ONLINE SUPPLEMENT
DISTINCT CLINICAL PHENOTYPES OF AIRWAYS DISEASE DEFINED BY
CLUSTER ANALYSIS
1
2
Mark Weatherall, 2Justin Travers, 2Philippa M Shirtcliffe, 2Suzanne E Marsh
Mathew V Williams, 1,3Michael R Nowitz, 2Sarah Aldington, 2,4Richard Beasley
1
2
University of Otago Wellington, Wellington, New Zealand
Medical Research Institute of New Zealand, Wellington, New Zealand
3
4
Pacific Radiology, Wellington, New Zealand
University of Southampton, Southampton, United Kingdom
Address for correspondence:
Professor Richard Beasley
Medical Research Institute of New Zealand
PO Box 10055, Wellington 6143, New Zealand
Telephone: +64-4-472 9199
Fax: +64-4-472 9224
Email: Richard.Beasley@mrinz.ac.nz
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METHODS
Detailed questionnaire
All participants completed a detailed written questionnaire compiled from a series of
validated questionnaires [1] administered by a trained interviewer in a standardized
manner.
Data obtained by the questionnaire included demographic information,
respiratory history and symptoms, smoking history, symptoms of allergy, family history,
occupational history, use of respiratory medications and use of health services for
respiratory problems. Subjects also completed the St George’s Respiratory Questionnaire
(SGRQ), a disease-specific quality of life assessment tool, validated in both asthma and
COPD [2]. The questionnaire is divided into three parts, measuring symptoms, activity
limitation and the social and emotional impact of disease. Overall scores range from 0 (no
effect on quality of life) to a maximum score of 100 (maximum perceived distress) and the
questionnaire is suitable for administration in healthy persons [3].
Pulmonary function testing
Pulmonary function tests were carried out on one site using two Jaeger Master Screen
Body volume-constant plethysmography units with pneumotachograph and diffusion unit
for spirometry and measurement of gas transfer (Masterlab 4.5 and 4.6 Erich-Jaeger,
Würzburg, Germany). Tests were undertaken by one of three trained operators (SA, SM,
MVW). Equipment was calibrated daily, prior to testing, for temperature, humidity and
barometric
pressure.
Volume
calibration
of
the
plethysmography
unit
and
pneumotachograph and gas calibration of the diffusion unit were carried out daily.
Subjects were requested to avoid carbonated drinks and caffeine for six hours prior to
testing. Subjects that had been prescribed inhaled medication were instructed not to use
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short acting bronchodilators for six hours prior to testing and to avoid long acting
bronchodilators for 36 hours prior to testing. Inhaled corticosteroids or other medication
was not altered. Subjects were required to refrain from smoking for two hours prior to
testing. Testing did not occur within three weeks of an upper or lower respiratory tract
infection (new or increased cough, sputum production, sore throat or nasal congestion).
Subjects over 120kg in weight were excluded due to the weight restriction of the CT
scanner.
All pulmonary function tests were carried out in accordance with American Thoracic
Society (ATS) and European Respiratory Society (ERS) criteria [4-6] and a nose clip was
worn for all tests. Airway resistance was measured during relaxed breathing at a rate of
approximately 0.5 Hz. Following a minimum of ten measurements of airway resistance
(Rtot) and attainment of a stable baseline representing functional residual capacity (FRC)
the shutter was closed thereby occluding breathing. Shutter closure was automatically
instigated at the start of inspiration and remained closed for two to three seconds during
which time the subject was instructed to continue with the motion of breathing, without
glottis closure, and thoracic gas volume (TGV) was calculated. A minimum of two
inspiratory/expiratory attempts was required. Following lifting of the shutter the subject
was instructed to breath out comfortably, maximally inspire and slowly expire to
completely empty for measurement of slow vital capacity (SVC). A minimum of three and
usually five measurements of TGV were carried out. Expiratory reserve capacity (ERV)
was measured from TGV to the point of maximum expiration and residual volume (RV)
was calculated from TGV – ERV. The total lung capacity (TLC) was calculated as SVC
(measured) + RV. The median value of TGV from correctly performed maneuvers and the
average value of airway resistance from 10 breaths was used to calculate specific airway
conductance (sGaw) and maximum values from individual maneuvers for ERV and SVC
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were used in the above calculations. A minimum of three acceptable spirometry
maneuvers were carried out with the best FEV1 and FVC selected for analysis. Maximal
mid expiratory flow rate (FEF25-75) was taken from the maneuver with the best
combination of FEV1 and FVC. For gas transfer measurement, washout and sample
volumes of 750 mls were used, except in subjects with COPD or asthma who were unable
to manage this, in which case a decrease to a minimum of 500mls was made. Gas transfer
measurements were expressed as raw values (DLCO) and corrected for alveolar volume
(DLCO/VA). All results were corrected for body temperature, atmospheric pressure and
water saturation (BTPS) and expressed as a percentage of predicated based on the
formulae of the ERS [7,8] except for FEV1/FVC which was expressed as an absolute ratio,
and sGaw which was expressed as a percentage of the lower limit of normal [9].
Measurements of lung volumes and spirometry were repeated 45 minutes after the
administration of 400mcg of Salbutamol (Ventolin™ GlaxoSmithKline) via a spacer
(Space Chamber™). Results were corrected to BTPS and expressed as a percentage of
predicated based on locally derived formulae [10] except for FEV1/FVC which was
expressed as an absolute ratio.
Following instruction in use of a peak flow meter (Breath Alert™, Medical Developments
International, Melbourne, Australia) subjects were asked to make twice daily (morning
and evening) peak flow readings over one week. Peak flow diaries were considered
satisfactory for analysis if twice daily readings were present for a minimum of four days.
Where a peak flow diary was not available subjects were assumed not to have peak flow
variability.
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CT scanning
Subjects were scanned using a single machine, (GE Prospeed, General Electric Medical
Systems, YMS, Japan) calibrated at weekly intervals using the manufacturers standard
phantom. Scans were obtained at full inspiration and no intravenous contrast was used. An
initial ‘scout film’ was used to identify the levels at which to acquire images. Three
images were obtained, one at each of the levels of 1cm above the aortic arch (Level 1),
1cm below the carina (Level 2) and 3cm above the top of the right hemi-diaphragm (Level
3)[11] with 1mm collimation, voltage of 120 kVp, 200 mAs. Scanning occurred in a
cranial to caudal direction with each image obtained during a separate breath hold each of
1.5 seconds duration. Images were reconstructed using high (GE bone) and low spatial
frequency (GE standard) resolution algorithms and the manufacturer’s ‘Density Mask’
program used to measure tissue density. Trachea and mainstem bronchi were excluded
from the measurements of lung area and lung tissue was separated from chest wall using a
density of -300 to -1200 HU to calculate the total area of lung tissue per slice. The areas of
tissue below the threshold of -950HU was expressed as a percentage of total lung area for
that slice (RA950).
Scans were independently examined for the presence of emphysema by two radiologists
(MN, AKT), blinded to the subjects’ clinical history. The radiological diagnosis of
emphysema was made if centrilobular low density areas, panlobular or paraseptal
emphysematous changes were present, and subjects were graded as having no evidence of
emphysema, changes suggestive of emphysema, or definite emphysema. In cases of
discordance, the images were viewed together and a consensus decision reached. Only
subjects with ‘definite’ emphysema were included as having macroscopic disease in
subsequent analyses.
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FENO measurement
FENO was measured by chemoluminescence using an online nitric oxide monitor (NIOX,
Aerocrine AB, Solna, Sweden), according to the 1999 ATS guidelines [12] and consistent
with the ATS guidelines published in 2005 [13]. Seated subjects exhaled fully then
inhaled ambient air through a nitric oxide scrubber to total lung capacity. Subjects then
exhaled against an automatically adjusting resistance to achieve a constant exhalation flow
rate of 50mls-1. Resistance was also adjusted so that an upper airway pressure of at least 5
cmH20 was maintained throughout exhalation, sufficient to close the velum and exclude
nasal air.
FENO measurements were taken from a stable plateau in exhaled nitric oxide
concentration of at least three seconds during an exhalation. Exhalations where flow rate
and plateau criteria were not met were deemed not acceptable for measurement. Repeated
exhalations were performed a maximum of six times to obtain three acceptable
measurements that agreed within 10%. The average of these three measurements was
used. The nitric oxide monitor was calibrated every 14 days or if the room temperature
changed by more than 5 degrees Celsius.
Measurements were made prior to other
pulmonary function testing. Subjects avoided eating for 1 hour, smoking tobacco for 2
hours, caffeine ingestion and short-acting bronchodilator use for 6 hours, long-acting
bronchodilator use for 36 hours, and antihistamine use for 72 hours prior to testing.
Subjects were not tested within three weeks of an upper or lower respiratory tract
infection.
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