A Revision of the 1990 Working Formulation
for the Classification of Pulmonary Allograft Rejection:
Lung Rejection Study Group (LRSG)

RECOMMENDATIONS:

1. Adequacy of specimens:

Transbronchial biopsy remains the mainstay of allograft evaluation.17,18 It was the uniform opinion of the LRSG that at least five pieces of alveolated lung parenchyma each containing bronchioles and greater than 100 air sacs are necessary to confidently grade acute and chronic rejection. In our experience, it may be necessary for the bronchoscopist to obtain more than five biopsies to provide this minimum number of adequate alveolated pieces. Furthermore, if bronchiolitis obliterans is a consideration, more than five pieces are frequently needed to sample the small bronchioles adequately. Specimens should be gently agitated in formalin to inflate the biopsy fragments and tenderly handled by histotechnicians to avoid crush artifacts, when embedding tissue in paraffin.

When appropriate sampling has not occurred it is essential to note in the pathology report that the biopsy findings may not be fully representative of the changes in the lung allograft.

2. Histology:

Histologic evaluation should include a minimum of: (1) sections from three levels of the paraffin block containing the specimens for hematoxylin and eosin stains (2) connective tissue stains to evaluate the submucosal fibrosis essential for the diagnosis of arteriosclerosis/bronchiolitis obliterans and (3) silver stains for fungi/pneumocystis. Beyond this minimum workup, individual investigators may augment their evaluation with a wide spectrum of histochemical, immunohistochemical and in-situ hybridization studies.

3. Review of clinical history and previous biopsies:

All biopsies should be studied by the pathologist with full knowledge of the native recipient disease and the results of the last biopsy and current bronchoalveolar lavage. In many instances this requires simultaneous review of prior slides with the current specimen. The biopsy should be reported in this context. Individual institutions may choose to utilize the term ongoing rejection for biopsies which remain unchanged from previous ones; resolving rejection for those biopsies where the mononuclear infiltrate is reduced in intensity and the number of activated cells has subsided as compared with a previous biopsy; or resolved rejection if the infiltrates have completely disappeared. In these contexts, it is recommended that a rejection score still be included.

4. Differential diagnosis of perivascular and interstitial infiltrates:

Perivascular and interstitial mononuclear infiltrates are not specific for acute rejection, and other conditions may simulate or mimic alloreactive injury.11,19 Differential considerations include:

  1. Cytomegalovirus pneumonitis
  2. Pneumocystis carinii pneumonia
  3. Post transplantation lymphoproliferative disease ranging from pneumonitis to active lymphoproliferative disorders20
  4. Bronchial associated lymphoid tissue, seen particularly in the submucosa and at the bifurcations of large airways, where it is well circumscribed, unassociated with epithelial injury or eosinophils and frequently accompanied by histiocytes containing particulate matter.
  5. Previous biopsy sites
  6. Recurrent primary disease, for example, sarcoidosis.
  7. Ischemia (preservation injury)

5. Differential diagnosis of airway inflammation:

There are many causes of airway inflammation and scarring of the airways, which may result in complete obliteration in some instances. The following conditions represent some non-rejection causes of lymphocytic bronchiolitis/bronchitis and bronchiolitis obliterans.2,3,21-24

  1. Infection - perhaps the most frequent cause of acute and chronic inflammation of the airways is low-grade infection of the respiratory tract, particularly viral, bacterial, mycoplasmal, fungal, and chlamydial in nature.
  2. Aspiration - because of the loss of cough reflex and tracheal sensitivity after the transplant procedure, patients with lung allografts are predisposed to recurrent aspiration; helpful features in diagnosing aspiration include the identification of exogenous material with an associated foreign body giant cell reaction within the airways, distal organizing pneumonia, and supportive radiologic studies.
  3. Granulation tissue reactions are common in the lung allograft and are manifested as intra-airway and intra-alveolar plugs of fibromyxoid connective tissue.20,25 These loose edematous polyps should be distinguished from the dense eosinophilic scarring of bronchiolitis obliterans. Particular settings in which these reactions occur are resolving infection (organizing pneumonia), aspiration, the proliferative phase of diffuse alveolar damage, active or resolving acute rejection reactions, and miscellaneous other conditions. Rare reports of idiopathic bronchiolitis obliterans organizing pneumonia (BOOP) in lung allografts are another potential cause.21
  4. A variety of nonspecific reactions may occur in the lung allograft. Although they usually do not cause diagnostic confusion with rejection, awareness of these injury patterns may be helpful in the evaluation of potential causes of graft dysfunction. Such reactions are listed in Table II.

TABLE II. Pathologic Alterations in the Pumonary Allograft - Nonrejection Related
1. Diffuse alveolar damage/patchy acute alveolar injury - usually a manifestation of ischemic/reperfusion injury or other peri-operative insults
2. Acute alveolar hemorrhage
3. Alveolar hemosiderosis - usually seen in a previous biopsy site, in resolved high grade acute rejection reactions where endothelial damage was significant, sites of prior infection, or in individuals with coagulation abnormalities
4. Post transplantation lymphoproliferative disorder
5. Recurrent native disease e.g. sarcoidosis, lymphangioleiomyomatosis, giant cell interstitial pneumonia
6. Smoker's macrophages in air spaces (if donor was a smoker or recipient continues/begins smoking).

6. Hyperacute Rejection:
The LRSG felt that a morphologic definition of hyperacute lung rejection was not possible at this point in time, and that it would require an integrated evaluation of clinical findings, histology, serologic studies, and immunofluorescence evaluation of allograft tissue.

7. Concomitant acute rejection and infection:

The LRSG recognized that the histologies of acute rejection and infection overlap, especially in cytomegalovirus and pneumocystis carinii pneumonitis.11,18 In some instances there may also be coexistent infection and rejection. In these cases, we continue to recommend that the pathologist indicate that he or she favors infection or rejection as the predominant process and that a follow-up biopsy be obtained after appropriate antimicrobial therapy, to adequately assess the presence of simultaneous rejection.

Some histologic features are so distinctive that they strongly suggest infection in the context of perivascular or interstitial mononuclear infiltrates.19 Granulomatous inflammation and punctate zones of necrosis are unusual in rejection, and should raise the possibility of mycobacterial, fungal or pneumocystis infection in the former, and herpes simplex or cytomegalovirus in the latter context. Pneumocystis and fungal disease can readily be diagnosed with silver stains as can mycobacterial infection by acid fast stains and culture. Viral infection may be confirmed by immunohistological studies or culture. Clues to cytomegalovirus infection include perivascular edema that outstrips the degree of mononuclear infiltration; disproportionate alveolar septal cellular infiltrates in comparison with perivascular cuffing; the presence of abundant neutrophils with the formation of microabscesses; marked atypia of alveolar pneumocytes; and acute airway inflammation. Finally, airspace consolidation or heavy interstitial infiltrates of eosinophils may suggest fungal infection.26

This revised working formulation for the classification of lung allograft rejection is a proposal that is intended to simplify the original classification scheme based on clinicopathologic study and empiric observations. What has been achieved is that the formulation has been stripped of some of its complexity, in terms of the numerous suffixes under acute rejection, the subtotal/total designations of bronchiolitis obliterans, and the presence of "vasculitis". The significance of airway inflammation without fibrosis, in the context of acute rejection, is unclear in the literature. In the 1995 proposal we encourage continued investigation of its significance. As part of this study, it may also be worthwhile to grade the intensity of the latter process, if desired by the institutional pathologist and the clinical care team. This flexibility was emphasized in the original proposal and should be noted once more - the working formulation scheme should not dissuade individuals from constructing their own local classifications, but should be used as a means of facilitating communication between clinicians, pathologists, disparate departments, and institutions. Furthermore, the system should always be used in the context of the individual patient, with the histopathology representing one piece of a therapeutic puzzle that includes symptomatology, physical and radiographic findings, pulmonary function studies, and microbiological culture results.


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