Broncboalveolar lavage using flexible fiberoptic bronchoscopy has emerged as a powerful investigative tool in

respiratory medicine. It has helped in better understanding of pathogenesis of various interstitial lung diseases

especially idiopathic pulmonary fibrosis and sarcoidosis. It has also been found useful in the diagnosis of various

infectious and non-infectious diseases of lung particularly in immunodeficient patients. Even though this technique

is used all over the world, it is still a research tool and more studies are required to exactly assess its value as a

diagnostic fool in clinical medicine. BAL is a safe procedure

Vijayan, V K

NIRT Institutional Repository

Current Diagnostic Status of Bronchoalveolar Lavage StudiesV.K. VijayanDeputy Director Cardio-pulmonary medicine Unit, TB Research Centre Indian Council of Medical ResearchBroncboalveolar lavage using flexible fiberoptic bronchoscopy has emerged as a powerful investigative tool inrespiratory medicine. It has helped in better understanding of pathogenesis of various interstitial lung diseasesespecially idiopathic pulmonary fibrosis and sarcoidosis. It has also been found useful in the diagnosis of variousinfectious and non-infectious diseases of lung particularly in immunodeficient patients.Even though this techniqueis used all over the world, it is still a research tool and more studies are required to exactly assess its value as adiagnostic fool in clinical medicine. BAL is a safe procedure.IntroductionBronchoalveolar lavage (BAL) using      a flexible fiberoptic bronchoscope hasemerged as an important investigativetool in pulmonology during the lastdecade1. BAL is a technique that allowsthe recovery of both cellular and noncellularcomponents from the epithelial surfaceof the lower respiratory tract and differsfrom bronchial washings which refer tothe aspiration of either secretions orsmall amounts of instilled saline from thelarge airways2. The first bronchoalveolarlavage was performed by Cantrell andcolleagues in 1973 in normal volunteers3.Subsequently it has become anacceptable procedure tosample thecontents of the lower respiratory tract4.The analysis of cellular and molecularcomponents of the alveolar epitheliallining fluid has helped to elucidate thepathogenetic mechanisms and also to assess the activity of interstitial and otherlung diseasess. This review deals mainlywith the methods and the role ofbronchoalveolar lavage in the diagnosisand management of various pulmonarydiseases.Method of bronchoalveolarlavage2,6 Bronchcalveolar lavage is performedwith a flexible fiberoptic bronchoscope(FOB) usually under local anesthesia2,6.Correspondence may be addressed to:Dr. V. K. Viayan,Cardio-pulmonary Medicine UnitTB Research CentreI.C.M.R.Madras 600 031It can also be performed in patientswith an oral or nasotracheal tube inposition by inserting the FOB throughthe tube, provided its diameter is greaterthan 6mm6. Patients can be pre- medicated with atropine (0.6mg IM). Localanesthesia is accomplished by sprayingof the nasal, oral,pharyngeal andlaryngeal areas with 4% lidocaine. Thetrachea, carina and bronchi can beanesthetized by direct instillation of1.5-2% lidocaine. BAL is a safeprocedure. Mild side-effects such ascoughing during lavage, fever and chillssome hours after lavage and transientalveolar infiltration 24 hours after theprocedure can occur in a proportion ofsubjects. Supplemental oxygen bynasal prongs, ear oximetry and ECG-monitoring are advised in patients withs vere underlying disease or othercritical conditions.In localized disease, the involvedsegment is lavaged. However, in diffuselung disease, the middle lobe or lingulahas been most commonly used asstandard sites; occasionally a lateral oranterior segment of a lower lobe isalso used. The bronchoscope iswedged in fourth or fifth order bronchi,taking care not to “overwedge” whichmay result in less fluid recovery. BAL isusually performed with buffered orunbuffered, pyrogen-free saline (isotonic0.9% NaCI) at room temperature orwarmed to body temperature (37°C). Thetotal volume instilled per site is 100 to150ml, and it is usually instilled as five20-ml aliquots or two or three 50-mlaliquots. The maximum total voIumeused (all sites combined) is usually nomore than 300 ml7. After each aliquoti  instilled, it is aspirated either by handsuction into the syringe or by normalclinical suction apparatus using negativepressures of 25-100mm Hg into aspecimen trap. There is no special time‘for lavage fluid to dwell in the lung beforeit is removed. However, the patient canbe encouraged to take one or twonormal breaths before the fluid iswithdrawn. The recovery from firstaliquot is generally poor.Thereafterthe return from subsequent aliquots is60 to 70% of the instilled volume innormal subjects and the overall recoveryis above 50 to 60%. Recovery of fluid isd creased in smokers and in mostpatients with diseased lungs, especiallyif airway obstruction exists. Afterbronchoscopy, the subject should beobserved for 4 to 6 hours until the effectsof local anesthesia have worn away.After measuring the recoveredvolume, the bronchoalveolar lavagefluid is strained through a sterile gauzeto remove the mucous particles. Totalcell count is done on a haemocytometerin the unconcentrated lavage fluid andexpressed both as the absolute numberof cells recovered by lavage; or asthe concentration of cells per ml ofrecovered lavage fluid.Cell viabilitycan be performed by the Trypan blueexclusion test. Total cell counts canalso be done after centrifugation.However, this may result in loss ofcells. Differential cell count can bed ne from millipore filter preparations,which can be stained with a modifiedhematoxylin- eosin method or with aPAP technique. Differential ceil countscan also be made from a cytocentrifugedcell preparation stained with Wright-51Giemsa. However, this methodunderestimates the percentage oflymphocytes, especially if their proportionis increased. The differential countsare made by viewing with a lightmicroscope, employing objectives ofx40 x 100. At least 300-500 cellsshould be counted. The cells recoveredfrom a normal non-smoking individualare alveolar macrophages, lymphocytes,neutrophils, eosinophils, mast cells and  occasionally plasma cells. A highpercentage of epithelial cells (>5%)is indicative of contamination withbronchial inflammatory cells. Special immunofluorescent andimmunocytochemical techniques using  monoclonal antibodies directed againstantigens present on the ceil surface oflymphocytes and/or monocytes/macrophages can be applied for bettercharacterization of the nature of thecells recovered by the BAL.The saline used for lavagesignificantly dilutes the epithelial liningfluid. Therefore, the soluble componentsin BAL cannot be quantitated becausea completely reliable denominator orreference substance is not yet available.Even though albumin, potassium,methylene blue and urea were triedpreviously, no ideal. denominator ormethod of calculation exists to quantitatethe dilution factor.                                  BAL in healthy volunteersCellular and acellular constituentsin bronchoalveolar lavage fluid (BALF)in non-smoking adults 8 are given inTable 1. In smokers, the cell yield is 3to 4 times greater. An increased numberof alveolar macrophages is recoveredfrom smokers and there may also be a slight increase(1-4%) in neutrophils. Inhealthy smokers, the proportion of BALT-helper cells was lower, T-suppressorcells higher and therefore, the helper -suppressor ratio is lower than that innon-smokers. BALF IgG was greater insmokers than in never-smokers9.diagnostic in some of the infectiousand non-infectious pulmonary diseases.I  some others BAL findings maycontribute to the diagnosis, although theyare not diagnostic.(a) Diseases in which BAL findingsare diagnostic(i) Infectious diseasesIn immunodeficient patients (eg.patients receiving immunosuppressivedrugs and with advanced humanimmunodeficient virus infection) withpulmonary infiltrates suggesting lowerrespiratory tract infection, BAL is a safeprocedure for making definitive aetiologicdiagnosis. In patients with HIV diseaseand suspected Pneumocystis cariniipneumonia, an induced sputumexamination should precedeBAL.The isolat ion of pathogenicmicroorganisms in lavage fluid can beconsidered diagnostic only if the isolatedorganism is not known to colonize therespiratory tract. BAL also can beuseful in immunocompetent patientsespecially with nosocomial pneumonia.BAL fluid should be analyzed forbacterial, fungal, opportunisticandviral infections. Lavage differential celldetermination shows a lymphocyticalveolitis in patients with HIVinfection. Infectious diseases that canbe diagnosed by isolation ofmicroorganisms from BAL fluid are shownin Table 2.(ii) Non-infectious diseaseEven though the major contributionof BAL was in understanding thepathogenesis of lung injury, repairand fibrosis especially in interstitial lungdisease, BAL findings are diagnosticin a few of the followingnon-infectious respiratory disorders (Table 2).(a) Alveolar proteinosisBAL fluid in alveolar proteinosis isTable 1Cel lu lar  and soluble const i tuents in BAL f lu id inn o n - s m o k i n g  a d u l t sCel l s           Total    Macrophages %   Lymphocytes %T cellsHelper (CDA)Suppressor (CDA)B cellsNeutrophils %Eosinophils %Basophils %Soluble factorsIgG, IgA    IgMC4, C3, factor BC5Mean ± SD)    10 to 15 x106 cells/dl of BAL fluid.84±1     11 ±1 6 2 ± 2  46±325±25 ± 2<1<1< 1+-+opaque and/or milky in appearance.Microscopic evaluation reveals largeacellular eosinophilic bodies against abackground of small eosinophilicgranules and amorphous debris. Oncombined Alcian blue - periodic acid-schiff (PAS) stain, there is predominantPAS staining of the proteinaceous materialwith a lack of significant Alcian bluestaining. Small lamellar bodies of wavyor regular periodicity, tubularmyelinstructures and myelin-likemultilamellated structures with electrondense central region are characteristicfindings in electron microscopy.carcinoma and lymphatic spread ofmetastatic cancer. The diagnostic yieldhad varied from 14 - 69% in various studies.A number of tumor markers have beenstudied, but none had proved to beiagnostic.Non-infectious diseases inwhich BAL may be helpful indiagnosis       (i) Pulmonary haemorrhageBAL fluid may be either bloody or(b) Histiocytosis X (Langerhan’scell granulomatosis)  Total 2 counts are increased inBALF and 90% of the patients withLangerhans  cell granulomatosis (LCG)are smokers The differential cell countreveals a high percentage of alveolarmacrophages, and a slight increaseof neutrophils and eosinophils. Thediagnostic feature is the demonstrationof Langerhans’ cells. These cellscontain characteristic pentalaminar plate-like cytoplasmic organelles andcytoplasmic inclusions variously referredto as Langerhans’ granules, Birbeck’sgranules or histiocytosis X bodies whichcan be demonstrated by electronmicroscopy. Langerhans’ cells alsoexpress CD a(T6) surface antigen. BAL    1.2.3.4.5.6.7.8.9.samples can be diagnostic if there aregreater than 5% of cells that react withOKT6 antibody.(c) Pulmonary malignanciesBronchoalveolar lavage of affectedorange - pink in colour in patients withpulmonary haemorrhage (PH) andhaemosiderosis. The total cellular countand alveolar macrophages are increasedin PH. Presence of haemosiderosis ladenalveolar macrophages may aid indiagnosis. Free red blood cells and redblood ceils in macrophages can also beobserved in patients with PH.(ii) Eosinophilic lung diseaseEosinophilic infiltrates in the lung canTable 2Infectious diseases in which isolation from BALF isd iagnos t i cPneumocystis cariniiToxoplasma gondiiStronglyoidesLegionellaHistoplasmaMycobacterium tuberculosisMycoplasmaInfluenzaRespiratory syncytial virusNon-infectious diseases in which analysis of BALFis  diagnostic                                                               1.    Alveolar proteinosis                                          2. Langerhans’ cell granulomatosis (Histocytosis X).3. Pulmonary malignanciessegments may permit a cytologicaldiagnosis of cancer. Bronchial and Non- infect ious diseases in which BALF f inding mayalveolar specimens can be processed inany one of the following procedures: 1.smears,   cytocentrifuge preparations,        2.membrane filter preparations and cellpellets embedded in paraffin. Processed 3.samples canI be stained with 4.Papanicolaou, Wright-Giemsa or 5.haematoxylin-eosin stains.be helpful  in  the diagnosisPulmonary haemorrhage6.Special stains using monoclonalantibodies for tumor markers can alsoEosinophilic lung diseasesOccupational lung diseases (berylliosis, asbestosis)                   Hypersensitivity pneumonitisIdiopathic pulmonary fibrosisSarcoidosisbe employed.The diagnostic yield of(Modified from American Thoracic Society Statement, Am Rev Respir Dis 1990; ,142:BAL is higher for bronchoalveolar cell    481-486)occur in a variety of disorders such asallergic broncho-pulmonary aspergillosis,tropical eosinophilia and eosinophilicpneumonia. Since eosinophils are notseen in lavage fluid of normal controls,any increase in eosinophils in BALFsuggests a pathologic process.Thehighest eosinophil counts are usuallypresent in tropical eosinophilia andeosinophilic pneumonia. A 5-10 foldincrease in total cells with a very higheosinophil count in BALF in our countrymay suggest tropical eosinophilia 10,11.(iii) Occupational lung diseasesBerylliosis and asbestosis are  two occupational lung diseases inwhich BAL may be helpful in thediagnosis. Berylliosis is a granulomatouslung disease that is clinically andhistologically similar to sarcoidosis, butthe inciting antigen is known in berylliosis.BALF shows macrophage-lymphocytealveolitis and most of the lymphocytesare helper T calls. Proliferative responseof broncho-alveolar lymphocytesespecially CD4 + T cell subset toberyllium salts can be utilized as adiagnostic test. BAL in asbestosis ischaracterized by several fold increasesin the numbers of alveolar macrophagesand by mild-to-moderate increases inneutrophils and lymphocytes. There is agood correlation between asbestos bodiesin BALF and in lung parenchyma. Oneasbestos body per mil. of lavage fluid isshown to be associated with more than1000 asbestos bodies per gram of driedlung parenchyma.(iv) Hypersensitivity pneumonitisHypersensitivity pneumonitis (HP),also known as extrinsic allergic alveolitis,is an interstitial lung disease sociatedwith repeated exposure to a wide rangeof inhaled organic dusts and relatedoccupational allergens. BAL studiesreveal an increase in the proportion andnumber of lymphocytes and majority oflymphocytes* are CD8+ lymphocytesresulting in a low CD4/CD8 ratio. Thepresence of BAL lymphocytescharacterised by CD3+/CD8+/CD56+CD57-/CD16- phenotype is highlysuggestive of Hp.(v) Idiopathic pulmonary fibrosisAlveolitis of activeidiopathicpulmonary fibrosis (IPF) is characterizedby three to four fold increase intotalnumbers of lung effector cells and thecells comprising alveolitis are mainlyalveolar macrophages andneutrophilswith smaller numbers oflymphocytesand eosinophils. The combination ofincreased neutrophils and eosinophilsoccur in about two-thirds of patients withIPF. Patients with lavage lymphocytosisrepresent an earlier cellular stage ininflammatory process of IPF andimproves with corticosteroids. However,lavage eosinophilia is associated withadvanced IPF and is unresponsive totreatment and lavage neutrophils donot reflect response to treatment.(vi) The alveolitis in sarcoidosis characterized by an increase in thenumber of T-lymphocytesandmacrophages in the alveolar structures.T-lymphocytes are mainly of helper/induced type, resulting in an elevation ofhelper to suppressor T-cell ratio.Inn appropriate clinical setting, BALFT-cell lymphocytosis and CD4 : CD8 ratiomore than 3.5 can be consistent withadiagnosis of sarcoidosis. More than20% 01*/07* macrophages in lavagefluid may also be helpful in the diagnosisif combined with other parameters. Anincrease in mast cells in BALF maysuggest initiation of fibrosis and thus is aprognostic sign.2. Therapeutic applications5Whole lung lavage (WLL) has beenfound to be useful in the treatment ofalveolarproteinosis. Mucus plugs canbe removed in patients with statusasthmaticus through a bronchosccpe afterthe instillation of saline or acetylcysteine.But, this procedure is associated with ahigh risk benefit ratio. WLL has beenused to remove the irritating dust in theacute form of silicosis in order toprevent irreversible damage. WLL hasalso been proposed in the treatment ofalveolar microlithiasis and exogenouslipoidisis.understanding the pathogenesis of variousinterstitial lung diseases and has beenfound to be useful in the dianogsis ofcertain pulmonary disorders, it is still aresearch tool and further studies arerequired before it can be recommendedfor general clinical use.References1.2.3.5.6.7.8.9.10.11.Rossi GA, Sacco O, Vassalio F andInnocenti LD. Bonchoalveolar lavageduring fiberoptic bronchoscopy: Whathas it brought to pulmonary medicine.Respiration 1988; 54 (Suppl - 1): 49-58.Reynolds HY. Bronchoalveolar lavage    Am Rev Respir Dis 1987; 135: 250-263.Cantrell ET, Warr GA, Busbee DL, MartinRR. Induction of arylhydrocarbonhydroxylase in human pulmonary-alveolar macrophages by cigarettesmoking. J Clin Invest 1973; 52:1881-4.4. American Thoracic Society. Clinicalrole of bronchoalveolar lavage in adultswith pulmonary disease. Am Rev RespirDis 1930; 142: 481-486.Clinical guidelines and indications forbronchoalveolar lavage (BAL): Report ofthe European Society of pneumonologyTask Group on BAL Eur Respir J 1990;3: 937-974.Technical recommendations andguidelines for bronchoalveolar lavage(BAL): Report of the European Society ofPneumonology Task Group on BAL. EurRespir J 1989; 2: 561-585.Crystal RG, Reynolds HY and KalicaAR. Bronchoalveolar lavage: The report,,of an international conference.Chest1966; 89:122-131.Daniel RP, Elias JA, Epstein MD andRossman MD. Bronchoalveolar lavage:Role in the pathogenesis, Diagnosis andManagement of interstitial lung diseases.Annals of Internal Medicine 1985;102:93-108.NHLBI - GNR Workshop on ‘Usefulnessand pespectives of Bronchoalvolarlavage*, Pharma June 13 -14, 1988.Respiration 1989; 56: 245 - 251.Vijayan VK, Sankaran K, Venkatesan Pand Prabhakar R. Effect ofdiethylcarbamazine on alveolitis oftropical eosinophilia. Respiration 1991;255 - 259.Pinkston P, Vijayan VK, Nutman TB et al.Acute tropical pulmonary eosinophilia  characterizationof the lower respiratorytracts inflammationand its response totreatment J Clin   Invest 1987; 80:216-2 2 5 .    

Current diagnostic status of bronchoalveolar lavage studies

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