Bronchoalveolar Lavage in the Normal Volunteer Subject* - Chest

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
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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)