A Randomised Controlled Trial of Intra-Operative Cell Salvage During Caesarean Section in Women Needing Blood Transfusion
Phase: NA
Conditions: Blood Transfusion
Sponsor: Wenzhou Medical University

 
0 
 
 
A Randomised Controlled Trial of Intraoperative Cell 
Salvageduring Caesarean Section in Women at Risk of 
Haemorrhage in China 
 
 
Keywords：Cell Salvage during Caesarean Section (CSCS) 
 
 
Protocol Version：beta 2.1 2017/3/12 
IRB Approval Date: 2017/3/19 
Translation Date: 2017/12/15 
 
 
 
 
 
 
 
 
 
Sponsors：Fang Gao, Qingquan Lian 
Principal investigators：Fang Gao, Qingquan Lian 
Major investigators：Ting Li, Mingping Hu 
 
 
 
Privacy Statement 
This document contains the clinical research ideas, data and other 
relevant information. All rights reserved by the Second Affiliated Hospital of 
Wenzhou Medical University. We allow the research members and ethics 
committees of participating research units, and other clinical research related 
institutions to read this document. Without the consent of the leader unit, 
please do not leak the program! 

 
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Contact information of test institution(s) and the respective 
major investigators:  
 
1. 
Institution Name: The Second Affiliated Hospital of Wenzhou Medical University 
Institution Address: 109 West Xueyuan Rd, Lucheng District, Wenzhou, Zhejiang 
 
Name of Principal Investigator: Ting Li 
Phone Number: 13587876896 
Email: liting1021@aliyun.com 
 
2. 
Institution Name: The First Affiliated Hospital of Wenzhou Medical University 
Institution Address: Anesthesia Department of The First Affiliated Hospital, Ouhai 
District, Wenzhou, Zhejiang 
 
Name of Principal Investigator: Xuzhong Xu 
Phone Number: 13706657799 
Email: xuzhong2263.net 
 
3. 
Institution Name: Wenzhou People’s Hospital 
Institution Address: 57 Canghou Street, Wenzhou, Zhejiang 
 
Name of Principal Investigator: Yanqin Wu 
Phone Number: 13858866092 
Email: wyq88533587@tom.com 
 
4. 
Institution Name: Taizhou Hospital of Zhejiang Province 
Institution Address: 150 Ximen Street, Linhai, Zhejiang 
 
Name of Principal Investigator: Mingcang Wang 
Phone Number: 13757616856 
Email: wangmc@tzhospital.com 
 
5. 
Institution Name: Lishui City People’s Hospital 
Institution Address: 15 Dazhong Street, Liandu District, Lishui, Zhejiang 
 
Name of Principal Investigator: Lihua Fan 
Phone Number: 18957090898 
Email: flh2222@163.com 
 
6. 
Institution Name: The Central Hospital of Lishui City 
Institution Address: 289 Kuocang Rd, Lishui, Zhejiang 
 
Name of Principal Investigator: Lipei Lei 
Phone Number: 13905883848 
Email: lei.lp@tom.com 
 
 

 
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Contact information of the research protocol author: 
 
1. 
Institution Name: The Second Affiliated Hospital of Wenzhou Medical University 
Institution Address：109 West Xueyuan Rd, Lucheng District, Wenzhou, Zhejiang 
Author Name: Ting Li 
Phone: 13587876896, 0577-88002990 
Email: liting1021@aliyun.com 
 
2. 
Institution Name: The Second Affiliated Hospital of Wenzhou Medical University 
Institution Address：109 West Xueyuan Rd, Lucheng District, Wenzhou, Zhejiang 
Author Name: Mingping Hu 
Phone: 13705880737, 0577-88002990 
Email: hmp2008mz@163.com 

 
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Statement of Principal Investigator: 
 
I hereby sign that I have read and understand this study, agreed the contents of the 
study, and will strictly follow all the provisions of this study, ‘Good Clinical Practice 
of Drugs’ and the implementation of relevant state laws and regulations. I will provide 
a copy of this document to all investigators who are responsible for this study, and 
discuss with them about the study and the related information to ensure that they are 
fully familiared with the drug we study, and how the study is conducted. 
 
 
Name of Principal Investigator: Qingquan Lian 
 
Signature of Principal Investigator: _________________ 
 
Date: ___________________________ 
 
 
 
Name of Principal Investigator: Fang Gao 
 
Signature of Principal Investigator: _________________ 
 
Date: ___________________________ 

 
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Statement of Statistical Analyst: 
 
I hereby sign that I have read and understand this study, agreed the contents of the 
study, and will strictly follow all the provisions of this study, ‘Good Clinical Practice 
of Drugs’ and the implementation of relevant state laws and regulations. I am 
responsible for the data management and statistical analysis of this clinical research. I 
will conscientiously perform the duties of statisticians, follow the relevant guidelines 
for clinical statistics, and ensure that the statistical results are real, scientific and 
standardized. 
 
 
Name of Statistical Analyst: Zuokai Xie 
 
 
Signature of Statistical Analyst: _________________ 
 
 
Date: ___________________________ 

 
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Statement of Major Investigator(s) of Test Institution(s): 
 
I have carefully read the study protocol, and have agreed to follow this protocol to perform this 
clinical study. 
 
1) I understand that the study protocol needs to be approved by the Ethics Committee, signed 
and approved before implementation. During the study, any change makes to this study 
protocol need to be approved by the Ethics Committee; any serious adverse event occurs 
in the study need to be promotly reported to the Ethics Committee. 
2) I know that according to the Regulations on ‘Good Clinical Practice of Drugs’ and the 
Helsinki Declaration (2000), before each patient's enrollment in the study, it is my 
responsibility to present a written form to give him/her or his/her designated representative 
a complete and overall introduction of the purpose, procudures and possible risks in this 
study. Patients should be aware that they have the right to withdraw from the study at any 
time. Each patient or relative must be given a written version of informed consent before 
being enrolled. It is my responsibility to have every patient receive the informed consent 
before enrolling the study and the informed consent should be retained for future reference 
as a clinical research document. 
3) I will enroll subjects who meet the criteria of this study. I will be responsible for making 
medical decisions related to clinical research, and promise that subjects will receive timely 
treatment once any adverse event occurs during the clinical trials. I know the requirement 
of correctly reporting serious adverse events, so I will be record and report these incidents 
based on these requirements. 
4) I promise that the data loaded into the case report form is accurate, complete, prompt and 
legal. I verify that the signature on each completed case report form indicates that I have 
carefully reviewed each page and took full responsibility for the relevant content. 
5) I accept the scrutiny or inspection of the CRA or CRAs sent or entrusted by the sponsor, 
and I accept the inspection of the drug regulatory authority to ensure the quality of the 
clinical trial. 
6) I know that the information in this trial is confidential. I hereby certify that I will not 
disclose any information in this study without the consent of the sponsor, except as required 
by the State Food and Drug Administration or other regulatory agencies. 
7) I will provide a resume before the the study starts, submit it to the Ethics Committee for 
reviewing, and may submit it to the drug regulatory authority. 
 
 
Name of major investigator: _________________ 
 
Signature of major investigator: _________________ 
 
Date: ___________________________ 

 
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Table 1：Proposal 
Title 
A Randomised Controlled Trial of Intraoperative Cell Salvage 
during Caesarean section in women at risk of 
haemorrhage in China 
Abbreviation 
Cell Salvage during Caesarean Section- CSCS 
Version 
Version 2.1 dated 2016/2/12 
Method 
A randomized, controlled, single-blind and multi-centre clinical 
intervention trial with cost-effective analysis. 
Trial Duration 
2 years recruitment 
6 months data analysis 
6 months write dissertation 
(3 years in total) 
Trial Center(s) 
6 Centers of Obstetric who are eligible for clinical trials and are 
eligible for intraoperative cell salvage: 
1. The Second Affiliated Hospital of Wenzhou Medical University 
2. The First Affiliated Hospital of Wenzhou Medical University 
3. Wenzhou People’s Hospital 
4. Taizhou Hospital of Zhejiang Province 
5. Lishui City People’s Hospital 
6. The Central Hospital of Lishui City 
Objectives 
• To determine the difference of blood loss postoperation in 
whether use of intraoperative cell salvage during caesarean 
section, or current practice of allogous transfusion. 
• To look into and compare whether there is any adverse 
effects or reaction after IOCS or allogous transfusion. 
• To determine if the routine use of IOCS during CS, in 
women at risk of haemorrhage, is cost effective in 
comparison to current practice. 
Estimated number 
of subjects 
120 cases 

 
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Objects 
Inclusion Criteria: 
• 18 years of age or elder 
• delivery by elective or emergency caesarean section with 
an identifiable increased risk of haemorrhage 
• ability to provide informed consent 
• needing for blood transfusion during CS 
Exclusion Criteria: 
• with blood transfusion history before the CS 
• Maternal preoperative Hemoglobin(Hb) < 70g/dL 
•  Maternal Rh(Rhesus Macacus) negetive blood type 
• Sickle cell disease 
• contraindicated to intraoperative cell salvage e.g. 
abdominal cancer, abdominal infection, intrauterine 
infection with blood transfusion history before the CS 
• Maternal 
preoperative 
(Prothrombin 
Time)PT 
or 
(activated partial thromboplastin time) APTT is 1.5 times 
greater than normal and above; or preoperative 
platelet(Plt) < 50*109L 
•  Cultural or social beliefs contraindicating blood 
transfushion, e.g. Jehovah’s witnesses18 years of age or 
elder 
• Significant antibodies making it difficult to find cross 
matched blood for transfusion 
• ability to provide informed consent 
• Joined other clinical trial within 3 months. 
• Investigator thinks maternal is not suitable to participate 
Statistical Method use safety dataset to analyze the security, and use Full Analysis 
Set(FAS) and Per Protocol Set(PPS) to analyze the efficacy 
accordingly. Use SAS9.13 for coding analysis 
 
 

 
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Contents 
1 
Introduction ............................................................................................................14 
1.1 
Background ...............................................................................................14 
1.2 
CSCS ..........................................................................................................14 
1.3 
Risk and Benefit of CSCS ......................................................................14 
1.3.1 Risk .......................................................................................... 11 
1.3.2 Benefit ..................................................................................... 12 
1.4 
Set Up .........................................................................................................14 
1.5 
Trial Feasibility ..........................................................................................14 
2 
Objectives and Design .......................................................................................14 
2.1 
Study Objects............................................................................................14 
2.1.1 Primary Objective .............................................................................14 
2.1.2 Seconday Objective .........................................................................14 
2.2 
Study Design .............................................................................................14 
3 
Subjects Screening .............................................................................................15 
Subjects: Woman who are admitted to a participating labor ward who fulfil all 
the following criteria will be eligible to be randomized. .......................................15 
3.1 
Eligible Criteria .........................................................................................15 
3.1.1 Inclusion Criteria ...............................................................................15 
3.1.2 Exclusion Criteria ..............................................................................15 
3.2 
Midway-Withdraw of Subjects ...............................................................16 
3.3 
Data Collection and Follow-up for Midway-Withdraw Subjects .....16 
4 
Study Procedure ...................................................................................................17 
4.1 
Flow Chart of Study .................................................................................17 
4.2 
Informed Consent Acquisition Process ...............................................17 
4.2.1 Elective Cesarean Section .............................................................17 
4.2.2 Emergency Cesarean Section .......................................................18 
4.3 
Patient Screening Process ....................................................................18 
4.4 
Randomization Process .........................................................................18 
4.5 
Methods of Minimizing Risk and Bias .................................................19 
4.6 
Preparation and Support for Medical Staff in the Trial ....................20 
4.7 
Treatment Plan .........................................................................................20 
4.8 
Procedure Table .......................................................................................21 
4.9 
Relevant Operating Plan ........................................................................22 
4.9.1 Assessment Method for Intraoperative and Postoperative 
haemorrhage in Obstetrics..........................................................................22 
4.9.2 Operation Precautions of Obstetric Cesarean Section ............23 
4.9.3 Anesthesia Cell Salvage Operation（See Appendix 3） ........23 
4.10 
About Early Termination of Trial ...........................................................23 
4.11 
About the Ending of Trial........................................................................23 
5 
Cell Salvage Equipment .....................................................................................24 
6 
Laboratory(Biochemical Test) .........................................................................24 
6.1 
Centers/Local Laboratory .......................................................................24 
6.2 
Sample Collection/Marking/Registration ............................................24 
6.3 
Sample Reception/Management/Responsibility ...............................24 
6.4 
Sample Analysis Process ......................................................................25 
6.5 
Sample Preservation Process(if needed) ...........................................25 
6.6 
Results Report and Registration Time ................................................25 
7 
Safety of Trial（Adverse Events） .................................................................26 

 
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7.1 
Definitions ..................................................................................................26 
7.1.1 Adverse Events（AE） ...................................................................26 
7.1.2 Severe Adverse Events（SAE） ..................................................26 
7.2 
Investigator’s Assessment of Adverse Events ..................................26 
7.2.1 Seriousness of Assessment ...........................................................26 
7.2.2 Causal Analysis.................................................................................27 
7.2.3 Urgency of Report ............................................................................27 
7.2.4 Severity of the Adverse Events .....................................................27 
7.3 
Record and Report of Adverse Event .................................................27 
7.4 
Record and Report of Severe Adverse Event ...................................27 
7.4.1 Expected Adverse Events ..............................................................28 
7.5 
Annual Safety Report ..............................................................................28 
8 
Statistical Analysis ..............................................................................................28 
8.1 
Estimation of Sample Size .....................................................................28 
8.1.1 
Sample Size Calculation ........................................................................28 
8.2 
Outcomes ..................................................................................................29 
8.2.1 Primary Outcomes ............................................................................29 
8.2.2 Secondary Outcomes ......................................................................30 
8.3 
Statistical Methods ..................................................................................30 
8.3.1 General Principle ..............................................................................30 
8.3.2 Significance Level.............................................................................30 
8.3.3 Hypothesis Test ................................................................................30 
8.3.4 Trial Population .................................................................................30 
9 
Data and Record Keeping .................................................................................31 
9.1 
Confidentiality Plan ..................................................................................31 
9.2 
Trial Required Documents .....................................................................32 
9.3 
Data Collection Process and its Regulatory Plan .....................33 
9.4 
Quality Control and Assurance .............................................................33 
10 
Trial Committees ...........................................................................................34 
10.1 
Exective Committee ........................................................................................34 
10.2 
Steering Committee ........................................................................................34 
10.3 
Operations Committee ...................................................................................34 
10.4 
Data Monitoring & Safety Monitoring Committee ....................................34 
11 
Publication Policy .........................................................................................34 
11.1 
Appendix 1 ..................................................................................................36 
11.2 
Appendix 2 ..................................................................................................37 
11.3 
Appendix 3 ..................................................................................................37 
Reference .......................................................................................................................39 
 

 
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Introduction 
1.1 Background 
Haemorrhage(excessive blood loss) is one of the major causes of maternal death. 
It is more likely to occur during caesarean section, especially for those maternal who 
have placenta accreta or placenta previa. 
Blood is a scarce and expensive resource. Hospitals in China at all levels have 
varying degrees of tension with blood. Due to the scarcity of blood and maternal’s 
risk of blood transfusions, it is common that mothers lose their blood transfusions in 
time, and result in postpartum anemia (low hemoglobin levels) in their mothers. A 
seriesof reports confirmed: anemia is a direct cause of maternal deaths1. Life-
threatening blood loss is an indication of emergency hysterectomy2. A retrospective 
analysis involving 24 hospitals shows that at present, the total domestic cesarean 
section rate is 57.84% (2005 ~ 2006) 3. Postoperative anemia will further prolong 
hospitalization, increase postoperative infection rates and delay the time of first 
mobilisation. Intraoperative cell salvage collects patients’ own lost blood, wash and 
filter into the patient's own blood circulation. It may help to reduce the incidence of 
postoperative anemia for patients and avoid prolonged hospitalization. Although it has 
been confirmed that using cell salvage technology in some operations can reduce 
allogeneic blood transfusion, since clinical concerns about the amniotic fluid 
contamination of the blood transfused by the mother, intraoperative cell salvage 
during caesarean section has not yet been fully validated. At present, the National 
Institute of Health and Clinical Excellence has developed guidelines which 
recommend to use intraoperative cell salvage in the event of excessive haemorrhage 
in emergency cesarean section. In addition, intraoperative cell salvage is also 
recommended by the Royal College of Obstetricians and Gynecologists and the 
American Society of Anesthesiologists. At present, only a few clinical cases in China 
have been reported for washing autologous blood transfusion for maternal 
hemorrhage. No relevant randomized controlled studies were found in the literature. 
The risks of allogeneic blood transfusion include: death from transfusion errors, acute 
transfusion reactions, fatal lung injury and infections4. Therefore, applying allogeneic 
blood transfusion to the healthy maternal need to be very cautious, and intraoperative 
cell salvage may possibly avoid these risks. Intraoperative cell salvage is a technique 
that can not only simultaneously reduce the need for allogeneic transfusions and 
prevent anemia, but also avoid bleeding-related serious complications. It has been 
shown to be well documented in non-cesarean sections, but current information is not 
able to demonstrate the safety and efficacy of autologous blood collection for 
bleeding during cesarean section, and have reported several cases of coagulation 
dysfunction associated with autologous blood collection.5. Therefore, it is necessary 
to carry out the monitoring of coagulation function and other checking of related 

 
11 
 
indexes for obstetric autologous blood transfusion patients6，7. In addition, scientific 
rigorous test is necessarily to be carried out to demonstrate the safety and 
effectiveness of intraoperative cell salvage in cesarean section. We plan to construct a 
randomized controlled multicenter study to evaluate the safety and efficacy of 
intraoperative cell salvage in caesarean section, and provide some statistics for 
application of autologous blood transfusion in obstetrics.   
 
1.2 CSCS 
Intraoperative cell salvage collects patient’s blood loss during surgery, and after 
separation purification and filtration, and then returns to the patient's own. This 
technique has been optimized in recent years, and it has been routinely used in 
departments which is at risk of heavy bleeding, such as cardiac surgery, orthopedics, 
hepatobiliary surgery and vascular surgery. British National Code of Practice is now 
only recommended to use of intraoperative cell salvage in the case of emergency 
cesarean section with heavy bleeding. However, in China, there’s no guidelines 
recommending to use of intraoperative cell salvage in the case of cesarean section, but 
some hospitals in China have now tried to perform intraoperative cell salvage when 
lack of suitable allogenic blood. However, there’s still a lack of evidence 
demonstrating the medical and economic benefits of intraoperative cell salvage for 
cesarean section. Regardless of amniotic fluid embolism, people generally think that 
intraoperative cell salvage technology may be superior to allogeneic blood. This 
protocol focuses on evaluating the difference in the efficacy of the two transfusion 
methods. In addition, this study will also compare the adverse reactions and economic 
benefits of two transfusion methods. Although currently the cost of intraoperative cell 
salvage is not low, compared with the cost of allogeneic blood transfusions and the 
extra cost of longer hospital stay and additional treatment, intraoperative cell salvage 
may reduce overall maternal medical costs. 
 
1.3 Risk and Benefit of CSCS 
1.3.1  Risk 
As mentioned earlier, cesarean section has been considered a contraindication to 
the use of intraoperative cell salvage, due to the theoretical risk of amniotic fluid 
embolism(an extremely serious, but very low (about one in 200) incidence of pregnancy 
and maternal complications. However, with the update of technology (new filter 
equipment), this risk is theoretically even lower. After testing the mothers who has been 
used cell salvage in cesarean section, we found that after the modernization of 
equipments, those salvaged, purified and returned blood in cesarean section is the same 

 
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as the normal maternal blood8，9. 
In addition, during autologous blood transfusion, in theory, the contamination of 
fetal red blood cell may cause fetal hemolysis in another pregnancy, but there is no 
evidence that autologous blood transfusion can increase future fetal hemolytic disease. 
About autologous blood transfusion and coagulation disorders, the general view is 
that autologous blood transfusion can wash out heparin and other anticoagulant 
substances, but there are also typical cases report that after autologous blood transfusion, 
abnormalities in coagulation index may happen. Since coagulation function and a 
variety of factors are related, in this trial, we will monitor some of the indicators. 
The risks of allogeneic blood transfusions include death from transfusion errors, 
acute transfusion reactions, fatal lung injury, and transfusion infections. 
Compared with the allogeneic transfusion group, there exsits potential or 
undetected adverse events to subjects themselves.  
1.3.2  Benefit 
Intraoperative cell salvage may reduce the demand for allogeneic transfusions, and 
therefore less transfusion reaction and infection would happen than allogeneic 
transfusions. Postoperative anemia is related to the prolonged hospitalization, the 
increased wound infection and the delayed recovery of the patient's activity. 
Intraoperative cell salvage allow those original blood that may be discarded goes back 
to the patient, which greatly reduces the incidence of postoperative anemia. 
 
1.4 Set-up 
Prior to the protocol development, a systematic review of the literature has been 
conducted. Prior to the pretest, a series of surveys of maternal and obstetricians and 
anesthesiologists has been conducted as well to understand their acceptance of 
intraoperative cell salvage. 
At the Second Affiliated Hospital of Wenzhou Medical University, we randomly 
invited 30 maternal to conduct a questionnaire survey. There are 29 successfully 
returned copies, and the response rate reached 96.7%. Data analysis shows that 75.86% 
of the maternal have some concerns on allogeneic blood transfusion, and 82.76% of the 
maternal have some concerns on intraoperative cell salvage. However, when maternals 
were asked, “if blood transfusion is needed during cesarean section, which method do 
you prefer?”, 13.79% of mothers are willing to receive intraoperative cell salvage, 
55.17% of mothers are willing to follow doctors’ decision, and only 31.03% of mothers 
are willing to accept allogeneic blood transfusion only. And 24.14% of the maternals 
clearly declare that they are willing to participate in our trials. Therefore, during the 
trial, there was some difficulty in obtaining subjects' informed consent and joining the 
trial, but there was still a certain acceptance rate. 

 
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At the Second Affiliated Hospital of Wenzhou Medical University, we randomly 
invited 30 obstetricians to conduct a questionnaire survey. 29 of the surveys were 
successfully returned with a response rate of 96.7%. Data analysis shows that 89.66% 
of obstetricians were affected during their work due to blood difficulties. 89.66% of 
obstetricians have heard of intraoperative cell salvage, and 41.38% of obstetricians 
have used this technique in their work. 72.41% of obstetricians think autologous cell 
salvage is more beneficial than allogeneic blood transfusion, 27.59% of doctors are not 
sure which method is more favorable, but none of the doctors think that allogeneic cell 
salvage is more favorable than autologous blood recovery. Meanwhile, 100% of the 
doctors think it is necessary to have some research on the safety and efficacy of the two 
transfusion methods. When asking “if the mother needs blood transfusion during the 
cesarean section, are you willing to let the mother participate in this study”, 48.28% of 
doctors are willing to, and 48.28% of doctors will be based on the condition. When 
asking the influence of postoperative bleeding according to the two transfusion methods, 
68.97% of doctors are not sure about it. The above data not only show the good 
acceptance of autologous cell salvage from obstetricians, but also show the value of 
this trial.  
At the Second Affiliated Hospital of Wenzhou Medical University, we randomly 
invited 30 anesthesiologists to conduct a questionnaire survey. All 30 of the surveys were 
successfully returned with a response rate of 100%. Data analysis shows that 76.67% of 
anesthesiologists were affected during their work due to blood difficulties. 26.67 % of 
anesthesiologists think autologous cell salvage is more beneficial than allogeneic blood 
transfusion, 66.67% of anesthesiologists are not sure which method is more favorable. 
The "uncertainty" is higher than obstetricians. Perhaps it is because anesthesiologists 
know more about intraoperative blood collection. When asking “if the mother needs 
blood transfusion during the cesarean section, are you willing to let the mother 
participate in this study”, compared to obstetricians, there are also 48.28% of 
anesthesiologists would like to, and 48.28% of anesthesiologists will be based on the 
condition. When asking the influence of postoperative bleeding according to the two 
transfusion methods, compared to obstetricians, 68.97% of anesthesiologists are not 
sure about it.  
1.5 Trial Feasibility 
After obtaining the approval of ethics committee, a randomized controlled trial of 
cell salvage in cesarean section will be conducted at the Second Affiliated Hospital of 
Wenzhou Medical University. This pretest will help to adjust the protocol and operation, 
test the effectiveness of the data collection method, and the acceptability of the test 
design.  
 
 

 
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2 Objectives and Design 
A randomized, controlled, single-blind and multi-centre clinical trial 
2.1 Study Objects 
2.1.1  Primary Objective 
 
Blood loss of the subjects in 5 days post operation 
2.1.2  Seconday Objective 
 
PT/APTT, Hct and Hb in first and fifth day after surgery 
 
the number of units of donor blood transfused 
 
transfusion-related adverse reactions 
 
postoperative complications: amniotic fluid embolism, sepsis, acute 
respiratory distress syndrome, disseminated intravascular coagulation, wound 
infection, acute hemolysis, etc. 
 
mortality during hospital stay 
 
time to first mobilisation,  
 
duration of hospital stay 
 
intraoperative salvaged cell; before and after cesarean section, maternal‘s 
squamous epithelial cells, fetal red blood cells, serum potassium, free hemoglobin. 
 
Before and after blood transfusion, maternal’s electrolytes, PH value, fetal 
red blood cells, serum potassium, free hemoglobin 
 
Total cost in hospital 
 
Second Stage: Follow-up when pregnancy again:  
 
Neonatal hemolytic disease（Appendix 2） 
Determine blood type, antibody, and (Immunoglobulin G)IgG titer  
2.2 Study Design 
A randomized, controlled, single-blind and multi-centre clinical trial with cost-
effective analysis. 

 
15 
 
3 Subjects Screening 
Subjects: Woman who are admitted to a participating labor ward who fulfil all the 
following criteria will be eligible to be randomized. 
 
3.1 Eligible Criteria 
3.1.1  Inclusion Criteria 
• 18 years of age or elder 
• admitted to a participating labor ward 
• delivery by elective or emergency caesarean section with an identifiable 
increased risk of haemorrhage 
• ability to provide informed consent 
• needing for blood transfusion during CS (according to blood transfusion 
guidelines [Appendix 1], obstetrician or anesthesiologist's advice) 
3.1.2  Exclusion Criteria 
• with blood transfusion history before the CS 
• Maternal preoperative Hemoglobin(Hb) < 70g/dL 
•  Maternal Rh negetive blood type 
• Sickle cell disease 
• previous Caesarean section or breech history 
• contraindicated to intraoperative cell salvage e.g. abdominal cancer, 
abdominal infection, intrauterine infection with blood transfusion history 
before the CS 
•  Cultural or social beliefs contraindicating blood transfushion, e.g. Jehovah’s 
witnesses18 years of age or elder 
• Significant antibodies making it difficult to find cross matched blood for 
transfusion 
• admitted to a participating labor ward 
• delivery by elective or emergency caesarean section with an identifiable 
increased risk of haemorrhage 
• ability to provide informed consent 
• Maternal preoperative PT or APTT is 1.5 times greater than normal and above; 
or preoperative platelet(Plt) < 50*109L 
• Joined other clinical trial within 3 months. 

 
16 
 
• Investigator thinks maternal is not suitable to participate 
3.2 Midway-Withdraw of Subjects 
After completing randomization, the subjects will be considered as midway-
withdraw from the study if: 
 
does not meet the inclusion criteria; 
 
for safety issue, one of the transfusion methods must be applied; 
 
subjects is no longer able to agree to participate the trial; 
 
subjects withdraw their consent form during the trial or during data collection; 
 
subjects no longer need cesarean section. 
3.3 Data Collection and Follow-up for Midway-Withdraw 
Subjects 
If the investigator or the clinician or medical team providing the medical service 
believes that the subject needs to withdraw from the trial, it must be accepted from the 
medical point of view. It is the participants’ freedom to determine whether continue 
follow-up after surgery or not. However, it is unavoidable that those midway-withdraw 
subjects will lead to biased outcome and the efficacy of the clinical trials will be worsen. 
Investigators need to collect and record the reasons and adverse events to any violation 
of the protocol as far as possible. The follow-up of maternal is also needed for safety 
issues. Maternal is still free to quit the program after completing the randomization. We 
will have to keep a record for the quit. Meanwhile, whether maternal agrees to be 
followed is also necessary to be recorded, if the participant decides to withdraw the 
study.If the subject explicitly requests to return all the data records, their decisions 
should be respected and be recored to the final study form. When maternal is unable to 
obtain the informed consent during the trial, it turns to her legal representative’s 
decision of whether continue or not. 
 
 

 
17 
 
4 Study Procedure 
4.1 Flow Chart of Study 
 
4.2 Informed Consent Acquisition Process 
4.2.1 Elective Cesarean Section 
Selected maternal in cesarean section will receive detailed information. They will 
Inclusion, exlucsion criteria review 
Obstetrician Agreement 
Subject Informed 
Consent Form 
Obstetrician Agreement 
Intraoperative blood transfusion? 
Exclude 
Yes 
No 
Trial Group： 
Intraoperative Cell Salvage 
Control Group：donor blood 
transfusion. 
Yes 
No 
Randomization 
Calculation of blood loss and other related parameters 
Follow-up 
Statistical Analysis 

 
18 
 
also have the opportunity to ask questions after the appointment of surgery. The paper 
version informed consent will also be obtained on the day of admission to the ward or 
the day of operation. The informed consent forms need to make it clear that they can 
withdraw from the trial at any time for any reason, without discrimination and without 
any justification for their withdrawal from the trial. Randomizaion will be carried out 
on the day of operation. 
 
4.2.2  Emergency Cesarean Section 
For independent randomized controlled trials of intraoperative cell salvage, it is a 
huge challenge to obtain informed consent during emergency cesarean section. 
Subjects in this group will be a special category among all participants. They will 
obtain particular benefits form this technology, therefore their participation is very 
important. 
Emergency cesarean section also requires a paper informed consent form. The 
informed consent forms also need to make it clear that they can withdraw from the trial 
at any time for any reason, without discrimination and without any justification for their 
withdrawal from the trial. Randomizaion will be carried out on the day of operation. 
Mostly, when there’s no feral distress during the emergency cesarean section, 
anticipate adequate time will be required for routine anesthesia. Therefore, after 
patients’ decision of having a cesarean section, there is still some time to obtain 
informed consent of the paper. 
For safety, in this trial, we do not accept the oral informed consent and the case 
that re-sign the informed consent after the operation. 
 
4.3 Patient Screening Process 
Researchers need to understand the situation of new hospitalized maternal, such 
as the presence of potential subjects. Researchers also need  to read their medical 
case reports in detail, and then confirm the inclusion criteria and exclusion criteria. 
When all the criteria meet, we need to get informed consent from potential subjects, 
and informed consent needs to be obtained prior to surgery. After receiving the 
informed consent, randomization will be performed directly in the delivery room at 
the time of preparation (in order to provide enough consideration to maternal time) 
and subjects will be assigned to the respective groups based on randomization results. 
The screening process needs to be recorded in the patient enrollment form. 
4.4 Randomization Process 
Once subjects are confirmed through all the criteria and have received theinformed 

 
19 
 
consent, they can be randomized to either the treatment group(Caesarean section with 
IOCS) or control group(Caesarean section without IOCS).The randomization will be 
proceed in operation if the patient needs blood tranfusion. 
Randomization (ratio:1:1) is used through a network-based randomization system, 
which follows SOP. We use central dynamic randomization system. 
The randomization will be stratified by 5 conditions: 
1. 
centers 
2. 
type of cesarean section（Emergency/Elective） 
3. 
whether there exist abnormality in placental 
4. 
number of fetus (twins or more) 
5. 
whether the ASA level is in I – II? 
The randomization system will be tested and validated before the trial starts and 
will be supervised by the leader center’s CRA and statistical expert throughout the 
trial.Once the subjects have been successfully grouped, they will be recorded in 
theenrollment form. 
Each test center has their independent account and password for randonmization 
system. The account number and password are authorized to the relevant staff by the 
major researchers in each center. After the staff has filled in the subject’s ID and details 
into the randomization system, the system will display the grouping result. 
4.5 Methods of Minimizing Risk and Bias 
Using a third party to hide the grouping will help reduce the errors caused by 
selection. The randomization will be proceed in operation, and the staff should set 
blinding to the subjects. 
Operational errors could result in change in blood transfusion rate.To minimize this 
risk, we need to make sure that all operating rooms and recovery wards in each center 
use an unified blood transfusion plan. Each medical team within each center should 
have same start-up criterion for setting up blood transfusion. Before participating in the 
trial, each center should provide a blood transfusion plan to the leader center for record. 
Some centers would use a prespecified value of Hb to determine whether blood 
transfusion is needed and this value should be unified among each center’s medical 
team. If any of the following sympotoms presents, such as inconscious, dyspnoea, 
exhaustion, which could clearly indicate level of red cell, blood transfusion is needed. 
Maternal mobility and neonatal care after cesarean section are different from the 
postoperation rehabilitation activity of most bedridden patients. According to the 
practical issues, since the clinicians may have different point of view, it is allowed to 
have little variation between each center. It represents the difference in reality cross the 
clinic, so it will not increase the bias. 
 Postpartum ward medical staff should use an unified blood transfusion plan as 
far as possible, according to the postoperative Hb levels and maternal symptoms. Do 
not deliberately observe the grouping of subjects. 

 
20 
 
4.6 Preparation and Support for Medical Staff in the Trial 
For obstetric, operating room nurses, obstetricians and anesthesiologists , who 
involve in the study, the application of intraoperative cell salvage during caesarean 
section surgery would be an extra workload. Especially in emergency caesarean section 
surgery, it would be more challenging to test how skilled clinicians are. Adequate 
training of all medical staff and the use of intraoperative cell salvage machine are very 
important to the  success of trials. Intraoperative cell salvage needs to have been on-
going at all participating centers, and many operating room staff should be highly-
skilled in use of technology. Prior to the start of the trial, each center will need to 
provide a list of staff, who would involve in intraoperative cell salvage operations and 
have been assessed by the main investigators of each center. During the inspection, the 
CRA will assess the standardization of operations (SOP) of relevant staff. 
4.7 Treatment Plan 
According to local transfusion guidelines and medical staff’s opinion (participant’s 
Hb is lower than 70g/L before transfusion, or Hb is between 70~100g/L, depending on 
transfusion plan and medical staff.), women will be randomly allocated to either: 
1. 
Caesarean section with IOCS(treatment group） 
2. 
Caesarean section without IOCS. eg. donor blood transfusion(control group） 
Theoretically, intraoperative blood transfusion need to be based on the following 
formula: 
the volumn of blood transfusion needed(ml) * Hb of transfusion samples = (Hb2-
Hb1) * blood volumn,  
where Hb1:actual Hb; Hb2: target Hb(100g/L） 
blood volumn = 100ml/kg10*weight 
Either in treatment group or control group,the difference between the actual 
volumn of blood transfusion and the theoretical volumn should be controlled within 
±15% (in terms of mass of Hb). 
a. IOCS group 
Eg.: Maternal actual Hb = 60g/L, weight = 60kg, Hb of blood sample = 200g/L 
Volumn of blood transfusion needed = 1200ml, range of blood transfusions = 
1200*(1±15%), 1020-1380ml; If there’s insufficient volumn of self blood for five-
minutes’ infusion, then we use formula to calulate the volumn of blood transfusion 
needed. After five-minutes’ infusion by self blood, if participant’s Hb is still lower than 
70g/L before transfusion, or Hb is between 70~100g/L, according to transfusion 
guidelines and medical staff’s opinion,  we continue to use formula to calulate the 
volumn of blood transfusion needed. 
b. Donor blood transfusion group 
Eg.: Maternal actual Hb = 60g/L, weight = 60kg, assumed Hb of allogeneic red cell 
is 240g/L11，per U red cell is around 200ml. 

 
21 
 
 
Volumn of blood transfusion needed = 1000ml，range of blood transfusions = 
1000*(1±15%), 850-1150ml，around 4.25-5.75U; 4.5U red blood cell allogeneic blood 
can be requested from the blood bank. Following the concept of using of blood 
economically, we should take as less suitable blood as possible. If needed, we can 
communicate with the transfusion department. After five-minutes’ infusion, we 
measure maternal’s Hb again. If participant’s Hb is still lower than 70g/L before 
transfusion, or Hb is between 70~100g/L, according to transfusion guidelines and 
medical staff’s opinion,  we continue to use formula to calulate the volumn of blood 
transfusion needed. 
    When emergency life-threatening bleeding events happen in donor blood 
transfusion group (control group), based on the actual situation, clinical staff there can  
take the most reasonable step for mothers, including the intraoperative cell salvage. We 
expect the probability of this situation to be extremely low. 
4.8 Procedure Table 
Preoperati
ve 
Intrao
perativ
e 
Postoper
ative 
Hospital 
Discharg
e 
Long-
term 
Follow-
up 
Eligible criterion and Grouping 
Inclusion, exlucsion criteria 
review 
X 
 
 
 
 
Informed Consent Acquisition 
X 
 
 
 
 
Record demographic information, 
contact info, diagnosis, type of 
surgery, cesarean section indication, 
birth history, preoperative blood 
loss, comorbidities (past history), 
blood group ABO, and other 
abnormalities 
X 
 
 
 
 
Randomization 
（Dynamic Randomized） 
 
X 
 
 
 
Blood Transfusion 
 
Intraoperative blood transfusion 
and other blood products 
 
X 
X 
 
 
Intraoperative autologous blood 
transfusion, and related data 
 
X 
 
 
 
Maternal 
 
Intraoperative anesthesia 
operation time, special 
circumstances (such as 
hypotension), urine output 
 
X 
 
 
 

 
22 
 
Intraoperative and postoperative 
daily bleeding and transfusion, 
infusion volume (crystal, gel), 
blood-related drugs 
 
X 
X 
 
 
The highest body temperature, 
Hb / Hct / Plt, PT / APTT, ALT / 
AST / total G / D and renal function 
(creatinine and urea) 
X 
X 
X 
 
 
Postoperative bleeding 
(drainage, bleeding, lochia, etc.) 
 
 
X 
 
 
Specific causes of postoperative 
bleeding (uterine inertia, placental 
conditions, soft birth canal 
laceration situation) 
 
 
X 
 
 
Test of blood component in 
recover blood and maternal blood 
fetal  
 
X
（Spec
imen 
only） 
X
（Speci
men 
only） 
 
 
Transfusion reaction 
 
X 
X 
 
 
Other hospitalization 
Time to first mobilisation 
 
 
X 
 
 
Duration of hospital stay 
 
 
 
X 
 
Intraoperative and postoperative 
adverse events 
 
X 
X 
 
 
Reoperation caused by bleeding 
 
 
X 
 
 
   Inpatient mortality 
 
 
 
X 
 
Overall medical expenses 
 
 
 
X 
 
Tracking for hemolytic disease 
in future birth 
 
 
 
 
X 
 
4.9 Relevant Operating Plan 
4.9.1  Assessment 
Method 
for 
Intraoperative 
and 
Postoperative haemorrhage in Obstetrics 
 Assessment Method for Intraoperative blood loss in Cesarean Section 
 Weighing Method: blood loss (ml) ≈ (weight of gauze with blood – weight of 
dry gauze) /1.05 
 Measuring Cup Method: used to measure amount of blood in a curved plate; 
blood loss ml=Volume of measuring cup (blood clots crush as much as 
possible). 
 Intraoperative blood loss = Recycling machine blood volume (recovery 
machine capacity - flushing, washing capacity) + Placental bleeding (using 
measuring cup to measure the amount of blood flow into the curved plate by 

 
23 
 
squeezing the placenta) + blood volumn of cleaning the vagina (bleeding with 
weighing method, blood cluts with measuring cup method) + blood volumn of 
gauze (bleeding with weighing method, blood cluts with measuring cup 
method)  
 Postoperative blood loss = blood volume of postpartum sterile mattress + 
blood volumn of gauze (bleeding with weighing method, blood cluts with 
measuring cup method) + volumn of shed blood（using measuring cup） 
 
4.9.2  Operation Precautions of Obstetric Cesarean Section 
In cesarean section, the absorption of amniotic fluid should be sufficient, so that 
the residual of intrauterine amniotic fluid can be minimized. When cut the umbilical 
cord, cord blood should be guaranteed not to enter the uterine cavity. Any action that 
makes fetal bleeding should be avoid. 
Prepare two sets of suction equipment, one for amniotic fluid and other. 
4.9.3 Anesthesia Cell Salvage Operation（See Appendix 3） 
4.10 About Early Termination of Trial 
If the blood collection machine manufacturer issues an important safety notice, 
then we will temporarily stop the trial. If the ethics committee, the national clinical trial 
management agency, or the funders need to suspend the trial due to the consideration 
for subjects’maximum benefit or the trial itself, then the principal investigator will 
receive a written notice, and stop the trial. Usually the concerns will involve safety, 
efficacy of the trial, violations of relevant provisions and protocol, etc. 
 
4.11 About the Ending of Trial 
    The ending time of Phase I in this trial is defined as the date that the last  subject 
discharges from hospital. The ending time of Phase II in this trial is defined as when 
the last subject exceeded the childbearing age and has been followed for 10 years. At 
the end of trial, we will notify the Ethics Committee. The final study report will be 
completed within six months after the end of trial. 

 
24 
 
5 Cell Salvage Equipment 
5.1 Blood Collection Machine （National certified 
washing cell salvage machine） 
5.2 Leucocyte Filter 
6 Laboratory(Biochemical Test) 
6.1 Centers/Local Laboratory 
The test is basically conducted in the local laboratory. Only part of the blood samples 
will be sent to the Second Affiliated Hospital of Wenzhou Medical University (Central 
Laboratory) for testing (eg, fetal components from maternal and cell salvage). It can be 
raised individually if anything else needs to be submited to the Central Laboratory. 
6.2  Sample Collection/Marking/Registration 
Sample:  
a. In operating roon, take the peripheral blood of maternal before anesthesia and after 
transfusion, 8ml respectively; 
b. self-salvaged blood, 8ml. 
Using EDTA or other anticoagulant whole blood, samples from other hospitals can be 
placed in a refrigerator at 4 ℃, prepare within 48h. 
Symboling and registering the samples with the unique study number. 
 
6.3  Sample Reception/Management/Responsibility 
Before sample processing, all samples need to be checked in by laboratory 
personnel. Once any error is found, it will be returned to the sample collector or research 
team. 
 

 
25 
 
6.4  Sample Analysis Process 
6.4.1. Set 8ml blood sample with NH4Cl lysis solution for 30min (Due to the large 
amount required and several bottles of commercial hemolysin may be required, it is 
preferred to formulate NH3CL by self.). The sample is centrifuged at 2000 rpm for 5 
minutes at room temperature and the supernatant is discarded; 
6.4.2. Use perfix-nc kit, punch and fix. (The usage reference of perfix-nc kit is 
specified in kit instructions); 
6.4.3. Antibody, CD45PECY7 / CD71-PE / HBF-FITC / 7-AAD (30ul / antibody) is added 
and incubated for 20 minutes; 
6.4.4. Use up-flow cytometer to circle non-cellular impurity groups, calculate the 
value of fluorescence; 
6.4.5. Use upstream cytometer to sort and select cells that expressing CD45- / 
CD71 + / HBF + / 7-AAD + at a rate of 30,000 cells per second or above (Due to large 
background mass of the sample cells, the time required to sort each sample at an 
effective sorting rate of 25-30 thousands cells per second is between 1 and 1.5 hours, 
so we need to ensure that cells are not lost and are complete, and try to improve the 
effective sorting speed); 
6.4.6. Receive and accept the sorted fetal with nucleated red blood cells in the 100 
ulPCR centrifuge tube; 
6.4.7. Fetal has nucleated red blood cell count. 
6.5  Sample Preservation Process(if needed) 
Keep autologous blood samples until 3 years after the end of the study  
6.6  Results Report and Registration Time 
All sample results need to be truthfully recorded in the original record sheet and ECRF. 
If needed, the test results can be printed to the original record sheet. 

 
26 
 
7 Safety of Trial（Adverse Events） 
7.1  Definitions 
7.1.1  Adverse Events（AE） 
An adverse event (AE) is any untoward medical occurrence in a patient or clinical 
investigation subject administered a pharmaceutical product and which does not 
necessarily have a causal relationship with this treatment. An adverse event can 
therefore be any unfavourable and unintended sign, an abnormal laboratory finding, 
symptom, or disease temporally associated with the use of a medicinal (investigational) 
product.  
 
7.1.2  Severe Adverse Events（SAE） 
In trials, if any untoward medical occurrence, it is defined as severe adverse event. 
 
death of maternal or fetus (note: death is a termination indicator, not an event) 
 
Is life-threatening 
 
after caesarean section，causes prolongation of existing hospitalization for 
more than 7 days by adverse event 
 
results in persistent or significant disability/incapacity 
 
fetus has congenital abnormalities or defects 
 
investigators think it is a severe medical event 
 
7.2 Investigator’s Assessment of Adverse Events 
7.2.1  Seriousness of Assessment 
The principal investigators at each center are responsible for participants’ medical 
treatment. If the principal investigator is unable to observe the abnormality, the 
authorized physician in this test team is responsible for assessing the severity of the 
adverse events as described in 7.2.4. 

 
27 
 
7.2.2  Causal Analysis 
Investigators must assess the causal relationship between all adverse events and the 
trial. 
7.2.3  Urgency of Report 
Investigators must assess the prognosis of all severe adverse events. If the cause of 
this serious adverse event is unpredictable related to the trial, it must be reported 
immediately. 
7.2.4  Severity of the Adverse Events 
According to the criteria described below, investigators need to assess the severity 
of the adverse events. The severity grades of the event can not be confused with the 
word ‘serious’ in a serious adverse event(SAE). ‘Severity’ is defined based on the 
patient / event outcome criteria. 
 General: observed discomfort but does not affect subject's daily life 
 Moderate: the level of discomfort is sufficient to affect or reduce the level 
of activity of a normal person 
 Serious: completely unable to carry out daily activities, and seriously 
affected the normal life. 
7.3 Record and Report of Adverse Event 
If the adverse event is not rated as a serious adverse event, then the adverse event 
needs to be recorded in the appropriate place or adverse event testing registration form 
for participant’s record. The research team also needs to track the progress of the 
participants. 
7.4  Record and Report of Severe Adverse Event 
All serious adverse events found in the trial or reported by the participants 
themselves, whether related to the trial or not, need to be recorded in the participants' 
case report forms and adverse event registration forms. All serious adverse events need 
to be followed until a new solution appears or the event is stable. Investigators need to 
provide follow-up information. All serious adverse events that are related to the 
withdrawal of the participants or happen at the end of the trial, need to be tracked until 
a satisfactory solution has been found. 
After cesarean section, due to adverse events resulting in the longest hospital stay 
than 7 days, we need to be considered as prolonged hospitalization, and recorded in the 

 
28 
 
serious adverse events record form or adverse events record form. 
According to local regulations, in each center, it is the major investigators’ 
responsibility to report serious adverse events to the principal investigators and their 
local authorities. All serious adverse events need to be reported to the principal 
investigator within 24 hours of occurrence. Within 24 hours, PI needs to give feedback 
to quality control managers and sponsors of the Practical Clinical Trial Unit (PCTU) 
about all relevant or unanticipated serious adverse events. Within 15 days, or within the 
time as requested by the sponsor or PCTU, PI is required to report a serious adverse 
event to the General Ethical Comittee. 
7.4.1  Expected Adverse Events 
Although no serious adverse events are expected, it is still possible. 
Risks of trial operation include: 
 
Maternal exposed to baby's blood 
 
Amniotic embolism 
 
Severe hypotension 
 
Transfusion reaction 
    For participants, the frequency of these risks is unlikely to increase substantially. 
Based on the criteria for a serious adverse event, if these risks meet the criteria for a 
serious adverse event, they are also reported as serious and unexpected adverse events. 
 
7.5 Annual Safety Report 
Principal investigators need to send an annual safety report to the Ethics 
Committee (MREC). The principal investigator needs to report annually to the Ethics 
Committee about all relevant and unexpected serious adverse events in tabular form 
with time order. Meanwhile, this form is reported to Data monitoring & Safety 
monitoring committee and Trial Steering Committee every 6-12 months. 
8 Statistical Analysis 
8.1 Estimation of Sample Size 
8.1.1 Sample Size Calculation 
In this study, we use data from control group (donor blood transfusion) to estimate 
the sample size, and then use 1: 1 ratio to calculate the sample size of IOCS sample. 

 
29 
 
The calculation is shown below: 
A pilot study has commenced at the The 2nd Affiliated Hospital of Wenzhou 
Medical University, Zhejiang Province, China, involving women having Caesarean 
sections and the need for red cell transfusion. We collected a samle of 50 from the study 
and use the following foemula to estimate the blood loss after te caesarean section:  
Blood loss = BV * (Hctfirst day post operation – Hctxth day post operation) + Vt 
where BV is the blood volume, Vt is the volumn of red cell (unit: ml), and Hct has a 
unit with decimal places. 
1. Sample Statistics for Donor Blood 
Transfution 
N 
effective 
48 
missing 
0 
mean 
444 
median 
274 
SD 
709 
minimum 
0 
maximum 
4347 
percentiles 
25 
0 
50 
274 
75 
700 
   
2. Sample Size Estimation Based On Population Mean 
 
If t0.05=1.96, S=709, δ=200, the estimated minimum sample size is 50. 
3. Thus, according to ratio 1:1, the sample size of IOCS group is also 50.  
4. Taking into account the unpredictable factors in the experiment that cause the loss 
of sample size, we recommend to use 1.2 times the minimum sample size as the 
sample minimum sample size. Therefore, according to ratio 1:1 between treatment 
group and control group, the total sample size would be 120 (where 60 for treatment 
group, and 60 for control group).  
8.2  Outcomes 
8.2.1  Primary Outcomes 
 
Blood loss post operation 
Hb and Hct in first and fifth day after surgery. 
The direct measurement of is the accurate volume of blood loss in major 
haemorrhage is difficult. Alternatives such as haemoglobin (Hb) or hematocrit (Hct) 

 
30 
 
are more frequently used and blood loss can be calculated using these parametres. 
Since Hct or Hb will be recorded as per standard practice during and postoperative 
in the hospital. 
In this proposed trial, we are planning that Hb and Hct in the first and fifth day 
after the surgery as the primary outcome to reflect the blood loss. 
8.2.2  Secondary Outcomes 
 PT/APTT, Hct and Hb in first and fifth day after surgery 
 the number of units of donor blood transfused 
 mean fall in serum haemoglobin level  
 maternal morbidity resulting from postoperative anaemia 
 time to first mobilisation,  
 duration of hospital stay 
 immediate multidimensional fatigue inventory. 
 Total cost in hospital 
 
Second Stage: Follow-up when pregnancy again:  
Neonatal hemolytic disease（Appendix 2） 
Determine blood type, antibody, and IgG titer） 
8.3 Statistical Methods 
8.3.1  General Principle 
Using SAS 9.13 for statistical analysis. All statistical tests should be two-sided. 
If p-value is less or equal to 0.05, then it is considered to be statistically significant. 
 
Variables data are described by mean±standard deviation, median, maximum 
and minimum. Use paired t-test to compare intra-group variations with respect to 
baseline and use ANOVA or Wilcoxon rank-sum test to compare variations 
between groups. 
 
Attributes data are descreibed as frequency (percentage). Between group 
comparison of percentages is tested by Fisher's exact test 
8.3.2 Significance Level 
Use superiority test to compare the primary objective between groups. The 
significance level α= 0.05 (two-sided). 

 
31 
 
8.3.3 Hypothesis Test 
Primary objective: The amount of blood lose post operation: 
H0: HR = 
λT
λC < 1,  H1: HR = 
λT
λC ≥ 1 
α= 0.05 (two-sided). 
By Log-Rank test, if p-value is less or equal to 0.05, and the median amount 
of blood lose in treatment group is less than in control group, then the superiority 
of the treatment group relative to the control group holds. 
Based on the Cox Proportional hazards model, hazard ratios and the 95% 
confidence interval (CI), if the lower bound of the CI is greater than 1, then the 
efficacy of the treatment group is better than that of the control group. 
8.3.4 Trial Population 
8.3.4.1 
Distribution of subjects 
 
Situation of subjects enrolling in the group and the completion 
of trail: frequency, percentage; 
 
Description of the proportion of diagnosed cases in each 
group. 
8.3.4.2 
Demographic 
characteristics, 
disease 
characteristics and medical history 
Statistically describe indicators, compare between groups to assess 
the equivalence between the groups 
 
age; 
 
height, weight; 
 
related medical history. 
 
9 Data and Record Keeping 
9.1  Confidentiality Plan 
    Any research-related documents must be filed with the research center and 
kept by the investigator. Patient-related documents are filed in accordance with 
the relevant laws of China. All research related documents must be kept for at 
least 15 years after the study ends. After this time limit expired, investigators 
must obtain the consent of the Second Affiliated Hospital of Wenzhou Medical 
University before discarding any documents. All patient-related data, including 
identification information and all personal medical information, should be treated 
as confidential documents. Data verification procedures should be based on the 
most stringent methods of confidentiality. Prior to obtaining the informed 

 
32 
 
consent, each patient should be provided with the confidentiality of the personal 
data, the original data verification, inspection, electronic data processing and file 
transfer procedures. During the trial, Study team members should keep any 
relevant information and results confidential. It is strictly forbidden to disclose 
any of these information to anyone not participating in this study. After the 
completion of the trial, all case report forms shall be filled in, reviewed and 
signed by the investigators and ombudsmen in accordance with the data 
management requirements of this study and kept together with the ‘informed 
consent’. 
 
9.2 Trial Required Documents 
Serial 
No. 
Name of Document 
Format of 
document 
D1 
Trial Preparation Stage 
D1.1 
Proposal 
D1.2 
Standardization of Operations
（SOP） 
D1.3 
Original Record Form 
D1.4 
CRF and its Manual 
D1.5 
Randomization System and its 
Manual 
D1.6 
Informed Consent 
D1.7 
Subjects’ Recruitment Ads 
D1.8 
Project Contract 
D1.9 
Table of Ethics Committee Members 
D1.10 Ethical Approval 
D1.11 Survey Questionnaire of Test Unit 
D1.12 Multi-center Cooperation Agreement 
D1.13 Resumes of Investigators 
D1.14 Investigators’ Work Division and 
Signature Proofs 
D1.15 Copy of GCP Certificate and 
Registration Form 
D1.16 Participants’ Screening Registration 
Form 
D1.17 Participants’ Follow-up Registration 
Form 
D1.18 Research Tools (scales, etc.) 
D1.19 Follow-up Letter to Subjects 
D2 
Trial Stage 

 
33 
 
D2.1 
Revision and Record of Documents 
D2.2 
Meeting Munites 
D2.3 
Signed Informed Consent 
D2.4 
Filled Original Record Form 
D2.5 
Filled CRF 
D2.6 
Filled Screening Form of Subjects 
D2.7 
Filled Follow-up Form of Subjects 
D2.8 
Report Record of Adverse Event 
D2.9 
Midterm Report 
D2.10 Audit Report 
9.3 Data Collection Process and its Regulatory Plan 
Data is collected by related personnels. The data will be entered by researchers 
twice (stage I: hospitalization, stage II: follow-up) and the CRAs will check the 
authenticity and traceability of the data. If you have questions to fill in, send a 
questionnaire to the researchers, and answer it in writing. The whole process of the 
study was supervised by the supervisory staff: the participants' informed consent, 
screening and inclusion, correct and single-blind randomization, correct entry of case 
report forms and confirmation of all adverse events were recorded. 
After the database is established, and information, data is reviewed and confirmed 
correct by CRA, then the data should be locked, and the locked data files are no longer 
allowed to make change. The data will be analyzed according to the statistical methods. 
 
9.4   Quality Control and Assurance 
The responsibilities and division of labor among relevant personnel should be 
clearly defined and it should be ensured that the data is correctly collected, 
randomized and single-blinded properly. We should obtain the subjects’ full 
informed consent and understanding so that we could improve the subject's 
compliance, and ensure the participants' perioperative information and 
postoperative follow-up information is authentic and complete. 
Regular audits will be conducted, including non-scheduled on-site inspections 
and telephone inspections before and during the study. Data will be entered twice to 
reduce errors in data entry. An independent data security committee will be 
established to audit data analysis. The clinical trial will be registered to make it 
transparent. 
 

 
34 
 
10 Trial Committees 
This Study consists of main investigators, CRC and other researchers from each 
research center. 
10.1 Exective Committee 
Chair: Fang Gao, Qingquan Lian 
Members: Ting Li, Zuokai Xie, Joyce Yeung, Teresa Melody and one member 
from each of other related centers 
10.2 Steering Committee 
Chair: Mingping Hu, Yuhuan Wang, Wangning Shangguan, Jun Li, Ting Li, 
Fang Gao, Zuokai Xie, Joyce Yeung, Teresa Melody 
10.3 Operations Committee 
Chair: Mingping Hu, Ting Li 
Members: Zuokai Xie, Chenchen Jiang, Yan Zhang, Yi Wang, members from 
other centers 
 
10.4 Data 
Monitoring 
& 
Safety 
Monitoring 
Committee 
Chair:  Jingwei Zheng 
Members: Han Lin, Weihe Zhou 
 
11 Publication Policy 
Intellectual Property states that this statement is formulated in accordance with 
the ‘Copyright Law of the People's Republic of China’. The copyrights of this 
protocol and intellectual property are retained by the authors, with first publication 

 
35 
 
rights granted to the Second Affiliated Hospital of Wenzhou Medical University. 
During the study, investigators in each center are required to report the research data 
to the Second Affiliated Hospital of Wenzhou Medical Universit. They shall not 
privately disclose and publish the data in any forms during this trial. This trial 
respects all the contribution of collaborators. After the research center announces that 
the study ends, through consultation, the names and order of participating units will 
be determined, generally according to the number of selected patients, amount of 
work through the study and so on. Anyone who violates the above declaration will be 
held liable according to law.

 
36 
 
Appendix 
11.1  Appendix 1 
Intraoperative and Postoperative transfusion Plan in 
Caesarean sections 
1. Flowchart of Red Cell Transfusion Plan 
 
2. Platelet (PLT) 
1)．PLT > 100*109/L, could have no blood transfusion; 
2)．PLT < 50*109/L，should consider to hanve blood transfusion (Considering the 
physiology of maternal hypercoagulability, the obstetrician decides whether or not 
to transfuse according to the condition or the bleeding condition of the wound); 
3)．PLT is between 50-100*109/L, and it will be decided based on whether there is 
spontaneous bleeding or wound bleeding; 
4)．If there’s uncontrolled bleeding occurs during operation and PLT function is 
determined to be impaired, then transfusions of PLT has no ceiling restriction. 
3. Fresh Frozen Plasma 
For patients who lack of clotting factor 
Hb: 70-100g/L 
Decide by obstetric 
and anesthesiologist 
Hb > 100g/L 
No blood 
transfusion 
Hb < 70g/L 
Should consider 
transfuse concentrated 
red cells 
Preoperative 
symptomatic patients 
with refractory anemia 
in cardiac function Ⅲ-
Ⅳ level, heart disease 
(congestive heart 
failure, angina) and 
ineffective to iron folic 
acid vitamin B 
treatment 
Patients with Hb less 
than 80 g / L and with 
symptoms (chest pain, 
orthostatic hypotension, 
tachycardia that is 
unresponsive to fluid 
resuscitation or 
congestive heart failure) 
should consider 
transfusing red cells 
Patients with 
Preoperative cardiac 
insufficiency, severe 
hypotension or 
increased metabolic 
rate, should maintain 
a relatively high Hb 
level (80-100g / L) 

 
37 
 
1)． PT or APTT greater than normal 1.5 times or INR > 2.0, diffuse wound 
bleeding; 
2)．Patients with acute bleeding, transfuse a large amount of whole blood or red 
blood cells (amount of bleeding, or amount of transfusion is equivalent to the 
patient’s own blood volumn). 
3） There’s advanced or acquired coagulation disorders in history or clinical 
performance. 
4） Acute anti-coagulation effects for warfarin 
 Each unit is equivalent to the amount of plasma in 200ml of fresh whole blood, and 
it can increase about 2% -3% of clotting factor for adults. It needs to be based on 
clinical symptoms and monitoring results to adjust the dose when apply 
4. Cryoprecipitate  
Cryoprecipitate is generally used when bleeding and fibrinogen < 800 ㎎/L, and 
other treatments are performed according to the experience of obstetrician physicians. 
 
 
11.2  Appendix 2 
 
Diagnostic Criteria 
If the following conditions meet, it can be diagnosed as neonatal ABO hemolytic 
disease: 
① ABO blood group incompatibility between mother and child; 
② Direct anti-human globulin test and/or Antibody release test result(s) in positive; 
③ Early pathological jaundice and / or Anemia (Hb≤170g / L) and / or Hemolysis 
(Percentage of Reticulocyte: Full-term children ≥ 6%; Preterm children ≥ 10%). 
11.3  Appendix 3 
Procedures of Intraoperative Cell Salvage  
Preparation of Equipment: 
a) 
Cell salvage machines and consumables. 
b) 
Heparin Anticoagulant: 500ml saline +3 heparin sodium injection for 
preparation of heparin saline.  
c) 
Prepare two sets of suction device, one for intraoperative cell salvage suction, 
one for amniotic fluid, irrigation fluid, etc. 
d) 
Large amount of saline for washing autologous blood. 
2. 
Prepare two sets of suction device before operation; one for attracting amniotic 
fluid, irrigation fluid during operationand one for intraoperative cell salvage 

 
38 
 
suction, only for bleeding during operation. 
3. 
Install the suction tube and blood reservoir first, and connect heparin anticoagulant 
fluid; Adjust the attraction pressure to the lowest available level (generally about 
20.0kPa)  
4. 
Pre-fill 200ml heparin saline into the blood storage tank, control the drop rate of 
heparin saline to 1-2 drops / second, and during the operation, adjust the drop rate 
of heparin saline according to the bleeding rate. When absorb blood, we should 
try to put the head of suction head below the surface. Whether or not recycle the 
blood on gauze during the operation is determined by each research center. 
5. 
When the blood transfusion indicator is reached, or if the doctor thinks it is 
necessary to transfuse, install a centrifuge cup (Thus, generally use manual mode). 
Once start to centrifuges the blood, the centrifuge cup speed should set to 300ml / 
min, and adjust to 500ml / min in emergency. After the centrifugal pump is full, 
begin to wash. Use 0.9% saline for washing only. Do not use any other types of 
liquids for washing, such as Ringer’s solution. The amount of liquid for washing 
should be about 8-10 times the total amount of centrifuge cup. The amount of 
washing liquid can increase according to real situation, but can not decrease. When 
finishing washing, the blood should packed into the blood storage bag. 
6. 
When using the WBC filter, please do not squeeze blood bags, let it filter by 
gravity. 

 
39 
 
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