767
Clinical Science (1991)81,7G7-775
Variability in the measurement of nebulized aerosol
deposition in man
S. H. L. THOMAS, M. J. O'DOHERTY", C. J. PAGE* AND T. 0. NUNAN"
Division of Pharmacological Sciences and Toxicology, United Medical and Dental Schools (St Thomas' Campus), London, and
*Department of Nuclear Medicine, St Thomas' Hospital, London
(Received 25 February15 June 1991; accepted 25 July 1991)
SUMMARY
1. This study was performed to determine the variability of two different scintigraphic methods of measuring pulmonary aerosol deposition, and to examine
nebulizer particle size and drug output as potential
sources of this variability.
2. A radioaerosol was produced from a 3 ml solution
of yymTc-labelled'colloidal human serum albumin (0.05
mg, 37 MBq) using a standard jet nebulizer and air compressor. This was inhaled on three separate occasions by
nine healthy male subjects. On one of these occasions, a
further inhalation was performed to assess immediate
repeatability using increased Y 9 mactivity
T ~ (92 MBq).
3. Intrapulmonary aerosol deposition was measured
with a y-camera and was corrected for tissue attenuation
and geometric distribution by using two different
methods.
4. Estimated mean pulmonary deposition was 4.3% of
the nebulizer dose using a lung phantom correction
method, and 6.1% using a tissue attenuation method. For
these two methods respectively variability between subjects (coefficient of variation) was 54 and 47%. For both
methods, within-subject variability (coefficient of variation) was 37% between occasions and 23% within
occasions.
5. The particle-size output of several nebulizers was
highly reproducible (coefficient of variation < 4%), but
the nebulizer mass and radionuclide output of two nebulizers was more variable (coefficient of variation 5-19%),
and appeared to be an important contributor to the variability in pulmonary aerosol deposition.
6. The data presented here for pulmonary deposition,
used with appropriate power statistics formulae, can be
used to estimate the sample sizes required for comparative studies of lung aerosol deposition.
Correspondence: Dr S. H. L. Thomas, Wolfson Unit, Department of Clinical Pharmacology, Claremont Place, Newcastle
upon Tyne NE1 4LP, U.K.
Key words: aerosols, nebulizers, pulmonary deposition,
repeatability, variability.
Abbreviations: b-n, between nebulizer; b-s, between subject; CV, coefficient of variation; HD, high-dose scan;
HSA, human serum albumin; LD, low-dose scan; w-n,
within nebulizer; w-s, b-o, within subject and between
occasion; w-s, w-0, within subject and within occasion.
INTRODUCTION
The use of nebulized drug aerosols as a method of drug
delivery is increasing. T h e value of inhaled bronchodilator and steroid aerosols is well established. More
recently, other drug aerosols have been used successfully,
including anti-pseudomonal agents [ 11, pentamidine [2],
anti-viral drugs [3] and amiloride [4]. While the successful
delivery of bronchodilator drugs to their site of action
within the lung can be inferred from their immediate
effect on respiratory function tests, this is often not the
case for other nebulized drugs. As inadequate deposition
in the lungs is likely to result in a disappointing therapeutic response, it is important that the pulmonary
deposition of nebulized drugs is measured so that the
optimum methods for administration can be determined.
This can be achieved by adding radiolabelled drug to the
nebulizer solution and measuring the resulting radioactivity in the lungs with a y-camera. If a suitable radiolabelled drug analogue is not available, then it may be
possible to use a suitable indirect radionuclide marker for
the drug under study [5-71. From the activity detected in
the chest it is possible to estimate drug deposition in the
lungs as a whole and in defined regions of interest.
Although several authors have shown substantial variability between subjects in scintigraphic measurements of
lung aerosol deposition [8-131, the repeatability of these
measurements within individual subjects within and
between occasions has not been examined previously.
This information would be useful, as the inter- and intra-
768
S. H. L. Thomas et al.
subject coefficients of variation (CVs) of the
measurement, used with appropriate power statistics
formulae [14], allow the accurate estimation of the patient
numbers required for comparative trials of nebulizer
administration methods. This study was therefore
designed to determine the variability and repeatability of
two scintigraphic methods of measuring lung aerosol
deposition. For one of these tissue attenuation was estimated individually for each subject using a flood source,
and for the other a uniform correction derived from a
lung phantom study was used for all subjects. In addition,
nebulizer output was also examined as a potential source
of variability in aerosol deposition.
METHODS
Nebulizer output
A series of experiments was performed to determine
the variability of the output of nebulizers. This was
measured as the particle-size output, including the mass
median diameter and span, and as the mass or radionuclide output of the nebulizer. All of these experiments
were performed at a constant temperature and humidity.
Particle-size characteristics were measured with a
Malvern Master laser diffraction particle-sizer (Malvern
Scientific Instruments Ltd, Malvern, Worcs, U.K.)
employing model-independent calculations. The effect of
different nebulizer systems on particle-size output and
variability was determined for seven nebulizers (see Table
1) when used with a solution of 300 mg of pentamidine
isethionate in 6 ml of saline (150 mmol/l NaCI). In a
second series of experiments, eight different drug
solutions (see Table 2) were nebulized using a single make
of jet nebulizer, the System 22 Acorn (Medic-Aid Ltd,
Pagham, Sussex, U.K.). For all of these experiments,
measurements were made using three different nebulizers
and for each of these triplicate measurements were made
(total of nine measurements). This allowed calculation of
within-nebulizer (intra-nebulizer, CV,,.,) and between
nebulizers (inter-nebulizer, cvh.,) Cvs. It was only
possible to measure the CV,., for the ultrasonic
nebulizers as only one example of each was available. Two
air compressors were used to drive the jet nebulizers.
Using a System 22 Acorn nebulizer the A F P Medical
compressor (AFP Medical, Rugby, U.K.) produces a gas
flow rate of 7.2 litres/min at a pressure of 15.5 p.s.i. (1.07
bar), whereas the CR 6 0 compressor (Medic-Aid) gives a
flow of 8.5 litres/min at a pressure of 19.5 p.s.i. (1.35 bar).
The output of these compressors was not measured
during these experiments.
The variability of nebulizer mass output was studied by
using two methods. The output of a 3 ml nebulizer
volume of distilled water was measured when nebulized
by two different makes of nebulizer, the System 22 Acorn
and the Micro-cirrus (Lutersurgical Ltd, Twickenham,
Middx., U.K.). Nebulization was considered to be
complete when a period of 2 min elapsed without the
nebulizer producing aerosol. Nine examples of each
nebulizer were studied, each on three separate occasions.
A micropipette was used to add 3 ml of distilled water to
each nebulizer. By weighing the nebulizer before filling
and before and after nebulization, the accuracy of the
nebulizer fill could be checked, and the nebulized weight
of distilled water could be calculated. From these results
the CV,,,., and CV., could be derived. Because a proportion of the observed weight loss is caused by evaporation
of water rather than nebulization, this method may overestimate the output of any drug dissolved in solution [15].
The same experimental protocol was therefore repeated
using nine nebulizers of each make, but on this occasion
measuring the output of the radionuclide tracer ""'Tclabelled colloidal human serum albumin ("""'Tc-HSA,
Ventocoll; Solco Basle U.K. Ltd, High Wycombe, Bucks,
U.K.). This was achieved by adding 37 MBq (50 pug) of
"'"Tc-HSA in 3 ml of saline to each nebulizer. On this
occasion, the nebulizer was filled with a standard 5 ml
syringe. The nebulizer was then connected in series to a
low-impedance microbiological filter (Pall, Biomedical
Ltd, Portsmouth, Hants, U.K.) which has previously been
shown to capture over 99.9% of the aerosol particles of
over 0.2 p m diameter [16]. The activity of the nebulizer
and filter were measured before and after nebulization by
using a y-camera, and the results were used to calculate
the output of nebulized '""'Tc-HSA.
Pulmonary aerosol deposition
The pulmonary deposition of a nebulized radioaerosol
was measured in nine healthy male non-smokers after
cthical approval had been obtained from the ethics
committee of the West Lambeth Health District. Each
subject gave informed consent, and was studied on three
occasions separated by a period of at least 1 week. At
their first attendance, their height, weight and lung spirometry were recorded. The chest depth, measured using
calipers as the anteroposterior dimension of the chest at
the level of the xiphisternum at functional residual
capacity, was recorded and the respiratory minute volume
was measured over a period of 5 min using a Wrights
respirometer (Siemens, Sunbury on Thames, Middx.,
U.K.). The subjects were then seated with their backs
resting against a y-camera (large field of view IGE Maxi
11, I.G.E. Medical Systems Ltd, St Albans, Herts, U.K.)
with a high-sensitivity collimator. Aerosol was delivered
using a System 22 Acorn nebulizer containing 37 MBq
(50 pg) of y'mTc-HSA in a solution volume of 3 ml and
driven by a CR 60 air compressor (Medic-Aid). For their
three experiments each subject used the same nebulizer
unit, which was carefully cleaned and dried between each
use. The subjects breathed normally from the nebulizer
mouthpiece for a period of 20 min and during this time
dynamic scans of the lungs (posterior projection) were
made in 15 s counting frames. This period started as the
nebulizer was activated. After aerosol inhalation, static
scans (100 s) were taken of the lungs, abdomen (anterior
and posterior views), administration apparatus, nebulizer
and expiration port filter. The activity in the nebulizer was
measured before and after nebulization with the y-camera
Variability in nebulized aerosol deposition
(acquisition time 10 s) and in an ionization chamber.
Assuming a maximum deposition of 10% of the nebulizer
dose and using the manufacturer's data, the radiation
dose was approximately 0.017 mSv (whole body), 0.45
mSv (lung), 0.16 mSv (marrow) and 0.016 mSv (stomach)
for a 37 MBq nebulizer dose.
The lung outline was drawn following the contour of
the static Y Y mimage,
T ~ which represented 10% of the
maximum activity pixel. This contour was chosen as it lies
on the lowest point of the rapid decline in lung counts at
the lung edge and because it gives clear separation of the
medial lung edges. Pulmonary regions of interest were
defined by dividing the lungs into three parts of equal
height (upper, middle and lower), and into central
(defined as a rectangle over the middle third of the medial
border of the lung and extending halfway across the lung)
and peripheral (remainder of the lung) regions.
Immediately after one of the three aerosol inhalations
described above, a second aerosol inhalation was performed so that the immediate repeatability of the
measurements could be assessed as the within-subject
within-occasion CV (CV,., ,-J. Out of necessity, a different nebulizer unit of the same make and batch was used.
For the second inhalation a higher radionuclide dose was
used (92 MBq, 'high-dose scan', HD); other than this the
experimental conditions and protocol were identical. The
counts from the original experiment ('low dose scan', LD)
were subtracted from the counts obtained in this repeat
experiment before lung deposition was calculated.
It was necessary to correct the measured intrapulmonary activity for the effect of geometric distribution
and tissue attenuation in order to derive accurate estimates of the absolute deposition of aerosol in the lung.
Two different methods were employed. For one, a simple
lung phantom was used to produce a single correction
which was used for all the subjects ('phantom method').
For the other, tissue attenuation was estimated for each
subject individually using a flood source ('tissue attenuation method') so that individual correction factors were
derived for each subject.
Phantom method. This was similar to the modification
by Newman et al. [5] of the method of Ruffin et al. [17]. A
perspex cylinder, 30 cm in diameter and 3 cm deep and
filled with a solution of 37 MBq of Yy"Tc-HSA,was used
as a lung phantom. This cylinder was placed 10 cm in
front of the y-camera and 2 cm of 'tissue equivalent'
material called 'mix-D was placed between the YYmTc
activity and the camera to represent the tissue attenuation
between the intrapulmonary activity and the camera. The
counts detected by the camera ( x ) were compared with
the counts detected when the same activity was placed in
3 ml of solution in the nebulizer ( y ) . From these, a
geometric correction factor was obtained (y/x), which
allowed estimation of true intrapulmonary activity from
the measured geometric mean [,/(anterior X posterior)]
counts, taking into account the tissue attenuation which
was assumed to be equivalent to a 2 cm thickness of mixD:
769
True intrapulmonary activity
= measured
intrapulmonary activity x correction factor
Tissue attenuation method. A perspex cylinder, 30
cm in diameter and 3 cm deep and filled with a solution of
37 MBq of yymTc-HSA,was used as a flood source. To
measure tissue attenuation, each subject sat with his back
to the y-camera and the flood source was placed over the
front of the chest. Counts detected with the subject seated
between flood and camera in this way ( I ) were compared
with the counts obtained with the flood held at the same
distance from the camera but with the subject removed
( Zo). These are related by the formula:
I = I 0e-P*
where p is the broad-beam tissue attenuation coefficient
and x is the tissue depth [ 181. Measured geometric mean
lung counts, corrected using this formula, can be related
to the initial nebulizer activity if the camera sensitivity is
known. This was obtained by placing a thin sheet of polythene over the camera face. On top of this was placed a 30
cm diameter piece of absorbent paper soaked with "'"Tc
of known activity (measured in MBq). The counts
detected by the camera (c.P.s.)could then be related to the
Y y mactivity
T~
to derive a camera sensitivity factor (c.p.s./
MBq) [ 191. Intrapulmonary YYmTc
deposition can thus be
calculated using the formula [20]:
Deposition ("%)
)
geometric mean lung counts x e(""lz)
x 100
= (sensitivity factor x initial nebulizer activity
Results from these experiments were used to calculate the
between subjects (inter-subject, cvb.,)and within subjects
(intra-subject) and between occasions ( CV,-, b.o) CVs. As
the flood transmission factor was different for each
subject, the cvb., will be different for the two methods of
geometric correction. Within-subject variability was not
affected by the method of correction as for each individual the correction factor was the same.
Because of the short half-life involved, the Y Y mflood
T~
source had to be made freshly on each occasion. As this
was inconvenient, the tissue attenuation experiments were
also carried out employing a 57C0 (half-life 270 days,
energy 122 keV) flood source which could be used
repeatedly. This was selected because the energy involved
is similar to that of yYmTc
(half-life 6 h, energy 140 keV).
Z o / Z values obtained using each flood source were compared and were used to calculate pulmonary aerosol
deposition.
Statistical methods
The variability of each of the methods studied here was
expressed as the CV. This was obtained using a two-way
(subjects or nebulizers and occasions) analysis of
variance. Within- and between-subject/nebulizers CVs
were calculated as the square root of the appropriate
mean square divided by the mean value and expressed as
S. H. L. Thomas et al.
770
Table 1. Variability of particle-size output from a solution of pentamidine isethionate (300 mg in
6 ml) using seven different nebulizers
Variability is expressed as CV,,,., and CV,,.,, and these values are compared by using the F-ratio.
The jet nebulizers were driven by an A F P air compressor. Manufacturers: 'Medic-Aid, ?Marquest
Medical Products Inc., Englewood, NJ, U.S.A., 3Medix Ltd, Lutterworth, Leics, U.K., 'System
Assistance Medical, Villeneuve-sur-Lot, France. Statistical significance: * P < 0.05 comparing
CV,-, and CV,.,.
Mass median diameter
Span
Mean
(Pm)
CV,,."
("/.)
CV,.,
("10)
F-ratio
Jet nebulizers
System 22 Acorn'
System 22 Mizer'
Mizerf Optimist 2'
Respirgard 112
Centhist?
5.4
5.2
2.1
2.1
2.1
0.9
0.8
1.6
1.0
1.6
2.4
2.6
1.3
1.7
2.0
7.1*
10.6*
1.5
2.9
1.6
2.2
2.0
2.0
1.8
1.9
Ultrasonic nebulizers
Fisoneb'
Samsonic'
5.8
4.6
0.6
0.2
-
-
1.7
2.0
Mean
(P-4
-
-
CV,,."
CV,.,
("10)
I;'-ratio
0.6
0.9
0.9
1.0
1.2
1.5
1.5
1.5
1.6
2.4
6.2*
2.8
2.8
2.6
4.0
0.3
1.2
-
-
("4
Table 2. Variability of particle-size output from various solutions using a System 22 Acorn
nebulizer (Medic-Aid) and CR 60 air compressor
Variability is expressed as CV,., and CV,., and these values are compared by using the F-ratio.
Statistical significance: * P < 0.05, **P< 0.01 comparing CV,-, and cvh-,.
Drug, solution volume
Saline-HSA, 3 ml
Amiloride 6 mg, 3 ml
Gentamicin 80 mg, 2 ml
Tobramicin 80 mg, 2 ml
Carbenicillin 1 g, 3.5 ml
Ceftazidime 1 g, 3.5 ml
Colistin 1 M-unit, 3.5 ml
Mass median diameter
Span
Mean
(,urn)
CV,.,
("10)
CV,,.,,
("/.)
F-ratio
4.9
4.0
4.2
3.7
3.7
3.9
3.1
0.8
0.8
0.7
0.7
1.7
1.0
0.6
2.1
2.5
2.3
2.4
2.7
1.7
3.0
6.9*
9.8**
10.8**
11.7**
2.5
2.9
25.0**
a percentage. Where appropriate, these CVs have been
compared for statistical significance by calculation of the
F-ratio. Immediate repeatability was also assessed by
using the method of Bland & Altman [21].
RESULTS
The variability of nebulizer particle-size output is shown
in Tables 1 and 2. For each individual nebulizer, particlesize output and span were very consistent (CV,,., < 2%),
and variability between different nebulizers of the same
make was also small (CV,,., < 4%). Variability was similar
for each of the seven types of nebulizer studied when used
with pentamidine solution (Table 1)and for each of seven
drug solutions nebulized using a System 22 Acorn
nebulizer (Table 2).
The variability of nebulizer mass and radionuclide output is shown in Table 3. Addition of distilled water by
micropipette was highly reproducible (CV< lob). CVs of
nebulizer output, measured as weight loss, were less than
Mean
CV,.,
(70)
CV,.,
("10)
F-ratio
(w4
2.5
2.3
2.6
2.3
3.2
3.0
2.7
0.3
0.3
1.1
0.3
1.3
0.9
1.6
3.7
2.4
2.7
1.1
2.6
2.4
2.0
152.1**
64.0**
6.0*
13.4**
4.0
7.1*
1.6
11% both within and between nebulizers of the same
make. Addition of yymTc-HSAsolution using a standard
5 ml syringe was less reproducible than addition by
micropipette, but CVs for nebulizer output were similar
for "'"Tc-HSA and distilled water. Inter-nebulizer variability was similar to intra-nebulizer variability. Output of
distilled water was larger than output of 'IY"'Tc-HSA
( P < 0.001), as was expected. The output of the System 22
Acorn was larger than that of the Micro-cirrus, although
this difference was only significant for distilled water
output.
The anthropomorphic and lung function data of the
nine male subjects taking part in the studies of pulmonary
aerosol deposition is shown in Table 4. Mean deposition
values and between-subject CVs calculated for each of the
two methods for estimating pulmonary deposition from
the y Y m T
activity
~
detected within the lungs (see the
Methods section) are shown in Table 5. As would be
expected, slightly lower values for total deposition were
found using the 57C0 compared with the y Y m flood
T~
771
Variability in nebulized aerosol deposition
Table 3. Variability of output of System 22 Acorn and Micro-cirrus nebulizers (as a percentage of
initial nebulizer weight or dose) from a nominal 3 ml nebulizer fill using a CR60 air compressor:
(a) output of distilled water, and (b) output of 99mTc-HSA
Variability is expressed as CV,., and CV,,-,, and these values are compared by using the F-ratio.
Statistical significance: * P < 0.05, **P < 0.01 comparing CVw-, and CV,,; t P < 0.001 for System
22 Acorn versus Micro-cirrus output.
System 22 Acorn
Micro-cirrus
(a) Distilled water
Starting solution wt. (g)
Nebulizer output by wt. (YV)
3.02
59.6
(b) ""Tc-HSA
Starting solution wt. (g)
3.04
Nebulizer output measured as
yymTcactivity
Loss from nebulizer (YO) 44.5
Recovery from filter (YO) 44.3
0.3
9.1
1.0
1.1
3.02
5l.Ot
1.0
4.8
0.4
10.6
1.1
1.0
1.2
3.04
2.7
1.7
2.0
5.9
15.1
8.2
5.4
1.93
7.2**
9.5
8.3
8.0
4.7
1.4
3.1*
Table 4. Details of nine healthy male subjects undergoing
measurement of pulmonary aerosol deposition
Abbreviations: FEV,, forced expiratory volume in 1 s;
FVC, forced vital capacity; PEFR, peak expiratory flow
rate; RMV, respiratory minute volume.
Mean rf: SD
Age (years)
35 k 7.5
184k6
Height (cm)
84+6
Weight (kg)
23k2
Chest depth (cm)
FEV,
4.57 rf: 0.41
litres
1 0 5 k 13
o/o of predicted
FVC
5.40 k 0.62
litres
102 k 16
% of predicted
PEFR
651k57
ml/min
102 k 9.4
o/o of predicted
9.6 k 1.7
RMV (litres)
Transmission factor ( I , , / [ )
(both lungs)
Y9mTc
3.9 k 0.6
57c~
3.6 k 0.7
6.2*
4.9*
0.3
8.7
Range
CV ("/o)
28-5 1
178-198
75-95
20-27
21
3
8
10
3.85-5.06
89- 135
12
12
4.68-6.50
86-134
12
15
520-724
82-113
8.0-12.4
9
9
17
3.0-4.7
2.7-4.5
15
20
sources, but the error involved was less than 5%. The
phantom method produced lower values for intrapulmonary deposition. This discrepancy was related to the 2 cm
thickness of mix-D used in the construction of the lung
phantom. This underestimated the actual tissue attenuation involved as the Z,/Z value for the phantom was 2.8,
compared with the mean value of the subjects of 3.9
(Table 4). To construct a phantom with equivalent tissue
attenuation to the mean value for our subjects required a
7.5 cm thickness of mix-D. Using a geometric correction
derived from this phantom gave a mean total pulmonary
deposition of 6.O%, almost identical with that calculated
using the y Y mtissue
T ~ attenuation method.
43.6
43.5
Between-subject variability in total pulmonary aerosol
deposition was high, but was slightly less marked for the
tissue attenuation method (CV 47%), which uses a correction factor derived separately for each individual, than for
the phantom method (CV 54%, F-ratio 1.32, P = n o t
significant). For individual lung regions similar statistically
insignificant differences were also observed (Table 5).
Within-subject variability (Table 6) was less marked than
between subject variability. This was not significant for
total deposition (CV,,, b-o 37%, F= 1.61, P = n o t significant), but in spite of the small sample size significant
differences were seen in some individual lung regions
(Table 6). CVs for nebulizer output in these experiments
were 12.5% (Cvb.,) and 15.8% (CV,.,. b.o).
The immediate repeatability of aerosol deposition
measurements can be calculated by comparing results
obtained from the HD with those found immediately
before using the LD. As these are calculated withinsubject, the values are equivalent for each method of
correction used. Mean values for total lung deposition
were similar for the HD and the LD. CVs are shown in
Table 6 and demonstrate that aerosol deposition is more
repeatable within than between occasions, with a CV,,, w-u
of 23% for total lung deposition, compared with the value
of 37% obtained between occasions (F-ratio = 2.59,
P< 0.05). Similar differences were observed for individual lung regions (Table 6). The within-occasion variability
is shown graphically in Fig. 1. Applying Bland-Altman
analysis [21], the mean bias between the two LDs and
HDs is 0.49% (95% confidence interval - 1.34 to
+ 1.83%) with 95%limits of agreement of - 2.99 ( - 5.31
to + 0.67%) and 3.97 (1.65 to 6.28%). It is clear, however, that discrepancies between the two scans increased
with higher deposition values (Fig. l a ) so log transformation of the data is required (Fig. 16). This gives limits of
agreement (log LD-log HD) of -0.69 and +0.49, thus
95% of HD values are expected to lie within the range of
50% below to 64% above the corresponding LD value.
+
772
S . H. L. Thomas et al.
Table 5. Scintigraphic measurements of total and regional pulmonary aerosol deposition,
expressed as the percentage of the initial nebulizer dose, and their CV,.,
Values were obtained by using a lung phantom with 2 cm of tissue equivalent material, and by a
tissue attenuation method using ')9mTcand 57C0flood sources (see the text).
Phantom method
Mean
Tissue attenuation method
cv,., (Yo)
"'"Tc flood
57C0flood
Mean
Cvh.5 (Yo)
Mean
cvh., ("10)
~
Total
4.3
54
6.1
47
5.9
48
Right lung
Total
Central
Peripheral
Upper
Middle
Lower
2.3
0.38
1.63
0.40
1.11
0.90
54
69
53
64
55
52
3.2
0.57
2.63
0.57
1.52
1.39
47
57
45
60
50
46
3.1
0.52
2.58
0.53
1.44
1.32
48
61
47
58
49
50
Left lung
Total
Central
Peripheral
Upper
Middle
Lower
2.0
0.38
1.39
0.36
1.06
0.7 1
57
63
53
40
54
66
2.9
0.6 I
2.28
0.53
1.50
1.18
50
57
50
39
47
2.8
0.53
2.24
0.48
1.38
I .09
50
56
49
32
58
65
DISCUSS I 0 N
Information on the variability of aerosol deposition in the
lung is important as it is required for the power statistics
formulae that are used to calculate the sample sizes
needed for comparisons of aerosol inhalation methods. If
the sources of variability are known, it may be possible to
reduce or eliminate some of these and thus reduce the
measured variability and improve the power or reduce the
required sample size of such trials. It is also probable that
reductions in aerosol deposition variability will cause a
reduction in the variability of the clinical response to
nebulized drug aerosols.
In these experiments we have estimated pulmonary
deposition of a radiolabelled aerosol using two different
methods to correct for the effects of geometric distribution and attenuation of counts. Using the simple lung
phantom method, a total deposition of 4.3% of the
nebulizer dose was estimated, whereas the more timeconsuming flood correction method gave a value of 6.1%.
While both values are within the range previously
reported for nebulized aerosol deposition [22], we have
shown that the amount of 'tissue' attenuation involved in
the phantom studies was too low. If this method is used to
estimate absolute pulmonary deposition, then the lung
phantom should include 7.5 cm of mix-D, which produces
a broad beam attenuation similar to the mean value for
our subjects. These findings are consistent with those of
Forge et al. [23] who showed that a 2 cm lung phantom
method underestimated true deposition by about 40%,
whereas the tissue attenuation method may overestimate
deposition by about 6%.
61
We have shown that there is considerable variability in
aerosol deposition between subjects. Our estimates of 47
and 54% for total pulmonary deposition, depending on
the method used, are consistent with our previous results
for deposition of aerosolized pentamidine in patients with
acquired immunodeficiency syndrome (CV,-, 37-72%)
[12, 131 and amiloride in adult patients with cystic fibrosis
(CV,-, 42-51%) [24], and with other reports from which
CV,., values of 48-60% can be obtained [9-111.
Possible contributors to this between-subject variability
include statistical counting errors for y-emissions and
variations in nebulizer output, which in turn may be
dependent on the variability in driving gas flow rate, relative humidity and temperature. Deposition may also vary
with respiratory pattern and lung size, and these in turn
are related to age and body proportions. The extent and
severity of lung disease is also expected to contribute.
Random variation of y-emissions complies with a
Poisson distribution and the maximum error involved is
the square root of the mean counts divided by the mean
counts [ 181. For total pulmonary deposition the detected
activity using the methods described above is usually over
50 000 counts and the random error is therefore
unimportant ( < 0.5%). For individual lung regions the
error may be as high as 2%, but this is still only a small
fraction of the total variability.
Limited previous data on the variability of nebulizer
output is available. The particle size output affects the
amount and distribution of deposition within the lung [25]
and the response to aerosol therapy [26]. Under strictly
controlled laboratory conditions we found particle-size
Variability in nebulized aerosol deposition
Table 6. Within-subject variability for scintigraphic
measurement of aerosol deposition
CVw-s,b-ovalues were obtained from experiments
performed three times using nine subjects, and CVw.,,w-o
values from LD and HD inhalations performed on the
same occasion (see the text). F-ratios are shown comparing (a) CVw-s,b-o
with CV,., (yymTcflood corrected tissue
attenuation method, Table 5), and (b) CVw-s,b-o with
CVW-,,-,. Statistical significance: * P < 0.05, **P< 0.01.
i2
i
37
1.6
23
2.6*
Right lung
Total
Central
Peripheral
Upper
Middle
Lower
38
45
32
34
36
31
1.5
1.6
2.0
3.1*
1.9
2.2
16
27
13
22
18
9
5.6**
2.8*
6.0**
2.4
3.6**
11.9**
Left lung
Total
Central
Peripheral
Upper
Middle
Lower
Nebulizer output?
35
42
40
42
54
35
15
2.0
1.8
1.6
1.2
1.3
3.0*
-
30
13
34
37
29
31
19
1.4
10.4**
1.4
1.3
3.5*
1.3
-
?Separate nebulizers were used for each subject and for each
experiment.
output (mass median diameter) to be very consistent over
a wide range of nebulizers and drug solutions. Others,
however, have found larger variability, for example CVb-,
values of 4-35% can be calculated from previously
published data [27,28]. T h e mass or radionuclide output
of the nebulizers was more variable than the particle-size
output. Even using strictly controlled conditions, the
variability of nebulizer output was appreciable, and
within-nebulizer (between occasion) variability was
similar (CV,., 5-1 5%) to the between nebulizer variability
(CVb-, 5-10%). A major determinant of nebulizer output
is the amount of nebulizer fluid trapped on the nebulizer
baffles. This appears to be intrinsically unpredictable and
variable. For an individual nebulizer variability in output
may also be produced by retained drug particles within
the working parts, enlargement of the jet holes by the flow
of compressed gas, small distortions caused by repeated
use and cleaning and by movement of the nebulizer components in relation to each other. Other authors have also
found considerable variability of nebulizer output, and
their data can be used to derive CVs of 3-38% [15,
27-29]. The use of nebulizers from different batches, not
done in our experiments, may cause a further increase in
the variability of nebulizer output and thus pulmonary
aerosol deposition. Further variability may be introduced
by differences in the output of air compressors. As well as
d
+
2
s
d
-3
-4
-5
d-2s
0
Total pulmonary
deposition
773
2
4
6
8
10
(LD+ HD)/2
-3
1
0
1
5
I
3
(log LD +log HD)/2
Fig. 1. Immediate repeatability of scintigraphic measures
of total pulmonary aerosol deposition ("/o of initial
nebulizer dose) by Bland-Altman [21] analysis. Discrepancies between LD and HD results plotted against
mean values for raw ( a ) and log-transformed ( b )data. d
and d + 2 s indicate mean bias and 95% limits of agreement (see the text).
random differences between different compressors of the
same make, within-compressor variability may be affected
by the air temperature and humidity and by the motor
temperature which in turn will depend on the time the
compressor is left running. Variability in the output of the
compressors used in these studies was not measured, so it
is unclear what contribution this made to the overall variability in aerosol output, but as particle sizes are affected
by gas flow rate [30] and were shown to be very consistent
we suspect that this contribution was small.
T h e importance of breathing pattern is well known.
Almost half the variability in aerosol deposition between
subjects can be explained by differences in the amount of
aerosol actually inhaled [lo] and this depends on the
pattern of breathing. Increases in respiratory minute
volume up to values of 10-15 litres/min are associated
with an initial increase in deposition, but above this level
deposition falls off [S].The duration of the pause between
inspiration and expiration has been shown to be
important for the single-breath studies which are used for
histamine challenge testing: an increase from 5 to 15 s
reduced the CV,, of aerosol deposition from 104%to 5%
774
S. H. L. Thomas et al.
[31].Variability in the duration of this pause is also likely
to affect the variability of aerosol deposition during continuous tidal breathing, although not to the same extent.
Use of controlled breathing protocols has reduced CV,.,
from 60% to less than 20% in two previous studies [8, 91.
Aerosol deposition may also be affected by respiratory
rate and tidal volume, although there is no published data
to show the effects of these factors on the variability of
measurements. While variability in respiratory pattern is
clearly an important contributor to the variability in
aerosol deposition, we have chosen not to attempt to control breathing as we wished to study aerosol deposition
under conditions which are representative of routine
clinical practice. Too few subjects were involved in these
experiments to be able to draw any conclusions on the
relationships between body size, respiratory minute
volume, respiratory rate and aerosol deposition.
There is little published evidence relating aerosol
deposition to body size. It is to be expected that larger
subjects will have increased respiratory minute volumes.
As a result they inhale a larger proportion of the aerosol
generated by the nebulizer and pulmonary aerosol
deposition is increased [32]. In support of this hypothesis,
Alderson et a/. [33] showed an increase in aerosol deposition with age in children with cystic fibrosis; however, they
were unable to demonstrate a relationship with tidal
volume or respiratory rate. Because a face mask was used,
it is possible that age-related differences in the use of nose
breathing and nasal passage resistance were responsible.
Body size may also affect measured aerosol deposition, as
the size and thickness of the chest will affect the geometric
distribution of radionuclide activity and the amount of
tissue attenuation. Using a modification of the geometric
correction described by Ruffin et a/. [17] (the 'phantom
method') the same correction factor was applied to all the
subjects so there is no compensation for these body-sizerelated factors. When transmission data collected during
flood studies is used in the derivation of the correction
factor ('tissue attenuation method') the dimensions and
thickness of the chest affect the result which is therefore
different for each subject. We have shown that the use of
this individually derived correction gives a small reduction in between-subject variability in measured aerosol
deposition. However, the method is more elaborate and
time-consuming, particularly as a fresh flood must be
made each day because of the short half-life of yymTc.We
were able to show that use of the more convenient 57C0
flood will give similar results, at least in subjects with
chest dimensions similar to those who took part in our
studies.
Our results have shown that variability in measured
pulmonary aerosol deposition is less within than between
subjects, presumably because body dimensions are constant, and variation in breathing pattern is less marked.
Nebulizer output remains variable even though the same
nebulizer unit was used for each individual subject. Variability was even less when measurements were made
within the same occasion, in spite of the fact that out of
necessity a different nebulizer unit and ""'Tc-HSA dose
was used for each inhalation. This is an important
observation as this experimental protocol conveniently
compares two methods of inhalation on a single occasion.
This is particularly appropriate for experiments where the
characteristics of respiration are likely to change, for
example in patients recovering from illness. Furthermore,
a reduced number of subjects would be needed to detect
significant differences in pulmonary deposition. The disadvantages are that a larger total radiation dose is needed
and that pulmonary clearance of the first low dose of
""'Tc-HSA taking place during the second H D may lead
to an underestimation of lung deposition. However, this is
unlikely to produce an important error because 9YmT~HSA clearance occurs by mucociliary clearance, which is
very slow. In support of this contention no significant bias
was obscrved when comparing results from the HDs and
LDs. The variability in nebulized aerosol deposition
measured in this way is similar to the previously reported
within-subject within-occasion variability for deposition
of inhaled aerosols using the single inhalation protocol
employed for histamine challenge testing [31].
The use of statistical power calculation formulae is
recommended for calculating sample size and power
before embarking on comparative trials. Such formulae
use an estimate of the difference between groups deemed
to be of sufficient importance to be worth detecting, the
power required to detect this difference, a pre-specified
significance level, and an estimate of the SD of the data.
The SD of the data can be derived from the appropriate
CV by multiplying by the expected mean value. For example, to detect a significant difference at the P<O.O5
level with 80% certainty ( a = 0.05, /3 = 0.80) between two
types of nebulizer, one of which produces a mean deposition of 5% and the other 7.5%, would require a sample
size of 19 subjects in each group for an unpaired comparison (CV 54%, SD 2.7%). For comparisons done within
subject, the CVs, SDS and required sample sizes are
smaller. The sample sizes required are nine for studies
done on separate occasions (CV 37%, SD 1.85) and four
(CV 23%, SD 1.15) for studies done on the same occasion
[ 141. Because of the increased variability involved, larger
sample sizes will be needed to detect significant differences in regional deposition. Trials comparing three or
more nebulizers would also require larger numbers and
for these studies the LD-HD method would not be
appropriate.
CONCLUSION
These experiments have shown that the variability in
scintigraphic measurements of pulmonary aerosol deposition is considerable both between and within subjects.
Several ways of minimizing this variability are suggested
by the results. By using the same nebulizer unit for each
subject, the variability in particle-size output is reduced;
however, the variability in mass output of drug is not
improved. Thus the advantage of this approach does not
outweigh the practical disadvantages, including the need
for cleaning and sterilizing. Variability might also be
reduced by using subjects of similar build and by controlling the breathing pattern, but as this would not be a
Variability in nebulized aerosol deposition
reflection of normal clinical practice the results of trials
using such manoeuvres may not be applicable to the clinical situation. Correction of data for tissue attenuation
gives a small but worthwhile reduction in between-subject
variability. Within-subject variability is less marked still,
so repeated measurements performed within the same
individuals will be more sensitive in detecting differences
in deposition. For paired comparisons of two methods of
aerosol inhalation the LD-HD method described above is
the method of choice because it is most reproducible,
measurements are completed quickly and changes in the
pattern of respiration are unlikely to occur during the
experimental period.
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