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Diagnosis of Pulmonary Alveolar Proteinosis
Usefulness of Papanicolaou-Stained Smears of Bronchoalveolar Lavage Fluid
Chung-Wei Chou, MD;
Fang-Chi Lin, MD;
Su-Mei Tung, BS;
Rong-Dih Liou, BN;
Shi-Chuan Chang, MD, PhD
Arch Intern Med. 2001;161:562-566.
ABSTRACT
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Background The globules (stained green, orange, or orange in the center coated
with a green rim) seen in Papanicolaou-stained smears of bronchoalveolar lavage
fluid are suggested to be characteristic of pulmonary alveolar proteinosis
(PAP).
Objective To evaluate the usefulness of Papanicolaou-stained smears of bronchoalveolar
lavage fluid in aiding a diagnosis of PAP.
Methods Papanicolaou-stained smears of bronchoalveolar lavage fluid obtained
from 7 patients (5 idiopathic, 2 secondary) with PAP were evaluated. To serve
as controls, the smears of 11 normal subjects and 128 patients with other
pulmonary disorders were also examined. The findings on the presence and number
of globules were recorded. To differentiate PAP from other pulmonary disorders,
the highest globule value obtained from the control group was chosen as the
cutoff point.
Results The characteristic globules were not found in normal subjects and only
found in 6 of 128 patients with other pulmonary disorders. Their clinical
diagnoses were Sjögren syndrome in 2 cases; polymyositis, idiopathic
pulmonary fibrosis, asbestosis, and hypersensitivity pneumonitis in 1 case
each. The numbers of globules in these 6 patients were 1, 3, 17, 7, 3, and
2. In contrast, more than 100 globules were found in all patients with PAP.
The number of globules was highly sensitive and specific in aiding a diagnosis
of PAP when the cutoff value was set at 18.
Conclusion The globules seen in Papanicolaou-stained smears of bronchoalveolar
lavage fluid may be valuable in aiding a diagnosis of PAP, especially when
the number of globules is more than 18.
INTRODUCTION
PULMONARY alveolar proteinosis (PAP) is a rare disease first described
by Rosen et al1 in 1958. It is characterized
by deposition within the air spaces of granular extracellular material composed
of protein and lipids that is periodic acid–Schiff (PAS)-positive and
diastase resistant.2, 3 The onset
of PAP is usually insidious and the initial symptoms are nonspecific, often
causing a significant delay in the diagnosis. Patients with PAP may progress
rapidly into severe respiratory distress. Therefore, a prompt diagnosis with
the initiation of appropriate therapy with bronchoalveolar lavage (BAL) has
clinical merit.
A diagnosis of PAP is based mainly on histopathologic findings obtained
with transbronchial lung biopsy (TBLB) or open lung biopsy specimens. However,
TBLB specimens are often so small that they do not provide specific evidence.
In a 1998 article,4 the diagnostic yield of
a TBLB specimen was only 29% in 68 patients with PAP. Bronchoalveolar lavage
has proved valuable in providing a diagnosis of PAP as reported in some articles5, 6, 7, 8, 9, 10;
however, ultrastructural examination of BAL fluid (BALF) specimens and/or
specific stains such as PAS and combined alcian blue and PAS stains are usually
required.6, 8, 9, 10, 11
Clinically, it is impractical to process all routine BALF specimens for electron
microscopy or special stains.
Cytologic findings of BALF specimens from patients with PAP have been
reported,5, 6, 7, 8, 9, 10
but the results of Papanicolaou-stained smears are insufficient for a definite
diagnosis. A 1997 study9 suggested that a routinely
processed cytologic smear of BALF with a Papanicolaou stain could be valuable
in providing a diagnosis of PAP. The globules (stained green, orange, or orange
in the center coated with a green rim) seen in Papanicolaou-stained smears
as seen under a light microscope are the multilamellated structures characteristic
of PAP as seen under an electron microscope and can be used as diagnostic
evidence of PAP. To evaluate the usefulness of Papanicolaou-stained cytologic
smears of BALF in aiding a diagnosis of PAP, 7 patients with PAP, 11 healthy
subjects, and 128 patients with other pulmonary disorders were studied.
PATIENTS AND METHODS
From February 1997 to April 2000, 7 (5 idiopathic, 2 secondary) consecutive
patients with PAP were included in this study. The diagnosis of PAP was established
by pathologic examination of TBLB specimens with or without electron microscopy
in 7 patients. In all 7 patients, BAL was performed to show the presence of
PAS-positive intra-alveolar material. The clinical information about the patients
with PAP is given in Table 1.
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Table 1. Clinical Data of 7 Patients With Pulmonary Alveolar Proteinosis*
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Bronchoalveolar lavage was performed prior to bronchial brushing or
TBLB through a fiberoptic bronchoscope (model BF 20 or P20; Olympus, Tokyo,
Japan) wedged in a segmental bronchus of the right middle lobe or lingula
or other appropriate location, using 3 aliquots of a 50-mL sterile isotonic
sodium chloride solution. Aspirates were pooled into a siliconized container
and kept on ice during transport. Part of the retrieved BALF was subjected
to Papanicolaou and Riu staining. Some slides were stored for subsequent staining
by PAS and combined alcian blue and PAS.
To serve as controls, Papanicolaou-stained cytologic smears of BALF
specimens from 11 healthy subjects and 128 patients with other pulmonary disorders
were studied. To avoid bias, BAL was done by one of us (S.-C.C.) following
the aforementioned protocol and the retrieved BALF was processed using the
same protocol. Those with unknown entities of their pulmonary disorders were
excluded. The clinical diagnoses of 128 patients with other pulmonary disorders
are summarized in Table 2.
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Table 2. Clinical Diagnoses of 128 Patients With Various Pulmonary
Disorders
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After a demonstration of characteristic globules seen in a Papanicolaou-stained
smear of BALF specimen from the reported case, all Papanicolaou-stained smears
were screened first by cytologic technicians (S.-M.T. and R.-D.L.), then were
read separately by 2 physicians (C.-W.C. and F.-C.L.). Both cytologists and
physicians were blind to the final diagnoses when they evaluated the smears.
The findings on the presence and number of globules were recorded separately
and then discussed, and the consensus findings were documented for the study.
When the 2 readers could not reach consensus, the case was presented to a
third expert reader (S.-C.C.), and the adjudicated reading became the final
one.
REPORT OF A CASE
A 40-year-old housewife was admitted to the hospital in February 1997
because of exertional dyspnea for 1 year and shortness of breath for 2 months.
She was found to have hypertension after pregnancy in 1990 and received antihypertensive
treatment as needed. For 3 years she also had type 2 diabetes mellitus treated
by diet only. One year prior to this admission, she began to experience exertional
dyspnea and was admitted to another hospital 4 months later. After a series
of examinations, idiopathic pulmonary fibrosis was diagnosed and the patient
underwent steroid treatment. Two months before the admission, productive cough
with whitish sputum, chest tightness, and shortness of breath developed in
addition to progression of exertional dyspnea. On admission, a frontal chest
radiograph revealed diffuse pulmonary infiltrates in both lungs, with lower
lung fields predominantly affected. Thoracic high-resolution computed tomography
revealed a diffuse ground-glass appearance in both lungs and a paved-stone
arrangement. The findings of radiographic studies were in keeping with those
of PAP. An elevated serum lactate dehydrogenase level (389 U/L) was noted.
Pulmonary function testing showed mildly restrictive ventilatory defect with
marked reduction of diffusing capacity for carbon monoxide (DLCO, 43 % of
predicted value). Results of arterial blood gas studies, measured while the
patient was breathing room air, were as follows: PaO2, 47.9 mm
Hg; PaCO2, 34.6 mm Hg; and pH, 7.412. The patient underwent bronchoscopy
with BAL and TBLB. Bronchoalveolar lavage was performed via a segmental bronchus
of the right middle lobe, and TBLB was done via the right lower lobe after
BAL. In gross appearance the retrieved lavage fluid was opaque and milky.
A Papanicolaou-stained cytologic smear of BALF showed numerous, dense, amorphous
globules stained green, orange, or orange in the center coated with a green
rim dispersed within a background of finely granular amorphous material devoid
of inflammatory cells (Figure 1,
A). These globules appeared to be dark blue in a Riu-stained12
(a modified Wright-stain) smear (Figure 1, B). Strong positive PAS staining was noted in a PAS-stained smear.
The combined alcian blue and PAS stain was performed later and showed globules
with strong PAS staining of the proteinaceous material with a lack of significant
alcian blue staining (Figure 1,
C). Pathologic examination of TBLB specimens evidenced a diagnosis of PAP.
Recognition of characteristic cytologic features of BALF specimens seen in
the Papanicolaou-stained smear from this patient with PAP prompted us to evaluate
the usefulness of Papanicolaou-stained smears of BALF in aiding a diagnosis
of PAP.
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A, Papanicolaou-stained smear of bronchoalveolar lavage fluid shows
the globules stained green, orange, or orange in the center coated with a
green rim (original magnification x400). B, The globules appear to be
dark blue in the Riu-stained smear (original magnification x400). C,
In the combined alcian blue and PAS (periodic acid–Schiff)-stained smear,
the globules show strong PAS staining with a lack of alcian blue staining
(original magnification x400).
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RESULTS
The cytologic features in 11 BAL samples from 7 patients with PAP were
similar. The gross appearance of the retrieved BALF was opaque and/or milky
in all specimens and sediment was abundant. The cells were poorly preserved
and abundant granular extracellular material was present. Total cell and differential
cell counts could not be measured in 9 specimens obtained from 5 patients
with idiopathic PAP.
Viewed under a light microscope, Papanicolaou-stained smears of BALF
obtained from patients with PAP contained abundant amorphous granular and
clumped extracellular material with a scarcity of cells. There were numerous
globules stained green, orange, or orange in the center coated with a green
rim interspersed in the amorphous material (Figure 1, A). The globules seen as solitary elements in Papanicolaou-stained
smears were more than 100 globules per slide in all 7 patients with PAP. These
globules appeared to be dark blue in Riu-stained smears (Figure 1, B). The globules and the amorphous material were PAS positive.
Combined alcian blue and PAS-stained smears showed the globules with positive
PAS staining of the proteinaceous material with a lack of significant alcian
blue staining (Figure 1, C) in all
patients with PAP.
In contrast, the globules could not be found in Papanicolaou-stained
smears of BALF obtained from 11 healthy subjects. Furthermore, the globules
were only found in 6 of 128 patients with other pulmonary disorders. The clinical
diagnoses of the 6 patients were as follows: Sjögren syndrome in 2 cases
and polymyositis, idiopathic pulmonary fibrosis, asbestosis, and hypersensitivity
pneumonitis in 1 case each. The numbers of globules in these 6 patients were
1, 3, 17, 7, 3, and 2, respectively. Compared with those who had PAP, the
smears obtained from patients with other pulmonary disorders revealed fewer
globules, a small amount of extracellular material, and a greater number of
cells. Globules with positive PAS staining were not found in combined alcian
blue and PAS-stained smears of BALF obtained from all 6 patients with other
pulmonary disorders.
The highest globule value seen in Papanicolaou-stained smears of BALF
obtained from the control group was 17. To distinguish PAP from other pulmonary
disorders, the value of 18 globules was chosen as a cutoff point. Using this
cutoff point, the globules seen in Papanicolaou-stained smears seemed to be
highly sensitive and specific in aiding a diagnosis of PAP (Table 3).
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Table 3. The Number of Globules in Papanicolaou-Stained Smears of Bronchoalveolar
Lavage Fluid Specimens*
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COMMENT
Pulmonary alveolar proteinosis is a disease characterized by phospholipoprotein
material filling in the alveoli and terminal bronchial trees. Its diagnosis
is mainly based on histopathologic findings obtained with TBLB or open lung
biopsy specimens. However, the diagnostic yield of TBLB is usually unsatisfactory.3, 4 Open lung biopsy may fail to ensure
a diagnosis owing to sampling error and may not be without morbidity or mortality.
Compared with TBLB and/or open lung biopsy, BAL can sample a much wider area
and is safer for the patient.
With the advent of improved bronchoscopic techniques, BAL has become
the standard for both the diagnosis9, 13
and therapy3, 14, 15, 16
for PAP. The retrieved BALF of PAP is grossly opaque, milky white, or muddy.
In Papanicolaou-stained smears of BALF specimens, the globules stained green,
orange, or orange in the center with a green rim have been described to be
characteristic of PAP.6, 7, 8, 9
However, the characteristic cytologic findings in Papanicolaou-stained smears
are considered insufficient for a diagnosis of PAP and ultrastructural examination
of BALF is usually needed.6, 8, 9, 10, 11
Clinically, electron microscopy and special stains like PAS and combined alcian
blue and PAS were not routinely performed for all BALF specimens because a
diagnosis of PAP is not usually expected. This may explain in part why in
most of the reported cases the diagnosis of PAP was established by open lung
biopsy or often missed prior to autopsy.
It is of clinical significance if a routinely processed cytologic-staining
method like a Papanicolaou stain proves to be useful in providing a diagnosis
of PAP. The findings of a 1997 article9 confirmed
that the globules stained green, orange, or orange in the center coated with
a green rim seen in Papanicolaou-stained smears of BALF specimens were the
multilamellated structures, characteristic of PAP when viewed under an electron
microscope and concluded that these globules can be used as diagnostic evidence
of PAP.
At variance with the results of the study by Mikami et al,9
the findings in our study indicated that the globules were not exclusively
found in patients with PAP. The globules described in Papanicolaou-stained
smears of BALF could be found in 6 of 128 patients with other pulmonary disorders.
Their clinical diagnoses were Sjögren syndrome in 2 cases and polymyositis,
idiopathic pulmonary fibrosis, asbestosis, and hypersensitivity pneumonitis
in 1 case each. The number of globules in these 6 patients was 1, 3, 17, 7,
3, and 2, respectively. Of clinical interest, the globules with positive PAS
staining were not found in the combined alcian blue and PAS-stained smears
obtained from all 6 patients with other pulmonary disorders. This may be explained
in part by the scarcity of globules. Another explanation is that the globules
lack protein.
Compared with those who had other pulmonary disorders, the globules
were numerous and there were more than 100 globules per slide in all 7 patients
with idiopathic or secondary PAP. In addition, the presence of a large amount
of amorphous and/or granular extracellular material and a scarcity of cells
seemed to be valuable in distinguishing PAP from other pulmonary disorders.
Furthermore, globules with positive PAS staining seen in combined alcian blue
and PAS could be found in all 7 patients with PAP. These findings can be of
clinical significance in differentiating PAP from other pulmonary disorders
and in supporting a diagnosis of PAP.
It is important that a differential diagnosis be made between PAP and Pneumocystis carinii pneumonitis. In P carinii pneumonitis, the Papanicolaou-stained smears of BALF specimens
contain characteristic findings described as a foamy mass or foamy exudate.17, 18 The globules characteristic of PAP
can be easily differentiated from this foamy mass or exudate described in P carinii pneumonitis by Papanicolaou staining. When the
differentiation is difficult, combined alcian blue and PAS and Grocott methenamine
silver staining methods can be used to make a differential diagnosis.
Immunosuppression seems to play a pivotal role in the defective alveolar
clearance that subsequently leads to PAP.2
With the increased use of immunosuppressive and/or cytotoxic agents to treat
a variety of immune-related diseases, the improved long-term survival of the
organ transplants, and the improved outcome of the patients with different
malignant neoplasms undergoing chemotherapy, secondary PAP can be expected
to occur more frequently. It is important to have a high index of clinical
suspicion to make a diagnosis. The characteristic gross appearance of a retrieved
BALF specimen can be of help but may be easily missed owing to limited experience.
Special stains for detecting PAS-positive material in a BALF specimen may
not be done as needed. These may cause a significant delay in diagnosis or
underdiagnosis of PAP.7 The presence of numerous
globules in routinely processed Papanicolaou-stained smears of BALF specimens
can be valuable in aiding a diagnosis of PAP as described in this article.
A diagnosis of PAP can be further confirmed by PAS stain, combined alcian
blue and PAS stain, or electron microscopy of BALF specimens. To avoid an
unnecessary delay or miss in making a diagnosis of PAP, it is suggested to
reserve some slides of BALF specimens for the patient undergoing BAL.
CONCLUSIONS
Our results suggested that the globules (stained green, orange, or orange
in the center coated with a green rim) seen in Papanicolaou-stained smears
of a BALF specimen can be of significant value in making a diagnosis of PAP,
especially when the number of globules is more than 18. Other cytologic findings
on the presence of a large amount of granular and/or amorphous extracellular
material and the scarcity of cells may further support a diagnosis of PAP.
When diagnosis is difficult, special stains like PAS and combined alcian blue
and PAS stains or electron microscopy of BALF specimens may confirm the diagnosis
of PAP.
AUTHOR INFORMATION
Accepted for publication September 14, 2000.
From the Chest Department, Veterans General Hospital–Taipei (Drs
Chou, Lin, and Chang and Mss Tung and Liou), and the School of Medicine, National
Yang-Ming University (Dr Chang), Taipei, Taiwan.
Corresponding author: Shi-Chuan Chang, MD, Chest Department, Veterans
General Hospital–Taipei, 201 Section 2, Shih-Pai Road, Shih-Pai, Taipei,
Taiwan 112, Republic of China (e-mail: scchang{at}vghtpe.gov.tw).
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