced expiratory volume in 1 s (FEV1) and vital capacity (VC). Although spirometry has a sensitivity of greater than 60% for detecting infection or rejection of Grade A2 and higher, it cannot differentiate between the two . Furthermore, the usefulness of spirometry is diminished in single lung transplant recipients, as the dysfunction of the native lung confounds the pulmonary function test results .
Although in approximately half of the cases of acute rejection, chest X-ray studies are normal, ill-defined perihilar and lower lobe opacities, along with septal lines and pleural effusions, may be seen. Findings on CT scan might include ground-glass opacities, septal thickening, volume loss, nodules and consolidation, and pleural effusions. Infiltrates observed on imaging studies during the first week after lung transplantation are usually caused by the reimplantation response, i.e., reperfusion edema and other factors. Infiltrates that persist beyond the first week following transplantation suggest acute rejection or infection. However, although early, the authors of small studies have attempted to demonstrate the usefulness of chest X-rays and chest CT scans in the diagnosis of rejection, more recent data show a very low sensitivity for acute rejection (as low as 35%) and no discriminatory value between rejection and other processes .
Exhaled nitric oxide (NO) can also serve as a marker of lung injury; it is often increased in patients with lymphocytic bronchiolitis and acute rejection [28, 29, 30]. Furthermore, in a study of inert gas single-breath washout, the slope of alveolar plateau for helium had a sensitivity of 68% for acute rejection .
Although the presentation of the patient and several ancillary studies may suggest the presence of acute allograft rejection, none of these findings are specific. Therefore, tissue diagnosis is necessary for a definitive diagnosis. Histopathology of adequate lung biopsy samples obtained from transbronchial biopsy is currently the gold standard to assess lung allografts for rejection and to distinguish rejection from its clinical mimickers such as aspiration, infection, drug toxicity, and recurrent disease.
Recently, the transbronchial cryobiopsy technique was introduced which yields larger biopsies containing more alveoli, small airways, and veins and venules while exhibiting less procedural alveolar hemorrhage and crush artifact than conventional forceps transbronchial allograft biopsies [31, 32, 33]. Although cryobiopsies appear to be as safe as forceps biopsies, complications can occur which is one of the reasons that this technique has so far not been universally performed for this purpose .
Other lung tissue specimens from lung allografts include wedge biopsies, explants for retransplant, or autopsy specimens from lung transplant recipients. Wedge biopsies, although seldom obtained in clinical practice, and specimens from explants provide useful histopathologic insights into the etiology of lung allograft dysfunction in advanced stages following all possible medical interventions.
Morphologic Features of Cellular Rejection
Cellular alloreactive injury to the donor lung affects both the vasculature and the airways . Perivascular mononuclear cell infiltrates are the hallmark of acute cellular rejection. These infiltrates may be accompanied by subendothelial chronic inflammation (e.g., endotheliitis or intimitis) and also by lymphocytic bronchiolitis, which is characteristic of small airway rejection. The histologic changes are divided into grades based on intensity of the cellular infiltrate and the occurrence of an accompanying acute lung injury pattern.
In 1990, the ISHLT sponsored the Lung Rejection Study Group (LRSG), a workshop to develop a “working formulation” for the diagnosis of lung rejection by transbronchial biopsy . Since then the grading scheme has been revised twice, in 1996  and 2007 . The grading scheme is strictly pathologic, based on morphologic features recognized in transbronchial biopsies of the allograft. Clinical parameters are not considered.
Due to overlapping histologic features between acute rejection and infection, the grading scheme relies on the absence of concurrent infection. Furthermore, infection and rejection may occur together. Therefore, the LRSG recommends grading rejection only after the rigorous exclusion of infection .
Classification of cellular allograft rejection according to the 2007 revised ISHLT consensus classification of lung allograft rejection
Type of rejection
Normal pulmonary parenchyma
Occasional blood vessels are surrounded by a thin chronic mononuclear cell infiltrate
Multiple blood vessels are surrounded by a more prominent mononuclear cell infiltrate
Infiltrate confined to the perivascular adventitia
Endotheliitis may occur
Dense mononuclear cell infiltrates surround blood vessels and extend into interstitium
Eosinophils and occasional neutrophils common
Acute lung injury may be apparent
Diffuse perivascular, interstitial, and air space infiltrates of mononuclear cells
Prominent alveolar pneumocyte damage and endotheliitis
Intra-alveolar necrotic epithelial cells, macrophages, eosinophils, hemorrhage, and neutrophils may occur
Acute lung injury
Small airway inflammation—Lymphocytic bronchiolitis
Unremarkable small airways
B1Ra Low grade
Lymphocytes within the submucosa of the bronchioles
B2R High grade
Marked lymphocytic infiltrate of the airway epithelium and airway wall
Greater numbers of eosinophils and plasmacytoid cells
Epithelial damage including necrosis, metaplasia, and marked intraepithelial lymphocytic infiltration
Epithelial ulceration, fibrinopurulent exudate, cellular debris, and neutrophils can occur
Grading hampered by lack of definite small airways, presence of infection, tangential cutting, artifact, etc.
Chronic airway rejection—Obliterative bronchiolitis
Small airways similar in size to the accompanying artery
Fibrosis in the wall of small airways
Chronic vascular rejection
No arterial or venous changes
Pulmonary arteries and/or veins are thickened by fibrointimal connective tissue
2007 ISHLT Revised Consensus Classification of Lung Allograft Rejection
Acute Rejection: A Grade
Acute rejection is defined by the presence of perivascular mononuclear cell infiltrates with or without endotheliitis . With progression, this infiltrate becomes more widespread and extends into the alveolar septa and, subsequently, into the alveoli. The majority of the mononuclear cells in acute rejection are T cells, although a few studies have described increased populations of B cells or eosinophils [34, 41, 42]. The histologic features of rejection are summarized in Table 13.1.
No Acute Rejection (ISHLT Grade A0)
Features of acute cellular rejection are lacking, although the biopsy may not be entirely normal.
Minimal Acute Rejection (ISHLT Grade A1)
Mild Acute Rejection (ISHLT Grade A2)
Moderate Acute Rejection (ISHLT Grade A3)
Severe Acute Rejection (ISHLT Grade A4)
Protocol surveillance biopsies of lung allografts are performed in many institutions. Even though these patients are in general asymptomatic and clinically stable, one study showed that 39% of surveillance biopsies reveal acute cellular rejection with 43% showing features of minimal rejection, 49% mild rejection, and 8% moderate rejection . A more recent prospective study identified morphologic findings of acute cellular rejection only in 6% of surveillance biopsies , while a retrospective study of 592 surveillance biopsies taken within 400 days of transplantation revealed histologic findings of either acute cellular rejection or obliterative bronchiolitis in 31% of biopsies with 36% within the first 100 days and 25% between 100 and 400 days following transplantation .
Evidence suggests that acute cellular rejection is an important risk factor for the development of BOS . Indeed, studies have demonstrated an increased risk of BOS with single episodes, increased frequencies, and increased severity of acute cellular rejection. Moreover, patients with multiple episodes of even minimal acute cellular rejection were shown to be at increased risk for BOS , and yet a single episode of minimal acute rejection without recurrence or subsequent progression to a higher grade has been identified as an independent significant predictor of BOS . Because of these findings, patients who are asymptomatic but are found to have acute cellular rejection (even minimal acute cellular rejection) on a surveillance allograft biopsy might be treated accordingly. However, several centers do not utilize surveillance transbronchial lung biopsies and/or treat asymptomatic patients with no clinical evidence of allograft dysfunction. Prospective well-designed clinical studies are needed to provide evidence to support surveillance transbronchial lung biopsies and therapeutic interventions.
Small Airway Inflammation: Lymphocytic Bronchiolitis —B Grade
This grade applies only to small airways such as terminal or respiratory bronchioles. Bronchi, if present, should be described separately. It is important to mention in the pathology report whether or not small airways are present. If no small airways are identified or the biopsy has obvious infection, the grade “BX” should be used. The R behind grades 1 and 2 denotes the revised 2007 version.
No Airway Inflammation (ISHLT Grade B0)
The small airways appear unremarkable without evidence of bronchiolar inflammation.
Low-Grade Small Airway Inflammation (ISHLT Grade B1R)
High-Grade Small Airway Inflammation (ISHLT Grade B2R)
Ungradeable Small Airway Inflammation (ISHLT Grade BX)
Small airways might not be evaluable for several reasons including lack of small airways due to sampling problems, infection, tangential cutting, artifact, etc. In patients who are known to have an infection that could cause lymphocytic bronchiolitis, the allograft biopsy should also be classified as ungradeable for small airway rejection.
Chronic Airway Rejection: Obliterative Bronchiolitis —C Grade
Chronic airway rejection is restricted to submucosal and intraluminal scarring of small airways including terminal and respiratory bronchioles. When large tissue sections of the lung are examined, obliterative bronchiolitis may be recognized as a panlobar process but is usually patchy.
No Chronic Airway Rejection (ISHLT Grade C0)
The small airways appear similar in size to the accompanying artery with a ragged inner surface. Fibrosis is not present.
Chronic Airway Rejection (ISHLT Grade C1)
Obliterative bronchiolitis is only infrequently identified in lung allografts by transbronchial biopsy, and the sensitivity of this morphologic finding for the presence of chronic rejection is only between 15 and 28% [48, 49, 50]. In a recent study, all seven conventional transbronchial biopsies that were included from patients clinically known to have BOS , the clinical equivalent to morphologic obliterative bronchiolitis, failed to reveal morphologic findings of obliterative bronchiolitis . Although cryobiopsies contained more small airways, all nine cryobiopsies that were also included in that study from patients with clinically proven BOS did not reveal obliterative bronchiolitis in the tissue . This low sensitivity is largely due to sampling and its patchy nature. Therefore, BOS is used and more reliable for the clinical assessment of chronic airway rejection. BOS is calculated as <80% FEV1 in at least two consecutive lung function tests of the patient’s maximum FEV1 posttransplantation . Despite the low sensitivity of transbronchial biopsies for obliterative bronchiolitis, the specificity of this morphologic finding in an allograft biopsy is high, ranging from 75 to 94% [49, 50]. Therefore, an attempt to diagnose obliterative bronchiolitis should be made in lung allograft biopsies.
Chronic Vascular Rejection: D Grade
No Chronic Vascular Rejection (ISHLT Grade D0)
The pulmonary arteries appear of a similar size as the accompanying airways. The intima is slender and the media not thickened.
Chronic Vascular Rejection (ISHLT Grade D1)
Chronic vascular rejection rarely is identified on biopsies since they usually lack vessels of sufficient size. Wedge biopsies, explants, or autopsy material may reveal it. Therefore, according to the ISHLT, the D grade of rejection is not applicable to allograft transbronchial biopsies. Although cryobiopsies contain a higher number of venules and small veins, in a recent small study, no difference was found in the number of cases with possible vascular rejection when compared to transbronchial biopsies .
Mimickers of Cellular Rejection
Mimickers of severe acute rejection include conditions that might present with acute lung injury or diffuse alveolar damage. These conditions include infection, drug toxicity, aspiration, AMR, or harvest/reperfusion injury. The presence of perivascular inflammation is helpful in establishing the diagnosis of rejection. However, perivascular inflammation is not entirely specific for acute rejection, and many other conditions may simulate or mimic alloreactive lung injury .
Marked perivascular and/or peribronchiolar mononuclear infiltrates might also raise the possibility of posttransplantation lymphoproliferative disease (PTLD), and in such cases, an appropriate workup should be performed, including doing studies for Epstein-Barr virus, which is ubiquitous in PTLD. Further differential diagnosis of perivascular and interstitial infiltrates include recurrent primary diseases.
Originally recognized in kidney transplant patients who presented with acute allograft rejection, anti-donor antibodies, and poor prognosis , AMR is now well established in kidney and heart allografts. In lung transplantation, AMR is still an evolving concept but likely explains acute and chronic graft dysfunction/failure in a subset of patients. Evidence suggests that AMR occurs due to circulating antibodies that are either (1) preformed because of pregnancy, blood transfusion, or previous organ transplantation or (2) arise de novo after transplantation due to HLA mismatch. Furthermore, the recent development of very sensitive and specific solid-phase flow cytometry and Luminex-based methodologies has allowed for more accurate detection of antibody specificities in sensitized recipients, and it has become clear that more patients than previously expected have or develop preformed anti-HLA antibodies. Immune stimulation by prior infections or autoimmunity may also contribute to the development of antibodies in those patients with no identifiable risk factors.
Overall, these preexisting or de novo antibodies can react with donor antigens, leading to immediate graft loss (hyperacute rejection), accelerated humoral rejection, and/or BOS . In addition, recent studies have consistently demonstrated an increased incidence of acute rejection (a threefold increase in one study) , persistent rejection, increased BOS , or worse overall survival  in patients with anti-HLA antibodies. This effect is seen both with pretransplant HLA sensitization and with the development of de novo anti-HLA donor-specific antibodies after transplantation .
About 10–15% of lung transplant recipients are pre-sensitized to HLA antigens .
Even though “unacceptable antigens” are avoided during the virtual crossmatch, patients with positive pretransplant PRA are at higher risk for posttransplant complications. Their posttransplant PRA can stay stable or increase via generation of either donor-specific or non-donor-specific anti-HLA antibodies. Similarly, patients that had negative PRA screening tests before transplantation can develop de novo non-donor-specific or donor-specific anti-HLA antibodies after transplantation.
The mechanisms by which antibodies promote lung allograft injury remain poorly understood. Antibody binding to allo-HLA or other endothelial or epithelial targets in the lung allograft can activate the complement cascade. Complement deposits lead to endothelial cell injury, production of proinflammatory molecules, and recruitment of inflammatory cells. Complement-independent antibody-mediated mechanisms can also induce endothelial cell activation without cell injury, leading to increased gene expression and subsequent proliferation . Furthermore, as demonstrated by in vitro studies, anti-HLA antibodies can cause proliferation of airway epithelial cells as well, producing fibroblast-stimulating growth factors , potentially contributing to the generation of obliterative bronchiolitis.
Although the diagnosis of AMR in lung allograft biopsies remains challenging, when the triple test criteria are met (graft dysfunction, positive panel reactive antibodies, and evidence of complement deposition in the graft), the disease can be life-threatening, and prognosis can be poor. Although the optimal treatment of AMR in the lung is currently not known due to the lack of clinical trials, treatment is typically comprised of plasmapheresis, possibly intravenous immunoglobulin (IVIG), and medications such as rituximab and bortezomib, among others. As such, the associated histopathologic and clinical parameters are the subject of intense investigation. Deposition of complement 4d (C4d) , a complement split product, on the capillary endothelium has been suggested as a surrogate marker for AMR in heart, kidney, and pancreas transplants [62, 63, 64, 65, 66, 67, 68, 69, 70, 71]. However, the role of C4d deposition in the diagnosis of AMR in lung allografts is still unclear. Moreover, reproducibility of C4d deposition in allograft lung TBBx is problematic, even among pathologists who routinely evaluate C4d in lung allograft biopsies . Furthermore, there are currently no specific or sensitive morphologic features of AMR in lung allografts, although some features that are more commonly identified in these patients have emerged in some recent studies . Studies have attempted to evaluate immunoglobulins (Ig) and complement deposits in the subendothelial space. Septal capillary deposits of Igs and complement products such as C1q, C3d, C4d, and C5b-9 have been described in association with anti-HLA antibodies [74, 75] as well as allograft dysfunction and BOS [76, 77]. However, except for C4d and in some institutions C3d, these studies have in general not been implemented for the workup of lung transplant biopsies for possible AMR. One of the reasons for the difficulties in lung is the relatively high background that is encountered in immunohistochemical as well as immunofluorescence studies. Often, C4d binds to the vascular elastic lamina or shows other non-specific binding such as intracapillary serum. Staining is commonly only focal, and, therefore, sensitivity and specificity have not been established. Only linear, continuous luminal endothelial staining of capillaries, arterioles, and/or venules by C4d should be interpreted as positive. In addition, C4d is not specific to AMR but also can be seen in infection, and harvest/reperfusion injury, or any process that is associated with complement activation.
Box 13.1 Histomorphologic and Clinical Indications for Immunopathologic Evaluation (C4d Staining) of Lung Allograft Biopsies
Neutrophilic septal margination
High-grade acute cellular rejection (≥ ISHLT Grade A3)
Persistent/recurrent acute cellular rejection (any ISHLT A grade)
Acute lung injury with or without diffuse alveolar damage
High-grade lymphocytic bronchiolitis (ISHLT Grade B2R)
Persistent low-grade lymphocytic bronchiolitis (ISHLT Grade B1R)
Obliterative bronchiolitis/chronic airways rejection (ISHLT Grade C1)
Arteritis in the absence of infection or cellular rejection
Graft dysfunction without morphologic explanation
Any histologic findings in setting of de novo DSA positivity
Used with permission of Elsevier from Berry G, Burke M, Andersen C, Angelini A, Bruneval P, Calbrese F, Fishbein MC, Goddard M, Leone O, Maleszewski J, et al. Pathology of pulmonary antibody-mediated rejection: 2012 update from the Pathology Council of the ISHLT. The Journal of heart and lung transplantation 2013; 32:14–21
Staging of antibody-mediated rejection as proposed by the International Society for Heart and Lung Transplantation
Clinical antibody-mediated rejection
Definite clinical AMRa
Histology suggestive of AMR
Other causes of graft dysfunction were excluded except ACRc which can occur concurrently
Probable clinical AMR
Two of the following 3 criteria:
Histology suggestive of AMR
When all 3 diagnostic criteria are identified, this grade can be applied even if infection or ACR is also present
Possible clinical AMR
One of the following 3 criteria:
Histology suggestive of AMR
When 2 diagnostic criteria are identified, this grade can be applied even if infection or ACR is also present
Subclinical antibody-mediated rejection
Histologic criteria of AMR identified on surveillance transbronchial biopsy with or without
No allograft dysfunction
Recently, Wallace and colleagues reported findings of the Banff study of the pathology of allograft lungs with DSA . Nine experienced lung transplant pathologists from multiple institutions performed digital slide interpretation to study transbronchial biopsy specimens from patients with known antibody status (established within 30 days of biopsy) and negative infectious workup. The study demonstrated that biopsies from patients with DSA more commonly showed morphologic features of acute lung injury with or without diffuse alveolar damage than biopsies from patients with non-DSA or no circulating antibodies. Endotheliitis was more common in patients with DSA than patients without circulating antibodies. However, there was no difference in occurrence of endotheliitis between biopsies from patients with circulating non-DSA vs DSA or non-DSA vs no circulating antibodies. Specimens associated with DSA had a significant higher frequency of capillary inflammation, including neutrophilic margination, increased neutrophils, or capillaritis with karyorrhexis than patients with non-DSA or no circulating antibodies. C4d staining was positive in less than 50% of capillaries in 14% of biopsies and in more than 50% of capillaries in 7% of biopsies. While there was no difference between the groups in biopsies with <50% staining, biopsies with DSA more often had over 50% capillaries staining for C4d than biopsies without any circulating antibodies. There were no significant differences identified between HLA classes of the DSA and any of the evaluated pathologic findings. Taken together, this study identified capillary inflammation, acute lung injury, and endotheliitis as morphologic features in lung allograft biopsies that correlate with the presence of circulating DSA. However, none of these histopathologic features were specific to patients with DSA. Morphologic findings of acute lung injury with diffuse alveolar damage had the highest odds ratio for the presence of circulating DSA. This study also cautioned the usefulness of C4d immunohistochemical stain for the diagnosis of AMR in lung allografts because of its infrequent diffuse positivity. Although the study shows that some morphologic features correlate with the presence of circulating DSA and, therefore, might be histopathologic markers to at least suggest the possibility of AMR, the reproducibility of these morphologic features is quite problematic even among experienced lung transplant pathologists. In fact, the interobserver reproducibility kappa values ranged between 0.14 and 0.4, indicating a less than a chance to moderate agreement. The lowest agreement was noted for suspicion for aspiration (median kappa, 0.14) and the highest for acute cellular rejection, alveolar hemosiderosis, and C4d staining (median kappa, 0.4, all).
Although a definite diagnosis of AMR seems to elude pathologic interpretation at the current time, in a fully contextualized clinical environment, the findings from the biopsy specimen may aid the clinician to make a reasonable diagnosis of AMR if other relevant clinical and serologic features are present. The proposed “triple test”  of clinical features, serologic evidence of DSA, and pathologic findings supportive of AMR including capillary inflammation, acute lung injury with or without diffuse alveolar damage, and endotheliitis may currently be the best guide to the diagnosis of AMR.
There is no IHSLT recommendation at this time regarding the coexistence of AMR and acute rejection, but it clearly does occur.
Hyperacute rejection is a severe form of AMR mediated by preexisting antibodies to ABO blood groups, HLA class I or II, or other antigens on graft vascular endothelial cells. This rejection occurs within minutes to a few hours after the transplanted organ begins to be perfused. As in any form of AMR, the preexisting antibodies can result from previous pregnancies, blood transfusions, or previous transplant, and their binding to donor antigens provokes complement and cytokine activation resulting in endothelial cell damage and platelet activation with subsequent vascular thrombosis and graft destruction. The outcome is commonly fatal.
In hyperacute rejection, lungs are edematous, cyanotic, and heavy, have a firm consistency, lack crepitation, and show red hepatization [80, 81, 82, 83]. The cut surface reveals patchy poorly defined areas of hemorrhagic consolidation. Anastomoses are intact and typically widely patent. Histologically, alveolar hemorrhage, platelet and fibrin thrombi, neutrophilic infiltration, necrosis of vessel walls, and diffuse alveolar damage are observed [76, 77, 78, 79, 80, 83, 84]. C4d deposition has been described.
Although hyperacute rejection is a well-known complication in kidney and heart transplantations, in lung transplantation, it appears to be rather rare with only eight cases reported. Six patients died within 1 h and 13 days after transplantation [80, 81, 82, 83, 84, 85]. Only two patients survived [86, 87]. One of these two patients was treated with plasmapheresis, antithymocyte globulin, and cyclophosphamide immediately after hyperacute rejection was diagnosed . The other patient was highly presensitized when he underwent double lung transplantation . This patient was treated with multiple plasma exchanges and intravenous immunoglobulin pre- and posttransplantation together with posttransplant rituximab and bortezomib and later with anti-C5 antibody and eculizumab. Although in pretransplant, panel reactive antibodies (PRAs) were negative in four of the eight reported patients, crossmatch was positive in all reported cases.
Collectively, although hyperacute rejection is rare after lung transplantation, one should keep this reaction in mind given that false-negative PRAs may occur and pretransplantation crossmatch is not often possible .
At least five pieces of well-expanded alveolated parenchyma are required for adequate evaluation of a transbronchial lung allograft biopsy specimen for acute rejection by the LRSG . This specimen requirement was based on the “uniform opinion of the consensus meeting.” To ensure that the minimum number of required pieces of alveolated lung parenchyma is available for pathology review, it is recommended that the bronchoscopist needs to take more than five pieces. Even more pieces might be necessary to provide small airways for review. Interestingly, a prospective 12-month single-operator study by Scott and colleagues  including 219 transbronchial allograft biopsies with 6 to 56 samples per procedure (mean 17.3 samples per procedure) taken from 3 lobes (or 2 lobes and the lingula of 1 lung) of 54 heart-lung transplant and 2 single lung transplant recipients revealed a sensitivity of 94% and a specificity of 90% for identification of rejection by histopathology. This study estimated that 18 samples per procedure are needed to have a 95% confidence of finding rejection. Therefore, false-negative results due to patchy distribution of acute rejection are likely not uncommon. The absence of histologic and immunophenotypic features of acute rejection or antibody-mediated rejection requires clinicopathologic correlation as a negative biopsy does not necessary rule out rejection. Furthermore, the bronchoscopist should be familiar with imaging studies, especially high resolution computed tomography studies if available, and aim to sample radiologically abnormal bronchopulmonary segments. If such imaging was not recently performed or the results are normal, then samples should be obtained from different lobes to try to minimize sampling error.
Specimens should be gently agitated in formalin to open up the alveoli. There is currently no recommendation for cryobiopsies. In a recent study using cryobiopsies to evaluate rejection in lung allografts, a median of three pieces provided twice as many alveoli and small airways than a median of ten pieces by conventional forceps biopsy .
The ISHLT recommends a minimum of three levels from the paraffin block for hematoxylin and eosin (H&E) staining for histologic examination . In addition, “connective tissue stains” such as trichrome or Verhoeff-Van Gieson (VVG) stain are recommended to evaluate airways for the presence of submucosal fibrosis and vessels for graft vascular disease. Stains for microorganisms including Gomori-Grocott methenamine silver stain (GMS) and acid fast bacilli (AFB) may be added. While silver stains are routinely performed on lung allograft biopsies in some institutions, they are currently not mandated by the LRSG because many microbiologic, serologic, and molecular techniques are available and used to identify infections in these patients [34, 89]. BAL may be performed at the time of biopsy and is useful for the exclusion of infection but currently has no clinical role in the diagnosis of acute rejection.
The transbronchial allograft biopsy is currently the gold standard to evaluate the graft for cellular rejection and to exclude its clinical mimickers in lung transplant patients. When reviewing transbronchial biopsy material of these patients, attention must be paid not only to features of rejection but also to its morphologic mimickers, especially infection, PTLD, and abnormal drug effect. Before a diagnosis of acute cellular rejection can be rendered, an infectious process should be excluded by using stains for microorganisms and/or clinical tests including cultures of BAL and/or tissue and serology. While studies to identify histopathologic and immunophenotypic features of AMR are evolving, there are currently no specific morphologic findings, and clinical and serologic correlations are required for the diagnosis. Prospective, well-designed long-term studies with longitudinal data of therapeutic intervention of ACR on histopathology in totally asymptomatic patients with no physiological or HRCT evidence of allograft dysfunction are needed to determine the clinical significance and relevance of such interventions.
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