Single-arm Multicenter Clinical Trial of Blood Transfusion From RhD+ Donors to Asian-type DEL Recipients
Phase: NA
Conditions: Blood Transfusion, Rhesus Isoimmunisation Due to Anti-D, Blood Group Antigen Abnormality
Sponsor: Guangzhou Blood Center

 
Version number: GZXY.RhD-HPT-V0.1-CTP 
Version Date: September 5, 2016 
 
A multi-center, single-arm clinical trial for exploring the safety and effectiveness of RhD+ blood 
transfusion in Chinese "Asia type" DEL patients 
 
Institute in charge of research: Guangzhou Blood Center 
Address: 31 Luyuan Road, Yuexiu District, Guangzhou, China 
 
Statistical analysis Institute:  
*School of Public Health, Southern Medical University 
Address: 1023 Shatai South Road, Baiyun District, Guangzhou, China 
*Guangdong Provincial Institute of Biological Products and Materia Medica 
Address: No. 200, Xipengling Road, Jiahe Hebian, Baiyun District, Guangzhou, China 
 
 
 
 
                                                                                         
Summary of previous version changes 
Version number 
Chapter with change 
Reason for revision 
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Declaration 
This trial will be carried out in accordance with the Drug Clinical Trial Quality Management Standards and 
Guidelines for Quality Management of Vaccine Clinical Trials (tentative standard) (Administrative Order No. 28). 
The investigators who participating the clinical trials and the staffs who are responsible for performing, 
managing, and supervising the clinical trials, need to receive the training about the protocol. 
The trial protocol, informed consent for blood transfusion treatment and all relevant files need to be 
submitted to the ethics committees for approval. All amendments of the plan must be reviewed and approved by 
the ethics committee before implementation. Before the subjects are selected, the review approval of protocol 
ethics needs to be obtained. 
 
Privacy statement 
This research protocol is confidential, and it is provided to the medical experts related to this research, the 
researchers participating in this research and other research-related staffs, and the medical institutions, the ethics 
committees, and other cooperated research organizations that undertake this research. Without the prior written 
consent of the sponsor, no part of this research protocol could be disclosed to the third parties. In addition, 
publishing of part or all results of this clinical study need to be approved by the sponsor. 
 

 
Ethical statement 
& The documents need to be followed: 
The trial must comply with all SOPs related to the clinical trial established by the Guangzhou Blood Center, and 
follow the design and regulations of this protocol. The trial protocol was designed to follow the documents below: 
 
Management Practices for the Quality Control of Drug Clinical Trials 
 
The Helsinki Declaration 
 
Technical Specifications for Clinical Blood Transfusion 
 
Technical Guidelines for Vaccine Clinical Trials 
 
Guiding Principles for Quality Management of Vaccine Clinical Trials (tentative standard) 
 
Regulations on the Management of Serious Adverse Events in Vaccine Clinical Trials (tentative standard) 
 
& Institutional Review Board and Ethics Committee 
The trial protocol needs to be reviewed and approved by the Guangzhou Blood Center Clinical Trial Ethics 
Committee before implementation. During the review, the sponsor and researcher should provide the ethics 
committee with the documents required, such as the clinical trial protocol, informed consent for blood transfusion 
treatment, and case report form and so on. This clinical trial must comply with the Helsinki Declaration, 
Management Practices for the Quality Control of Drug Clinical Trials issued by the National Medical Products 
Administration (NMPA) and related regulations in China. Before the trial begins, this study must be approved by the 
clinical trial ethics committee of the Guangzhou Blood Center. Any changes of the protocol must be approved by 
the ethics committee. The researchers are responsible for submitting the trial report regularly in accordance with 
the relevant requirements of the ethics committee. 
 
& The informed consent of transfusion therapy 
Before each possible subject participates in any activities related to the trial, the investigator must explain to 
the subject in details about the nature, purpose, relevant procedures, expected time, potential risks and benefit 
and any discomfort that may arise. Each subject must know that participation in the trial is voluntary and that he/she 
can withdraw from the trial and withdraw informed consent for blood transfusion treatment at any time without 
affecting his/her subsequent treatment or relationship with the responsible physician. 
After explaining the major content of the trial, the investigator has been convinced that each subject who will 
participate in the trial has knowledge of the trial and each subject who will participate in the trial should be required 
to sign the informed consent form for the transfusion, date, and contact information. Subjects should read and 
consider carefully before signing the informed consent of blood transfusion treatment. 
 
 
 

 
Table of Contents 
Abbreviations Table ........................................................................................................................... 1 
Protocol Abstract ............................................................................................................................... 2 
Protocol Text ..................................................................................................................................... 4 
I. Research Background ..................................................................................................................... 4 
II. Purpose of the Tial ........................................................................................................................ 6 
III. Design of the trial ......................................................................................................................... 6 
IV. Selection of Subjects .................................................................................................................... 7 
(I) Inclusion criteria ................................................................................................................... 7 
(II) Exclusion criteria .................................................................................................................. 7 
(III) Rejection criteria ................................................................................................................. 7 
(IV) Dropout criteria .................................................................................................................. 7 
(V) Criteria for trial termination ................................................................................................ 8 
V. Experimental Interventions ........................................................................................................... 8 
VI. Observation Index ........................................................................................................................ 9 
(I) Demographics ....................................................................................................................... 9 
(II) Subject characteristics ......................................................................................................... 9 
(III) Safety observation indicators ........................................................................................... 10 
(IV) Observation index of curative effects ............................................................................... 11 
(V) Concurrent treatment ....................................................................................................... 11 
VII. Evaluation Index ........................................................................................................................ 11 
(I) Main indicators for evaluation of the effectiveness ........................................................... 11 
(II) Indicators for evaluation of the safety of the trial ............................................................. 11 
VIII. Steps of Clinical Trial ................................................................................................................ 12 
(I) Phase I of Visiting: -7~ 0 days before transfusion ............................................................... 12 
(II) Phase II of Visiting：Blood transfusion treatment period（0~1 days） ........................... 13 
(III) Phase III of Visiting：1 day to 1 month after blood transfusion ...................................... 13 
(IV) Phase IV of Visiting：1 month to 12 months after blood transfusion ............................. 13 
(V) Phase V of Visiting：over 12 months after blood transfusion .......................................... 13 
IX. Adverse Events, Serious Adverse Events .................................................................................... 14 
(I) Definition ............................................................................................................................ 14 
(II) Grading criteria for the adverse events ............................................................................. 15 
(III) Evaluation of the correlation between adverse events and research interventions ........ 15 
(IV) Adverse event management ............................................................................................. 16 
(V) Follow-up of adverse events .............................................................................................. 18 
X. Quality Control of Clinical Trials .................................................................................................. 18 
(I) Quality control measures .................................................................................................... 18 
(II) Training of the researcher .................................................................................................. 19 
(III) Measures to improve patient’s compliance...................................................................... 19 
(IV) Monitoring of clinical research ......................................................................................... 19 
(V) Inspection of clinical research ........................................................................................... 20 
XI. Data Management ..................................................................................................................... 20 
(I) Data Entry and Modification ............................................................................................... 20 

 
(II) Data Locking ....................................................................................................................... 20 
XII. Statistical Analysis ..................................................................................................................... 20 
(I) Population for statistical analysis ........................................................................................ 20 
(II) Statistical analysis methods ............................................................................................... 21 
(III) Analysis software .............................................................................................................. 22 
XIII. Preservation of the orginal data............................................................................................... 22 
XIV. References ................................................................................................................................ 23 
XV. Flowchart of Clinical Research................................................................................................... 25 
 

1 
 
Abbreviations Table 
Acronyms 
Full name 
AE 
Adverse Event 
ALP 
alkaline phosphatase 
ALT 
Alanine aminotransferase 
AST 
Aspartate aminotransferase 
BLD 
Urine occult blood 
BUN 
Blood urea nitrogen 
Cr 
Creatinine 
CRF 
Case Report Form 
DBIL 
Direct bilirubin 
ECG 
Electrocardiogram 
GLU 
Fasting blood glucose 
Hb 
Hemoglobin 
Hct 
Hematokrit 
LEU 
leukocyte 
NEUT% 
Neutrophil ratio 
NMPA 
National Medical Products Administration 
PLT 
Platelet 
PRO 
Proteinuria 
RBC 
Red Blood Cell 
SS 
Safety Data Set 
SAE 
Serious Adverse Reaction 
TBIL 
Total bilirubin 
TP 
Total protein 
WBC 
White Blood Cell 
γ-GT 
γ-glutamyl transpeptidase 

2 
 
Protocol Abstract 
Name of the 
trial 
A multi-center, single-arm clinical trial for exploring the safety and 
effectiveness of RhD+ blood transfusion in Chinese "Asia type" DEL patients 
Sponsor 
Guangzhou Blood Center 
Purpose of the 
trial 
To explore the safety and effectiveness of RhD+ blood transfusion in Chinese 
“Asia type” DEL patients to lay the foundation for establishing a new blood 
transfusion strategy for “Asia type" DEL patients. 
Design of the 
trial 
A multi-center and single-arm trial  
Target 
indication 
Patients with “Asia type” DEL blood group who need transfusion therapy in 
clinic 
Sample size 
“Asia type” DEL is one of rare blood groups with a distribution frequency of 
0.75-1‰. So, to trying to collect as many samples as possible according to the 
inclusion criteria in this study. 
Study 
population 
Inclusion criteria: 
1. Patients identified as "Asia type" DEL blood group; 
2. Male patients, or female patients ≥49 years of age, or female patients with 
severe illness and no further pregnancy plan; 
3. Patients with need for blood transfusion in line with the guidelines for 
internal or surgical and traumatic blood transfusion: 
* In line with the guidelines for internal blood transfusion of the Technical 
Specifications for Clinical Blood Transfusion, red blood cell transfusion is 
considered for the patients with chronic anemia caused by excessive 
destruction of red blood cells, loss or production disorders with hypoxic 
symptoms; hemoglobin <60g / L or hematocrit <0.2; 
* In line with the guidelines for surgical and traumatic blood transfusion of 
the Technical Specifications for Clinical Blood Transfusion, the red blood cell 
transfusion is considered for the patients who need to increase blood oxygen 
carrying capacity with normal or corrected blood volume; hemoglobin <70g/L; 
4. Signing the informed consent voluntarily for blood transfusion treatment 
before the trial. Patients and/or their legal representatives have ability to fully 
understand the trial content, process and possible adverse reactions, and 
enable patients to comply with the visiting and inspections specified in the 
protocol. 
Exclusion criteria: 
1. Patients who had a transfusion reverse reaction in a previous transfusion; 
2. Patients had allergies, immunodeficiency, and immunosuppression 
treatment; 
3. Patients needed blood transfusion for acute blood loss such as trauma; 

3 
 
4. Female patients had positive result of pregnancy test; 
5. Patients had conscious dysfunction and severe mental illness; 
6. Patients were judged unsuitable for the study by the investigator. 
Intervention 
method 
When the transfusion departments of the participating hospitals encounter 
the patients with the rare primary RhD negative blood group who needed to 
apply for blood transfusion in non-emergency situations, the application form 
and blood samples of the patients were sent to the reference lab of 
Guangzhou Blood Center for "Asia type" DEL blood group typing. The patients 
identified as "Asia type" DEL blood group and meeting the inclusion criteria 
will be provided the compatible RhD+ blood with the same ABO type after 
cross-matching test by Guangzhou Blood Center. RhD+ blood transfusion for 
the patients with "Asia type" DEL blood group was performed in the 
cooperated hospitals in line with the Technical Specifications for Clinical Blood 
Transfusion. 
Evaluation 
index for 
effectiveness  
Hemoglobin concentration (Hb); Hematocrit (Hct) etc. 
Evaluation 
index for 
safety 
1. Incidence of anti-D alloimmunization; 
2. Incidence of adverse blood transfusion reactions; 
3. Observation of vital signs: temperature, heart rate, breathing, blood 
pressure etc.; 
4. Laboratory testing; 
5. Incidence and severity of other adverse events. 
Visiting time 
Phase I of Visiting: -7~ 0 days before transfusion;  
Phase II of Visiting：Blood transfusion treatment period（0~1 days）; 
 
Phase III of Visiting：1 day to 1 month after blood transfusion;  
Phase IV of Visiting：1 month to 12 months after blood transfusion (based on 
the actual return visit in the hospital or follow-up at home) ; 
Phase V of Visiting：Over 12 months after blood transfusion. 
 
Statistical 
Analysis 
As the “Asia type” DEL is a rare blood group with a frequency of around 0.75-
1‰ in East and Southeast Asian population, it is not feasible to collect the 
enough samples to conduct the common statistical analysis. So, the 
descriptive statistics was regarded as the principal analysis method in this 
study. Continuous variables are described by number of cases, mean, standard 
deviation, median, minimum, and maximum; categorical variables are 
summarized by frequency and percentage. Statistical analysis is conducted 
using SAS9.4 statistical software. 
Number of 
research 
centers 
A total of 9 hospitals participate the trial. 
Progress of 
the trial 
The trial is expected to be completed within 48 months after beginning. 

4 
 
 
Protocol · Text 
I. Research Background 
Alloanti-D immunization against RhD blood group antigens in RhD-negative (D–) individuals 
can cause severe adverse transfusion reactions and fetal and neonatal hemolytic disease (HDFN). 
Therefore, routine RhD blood group testing and RhD matched blood transfusion strategies have 
been adopted in the most countries of the world. 
The D– phenotype is more common in white (15-17%) and African (3-5%) populations, but is 
rare in the Asian population (0.3-0.4%), which led to supply shortage of D– blood in Asian countries. 
In recent years, a series of studies have shown that 9–30% of the population with serologically 
apparent D– phenotype actually belong to a variant blood type called DEL (RhD elution), not the 
true D– phenotype in East and Southeast Asia, including China [1-7], South Korea [8, 9], Japan [10], 
and Thai [11], which is very rare among white and black races. DEL is a very weak form of expression 
of RhD antigen, that is, there is a very small amount of RhD blood group antigen expressing on the 
surface of red blood cells (RBC). It will not cause an effective agglutination reaction in conventional 
RhD blood group typing, so it will be detected as the rare D– blood group. Among the traditional 
serological methods, only adsorption/elution experiments can detect the DEL phenotype, but this 
method is time-consuming and reagent-intensive, and has high technical requirements for the 
operators, so it is not be applied as a routine clinical testing. In East Asia and Southeast Asia 
including China, the patients with the DEL phenotype are always clinically detected as the rare D–
patients and receive the rare D– blood transfusion to prevent alloanti-D immunization. 
The DEL phenotype has many different molecular genetic backgrounds with strong genetic 
heterogeneity. So far, more than 40 alleles have been reported to relate to the DEL phenotype, but 
RHD*1227A (RHD*01EL.01) is the predominant allele (>99%) distributed in the DEL individuals 
among East and Southeast Asian populations [1, 3, 6, 8-12]. Therefore, DEL with this allele is called 
the "Asia type" DEL blood group [13]. The DEL blood group can be divided into two main types 
according to whether the epitopes are intact and whether anti-D alloimmunization could occur 
[14]. One type is the "partial DEL" phenotype with missing D epitopes and anti-D alloimmunization 
can occur under the stimulation of D+ red blood cells. The "partial DEL" patients should be treated 
as D– rare patient in clinic to receive D– blood transfusion. The other is the “complete DEL" 

5 
 
phenotype with complete D antigenic determinants (with the same D antigen structure comparing 
with normal D+ antigen), but with low number of antigen expression. It will not cause anti-D 
alloimmunization under the stimulation of intact D+ antigen, so it is not necessary to treat it as the 
rare D– blood patients in clinic. 
In recent years, more and more experimental and clinical data show that the most common 
"Asia type" DEL in Asian populations has the characteristics of "complete DEL". First, in several 
independent studies, the adsorption/elution experiments did not detect the absence of D antigen 
determinant in "Asia type" individuals [2, 14]. In addition, the synonymous mutation in exon 9 of 
the RHD gene (c.1227G> A, p.K409K) defined the "Asia type" DEL allele have been shown to cause 
abnormal mRNA splicing in molecular mechanisms studies. It resulted in a series of abnormal 
mRNA transcripts lacking exon 9, but further Mini Splicing function analysis in vitro found that the 
"Asia type" DEL allele express extremely low abundance transcripts including complete exon 9. This 
transcript may express a very low density of normal D antigen, thus forming the "Asia type" DEL 
phenotype [15]. Most importantly, several prospective and retrospective studies on observation of 
"Asia type" DEL pregnant women have shown that none of them developed alloanti-D. These 
studies involved the data from at least a thousand of "Asia type" DEL pregnant women (including 
more than 600 cases studied in our study and hundreds of samples included in several other 
studies) [6, 13, 16, 17] in China, which covering more than one million of people. In contrast, in the 
true D– Chinese population excluding “Asia type" DEL, the frequency of alloanti-D immunity ranges 
from 8.5% to 40% [13, 17]. Therefore, clinical data show that in the case of “Asia type" DEL Chinese 
pregnant women with high probability of having a normal RhD-positive fetus (>95%), alloanti-D 
will not be produced by D+ red blood cells from the fetus and newborn during pregnancy and 
childbirth. 
Based on the results of previous research, it is extremely urgent to conduct a clinical trial of 
D+ blood transfusion in “Asia type" DEL patients, and to follow up in detail to obtain the anti-D 
immunization data in transfusion patients. This study is essential to set up a new blood transfusion 
strategy for “Asia type" DEL patient. In South Korea, two cases of “Asia type" DEL patients have 
been reported to receiving D+ blood transfusion, but still lacking of systematic clinical trial data to 
make the conclusion [18]. Therefore, the Korean National Blood Transfusion Guidelines (KNGBT) 
concludes that now there is not enough direct evidence to prove the safety of “Asia type" DEL 

6 
 
patients receive D+ blood transfusion, and D+ blood transfusion is only allowed in the condition of 
emergency or lacking of D– blood supply for “Asia type" DEL patients [18]. In summary, this clinical 
study has important practical significance and can provide basic evidence whether anti-D 
alloimmunization will occur in “Asia type" DEL patients with ordinary D+ blood transfusion. If a new 
strategy of D+ blood transfusion in “Asia type" DEL patients can be established in the future, that 
is, the routine “Asia type" DEL testing in patients with a primary D– phenotype could be performed, 
and then to treat the “Asia type" DEL patients as ordinary D+ patients, rather than the rare D– 
patients in clinic. It will save at least 25% of rare D– blood in East and Southeast Asian countries, 
and it will also prevent “Asia type" DEL pregnant women receive anti-D immunoglobulin injection 
and other medical intervention/monitoring to prevent anti-D associated fetal neonatal hemolytic 
disease, which also could save the precious blood product anti-D immunoglobulin that derived 
from the sera from the D– volunteers who are sensitized. 
 
II. Purpose of the trial 
To explore the safety and effectiveness of RhD+ blood transfusion in Chinese “Asia type” DEL 
patients to lay the foundation for establishing a new blood transfusion strategy for “Asia type" DEL 
patients. 
 
III. Design of the trial 
The aim of this study is to analyze whether anti-D alloimmunization will occur in “Asia type" 
DEL patients after D+ blood transfusion, and then to compare the results with the data from the 
true D– patients received D+ blood transfusion to determine the “Asia type" DEL patients should 
be treated as D+ or D– patients in the future. The ideal study control group is the incidence of anti-
D alloimmunization of true D– patients receiving D+ blood transfusion. But for ethical reasons 
(according to the requirements of Technical Specifications for Clinical Blood Transfusion, true D– 
patients can only be transfused with D+ blood in urgent situation or lacking of D– blood supply), 
and it is not appropriate to set up the control group (to transfuse D+ blood in true D– patients in 
non-emergency situations). Although the summarized rate of alloanti-D occurrence (22.9%, 53/231) 
[19-22] in true D– patients transfused with D+ blood in previous reported studies could be used as 
the control for the statistical analysis including sample size calculation, our study could not satisfy 

7 
 
the demands as the “Asia type” DEL transfusion satisfied the inclusion criteria belong to a rare 
event because of the rare distribution of “Asia type” DEL in our population (0.75-1‰). In theory, 
more than 2000 “Asia type” cases receiving D+ blood transfusion, which covering around fifty 
millions population, are necessary to compare the difference of alloanti-D occurrence between 
the “Asia type” DEL patients group in our study and the true D– control patients group with 
similar disease categories receiving D+ RBCs transfusion. It is not feasible in practice for this 
study. 
Therefore, a multi-center, single-arm clinical trial was performed in this study. When any 
hospital in Guangzhou city applies for D– blood supply in a non-emergency situation, routine “Asia 
type" DEL typing, antibody screening and identification, and cross-matching test for “Asia type" 
DEL patients were conducted in the reference lab of Guangzhou Blood Center. The “Asia type" DEL 
patients meeting the inclusion criteria were recruited in each cooperative hospital. After the 
informed consent for D+ blood transfusion was obtained from the patient or family members, 
cross-matched D+ blood was provided by Guangzhou Blood Center. D+ blood transfusion was 
performed at the corresponding hospital according to Technical Specifications for Clinical Blood 
Transfusion [23] and Technical Guidelines for Vaccine Clinical Trials [24].   
 
IV. Selection of Subjects  
(I) Inclusion criteria (same to the Protocol Abstract) 
(II) Exclusion criteria (same to the Protocol Abstract) 
(III) Rejection criteria 
1. Cases that did not meet the inclusion criteria or belong to one of the exclusion criteria after 
enrollment; 
2. Cases who did not receive blood transfusion finally in clinic after enrollment. 
 
(IV) Dropout criteria 
1. All patients, who signed the informed consent of blood transfusion, meet the inclusion 
criteria and enrolled, but failed to complete the follow-up observation in accordance with the 
protocol requirements; 
2. The subject requested to withdraw from the trial; 

8 
 
3. In the trial, if the subject has an adverse event that prevents him/her from completing the 
clinical trial, the investigator should record the process and outcome of the adverse event in 
detail, and make a judgment on the cause and effect of the trial. Cases dropout due to adverse 
events should be included in the statistical analysis of adverse events, but were not included in 
the evaluation of efficacy; 
4. The subject get Lost for follow-up after receiving transfusion treatment; 
5. The data of the CRF table is incomplete or cannot be traced to the source, which affects the 
evaluation of efficacy and safety. 
 
 
(V) Criteria for termination of the clinical trial 
Study termination means that the clinical study has not been completed as planned, and 
all studies have been stopped halfway. The purpose of the study termination is to protect the 
rights and interests of the subjects, ensure the quality of the trial, and avoid unnecessary 
financial losses. 
1. Serious safety problems occurred during the study, and the study should be suspended timely; 
2. It is found that there are major mistakes in the clinical protocol in the study, and it is difficult 
to evaluate the treatment effect; 
3. The research was stopped due to the requirements for funding and management; 
4. The administrative department revokes the test, etc.; 
When this occurs, the investigator needs to inform the subjects before stopping the trial. 
 
V. Experimental intervention 
When any hospital in Guangzhou city applies for D– blood supply in a non-emergency 
situation, the application and the patient's blood sample will be sent to the Reference laboratory 
of Guangzhou Center for “Asia type" DEL routine testing. For the “Asia type" DEL patients identified, 
the patients who meet the inclusion criteria will be enrolled in the hospital. After the patient or 
family members signed the informed consent for blood transfusion treatment, the matching D+ 
blood will be provided by Guangzhou Blood Center. According to the clinical blood transfusion 
guidelines, the blood transfusion for “Asia type" DEL patients was conducted in the corresponding 
hospital. According to the Technical Specifications for Clinical Blood Transfusion, the registration of 

9 
 
blood out-put and in-put, checking, and issuing should be carefully done in the blood transfusion 
departments or blood banks of all participating hospitals and the blood should be stored and 
transported in the temperature of 4 ± 2 ° C. 
1. Before blood transfusion, two medical staffs should check the cross-matching report, the label 
of the blood bag, and check whether the blood bag is damaged or leaking and whether the blood 
color is normal. When all of these have been confirmed to be normal, the transfusion could start. 
2. During the blood transfusion, two medical staffs will bring the medical records to the patient's 
bed to check the patient's name, gender, age, blood type, medical record number, 
outpatient/ward room, bed number, etc., and confirm that the blood matches the cross-matching 
report. After checking the blood again, the blood transfusion begins using a transfusion device 
that meets the standards. 
3. The blood should be transfused as soon as possible after taken back and not be stored. Before 
transfusion, to gently mix the ingredients in the blood bag and avoid violent shaking. No other 
drugs are permitted be added into the blood. If the dilution is needed, only saline for intravenous 
injection can be used. 
4. Flushing the vessel with the intravenous saline before and after blood transfusion. When the 
patient receives continuous transfusion with the blood from different donors, after the previous 
bag of blood has been transfused, the transfusion set should be flushed with the intravenous 
saline, and then the next bag of blood could be continuously transfused. 
5. During the process of blood transfusion, the speed of transfusion should be slow in the beginning 
and then faster, adjust the infusion rate according to the condition and age of the patient. It 
should be closely observed the recipients for adverse transfusion reactions. If abnormal 
conditions occur, the patient should be treated in time. 
 
VI. Observation index 
(I) Demographics 
To collecting the ID information, date of birth, gender, and telephone number of the patient. 
(II) Subject characteristics 
The source of the patient, case number, chief complaint, current medical history, clinical diagnosis, 
blood type, pregnancy-labor history, history of infectious diseases, blood transfusion history, 

10 
 
allergy history, and family history. 
(III) Safety observation indicators 
During the screening period, the laboratory test results within 3 days before the informed 
consent is signed are acceptable, and the method for laboratory tests are not limited and the 
results can be traced. For any abnormal symptoms, signs, laboratory test results (except 
pregnancy test results) in clinical research, no matter the degree of severity or the relation to 
treatment and the authorized investigator should determine that a repeated test is necessary or 
not. If necessary, the patients should be asked for regular visits, and the characters and treatment 
history should be recorded in detail. The follow-up should be continuing until the patient recover 
to the normal or baseline levels, or the investigator could reasonably explain the abnormality and 
do not think the follow-up is necessary. 
The main safety observation indicators shown below: 
1. The incidence of anti-D alloimmunization; 
2. The Incidence of adverse blood transfusion reactions; 
It is necessary to monitor the whole process of blood transfusion, and record the specific 
manifestations of adverse reactions of blood transfusion in detail. 
3. Observation of vital signs 
To monitoring the heart rate, breathing, blood pressure, and temperature. To measure blood 
pressure while resting. 
4. Laboratory testing 
1) Blood routine: white blood cells (WBC), neutrophils (NEUT), hemoglobin (Hb), hematocrit (Hct), 
platelets (PLT); 
2) Four items for infections detection; 
3) Liver function testing: alanine aminotransferase (ALT), aspartate aminotransferase (AST), total 
bilirubin (TBIL), direct bilirubin (DBIL), total plasma protein (TP), plasma albumin (ALB); 
4) Renal function testing: Urea, creatinine (Cr); 
5) Electrolyte inspection: potassium (K+), sodium (Na+), calcium (Ca2+), phosphorus (P3+), 
magnesium (Mg2+); 
6) Examination of coagulation function: prothrombin time (PT), activated partial thromboplastin 
time (APTT), thrombin time (TT), fibrinogen (FIB); 

11 
 
7) Urine routine; 
8) Stool routine, occult blood test. 
5. Pregnancy test: The women patients of childbearing age who are judged to need a pregnancy 
test based on the actual situation need to obtain a negative result to rule out pregnancy before 
enrollment. The women patients who reach menopause, or are undergoing menstruation at the 
time of the test, or have undergone birth control surgery do not need to have a pregnancy test, 
but need to record the reason for not doing so. 
6. Observation and recording of the adverse events 
It is necessary to monitor and record the occurrence of the adverse events and its development 
in detail throughout the clinical trial. 
(IV) Observation index of curative effects 
To record the changes in hemoglobin concentration and hematocrit before and after blood 
transfusion. 
(V) Concomitant treatment 
During the study, all concomitant treatments or combined medications (from the beginning 
of the signed informed consent to the end of the trial) must be recorded in detail in the original 
medical record, including the intervention name, intervention purpose, dosage form, single dose, 
dosage unit, frequency of use, route of administration, start date, end date, and whether it is still 
being used. 
For the combined treatment in the clinical research process, the corresponding analysis and 
recording should be done, especially the combined treatment in the event of adverse events, 
recorded and reported in a timely manner. 
 
VII. Evaluation Index 
(I) Main indicators for evaluation of the effectiveness  
Hemoglobin concentration and hematocrit. 
(II) The indicators for evaluation of the safety of the trial  
1. Incidence of anti-D alloimmunization; 
2. Transfusion r adverse reaction; 
3. Vital signs: temperature, heart rate, breathing, blood pressure; 

12 
 
4. Blood routine (WBC, NEUT, Hb, Hct, PLT), renal function (Urea, Cr), liver function (ALT, AST, TBIL, 
DBIL, TP, ALB), electrolytes (K+, Na+, Ca2+, P3+, Mg2+) before and after transfusion; 
5. Incidence and severity of adverse events. 
 
VIII. Steps of Clinical trial 
(I) Phase I of Visiting: -7 ~ 0 days before transfusion 
1. To explain the research purpose, methods, and requirements to the subjects, and to sign the 
informed consent for blood transfusion treatment by the two parties jointly; 
2. To Collecting the demographics, characteristics, past/concomitant medical history of the 
subjects, and to conduct detailed physical examinations, and measure vital signs (including heart 
rate, breathing, blood pressure, body temperature; to measure blood pressure in a resting state); 
3. Laboratory testing: 
1) "Asia type" DEL typing; 
2) Cross-matching test (only for the "Asia type" DEL patients); 
3) Blood routine: white blood cells (WBC), neutrophils (NEUT), hemoglobin (Hb), hematocrit (Hct), 
platelets (PLT); 
4) Four items for infections detection; 
5) Liver function testing: alanine aminotransferase (ALT), aspartate aminotransferase (AST), total 
bilirubin (TBIL), direct bilirubin (DBIL), total plasma protein (TP), plasma albumin (ALB); 
6) Renal function testing: Urea, creatinine (Cr); 
7) Examination of coagulation function: prothrombin time (PT), activated partial thromboplastin 
time (APTT), thrombin time (TT), fibrinogen (FIB); 
8) Urine routine; 
9) Stool routine, occult blood test; 
10) Pregnancy test: The women patients who reach menopause, or are undergoing menstruation 
at the time of the test, or have undergone birth control surgery do not need to have a pregnancy 
test, but need to record the reason for not doing so. 
11) Electrocardiogram. 
4. Combined treatment/medication; 
5. Evaluation of the patients for participation of this study based on inclusion and exclusion 

13 
 
criteria. 
(II) Phase II of Visiting：Blood transfusion treatment period（0~1 days） 
1. To record the detail of transfusion, including the time of transfusion start and end, the type of 
transfusion, the volume of transfusion, and to observe the adverse reaction during the 
transfusion treatment; 
2. Observation of vital signs: temperature, heart rate, breathing, blood pressure; 
3. Observation and recording of adverse events; 
4. Combined treatment/medication. 
(III) Phase III of Visiting：1 day to 1 month after blood transfusion 
1. Physical examination, observation of vital signs (including heart rate, breathing, blood pressure, 
body temperature and to measure blood pressure in a resting state); 
2. Laboratory testing: 
1) Detection of anti-D immune response (using antibody screening test); 
2) Blood routine: including white blood cells (WBC), neutrophils (NEUT), hemoglobin (Hb), 
hematocrit (Hct), platelets (PLT); 
3. Observation and recording of adverse events; 
4. Combined treatment/medication. 
(IV) Phase IV of Visiting：1 month to 12 months after blood transfusion  
1. Laboratory testing: 
1) Detection of anti-D immune response (using antibody screening test); 
2) "Asia type" DEL blood group typing. 
2. Observation and recording of adverse events. 
(V) Phase V of Visiting：Over 12 months after blood transfusion 
1. Laboratory testing: 
1) Anti-D immune response test (using antibody screening test); 
2) "Asia type" DEL blood group typing. 
2. Observation and recording of adverse events. 
 
IX. Adverse events, serious adverse events 
The clinical trials of blood transfusion during the study period shall strictly abide the 

14 
 
Management Practices for the Quality Control of Drug Clinical Trials (GCP) [25], and the Guiding 
Principles for Quality Management of Vaccine Clinical Trials (tentative standard) [26] and comply 
with relevant regulatory requirements. Adverse events that occur in clinical trials need to be 
monitored by relevant regulatory authorities and relevant parties (registered applicants, ethics 
committees, and researchers), and to be reported in accordance with the Regulations on the 
Management of Serious Adverse Events in Vaccine Clinical Trials (tentative standard). The 
researchers should strictly comply with the GCP principles and closely observe the occurrence of 
adverse events in each subject. All adverse events should be fully recorded in the CRF form, 
including symptoms, time of occurrence, severity (mild, moderate, severe), duration time, 
measures taken and condition of recovery. The researchers make a judgement about the 
relationship between the adverse events and research methods according to prescribed standards. 
The researcher should inform the subject or his legal guardian that he or she should promptly 
notify the doctor of any health problems so that the adverse events can be recorded or handled in 
a timely manner. 
(I) Definition 
Adverse Event (AE): An adverse medical event that occurs after a clinical trial subject received 
a blood transfusion treatment, but it does not necessarily have a causal relationship with the 
treatment. 
Serious adverse event (SAE) refers to the medical events that require hospitalization or 
prolonged hospital stay, disability, affecting work ability, threatening the life or death, and resulting 
in congenital malformations during the clinical trial. These include the following unexpected 
medical events: 
 
Events leading to death; 
 
Life-threatening event (defined as the subject has risk of death at the time of the event); 
 
Events requiring hospitalization or extension of hospitalization; 
 
Events that can cause permanent or severe disability/dysfunction; 
 
Congenital anomalies or birth defects; 
 
Causing other medical events and have the conditions listed above in the condition of lacking 
of treatment. 
(II) Grading criteria for the adverse events 

15 
 
When filling out the adverse event form, the researchers will use "mild, moderate, and 
severe" to describe the extent of the adverse event. To harmonize the standards, the levels of 
incidents are classified as follows: 
 
Level 1: Mild, short-term (less than 48 hours) or minor discomfort, no need for treatment; 
 
Level 2: Moderate, Mild or moderately restricted movement, may require medical attention, 
no or only need for minor treatment; 
 
Level 3: Severe, movement is significantly limited, requiring medical attention and treatment, 
and may require hospitalization; 
 
Level 4: Severe or potential life threatening, severely restricted movement, requiring 
treatment, emergency or hospitalization. 
The grading of the clinical observation indicators (local manifestation, vital signs, and systemic 
manifestation), laboratory testing indicators (blood biochemistry, blood routine, and urine routine), 
the clinical abnormalities, and the adverse events in the trial can refer to the Guideline of the 
Grading Standard for the Adverse Events in Preventive Vaccine Clinical Trials. 
(III) Evaluation of the correlation between adverse events and research interventions 
The researcher should conduct a comprehensive analysis according to the specific 
circumstance of adverse events of the subject and the subject's previous medical history, 
concomitant disease, and concomitant medication, to determine the relationship between the 
adverse event and the drug (product or treatment) with reference to the Manual of Adverse Drug 
Reaction Reporting and Monitoring issued by the State Drug Administration in November 2012 and 
the Administrative Measures for Adverse Drug Reaction Reporting and Monitoring implemented 
on July 01, 2011 (Order of the Ministry of Health No. 81). As far as possible, it should try to 
determine the relationship between the adverse events (including serious adverse events) and the 
drug (product or treatment). If it is determined to be related, no matter the conclusion is to be 
positively related, likely to be related, or may be related, all these will be regarded as adverse 
reactions caused by the intervention, and the incidence of adverse reactions will be counted. 
The five principles for evaluating the correlation between the intervention methods and 
adverse reactions: First, is there a reasonable time relationship between the intervention methods 
and the occurrence of adverse reactions/events? Second, does the response conform to the type 
of adverse reactions known to the intervention method? Third, does the reaction disappear or 
alleviate after the intervention method is stopped? Four, does the same reaction/event occur again 
when the method is used again? Five, can the adverse reaction/event be explained by the effect of 

16 
 
the combined medication/treatment, the progress of the patient's condition, and the impact of 
other treatments? 
According to the above five principles, the relevance evaluation is classified into 6 levels of 
positive, very likely, possible, possibly irrelevant, to be evaluated, and unable to be evaluated. 
Principles 
Results 
① 
② 
③ 
④ 
⑤ 
Positive 
＋ 
＋ 
＋ 
＋ 
－ 
Very likely 
＋ 
＋ 
＋ 
？ 
－ 
Possible 
＋ 
± 
±？ 
？ 
±？ 
Possibly irrelevant 
－ 
－ 
±？ 
？ 
±？ 
To be evaluated 
With a need to supplement evidence to evaluate 
Unable to be 
evaluated 
The necessary materials for evaluation are not available 
Note: ‘+’ means positive; ’－‘ means negative; ‘±’ means difficult to affirm or negative; ‘？’ means 
unknown 
(IV) Adverse events management 
Any adverse event, such as discomfort and abnormal laboratory tests of the subject, should 
be treated seriously, analyzed carefully, and immediate measures should be taken to protect the 
safety of the subjects. 
1. Handling procedure 
The adverse events occurred during the trial should be recorded in the CRF forms in terms of 
type, degree, occurrence time, duration, treatment measures, treatment process and outcome, 
etc. And to evaluate the relevance of the trial interventions with the reverse events based on the 
comprehensive consideration of comorbidities and combined medications and then recorded in 
detail by the physician. The investigator should follow up the adverse event until the symptoms 
disappear or stabilize. For the more serious adverse events, it should be followed up for a longer 
period, and the follow-up ways can be inpatient, outpatient, telephone, etc. The adverse events 
happened at the end of the study need to be followed up within one month after the study end. It 
should follow up the cases withdrawn due to adverse events and adverse reactions, and to record 
the continuity, outcome, and disappearance in detail. 
If the testing results for safety detection (blood routine, urine routine, stool routine, ECG, liver 

17 
 
function, kidney function) are abnormal after treatment, they must be retested at an appropriate 
time, truthfully recorded, and comprehensively analyzed to determine whether they are related to 
the intervention. 
2. Handling of the serious adverse events 
When there is a serious adverse event in a clinical trial, the researcher should immediately 
take appropriate treatment measures for the subject and report it to the clinical trial management 
department of the institution and then to notify the sponsor with a writing report referring to the 
Manual of Adverse Drug Reaction Reporting and Monitoring issued in November 2012 and the 
Administrative Measures for Adverse Drug Reaction Reporting and Monitoring implemented on 
July 01, 2011 (Order of the Ministry of Health No. 81), and The Regulations on the Management of 
Serious Adverse Events in Vaccine Clinical Trials (tentative standard) issued on the January 17, 2014. 
The clinical trial management department shall report to the corresponding ethics committee and 
the food and drug supervision and administration department and the health authority of the 
province, autonomous region, and municipality where the clinical trial institution is located within 
24 hours. For the death case, clinical trial agencies and researchers should provide all the 
information they need to the ethics committee and sponsors. 
The researcher should record all adverse events and method defects found during the clinical 
trial, and work with the sponsor to analyze the cause of the event, to form a written analysis report, 
and to put forward opinions on continuing, suspending or terminating the trial. Then, to submit 
the report to the ethics committee for review by the clinical trial management department of the 
clinical trial institution. 
For the serious adverse events and deficiencies in treatment methods that may lead to serious 
clinical consequences, the sponsor should report to the clinical trial supervision and management 
department and the health authority within 5 working days of being informed, and should also 
report at the same time to other clinical trial participating institution and the investigator. It shall 
also promptly notify the ethics committee of the clinical trial institution through their medical 
clinical trial management department. 
In this trial, please report the serious adverse event report to the National Medical Products 
Administration, the National Health and Family Planning Commission in a timely manner according 
to the following contact information, and notify the research institution regularly. 

18 
 
The investigator must complete a Serious Adverse Event Report Form, and should record the 
time, the way, and to whom to report the serious adverse events in the original data. The sponsor 
guarantees it to satisfy all reporting requirements of laws and regulations. 
The institute to report 
Contact 
department 
/person 
Telephone  
National 
Medical 
Products 
Administration（NMPA） 
Registration 
department 
010-88330732/010-
88330742 
Institute of clinical blood transfusion, 
Guangzhou Blood Center 
Yongshui Fu 
020-83595049 
In case of any serious adverse event unrelated to the trial method, the researchers shall 
withdraw from the clinical trial when they think it is necessary or the subject or his legal guardian 
requests to withdraw from the trial, and the withdrawal case shall complete the last laboratory 
examination. 
（V） Follow-up of the adverse events 
All adverse events should be followed up to the normal or baseline level, or the investigator 
can reasonably explain the abnormality and think that no follow-up is necessary to ensure the 
safety of the subjects. According to the type and severity of the adverse reactions, the follow-up 
methods can be selected as inpatient, outpatient, home visiting, and telephone visiting and other 
ways. 
 
X. Quality control of the clinical trial 
（I） Quality control measures  
Both the sponsor and the investigator should adopt Standard Operating Procedure (SOP) to 
ensure the quality control and the implementation of quality assurance system of the clinical 
research. All the observation results and findings in the clinical research should be verified to 
ensure the reliability of the data and the conclusions in the clinical research origin from the original 
data. Quality control must be adopted at every stage of data processing to ensure that all data are 
reliable and processed correctly. 

19 
 
（II） Training of researchers 
Before the formal starting of clinical research, the responsibility and attitude of the 
researchers participating in the clinical research must be emphasized firstly. The person in charge 
of the trial shall train the researchers on the experimental scheme, unify the understanding, be 
familiar with the collection methods and procedures, and understand the special requirements of 
the research project, so as to improve the internal observation consistency and inter observer 
consistency of the clinical research data collectors and ensure the reliability of the research 
conclusion. At the same time, the researchers are required to strictly implement SOP in the whole 
process of clinical research to ensure the implementation of quality control measures in clinical 
research and improve the quality of clinical research and case report form. 
（III） The measures to improve patients' compliance 
1. The investigator should carefully implement the informed consent of transfusion treatment 
to make the patient or his legal guardian fully understand the requirements of the trial and 
cooperate well. The sponsor provides free trial intervention and related laboratory tests. 
2. To monitor the compliance of the patient during the trial. 
（IV） The monitoring of clinical research 
The sponsor authorizes Guangzhou Haibote Pharmaceutical Technology Co., Ltd. to appoint 
qualified supervisors to conduct regular on-site monitoring visits in the experimental hospitals to 
ensure that all contents of the research plan are strictly observed and the original data are checked 
to ensure that they are consistent with the contents on the CRF. The appointed supervisor shall 
carry out the audit according to the requirements of GCP, and write the audit report, which includes: 
1. Whether the clinical trial institute has appropriate conditions; 
2. The patient's informed process complies with the declaration and regulations of Helsinki; 
3. The implementation of the trial plan; 
4. The protection measures for patients during the trial; 
5. To confirm that all data records and reports are correct and complete; 
6. To confirm that all adverse events are recorded and serious adverse events are reported 
and recorded within the specified time; 
7. To verify that the test method is carried out in accordance with the relevant laws and 
regulations, and have the corresponding records; 

20 
 
8. Including the supervision date, time, the name of the supervisor, the problems found during 
the supervision and handling methods, etc. 
（V） The inspection of clinical research 
The clinical trial supervision and management department and the sponsor could authorize 
the auditors to carry out systematic inspection on the clinical study to determine whether the 
implementation of the trial is consistent with the trial scheme and whether the reported data is 
consistent with the records of all clinical participating institutes, that is, whether the data reported 
or recorded in the case report form is the same with the medical record or other original records. 
The audit shall be performed by the person not directly involved in the clinical study. 
 
XI. Data management 
（I） Data entry and modification 
The leading institute of the trial is responsible for the data entry and management. For the 
questions in the case report form, the data manager will send the questions to the researcher 
through the supervisor, and the researcher shall answer and return them as soon as possible. The 
data manager will modify, confirm and input the data according to the researcher's answers, and 
send the DRQ again if necessary. 
（II） Data locking 
After all doubts are solved and the established database is confirmed to be correct, the data 
manager should write the data management audit report and lock the audited data. The locked 
data file and statistical plan will not be changed. 
 
XII. Statistical analysis 
（I） The population for statistical analysis  
The population for analysis includes safety analysis set (SS) and full analysis set (FAS). 
1. The Safety analysis population: the subjects receiving at least one time of blood transfusion. 
2. Full analysis population. 
（II） Statistical analysis method 
1. Demographic data and baseline analysis 
* Analysis according to FAS; 

21 
 
* Descriptive demographic data and other baseline characteristic values; 
* To calculate the number of cases, mean value, standard deviation, median, minimum and 
maximum for the continuous variables; 
* To calculate the frequency for the count and grade data. 
2. Safety analysis 
The SS population was used for safety analysis, and safety indexes included adverse events, 
laboratory examination, vital signs, etc. To summarize the data by stages and units. The adverse 
events will be aggregated. 
* Adverse events 
All adverse events will be coded with MedDRA. 
According to NCI CTCAE V5.0, the incidence (frequency) and severity distribution of AE, TEAE, 
SAE and AESI were summarized according to SOC and PT in MedDRA code. 
The subjects who terminated the treatment due to the adverse events, the subjects who had 
SAE and the dead subjects will be listed (at least including the start date, end date, severity, 
relationship with trial intervention, measures taken, outcome of AE). 
* Laboratory examination 
Blood routine and biochemical indexes were used to describe the measurement value and 
change value before and after treatment with mean ± standard deviation, maximum value, 
minimum value and median. And the normal and abnormal changes before and after treatment 
were described with cross classification table. 
Urine routine: use cross classification table to describe the normal and abnormal changes 
before and after treatment. 
The proportion of the subjects with abnormal and clinically significant changes will be 
described, in which whether the abnormal has clinical significance is judged by the researchers. 
The list of subjects with abnormal changes (with or without clinical significance) after 
treatment. 
* ECG examination 
Descriptive statistical ECG indicators compared with the baseline changes, using a cross 
classification table to describe the normal and abnormal changes before and after treatment, and 
a list of data will be provided. 

22 
 
* Vital signs, physical examination and other safety related examinations 
The examination results of vital signs and changes compared with the baseline will be 
described statistically. 
The subjects with abnormal changes in physical examination compared with baseline were 
described in the form of list. 
（III） The software for analysis 
SAS9.4 is used for the statistics analysis of the general data. 
 
XIII. Preservation of the original data 
The original data are kept in each clinical trial institution. The data kept by the researchers 
include the trial protocol and its amendment, the informed consent (form), the approval document 
of ethics committee, the member form of ethics committee, the original outpatient or inpatient 
medical records, the case report form and the research medical record (filled in, signed and dated), 
and the original serious adverse event report sent by the researcher to the sponsor. The 
researchers keep all research data, including confirmation of all patients that can effectively check 
different records, such as the original records of the hospital, all original informed consent signed 
by the patients, all case observation forms, research medical records, detailed records of 
transfusion treatment, etc. The researchers should keep the clinical trial data for at least 5 years 
after the termination of clinical trial. 
 
 

23 
 
XIV. Reference 
[1] Chen Q, Li M, Li M, et al. Molecular basis of weak D and DEL in Han population in Anhui 
Province, China. Chin Med J (Engl). 2012;125(18):3251-3255.  
[2] Gu J, Sun AY, Wang XD, et al. Analysis of density and epitopes of D antigen on the surface of 
erythrocytes from DEL phenotypic individuals carrying the RHD1227A allele. Blood Transfus. 
2014;12(2):244-249. doi: 210.2450/2013.0091-2413.  
[3] Li Q, Hou L, Guo ZH, Ye LY, Yue DQ, Zhu ZY. Molecular basis of the RHD gene in blood donors 
with DEL phenotypes in Shanghai. Vox Sang. 2009;97(2):139-146. doi: 110.1111/j.1423-
0410.2009.01181.x.  
[4] Shao CP, Maas JH, Su YQ, Kohler M, Legler TJ. Molecular background of Rh D-positive, D-
negative, D(el) and weak D phenotypes in Chinese. Vox Sang. 2002;83(2):156-161.  
[5] Xu Q, Grootkerk-Tax MG, Maaskant-van Wijk PA, van der Schoot CE. Systemic analysis and 
zygosity determination of the RHD gene in a D-negative Chinese Han population reveals a 
novel D-negative RHD gene. Vox Sang. 2005;88(1):35-40. doi: 10.1111/j.1423-
0410.2005.00584.x.  
[6] Xu W, Zhu M, Wang BL, Su H, Wang M. Prospective evaluation of a transfusion policy of RhD-
positive red blood cells into DEL patients in China. Transfus Med Hemother. 2015;42(1):15-21. 
doi: 10.1159/000370217.  
[7] Ye SH, Wu DZ, Wang MN, et al. A comprehensive investigation of RHD polymorphisms in the 
Chinese 
Han 
population 
in 
Xi'an. 
Blood 
Transfus. 
2014;12(3):396-404. 
doi: 
310.2450/2013.0121-2413.  
[8] Kim JY, Kim SY, Kim CA, Yon GS, Park SS. Molecular characterization of D- Korean persons: 
development of a diagnostic strategy. Transfusion. 2005;45(3):345-352. doi: 310.1111/j.1537-
2995.2005.04311.x.  
[9] Luettringhaus TA, Cho D, Ryang DW, Flegel WA. An easy RHD genotyping strategy for D- East 
Asian persons applied to Korean blood donors. Transfusion. 2006;46(12):2128-2137. doi: 
2110.1111/j.1537-2995.2006.01042.x.  
[10] Ogasawara K, Suzuki Y, Sasaki K, et al. Molecular basis for D- Japanese: identification of novel 
DEL and D- alleles. Vox Sang. 2015;109(4):359-365. doi: 310.1111/vox.12290.  
[11] Srijinda S, Suwanasophon C, Visawapoka U, Pongsavee M. RhC Phenotyping, 
Adsorption/Elution Test, and SSP-PCR: The Combined Test for D-Elute Phenotype Screening in 
Thai 
RhD-Negative 
Blood 
Donors. 
ISRN 
hematology. 
2012;2012:358316. 
doi: 
358310.355402/352012/358316.  
[12] Chen JC, Lin TM, Chen YL, Wang YH, Jin YT, Yue CT. RHD 1227A is an important genetic marker 

24 
 
for RhD(el) individuals. Am J Clin Pathol. 2004;122(2):193-198. doi: 110.1309/1303XMF-
1302NV1305-1707T-JE1307X  
[13] Shao CP. Transfusion of RhD-positive blood in "Asia type" DEL recipients. N Engl J Med. 
2010;362(5):472-473. doi:  410.1056/NEJMc0909552  
[14] Kormoczi GF, Gassner C, Shao CP, Uchikawa M, Legler TJ. A comprehensive analysis of DEL 
types: partial DEL individuals are prone to anti-D alloimmunization. Transfusion. 
2005;45(10):1561-1567. doi: 1510.1111/j.1537-2995.2005.00584.x  
[15] Fichou Y, Gehannin P, Corre M, et al. Extensive functional analyses of RHD splice site variants: 
Insights into the potential role of splicing in the physiology of Rh. Transfusion. 2015;55(6 Pt 
2):1432-1443. doi: 1410.1111/trf.13083.  
[16] Wang M, Wang BL, Xu W, et al. Anti-D alloimmunisation in pregnant women with DEL 
phenotype in China. Transfus Med. 2015;25(3):163-169. doi 110.1111/tme.12211.  
[17] Wang QP, Dong GT, Wang XD, et al. An investigation of secondary anti-D immunisation among 
phenotypically RhD-negative individuals in the Chinese population. Blood Transfus. 
2014;12(2):238-243. doi: 210.2450/2013.0184-2412  
[18] Choi S, Chun S, Seo JY, Yang JH, Cho D. Planned Transfusion of D-Positive Blood Components 
in an Asia Type DEL Patient: Proposed Modification of the Korean National Guidelines for 
Blood Transfusion. Annals of laboratory medicine. 2019;39(1):102-104. 
doi:110.3343/alm.2019.3339.3341.3102.  
[19] Baldwin ML, Ness PM, Scott D, Braine H, Kickler TS. Alloimmunization to D antigen and HLA 
in D-negative immunosuppressed oncology patients. Transfusion. 1988;28(4):330-333. 
doi:310.1046/j.1537-2995.1988.28488265260.x.  
[20] Boctor FN, Ali NM, Mohandas K, Uehlinger J. Absence of D- alloimmunization in AIDS 
patients receiving D-mismatched RBCs. Transfusion. 2003;43(2):173-176. 
doi:110.1046/j.1537-2995.2003.00289.x.  
[21] Frohn C, Dumbgen L, Brand JM, Gorg S, Luhm J, Kirchner H. Probability of anti-D 
development in D- patients receiving D+ RBCs. Transfusion. 2003;43(7):893-898.  
[22] Yazer MH, Triulzi DJ. Detection of anti-D in D- recipients transfused with D+ red blood cells. 
Transfusion. 2007;47(12):2197-2201. 
[23] No.184 [2000]. Technical Specifications for Clinical Blood Transfusion. 
[24] NMPA. Technical Guidelines for Vaccine Clinical Trials [20] 2004.12 
[25] NMPA. Management Practices for the Quality Control of Drug Clinical Trials 2016.12 
[26] NMPA. Guiding Principles for Quality Management of Vaccine Clinical Trials (tentative 
standard) 2013.1 
 

25 
 
XV. The flow chart of the Clinical research  
 
Stage 1 
Stage 2 
Stage 3  
Stage 4 
Stage 5 
Visiting time 
-7~ 0 Days 
before 
transfusion 
Blood 
transfusion 
treatment 
period
（0~1days） 
1 day to 1 
month 
after blood 
transfusion 
1 month to 
12 months 
after blood 
transfusion 
Over 12 
months 
after blood 
transfusion 
Informed consent 
signed 
● 
 
 
 
 
Basic information 
collection 
● 
 
 
 
 
Physical 
examination 
● 
 
● 
● 
● 
Vital signs 
● 
● 
● 
 
 
Blood routine 
● 
 
● 
 
 
Urine routine 
● 
 
 
 
 
Stool routine + 
Occult Blood ab 
● 
 
 
 
 
Liver function a 
● 
 
 
 
 
Renal function a 
● 
 
 
 
 
Coagulation 
function a 
● 
 
 
 
 
Four items for 
infections detection 
● 
 
 
 
 
Electrocardiogram a 
● 
 
 
 
 
HCG test b 
● 
 
 
 
 
Genotyping of “Asia 
type” DEL 
● 
 
 
● 
● 
Detection of 
alloanti-D 
● 
 
● 
● 
● 
Cross-matching test 
c 
● 
 
 
 
 
Review of the 
inclusion and 
exclusion criteria 
● 
 
 
 
 
Recording of 
adverse events 
 
● 
● 
● 
● 

26 
 
Recording of 
combined 
treatment 
● 
● 
● 
● 
● 
CRF reviewed by 
the person in 
charge 
 
 
 
 
● 
CRF reviewed by 
the inspector 
 
 
 
 
● 
 
Note：a In case of any abnormality of the patients during the follow-up, the authorized 
investigator shall determine whether re-examination is necessary. If necessary, the investigator 
shall be instructed to make a timely return visit, record in detail, and follow-up to the normal or 
baseline level, or the investigator can reasonably explain the abnormality and think that no 
follow-up is necessary; 
b Optional, depending on the clinical situation; 
c “Asia type” DEL patients will be involved.