In this survey, we adopted a multi-stage, stratified, and random cluster sampling method to ensure that the survey was economic and effective, that the precision of results was over 95%, that samples were representative and that sampling was feasible. The participants aged from 7 to 79, varying from workers, farmers, officials, teachers, students to others. According to the general division method of the China Population Information Research Center, and the WHO age grouping standard, the participants of the survey were divided into four age groups: children and teenagers (aged 7~18), young adults (aged 19~44), middle-aged adults (aged 45~59) and the elderly (aged over 60). By means of random sampling, we carried out the investigations in 10 cities and rural areas in Hebei, Zhejiang and Guangxi province. We also investigated teenagers from Beijing.

1.1 Samples and Sampling Method

1.1.1 Sampling principles: We must adopt a multi-stage, stratified, and random cluster sampling method to ensure that the survey is economic and effective, that the precision of results is over 95%, that samples nationwide are representative, and that sampling is feasible and reasonable.

1.1.2 Sample Size

The minimum sample size determination and calculation method is shown as follows:

The permissible error is:

 

1.2 Sampling Methods

 

1.2.1 Distribution of sample size: the minimum sample size required for the common physical examination (such as physique) was 3,200 persons for every survey site.

1.2.2 Minimum sample size for laboratory examination: There are some common differences in the physiological constants of human bodies at different ages (divided into: teenagers, the young, the middle-aged and the elderly). To ensure that the laboratory testing indexes could be used as normal reference values, 20% of surveyed participants in each group were select to have the laboratory examination.

1.2.3 Minimum sample size for the respiratory function testing:  respiratory function are closely associated with height and weight. It is not completely proportional to age. Therefore, the participants were divided into groups of school age (aged 7-18) and an adult group (aged over 18). We randomly sampled 50% of those who also participated in laboratory tests .

 

1.3 Quality Inspection and Control

It is vital to control non-sampling errors in the process of each survey, especially at survey sites. During this survey, all completed questionnaires and health forms were  checked by the research group. It had been shown that 98% of these forms were qualified .  And only qualified forms were input into the database.

1.3.1 On-Site Quality Control

We made the same survey questionnaire for different survey sites and then numbered the different survey forms together. Investigators carefully conducted tests and wrote down related information. Professional staff was responsible for verifying the data on site. 

1)Anthropometric measurement and blood pressure determination were conducted by staffs who were specially trained. And each staff’s duty was fixed in each survey site.

2)Instruments used for physique measurement and blood pressure determination were calibrated on site every day to ensure these instruments worked at the same condition;

3)We sampled the completed survey forms everyday to identify whether they are qualified. So we could deal with existing problems timely;

4)The cooperation organizations had a clear division of work, and established a mutual supervision mechanism, which guaranteed the success of this project.

1.3.2Data Input Quality Control

To make sure the input data is accurate, two different persons simultaneously input data into computer. Then special software was used to check the similarity, logicality boundary values of the two set of data. If any logic errors exist, the input data were corrected according to the original survey forms.

1.4 Basic Information Questionnaire

First, with the help of professional investigators, surveyed participants should fill out the questionnaires The participants must meet the following requirements:

1)The investigators should receive strict training. First, they should read the contents of survey forms carefully. Then they were taught the meaning of survey forms and the needed inquiry methods. New staff should be trained before the job.

2)The investigators should ask participants all designed questions in the survey forms . They should also provide proper explanations if someone fails to understand any question due to his/her educational level or language barriers.

3)The survey items are inner-connected, and data quality is important to conclusions of the whole analysis. So we must take every item seriously to obtain needed data.

1.4.1 Entry Items

1)Surveyed participants should write down their names and some basic information (province and city, urban or rural area, gender, nationality, birth date, age and occupation etc.), which serves as grouped options . Then the investigators verify the information. Or the participants ask the investigators to fill in forms.

2)Information, like province and city, urban or rural , and survey numbers, should be uniformly filled out by the investigators.

Urban or Rural: depends on the birthplace. Here, “urban” refers to those with urban resident registration; “rural” refers to those with rural resident registration.

Birth Date: this should be filled out according to the Gregorian calendar (solar calendar). The lunar-calendar date should only be used if someone don’t know the solar-calendar date .

Age: use the chronological age.

For those who have celebrated their birthday that year: Age = the year of survey – the year of birth

For those who haven’t celebrated their birthday that year: Age = (the year of survey – the year of birth) – 1

Past medical history (PMH): any disease diagnosed and confirmed by doctors

1.4.2 Physical Examination Items

The physical examination should be made by physicians who have some clinical experience. In every survey site, there should be more than one physician in-charge to instruct the operation .

Instruments: diagnostic couch, stethoscope, sphygmomanometer and electrocardiograph etc.

Examination method: Physicians should ask participants information about PMH with some methods and make a physique examination with physical methods.

The main purpose of the internal medicine examination is to perform a health screening to choose normal persons for the physical examination. According to the PMH and physical examination information, someone should not be permitted to take the physical tests under any of the following circumstances .

1)People with heart, lung, liver or kidney disorders

2)People with abnormal physical growths

3)People who take calcium supplements or have diabetes

4)People with any acute diseases or those who have had a fever or cold within the past 15 days

 

1.5 General Health Inspection on Growth and Development

1.5.1Height

The testing group designates two trained persons to perform the test. One person measures the height and the other writes down the result .

Instrument: stadiometer . This instrument should be calibrated to the zero point before use. A steel ruler should be used to check whether the height of the red line on the sliding horizontal headpiece is 10 cm. Staff should also check whether the ruler is vertical, whether the joint is tight and the parts are in good condition. If one of these problems happens, staff should deal with them timely.

Testing method: Participant stand on the basic board of the stadiometer without shoes. The upper arms should naturally hang down. The two heels should be close together with the tiptoes apart, and the sacrum and two shoulders should be placed close to the column. The body should be naturally straight with head upright so that the top tragus and the lower orbit eyelid margin could be in the same horizon. The investigator should stand on the right side of participants and adjust the horizontal headpiece carefully to let it rest on the top of the head. A staff should read the number with two eyelids and the headpiece at the same horizon. He /she repeats the numbers and then record them. The values should be in cm and accurate to one decimal place. The test error should not exceed 0.5cm.

Considerations:

1)The manufacturers of stadiometers must have a certification issued by State Metrology Bureau. And the stadiometers should also have a quality certification provied by the manufacturers.

2)The stadiometers should be placed against a wall on a flat surface. The ruler should face towards a light source.

3)Follow the measuring gesture requirements mentioned above

4)Staff should make their eyes in the same horizon with the headpiece when reading. And staff should make some adjustment if it’s necessary.

5)The headpiece should be placed appropriately on the top of the head of the participant.

6)In order to avoid damage, the staff should gently move the headpiece to a safe height immediately after reading.

1.5.2Weight

The testing group will designate special persons (trained) to make the measurements.

Instrument: lever balance. Staff should check whether the lever balance is accurate and sensitive before use.

Accuracy testing method: weigh the spare 10kg, 20kg and 30kg weights (or replace them with calibrated objects of equal weight). This must be done to check whether the differences between the indications and the standard weights are within the permissible range. The error here should not exceed 0.1% (namely, the error per hundred kg must be less than 0.1 kg).

Sensitivity testing method: place a 100g weight and observe the change in the graduated scale. If the scale raises 3 mm - or the vernier moves forward by 0.1kg but the scale still keeps horizontal, it means the lever balance is qualified.

Testing method: the lever balance should be placed on a flat surface during testing. Staff should adjust it to the zero point where the graduated scale is horizontal. Men should only wear short trousers while women wear short trousers and T-shirts. They should stand on the center of the balance.. Staff should place the proper weights and move the vernier until the graduated scale is balanced. The value must be in kg and accurate to one decimal place. The measurement error here should not exceed 0.1kg.

Considerations:

1)The manufacturer of the beam balance should have a quality certificate issued by the State Metrology Bureau. The beam balance being used should also have a quality certificate issued by the manufacturer.

2)The beam balance should be placed on a flat surface.

3)The beam balance should be calibrated before use every day.

4)The measurement organizations should be in kg and accurate to one decimal place (round off).

1.5.3Blood Pressure

Blood pressure includes the indexes of systolic and diastolic pressure. The testing group will designate special staffs to conduct the testing.

Instrument: vertical mercury sphygmomanometer. Before measurement, staff should check whether the mercury column is at the zero position. If it is not, calibrate it with a standard sphygmomanometer. Meanwhile, observe whether there are any bubbles in the mercury column. If any are found, eliminate them. The sphygmomanometer should be placed flat and the cuff should cover 1/2---2/3 of the participant’s upper arm during testing.

Testing method: the participant should sit on the right side of the tester with his/her right arm naturally stretched out on the table. Sphygmomanometer’s zero position should be horizontal to the positons participant’s heart and right arm cuff. Wind the cuff smoothly to a proper tightness. The cubital fossa should be fully exposed. Place the stethoscope head on the radial artery. The stethoscope head should closely touch the skin, but staff shouldn’t press it too hard or place it under the cuff. Then, pump air into it so that the mercury column rise rapidly until the beat of radial artery can no longer be heard. Then, make it rise by 20~3 0mmHg. then slowly deflate the cuff. Preferably, it should fall by 2~3 mmHg every beat after hearing the systolic pressure. The height of the mercury column is the systolic pressure when the pulse beating sound is first heard. Keep deflating, and the beating sound will change. The height of the mercury column is the diastolic pressure when the beating sound disappears. Blood pressure testing is preferably finished only once. Staff can also retest blood pressure. Respectively record the systolic and diastolic pressures in mmHg on the health form.

Considerations:

1)Participants should not take strenuous exercise within 1~2hours before undergoing the test.

2)Participants should sit in a comfortable way for over 10 minutes to eliminate stress and stabilize their mood.

3)During the blood pressure testing, the upper arm should not be restricted by a tight sleeve.

4)If blood pressure values need to be read repeatedly, make sure the mercury column falls to the zero position.

5)Results should be recorded in mmHg. Round-off the numbers and respectively record them.

1.5.4 Internal Checkup

 

1.6 Circulatory System Function (Cardiac Function) Testing

1.6.1Testing Instruments

1)BioZ.comTM digitized noninvasive hemodynamic monitoring system, BioZ.com cardio dynamics (American Cardiodynamics International Corporation). This instrument is used for the surveys in Hebei, Zhejiang and Guangxi provinces.

2)NCCOM-3 non-invasive cardiovascular monitor (American Bomed Medical Mfg. Ltd). This instrument is used for the surveys in Beijing.

1.6.2Testing Indexes and Implications

1)    HR, Heart Rate: the number of heartbeats per minute (beat/min).

2)    SBP/DBP, Systolic Blood Pressure/Diastolic Blood Pressure: SBP, which is the top number, measures the pressure in the arteries when the heart beats (when the heart muscle contracts) .DBP，which is the bottom number, measures the pressure in the arteries between heartbeats (when the heart muscle is resting between beats and refilling with blood) .

3)    CI, Cardiac Index: CI relates the  HYPERLINK "http://en.wikipedia.org/wiki/Cardiac_output" \o "Cardiac output" cardiac output (CO) from left ventricle in one minute to body surface area (BSA) when someone is fasting and at rest. (L/min·m2).

4)    CO: Cardiac Output is the volume of blood being pumped by the heart, in particular by a left or right ventricle in the time interval of one minute  (L/min).

5)    SI, Stroke Index: the stroke volume to body surface area (ml/beat· m2).

6)    SV: Stroke Volume is the volume of bloodblood pumped from one  HYPERLINK "http://en.wikipedia.org/wiki/Ventricles_of_the_heart" \o "Ventricles of the heart" ventricle of the  HYPERLINK "http://en.wikipedia.org/wiki/Heart" \o "Heart" heart with each beat.  (ml)

7)    SVR: Systemic Vascular Resistance: also called as afterload.  It is the resistance to flow that must be overcome to push blood through the peripheral circulation system. It is related to blood viscosity and vasomotoricity etc.

8)    LCW: Left Cardiac Work is an indicator of the amount of work the left ventricle must perform to pump blood each minute  (Kg ·m/min).

9)    LVET, Left Ventricular Ejection Time is the time interval from the opening to the closing of the aortic valve (mechanical systole)  (msec).

1.6.3Testing Methods and Operation Steps

1) Instrument Testing Operation Steps

a)    First, connect the instrument with the sensors, the blood pressure cuff and the cables.

b)    Turn on the instrument.

c)    Enter the ID number, name, height (cm), weight (kg), gender, age and relevant other data into the display menu.

d)    Begin the test only after confirming the information.

2) Operation Steps

a)    Clean two sides of the neck and chest of participants with alcohol.

b)    Participants should lie down.

c)    Place the four pairs of electrodes (white, black, red and green) in proper positions. First, place the white electrodes on the both sides of the bottom of the neck and the black electrodes 5cm away from the white ones. Second, place the red electrodes on the xiphoid of both sides of the chest along the mid-axillary line and the green electrodes 5cm away from the red ones.

d)    Wind the blood pressure cuff on the participant’s upper right arm appropriately.

1.6.4Factors of Testing and Considerations

1)    The participants should lie down in a comfortable way to relax themselves, and should reduce the exercise of the chest.

2)    Make sure the skin where the pads are applied is clean and dry to avoid the influence on signal transmission.

3)    Make sure the electrodes are within the validity term before testing. Also make sure the conductive paste is wet enough.

4)    The four pairs of electrodes must be correctly placed. Each pair of electrodes should be placed opposite to each other. Or it will affect the testing results.

 

1.7 Blood Biochemistry Testing

1.7.1 Testing Component Description: the blood biochemistry profile includes serum protein, blood lipids, sugar, enzyme and inorganic elements, etc. The specific analytes are shown as follows: Total protein, TP; Albumin, ALB; A/G; Total cholesterol, TC; Triglyceride, TG; High density lipoprotein cholesterol, HDL-C; Low density lipoprotein cholesterol, LDL-C; Glucose, Glu; Alanine aminotransferase, ALT; Aspartate aminotransferase, AST; Hepatitis B antigen HBsAg; Alkaline phosphatase, ALP; Total calcium, TCa2+; and inorganic phosphate, IP.

 

1.7.2 Testing Instruments: The analyses are performed with 2700 Olympus biochemical autoanalyzer. The machine is operated according to the instrument and kit instructions and routine internal quality control is performed.

1.7.3 Testing methods: Blood is collected from the survey participant’s forearm venous after they have fasted for 12 hours. Place it at room temperature(20~30℃) for 45 minutes and then centrifuge it .Then extract the serum and place it at the temperature of -20℃ for testing. Make sure that the sample has no hemolysis or liposolution and test it as soon as possible. The serum should be stored at the temperature of -70℃if it cannot be analyzed right now. The specific methods to be used are shown as follows:

1)    Serum protein determination: including TP and Alb determination. Calculate A/G. TP: biuret method. Alb: BCG method.

2)    Glucose determination: Because at the room temperature glucose is degraded rapidly, staff should place it at the temperature of 4℃ and test it within 4 hours with GOPOD.

3)    Inorganic element determination: TCa2+ determination: colorimetry; IP: colorimetry.

4)    Enzymatic assay: ALT: velocity method; AST: velocity method; ALP: p-nitrophenyl method.

5)    Blood lipid determination: TC and TG: enzymic method; HDL-C and LDL-C: homogeneous assay.

 

1.8 Respiratory System Function (Pulmonary Function) Testing

1.8.1 Testing Instruments: Scope Rotary portable computerized respiratory function instrument, Model: MasterScreen Rotry, Software version: Lab4.52, Manufacturer: Jaeger. This instrument uses turbine sensor which takes photoelectric encoder sensing technology.

1.8.2 Parameters Definitions: The parameters of lung function can divided into two groups: the parameters of lung volume and the parameters of pulmonary ventilation function .

Parameters of Lung volume: lung volume is the capacity of air that the lung can contain. It is variable as the lung expands or retracts to exchange gases. Lung volume parameters usually refer to the changes that occur in the respiratory air volume during a single breath while the patient is in the rest. The following five parameters can be measured directly with the respiratory function instrument:

1)    Tidal volume (VT): the volume of air moved into or out of the lungs during quiet breathing

2)    Breathing frequency (BF): the number of breaths per minute

3)    Expiratory reserve volume (ERV): the maximal volume of air that can be exhaled from the end-expiratory position.

4)    Inspiratory capacity (IC): the maximum volume of air that is inhaled after normal exhalation.

5)    Vital capacity (VC): the volume of air breathed out after the deepest inhalation.

Parameters of pulmonary ventilation function: the respiratory muscle drives the thorax to expand or retract. This changes the lung volume to produce ventilation. Normal pulmonary ventilation function depends on three elements, namely: respiratory muscle function, thorax and pulmonary elasticity, and the smoothness of the respiratory tract. The following indexes should be tested:

1)    Minute ventilation (MV): the volume of air inhaled or exhaled per minute while the survey participant is in the rest.

2)    Forced vital capacity (FVC): the amount of air a person can expire after a maximum inspiration.

3)    Forced expiratory volume in one second (FEV1): the volume forcedly exhaled at the end of the first second after a deep inhalation.

4)    FEV1/FVC: the ratio of FEV1 to FAVC.

5)    Maximal breathing capacity (MBC): the volume of air that is breathed per unit of time when a person breathes as deeply and quickly as possible.

6)    Parameter in the pulmonary ventilation function reflecting small airway function: this tests the velocity of airflow passing through the airway during a forced exhalation.

7)    Peak expiratory flow (PEF): the highest forced expiratory flow measured with a peak flow meter.

8)    FEF25%: forced expiratory flow related to25% of the forced vital capacity.

9)    FEF50%: forced expiratory flowrelated to 50% of the forced vital capacity.

10)  FEF75%: forced expiratory flow related to75% of the forced vital capacity.

11)  Maximal mid-expiratory flow (MMEF75/25%): it relates the average flow between FEF 25% and FEF 75%.

1.8.3 Testing Methods

Referring to the technologies and approaches in the National Pulmonary Function Normal Value Compilations, staff should train participants according to testing requirements, to make sure that the testing results are accurate and reliable. The details are shown as follows:

Preparation before Testing:

1)    Before testing: the participants should relax themselves for 5-15 minutes.

2)    Participants should sit down in a comfortable way with their mouths parallel to the nozzle of the instrument. The survey participants should take the nozzle into their mouth and fully cover it with their mouth (make sure all of the air passes through the nozzle without any leakage when breathing).

3) The participants should pinch the nose (with a nose clip), and keep the abovementioned posture so that all gases can go through the instrument while the participant inhales or exhales.

Begin the following tests:

1) Spir-Flow: Staff can draw on data from participants when they sit in the way mentioned above and breathe stably (after about 12 breaths). Firstly, participants should breathe normally for 1-2 times and exhale as possible as they can (try to exhale the air in their lungs). Secondly, they should inhale to TLC (to inhale air as much as possible). Then, they exhale to their residual capacity. This phase of test can be finished while participants breathe normally where the respiratory wave aligns to the sampling line.

2) Flow-Volume: this test can be conducted after the first test. Ask participants to breathe normally. When the respiratory wave is stable enough (after about 12 breaths),participants should exhale normally to their residual capacity and then inhale to TLC. Finally, they force all of the air out to their residual capacity. Then the test is finished. During this test, participants should inhale and exhale as possible as they can to meet the test requirements.

3) MVV: After finishing these two tests, participants can take a rest. Then staff should explain to them in detail about how to conduct the MVV test. Here, participants should breathe with a maximum pulmonary ventilation volume as rapidly as possible (like gasping after a 100m sprint). The rate of breathing is usually considered to be once per second.

1.8.4 Considerations

1)    During the lung volume tests, staff have to relax participants as possible as they can. For example, the staff can speak calmly and compassionately to calm participant down and to encourage them to cooperate etc.

2)    Start the test after participants breathe normally. Record only when the respiratory wave is stable with over 10 even wave lines, when they inhale and exhale about 20±5 beats/minute, and the instrument permits.

3)    In terms of pulmonary ventilation function determination, participants should take the test at least three times (or more) until they have met all requirements. At this point, record the best value.

 

1.9 Skeletal System (Calcaneus Ultrasound Bone Mineral Density) Testing

1.9.1 Testing Instrument: ACHILLES EXPRESS Calcaneus Ultrasound Bone Densitometer manufactured by US Lunar Co., Ltd., software version: 4.1X.

1.9.2 Testing principle: the instrument is an ultrasonic receiver. One end of the probe emits ultrasound waves. These waves penetrate through the skin, enter into the bone, and then spread along the axis until they reach the other end of the probe. The transmission process is processed with a computer to calculate the speed at which the ultrasound passes through the bone (SOS, organization: m/sec), as well as the  attenuation of ultrasonic frequency (BUA, unit: dB/MHz). As changes that occur when an ultrasound passes through bones are connected to the chemical composition and structure of the attenuate tissue, the results here can provide not only information on bone density, but also additional information on bone strength (including micro-structure, elasticity and brittleness, etc). To make the testing results available, the Achilles Express Determinator combines SOS and BUA values into a clinical metric——Stiffness Index (SI).

1.9.3 Testing Locations and Significance: select the calcaneus (heel bone) for the ultrasonic bone density determination. The calcaneus is a weight-bearing bone. 75-90% of the calcaneus is comprised of trabecular bones which reacts much more rapidly to bone changes caused by age, disease and treatment. Additionally, changes in calcaneus density are significantly related with fracture. Therefore, the risk of fracture can be evaluated via the determination of calcaneus density. This is the reason why we chose the calcaneus to carry out QUS determination.

1.9.4 Testing Indexes and Definitions: Stiffness index (SI): it is a clinical metric comprised of the SOS and the BUA, mainly reflecting bone stiffness and hardness. Its accuracy errors here are far lower than that ofo a single variable. The formula used is SI＝(0.67×BUA+0.28×SOS)-420. This will automatically be calculated by the instrument.

1.9.5 Testing Methods and Operation Steps

1) Instrument Testing Operation Steps

a)    First, connect the instrument to the computer with the cables.

b)    Turn on the instrument, and then carry out quality control on the instrument according to the quality assurance procedures.  Then participants can take tests.

c)    Enter the information of participants according to the display menu, such as the number, name, gender, age, height and weight .

2) Respondent Testing Operation Steps

a)    Clean the two sides of ankle joint of the right foot and the entire heel of the participant with alcohol so as to remove residual grease on the skin.

b)    Evenly apply the water-soluble ultrasonic coupling gel onto the two sides of ankle joint of the right foot and the entire heel of the participant.

c)    Place the right foot into the foot adjuster and begin the test.

1.9.6 Considerations

1)    Perform all quality assurance steps every day before beginning the testing;

2)    Carry out complete low-level disinfection procedures between every two participants;

3)    Make sure that the participant’s right foot is correctly positioned.

4)    During the test, the participant’s right foot should be fixed so as not to affect the ultrasonic measurements.

 

Related surveys can provide correct and reliable data for us to broaden the database,which involves human body’s physical constants. Such database can help us know the changes and rules related to our growth, development and senescence .

1.To determine the physique phenotype and reference values of physiological constants which can reflect how well the important internal organs work, targeting urban and rural Chinese residents different from ages, genders, and nationalities, and provinces (cities).

2. To establish a monitoring network for the physiological surveys, which target children and teenagers (aged 6 -18), young adults (aged 19-44), middle-aged adults (aged 45-64) and the elderly (aged more than 65), And update data, standardize and normalize the testing system, train the team, and work out a long-term plan.

According to the territorial, ethnic, and economic features of China, we selected the following 6 provinces: Inner Mongolia Autonomous Region, Heilongjiang Province, Ningxia Hui Autonomous Region, Sichuan Province, Yunnan Province, and Hunan Province.

1.1 General sampling principles:

Following the statistical sampling principles, we must adopt a multi-stage, stratified, and random cluster sampling method to ensure in surveyed areas the survey economic and effective, that the precision of results is over 95%, that samples nationwide are representative, and that sampling is feasible and reasonable. It targeted people who aged more than 6. According to WHO standards, they are divided into 4 age groups, and then sub-divided again according to the research features associated with different disciplines:

1)    Children and teenagers (6-18): children (6-12) and teenagers (13-18);

2)    Young Adults (19-44): 19-2, 27-34, 35-44;

3)    Middle-aged Adults (45-64): 45-54, 55-64;

4)    The Elderly (65 or above)

 

1.2 Primary Sampling

1.2.1 Primary Sampling Principles

According to the PMH and the physical examination results, the following people should not take part in the physical examinations:

1)    People with heart, lung, liver or kidney disorders

2)    People with abnormal physical growths

3)    People with acute diseases or those who have suffered a fever or cold within the past 15 days

1.2.2 Survey Items

1)    Carry out surveys on demographic indicators, habits and customs, and health condition  clinical internal and external checkups via a questionnaire to sample participants;

2)    Physical phenotype: height, girth, weight and skin-fold thickness etc.;

3)    Complete blood cell count;

4)    ECG

 

1.3 Secondary Sampling

1.3.1 Secondary Sampling Principles:

1) Those who have any of the following 12 diseases and are clinically diagnosed should be excluded  : respiratory system diseases; cardiovascular diseases; Rheumatic immune diseases; physicochemical diseases; digestive system diseases; Endocrine, nutritional and metabolic diseases; blood diseases; nervous system diseases; mental diseases; urinary system diseases; osteoarthropathy; malignant tumors

2) Those whose BMI is over 28 should be excluded.

1.3.2 Examination Items

1) Blood System:

a)    Liver function: TP, ALB, A/G, ALT, AST, GTT, ALP and LD.

b)    Renal function: Ca, P, Glu, BUN, Cr and UA.

c)    Blood fat: CHO, TG, HDL-C and LDL-C.

d)    Serum enzymes: AMY and CK.

2) Immune System

Testing indexes: IgG, IgA and IgM.

3) Circulatory System

In terms of cardiac function test, we adopted non-invasive technology to determine TEB. The hemodynamic parameters were determined with TEB and ECG signals.

Testing parameters: 15 parameters including HR; SBP/DBP; CI; CO; SI; SV; SVR; LCW; LVET; EVI; etc.

4) Respiratory System

Pulmonary function test with a portable computerized respiratory function instrument

Measuring indexes: 15 parameters, including: VT; BF; MV; ERV; IC; VC; FVC; FEV1; FEV1/FVC%; PEF; FEF25%; FEF50%; FEF75%; MMEF75/25; MBC or MVV;

 

1.4 Determination of Sample Size:

The effective sample size for every surveyed province is 15,000 persons.Every province includes 4 urban and rural survey sites with 3,500 persons in each site. The secondary sample size is 33.6% of the effective sample size.

On-site surveys were organized and implemented together by the central and local departments of health, the Centers for Disease Control and Prevention, and various medical schools. The on-site organization and coordination was jointly performed by the related departments from the surveyed provinces.

1.5 Establish a leading institution related to survey organization, implementation, and quality control.

1)    Establish an expert advisory committee, which give related suggestions about the project on the design, task division, contents, planning, deployment, implementation and organization, as well as the research team.

2)    Establish an expert committee on quality control, which is comprised of the related experts and technicians from the major sponsor of the project and other involved organizations. Its duties are: to work out general and specific quality control plans; to decide quality control approaches for the survey; to uniform survey methods and standards; to organize technical instructions for on-site surveys in different provinces, and to take charge of quality control of the whole survey.

3)    Establish an on-site work committee, which consists of leaders and professionals from the major sponsor of the project and other involved organizations who are in charge of on-site organization and implementation. According to the purpose and requirements of the project, the committee needs to train investigators and design work processes  They should also implement and conduct quality supervision over the on-site surveys.

 

1.6 Preparation for On-Site Survey Implementation

1) Survey teams : each survey team consists of about 15 professionals dispatched by the major sponsor of the project  (mainly responsible for finishing on-site organization, research on physique and the functions of main organ systems, and quality control, etc.); a survey team consists of  30-50 investigators from other involved organizations (in charge of on-site organization, physical examination, blood sampling and processing, other testing, and logistics etc.) ;

2) Staff training: the On-site work committee is responsible to train investigators at different levels. These people should all be qualified before starting their work;

3) The same workbooks available: the workbooks should have details of the operation processes, technical requirements, quality control methods, and the implementation considerations for different surveys;

4) The same survey tools available: staffs should provide the same survey forms and original data record sheets to participants; the weighing scale, height meter, sphygmomanometer and measuring scale for waistline, chest circumference and hipline should also all be the same in type; all materials and lab materials required by the site should be provided uniformly (such as blood lancets, syringes, the anticoagulation centrifuge tube, and centrifugal machine etc); they should also provide key equipment directly affecting the measurement accuracy uniformly.

Testing Methods, Equipment, and Quality Control

1.7 Testing of Physique Phenotype of Human Growth and Development

1.7.1 Clinical physique examination: first, carry out all internal checkups to exclude those who have any of the following 16 diseases, symptoms, or conditions: respiratory system diseases, cardiovascular diseases, malignant tumors, cerebrovascular diseases, diabetes, gynecologic diseases, infectious diseases, tuberculosis, urinary system diseases, rheumatism, thyroid diseases, digestive system diseases, allergic immune diseases, people with abnormal physical growths or those taking calcium supplements, as well as people with any other acute disease or those who have suffered a fever or cold within the past 15 days.

1.7.2 Growth and Development Testing: this includes height, weight, sitting height, chest circumference, waistline, hipline, blood pressure (SBP and DBP) and heart rate.

1.7.3 Body Composition Testing

1)    Testing indexes: basal metabolic rate, fat mass, fat percentage, lean body mass, BMI, total body water, and electrical impedance.

2)    Data collected in the database includes: basal metabolic rate, fat mass, lean body mass, total body water and electrical impedance.

3)    Instrument and methodology: Biodynamics BI-310 Body Composition Analyzer (US Biodynamics Corporation).

4)    Principle: to calculate the percentage and weight of different compositions in the human body by use of the impendence (bio-impedance). This is produced by the human body when it reacts to a harmless, low current sended by the analyzer.

5)    Testing Procedures and Considerations:

Steps: ① The participant lies on the back with their body relaxed. ② The participant should expose the right wrist and place an electrode near to the wrist. The edge of the electrode should be placed at the crease between the hand and wrist; the tester should place another electrode on the back center of the same hand, close to metacarpophalangeal joints. ③ Place an electrode close to the ankle of the participants. The edge of the electrode should be placed at the crease between the foot and leg; the tester should place another electrode on the back center of the same foot, close to the base of the toes. ④ Connect the instrument to the induction line and the electrodes. ⑤Respectively input the participant’s ID number, gender, age, height and weight etc. according to the display menu.⑥ Begin the  test only after confirmation.

6)    Considerations: ① No drinking within 24h before the test; No eating or exercise within 4h before the test;participants should not take diuretics before the test. ② The participants lie on their back and relex themselves. Participants should place their hands away from both sides of the body at least 15cm; their feet should be separated  with a distance of more than15 cm. They should fully expose the right ankle and wrist without any nylon fabrics. ③Before tests ,testers should check whether the electrodes are effective and they are placed and connected correctly.

1.7.4 Quality Control

1) Quality Control of On-site Survey

a)    Personnel training: surveyors should all be uniformly trained according to the training workbooks who are responsible for the clinical physical examination, physique testing, and system organ function testing .

b)    Optimize the on-site investigation recording procedures: When each investigation and all critical steps are finished , the operators must sign for better supervision and recording.

c)    Testing equipment quality control: All of the instruments should be calibrated everyday. The related data should be recorded in the worksheet.

d)    Data accuracy:  Staff should sample the finished questionnaires everyday to check the quality. If errors exist, correct them timely.

e)    Every staff performs his/her own duty with mutual supervision and timely coordination.

f)    Height, weight, and blood pressure are all measured with instruments. For example, the weighing scale, height meter and sphygmomanometer should all be of a unified model. Please refer to the survey workbooks for the details of the measuring standards and requirements . BWH measurements are taken with a plastic measuring tape lined with nylon wire. This tape should be calibrated with steel tap before use. The error per meter should not exceed 0.2cm.

g)    Train the instrument operators uniformly and the operators need to be fixed.

h)    Purchase testing instruments of the same model and brand. Compare several instruments before testing to reduce measuring errors among the instruments as much as possible.

2)    Data input Quality Control    

To make sure data input is accurate, all data should be inputted twice using professional software (Epidata). Data administrators must check whether the two sets of data are the same and logical and boundary values with computers , and correct any erroneous records according to the verification results and questionnaires. After the data input,, special members should review the data classified by the survey contents. Feedback suspicious records and check them compared with the original forms according to the unified review principles. Finally,  a certified database is available.

 

1.8 Methods and Materials for Complete Blood Cell Count Testing

1.8.1 Participants preparations

1)    Participants should have a balanced diet at least 3 days but 1 week better before the test (i.e. Participants should follow their normal lifestyle and have meals regularly without too much or too less food. Don’t eat food that is too greasy or that contains too much protein.).

2)    Try to stop taking any drugs (common drugs and various therapeutic medicines such as vitamins and aspirin) at least 1 week before testing. If not,, they must explain this to their physical examiners.

3)    Don’t drink alcohol or any beverage containing alcohol for one week before the physical examination.

4)    Draw blood in the early morning under fasting conditions. The fasting time should ideally be between 8-12h ,and it cannot be over 14h or less than 8h. During the fast, participants can only drink water .

5)    Don’t do strenuous exercise within 24h before blood collection (such as, bodybuilding and heavy physical labor).

6)    Participants should try to relax themselvesbefore blood collection. Those who rush to the blood collection station should rest for at least 15min before blood collection. Allparticipants should sit for 5min before blood collection.

1.8.2 Instruments and Components

Japan Sysmex XT-1800i Automatic Hematology Analyzer and originally packed components

1.8.3      Calibration and Quality Control

1)    The technicians of the factory should calibrate the instruments’ laser by the factory standards with a micro-particle calibrator after installment.

2)    Calibrate all instruments used on the project with their originally packed calibrators (SCS-1000, lot number: 92090525).

3)   Daily quality control: use the originally packed sysmex quality control instrument e-CHECK, check three levels a day. Lot number: 81470801, 81470802, 81470803, 82030801, 82030802 and 82030803.

4)    Make comparison of the test results of 20 fresh blood samples respectively obtained from the instruments installed and debugged on the site and the same instruments at the Clinical Laboratory of Peking Union Medical College Hospital. The comparison standards should follow the qualification judgment that the Clinical Laboratory of Peking Union Medical College Hospital uses for comparison results, namely, WBC≤ 7.5 %, RBC≤ 3%, Hb≤ 3.5%, HCT≤ 3% and PLT≤ 12.5 %.

5)    Immediately shake all collected EDTA-K2 anticoagulation samples (shake gently about 8 times upside down) to avoid blood coagulation. Be careful to avoid hemolysis caused by violent or extended shaking. Keep the samples at room temperature and finish the machine testing for all samples within 8h.

1.8.4 Data Screening Principle - Exclusion criteria: test results exceeding ±3s.

 

1.9 ECG Observation Methodology

1.9.1      ECG Testing Instruments and Methods

The participant should sit calmly for at least 5 minutes before undergoing an ECG test. They should fully expose the body parts where the electrodes are to be placed. Place the routine 12 leads according to international standards. The ECG survey used a Philips PageWriter Trim II Digital 12-Lead Standard Synchronous Electrocardiograph, with a frequency range of 0.05-150Hz. The digital sampling frequency here was 500Hz/lead and the least significant bit was 5 μV. ECG records use the public XML format to store 10 second ECG data. The ECG data acquired in this survey meeted  the standard synchronous 12-lead digital ECG collection standard recommended by both the AHA and the digital ECG database standard established by the NHLBI.

In the description for ECG parameters related to population distribution, we excluded participants diagnosed by on-site physicians as atrial flutter, atrial fibrillation, complete bundle branch blockage (diagnosed as bundle branch blockage and QRS durations>120ms) those with pacemaker ECG and FVPB, and those whose ECG signals are of poor quality.

1.9.2 Main ECG Parameters

The main ECG parameters are obtained through the Philips PH09 algorithm. The parameters are extracted from the Philips ECG XML documents with a self-programmed PERL script. These extracted parameters include: heart rate, PR interval, QT interval, QTc (Bazett correction), QRS duration, P-wave axis, QRS axis, T-wave axis and voltage values of RaVL, SV3, SV1, RV5 and RV6. The Cornell index is the sum of the RaVL and the SV3. The Sokolow-Lyon index is the sum of the SV1 and the RV5 or RV6 (whichever is higher).

 

1.10 Blood Bio-Chemical Testing Methods and Materials

1.10.1 Preparation of participants:        Participants should follow their normal lifestyle and have a balanced diet at least 3 days but 1 week better before the test . Participants should have meals regularly without too much or too less food. Don’t eat food that is too greasy or that contains too much protein.)Don’t drink alcohol or any beverage containing alcohol for one week before the physical examination. Participants should stop taking all drugs (common drugs and various therapeutic medicines such as vitamins and aspirin) at least 1 week before the test. If not, they must report this to the physical examiners. Blood should be drawn in the early morning after participants have lasted for 8-12h. Participants should not partake in any strenuous exercise within 24h before blood collection (such as bodybuilding and heavy physical labor).Participants should try to rid themselves of nervousness and excitement before  blood collection.

1.10.2 Sample Collection and Transportation:   the participant should sit calmly for 5min before blood collection. Blood should be drawn from the participant’s cubital veins with vacuum blood drawing technology. Blood collection and disinfection should follow the routine operation procedures. Venipuncture should be correct and gentle so as to avoid insert and with drawl the needle repeatedly in cases of blood hemolysis. Tourniquet should be removed after 1 minute (it is better to loosen the tourniquet as soon as the needle is inserted into the blood vessel). Blood collection sequence: first use the red-capped tube (containing separation gel) and then use the purple-capped one. The blood collection volume should be as accurate as possible (the error here should be no more than 0.5ml).

After collection, operators should place the red-capped tube at room temperature for 15-30min (10-15min is enough if the room temperature exceeds 26℃). Centrifuge the blood with 3000g power at a temperature of 20℃ for 10min (it needs to calculate the corresponding centrifugal speed which is usually 3000rpm). After centrifugation, place the samples at the temperature of 2-8℃ within 30min. Always keep the samples away from light during storage.

Serum can be transported within a reliable sample delivery box. Try to keep the samples at a low temperature during transportation. Move the samples from a 2-8℃ refrigerator into the sample delivery box before transportation. Fill up the sample delivery box with dry ice (not less than 1/2 of the box) or ice bags around the samples. The samples should be separated from the dry ice or ice bag with foam to avoid direct contact. The transportation time should not exceed 16h with the maximum of 24h. There should be clear labels on the delivery box to prevent it being turned upside down or sideways. If the delivery time exceeds 24h, the sample should be rejected and this must be recorded. After the samples are sent to the testing center, the tester should open the box and make sure that the samples have no inverse, leaks, or cross contamination. The tester should then make related records and confirm that the sample box temperature has been low and the refrigeration has been effective.

1.10.3 Instruments and Reagents

Item	Determination Method	Testing Instrument	Reagent	Calibrator	Quality Control
A	Bromocresol Green	Olympus AU2700	Olympus	Olympus	Olympus
ALT	Velocity Method	Olympus AU2700	Olympus	Olympus	Olympus
AST	Velocity Method	Olympus AU2700	Olympus	Olympus	Olympus
TP	Biuret	Olympus AU2700	Olympus	Olympus	Olympus
GGT	Velocity Method	Olympus AU2700	Olympus	Olympus	Olympus
LD	Velocity Method	Olympus AU2700	Olympus	Olympus	Olympus
Cr	Enzyme Method	Olympus AU2700	Daiichi	Randox	Randox
UA	Urate Oxidase Indirect Peroxidase Couple	Olympus AU2700	Olympus	Olympus	Olympus
Glu	Hexokinase Method	Olympus AU2700	Olympus	Olympus	Olympus
CK	Velocity Method	Olympus AU2700	Olympus	Olympus	Olympus
TC	COD Method	Olympus AU2700	Daiichi	Daiichi	Randox
TG	GPO-POD Method	Olympus AU2700	Daiichi	Daiichi	Randox
HDL-c	Direct Determination	Olympus AU2700	Daiichi化	Daiichi	Randox
LDL-c	Direct Determination	Olympus AU2700	Daiichi	Daiichi	Randox
BUN	Urease UV Velocity Method	Olympus AU2700	Olympus	Olympus	Olympus
Ca	Arsenazo Colorimetry	Olympus AU2700	Olympus	Olympus	Olympus
P	Phosphomolybdic Acid Colorimetry	Olympus AU2700	Olympus	Olympus	Olympus
ALP	Velocity Method	Olympus AU2700	Olympus	Olympus	Olympus
Amy	CNP-G3	Siemens Dimension Rxl	Siemens	Siemens	Randox
IgA	Rate Nephelometry	Siemens BNⅡ	Siemens	Siemens	Siemens
IgG	Rate Nephelometry	Siemens BNⅡ	Siemens	Siemens	Siemens
IgM	Rate Nephelometry	Siemens BNⅡ	Siemens	Siemens	Siemens

 

1.10.4 Calibration and Quality Control 

Quality Assurance: According to related CLSL documents, all of the instruments and reagents involved in this experiment should pass the performance assessment experiments on calibration verification, sensitivity, precision, methodological comparison, deviation assessment and reference range verification. All involed items should meet the corresponding quality assessment plan from different labs and have complete PT Non-Compliance Processing Procedures. This will help us to correctly evaluate and analyze experimental data in case any problem exists. During the experiment, compare 40 samples from Peking Union Medical College Hospital .

Calibration and Internal Quality Control: Operators should calibrate the instruments and related  calibrators before the experiment to ensure all of the instruments are in a good state. Regular chemical internal quality control uses two-level quality control materials and immune globulin uses a three-level one. Finish quality control once within 1h before the test. If it is out of control, it should be processed before testing. Make another quality control assessment after testing and analyze whether the daily results are reliable if the results don’t align to requirements. Test again if necessary, and record the process.

Sample testing: Testers should know numbering rules clearly before the tests and test all samples within the given time. Re-check if the results are suspicious (such as abnormal indications on the instruments). If not, choose1-2 samples/lot randomly. Strict process management and a good recording system can ensure calibration, testing, record keeping and inappropriate processing in the lab is closely controlled, which contribute to accurate and reliable results.

1.10.5 Methodological Comparison 

To ensure the consistency of bio-chemical and immune testing results from different sites, each site should compare the related results.. As the time to start surveys varies between survey sites (only two different provincial sites every year) and the laboratories used for tests can only be determined before the survey. it is impossible to compare test results from different sites at the same time. Therefore, we compared results of the survey sites with that of the Clinical Laboratory at Peking Union Medical College Hospital. This is done before and after surveys in the sites. We identified .the number of samples for comparison and the evaluation standard ,referring to the CLSI EP9A document. When summarizing all data after the project ends, we will adjust the related data or eliminate some specific items whose comparison results are unsatisfactory. Meanwhile, we will not adjust the data summary from each site.

1.10.6  Data Screening Principle 

Input the blood bio-chemical index testing data into a database twice by different operators and eliminate the data whose BMI>28. Screen the data between ±3 swhich could be used in the population distribution tables, mean tables, and percentiles tables for different indexes after logic verification.

 

1.11 Methods and Steps for Circulatory System Function Testing

1.11.1    Testing Methods

The BioZ.comTM Digital Non-Invasive Hemodynamic Monitoring System (USA Cardiodynamics) used in this survey is based on the development of bio-simulation technology. It uses non-invasive technology to determine TEB and then determines the hemodynamic parameters with the TEB and ECG signals:

1)    HR （heart rate）

2)    SBP/DBP: systolic blood pressure/ diastolic blood pressure (mmHg).

3)    MAP: mean arterial pressure (mmHg)

4)    CI: cardiac index (L/min·m2).

5)    CO: cardiac output (L/min).

6)    SI: stroke index (ml/beat·m2).

7)    SV: stroke volume (ml).

8)    SVR: systemic vascular resistance (dyne·sec·cm-5).

9)    SVRI: systemic vascular resistance index) (dyne·sec·cm-5·m-2)

10)  LCW: left cardiac work (kg·m).

11)  LCWI: left cardiac work index) (kg·m/m2)

12)  PEP: pre-ejection period (s)

13)  TFC: thoracic fluid content (1/KΩ)

14)  LVET: left ventricular ejection time (s).

 

1.11.2 Testing Steps

1.11.2.1 Instrument Operation Steps

a)    First, connect the instrument to the sensors, blood pressure cuff with the cables.

b)    Open the instrument's power switch and push it to the “OK” position

c)    Enter the ID number, name, height (cm), weight (kg), gender, age and other relevant data in the display menu.

1.11.2.2 Test Operation Steps

a)    Clean both sides of the neck and the chest of the participants with alcohol

b)    The participant should lie down.

c)    Place the four pairs of electrodes (white, black, red and green) in their proper positions. First, place the white electrodes on the both sides of the bottom neck and the black electrodes 5cm away from the white ones. Second, place the red electrodes on the xiphoid of both sides of the chest along the mid-axillary line. Finally, place the green electrodes 5cm away from the red ones.

d)    Wind the blood pressure cuff around the participant’s upper right arm properly.

e)    After confirmation, begin the test

 

1.12 Methods and Preparation for Respiratory System Function Testing

The participants should sit calmly to relax themselves and then take the test. Before the test, the tester will explain the procedure to the participant and show them how to adopt  the correct testing methods.

1.12.1 Testing Instruments

1)    Testing Instrument: Master Scope Portable Respiratory Function Instrument, Model: Master Scope, Software version: Lab5.10, Manufacturer: Jaeger. The sensor is a differential pressure sensor.

2)    Instrument Preparation: calibrate the instrument every day according to the local temperature, humidity, atmospheric pressure, and altitude .. The instrument should also be calibrated with a standard volume calibrator. Prepare backup screens for the sensor, replace ,clean and disinfect the screen timely (as required) after use.

1.12.2 Quality Control

1)    Standardize the testing process according to the criterion and testing procedures recommended by the ATS.

2)    Testers must be trained strictly to, control the quality of the test. and standardize the testing method.

3)    Testers need to be fixed .

1.12.3 Population Sampling Standard: surveyors sample 25-35% of all people who pass the physical examination to test pulmonary functions. The standards for passing the physical examination are shown as follows: no PMH on main organs, no abnormality found in the cardiopulmonary auscultation, normal blood pressure; normal liver and renal function, normal blood glucose and normal results in the routine blood examination; no abnormalities in the ECG and cardiac function examination.

1.12.4 Data Screening Principles

1)    Exclude participants whose ratio of the measured value of the lung capacity to the predictive value is less than 80%.

2)    Indexes reflecting ventilation function: exclude participants if two of their FEF25%, FEF50% and FEF75% numbers are lower than 80%.

3)    Exclude aparticipants who are uncooperative during the test or who are not qualified according to  the ATS Acceptance Criteria.

 

To establish physique and health parameters/standards for different regions and nationalities

Samples are selected from Han ethnic group and some minorities in Guizhou province, Xinjiang Uygur Autonomous Region, Inner Mongolia Autonomous Region, Heilongjiang province and Yunnan province.

1.1 Sampling principle:

males and females aged from 20 to 80 from natural population. The samples should cover both urban and rural areas, different genders, and people with different occupations.

1.2  Organization and Implementation：

1.3 Field Quality Control

1) Staff training: field staff should be uniformly trained to carry out physical examinations, physique tests and function tests of systems and organs according to the training manual;

2) Complete procedures to record field investigation: Operators must sign their names when each investigation and its critical steps accomplished.

3) Quality control for the testing equipment: All instruments should be calibrated timely as needed and recorded in the log book.

4) Data accuracy: Questionnaires should be verified everyday for omissions and incorrect fillings so as to be corrected timely.

1.4 Laboratory Quality Control

1) Determination of each index should be unified and made by fixed staff.

2) Quality control should be undertaken for every batch of test results. Results should be retested and compared timely to ensure the accuracy of each test.

1.5 Data Input Quality Control

Data should be inputted by two staff members using the double entry method. Data administrators should carry out consistency and logic checks on the input data and correct errors according to the check result.

1.6 General Physical Examination

1.6.1 Body Temperature

Human auxillary temperature should be taken with Omron electronic thermometer.  Press ON/OFF button and electronic thermometer will begin to work. When “L°C” and “°C” blink on the screen, this indicates that the thermometer is ready. Put the thermometer in the center of the armpit, turn it so that the screen is facing inward, and gently hold inward with the arm to fix the thermometer securely under the arm. The angle between the thermometer and the arm should be 35-45°. When the thermometer makes a “beep-beep-beep” noise three times, the measurement has been finished. Take out the thermometer to read the result and then press ON/OFF button to turn it off.

Note:

① When the thermometer starts operating, if no “L°C” appears (and it is not an error case), then simply measure the body temperature as usual.

② Keep the armpit folded close to the body for more than 5 minutes before measuring so that the result can be more accurate.

③ Errors will be made if you measure the temperature immediately after the survey participant stands up, exercises, bathes or eats. You should wait at least 30 minutes after these activities before measuring the body temperature; having heavy sweat under arm or covering body with a blanket for a long time will cause measuring errors. Armpit sweat should be wiped before measuring.

1.6.2 Sitting Height

Sit on the seat board of the sitting height gauge. Place the sacrum and both shoulders close to the pillar. The body should be naturally straight with head upright and eyes looking forward horizontally in order to keep the top tragus and lower orbit in the horizontal position. The arms should hang down naturally, and the hands should not press against the board. Keep the two legs close together with both feet flat on the floor. The thighs should be parallel to the floor and rectangular to the shanks (appropriately adjust the pedal height to maintain this correct measuring pose according to the shank length of each survey participant). The investigator should stand on the right side of the survey participant and slide the horizontal plate down along the pillar to the head of the survey participant. Place the two eyes and the plate in the horizontal position for reading. The results are to be recorded in cm and be made accurate to one decimal place.

Note: when measured, the survey participant should first bend down to allow the sacrum to be as close to the pillar as possible. Then, the survey participant should sit down in order to keep the test position accurate.

1.6.3 Height

The survey participant should remove all hats and other head-ware. They should also allow any hair or braids to hang below the top of the head and keep their feet bare. Then, the survey participant should stand close to the column of the instrument with their heels together. The forefeet should be placed 60° apart with the toes splayed and arms naturally hanging down. Make sure the head, shoulder, hips and heels are close to the instrument. Keep the ear canals and lower orbit in the same horizontal position. Press the measurement film to force the head. Make the survey participant stand as straight as possible and breathe deeply.

1.6.4 Weight

Body weight should be measured with the TANITA BC-420. The survey participant should remove heavy coats, wear only light clothing, keep their feet bare and stand upright on the electrode.

1.6.5 Waistline and hipline

1) Waistline: the survey participant should stand naturally with their shoulders relaxed and arms folded across their chest. The investigator should face to the survey participant and make a circle with the measuring tape around the area which is 0.5-1cm above the umbilicus (obese survey participants should be measured around the widest part of their waist). The tightness of the tape around the waist should be appropriate (the skin should not dimple around the tape). The numerical value intersected by the “0” on the tape is to be the measured value.

2) Hipline: the survey participant should stand naturally with their shoulders relaxed and arms folded across their chest. The investigator should face to the survey participant and make a circle with the measuring tape around the most protrusive part of the gluteus maximus. The tightness of the tape around the hip should be appropriate (the skin should not dimple around the tape). The numerical value intersected by the “0” on the tape is to be the measured value.

Note: when measuring the waist and hip circumferences, the investigator should strictly control the tightness of the tape. The survey participants should not expand or contract their stomachs during the measurement process.

1.6.6 Vertical Jump

Open the power switch on the vertical jump gauge. Press “button”, and then the flash signal will appear on the display screen. The buzzer should sound, indicating that the gauge has entered its working state. The survey participant should jump on the springboard with their feet naturally apart and in an upright posture. They should then be ready to begin the testing. Start the test when the screen shows “0.0”. The survey participant should squat on his/her knees, try their best to backswing his/her arms, then quickly swing the arms forward and push up legs at the same time so as to jump straight up. The screen will display measured values after the survey participant falls back to the board. Two jumps from the survey participant should be tested and recorded. The values are to be recorded in cm and must be accurate to one decimal place.

Note: If the fuse of the vertical jump gauge is blown and needs to be replaced (fuse specification: F02A, 250V), first unscrew the black knob behind the screen which displays the data. Then, remove the blown fuse and insert the end of the new fuse engraved with figures into the black knob. Then, securely tighten the black knob.

1.6.7 Grip Strength

The survey participant should keep their feet naturally apart and remain in an upright posture with their arms naturally down and their palms inward. The survey participant should grip the handle of the grip exerciser with one hand as hard as possible (the handle should not touch the clothing or the body at any time). Squeeze with the hand twice and take the maximum value. This value should be recorded in kilograms and be accurate to one decimal place. Before testing, the survey participant may adjust the grip distance of the exerciser handle to an appropriate size according to his/her palm size.

1.6.8 Sit and Reach

Turn on the power switch and push the cursor to the proximal rail. When the screen shows “-20.0cm”or less, it has come into its working state. While facing the instrument, the survey participant should sit on the mat with their legs straight forward and heels together. They should then pedal the tester damper with their tiptoes naturally apart. Adjust the rail height in order to make the survey participant’s tiptoes flush with the lower edge of the cursor. While testing, the survey participant should keep the hands together, palms down horizontally, knees unbent, and the upper body bent. Push the cursor smoothly forward with both middle fingertips until it cannot be pushed any further. At this time, the value on the screen will be the measured value. Test and record this process twice. The values are to be in cm and must be accurate to one decimal place.

Note: the survey participant should be well prepared before testing. The arms should never suddenly vibrate forward. Do not push the cursor with one hand. The knees cannot be bent. The investigator should push the cursor to the proximal position of the rail before each test. The investigator should accurately record  the “+” and “-” of the survey participant’s measured values. If the measured value is less than “-20.0cm”, the result should be recorded as “-20.0cm”. The fuse replacement process for the sit and reach instrument is same as the vertical jump instrument.

1.6.9 Blood Pressure and Heart Rate

The survey participant should not drink alcohol or coffee, smoke, or eat food before the test. The survey participant should sit, lean back in the seat, and remain resting in this position for 5 minutes. The right arm should be bent slightly with the palm facing up. The midpoint of the upper right arm should be at the same level as the heart.

Take measurements with the Omron electronic sphygmomanometer. First, ask the survey participant to take off their coat to expose their upper right arm. The arm should not be oppressed by the sleeves in any way. After sitting, ask the survey participant not to cross his/her legs. Wind the cuff closely around the upper arm. The middle part of the air sac should just hold down the brachial artery. The lower edge of the air sac should be about 2cm above the elbow. Measure the blood pressure of the same arm three times at one minute intervals.

Heart rate: record the heart rate during the last electronic sphygmomanometer measurement.

1.7 Body Composition

1.7.1 Operation procedures for the BC-420 body composition analyzer

1) Switch on the power, connect the data line, and open the switch.

2) Run “MDApp” on the computer. Then, enter the user name and password (both the initial user name and password are: admin), press the “Login” button in order to enter the menu screen.

3) Click the “Instrument Connection Settings” button in the main menu in order to enter the “Instrument Connection Settings” screen. Content in the drop-down box “Serial Port” is the serial port being used by the computer. Click “Connection Test” to perform a connection test. The connection test is successful when the screen shows “OK”. Press “Confirm” and the update prompt will appear; if “Yes” is selected, all of the original saved settings will be updated. Click “Return” and the instrument connection setting screen will then be closed.

4) Click “Member Management” in the main menu in order to enter the member management screen. Click “Add” and a member information input screen will appear. Check “Manual Input” and enter the member ID (for example: 520123). The ID number requires a second entry and this is entered in the contact box. Age will be automatically generated according to the input birth date. Complete all other information according to the actual situation. Click “Save” after completion. Click “Enter Test” in order to enter the test screen.

5) Click “Test” and the computer will send the information entered on the screen to the machine. Let the survey participant stand on the electrodes (remove socks; apply a 0.5% saline solution to the electrodes if the socks cannot be removed or if the feet cocoon is thick). Survey participants should stand on the electrodes after the prompt from the computer or after the “step on” light of the instrument begins to flash. When the computer notifies that the test is finished, click “Return” and ask the survey participant to leave. Click “Print” and all of the test data for this survey participant will appear.

1.7.2 Bulk Data Export

1) Run “MDApp” on the computer. Enter the user name and password (both the initial user name and password are: admin). Click the “Login” button in order to enter the menu screen.

2) Click “Data Management” to enter the data search screen. Select the retrieval mode as required (by ID, registration date, name, test date, age and gender). Click “Retrieve” in order to obtain the corresponding data. Directly click “Retrieve” and the dialog box and “whether to retrieve all the data” appears. Then, click “Yes” in order to obtain all the data.

3) Click “EXCEL” to automatically generate Excel forms.

4) The Excel forms are uniformly named as: "BCA + Date"

1.8 ECG

Performing the 12-lead synchronous resting electrocardiogram measurement

1) Preparation

① Materials required for preparation: conductive paste, soapy water (used to clean dirt off the skin of some survey participants as required), dry cotton ball.

② Keep the room warm (not less than 18°C) in order to avoid electromyography disturbance caused by cold temperatures.

③ The width of the test bed should be no narrower than 80cm in order to avoid any electromyography disturbance created by tension.

④ All interpretation work should be done well in advance in order to eliminate nervousness and keep the survey participant’s breathing quiet.

2) Operation Steps

① Clean the skin with soapy water if the location where the electrodes are placed is dirty.

② Rub the skin where the electrodes are placed with the conductive paste after the skin has dried. Note: the conductive paste should never be placed directly on the electrodes.

③ Correctly place the routine lead and electrodes (drawing enclosed) according to international standards. The lead includes the limb lead (RA/R, LA/L, RL/N, LL/F) and the precordial leads (V1 / C1, V2/C2, V3/C3, V4/C4, V5/C5, V6/C6). The limb lead is placed on the flat and fleshy arm. The limb lead electrodes on both sides should be the same distance to the heart. The electrode placement and body surface marker searching methods are as follows:

Figure 1 Electrode placement

Figure Description

Lead	Position
	Right leg (on the lower leg, midway between the knee and ankle) (black)
	Left leg (on the lower leg, midway between the knee and ankle) (green)
	Right arm (inside) (red)
	Left arm (inside) (yellow)
	Right sternum, the fourth intercostal space (red)
	Left margin of the sternum, the fourth intercostal space (yellow)
	The midpoint between V2 and V4 (green)
	The fifth intercostal space on the mid-clavicular line (brown)
	At the same level as V4, on the anterior axillary line, between V4 and V6 (black)
	At the same level as V4, on the mid-axillary line

④ Females with sagging breasts should lift the breast, and place the V3, V4 and V5 electrodes on the chest wall at the lower edge of the breast rather than directly on the breast.

⑤ The recording length for each lead should not be less than 10s.

⑥ Make sure that the number and name of each survey participant on the digital electrocardiograph is correct. Save the ECG information and general information of the survey participants on the mobile hard disk.

3) Precautions

① Never replace the conductive paste with alcohol (or water).

② The conductive paste should only be applied to the skin where electrodes are placed after the skin is dried. No soapy residue should remain.

③ Never place the left and right lower limb electrodes on the same lower limb for convenience.

1.9 Heart Function

1.9.1 Test Method

The determination of cardiac function level is reflected by different functional parameters. The testing for functional parameters is mainly divided into two measurement techniques: invasive and non-invasive. The instrument used in this survey is the BioZ.comTM digitized noninvasive hemodynamic monitoring system (U.S. Cardiodynamics Inc). This machine is based on the development of bio-simulation technology. It can determine thoracic electrical bioimpedance (TEB) with a noninvasive technique, and then further determine the relevant hemodynamic parameters after processing the thoracic electrical impedance and ECG signals with the use of computer technology.

1)    Heart rate (HR)

2)    Systolic blood pressure (SBP)

3)    Diastolic blood pressure (DBP)

4)    Mean blood pressure (MBP)

5)    Cardiac index (CI)

6)    Cardiac output (CO)

7)    Stroke volume (SV)

8)    Stroke index (SI)

9)    Systemic vascular resistance (SVR)

10)  Systemic vascular resistance index (SVRI)

11)  Left ventricular ejection time (LVET)

1.9.2 Procedures

1) Instrument operation steps

① First, connect the instrument to the sensors, blood pressure cuff, and cable.

② Open the instrument's power switch and push it to the “OK” position.

③ Enter the ID number, name, height (cm), weight (kg), gender, age, and all other relevant data as soon as the display menu appears.

2) Inspection Procedure

① Clean the bilateral neck and bilateral chest of the survey participants with alcohol.

② The survey participants should be in a horizontal position.

③ Place the four pairs of electrodes (white, black, red and green) in their respective locations. First, place the white electrodes on both sides of the bottom of the neck and the black electrodes on the adjacent upper sides. Second, place the red electrodes on the xiphoid of both sides of the chest along the mid-axillary line. Then, place the green electrodes on the adjacent lower sides. Make sure to pay attention to the left and right logos of the sensors when operating.

④ Wind the blood pressure cuff on the survey participant’s upper right arm to a suitable tension.

⑤ Test after confirmation. The data will be automatically recorded.

⑥ After the data appears, click “Finish”.

2.0 Lung Function

A lung function test can be used to determine whether survey participants have a respiratory dysfunction, the nature and degree of dysfunction, and to diagnose some lung diseases. A lung function test can also serve as an important index of curative effect judgment in order to guide and evaluate clinical treatment. Moreover, lung function measurements at the preoperative stage of thoracic surgery are helpful for judging the potential risks of the operation. In the field of labor hygiene and occupational disease, lung function tests can also be used to understand the impact of work environments on lung function and to make labor safety assessments.

2.0.1 Test Method

Lung function testing involves medical measurement testing techniques that are used to determine respiratory capacity, flow rates, and pressure. This helps medical professionals to understand the functional status of respiratory organs and tissues. For this study, the lung function testers - German Jaeger (Model: MasterScreen Rotary, Software version: Lab5.10) have been adopted. The system is fully compliant and supports all relevant lung function testing standards of the ERS and the ATS 2005 (European Respiratory Society and the American Thoracic Society).

2.0.2 Lung Function Test Operation Procedures

1) Start the instrument

① In the off state, connect all testing equipment according to the schematic diagram which can be found on the computer casing.

② Turn on the computer and click "AB" to start the software.

2) Calibration

① Click "calibration programs" to start calibration.

② Click "Ambient conditions" and input the environmental parameters in the form according to the readings found on the thermometer & hygrometer and the pressure gauge.

③ Click "volume calibration" and make sure that the sensor and calibration tube is correctly connected. Click "setting" and then in the pop-up window set the value of “purple volume” to 3.00. Click “OK”, click “Calibration” on the upper left corner in order to place the sensor mouth vertically. Then click OK. Push and pull the tube until the predictive value appears on the right screen. Now the calibration will automatically end. Press F12 to exit the calibration system. A dialog box indicating whether to save the settings will pop up, click “OK”.

④ Click " survey participant data" in order to create new survey participant information and enter each item as required. Enter the survey participant’s name (Pinyin) in the “last name” box. In order to ensure the accuracy of the ID number, enter the ID number both in the “first name” and “identification” and then click F12 to return to the test interface.

3) Determining slow lung capacity

① Click “Spirometry Flow-volume” and operate according to the requirements of the pop-up dialog box. Then, click “OK” in the pop-up dialog box.

② Apply the seat, nose clip, and mouth piece to the survey participant. The survey participant should breathe quietly at least 5 times. Click F2 when the two quality control indicating columns dvT and dFRc on the right screen simultaneously appear as green. Let the survey participant deeply exhale to their residual capacity, then they should inhale deeply to their total lung capacity, and then finally exhale deeply once again to their residual capacity. Press F7 to stop and save when restoring normal tidal breathing.

③ Observe the data quality control and press F2 in order to begin new testing again. Repeat until at least 3 qualified tests have been obtained. The repeatability of this process should also meet the standards.

④ Quality Control and Repeatability Requirements:

No expiratory platform;

Errors from the first three lung capacity measurements at the end of tidal breathing before VC: <100ml;

Repeat the measurement at least 3 times. The VCmax difference should be < 5% or 0.2 L.

4) Determining Forced Vital Capacity

① Click F3 and start the forced vital capacity test.

② The survey participant should calmly breathe in and out at least 5 times. Let the survey participant quickly inhale to their total lung capacity, and then immediately exhale to their residual capacity with a maximum of explosive power. The two quality control indication columns TimeEx and EoT on the upper right screen should both appear as green. Then, have the survey participant deeply inhale to their total lung capacity and then restore their normal breathing. Press F7 to stop.

③ Observe the data quality control and press F3 in order to begin new testing. Repeat until at least 3 qualified tests have been obtained. The repeatability should also meet the standards.

④ Single Test Standard:

a) When the survey participant reaches their maximum expiratory effort, PEF peak will rapidly appear and the extrapolation capacity should be less than 5% FVC or 0.15L.

b) The descending curve should be smooth. The survey participant should exhale for at least 6 seconds. If the expiratory time is less than 6 seconds, the expiratory phase platform should appear in its time-volume curve and the time should be more than 2 seconds.

c) The expiration must have no interruption, no coughing, no leakage, and the tongue should never block the mouthparts. There should also be no glottis closure as this could affect the test.

⑤Repeatability Standard:

a) Repeat this measurement at least 3 times (generally no more than 8 times).

b) The difference between the optimal values and sub-optimal values for FEV1 and FVC should be less than 0.2L in acceptable tests.

c) The difference between the optimal values and sub-optimal values for PEF should be less than 0.67L/s in acceptable tests.

⑥ Result reporting: report the maximum values for FVC, FEV1 and FEV6. The maximum value of MMEF 75-25 will be the sum of FVC + FEV1, and the best value will be the measured value of the smooth curve.

5) Maximum Voluntary Ventilation Parameter:

① Click F4 and have the survey participant breathe quickly and deeply.

② A respiratory rate standard according to the ATS / ERS is: 90 to 110 beats /min.

6) Repeat the above steps when testing the next survey participant.

7) Bulk export of the test result data

① Click “SQL Export” at the upper right corner. Then, click “Next” in order to create a folder for the data to be exported to, and then choose the date range for the output data.

② Click “DB Export to Excel”, and then click “Import Data”. Finally, click “Export Data” to convert the exported data into Excel format.

2.1 Blood Biochemistry Test

2.1.1 Blood Collector Responsibilities:

The regulation team will designate one responsible person and the local team will designate one coordinator. Total number of blood collectors: 5-6.

1) Prepare blood for collection, and then manage and check the blood collection materials.

2) Complete all necessary blood collection in the morning. Note that all the operations should be performed in strict accordance with the operating procedures - especially the mixing process.

3) Two people will prepare the required materials for the next day during the afternoon. They will affix all appropriate labels on the blood collection tubes (blood RT label).

2.1.2 Responsibilities of the people transferring and separating the blood samples:

Serum is to be centrifugally separated by 1-2 people. These will be the transferring people:

1) Team members should arrive at the blood collection location (physical examination center) every morning.

2) Centrifuge one batch of blood biochemical samples every 30mins. Meanwhile, routine blood samples should be sent to the blood routine test room.

3) Divide the centrifuged blood biochemical samples into two parts. These should then be stored in the tubes under the cold chain.

4) Transfer the frozen samples for storage below -70°C for cryopreservation within 8 hours. Fill out the necessary sample transferring form.

5) Prepare the labels that will be used for the storage tubes the following day.

6) Pack the samples to be shipped to Beijing under cold chain.

2.1.3 Blood Sample Tester Responsibilities and Precautions:

One person will be in charge of the routine blood testing. There will also be one routine blood test assistant. 1-2 persons with inspection backgrounds should be added if blood typing is required.

1) Prepare instruments and required materials every morning.

2) Carry out quality control (once before work and once after work) every day. All of the original quality control data should be retained.

3) Fill out the relevant blood sample detection conditions in the transferring form.

4) When the samples arrive at the labs, organize them by number and sort.

5) Begin the test.

6) Enter the unique sample number.

7) If a detection delay occurs due to various conditions, promptly report this delay to the relevant personnel at the research group.

8) Print all of the test results (in duplicate) and hand them over to the designated person at the research group.

9) Submit all of the original data to the designated person at the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, within 15 days after the completion of all filed experiments.

2.1.4 Required biochemical and blood testing quality control personnel: 1 to 2

1) Organize comparative experiments and determine the correlation coefficient.

2) Evaluate all relevant software and hardware conditions in the laboratory.

3) On-site inspection: twice

4) Train for and simulate the on-site process.

5) Summarize and evaluate the sample transferring forms and internal quality control.

2.2 Questionnaire Survey

Affix the numbers to the new questionnaires before beginning the investigation. Personnel assignment, questionnaire filling, collection, sorting, and storing should all be properly performed during the investigation.