Vet Clin Equine 24 (2008) 423–435

Bronchoalveolar Lavage: Sampling

Technique and Guidelines for Cytologic
Preparation and Interpretation
Andrew M. Hoffman, DVM, DVSc
Tufts University, Cummings School of Veterinary Medicine, 200 Westboro Road,
North Grafton, MA 01539, USA

Bronchoalveolar lavage (BAL) is a method for the recovery of respiratory

secretions that line the peripheral airways and alveoli. The technique was
first performed by Dr. Laurent Viel at the University of Guelph in Ontario
in the late 1970s in an effort to better characterize lower airway disease in
horses. Specific indications for the harvesting of respiratory secretions using
BAL are the diagnosis of inflammatory airway disease and heaves, in the ab-
sence of a clear diagnosis using other means [1–3]. Radiography [4], endos-
copy, and tracheal wash (TW) cytology are insufficient for the diagnosis of
inflammatory airway disease (IAD), which has led to the more widespread
use of BAL. The technique of BAL has been used extensively to characterize
the prevalence and clinical consequences of airway inflammation in horses
[5–8] and to disentangle the temporal aspects of inflammatory mediators
(cytokines, myeloperoxidases, proteases/antiproteases) in heaves [1,9–19]
and IAD [19–22], as well as potential therapies for this condition [23,24].
The analysis of BAL fluid has also facilitated the quantification of blood
in the airways of horses suspected of exercise-induced pulmonary hemor-
rhage (EIPH) and the assessment of various risk factors (eg, exercise inten-
sity, incline) and potential therapies (eg, corticosteroids, endothelin A
antagonist, furosemide, nasal strips) for this condition [10,25–33]. However,
there is more widespread agreement that BAL is useful to characterize
nucleated cell differential counts, than the quantity of red blood cells shed
in the airways.
Overall BAL is considered very safe and sufficiently sensitive to detect
inflammation at the cytologic level. The good correlation between BAL dif-
ferential cell counts and exercise-induced hypoxemia or lactic acidosis [6,8],

E-mail address: andrew.hoffman@tufts.edu

0749-0739/08/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.cveq.2008.04.003 vetequine.theclinics.com
424 HOFFMAN

airway obstruction [7], or airway responsiveness [34] attests to the relevance

of BAL cytology to the structure and function of the equine airways. Thus,
an important advantage of BAL over tracheal wash cytology is that BAL
cytology relates well to the clinical signs and pathophysiologic conse-
quences of IAD [6,7,35], and this observation has led a panel of experts
to incorporate BAL rather than TW cytologic findings in the definition
of IAD [1].

Sampling technique
Past studies have examined the reproducibility and confounding influ-
ences of site, lavage volume, and prior BAL on the results of BAL cytology
and lung function. The BAL procedure had either no effect on subsequent
BAL cytology [36] or a transient (for 48 hours) increase in neutrophils
[37]. Exercise did not alter BAL cytology [36] although it is customary to
perform BAL after exercise rather than before, because mucus can be
observed more readily at this time. Although most cell types did not show
a difference in samples from the left and right lungs, there were more
mast cells in those from the left lung, and mast cells in particular were
enriched in lower volume samples [38]. In our hands, the use of higher
volume lavage (300–500 mL) overcomes these inconsistencies, probably
because of the greater surface area sampled.
There is no evidence that BAL causes any significant disturbance in lung
function; indeed, in one study, the BAL procedure was associated with an
improvement in airway function, which they attributed to a removal of mu-
cus [39]. Furthermore, there was no evidence that prior pulmonary function
testing, including broncho-provocation with histamine, altered BAL cytol-
ogy or measured cytokine levels (Gillian Perkins, personal communication,
2008).
The BAL provides consistent cytologic differential cell counts if the tech-
nique is deployed in a consistent fashion. The most reproducible results can
be obtained if the horse is properly sedated, the sampling device is properly
wedged, and a large volume of isotonic fluid is used.
BAL should never be performed in a client-owned horse that is exhibiting
an exacerbation of heaves, markedly tachypneic or dyspneic, cyanotic,
weak, hypotensive, showing abnormal cardiac arrhythmias, or has signifi-
cant hypoxemia or a paroxysmal cough. It is also contraindicated to per-
form the BAL immediately before exercise, because a transient loss of
regional lung compliance and gas exchange in that segment is to be
expected. In our experience, horses should be banned from exercise for
24 hours, after which time submaximal exercise can be resumed, and after
48 to 72 hours maximal exercise can be undertaken. Bronchodilation before
BAL reduces the risk of a bronchoconstriction or cough during the proce-
dure and, importantly, for several hours afterwards, although this has not
been studied systematically as it has for antitussives. The most rapid and
 BRONCHOALVEOLAR LAVAGE 425

efficient method to deliver bronchodilator is by inhalation. At the time of

writing, we use albuterol (4 puffs, 360 mg) or ipratropium bromide (4 puffs,
84 mg) using a compact aerosol delivery device (AeroHippusR, Trudell
Medical International, London, Ontario, Canada) approximately 10 min-
utes before BAL.
The BAL procedure cannot be performed safely without chemical re-
straint. It is customary to use xylazine (0.5–0.75 mg/kg intravenously [IV])
or detomidine (0.005–0.01 mg/kg IV) with or without the addition of butor-
phanol (0.02–0.04 mg/kg IV) before introduction of the instrument. Butor-
phanol significantly reduced coughing associated with BAL in one study
[40]. A twitch is applied if needed.
It is helpful to warn owners that their horse will likely cough and gag vig-
orously during the procedure, which can be performed with a BAL tube (eg,
VBAL30, Bivona tube, outside diameter 11 mm, 244 cm length, Smith Med-
ical) or a long (2–3 m) endoscope. The tube or endoscope is passaged much
in the fashion of a stomach tube via the ventral meatus to the nasopharynx.
It is useful when using a BAL tube, to premeasure this distance (nare to the
medial canthus of the eye) before passing the tube to this level. To further
reduce coughing, inject 30 mL of warmed 0.66% lidocaine [40] (without
epinephrine) onto the glottis. Stretch the head horizontally, and passage
the tube or endoscope gently but swiftly into the trachea, the path of least
resistance. A few coughs are normally evoked at this point, and there should
be little to no swallowing efforts. The position of the tube can be verified
further as in the trachea by the ability to aspirate air (this would not be pos-
sible if the tube was in the esophagus), the lack of swallowing efforts, and
the failure to visualize or palpate the tube in the esophagus. The injection
of lidocaine solution may also evoke a transient coughing reflex if the
tube is in the trachea.
Next, reposition the head in a more relaxed position for further passage
of the instrument. Observations concerning the nature of mucus are made, if
an endoscope is used. Then, inject an additional 30 to 60 mL of lidocaine
solution (0.66%) for further anesthesia, unless a TW is intended. Generally,
one would only perform a TW in conjunction with BAL if infection was sus-
pected or for research purposes. We have also used both techniques if a local
inflammatory process of the trachea is suspected as a cause of cough.
Cytology of BAL and TW correlate poorly [41–43], and BAL is a more
sensitive technique to detect peripheral airway inflammation, ie, IAD. The
TW will not interfere with the BAL cytology, but the BAL procedure often
will interfere with a clean TW. Next, the instrument (tube or endoscope) is
passed until it wedges in the most distal bronchus, which is usually the
fourth or fifth generation subsegment of the caudal lobe. If using a cuffed
tube, the cuff is inflated with around 5 mL of air to seal the airway. Addi-
tional lidocaine solution (10–20 mL, 0.66%) can be administered at this
time but is usually unnecessary. Once the horse is settled, the fluid can be
instilled. According to the consensus statement by the American College
426 HOFFMAN

of Veterinary Internal Medicine panel on IAD [1] and past reviews on this
subject [3], it is recommended to use between 300 and 500 mL of warm
(w37 C) sterile physiologic saline (PSS) or phosphate-buffered saline
(PBS) for instillation, to obtain consistent results that are comparable to
published reference ranges. In our institution, we routinely use 500 mL
PSS (w37 C) to sample the widest area. One study refutes the need for
higher volumes, showing that sequential aliquots of saline (100 mL
each) did not significantly differ from a pooled sample (300 mL), but
mast cell percentages were higher in the first aliquot [44]. Until the clinical
significance of this finding is resolved, we continue to use a larger volume
and pool all aliquots for evaluation. This also limits the error associated
with the delivery or recovery of any one single aliquot.
 BRONCHOALVEOLAR LAVAGE 427

With regard to the safety of BAL, the average (500 kg) horse’s lung vol-
ume at end-expiration contains 30 L of air [45]; thus, the instillation of
500 mL of fluid is trivial in terms of gas exchange, assuming the fluid
does not leak (ie, the airway is sealed). The fluid is instilled through a solu-
tion administration set plugged into a saline bottle on the proximal end and
via stopcock into the channel of the BAL tube at the distal end. The bottle is
inverted, the stopcock opened to the BAL tube, and the fluid is pushed into
the airway by pressure exerted using a pressure bulb. Gravity is too slow, so
an alternative to a pressure bulb (for a stiff plastic bottle) or pressure bag
(for a collapsible bag) is to use large syringes that are prefilled with saline.
Half (250 mL) of the sample is injected, followed by collection through
the side port of the stopcock, using gentle suction applied with a suction
pump (and sterile collection trap for the sample) or the same syringes
used for injection. The first sample is collected when the return begins to
slow down. The second bolus of fluid can be injected and collected in the
same fashion. The proportion of fluid recovered, and the amount of foam,
is generally greater for the second bolus (60%–70% versus 30%–40%). If,
during the instillation period, the fluid leaks back into the trachea, it means
that the tube or endoscope is not wedged securely. It will be readily coughed
up assuming the horse maintains a cough reflex. With heavy sedation, the
cough reflex can be impaired. In this rare case in which BAL fluid has leaked
back into the trachea and the horse does not cough, simply drop the head to
empty the trachea. Out of thousands of BAL procedures, we have rarely
ever experienced this problem or any adverse effect, and the literature sup-
ports the safety of this method.
:

Fig. 1. (A) An 8-year-old Quarter horse gelding barrel racer: numerous hemosiderophages
(arrow) and an increased percentage of neutrophils (15%). (B) A13-year-old Quarter horse geld-
ing with persistent cough: 24% neutrophils, fibrillar mucus. (C) A12-year-old Irish Warmblood;
progressive exercise intolerance: 12% neutrophils and silica particles within giant cells and mac-
rophages, suggesting silicosis. (D) A 12-year-old Thoroughbred gelding eventer with exercise in-
tolerance: mastocytosis. (E) Fungal hyphae (incidental finding). (F) Cellulose fibers inspired as
contaminant of airways (incidental finding). (G) A 5-year-old Standardbred gelding racehorse
with poor performance: 4% eosinophils. (H) A 22-year-old horse with tachypnea and fever:
Pneumocystis jiroveci (formerly Pneumocystis carinii) spores within their ascus cyst (Toluidine
Blue stain). (I) Horse with loss of performance, poor recovery from exercise, and slight tachyp-
nea at rest: inorganic particulates phagocytosed by alveolar macrophages. (J) A 4-year-old
Quarter horse gelding barrel racer with reduced performance: 6% mast cells (arrow); rare hemo-
siderophages, indicating EIPH (Toluidine Blue stain). (K) A 6-year-old Thoroughbred mare
with chronic cough and recent loss of stamina: eosinophils (7%); ciliated epithelial cells (arrow).
(L) Neutrophil-rich sample; neutrophils in high density are localized to aggregates of mucus
(right). Adjacent patch of mononuclear cells in amorphous mucus. (M) Ciliocytophthoria:
apical end of ciliated epithelial cell with cilia attached to terminal bar; observed after viral in-
fection. (N) Horse with chronic inflammatory airway disease: giant cell (arrow), fibrillar mucus,
and nonlytic neutrophils. (O) Horse with EIPH: eosinophilia (4%) and hemosiderophages.
428 HOFFMAN

Handling and processing of the sample

The sample should be examined grossly for the presence of foam (surfac-
tant) and turbidity (cells). A clear sample without foam is probably not
a wedged BAL, rather the sample was inadvertently obtained from the large
airways or trachea. Nevertheless, such samples should be evaluated cytolog-
ically, in case bronchoalveolar cells are present. Because there are so many
cells (105-6/mL) in the sample, there is no immediate concern about losing
cells from adherence to collection vessels or to each other, but it is best to
take certain precautions. A subsample (10 mL) should be set aside on ice
or in a refrigerator for the least bacterial overgrowth and best viability
 BRONCHOALVEOLAR LAVAGE 429

and morphology, although the differential cell counts appear to be stable for
up to 72 hours at 4 C to 38 C [46]. It is preferable to avoid fixation of the
sample (eg, with formalin or alcohol), unless the sample is not going to be
refrigerated, because fixation can alter morphology with certain stains, eg,
Leishman’s [46]. The use of tubes containing EDTA will reduce cell clump-
ing but is unlikely to improve cell morphology or differential cell counts. No
studies have examined the benefit of added serum, but it is likely that this is
unnecessary for the short term in refrigerated samples in which cell homeo-
stasis is less demanding. In sum, the shorter the period of time between sam-
pling and centrifugation the better, but if the sample has to sit, it is best to
refrigerate it without fixation.
The sample is then centrifuged using a cytocentrifuge (200–600 mL per
slide) or table-top centrifuge (10 mL total). Cells are fragile, so spinning
at less than 300g by either method is recommended. Each centrifuge has
a different RPM relative to ‘‘g’’ so it is important to look up the reference
curve and keep the ‘‘g’’ low no matter what the RPM. If you are going to
prepare a smear, the sample is simply spun down for 10 minutes, and the
supernatant poured off. The remaining pellet and fluid is gently mixed by
flicking the outside of the bottom of the tube until the contents appear ho-
mogeneous (cloudy). A transfer pipette is used to remove about 250 mL (1
gradation on a standard transfer pipette) to make the smear. Gentle smear-
ing in a similar fashion to blood smears is used to spread the sample.
The most important aspects of smear preparation are: (1) to avoid undue
pressure when smearing, which will rupture cells and (2) rapid air drying to
avoid crenation and distortion of morphology. Use a fan to dry the slides
whenever possible. Otherwise dry them manually by waving the slides in the
air. If the slides are to be sent to a laboratory for evaluation, it is useful to stain
:

Fig. 2. (A) Cluster of ciliated cells is observed in low numbers in normal BAL. (B) Curschmann’s
spiral (plug of mucus) in horse with long-standing inflammatory airway disease. (C) Horse with
chronic neutrophilic inflammatory airway disease underlying intermittent cough and loss of
stamina: very dense fibrillar mucus. (D) Giant cell containing refractile bodies (silica), many
degenerate macrophages, and epithelial cells; amorphous mucus layer. (E) Cluster of alveolar
macrophages containing irregular silica fragments, visible on bright-field microscopy. (F) Cluster
of alveolar macrophages containing silica fragments that appear refractile under polarized con-
trast illumination. (G) Neutrophils (red arrow) in layer of mucus, containing degenerate epithelial
cells (black arrow). (H) Horse with exercise intolerance: globule leukocyte (arrow) among alveolar
macrophages, lymphocytes, and a few neutrophils (Toluidine Blue stain). (I) A 3-year-old Stan-
dardbred racehorse stallion with a decline in performance: mast cell (MC) and eosinophils (EOS).
(J) Amorphous body. (K) Horse with chronic cough and exercise intolerance: clear plastic parti-
cles (0.5  5-10 mm), copious amorphous mucus. (L) Horse with reduced performance: cellulose
fragments (C) and numerous basophilic particulates engulfed by alveolar macrophages. (M)
Horse with a recent history of exercise intolerance: mast cells (MC) lodged in mucus, showing typ-
ical metachromatic (purple) staining with Toluidine Blue O (Tolonium Chloride). (N) A 12-year-
old Warmblood dressage mare with IAD: chronic cough associated with neutrophilia. (O) Horse
with a history of ‘‘seasonal’’ cough spanning several years: neutrophilic inflammation.
430 HOFFMAN

one slide with Diff-Quik for cursory inspection in your laboratory, to assure
that the sample contains many cells and that the cells are not smudged.
Some laboratories will accept fluid samples. If you plan to send fluid samples,
protect them with an ice pack and ship them overnight if possible.
BAL cytology can be interpreted readily from either cytocentrifuged or
smeared sample pellets. Certain cells (macrophages, mast cells, eosinophils)
are more prevalent on differential counts when using the cytocentrifuge ver-
sus smear technique [47]. Hence, a significantly higher proportion of mast
cell can be obtained from low volume, first aliquot samples (see above) pre-
pared by cytocentrifuge, and this may create disparities between results.
 BRONCHOALVEOLAR LAVAGE 431

Each practice should develop a standard method and personal reference

ranges if processing the samples in-house. The smear technique is more com-
mon in equine practice and yields an equally valid diagnostic method [47].
The problem with the smear method is that cells are rounder, smaller, and
darker, making their morphologic characteristics less distinct. Care must
be taken not to overstain the samples or to prepare them too thick, because
this will greatly obscure the recognition of cell types and the evaluation of
cell contents (eg, particulates, mold, bacteria, silica).
Cytocentrifuge or smear slides can be stained with a variety of commer-
cial stains, including Diff-Quik (modified Romanowsky), Wright’s-Giemsa,
May-Grunwald-Giemsa, and Leishman’s stain. Diff-Quik generally is the
easiest to use in the practice environment. The drawback associated with
Diff-Quik is the potential for poor staining of mast cell granules, making
the enumeration of mast cells dependent on cell morphology, which is inher-
ently difficult [48]. Hence, an additional stain is required. Toluidine Blue had
the greatest sensitivity for the detection of mast cells when compared with
the aforementioned stains [48]. In our laboratory, we use Diff-Quik for dif-
ferential cell counts (minus mast cells) and Toluidine Blue (30 minutes) for
mast cell differential counts, which are enumerated separately. The fixation
of air-dried smears for Toluidine Blue stain is the same as for Diff-Quik,
adding convenience to this process. Although using a second stain to count
mast cells adds time to the cytologic assessment, the diagnosis of IAD asso-
ciated with mast cells can be made more confidently. In general, we count
400 cells per cell differential count, using a bright-field microscope at 630
to 1000 x magnification.
:

Fig. 3. (A) Horse with reduced exercise tolerance and increased interstitial densities on radiog-
raphy: giant cell packed with particulates from environmental exposure. (B) Refractile particu-
lates, likely silica mixed with darker staining particulates of unknown origin. Silica is commonly
found in conjunction with other inorganic or organic particles. (C) Squamous epithelial cells
coated with bacteria. These cells originate in the oropharynx. Their presence in BAL is indica-
tive of aspiration or contamination of the instrument channel during passage. (D) Horse with
fever, purulent nasal discharge, and alveolar-interstitial infiltrates: evidence of Pneumocystis jir-
oveci spores within ascus cysts associated with predominantly mononuclear inflammation in
a thick amorphous (disorganized) mucus layer. (E) Smoke inhalation. (F) Horse with a recent
history of cough and decreased athletic performance: inhaled plastic fibers of many colors. (G)
Mixed mast cell and neutrophil inflammation (Toluidine Blue stain). (H) Marked increase in the
percentage of mast cells (Toluidine Blue stain). (I) Giant cell containing mold spore. BAL cy-
tology is a window on the environment of the horse. The frequency of mold spores is an indi-
cator of particulate exposure from forage. (J) Normal BAL cytology. (K) Recent EIPH, with
engulfed and free red blood cells and early transition of hemoglobin to hemosiderin. L. Horse
with moderate EIPH: intense staining of hemosiderin in the majority of alveolar macrophages.
(M) Horse in which the BAL tube was not wedged properly: ciliated epithelial cells. (N) Horse
with chronic IAD: fibrillar mucus containing the contents of goblet cells. (O) Horse with
chronic cough, without heaves: neutrophilic inflammation.
432 HOFFMAN

Cytologic interpretation
Interpretation of cytology requires experience with equine BAL samples,
but the enumeration of differential cell counts can be learned quickly, be-
cause the cell types in well-prepared samples are distinct. More experience
is required to interpret samples with less than ideal morphology and with
more obscure diagnoses.
Reference values for BAL cytologic differential cell count of the horse,
based on our experience and a variety of studies that have compared
BAL cytology in normal to abnormal horses [1,3,6–8,22,26,35,49–58], is
as follows: macrophages 50% to 70%, lymphocytes 30% to 50%, neutro-
phils !5%, mast cells !2%, and eosinophils !0.1%. Epithelial cells gen-
erally are rare, although a few nonciliated bronchial epithelial or goblet cells
may be observed.
Figs. 1–3 show some common cytologic findings in clinical cases. Unless
otherwise stated, stain is Diff-Quik and magnification is 630x or 1000x.

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