Revision of the 1996 Working Formulation for the Standardization
of Nomenclature in the Diagnosis of Lung Rejection
GENERAL RECOMMENDATIONS
Adequacy of Specimens
Transbronchial biopsy has been the mainstay of lung
allograft evaluation. It was again the uniform opinion of
the consensus meeting that at least five pieces of
well-expanded alveolated lung parenchyma are required
for an assessment of acute rejection. The bronchoscopist
may need to submit more than five biopsies
to provide this minimum number of adequately alveolated
pieces, and possibly further biopsies if small
bronchioles are required to be present. A strip of
bronchus may be attached to the alveolated parenchyma
and this should be distinguished from bronchiolar
tissue. Specimens can be gently agitated in formalin
to inflate the fragments and require tender handling in
the laboratory to avoid crush artifacts that can render
interpretation difficult or nearly impossible.
Histologic Examination
Histologic examination should include a minimum of
sections from three levels of the paraffin block for
hematoxylin and eosin (H&E) staining with connective
tissue stains to evaluate any sub-mucosal fibrosis, essential
for the diagnosis of bronchiolitis obliterans and
arteriosclerosis. Silver stains can be performed for
fungi, including pneumocystis, but have not been absolutely
mandated by the group in view of the numerous
microbiologic, serologic and molecular techniques
presently in use for the diagnosis of opportunistic
infections in these patients. Beyond this minimum H&E and connective tissue stain work-up, investigators may
wish to augment their evaluation with histochemical,
immunohistochemical and in situ hybridization studies.
Bronchoalveolar lavage may be performed at the time of
biopsy and is useful for the exclusion of infection and
for research investigations, but it has no clinical role in
the diagnosis of acute rejection.
Differential Diagnosis of Perivascular and Interstitial Infiltrates
Perivascular mononuclear infiltrates are not specific for
acute rejection and many other conditions may simulate
or mimic alloreactive lung injury.27 Differential diagnostic
considerations include cytomegalovirus pneumonitis,
Pneumocystis jiroveci (previously P carinii) pneumonia
and post-transplantation lymphoproliferative disease,
which can itself range from pneumonitis to active
lymphoproliferation with tumor nodules. These conditions
have been described elsewhere. Cytomegalovirus
(CMV) pneumonitis often shows disproportionate alveolar
septal cellular infiltrates as compared with any
perivascular cuffing, and may include perivascular
edema. In addition to infected cells with intranuclear
and intracytoplasmic viral inclusions, the presence of
abundant neutrophils with the formation of microabscesses
and marked atypia of alveolar pneumocytes
may also contribute to the diagnosis.
Molecular and serologic methods for monitoring and
diagnosing CMV disease are also extremely helpful in
suggesting the diagnosis. Transbronchial biopsy, however,
remains the only standard for assessing concomitant
CMV infection/pneumonitis and acute rejection.
Although pneumocystis can exactly mimic acute rejection
with perivascular and interstitial infiltrates, it can
also manifest atypical histologic reactions, including
granulomatous inflammation, diffuse alveolar damage
and foci of necrosis. Granulomatous inflammation is not
a feature of acute rejection and should always raise the
possibility of mycobacterial or fungal, including pneumocystis,
infection. Punctate zones of necrosis should also
raise the possibility of mycobacteria, fungi or herpesvirus
infections rather than acute rejection. Further differential
diagnoses of perivascular and interstitial infiltrates include
recurrent primary disease such as sarcoidosis and, in the
early post-transplant period, reperfusion injury, although
the latter is more often associated with neutrophils and
evidence of acute lung injury.
OTHER NON-REJECTION BIOPSY FINDINGS
Aspiration
The pulmonary allograft is not protected by a cough
reflex and patients are highly predisposed to recurrent
aspiration. Helpful features in making this diagnosis
include the identification of exogenous material with
associated foreign-body giant-cell reaction within the airways and parenchyma (Table 3). Large lipid droplets
and/or macrophages with large vacuoles are helpful
markers of aspiration. Distal organizing pneumonia can
also be seen. Since the last revision of lung rejection
grading, aspiration has emerged as a significant cause of
chronic allograft dysfunction, which may be ameliorated
by treatment.28,29 It can occur early or late after
transplantation and is therefore within the differential
diagnosis throughout the post-operative period.
Organizing Pneumonia
Organizing pneumonia with intra-alveolar fibromyxoid
tissue associated with variable interstitial inflammation
is another common finding in biopsies from lung allografts.
30 It can occur in a variety of clinical contexts and
requires microbiologic correlation where infection is
suspected. Organizing pneumonia can be seen as a
sub-acute form of infectious lung damage. Patchy organizing
pneumonia may also represent reperfusion/ischemic
injury where there may have been evidence of
primary graft failure. The histologic pattern of organizing
pneumonia can also be seen in association with
acute rejection of Grade A3 severity and greater where
there is alveolar extension of the acute inflammatory
response with subsequent organization. Idiopathic/
cryptogenic organizing pneumonia can also manifest
identical histologic features in biopsies from a lung
transplant recipient, but many other causes must be
excluded before the reaction is attributed to an idiopathic
origin.
Large Airway Inflammation
The importance of distinguishing large and small airways
inflammation was again the subject of much
discussion and dissent.7,31 No definite evidence was
produced to support a separation of small and large
airway inflammation as useful in the diagnosis of acute
rejection. Large airway inflammation is most commonly
associated with infection and aspiration (see earlier).
Scarring can be seen in the large airway in addition to
the bronchiolar scarring of bronchiolitis obliterans, but
this feature is regarded as so non-specific as to not warrant a separate comment. However, the presence of
large airway scarring, like the presence of intra-alveolar,
foamy macrophages, can alert the pathologist to the
possibility of obliterative bronchiolitis and the need to
examine further sections.
Bronchus-associated Lymphoid Tissue
Bronchus-associated lymphoid tissue consists of subepithelial
mucosal lymphoid follicles that are distributed
along the distal bronchi and bronchioles. It is
scattered throughout the lung in adults, tending to be
most prominent at the bifurcation points of airways.
The lymphoid follicles contain mainly B lymphocytes
and normally lack true germinal centers. These follicles
are associated with specialized bronchial and bronchiolar
epithelium, which is composed of modified cuboidal,
non-ciliated, non-mucinous cells allowing for the
trans-epithelial migration of antigens and cells.32 Attention
to these histologic features and recognition of the
often prominent vascularity should enable distinction
to be made between bronchus-associated lymphoid
tissue (BALT) and rejection-related airway inflammation.
32,33 BALT is often well circumscribed and may
contain macrophages with particulate matter. There
should be no evidence of epithelial injury, neutrophils
or eosinophils in a BALT collection. BALT aggregates
can trail off into fibrovascular septa and should not be
confused with perivascular or interstitial infiltrates.
Smokers'-type Respiratory Bronchiolitis
In respiratory (smokers') bronchiolitis, biopsies show
an accumulation of tan-colored alveolar macrophages
around respiratory bronchioles. Macrophages may contain
flecks of brown or black material and show Prussian
blue positivity. There may be associated interstitial
thickening and variable accompanying chronic inflammation.
There may be other features of chronic obstructive
pulmonary disease with goblet-cell metaplasia, mucostasis
and bronchiolar metaplasia. This appearance
should be distinguished from rejection-related inflammation
and BALT. The incidence of smokers'-type respiratory
bronchiolitis in transbronchial biopsies from
lung transplants has increased with the expansion of
the donor pool to include smokers' organs. Occasionally,
dust macules/nodules are seen of donor origin. The
persistence of smokers' macrophages in the donor lung
should not be confused with recipient smoking.
Alveolar Septal Fibrosis
Some members of the consensus group had observed
fibrotic thickening of the alveolar septal walls in transbronchial
biopsies from pulmonary allografts and noted
the clinical entity of upper-lobe fibrosis, which has
been described as a newly identified late-onset complication
after lung transplantation.34,35 However, due to the lack of specificity and the difficulty in interpretation
of interstitial fibrosis in transbronchial biopsy specimens
it was considered to be an unhelpful observation.
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