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Evaluation of Respiratory Inductive Plethysmography : Accuracy for Analysis of Respiratory Waveforms Pierre-Yves Carry, Pierre Baconnier, Andre Eberhard, Pierre Cotte and Gila Benchetrit Chest 1997;111;910-915 DOI 10.1378/chest.111.4.910 The online version of this article, along with updated information and services can be found online on the World Wide Web at: http://chestjournal.chestpubs.org/content/111/4/910 CHEST is the official journal of the American College of Chest Physicians. It has been published monthly since 1935. Copyright 1997 by the American College of Chest Physicians, 3300 Dundee Road, Northbrook, IL 60062. All rights reserved. No part of this article or PDF may be reproduced or distributed without the prior written permission of the copyright holder. (http://chestjournal.chestpubs.org/site/misc/reprints.xhtml) ISSN:0012-3692 Downloaded from chestjournal.chestpubs.org at UJF-INP Grenoble SICD1 on June 3, 2010 1997 by the American College of Chest Physicians Respiratory Inductive Plethysmography* Evaluation of Accuracy for Analysis of Respiratory Waveforms Pierre-Yves Carry, MD, PhD; Pierre Baconnier, Pierre Cotte; and Gila Benchetrit, MD, PhD PhD; Andre Eberhard, PhD; Objective: To assess the accuracy of respiratory inductive plethysmography (RIP) waveforms to those obtained with whole body plethysmograph (BP) as this device gives a plethysmographic signal and a pneumotachograph (PNT). Design: Randomized controlled trial. Setting: Physiologic laboratory in a university hospital. Participants: Eleven subjects from the laboratory staff. study was achieved during four consecutive periods in subjects breathing through different added resistive loads. Using the least square method spontaneously calibration, two RIP waveforms, VRiP.BP(t) and VRiP.PNT(t), were simultaneously calculated with coefficients obtained from BP and from PNT volume waveforms, respectively VBP(t) and VPNT(t). For each recording, to compare volume waveforms, we calculated their differences in term of distances, Drip-bp and Drip-pnt, between the normalized RIP volume signal (respectively, VRiP.BP[t] and VRiP.PNT[t]) and its normalized reference (respectively, VBP[t] and VPNT[t]). We also calculated the distance Dpnt-bp between the two normalized references VBP(t) and VPNT(t). Results: No significant effect of load or time on the distance occurred. Including all the appears significantly lower than both the mean recordings, the mean distance Drip-bp (3.4± 1.1%) distance Drip-pnt (4,5±1.3%; p<0.04) and the mean distance Dpnt-bp (4.6±0.9%; p<0.008). For each period or load level, Drip-bp appears to be lower than Drip-pnt and Dpnt-bp. Conclusion: The RIP seems reasonably accurate for analysis of respiratory waveform while (CHEST 1997; 111:910-15) subjects subsequently breathe against resistive loads. Interventions: This and Key words: body plethysmography; respiratory inductive plethysmography; volume waveform Abbreviations: ABD=abdomen; AN OVA=analysis of variance; BP=body plethysmograph; Dpnt-bp=distance be¬ tween PNT waveform and body plethysmograph waveform; Drip-bp=distance between RIP waveform calibrated with between RIP waveform calibrated with body plethysmographandand body plethysmograph waveform; Drip-pnt=distance waveform; FetC02=fractional end-tidal C02; Pbody=pressure measured pneumotachograph pneumotachograph a pressure transducer; PNT=pneumotachograph; RC=rib cage; RIP=respiratoiy inductive plethysmograph; by R4.7=resistive ventilatory load of 4.7 cm H20/L/s; R7.5=resistive ventilatory load of 7.5 cm H20/L/s; V=flow at mouth; VBP(t)= waveform from body plethysmographic signal; VBP(t)= normalized waveform from body plethysmographic pneumotachograph signal; VPNT(t)=normalized waveform from pneumotachograph signal; VPNT(t) waveform from VRiP.BP(t)=waveform from RIP signal calibrated with body plethysmograph; VRip.BP(t)=normalized waveform signal; from RIP signal calibrated with body plethysmograph; VRiP.PNT(t) ^waveform from RIP signal calibrated with VRiP.PNT(t)=normalized waveform from RIP signal calibrated with pneumotachograph; VT=tidal pneumotachograph; volume = T^\ ifferent circumstances in anesthesia and intencare can modify respiratory drive.1 Re¬ Renchetrit et al,2 using a quantitative analysis cently, of respiratory waveforms, demonstrated that primary variables (frequency, mean inspiratory flow rate, and fractional inspiratory time) commonly used for mon¬ itoring of breathing pattern are not sensitive enough to detect respiratory personality differences. One -*-^ sive Grenoble, PRETA-TIMC/ Supported by the University Hospital of Lyon (Dr. Carry). *From the Faculte de Medecine de I MAG, La Tranche, France. Manuscript received January 23, 1996; revision accepted October 2. 910 might expect that the monitoring of respiratory pattern via such a waveform analysis could help in detecting respiratory drive changes in anesthesia and intensive care. The quantitative analysis of respiratory waveforms developed by Renchetrit et al2 (harmonic or Fourier analysis) relies on the precise measurement of air¬ flow profile. In the intubated patient, this analysis of breathing pattern is easy to perform by attaching a spirometer or a pneumotachograph (PNT) to the patient's endotracheal tube. During recovery from anesthesia or during weaning from mechanical venClinical Downloaded from chestjournal.chestpubs.org at UJF-INP Grenoble SICD1 on June 3, 2010 1997 by the American College of Chest Physicians Investigations tilation, problems arise because these devices, re¬ direct connection to the patient's airway, quiring demonstrate a low tolerance.3 In addition, the use of alterations in the mouthpiece produces spurious breathing pattern, causing tidal volume (Vt) to increase and respiratory frequency to decrease.47 Other devices, such as body plethysmograph (RP), the measurements of respired volumes, are allowing not convenient for clinical monitoring, particularly a during a prolonged period or during sleep. Conse¬ quently, devices have been developed to measure ventilation directly. Respiratory inductive plethysmography (RIP) is the most widely accepted method for quantitative and qualitative noninvasive respiratory measure¬ ments.810 When correctly calibrated, the RIP allows the measurement of volume and time components of the breathing cycle as well as the relative participa¬ tion of thorax and abdomen to this cycle. To our knowledge, the study of Stromberg et al11 is the only one that investigates the reliability of the time course profile of RIP as compared to that of the PNT. While studying the influence of RIP accuracy on the respiratory phase chosen for the calibration, these authors observed that RIP underestimated lung volume at the start of inspiration and overesti¬ mated lung volume at the end of inspiration. They observed a similar tendency during expiration. As RIP is supposed to give a plethysmographic waveform,12 we undertook the present work to assess the accuracy of RIP waveform by comparing it with from RP. the volume waveform obtained the whole We also carried out the same comparison with the PNT as this device is commonly used for breathing pattern analysis.2 The waveforms of the RIP volume calibrated, respectively, with a RP and a PNT were of the volume ob¬ compared with the waveforms RP and with PNT. This study was achieved in subjects breathing spontane¬ added resistive loads to simulate ously and through diseases. tained, respectively, with respiratory Materials and Methods Subjects Eleven healthy subjects (eight male, three female) without histoiy of respiratory disease, recruited from the laboratory staff, volunteered for RIP validation against BP and PNT. The protocol was approved by the local ethics committee and all subjects gave informed consent. The characteristics of these subjects are presented in Table 1. Apparatus Measurements were made with the subjects seated in a 690-L barometric whole BP. This device (Pulmostar SMB; Table Subject/Sex/Age, 1.Subject Characteristics* Weight, vr l/M/27 2/M/46 3/M/23 4/M/38 5/M/28 6/F/24 7/F/25 8/F/33 9/M/37 10/M/45 ll/M/47 *Ve=minute ventilation; 68 90 77 92 65 54 55 57 68 75 72 Ve, F, FetC02 L/min /min % 11.7 12.2 12.5 12.7 8.7 10 14 14 18 10 19 11 11 8 8 13 5.0 4.5 12.0 9.1 7.9 7.1 9.0 12.1 4.4 4.5 5.1 4.6 3.9 5.5 4.6 5.5 4.0 F=respiratory frequency. Geneva, Switzerland), with high-frequency response, allowed the measurements of rapid volume changes.13 A pressure transducer measured the pressure (Pbody) of gas compressed in BP by pulmonary volume changes, giving after calibration (see below) a first volume waveform VBP(t). During protocol, absence of gas leaks in the BP was checked by measuring the stability of Pbody during a voluntary apnea. Stability of BP temperature during each recording was checked by the aver¬ age stability of Pbody signal (no drift). A heated PNT (Fleisch; Lausanne, Switzerland) inserted in the front wall of the BP measured the flow at mouth (V). The zero flow of this channel was carefully set at the beginning of each protocol but was not readjusted afterward. The volume waveform from PNT, VPNT(t), was obtained by numerical integration of flow. After zero setting, BP and PNT were calibrated with a 1-L syringe before each procedure.14 The coefficient of calibration for each signal was determined from five syringe maneuvers. The rib cage (RC) and abdominal (ABD) displacements were measured using a direct current-coupled RIP. This device con¬ sists of two belts, to which wavy coated wires are attached, that encircle the RC and the ABD. A garment incorporating the two coils was developed in our laboratory. It consists of a sleeveless jacket, made of a special fabric with texture allowing horizontal wiredrawing only.15 During each experiment, the subject had his or her back applied to a wall inside the BP and was instructed to avoid any to prevent interference of changes in spinal changes in position attitude with RIP calibration. This calibration is based on the assumption that the respiratory system behaves with two degrees of freedom motion16 such that the change in lung volume (Vrip) is the sum of the volume changes of the rib cage (Vrc) and abdominal (Vabd) compartments. Vrc and Vabd are expressed in terms of RC and ABD signals by the volume-motion coeffi¬ cients a and b: VRiP=a-RC+b-ABD. The a and b coefficients are obtained by the least squares calibration procedure.17 Two RIP waveforms (VRiP.BP[t] and VRiP.PNT[t]) are simultaneously calculated with coefficients ob¬ tained respectively from BP volume waveform (VBPft]) and from PNT volume waveform (VPNT[t]). The a and b coefficients were calculated from the data recordings from each situation, thus allowing comparisons based on the same source data. All signals (Pbody, V, ABD, and RC) were recorded on a computer (Macintosh Ilci) equipped with a 12-bit analogue-todigital converter (MacAdios; G.W. Instruments; Boston) and each signal sampled at 32 Hz (softwares are written in Think Pascal; Symantec SARL, Surenes, France). CHEST / 111 / 4 / APRIL, 1997 Downloaded from chestjournal.chestpubs.org at UJF-INP Grenoble SICD1 on June 3, 2010 1997 by the American College of Chest Physicians 911 Experimental Protocol The study was conducted while the subjects, seated in the BP with the RIP body jacket, wore noseclips and breathed through the mouthpiece connected to the PNT (palms on the cheeks) (Fig 1). To analyze the effects of load level on RIP volume waveforms, the subjects breathed successively through different load levels. The resistance of the BP-mouthpiece-PNT system was 2.0 cm H20/L/s; the first and last measurements were obtained when subjects breathed through this equipment (control load level, R2.0). The addition of two different resistive ventilatory loads, respectively, 2.7 and 5.5 cm H20/L/s, to the outer part of PNT provided two other levels of measurements (R4.7 and R7.5). Linearity of these different resistances was checked by plotting pressure at mouth vs V during tidal breathing. Additional deadspace induced by added resistances was negligible. After a 15-min temperature stabilization period, the instruments were calibrated. Each subject underwent four consecutive periods of 92-s recordings: control (PI); first added resistive load (P2); second resistive load (P3); control (P4). The subjects were allowed to choose their own breathing pattern. The order of R4.7 and R7.5 were at random. Each subject underwent then one of the following load sequences: (R2.0 R4.7 R7.5 R2.0) or (R2.0 R7.5 R4.7 R2.0). For each period, recordings were undertaken once fractional endtidal C02 (FetC02) was in steady state. FetC02 was measured by a capnograph (Engstrom Eliza; Bromma, Sweden) through a side port of the mouthpiece (this side port was closed during recording periods). Data Analysis The primary signals V, Pbody, ABD, and RC were simulta¬ neously sampled and recorded on the microprocessor system during periods of 92 s (which corresponds to 2,944 points at 32 Hz sampling frequency for each signal). The accuracy of the two RIP volume waveforms (VRiP.BP[t] and VRiP.PNTft]) during the four different periods was estimated in the following way. We compared the closeness of VRiP.BP(t) and Vrip.pnt(0 with the corresponding reference waveform, respectively, VBP(t) and VpNT(t). To compare the waveforms of these four signals, we had to eliminate any discrepancy due to baseline or to amplitude difference. These conditions were realized by normalizing each signal waveform (ie, mean value of each waveform at zero, and SD at 1) (Fig 2). For each recording, we calculated the mean square difference A2 between each normalized RIP waveform (VRiP.Bp[t] or VRiP.PNT[t]) and its normalized reference (VBP[t] or VPNT[t]) on the whole recordings (2,944 points): ARiP-BP2=(l/N)*X[YRiP-BP(i)-VBP(i)]2 (i=l to N), ARIP-PNT2=(l/N)*XKRIP-PNT(i)~VPNT(i)]2 (i=l to N) (N 2,944). = To have a comparison between the reference signals, we also calculated the mean square difference between the normalized signals VBp(t) and VPNT(t): APNT-BP2=(l/N)*^][VPNT(i)-VBP(i)]2 (i=l to N) (N=2,944). As waveforms are normalized, 95% of the values lie between .2 and +2, then a near maximum value for A2 is equal to 16 (a situation in which each sample pair is 2 SDs apart). The square root of A^ being equivalent of an euclidean distance, we called D this distance between each waveform and its reference, expressed in percentage of this maximum, respectively: Drip-bp=100 /vy\yv Pbody ^^/WVTVrc Figure 1. The system used in the present study, showing the at the mouth from PNT; (2) Pbody from whole following:RC(1)andflowABD BP; (3) signals from RIP. 912 16 and Drip-pnt=100 Arip-pnt 16 We also calculated the distance Dpnt-bp between PNT and BP waveforms: Dpnt-bp 100 Apnt-bp2 = 16 Statistics The effects of load level and time on distances were analyzed using analysis of variance (ANOVA). The paired t tests were used compare the global mean values of distances. The level of statistical significance was taken as p<0.05. to PSL <vw Flow t5p. Arip-bp2 Results ANOVA shows that the load level has effect on the no signifi¬ distances (Drip-bp, Drippnt, or Dpnt-bp) (Fig 3). However, mean distances were similar whatever the period: ANOVA did not evidence any statistical difference (Fig 4). The mean values of the three distances for each subject are gathered in Table 2. Including all the recordings, the statistical analysis exhibits that the mean distance Drip-bp (3.4±1.1%) appears signifi¬ cantly lower than both the mean distance Drip-pnt (4.5±1.3%; p<0.04) and the mean distance Dpnt-bp (4.6±0.9%; p<0.008). No statistical differ¬ ence appears between Drip-pnt and Dpnt-bp cant mean (p=0.63). Clinical Downloaded from chestjournal.chestpubs.org at UJF-INP Grenoble SICD1 on June 3, 2010 1997 by the American College of Chest Physicians Investigations VRIP.PNT(t) VRIP.BP(t) VpNT(t) VBP(t) B 1.5 SD VRIP.BP(t) YBP(t) -1.5 SD (A) The four waveforms Vbp(0, VPNT(t), VRiP.BP(t), and VRiP.PNT(t) for one breath during recording period in a representative subject; (B) normalized curves VBP(t) and VRiP.BP(t) waveforms from this breath showing that they almost overlap each other; (C) part of VBP(t) and VRlP.BP(t) waveforms has been enlarged and the distance for one sample (VRiP.BP[i] Vbp[i]) is illustrated (see text for commentary). Figure 2. a Discussion The purpose of the present study was to test the that the RIP volume waveform is as hypothesis accurate as that obtained from RP and from PNT. To confirm this hypothesis, we analyzed the stability of the different signals during the recording period and ? demonstrated that the measurements from the RIP, RP, and PNT were comparable. Following the initial work of Konno and Mead,16 several investigators proposed RIP as a method to RC-ARD motions. In clinical situations, the analyse RIP generally is used for continuous noninvasive monitoring.18 These different utilizations of RIP R2.0 R7.5 D PNT-BP Dpnt-bp Drip-bp Figure 3. Mean distances Drip-bp, Drip-pnt, and Dpnt-bp for all subjects while breathing against the three different load levels (R2.0, R4.7, and R7.5) (see "Experimental Protocol" section for more details). Results are expressed in percent; vertical bars indicate SD. D RIP-BP D RIP-PNT Drip-pnt Figure 4. Mean distances Drip-bp, Drip-pnt, and Dpnt-bp for all subjects during the successive periods (PI control, = P2=resistive load R4.7 or R7.5 [random], P3 resistive load R7.5 or R4.7 [random], P4=control) (see "Experimental Protocol" = section for more details). Results vertical bars indicate SD. are expressed in percent; CHEST/111 IM APRIL, 1997 Downloaded from chestjournal.chestpubs.org at UJF-INP Grenoble SICD1 on June 3, 2010 1997 by the American College of Chest Physicians 913 Table 2.Mean Values of Distance for Each Subjects 9+ 10 11 + Mean Drip-bp Drip-pnt 4.6 2.8 2.6 4.5 4.6 3.8 5.0 ± 1.0 7.0 ± 0.4 2.6 ± 0.7 5.6 ± 2.0 4.2 ± 1.8 5.8 ± 2.2 3.7 ± 1.2 5.0 ± 1.3 4.1 ± 1.5 3.0 ± 0.4 3.4 ± 0,5 4.5 ± 1.3* ± ± ± ± ± ± 2.7 ± 2.2 ± 4.7 ± 1.0 0.6 0.2 0.7 1.6 1.5 0.3 0.1 0.8 1.8 ±0.1 2.5 ± 0.3 3.4 ± 1.1 Subject* Dpnt-bp 4.0 6.8 3.4 4.3 5.3 5.3 ± ± ± ± ± ± 4.4 ± 5.1 4.6 4.4 3.5 4.6 ± ± ± ± ± 0.9 0.7 0.2 1.4 1.7 2.4 0.7 1.2 0.4 0.3 0.8 0.9n *Data include all the situations and all subjects. Plus sign indicates subjects for whom R7.5 was imposed before R4.7. Drip-bp distance between BP and RIP calibrated with BP; Drip-pnt, distance between PNT with RIP calibrated with PNT; Dpnt-bp distance between BP and PNT. Results are expressed in percent ± SD. +p<0.05 vs Drip-bp. 'Not significant vs Drip-pnt. assume (1) a minimum drift of the RIP signal and (2) the stability of calibration during the time of record¬ ing. Such assumptions must also be testified for other comparative methods. Using current RIP tech¬ transducers placed on different-shaped nology with Watson et al19 confirmed the baseline sta¬ models, RIP. Two studies demonstrated that the RIP of bility calibration was well maintained in normal sub¬ jects.20-21 Hudgel et al,22 using inductance vest in COPD patients, showed that calibration changed after 240 min. minimally Refore measurements, the RIP must be cali¬ brated. As noted by Sartene et al,23 the choice of the calibration method depends on experimental or clin¬ ical conditions. Among the different methods,161724 Chadha et al17 proposed the least squares method as the most accurate during long-term trial when in body posture and/or Vt cannot be con¬ change trolled. Tobin et al25 confirmed that the least squares method provides the most reliable measurements However, the changes of breathings. during loaded after and patient movements calibration position could possibly have an adverse effect on satisfactory measurements.11-26 In our study, each subject seated in the RP was instructed to avoid any changes in position and to breathe through the mouthpiece, on the cheeks during recording to prevent palms excessive interference of movements.7 When the waveform signal was obtained from RP, the drift is essentially associated with the variations of temperature and humidity in the box. Retween each recording, the subjects breathed without con¬ nection with the mouthpiece. In these conditions, no drift of plethysmographic signal was observed during 914 the recording periods. To obtain the correct V offset during steady-state period, we hypothesized that the end-expiratory volume is the same at the beginning and at the end of each level of recording as proposed Peslin et al.27 byThe comparison of the waveforms of the different needs the stability of the transducers and elec¬ signals tronics used. In addition, if one needs to quantify the RC-ARD motion changes with RIP, one has to dem¬ onstrate the reliability of the time course of the signal. The frequency responses of measurement devices must also be compatible. The pressure-compensated vol¬ ume-displacement RP has a frequency response com¬ with that of the PNT.28 Roynton et al29 dem¬ patible onstrated in dogs that the sum of the RC and ARD band signals underestimated determined volume above 8 Hz. A frequency spectral analysis of the change three signals demonstrated that frequency spectra were very similar up to 10 Hz, except for RIP which exhibits higher cardiogenic oscillations (between 1 and 1.5 Hz). This can only be responsible for a parallel increase of Drip-bp and Drip-pnt relative to Dpnt-bp and can¬ not explain the observed difference between Drip-bp and Drip-pnt. We then think that the lower degree of damping of cardiogenic oscillations found in RIP does not influence the results of the present study. In our study, we demonstrated the closeness of RIP waveform VRiP.BP(t) and VRlP.PNT(t) with the corresponding reference waveform, respectively, VBP(t) and VPNT(t). Our results suggest that the RIP frequency response is comparable to that of the PNT and RP. Finally, paired t tests applied to Vt obtained from the four different volume measurement methods exhibit no statistical difference between methods. This confirms the accu¬ rate calibrations of the three devices. Currently, the RC and ARD coefficients were obtained from RIP calibrated with flow signal or volume signal.7'2326 The qualitative comparison of simultaneous volume waveforms from RIP and spi¬ rometer, in normal subjects breathing through low levels of resistances, exhibited no difference.21 We calibrated RIP against PNT as currently performed. Simultaneously, RIP was also calibrated against RP as these two measurement methods are based on the same physical property, volume displacement. In our study, the comparison of waveforms exhibits that the RIP signal is closer to the RP signal than to the PNT signal. This confirms that RIP is a good plethysmo¬ graph that is possibly suitable for use during pulmo¬ nary rehabilitation.14 Comparing the mean distances Drip-bp and Drip-pnt, we demonstrated no difference whatever the periods or added resistances used. Stromberg et al,11 comparing RIP and PNT measurements, showed that the RIP underestimated volume profile at the start of inspiration or expiration and overestiClinical Downloaded from chestjournal.chestpubs.org at UJF-INP Grenoble SICD1 on June 3, 2010 1997 by the American College of Chest Physicians Investigations mated it at the end of inspiration or expiration. They attributed these phase differences to the fact that the movements of the RC and the ARD are not linearly related to the lung volume. This discrepancy may be by the physical approach. The phase interpreted differences observed between volume displacement (measured by RIP) and air displacement (measured PNT) may be attributed to gas compression which by is similarly observed when volume displacement (measured by RP) and air displacement at mouth are used to assess airway resistances.25'30 The comparison between different signals was carried out in subjects breathing against different resistive loads. These resistive loads were lower than the values inducing respiratory muscle fatigue pro¬ posed by Tobin et al.25 However, the resistive levels obtained by added resistances were chosen to be the values of airway resistances comparable with in some human diseases.9 The state during steady values of resistances were calculated with RP using a reference method to measure resistances.31 In conclusion, the results of this study indicate that the RIP seems reasonably accurate for analysis of respiratory waveform while subjects subsequently breathe against resistive loads. This confirms that RIP can be considered as a good plethysmograph. If this were to be also true in clinical situations, one would then get a precise and noninvasive tool of chest wall motion monitoring. ACKNOWLEDGMENTS: We gratefully acknowledge the coop¬ eration of the staff of PRETA laboratory and V. 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J Clin Invest 1956; 35:327-35 31 Dubois CHEST/111 /4/APRIL, 1997 Downloaded from chestjournal.chestpubs.org at UJF-INP Grenoble SICD1 on June 3, 2010 1997 by the American College of Chest Physicians 915 Evaluation of Respiratory Inductive Plethysmography : Accuracy for Analysis of Respiratory Waveforms Pierre-Yves Carry, Pierre Baconnier, Andre Eberhard, Pierre Cotte and Gila Benchetrit Chest 1997;111; 910-915 DOI 10.1378/chest.111.4.910 This information is current as of June 3, 2010 Updated Information & Services Updated Information and services can be found at: http://chestjournal.chestpubs.org/content/111/4/9 10 Citations This article has been cited by 6 HighWire-hosted articles: http://chestjournal.chestpubs.org/content/111/4/9 10#related-urls Permissions & Licensing Information about reproducing this article in parts (figures, tables) or in its entirety can be found online at: http://www.chestpubs.org/site/misc/reprints.xhtml Reprints Information about ordering reprints can be found online: http://www.chestpubs.org/site/misc/reprints.xhtml Citation Alerts Receive free e-mail alerts when new articles cite this article. 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