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The utility of bronchoalveolar lavage in the evaluation of interstitial lung diseases: A clinicopathological perspective
Accepted Manuscript
THE Utility of bronchoalveolar lavage in THE EVALUATION OF interstitial lung diseaseS: A CLINICOPATHOLOGICAL PERSPECTIVE Houda Gharsalli MD , Mouna Mlika MD , Imen Sahnoun MD , Sonia Maalej MD , Douik El Gharbi Leila MD , Faouzi El Mezni MD PII: DOI: Reference:
S0740-2570(18)30051-0 https://doi.org/10.1053/j.semdp.2018.08.003 YSDIA 50566
To appear in:
Seminars in Diagnostic Pathology
Please cite this article as: Houda Gharsalli MD , Mouna Mlika MD , Imen Sahnoun MD , Sonia Maalej MD , Douik El Gharbi Leila MD , Faouzi El Mezni MD , THE Utility of bronchoalveolar lavage in THE EVALUATION OF interstitial lung diseaseS: A CLINICOPATHOLOGICAL PERSPECTIVE, Seminars in Diagnostic Pathology (2018), doi: https://doi.org/10.1053/j.semdp.2018.08.003
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ACCEPTED MANUSCRIPT THE UTILITY OF BRONCHOALVEOLAR LAVAGE IN THE EVALUATION OF INTERSTITIAL LUNG DISEASES: A CLINICOPATHOLOGICAL PERSPECTIVE
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, Mouna Mlika, MD , Imen Sahnoun, MD , Sonia Maalej, MD 1,3 2,3 Gharbi Leila, MD , Faouzi El Mezni, MD
1- Department of Pulmonology Department, A.Mami Hospital, Tunis, Tunisia 2- Department of Pathology, Abderrahman Mami Hospital, Ariana, Tunis, Tunisia
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3- University Tunis El Manar, Tunis,Tunisia
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, Douik El
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Houda Gharsalli, MD
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Abstract
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Bronchoalveolar lavage (BAL) is a noninvasive and well-tolerated procedure that is performed with a fiberoptic bronchoscope in the wedged position within a selected bronchopulmonary segment. After it was introduced to clinical practice, BAL rapidly gained acceptance in a large number of centers as a procedure that could be applied to the clinical evaluation of patients with various pulmonary disorders, especially the group of interstitial lung diseases (ILD). Cytological and flow cytometric analysis of BAL fluid in ILD is done with knowledge of the clinical presentation and radiological findings. BAL typically reveals variations in the types and numbers of nucleated immune cells and acellular components in patients with ILD, which differ from those seen in normal control subjects. Many clinicians currently use this technique as a guide in the differential diagnoses of ILD; it can also be used to monitor the course of disease and possible response to therapeutic interventions. This article summarizes current clinicopathological information concerning the use of BAL by pulmonologists and pathologists.
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Key Words: Bronchoalveolar lavage; interstitial lung diseases; flow cytometry; genomics; proteomics
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Abbreviations
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BAL : Bronchoalveolar lavage ILD : Interstitial lung diseases PPFE : Pleuroparenchymal fibroelastosis IIP : Idiopathic interstitial pneumonias IP : Interstitial pneumonias NSIP : Nonspecific interstitial pneumonia COP : Cryptogenic organizing pneumonia ARDS : Acute respiratory distress syndrome DAD : Diffuse alveolar damage PLCH : Pulmonary Langerhans cell histiocytosis DIP: Desquamative interstitial pneumonia RBILD : Respiratory bronchiolitis interstitial lung disease HLA: human leucocyte antigen NKT : Natural Killer T TSLP : Thymic stromal lymphopoietin IPF : idiopathic pulmonary fibrosis AM: alveolar macrophage Eos : eosinophils Lymph : lymphocytes Neut :neutrophils MCP-1/CCL2 : monocyte chemoattractant protein-1 , TARC/CCL17 : thymus- and activation-regulated chemokine MDC/CCL22 : macrophage-derived chemokine
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Bronchoalveolar lavage (BAL) is a noninvasive and well-tolerated procedure that is performed with a fiberoptic bronchoscope in the wedged position within a selected bronchopulmonary segment [1].
After it was introduced to clinical practice, BAL rapidly
gained acceptance in a large number of centers as a procedure that could be applied to the clinical evaluation of patients with various pulmonary disorders, especially the group of interstitial lung diseases (ILD) [1,2,3,4]. Cytological and flow cytometric analysis of BAL fluid in ILD is done with knowledge of the clinical presentation and radiological findings. BAL
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typically reveals variations in the types and numbers of nucleated immune cells and acellular components in patients with ILD, which differ from those seen in normal control subjects. Many clinicians currently use this technique as a guide in the differential diagnoses of ILD [1]. In the past, the role of BAL in the diagnosis and management of ILD was the subject of controversy. This article reviews the current status of that topic.
ILD represent a diverse group of disorders that are characterized by alveolar and
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interstitial damage in the lungs, with variable degrees of pulmonary inflammation (usually coupled with fibrosis), decreased pulmonary capacity, and impaired gas exchange [5]. Most forms of ILD
are chronic [1]. ILD can be attributed to both known and unknown etiologies [6]. ILD with a defined cause include pneumoconiosis, hypersensitivity pneumonitis (HP), iatrogenic (e.g., postradiation), and postinfectious ILD.
ILD with unknown causes are represented by interstitial
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pulmonary disease that is seen in the context of systemic diseases, such as those related to connective tissue disorders [7], recently-described forms of interstitial pneumonia with
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autoimmune features [8], and idiopathic interstitial pneumonias (IIP).
Thoracic
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Respiratory
Society
(ATS/ERS)
classification of IIP was updated in 2013 [9] (Table1). Most main entities were preserved,
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major IIP were distinguished from rare IIP, and the category of “unclassifiable IIP” was introduced because of the acknowledgment that a definitive clinicopathologic diagnosis is not Moreover, the existence of idiopathic pleuroparenchymal
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always achievable [9,10].
fibroelastosis (PPFE) as a specific entity was confirmed. Within the group of major IIP, categorization into chronic fibrosing interstitial pneumonias (IP), smoking-related IP, and
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acute/ subacute IP was introduced [10]. DIAGNOSTIC UTILITY OF BAL The diagnostic value of BAL in discriminating between different forms of ILD is still a
challenging issue [4]. However, when results of this technique are interpreted in the context of the clinical presentation and radiological data, BAL can definitely be a useful diagnostic tool [11,12,13]. Its diagnostic utility is based on several features.
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Appearance of the BAL fluid
The appearance of BAL fluid at the time of procurement can provide diagnostic information and important clues concerning the cause of ILD. The presence of blood, with a progressive increase in the intensity of bloody discoloration in the retrieved BAL fluid with sequential aliquots during the BAL procedure
(Figure
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strongly suggests a
pulmonary/alveolar hemorrhage syndrome [14]. If the BAL fluid is grossly cloudy (’milky’) with a light brown or beige color and whitish flocculent material that settles to the bottom of a
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specimen container, the diagnosis of pulmonary alveolar proteinosis is suggested [15]. When cloudy material is present but low-speed centrifugation clears the fluid, the diagnosis of pulmonary microlithiasis can be suspected [16]. If oily material layers out on the top of the BAL fluid, the presence of lipoid pneumonia is likely. The presence of a black cell pellet after centrifugation suggests that the subject is a tobacco smoker or has had other exposure to significant amounts of carbonaceous material in the inhaled air. BAL cell pattern
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Cell pattern analysis is an important component of the evaluation of BAL fluid [17,18]. In the American Thoracic Society clinical practice guideline from 2012, concerning the use of BAL cellular analysis in ILD [1], it was stated that BAL cellular analysis-- including total and differential counts-- may be a useful adjunct in the diagnostic evaluation of patients with
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suspected ILD [1]. A differential cell count of lymphocytes, neutrophils, eosinophils, and mast cells is recommended. The remaining sample may be used for microbiological, virological,
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and cytologic evaluation, as clinically indicated. Those are important additional tests to consider, because infections and selected neoplasms can masquerade as ILD or coexist with them [1].
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The reason for routine cellular analysis of BAL specimens in cases of suspected ILD is that identification or exclusion of a predominantly inflammatory cellular pattern (increased
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lymphocytes, eosinophils, and/or neutrophils) can support a specific type of ILD or help narrow the differential diagnosis when considered in the context of the clinical and radiological findings [1]. Nevertheless, it should also be understood that a normal BAL
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differential cell profile does not exclude microscopic abnormalities in the lung tissue [1]. BAL samples obtained from healthy, never-smoking individuals should contain, on
average, a majority of alveolar macrophages (80 to 90%), some lymphocytes (5 to 15%) and very few neutrophils (≤3%) or eosinophils (<1%) (Figure 2). BAL cell differential counts and total cell counts from distant ex-smokers should be similar to those of never-smokers, but active smokers usually show a significantly increased total BAL cell count as well as total macrophages and neutrophils per ml of BAL fluid. However, the BAL differential cell count for smokers does not appear to vary significantly from that of never-smokers or distant exsmokers [19,20]. Elderly subjects appear to have increased percentages of lymphocytes
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and neutrophils in their differential cell counts, suggesting that advanced age alone may affect BAL results [21]. BAL patterns of nucleated immune cells that deviate from those observed in normal individuals indicate the presence of an infiltrative/inflammatory process that has perturbed the lung parenchyma [22]. Various reports have suggested that reasonable thresholds for increased cell differential counts are >15% for lymphocytes, >3% for neutrophils, and >1% for eosinophils. Increased percentages those inflammatory elements usually correlate with a
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disease process in general, but do not always indicate a specific diagnosis [23]. Table 2 presents information on disorders that associated with increased percentages of BAL cell types in ILD.
Specific white blood cell differential patterns can prove useful in evaluating patients with ILD. A BAL lymphocyte differential count exceeding 25% is likely to be caused by ILD
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associated with granuloma formation, (e.g., sarcoidosis and HP), or drug toxicity, if other possibilities such as mycobacterial or fungal infection are excluded [1]. Welker et al. showed that the likelihood of sarcoidosis increased from 33.7% to 68.1% when lymphocyte numbers were in the range of 30–50% and granulocyte numbers were low [24].
Extreme
lymphocytosis, especially with differential counts ≥ 50%, combined with a plausible exposure
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history to an inciting antigen, strongly suggests a diagnosis of HP. Lymphocyte counts also reflect the different histopathologic patterns seen in ILD, with the highest counts present in organizing pneumonia and cellular NSIP (Figure 3), and the lowest (but still increased)
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counts in disorders with a usual interstitial pneumonia-like pattern [25,26]. This information has particular importance in cases of chronic HP which present with an HRCT image that
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suggests usual interstitial pneumonia [25,26,27,28,29]. An eosinophil differential count of > 25% is highly diagnostic of eosinophil-mediated
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lung diseases in the appropriate clinical setting [1] (Figure 4). A predominance of macrophages containing smoking-related inclusions (Figure 5), with insignificant increases in other cell types, is consistent with smoking-related ILD such as desquamative interstitial
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pneumonia (DIP), respiratory bronchiolitis interstitial lung disease (RBILD), or pulmonary Langerhans cell histiocytosis (PLCH) [1]. Additional tests to identify and quantify Langerhans cells in the BAL specimen may be useful in narrowing the differential diagnosis. The presence of hemosiderin-laden alveolar macrophages in BAL fluid is compatible with chronic or occult alveolar hemorrhage syndromes, resulting in pulmonary hemosiderosis or diffuse alveolar damage (Figure 6) [1,30]. -
Diagnosis based on flow cytometry
In some instances, it may be desirable to enumerate the percentage of T cells, T cell subsets, or other specific cellular populations. Analyses for T cell subsets by flow cytometry
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ca be done with reference to T helper (CD4+) versus T suppressor (CD8+) phenotypes, using antibodies directed against those two lymphoid antigens [23]. If Langerhans’ histiocytosis of lung is suspected, it is desirable to assess the presence of CD1a-bearing cells cytometrically [23]. Lymphocyte subset analysis is not recommended routinely, but it is appropriate if a possible lymphocytic disease is suspected or initial BAL findings identify lymphocytosis [1]. In the study by Lee et al., routine analysis of lymphocyte subsets in BALs from 69 cases of
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ILD did not provide any meaningful information regarding the differential diagnosis of ILD [31]. Assessment of the CD4/CD8 ratio could be considered in the presence of ≥15% lymphocytes [1]. In other instances, however, that procedure is unlikely to be advantageous [1]. Several previous studies have found correlations of the CD4+/CD8+ T lymphocyte ratio with specific diagnoses such as sarcoidosis [24,32,33] and HP [34,35,36].
Nevertheless, because of its variability, the diagnostic value of the CD4+/CD8+ ratio has
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been debated [31,37,38,39]. Increased CD4 and decreased CD8 counts with an increased CD4/CD8 ratio are suggestive of sarcoidosis [40,41]. The cut-off for the CD4/CD8 ratio ranges from 3.5 to 4, with sensitivity and specificity figures of 52% to 59% and 94% to 96%, respectively [32,40]. A recent meta-analysis provided evidence that the determination of the CD4/CD8 ratio can assist in the diagnosis of sarcoidosis in concert with a typical
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clinicoradiological presentation of that disease [42]. However, the ratio is not specific or selective enough to use on its own in this context. It may be normal or even subnormal in
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~50% of patients with sarcoidosis [43]. The ratio is frequently high in patients with Löfgren’s syndrome and acute sarcoidosis, but it is usually normal in inactive sarcoidosis [44]. In addition, it may be increased in normal elderly subjects [45]. CD4-positive BAL lymphocytes
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may also be seen in methotrexate-induced pneumonitis and chronic berylliosis [46]. The BAL lymphocytosis in HP is often dominated by CD8+ T cells, resulting in an
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inverted CD4/CD8 ratio with mean values of 0.5–1.5, together with an increase in relative numbers of mast cells and neutrophils. Foamy alveolar macrophages are also present [44,45]. Considerable overlap is seen between the BAL profiles of sarcoidosis and HP, with
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an increased CD4/CD8 ratio in some patients with HP and variable lymphocyte phenotypes that depend on the causative antigens [47,48]. Several studies have evaluated the utility of integrin CD103, expressed on CD4+ T
lymphocytes in BAL, as a putative marker for sarcoidosis, with controversial conclusions [41,49,50,51,52]. That integrin is present on CD4+ lymphocytes in the bronchial mucosa, but not on peripheral blood lymphocytes. CD4+ cells in pulmonary sarcoidosis are hypothesized to originate from a redistribution from the peripheral blood to the lung, and therefore do not bear the CD103 integrin [52].
A low CD103+ percentage among BAL fluid CD4+
lymphocytes has therefore been suggested as a possible discriminative marker between
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sarcoidosis and ILD. A high CD4+/CD8+ ratio, combined with a low CD103+CD4+/CD4+ ratio, has been also suggested as another possible diagnostic indicator for sarcoidosis [49,50]. Heron et al. studied 56 patients with sarcoidosis, and concluded that the combined use of the CD103+ CD4+/CD4+ ratio (<0.2), with either the BAL CD4+/CD8+ ratio (>3) or the relative BAL/peripheral blood CD4+/CD8+ ratio (>2), could identify sarcoidosis with a sensitivity of 66% and a specificity of 89%. However another recent study found that this ratio did not accurately discriminate between sarcoidosis and other causes of lymphocytic
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alveolitis, either alone or in combination with the CD4+/CD8+ ratio [52]. Lymphocyte subsets of HLA-DR+ CD8+ T-cells and natural killer T-cells in BAL fluid have also been studied as possible markers for the diagnostic separation of sarcoidosis and HP. In fact, activated BAL fluid lymphocytes in both sarcoidosis and HP express human leucocyte antigen (HLA)-DR [53]. HLA-DR expression on CD8+ lymphocytes appears to be lower in sarcoidosis than in HP [54]. Natural killer-T (NKT) cells, which express CD56 and/or CD16 in
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addition to T cell receptors, are seen in lesser numbers in the BAL of patients with sarcoidosis, compared with HP [55].
Flow cytometry can be helpful in the diagnosis of other entities as well. For example, PLCH is supported by elevated levels of CD1a labeling (>5%) of cells in the BAL fluid, if clinical data and imaging are also consistent with that diagnosis [53].
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- Diagnosis based on cytokine assessment
BAL can be an informative tool in understanding the immunopathology of various lung
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diseases by quantifying the levels of cytokines that are present, in addition to various inflammatory cells [56]. Several studies have suggested that BAL cytokines and chemokines could have a role in the differential diagnosis of ILD. Thymic stromal lymphopoietin (TSLP)
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and IL-33 were assessed in BAL fluid in a recent analysis [57]. Levels of those moieties were significantly higher in cases of idiopathic pulmonary fibrosis (IPF) than in normal controls and
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patients with other lung diseases. Cai et al. evaluated CCL18, which is a member of the CC chemokine family; in paired serum and BAL fluid specimens [58]. The concentration of that marker was significantly increased in cases of HP, compared with IPF, RBILD, DIP, and
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COP [58]. Table 3 summarizes situations in which BAL can play a role in diagnosis of ILD. It may help to avoid lung biopsies in diseases such as pulmonary alveolar proteinosis, alveolar hemorrhage, eosinophilic pneumonia, and PLCH (Figure 7) [56,59]. In a large cohort of cases (N=3118) that were investigated for the predictive diagnostic value of BAL, BAL cell counts were suggested as the most useful analyte for the diagnosis of relatively common entities such as sarcoidosis, as opposed to relatively rare forms of ILD [24]. Typical cellular BAL findings in (IIP) are listed in Table 4.
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THE UTILITY OF BAL IN SCREENING FOR INFECTION Infection can present acutely or subacutely with diffuse lung infiltrates, and it may also coexist with noninfectious ILD [13]. Because fungal and mycobacterial infections can particularly masquerade as ILD or coexist with them, BAL fluid should be examined for those infectious agents, as clinically indicated, in the evaluation of diffuse ILD [1]. THE UTILITY OF GENOMIC & PROTEOMIC CHARACTERIZATION OF BAL SPECIMENS Genomic and proteomic characterization of BAL specimens (both cellular and soluble
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components) may change the utility of BAL in making an accurate diagnosis of ILD in the future. It could also have a role in choosing and implementing potentially effective therapies, monitoring disease activity, and assessing the effect of therapeutic pharmacologic interventions [13]. Determinations of gene and protein expression patterns in BAL samples over time can potentially identify key molecules that are involved in the initiation and progression of different ILD.
In addition, they represent another possible way to make
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diagnoses of specific ILDs without resorting to lung biopsies, and could be utilized to indicate targets for new and effective therapies [13].
Microarray genetic analysis can delineate characteristic gene expression patterns in specific ILD.
For example, a global analytic approach has identified genes that show
increased expression in IPF [61]. Thonhofer et al. [62] found that stimulated BAL cells from
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patients with sarcoidosis displayed up- or down- regulation of 1,000 genes.They included selective upregulation of B-MYB, a potent growth factor for lymphocytes and regulator of
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apoptosis, and FABP4, a regulator of lipid metabolism and arachidonic acid uptake by macrophages. Gene expression patterns may distinguish one ILD from another. Selman et al. [63] validated that concept by demonstrating that the patterns seen in patients with IPF
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differed from those with HP.
A proteomic approach to the analysis of BAL could also contribute toe the diagnosis
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and management of ILD [64]. This technique involves the identifiication of all proteins that are present in the lavage specimens. The development of leading-edge technologies has benefited proteomic research in this area. It appears to now be in an exponential growth
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phase [65] offering new perspectives for clinical application. Several studies have compared BAL proteomic profiles
and demonstrated a potential for better understanding of the
biological mechanism of ILD.
Early investigations that used electrophoretic techniques in
that context were able to show differences between IPF and sarcoidosis [66,67] and later studies using two-dimensional (2D) electrophoretic techniques allowed for enhanced fingerprinting of digested proteins from BAL supernatant fluids. They demonstrated different profiles for IPF, sarcoidosis, and HP [68,69]. More recently, Rottoli et al. [70] used 2D electrophoresis to construct protein maps from BAL fluid specimens, documenting differences between IPF, systemic sclerosis, and sarcoidosis.
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PROGNOSTIC UTILITY OF BAL ANALYSIS ILD are a heterogenous group that includes clinical entities with differing prognoses [1,2,3]. BAL findings at the time of diagnosis have been reported to reflect the severity of disease and predict the likelihood of its progression. As an example, the number of neutrophils in BAL specimens over time has been correlated with disease severity and prognosis for both HP [71,72] and IPF [73]. Increased numbers of eosinophils in BAL samples have also been associated with
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more severe disease and worsened prognosis in IPF [74,75]. Similarly, an increased neutrophil count in BAL may be associated with an unfavorable outcome in newly diagnosed patients with sarcoidosis and it may indicate the need for active treatment [49,76]. Ozdemir et al. [77] showed that high CD95 expression by BAL lymphocytes predicts a chronic course in patients with sarcoidosis. The role of chemokines and cytokines as markers reflecting the severity of ILD and predictors of outcome has been studied
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extensively. Shinoda et al. [78] concluded that elevated levels of monocyte chemoattractant protein-1 (MCP-1/CCL2), thymus- and activation-regulated chemokine (TARC/CCL17)
and
macrophage-derived chemokine (MDC/CCL22) in BAL fluid could predict a poor outcome in patients with IPF. Schmidt et al. [79] analyzed soluble mediators in BAL fluid from patients with systemic sclerosis. The highly sensitive Bioplex assay, which allows for the detection of High cytokine and chemokine
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cytokines without any manipulation of BAL fluid, was used.
levels (CCL2, IL-7, IL-8 and IL-4) were associated with severe ILD in systemic sclerosis and
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also with clinical deterioration [79].
THE UTILITY OF BAL IN THE CLINICAL MANAGEMENT OF ILD With the exception of whole lung lavage as a therapeutic invention for pulmonary
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alveolar proteinosis, [80], a role for BAL in the treatment of ILD (e.g., in directing or monitoring pharmacological therapies) has not been clearly established [81]. One application
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of BAL in management is its use to evaluate sudden changes in symptoms and lung function. Several adverse events may occur in the course of all ILD, including respiratory infection (Figure 8), drug reactions, hemorrhage, or an acute exacerbation of the disease process.
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BAL may play an important role in identifying the specific cause of disease exacerbation [81]. Importantly, BAL itself has only rarely been reported to cause worsening of pulmonary function or progression of ILD [82,83]. CONCLUSIONS Despite the presence of some controversies, BAL should be regarded as a very useful tool in the clinical management of ILD. The diagnostic value of this procedure in discriminating between various ILD is still challenging. BAL fluid analysis is not a stand-alone diagnostic test and correlation with clinical and radiological information is necessary. That is
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why the definitive diagnosis of ILD should be discussed in multidisciplinary meetings after integration of BAL findings and other diagnostic data. Genomic and proteomic analysis of
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BAL specimens is a promising area for the diagnosis and treatment of ILD.
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44. Ohshimo S, Guzman J, Costabel U, Bonella F. Differential diagnosis of granulomatous lung disease: clues and pitfalls: Number 4 in the Series “Pathology for the clinician” Edited by Peter Dorfmüller and Alberto Cavazza. Eur Respir Rev. 2017; 26(145):170012. 45. Meyer K, Soergel P. Variation of bronchoalveolar lymphocyte phenotypes with age in the physiologically normal human lung. Thorax. 1999; 54(8):697–700. 46. Schnabel A, Richter C, Bauerfeind S, Gross WL. Bronchoalveolar lavage cell profile in methotrexate induced pneumonitis. Thorax. 1997; 52(4):377–9.
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48. Ando M, Konishi K, Yoneda R, Tamura M. Difference in the phenotypes of bronchoalveolar lavage lymphocytes in patients with summer-type hypersensitivity pneumonitis, farmer’s lung, ventilation pneumonitis, and bird fancier’s lung: report of a nationwide epidemiologic study in Japan. J Allergy Clin Immunol. 1991; 87(5):1002–9.
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50. Heron M, Slieker WAT, Zanen P, van Lochem EG, Hooijkaas H, van den Bosch JMM, et al. Evaluation of CD103 as a cellular marker for the diagnosis of pulmonary sarcoidosis. Clin Immunol. 2008; 126(3):338–44. 51. Mota PC, Morais A, Palmares C, Beltrão M, Melo N, Santos AC, et al. Diagnostic value of CD103 expression in bronchoalveolar lymphocytes in sarcoidosis. Respir Med. 2012; 106(7):1014–20. 52. Bretagne L, Diatta I-D, Faouzi M, Nobile A, Bongiovanni M, Nicod LP, et al. Diagnostic Value of the CD103+CD4+/CD4+ Ratio to Differentiate Sarcoidosis from Other Causes of Lymphocytic Alveolitis. Respiration. 2016; 91(6):486–96.
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56. Dua K, Shukla SD, Hansbro PM. Aspiration techniques for bronchoalveolar lavage in translational respiratory research: Paving the way to develop novel therapeutic moieties. J Biol Methods. 2017; 4(3):e73.
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57. Lee J-U, Chang HS, Lee HJ, Jung CA, Bae DJ, Song HJ, et al. Upregulation of interleukin-33 and thymic stromal lymphopoietin levels in the lungs of idiopathic pulmonary fibrosis. BMC Pulm Med. 2017; 17(1):39. 58. Cai M, Bonella F, He X, Sixt SU, Sarria R, Guzman J, et al. CCL18 in serum, BAL fluid and alveolar macrophage culture supernatant in interstitial lung diseases. Respir Med. 2013; 107(9):1444–52.
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59. Costabel U, Guzman J. Bronchoalveolar lavage in interstitial lung disease. Curr Opin
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61. Agostini C, Miorin M, Semenzato G. Gene expression profile analysis by DNA microarrays: a new approach to assess functional genomics in diseases. Sarcoidosis Vasc Diffuse Lung Dis Off J WASOG. 2002; 19(1):5–9.
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62. Thonhofer R, Maercker C, Popper HH. Expression of sarcoidosis related genes in lung lavage cells. Sarcoidosis Vasc Diffuse Lung Dis Off J WASOG. 2002; 19(1):59–65.
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63. Selman M, Pardo A, Barrera L, Estrada A, Watson SR, Wilson K, et al. Gene Expression Profiles Distinguish Idiopathic Pulmonary Fibrosis from Hypersensitivity Pneumonitis. Am J Respir Crit Care Med. 2006; 173(2):188–98. 64. Bowler RP, Ellison MC, Reisdorph N. Proteomics in Pulmonary Medicine. Chest. 2006; 130(2):567–74. 65. Hirsch J, Hansen KC, Burlingame AL, Matthay MA. Proteomics: current techniques and potential applications to lung disease. Am J Physiol-Lung Cell Mol Physiol. 2004; 287(1):L1–23.
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66. Lenz A-G, Meyer B, Costabel U, Maier K. Bronchoalveolar lavage fluid proteins in human lung disease: Analysis by two-dimensional electrophoresis. Electrophoresis. 1993; 14(1):242–4. 67. Uebelhoer M, Bewig B, Oldigs M, Nowak D, Magnussen H, Petermann W, et al. Protein profile in bronchoalveolar lavage fluid from patients with sarcoidosis and idiopathic pulmonary fibrosis as revealed by SDS-PAGE electrophoresis and western blot analysis. Scand J Clin Lab Invest. 1993; 53(6):617–23.
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68. Wattiez R, Hermans C, Cruyt C, Bernard A, Falmagne P. Human bronchoalveolar lavage fluid protein two-dimensional database: Study of interstitial lung diseases. Electrophoresis. 2000; 21(13):2703–12. 69. Magi B, Bini L, Perari MG, Fossi A, Sanchez J-C, Hochstrasser D, et al. Bronchoalveolar lavage fluid protein composition in patients with sarcoidosis and idiopathic pulmonary fibrosis: a two-dimensional electrophoretic study. Electrophoresis. 2002; 23(19):3434–44.
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70. Rottoli P, Magi B, Perari MG, Liberatori S, Nikiforakis N, Bargagli E, et al. Cytokine profile and proteome analysis in bronchoalveolar lavage of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis and idiopathic pulmonary fibrosis. PROTEOMICS.2005; 5(5):1423–30.
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71. Haslam PL, Dewar A, Butchers P, Primett ZS, Newman-Taylor A, TurnerWarwick M. Mast Cells, Atypical Lymphocytes, and Neutrophils in Bronchoalveolar Lavage in Extrinsic Allergic Alveolitis. Am Rev Respir Dis. 1987; 135(1):35–47.
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72. Drent M, van Velzen-Blad H, Diamant M, Wagenaar SS, Hoogsteden HC, van den Bosch JM. Bronchoalveolar lavage in extrinsic allergic alveolitis: effect of time elapsed since antigen exposure. Eur Respir J. 1993; 6(9):1276–81.
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73. Lynch JP, Standiford TJ, Rolfe MW, Kunkel SL, Strieter RM. Neutrophilic Alveolitis in Idiopathic Pulmonary Fibrosis: The Role of Interleukin-8. Am Rev Respir Dis. 1992; 145(6):1433–9.
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74. Watters LC, Schwarz MI, Cherniack RM, Waldron JA, Dunn TL, Stanford RE, et al. Idiopathic Pulmonary Fibrosis. Am Rev Respir Dis. 1987; 135(3):696–704.
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75. Peterson MW, Monick M, Hunninghake GW. Prognostic Role of Eosinophils in Pulmonary Fibrosis. Chest. 1987; 92(1):51–6. 76. Ziegenhagen MW, Rothe ME, Schlaak M, Müller-Quernheim J. Bronchoalveolar and serological parameters reflecting the severity of sarcoidosis. Eur Respir J. 2003; 21(3):407–13. 77. Ozdemir OK, Celik G, Dalva K, Ulger F, Elhan A, Beksac M. High CD95 expression of BAL lymphocytes predicts chronic course in patients with sarcoidosis. Respirology. 2007; 12(6):869–73. 78. Shinoda H, Tasaka S, Fujishima S, Yamasawa W, Miyamoto K, Nakano Y, et al. Elevated CC Chemokine Level in Bronchoalveolar Lavage Fluid Is Predictive of
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a Poor Outcome of Idiopathic Pulmonary Fibrosis. Respiration. 2009; 78(3):285– 92. 79. Schmidt K, Martinez-Gamboa L, Meier S, Witt C, Meisel C, Hanitsch LG, et al. Bronchoalveoloar lavage fluid cytokines and chemokines as markers and predictors for the outcome of interstitial lung disease in systemic sclerosis patients. Arthritis Res Ther. 2009; 11:R111.
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80. Shah P, Hansell D, Lawson P, Reid K, Morgan C. Pulmonary alveolar proteinosis: clinical aspects and current concepts on pathogenesis. Thorax. 2000; 55(1):67–77. 81. Meyer KC. Bronchoalveolar Lavage as a Diagnostic Tool. Semin Respir Crit Care Med. 2007; 28(5):546–60.
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82. Hiwatari N, Shimura S, Takishima T, Shirato K. Bronchoalveolar Lavage as a Possible Cause of Acute Exacerbation in Idiopathic Pulmonary Fibrosis Patients. Tohoku J Exp Med. 1994; 174(4):379–86.
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83. Kim DS, Park JH, Park BK, Lee JS, Nicholson AG, Colby T. Acute exacerbation of idiopathic pulmonary fibrosis: frequency and clinical features. Eur Respir J. 2006; 27(1):143–50.
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Tables (See separate document) Table 1: Revised American Thoracic Society/European Respiratory Society classification and categorization of idiopathic interstitial pneumonias [9,10]. Table 2: Disorders associated with increased percentage of specific BAL cell types in interstitial lung diseases [1].
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Table 3: Settings in which BAL can play a role in diagnosis of ILD [52,60].
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Table 4: Usual BAL Cell Patterns in idiopathic interstitial diseases [1].
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Figure Legends
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Figure 1: Gross appearance of sequential bronchoalveolar lavage (BAL) specimens in a case of alveolar hemorrhage syndrome. An increasing level of erythrocyte content is seen
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between the first (left) and the last (right) aliquot.
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Figure 2: BAL specimens in normal, never-smoking individuals consist almost entirely of
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alveolar macrophages (left). The histology of the lung is shown on the right.
Figure 3: Diseases featuring lymphocytic infiltration of the lung parenchyma are associated with lymphocytosis in BAL specimens (left). nonspecific interstitial pneumonia (right).
This example is from a case of cellular
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Figure 4:
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Numerous eosinophils in a BAL sample (left) are seen in this case of acute
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eosinophilic pneumonia (right).
Figure 5: Alveolar histiocytes are laden with carbonaceous inclusions (left) in a case of smoking-related lung disease (respiratory bronchiolitis-interstitial lung disease—right).
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Figure 6: Dense deposits of hemosiderin in alveolar macrophages are present in a BAL sample stained with the Prussian blue method (left). The patient had idiopathic pulmonary
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hemosiderosis (right).
Figure 7: Numerous histiocytes with folded nuclear contours are present together with eosinophils in a BAL specimen from a case of pulmonary Langerhans cell histiocytosis (left). The histologic image of that disease is shown on the right.
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Figure 8: Innumerable neutrophils are present in a BAL specimen taken from a patient with
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acute bronchopneumonia. Intense tissue neutrophilia is present (right).
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Table 1: Revised American Thoracic Society/European Respiratory Society classification and categorization of idiopathic interstitial pneumonias [9,10].
Major idiopathic interstitial pneumonias Chronic fibrosing IP
Idiopathic non-specific interstitial pneumonia
Smoking-related IP Respiratory bronchiolitis interstitial lung disease
Acute/ subacute IP Cryptogenic organizing pneumonia Acute interstitial pneumonia
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Rare idiopathic interstitial pneumonias
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Desquamative interstitial pneumonia
Idiopathic lymphoid interstitial pneumonia
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Idiopathic pleuroparenchymal fibroelastosis
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Unclassifiable idiopathic interstitial pneumonias
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Idiopathic pulmonary fibrosis
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Table 2: Disorders associated with increased percentage of specific BAL cell types in interstitial lung diseases [1].
Neutrophilic
Eosinophilic
cellular pattern
cellular pattern
cellular pattern
> 15% lymphocytes
> 3% neutrophils
> 1% eosinophils
- Sarcoidosis
- Collagen vascular disease
- Eosinophilic pneumonias
- Nonspecific interstitial
- Idiopathic pulmonary fibrosis
- Drug-induced pneumonitis
pneumonia (NSIP)
- Aspiration pneumonia
- Hypersensitivity pneumonitis
- Infection : bacterial, fungal
- Drug-induced pneumonitis
- Bronchitis
- Collagen vascular disease
- Asbestosis
- Radiation pneumonitis
- Acute respiratory distress
- Cryptogenic organizing
syndrome (ARDS)
- Diffuse alveolar damage (DAD)
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- Lymphoproliferative disorders
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pneumonia (COP)
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Lymphocytic
- Bone marrow transplant - Asthma, bronchitis - Churg-Strauss syndrome - Allergic bronchopulmonary aspergillosis,
- Bacterial, fungal, helminthic, Pneumocystis infection - Hodgkin’s disease
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Table 3: Settings in which BAL can play a role in diagnosis of ILD [52,60]. BAL findings that are highly suggestive of specific types of ILD Diffuse alveolar
lavages/hemosiderin positive alveolar macrophage
hemorrhage
Milky fluid with positive periodic acid Schiff staining and amorphous acellular debris
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Progressive increase in bloody fluid return with sequential
Pulmonary alveolar proteinosis
Lymphocytosis ( > 25%)
Granulomatous disease:
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sarcoidosis,
Eosinophilia (> 25%)
Hypersensitivity pneumonitis or chronic beryllium disease
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Virtually diagnosis of acute or chronic eosinophilic pneumonia
Acute hypersensitivity
and > 3% neutrophils
pneumonitis
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A cell differential count of> 1% mast cells, >50% lymphocytes
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A predominance of macrophages containing smoking-related
Smoking-related ILDs
inclusions with no or minor increases in other cell types
(DIP, RBILD, or PLCH
CD4/CD8 >4
Sarcoidosis
CD1a positive cells ≥5%/Birbeck granules in macrophages
Langerhans’cell
( electron microscopy)
histiocytosis
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Positive lymphocyte transformation test to specific beryllium
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Chronic beryllium disease
antigen Asbestosis
Dust particles by polarized microscope
Silicosis
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Ferruginous bodies
Lipid-laden macrophages (oil-red-O-stain)
Lipoid pneumonia/chronic microaspiration
Definition of abbreviations: ILDs = interstitial lung disease, RBILD = respiratory bronchiolitis interstitial lung disease ,
DIP =desquamative interstitial pneumonia, PLCH = pulmonary
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Langerhans cell histiocytosis
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Table 4: Usual BAL Cell Pattern in idiopathic interstitial diseases[1]. Usual BAL Cell Pattern ↑AM, ↑ Neut , +/-↑ Eos
Idiopathic pulmonary fibrosis (UIP histopathology)
↑ AM, ↑ Lymph, ↑ Neut
Nonspecific interstitial
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pneumonia (NSIP)
↑↑ AM (heavily pigmented)
Desquamative interstitial pneumonia (DIP)
↑↑ AM (heavily pigmented)
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Respiratory bronchiolitis with interstitial lung disease (RB/ILD)
↑AM, ↑ Neut , +/-↑ Eos
Cryptogenic organizing
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Pneumonia
(EP)
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pneumonia (LIP)
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Lymphocytic interstitial
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Eosinophilic pneumonia
↑↑ Eos
↑↑ Lymph
Definition of abbreviations: BAL: bronchoalveolar lavage, AM: alveolar macrophage; Eos
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:eosinophils; Lymph : lymphocytes; Neut :neutrophils
THE Utility of bronchoalveolar lavage in THE EVALUATION OF interstitial lung diseaseS: A CLINICOPATHOLOGICAL PERSPECTIVE Houda Gharsalli MD , Mouna Mlika MD , Imen Sahnoun MD , Sonia Maalej MD , Douik El Gharbi Leila MD , Faouzi El Mezni MD PII: DOI: Reference:
S0740-2570(18)30051-0 https://doi.org/10.1053/j.semdp.2018.08.003 YSDIA 50566
To appear in:
Seminars in Diagnostic Pathology
Please cite this article as: Houda Gharsalli MD , Mouna Mlika MD , Imen Sahnoun MD , Sonia Maalej MD , Douik El Gharbi Leila MD , Faouzi El Mezni MD , THE Utility of bronchoalveolar lavage in THE EVALUATION OF interstitial lung diseaseS: A CLINICOPATHOLOGICAL PERSPECTIVE, Seminars in Diagnostic Pathology (2018), doi: https://doi.org/10.1053/j.semdp.2018.08.003
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ACCEPTED MANUSCRIPT THE UTILITY OF BRONCHOALVEOLAR LAVAGE IN THE EVALUATION OF INTERSTITIAL LUNG DISEASES: A CLINICOPATHOLOGICAL PERSPECTIVE
1,3
2,3
1,3
, Mouna Mlika, MD , Imen Sahnoun, MD , Sonia Maalej, MD 1,3 2,3 Gharbi Leila, MD , Faouzi El Mezni, MD
1- Department of Pulmonology Department, A.Mami Hospital, Tunis, Tunisia 2- Department of Pathology, Abderrahman Mami Hospital, Ariana, Tunis, Tunisia
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3- University Tunis El Manar, Tunis,Tunisia
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, Douik El
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Houda Gharsalli, MD
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Abstract
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Bronchoalveolar lavage (BAL) is a noninvasive and well-tolerated procedure that is performed with a fiberoptic bronchoscope in the wedged position within a selected bronchopulmonary segment. After it was introduced to clinical practice, BAL rapidly gained acceptance in a large number of centers as a procedure that could be applied to the clinical evaluation of patients with various pulmonary disorders, especially the group of interstitial lung diseases (ILD). Cytological and flow cytometric analysis of BAL fluid in ILD is done with knowledge of the clinical presentation and radiological findings. BAL typically reveals variations in the types and numbers of nucleated immune cells and acellular components in patients with ILD, which differ from those seen in normal control subjects. Many clinicians currently use this technique as a guide in the differential diagnoses of ILD; it can also be used to monitor the course of disease and possible response to therapeutic interventions. This article summarizes current clinicopathological information concerning the use of BAL by pulmonologists and pathologists.
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Key Words: Bronchoalveolar lavage; interstitial lung diseases; flow cytometry; genomics; proteomics
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Abbreviations
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BAL : Bronchoalveolar lavage ILD : Interstitial lung diseases PPFE : Pleuroparenchymal fibroelastosis IIP : Idiopathic interstitial pneumonias IP : Interstitial pneumonias NSIP : Nonspecific interstitial pneumonia COP : Cryptogenic organizing pneumonia ARDS : Acute respiratory distress syndrome DAD : Diffuse alveolar damage PLCH : Pulmonary Langerhans cell histiocytosis DIP: Desquamative interstitial pneumonia RBILD : Respiratory bronchiolitis interstitial lung disease HLA: human leucocyte antigen NKT : Natural Killer T TSLP : Thymic stromal lymphopoietin IPF : idiopathic pulmonary fibrosis AM: alveolar macrophage Eos : eosinophils Lymph : lymphocytes Neut :neutrophils MCP-1/CCL2 : monocyte chemoattractant protein-1 , TARC/CCL17 : thymus- and activation-regulated chemokine MDC/CCL22 : macrophage-derived chemokine
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Bronchoalveolar lavage (BAL) is a noninvasive and well-tolerated procedure that is performed with a fiberoptic bronchoscope in the wedged position within a selected bronchopulmonary segment [1].
After it was introduced to clinical practice, BAL rapidly
gained acceptance in a large number of centers as a procedure that could be applied to the clinical evaluation of patients with various pulmonary disorders, especially the group of interstitial lung diseases (ILD) [1,2,3,4]. Cytological and flow cytometric analysis of BAL fluid in ILD is done with knowledge of the clinical presentation and radiological findings. BAL
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typically reveals variations in the types and numbers of nucleated immune cells and acellular components in patients with ILD, which differ from those seen in normal control subjects. Many clinicians currently use this technique as a guide in the differential diagnoses of ILD [1]. In the past, the role of BAL in the diagnosis and management of ILD was the subject of controversy. This article reviews the current status of that topic.
ILD represent a diverse group of disorders that are characterized by alveolar and
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interstitial damage in the lungs, with variable degrees of pulmonary inflammation (usually coupled with fibrosis), decreased pulmonary capacity, and impaired gas exchange [5]. Most forms of ILD
are chronic [1]. ILD can be attributed to both known and unknown etiologies [6]. ILD with a defined cause include pneumoconiosis, hypersensitivity pneumonitis (HP), iatrogenic (e.g., postradiation), and postinfectious ILD.
ILD with unknown causes are represented by interstitial
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pulmonary disease that is seen in the context of systemic diseases, such as those related to connective tissue disorders [7], recently-described forms of interstitial pneumonia with
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autoimmune features [8], and idiopathic interstitial pneumonias (IIP).
Thoracic
Society/European
Respiratory
Society
(ATS/ERS)
classification of IIP was updated in 2013 [9] (Table1). Most main entities were preserved,
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major IIP were distinguished from rare IIP, and the category of “unclassifiable IIP” was introduced because of the acknowledgment that a definitive clinicopathologic diagnosis is not Moreover, the existence of idiopathic pleuroparenchymal
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always achievable [9,10].
fibroelastosis (PPFE) as a specific entity was confirmed. Within the group of major IIP, categorization into chronic fibrosing interstitial pneumonias (IP), smoking-related IP, and
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acute/ subacute IP was introduced [10]. DIAGNOSTIC UTILITY OF BAL The diagnostic value of BAL in discriminating between different forms of ILD is still a
challenging issue [4]. However, when results of this technique are interpreted in the context of the clinical presentation and radiological data, BAL can definitely be a useful diagnostic tool [11,12,13]. Its diagnostic utility is based on several features.
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Appearance of the BAL fluid
The appearance of BAL fluid at the time of procurement can provide diagnostic information and important clues concerning the cause of ILD. The presence of blood, with a progressive increase in the intensity of bloody discoloration in the retrieved BAL fluid with sequential aliquots during the BAL procedure
(Figure
1),
strongly suggests a
pulmonary/alveolar hemorrhage syndrome [14]. If the BAL fluid is grossly cloudy (’milky’) with a light brown or beige color and whitish flocculent material that settles to the bottom of a
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specimen container, the diagnosis of pulmonary alveolar proteinosis is suggested [15]. When cloudy material is present but low-speed centrifugation clears the fluid, the diagnosis of pulmonary microlithiasis can be suspected [16]. If oily material layers out on the top of the BAL fluid, the presence of lipoid pneumonia is likely. The presence of a black cell pellet after centrifugation suggests that the subject is a tobacco smoker or has had other exposure to significant amounts of carbonaceous material in the inhaled air. BAL cell pattern
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Cell pattern analysis is an important component of the evaluation of BAL fluid [17,18]. In the American Thoracic Society clinical practice guideline from 2012, concerning the use of BAL cellular analysis in ILD [1], it was stated that BAL cellular analysis-- including total and differential counts-- may be a useful adjunct in the diagnostic evaluation of patients with
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suspected ILD [1]. A differential cell count of lymphocytes, neutrophils, eosinophils, and mast cells is recommended. The remaining sample may be used for microbiological, virological,
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and cytologic evaluation, as clinically indicated. Those are important additional tests to consider, because infections and selected neoplasms can masquerade as ILD or coexist with them [1].
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The reason for routine cellular analysis of BAL specimens in cases of suspected ILD is that identification or exclusion of a predominantly inflammatory cellular pattern (increased
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lymphocytes, eosinophils, and/or neutrophils) can support a specific type of ILD or help narrow the differential diagnosis when considered in the context of the clinical and radiological findings [1]. Nevertheless, it should also be understood that a normal BAL
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differential cell profile does not exclude microscopic abnormalities in the lung tissue [1]. BAL samples obtained from healthy, never-smoking individuals should contain, on
average, a majority of alveolar macrophages (80 to 90%), some lymphocytes (5 to 15%) and very few neutrophils (≤3%) or eosinophils (<1%) (Figure 2). BAL cell differential counts and total cell counts from distant ex-smokers should be similar to those of never-smokers, but active smokers usually show a significantly increased total BAL cell count as well as total macrophages and neutrophils per ml of BAL fluid. However, the BAL differential cell count for smokers does not appear to vary significantly from that of never-smokers or distant exsmokers [19,20]. Elderly subjects appear to have increased percentages of lymphocytes
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and neutrophils in their differential cell counts, suggesting that advanced age alone may affect BAL results [21]. BAL patterns of nucleated immune cells that deviate from those observed in normal individuals indicate the presence of an infiltrative/inflammatory process that has perturbed the lung parenchyma [22]. Various reports have suggested that reasonable thresholds for increased cell differential counts are >15% for lymphocytes, >3% for neutrophils, and >1% for eosinophils. Increased percentages those inflammatory elements usually correlate with a
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disease process in general, but do not always indicate a specific diagnosis [23]. Table 2 presents information on disorders that associated with increased percentages of BAL cell types in ILD.
Specific white blood cell differential patterns can prove useful in evaluating patients with ILD. A BAL lymphocyte differential count exceeding 25% is likely to be caused by ILD
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associated with granuloma formation, (e.g., sarcoidosis and HP), or drug toxicity, if other possibilities such as mycobacterial or fungal infection are excluded [1]. Welker et al. showed that the likelihood of sarcoidosis increased from 33.7% to 68.1% when lymphocyte numbers were in the range of 30–50% and granulocyte numbers were low [24].
Extreme
lymphocytosis, especially with differential counts ≥ 50%, combined with a plausible exposure
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history to an inciting antigen, strongly suggests a diagnosis of HP. Lymphocyte counts also reflect the different histopathologic patterns seen in ILD, with the highest counts present in organizing pneumonia and cellular NSIP (Figure 3), and the lowest (but still increased)
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counts in disorders with a usual interstitial pneumonia-like pattern [25,26]. This information has particular importance in cases of chronic HP which present with an HRCT image that
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suggests usual interstitial pneumonia [25,26,27,28,29]. An eosinophil differential count of > 25% is highly diagnostic of eosinophil-mediated
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lung diseases in the appropriate clinical setting [1] (Figure 4). A predominance of macrophages containing smoking-related inclusions (Figure 5), with insignificant increases in other cell types, is consistent with smoking-related ILD such as desquamative interstitial
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pneumonia (DIP), respiratory bronchiolitis interstitial lung disease (RBILD), or pulmonary Langerhans cell histiocytosis (PLCH) [1]. Additional tests to identify and quantify Langerhans cells in the BAL specimen may be useful in narrowing the differential diagnosis. The presence of hemosiderin-laden alveolar macrophages in BAL fluid is compatible with chronic or occult alveolar hemorrhage syndromes, resulting in pulmonary hemosiderosis or diffuse alveolar damage (Figure 6) [1,30]. -
Diagnosis based on flow cytometry
In some instances, it may be desirable to enumerate the percentage of T cells, T cell subsets, or other specific cellular populations. Analyses for T cell subsets by flow cytometry
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ca be done with reference to T helper (CD4+) versus T suppressor (CD8+) phenotypes, using antibodies directed against those two lymphoid antigens [23]. If Langerhans’ histiocytosis of lung is suspected, it is desirable to assess the presence of CD1a-bearing cells cytometrically [23]. Lymphocyte subset analysis is not recommended routinely, but it is appropriate if a possible lymphocytic disease is suspected or initial BAL findings identify lymphocytosis [1]. In the study by Lee et al., routine analysis of lymphocyte subsets in BALs from 69 cases of
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ILD did not provide any meaningful information regarding the differential diagnosis of ILD [31]. Assessment of the CD4/CD8 ratio could be considered in the presence of ≥15% lymphocytes [1]. In other instances, however, that procedure is unlikely to be advantageous [1]. Several previous studies have found correlations of the CD4+/CD8+ T lymphocyte ratio with specific diagnoses such as sarcoidosis [24,32,33] and HP [34,35,36].
Nevertheless, because of its variability, the diagnostic value of the CD4+/CD8+ ratio has
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been debated [31,37,38,39]. Increased CD4 and decreased CD8 counts with an increased CD4/CD8 ratio are suggestive of sarcoidosis [40,41]. The cut-off for the CD4/CD8 ratio ranges from 3.5 to 4, with sensitivity and specificity figures of 52% to 59% and 94% to 96%, respectively [32,40]. A recent meta-analysis provided evidence that the determination of the CD4/CD8 ratio can assist in the diagnosis of sarcoidosis in concert with a typical
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clinicoradiological presentation of that disease [42]. However, the ratio is not specific or selective enough to use on its own in this context. It may be normal or even subnormal in
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~50% of patients with sarcoidosis [43]. The ratio is frequently high in patients with Löfgren’s syndrome and acute sarcoidosis, but it is usually normal in inactive sarcoidosis [44]. In addition, it may be increased in normal elderly subjects [45]. CD4-positive BAL lymphocytes
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may also be seen in methotrexate-induced pneumonitis and chronic berylliosis [46]. The BAL lymphocytosis in HP is often dominated by CD8+ T cells, resulting in an
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inverted CD4/CD8 ratio with mean values of 0.5–1.5, together with an increase in relative numbers of mast cells and neutrophils. Foamy alveolar macrophages are also present [44,45]. Considerable overlap is seen between the BAL profiles of sarcoidosis and HP, with
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an increased CD4/CD8 ratio in some patients with HP and variable lymphocyte phenotypes that depend on the causative antigens [47,48]. Several studies have evaluated the utility of integrin CD103, expressed on CD4+ T
lymphocytes in BAL, as a putative marker for sarcoidosis, with controversial conclusions [41,49,50,51,52]. That integrin is present on CD4+ lymphocytes in the bronchial mucosa, but not on peripheral blood lymphocytes. CD4+ cells in pulmonary sarcoidosis are hypothesized to originate from a redistribution from the peripheral blood to the lung, and therefore do not bear the CD103 integrin [52].
A low CD103+ percentage among BAL fluid CD4+
lymphocytes has therefore been suggested as a possible discriminative marker between
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sarcoidosis and ILD. A high CD4+/CD8+ ratio, combined with a low CD103+CD4+/CD4+ ratio, has been also suggested as another possible diagnostic indicator for sarcoidosis [49,50]. Heron et al. studied 56 patients with sarcoidosis, and concluded that the combined use of the CD103+ CD4+/CD4+ ratio (<0.2), with either the BAL CD4+/CD8+ ratio (>3) or the relative BAL/peripheral blood CD4+/CD8+ ratio (>2), could identify sarcoidosis with a sensitivity of 66% and a specificity of 89%. However another recent study found that this ratio did not accurately discriminate between sarcoidosis and other causes of lymphocytic
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alveolitis, either alone or in combination with the CD4+/CD8+ ratio [52]. Lymphocyte subsets of HLA-DR+ CD8+ T-cells and natural killer T-cells in BAL fluid have also been studied as possible markers for the diagnostic separation of sarcoidosis and HP. In fact, activated BAL fluid lymphocytes in both sarcoidosis and HP express human leucocyte antigen (HLA)-DR [53]. HLA-DR expression on CD8+ lymphocytes appears to be lower in sarcoidosis than in HP [54]. Natural killer-T (NKT) cells, which express CD56 and/or CD16 in
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addition to T cell receptors, are seen in lesser numbers in the BAL of patients with sarcoidosis, compared with HP [55].
Flow cytometry can be helpful in the diagnosis of other entities as well. For example, PLCH is supported by elevated levels of CD1a labeling (>5%) of cells in the BAL fluid, if clinical data and imaging are also consistent with that diagnosis [53].
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- Diagnosis based on cytokine assessment
BAL can be an informative tool in understanding the immunopathology of various lung
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diseases by quantifying the levels of cytokines that are present, in addition to various inflammatory cells [56]. Several studies have suggested that BAL cytokines and chemokines could have a role in the differential diagnosis of ILD. Thymic stromal lymphopoietin (TSLP)
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and IL-33 were assessed in BAL fluid in a recent analysis [57]. Levels of those moieties were significantly higher in cases of idiopathic pulmonary fibrosis (IPF) than in normal controls and
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patients with other lung diseases. Cai et al. evaluated CCL18, which is a member of the CC chemokine family; in paired serum and BAL fluid specimens [58]. The concentration of that marker was significantly increased in cases of HP, compared with IPF, RBILD, DIP, and
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COP [58]. Table 3 summarizes situations in which BAL can play a role in diagnosis of ILD. It may help to avoid lung biopsies in diseases such as pulmonary alveolar proteinosis, alveolar hemorrhage, eosinophilic pneumonia, and PLCH (Figure 7) [56,59]. In a large cohort of cases (N=3118) that were investigated for the predictive diagnostic value of BAL, BAL cell counts were suggested as the most useful analyte for the diagnosis of relatively common entities such as sarcoidosis, as opposed to relatively rare forms of ILD [24]. Typical cellular BAL findings in (IIP) are listed in Table 4.
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THE UTILITY OF BAL IN SCREENING FOR INFECTION Infection can present acutely or subacutely with diffuse lung infiltrates, and it may also coexist with noninfectious ILD [13]. Because fungal and mycobacterial infections can particularly masquerade as ILD or coexist with them, BAL fluid should be examined for those infectious agents, as clinically indicated, in the evaluation of diffuse ILD [1]. THE UTILITY OF GENOMIC & PROTEOMIC CHARACTERIZATION OF BAL SPECIMENS Genomic and proteomic characterization of BAL specimens (both cellular and soluble
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components) may change the utility of BAL in making an accurate diagnosis of ILD in the future. It could also have a role in choosing and implementing potentially effective therapies, monitoring disease activity, and assessing the effect of therapeutic pharmacologic interventions [13]. Determinations of gene and protein expression patterns in BAL samples over time can potentially identify key molecules that are involved in the initiation and progression of different ILD.
In addition, they represent another possible way to make
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diagnoses of specific ILDs without resorting to lung biopsies, and could be utilized to indicate targets for new and effective therapies [13].
Microarray genetic analysis can delineate characteristic gene expression patterns in specific ILD.
For example, a global analytic approach has identified genes that show
increased expression in IPF [61]. Thonhofer et al. [62] found that stimulated BAL cells from
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patients with sarcoidosis displayed up- or down- regulation of 1,000 genes.They included selective upregulation of B-MYB, a potent growth factor for lymphocytes and regulator of
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apoptosis, and FABP4, a regulator of lipid metabolism and arachidonic acid uptake by macrophages. Gene expression patterns may distinguish one ILD from another. Selman et al. [63] validated that concept by demonstrating that the patterns seen in patients with IPF
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differed from those with HP.
A proteomic approach to the analysis of BAL could also contribute toe the diagnosis
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and management of ILD [64]. This technique involves the identifiication of all proteins that are present in the lavage specimens. The development of leading-edge technologies has benefited proteomic research in this area. It appears to now be in an exponential growth
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phase [65] offering new perspectives for clinical application. Several studies have compared BAL proteomic profiles
and demonstrated a potential for better understanding of the
biological mechanism of ILD.
Early investigations that used electrophoretic techniques in
that context were able to show differences between IPF and sarcoidosis [66,67] and later studies using two-dimensional (2D) electrophoretic techniques allowed for enhanced fingerprinting of digested proteins from BAL supernatant fluids. They demonstrated different profiles for IPF, sarcoidosis, and HP [68,69]. More recently, Rottoli et al. [70] used 2D electrophoresis to construct protein maps from BAL fluid specimens, documenting differences between IPF, systemic sclerosis, and sarcoidosis.
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PROGNOSTIC UTILITY OF BAL ANALYSIS ILD are a heterogenous group that includes clinical entities with differing prognoses [1,2,3]. BAL findings at the time of diagnosis have been reported to reflect the severity of disease and predict the likelihood of its progression. As an example, the number of neutrophils in BAL specimens over time has been correlated with disease severity and prognosis for both HP [71,72] and IPF [73]. Increased numbers of eosinophils in BAL samples have also been associated with
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more severe disease and worsened prognosis in IPF [74,75]. Similarly, an increased neutrophil count in BAL may be associated with an unfavorable outcome in newly diagnosed patients with sarcoidosis and it may indicate the need for active treatment [49,76]. Ozdemir et al. [77] showed that high CD95 expression by BAL lymphocytes predicts a chronic course in patients with sarcoidosis. The role of chemokines and cytokines as markers reflecting the severity of ILD and predictors of outcome has been studied
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extensively. Shinoda et al. [78] concluded that elevated levels of monocyte chemoattractant protein-1 (MCP-1/CCL2), thymus- and activation-regulated chemokine (TARC/CCL17)
and
macrophage-derived chemokine (MDC/CCL22) in BAL fluid could predict a poor outcome in patients with IPF. Schmidt et al. [79] analyzed soluble mediators in BAL fluid from patients with systemic sclerosis. The highly sensitive Bioplex assay, which allows for the detection of High cytokine and chemokine
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cytokines without any manipulation of BAL fluid, was used.
levels (CCL2, IL-7, IL-8 and IL-4) were associated with severe ILD in systemic sclerosis and
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also with clinical deterioration [79].
THE UTILITY OF BAL IN THE CLINICAL MANAGEMENT OF ILD With the exception of whole lung lavage as a therapeutic invention for pulmonary
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alveolar proteinosis, [80], a role for BAL in the treatment of ILD (e.g., in directing or monitoring pharmacological therapies) has not been clearly established [81]. One application
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of BAL in management is its use to evaluate sudden changes in symptoms and lung function. Several adverse events may occur in the course of all ILD, including respiratory infection (Figure 8), drug reactions, hemorrhage, or an acute exacerbation of the disease process.
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BAL may play an important role in identifying the specific cause of disease exacerbation [81]. Importantly, BAL itself has only rarely been reported to cause worsening of pulmonary function or progression of ILD [82,83]. CONCLUSIONS Despite the presence of some controversies, BAL should be regarded as a very useful tool in the clinical management of ILD. The diagnostic value of this procedure in discriminating between various ILD is still challenging. BAL fluid analysis is not a stand-alone diagnostic test and correlation with clinical and radiological information is necessary. That is
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why the definitive diagnosis of ILD should be discussed in multidisciplinary meetings after integration of BAL findings and other diagnostic data. Genomic and proteomic analysis of
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BAL specimens is a promising area for the diagnosis and treatment of ILD.
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75. Peterson MW, Monick M, Hunninghake GW. Prognostic Role of Eosinophils in Pulmonary Fibrosis. Chest. 1987; 92(1):51–6. 76. Ziegenhagen MW, Rothe ME, Schlaak M, Müller-Quernheim J. Bronchoalveolar and serological parameters reflecting the severity of sarcoidosis. Eur Respir J. 2003; 21(3):407–13. 77. Ozdemir OK, Celik G, Dalva K, Ulger F, Elhan A, Beksac M. High CD95 expression of BAL lymphocytes predicts chronic course in patients with sarcoidosis. Respirology. 2007; 12(6):869–73. 78. Shinoda H, Tasaka S, Fujishima S, Yamasawa W, Miyamoto K, Nakano Y, et al. Elevated CC Chemokine Level in Bronchoalveolar Lavage Fluid Is Predictive of
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a Poor Outcome of Idiopathic Pulmonary Fibrosis. Respiration. 2009; 78(3):285– 92. 79. Schmidt K, Martinez-Gamboa L, Meier S, Witt C, Meisel C, Hanitsch LG, et al. Bronchoalveoloar lavage fluid cytokines and chemokines as markers and predictors for the outcome of interstitial lung disease in systemic sclerosis patients. Arthritis Res Ther. 2009; 11:R111.
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80. Shah P, Hansell D, Lawson P, Reid K, Morgan C. Pulmonary alveolar proteinosis: clinical aspects and current concepts on pathogenesis. Thorax. 2000; 55(1):67–77. 81. Meyer KC. Bronchoalveolar Lavage as a Diagnostic Tool. Semin Respir Crit Care Med. 2007; 28(5):546–60.
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82. Hiwatari N, Shimura S, Takishima T, Shirato K. Bronchoalveolar Lavage as a Possible Cause of Acute Exacerbation in Idiopathic Pulmonary Fibrosis Patients. Tohoku J Exp Med. 1994; 174(4):379–86.
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83. Kim DS, Park JH, Park BK, Lee JS, Nicholson AG, Colby T. Acute exacerbation of idiopathic pulmonary fibrosis: frequency and clinical features. Eur Respir J. 2006; 27(1):143–50.
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Tables (See separate document) Table 1: Revised American Thoracic Society/European Respiratory Society classification and categorization of idiopathic interstitial pneumonias [9,10]. Table 2: Disorders associated with increased percentage of specific BAL cell types in interstitial lung diseases [1].
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Table 3: Settings in which BAL can play a role in diagnosis of ILD [52,60].
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Table 4: Usual BAL Cell Patterns in idiopathic interstitial diseases [1].
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Figure Legends
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Figure 1: Gross appearance of sequential bronchoalveolar lavage (BAL) specimens in a case of alveolar hemorrhage syndrome. An increasing level of erythrocyte content is seen
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between the first (left) and the last (right) aliquot.
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Figure 2: BAL specimens in normal, never-smoking individuals consist almost entirely of
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alveolar macrophages (left). The histology of the lung is shown on the right.
Figure 3: Diseases featuring lymphocytic infiltration of the lung parenchyma are associated with lymphocytosis in BAL specimens (left). nonspecific interstitial pneumonia (right).
This example is from a case of cellular
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Figure 4:
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Numerous eosinophils in a BAL sample (left) are seen in this case of acute
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eosinophilic pneumonia (right).
Figure 5: Alveolar histiocytes are laden with carbonaceous inclusions (left) in a case of smoking-related lung disease (respiratory bronchiolitis-interstitial lung disease—right).
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Figure 6: Dense deposits of hemosiderin in alveolar macrophages are present in a BAL sample stained with the Prussian blue method (left). The patient had idiopathic pulmonary
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hemosiderosis (right).
Figure 7: Numerous histiocytes with folded nuclear contours are present together with eosinophils in a BAL specimen from a case of pulmonary Langerhans cell histiocytosis (left). The histologic image of that disease is shown on the right.
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Figure 8: Innumerable neutrophils are present in a BAL specimen taken from a patient with
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acute bronchopneumonia. Intense tissue neutrophilia is present (right).
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Table 1: Revised American Thoracic Society/European Respiratory Society classification and categorization of idiopathic interstitial pneumonias [9,10].
Major idiopathic interstitial pneumonias Chronic fibrosing IP
Idiopathic non-specific interstitial pneumonia
Smoking-related IP Respiratory bronchiolitis interstitial lung disease
Acute/ subacute IP Cryptogenic organizing pneumonia Acute interstitial pneumonia
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Rare idiopathic interstitial pneumonias
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Desquamative interstitial pneumonia
Idiopathic lymphoid interstitial pneumonia
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Idiopathic pleuroparenchymal fibroelastosis
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Unclassifiable idiopathic interstitial pneumonias
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Idiopathic pulmonary fibrosis
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Table 2: Disorders associated with increased percentage of specific BAL cell types in interstitial lung diseases [1].
Neutrophilic
Eosinophilic
cellular pattern
cellular pattern
cellular pattern
> 15% lymphocytes
> 3% neutrophils
> 1% eosinophils
- Sarcoidosis
- Collagen vascular disease
- Eosinophilic pneumonias
- Nonspecific interstitial
- Idiopathic pulmonary fibrosis
- Drug-induced pneumonitis
pneumonia (NSIP)
- Aspiration pneumonia
- Hypersensitivity pneumonitis
- Infection : bacterial, fungal
- Drug-induced pneumonitis
- Bronchitis
- Collagen vascular disease
- Asbestosis
- Radiation pneumonitis
- Acute respiratory distress
- Cryptogenic organizing
syndrome (ARDS)
- Diffuse alveolar damage (DAD)
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- Lymphoproliferative disorders
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pneumonia (COP)
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Lymphocytic
- Bone marrow transplant - Asthma, bronchitis - Churg-Strauss syndrome - Allergic bronchopulmonary aspergillosis,
- Bacterial, fungal, helminthic, Pneumocystis infection - Hodgkin’s disease
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Table 3: Settings in which BAL can play a role in diagnosis of ILD [52,60]. BAL findings that are highly suggestive of specific types of ILD Diffuse alveolar
lavages/hemosiderin positive alveolar macrophage
hemorrhage
Milky fluid with positive periodic acid Schiff staining and amorphous acellular debris
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Progressive increase in bloody fluid return with sequential
Pulmonary alveolar proteinosis
Lymphocytosis ( > 25%)
Granulomatous disease:
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sarcoidosis,
Eosinophilia (> 25%)
Hypersensitivity pneumonitis or chronic beryllium disease
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Virtually diagnosis of acute or chronic eosinophilic pneumonia
Acute hypersensitivity
and > 3% neutrophils
pneumonitis
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A cell differential count of> 1% mast cells, >50% lymphocytes
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A predominance of macrophages containing smoking-related
Smoking-related ILDs
inclusions with no or minor increases in other cell types
(DIP, RBILD, or PLCH
CD4/CD8 >4
Sarcoidosis
CD1a positive cells ≥5%/Birbeck granules in macrophages
Langerhans’cell
( electron microscopy)
histiocytosis
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Positive lymphocyte transformation test to specific beryllium
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Chronic beryllium disease
antigen Asbestosis
Dust particles by polarized microscope
Silicosis
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Ferruginous bodies
Lipid-laden macrophages (oil-red-O-stain)
Lipoid pneumonia/chronic microaspiration
Definition of abbreviations: ILDs = interstitial lung disease, RBILD = respiratory bronchiolitis interstitial lung disease ,
DIP =desquamative interstitial pneumonia, PLCH = pulmonary
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Langerhans cell histiocytosis
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Table 4: Usual BAL Cell Pattern in idiopathic interstitial diseases[1]. Usual BAL Cell Pattern ↑AM, ↑ Neut , +/-↑ Eos
Idiopathic pulmonary fibrosis (UIP histopathology)
↑ AM, ↑ Lymph, ↑ Neut
Nonspecific interstitial
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pneumonia (NSIP)
↑↑ AM (heavily pigmented)
Desquamative interstitial pneumonia (DIP)
↑↑ AM (heavily pigmented)
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Respiratory bronchiolitis with interstitial lung disease (RB/ILD)
↑AM, ↑ Neut , +/-↑ Eos
Cryptogenic organizing
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Pneumonia
(EP)
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pneumonia (LIP)
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Lymphocytic interstitial
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↑↑ Eos
↑↑ Lymph
Definition of abbreviations: BAL: bronchoalveolar lavage, AM: alveolar macrophage; Eos
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:eosinophils; Lymph : lymphocytes; Neut :neutrophils