APPENDIX B
Bronchoalveolar Lavage
Analyzing specimens obtained by bronchoalveolar lavage
(BAL) is a method for obtaining cellular, immunologic, and
microbiologic information from the lower respiratory tract.
BAL is particularly useful in evaluating immunocompromised
patients, interstitial lung disease (infectious, non-infectious,
immunologic, or malignant), airway diseases, suspected alve-
olar hemorrhage, pulmonary alveolar proteinosis, Langerhans
cell histiocytosis, and dust exposure. It is often used in con-
junction with high-resolution computerized tomography
(HRCT), medical history, and physical examination to deter-
mine the need for a surgical biopsy.
During bronchoscopy, a 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. Optimal targets are
areas of alveolar ground glass opacity, more prominent nodular
profusion, or fine reticulation.1 The segment lavaged should
be recorded on the requisition form. Aliquots of sterile normal
saline are instilled into the alveolar spaces through the bron-
choscope to mix with the bronchial contents and are aspirated
for cellular examination and culture. The instillation volume
is between 100 and 300 mL of sterile saline in 20- to 50-mL
aliquots.2 The first aliquot is discarded, the remaining aliquots
are either sent individually for analysis or pooled for further
analysis.2 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.
Keep specimens at room temperature during transport to
the laboratory and process them immediately. When delivery
to the laboratory is delayed for longer than 30 minutes, trans-
port the specimens on ice (4°C).1 Specimens that will not be
analyzed immediately should be centrifuged, the cells resus-
pended in a nutrient-supplemented medium, and refrigerated
at 4°C for up to 24 hours.1 Specimens are unacceptable for
testing after 24 hours. Cell counts should be performed within
1 hour or are stable for up to 3 hours if the fluid is in a
nutrient-supplemented medium.2 Samples can be filtered
through loose gauze (50 to 70 µm nylon filter) to remove
mucus, phlegm, and dust.
Diagnostic tests on BAL fluid include a cell count with dif-
ferential, microbiologic studies, and cytopathology. A macro-
scopic 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.
A bloody BAL fluid with increasing intensities during the
sequential aliquots indicates acute diffuse alveolar hemorrhage,
whereas orange-red BAL fluid is the result of an older hemor-
rhagic syndrome and would be evaluated for intracellular iron
content by cytochemistry.2 A milky or light brown-beige color
BAL fluid indicates an accumulation of phospholipid-protein
complexes derived from pulmonary surfactant in the lung
alveoli and strongly suggests pulmonary alveolar proteinosis.
The BAL fluid should be centrifuged if it looks milky.2 The
presence of clots should be noted. The fluid volume is meas-
ured and cell counts and differential counts are performed.
White and Red Blood Cell
Counts
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. Counts also can be performed with certain
automated cell counters as designated by the manufacturer.
When cell concentration is less than the automated instrument’s
linearity specifications, hemocytometry should be used.2
If a hemocytometer is used, WBC counts may be diluted
using the BMP LeukoChek system to facilitate counting. A
BMP LeukoChek system is available with a 1 to 100 dilution
of ammonium oxalate to 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 hema-
cytometer and calculate the average of the two sides. Using
the following formula, calculate the WBC count using the
following formula:3
average number of cells dilution factor 10
WBC/cmm =
9 squares
RBC counts may be diluted with isotonic saline using an
MLA pipette. Plate the fluid on a hemocytometer and allow it
to settle for 5 minutes. Count both sides of the hemocytometer
and use the following formula to calculate the RBC/cmm:3
number of cells dilution factor 10
RBC/cmm =
Number of squares counted
Cells must be evenly distributed over the hemocytometer
surface. For a leukocyte count within the reference range, there
should be no more than a 15-cell difference between the high-
est and lowest total number of cells found among the squares
counted. For a RBC count, there should be no more than a
30-cell difference between the highest and lowest total number
of cells found among the squares counted. Total cells counted
293
294 Appendix B | Bronchoalveolar Lavage
on each side of the counting chamber should agree within 10%
of each other. Counts that do not meet this standard should
not be reported. Mix the specimen well and repeat the count.
If clumps of cells or clots are present, note on the requisition
that the “cell count may be inaccurate due to clumps of
cells and/or clots.” BAL body fluids may not be counted on
the Sysmex instrumentation because of the varied types of
cells present.3
Leukocytes
Evaluating the predominant inflammatory cellular pattern pro-
vides valuable information to the clinician in determining a
differential diagnosis. The morphologic appearance of cells and
particles, such as the morphology of macrophages in extrinsic
allergic alveolitis and sarcoidosis, or the detection of dust par-
ticles in occupational exposure conditions, provides diagnostic
information.2
Differential slides are prepared by cytocentrifugation
using routine procedures with staining (Wright-Giemsa or
May Grunwald-Giemsa) and at least 300 cells but often 500 to
1000 cells are counted and classified.4 Cells seen in BAL
fluid include macrophages, lymphocytes, ratio of CD4+ and
CD8+ lymphocytes (CD4/CD8 ratio), neutrophils, eosinophils,
ciliated columnar bronchial epithelial cells, and squamous
epithelial cells.
Macrophages, often containing a variety of phagocytized
material, are the cells most frequently seen, in numbers ranging
from 56% to 80% (Fig. B–1). Phagocytized material includes
hemosiderin; golden, brown, or black pigment inclusions; or
foamy cells. A predominance of macrophages containing
smoking-related inclusions suggests smoking-related intersti-
tial lung disease or pulmonary Langerhans cell histiocytosis.1
Lymphocytes, normally constituting 1% to 15% of the cell
population, are increased in interstitial lung disease, drug re-
actions, pulmonary lymphoma, and nonbacterial infections. A
lymphocyte differential count equal to or greater than 25%
suggests granulomatous lung disease, whereas a lymphocyte
differential count greater than 50% suggests hypersensitivity
pneumonitis or nonspecific interstitial pneumonia.1 The ratio
Figure B–1 Bronchoalveolar lavage: Normal macrophages and lym-
phocytes (×1000).
of CD4 to CD8 lymphocytes further defines the disease
process. An elevated CD4/CD8 indicates sarcoidosis or con-
nective tissue disorders. A normal CD4/CD8 is associated
with tuberculosis or malignancies, whereas a low CD4/CD8 in-
dicates hypersensitivity pneumonitis, silicosis, drug-induced
disease, or HIV infection. Immunologic analysis is performed
by flow cytometry.
Neutrophils are the primary granulocyte seen, with a
normal value of less than 3%. They are elevated in cigarette
smokers, and in cases of bronchopneumonia, toxin exposure,
and diffuse alveolar damage. A neutrophil count equal to or
greater than 50% strongly suggests acute lung injury, aspiration
pneumonia, or suppurative infection.1
Eosinophils, usually less than 1% to 2% of the total cells,
are elevated in asthma, drug-induced lung disease, infections
(parasitic, mycobacterial, or fungal), hypersensitivity, pneu-
monitis, and eosinophilic pneumonia. An eosinophil differen-
tial count greater than or equal to 25% diagnoses eosinophilic
lung disease.1
A mast cell differential count greater than 1% combined
with a lymphocyte count greater than 50% and a neutrophil
count greater than 3% strongly suggests hypersensitivity
pneumonitis.1
Erythrocytes
The presence of erythrocytes indicates an acute alveolar hem-
orrhage. Phagocytosed erythrocytes suggest that an alveolar
hemorrhage has occurred within the past 48 hours, whereas
hemosiderin-laden macrophages indicate an alveolar hemor-
rhage older than 48 hours.
Epithelial Cells
Ciliated columnar bronchial epithelial cells are more numer-
ous in bronchial wash specimens than in bronchial lavage
specimens because of the more vigorous washing technique.
In a lavage specimen, these cells normally range from 4% to
17% (Fig. B–2).
Figure B–2 Bronchoalveolar lavage: Ciliated bronchial epithelial cells;
notice the eosinophilic bar (×1000).

Fungi, Viruses, and Bacteria
Fungal elements and viral inclusions may also be observed
in respiratory specimens. Organisms identified include Pneu-
mocystis carinii, Toxoplasma gondii, Strongyloides stercoralis,
Legionella pneumophila, Cryptococcus neoformans, Histoplasma
capsulatum, Mycobacterium tuberculosis, Mycoplasma pneumo-
niae, influenza A and B viruses, and respiratory syncytial
virus. Quantitative or semiquantitative cultures are useful
for ventilator-associated pneumonia and can diagnose the
infection if the organism is identified. 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 diagnos-
tic test for P. carinii in immunocompromised patients. With
P. carinii, characteristic amorphous material is seen microscop-
ically under low power and organisms are visible under high
power (Figs. B–3 and B–4).5 C. neoformans has become a
significant opportunistic pathogen in patients with AIDS. A
diagnosis of pulmonary cryptococcosis can be made by
demonstrating a positive cryptococcal antigen in respiratory
specimens exhibiting yeast cells that morphologically resemble
C. neoformans. The extent of the cryptococcal infection corre-
lates with the antigen titer.
Figure B–3 Bronchoalveolar lavage: Amorphous material associated
with P. carinii when examined under low power (×100).
Appendix B | Bronchoalveolar Lavage 295
Figure B–4 Bronchoalveolar lavage: Characteristic cup-shaped organ-
isms indicating P. carinii (×1000).
Cytology
Cytologic studies include observing sulfur granules (actino-
mycetes), hemosiderin-laden macrophages, Langerhans cells,
cytomegalic cells, fat droplets seen in fat embolism with an
Oil Red O stain, and lipid-laden alveolar macrophages using a
Sudan III stain. Periodic acid Schiff staining or Oil Red O stain-
ing may be useful in diagnosing pulmonary alveolar proteinosis
or aspiration.1 Dust particle inclusions indicate pneumoco-
nioses or asbestos exposure. Specimens are evaluated by a
pathologist in cytology whenever malignancy is suspected.
References
1. Meyer, KC, Raghu, G, Baughman, RP, et al.: An official American
Thoracic Society clinical practice guideline: The clinical utility of
bronchoalveolar lavage cellular analysis in interstitial lung disease.
Am J Respir Crit Care Med 185(9):1004–1014, May 1, 2012.
doi:10.1164/rccm.201202-0320ST.
2. Clinical and Laboratory Standards Institute: Body Fluid Analysis
for Cellular Composition; Approved Guideline. CLSI document
H56-A. Clinical and Laboratory Standards Institute. Wayne, PA
2006.
3. Bronchoalveolar Lavage, BAL Cell Count and Differential.
Methodist Hospital: Clinical Laboratory Procedure Manual.
Omaha, NE, January 5, 2012.
4. Jacobs, JA, DeBrauwer, EI, et al: Accuracy and precision of
quantitative calibrated loops in transfer of bronchoalveolar
lavage fluid. J Clin Micro 38(6):2117–2121, 2000.
5. Linder, J: Bronchoalveolar Lavage. ASCP, Chicago, 1988.