Introduction

An interstitial lung disease is a heterogeneous group of disorders with different clinical-radiological presentations and evolution. Diagnosis of these diseases is usually based on medical history, laboratory tests, pulmonary function tests, and CT scans. Bronchoscopic techniques are required when these data alone are inconclusive and tissue or alveolar uid is necessary for a defnitive diagnosis or for therapeutic decisions [1]. Bronchoscopic procedures involved in the diagnostic process of diffuse parenchymal lung diseases include bronchoalveolar lavage (BAL), conventional transbronchial lung biopsy (TBB), and transbronchial lung cryobiopsy (TLCB).

Bronchoalveolar Lavage (BAL)

Technique

BAL is a minimally invasive procedure, well tolerated, and easily performed with a exible bronchoscope introduced through the mouth or nose in a wedge position (in a segmental or sub-segmental bronchus). Sterile normal saline, usually 100–300 mL divided into 3–6 multiple aliquots, is instilled into a subsegment of the lung; after the instillation of each aliquot, instilled saline has to be retrieved using negative pressure (<100 mmHg) and volume retrieved should be in total >5% (optimal >30%) [2]. The recovered uid should be collected into a container to which cells are poorly adherent and quickly send to the laboratory to enable processing

C. Ravaglia · S. Puglisi · C. Gurioli · F. Sultani · A. Arcadu Thoracic Diseases Department, G.B. Morgagni - L. Pierantoni Hospital, Forlì, Italy

e-mail: fabio.sultani@auslromagna.it

V. Poletti (*)

Thoracic Diseases Department, G.B. Morgagni - L. Pierantoni Hospital, Forlì, Italy

Department of Respiratory Diseases and Allergy, Aarhus University Hospital, Aarhus, Denmark

within 1 h (BAL cells deteriorate rapidly in saline). The BAL sample will be divided into measured aliquots, for example, more than 20 mL for BAL cytology/ ow cytometry, 10 mL for microbiology, and, if needed, 20 mL for electron microscopy (Table 30.1). Normal values of cytological profle are reported in (Table 30.2). A variety of stains may be used in the diagnostic interpretation of BAL. It is possible to differentiate blood cell types by employing the Wright Giemsa stain or the more commonly used Diff Quick stain which requires shorten staining time but does not stain mast cells. For infectious conditions, the Wright Giemsa stain can be used to detect clusters of the pneumocystis in Pneumocystis jirovecii Pneumonia, and confrmed by using the silver stain. The Giemsa stain is also useful to identify intracellular organisms, such as in bacterial pneumonia (Table 30.3).

Table 30.1  Technical recommendations to perform BAL

1.Perform BAL under local anesthesia with lidocaine and use pre-medication with a sedative compound

2.Decide the appropriate site to perform BAL and gently wedge the tip of the bronchoscope into the selected subsegmental bronchus

3.Sequentially introduce and aspirate standard aliquots of saline (total volume should be no less than 100 mL and should not exceed 300 mL).

4.For an optimal sampling of distal airspaces, the volume retrieved should be in total >5% (optimal >30%), so that a minimal volume of 5 mL is required for BAL cellular analyses

5.Send the BAL sample to the laboratory within 1 h because BAL cells deteriorate rapidly in saline

Table 30.2  Cellular profle of bronchoalveolar lavage uid: normal values

Table 30.3  Useful stainings of BAL

BAL is a relatively low-risk technique and the most frequently observed side effects are self-limited fever and hypoxia, bronchospasm. Rarely hypotension, pneumothorax, bleeding, can be observed in critically ill patients. Finally acute exacerbations of idiopathic pulmonary fbrosis (IPF) within 30 days after the procedure has been reported [3].

Interpretation

The diagnostic value of BAL in discriminating between different forms of ILD is still a challenging issue. Analysis of BAL cell counts, cytology, and culture provides insights into immunologic, in ammatory, neoplastic, and infectious processes occurring at the alveolar level. BAL results should be interpreted in the context of the clinical and radiological presentation on the basis of several features such as the appearance and the cell pattern analysis of BAL uid (BALF).

The appearance of BALF can provide diagnostic information. The presence of blood, with a progressive increase in

the intensity of bloody discoloration in the retrieved BALF with sequential aliquots during the BAL procedure suggests a pulmonary alveolar hemorrhage syndrome [4]. If the BALF is grossly cloudy (“milky”) or light brown to whitish, cloudy appearance the diagnosis of pulmonary alveolar proteinosis is suggested [5]. Cytological features may be considered diagnostic in alveolar proteinosis, exogenous lipoidic pneumonia, diffuse alveolar damage or may confrm a professional exposure to asbestos (Fig. 30.1).

In the American Thoracic Society clinical practice guideline, concerning the role of BAL cellular analysis in the diagnosis of ILDs, it was stated that BAL cellular analysis may add more information in the diagnostic evaluation of patients with suspected ILD. A differential cell count of lymphocytes, neutrophils, eosinophils, and mast cells is recommended to identify an in ammatory cellular pattern (increased lymphocytes, eosinophils, and/or neutrophils) orienting the diagnosis through a specifc type of ILD. BAL samples obtained from healthy, never-smoking individuals should contain, on average, a majority of alveolar macrophages (80–90%), some lymphocytes (5–15%) and very few neutrophils (≤3%) or eosinophils (<1%); smoking increases the absolute number of BAL macrophages and neutrophils. When neutrophil counts are very high it is important to check for intracellular bacteria, which can indicate active bacterial pneumonia.

In ILD patients a variety of changes in the relative and absolute numbers of individual cell constituents have been described. Usually, these changes are nonspecifc, but occasionally, the pattern is suffciently characteristic to guide the differential diagnosis [6].

Increased lymphocytes (>25%) can be associated with granuloma formation (such as sarcoidosis and hypersensitivity pneumonitis) or drug toxicity. BAL lymphocytosis can also be observed in other diseases, such as cryptogenic-­ organizing pneumonia (COP) secondary organizing pneumonia, lymphocytic interstitial pneumonia (LIP), cellular NSIP (non-specifc interstitial pneumonia), or lymphoproliferative disorders [1]. The T-lymphocyte component can be sub-categorized by ow cytometry with reference to T helper (CD4+) versus T suppressor (CD8+) phenotypes, using antibodies directed against those two lymphoid antigens. Assessment of the CD4/CD8 ratio should not be performed routinely but only in the presence of ≥15% lymphocytes [6]. Usually, a CD4+/CD8+ ratio in BAL from clinically healthy, younger adults usually is 1.0–3.5 (average values 1.5–2.0). CD4+/CD8+ ratio >3.5 combined with BAL lymphocytosis and normal neutrophil count is relatively specifc for sarcoidosis, but sensitivity is low so that it is possible to have sarcoid patients with the normal ratio in almost 50% of cases

Fig. 30.1  (a) Periodic Acid Schiff staining in BAL confrming pulmonary alveolar proteinosis (PAP); (b) asbestos fber in bronchoalveolar lavage; (c) lipid-laden alveolar macrophages marker of aspiration; (d)

diffuse alveolar damage (DAD): the epithelial component displays various degrees of nuclear atypia. (Source: Pathology Department, G.B. Morgagni – L. Pierantoni Hospital, Forlì, Italy)

[7]. The CD4/CD8 ratio is usually decreased in subacute HP (ratio <1) [1, 8], it can be increased in normal elderly subjects [9]. The BAL lymphocytosis in HP is often dominated by CD8+ T cells, resulting in an inverted CD4/CD8 ratio with mean values of 0.5–1.5, together with an increase in relative numbers of mast cells and neutrophils [10]. 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 [11, 12].

Increased number of mast cells (1–2%) have been associated with HP, drug reactions, sarcoidosis, ILD associated with collagen vascular disease, IPF, COP and malignancy. A more prominent “lymphocytic in ammation” on BAL is frequently present in NSIP patients with clinical/radiological characteristics suggestive of organizing pneumonia (OP),

which usually have a better outcome and treatment response. NSIP with more prominent “fbrotic changes,” that is characterized by no lymphocytosis on BAL, is associated with a worse outcome [13].

Eosinophil differential cell counts >25% are likely to be caused by eosinophilic lung disease, especially eosinophilic pneumonia [14], although eosinophilia may be present in desquamative interstitial pneumonia (DIP) [1]. In acute eosinophilic pneumonia type II dysplastic/reactive pneumocytes may be also detected [1].

A predominance of macrophages containing smoking-­ related inclusions, within signifcant increases in other cell types such as eosinophils, is consistent with smoking-related ILD such as desquamative interstitial pneumonia (DIP) or pulmonary Langerhans cell histiocytosis (PLCH) [1, 15]. In respiratory bronchiolitis eosinophils are not increased.