Lung Rejection Study Group


Revision of the 1996 Working Formulation for the Standardization of Nomenclature in the Diagnosis of Lung Rejection


Histologic Grading of Pulmonary Allograft Rejection

TABLE 1. Revised Working Formulation for Classification and Grading of Pulmonary Allograft Rejection


A. Acute Rejection B. Airway inflammation - lymphocytic bronchitis/bronchiolitis *
Grade 0 - None Grade 0 - None
Grade 1 - Minimal Grade 1R - Low Grade
Grade 2 - Mild Grade 2R - High Grade
Grade 3 - Moderate Grade X - Ungradeable
C. Chronic airway rejection - obliterative bronchiolitis
0 - Absent
1 - Present
D. Chronic vascular rejection - accelerated graft vascular sclerosis

* "R" denotes revised grade to avoid confusion with 1996.

The histopathology task force again recognized that alloreactive injury to the donor can affect both the vasculature and the airways in acute and chronic rejection. Acute rejection is characterized by perivascular mononuclear cell infiltrates, which may be accompanied by sub-endothelial infiltration, so-called endothelialitis or intimitis, and also by lymphocytic bronchitis and bronchiolitis.1,2,7 However, chronic rejection is manifest by fibrous scarring, which is often dense and eosinophilic, involving the bronchioles and sometimes associated with accelerated fibrointimal changes affecting pulmonary arteries and veins. As in the original and revised classifications, the histologic changes have been divided into grades based on the intensity of the cellular infiltrate and the presence or absence of fibrosis. The presence of presumed irreversible dense eosinophilic hyaline fibrosis in airways and vessels remains the key histologic discriminator between acute and chronic rejection of the lung.

A. ACUTE REJECTION

A diagnosis of acute rejection is based exclusively on the presence of perivascular and interstitial mononuclear cell infiltrates. The intensity of the perivascular mononuclear cell cuffs and the distribution of the mononuclear cells, including extension beyond the vascular adventitia into adjacent alveolar septa, form the basis of the histologic grade. Acute rejection usually affects more than one vessel (particularly in adequate transbronchial biopsy samples) but is occasionally seen as a solitary perivascular infiltrate. This finding should be evaluated with the same criteria as those applied to multiple infiltrates as outlined in what follows. In the setting of multiple foci of rejection, the grade reflects the most advanced pattern of rejection rather than the predominant pattern. The infiltrates surrounding small vessels in the sub-mucosa of airways are again interpreted as part of the spectrum of airway inflammation rather than being diagnostic of acute rejection, Grade A.

GRADE A0 (No Acute Rejection)

In Grade A0 acute rejection, normal pulmonary parenchyma is present without evidence of mononuclear cell infiltration, hemorrhage or necrosis.

Figure 1. Minimal acute cellular rejection (A1). The characteristic feature of minimal acute cellular rejection is circumferential infiltration of the perivascular interstitium by mononuclear cell inflammatory infiltrate. This typically involves the small veins and consists of scattered mononuclear cells within loose perivascular connective tissue. No significant expansion of the perivascular interstitium or extension of mononuclear cells into adjacent alveolar septa is present. Haematoxylin and eosin (H&E).
Figure 2. Minimal acute cellular rejection (A1). A sparse mononuclear cell infiltrate is present in the perivascular zones in this example of A1 minimal rejection at right. At left is a nonspecific mononuclear inflammatory infiltrate which fails to show the circumferential cuffing of vessels or the density of mononuclear cells that is sufficient for a diagnosis of minimal acute cellular rejection. H&E.
Figure 3. Minimal acute cellular rejection (A1). In this example of A1 rejection, the vessel at right displays a perivascular mononuclear infiltrate which in one segment of this vessel appears to be circumferential therefore warranting a diagnosis of minimal acute rejection. At left the vessel contains an ill-defined noncircumferential infiltrate of mononuclear cells of low intensity which would be regarded as a nonspecific morphologic finding. H&E.


GRADE A1 (Minimal Acute Rejection)
In Grade A1 acute rejection, there are scattered, infrequent perivascular mononuclear infiltrates in alveolated lung parenchyma (Figures 1, 2 and 3). Blood vessels, particularly venules, are cuffed by small, round plasmacytoid and transformed lymphocytes forming a ring of two or three cells in thickness within the perivascular adventitia. This cuffing may be loose or compact and is generally circumferential. Eosinophils and endothelialitis are not present. The previous grading schemes suggest that these minimal infiltrates are not obvious at low magnification, but it was believed that this criterion can be misleading. Grade A1 infiltrates can be seen at scanning magnification if the specimen is adequately alveolated and free from artifact. The consensus was that evidence of infrequent perivascular infiltrates at low-power (scanning) magnification is not a reliable discriminator between Grade A1 and A2 acute rejection.

Figure 4. Mild acute cellular rejection (A2). In mild acute cellular rejection, the perivascular interstitium of small vessels, venules and arterioles, demonstrates significant circumferential expansion of the perivascular interstitium by mononuclear inflammatory infiltrate. The infiltrate consists largely of mononuclear cells with occasional activated lymphocytes and plasmacytoid lymphocytes. The mononuclear inflammatory infiltrate within the perivascular zones may be accompanied by alveolar macrophages. No infiltration of adjacent alveolar septa by the mononuclear infiltrate is present. H&E.
Figure 5. Mild acute cellular rejection (A2). In this example of mild acute cellular rejection, a tortuous small vessel in the lung parenchyma is cuffed by an inflammatory cell infiltrate which expands and tracks along the perivascular interstitium. The infiltrate remains associated with the perivascular interstitium without infiltration or expansion of adjacent alveolar septa by mononuclear cells. Subendothelial lymphocytic infiltration is also noted in upper right and such endothelialitis is a common finding in mild acute cellular rejection. H&E.
Figure 6. Borderline minimal-mild acute cellular rejection (A1-A2). In this example, a blood vessel is cut along its long axis. For the most part, the lymphoplasmacytic infiltrate is loosely distributed within the perivascular connective tissue. In only one focus is there expansion of the perivascular interstitium by the mononuclear infiltrate and therefore this case would be classified as mild acute cellular rejection even though the majority of this vessel shows minimal changes. H&E.
Figure 7. Borderline minimal-mild acute cellular rejection (A1-A2). This solitary perivascular infiltrate is associated with endothelial cell hyperplasia and expansion of the perivascular interstitium by mononuclear cells. The expansion, however, is rather slight and not pronounced and such a case would fall along the borderline of minimal-mild acute cellular rejection. H&E.


GRADE A2 (mild acute rejection)
In Grade A2 mild rejection, more frequent perivascular mononuclear infiltrates are seen surrounding venules and arterioles and are readily recognizable at low magnification (Figures 4, 5, 6 and 7). They may be densely compacted or loose. These infiltrates usually consist of a mixture of small, round lymphocytes, activated lymphocytes, plasmacytoid lymphocytes, macrophages and eosinophils. Eosinophils are not a feature of Grade A1 minimal rejection. In Grade A2 rejection there is frequently sub-endothelial infiltration by mononuclear cells, which may be associated with hyperplastic or regenerative changes in the endothelium, that is, endothelialitis. In making the distinction between Grade A2 and higher grade acute rejection it is important to note that the perivascular interstitium can be expanded by mononuclear cells in A2 rejection but there is no obvious infiltration by mononuclear cells into the adjacent alveolar septa or air spaces. Concurrent lymphocytic bronchiolitis (see later) may be seen in association with mild acute rejection (Grade A2), but is less common with minimal acute rejection (Grade A1). Mild acute rejection is therefore distinguished from minimal acute rejection by the presence of unequivocal mononuclear infiltrates, which are more easily identified at scanning magnification. In addition, endothelialitis, the presence of eosinophils and co-existent airway inflammation favor mild Grade A2 over minimal A1 acute rejection.

Figure 8. Moderate acute cellular rejection (A3). At scanning power, the perivascular mononuclear inflammatory infiltrate within the lung is easily identified. In addition, mononuclear cells are seen to percolate from the perivascular interstitium of small vessels into the alveolar septa where they are accompanied by alveolar pneumocyte hyperplasia. Mononuclear cells percolate into the perivascular airspaces where they are accompanied by a pronounced intraalveolar macrophage reaction. H&E.
Figure 9. Moderate acute cellular rejection (A3). In this transbronchial biopsy, the lung parenchyma appears rather collapsed and atelectatic and yet, the perivascular mononuclear inflammatory infiltrate, with plasmacytoid lymphocytes and occasional eosinophils, expands the perivascular interstitium and extends into alveolar septa resulting in an "interstitial pneumonitis". Alveolar pneumocytes are prominent and endothelialitis is readily identified. H&E.
Figure 10. Moderate acute cellular rejection (A3). The characteristic feature of moderate acute cellular rejection is the expansion of the perivascular interstitium by mononuclear cells and the extension of the same cells into adjacent perivascular alveolar septa. Such cells may extend into the airspaces resulting in collections of macrophages and lymphocytes within alveoli. H&E.
Figure 11. Moderate acute cellular rejection (A3). In almost all cases of moderate acute cellular rejection, subendothelial infiltrates of small round and plasmacytoid lymphocytes are characteristic, often accompanied by eosinophils i.e. endotheliitis or endothelialitis. H&E.


GRADE A3 (moderate acute rejection)
Grade A3 acute rejection shows easily recognizable cuffing of venules and arterioles by dense perivascular mononuclear cell infiltrates, which are commonly associated with endothelialitis (Figures 8, 9, 10 and 11). Eosinophils and even occasional neutrophils are common. This grade is defined by the extension of the inflammatory cell infiltrate into perivascular and peribronchiolar alveolar septa and airspaces, which may be associated with collections of intra-alveolar macrophages in the zones of septal infiltration and Type 2 alveolar cell hyperplasia. The interstitial infiltration can take the form of cells percolating singly into alveolar walls or more sheet-like infiltration with corresponding expansion of the septa. There is continuity with the perivascular infiltrates. True interstitial infiltration characterizing moderate acute rejection should be distinguished from the expansion of the potential space of the perivascular adventitia in mild acute rejection.

Figure 12. Severe acute cellular rejection (A4). At low magnification, the perivascular spaces and alveolar septa are expanded by a mononuclear inflammatory infiltrate, which paradoxically seems less intense than that seen in moderate acute cellular rejection. Percolation of mononuclear cells into the alveolar septa is readily identified and in severe rejection is accompanied by such pronounced alveolar pneumocyte injury that airspace fibrin and hyaline membranes form, with varying degrees of organization. This is accompanied by a nonspecific neutrophilic infiltrate. Such injury to the alveolar septa with fibrin exudation and neutrophil infiltration is characteristic of severe acute cellular rejection. Endothelialitis is almost uniformly seen in these cases. H&E.
Figure 13. Severe acute cellular rejection (A4). In this example of severe acute rejection, perivascular mononuclear infiltrates are seen surrounding a small vessel at upper left and within alveolar septa. Injury to alveolar septa has resulted in hemorrhage and airspace fibrin undergoing varying degrees of organization. H&E.
Figure 10. Moderate acute cellular rejection (A3). The characteristic feature of moderate acute cellular rejection is the expansion of the perivascular interstitium by mononuclear cells and the extension of the same cells into adjacent perivascular alveolar septa. Such cells may extend into the airspaces resulting in collections of macrophages and lymphocytes within alveoli. H&E.


GRADE A4 (severe acute rejection)
In Grade A4 severe rejection there are diffuse perivascular, interstitial and air-space infiltrates of mononuclear cells with prominent alveolar pneumocyte damage and endothelialitis (Figures 12, 13 and 14). These may be associated with intra-alveolar necrotic epithelial cells, macrophages, hyaline membranes, hemorrhage and neutrophils. There may be associated parenchymal necrosis, infarction or necrotizing vasculitis, although these features are more evident on surgical rather than transbronchial lung biopsies. There may be a paradoxical diminution of perivascular infiltrates as cells extend into alveolar septa and spaces where they are admixed with macrophages.

Grade A4 acute rejection must be distinguished from post-transplantation acute lung injury by the presence of numerous perivascular and interstitial mononuclear cells, which are not a feature of reperfusion-related damage.

In summary, the diagnosis of acute rejection is based on the presence of perivascular and interstitial mononuclear cell infiltrates. After much debate about the merits or otherwise of collapsing the A1 to A4 grades into fewer grades, the consensus was to retain the existing 5-point system while recognizing that, in most pathologists' experience, Grade A4 is uncommon. The nature of the tissue damage in Grade A4, however, was identified as having a potential relationship with an antibody-mediated form of acute rejection (see later) and therefore potentially useful in contributing to further understanding of lung rejection in the future, albeit in an infrequently diagnosed grade. The histopathology task force also recommended that perivascular infiltrates related to acute rejection should be truly circumferential and that incomplete vascular cuffing is unlikely to represent acute rejection. It is advised that further samples, deeper serials or levels into the tissue block should be obtained when the infiltrates are equivocal to discriminate both between rejection and non-rejection pathology and between the various grades of acute cellular rejection.

The participants also noted that the transbronchial biopsy diagnosis of acute rejection represents but one component of an integrated approach to the assessment of lung allograft recipients. The diagnosis of acute lung rejection therefore requires integration with clinical and particularly microbiologic data.8 In relation to the treatment of acute rejection the task force noted that different clinical groups have different therapeutic algorithms and that, since the 1996 working formulation, the potential long-term significance of Grade A1 minimal acute rejection has emerged.9-11 It was decided to retain this minimal grade for further evaluation in light of better guidance for its recognition.




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