Evaluation of quantitative aerosol techniques for use in bronchoprovocation studies

Evaluation of quantitative aerosol techniques for use in bronchoprovocation studies Matthew

S. Messina,

M.D., and Gerald

C. Smaldone,

M.D., Ph.D.

Stony Brook, N. Y.

To investigate airway physiology by use of inhaled aerosols, it is frequently necessaryto measure the actual amount of material deposited on the airway wall as well as the site of particle deposition. To satisfy these needs, radiolabeled aerosols and gamma camera techniques have been used to measure regional deposition of inhaled particles. To make quantitative measurements of the amount deposited, previous investigators have used a “phantom” technique to indirectly calibrate the gamma camera for the attenuation of gamma rays through the lungs and chest wall. For this calibration, the phantom is a simulated lung containing a known amount of radioactivity. Radioactive counts emitted from the phantom are assumed to be attenuated in the same manner as the intact human lung. The present article describesa technique to determine directly the amount of inhaled aerosol deposited in the lung and simultaneously to calibrate the gamma camerafor each individual subject. We used right angle light scattering and a gamma camera to measure individual values of the deposition fraction (OF) of inhaled aerosol deposited in the lung and the ‘coeficient of attenuation (AC) of gamma rays in normal and obstructed lungs of human subjects. Radiolabeled monodisperseaerosols 1 and 2 pm in diameter were used. Knowledge of the activity of the inhaled aerosol (microcurie per liter), the volume inhaled, and the measured DF determined each subject’s AC (counts per minute per microcurie), DF varied by an order of magnitude in normal (0.04 to 0.48) and obstructed (0.16 to 0.75) of subjects. AC also varied signt$cantly in normal subjects (124 to 790) and patients (112 to 373). Factors important in the observed variation in values of AC included body surface area and regional deposition pattern. Our results suggest that becauseof individual variation in thoracic geometry and deposition pattern, the use of single value phantom calculations may not accurately reflect the amount of aerosol deposited in the lung, We suggest that attempts to correlate changes in pulmonary function with quantitative inhalation challenge require direct determination of the dose entering the lung for each individual patient. (J ALLERGY CLIN IMMJNOL

75:252-7, 1985.)

Aerosols have been used to study airway physiology and pharmacology in animals and human subjects.‘, * For example, in human asthma, investigators have used increased reactivity to inhaled methacholine or histamine as an integral part of the definition of the disease. 3,4 Correct interpretation of the physiologic effects of deposited aerosol requires an accurate determination

From tbe pulmonary Disease.Division, Department of Medicine, State University of New York at Stony Brook, Stony Brook, N. Y. Supported by grants HLOO461, AI16337, and ES07088-03 from the National Heart, Lung, and Blood Institute. Received for publication Sept. 2, 1983. Accepted for publication June 19, 1984. Reprint requests: Gerald Smaldone, M.D., Division of Pulmonary Medicine, Dept. of Medicine, SUNY at Stony Brook, Stony Brook, NY 11794.

252

Abbreviations used A: AC: COPD: DF: N* N::I TC: V: +: NS:

Radioactivity of the aerosol in microcurie Per liter Attenuation coefficient Chronic obstructive pulmonary disease Deposition fraction Number of particles exhaled Number of particles inhaled Transmission coefficient Volume inhaled Flow Not significant

of the dose of the material deposited on the airways. Conventional techniques such as an automatic .airmetering device’ or simple nebulization do not measure the DF of inhaled material on the airway wall.

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This fraction may vary greatly from one individual to the next and may even vary during the same experi-

aerosol

bronchoprowcation

253

Plexigbss plern, 0.7Scm x 3&m containing t !s Ci TeesDispersed uniformly

ment as airway dynamics is altered.16 Recognition of this problem has led to indirect attempts to determine the DF of particles in the lung by use of radiolabeled aerosols and a gamma camera.6-1”With this technique a known quantity of radioactive material contained in a lung model called a “phantom” is presented to the

gamma camera. If the geometry of the phantom accurately simulates the lung, the observed counts divided by the known amount of radioactivity in the

phantom will calibrate the camera for all subjects. This calibration should acount for the attenuation of gamma rays by the lungs, chest wall, and camera and is called an AC. Once it is calibrated, the gamma camera can measure quantitative and regional aerosol deposition on the airways in human subjects and animals.

Although this technique has advantages, the basic

FIG. 1. Diagram of phantom lung.

assumption that the geometry of the phantom repre-

sents that of the human lung remains untested and appears contrary to basic principles in radiation physics. Subject-to-subject variation in thoracic geometry including anthropomorphic differences in chest wall

thickness suggests that a single value phantom calculation applied to all subjects may be an oversim-

plification. ’ ‘I ” Although the literature contains many theoretic and experimental assessments of thoracic geometry, 13,‘4 there are no studies that demonstrate the predictive values of these theories in determining the actual attenuation of activity deposited via inhaled aerosols. For this reason we developed a method for

measuring individual ACs in human subjects as well as precise DFs for inhaled aerosols under varied physiologic conditions. The present study combined light scattering techniques together with gamma camera imaging to measure directly the fraction of inhaled aerosol retained by the lung, its site of deposition, and to calculate the specific AC of each human subject. We

found marked variation in values of these individually measured DFs and ACs suggesting that the use of a

fixed AC derived from a single phantom measurement may misrepresent the dose of pharmacologic agent delivered to the lungs.

METHODS Gamma cameraACs are measuredby dividing the counts obtained from the scintillation scannerby a known quantity of radioactivity placed before the collimator as in the formula: AC =

cpm radioactivity in the lung

air represents one half the lung volume and is separated from the collimator by 2.5 cm of water simulating the chest wall. The counts that are obtained on Hooding the camera are divided by the radioactivity of the source to determine the AC. Instead of using a source of known radio&iv&y and a simulated lung, we measured the actual radioactivity deposited in the lungs of our subjects and divided this value by the counts obtained on the gamma camera. The radioactivity was delivered by monodisperseoil aerosols I and 2 brn in diameter labeled with %Tc human serum albumin. The particles (diethylhexyl sebacate) were generated in a modified Sinclair-LaMer generatoP and were inhaled by subjects seated in front of a gamma camera (Picker Dyna Camera 4, Fig. 2). A low energy, parallel-hole collimator was used, and the camera was set with a 20% window around a peak energy of 140 keV. The camera was peaked regularly and demonstratedno change in sensitivitybetween measurement intervals. A Tyndallometer was used to measure the concentration of aeros inhaled and exhaled at the mouth This device, used by many previous investigators, 16-*’consistsof a photomultiplier tube oriented 90° to a light source. As monodisperse aerosol traversesthe system, the scatteredlight measuredby the photomLlltiplier tube is directly proportional to the aerosol concentration. This systemhas generally been used to measurelung deposition of nonradioactive particles. Recent work by ltoh et al.‘” by use of radioactive monodisperseparticles in dogs compared the DF as measured by the Tyndallometer to that measured by two other methods: gamma camera ACs and fractional inspiratory and expiratory filtration. These techniqueswere demonstrated to correlate closely with one another fr = 0.960, p < 0.02).

(1)

For example, in the “phantom” technique (Fig. 1) a known quantity of radioactivity informly dispersedin a plexiglass plate is placed 9.5 cm away from the camera’; 7.5 cm of

In our experiments (Fig. 2), V was measuredby a pneumotacbograph placed in serieswith the ~~~~~~, and this signal was integrated to measure tidal V. N, and N,, were calculated for each breath by integrat.&g the product of the concentration and V over time:

254

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FIG. 2. Sketch

TABLE

of experimental

apparatus

and

glossary

of terms

and

equations.

Weight

(lb)

I. Age Normal subjects 0. N.

G. D. R. H. W. M. K. S. F. B. M. C. COPD patients H. S. H. M. B. H. A. T. R. P. G. T. s. s.

(yrl

Sex

Height

(in]

SSA

(m*)

46 65 60 62 59 38 23

M M F M M M M

12 65 67 72 71 68.5 74

196 195 175 210 132 197 195

2.15 1.96 1.89 2.16 1.78 2.06 2.15

58 61 47 64 54 65 56

M M M M M M M

67.5 74 73 67 68 65.5 63

135 132 148 142 149 130 177

1.73 1.82 1.90 1.74 1.82 1.65 1.88

BSA = Body surfacearea. N = j- (C . V)dt (2) where N can be Ni. or N,,. The DF, the fraction of particles inhaled that actually deposited in the lung, was defined by equation 3: DF

=

8Nn

-

ZNx

8N”

(3)

for all breaths. The A was measured by sampling aerosol from the inhalation line (Fig. 2). Multiplying this radioactivity by V and DF determined the radioactivity deposited in the lung: radioactivity in the lung = A . V * (DF)

(4)

This deposited radioactivity was then substituted into equation 1 to determine the AC for each subject. There was no demonstrable pharyngeal deposition for these subjects inhaling 1 to 2 Frn of aerosols at rest. This was carefully

examined by scanning the pharynx and stomach during and immediately after deposition. Before deposition of aerosolan equilibrium “‘xenon scan was performed to outline the lung parenchyma. This study allowed the creation of suitable regions of interest to quantitate the deposition pattern of the inhaled aerosol. Seven normal subjects and seven patients with COPD were studied (Table I). Each group was defined by their smoking history, spirogram, and flow-volume curves. All normal subjects had a negative smoking history, FEV, higher than 65% of the FVC, and a tidal flow-volume loop much less than their forced expiratory loop at functional residual capacity. COPD patients universally had a history of excessivesmoking, FEV, less than 60% of FVC, and a forced expiratory flow-volume loop superimposedon their expiratory tidal flow-volume loop. In order to illustrate the anthropomorphic effects on transmission of radioactivity, a parallel study was performed. A disc-shaped source of radioactivity (%Tc similar to the

‘JOLUME 75 NUMBER 2

Quantitative

TABLE II. ---.

aerosol broncbopr~v~~c~t~on 255

~Normal subjects AC (cpm/pCi)

DF

COPD patients .__” -~ ~-..--AC (cpm/pCi) DF I pm

1 Pm 0.

N.

G. D. R. H. Mean -t SD W. M. K. S. F. B. M. C. Mean t SD All normal subjects: 271 k 233 All subjects: Mean ?I SD

TABLE ltt. *Tc

136 224 124 159 160 -c 45

2 km

0.04 0.08 0.20 0.11 ? 0.08

H. S. H. M. B. H.

373 229 277 293 t 73

0.04 0.25 0.47 0.48 0.31 2 0.21 All patients:

A. T. R. P. G. T. s. s.

213 228 112 119 168 ? 61 0.43 -t 0.20

AC

i!.l?,X

0.54 0.3.~ i- 0.19 2

Pm

DF 0.33 * 0.22

256 IL 172

~)..W 0.4 I 0.55 ik.7.5

O.5U 3: 0.19

---

TCs for normal human subjects Age (yr)

Sex

Height (in)

D. R. G. s.

17 35 28 32 34 45

F M M M M M

67 69 71 71 72 71

M. M. M. S. 1. R.

222 k 90

il. I6

0.22 t 0.19

SUbiQct

E. L.

BSA

790 236 228 418 k 322

uwBht

139 165 165 185 210 235

fib)

MM WI

1.72 1.90 1.92 2.03 2.17 2.23 Mean ‘-c SD

TC

0.272 0.258 0.211 0.217 0.138 0.129 0.204 5 0.060

= Body surfacearea

phantom in Fig. 1) was placed a fixed distance from the camera. The same collimator and window were used. After recording the activity of the source, different normal subjects were seatedbetween the source and the camera, and repeat scanswere obtained of the sourceactivity transmitted through the subject. The transmitted activity over the lung divided by the activity of the source defined a TC specific for each subject studied.

RESULTS The ACs measured for normal and obstructed subjects and patients arc listed in Table II. Values for the normal airways in subjects varied from 124 to 790 cpm/pCi with a mean and standard deviation of 271 & 233 cpm/pCi. For the obstructed airways in patients, values ranged from 112 to 373 cprn/pCi with a mean and standard deviation of 222 k 90 cpm/pCi. The average value for all measurements was 256 + 172 cpm/pXi. There were no statistically significant differences in measured ACs

between normal and obstructed airways in subjects and between subjects breathing 1 OF 2 pm particles. The measured fractions of deposited aemsul arealso listed in Table II. Marked variation in Mb grq~ was found. Values for normal s&j&% mn@d from 0.04 to 0.48 wi* a mean and etch deviatin of 0.22 + 0.19. Deposition for the ob&Wed lur@ in subjects ranged from 0.16 to 0.75 with a mean md standard deviation of 0.43 & 0.20. In coNrast ta the AC data, there was a statistically a~~~~ Mkmnce in average DF between Mrmal aad airways in subjects when the data for both 1 and 2 @m particles are grouped together. There was no sig@icant correlation between ACs and DFs for any of the groups studied. As expected from previous ~tudi~s,‘~~~~ the patients with obstructive lung disease had a ma&ed atral deposition pattern when this p&em was compmd to the peripberal pattern observed in the rmrmal subjects.

256

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I 1.0

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I

2.0 BODY SURFACE AREA (m21 1.5

I

2.5

FIG. 3. AC (CPM/&i) vs. body surface area (m*): normal subjects IX); patients (0). Calculations for the indicated linear regression do not include the circled extreme values (see text).

There was no orophatyngeal deposition of aerosol in any of our subjects. Measured values for the transmission data are listed in Table III. Values ranged from 0.277 to 0.129 with a mean & standard deviation 0.204 + 0.060 for the six subjects studied, all free of lung disease. Generally, as the body surface area of a subject increased, the transmitted radioactivity decreased. DISCUSSION The purpose of our article was to assessthe ability of a single value phantom-determined AC to measure lung deposition of inhaled aerosols. This was done by independently measuring DF and concomitantly measuring the individual AC in a given subject. As long as the measured DF is accurate, the hypothesis will be validly tested independent of particle size, breathing pattern, or even the presence of disease. We deliberately varied these parameters and looked at obstructed airways in patients to simulate clinical testing as reported by most ,investigators. lo We found that the AC varied to such an extent that no single value derived from a phantom measurement could be used to determine the fraction of aerosol deposited on the airway wall. For example, if we accepted the single value obtained from the phantom of 250 cpm/&i,g the DFs for the last two subjects listed in Table II,

when they are calculated by this technique, would be 0.30 for M. C. and 0.36 for S. S. This is markedly different from the actual measured values of 0.48 and 0.75, respectively. Phantom measurements are frequently used in the literature as approximations of lung geometry to test theories of gamma ray attenuationI but principles of radiation physics predict intersubject variability because of basic differences in chest wall density and variations in distance of the source from the camera. ‘I, ‘* This predicted variation between subjects is demonstrated by our TC data. The individual values range from 0.272 to 0.129 and correlate closely with body surface area, r = -0.906, p < 0.02. Supporting these concepts, Acevedo et al.*‘j measured perfusion scans by use of 99mT~macroaggregates in human subjects. The image of these scans appears similar to the aerosol image obtained in our normal subjects, i.e., a uniform pattern representing the lung parenchyma as outlined by ‘33xenon inhalation. With the use of their perfusion data, calculated ACs varied significantly (54.8 + 13.5 cpm/@Zi) and correlated closely with anthropomorphic predictions when these were related to body surface area (r = - 0.84, p < 0.01). The difference in the absolute magnitude of their average value when their value was compared to our average value for AC can be accounted for by differences in collimation. For our data a parallel hole, low-energy collimator was used, whereas Acevedo et al. used a parallel hole medium-energy collimator. Therefore, although previous investigators have used single value ACs to calculate the quantity of aerosol deposited,6-‘0* I3 a consideration of basic principles in radiation dosimetry, tissue density and chest wall configuration would predict significant intersubject variation in individual AC. However, these illustrations do not explain all our measured variation. Fig. 3 demonstrates a poor correlation between body surface area and ACs for all our subjects (r = -0.243,~ = NS). There does appear to be some indication of a relationship with body surface area since a much better correlation is observed if the two extreme values (circled in Fig. 3) are eliminated (r = - 0.647, p = NS). An important factor contributing to the correlation difference between a clearly anthropomorphic measurement (TC) and the aerosol-determined AC may be the pattern of aerosol deposition in normal versus obstructed objects. For purposes of discussion, again leaving out the two extreme values, our normal subjects with a uniform peripheral pattern of aerosol deposition had the best correlation with body surface area (r = -0.807, p < 0.05). This correlation was also similar to that obtained by Acevedo et al. (r = - 0.84,

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p < 0.0 1) as demonstrated above. The obstructed subjects, v limited during quiet breathing, all had a central deposition pattern and correlated poorly (r = -0.486, p = NS). Therefore, differences in the uniformity of the deposition pattern between subjects in the obstructed airway group combined with factors illustrated above could account for the differences in correlation between these groups. The measured fraction of deposited aerosol also varied greatly from subject to subject. This was expected because at least two factors known to affect aerosol deposition, tidal V and frequency of respiration . ” were not controlled in order to simulate techniques used by other investigators in this field.‘, *, 5-‘0 Another factor known to increase deposition as well as alter the distribution pattern of deposited particles is the dynamic compression of central airways during expiration.22-‘5 Enhanced deposition of particles occurs at sites of flow-limitation that form in the central airways, severely obstructed, in patients during tidal breathing. This may explain the significantly elevated DF and central pattern of deposition observed in obstructed airways in our group. The details of this observation are the subject of ongoing studies. Therefore, dose-response measurements of airway reactivity to inhaled aerosols require the direct measurement of aerosol deposited on the airway wall, as well as a determination of the site of deposition. Since the DF varies markedly between subjects, the deposited dose cannot be accurately estimated from the inhaled dose. Furthermore, a single AC cannot be used for different subjects to calculate indirectly the deposition fraction. Our data suggest that variation in body surface area, pattern of aerosol deposition, and complex factors involving thoracic geometry are great enough to require the individual measurement of ACs matched for deposition pattern if this technique is to be useful in quantitating particle deposition. REFERENCES 1. Rosenthal RR: The emerging role of bronchoprovocation. J ALLERGYCLANIMMLJNOL64564, 1979 2. Chai H: Antigen and methacholine challenge in children with asthma. J ALLERGYCLIN IMMUNOL64565, 1979 3. Spector S, Farr R: Bronchial inhalational challenge with antigens. J ALLERGYCLIN IMMUNOL64:580, 1979 4. Townley RG, Bentra AK, Nair NM, Brodkey FC, WattGD, Burke BS: Methacholine inhalational challenge studies. J ALLERGY CLIN IMMUNOL64569, 1979 5. Chai H, Farr RS, Froehlich LA, Mathisen DA, McLean JA, Rosenthal RR, Scheffer AL, Spector SL, Townley RG: Standardization of bronchial inhalational challenge procedures. J ALLERGYCLIN IMMUNOL56:323, 1975 6. Ruffin RE, Kenworthy MC, Newhouse MT: Response of pa-

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