Basic principles and technique of bronchoalveolar lavage

INTRODUCTION — Bronchoalveolar lavage (BAL), performed during flexible bronchoscopy, has gained widespread acceptance as a minimally invasive method that provides important information about immunologic, inflammatory, and infectious processes taking place at the alveolar level [1]. Studies comparing BAL cellular constituents to cells obtained from open lung biopsy have shown that the cell types and their state of activation are similar with either collection method.

This review will consider the basic principles and technique of BAL. The utility of this procedure in individual diseases is discussed in the appropriate topic reviews. The use of preprocedure medication, topical anesthesia of the upper airway, and techniques for the passage of the bronchoscope into the lung are discussed elsewhere. (See "Flexible bronchoscopy in adults: Overview" and "Flexible bronchoscopy in adults: Indications and contraindications" and "Flexible bronchoscopy in adults: Preparation, procedural technique, and complications" and "Flexible bronchoscopy in adults: Associated diagnostic and therapeutic procedures".)

PATIENT SELECTION — Bronchoalveolar lavage (BAL) has been widely used in patients with a variety of lung diseases [2]. Advantages with this technique include its noninvasive nature and ability to readily sample alveolar contents.

As a result, it has an important role in management of diffuse lung diseases for the following reasons [3,4]:

●It is an excellent method to obtain specimens to rule out opportunistic infections in immunocompromised hosts [5]

●It can provide clues to the correct diagnosis of certain diseases (eg, acute eosinophilic pneumonia and diffuse alveolar hemorrhage) (see "Idiopathic acute eosinophilic pneumonia" and "The diffuse alveolar hemorrhage syndromes")

●In some cases, it permits assessment of the stage of disease and potential responsiveness to therapy

There are no absolute contraindications to the performance of BAL beyond those commonly associated with bronchoscopy (table 1) [6,7]. (See "Flexible bronchoscopy in adults: Indications and contraindications".)

TECHNIQUE — Bronchoalveolar lavage (BAL) is performed following general inspection of the tracheobronchial tree and before biopsy or brushing [8]. This sequence minimizes the likelihood that iatrogenic bleeding will alter the concentration of cellular and protein components. The usual procedures for patient preparation, sedation, and monitoring for flexible bronchoscopy should be followed. (See "Flexible bronchoscopy in adults: Preparation, procedural technique, and complications".)

BAL differs from bronchial washings, in which secretions are aspirated from large airways directly or following the instillation of 10 to 30 mL of saline.

Whole lung lavage is a different procedure that is uniquely used as therapy for pulmonary alveolar proteinosis; 30 to 50 liters of sterile saline are delivered via a double lumen endotracheal tube under general anesthesia. (See "Causes, clinical manifestations, and diagnosis of pulmonary alveolar proteinosis in adults".)

Optimal site — Little information exists regarding the optimal site in the lung for BAL, although a good general rule is to perform the lavage where the disease is most prominent radiographically [8]. In localized disease, lavage of the involved segment is more likely to yield the best results. In diffuse disease, the right middle lobe or lingula is lavaged most commonly because, when the patient is supine, the anatomy favors maximal recovery of fluid and cells from these sites. If anatomic or technical difficulties are encountered, either the superior or anterior segment of a lower lobe may be used.

Lavage in one site is usually adequate, especially if a cumulative volume of 100 mL or greater is instilled. A lavage volume of 100 mL samples approximately one million alveoli (1.5 to 3 percent of the lung). It is thought that such a sample provides a representative picture of the inflammatory and immune processes in the alveoli regardless of the site of the lavage [9]. Nevertheless, some centers routinely instill 100 mL of fluid into each of two or three different areas. Such an approach may be particularly useful when there is marked radiographic heterogeneity in the disease process. The lavage site(s) should always be recorded in the bronchoscopy report.

Fluid instillation — Once the lavage site has been chosen, the bronchoscope is advanced into a subsegmental bronchus until the lumen is occluded; this is referred to as the "wedged" position. Care must be taken to avoid "over wedging" the bronchoscope, since this can result in additional trauma to the airway and diminish fluid recovery. A good wedge position is usually confirmed by noting slight airway collapse when gentle suction is applied. A poor wedge position allows leakage of lavage fluid around the bronchoscope, leading to cough soon after the fluid is instilled. Optimum fluid recovery occurs when the bronchoscope completely occludes the bronchial lumen of a third or fourth bronchial subsegment (figure 1) [8].

Sterile buffered or unbuffered saline (commercial 0.9 percent NaCl without additives for intravenous use) is used as the instillate. Prewarming the lavage fluid to 37ºC has been suggested to decrease coughing and to increase the cellular yield, but most groups continue to use room temperature saline.

The total volume of fluid to be instilled has not been standardized. BAL typically involves the delivery of a total of 100 to 240 mL of fluid in 20 to 60 mL aliquots; larger volumes are occasionally used for research purposes. We usually use two to three sequential aliquots of 40 to 60 mL each.

Using tubing with an inline clamp or three way stopcock, a saline-filled, 50 to 60 mL syringe is attached to the side port of the bronchoscope. The first aliquot of saline is instilled slowly and steadily [10].

Fluid recovery — After the first aliquot of saline is infused, it is recovered immediately into the same syringe by gentle continuous hand suction [2,11]. Suction should be gentle enough that visible airway collapse does not occur. In patients with marked airway collapse despite gentle suction, the suctioning process should be slower, and discontinuous suction should be used to maximize fluid retrieval. When no further fluid can be aspirated, the clamp or stopcock is closed and the syringe (but not the tubing) is removed. The second saline filled syringe is attached to the tubing and the procedure is repeated. Following these steps, a third lavage is completed, if desired.

In patients with suspected alveolar hemorrhage, three sequential lavages are performed at the same site. The recovered samples are visually inspected for any increase in hemorrhagic appearance from the first syringe to the third. (See "The diffuse alveolar hemorrhage syndromes".)

There is no established dwell time for the fluid to remain before it is suctioned back. We feel that the cellular and noncellular yield is improved by having the patient take several breaths before the fluid is retrieved, though some have raised concern about breaking the wedged position by adding this step. The total duration of lavage after wedging the bronchoscope should take approximately 5 to 10 minutes.

Lavage fluid recovery is influenced by several factors [12]:

●It increases with larger volumes instilled. The first aliquot of fluid tends to be poorly recovered (<20 percent); with subsequent aliquots, the return increases, such that 40 to 70 percent of the total instilled volume is recovered.

●Small instilled volumes (less than 100 mL) increase the likelihood of contamination of lavage fluid by mucus and large airway cells, rather than sampling of distal (alveolar) sites.

●A history of current or past smoking or the presence of chronic obstructive pulmonary disease (COPD) significantly decreases the fluid recovery rate [13]. The average BAL fluid recovery in normal healthy nonsmokers is between 50 to 80 percent of the instilled volume, compared with recovery as low as 20 to 30 percent in otherwise healthy smokers.

●Fluid recovery decreases with increasing age.

The lavage procedure should be terminated if the instilled volume exceeds recovery by more than 100 mL, or if the patient experiences substantial distress (eg, excessive coughing) or falling oxygen saturation requiring increased oxygen supplementation [8].

Monitoring — Flexible bronchoscopy is performed under conscious sedation with monitoring of blood pressure, heart rate, cardiac rhythm, respiratory rate, and pulse oximetry. BAL is rarely complicated by transient hypoxemia due to residual fluid in the alveoli. Thus, all patients should breathe supplemental oxygen during the procedure, titrated to maintain the oxygen saturation above 92 percent. In patients with borderline oxygenation, elective preprocedure intubation and ventilation during the procedure should be considered, as oxygenation will likely deteriorate during the BAL. (See "Flexible bronchoscopy in adults: Preparation, procedural technique, and complications".)

PROCESSING THE LAVAGE SPECIMEN — The recovered lavage fluid should be pooled into a single container (siliconized glass or non-cell adherent plastic) and the total volume measured. Controversy exists about whether to include the first aliquot of fluid recovered with the remainder; this is more of an issue when the specimen is being processed for cell counts in patients with interstitial lung disease than when evaluating for infection. The first aliquot is usually a small volume and represents a disproportionate amount of bronchial airway material (epithelial cells and neutrophils) compared to alveolar fluid. Our practice is to pool all of the recovered bronchoalveolar lavage (BAL) fluid and to ensure that the pooled fluid is well-mixed.

BAL fluid generally should be transported to the laboratory on ice, but can be stored or transported at room temperature if processing will occur in less than one hour. Cells in BAL fluid remain viable for up to four hours when stored at 25°C. In general, BAL samples should be processed immediately for culture; however, storage up to 24 hours at 4°C does not result in loss of diagnostic accuracy of microbiologic culture [14].

CELL COUNTING — Cell counting of BAL fluid is useful in the evaluation of certain interstitial lung diseases, such as the eosinophilic pneumonias, hypersensitivity pneumonitis, sarcoidosis, and drug-induced lung disease. (See "Role of bronchoalveolar lavage in diagnosis of interstitial lung disease".)

Initial processing — A number of steps are involved in the optimal processing and examination of BAL samples. The lavage fluid frequently contains large amounts of mucus. When cell counts will be performed in the evaluation of interstitial lung disease, filtration of the fluid through sterile cotton gauze or nylon mesh is often performed to prevent the mixing of mucus with the cell pellet after centrifugation; filtration also results in the preferential removal of bronchial epithelial cells. An aliquot of the fluid and cells should be stored in case future testing is required.

Total cell counts — The total number of leukocytes recovered by lavage is determined by examination of a sample of the pooled fluid with a hemocytometer. The cell counts are most accurate when done on the original, pooled sample before any washing procedure has been performed; washing results in a loss of total cells and a decrease in the cell viability.

The total white cell count is usually expressed both as the total number of cells recovered per lavage and as the concentration of cells per mL of recovered fluid. Red blood cells and epithelial cells should be enumerated.

Differential cell counts — Differential cell counts are used to determine whether the patient has an eosinophil or lymphocyte predominant pattern of diffuse lung disease. Cell counts are most frequently determined on slides prepared either by cytocentrifugation or filtration. These procedures are operator-dependent and should be performed by an experienced and properly trained cytotechnologist.

With either technique, at least 8 to 10 slides are prepared. The differential counts are performed with a light microscope employing 25x and 40x objectives. Counts are made from random fields of 200 to 500 cells. Esterase staining is often employed to distinguish immature macrophages from large lymphocytes. The number of ciliated or squamous epithelial cells should be noted, but they are not included in the differential count.

●Cytocentrifugation – Cytocentrifugation is the more commonly used method for obtaining a differential cell count; as the name implies, it collects cells by virtue of their outward migration when centrifuged [15]. Differential counting is commonly performed on air-dried (15 to 30 minutes) May-Grunwald-Giemsa or Wright-Giemsa stained preparations. The specimens have less distortion of cell structure and less cell loss (especially the selective loss of lymphocytes) when the "neat" fluid, ie, the original sample before centrifugation and resuspension, is used for the cytocentrifuge preparation.

Advantages of this technique include lower cost than filtration methods and the fact that cytocentrifugation slides permit detection of more subtle cytologic features which can occasionally aid diagnosis. Special stains for iron, inorganic dust, malignant cells, and microorganisms may be performed if desired.

●Filtration – With the filtration method, differential cell counts are performed after trapping cells on a filter. The cells are then stained with hematoxylin and eosin or the modified Papanicolaou technique [16]. This method offers two advantages. First, cell retention by the filter is high, providing a more accurate representation of the cell types and numbers recovered by lavage. Second, these slides can be stored indefinitely without significant loss of cell detail or staining. The major disadvantages of the filter preparations are that they may underestimate the number of neutrophils, and that the method is considerably more time consuming and costly than cytocentrifuge preparation.

Adequacy of BAL — No clear criteria exist for determining the adequacy of the BAL specimen. Unsatisfactory specimens for cell counts for evaluating interstitial lung disease:

●Contain fewer than two million total cells

●Have fewer than 10 alveolar macrophages per high power field

●Contain excessive numbers of epithelial cells, either showing morphologic degenerative changes or exceeding the number of alveolar macrophages present

●Contain a mucopurulent exudate of polymorphonuclear cells

●Contain excessive red blood cells due to trauma during the procedure

●Contain degenerative changes or laboratory artifacts obscuring cell identity

Data expression — The most common method is to present cell differential data in the same manner used for peripheral blood, ie, each white cell type is given as a percentage of the total white cells recovered. The advantage of this method is that it provides the data in a fashion that is familiar. A disadvantage is that the numbers presented are proportions of the total and, as such, changes in the absolute numbers of a single cell line necessarily affect the reported percentages of all cell lines.

With the total cell method, the differential is combined with the total cell count to quantify the absolute numbers of each cell type per volume of fluid recovered. The advantage of this method is that it also gives insight into the total number of effector cells present in the alveolar structures. Unfortunately, such quantification is most meaningful if standardized lavage volumes are instilled and recovered. At present, such standardization is not possible, and the differential percentage count of total BAL cells is the more commonly used approach. However, until clear standards are established, both methods should be used and reported by the cytology laboratory.

Noncellular components of BAL — The usefulness of routine measurement of the noncellular components of BAL in clinical practice remains to be elucidated. It has been extremely difficult to determine what proportion of the fluid recovered by lavage represents alveolar fluid from the distal airways and alveoli, so-called epithelial lining fluid (ELF). Several approaches have been proposed to determine the volume of epithelial lining fluid recovered by BAL, including quantitation of albumin, total lavage protein, potassium, methylene blue, and urea. However, none of these have become standard practice. In the future, proteomic analysis of ELF may become an attractive method to examine for changes in protein expression and secretion during the course of pulmonary disorders [17,18].

Lymphocyte subpopulations and immunohistochemistry — Application of immunofluorescent and immunocytochemical techniques to the analysis of cells recovered from BAL is helpful in the diagnosis of certain interstitial lung diseases and pulmonary lymphomas [19-21]. (See "Role of bronchoalveolar lavage in diagnosis of interstitial lung disease".)

Lymphocyte subpopulations (eg, CD3, CD4, CD8) can be assessed using immunofluorescent labeled monoclonal antibodies. After incubation with these antibodies, the cells are passed through a flow cytometer for counting and assessment of polyclonality. The CD4 to CD8 ratio can then be calculated. The advantage of flow cytometry over hand counting of immunohistochemical stained cells on cytocentrifuge prepared slides is the greater number of cells counted by flow cytometry.

Flow cytometry can also be used to identify monoclonal populations of lymphocytes that indicate lymphomatous or leukemic infiltrates.

When pulmonary Langerhans cell histiocytosis (PLCH) is suspected (middle and upper lung zone predominant nodular and cystic lesions), staining for CD-1a, S-100 protein, or CD207 (langerin) can be diagnostic [22-24]. (See "Pulmonary Langerhans cell histiocytosis", section on 'Flexible bronchoscopy' and "Role of bronchoalveolar lavage in diagnosis of interstitial lung disease", section on 'Other useful BAL findings' and "Clinical manifestations, pathologic features, and diagnosis of Langerhans cell histiocytosis", section on 'Pathologic features'.)

Normal BAL cell counts — Bronchoalveolar lavage (BAL) performed in healthy volunteers has permitted determination of the normal composition of BAL fluid [8,25,26]. The average number of cells recovered in healthy nonsmoking adults ranges from approximately 100 to 150 thousand per mL of lavage fluid recovered. The proportions of the different cell types in normal nonsmoking adults are provided in the table (table 2).

●Smoking – Smoking increases the number of cells recovered by a factor of four to six, largely due to an increase in the number of macrophages [25,27]. Additional changes in the cell populations are associated with smoking as noted in the tables: never smokers (table 2); former smokers (table 3); and current smokers (table 4) [25].

MICROBIOLOGIC ANALYSIS — Depending on the clinical situation, samples may be sent for a variety of microbiologic analyses [28]. As examples:

●Routine qualitative or quantitative bacterial culture (see "Clinical presentation and diagnostic evaluation of ventilator-associated pneumonia", section on 'Diagnostic evaluation')

●Direct fluorescent antigen (DFA) testing for Legionella species (see "Clinical manifestations and diagnosis of Legionella infection")

●Legionella culture (see "Clinical manifestations and diagnosis of Legionella infection")

●Nocardia, actinomycosis, mycobacterial, fungal, and viral culture

●Galactomannan enzyme immunoassay for fungal antigen detection (see "Diagnosis of invasive aspergillosis", section on 'Bronchoalveolar lavage fluid')

●Shell vial immunohistochemical assay or direct staining of bronchoalveolar lavage (BAL) macrophages with monoclonal antibodies for cytomegalovirus pneumonia (see "Overview of diagnostic tests for cytomegalovirus infection")

●Fluorescent or silver stain for Pneumocystis jirovecii (see "Epidemiology, clinical presentation, and diagnosis of Pneumocystis pulmonary infection in patients with HIV" and "Epidemiology, clinical manifestations, and diagnosis of Pneumocystis pneumonia in patients without HIV")

●Metagenomic next-generation sequencing of BAL fluid has promise as an adjunct to conventional microbiologic tests in the diagnosis of suspected pneumonia, especially in immunocompromised patients [29].

CYTOLOGIC ANALYSIS — Cytologic analysis of bronchoalveolar lavage (BAL) fluid may be helpful in evaluating some lung malignancies (eg, lymphangitic carcinomatosis) and diffuse lung diseases [30-34]. (See "Role of bronchoalveolar lavage in diagnosis of interstitial lung disease".)

After pooling of the total recovered lavage fluid (see 'Processing the lavage specimen' above), a sample is removed and placed in cytology preservative solution and transported to the cytology laboratory.

●Papanicolaou staining is typically used for detecting tumor cells and viral inclusion bodies.

●Periodic acid-Schiff (PAS) staining is used to identify PAS-positive lipoproteinaceous material in the distal air spaces and macrophages in pulmonary alveolar proteinosis. (See "Causes, clinical manifestations, and diagnosis of pulmonary alveolar proteinosis in adults", section on 'Diagnosis'.)

●Prussian blue staining for hemosiderin detects macrophages that have taken up red cells during chronic bleeding, as in diffuse alveolar hemorrhage. When greater than 20 percent of 200 macrophages stain positive for hemosiderin, a diagnosis of diffuse alveolar hemorrhage is usually made [35,36]. (See "The diffuse alveolar hemorrhage syndromes", section on 'Bronchoalveolar lavage'.)

●CD 1a immunostaining identifies Langerhans cells; greater than 5 percent positivity enables a diagnosis of pulmonary Langerhans cell histiocytosis. (See "Pulmonary Langerhans cell histiocytosis", section on 'Flexible bronchoscopy'.)

BERYLLIUM LYMPHOCYTE PROLIFERATION TEST — When chronic beryllium disease is suspected, a beryllium lymphocyte proliferation test (BeLPT) can be performed on BAL fluid [37]. The BeLPT requires more cells than are usually obtained during BAL, so four successive 60 mL lavages (total instilled volume of 240 mL) are performed. (See "Chronic beryllium disease (berylliosis)", section on 'Bronchoalveolar lavage'.)

ADVERSE EFFECTS — Flexible bronchoscopy with bronchoalveolar lavage is usually well tolerated. Rarely, BAL is complicated by fever, bronchoconstriction, transient hypoxemia or pneumothorax [6,7,38]. However, pneumothorax is rare enough that postprocedure chest radiographs are usually not obtained unless the clinical status of the patient has deteriorated. (See "Flexible bronchoscopy in adults: Overview" and "Flexible bronchoscopy in adults: Preparation, procedural technique, and complications", section on 'Complications'.)

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Interstitial lung disease".)

SUMMARY AND RECOMMENDATIONS

●Bronchoalveolar lavage (BAL), performed during flexible bronchoscopy, is a minimally invasive technique for evaluating the immunologic, inflammatory, and infectious processes taking place at the alveolar level in diffuse lung disease. (See 'Introduction' above.)

●BAL is an excellent technique for evaluating opportunistic infections in immunocompromised hosts. (See 'Patient selection' above.)

●There are no absolute contraindications to the performance of BAL beyond those commonly associated with bronchoscopy (table 1). (See 'Patient selection' above.)

●In localized disease, lavage of the involved segment is most likely to yield the best results. In diffuse disease, the right middle lobe or lingula is often chosen to optimize fluid recovery. (See 'Technique' above.)

●The total volume of fluid to be instilled during BAL has not been standardized. We usually use two to three sequential aliquots of 40 to 60 mL each. (See 'Fluid instillation' above.)

●A number of steps are involved in the optimal processing and examination of BAL samples for total and differential cell counting. (See 'Cell counting' above.)

●Lymphocyte subpopulations (eg, CD3, CD4, CD8) can be assessed using immunofluorescent labeled monoclonal antibodies and either flow cytometry or hand counting of a slide from a cytocentrifuge specimen. (See 'Lymphocyte subpopulations and immunohistochemistry' above.)

●Depending on the clinical situation, samples of BAL fluid may be sent for a variety of microbiologic analyses, including bacterial, viral and fungal cultures, and also direct fluorescent antigen (DFA) staining for Legionella or Pneumocystis jirovecii. (See 'Microbiologic analysis' above.)

●Cytology of BAL fluid may be of use in the evaluation of some diffuse neoplasms affecting the lung (eg, bronchoalveolar carcinoma, lymphangitic carcinomatosis, and lymphoma) and in the evaluation of some diffuse lung diseases (eg, pulmonary alveolar proteinosis). (See 'Cytologic analysis' above.)