Investigating the Health Benefits of Indoor Air Filtration Among Children: a Randomized Crossover Trial in Jiaozuo, China
Phase: NA
Conditions: Protective Effect of Air Purifier on Children's Health
Sponsor: Tiantian Li

 
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Official Title: The Health Benefits of Indoor Air Filtration Among 
Children 
NCT Number: NCT04835337 
Document Date: 2024-09-12  
 

 
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Protocol Synopsis 
Title 
The Health Benefits of Indoor Air Filtration Among Children 
Intervention 
Classroom and household air purifiers 
Study Objectives 
This study aims to explore the health benefits of air purifiers on children's health. 
Study Design 
The study is a randomized, controlled, double-blind, crossover trial of a multi-
setting air purification intervention. 
Primary Endpoint(s) 
The primary outcomes are cardio-respiratory function including lung function, 
airway inflammation, blood pressure, heart rate variability, electrocardiograph, 
academic performances including standardized scores and relative ranking of 
scores.  
Secondary Endpoint(s)  
The secondary outcomes are metabolites in exhaled breath condensate, serum 
protein, cognitive function measured by Wisconsin Card Sorting Test, Attention 
Network Test, and N-BACK Test, other omics and targeted biomarkers of interest 
(e.g., CRP, 8-OHdG, etc). 
Accrual Objective 
105 
Study Duration 
9 months (from April 2021 to December 2021) 
Treatment Description 
Participants are divided into an intervention group and a control group according 
to the principle of cluster randomization. The intervention group is equipped with 
true-particulate air purifiers and the control group is equipped with sham-
purification devices in households and school classrooms. 
Inclusion Criteria 
(i) Fourth-grade students (10–12 years old) in primary school; (ii) no plans to 
change schools during the current school year; (iii) those who participate 
voluntarily and are able to undergo questionnaires and health checks; and (iv) 
those who have good compliance and accept the installation of an air purifier and 
PM2.5 monitoring equipment at home.  
Exclusion Criteria for Individual 
(i) transferring schools expected during the study period; (ii) having a chronic 
disease diagnosed by a doctor within the past year. 

 
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Glossary of Abbreviations ........................................................................................................................ 5 
1. Background and Rationale.................................................................................................................... 6 
2. Study Objectives ................................................................................................................................... 6 
3. Study Design ........................................................................................................................................ 6 
3.1 Description of Study Design ............................................................................................................... 6 
3.2 Primary and Secondary Outcomes...................................................................................................... 6 
3.3 Stratification, Randomization, and Blinding ...................................................................................... 6 
4. Participants and Sites ............................................................................................................................ 7 
4.1 Inclusion Criteria ................................................................................................................................ 7 
4.2 Exclusion Criteria for Participant ....................................................................................................... 7 
4.3 Sites .................................................................................................................................................... 7 
5. Known and Potential Risks and Benefits to Participants ...................................................................... 7 
6. Intervention .......................................................................................................................................... 7 
6.1 Intervention ........................................................................................................................................ 7 
6.2 Washout Period ................................................................................................................................... 8 
6.3 Biological sample collection and testing ............................................................................................ 8 
6.4 Criteria for discontinuing or modifying .............................................................................................. 8 
6.5 Strategies for adherence...................................................................................................................... 8 
6.6 Permitted or prohibited interventions ................................................................................................. 8 
7. Study Procedures .................................................................................................................................. 8 
7.1 Enrollment .......................................................................................................................................... 8 
7.2 Randomization .................................................................................................................................... 8 
7.3 Environmental exposure measurement ............................................................................................... 8 
7.4 Questionnaire ...................................................................................................................................... 9 
7.5 Physical Examination ......................................................................................................................... 9 
7.5.1 Height measurement ........................................................................................................................ 9 
7.5.2 Weight measurement ....................................................................................................................... 9 
7.5.3 Waist circumference measurement .................................................................................................. 9 
7.5.4 Hip circumference measurement ..................................................................................................... 9 
7.6 Respiratory health measurements ....................................................................................................... 9 
7.7 Blood pressure measurement ............................................................................................................ 10 
7.8 Heart rate variability measurements ................................................................................................. 10 
7.9 Electrocardiograph measurements .................................................................................................... 10 
7.10 Academic achievement measurements ........................................................................................... 10 
7.11 Cognitive executive function measurements .................................................................................. 10 
7.11.1 The Attention Network Test ......................................................................................................... 10 
7.11.2 The Wisconsin Card Sorting Test ................................................................................................. 11 
7.11.3 The N-BACK Test ....................................................................................................................... 11 
8.Statistical Considerations and Analytical Plan .................................................................................... 11 
8.1 Sample size calculation .................................................................................................................... 11 
8.2 Analytical models ............................................................................................................................. 12 
8.3 Analytical tools ................................................................................................................................. 12 
9. Criteria for Participant and Study Completion and Premature Study Termination ............................. 12 
9.1 Participant Completion ..................................................................................................................... 12 

 
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9.2 Participant Stopping Rules and Withdrawal Criteria ........................................................................ 12 
10. Protocol Deviations .......................................................................................................................... 12 
11. Ethical Considerations ...................................................................................................................... 12 
12. Quality Control and Quality Assurance ............................................................................................ 12 
12.1 Quality control during site surveys ................................................................................................. 12 
12.2 Quality control of questionnaires.................................................................................................... 13 
12.3 Quality control of physical examinations and on-site laboratory tests ........................................... 13 
References .............................................................................................................................................. 14 
 
 
 

 
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Glossary of Abbreviations 
FEV1 
Forced expiratory volume in the first second 
FVC 
Forced vital capacity 
PEF 
Peak expiratory flow 
FEF25-75% 
Forced expiratory flow at 25–75% of FVC 
MEF75% 
Maximal expiratory flow at 75% of FVC 
MEF50% 
Maximal expiratory flow at 50% of FVC 
MEF25% 
Maximal expiratory flow at 25% of FVC 
FeNO 
Fractional exhaled nitric oxide 
FeCO 
Fractional exhaled carbon monoxide 
COHb 
Carboxyhemoglobin 
TC 
Total correct  
%TC 
Percentage total correct 
TE 
Total errors 
%TE 
Percentage total errors 
PR 
Perseverative responses 
%PR 
Percentage perseverative responses 
PE 
Perseverative errors 
%PE 
Percentage perseverative errors 
NPE 
Nonperseverative errors 
%NPE 
Percentage nonperseverative errors 
CLR 
Conceptual level responses 
%CLR 
Percentage conceptual level responses 
CC 
Categories completed 
FM 
Failure to maintain set 
SBP 
Systolic blood pressure 
DBP 
Diastolic blood pressure 
MAP 
Mean arterial pressure 
PP 
Pulse pressure 
SDNN 
Standard deviation of all normal-to-normal intervals 
rMSSD 
Root mean square of successive differences between adjacent normal cycles 
pNN50 
Percentage of adjacent NN interval differences greater than 50 ms 
VLF 
Very low frequency 
LF 
Low frequency 
HF 
High frequency 
LF/HF 
Ratio of LF to HF 
PR interval 
The interval of the beginning of the P wave to the beginning of the QRS complex 
QRS duration 
The interval of the beginning of Q wave to the end of the S wave 
QT interval 
The interval from the onset of the QRS complex to the end of the T wave 
QTc interval 
The QT correction for rate 
RV5 
Amplitude of the R wave in lead V5 
SV1 
S-wave depth in lead V1 
RV5+SV1 
The sum of the R-wave amplitude in lead V5 and the S-wave amplitude in lead V1 

 
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1. Background and Rationale  
Previous studies have suggested that fine particulate matter (PM2.5) has adverse effects on children’s 
respiratory, cardiovascular, and cognitive health. However, it is not clear whether reduced PM2.5 
concentration by indoor air purifiers is beneficial for the multi-system health of school-age children. 
2. Study Objectives 
This study aims to explore the health benefits of air purifiers on children's health. 
3. Study Design  
3.1 Description of Study Design  
A cluster-randomized, double-blind, crossover trial of a multi-setting (classroom and bedroom) air 
purification intervention is conducted in Mengzhou City, Henan Province, China, from April 2021 to 
December 2021. Children aged 10–12 years old (fourth grade) are recruited as subjects. Participants are 
divided into an intervention group and a control group according to the principle of cluster randomization. 
The intervention group is equipped with true-particulate air purifiers and the control group is equipped 
with sham-purification devices both in bedrooms and school classrooms. The two groups receive 
alternating treatments of true or sham purification in a random order, with each study period separated 
by a washout period of over two months. A linear mixed-effects model is used to explore the effects of 
purification intervention. Ambient PM2.5 and indoor PM2.5 exposure concentrations are monitored in the 
study. 
3.2 Primary and Secondary Outcomes  
The primary outcomes for respiratory health are lung function indicators, including forced expiratory 
volume in 1 s (FEV1), forced vital capacity (FVC), FEV1/FVC ratio, peak expiratory flow (PEF), forced 
expiratory flow at 25–75% of FVC (FEF25-75%), maximal expiratory flow at 75% of FVC (MEF75%), 
maximal expiratory flow at 50% of FVC (MEF50%), and maximal expiratory flow at 25% of FVC 
(MEF25%); changes of FeNO; Changes of FeCO. The primary outcomes for cardiovascular health are 
blood pressure indicators, including systolic blood pressure (SBP), diastolic blood pressure (DBP), mean 
arterial pressure (MAP) and pulse pressure (PP); heart rate variability indicators, including standard 
deviation of all normal-to-normal intervals (SDNN), the root mean square of successive differences 
between adjacent normal cycles (rMSSD), the percentage of adjacent NN interval differences greater 
than 50 ms (pNN50), very low frequency (VLF), low frequency (LF), high frequency (HF), the ratio of 
LF to HF (LF/HF); and electrocardiograph parameters, including the interval of the beginning of the P 
wave to the beginning of the QRS complex (PR interval), the interval of the beginning of Q wave to the 
end of the S wave (QRS duration) , the interval from the onset of the QRS complex to the end of the T 
wave (QT interval), the QT correction for rate (QTc interval), amplitude of the R wave in lead V5 (RV5), 
S-wave depth in lead V1 (SV1) and the sum of the R-wave amplitude in lead V5 and the S-wave 
amplitude in lead V1 (RV5+SV1). The primary outcomes for cognitive health are academic performances, 
including standardized scores and relative ranking of scores. 
The secondary outcomes include the metabolites in exhaled breath condensate, serum protein, the 
indicators measured by the Wisconsin Card Sorting Test, Attention Network Test, and N-BACK Test. 
Furthermore, other omics and targeted biomarkers of interest (e.g., CRP, 8-OHdG, etc) are also 
considered. 
 
3.3 Stratification, Randomization, and Blinding 
Students are allocated by class cluster randomization to the intervention group or the control group. The 
intervention assignment is handed over to independent trained professionals, who do not participate in 

 
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any other processes of the trial. Confidentiality of the intervention is maintained for the researcher and 
participants to achieve double blindness. 
4. Participants and Sites 
4.1 Inclusion Criteria  
Inclusion rules for the study participants are: 
1. Fourth-grade students (10–12 years old) in primary school 
2. No plans to change schools during the current school year 
3. Those who participate voluntarily and are able to undergo questionnaires and health checks 
4. Those who have good compliance and accept the installation of air purifiers and PM2.5 monitoring 
equipment at home 
4.2 Exclusion Criteria for Participant  
The exclusion criteria are as follows:  
1. Expecting to transfer schools during the study period  
2. Having a chronic disease (including asthma, childhood diabetes, childhood hypertension, behavior-
related diseases, etc.) diagnosed by a doctor within the past year. 
4.3 Sites  
In 2020, the average annual PM2.5 concentration of 56 μg/m3 in Jiaozuo City still exceed the annual 
average PM2.5 limit of 35 μg/m3 1. In addition, there are various sources of air pollution in Jiaozuo, 
including agricultural, industrial and transportation sources. Therefore, Jiaozuo City, Henan Province is 
selected as the study area. 
5. Known and Potential Risks and Benefits to Participants 
The medical examination is carried out by professional staff and is demonstrated and explained before 
the measurements, which does not cause health risks and discomfort. All information and the samples 
tested about the subject are kept strictly confidential and are only used for this project. Participation in 
the survey is voluntary, and the rights of respondents who do not agree to participate in this survey, or 
withdraw at any time after the start of the survey, are not affected in any way. 
Respondents receive a feedback report on the results of the medical examination, an air pollution 
exposure monitoring report, personalized protection advice, and a small gift to thank for participating. 
6. Intervention 
6.1 Intervention 
The intervention group is equipped with purifiers with true-purification (i.e., with high-efficiency 
particulate air (HEPA) filter), and the control group with sham-purification purifiers (without HEPA 
filter), which are installed in the classrooms and households of both groups. In the school environment, 
an air purification efficiency of 850 m3/h (LEXY, KJ901) and a fresh air ventilation with the purification 
efficiency of 320 m3/h (AIRTAO, AT320) are placed in the front of the classroom and the air purification 
equipment is the same as the back of classroom. In the home environment, one air purifier with the 
purification efficiency of 220 m3/h (Haier, KJ210F- A180A) is installed in each of the children's bedroom.  
Two different treatments are employed, including true-purification and sham-purification, in random 
order with a washout period of over 2 months. The air purification intervention is performed by trained 
professionals on and off at regular intervals each day to ensure that the air purification unit is on during 
the time the children are in the indoor environment. In the event of offline situations such as those caused 
by poor internet access, the survey respondents are contacted and communicated with promptly. A health 
examination is conducted at the beginning and end of each study period, and a questionnaire is used to 
collect basic information about the respondents. We also measured the environmental factors including 

 
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PM2.5 concentration, temperature, and relative humidity. 24 time-weighted mean PM2.5 concentrations 
are calculated as personal daily exposures for further analysis. 
6.2 Washout Period 
A total of four surveys are conducted during the study, and the two sets of purifiers are swapped before 
the third survey to achieve the purpose of elution. 
6.3 Biological sample collection and testing  
The trained medical staff collect exhaled breath condensate (EBC) before and at the end of each 
intervention period. EBC samples are stored in a -80°C refrigerator before metabolomics analysis. After 
the cardiovascular indicator measurements, medical staff collect 6 ml peripheral venous blood. Children 
are instructed to fast before the blood collection. The blood samples are heated in a water bath and 
centrifuged, after which the upper serum is extracted in EP tubes and stored in the -80°C freezer before 
proteomics testing and analysis. Urine, saliva, oral cells, oral swabs, nasal swabs, skin swabs, nails and 
feces samples are collected during each visit and stored in the -80°C refrigerator before laboratory testing. 
6.4 Criteria for discontinuing or modifying  
1. The intervention is ended if it is disruptive to the classroom. 
2. The participation is voluntary and participants can be withdrawn at any time. Their rights are not 
affected in any way. 
6.5 Strategies for adherence 
During the investigation, all tests are conducted by professionally trained medical personnel and all 
computer-based tests are conducted for children of that age and do not cause any adverse effects. 
The physical examination report and environmental test report are returned timely so that the survey 
subjects can better understand their own health status and environmental exposure, deepen their 
understanding of the test and better cooperate. 
6.6 Permitted or prohibited interventions 
During the intervention study period, it is recommended to have less window opening ventilation to 
ensure the effectiveness of the air purification intervention. If the subject takes medication regularly, it 
can be carried out normally according to the doctor's instructions. 
7. Study Procedures 
7.1 Enrollment 
The research subjects are surveyed through the screening questionnaire, combined with the above 
inclusion and exclusion criteria, and the research subjects with good compliance and willingness to 
complete the study according to the requirements of the project are selected. 
7.2 Randomization 
Students are allocated by class cluster randomization to the intervention group or the control group. The 
intervention assignment is handed over to independent trained professional, who does not participate in 
any other processes of the trial. Confidentiality of the intervention is maintained for the researcher and 
the survey participants to achieve double blindness. 
7.3 Environmental exposure measurement 
Exposure assessment of PM2.5 is measured based on the daily activity pattern of students, including 
indoor measurement (residence and classroom) and ambient measurement during their commute to 
school. Indoor PM2.5 exposure data is collected by installing Hike B3-L2 (Beijing Haike Zhidong 
Technology Development Co. LTD, China) in the study children's bedrooms and classrooms. The 
monitors are installed on a platform at least 1m from the purifier and approximately 1.5m above the floor. 
The monitors collect ambient PM2.5 concentrations every five minutes, and we collect real-time PM2.5 

 
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exposure levels in bedrooms and classrooms for the duration of the study. Secondly, real-time outdoor 
PM2.5 data is collected by installing a micro atmospheric environment monitoring station (Capri 
Environmental Technology Ltd., Shenzhen, Guangzhou) on the roof of the school. Meanwhile, this micro 
atmospheric environment monitoring station also monitors and collects outdoor concentrations of PM10 
(μg/m3), NO2 (μg/m3), SO2 (μg/m3), CO (mg/m3), and O3 (μg/m3). Finally, twenty-four-hour time-
weighted mean PM2.5 concentrations are calculated as personal daily exposures based on PM2.5 
concentrations in different environments and twenty-four-hour time-activity patterns of survey 
respondents. The monitoring particle concentration data has undergone quality control and calibration 
following the previous approach 2. Temperature and relative humidity are collected via the HOBO® MX 
Temp/RH Data Logger MX1101 (Onset, USA). 
7.4 Questionnaire 
The questionnaire is in the form of an electronic questionnaire (questionnaire.com). Professional staff 
train and qualify classroom teachers to supervise parents and students to complete the parent 
questionnaire, student questionnaire and the 24h time-activity questionnaire. Information collected 
during the baseline survey does not have to be collected for the other surveys if there is no significant 
change during the other surveys. 
The parent questionnaires are conducted on the parents of the students, and the survey included basic 
family information, living environment, risk perception, and child evaluation scale (parent volume). The 
children's questionnaires are conducted on students, and the survey included risk perceptions and the 
Children's Evaluation Scale (Children's Volume). The 24h time-activity questionnaires are conducted on 
students, documenting their activity patterns and diet behaviors. 
7.5 Physical Examination 
7.5.1 Height measurement 
Height measurement is conducted by using a tape measure fixed on the wall. Before the measurement, 
the subjects are required to take off their shoes, hats and outerwear, stand on the pedal and maintain an 
upright posture. After the measurement is completed, the accurate measurement results are filled into the 
physical examination form. 
7.5.2 Weight measurement 
Weight measurement is performed using an electronic weight scale. Measurements are taken in a quiet, 
spacious environment with a level and firm floor. After the measurement is completed, the results are 
entered into the medical examination form in a timely manner. 
7.5.3 Waist circumference measurement 
Waist circumference is measured using a waist tape. The subject stands upright, with the abdomen 
relaxed, arms hanging down naturally, feet combined and weighted evenly, exposing the abdominal skin. 
After completing the measurement, the results are entered into the physical examination form. 
7.5.4 Hip circumference measurement 
Hip circumference measurement is performed using a waist circumference tape. The soft ruler is lightly 
pressed against the skin, passing through the highest point of the buttocks and circling around the body. 
Measurements are carried out twice, with a difference of no more than 1 cm, and the average value should 
be recorded in the physical examination form. 
7.6 Respiratory health measurements 
Pulmonary function measures are performed by a professional medical device. Before the pulmonary 
function test, subjects practiced several times with the guidance of professional staff. During the 
examination, each subject is in a sitting position clamp the nose clip, and repeat the test, with the best 

 
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result as the criterion. The indicators include FEV1, FVC, FEV1/FVC, PEF, FEF25-75%, MEF75%, MEF50%, 
MEF25%. A NIOX VERO Sensor is used to measure fractional exhaled nitric oxide (FeNO) as a biomarker 
for airway inflammation. After deep breathing, the subjects gently inhaled into the device. The instrument 
shows the FeNO level of the subjects. Use PICO to measure fractional exhaled carbon monoxide (FeCO) 
as a biomarker. After deep breathing and holding the breath for 15 seconds, the subjects gently inhaled 
into the device. The instrument shows the FeCO and COHb level of the subjects. 
7.7 Blood pressure measurement 
After a 5-minute rest in a quiet environment, the child-appropriate BP cuff is wrapped around the left 
upper arm of the children to measure BP using the Omron electronic sphygmomanometers (OMROM, 
J751). BP is measured at 2-minute intervals, with a total of three measurements taken. If the difference 
in BP values between the last two measurements exceeds 5 mmHg, additional measurements are 
performed. Each child is allowed to measure BP at least three times and up to five times. The SBP, DBP 
and heart rate are recorded; the mean arterial pressure (MAP) and pulse pressure (PP) are calculated. The 
HOBO thermo-hygrometer is set up in the medical examination room to monitor the temperature and 
relative humidity. 
7.8 Heart rate variability measurements 
A 3-minute comprehensive assessment is conducted to measure HRV using the handheld 
electrocardiographic recorder-CarePatch (ECG-H01, Hangzhou Proton Technology Co., Ltd., China). 
The participants are required to rest quietly for 2 minutes before the measurement. The professional staff 
activate the Bluetooth on the terminal device (Thinkpad) and enter the “CarePatch” (a WeChat Mini 
Program), then guide the children to hold the electrodes with their thumbs at moderate pressure for a “3-
minute comprehensive examination”. During this process, children are prohibited from speaking or 
physical activity. 3 time-domain indicators, including standard deviation of all normal-to-normal 
intervals (SDNN), the root mean square of successive differences between adjacent normal cycles 
(rMSSD), the percentage of adjacent NN interval differences greater than 50 ms (pNN50); and 4 
frequency-domain indicators, including very low frequency (VLF), low frequency (LF), high frequency 
(HF), the ratio of LF to HF (LF/HF) are selected. 
7.9 Electrocardiograph measurements 
The staff use a 12-lead electrocardiogram (ECG) to record the values of PR interval (the interval of the 
beginning of the P wave to the beginning of the QRS complex), QRS duration (the interval of the 
beginning of Q wave to the end of the S wave), QT interval (the interval from the onset of the QRS 
complex to the end of the T wave), QTc interval (the QT correction for rate), RV5 (amplitude of the R 
wave in lead V5), SV1 (S-wave depth in lead V1), and RV5+SV1 (the sum of the R-wave amplitude in 
lead V5 and the S-wave amplitude in lead V1). 
7.10 Academic achievement measurements 
Academic achievement is collected at the beginning and end of each study period. The evaluation of 
academic performance consisted of standardized scores (maths, chinese and total) and the relative 
ranking of the scores within the grade. Standardized grades are converted from raw grades by z-scores 
according to the number of exams and class in order to eliminate differences due to different exams; 
rankings are converted from place rankings of raw grades, reflecting the relative value of grades. 
7.11 Cognitive executive function measurements 
7.11.1 The Attention Network Test  
The Attention Network Test (ANT) is used to measure an individual's ability to acquire and maintain a 
state of alertness to a certain type of information or target, selective attention to externally useful 

 
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information, and processing of conflicting information. The ANT is a computer-based, annotated test that 
is typically used to measure different behavioral aspects of attention, and it is based on the Attention 
Network Theory. 
The ANT is divided into two phases: a practice and a formal game. The practice phase consists of 10 
questions that are used to familiarize with the operation. After completing the exercise, the system 
prompts the start of the formal game. The formal game is divided into three parts, with the option of 
resting or continuing directly between each part. In this test, the subject's task is to feed a hungry little 
fish located right in the middle by judging the direction of its mouth. When the mouth of the small fish 
is facing left, press the left button; when the mouth of the small fish is facing right, press the right button. 
During the test, the subject is asked to look intently at the gaze point (+) on the screen as it will indicate 
where the minnow is likely to appear. Also, watch out for the left and right buttons to show clear positions 
on the screen. 
7.11.2 The Wisconsin Card Sorting Test 
The Wisconsin Card Sorting Test (WCST) is used to measure children's cognitive executive function. 
WCST is a widely used executive function task proposed by Berg (1948) to assess abstract reasoning 
and cognitive change in the context of changing environmental contingencies and the ability to cope with 
changing environmental contingencies3-5. 
The study uses computerized WCST software (Kunming Quanlu Technology Co., Ltd) and is 
administered to children by professional investigators in a quiet classroom to minimize external 
distractions. WCST consist of four key cards and one hundred and twenty-eight stimulus cards which 
depicted four shapes, four colors and four numbers in response. The 4 key cards with the upper features 
are placed in front of the subject in order from left to right. The stimulus cards are then presented to the 
subject one at a time and the subject is instructed to match the stimulus card with one of the key cards 
using either the pictorial, color, or number consistency rules. Subjects are only told whether each 
response is correct or incorrect. The matching rule is changed once the subject has successfully matched 
the stimulus card to the key card in succession using the matching rule. The WCST underwent extensive 
set transfer (i.e., matching rule) across the three categories (color, pictorial and numerical). 
7.11.3 The N-BACK Test 
The N-back test is a classic working memory task used to assess an individual's working memory 
capacity and attentional control. The N-back test consists of a 1-back and a 2-back, and each level of the 
test includes three tests: a colour contrast, a shape contrast, and a position contrast, so there are a total of 
six tests, namely the 1-back colour test, the 1-back shape test, the 1- back position test, 2-back colour 
test, 2-back colour test, and 2-back colour test. In this test, the subject's task is to compare the position, 
colour, or shape of the currently displayed stimulus card with that of a stimulus card in a previous or 
alternate position. If they are consistent, click the "Confirm" button on the right side of the interface; if 
they are not consistent, do not click any button. Subjects are asked to focus their attention, constantly 
update and maintain the stimulus information, and make accurate judgments within a short period of 
time, this task is designed to examine the short-term memory ability of the subjects as well as their ability 
to update and maintain the information. 
8. Statistical Considerations and Analytical Plan 
8.1 Sample size calculation 
In this randomized, double-blind crossover study, the sample size is determined according to the relative 
literature on respiratory outcome6-8, cardiovascular outcome9-10, and cognitive function outcome11 and 
confirmed in PASS15.0 software with the statistical significance of 0.05. Combined with the supporting 

 
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evidence and the requirement of sample size calculated in PASS15.0, and considering the typical number 
of students in each class and the possible withdrawal, we ultimately plan to recruit 100 children for this 
study. 
8.2 Analytical models 
We use a linear mixed-effects model to estimate the effects of air purification intervention or per 
interquartile range (IQR) decrease in personal PM2.5 exposure on several functional indicators and 
targeted biomarkers of children’s health. To account for the impact of varied outcome levels at baseline, 
the absolute or relative change in outcome level is calculated as the difference between post-intervention 
and pre-intervention, comparing the change in health outcomes between the intervention and control 
groups after the intervention. We further adjusted for the confounders including sex, age, body mass 
index (BMI, kg/m2), temperature (℃), relative humidity (%), etc. A random intercept for each participant 
is included in the models to account for the repeated measurements.  
In addition, we plan to perform metabolomics analysis of EBC samples using ultra-high performance 
liquid chromatography-tandem mass spectrometry and proteomic analysis of serum samples with 4D-
label free technology. Besides, other omics analyses and targeted biomarkers of interest are planned to 
further explore the potential biological mechanisms. 
8.3 Analytical tools 
All statistical analyses are carried out in R software. Statistical tests are two-sided, and p < 0.05 is 
considered statistically significant. 
9. Criteria for Participant and Study Completion and Premature Study Termination 
9.1 Participant Completion 
Study participants who meet the inclusion-exclusion criteria and complete all interventions during the 
study. 
9.2 Participant Stopping Rules and Withdrawal Criteria  
Subjects may terminate the study early for the following reasons:  
1. Study participants may voluntarily withdraw at any point in the course of the study.  
2. Subject "lost to follow-up"  
3. Participant died.  
4. If any clinical adverse events, other medical conditions, or circumstances occur that make it not in 
the best interest of the subject to continue participating in the study, the study subject will discontinue 
further study intervention.  
5. The intervention is determined to be too disruptive in the classroom or school, and the school or 
classroom staff requested to withdraw from the study. 
10. Protocol Deviations  
The investigator conducts the study in strict accordance with the protocol and does not allow deviations 
from the protocol. In the event of any changes, disagreements, or deviations during the course of the 
study, corrective actions are developed by the site and implemented immediately. 
11. Ethical Considerations 
Participation in the trial is voluntary and the subject may withdraw from the study at any time for any 
reason. Subjects (or their legal representatives) participating in the trial are issued and read an informed 
consent form, signed and dated informed consent form prior to any study procedures, and the informed 
consent materials are presented in the participant's primary language. 
12. Quality Control and Quality Assurance  
12.1 Quality control during site surveys 

 
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Before the on-site investigation, each technical person responsible for exposure monitoring, 
questionnaire survey and computer testing should participate in technical training and pass the training 
assessment. Each technical person should be familiar with the division of labor and tasks and 
responsibilities. During the on-site investigation, all technical personnel should carry out various tasks 
in strict accordance with the investigation process and operating procedures. When encounter a problem, 
one should report it to the relevant person in charge and ask for help as soon as possible. The replacement 
of research subjects and monitoring instruments should be recorded, and the monitoring data obtained 
should be handed in a timely manner, and the data collector should check the quality of the monitoring 
data in a timely manner, and feedback to the investigator for correction if necessary. 
12.2 Quality control of questionnaires 
First of all, make sure that the questionnaire is filled in on the day of the physical examination, and the 
questionnaire quality review should be completed on the day of the questionnaire survey. Each technical 
person shall review whether the questionnaire filled in by the respondent assigned to him is complete 
and correct, ensure that the completion date is correct, and exchange the audit with other technical 
personnel after completing the audit within the group, and the specific audit content includes, whether 
the information of the respondent is complete, whether there are outliers, the unit is not uniform, the 
logic is wrong, and the phenomenon of blind filling and filling in is random. If the subjects find a problem, 
they should promptly report to the school teacher, ask them to verify and correct the information, or 
contact the parents to make corrections. Research group sent technical personnel to supervise and provide 
technical support for the investigation site. 
12.3 Quality control of physical examinations and on-site laboratory tests 
Laboratory tests use blank samples for quality control. 
 
 

 
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