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ABSTRACT
INTRODUCTION
Since the 1980s, sputum induction by in-
halation of hypertonic saline has been success-
fully used for diagnosing Pneumocystis carinii
pneumonia in patients infected with HIV.
Pitchenik et al. (1986) showed that, with 5%
hypertonic saline administered via an ultrasonic
nebulizer for 10 or 20 minutes, sputum could
be induced in the majority of patients with aids
and in patients with Pneumocystis carinii pneu-
monia.
1
Pin et al. (1992) adapted the method
for use in asthmatic subjects, and this was the
first study to attempt to use induced sputum
for examining the inflammatory response in
asthma.
2
In recent years, sputum induction by
hypertonic saline and its subsequent process-
ing has been refined as a noninvasive research
tool providing important information about in-
flammatory events in the lower airways. In con-
clusion, the ability to study inflammation has
changed considerably with the development of
this technique as a research tool and increas-
ingly as a clinical tool.
3
Induced sputum has
been used for studying various illnesses: asthma,
chronic obstructive pulmonary disease, tuber-
culosis, Pneumocystis carinii pneumonia, cystic
fibrosis, lung cancer and chronic cough.
Induced sputum has several advantages
over other techniques. Bronchoscopy is an in-
vasive procedure and is not easily applicable on
a large scale in follow-up studies. Sputum analy-
sis might be an alternative to this, for obtain-
ing airway secretions that may potentially be
used for monitoring airway inflammation.
4
Although fiberoptic bronchoscopy with trans-
bronchial biopsy, bronchial brushing and
bronchoalveolar lavage are relatively safe pro-
cedures, they still entail some morbidity and
are relatively unpleasant and expensive proce-
dures compared with sputum induction.
1
With
sputum induction, samples can be obtained
from the lower airways with minimal discom-
fort to the patient. Bronchoscopy allows sam-
pling of the cells and mediators in the airway
lumens by means of bronchoalveolar lavage and
enables biopsy of the mucosal tissue. The com-
bined information thus obtained is therefore
superior to that of sputum alone. However,
bronchoalveolar lavage samples only distinguish
lung segments that are distal to the bronchus
into which the bronchoscope is wedged. Fur-
thermore, significant mixing of distal, alveolar
and proximal bronchial compartments occurs.
The mediators are usually diluted in the large
volumes of physiological saline solution used
in the washing, and some exchange with the
blood compartment is inevitable. In contrast,
induced sputum probably provides a more rep-
resentative sample of several proximal airways,
although with prolonged induction the distal
parts can also be sampled, as evident from in-
creased numbers of macrophages from the al-
veolar compartment. The comparison between
bronchoscopy and induced sputum can be
seen in Table 1.
CLINICAL APPLICATIONS
OF INDUCED SPUTUM
Asthma
In clinical practice, it is difficult to assess
airway inflammation and the effects of medi-
cation on such inflammation. Subjective as-
sessment of symptoms is always difficult and
has often been found to be unsatisfactory for
• Marcos Eduardo Scheicher
• João Terra Filho
• Elcio Oliveira Vianna
Sputum induction:
review of literature and
proposal for a protocol
Pulmonary Division, Department of Medicine, Faculdade de Medicina de
Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo,
Brazil
Since the 1980s, sputum induction by inhala-
tion of hypertonic saline has been successfully
used for diagnosing
Pneumocystis carinii
pneu-
monia in patients infected with HIV. In recent
years, sputum induction and its subsequent
processing has been refined as a noninvasive
research tool providing important information
about inflammatory events in the lower airways,
and it has been used for studying various ill-
nesses. In asthma, one application is to use spu-
tum inflammatory indices to increase our under-
standing of complex relationships between in-
flammatory cells, mediators, and cytokine mecha-
nisms. In chronic obstructive pulmonary disease,
sputum assessment could be used as a screen-
ing test before deciding on long-term corticoster-
oid treatment. In tuberculosis, sputum induction
is a valuable diagnostic tool for HIV-seropositive
patients who do not produce sputum. Sputum
induction appears to be a relatively safe,
noninvasive means of obtaining airway secre-
tions from subjects
with cystic fibrosis, especially
from those who do not normally produce spu-
tum. Moreover, sputum induction can also be
used in chronic cough and lung cancer. Gener-
ally, induction is performed through ultrasonic
nebulizers, using hypertonic saline. It is recom-
mended that sputum be processed as soon as
possible, with complete homogenization by the
use of dithiothreitol. We have also shown in this
article an example of a protocol for inducing
and processing sputum employing a nebulizer
produced in Brazil.
KEY WORDS: Sputum. Asthma. Cytological tech-
niques. Tuberculosis. Cystic fibrosis.
Review Article
Sao Paulo Med J 2003; 121(5):213-219.
São Paulo Medical Journal — Revista Paulista de Medicina
214
monitoring asthma severity.
5
Nonetheless, the
regular use of peak flow measurements has
been shown to improve asthma control, peak
flow rates and diurnal peak flow relapse. Meas-
urements of the levels of exhaled gases such as
nitric oxide may be useful, but more data are
needed to fully evaluate the importance of
such markers in assessing airway inflamma-
tion in asthma, especially since nitric oxide
can be produced in large amounts in paranasal
sinuses and the stomach.
5
Asthma is commonly associated with spu-
tum eosinophilia. Up to 80% of corticoster-
oid-naive subjects and more than 50% of cor-
ticosteroid-treated subjects with currently
symptomatic asthma have a sputum eosinophil
count that is outside of the normal range. The
validity of a high sputum eosinophil count for
the identification of asthma is better than peak
expiratory flow measurement.
6
The short-term response to inhaled
corticosteroids differs markedly according to
the sputum eosinophil count, with little evi-
dence of improvement in symptoms and air-
way responsiveness in subjects with a baseline
sputum eosinophil count of less than 3%.
These findings suggest that measuring the
underlying airway inflammation might pro-
vide a better guide as to the need for corticos-
teroid treatment than assessment of functional
abnormality.
6
Occupational asthma is associated with an
increase in sputum eosinophilia. There is some
evidence that sputum eosinophil counts in-
crease during workplace exposure in subjects
with occupational asthma.
6
One obvious application of sputum induc-
tion is to use sputum inflammatory indices to
increase our understanding of complex relation-
ships between inflammatory cells, mediators
and cytokine mechanisms in asthma. The spu-
tum fluid phase seems to be suitable for meas-
uring eosinophil cationic protein, some
cytokines and histamine. Assessment of airway
inflammation using sputum could also be used
for evaluating the effects of drugs on asthmatic
airway inflammation and relating their anti-
inflammatory effect to the effects on symptoms
and disordered airway function.
Chronic obstructive pulmonary disease
Chronic obstructive pulmonary disease is
a clinical entity that is characterized by the
presence of blockage or chronic limitation of
the airflow that presents slow and irreversible
progression. The origin of such alterations is
the pulmonary combination of chronic bron-
chitis and emphysema. The pathophysiology
of chronic obstructive pulmonary disease in-
volves an inflammatory disorder characterized
by neutrophilic
inflammation in airway secre-
tions, with the presence of macrophages
and
lymphocytes on airway tissue.
Bronchoscopic
investigations are
often not possible due to
disease severity.
7
Thus, sputum induction is a
valuable tool for pathophysiology studies. The
sputum neutrophil count is usually high, and
the neutrophil count can be correlated with a
reduction in forced
expiratory volume in one
second (FEV
1
) and the rate of decline in FEV
1
,
thus suggesting that neutrophilic airway in-
flammation is functionally important.
Peleman et al. (1999) studied the cellular com-
position of induced sputum in chronic ob-
structive pulmonary disease and found marked
sputum neutrophilia.
8
Despite its nonspecific nature, the early
inflammatory response
to cigarette smoke is
probably crucial to the development of
subse-
quent tissue damage and disease in suscepti-
ble individuals.
Neutrophils and macrophages
can potentially produce large quantities
of
proteases, of which the various elastase en-
zymes have attracted
the most attention as
likely causes of the loss of elastic recoil
and
destruction of elastic fibers in the lung paren-
chyma.
Indeed, lung specimens from patients
with panlobular emphysema
have a signifi-
cantly decreased elastin content.
9
Confalonieri et al. (1998) studied the ef-
fects of two months of treatment with inhaled
beclomethasone
dipropionate (1,500 µg/day)
on bronchial inflammation in patients with
stable, mild to moderate chronic obstructive
pulmonary disease, by using sputum induc-
tion. They found that the number of neu-
trophils present in induced
sputum samples
decreased after treatment.
10
Moreover, a short
course of oral glucocorticoid therapy
has been
demonstrated to improve pulmonary function
in some
patients with chronic obstructive pul-
monary disease, but not all.
11
In a recent prospective trial, Borbeau et al.
(1998) showed that, in a group of 140 chronic
obstructive pulmonary disease patients, 19
(13.5%) responded to the two-week treatment
with 40 mg prednisone daily. Response to treat-
ment was defined as a 15% improvement in
FEV
1.
12
Also employing FEV
1
to assess response,
Mendella et al. (1982) showed that 17% of
chronic obstructive pulmonary disease cases
were considered responsive to a course of 32
mg/d methylprednisolone for two weeks.
13
Furthermore, Brazilian authors studying spu-
tum eosinophilia in smokers have found that
eosinophilic inflammation can occur in smok-
ers with or without chronic airflow limitation
Table 1. Comparison of the advantages and disadvantages of bronchoscopy and induced sputum
Advantages Disadvantages
Bronchoscopy • Allows biopsy and BAL: samples can be • Requires trained personnel and expensive equipment
obtained from mucosal tissue, and cells and
mediators from the airway lumen • Invasive
• Provides information on structural changes • There is mixing of content from alveolar and
(relating to epithelium, basement membrane and laminae) bronchial compartments
• Can be followed by immunohistochemistry, • Can be contaminated with blood
in situ
hybridization, or electron microscopy
• Can be followed by segmental allergen challenge • Biopsy samples can only be obtained from the larger
• Allows the use of bronchial wash (cells for
in vitro
study) airways and cell count reproducibility is low
Induced sputum • Relatively non-invasive • Risk of bronchoconstriction
• Allows samples to be obtained from • Success rate around 80%
several proximal airways
• Can be undertaken repeatedly • Processing methods fairly laborious
• Safe even in cases of severe disease
• No expensive equipment required • Results not available immediately
• Allows study of large patient populations
BAL: bronchoalveolar lavage.
Sao Paulo Med J 2003; 121(5):213-219.
São Paulo Medical Journal — Revista Paulista de Medicina
215
(chronic obstructive pulmonary disease) and
that sputum eosinophilia may predict those
patients who will benefit from steroid therapy.
14
Fujimoto et al. (1999) investigated the influ-
ence of glucocorticoid in the reversibility of
eosinophilic inflammation in patients with
pulmonary emphysema. They found that the
reversibility of
airway obstruction following the
treatment could be correlated with the eosi-
nophil count in the induced sputum, and that
the treatment significantly reduced eosinophil
count and eosinophil mediators.
In addition,
patients who did not show improvement in
FEV
1
, had lower baseline
eosinophil counts.
11
In conclusion, sputum assessment could
be used as a screening test before deciding on
long-term corticosteroid treatment in chronic
obstructive pulmonary disease.
Cough
Chronic cough is associated with pre-
dominant sputum neutrophilia, but up to
40% of subjects with cough have a sputum
eosinophil count of more than 3%. Patients
with cough and sputum eosinophilia exhibit
an objective response to corticosteroid treat-
ment that occurs in parallel with a treatment-
associated fall in the sputum eosinophil count.
In contrast, patients without sputum eosi-
nophilia do not respond.
6
Tuberculosis
Pulmonary tuberculosis remains one of
the most important health problems in the
world.
15
The World Health Organization rec-
ommends the detection of acid-fast bacilli in
respiratory specimens as the initial approach
to the diagnosis of tuberculosis.
16
However,
this method has low sensitivity and has little
value in patients who cannot produce sputum
spontaneously. In Brazil, with an estimated
annual prevalence of 129,000 cases, approxi-
mately 22% of adult HIV-seronegative pa-
tients with suspected tuberculosis do not pro-
duce sputum spontaneously, or have negative
acid-fast bacilli smears.
17
Thus, the diagnosis
of tuberculosis in these patients is difficult,
and in most cases they are treated empirically
on the basis of clinical and chest radiographic
findings. However, empirical therapy may re-
sult in unnecessary cost and toxicity. Moreo-
ver, HIV-seropositive patients who do not
produce sputum often undergo expensive and
more invasive procedures.
17
Thus, sputum
induction is a valuable tool for diagnosing
pulmonary tuberculosis.
Conde et al. (2000) compared sputum in-
duction with fiberoptic bronchoscopy in the
diagnosis of tuberculosis in a reference center
in Rio de Janeiro, Brazil. They found that spu-
tum induction is a safe procedure with high
diagnostic yield and high agreement with the
results from fiberoptic bronchoscopy, for the
diagnosis of tuberculosis in HIV-seronegative
and HIV-seropositive patients. In localities
where fiberoptic bronchoscopy is not readily
available, and as part of the work-up of sus-
pected tuberculosis prior to bronchoscopy, in-
duced sputum offers an alternative or addi-
tional approach to the diagnosis of sputum
smear-negative tuberculosis, and would en-
hance diagnostic sensitivity in resource-poor
areas.
17
Anderson et al. (1995) compared spu-
tum induction to fiberoptic bronchoscopy in
the diagnosis of pulmonary tuberculosis in
immunocompromised patients and found
that sputum induction was well-tolerated,
low-cost and provided the same, if not bet-
ter, diagnostic yield compared with bron-
choscopy in the diagnosis of smear-negative
pulmonary tuberculosis.
18
Bacteriological confirmation of pulmo-
nary tuberculosis in infants and children re-
mains difficult. Older children can produce
or be induced to produce
sputum. However,
there are no reports of its use in infants or
children
younger than 3
years of age.
Gastric lavage is regarded as the standard
procedure for obtaining specimens for stain-
ing and culture of Mycobacterium tuberculosis
in younger children, because they swallow
their sputum and do not
expectorate. But spu-
tum induction can be effectively performed
and is well tolerated and safe, even in infants.
Zar et al. (2000) compared induced sputum
and gastric lavage for the isolation of M. tu-
berculosis in both
HIV-infected and uninfected
infants and children and found that induced
sputum is better than gastric lavage.
19
The
bacteriological
yield from sputum or gastric
lavage in cases of pulmonary tuberculosis does
not differ
according to HIV status. The use of
induced sputum should be considered as a
first-line investigation in children suspected
of having pulmonary
tuberculosis, especially
in circumstances in which a culture-confirmed
diagnosis needs to be vigorously sought (such
as when the source
of the case is unknown,
drug resistance is suspected, or cutaneous al-
lergy
occurs).
19
Cystic fibrosis
Cystic fibrosis is a hereditary disease of
autosomal recessive transmission also known
as mucoviscidosis or cystic fibrosis of the pan-
creas. The fundamental abnormality consists
of the production of abnormal secretions from
a variety of exocrine glands. It is chiefly a dis-
ease of infants and children, although adult
cases are being recognized with greater fre-
quency. There is no sex predominance. In-
volvement of the lungs usually is manifested
clinically by recurrent chest infections (Pseu-
domonas aeruginosa, Staphylococcus aureus,
Haemophilus influenzae) that are associated
with wheezing, dyspnea, productive cough,
and hemoptysis, as a result of bronchiectasis.
Respiratory insufficiency and cor pulmonale
develop frequently in the later stages of the
disease. The lack of pancreatic enzymes results
in poor digestion, particularly of fat.
20
Inflam-
mation begins at an early age, even in the ab-
sence of concomitant infection, and persists
and progresses throughout life, ultimately
leading to lung destruction. Quantitative
measurements of infection and inflammation
are
therefore important in disease staging and
new treatment evaluation.
21
Each of the current techniques
used for
defining the microbiology and inflammatory
response of the
cystic fibrosis airway has nota-
ble limitations. Expectorated sputum provides
an accurate measure of infection and inflam-
mation in the lower
airways, but many chil-
dren with cystic fibrosis are unable to sponta-
neously expectorate
sputum.
21
Fiberoptic bron-
choscopy with bronchoalveolar lavage
is inva-
sive, risky and costly. Serial bronchoalveolar
lavages are particularly difficult
to perform.
Furthermore, lavage generally samples only one
or two
segments of the lung, thereby possibly
limiting the detection of infection.
Oropharyn-
geal cultures, commonly used in young chil-
dren with cystic fibrosis
who are not capable of
expectorating, do not reliably predict
the pres-
ence of lower airway pathogens, lack sensitiv-
ity
for identifying Pseudomonas aeruginosa and
Staphylococcus aureus, and provide no informa-
tion about
inflammation.
21
In older children who cannot spontane-
ously expectorate sputum, induction can be a
helpful diagnostic tool.
Yet this diagnostic tool
has received very little attention in
cystic fi-
brosis. Recently, sputum induction was com-
pared with spontaneously expectorated
spu-
tum and bronchoalveolar lavage in 10 adults
with cystic fibrosis.
22
Induced sputum was well
tolerated by and preferred over broncho-
alveolar lavage by all subjects, and resulted in
larger sample volumes than for regular spu-
tum samples.
22
Induced sputum yielded simi-
lar cell numbers and similar detection rates
for bacterial
pathogens.
22
Sputum induction
appears to be a relatively safe, noninvasive
means of obtaining airway secretions from
subjects
with cystic fibrosis, especially from
those who do not normally produce sputum.
21
Sao Paulo Med J 2003; 121(5):213-219.
São Paulo Medical Journal — Revista Paulista de Medicina
216
is suspected.
25
Kirsch et al. (1990), studying
62 patients with possible aids-associated
Pneumocystis carinii pneumonia to determine
the diagnostic usefulness of sputum analysis,
found that sputum analysis is a sensitive, spe-
cific, rapid and low-cost technique for the di-
agnosis of Pneumocystis carinii pneumonia.
24
METHODS FOR SPUTUM
INDUCTION AND PROCESSING
Induction
Ultrasonic nebulizers are recommended
for sputum inducing since other nebulizers
do not usually have sufficient saline aerosol
output. Spirometry is necessary to assess the
baseline airway caliber and avoid excessive
bronchoconstriction during saline inhalation.
Spirometers are preferable to peak flow me-
ters because of the greater sensitivity of FEV
1
in detecting induced bronchoconstriction.
Sputum induction requires a high degree of
cooperation from the patient. The procedure
should be conducted by an experienced tech-
nician under the supervision of an experi-
enced physician.
26
Because hypertonic saline causes bron-
choconstriction in asthmatic subjects,
27
pre-
treatment with a short-acting beta-2 agonist is
recommended as the standard procedure.
2,28
Salbutamol, usually 200-400 mmg, i.e. 2-4
puffs from a standard metered-dose inhaler, has
generally been used for pretreatment.
26
The concentration of the saline used for
sputum induction has ranged from 0.9 to
7%.
28-30
The concentration may be changed
during the procedure, starting with 3% and
subsequently increasing to 4 or 5%.
2,31
Hyper-
tonic saline solution is reportedly more effec-
tive than isotonic saline in inducing sputum.
32
Different compartments of the respiratory
tract are sampled at different time-points dur-
ing induction, i.e. central airways are sampled
early, whereas peripheral airways and alveoli
are sampled later. Shorter inhalation times (15-
20 min) appear to have feasibility and success
rates that are similar to those of longer inha-
lation times (30 min). The consensus is to use
cumulative nebulization duration of 15-20
minutes.
26
The procedures for induction can
be seen in Figure 1.
Sputum Processing
It is recommended that sputum be proc-
essed as soon as possible or within two hours,
in order to ensure optimum cell counting and
staining.
33,34
Complete homogenization is im-
portant and can be achieved by the use of
dithiothreitol (DTT). Cells that are incom-
Airway inflammation and infection are sig-
nificantly increased
in both non-expectorat-
ing and expectorating children with cystic fi-
brosis, in
comparison with healthy children.
21
Also, induced sputum samples appear
to be
comparable to spontaneously expectorated
samples in describing
both inflammation and
infection in the cystic fibrosis
airway.
21
Induced
sputum
differs from spontaneous sputum by
having a higher number of viable
cells and less
squamous cell contamination.
22
Lung Cancer
With regard to lung cancer, identification
of early (or pre-symptomatic) lung cancer in
smokers is considered the best strategy for
preventing this disease.
5
But cytological exami-
nation of sputum has been shown to lead to
lung cancer detection at an earlier stage,
thereby resulting in an improved five-year sur-
vival rate. Recent studies of sputum specimens
and clinical data linking specimens to lung
cancer outcomes may make it possible to de-
termine molecular diagnoses of cancer several
years before its clinical presentation. This has
become possible through the use of tests to
evaluate altered gene expression, including
specific oncogene activation and tumor sup-
pressor gene detection, as well as genomic in-
stability and abnormal methylation. Such
studies clearly indicate that good sputum sam-
ples ought to allow complicated genetic analy-
sis to be performed, thus providing further
impetus for considering the induced sputum
technique as a tool for lung cancer screening.
5
Pneumocystis
Carinii Pneumonia
Pneumocystis carinii pneumonia remains
a significant cause of morbidity and mortal-
ity in HIV-infected individuals, causing clini-
cally apparent pneumonia virtually exclu-
sively in immunosuppressed patients. The
clinical presentation is characterized by fe-
ver, shortness of breath, substernal tightness,
and nonproductive cough. Especially in HIV-
infected patients, the symptoms can be rela-
tively mild and slowly progressive, which may
delay diagnosis.
23
Transbronchial biopsy and
bronchoalveolar lavage have been shown to
have 98 to 100% yield and 92 to 100% nega-
tive predictive value for the diagnosis of
Pneumocystis carinii pneumonia. Although
these are considered to be the gold standard,
they still entail some morbidity and are rela-
tively expensive procedures.
24
Thus, induced
sputum may have a role in diagnosing
Pneumocystis carinii pneumonia.
It was reported in the mid-1980s that the
examination of sputum induced by the inha-
lation of hypertonic saline solution was fre-
quently diagnostic for Pneumocystis carinii
pneumonia.
1
Since then, this diagnostic
method has generally become the first em-
ployed when Pneumocystis carinii pneumonia
FEV
1
= forced expiratory volume in one second.
Measure FEV
1
↓
short-acting beta-2 agonist
↓
remeasure FEV
1
after 20 min
↓
administer saline solution using ultrasonic nebulizer
< 10% fall in FEV
1
↓
blow nose, rinse mouth and swallow water → expectorate sputum
> 10%, < 20% fall in FEV
1
→ repeat previous inhalation
> 20% fall in FEV
1
, or
troublesome symptoms
→ discontinue
remeasure FEV
1
Sao Paulo Med J 2003; 121(5):213-219.
Figure 1. Method for sputum induction.
São Paulo Medical Journal — Revista Paulista de Medicina
217
pletely released from mucus tend to stain darkly,
making correct identification difficult. DTT
(0.1%) has been shown to be more effective
for dispersing cells than phosphate-buffered
saline (PBS), and has no adverse effects on cell
counts. The volume of mucolytic agents used
during the processing of all the expectorated
sputum, although fixed at 1:1, is variable in
relation to the sputum/saliva ratio, which is an
unknown.
34,35
Filtration through a 48-µm ny-
lon mesh is commonly used to remove mucus
and debris, and is strongly recommended.
36
Centrifugation is necessary to separate sputum
cells from the fluid phase. Centrifugal forces
used in studies to date have ranged from 300
to 1,500 xg and the duration of centrifuga-
tion from 5 to 10 minutes. The total cell count
is performed manually using a hemo-
cytometer, and cell viability is determined by
the trypan blue exclusion method.
34,37
Fluid
phase storage temperatures used have ranged
from –20 to –70º C.
36
Preparation of cytospins with an optimum
number of cells (40-60 x 10
3
cells) provides a
more accurate estimate of cell distribution
than smears.
Cytocentrifugation speeds range
from 10 to 51 xg (using a cytocentrifuge), with
the most common conditions being 22 xg for
6 minutes.
37,38
Cytospin staining for differen-
tial cell counts can be achieved using either
Wright or Giemsa staining. The differential
cell count is determined by counting a mini-
mum of 400 non-squamous cells, and is re-
ported as the relative numbers of eosinophils,
neutrophils, macrophages, lymphocytes and
bronchial epithelial cells, expressed as a per-
centage of total non-squamous cells. The sq-
uamous cell percentage should always be re-
ported separately.
36
The processing procedures
can seen be in Figure 2.
Objective quantitative
analysis of cells in sputum
Manual differential counting of sputum
cytospins is tedious to perform and, although
based on objective morphological criteria,
observer variability makes more objective as-
saying desirable. The laser scanning cytom-
eter is a novel microscope-linked and compu-
ter-operated instrument that measures fluo-
rescence and optically scans cells labeled with
fluorescent probes on a microscope slide.
39
EXAMPLE OF PROTOCOL
USED IN BRAZIL
The protocol for inducing and process-
ing sputum used by the Pulmonary Division,
Medical School of Ribeirão Preto, Univer-
sidade de São Paulo, is as follows.
Nebulizer
We compared two ultrasonic nebulizers
for sputum induction, one Brazilian and the
other imported, in terms of safety (bronchos-
pasm risk) and yield (sputum volume and cell
numbers).
40
Patients with mild or moderate
asthma formed two groups that underwent
sputum induction by inhalation of NaCl 4.5%
for 20 minutes. Peak flow measurements were
done before induction and every five minutes
during induction. Group 1 used the Icel
nebulizer (Figure 3) (Evolusonic 1000BR, Icel,
São Paulo, SP, Brazil), a low-output nebulizer
whose measured output rate was 1 ml/min (n
= 9). Group 2 used the DeVilbiss nebulizer
(Figure 4) (Ultra-Neb 2000, DeVilbiss-Sun-
rise Medical, Somerset, Pennsylvania, United
States of America), a high-output nebulizer
with a rate of 2.5 ml/min (n = 9).
The results can be seen in Table 2.
We concluded that the much greater spu-
tum induction seen in Group 2 might be a
consequence of the higher output rate pro-
vided by the nebulizer used in that group
Figure 3. Icel ultrasonic nebulizer (model Evolusonic
1000BR).
Figure 4. DeVilbiss ultrasonic nebulizer (model Ultra-Neb 2000).
TCC: total cell count; DCC: differential cell count; DTT: dithiothreitol.
Figure 2. Sputum processing method
Pour entire expectorate into pre-weighed polystyrene tube and weigh → add equal volume of DTT (0.1%) →
aspirate and dispense several times using disposable pipette and agitate in a vortex mixer → place in
agitated water bath or rocker for 15 min at either 22 or 37º C → filter through 48 µm mesh into pre-weighed
conical tube → weigh filtrate → perform manual TCC and assess viability → calculate TCC per millimeter for
entire expectorate → prepare cytospins and stain using Wright or Giemsa method → perform DCC ≥ 400
non-squamous cells
Sao Paulo Med J 2003; 121(5):213-219.
São Paulo Medical Journal — Revista Paulista de Medicina
218
1. Pitchenik AE, Ganjei P, Torres A, Evans DA, Rubin E, Baier H.
Sputum examination for the diagnosis of Pneumocystis carinii
pneumonia in the acquired immunodeficiency syndrome. Am
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(DeVilbiss). On the basis of this conclusion,
we have been utilizing the high-output
nebulizer (DeVilbiss) for routine induction.
However, in cases of severe asthma, we may
prefer low output so as to decrease the risk of
bronchospasm. Moreover, the high cost of the
imported equipment may lead us to use the
Brazilian nebulizer, which is also effective in
inducing sputum.
Induction Method
Measure peak flow. Apply 2 to 4 puffs of
salbutamol (200-400 µg). After 20 minutes,
measure peak flow and calculate the critical
peak flow (fall of 10%). Administer NaCl
4.5%, using a ultrasonic nebulizer like
DeVilbiss. Interrupt this every 5 minutes, to
discard saliva and collect sputum in a specific
tube. Total induction time is 20 minutes, or
Tabela 2. Comparison between two different ultrasonic
nebulizers in sputum induction (n = 18)
Icel* DeVilbiss** P (t test)
Fall in peak flow 11.65 ± 3.8 19.9 ± 3.6 0.14
Sputum volume (ml) 4.34 ± 1.2 13.2 ± 3.2* 0.02
Total cell count (million) 4.4 ± 1.0 14.6 ± 4.3* 0.03
Cells/ml (million) 1.64 ± 0.3 1.33 ± 0.3 0.49
Cell viability (%) 82.3 ± 3.5 83.4 ± 2.7 0.80
* Evolusonic 1000BR, Icel, Brazil; ** Ultra-Neb 2000, DeVilbiss-Sunrise Medical, United States of America.
PBS. Prepare cytospins: 75 µl/cups to spin for
60 seconds at 1,000 rpm (in cytocentrifuge).
Stain the slides using the May-Grunwald and
Giemsa methods.
FUTURE DIRECTIONS
The identification of biomarkers that al-
low early diagnosis, monitoring and optimi-
zation of lung disease therapy is one of the
most ambitious goals in respiratory medicine.
The induced sputum technique allows sam-
pling of the airways in a noninvasive fashion
and thus offers a unique opportunity for iden-
tifying biomarkers of potential clinical use in
respiratory medicine. It is hoped that, in the
future, induced sputum will provide clinicians
with useful markers that can be used routinely
for performing more accurate and, ideally,
more rapid determination of disease pheno-
types in many lung diseases.
The hope is that the induced sputum tech-
nique will provide a simple and cost-effective
tool for monitoring airway inflammation in
the clinical setting, an approach that was pre-
cluded by previous techniques such as bron-
chial biopsy and bronchoalveolar lavage. In
addition, since the induced sputum technique
enables regular monitoring of inflammation,
it will be of great help in assessing the anti-
inflammatory potential of new treatments.
less if peak flow falls to the critical value.
Processing Method
Weigh the sputum. Add an equal volume
of DTT. Aspirate and dispense several times
using a disposable pipette, and agitate in a
vortex mixer. Agitate in a water bath at 37º C
(150 cycles/min) for 15 minutes, with aspira-
tion every 5 minutes for homogenization.
Centrifuge at 750 g for 10 minutes, with me-
dium acceleration/deceleration. Remove the
supernatant using a micropipette and store it
at –70º C. Resuspend the cell pellet in 1 ml
of PBS. Determine cell viability by means of
the trypan blue exclusion method in a
Newbauer chamber (non-viable cells are
stained blue). Perform the total count in the
Newbauer chamber after Turk staining. Ad-
just the cell solution to 0.5 million cells/ml of
Sao Paulo Med J 2003; 121(5):213-219.
São Paulo Medical Journal — Revista Paulista de Medicina
219
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Available from: URL: http://www.abstracts2view.com/atsall/
search.php?search=do&intMaxHits=10&where%5B%5D
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Indução de escarro: revisão de literatura e pro-
posta de protocolo
Desde a década de 80, a indução de escarro
pela inalação de solução salina hipertônica
tem sido usada com sucesso para diagnosti-
car pneumonia por Pneumocystis carinii em
pacientes infectados pelo HIV. Em anos re-
centes, a indução de escarro e seu subseqüente
processamento foram refinados como uma
ferramenta de pesquisa não invasiva que for-
nece informações importantes sobre eventos
inflamatórios nas pequenas vias aéreas, sen-
do usada para estudar várias doenças. Na
asma, uma aplicação é para o uso de índices
inflamatórios do escarro para aumentar nos-
sa compreensão de complexos relacionamen-
tos entre célula inflamatória, mediador e
mecanismos das citocinas. Na doença pulmo-
nar obstrutiva crônica, a avaliação do escarro
pode ser usada como um teste de seleção an-
tes de se decidir pelo tratamento a longo pra-
RESUMO
Marcos Eduardo Scheicher, BSc (PT), MSc. Faculdade
de Medicina de Ribeirão Preto, Universidade de São Paulo,
Ribeirão Preto, São Paulo, Brazil.
João Terra Filho, MD, PhD. Faculdade de Medicina de
Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto,
São Paulo, Brazil.
Elcio Oliveira Vianna, MD, PhD. Faculdade de Medicina
de Ribeirão Preto, Universidade de São Paulo, Ribeirão
Preto, São Paulo, Brazil.
Sources of funding: Fapesp — Process nº. 02/02194-2
Conflict of interest: Not declared
Date of first submission: April 16, 2003
Last received: April 16, 2003
Accepted: May 30, 2003
Address for correspondence
Marcos Eduardo Scheicher
Divisão de Pneumologia, Hospital das Clínicas
Faculdade de Medicina de Ribeirão Preto — USP
Av. Bandeirantes, 3900
Ribeirão Preto/SP — Brasil — CEP 14048-900
Tel. (+55 16) 602-2631 — Fax (+55 16) 633-6695
E-mail: mscheicher@bol.com.br
COPYRIGHT © 2003, Associação Paulista de Medicina
Publishing information
zo com corticosteróide. Na tuberculose, a
indução do escarro é uma ferramenta valiosa
para o diagnóstico nos pacientes HIV-
soropositivos que não produzem escarro. A
indução de escarro parece ser um meio rela-
tivamente seguro, não-invasivo de se obter
secreções das vias aéreas de sujeitos com
fibrose cística, especialmente aqueles que não
produzem normalmente o escarro. Além dis-
so, a indução do escarro também pode ser
usada na tosse crônica e no câncer de pul-
mão. Geralmente, a indução é executada atra-
vés dos nebulizadores ultra-sônicos, usando
solução salina hipertônica. Recomenda-se que
o escarro seja processado o mais cedo possí-
vel, com homogeneização completa pelo uso
de ditiotreitol. Mostramos também neste ar-
tigo um exemplo de protocolo que emprega
um nebulizador de fabricação nacional para
induzir o escarro.
PALAVRAS-CHAVE: Escarro. Asma. Técnicas
citológicas. Tuberculose. Fibrose cística.
Sao Paulo Med J 2003; 121(5):213-219.
