SEMINAR 1Lecturer: Ma’am, Jhastine Elmira F.
Umali
BRONCHOALVEOLAR LAVAGE
BRONCHOALVEOLAR LAVAGE (BAL)
● A procedure done during a bronchoscopy
○ Bronchoscopy – a procedure that allows the
physician/healthcare provider to look at a
patient’s lungs by using a thin, lighted tube
called bronchoscope
■ It will help the physician to
diagnose and treat certain lung
diseases
○ Bronchoscope – The bronchoscope is put in
the nose or mouth. It is moved down the
throat and windpipe (trachea), and into the
airways.
● Also known as, bronchoalveolar washing
● Method for obtaining cellular, immunologic
and microbiologic information from the lower
respiratory tract.
○ BAL is used to collect sample from the lungs
for testing
● Useful in evaluating immunocompromised
patients, interstitial lung disease, airway
diseases, suspected alveolar hemorrhage,
pulmonary alveolar proteinosis, Langerhans
cell histiocytosis and dust exposure.
○ All of the following disease may be evaluated
with the use of a bronchoalveolar lavage
(BAL) fluid
● It is often used in conjunction with high-
resolution computerized tomography
(HRCT), medical history and physical
examination to determine the need for a
surgical biopsy.
○ HRCT – a high resolution CT scan
BRONCHOSCOPY
● Fiber-optic bronchoscope is guided into a
selected bronchopulmonary segment,
usually the right middle or lingular lobe.
○ However, target areas are better defined
using HRCT before the procedure.
● Aliquots of sterile normal saline are instilled
into the alveolar spaces through the
SEMR411 | Bronchoalveolar Lavage 1
Transcriber: Almazan, Diana R.
bronchoscope to mix with the bronchial
contents and are aspirated for cellular
examination and culture.
○ During the procedure, a saline solution is put
into the bronchoscope to wash the airways
and capture the fluid sample
● The instillation volume is between 100 and
300 mL of sterile saline in 20- to 50-mL
aliquots.
● The first aliquot is discarded, the remaining
aliquots are either sent individually for
analysis or pooled for further analysis.
● The desired fluid volume for analysis is 10 to
20 mL (minimal volume is 5 mL).
● Optimal sampling retrieves greater than 30%
with a typical recovery range of 50% to 70%.
● Low-volume recovery (less than 25%)
caused by fluid retention in the lung may
appear in chronic obstructive lung diseases
and should be noted on the requisition form.
STORAGE AND TRANSPORT
● Keep specimens at room temperature during
transport to the laboratory and process them
immediately.
● Transport the specimens on ice (4⁰C) if the
delivery is delayed for longer than 30
minutes.
● Specimens that will not be analyzed
immediately should be centrifuged, the cells
resuspended in a nutrient-supplemented
medium, and refrigerated at 4℃ for up to 24
hours.
● Specimens are unacceptable for testing after
24 hours (even if preserved or not)
● Cell counts should be performed within 1
hour (if not suspended in nutrient-
supplemented medium)
○ Stable for up to 3 hours if the fluid is in a
nutrient-supplemented medium.
● Specimens can be filtered through loose
gauze (50 to 70 µm nylon filter) to remove
mucus, phlegm and dust.
SEMINAR 1Lecturer: Ma’am, Jhastine Elmira F. Umali
MACROSCOPIC OBSERVATION
● Diagnostic tests on BAL fluid include a cell
count with differential, microbiologic studies,
and cytopathology.
● A macroscopic observation is recorded
describing the color and clarity of the
specimen. The appearance of the BAL fluid
can provide valuable diagnostic information.
● BAL fluid color can be clear (colorless), milky
white, light brown-beige and red.
● BAL fluid clarity may be described as clear,
hazy, cloudy or turbid.
● The BAL fluid should be centrifuged if it looks
milky.
● The presence of clots should be noted.
● Fluid volume is measured, cell counts and
differential counts are performed.
○ Fluid volume is significant. Because
when there is a low volume recovery, it is
possibly related with chronic obstructive
lung disease.
Macroscopic Indication:
Appearance
Bloody Acute diffuse alveolar
hemorrhage
Orange-red Older hemorrhagic
syndrome
Milky or Light brown- Pulmonary alveolar
beige proteinosis
● A bloody BAL fluid with increasing
intensities during the sequential aliquots
indicates acute diffuse alveolar hemorrhage.
● An orange-red BAL fluid is the result of an
older hemorrhagic syndrome and would be
evaluated for intracellular iron content by
cytochemistry.
● A milky or light brown-beige color BAL
fluid indicates an accumulation of
phospholipid-protein complexes derived
from pulmonary surfactant in the lung alveoli
SEMR411 | Bronchoalveolar Lavage
Transcriber: Almazan, Diana R.
and strongly suggests pulmonary alveolar
proteinosis.
CELL COUNTING
● WBC and RBC counts are performed on a
hemocytometer.
○ White blood cell (WBC) and red blood cell
(RBC) counts are performed on BAL and may
be diluted to facilitate counting using a
hemocytometer.
● Cell viability can be determined by adding
Trypan blue.
○ Trypan Blue – an azo dye used as a vital
stain to selectively color the dead
tissues/cells blue.
■ It turns dead cells blue and viable cells
unstained.
■ Live cells/tissues have intact cell
membranes so it is not colored by the
trypan blue.
● WBC counts may be diluted using BMP
LeukoChek system.
● BMP LeukoChek system is available with
with a 1 to 100 dilution of ammonium oxalate
– lyse the RBCs
○ When the RBCs have lysed and the solution
is clear, plate the fluid on a hemocytometer
and allow the cells to settle for 5 minutes.
○ Count all cells in the 18 squares on both sides
of the hemacytometer and calculate the
average of the two sides.
● RBC counts may be diluted with isotonic
saline using an MLA pipette.
● Aside from using a hemacytometer, the cell
counts on BAL fluid can also be performed
with certain automated cell counters as
designated by the manufacturer.
BMP LEUKOCHECK SYSTEM
The BMP LeukoChek is a single use test for the
microscopic counting of leukocytes and platelets in
whole blood and body fluids.
2
SEMINAR 1Lecturer: Ma’am, Jhastine Elmira F. Umali
Transcriber: Almazan, Diana R.

Procedure: (NOTE: Instead of blood, BAL will be 8. Discard the first 3 or 4 drops and then expel
used) the reservoir solution into a hemocytometer
Whole blood (20 µL) is added to the BMP with a Neubauer grid for the counting of
LeukoChek reservoir from either a fingerstick and/or leukocytes and platelets.
from a well-mixed anticoagulated tube of whole 9. A leukocyte count is performed under 100x
blood. The capillary tube provided by BMP fills via total magnification. Leukocytes are counted
capillary action to exactly 20 µL of whole blood. Each in all nine large squares of the counting
reservoir contains 1.98 mL of a 1% buffered chamber. Add 10% to the total number of
ammonium oxalate solution. this provides for a ratio cells counted and then multiply this value by
of 1 to 100, sample to total volume. 100 to get the total leukocyte count. Please
1. Puncture the cap diaphragm with the follow your current calculation method.
protective shield on the pipette assembly. *For example, if 60 cells are counted the total
2. Remove the shield and fill the tube with count would be 60 + 6 x 100 which would
whole blood from either a fingerstick and/or represent a total count of 6,600 leukocytes
from a tube of whole blood. Be sure that the per cubic mm.
capillary tube fills completely. When the 10. A platelet count is performed under 400x
blood reaches the end of the capillary tube it total magnification; platelets are counted in
will stop automatically, filling the tube with 20 all 25 squares in the large centerpiece.
µL of whole blood. Gently wipe excess blood Multiply platelets by 1000 to get a total
from the outside of the capillary tube. platelet count. Perform all counts within three
3. Squeeze reservoir slightly to expel a volume hours.
of air. Maintain pressure on the reservoir
while inserting the pipette with whole blood
into the reservoir. Be sure to simultaneously
cover the top opening of the capillary tube
holder.
4. Release pressure from the reservoir and
then from the capillary tube opening. This will
cause the whole blood to be drawn into the
diluent.
5. Gently squeeze the reservoir to rinse the
capillary tube taking care not to expel any
mixture of diluent and whole blood through
the top of the opening of the inserted capillary
tube holder. Place finger over the top of the
opening of the capillary tube holder and
gently invert the reservoir several times to
ensure proper mixing.
6. Wait 10 minutes before attempting to count
leukocytes and platelets. CELL COUNTING
7. Remove pipette assembly, invert and seat
the assembly in a reverse position into the ● Cells must be evenly distributed over the
top of the cap. This changes the pipette hemocytometer surface/slide.
assembly from a collection device to a ● Leukocyte count: No more than 15-cell
dropper. difference between the highest and lowest

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SEMINAR 1Lecturer: Ma’am, Jhastine Elmira F. Umali
Transcriber: Almazan, Diana R.

total number of cells found among the

squares counted. CELLS NORMAL CLINICAL SIGNIFICANCE
● RBC count: No more than 30-cell difference VALUE
between the highest and lowest total number Macrophages 56% - 80% Smoking-related inclusions:
of cells found among the squares counted. Smoking-related interstitial lung
● Total cells counted on each side of the disease, Pulmonary Langerhans
cell histiocytosis
counting chamber should agree within 10%
of each other. Lymphocytes 1% - 15% Increased: Interstitial lung
disease, Drug reactions,
○ Get the difference of the values of each side
Pulmonary lymphoma,
of the counting chambers, then divide it by Nonbacterial infections
the average of the 2 values, then multiply ≥ 25%: Granulomatous lung
the number times 100 disease
>50%: Hypersensitivity
● Counts that do not meet this standard should pneumonitis, Nonspecific
not be reported. Mix the specimen well and interstitial pneumonia
↑ CD4/CD8: Sarcoidosis,
repeat the count. Connective tissue disorders
● Clumps or clots present: “cell count may be Normal CD4/CD8: Tuberculosis,
Malignancies
inaccurate due to clumps of cells and/or ↓ CD4/CD8: Hypersensitivity
clots” pneumonitis, Silicosis, Drug-
○ This should be noted on the requisition slip. induced disease, HIV infection

Neutrophils < 3% Increase: Cigarette smokers,

WBC DIFFERENTIAL COUNT Bronchopneumonia, Toxin
exposure, Diffuse alveolar
damage
● Evaluating the predominant inflammatory ≥ 50%: Acute lung injury,
cellular pattern provides valuable information Aspiration pneumonia,
Suppurative infection
to the clinician in determining a differential
diagnosis. Eosinophils < 1-2% Increase: Asthma, Drug-induced
lung disease, Infections,
● The morphologic appearance of cells and
Hypersensitivity pneumonitis,
particles, such as the morphology of Eosinophilic pneumonia
macrophages in extrinsic allergic alveolitis ≥ 25%: Eosinophilic lung disease
and sarcoidosis, or the detection of dust Mast Cell < 1% > 1%, Lymphocyte >50%,
particles in occupational exposure Neutrophil >3%: Hypersensitivity
pneumonitis
conditions, provides diagnostic information.
● Differential slides are prepared by ● Macrophages, often containing a variety of
cytocentrifugation using routine procedures phagocytized material.
with staining. o Phagocytized material includes
● Stains used: Wright-Giemsa or May hemosiderin; golden, brown, or black
Grunwald-Giemsa pigment inclusions; or foamy cells.
● At least 300 cells (but often 500 to 1000 cells) ● Neutrophils are the primary granulocyte seen
are counted and classified. in BAL fluid.
● Cells seen in BAL fluid include
macrophages, lymphocytes, ratio of CD4+ ERYTHROCYTES
and CD8+ lymphocytes (CD4/CD8 ratio),
neutrophils, eosinophils, ciliated columnar ● Presence of erythrocytes indicates an acute
bronchial epithelial cells, and squamous alveolar hemorrhage.
epithelial cells.

SEMR411 | Bronchoalveolar Lavage 4

SEMINAR 1Lecturer: Ma’am, Jhastine Elmira F. Umali
Transcriber: Almazan, Diana R.

● Phagocytosed erythrocytes suggest alveolar ○ A diagnosis of pulmonary cryptococcosis can

hemorrhage has occurred within the past 48 be made by demonstrating a positive
hours. cryptococcal antigen in respiratory
● Hemosiderin-laden macrophages indicate an specimens exhibiting yeast cells that
morphologically resemble C. neoformans.
alveolar hemorrhage longer or older than 48
hours.
EPITHELIAL CELLS
CYTOLOGY
● Ciliated columnar bronchial epithelial cells
are more numerous in bronchial wash ● Cytologic studies include observing sulfur
specimens than in bronchial lavage granules (actinomycetes), hemosiderin-
specimens. laden macrophages, Langerhans cells,
○ Bronchial wash specimen – more cytomegalic cells
numerous because of a more vigorous ● Fat droplets seen in fat embolism with an Oil
washing technique Red O stain and lipid-laden alveolar
● Normal range: 4% to 17% macrophages using a Sudan III.
● Period acid Schiff staining or Oil Red O
FUNGI, VIRUSES, BACTERIA staining may be useful in diagnosing
pulmonary alveolar proteinosis or aspiration.
● Fungal elements and viral inclusions may ● Dust particle inclusions indicate
also be observed in respiratory specimens. pneumoconiosis or asbestos exposure.
● Organisms identified include Pneumocystis ● Specimens are evaluated by a pathologist in
carinii, Toxoplasma gondii, Strongyloides cytology whenever malignancy is suspected.
stercoralis, Legionella pneumophila,
Cryptococcus neoformans, Histoplasma
capsulatum, Mycobacterium tuberculosis,
Mycoplasma pneumoniae, influenza A and B
viruses, and respiratory syncytial virus.
● With the increasing concern about
nosocomial infections and antibiotic-resistant
microorganisms, BAL is more frequently
performed on ventilator-assisted patients to
detect infection and monitor antibiotic
therapy.
● Bronchoalveolar lavage is becoming an
important diagnostic test for Pneumocystis
carinii in immunocompromised patients.
○ With P. carinii, characteristic amorphous
material is seen microscopically under low
power and organisms are visible under high
power
● Cryptococcus neoformans has become a
significant opportunistic pathogen in patients
with AIDS.

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