DOI: https://doi.org/10.21203/rs.3.rs-99501/v1
Background: Impulse oscillometry (IOS) is a non-invasive pulmonary function test for measuring respiratory impedance. Available reference equations of IOS indices for adults are limited. The aim of this study was to develop reference equations of IOS indices for Chinese adults.
Methods: In a multicentral, cross-sessional study of impulse oscillometry in Chinese adults, IOS data from healthy subjects were collected from 19 general hospitals across China was between 2016 and 2018. IOS measurements were conducted in accordance with recommendations of the European Respiratory Society. Multiple linear regression was performed to develop sex-specific reference equations of IOS indices.
Results: IOS measurements were performed in 1318 subjects, of which 567 subjects were defined as healthy normal individuals with acceptable IOS data and were included in the final analysis. Reference equations and limits of normal (LLN/ULN) of IOS indices were developed separately for males and females. Height but not age was shown to be the most influential contributor to IOS indices. The reference equations currently used in lung function laboratories predicted higher R5 and X5. Normal ranges of R5 and X5 recommended by the equipment manufacturer were clearly different from the ULN/LLN derived from the reference equations.
Conclusions: Reference equations of IOS indices for Chinese adults from a wide region were provided in this study. It is necessary to update new IOS reference equations and adopt ULN/LLN as normal ranges of IOS indices.
Clinical Trial Registration: This study was registered at www.clinicaltrials.gov as part of a larger study NCT03467880.
Impulse oscillometry is a non-invasive method of measuring respiratory impedance based on the forced oscillation technique (FOT) [1]. IOS is regarded as a complementary tool to conventional lung function tests due to its minimal demand for cooperation and sensitivity in the evaluation of small airway function [2]. Reports of IOS as a useful tool in the assessment of asthma [3–6], chronic pulmonary obstructive diseases [7] and bronchiectasis [8] have increased its application in research and clinical settings.
In accordance with other lung function tests, choosing optimal reference values is crucial for the interpretation of IOS. The first and also the most widely used reference equation of respiratory impedance with IOS is the one proposed by Vogel et al. in 1994 [9]. The sample population of Vogel’s equations was from a German industrial city that suffered from air pollution and included smokers, which obviously did not meet the American Thoracic Society (ATS) recommended criteria for the data source of lung function reference values [10]. Furthermore, the study only used age as predictor of the respiratory impedance, where later studies [11–17] have demonstrated that respiratory impedance is more associated with height and weight rather than age. This may have an impact on the predictive value and normal ranges of IOS indices, thus decreasing the capacity for IOS to identify respiratory abnormality. Although some studies had focused on developing new reference equations of respiratory impedance by FOT or IOS, the available reference equations of IOS indices for adults are limited [11–16, 18–21]. The lack of appropriate reference equations and normal ranges of IOS have hindered the application of IOS in clinical practice.
The aim of the present study was to develop reference equations of IOS indices that: 1) are based on data from healthy Chinese adults collected a wide region and under standardized quality control; 2) provide normal limits of IOS indices with up-to-date criteria for clinical use.
The study of impulse oscillometry in Chinese was a multicenter, cross-sectional and observational, and collected IOS and spirometry reports from healthy subjects and patients with respiratory diseases in 20 general hospitals from 15 provincial regions throughout China (details of these hospitals are shown in Additional File 2). IOS data from healthy subjects were used to develop reference equations of IOS in the present study.
Subjects were recruited mainly from individuals who have regular check-ups in hospitals, volunteer students at colleges and the relatives of hospital patients. Self-reported questionnaire was used to collect medical history, history of smoking and occupational exposures, respiratory symptoms, and results of chest radiography within the last 6 months. Subjects who met the following criteria were included as healthy subjects: no history of smoking or smoked < 100 cigarettes in their lifetime; no occupational exposures; no respiratory tract infections in the last 4 weeks; no chronic or recurrent respiratory symptoms including cough, expectoration, wheezing, or shortness of breath; reported no severe cardiopulmonary diseases or systematic diseases. Those who had abnormalities on chest radiography or spirometry were excluded.
A Masterscreen IOS device (CareFusion, Hoechberg, Germany) was used for IOS measurements in this study. IOS measurements were conducted following the official technical recommendations for FOT from the European Respiratory Society (ERS) [22]. Verification of impedance was performed daily and a criterion of error ≤ 10% or 0.1 kPa·s·L− 1 was adopted. Measurements were performed in the sitting position with head in a neutral or slightly extended position, and the nose was closed by a nose clip. The cheeks were firmly supported by hands to reduce the effects of upper airway shunt. The subject was instructed to breath quietly at functional residual capacity level for 45–60 s. Measurements were repeated until 3 acceptable measurements were achieved, and the average of the 3 measurements was used for analysis. An acceptable measurement had a data acquisition of at least 30 s and included 5 normal breaths without obvious artifacts like spikes in Z-time tracing or drifts in the volume-time tracing. The coherence of each measurement was ≥ 0.8 at 5 Hz and ≥ 0.9 at 20 Hz. Unacceptable data were excluded from the analysis.
Based on the fundamentals of FOT, IOS measures respiratory impedance (Z) by superimposing pressure wave on the normal breathing [1]. Z includes resistance (R) and reactance (X). R represents the resistive properties of respiratory system, and X represents the capacitive and inert properties of respiratory system. IOS indices analyzed in this study included R and X at different frequencies (5–35 Hz), the difference between and R5 and R20 (R5–R20), resonant frequency (fres), and low-frequency reactance area (AX). The key indices for the normal limits analysis were R5 and X5, which are respectively recognized as total resistance and capacitance of respiratory system.
Spirometry (CareFusion, Hoechberg, Germany) was performed immediately after the IOS measurements as per the ATS/ERS guidelines for spirometry [23]. If spirometry was performed before the IOS, at least 3 min of rest was allowed for rest [22]. Three acceptable measurements that met the quality control criteria of the ATS/ERS guidelines [24] were acquired and the best one was used in the analysis. Spirometric indices analyzed in this study were FEV1 (forced expiratory volume in the first second), FVC (forced vital capacity), FEV1/FVC and MMEF (maximal mid-expiratory flow). Reference values of spirometry were derived from the study of reference values for spirometry in Chinese aged 4–80 years [25].
As IOS indices exhibited skewness distribution, the results were presented as median with interquartile range. Mann-Whitney U tests were performed for the comparison of continuous indices. A P-values < 0.05 was considered statistically significant.
Reference equations were calculated separately for males and females using multiple linear regression analysis. Scatter plots (see Additional File 1) were drawn to observe the linear relationship between IOS indices and predictor variables. Normal P-P plots and residual plots were drawn to examine the normality and equal variance of the residuals. As the residuals of fres and AX only displayed normality and equal variance after fres and AX were log transformed, thus fres and AX were calculated as log10 transformation (lgfres and lgAX) in the equations. Predictor variables (height, weight and age) were selected using the stepwise method, in which predictors would enter the model if P < 0.05 and were removed if P > 0.10. Fitness of the model was assessed by the coefficient of determination (R2). Normal limits of IOS indices were calculated as followed: ULN (upper limit of normal) of R = predictive value + 1.645 × RSD (residual standard deviation), LLN (lower limit of normal) of X = predictive values–1.645 × RSD.
In this study, a total number of 1318 of subjects were recruited and finished the IOS measurements between 2016 and 2018 in 19 hospitals across China, 567 subjects from 13 hospitals were included in the final analysis (see Fig. 1 and a-Table 1 in Additional File 3). The baseline characteristics are presented in Table 1 and Fig. 2. Height range is 154–186 cm in males and 142–176 cm in females. The spirometric indices FEV1, FVC, FEV1/FVC and MMEF from the analyzed population were all within normal limits.
| Male | Female | P-value | |
|---|---|---|---|
| (N = 270) | (N = 297) | ||
| Age (years) | 36.0 (23.8) | 34.7 (23.0) | 0.065 |
| Height (cm) | 170.0 (9.0) | 159.0 (7.0) | < 0.001 |
| Weight (kg) | 69.0 (15.1) | 56.0 (12.0) | < 0.001 |
| BMI (kg/m2) | 23.7 (4.4) | 21.9 (4.4) | < 0.001 |
| FEV1 (L) | 3.88 (0.87) | 2.86 (0.55) | < 0.001 |
| FEV1 z score | 0.28 (1.24) | 0.16 (1.16) | 0.213 |
| FVC (L) | 4.66 (0.99) | 3.34 (0.62) | < 0.001 |
| FVC z score | 0.23 (1.31) | 0.17 (1.27) | 0.211 |
| FEV1/FVC | 0.83 (0.07) | 0.84 (0.07) | 0.002 |
| FEV1/FVC z score | -0.05 (1.10) | -0.05 (1.17) | 0.839 |
| MMEF (L/s) | 3.86 (1.51) | 2.95 (1.21) | < 0.001 |
| MMEF z score | 0.12 (1.23) | 0.05 (1.26) | 0.461 |
| Data are presented as median (interquartile range). BMI: body mass index, FEV1: forced expiratory volume in the first second, FVC: forced vital capacity, MMEF: maximum mid-expiratory flow. | |||
For the whole population, the median (interquartile range) of R5 was 0.29 (0.09) kPa·s·L− 1, R20 was 0.27 (0.08) kPa·s·L− 1, and X5 was − 0.10 (0.04) kPa·s·L− 1. Resistance at all frequencies in females were significantly higher than in males, while reactance at all frequencies (except for X15) were more negative in females than in males (details are shown in a-Table 2 in Additional File 3).
Results of the reference equations for main IOS indices are shown in Table 2 (complete results of other IOS indices are shown in a-Table 3 and a-Table 4 in Additional File 3). Except for R35 in males, all IOS indices in this study derived significant reference equations with height and/or weight as predictors. R2 of the equations ranged from 0.0154 to 0.250 and tended to be smaller in equations of the impedance at higher frequencies. Shunt effect of the upper airway may account for the less fitness of models of impedance at high frequencies.
| Equations | RSD | R2 | ||
|---|---|---|---|---|
| Z5 | M | 0.6811–0.0032 × H + 0.0019 × W | 0.0415 | 0.1877 |
| F | 0.9110–0.0042 × H + 0.0023 × W – 0.0008 × A | 0.0493 | 0.1673 | |
| R5 | M | 0.6275–0.0030 × H + 0.0019 × W | 0.042 | 0.1789 |
| F | 0.8103–0.0038 × H + 0.0024 × W–0.0005 × A | 0.0491 | 0.1606 | |
| R20 | M | 0.5038–0.0019 × H + 0.0010 × W – 0.0004 × A | 0.0401 | 0.0668 |
| F | 0.5042–0.0013 × H | 0.0442 | 0.0282 | |
| R5–R20 | M | 0.2485–0.0018 × H + 0.0010 × W | 0.0205 | 0.2249 |
| F | 0.2360–0.0019 × H + 0.0017 × W | 0.0306 | 0.1865 | |
| X5 | M | –0.3100 + 0.0013 × H + 0.0002 × A | 0.0195 | 0.1105 |
| F | –0.3605 + 0.0015 × H + 0.0004 × A | 0.0236 | 0.1233 | |
| lgfres | M | 1.9238–0.0068 × H + 0.0033 × W | 0.0801 | 0.1963 |
| F | 1.8261–0.0067 × H + 0.0051 × W | 0.0805 | 0.2505 | |
| lgAX | M | 1.3268–0.0142 × H + 0.0043 × W | 0.2124 | 0.1179 |
| F | 1.6639–0.0166 × H + 0.0089 × W – 0.0029 × A | 0.2037 | 0.154 | |
Z5: Total respiratory impedance at 5 Hz; R5: resistance at 5 Hz; R20: resistance at 20 Hz; R5–R20: R5 minus R20; X5: reactance at 5 Hz; AX: low-frequency reactance area; fres: resonant frequency. R2: coefficient of determination; RSD: residual standard deviation.
In the equations of most IOS indices, height was shown to be the most influential predictor as it contributed the largest R2 changes in the prediction models. Height was negatively associated with R, fres and AX, and positively associated with X. On the contrary, weight showed a positive association with R, fres and AX, and negative association with X. Age was shown to be predictor to some indices including X5, X25, and X35 in both genders and Z5, R5, and lgAX in females (see a-Table 3 and a-Table 4 in Additional File 3). However, the scatter plots (Additional File 1) between these indices and age did not display notable linear relationships.
Sex-specific reference equations for respiratory impedance were developed based on large-scale data from healthy Chinese adults from a wide region in a multicenter IOS study.
As pulmonary function is associated with physiological changes during growth and aging, reference equations of pulmonary function commonly include anthropometric variables such as sex, age, height and weight, in order to justify the contributory effects of these factors to lung function. Our study found that height was the most influential contributor to respiratory impedance measured with IOS, with that taller individuals had higher R and less negative X. This finding is consistent with the previous studies [11–17, 19, 21, 26]. As shown in Table 3, though the coefficients or forms of the predictors in the IOS equations were different, the tendency of the effect of predictors in most equations remained the same; that is, height was negatively correlated with R5 and R20, and positively correlated with X5. The association between height and respiratory resistance can be explained by the effect that height contributes to the diameter of the airways and lung volume. This could also explain the discrepancies of respiratory impedance between males and females. In contrast, weight displayed a positive association with R. The decreased lung volume and ventilation heterogeneity reported in the obese subjects [27, 28] may account for the higher respiratory resistance in the obese. However, the mechanism behind how body weight affects respiratory impedance in subjects with normal weight is still not clear since most studies have focused on overweight or obese individuals. Although age did not appear to be a marked contributor to R in adults in our study, a negative dependence on age for R has been reported in studies of children and adolescences [29, 30]. Thus, the effect of age on R may be related to the rapid physiological changes during growth, especially on the growth of the respiratory system in children and adolescences.
| Equations | Area | N | F/M | Age range (years) | Ethnicity | Predictors | ||||
|---|---|---|---|---|---|---|---|---|---|---|
| R5 | R20 | X5 | fres | |||||||
| Present study | China | 567 | 1.10 | 18–82 | 99.3% Han | M | -H, W | -H, W | H, A | -H, W |
| F | -H, W, -A | -H, | H, A | -H, W | ||||||
| Vogel et al. [9], 1994 | Germany | 506 | 0.70 | 18–69 | NA | M | A | A | -A | NA |
| F | A | A | -A | NA | ||||||
| Zhao et al. [11], 2002 | Xinjiang, China | 457 | 0.80 | 16–81 | Han, Uygur | M | W, -H | W, -H, -A | H | W, -H |
| F | W, -H | -H, -A | H, W | W, -H | ||||||
| Fan et al. [12], 2005 | Kunming, China | 185 | 0.73 | 19–68 | NA | M | -W *H | -W *H | NA | -eH |
| F | -eW | -eW | NA | -LogH, LogW | ||||||
| Satomi et al. [13], 2005 | Japan | 166 | 1.40 | 20–83 | NA | -LogH | -LogH | LogH, -A | ||
| Ni et al. [14], 2006 | Nantong, China | 120 | 0.69 | 20–79 | Han | M | -H, W, A | -H, W, A | H, W, A | H, W, A |
| F | H, W, A | H, W, -A | H, W, A | H, W, A | ||||||
| Newbury et al. [15], 2008 | Australia | 125 | 1.12 | 25–74 | Caucasian | M | -H, W, -A | -H, W, -A | H, A | NA |
| F | -H, A | -H, A | H, -A | NA | ||||||
| Wang et al. [16], 2011 | Shenyang, China | 100 | 0.69 | 19–80 | NA | M | -lgH, -lgA | -H2, -eA | -A2 | A2 |
| F | -lgH | -H2 | H2, -eA | -lgH, A*W | ||||||
| Li et al. [26], 2012 | Lanzhou, China | 920 | 1.04 | > 18 | NA | M | -lgH, W | -H, W | -A2 | -H, W |
| F | -H, W | W | -A2 | -H, W | ||||||
| Schulz et al. [18], 2013 | Germany | 397 | 1.58 | 45–85 | Caucasian | M | -H, W, -A | -H, W, -A | H, -W, -A | -H, W, A |
| F | -H, W, -A | -H, W, -A | H, -W, -A | -H, W, A | ||||||
| Zhang et al. [19], 2015 | Macau, China | 362 | 1.02 | 18–78 | Han | M | W, -H | -H | H | A, -H |
| F | CW, -W, A | -H | H, A | A, CW, -W | ||||||
| Shu et al. [20], 2016 | Jianghan Plain, China | 431 | 1.03 | 18–79 | NA | M | -AH, A, -A2, eW, -eA | -AH, A, -A2, -lgA | A*H, -A | -H2, W2 |
| F | -H2, W, -eW | W, -H2 | H2 | W, -H2, -eW | ||||||
| N: the number of the study sample; F/M: ratio of female subjects to male subjects; R5: resistance at 5 Hz; R20: resistance at 20 Hz; X5: reactance at 5 Hz; fres: resonant frequency; NA: information was not available in the published paper; H, W, A respectively indicate height, weight and age as predictors of the equations, “-” indicates a negative effect of the predictor. M: equations for males; F: equations for female. | ||||||||||
Table 3 (See the end of the document text file)
Reference equations of respiratory impedance with IOS
Figure 3 displays the comparisons of reference values of R5 and X5 produced by different IOS reference equations. Since age is shown to have little impact on respiratory impedance in adults, it is no surprise that marked differences of predictive values were found between the Vogel’s equations and the equations from other three representative studies (Newbury et al. [15], Schulz et al. [18] and the present study), as age is the only predictor in Vogel’s equations [9]. Given that many lung function laboratories are still using Vogel’s equations, it is important for the physicians to note that Vogel’s equations predict higher R5 and X5 than other equations, especially for X5, and the differences are greater in tall subjects for R5 and in short subjects for X5. Undoubtedly, developing a more appropriate equation is imperative.
As summarized in Table 3, since Vogel’s equations were developed, 10 studies have developed new equations of IOS indices in adults, and 7 of these were from China. However, most of these studies have limitations such as lack of quality control of IOS data or small sample sizes (Fan et al. [12], Satomi et al. [13], Newbury et al. [15], Ni et al. [14], Wang et al. [16]). Among these studies, only 6 studies had mentioned the number of IOS measurements for each subject, only 4 studies (Wang et al. [16], Schulz et al. [18], Zhang et al. [19], Shu et al. [20]) had mentioned the requirements of repeatability, and 3 studies (Satomi et al. [13], Newbury et al. [15], Schulz et al. [18]) had mentioned the acceptable criteria for the IOS measurements. As the variations of IOS indices are greater than spirometric indices [31], multiple measurements and strict quality control are particularly important in IOS measurements to ensure the repeatability and reliability of the data. Regarding sample size, studies from Satomi et al. [13] and Newbury et al. [15] were based on small sample sizes, with 166 and 125 subjects, respectively. This may decrease the reliability and applicability of their equations as a study have shown that at least 150 males and 150 females are required to validate reference equations of lung function tests in individual laboratories [32]. Also, Satomi’s equation did not take sex into account, whereas sex-related differences in IOS indices have been reported in the present and former studies [21]. Schulz’s study [18] was based on data from a relatively large sample size and clear quality control criteria. Similar values of R5 and X5 produced by Schulz’s study and the present study in Fig. 3 provide evidence of the reliability of our reference equations.
Although 7 studies from China have developed reference equations of IOS indices [11, 12, 14, 16, 19, 20, 26] (Table 3), all of these studies were based on local sample populations, which may be less representative of the whole population of China, as China is a country with large territory and population. Heterogeneity in the inclusion criteria of participants and quality control also hinder the integration of these databases. Our study was a multicenter study that included data from a wide region across China, with uniform inclusion criteria and standardized quality control. Therefore, this study is more representative of the general population and produced more reliable data.
Despites the fact that ERS had published official recommendations for the application of FOT in the clinical practice [22],[33], there are no acknowledged criteria for the normal ranges of respiratory impedance with FOT or IOS, probably due to the lack of systematic studies concerning on normative values of respiratory impedance. The IOS equipment manufacturer recommends using 150% of the predicted value as the normal limit for R5 and R20, and predicted value minus 0.2 kPa·s·L− 1 as the normal limit of X5. The former was derived from reports from a bronchial challenge showing that a 20% decrease in FEV1 was comparable to a 50% increase in airway resistance [1]. However, ATS guidelines for pulmonary function tests reported in 2017 have recommended using LLN/ULN as the criteria of abnormal of pulmonary function [10]. As is shown in Fig. 4, predicted X5 minus 0.2 kPa·s·L− 1 (X5P–0.2 kPa·s·L− 1) was significantly more negative than the LLN of X5, regardless of the equation used. The 150% of predicted R5 (150%R5P) was much higher than the ULN of R5 produced by our equations, and marked differences were also shown in the comparison of ULN of R5 and 150%R5P produced by Newbury’ equations in males and Schulz’s equations in females. The above differences between the ULN/LLN and the normal limits currently used in laboratories will apparently increase the risk of misdiagnosis. Under the increasing application of IOS in clinical practice, it is necessary to update new equations and normal ranges of IOS. The validation of our new equations and normal ranges of IOS in patients with respiratory diseases will be further analyzed and discussed in our later reports.
There were limitations to the present study. First, due to the practical limitations, our study population was not a random sample and may be less representative of the whole healthy population. Nevertheless, multicenter sources of data and strict inclusive criteria for healthy subjects in this study provide a guarantee of the representativeness of a healthy population. To date, our equations are the most representative and reliable for healthy Chinese adults. Second, our equations are based on the data of Chinese population, its use in other populations or ethnicities may be limited. However, we believe that these data may be a foundation or promotion to the development of multiethnic reference equations of IOS in the future, and our findings about the inappropriateness of the current normal ranges of IOS indices may provide evidences for the update of the internationally technical standards. Third, as the IOS data from children and adolescents in multicenter study of impulse oscillometry in Chinese were not enough to develop reference equations, we failed to develop continuous reference equations with a full age range. Studies containing a larger number of healthy children and adolescents with a randomized sample are needed in the future.
In summary, based on the data of a large-scale healthy population in the multicenter IOS study in China, we developed new reliable reference equations of respiratory impedance with IOS. Also, we found that the normal ranges of IOS indices wildly used in laboratories were clearly different from the ULN/LLN derived from the reference equations. It is necessary to update new IOS reference equations and adopt ULN/LLN as normal ranges of IOS indices for better use of IOS in clinical practice.
| Abbreviation | Full definition |
|---|---|
| ATS | American Thoracic Society |
| AX | Low-frequency Reactance Area. |
| BMI | Body Mass Index |
| ERS | European Respiratory Society |
| FEV1 | Forced Expiratory Volume in the First Second |
| FOT | Forced Oscillation Technique |
| fres | Resonant Frequency |
| FVC | Forced Vital Capacity |
| IOS | Impulse Oscillometry |
| LLN | Lower Limit of Normal |
| MMEF | Maximum Mid-expiratory Flow |
| Pred | Predictive value |
| R5 | Resistance at 5 Hz |
| R10 | Resistance at 10 Hz |
| R15 | Resistance at 15 Hz |
| R20 | Resistance at 20 Hz |
| R25 | Resistance at 25 Hz |
| R35 | Resistance at 35 Hz |
| R | Resistance |
| R2 | Coefficient of Determination |
| RSD | Residual Standard Deviation |
| ULN | Upper Limit of Normal |
| X5 | Reactance at 5 Hz |
| X10 | Reactance at 10 Hz |
| X15 | Reactance at 15 Hz |
| X20 | Reactance at 20 Hz |
| X25 | Reactance at 25 Hz |
| X35 | Reactance at 35 Hz |
| X | Reactance |
| Z | Impedance |
| 95%CI | 95% Confidence Interval |
Ethics approval and consent to participate
This study was approved by the medical ethics committee of the First Affiliated Hospital of Guangzhou Medical University - approval: MEC-2015-37.
Consent for publication
All subjects in this study gave their written informed consent.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Competing interests
The authors declare that they have no competing interests
Funding
This study is funded by the National Key Research and Develop Program of China (No. 2018YFC1311900), Science and Technology Planning Project of Guangdong Province (NO. 2014A020212358) and the National Science and Technology Support Plan (NO. 2015BAI12B10).
Authors' contributions：
YG and J-PZ conceive and design the study. XLL, JD, LD, WH, JML, YL, YP, BRZ and TW collected the data. XLL analyzed the data and draft manuscript drafting. YG, WJG, JPZ, JD, LD, WH, JML, YL, YP, BRZ and TW critically review and revision of the manuscript. All authors have read and approved the final manuscript.
Acknowledgements
The authors wish to thank to the following people and centers for offering data to this manuscript: Tie-Ying Sun and Yi-Meng Yang (Beijing Hospital); Li Xiang (Beijing Children’s Hospital, Capital Medical University); Cheng Zhang (Guizhou Provincial People's Hospital); Gui-Hua Zhao (Henan Provincial People's Hospital); Ming-Juan Zhou (Guangdong Provincial Hospital of Traditional Chinese Medicine); Chun-Yan Zheng and De-Xiang Wang (Qilu Hospital of Shandong University); Ning Zhou (Tianjin Medical University General Hospital); Xu-Hua Zhang (General Hospital of Ningxia Medical University); Xiao-Ju Liu and Hai-Rong Bao (The First Hospital of Lanzhou University); Xin Chen (Zhujiang Hospital of Southern Medical University).