Broncho-Alveolar Broncho-Alveolar Lavage Fluid Lavage Fluid

AnalysisAnalysis

By Dr Uttam Kumar DasBy Dr Uttam Kumar DasPGT Dept of Pathology

BSMC Bankura05.03.2014

Introduction:BAL is performed with the FOB in a wedge position within the selected broncho-pulmonary segment.

The total instilled volume of normal saline should be from 100-300ml, repeated 2 to 6 times with 20-50ml saline each.

To obtain an adequate specimen 40-60 mL (usually 40-70% recovery of the total instillate) must be drawn back.

Aspirates and washings provide information on the status of the respiratory tract in small bronchi beyond reach of the bronchoscopic brush.

Area That Is Lavaged Procedures were usually performed in the

right middle lobe or lingula

But lavage can be done in the most affected areas of the lung

(In evaluating BAL in patients with Pneumocystis jiroveciipneumonia, it was found that lavage in the upper lobeshad a higher yield than the traditional right middle lobeor lingula)

Handling of Aspirated Fluid At the time of the lavage cells should be

stored in silicone-coated or similar containers

Cell counts should probably be made on unfiltered, unwashed, and unconcentrated

samples (If concentration is performed, the method should be specified)

Centrifugation to concentrate proteins and cells can lead to loss of cells

Washing the cells can change the differential count considerably

Satisfactory Sample1. A total of 2×106 cells is considered a minimum

requirement

2. Furthermore, more than 10 macrophages should be present in a high-powered microscopic field

3. Degenerative changes should cover less than 20% of the specimen area on the slide

4. If the number of squamous epithelial cells, bronchial cells, RBCs, or inflammatory cells exceeds that of macrophages, the specimen is considered unsatisfactory

The Storage of Fluid Cells stored at 4< C can be analyzed up to 24

hours after the procedure without significant changes in the count and differentials

Certain proteins may be temperature sensitive and the samples may need to be stored at -80< C

Correcting for Bronchoalveolar Lavage Dilution Instilled fluid is mixed with the endogenous fluid

in the alveoli

Alveolar space is also in contact with a vascular space-So water and solutes can transfer into the alveolar space

This process leads to the uncertainty of any measurement of the concentration of any material in the alveolar space

Solution

One method has been to report per mL ofaspirated fluid.

Using this correction method has allowed clinicians to quantitate the number of bacteria in the alveolar space and to therefore diagnose bacterial pneumonia

Unsatisfactory BAL specimen that shows squamous epithelial cells (large cells) and degenerating columnar epithelial cells (arrow)

Steps in Handling Cellular Population of Bronchoalveolar Lavage Fluid

Cellular Staining

Papanicolaou Stain: -Detect Cancer & Infection -Not good at differentiating between

inflammatory cells

Toluidine blue staining: -Mast cells are better seen

Wright-Giemsa stain: -Good at differentiating between inflammatory

cells

Diff-Quik (modification of the Wright-Giemsa stain): -Is a rapid method allowing staining of the slide

within a few minutes

Limitations: -The cells must be adequately adhered to the slide

prior to fixation -Some cells are underestimated by these

techniques

Oil red O stain: -In fat embolism

Fat and Lipid stain (e.g. Sudan III): -Lipoid pneumonia (aspiration) Lipid-laden alveolar macrophage index >

100 (Sensitivity of 100%, Specificity 57%)

Periodic acid-Schiff (PAS): -Pulmonary alveolar proteinosis

Other stains

KOH preparation: Fungal

Auramine-rhodamine or Ziehl-Neelson: Mycobacterial

Modified acid fast stain (Kinyoun): Nocardia Silver methenamine: Pneumocystis jirovecii

pneumonia, fungal Direct fluorescent antibody testing (DFA) for

Legionella

Number of Cells Counted

De Brauwer et al determined that between 300 and 500 cells counted provided a good representation of the number of nucleated cells for a BAL sample

Different cell types in respiratory tract Upper respiratory tract Ciliated pseudostratified columnar cells Squamous cells

Trachea and bronchi Peudostratified Ciliated columnar cells Goblet cells

Terminal bronchioles Low columnar or cuboidal-may be ciliated

Club cells (Clara cells)-nonciliated, secretory cuboidal cells

Alveoli Type I pneumocytes-simple squamous alveolar cells

Type-II pneumocytes-great alveolar cells

Dust Cell-in the alveoli

Alveolar macrophages- in the connective tissue of alveolar walls or interalveolar

septa

General indications for BAL:

-Non-resolving pneumonia

- Diffuse lung infiltrates (interstitial

and/or alveolar)

- Infiltrates in an immunocompromised

host

- Suspected alveolar hemorrhage

- Quantitative cultures for VAP

- Exclusion of diagnosable conditions by

BAL

- Research

Gross examination-

Pulmonary alveolar proteinosis

-Opaque or translucent brownish or sandy colored fluid

-Sediments out into two layers if left to sit

Alveolar hemorrhage

-Sequentially more hemorrhagic with each aliquot

Amorphous, predominantly acellular debris (pulmonary alveolar proteinosis)

Papanicolaou stain- foamy proteinaceous alveolar cast

Alveolar macrophages

Normal >80%

Decreased in

Sarcoidosis (to 55% or less)

Cell count and differential count

Predominance of alveolar macrophages in BAL from a normal subject

This photomicrograph shows an asbestos body under higher magnification, surrounded by alveolar macrophages

Neutrophils (Normal <3%): Nonspecific, but suggests active alveolitis

Increased in: ARDS Connective tissue disorders Idiopathic pulmonary fibrosis Infection Pneumoconiosis Wegener's granulomatosis

BAL neutrophil predomnance with intracellular bacteria

Eosinophils (Normal <1-2%) Low to Moderate Eosinophilia (5-20%):

Drug induced lung disease Minocycline Nitrofurantoin Penicillin

Infections Parasitic Mycobacterial Fungal

Bronchial Asthma

Malignancies (infrequently)

Other interstitial pneumonias occasionally (BOOP or COP, IPF/UIP, ILD associated with Connective tissue disorders, Sarcoidosis)

BAL eosinophilia

Moderate to Marked Eosinophilia (>20%):

Allergic bronchopulmonary aspergillosis

Acute eosinophilic pneumonia

Churg-Strauss syndrome

Chronic eosinophilic pneumonia

Idiopathic hypereosinophilic syndrome

Lymphocytes (Normal <15%) Normal CD4/CD8 (0.9-2.5:1): Tuberculosis Malignancies

Low CD4/CD8: Hypersensitivity Pneumonitis Silicosis Drug-induced lung disease HIV infection BOOP (COP)

Elevated CD4/CD8: Active sarcoidosis (>4:1 up to 10:1)AsbestosisBerylliosisCrohn's diseaseConnective tissue disordersSometimes in normal persons (inc. with

age)

BAL Lymphocytosis

Erythrocytes

◦ Elevated erythrocyte count - early sign of

alveolar hemorrhage (first several hours)

◦ Phagocytosed erythrocytes - alveolar

hemorrhage within 48 hrs

◦ Hemosiderin laden macrophages - alveolar

hemorrhage > 48hrs

Foamy macrophages: Non specific finding May be seen in amiodarone use

Malignancies (sensitivity ranges from 35% to 70%) ◦ Lymphangitic carcinomatosis ◦ Lymphoma ◦ Bronchoalveolar carcinoma and other primary lung

malignancies◦ Extrapulmonary malignancies

Hemosiderin Laden Macrophages: 20% is highly specific and sensitive for alveolar

hemorrhage

Langerhans cells >5% suggestive of Pulmonary Langerhans cell

histiocytosis

Cytomegalic cells Viral pneumonias (cytomegalovirus, herpes)

Sulfur granules: Actinomycetes

Microbiology

Cultures

Polymerase chain reaction (PCR) TB and

others

Quantitative or semi-quantitative

cultures VAP

GMS BAL fluid showing round to cup shaped cysts of Pneumocystis

Pap-stained BAL fluid demonstrating large, retractile yeast forms of Blastomyces dermatiditis (400X)

Pap-stained BAL fluid showing variably-sized, round yeast forms of Cryptococcus neoformans (1000X)

Wright-stained BAL fluid demonstrating intracellular yeast forms of Histoplasma capsulatum

Wright-stained BAL fluid demonstrating oblong, budding yeast forms with pseudohyphae (1000X)

Complications/Adverse events:

No complications in up to 95%

Cough

Transient fever (2.5%)

Transient chills and myalgias

Transient infiltrates in most (resolves in 24 hours)

Bronchospasm (<1%)

Transient fall of lung function

Transient decrease in baseline PaO2

In patients with already severely compromised respiratory status, the loss of lung function may necessitate the need for Mechanical Ventilation

Pulmonary alveolar microlithiasis

Calcospherites can be demonstrated in BAL fluid

(one of the tiny round bodies formed during calcification by chemical union of calcium particles and albuminous matter of cells)

Thank You