- Research article
- Open Access
- Open Peer Review
Noninvasive assessment of asthma severity using pulse oximeter plethysmograph estimate of pulsus paradoxus physiology
© Arnold et al; licensee BioMed Central Ltd. 2010
- Received: 20 November 2009
- Accepted: 29 March 2010
- Published: 29 March 2010
Pulsus paradoxus estimated by dynamic change in area under the oximeter plethysmograph waveform (PEP) might provide a measure of acute asthma severity. Our primary objective was to determine how well PEP correlates with forced expiratory volume in 1-second (%FEV1) (criterion validity) and change of %FEV1 (responsiveness) during treatment in pediatric patients with acute asthma exacerbations.
We prospectively studied subjects 5 to 17 years of age with asthma exacerbations. PEP, %FEV1, airway resistance and accessory muscle use were recorded at baseline and at 2 and 4 hours after initiation of corticosteroid and bronchodilator treatments. Statistical associations were tested with Pearson or Spearman rank correlations, logistic regression using generalized estimating equations, or Wilcoxon rank sum tests.
We studied 219 subjects (median age 9 years; male 62%; African-American 56%). Correlation of PEP with %FEV1 demonstrated criterion validity (r = - 0.44, 95% confidence interval [CI], - 0.56 to - 0.30) and responsiveness at 2 hours (r = - 0.31, 95% CI, - 0.50 to - 0.09) and 4 hours (r = - 0.38, 95% CI, - 0.62 to - 0.07). PEP also correlated with airway resistance at baseline (r = 0.28 for ages 5 to 10; r = 0.45 for ages 10 to 17), but not with change over time. PEP was associated with accessory muscle use (OR 1.16, 95% CI, 1.11 to 1.21, P < 0.0001).
PEP demonstrates criterion validity and responsiveness in correlations with %FEV1. PEP correlates with airway resistance at baseline and is associated with accessory muscle use at baseline and at 2 and 4 hours after initiation of treatment. Incorporation of this technology into contemporary pulse oximeters may provide clinicians improved parameters with which to make clinical assessments of asthma severity and response to treatment, particularly in patients who cannot perform spirometry because of young age or severity of illness. It might also allow for earlier recognition and improved management of other disorders leading to elevated pulsus paradoxus.
- Airway Resistance
- Asthma Exacerbation
- Acute Asthma
- Acute Asthma Severity
- Accessory Muscle
Clinicians have few objective measures to evaluate acute asthma severity and are likely to under-treat these episodes[1–6]. A severity measure should correlate with an accepted criterion standard (criterion validity) and quantify clinically important changes of this standard over time (responsiveness).
Spirometry is the criterion standard for assessing the severity of airway obstruction (% predicted FEV1, %FEV1) but is effort dependent and not available in most acute care settings[8, 9]. Airway resistance is another measure of lung function. Portable devices for measurement of airway resistance by the interrupter technique (Rint) are available and require only tidal breathing. Rint has been demonstrated to correlate with %FEV1 and specific airway resistance by body box plethysmography[10–13].
Accessory muscle use has been shown to be associated with a clinically meaningful decrease in %FEV1[14, 15]. However, though accessory muscle use gives an indication of work of breathing, it does not provide a precise measure of airflow limitation.
Measurement of pulsus paradoxus (PP) during acute asthma exacerbations is currently recommended by national and international guidelines [16, 17]. However, manual determination of PP is difficult, particularly in the tachypneic patient and in noisy clinical environments[18–21]. More than 98% of providers do not use this measurement at the bedside.
The pulse oximeter plethysmograph waveform closely mirrors radial artery Doppler waveforms, and previous investigations have provided evidence supporting the use of oximeter plethysmograph waveform data to estimate PP[23–28]. Developing methods to objectively quantify plethysmograph waveform data is clinically relevant as it might provide a non-invasive and continuous estimate of the severity of airway obstruction or other physiologic disturbances contributing to PP.
Our primary objective was to determine how well a mathematic model for plethysmograph estimation of pulsus paradoxus physiology (PEP) correlates with %FEV1 (criterion validity) and change of this criterion standard (responsiveness) during treatment in pediatric patients with acute asthma exacerbations. Secondary objectives were to determine the correlation of PEP with airway resistance and accessory muscle use.
Study design and study population
We enrolled a prospective convenience sample of subjects ages 5 to 17 years with doctor-diagnosed asthma, signs or symptoms of an asthma exacerbation, (cough, dyspnea, shortness of breath, wheezing and/or chest pain), and need for treatment with systemic corticosteroid (CCS) and inhaled albuterol as determined by the pediatric emergency medicine attending. The setting was an urban, academic, tertiary care children's hospital emergency department (PED). We excluded patients with pneumonia by clinical or radiographic criteria. Enrollment hours were 7 am to 10 pm weekdays and approximately every 3rd weekend day. The Vanderbilt University Institutional Review Board approved the study protocol and a waiver of immediate informed consent such that baseline variables could be obtained prior to the informed consent process.
Study protocol and measurements
All study data was collected by the principal investigator (DHA) or research assistant (DJR). The principal investigator trained the research assistant in the study protocol, and both individuals received training in portable spirometry performance by pediatric pulmonary function technicians. The investigators were not masked to study data during data acquisition.
At enrollment we recorded demographic information, medical history, family asthma history, asthma medication use, asthma symptom history, and Global Initiative for Asthma (GINA) chronic asthma control. All other variables were obtained prior to administration of CCS (baseline) and bronchodilator treatments and again at 2 and 4 hours after CCS administration if the subject remained in the PED at that time.
We applied the oximeter sensor to a finger for PEP data acquisition. The subject was asked to keep the hand still and to not talk. This was necessary because the unprocessed IR signal is subject to movement artifact. We acquired PEP data over a minimum of 5 minutes to allow the graphical output of PEP to stabilize. The PEP value at the end of this period was recorded and is expressed as a percent value.
We used %FEV1 as the criterion standard to assess the diagnostic accuracy of PEP and used airway resistance and accessory muscle use as secondary severity measures. Accessory muscle use was defined as any visible use of the scalene, sternocleidomastoid, suprasternal, intercostal or subcostal muscles.
We measured airway resistance using a MicroDirect MRT6000 module (Micro Medical, Kent, England). This measurement was made prior to spirometry because the forced vital capacity maneuvers for spirometry can temporarily alter airway tone and airway resistance measures. We applied a nose clip and instructed the subject to breathe comfortably while supporting the cheeks and submental tissue and extending the neck slightly. Five measurements during exhalation were made and the median value recorded. The device calculates % predicted values for subjects ages 5 to 10 years (%Rint) using the McKenzie standards and outputs absolute values for subjects 11 years of age and above (aRint).
We used a MicroDirect MicroLoop spirometer for %FEV1 determination. After applying a nose clip we instructed each subject to perform forced vital capacity maneuvers in accordance with American Thoracic Society (ATS) 1994 spirometry standards.9 %FEV1 was calculated based on Knudson standards[34, 35].
Some subjects could not perform 3 forced vital capacity maneuvers for each trial in accordance with ATS criteria because of the severity of acute asthma or young age. However, some of these trials included one or two maneuvers with acceptable flow-volume and volume-time curves.
A pulmonary function test oversight committee reviewed these non-ATS trials to determine if any of the data should be included in the analysis. This committee included a pulmonary physiologist and a pediatric pulmonary function lab technician (RRT). Each member recorded their determination whether a non-ATS trial should be retained for analysis based on the flow-volume and volume-time curves. Committee members were blinded to all other subject data and to the other member's determination. A non-ATS trial was retained for data analysis if both members independently determined that it should be retained.
We considered a correlation coefficient of 0.30 or greater to be clinically relevant based on the study of Wright and colleagues in which measurement of PP calculated from change in height of finger arterial pressure monitor waveforms was correlated with %PEF (r = - 0.31). A sample size of 82 subjects would enable us to detect a correlation coefficient of 0.35 or greater between PEP and %FEV1 values with 90% (β = .010) power and a two-sided significance level of 0.05 (α = 0.05). We set our sample size at a minimum of twice this calculated value in order to account for incomplete data. We anticipated that we would achieve the necessary sample size over a 12 month period and chose this enrollment period to minimize spectrum bias in seasonal asthma etiology and severity.
Statistical analysis and data management
Descriptive statistics are presented as mean (SD) or median (IQR), as appropriate. We compared our study sample to the patient population ages 5 to 17 years seen in the PED during the study period with a final diagnosis code (ICD 493) of asthma exacerbation. This population data was extracted from a database designed primarily for billing purposes. While not directly comparable to our study sample, this data would provide a sense of how representative our sample was of the overall population meeting study inclusion criteria.
Differences, proportions, and correlations are reported as point estimates, bounded by 95% confidence intervals (CI). Analyses involving airway resistance are done separately for subjects ages 5-10 (%Rint) and ages 11 to 17 (aRint) years. The internal validity of PEP versus %FEV1 and PEP versus airway resistance at baseline was assessed using the Pearson product-moment correlation coefficient. The strength of the relationship between the proportionate change in PEP and the proportionate change in %FEV1 or the proportionate change in airway resistance was assessed using the Spearman's rank correlation coefficient. The relationship between PEP and accessory muscle use (present/not present) was examined in two ways. First, a logistic regression model using generalized estimating equations to account for repeated measures on a subject was used to assess the relationship between PEP and accessory muscle use, adjusting for time and any interaction it may have with changes in PEP. Second, Wilcoxon Rank Sum tests were used at each time point to determine whether the distribution of PEP values differ between those with accessory muscle use and those without.
Subject and Study Population Demographic and Clinical Characteristics
PED Asthma Population*
(n = 1,060)
(n = 219)
Age, median (IQR)
8.9 (6.9 - 11.7)
9.0 (6.9 - 11.9)
5 (2 - 8)
Prior PICU admission
Prior endotracheal intubation for asthma
Disposition from pediatric ED
Discharge to home
Admit to floor bed
Admit to PICU
Predictor and Outcome Variable Measurements
(n = 219)
(n = 138)
(n = 58)
38 (31 - 44, 214)
35 (29 - 40, 136)
38 (31 - 43, 57)
61 (42 - 80, 148)
69 (52 - 82, 100)
63 (47 - 81, 46)
Age 5 to 10 yr‡
178 (142 - 241, 150)
143 (124 - 172, 93)
141 (124 - 161, 39)
Ages ≥ 11 yr§
0.71 (0.56 - 0.94, 67)
0.59 (0.44 - 0.75, 44)
0.68 (0.56 - 0.80, 19)
Access. M. Use¶
Associations with outcome measures
Correlations of Plethysmograph Estimate of Pulsus Paradoxus Physiology with %FEV1 and Airway Resistance
(r, 95% CI)
Baseline to 2 Hr change*
(r, 95% CI)
Baseline to 4 Hr change*
(r, 95% CI)
-0.44 (-0.56, -0.30)‡
-0.38 (-0.62, -0.07)
Age 5 - 10 yr†
0.28 (0.12, 0.42)‡
0.06 (-0.16, 0.28) ¶
0.19 (-0.13, 0.51) ¶
Ages ≥ 11 yr§
0.45 (0.23, 0.63)‡
0.21 (-0.13, 0.52) ¶
0.25 (-0.24, 0.66) ¶
A logistic regression model using generalized estimating equations was used to assess the relationship between accessory muscle use and PEP, adjusting for time. There was a statistically significant association of increased PEP and accessory muscle use (OR 1.16, 95% CI, 1.11 to 1.21, P < 0.0001). In addition, the model indicated that this association differed over time as seen by the significant interaction term (P < 0.0001). However, this result should be viewed cautiously given the small number of subjects who remained in the PED and had both 4 hour measurement of PEP and accessory muscle use at that time (n = 9). Wilcoxon Rank Sum tests were also used to assess the relationship of PEP with accessory muscle use at each time point. Test results for each time point across the groups were statistically significant at baseline (P < 0.0001) and 2 hours (P = 0.006), but not at 4 hours (P = 0.28). That this association was not statistically significant at 4 hours may be due to the small sample size at that time point (n = 57).
We found that the use of quantified pulse oximeter plethysmograph waveform data (PEP), a continuous, real-time and effort-independent measurement, correlates with %FEV1 (criterion validity, r = -0.44) as well as with proportionate changes in %FEV1 over the first 2 and 4 hours (responsiveness, r = - 0.31 and - 0.38) of acute asthma treatment in a PED. This compares favorably with the findings of Wright and colleagues who estimated PP based on change in waveform height and found correlation with %PEF (r = - 0.31). In our investigation we utilized %FEV1, the widely accepted criterion standard of acute asthma severity, in addition to secondary severity measures. We noted statistically significant correlations between PEP and airway resistance at baseline but not over time, and between PEP and accessory muscle use at baseline and over time.
There are limitations of this study. First, the three outcome measures may not fully reflect lung function, particularly in patients in significant respiratory distress. Spirometry is highly effort dependent. Although we ascertained the validity of each test by ATS criteria and review of flow-volume loops, some subjects may not have performed well due to respiratory distress or young age. Additionally, % predicted values of Rint for children ages 5 to 10 years of age are derived from a small sample (n = 236) of healthy children of four ethnicities and may not be a valid outcome measure. Second, the raw, unfiltered, unsmoothed IR light signal must be used for waveform analysis in order to fully capture AUC variability that estimates PP. Movement artifact was possible, and signal stabilization methods might be employed to minimize this artifact. Third, outliers were noted (figure 3), possibly from movement artifact or individual differences in the physiologic events influencing PEP. The effect of these outliers was to decrease correlations of PEP with the outcome measures. Fourth, we programmed the software to calculate ΔAUC by identifying the smallest and largest AUC in successive 3-second time intervals. If this interval is shorter than the respiratory cycle, calculated PEP will underestimate PP; the inverse will apply if the interval is longer than the respiratory cycle. The former appears to apply overall because the median respiratory rates were 25/min at baseline and 24/min at 2 and 4 hours. We recognize the need to incorporate into this evolving technology a method for respiratory rate detection that will then allow gating the interrogation interval more precisely with the respiratory cycle. Lastly, there was very little change in PEP, %FEV1 and Rint from 0 to 2 to 4 hours. This may have been because those subjects remaining in the PED at 2 and 4 hours were minimally improved as the reason for not being discharged and, thus, had little change in PEP as a group.
The results of this study indicate that PEP has criterion validity and responsiveness in pediatric patients with acute asthma exacerbations. Incorporation of this technology into contemporary pulse oximeters may provide clinicians improved parameters with which to make clinical assessments of asthma severity and response to treatment, particularly in patients who cannot perform spirometry because of young age or severity of illness. It might also allow for earlier recognition and improved management of other disorders leading to elevated pulsus paradoxus.
Dr. Arnold is Associate Professor of Pediatrics and Emergency Medicine, Monroe Carell Jr. Children's Hospital at Vanderbilt. Ms. Jenkins is Biostatistician III, Department of Biostatistics. Dr. Hartert is Associate Professor, Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine. All authors are at the Vanderbilt University School of Medicine, Nashville, TN, USA.
This research was supported by the National Institutes of Health [Grant K23 HL80005-01A2] (Dr. Arnold), [NCRR UL1 RR024975] (Vanderbilt CTSA/REDCap database), and NIAID [K24 AI77930] (Dr. Hartert).
- Spiteri MA, Cook DG, Clarke SW: Reliability of eliciting physical signs in examination of the chest. Lancet. 1988, 1: 873-875. 10.1016/S0140-6736(88)91613-3.View ArticlePubMedGoogle Scholar
- Holleman DR, Simel DL: Does the clinical examination predict airflow limitation?. JAMA. 1995, 273: 313-319. 10.1001/jama.273.4.313.View ArticlePubMedGoogle Scholar
- McFadden ER: Acute severe asthma. Am J Respir Crit Care Med. 2003, 168: 740-759. 10.1164/rccm.200208-902SO.View ArticlePubMedGoogle Scholar
- Wolfenden LL, Diette GB, Krishnan JA, Skinner EA, Steinwachs DM, Wu AW: Lower physician estimate of underlying asthma severity leads to undertreatment. Arch Intern Med. 2003, 163: 231-236. 10.1001/archinte.163.2.231.View ArticlePubMedGoogle Scholar
- Baxt WG: Prospective application of an asthma severity rule. Acad Emerg Med. 2002, 9: 868-869. 10.1111/j.1553-2712.2002.tb02185.x.Google Scholar
- Husted JA, Cook RJ, Farewell VT, Gladman DD: Methods for assessing responsiveness: a critical review and recommendations. J Clin Epidemiol. 2000, 53: 459-468. 10.1016/S0895-4356(99)00206-1.View ArticlePubMedGoogle Scholar
- Hulley SB, Martin JN, Cummings SR: Planning the Measurements: Precision and Accuracy. Designing Clinical Research. Edited by: Hulley ST, Cummings SR, Browner WS, Grady D, Hearst N, Newman TB. 2007, Philadelphia: Lippincott Williams & Wilkins, 37-49. 3Google Scholar
- Miller A: Lung function testing: selection of reference values and interpretative strategies. Am Rev Respir Dis. 1992, 146: 1368-1369.View ArticlePubMedGoogle Scholar
- Standardization of Spirometry, 1994 Update. American Thoracic Society. Am J Respir Crit Care Med. 1995, 152: 1107-1136.Google Scholar
- Bisgaard H, Klug B: Lung function measurement in awake young children. Eur Respir J. 1995, 8: 2067-2075. 10.1183/09031936.95.08122067.View ArticlePubMedGoogle Scholar
- Chowienczyk PJ, Lawson CP, Lane S, Johnson R, Wilson N, Silverman M, Cochrane GM: A flow interruption device for measurement of airway resistance. Eur Respir J. 1991, 4: 623-628.PubMedGoogle Scholar
- Bridge PD, Lee H, Silverman M: A portable device based on the interrupter technique to measure bronchodilator response in schoolchildren. Eur Respir J. 1996, 9: 1368-1373. 10.1183/09031936.96.09071368.View ArticlePubMedGoogle Scholar
- Oswald-Mammosser M, Charloux A, Donato L, Albrech C, Speich JP, Lampert E, Lonsdorfer J: Interrupter technique versus plethysmography for measurement of respiratory resistance in children with asthma or cystic fibrosis. Pediatr Pulmonol. 2000, 29: 213-220. 10.1002/(SICI)1099-0496(200003)29:3<213::AID-PPUL10>3.0.CO;2-N.View ArticlePubMedGoogle Scholar
- Kerem E, Canny G, Tibshirani R, Reisman J, Bentur L, Schuh S, Levison H: Clinical-physiologic correlations in acute asthma of childhood. Pediatrics. 1991, 87: 481-486.PubMedGoogle Scholar
- Arnold DH, Gebretsadik T, Minton PA, Higgins S, Hartert TV: Clinical measures associated with FEV1 in persons with asthma requiring hospital admission. Am J Emerg Med. 2007, 25: 425-429. 10.1016/j.ajem.2006.09.006.View ArticlePubMedGoogle Scholar
- Global Initiative for Asthma POMC: Global strategy for asthma management and prevention. 7 A.D. [http://www.ginasthma.com/Guidelineitem.asp??l1=2&l2=1&intId=60]
- Expert Panel Report 3: Guidelines for the Diagnosis and Management of Asthma. [http://www.nhlbi.nih.gov/guidelines/asthma/01_front.pdf]
- Jay GD, Onuma K, Davis R, Chen MH, Mansell A, Steele D: Analysis of physician ability in the measurement of pulsus paradoxus by sphygmomanometry. Chest. 2000, 118: 348-352. 10.1378/chest.118.2.348.View ArticlePubMedGoogle Scholar
- Galant SP, Groncy CE, Shaw KC: The value of pulsus paradoxus in assessing the child with status asthmaticus. Pediatrics. 1978, 61: 46-51.PubMedGoogle Scholar
- Shim C, Williams MH: Pulsus paradoxus in asthma. Lancet. 1978, 1: 530-531. 10.1016/S0140-6736(78)90553-6.View ArticlePubMedGoogle Scholar
- Pearson MG, Spence DP, Ryland I, Harrison BD: Value of pulsus paradoxus in assessing acute severe asthma. British Thoracic Society Standards of Care Committee. BMJ. 1993, 307: 659-10.1136/bmj.307.6905.659.View ArticlePubMedPubMed CentralGoogle Scholar
- Canny GJ, Reisman J, Healy R, Schwartz C, Petrou C, Rebuck AS, Levison H: Acute asthma: observations regarding the management of a pediatric emergency room. Pediatrics. 1989, 83: 507-512.PubMedGoogle Scholar
- Wisely NA, Cook LB: Arterial flow waveforms from pulse oximetry compared with measured Doppler flow waveforms apparatus. Anaesthesia. 2001, 56: 556-561. 10.1046/j.1365-2044.2001.01987.x.View ArticlePubMedGoogle Scholar
- Rayner J, Trespalacios F, Machan J, Potluri V, Brown G, Quattrucci LM, Jay GD: Continuous noninvasive measurement of pulsus paradoxus complements medical decision making in assessment of acute asthma severity. Chest. 2006, 130: 754-765. 10.1378/chest.130.3.754.View ArticlePubMedGoogle Scholar
- Arnold DH, Spiro DM, Desmond RA, Hagood JS: Estimation of airway obstruction using oximeter plethysmograph waveform data. Respir Res. 2005, 6 (65): 65-10.1186/1465-9921-6-65.View ArticlePubMedPubMed CentralGoogle Scholar
- Hartert TV, Wheeler AP, Sheller JR: Use of pulse oximetry to recognize severity of airflow obstruction in obstructive airway disease: correlation with pulsus paradoxus. Chest. 1999, 115: 475-481. 10.1378/chest.115.2.475.View ArticlePubMedGoogle Scholar
- Tamburro RF, Ring JC, Womback K: Detection of pulsus paradoxus associated with large pericardial effusions in pediatric patients by analysis of the pulse-oximetry waveform. Pediatrics. 2002, 109: 673-677. 10.1542/peds.109.4.673.View ArticlePubMedGoogle Scholar
- Frey B, Butt W: Pulse oximetry for assessment of pulsus paradoxus: a clinical study in children. Intensive Care Med. 1998, 24: 242-246. 10.1007/s001340050557.View ArticlePubMedGoogle Scholar
- Sanders DL, Gregg W, Aronsky D: Identifying asthma exacerbations in a pediatric emergency department: A feasibility study. Int J Med Inform. 2006, 76 (7): 557-64. 10.1016/j.ijmedinf.2006.03.003.View ArticlePubMedGoogle Scholar
- Arnold DH: Method and apparatus for measuring pulsus paradoxus. 10/228,641(6,869,402 B2). 3-22-2005Google Scholar
- Black j, Baxter-Jones ADG, Gordon J, Findlay AL, Helms PJ: Assessment of airway function in young children with asthma: comparison of spirometry, interrupter technique, and tidal flow by inductance plethsmography. Pediatr Pulmonol. 2004, 37: 548-553. 10.1002/ppul.20046.View ArticlePubMedGoogle Scholar
- Hadjikoumi I, Hassan A, Milner AD: Effects of respiratory timing and cheek support on resistance measurements, before and after bronchodilation in asthmatic children using the interrupter technique. Pediatr Pulmonol. 2003, 36: 495-501. 10.1002/ppul.10384.View ArticlePubMedGoogle Scholar
- McKenzie SA, Chan E, Dundas I, Bridge PD, Pao CS, Mylonopoulou M, Healy MJ: Airway resistance measured by the interrupter technique: normative data for 2-10 year olds of three ethnicities. Arch Dis Child. 2002, 87: 248-251. 10.1136/adc.87.3.248.View ArticlePubMedPubMed CentralGoogle Scholar
- Knudson RJ, Slatin RC, Lebowitz MD, Burrows B: The maximal expiratory flow-volume curve. Normal standards, variability, and effects of age. Am Rev Respir Dis. 1976, 113: 587-600.PubMedGoogle Scholar
- Sherrill DL, Lebowitz MD, Knudson RJ, Burrows B: Methodology for generating continuous prediction equations for pulmonary function measures. Comput Biomed Res. 1991, 24: 249-260. 10.1016/0010-4809(91)90047-Z.View ArticlePubMedGoogle Scholar
- Wright RO, Steele DW, Santucci KA, Natarajan R, Jay GD: Continuous, noninvasive measurement of pulsus paradoxus in patients with acute asthma. Arch Pediatr Adolesc Med. 1996, 150: 914-918.View ArticlePubMedGoogle Scholar
- R: A language and environment for statistical computing. [http://www.R-project.org]
- Harris PA, Taylor R, Thielke R, Payne J, Gonzalez N, Conde JG: Research electronic data capture (REDCap)--A metadata-driven methodology and workflow process for providing translational research informatics support. Journal of Biomedical Informatics. 2009, 42: 377-381. 10.1016/j.jbi.2008.08.010.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2466/10/17/prepub
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