A 3-month-old full term Hispanic infant girl presented with severe interstitial lung disease. She was
delivered by Cesarean section after an uneventful pregnancy and had no perinatal complications,
discharged on the fourth day of life. She presented initially at 10 weeks of age with respiratory
distress including nasal flaring, retractions, tachypnea, and hypoxia. She was afebrile without cough
and there were no concurrent illnesses in family members. Chest x-ray showed bilateral interstitial
infiltrates with consideration of bronchiolitis versus bilateral pneumonia. Chest CT showed heavy
ground-glass appearance consistent with interstitial infiltrates or alveolitis. She received a two-week
course of antibiotics and also steroids, bronchodilators, and supplemental oxygen during her course, but
her respiratory status continued to deteriorate. Other evaluation included an esophagram showing
frequent episodes of gastroesophageal reflux, normal echocardiogram, negative HIV serology, negative
cystic fibrosis mutation analysis, and a bronchoalveolar lavage with increased lipid-laden macrophages
and negative cultures. One month after initial presentation, she underwent an open lung biopsy.
Case 3 - Figure 6 -
Bronchoalveolar lavage (Wright-Giemsa). Amorphous proteinosis material appears basophilic on Giemsa stain.
Case 3 - Figure 7 -
Bronchoalveolar lavage (Wright-Giemsa). The cellular components included many alveolar macrophages including some with orangophilic droplets, as well as occasional reactive alveolar epithelial cells. Increased lipid-laden macrophages and occasional hemosiderin-laden macrophages were noted.
Case 3 - Figure 1 -
Lung biopsy (H&E). The alveolar architecture is altered with mild lobular remodeling and airspace enlargement.
Case 3 - Figure 2 -
Lung biopsy (H&E). Patchy areas of eosinophilic globular and granular proteinosis material fill the airspaces.
Case 3 - Figure 3 -
Lung biopsy (H&E). The alveolar proteinosis is accompanied by diffuse type II alveolar epithelial cell hyperplasia.
Case 3 - Figure 4 -
Lung biopsy (H&E). Scattered alveolar macrophages and occasional cholesterol clefts are present within areas of granular proteinosis material.
Case 3 - Figure 5 -
Lung biopsy (PAS). The alveolar material is PAS-positive, typical of alveolar proteinosis.
Case 3 - Figure 8 -
Lungs (Gross). At autopsy, the lungs showed subpleural stippled yellow material corresponding to alveolar lipoproteinosis material seen histologically.
Case 3 - Figure 9 -
Non-specific interstitial pneumonia pattern. Lung biopsy in 6 year old with chronic lung disease and ABCA3 mutations (H&E). Mild interstitial lymphocyte infiltrates with reactive alveolar epithelial hyperplasia, clusters of alveolar macrophages, and extension of airway smooth muscle into the lobular interstitium.
Case 3 - Figure 10 -
Non-specific interstitial pneumonia pattern. Lung biopsy in 6 year old with chronic lung disease and ABCA3 mutations (H&E). Subpleural cholesterol clefts and clusters of foamy macrophages associated with lobular remodeling, similar to endogenous lipoid pneumonia.
Case 3 - Figure 11 -
Non-specific interstitial pneumonia pattern. Lung biopsy in 6 year old with chronic lung disease and ABCA3 mutations (PAS). Rare PAS-positive alveolar material.
Case 3 - Figure 12 -
Chronic pneumonitis of infancy pattern. Lung biopsy in 3 month old infant with SP-C mutation (H&E). Infants with SP-C mutations tend to have more prominent lobular remodeling and extension of airway smooth muscle into the lobular interstitium. Cholesterol cleft formation may be prominent.
Case 3 - Figure 13 -
Chronic pneumonitis of infancy pattern. Lung biopsy in 3 month old infant with SP-C mutation (H&E). Alveolar proteinosis material is inconspicuous relative to SP-B or ABCA3 mutations.
Surfactant Dysfunction Disorder (Pulmonary Alveolar Proteinosis Pattern)
Acquired (Secondary) Pulmonary Alveolar Proteinosis
Pneumocystic Jiroveci Pneumonia
Endogenous Lipoid Pneumonia
Lung biopsy findings:
The lung biopsy shows diffuse parenchymal disease with mild lobular remodeling and diffuse, focally
pronounced, alveolar epithelial hyperplasia. The alveoli show mild variability in size with mild
widening of the interstitium by fibroblasts. Interstitial inflammatory cells are inconspicuous. Within
the alveolar spaces are diffusely scattered aggregates of foamy macrophages, granular eosinophilic
material, and globular dense eosinophilic material. A few cholesterol clefts are also noted admixed with
the granular material. Periodic acid Schiff (PAS) stains with and without diastase were positive in the
areas of eosinophilic material, confirming the presence of pulmonary alveolar proteinosis. The airways
and vasculature are unremarkable, as is the pleura. There was no evidence an inflammatory process or
aspiration. The diagnosis of pulmonary alveolar proteinosis consistent with surfactant dysfunction
disorder was made.
Additional studies and hospital course: Surfactant Dysfunction Disorders
Cytologic examination of bronchoalveolar lavage (BAL) fluid showed abundant alveolar macrophages and
amorphous basophilic material in the background on Giemsa stains. Increased lipid-laden macrophages and
occasional hemosiderin-laden macrophages were noted, as well as occasional reactive alveolar epithelial
cells. Surfactant protein analysis of the bronchoalveolar lavage fluid showed adequate amounts of
surfactant protein B (166 ng/mL) and surfactant protein A (18 mcg/mL). Analysis of the SP-B gene showed
no evidence of the most common mutation (121ins2), and sequencing of the SP-C gene also showed no
Her course was complicated by recurrent pneumothoraces and thoracostomy tube placement. Due to her
poor prognosis, ventilatory support was eventually withdrawn, and she died approximately five weeks after
initial presentation and eleven days after lung biopsy. At autopsy, the lungs were firm and heavy,
weighing 233.6 grams together (72.0 grams expected for age). The external surfaces showed areas of
speckled yellow material in the subpleural airspaces corresponding to alveolar proteinosis material. The
histologic features were similar to that seen on biopsy with a pattern of alveolar proteinosis and mild
lobular remodeling, as well as interstitial emphysema and focal hemorrhage in the left upper lobe.
Electron microscopy of lung tissue showed inadequate tissue preservation for evaluation of lamellar body
structure. Though the histologic features clearly demonstrated a pattern typical of inherited defects in
surfactant metabolism, the specific etiology remained unclear at that time.
Less than two years after autopsy, mutations in ABCA3 gene were described as a cause of fatal
surfactant deficiency in newborns. Sequencing of the ABCA3 gene was performed retrospectively in this
case and demonstrated two mutations: a single base insertion in exon 20 (1644insC) causing a frameshift
mutation and a missense mutation in exon 25 resulting in an amino acid substitution (P1263S),
definitively identifying this as the cause of surfactant dysfunction in this case.
Surfactant dysfunction disorders are a group of lung diseases occurring predominantly in infants and
children which are caused by inherited mutations in genes affecting surfactant metabolism. This group
now includes mutations in three known genes: surfactant protein B (SFTPB, chromosome 2p12-p11.2),
surfactant protein C (SFTPC, chromosome 8p21), and most recently ATP-binding cassette sub-family A member
3 (ABCA3, chromosome 16p13.3). Mutations in SP-B and ABCA3 genes are inherited in an autosomal recessive
fashion, while mutations in SP-C have an autosomal dominant inheritance pattern, manifesting as chronic
lung disease in successive generations. While mutations in these three genes explain the majority of
cases with findings consistent with surfactant dysfunction, a subset of cases with typical clinical and
histologic features remains unexplained, suggesting the presence of other unrecognized causative genes.
The histologic manifestations of surfactant dysfunction disorders in infancy include two major
patterns: pulmonary alveolar proteinosis and chronic pneumonitis infancy. Mutations in SP-B typically
result in a classic pulmonary alveolar proteinosis pattern with alveolar granular eosinophilic material
and little evidence of lobular remodeling. These children typically die in the neonatal period or early
infancy. In contrast, infants with mutations in SP-C gene typically show a pattern of chronic
pneumonitis of infancy, with less proteinosis material and more prominent cholesterol clefts and lobular
remodeling. SP-C mutations have also been recognized in some families as a cause of interstitial
pneumonia and pulmonary fibrosis in adults. Mutations in ABCA3 often result in the pulmonary alveolar
proteinosis pattern in infancy, but also may show a desquamative interstitial pneumonia (DIP) pattern in
some infants and non-specific interstitial pneumonia (NSIP) pattern in older children. While mutations
in SP-B have relatively uniform manifestations early in infancy, mutations in SP-C and ABCA3 appear to
have a wider clinical and histologic spectrum, which is likely age-dependent and perhaps also
mutation-dependent, though detailed genotype-phenotype correlations with large numbers of patients are
not available to date.
In many cases, the characteristic constellation of histologic features allows the surgical pathologist
to recognize a congenital surfactant dysfunction disorder. The diagnosis may be supported by use of
immunohistochemistry and/or electron microscopy. Immunohistochemistry using antibodies to SP-A, SP-B,
proSP-B, and proSP-C proteins has been applied, though it is not used for routine diagnostic purposes in
our institution. A similar immunohistochemical staining pattern has been described for both SP-B and
ABCA3 deficiency, characterized by robust SP-A and proSP-B staining in both alveolar epithelium and
intra-alveolar material, robust SP-C staining restricted to alveolar epithelial cells, and only weak SP-B
staining. In contrast, patients with SP-C deficiency have robust mature SP-B and proSP-B staining but
deficient proSP-C staining. Electron microscopy should be performed in cases suspected to represent
surfactant dysfunction disorders to document abnormal lamellar body structure associated with SP-B and
ABCA3 mutations. Patients with SP-B deficiency typically have deficient mature lamellar bodies with
increased multivesicular and multilamellated structures. Patients with ABCA3 mutations may have
distinctive electron-dense bodies associated with structures resembling abortive and condensed lamellar
bodies. These round dense bodies have been described as having a "fried-egg" appearance and, if present,
are highly characteristic of ABCA3 abnormalities. The lamellar bodies in patients with SP-C deficiency
are typically normal by ultrastructural examination. Definitive diagnosis of these disorders rests on
mutation analysis for SP-B, SP-C, and/or ABCA3 genes. This testing is most often performed on a blood
sample at the time of initial diagnostic evaluation, often performed preceding or concurrent with lung
biopsy. However, lung tissue may also be used. In this regard, appropriate triage of lung biopsy tissue
is a critical component of the diagnostic process. A protocol for handling pediatric lung biopsies
allows appropriate distribution of tissue for ancillary studies, if needed (see below for suggested
protocol). In the setting of infant biopsies, particularly those with clinical suspicion of a
surfactant disorder, priority should be given to retention of tissue in glutaraldehyde for electron
microscopy and snap freezing tissue for possible molecular studies.
ABCA3 and ATP-binding cassette transporters:
The ABCA3 gene encodes an ATP-binding cassette transporter which functions in lipid transport. It is
one of a family of ABC transporters involved in lipid metabolism. Mutations in several other ABC genes
are known to cause human disease, including CFTR/ABCC7 (cystic fibrosis), ABCB11 (progressive familial
intrahepatic cholestasis type 2), and ABCA1(Tangier disease). ABCA3 is found at high levels in the lung
and is expressed in type II alveolar epithelial cells, predominantly at the limiting membrane of the
lamellar bodies. The ABCA3 gene has been found to be upregulated after glucocorticoid administration in
animals. The precise function of ABCA3 in surfactant metabolism is not known, however similarity to
other ABC transporters would suggest a role in phospholipid transport and organization during lamellar
body formation. This concept is supported by abnormal lamellar bodies noted by ultrastructural
examination in patients with mutations in this gene (see below). A variety of mutations in ABCA3 gene
have been detected including nonsense and frameshift mutations. Homozygotes or compound heterozygotes
typically have severe respiratory disease and death in infancy. A single mutation with decreased but not
absent ABCA3 function in heterozygotes may explain prolonged survival in some cases.
Prognosis of the surfactant dysfunction disorders varies to some degree upon the gene affected.
Children with SP-B mutations typically die in the neonatal period, usually in the first weeks and months
of life. Similarly, babies with ABCA3 mutations often die in the neonatal period, although some cases
result in chronic lung disease in late childhood. Clinical presentation is somewhat later for
individuals with SP-C mutations and survival is variable. While some patients manifest with chronic
pneumonitis of infancy in the first several months of life, other individuals survive to adulthood with
chronic lung disease, often manifesting as either non-specific interstitial pneumonia or idiopathic
pulmonary fibrosis. There is no effective therapy for the inherited surfactant dysfunction disorders
other than supportive measures and lung transplantation. Administration of surfactant in the neonatal
period does not typically result in clinical improvement.
Future advances in this heterogenous set of diseases will likely come from increased understanding of
the biologic mechanisms of disease, recognition of other causative genes, and genotype-phenotype
correlation for further prognostication. The potential for these mutations to act as disease modifiers
also merits further investigation, for example in premature infants with chronic neonatal lung disease or
in older children with other forms of concurrent lung injury.
Acquired pulmonary alveolar proteinosis:
The principal differential diagnosis, particularly in older infants and children, is acquired
(secondary) pulmonary alveolar proteinosis. Acquired proteinosis occurs by two general mechanisms: (1)
presence of antibodies to GM-CSF, and (2) macrophage dysfunction. Cases of acquired alveolar proteinosis
may occur in patients with immunodeficiencies, leukemia, after chemotherapy, and associated with
infections. While inherited surfactant dysfunction disorders show widespread proteinosis, acquired
alveolar proteinosis tends to be patchy in its distribution and has a more even finely granular
appearance relative to the coarse globules often seen in the congenital surfactant disorders.
Another diagnostic consideration in the setting of alveolar proteinosis includes Pneumocystis jiroveci pneumonia, which is typically associated with similar
alveolar granular eosinophilic material. The alveolar material seen in the setting of Pneumocystis pneumonia typically has a more foamy or vacuolated appearance as
compared to the surfactant dysfunction disorders, and does not result in large globular protein,
cholesterol cleft formation, or the prominent alveolar epithelial hyperplasia associated with the
surfactant disorders. The diagnosis is typically easily clarified by silver stain for fungus. Clinical
history of immunodeficiency is also helpful, though Pneumocystis may be the
initial presenting finding in newly diagnosed congenital immunodeficiencies in infants.
Endogenous lipoid pneumonia:
Endogenous lipoid pneumonia refers to a pattern of foamy macrophages and cholesterol cleft formation
which occurs due to cell breakdown after lung injury or poor clearance of secretions and cellular
debris. In children, such "cholesterol granulomas" may result from aspiration, obstructive airway
disease, hemosiderosis or resolving hemorrhage, and quite often in the setting of chronic neonatal lung
disease. In the latter, it is thought that the architectural remodeling and enlargement of peripheral
airspaces results in stasis and poor clearance of secretions. PAS-positive proteinosis material and
diffuse marked alveolar epithelial cell hyperplasia is absent.
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Suggested Protocol for Handling Pediatric Lung Biopsies
Bacterial, fungal, acid fast, and viral cultures, as appropriate.
Several 1 mm pieces in glutaraldehyde for potential ultrastructural examination.
One section snap frozen without OCT for potential molecular studies.
One section injection inflated with OCT/sucrose mixture using a tuberculin syringe or other fine needle. Tissue is embedded in OCT and snap frozen at –70 C.
At least 50% of the tissue is slowly inflated with formalin by transpleural injection using a tuberculin syringe, until fully distended. Adequate expansion of alveolar parenchyma is critical for histologic assessment, particularly with regard to alveolar growth abnormalities and interstitial disease. The tissue is allowed to fix for at least 20 minutes and then serially sectioned perpendicular to the pleural edge for histologic sections. Sectioning perpendicular to the pleural edge allows proper orientation and assessment of differences in peripheral and more proximal airways and alveoli.
Note: If the wedge biopsy is small, tissue should be distributed as most appropriate for clinical working diagnosis. In cases of suspected surfactant disorders, priority should be given to electron microscopy and snap freezing tissue without OCT.
Molecular Diagnosis of Surfactant Dysfunction Disorders
Mutation testing available for: SP-B (clinical), SP-C (clinical), ABCA3 (research)
Dr. Lawrence M. Nogee and Dr. Garry Cutting
DNA Diagnostic Laboratory
Johns Hopkins Hospital
600 N. Wolfe Street
Baltimore, MD 21287-3916