Bronchoalveolar Lavage in the Normal Volunteer Subject* - Chest

Bronchoalveolar Lavage in the Normal
Volunteer Subject*
1. Technical Aspects and Intersubject Variability
David B. Ettensohn, M.D., F.C. C.P; Marilee f Jankowski, B.A., B. S.;
Pamela G. Duncan: and Peggy A. Lalor, B. S.
To investigate subject-to-subject variability in commonly
used parameters from bronchoalveolar lavage (BAL), we
analyzed the results of BAL from 78 consecutive normal
volunteer subjects. The BAL was performed using three,
40 ml aliquots of normal saline solution, in a lingular
subsegment. The same diameter bronchoscope (4.8 mm)
was used and BAL was performed and analyzed identically
in all subjects. While the percentage of lavageate returned
was relatively consistent from subject to subject (63.4 + 10.8
percent, mean ± SD), there was marked variability in the
number of cells obtained (7.3 ± 3.9 x 106). While low returns
were generally associated with a diminished cell recovery,
in the range of normal percentage returned, there was no
correlation with the number recovered. There was no
correlation of cell number or percent lavageate returned
ronchoalveolar lavage (BAL) is a well established,
valuable research tool with which to examine
alveolar lining cells and fluid phase constituents of the
human lung."2 In contrast, there have been mixed
opinions as to its clinical value3'4 except for the obvious
advantages BAL affords in the diagnosis of infectious
diseases in immunocompromised patients.56 While
there is some preliminary indication that BAL may be
useful for staging, prognosticating and following response
to therapy in some patients with interstitial
lung diseases,1-3'7'8 others have found it to be of little
clinical utility in the management of this group of
pulmonary diseases.4'9 Clinical application of BAL is
primarily hampered by variability in results. While
this may be due to disease processes in patient
subjects, methodologic differences employed by investigators
to obtain and process the lavage specimens
may also contribute to variability in results. Parameters
known to influence the composition of the lavageate
include the type and temperature of physiologic
fluid instilled,'° volume of the aliquot,"1 number of
*From the Department of Medicine, Division of Pulmonary Medicine,
Brown University Program in Medicine, Memorial Hospital
of Rhode Island, Pawtucket, and Brown University, Providence,
RI.
This study was supported in part by NIH grants HL-34116 and
RR05862.
Manuscript received November 12; revision accepted February 9.
Reprint requests: Dr Ettensohn, Division of Pulmonary Medicine,
The Memorial Hospital, Pawtucket, Rhode Island 02860
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with gender, height, or any lung volume (as determined on
pulmonary function tests). There was relatively little variability
in differential analysis of recovered cells. The BAL
was well tolerated by all subjects; the chest roentgenogram
was found to be of little utility in screening normal subjects,
but pulmonary function testing detected obstructive lung
disease in three potential subjects leading to their disqualification
as "normal." Our experience confirms the safety
of bronchoalveolar lavage in normal volunteer subjects. We
report the BAL results that can be expected for research
or clinical comparison using a protocol described in detail.
The variability of cell numbers from BAL, even under
these controlled conditions, precludes the routine use of
this measurement from BAL performed for clinical purposes.
aliquots,11, 2 and in which bronchopulmonary segment
the lavage is performed.'0 Another parameter which
may influence BAL results, which has not been
systematically investigated, is the variability inherent
in human subjects.
Data compiled from normal volunteer subjects serve
as the standard for BAL analysis in many laboratories.
Several groups have performed BAL on normal subjects
and have published values for percentage of
lavageate returned, cell numbers recovered, and differential
analysis.13 While the results of most studies
are similar, there have also been widely disparate
values reported for these BAL parameters.4"13 Much
of this variability may relate to the small number of
normal subjects studied by different groups and technical
differences in either the performance of BAL or
preparation of the cells for differential analysis. Incomplete
understanding of the extent of the variability
that is found in normal subjects limits the extrapolation
of findings to patients. Moreover, for investigational
purposes, it is difficult to know whether lavage results
from one group of normal volunteers is comparable to
those described by others. In the current study, we
describe our BAL technique in detail and report our
experience in the performance ofBAL and the analysis
of the specimens using identical methodology in 78
normal volunteer subjects.
CHEST / 94 / 2 / AUGUST, 1988 275

MATERIALS AND METHODS
Subjects
The study was completed over an interval of approximately five
years. Normal volunteer subjects were solicited for the study by
word of mouth and posted notices and were reimbursed for their
participation. Rigid criteria for inclusion in the study included the
following: (1) lack of history of pulmonary disease; (2) nonsmokers;
(3) normal posterior-anterior and lateral chest roentgenograms (if
none in the previous year); (4) normal, complete pulmonary function
tests; (5) normal heart and lung physical examination; (6) taking no
medication; and (7) no history of viral or other illness for several
weeks prior to the study Performance of BAL in volunteer subjects
was approved by the Committee for the Use of Human Subjects at
Memorial Hospital of Rhode Island and informed consent was
obtained from all subjects prior to study Seventy-eight subjects (44
men and 34 women), aged 20 to 36 years, participated in the study.
The average age of the subjects was 26.3. Several potential subjects
were excluded prior to pulmonary function testing on the basis of
historic evidence of lung disease, usually reactive airways disease.
One potential subject had an abnormal chest roentgenogram showing
bilateral hilar adenopathy and was thus disqualified from
participation. Three other subjects were disqualified for participation
in the study by virtue of evidence of obstructive lung disease
on pulmonary function testing despite giving no history of airways
disease. Appropriate local anesthesia could not be achieved in two
other subjects and consequently, bronchoscopy was terminated
prior to entering the trachea.
Bronchoscopy and BAL
Subjects were instructed to take nothing by mouth after midnight
prior to the bronchoscopy All bronchoscopies were performed
between 7:30 and 8:30 AM, by, or under the direct supervision of
one of the authors, (D. B. E.). An intravenous line with 5 percent
dextrose in water was established in an antecubital vein. Atropine,
0.6 mg, was administered immediately via this line. Cardiac rate
and rhythm were monitored electrocardiographically using precordial
chest leads. Oxygen, 2 liters per minute, per nasal cannulae
was administered for the first 25 bronchoscopies. However, use of
oxygen during subsequent bronchoscopies was discontinued since
ear oximetry showed no decrease of oxygen desaturation at any
point during the bronchoalveolar lavage procedure even when no
supplemental 02 was used, and the nasal cannulae were a major
source of discomfort for the subjects. There was no evidence that
the administration of 02 changed BAL results. Subjects were
maintained in a semirecumbent position throughout the procedure
in an adjustable, dental chair. Local anesthesia of the posterior
pharynx was achieved by an aerosolized spray of 2 percent xylocaine
delivered through an air driven atomizer. Subjects were instructed
to pant and then deeply inhale the aerosolized anesthetic. A
bronchoscope (external diameter of 4.8 mm) was then introduced
perorally or occasionally pernasally (three subjects required pernasal
route due to excessive gag using the peroral route). Once the
posterior portion of the tongue and epiglottis were visualized, 2
percent xylocaine was administered directly via the bronchoscope.
Adequate anesthesia was usually achieved with two, 2 ml aliquots
of xylocaine. The bronchoscope was then advanced and the larynx
and vocal cords were similarly anesthetized and then passed. One
milliliter of 2 percent xylocaine was then applied to the main carina,
the left main stem bronchus, the secondary carina, and at the
lingular orifice. The bronchoscope was then advanced into a B4 or
B5 lingular bronchus until it was completely wedged in such a
position as to allow visualization of the distal bronchus. Three
aliquots of40 ml of sterile normal saline solution at room temperature
were instilled through the bronchoscope and then suctioned back
(immediately after each instillation) into a sterile, 250 ml side-arm
276
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2, L
-15"10 TO BRONCHOSCOPE
-250
TO WALL SUCTION
FIGURE 1. Diagram of modification of a 250 ml filtration flask for
collection of fluid returned by suction of BAL. The entire unit can
withstand repeated steam autoclaving.
filtration flask adapted to function as a suction trap (see Fig 1). This
entire unit is steam autoclavable to provide sterility should the cells
be used later for tissue culture. Wall suction pressure was used but
was applied in variable strength, controlled by partial occlusion of
the suction port with the finger of the operator to permit sufficient
suction without collapsing the airway. Subjects were instructed to
take slow deep breaths following instillation and removal of the third
(and final) aliquot of saline solution. The bronchoscope was withdrawn
and the subject was observed for possible adverse reactions
(20 to 30 minutes) before being allowed to depart. Each was
encouraged to cough and to deep breathe for the remainder of the
day and to report any adverse effects.
Processing of the BAL Specimen
A total of IOOU penicillin g/ml and 100 ,ug streptomycin/ml were
added to the lavageate which was immediately decanted through
four layers of sterile cotton gauze into one or two polystyrene, 50
ml centrifuge tubes. The specimen was then centrifuged at 5OOg for
20 minutes at room temperature. The supernatant fluid was
decanted, aliquoted, and stored at - 700 C. The cell pellets were
resuspended with gentle pipetting with a plugged Pasteur pipette
in 3 ml phosphate buffered saline solution (PBS), pooled and brought
up to 50 ml by addition of PBS, and then re-centrifuged at 500 g
for 20 minutes. This washing step was repeated a total of three times
and the cells were then resuspended in 3 to 5 ml of culture medium.
A tenfold dilution of a 50 pl aliquot of this suspension was used to
count the cells on a hemocytometer. Viability of the cells was
determined by trypan blue dye exclusion. Cytocentrifuge preparations
were made by placing 100 to 200 pd of a twofold to threefold
dilution (ie, an approximate total of 1-2 x 105 cells) of the original
cell suspension in the cytocentrifuge cones and centrifuging (at 250
BAL in Normal Volunteer Subjects. 1. Technical Aspects (Ettensohn et al)

Table 1-BAL Analysis ofNormal Volunteer Subjects
No. %Return Cell No./
Group of of 120 ml Cell No. ml BAL
Studied Subj Age* BAL X 106 Fluid x 103 %ALVM0t %LY %PMN %EOS
All 78 26.3±3.6 63.4±10.8 7.28±3.9 93.7±45.1 95.1±2.9 3.9±2.4 0.7±0.8 0.17±.9
Subjects (20-36) (23.3-79.2) (0.69-23) (9.9-267) (84.2-99.3) (0.7-14.4) (0-3.2) (0-7.1)
Men 44 26.0+3.6 61.7+12.3 7.29+4.3 94.2±49.3 94.9+3.2 4.2+2.7 0.8±0.8 0.2±1
(20-36) (23.3-67.2) (0.69-23) (9.9-267) (84.2-99.3) (0. 7-14.4) (0-3) (0-7.1)
Women 34 26.6±3.6 64.5±7.3 7.25±3.4 93.5±40.5 95.5±2.4 3.7±1.9 0.7±0.8 0.1±0.5
(22-35) (50-79.2) (3.1-15.5) (3.7-200) (89.9-99.2) (0.8-9) (0-3.2) (0-2.6)
*All data presented as mean ± SD of the mean (range).
tAbbreviations used: AlvM0, alveolar macrophages; Ly, lymphocytes; PMN, neutrophils; EOS, eosionophils.
rpm for five minutes) the cells onto clean glass slides using filter
papers. These preparations were allowed to air dry and were then
stained by a modified Wright-Giemsa technique. A total of 500 to
1,000 cells were counted in the random field counting technique
and results reported as percentage of each cell type present. The
distinction between macrophages and lymphocytes was confirmed
by nonspecific esterase staining. Over the time course of the study,
the vast majority of differential analysis was performed by either
one of the authors (M.J.J. or PA.L.) each trained in the technique
of differential analysis by D. B. E.
Statistics
Student's t-test was used to assess for significant differences
between groups for the same parameter, and linear regression
analysis was used to determine correlation between two parameters.
A p55 percent) and when the entire group was analyzed,
there was no correlation between cell number
recovered and percentage lavageate returned
(r =0.27). Male subjects, as expected, had a greater
total lung capacity than female subjects (6.9 ± 0. 9L vs
5.2 ± . 8L, p

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OBSERVATIONS
FIGURE 3. Scattergrams of the percentage of A, alveolar macrophages;
B, lymphocytes; C, neutrophils; and D, eosinophils (as
determined by analysis of cytocentrifuge preparation of BAL cells
from 78 consecutive normal volunteer subjects).
extremely rare and were similarly not included in the
differential analysis.
Viability of BAL Cells
The viability (mean +±SD) of BAL cells was
91.4 8.6 percent with a range from 66 to 100 percent.
Viability of cells from seven subjects (9 percent) was
less than 80 percent, but there was no evident
explanation for the lower than normal cell viability
Safety and ComplicationslAdverse Reaction
There were no complications of any consequence
related to the performance of BAL in any subject.
Virtually all subjects noted mild transient light headedness
of 15 to 20 minute duration immediately
278
2
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0
±sd
-sd
following termination of the BAL. Approximately 50
percent of the subjects reported being more tired than
usual for up to 24 hours following the BAL. Subjects
were specifically requested to monitor for fever, but
only seven reported any temperature elevation and in
only one subject was the elevation >37.7°C. This
isolated subject noted onset offever (39.1C) and chills
approximately four hours after the procedure which
subsided over the next 24 hours without morbid
sequelae. No cardiac arrhythmias were noted.
DISCUSSION
There is considerable variation in the manner in
which BAL is performed and the specimen processed
in both clinical and research applications of BAL.
There is little evidence to support the use of one
technique over the other, and in all likelihood, if BAL
specimens are processed similarly in an individual
laboratory, results may be compared to the locally
established norms. However, there may be some
danger in comparing results obtained and published
by other investigators who may have used a different
size bronchoscope or processing techniques. More-
may also limit comparison of results of in vitro
functional assays in which BAL cells are used. The
purpose of the current study was not to promote or
espouse a particular method, but rather to report on
the subject-to-subject variability in BAL results found
in a relatively large population of normal subjects
when using rigidly controlled conditions.
All bronchoscopies and processing of the BAL
specimens were performed by or under the direct
supervision of one experienced individual, and all
BAL was performed using the same diameter bronchoscope,
the same type of fluid, the same relative
location (lingula), at the same time of day. The subject
pool was also controlled by several criteria. Despite
the uniformity of the BAL protocol, there was considerable
variability in both the percentage of return and
cell number obtained (Table 1) and with the exception
of BALs with low percentage return, there was no
correlation between cell number and percentage of
return. There was also no correlation of either value
with any lung volume. Similarly, there was no correlation
ofnumbers of lavage cells per milliliter lavageate
with any lung volume. Not surprisingly then, even
though female subjects had significantly smaller lung
volumes than males, there was no correlation between
cell numbers or percentage of return and the gender
of the lavage subject and no significant gender-related
differences in any lavage parameter. A possible explanation
of this observation may be that similar surface
area of lung was lavaged in all subjects irrespective of
lung size. Since the same diameter bronchoscope was
used for all subjects, it is likely that bronchi of nearly
BAL in Normal Volunteer Subjects. 1. Technical Aspects (Ettensohn et al)

identical diameter were lavaged. In animal studies,
analysis of bronchial casts indicate that the cross
sectional area of the bronchus is related to the number
of terminal units served by that bronchus. 14 However,
the wide variation in the number of cells recovered
from subject to stubject indicates that either this is a
subject-dependent parameter, or there are other,
noncontrollable factors that contribute to this variability
As illustrated in the companion study to the current
manuscript, there is limited support for the former
possibility 15 but no way to discount the latter. Since
greater than 25 percent of the cells may be lost by
washing the cells three times as in the current study'2
(necessary to remove the xylocaine to reduce interference
in functional, in vitro assays'6), it is possible that
greater or lesser loss of cells from subject to subject
may have contributed to the variability However,
similar BAL analyses by others" in which the cells
were not washed have yielded results similar to those
described herein.
In contrast to the variability noted in the percentage
of lavageate and cell number recovered, there was
marked uniformity in the differential analysis of the
recovered cells. This suggests consistency in both the
subject pool and observers performing the differential
analysis. On the basis of this present study, we have
established normal values for differential analysis of
lavage cells as delineated by the mean ±2 standard
deviations (ie, 95 percent confidence limits). These
are as follow: alveolar macrophages, >89 percent;
lymphocytes,

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Course in Allergy and Immunology
The Pacific Presbyterian Medical Center will present the 18th Course on Allergy and
Immunology on October 14 in San Francisco. For information, contact Herbert S. Kaufman,
M.D., 2340 Clay Street, Room 313, San Francisco 94115 (415:923-3441).
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BAL in Normal Volunteer Subjects. 1. Technical Aspects (Ettensohn et a/)