RH Genotype Matched Red Cells for Patients With Sickle Cell Disease and Anti-D
Phase: EARLY_PHASE1
Conditions: Sickle Cell Disease, Anti-D Antibodies
Sponsor: Children's Hospital of Philadelphia

 
Template version: May 28, 2014 
 
Title: 
RH genotype matched red cells for patients with sickle cell 
disease and anti-D 
Short Title 
RHD genotype matching for anti-D 
eIRB Number 
19-016566 
Protocol Date: 
July 28, 2022 v5 
Amendment 1 Date:  
Amendment 3 Date:  
Amendment 2 Date: 
Amendment 4 Date: 
 
Study Principal Investigator 
Stella T. Chou, MD 
Children’s Hospital of Philadelphia 
Abramson Research Building, Room 316D 
3615 Civic Center Boulevard 
Philadelphia, PA 19104 
Phone 215-285-1811 
email: chous@email.chop.edu 
 
 
 

  
 
 
 
 
 
 
 
ii 
TABLE OF CONTENTS 
TABLE OF CONTENTS ................................................................................................................................... II 
ABBREVIATIONS AND DEFINITIONS OF TERMS ................................................................................ IV 
ABSTRACT ......................................................................................................................................................... V 
PROTOCOL SYNOPSIS ................................................................................................................................ VII 
TABLE 1: SCHEDULE OF STUDY PROCEDURES .................................................................................. IX 
1 
BACKGROUND INFORMATION AND RATIONALE ........................................................................ 1 
NO INDEX ENTRIES FOUND.1.2 .. NAME AND DESCRIPTION OF INVESTIGATIONAL PRODUCT OR INTERVENTION
 
1 
1.3 
RELEVANT LITERATURE AND DATA ..................................................................................................... 1 
1.4 
COMPLIANCE STATEMENT .................................................................................................................... 3 
2 
STUDY OBJECTIVES ............................................................................................................................... 4 
2.1 
PRIMARY OBJECTIVE ............................................................................................................................ 4 
3 
INVESTIGATIONAL PLAN ..................................................................................................................... 4 
3.1 
GENERAL SCHEMA OF STUDY DESIGN .................................................................................................. 4 
3.1.1 
Screening Phase and Baseline Assessment ....................................................................... 4 
3.1.2 
Intervention visit ........................................................................................................................ 4 
3.1.3 
Follow-up Phase ....................................................................................................................... 5 
3.2 
STUDY DURATION, ENROLLMENT AND NUMBER OF SITES ................................................................... 5 
3.2.1 
Duration of Study Participation ............................................................................................... 5 
3.2.2 
Total Number of Study Sites/Total Number of Subjects Projected ................................... 5 
3.3 
STUDY POPULATION ............................................................................................................................. 5 
3.3.1 
Inclusion Criteria ....................................................................................................................... 5 
3.3.2 
Exclusion Criteria ...................................................................................................................... 6 
4 
STUDY PROCEDURES ............................................................................................................................. 6 
4.1 
SCREENING VISIT AND BASELINE ASSESSMENT .................................................................................... 6 
4.2 
STUDY TREATMENT PHASE ................................................................................................................... 6 
4.2.1 
Visit 1 .......................................................................................................................................... 8 
4.2.2 
Visit 2 .......................................................................................................................................... 8 
4.2.3 
Visit 3 plus .................................................................................................................................. 8 
4.3 
FOLLOW-UP PHASE ............................................................................................................................... 8 
4.3.1 
End of Study .............................................................................................................................. 8 
4.4 
UNSCHEDULED VISITS .......................................................................................................................... 9 
4.5 
SUBJECT COMPLETION/WITHDRAWAL .................................................................................................. 9 
4.5.1 
Early Termination Study Visit .................................................................................................. 9 
5 
STUDY EVALUATIONS AND MEASUREMENTS ............................................................................ 10 
5.1 
SCREENING AND MONITORING EVALUATIONS AND MEASUREMENTS ................................................ 10 
5.1.1 
Medical Record Review ......................................................................................................... 10 
5.1.2 
Physical Examination ............................................................................................................. 10 
5.1.3 
Vital Signs ................................................................................................................................ 10 
5.1.4 
Laboratory Evaluations .......................................................................................................... 10 
5.1.5 
Diagnostic Tests ...................................................................................................................... 11 
5.2 
SAFETY EVALUATION ......................................................................................................................... 11 
6 
STATISTICAL CONSIDERATIONS .................................................................................................... 12 
6.1 
PRIMARY ENDPOINT ............................................................................................................................ 12 
6.2 
STATISTICAL METHODS ...................................................................................................................... 12 

 
 
 
 
 
 
 
 
iii 
6.2.1 
Baseline Data .......................................................................................................................... 12 
6.2.2 
Efficacy Analysis ..................................................................................................................... 12 
6.2.3 
Safety Analysis ........................................................................................................................ 12 
6.3 
SAMPLE SIZE AND POWER ................................................................................................................... 12 
6.4 
INTERIM ANALYSIS ............................................................................................................................. 12 
7 
STUDY INTERVENTION ....................................................................................................................... 13 
7.1 
DESCRIPTION ...................................................................................................................................... 13 
7.1.1 
Packaging ................................................................................................................................ 13 
7.1.2 
Labeling .................................................................................................................................... 13 
7.1.3 
Dosing ....................................................................................................................................... 13 
7.1.4 
Treatment Compliance and Adherence .............................................................................. 13 
7.1.5 
Transfusion Product Accountability ..................................................................................... 13 
8 
SAFETY MANAGEMENT ...................................................................................................................... 14 
8.1 
CLINICAL ADVERSE EVENTS ............................................................................................................... 14 
8.2 
ADVERSE EVENT REPORTING ............................................................................................................. 14 
8.3 
DEFINITION OF AN ADVERSE EVENT ................................................................................................... 14 
8.4 
DEFINITION OF A SERIOUS ADVERSE EVENT (SAE) ............................................................................ 15 
8.4.1 
Relationship of SAE to study drug or other intervention ................................................... 15 
8.5 
IRB/IEC NOTIFICATION OF SAES AND OTHER UNANTICIPATED PROBLEMS ...................................... 15 
8.5.1 
Follow-up report ...................................................................................................................... 16 
8.6 
INVESTIGATOR REPORTING OF A SERIOUS ADVERSE EVENT TO SPONSOR .......................................... 16 
8.7 
MEDICAL EMERGENCIES ..................................................................................................................... 16 
9 
STUDY ADMINISTRATION .................................................................................................................. 17 
9.1 
DATA COLLECTION AND MANAGEMENT ............................................................................................. 17 
9.2 
CONFIDENTIALITY ............................................................................................................................... 17 
9.3 
REGULATORY AND ETHICAL CONSIDERATIONS .................................................................................. 17 
9.3.1 
Data and Safety Monitoring Plan ......................................................................................... 17 
9.3.2 
Risk Assessment .................................................................................................................... 18 
9.3.3 
Potential Benefits of Trial Participation ............................................................................... 19 
9.3.4 
Risk-Benefit Assessment ....................................................................................................... 19 
9.4 
RECRUITMENT STRATEGY ................................................................................................................... 19 
9.5 
INFORMED CONSENT/ASSENT AND HIPAA AUTHORIZATION ............................................................. 19 
9.6 
PAYMENT TO SUBJECTS/FAMILIES ...................................................................................................... 19 
10 
PUBLICATION ................................................................................................................................ 20 
11 
REFERENCES ................................................................................................................................. 20 
  

  
 
 
 
 
 
 
 
iv 
ABBREVIATIONS AND DEFINITIONS OF TERMS 
°C 
 
Degrees centigrade 
AE 
 
Adverse event 
CBC 
 
Complete blood count 
DHTR 
 
Delayed hemolytic transfusion reaction 
Hgb 
 
Hemoglobin 
PI 
 
Principal investigator 
RBC 
 
Red blood cells 
SCD 
 
Sickle cell disease 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

  
 
 
 
 
 
 
 
v 
ABSTRACT 
Context:   
Red blood cell transfusion remains a critical therapy for patients with sickle cell disease 
(SCD). A major problem is the high rate of alloimmunization (antibody formation against 
transfused red cells) that occurs in patients with SCD. Recent studies performed by our 
group and others demonstrate RH genetic variants in patients and donors is a major risk 
factor leading to Rh alloimmunization. Anti-D formation in D+ patients occurs frequently, 
and once identified, providing D- cells for all subsequent transfusions can be challenging. 
These anti-D antibodies in D+ patients suggest exposure to different or variant D protein on 
donor cells. We will test whether transfusion of patients with anti-D with RHD genotyped 
matched red cells is feasible, safe and can decrease D- donor unit demand.  
Objectives:  
Primary: we will prospectively study whether provision of RHD genotype and C, E, K 
matched red cells to chronically transfused patients with a history of anti-D is feasible and 
safe. 
Study Design:  
This is a pilot study to evaluate the feasibility and safety of providing RH genotype matched 
D+ RBCs to chronically transfused patients with SCD who type D+ but have formed anti-D 
and are currently transfused with D- RBC units. 
Setting/Participants: 
The study setting will be the apheresis unit, the outpatient day hospital or the inpatient units 
at the Children’s Hospital of Philadelphia.  
There will be two sites: The Children’s Hospital of Philadelphia, New York Blood Center.  
We will recruit 20 subjects overall from The Children’s Hospital of Philadelphia. The first 5 
subjects enrolled will be adults.  
Inclusion Criteria include: 
• 
Diagnosis of SCD, all genotypes 
• 
Require a period of chronic red cell transfusion therapy 
• 
Require a minimum of 3 units per transfusion visit 
• 
History of anti-D (anti-D not detectable at screening visit and past 6 months) 
• 
RH genotype predicts D+ expression  
 
Study Interventions and Measures:  
We will provide one red cell unit of D+ RH genotype matched RBCs at the first transfusion 
study visit. The remainder of units will be provided per clinical standard of care, i.e. D-, 
CEK-matched, and negative for all other antigens the patient is alloimmunized against. If 
laboratory monitoring shows no reappearance of anti-D and no signs of increased red cell 
hemolysis, the patient will receive one unit of D+ RH genotype matched RBCs at the 2nd 
transfusion study visit, and if tolerated, D+ red cell exposures will increase by one unit per 
study visit until all units required are D+. 

  
 
 
 
 
 
 
 
vi 
For subjects with a history of stroke/recurrent transient ischemic attack or other indication 
who require tight control of Hb S and RH genotyped blood is not available, standard of care 
serologic matched blood would be administered rather than delaying transfusion and risking 
higher Hb S level. 
We will collect a pre-transfusion sample to prospectively store plasma on all study subjects.  
Study participants will return 5-12 days post-transfusion for a CBC, reticulocyte count, hgb 
quantification, bilirubin level, and antibody screen to evaluate for anti-D reappearance or 
red cell hemolysis. 
Main study outcome measures are feasibility of identifying sufficient RH genotype matched 
units (identifying sufficient RH genotype match red cells without delays in transfusion), and 
safety (no anti-D reappearance or evidence of hemolysis of transfused red cells).  
 

 
 
 
 
 
 
 
 
 
 
vii 
PROTOCOL SYNOPSIS 
Study Title 
RH genotype matched red cells for patients with sickle cell disease 
and anti-D 
Funder 
National Institutes of Health 
Clinical Phase 
Pilot 
Study Rationale 
Patients with SCD exhibit significantly increased rates of 
alloimmunization compared to other chronically transfused 
populations, and antibodies formed against Rh are the most 
common, despite provision of standard serologic Rh matched red 
cells. Recent studies reveal RH gene variation is common in 
patients with SCD and in African-American blood donors, and 
contributes to Rh alloimmunization. Many D+ patients have formed 
anti-D, and now require D- units. These anti-D suggest exposure to 
different or variant D antigen on donor cells. We will test whether 
providing RH genotype matched D+ RBCs to chronically transfused 
patients with SCD is feasible, safe, and can lessen demand for D- 
RBC units. 
Study Objective(s) 
Primary  
• 
To determine whether provision of RHD genotype and C, E, K 
matched red cells to chronically transfused D+ patients with a 
history of anti-D is feasible and safe 
Test Article(s) 
 
RH genotyped D+ donor red cell units will be matched to the 
patient’s RH genotype. 
Study Design 
 
This is a pilot study to evaluate the feasibility and safety of 
providing RH genotype matched D+ RBCs to patients with SCD 
who type D+ but have formed anti-D and currently require D- RBC 
units. We will identify D+ patients with a historical anti-D that is not 
currently detectable, and on a chronic red cell exchange program. 
RH genotyped donor units will be obtained from the New York 
Blood Center. We will transfuse one D+ unit matched by RHD 
genotype at the first two study visits and increase D+ units 
incrementally with subsequent transfusion visits.  We will determine 
whether sufficient RH matched donor units can be identified for the 
patient’s RH genotype (identifying sufficient RH genotype match 
red cells without delays in transfusion), and its safety (no anti-D 
reappearance or evidence of increased red cell hemolysis).  
For subjects with a history of stroke/recurrent transient ischemic 
attack or other indication who require tight control of Hb S and RH 
genotyped blood is not available, standard of care serologic 
matched blood would be administered rather than delaying 
transfusion and risking higher Hb S level. 
For all subjects, standard of care serologic matched blood would be 
administered rather than delaying transfusion beyond 7 days. 

 
 
 
 
 
 
 
 
 
 
viii 
We will enroll at least 5 adults to collect safety and preliminary 
efficacy data, before enrolling children to the study.   
Subject Population 
key criteria for Inclusion 
and Exclusion: 
Inclusion Criteria 
1. Subjects age > 8 years old 
2. Diagnosis of SCD, all genotypes 
3. Require chronic red cell transfusion therapy 
4. History of anti-D 
5. RH genotype predicts D+ expression 
Exclusion Criteria 
1. Rare RH genotype that would preclude sufficient RBC 
units 
2. Antigen negative requirements due to alloimmunization 
that would preclude sufficient RBC units 
Number of Subjects  
 
Total Number of Subjects = 20 
Total Number at CHOP = 20 
Total Number of Sites = 2 
Study Duration 
Each subject’s participation will last up to 2 years depending on 
how many red cells units are transfused per visit. 
The entire study is expected to last 5 years. 
Study Phases 
Screening 
Study Treatment 
Follow-Up   
(1) Screening: screening for eligibility and obtaining consent  
(2) Intervention: study intervention of D+ RH genotype matched red 
cell units for transfusion for patients with SCD and history of anti-D 
(3) Follow-up: monitor feasibility of identifying sufficient RH 
genotype matched red cell units and formation of Rh antibodies by 
ongoing chart review 
Safety Evaluations 
Study participants will return 5-12 days post-transfusion for a CBC, 
reticulocyte count, hgb quantification, bilirubin level, and antibody 
screen to evaluate for anti-D reappearance or red cell hemolysis. 
Statistical and Analytic 
Plan 
This a pilot feasibility and safety study that will evaluate qualitative 
measures and determine whether adequate RH genotyped units 
can be identified for chronically transfused patients with SCD in a 
timely fashion, and whether patients experience anti-D 
reappearance and/or red cell hemolysis of transfused cells. 
DATA AND SAFETY 
MONITORING PLAN 
The PI will be responsible for data quality management and 
ongoing assessment of safety. 

 
ix  
 
 
 
 
 
 
 
TABLE 1: SCHEDULE OF STUDY PROCEDURES  
 
Study Phase 
Screening and 
baseline assessment 
Intervention visit 
Visit Number 
1 
2+ 
Informed Consent/Assent 
X 
 
Review Inclusion/Exclusion 
Criteria 
X 
 
Demographics/Medical History 
X 
 
Interim Medical History 
 
X 
Physical Examination 
X 
 
Vital Signs 
X 
X 
Review of Height and Weight 
X 
X 
Medication review 
X 
X 
Clinical Laboratory Review  
X 
X 
Blood draw for study sample 
 
X 
Transfusion of RH genotype 
matched red cell units 
 
X 
Blood draw for follow-up labs 
5-12 days after transfusion 
 
X 
Adverse Event Assessment 
 
X 

 
page x 
  
 
 
 

 
  
 
 
 
1 
1 
BACKGROUND INFORMATION AND RATIONALE 
1.1 
Introduction 
Red blood cell transfusion remains a life-saving therapy for patients with sickle cell disease 
(SCD).  A major problem is the high rate of alloimmunization (antibody formation against 
transfused red cells) that occurs in transfused patients with SCD. Alloimmunization leads to 
delays in care, increases costs, and makes transfusion therapy unsafe and impossible for 
some patients.  The most common antibodies formed by patients with SCD are directed 
against the Rh blood group system.  Genetic diversity in Rh antigens in patients and blood 
donors of African descent contributes to the high incidence and complexity of antibodies 
found in patients with SCD.1  Recent studies performed by our group and others demonstrate 
RH genetic variants in patients and donors is a major risk factor leading to Rh 
alloimmunization.  Routine blood bank assays cannot identify these variants, so although Rh-
matched red cells by standard serologic assays are transfused to patients with SCD, they 
would only be truly Rh-matched with DNA-based matching. Many D+ patients have formed 
anti-D, and now require D- units, resulting in new challenges. These anti-D suggest exposure 
to different or variant D protein on donor cells. The major goal of this study is to perform pilot 
clinical studies to test whether providing RH genotype matched D+ RBCs to chronically 
transfused patients with SCD and anti-D is feasible, safe, and can lessen demand for D- RBC 
units.  RH genotype matched red cells are not currently available for transfusion outside this 
study except for rare cases. We will determine the feasibility of identifying adequate genotype 
matched donor units in real clinical practice, identify barriers, and prospectively monitor 
patients for anti-D reappearance or signs of hemolysis. 
1.2 
Name and Description of Investigational Product or Intervention 
Chronically transfused patients with SCD and anti-D will receive D+ RH genotyped matched 
red cell units for transfusion in addition to standard C, E, and K antigen matching and being 
hemoglobin S negative, which is our institutional standard of care for patients with SCD. RH 
genotyping of donor units will be performed by the NYBC Immunogenetics laboratory, led by 
Dr. Connie Westhoff, co-investigator.   
1.3 
Relevant Literature and Data 
Alloimmunization is a major complication of blood transfusion in patients with SCD 
One major explanation for high alloimmunization rates in patients with SCD is the disparate 
distribution of RBC antigens between patients who are primarily of African ancestry, and 
blood donors of non-African ethnic backgrounds.2  The frequency of C, E, K, Jkb, Fya, Lea 
and S antigens is significantly lower in individuals of African descent compared to blood 
donors who are primarily of European descent.  Outside of ABO, the Rh blood group system 
is the most immunogenic.  Since sensitization to Rh antigens (D, C, c, E, e) and to K (a Kell 
system antigen) comprise a majority of the RBC antibodies encountered in patients with SCD, 
consensus guidelines including the 2014 NHLBI expert panel report, recommend provision 
of C, E, K-matched RBCs to this patient population.3  Transfusion with RBC units from African 
American donors with the same ethnic background, who are more likely to have similar blood 
group antigen profiles, has also been suggested to mitigate exposure to foreign antigens that 
cause high rates of alloimmunization. This approach has been combined with C, E, K 
matching for over two decades at The Children’s Hospital of Philadelphia (CHOP).  
RBC alloimmunization despite transfusion from Rh matched minority donors  

 
  
 
 
 
2 
We performed a 15-year retrospective analysis to assess antigen matching for D, C, E, K and 
transfusion from African American donors on alloimmunization rates, antibody specificity, and 
clinical significance in pediatric and young adult patients with SCD (featured article in 
BLOOD).1 Our study included 182 patients who received a total of 44,482 RBC units. 
Surprisingly, we found 58% of 123 chronic and 15% of 59 episodically transfused patients 
were alloimmunized (median units transfused per patient was 230 and 3, respectively).  Two-
thirds of the antibodies formed were directed against the same Rh blood group antigens that 
were targeted for prevention (D, E, e, C, c, Fig. 1A).  Notably, the majority of Rh antibodies 
occurred in patients whose red cells were phenotypically positive for the corresponding Rh 
antigen and would not be expected to form antibodies to a “self” antigen. Anti-D was identified 
in 29 patients whose cells all typed D+ (Fig. 1B).  Overall, one-third of these antibodies were 
clinically significant with lower hemoglobin (hgb) or % hgb S levels than their baseline pre-
transfusion values, suggestive that the patient hemolyzed the transfused RBCs. 
Genetic analysis of the two genes that encode the Rh antigens, RHD and RHCE, revealed 
that >85% of our patients carry variant alleles that result in loss or alteration of Rh antigenic 
epitopes.1 We subsequently showed that African American donors share a similar degree of 
RH genetic variation.4  Variant Rh antigens are not detected by traditional blood bank tests 
which identify only the common Rh antigens (D,C,c,E,e).  Consequently, patients with SCD 
are at risk of Rh antibody formation when exposed to conventional Rh antigens on donor red 
cells,1 or other foreign Rh variants on African American donor cells.4  This problem is unique 
to patients with SCD who are primarily of African Black ethnicity, as RH variation occurs in 
<3% of individuals from other ethnic populations, but up to 85% of Blacks. For anti-D, we 
identified numerous D+ patients with at least one conventional RHD allele, yet still formed 
anti-D antibodies, suggesting an immune response to variant D exposure from African 
American donor units. 
 
RH genes and proteins 
The Rh system is the most complex of all blood group systems and includes greater than 50 
different antigens encompassing polymorphic epitopes, but standard serologic “Rh typing” of 
red cells tests for the five common antigens in all populations (D,C,c,E,e). The Rh proteins 
are encoded by two genes: RHD encodes the D antigen and RHCE encodes the CE antigens 
in various combinations (ce, cE, Ce, CE) (Fig. 2).  Individuals with D antigen expressed are 
“Rh positive” and the absence of D, usually due to RHD gene deletion, are “Rh negative.” 
The highest incidence of “Rh negative” (~15%) occurs in those of European ancestry. The 
RH genes are inherited as a haplotype, and allele frequencies differ in various populations.  
 
Figure 1. Alloimmunization in 182 patients with SCD transfused with D,C,E,K-matched RBCs from 
minority donors. A. 146 antibodies were identified and 70% had Rh specificity. B. 56 Rh antibodies occurred 
in patients whose RBCs typed positive for that antigen (sometimes mistaken for “autoantibodies”), and 35 were 
identified in antigen-negative patients despite transfusion with Rh-matched RBCs (i.e. negative for that antigen). 
A
B

 
  
 
 
 
3 
Rh variants encode “partial” Rh antigens undetectable with current typing reagents 
RHD and RHCE genetic variants are frequent in individuals of African ethnicity and result in 
altered epitopes often termed “partial” Rh antigens because they lack common epitopes.  
Patients with variant RH who lack commonly encoded epitopes are at risk of antibody 
production if exposed to these Rh epitopes via transfusion or pregnancy. Thus, D+ individuals 
with “partial D” antigen may form anti-D (to the epitopes of D they lack) when exposed to 
conventional D antigen.1 For example, RHD*DAU4 encodes a protein in which lysine 
replaces glutamic acid at amino acid position 233 resulting in loss or alteration of one or more 
common RhD epitopes (Fig. 2).  Variant RHCE alleles encoding “partial C, c, or e antigens” 
occurs frequently in African Blacks, and their RBCs often lack high prevalence Rh antigenic 
epitopes, such as hrB and hrS, and express novel antigenic epitopes (V, VS).  The relatively 
common altered allele, RHCE*ce(733G), encodes a new antigen VS and loss of the high 
prevalence antigen hrB (Fig. 2).  We demonstrated that variant RHD or RHCE contributes to 
Rh alloimmunization and delayed transfusion reactions in patients with SCD.1,5  Inheritance 
of variant RH alleles explained ~1/3 of antibodies, but the remainder were found in patients 
with the corresponding conventional alleles or in patients who were C- or E-negative and had 
received donor units typed as negative.  These observations suggested that Rh antibodies 
are not only a result of inheriting variant RH alleles but may also be the result of variant or 
altered epitopes on African American donor red cells.4  However, recruitment of African  
American 
donors 
is 
important to identify an 
adequate supply of CEK 
negative units. Genotype 
matching 
of 
donors 
to 
patients may be feasible in 
the future, but the cost and 
infrastructure of genotyped 
red cells for patients with 
SCD is currently prohibitive. 
With improved sequencing 
approaches, we anticipate 
cost will not remain a 
longstanding 
challenge. 
Feasibility of RH genotyping 
for patients with SCD is an 
important first step.  
In a recent publication from Brazil, 11 patients with SCD requiring chronic transfusion who 
typed D+ and had a history of anti-D, were switched from D- to D+ units after RH genotyping 
revealed at least one conventional RHD allele.6 The D+ donor units were not RH genotype 
matched, so they only switched patients who had at least one conventional RHD allele. The 
authors reported that no patient exhibited adverse effects or hemolytic transfusion reactions 
to transfusion with D+ red cells, and over 200 D- units remained in the inventory for 
emergencies or transfusion of D- individuals on an annual basis. 
1.4 
Compliance Statement 
This study will be conducted in full accordance of all applicable Children’s Hospital of 
Philadelphia Research Policies and Procedures and all applicable Federal and state laws 
and regulations including 45 CFR 46, and the Good Clinical Practice: Consolidated Guideline 
approved by the International Conference on Harmonisation (ICH). All episodes of 
noncompliance will be documented. 
Figure 2. Rh proteins. Predicted 12 transmembrane RhD and RhCE 
proteins (left). The amino acid differences between conventional RhD 
and RhCE are shown as red circles. Positions 103 and 226 in RhCE are 
critical for C or c and E or e expression. Yellow circles depict amino acid 
changes on variant Rh proteins DAU4 and ce(733G) (right). 

 
  
 
 
 
4 
The investigators will perform the study in accordance with this protocol, will obtain consent 
and assent, and will report unanticipated problems involving risks to subjects or others in 
accordance with The Children’s Hospital of Philadelphia IRB Policies and Procedures and all 
federal requirements. Collection, recording, and reporting of data will be accurate and will 
ensure the privacy, health, and welfare of research subjects during and after the study.  
2 
STUDY OBJECTIVES 
The purpose of the study is to determine the feasibility, safety and challenges of transfusing 
RHD genotype matched donor red cells for D+ patients with SCD and history of anti-D. 
2.1 
Primary Objective 
The primary objective of this study is to determine the feasibility and safety of RH genotype 
matched red cells (intervention) for chronically transfused D+ patients with SCD and history 
of anti-D.  We will determine whether sufficient RHD genotyped units can be matched to the 
patient’s own RH genotype, and whether re-exposure to D+ units that are RHD genotype 
matched is safe, as monitored by anti-D reappearance or signs of hemolysis.   
3 
INVESTIGATIONAL PLAN 
3.1 
General Schema of Study Design 
There will be a screening and baseline assessment, and a treatment phase.  See Table 1 for 
an overview of the study visits and procedures. 
We will enroll at least 5 adults to collect safety and preliminary efficacy data, before enrolling 
children to the study. The data gathered will be submitted to the IRB via an amendment to 
be reviewed by the convened board. We will obtain IRB approval before opening enrollment 
to children.   
3.1.1 Screening Phase and Baseline Assessment 
Potential subjects will be screened using the protocol inclusion and exclusion criteria. We will 
identify D+ patients with a historical anti-D that is not currently detectable, and on a chronic 
transfusion program (by apheresis preferably). Eligible subjects will be approached in the 
hematology clinic, the Apheresis Unit, the Day Hospital (where patients receive straight 
transfusions), or the inpatient units at The Children’s Hospital of Philadelphia.  
Subject/parental/guardian permission (informed consent) and, if applicable, child assent, will 
be obtained prior to any study related procedures being performed, including discontinuation 
of current therapy.   
A baseline assessment will be performed at the time of a clinic, day hospital transfusion, or 
apheresis visit. At the baseline assessment, one of the clinical study team members will 
obtain a medical history, physical exam, and review pertinent laboratory studies. 
3.1.2 Intervention visit 
Intervention visits will occur at the time of each of the patient’s scheduled transfusion visits.  
Typically, patients requiring chronic transfusion therapy present for transfusion every 3-5 
weeks to maintain their target hemoglobin S level.  
At these intervention visits, a member of the research team will obtain an interim medical 
history, as well as review clinically obtained vital signs and laboratory evaluations. At the time 

 
  
 
 
 
5 
of pre-transfusion blood draw, IV placement or central line access, 5-10 ml of blood will be 
drawn for study evaluations (for additional antibody evaluation as needed). Patients will 
receive their transfusions as ordered for their clinical indication, but will receive red cell units 
from the blood bank that are RH genotype matched in addition to standard C, E, and K 
antigen matching. Other red cell processing such as irradiation, leukoreduction and/or 
washing will occur as clinically indicated. We will transfuse one D+ unit matched by RHD 
genotype at the first study visit, increase D+ units incrementally with subsequent transfusion 
visits, and monitor for anti-D re-demonstration and signs of hemolysis or other adverse 
effects. Adverse events will be assessed at each of these intervention visits. The next 
transfusion and intervention visit will be scheduled prior to the subject’s discharge from their 
transfusion visit. 
A laboratory visit will be scheduled 5 to 12 days after each transfusion to screen for antibodies 
and laboratory signs of increased red cell hemolysis from baseline.  
If there is an inability to identify sufficient RH genotype matched units, standard of care 
serologic matched red cells will be administered rather than delaying transfusion for more 
than 7 days.   
3.1.3 Follow-up Phase 
The follow-up phase involves medical chart review and will continue for up to 6 months after 
the subject’s last transfusion and intervention visit.   
3.2 
Study Duration, Enrollment and Number of Sites 
3.2.1 Duration of Study Participation 
The study duration per subject will be up to 2 years, with up to 1 day of screening and 
baseline assessment, up to 1.5 years of Intervention visits, and 6 months follow-up. 
3.2.2 Total Number of Study Sites/Total Number of Subjects Projected 
The study will be conducted at 2 investigative sites in the United States. The Children’s 
Hospital of Philadelphia, and The New York Blood Center (no subject enrollment, providing 
RH genotyped red cell units and engaged in research activities). 
Recruitment will stop when approximately 20 subjects are enrolled. It is expected that 
approximately 20 subjects will be enrolled to produce 18 evaluable subjects.  
3.3 
Study Population 
We will enroll at least 5 adults to collect safety and preliminary efficacy data, before 
enrolling children to the study 
3.3.1 Inclusion Criteria 
1) Males or females age > 8 years.   
2) Diagnosis of SCD, all genotypes 
 
3) Requires chronic red cell transfusion therapy 
 
4) Requires a minimum of 3 units per transfusion visit 
 

 
  
 
 
 
6 
5) History of anti-D 
 
6) RH genotype predicts D+ expression 
 
7) Anti-D not detectable at screening visit and past 6 months 
3.3.2 Exclusion Criteria 
1) Rare RH genotype that would preclude sufficient RBC units 
2) Antigen negative requirements due to alloimmunization that would preclude sufficient 
RBC units 
3) Parents/guardians or subjects who, in the opinion of the Investigator, may be non-
compliant with study schedules or procedures. 
Subjects that do not meet all of the enrollment criteria may not be enrolled. Any violations of 
these criteria must be reported in accordance with IRB Policies and Procedures.  
For subjects with a history of stroke/recurrent transient ischemic attack or other indication 
who require tight control of Hb S and RH genotyped blood is not available, standard of care 
serologic matched blood would be administered rather than delaying transfusion and risking 
higher Hb S level. 
4 
STUDY PROCEDURES 
4.1 
Screening Visit and Baseline Assessment 
The screening visit and baseline assessment will occur on the same day with the following 
procedures: 
• 
Informed consent 
• 
Review inclusion/exclusion criteria 
• 
Demographics/medical history 
• 
Medication review 
• 
Physical exam 
• 
Vital signs 
• 
Laboratory test review 
• 
Medical Record Review 
4.2 
Study Treatment Phase 
Intervention visits will be scheduled with each of the subject’s transfusion visits as per the 
subject’s clinical indication for chronic transfusion therapy. We will provide one unit of D+ RH 
genotype matched RBCs at the first transfusion study visit (Figure 3). The remainder of units 
will be D-, CEK-matched, and negative for all other antigens the patient is alloimmunized 

 
  
 
 
 
7 
against. In this manner, should anti-D be re-stimulated, only one unit of the 3 to 11 units 
required for red cell exchange will be at risk for hemolysis, and the patient would not become  
 
Figure 3. Example transfusion protocol for D+ patient with history of anti-D that is not currently 
detectable, and requires 6 units per RBC exchange every 3 weeks. One D+ unit will be matched by RHD 
genotype and transfused at the first two study visits, and an increasing number of D+ units will be issued at 
consecutive RBC exchange visits. U, unit. Triangle denotes a study visit for laboratory studies including an 
antibody screen, CBC, hemoglobin quantification and bilirubin level 
acutely anemic. If laboratory monitoring shows no anti-D re-appearance or signs of increased 
hemolysis, the patient will receive one unit of D+ RH genotype matched RBCs at the 2nd 
transfusion study visit, and again if no anti-D re-appearance or signs of increased hemolysis,  
we will increase by one D+ unit per study visit until all units are D+. If a patient re-
demonstrates anti-D, we will end that subject’s participation and he/she will resume 
transfusions with D negative RBC units.  
All patients with SCD on chronic transfusion therapy have a pre-transfusion complete blood 
count (CBC), Hgb quantification to determine %hgb A and S levels, and an antibody screen. 
We will collect a pre-transfusion sample to prospectively store plasma on all study subjects. 
Patients undergoing chronic red cell exchange will have an immediate post-procedure CBC 
and hgb quantification as standard of care. Study participants will return 5-12 days post-
transfusion for a CBC/reticulocyte count, hgb quantification, bilirubin level, and antibody 
screen to evaluate for anti-D recrudescence or red cell hemolysis (Table 2). 
Table 2. Laboratory monitoring 
 
Pre-transfusion 
Immediate Post-
transfusion 
5-12 days Post-
transfusion 
Complete blood count 
ü 
ü 
ü 
Hemoglobin quantification 
ü 
ü 
ü 
Reticulocyte count 
ü 
 
ü 
Antibody screen/identification 
ü 
 
ü 
Bilirubin (total, unconjugated) 
 
 
ü 
Creatinine 
 
 
ü 
Plasma sample frozen 
ü 
 
ü 
Donor red cells frozen 
ü 
 
 
Any time a patient shows a re-appearance of anti-D on an antibody screen, the medical 
monitor will be notified, and all safety data will be reviewed. As stated above, if a subject re-
demonstrates anti-D, we will end that subject’s participation and he/she will resume 
transfusions with D negative RBC units. 
If any subject shows signs of transfused red cell hemolyis, with or without an anti-D identified 
on the antibody screen, the medical monitor will be notified, and all safety data will be 
reviewed.  
Week
1     2     3     4     5     6     7     8     9   10   11   12   13   14   15   16   17   18   19   20   21   
1U D+
5U D-
2U D+
4U D-
1U D+
5U D-
3U D+
3U D-
4U D+
2U D-
5U D+
1U D-
6U D+
0U D-

 
  
 
 
 
8 
Study stopping criteria include: 
1) Two of the first 5 enrolled subjects, or after the first 5 enrolled subjects, greater than 
30% of enrolled patients, develop re-appearance of anti-D 
2) Any subject with anti-D re-appearance and clinical or laboratory evidence of 
transfused red cell hemolysis (dark urine, increased scleral icterus, exacerbated 
anemia, higher Hb S level than expected based on timing from last transfusion 
If either of these criteria occur, the study will be stopped, the safety medical monitor will 
review all clinical and laboratory evidence to determine safety risks, and will meet with the 
investigative team to determine a safe course of action. The IRB would be notified and the 
study procedures would be suspended until approval of the IRB.  
4.2.1 Visit 1 
At each intervention visit the following procedures and measurements include:   
• 
Interim medical history 
• 
Medication review 
• 
Review of vital signs 
• 
Review of clinical laboratory evaluation 
• 
Peripheral blood sample collection  
• 
Transfusion of 1 unit D+ RH genotype matched red cell units. Remainder of units 
transfused will be serologic CEK matched red cells that are D negative.  
• 
Adverse event assessment 
4.2.2 Visit 2 
• 
Laboratory tests (see table 2) 
• 
Assess possible adverse events  
4.2.3 Visit 3 plus 
• 
Same as Visit 1, keep repeating until all units transfused are D+ or anti-D 
reappearance 
4.3 
Follow-up Phase  
The follow-up phase involves medical chart review and will continue for up to 6 months after 
the subject’s last transfusion and intervention visit. The medical chart review will abstract data 
regarding the transfusion visit, as well as blood bank evaluations including antibody screens 
and antibody identification. 
4.3.1 End of Study 
There are no special procedures for the end of study participation.  

 
  
 
 
 
9 
4.4 
Unscheduled Visits 
We do not anticipate unscheduled visits but these will be recorded. If a patient presents to 
Emergency Department, or is admitted to the Inpatient Hospital service, and requires 
transfusion, we will order D- red cells matched for C, E, and K by standard serologic methods 
which is the standard of care.  
4.5 
Subject Completion/Withdrawal 
Subjects may withdraw from the study at any time without prejudice to their care.  They may 
also be discontinued from the study at the discretion of the Investigator for lack of adherence 
to study treatment or visit schedules, or AEs.  The Investigator may also withdraw subjects 
who violate the study plan, or to protect the subject for reasons of safety or for administrative 
reasons.  It will be documented whether or not each subject completes the clinical study. If 
the Investigator becomes aware of any serious, related adverse events after the subject 
completes or withdraws from the study, they will be recorded in the source documents and 
on the CRF.  
4.5.1 Early Termination Study Visit 
Subjects who withdraw from the study will have all procedures enumerated for the last 
transfusion/intervention as the early termination visit.  

 
  
 
 
 
10 
5 
STUDY EVALUATIONS AND MEASUREMENTS 
5.1 
Screening and Monitoring Evaluations and Measurements 
5.1.1 Medical Record Review 
The following variables will be abstracted from the medical chart (paper or electronic). 
• 
Date of birth 
• 
Height and weight 
• 
Vital signs (temperature, blood pressure, heart rate, respiratory rate) 
• 
Medications 
• 
Medical history including SCD clinical course, complications, and indication for 
chronic red cell transfusion therapy 
• 
Transfusion and blood bank history including date of first transfusion, number of 
lifetime red cell exposures, alloimmunization history, antibody evaluations, history of 
transfusion reactions, RH genotype, extended red cell antigen profiles 
• 
Additional clinical laboratory studies including complete blood counts, reticulocyte 
counts, antibody evaluations, direct antiglobulin test, chemistry panels, lactate 
dehydrogenase levels  
5.1.2 Physical Examination 
A physical examination will be performed for the baseline assessment. 
5.1.3 Vital Signs 
Vital signs (temperature, blood pressure, heart rate, respiratory rate) will be measured as 
part of clinical care. The study team will review and record the vital signs from the chart. 
5.1.4 Laboratory Evaluations 
The following laboratory evaluations are obtained as part of routine clinic and transfusion 
visits, which the study team will review and record from the patient’s chart.  
5.1.4.1 Table: Clinical Laboratory Tests 
Category 
Tests 
Hematology 
Hemoglobin, hematocrit, platelet count, WBC +/- differential, 
reticulocyte count, hemoglobin quantification 
Liver function tests 
AST, ALT, total bilirubin (total and unconjugated) 
Renal function tests 
BUN, creatinine 
Chemistry tests 
Lactate dehydrogenase  
Blood bank tests 
Type and screen, compatibility testing, antibody 
identification, direct antiglobulin test, RH genotype, 
extended red cell antigen profile 

 
  
 
 
 
11 
5.1.4.2 Table: Study Laboratory Tests 5-12 Days After Transfusion 
Category 
Tests 
Hematology 
Hemoglobin, hematocrit, platelet count, WBC +/- differential, 
reticulocyte count, hemoglobin quantification 
Liver function tests 
Bilirubin (total and unconjugated) 
Renal function tests 
BUN, creatinine 
Blood bank tests 
Antibody screen, antibody identification, +/- direct 
antiglobulin test 
 
5.1.5 Diagnostic Tests 
Pre-transfusion and post-transfusion laboratory studies will be monitored for anti-D re-
appearance or signs of increased hemolysis (i.e. lower hemoglobin/hematocrit, lower hgb A 
and higher hgb S level, elevated bilirubin).   
5.2 
Safety Evaluation 
Subject safety will be monitored by adverse events, vital signs, anti-D reappearance and 
other laboratory data (complete blood count, hemoglobin quantifications, red cell antibody 
evaluations). It will also include evaluation for any delays in transfusion of red cells. If a patient 
re-demonstrates anti-D, we will end that subject’s participation and he/she will resume 
transfusions with D negative RBC units. 
We will have a medical monitor independent of the study team who is familiar with SCD and 
transfusion, and blood bank procedures. The medical monitor will review safety data, 
including antibody screens for anti-D reappearance, and other laboratory studies, i.e blood 
counts and hemoglobin quantifications, for signs of transfused red cell hemolysis. Details 
about the untis transfused, including the RH genotype, and the red cell antigen profile, will 
be reviewed. 

 
  
 
 
 
12 
6 
STATISTICAL CONSIDERATIONS 
This is a pilot study to determine the feasibility and safety of providing RH genotype matched 
D+ red cells to patients with SCD and anti-D who require chronic transfusion therapy.   
6.1 
Primary Endpoint 
The primary endpoint is the feasibility and safety of providing RH genotype matched D+ red 
cells for patients with SCD and history of anti-D who require chronic transfusion. RH genotype 
matched red cells are currently not available clinically except for rare circumstances.  
Descriptive, qualitative data, i.e. anti-D reappearance and signs and symptoms of increased 
red cell hemolysis, or delays in transfusion, will be collected to inform future multi-center 
studies of RH genotype matched red cells designed to test the effectiveness of providing RH 
genotype matched D+ red cells for patients with SCD and history of anti-D. 
Given that there is donor center scalability with RH genotyping if more than one institution 
was requesting RH genotype matched red cells, we would consider this matching strategy 
feasible if provision of matched units were possible in > 85% of the transfusion study visits 
without a delay in transfusion. 
6.2 
Statistical Methods 
6.2.1 Baseline Data  
Baseline and demographic characteristics will be summarized by standard descriptive 
summaries (e.g. means and standard deviations for continuous variables such as age and 
percentages for categorical variables such as gender). 
6.2.2 Efficacy Analysis 
The primary efficacy endpoints will be anti-D re-appearance, signs of increased hemolysis, 
and timely issue of units for transfusion.  These will be descriptive data.    
6.2.3 Safety Analysis 
All subjects entered into the study at Visit 1 will be included in the safety analysis. The 
frequencies of AEs by type, severity and relationship to study intervention will be 
summarized. SAEs (if any) will be described in detail. The analysis will be a qualitative 
description of AEs. 
6.3 
Sample Size and Power 
The sample size for this feasibility and safety study is based on our current chronically 
transfused patient population who receive apheresis and have a historical anti-D, which is 
approximately 20-25 active patients. We expect an additional 2-3 eligible patients per year. 
Therefore, we anticipate that with ~20 subjects will enroll over the 5 years of the study.  
6.4 
Interim Analysis 
If a patient re-demonstrates anti-D, we will end that subject’s participation and he/she will 
resume transfusions with D negative RBC units. If a patient’s genotype precludes readily 
identified RH genotype matched RBC units and a delay in transfusion is documented in 3 
consecutive visits, we will consider termination of study participation for that individual. 
 

 
  
 
 
 
13 
7 
STUDY INTERVENTION 
7.1 
Description 
The study intervention is provision of RH genotype matched D+ red cells for transfusion in 
chronically transfused patients with SCD and history of anti-D. All other clinical care will 
remain as usual, including the decision to transfuse, timing of transfusions, and the quantity 
of red cells to transfuse.  Red cells will be ordered from NY Blood Center using a secure 
online ordering system, per usual clinical practice guidelines established at CHOP for patients 
with SCD. Specifically, serologic C, E, and K antigen matched donor units are ordered. All 
units are leukoreduced, CMV-safe, and irradiated per CHOP policy. The CHOP blood bank 
aims to issue < 21 day old for all patients with hemoglobinopathies, particularly for those 
chronically transfused.  
The study intervention is D+ donor units that are RH genotyped by the blood supplier, and 
will be matched according to the patient’s RH genotype. RH genotyping assays are not FDA 
approved, but are performed in an American Association of Blood Banks (AABB) accredited 
and CLIA certified lab, and validated for sensitivity, specificity, reproducibility and 
repeatability by NY State regulations as laboratory developed tests (LDTs). There are no 
current FDA approved RH genotyping assays that distinguish Rh variants from conventional 
Rh antigens. In fact, many assays including the RH genotyping that are currently used 
clinically to aid in antibody identification and red cell matching for patients with SCD are not 
FDA approved. 
7.1.1 Packaging 
Red cell units will have the typical packaging from the blood supplier. 
7.1.2 Labeling 
Red cell unit labels will have the typical labels from the blood supplier but the donor unit ID 
will be linked to RH genotype data. 
7.1.3 Dosing 
The volume of red cells ordered for transfusion will be based solely on the clinician order. 
Typically, patients receive between 2 and 11 units of RBCs for a red cell exchange, 
depending on the pre-transfusion hgb and hgb S level. 
7.1.4 Treatment Compliance and Adherence 
Compliance with transfusion visits will be monitored. Termination of study participation will 
be considered for patients who miss multiple scheduled transfusion visits. 
7.1.5 Transfusion Product Accountability 
Adequate records of study red cell unit receipt and disposition will be maintained by the 
CHOP Blood Bank including blood orders, dispensing records, and disposition forms, which 
is standard for all blood products issued. These will be examined during the course of the 
study.  The study coordinator will extract this data for study records. The purpose of these 
records is to ensure regulatory authorities and the Sponsor that the investigational product is 
accounted for.  Should a patient miss their appointment, the blood may be issued to non-
study individuals at the discretion of the investigators. 

 
  
 
 
 
14 
8 
SAFETY MANAGEMENT 
8.1 
Clinical Adverse Events 
Clinical adverse events (AEs) will be monitored throughout the study.  
We will have a medical monitor independent of the study team who is familiar with SCD and 
transfusion, and blood bank procedures. The medical monitor will review safety data, 
including antibody screens for anti-D reappearance, and other laboratory studies, i.e blood 
counts and hemoglobin quantifications, for signs of transfused red cell hemolysis. Details 
about the untis transfused, including the RH genotype, and the red cell antigen profile, will 
be reviewed.  
Any time a patient shows a re-appearance of anti-D on an antibody screen, the medical 
monitor will be notified, and all safety data will be reviewed. As stated above, if a subject re-
demonstrates anti-D, we will end that subject’s participation and he/she will resume 
transfusions with D negative RBC units. 
If any subject shows signs of transfused red cell hemolyis, with or without an anti-D identified 
on the antibody screen, the medical monitor will be notified, and all safety data will be 
reviewed.  
Study stopping criteria include: 
1) Three of the first 5 enrolled subjects, or greater than 50% of enrolled patients, 
develop re-appearance of anti-D 
2) Any subject with anti-D re-appearance and clinical or laboratory evidence of 
transfused red cell hemolysis (dark urine, increased scleral icterus, exacerbated 
anemia, higher Hb S level than expected based on timing from last transfusion 
If either of these criteria occur, the study will be stopped, the safety medical monitor wll review 
all clinical and laboratory evidence to determine safety risks, and will meet with the 
investigative team to determine a safe course of action. The IRB would be notified and the 
study procedures would be suspended until approval of the IRB. 
8.2 
Adverse Event Reporting 
Unanticipated problems related to the research involving risks to subjects or others that occur 
during the course of this study (including SAEs) will be reported to the IRB in accordance 
with CHOP IRB SOP 408: Unanticipated Problems Involving Risks to Subjects. AEs that are 
not serious but that are notable and could involve risks to subjects will be summarized in 
narrative or other format and submitted to the IRB at the time of continuing review.   
8.3 
Definition of an Adverse Event 
An adverse event is any untoward medical occurrence in a subject who has received an 
intervention (drug, biologic, or other intervention).  The occurrence does not necessarily have 
to have a causal relationship with the treatment.  An AE can therefore be any unfavorable or 
unintended sign (including an abnormal laboratory finding, for example), symptom, or disease 
temporally associated with the use of a medicinal product, whether or not considered related 
to the medicinal product. 

 
  
 
 
 
15 
All AEs (including serious AEs) will be noted in the study records and on the case report form 
with a full description including the nature, date and time of onset, determination of non-
serious versus serious, intensity (mild, moderate, severe), duration, causality, and outcome 
of the event. 
8.4 
Definition of a Serious Adverse Event (SAE) 
An SAE is any adverse drug experience occurring at any dose that results in any of the 
following outcomes:  
• 
death, 
• 
a life-threatening event (at risk of death at the time of the event),  
• 
requires inpatient hospitalization or prolongation of existing hospitalization, 
• 
a persistent or significant disability/incapacity, or 
• 
a congenital anomaly/birth defect in the offspring of a subject.   
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse drug event when, based upon 
appropriate medical judgment, they may jeopardize the subject and may require medical or 
surgical intervention to prevent one of the outcomes listed in this definition. 
A distinction should be drawn between serious and severe AEs.  A severe AE is a major 
event of its type.  A severe AE does not necessarily need to be considered serious.  For 
example, nausea which persists for several hours may be considered severe nausea, but 
would not be an SAE.  On the other hand, a stroke that results in only a limited degree of 
disability may be considered a mild stroke, but would be an SAE.  
8.4.1 Relationship of SAE to study drug or other intervention 
The relationship of each SAE to the study intervention should be characterized using one of 
the following terms in accordance with CHOP IRB Guidelines: definitely, probably, possibly, 
unlikely or unrelated.  
8.5 
IRB/IEC Notification of SAEs and Other Unanticipated Problems 
The Investigator will promptly notify the IRB of all on-site unanticipated, serious Adverse 
Events that are related to the research activity. Other unanticipated problems related to the 
research involving risk to subjects or others will also be reported promptly. Written reports 
will be filed using the eIRB system and in accordance with the timeline below. External 
SAEs that are both unexpected and related to the study intervention will be reported 
promptly after the investigator receives the report.   
Type of Unanticipated 
Problem 
Initial Notification  
(Phone, Email, Fax) 
Written Report 
Internal (on-site) SAEs 
Death or Life 
Threatening  
24 hours 
Within 2 calendar days 
Internal (on-site) SAEs 
7 days 
Within 7 business days 

 
  
 
 
 
16 
All other SAEs 
Unanticipated Problems 
Related to Research 
7 days  
Within 7 business days 
All other AEs 
N/A 
Brief Summary of important 
AEs may be reported at 
time of continuing review 
8.5.1 Follow-up report 
If an SAE has not resolved at the time of the initial report and new information arises that 
changes the investigator’s assessment of the event, a follow-up report including all relevant 
new or reassessed information (e.g., concomitant medication, medical history) will be 
submitted to the IRB. The investigator is responsible for ensuring that all SAE are followed 
until either resolved or stable.  
8.6 
Investigator Reporting of a Serious Adverse Event to Sponsor 
Reporting will be consistent with regulatory requirements.  
8.7 
Medical Emergencies 
Describe any plans or procedures for taking care of medical emergencies that might develop 
during the course of the study. Should a patient demonstrate anti-D reappearance that is 
associated with increased hemolysis, he/she will be treated with immunosuppression 
congruent to the degree of anemia and hemolysis (i.e. oral steroids, IV steroids, and/or IVIg 
depending on severity). If moderate to severe hemolytic transfusion reaction, he/she may 
need to be admitted to the hospital and may require transfusion of D- RBC units. 

 
  
 
 
 
17 
9 
STUDY ADMINISTRATION 
9.1 
Data Collection and Management 
Primary records (source documents) and data abstraction forms for entering the data into our 
study database (Filemaker) will be used.  The plan is consistent with CHOP Policy A-3-6: 
Acceptable Use of Technology Resources that defines the requirements for encryption and 
security of computer systems, and addresses the following: 
1. Confidentiality.  Confidentiality of the data will be ensured with the use of password-
protected files. Specifically, a Filemaker database is hosted on the CHOP research 
network and password protected such that only the study team members have 
access. Data may be exported into Excel files, which can be both coded with the UPIN 
and a separate master list, and password-protected. Blood ordering is performed via 
a secure online blood ordering system hosted by the blood supplier (NY Blood 
Center).   
2. Security.  Data is backed up intermittently as a copy of the password-protected file on 
the PI’s office computer and the original is hosted on one of the Hospital’s secure 
servers for research community. 
3. Anonymization, de-identification or destruction.  Analyses from this study will be 
conducted over many years and we would like to retain PHI to link the samples with 
the subjects. The identifiers and data will be retained per CHOP Data Retention Policy 
A-3-9 for Human subjects research that is greater than minimal risk.   
9.2 
Confidentiality 
All data and records generated during this study will be kept confidential in accordance with 
Institutional policies and HIPAA on subject privacy. The Investigator and other site personnel 
will not use such data and records for any purpose other than conducting the study. Subject 
confidentiality will be maintained by the use of password protected files that are accessible 
only to study personnel and regulatory agencies overseeing the research.  
No identifiable data will be used for future study without first obtaining IRB approval. The 
investigator will obtain a data use agreement between the provider (the PI) of the data and 
any recipient researchers (including others at CHOP) before sharing a limited dataset (PHI 
limited to dates and zip codes).  
9.3 
Regulatory and Ethical Considerations 
9.3.1 Data and Safety Monitoring Plan 
The study has greater than minimal risk. The study intervention of RH genotype matched 
D+ red cells to patients with SCD and anti-D provides a higher level of matching than 
current standard of care, but there is a risk of anti-D reappearance, increased red cell 
hemolysis and delay to transfusion for up to 7 days. If a patient re-demonstrates anti-D, we 
will end that subject’s participation and he/she will resume transfusions with D negative 
RBC units. In the case of acute need for transfusion, subjects may receive non-RH 
genotype matched red cells that would be matched by standard of care methods at the 
discretion of the attending physician, i.e. D-, CEK matched red cells.  Oversight for the 
emerging safety information will include the PI. Red cell units will continue to be issued per 
standard institutional blood bank policies. 

 
  
 
 
 
18 
We will have a medical monitor independent of the study team who is familiar with SCD and 
transfusion, and blood bank procedures. The medical monitor will review safety data, 
including antibody screens for anti-D reappearance, and other laboratory studies, including 
blood counts and hemoglobin quantifications, for signs of transfused red cell hemolysis. 
Details about the units transfused, including the RH genotype, and the red cell antigen profile, 
will be reviewed.  
Any time a patient shows a re-appearance of anti-D on an antibody screen, the medical 
monitor will be notified, and all safety data will be reviewed. As stated above, if a subject re-
demonstrates anti-D, we will end that subject’s participation and he/she will resume 
transfusions with D negative RBC units. 
If any subject shows signs of transfused red cell hemolyis, with or without an anti-D identified 
on the antibody screen, the medical monitor will be notified, and all safety data will be 
reviewed.  
Study stopping criteria include: 
1) Two of the first 5 enrolled subjects, or after the first 5 enrolled subjects, greater than 
30% of enrolled patients, develop re-appearance of anti-D 
2) Any subject with anti-D re-appearance and clinical or laboratory evidence of 
transfused red cell hemolysis (dark urine, increased scleral icterus, exacerbated 
anemia, higher Hb S level than expected based on timing from last transfusion 
If either of these criteria occur, the study will be stopped, the safety medical monitor will 
review all clinical and laboratory evidence to determine safety risks, and will meet with the 
investigative team to determine a safe course of action. The IRB would be notified and the 
study procedures would be suspended until approval of the IRB. 
The PI will monitor study progress, ensure subject safety, and the accuracy and security of 
the data at CHOP, and will report any adverse events in accordance with the FDA 
regulations as they pertain to blood products and IRB policies. 
9.3.2 Risk Assessment 
Risks are greater than minimal.  Providing D+ RBC units to a patient with history of anti-D 
may cause re-appearance of the anti-D with or without transfused red cell hemolysis. RH 
genotype matched red cells provide a higher level of matching, and thus the risk of anti-D 
reappearing is predicted to be very low. The study was also designed to minimize the risk of 
harm from anti-D reappearance by transfusing D+ RH genotype matched units for only 1 out 
of a typical 3 to 8 RBC units for the first two visits, and then titrating up by one unit with each 
subsequent transfusion. There is the possibility that transfusion would be delayed up to 7 
days to identify adequate RH genotype red cell units, this may cause inconvenience (waiting 
or rescheduling transfusion visit).  
There are risks involved with red cell transfusion, i.e. allergic reaction, infection, 
alloimmunization, but these risks are associated with routine clinical care and not a result of 
the research activities.  
The risks involved with a venipuncture are pain, bleeding or bruising at the venipuncture site.  

 
  
 
 
 
19 
9.3.3 Potential Benefits of Trial Participation 
Direct benefits to the study subject as a result of participation may be demonstrating that the 
patient can be transfused safely with D+ RH genotype matched red cells and no longer need 
to rely on D- red cell units, resulting in improved ability to identify compatible units. The 
indirect benefits are that all patients with SCD may receive safer transfusions in the future if 
we find RH genotype matching is feasible.  
9.3.4 Risk-Benefit Assessment 
RH genotype matched red cells are predicted to reduce Rh alloimmunization and result in 
safer transfusion products, thus, providing more benefit than risk. The risks of providing D+ 
units to D+ patients with history of an anti-D are greater than minimal due to risk of anti-D re-
appearance and potential for increased hemolysis, but the benefit of RH genotype matched 
red cells that provide a higher level of matching than standard clinical practice outweigh those 
risks.  
9.4 
Recruitment Strategy 
Patients will be recruited from the chronic transfusion program for SCD at the Children’s 
Hospital of Philadelphia. This will primarily include patients who receive their red cell 
transfusions erythrocytapheresis in the Apheresis Unit, but could include patients transfused 
by simple transfusion in the Day Hospital. Since the principal investigator is a pediatric 
hematologist and transfusion medicine specialist, she will refer her own patients as well as 
other care providers. All patients with SCD receiving chronic transfusion therapy will be 
screened for eligibility. If the prospective subjects are not patients of the investigator, one of 
the investigators or the research coordinator will approach the subjects and offer participation 
in the study in person during a transfusion visit. If recruitment is poor from the apheresis 
transfusion cohort at CHOP, patients receiving 2-3 units for simple red cell transfusion can 
be recruited from the Day Hospital. 
9.5 
Informed Consent/Assent and HIPAA Authorization 
All prospective subjects eligible for participation in this study will be enrolled after obtaining 
informed consent. The study coordinator or one of the investigators will obtain consent in the 
Hematology Clinic, Day Hospital, or Apheresis Unit.  The consent process will take place in 
the clinic exam room, or other private room where privacy is assured. The investigational 
nature of the study, the study objectives, procedures involved and the potential risks and 
benefits will be explained to the subject and/or guardian using lay language. Consent forms 
will be written in a well-organized format using direct language. We will explicitly state that 
the choice of study participation will not impact their access to clinical care.  Assent will be 
obtained from all children seven years and older before study enrollment when deemed 
appropriate by the investigator and the parent(s). Written informed combined consent/HIPAA 
authorization and assent (if applicable) will be obtained. Individuals with limited English 
proficiency will only be enrolled in person after obtaining written consent using the short form 
consent process.  
9.6 
Payment to Subjects/Families 
Subjects will be compensated a nominal amount for the inconvenience of participating in the 
study, which requires a return visit for laboratory studies.  Subjects will be compensated $50 
per laboratory only visit, since this is a separate visit outside of routine clinical care. For 
patients who encounter economic hardship to travel to and from CHOP for the laboratory only 

 
  
 
 
 
20 
visit despite the $50 compensation, we can offer reimbursement for travel for the follow up 
lab visits that are obtained 5-12 days after the transfusion since these are outside the visits 
needed for routine clinical care. Money will be added after each laboratory visit to a debit 
card.  
10 PUBLICATION 
The study investigators using standard publication guidelines will prepare manuscript(s) 
and abstract(s) from the data collected during this trial. 
11 REFERENCES 
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Chou ST, Jackson T, Vege S, Smith-Whitley K, Friedman DF, Westhoff CM. High 
prevalence of red blood alloimmunization in sickle cell disease despite transfusion 
from Rh-matched minority donors. Blood. 2013;122(6):1062-1071. 
2. 
Vichinsky EP, Earles A, Johnson RA, Hoag MS, Williams A, Lubin B. 
Alloimmunization in sickle cell anemia and transfusion of racially unmatched blood. 
N Engl J Med. 1990;322(23):1617-1621. 
3. 
Yawn BP, Buchanan GR, Afenyi-Annan AN, et al. Management of sickle cell 
disease: summary of the 2014 evidence-based report by expert panel members. 
Jama. 2014;312(10):1033-1048. 
4. 
Chou ST, Evans P, Vege S, et al. RH genotype matching for transfusion support in 
sickle cell disease. Blood. 2018;132(11):1198-1207. 
5. 
Coleman S, Westhoff CM, Friedman DF, Chou ST. Alloimmunization in patients with 
sickle cell disease and underrecognition of accompanying delayed hemolytic 
transfusion reactions. Transfusion. 2019. 
6. 
Dezan MR, Ribeiro IH, Oliveira VB, et al. RHD and RHCE genotyping by next-
generation sequencing is an effective strategy to identify molecular variants within 
sickle cell disease patients. Blood Cells Mol Dis. 2017;65:8-15.