Eosinophilic Globules in Bronchoalveolar Lavage Fluid of Patients With Systemic Sclerosis-Related Interstitial Lung Disease: A Diagnostically Useful, Previously Unreported Finding

Giulio Rossi, MD; Alessandro Andreani, MD; Paola Morandi, MD; Alessandro Marchioni, MD; Paolo Corradini, MD; Gaia Cappiello, MD; Monica Bortolotti, MD; Ardian Qosja, MD; Carlo Manzini, MD; Clodoveo Ferri, MD; Luca Richeldi, MD; Alberto Cavazza, MD


Am J Clin Pathol. 2008;130(6):927-933. 

In This Article


BAL examination is a minimally invasive method to analyze diseases involving the peripheral alveolar regions of the lungs.[3,4,5] In some cases, BAL has true diagnostic usefulness, obviating more invasive procedures. In particular, analysis of BAL samples can be a formidable diagnostic tool to detect infections, malignancies, and some ILDs (eg, Langerhans cell histiocytosis, AP, alveolar hemorrhage, eosinophilic pneumonia, and lipoid pneumonia).[6,7,8,9,16,17,18] In general, the interpretation of BAL results must always be integrated with clinical, laboratory, and imaging data.[3,4,5,10] In the setting of CVD-related ILDs, the differential cell count may be helpful in correlating inflammatory pattern shown by BAL analysis with various patterns and extent of lung involvement shown by HRCT, as well as in monitoring lung inflammation or excluding complications.[3,4,5] Welker at al[10] demonstrated that a BAL cell count in ILD can be particularly helpful in sarcoidosis and the HP and UIP patterns, but not in other ILD. In SSc, there are controversies as to whether a differential cell count in BAL samples (ie, for neutrophilia and eosinophilia) is a helpful tool for evaluating alveolitis and in identification of patients at high risk of progression of lung disease or for predicting response to therapy with cyclophosphamide.[11,12,13,14,15]

We observed, in a retrospective (14 cases) and prospective (4 cases) manner, the presence of eosinophilic globular deposits of amorphous material in a subset of BAL fluid samples from patients with ILD. These globular structures resulted in diastase-resistant, PAS+, green-colored trichrome staining and did not react with SP-A and collagen IV. It is interesting that the great majority of patients (16 cases) had SSc, whereas ILD with diffuse and severe reticular and honeycombing changes consisting of the UIP pattern (apparently idiopathic, then IPF) characterized the remaining 2 cases.

SSc is a multisystemic autoimmune disease of unknown etiology characterized by microangiopathy and excessive accumulation of extracellular collagen deposition in the skin and several internal organs.[19] In about 70% of patients, pulmonary involvement develops, characterized by ILD with HRCT features closely resembling, if not indistinguishable from, those observed in idiopathic ILD and by vasculopathy eventually leading to pulmonary hypertension.[20] In addition, ILD is the main cause of death in SSc.[21] Of note, a not insignificant rate of patients with apparently idiopathic ILD (particularly with an NSIP pattern) had instead an underlying unknown CVD, with pulmonary manifestations preceding the systemic symptoms.[22] ILD occurs in more than 80% of patients with SSc sine-scleroderma and may likely be confused with IPF.[23]

Previous ultrastructural studies by Harrison et al[24,25] on lung biopsy specimens from patients with SSc demonstrated important pulmonary damage involving epithelial and endothelial structures leading to interstitial edema and excessive collagen deposition in interstitium and alveoli. More recently, Andersen et al[26] found significantly higher levels of total and pro-matrix metalloproteinase-9 (MMP-9), a collagenase, in BAL fluid samples from patients with SSc-related ILD than in samples from patients with SSc without ILD and from healthy subjects. The authors then suggested that alveolar MMP-9 may have a role in lung remodeling, promoting lung fibrosis possibly through MMP-9 production by neutrophils.[26]

In our study, we identified eosinophilic globular structures of uncertain nature (but possibly consisting of collagen material as highlighted by the trichrome stain) in 16 of 50 BAL fluid samples from a subgroup of patients with SSc-related ILD. The presence of these globules was also observed in 2 patients with an apparently idiopathic UIP pattern shown by HRCT, but not in several other ILDs of different causes. In addition, eosinophilic globules in SSc were significantly associated with BAL neutrophilia and eosinophilia and with more severe ILD (honeycombing) shown by HRCT; both conditions characterize a clinicoradiologic subset of patients with extensive ILD in SSc.[12,13,14,15]

In some cases, the acellular material in the background of BAL fluid may be the key diagnostic component for a specific disease, as in pulmonary AP, diffuse alveolar hemorrhage, asbestosis, Pneumocystis infection, pulmonary alveolar microlithiasis (PAM), and amyloidosis. However, eosinophilic globular deposits described in this report need to be mainly differentiated from globular amorphous materials usually observed in AP Image 2A and Image 2B, Pneumocystis infection Image 2C and Image 2D, PAM, and pulmonary amyloidosis.[27,28,29,30,31,32]

A, Globular deposits of periodic acid–Schiff (PAS)+ material in a patient with alveolar proteinosis (AP) (×200). B, Globular deposits in AP are grossly granular and rounded but characterized by a poorly cellular, dirty background with PAS-diastase+ foamy macrophages (×200). C, Frothy, ill-defined material in Pneumocystis infection within a necrotic and inflammatory background (H&E, ×100). D, Silver methenamine staining highlights the presence of numerous round organisms consistent with Pneumocystis species (Grocott, ×100).

AP is a rare lung disease characterized by excessive accumulation of surfactant-derived phospholipids and proteins into the alveoli.[3,5,17,18,27,28,29] An autoimmune pathogenesis related to the presence of anti–granulocyte-macrophage colony-stimulating factor with consequent impaired surfactant phagocytic function of the alveolar macrophages has been suggested. The disease may be congenital, idiopathic, or secondary to several conditions (ie, dust inhalation, infections, and malignancies), frequently has a characteristic "crazy-paving" pattern shown by HRCT, and BAL fluid derived from patients with AP generally appears turbid and milky.[3,5,28]

BAL cytologic features are often diagnostic in these cases and show a dirty background with an amorphous PAS+ granular substance, foamy macrophages with intracellular inclusions, and acellular diastase-resistant PAS+ globules.[3,5,17,18,28,29,30] Apart from the different clinicoradiologic scenario, BAL fluid with eosinophilic globules described herein was grossly limpid, associated with an inflammatory component consisting of granulocytes and eosinophils, without a dirty background. In contrast with globules in AP, those reported here are negative for surfactant proteins.[3,5,17,18,28,29,30]

Eosinophilic globules did not stain with PE-10, a monoclonal antibody recognizing the SP-A (the most abundant protein in lung surfactant) but not SP-B, SP-C, or SP-D. Although this finding cannot entirely exclude that these globules might be related to surfactant, it seems a very unlikely possibility.

Frothy eosinophilic material with "bubbles" in an inflammatory-to-necrotic background is often observed in Pneumocystis infection, a disease occurring in association with the defective immunity in several underlying conditions, from AIDS to immunosuppressive therapies for malignancies, transplantation, or CVD.[9,30] However, isolated or aggregates of round, indented, or helmet-shaped organisms are commonly well evidenced by a methenamine silver stain (ie, Grocott) in the frothy material.[31]

PAM is another rare autosomal recessive heritable pulmonary disorder of uncertain pathogenesis in which mutations involving the SLC34A2 gene have been recently demonstrated.[32,33] PAM is characterized by formation of several microliths in the alveolar spaces and bilateral infiltrates with a sand-like or snowstorm micronodular appearance on radiographs and HRCT.[33] Several spherical to ovoid concentrically laminated microliths with radial striations, ranging from 250 µm to 1 mm, can also be observed in BAL samples.[32] Microliths are mainly composed of calcium and phosphorus and show positive staining with von Kossa stain.[32,33]

Amorphous eosinophilic materials may occasionally be present in pulmonary amyloidosis. However, Congo red stain highlights apple-green birefringence in polarized light.[34]

Finally, corpora amylacea should also be considered in the differential diagnosis. However, these incidental and clinically insignificant eosinophilic structures had a rounded to spherical form with concentric rings and radiating striations from the dark weakly polarized center to the periphery, also staining with PE-10.[35,36]

We first described in this study the presence of eosinophilic globular deposits of uncertain nature (possibly collagen material to be further characterized by immunocytochemical or ultrastructural analysis) in BAL fluid from a subset of patients with extensive and severe ILD, mainly related to SSc (16 of 18), but not in BAL samples from patients with other ILDs of different causes. Based on the presence of these globules, we prospectively suggested a clinically confirmed diagnosis of SSc with pulmonary ILD in 4 cases. In the SSc setting, a statistically significant correlation between the finding of globular deposits in BAL samples and radiologic identification of involvement of ILD was found. Although these preliminary observations need to be validated by further studies, pathologists should be aware that the finding of these eosinophilic globules in BAL samples may indicate an underlying severe SSc-related ILD and may be useful in suggesting this diagnostic possibility, which requires confirmation by integration with clinical and laboratory data.


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