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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