Red Blood Cell Survival Study: The Impact of Oxidative Stress on Erythrocyte Biology
Phase: PHASE2
Conditions: Sickle Cell Disease Without Crisis
Sponsor: University of North Carolina, Chapel Hill

 
 
The Impact of Oxidative Stress on Erythrocyte Biology 
 
 
 
 
NCT number NCT04028700 
Document Date 01/12/2023 
 

 
MCW Protocol No: PRO00035124 
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Version No.: 6 
 
 
Version Date: 01/12/2023 
 
 
 
 
 
 
 
 
CLINICAL STUDY PROTOCOL 
 
 
 
Red Blood Cell Survival Study: 
 
The Impact of Oxidative Stress on Erythocyte Biology 
 
Clinical Trials.gov Number:  NCT04028700 
 
 
Principal Investigator: Matthew S. Karafin, MD, MS 
 
 
 Version 6: 12 Jan 2023 
 
 
 
Proprietary and Confidential 
The information in this document is considered privileged and confidential, and may not be disclosed 
to others except to the extent necessary to obtain Institutional Review Board approval and  
informed consent, or as required by federal and state laws. Persons to whom this information is 
disclosed should be informed that this information is privileged and confidential and  
that it should not be further disclosed. 
 
 

 
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Version Date: 01/12/2023 
 
Title: The Impact of Oxidative Stress on Erythocyte Biology 
MCW Protocol No.: PRO00035124  
 
Principal Investigator:  
Name: Matthew S Karafin 
Title: Principal Investigator 
Institution: UNC at Chapel Hill 
Address: 101 Manning Dr, Chapel Hill, NC 
27514 
Telephone: (984)-974-1583 
Fax: 414-937-6833 
Email address: matthew.karafin@unc.edu 
 
  
Sub-investigator:  
Name: Jane Little 
Title: Co-Investigator 
Institution: UNC at Chapel Hill 
Address: 101 Manning Dr, Chapel Hill, NC 
27514 
Telephone: (919) 966-6735 
Fax:  
Email address: jane_little@med.unc.edu 
Sub-investigator:  
Name: Eric Smith 
Title: Co-Investigator 
Institution: Nuclear Medicine 
Address: 101 Manning Dr, Chapel Hill, NC 
27514 
Telephone:  
 Fax:  
Email address: eric_smith@med.unc.edu 
Primary Research Coordinator: 
Name: David Wichlan 
Title: Research Coordinator 
Institution:  UNC at Chapel Hill 
Address: 101 Manning Dr, Chapel Hill, NC 
27514 
Telephone: (919) 966-6876 
Fax:  
Email address: david_wichlan@med.unc.edu 
 
 
 
Funding Sponsor:  
Name: NHLBI R01 HL148151-01 
 
 
 
 
 

 
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Version Date: 01/12/2023 
 
VERSION HISTORY 
Version 1, Version Date 16JUL2019 
Initial submission of the protocol.  
Version 
Date 
1 
30 JUL 2019 
2 
25 FEB 2021 
3 
28 JUL 2021 
4 
12 Nov 2021 
5 
11 Feb 2022 
6 
12 Jan 2023 
 

 
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Version Date: 01/12/2023 
 
TABLE OF CONTENTS 
VERSION HISTORY ......................................................................................................................................... 3 
TABLE OF CONTENTS .................................................................................................................................... 4 
PROTOCOL SUMMARY ................................................................................................................................... 6 
LIST OF ABBREVIATIONS ............................................................................................................................ 11 
1 BACKGROUND ........................................................................................................................................... 12 
SICKLE CELL DISEASE AND RED BLOOD CELL TRANSFUSIONS ................................................... 12 
2 SPECIFIC AIMS ........................................................................................................................................... 13 
2.1 PRIMARY OBJECTIVE (AIM 1) ............................................................................................. 13 
2.2 SECONDARY OBJECTIVES (AIMS 2A-2D) ............................................................................. 13 
2.3 RATIONALE FOR THE OUTCOME MEASURES SELECTED ....................................................... 13 
3 STUDY DESIGN ........................................................................................................................................... 14 
3.1 GENERAL DESCRIPTION .................................................................................................... 14 
3.2 STUDY COMPLETION ......................................................................................................... 14 
4 SUBJECT PARTICIPATION, DISCONTINUATION, AND WITHDRAWAL .................................................. 14 
4.1 SUBJECT STATUS .............................................................................................................. 14 
4.2 PRESCREENING AND SCREENING LOG ................................................................................ 15 
4.3 CONSENT ......................................................................................................................... 15 
4.4 SCREENING PROCEDURES ................................................................................................. 15 
4.5 ELIGIBILITY CONFIRMATION ................................................................................................ 15 
4.6 STUDY ELIGIBILITY CRITERIA ............................................................................................. 15 
4.7 ENROLLMENT .................................................................................................................... 16 
4.8 UNIT SELECTION, IDENTIFICATION, AND STUDY ACTIVITIES .................................................. 16 
4.9  DISCONTINUATION OF STUDY TREATMENT, WITHDRAWAL, AND COMPLIANCE ....................... 20 
4.10  LOST TO FOLLOW-UP ...................................................................................................... 20 
4.11  ACCRUAL SUSPENSION AND CLOSURE............................................................................. 21 
4.12  END OF STUDY DEFINITION ............................................................................................. 21 
4.13  STUDY DISCONTINUATION AND CLOSURE ......................................................................... 21 
5 STUDY PLAN............................................................................................................................................... 21 
5.1 RBC UNIT ADMINISTRATION .............................................................................................. 21 
5.2 MONITORING SUBJECT COMPLIANCE .................................................................................. 22 
5.3 FOLLOW-UP PERIOD .......................................................................................................... 22 
6 DOSING DELAYS/DOSE MODIFICATIONS ................................................................................................ 22 
6.1 MONITORING AND TOXICITY MANAGEMENT ......................................................................... 22 
7 ADVERSE EVENTS: DEFINITIONS AND REPORTING REQUIREMENTS ................................................ 24 
7.1 DEFINITIONS ..................................................................................................................... 24 
7.2 KNOWN AES LIST .............................................................................................................. 27 
7.3 TIME PERIOD AND GRADE OF AE CAPTURE ........................................................................ 28 
7.4 MONITORING AND RECORDING AN ADVERSE EVENT ............................................................ 28 
8 INTERVENTION INFORMATION ................................................................................................................. 31 

 
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8.1 AGENT #1: CHROMIUM-LABELED RED CELLS ....................................................................... 31 
9 STATISTICAL CONSIDERATIONS ............................................................................................................. 32 
9.1 STUDY MEASURES ............................................................................................................ 32 
9.2 STUDY DESIGN ................................................................................................................. 32 
9.3 RANDOMIZATION, CONCEALMENT, AND BLINDING ................................................................ 33 
9.4 REPLACEMENT POLICY ...................................................................................................... 33 
9.5 ACCRUAL ESTIMATES ........................................................................................................ 33 
9.6 STATISTICAL ANALYSIS PLANS  .......................................................................................... 33 
9.7 INTERIM ANALYSES ........................................................................................................... 35 
9.8 ANALYSIS OF SAFETY DATA ............................................................................................... 35 
9.9 SAMPLE SIZE RATIONALE .................................................................................................. 35 
10 PLANS TO ENSURE DATA QUALITY ...................................................................................................... 36 
11 DATA AND SAFETY MONITORING PLAN (DSMP) .................................................................................. 37 
11.1 STUDY TEAM .................................................................................................................. 37 
11.2 QUALITY ASSURANCE ...................................................................................................... 37 
11.3 SMC .............................................................................................................................. 37 
12 REGULATORY COMPLIANCE, ETHICS AND STUDY MANAGEMENT ................................................... 39 
12.1 ETHICAL STANDARD ........................................................................................................ 39 
12.2 REGULATORY COMPLIANCE ............................................................................................. 39 
12.3 PRESTUDY DOCUMENTATION ........................................................................................... 39 
13.4  INSTITUTIONAL REVIEW BOARD ....................................................................................... 39 
13.5  SUBJECT CONFIDENTIALITY AND ACCESS TO SOURCE DOCUMENTS/DATA ......................... 40 
13.6  PROTECTION OF HUMAN SUBJECTS ................................................................................. 41 
13.7  CHANGES IN THE PROTOCOL ........................................................................................... 41 
13.8  INVESTIGATOR COMPLIANCE ........................................................................................... 41 
13 DATA HANDLING AND RECORD KEEPING ............................................................................................ 42 
13.1 OVERVIEW ...................................................................................................................... 42 
14.2  DATA MANAGEMENT RESPONSIBILITIES ........................................................................... 42 
14.4  SOURCE DOCUMENTS .................................................................................................... 43 
14.5  CASE REPORT FORMS .................................................................................................... 43 
14.6  STUDY RECORD RETENTION ........................................................................................... 44 
14.7  PUBLISHING DATA .......................................................................................................... 44 
REFERENCES ................................................................................................................................................ 45 

 
 
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PROTOCOL SUMMARY 
Title 
The Impact of Oxidative Stress on Erythrocyte Biology 
Principal 
Investigator  
Matthew S. Karafin MD, MS 
Target  Population Adults with sickle cell disease who receive RBC transfusions 
Primary     
Objective 
Aim 1.  Evaluate the relative effects of blood from a donor with 
G6PD-deficiency on 24-hour post-transfusion recovery (PTR) as 
measured by Chromium 51 (51CR).                                                       * 
Using a crossover experimental design, we will compare the two 
treatment regimens (G6PD-deficient vs. G6PD-normal donor units) in 
terms of the mean response on the 51CR scale.  The same approach will 
be used for Aims 2a – 2d. 
Secondary 
Objectives 
Aim 2a.  Evaluate the relative effects of blood from a donor with 
G6PD-deficiency on 4-week post-transfusion outcomes as measured 
by mean change in hemoglobin A.  
Aim 2b.   Evaluate the relative effects of blood from a donor with 
G6PD-deficiency on 4-week post-transfusion outcomes as measured 
by mean changes in twenty-three other clinically-relevant measures.   
Aim 2c.   Evaluate the relative effects of blood from a donor with 
G6PD-deficiency on the longitudinal series of metabolic profiles 
obtained from blood samples.  
Aim 2d.   Evaluate the relative effects of blood from a donor with 
G6PD-deficiency on longitudinal pain scores, infection symptoms, 
and antibiotic use. 
Outcome 
Measures 
For Aims 1and 2 the outcome measures of interest will be obtained from 
blood samples on 15 occasions within each of Period1 and Period2: 
At baseline prior to the infusion treatment.  Post-infusion at 5, 7.5, 10, 12.5, 
15, 30 minutes,  and at 1, 24, 48, 72 hours,  and at 1, 2, 3, 4 weeks. 
For Aim 1, the outcome measure is  24-hour 51CR-labeled PTR of RBCs.  
 
For Aim 2a, the outcomes are Hemoglobin A.  
 
For Aim 2b, the outcome variables are 26 clinically-relevant measures. 
 
For Aim 2c, the outcomes of interest are the metabolomic profile from blood 
samples obtained longitudinally.    
 
For Aim 2d, pain scores, occurrence of infection, and antibiotic use will be 
obtained via a daily 4-week diary for evaluation of pain on an ordinal scale 
(0,1,2,…,10), occurrence of infection symptoms, and use of antibiotics 
(type, duration, dose).   

 
 
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Study Design 
For this Phase II, single-blind, longitudinal controlled trial, the experimental 
design is a two-treatment, two-period, two-sequence cross-over design. 
The two treatment regimens are (A) infusion of G6PD-deficient donor units, 
and (B) infusion of G6PD-normal donor units.  Due to the scarcity of G6PD-
deficient units in the donor pool, we will exchange units that are available 
at the time of scheduled study exchange (likely normal units) and not utilize 
a randomization scheme. Each 4-week study exchange period will be 
separated from the second study exchange by a 4-month washout 
interval.  During the washout period, we will look for suitable units (likely 
G6PD-deficient) for the second study exchange. 
Sample 
N = 16 eligible patients will be enrolled 
Participation Time 
Each enrollee’s participation will last at least 6 months 
Enrollment Time 
Recruitment and enrollment of the participants will require 4 years  
 
 
 

 
 
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SCHEMA 
 
Figure 1: Study Schema: We will compare two treatment regimens via is a two-treatment, 
two-period, two-sequence cross-over design: CO(2,2,2). The two treatment regimens are (A) 
infusion of G6PD-deficient donor units, and (B) infusion of G6PD-normal donor units.   
The two sequences (AB and BA) will be assigned to the N=16 enrollees by RBC unit availability 
at the time of the first study exchange transfusion .   
The two 4-week periods will be separated by a 4-month washout interval. The outcome 
measures of interest are  24-hour 51CR-labeled PTR of RBCs, and Hemoglobin A evaluated 
before and 4 weeks after infusion. 
 
 

 
 
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Period/ 
Procedure 
 
 
 
 
Follow-up visits 
 
 
Study 
Day/Visit Day 
24-72 
hrs 
pre-
infusio
n 
Pre-
transfusio
n 
5,7.5,10,12.5,15, 
30 min post 
infusion 
1 hr post transfusion/ 
infusion 
 (+/-15 min) 
 
24hr post infusion 
(+/- 3 hours) 
48 hr 
post 
infusio
n (+/- 3 
hours) 
72hr 
post 
infusio
n (+/- 3 
hours) 
1week 
post 
infusio
n (+/- 1 
day) 
2week 
post 
infusio
n ( +/- 2 
days) 
3 week 
post 
infusio
n ( +/- 2 
days) 
4 week 
post 
infusio
n ( +/- 2 
days) 
Informed 
consent 
X 
 
 
 
 
 
 
 
 
 
 
Study Diary 
 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
AE assessment 
 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
Type and 
screen sample 
X 
 
 
 
 
 
 
 
 
 
 
Clinical 
procedures 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical exam 
 
X 
 
 
 
 
 
 
 
 
X 
Vital signs 
 
X 
 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
 
 
Midline 
Placement  
 
X 
 
 
 
 
 
 
 
 
 
Laboratory 
procedures 
 
 
 
 
 
 
 
 
 
 
 
 
 
CBC w/ Diff 3 
 
X 
 
X 
 
X 
x 
X 
X 
X 
X 
X 
Reticulocyte 
count/% 
 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
Hb profile 
 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
Haptoglobin 
(hapto) 
 
X 
 
X 
X 
 
 
X 
X 
X 
X 
Iron Panel 
 
X 
 
X 
 
 
 
 
X 
X 
X 
X 
Cell free 
hemoglobin 
 
X 
 
X 
 
X 
 
 
X 
X 
X 
X 

 
 
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Period/ 
Procedure 
 
 
 
 
Follow-up visits 
 
 
Study 
Day/Visit Day 
24-72 
hrs 
pre-
infusio
n 
Pre-
transfusio
n 
5,7.5,10,12.5,15, 
30 min post 
infusion 
1 hr post transfusion/ 
infusion 
 (+/-15 min) 
 
24hr post infusion 
(+/- 3 hours) 
48 hr 
post 
infusio
n (+/- 3 
hours) 
72hr 
post 
infusio
n (+/- 3 
hours) 
1week 
post 
infusio
n (+/- 1 
day) 
2week 
post 
infusio
n ( +/- 2 
days) 
3 week 
post 
infusio
n ( +/- 2 
days) 
4 week 
post 
infusio
n ( +/- 2 
days) 
Total, direct, 
indirect bilirubin 
 
X 
 
X 
 
X 
 
 
X 
X 
X 
X 
Labile plasma 
Iron (NTBI) 
 
X 
 
X 
 
X 
 
 
X 
X 
X 
X 
Ferritin & 
Transferrin 
saturation 
 
X 
 
X 
 
X 
 
 
X 
X 
X 
X 
hsC-reactive 
protein 
(HSCRP) 
 
X 
 
X 
 
X 
 
 
X 
X 
X 
X 
Myeloperoxidas
e (MPO) 
 
X 
 
X 
 
X 
 
 
X 
X 
X 
X 
Lactate 
dehydrogenase 
(LDH) 
 
X 
 
X 
 
X 
 
 
X 
X 
X 
X 
Patient 
Metabolomic 
Samples 
 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
RBC Unit blood 
donor sample 
 
X 
 
 
 
 
 
 
 
 
 
Subject 
Chromium 
samples 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Pregnancy test 
(urine) 
 
X 
 
 
 
 
 
 
 
 
 
 
 

 
 
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LIST OF ABBREVIATIONS   
AE 
adverse event 
CBCD 
complete blood cell (count) with differential 
CRC 
clinical research coordinator 
CRF 
case report form 
CR51 
CTCAE  
Chromium 51 
Common Terminology Criteria for Adverse Events 
DSMC 
Data and Safety Monitoring Committee 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
G6PD 
HbA 
HbS 
HCT 
Glucose 6 phosphate dehydrogenase 
Hemoglobin, type A 
Hemoglobin, type S 
hematocrit 
HGB 
hemoglobin 
IRB 
Institutional Review Board 
IV 
intravenous 
LDH 
lactate dehydrogenase 
LFT 
liver function test 
NTBI 
PTR 
Non-transferrin bound iron (synonym is LPI) 
post transfusion recovery 
RBC 
red blood cell 
SAE 
serious adverse event 
SCD 
sickle cell disease 
UNC 
WBC 
University of North Carolina 
white blood cell (count) 

 
 
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1 BACKGROUND 
Sickle Cell Disease and Red Blood Cell Transfusions 
For logistical purposes, RBCs are stored for up to 42 days in the United States, resulting in 
altered RBC biology (i.e., the “storage lesion”).8 Indeed, if the storage lesion does nothing else, 
it does decrease the effective dose of transfused RBCs by up to 25% in healthy autologous 
recipients, as mandated by FDA criteria.6,9 However, this is only an average; thus, some units 
store even worse than this.9 More importantly, this is also an underestimate, because stored 
RBCs do more poorly when transfused into sick patients,10 whereas FDA approval studies are 
performed in healthy volunteers. Although controversy exists as to whether stored RBC 
transfusions are dangerous in certain settings, there is consensus that iron overload from red 
blood cell transfusions is a very serious adverse outcome in chronically transfused, non-
bleeding patients (e.g., in SCD and thalassemia).11,12 Such patients can have significant 
morbidity, and even mortality, from iron overload, despite chelation therapy. As such, providing 
them with RBC units with better recovery and survival (post transfusion recovery, PTR), thereby 
transfusing fewer units and less iron, would be of substantial benefit.  
 
Of equal significance to improving understanding of the storage lesion, is realizing that it results 
from oxidant stress.8,13 As such, storage biology is also relevant for elucidating RBC oxidant 
stress mechanisms which are important for various diseases. G6PD deficiency, the most 
common human enzymopathy, affects >400 million humans worldwide and is a serious barrier 
to malaria treatment and eradication.14 The only drugs capable of completely eliminating 
Plasmodium vivax cannot be given to patients with G6PD deficiency, due to hemolysis in vivo 
from oxidant damage.14 It is well established that G6PD deficiency causes red cell hemolysis 
and anemia during oxidant stress. While this deficiency is critical to red cell biology and survival, 
blood donor centers currently do not screen for this deficiency, and about 1 in 10 RBC units 
provided to patients with SCD currently have this deficiency.46 The impact of these units on 
current transfusion protocols for patients with SCD is unknown. By better defining known 
oxidant stress pathways (e.g., G6PD deficiency), and also elucidating novel pathways, 
this project has broad significance to RBC oxidant biology, in general, in addition to its 
immediate relevance to RBC transfusion therapy. 
 
Study Rationale 
Mounting evidence suggests that stored G6PD-deficient RBCs have reduced transfusion 
quality. Thus, the PTR of stored autologous G6PD-deficient RBCs is modestly, but significantly, 
worse than that of G6PD-normal RBCs in healthy volunteers;78,79 however, the effects in actual 
patients have not been evaluated systematically. Patients with SCD are regularly transfused 
with units from G6PD-deficient donors, with an exposure incidence of around 10%. 
Nonetheless, case reports suggest that intra- and extra-vascular hemolysis can occur in 
patients transfused with G6PD deficient donor RBCs.45 Furthermore, in our observational study 
of children with SCD,80 transfusions of G6PD deficient RBCs reduced the persistence of HbA-
containing RBCs, reflected by increased post-transfusion HbS and reticulocyte levels. In adults 
with SCD, we would anticipate that these changes would still occur, but may be proportionally 
reduced due to differences in patient blood volumes. Because patients with SCD have 
increased iron, inflammation, and oxidant stress at baseline,81 we hypothesize that G6PD-
deficient RBCs will circulate more poorly in all patients with SCD than in healthy controls. 
Therefore, our pilot study described herein is critical to begin to define the clinical and biological 
impact associated with the frequent transfusions of G6PD-deficient RBCs in patients with SCD, 
who often require chronic transfusion therapy. 

 
 
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2 SPECIFIC AIMS 
2.1 Primary Objective (Aim 1) 
Aim 1. Evaluate the effects of blood from a donor with G6PD-deficiency on 24-hour post-
transfusion recovery (PTR) as measured by Chromium 51 (51CR).  
 
We hypothesize that, in contrast to the decreased PTR we found in healthy adult recipients, 
adults with SCD have a greater decrease in PTR when receiving older, stored RBCs from 
G6PD-deficient donors, as compared to G6PD-normal donors. We anticipate that the mean 
difference (i.e., the treatment effect) is larger than we observed in our previous study of healthy 
controls (6.8%).  
 
The Aim 1 investigation will be considered a success if the confidence interval estimate for the 
treatment effect (i.e., the mean difference in PTR between G6PD-deficient donors and G6PD-
normal donors) in SCD patients is not too wide.  With an n = 16 participants with complete data, 
with a hypothesized difference in PTR of 10%, the chance of drawing a sample of patients that 
would yield a p-value smaller than α = 0.05 is 99%. 
 
2.2   Secondary Objectives (Aims 2a-2d) 
Aim 2a.   Evaluate the relative effects of blood from a donor with G6PD-deficiency on 4-week 
post-transfusion outcomes as measured by mean change in hemoglobin A (HbA).  
Aim 2b.   Evaluate the relative effects of blood from a donor with G6PD-deficiency on 4-week 
post-transfusion outcomes as measured by mean changes in twenty-three clinically-relevant 
measures.    
Aim 2c.   Evaluate the relative effects of blood from a donor with G6PD-deficiency on the 
longitudinal series of metabolic profiles obtained from blood samples.  
Aim 2d.   Evaluate the relative effects of blood from a donor with G6PD-deficiency on 
longitudinal pain scores, infection symptoms, and antibiotic use. 
 
The investigations of Aims 2a-2d will be considered success if the confidence intervals for the 
treatment effects are not too wide. 
 
2.3 Rationale for the Outcome Measures Selected 
For Aim 1, the primary outcome (24-hour PTR) is the only in-vivo FDA approved method for 
guiding quality of red cell products.  This method has been used for multiple decades, and a 
large volume of literature can be applied to help interpret the proposed study findings. 
For Aim 2a, hemoglobin A is the leading clinically-relevant measure of interest.  
For Aims 2b and 2d, the selected secondary measures are fundamentally important clinical 
measures.    
For Aim 2c, we expect the metabolomic data to provide insights for generation of new 
hypotheses. 

 
 
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3 STUDY DESIGN 
3.1 General Description 
This is a phase II, single blind, crossover study in 16 adults with SCD who are  actively on a 
chronic transfusion protocol. Patients with SCD, >18-years old, will be recruited from the UNC 
Adult Sickle Cell Clinic. Subjects will receive 15-25 day old RBCs from sickle trait-negative, 
ABO-compatible (but not identical, if non-O), CEK-matched, cross-match compatible, G6PD-
deficient or G6PD-normal donors. Subjects will be blinded to infusion assignment. Subjects will 
be transfused 1 experimental unit of RBC per the UNC blood transfusion protocol as part of a 
routine red cell exchange. During the last 30-45 minutes (+/- 30 minutes) of the exchange, a 
50mL sterile sample will be removed from the experimental RBC unit, one aliquot used for 51Cr-
labeling and one for “omics” studies. Once the exchange is complete, the subject will then be 
rapidly infused with the 51Cr labeled aliquot per Nuclear Medicine’s protocol (Please refer to 
RDRC documents).  
 
Blood samples will be obtained pre-infusion, and at approximately 5, 7.5, 10, 12.5, 15, 30 
minutes; 1, 24, 48, 72 hours; and 1, 2, 3, 4 weeks post-infusion. We will measure PTR and long-
term lifespan of the donor RBCs, accounting for the expected elution of 51Cr using standard 
formulas. Hemolysis markers, CBCs, and HbA/S% levels will be measured at defined time 
points post-transfusion to explore correlations with the PTR. After a >120-day washout period 
(i.e., the RBC lifespan), the same procedures will again be performed on each subject, this time 
receiving RBCs from a donor of the alternative G6PD status (i.e., normal vs. deficient). During 
this time, the patient will remain on their clinical treatment protocols as ordered by their provider, 
and no changes to their clinical care will be made as a result of their participation in the study. 
 
3.2 Study Completion 
The study will reach completion once all 16 subjects have completed the study. This study is 
estimated to take 4 years to complete once the study opens to accrual. 
4 SUBJECT PARTICIPATION, DISCONTINUATION, AND 
WITHDRAWAL   
 
We will follow all UNC IRB requirements and policies regarding subject participation: 
 
4.1 Subject Status 
 
Subject statuses throughout the trial are defined as follows:   
 
• 
Prescreening: preconsent (subject considering trial or study staff considering patient for 
the trial per institutional recruitment methods). 
• 
Screening: period after consent, but prior to eligibility confirmation. 
• 
Consented: consented, prior to eligibility confirmation. 
• 
Eligible: the local investigator confirms all eligibly criteria apply. 

 
 
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• 
On study/enrolled: date eligibility is confirmed. 
• 
Off study: no additional subject data gathered. 
• 
Withdrawn: subject fully withdraws consent (i.e., refuses ALL follow-up) or is taken off 
study by the local principal investigator. 
 
4.2 Prescreening and Screening Log 
 
The UNC study principal investigator regularly reviews screen failure reasons to understand 
barriers to accrual and consider amending eligibility criteria. Screen failures are defined as 
participants who were considered for the trial to participate in the clinical trial with or without 
consent, but are not subsequently assigned to the study intervention or enrolled in the study. 
Prescreening and screening tracking will follow standard practice for research at UNC. 
 
4.3 Consent  
 
Investigators or their appropriate designees will identify potentially eligible subjects from their 
clinics, subject self-referrals, referrals from other clinicians, and/or other IRB-approved 
recruitment methods. No study conduct, including subject prescreening, can occur until after 
IRB approval. 
 
A written, signed informed consent form (ICF) and a Health Insurance Portability and 
Accountability Act (HIPAA) authorization must be obtained before any study-specific 
assessments are initiated. A signed ICF copy will be given to the subject and a copy will be filed 
in the medical record (per local IRB policies and SOPs). The original will be kept on file with the 
study records. UNC exchange and blood consent will also be obtained prior to any RBC 
transfusion activities, if not already up to date. 
 
4.4 Screening Procedures 
 
For women of childbearing potential, a negative pregnancy test must be obtained prior to RBC 
transfusion. Visit procedures that were performed as standard of care prior to consent (without 
the specific intent to make the subject eligible for the trial), may count toward screening tests 
and eligibility if they are within the screening window.  
 
 
 
4.5 Eligibility Confirmation 
All patient eligibility criteria will be confirmed by a trained study investigator prior to ordering any 
blood products for infusion.  
 
4.6 Study Eligibility Criteria 
*No waivers of protocol eligibility will be granted. 
 
Inclusion Criteria 
1. Age 18-60 years 
2. A diagnosis of sickle cell disease 
3. Steady state (no pain or baseline pain and ≥1 month from any hospital admission) 
4. Receiving chronic transfusions (i.e regular transfusion every 4-8 weeks). 

 
 
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Exclusion Criteria 
1. History of transfusion reactions not adequately managed by antihistamines 
2. No available crossmatch compatible red cell units 
3. Known G6PD deficiency 
4. Hepato- or splenomegaly 
5. Participation in another therapeutic trial 
6. Pregnant or nursing 
7. HIV positive 
8. At investigator discretion for uncontrolled inter-current illness or social situation limiting 
compliance with study requirements. 
9. Inability to speak and/or read English 
 
4.7 Enrollment 
Subject enrollment logistics are defined as follows: 
 
Patients ≥ 18 years of age with sickle cell disease will be identified through the adult outpatient 
sickle cell clinic at UNC through the sickle cell providers trained on the study protocol. 
 
Potentially eligible patients, as described in the previous paragraph, will be approached for 
study consent prior to their transfusion or infusion. Individual center scheduling practices will 
influence how this contact is arranged. Subjects who consent to the study will be assigned a 
study ID number and have their eligibility status determined. If the subject is eligible for the 
study based on the inclusion/exclusion criteria (section 4.7, 4.8), they will be enrolled in the 
study and will be considered for randomization (see sections 4.2 and 5.1). 
 
4.8 Unit Selection, Identification, and Study Activities  
At enrollment, basic features of patient medical and surgical histories (i.e. age, gender, past red 
cell transfusions, red cell phenotype) will be collected. The subject’s eligibility status and 
ABO/RBC phenotype will be entered into the electronic data capture system (EDC). If eligible, 
the subject’s first data of chromium labeling will be determined, and appropriate RBC units at 
the UNC transfusion service will be identified.  
 
At a minimum, potential RBC units will be selected from the appropriate RBC unit inventory to 
ensure that the unit is: 1) ABO compatible with the subject, 2) D, Cc, Ee, and Kk matched to the 
subject (rr, Ror, or RoRo phenotype), 3) stored in AS1, 4) sickle negative, 5) G6PD positive or 
negative based on testing. African black donor units will be preferred, but not required, for 
selection due to the known increased prevalence for G6PD deficiency. All other standard quality 
metrics for RBC units will be adhered to per standard blood bank service practice. 
 
For G6PD testing: Two sterile segment links from the units assigned for the subject’s red cell 
exchange identified by UNC will be codified and tested for G6PD enzyme activity in the clinical 
laboratory (McClendon Laboratories). Units identified as G6PD deficient/not deficient will be 
selected for use. Given the rarity of G6PD deficient units, these units will be selected first, if 
available. If not, a G6PD normal unit will be selected. Cross-matching for the unit with the 
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Only study staff with the appropriate security level will be able to access the arm assignment 
(G6PD deficient first, or G6PD normal first). Access to case report forms containing information 
about the RBC products sent for each subject will also be restricted at the site to the appropriate 
study staff. Clinical staff overseeing the subject’s participation in the trial, collecting data, and 
reporting the data into EDC will have access to the treatment arm assignment and information 
about the G6PD-status of the RBC products transfused.  
 
No alteration will be made to the labels on the RBC units. The expiration date, collection date, 
and any processing dates (e.g. irradiation dates) will not be obscured.  Trained personnel at the 
adult Translational Research Unit will verify product and patient identity according to hospital-
specific procedures. These personnel will be instructed to not divulge the patients’ unit type. 
Other ancillary staff (other than those actually infusing RBCs), will be instructed not to seek to 
identify the G6PD status of the products transfused. The subjects themselves will not be 
informed of the identity of the labeled unit and will also be instructed not to seek to identify the 
G6PD status of the products they are receiving. However, as the key components of this study 
are objective, inadvertent unblinding of the unit-status will not compromise the validity of 
the study data. 
 
Study Activities 
 
24-72 hours prior to the 51Cr-labeled RBC infusion: 
Draw 10mL of whole blood for type and screen  
 
 Day 1 
• 
Physical exam by principal investigator or designee. 
• 
Double-lumen Midline catheter placement by Interventional Radiology or VAT 
• 
Provide daily diary 
• 
Urine sample for urine pregnancy screen (females only) 
• 
Vital signs will be checked for eligibility (subjects who are pregnant, have SBP >180 or 
<90mm Hg, DBP >100 or <50mm Hg, Heart rate <50 or >100, Temperature >100.4 F or 
feeling ill, will not continue study activities, and will be evaluated to be rescheduled).   
• 
Informed consent for a blood transfusion, if not up to date 
• 
Draw 10 mL of whole blood for CBCD, reticulocyte count, and hemoglobin 
electrophoresis. 
• 
Draw 4 mL of whole blood for cell free hemoglobin. 
• 
Draw  15 mL of whole blood for Hapto, Iron panel, BILI T/D, HSCRP, LDH, Ferritin, Iron 
Saturation, MPO, and NTBI. 
• 
Clinic RN will transfuse the experimental RBC unit to the patient as the last unit of the 
red cell exchange or transfusion following hospital standard procedures 
• 
Toward the end of the exchange/transfusion (about 30-45 minutes prior to completion), 
draw 30 mL from the unit into a syringe for chromium labeling.  The syringe will be kept 
in a cooler once collected, and will be transported to Nuclear Medicine in the cooler for 
Chromium labeling once the transfusion is completed.  
• 
Draw 6 mL of whole blood for in vivo chromium measurement (baseline- pre infusion). 
• 
Infuse Chromium labeled blood into study subject. 
 
Day 1 – post infusion 
• 
Draw 6 mL of whole blood for chromium measurements will be taken at time 5, 7.5, 10, 
12.5, 15, 30 minutes, and 1 hour post labeled red cell infusion (samples for Omics will 
be taken from this tube).  

 
 
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• 
At 1 hour (+/- 15 minutes), draw 4 mL of whole blood for cell free hemoglobin. 
• 
At 1 hour (+/- 15 minutes) post-RBC transfusion, draw 10 mLfor CBCD, reticulocyte 
count, and hemoglobin electrophoresis.  
• 
At 1 hour (+/-), draw 15 mL Hapto, Iron panel, BILI T/D, HSCRP, LDH, Ferritin, Iron 
Saturation, MPO, NTBI. 
• 
Process and store samples for NTBI and metabolomics studies. 
 
24hrs - Post Infusion 
• 
Draw 6 mL of whole blood for chromium measurements taken at 24 hrs (+/3 hours) 
post infusion (samples for Omics will be taken from this tube).  
• 
Draw 4 mL of whole blood for cell free hemoglobin 
• 
Draw 10 mL of whole blood for CBCD, reticulocyte count, and hemoglobin 
electrophoresis.   
• 
Draw 15 mL of whole blood for Hapto, Iron panel, BILI TD, HSCRP, LDH, Ferritin, Iron 
Saturation, and MPO at 24 hrs (+/- 3 hours) post infusion. 
• 
Process and store samples for NTBI and metabolomics studies. 
• 
Review of daily diary 
 
48hrs - post infusion 
• 
Draw 6 mL of whole blood  for chromium measurements taken at 48 hrs (+/- 3 hours) 
post infusion (samples for Omics will be taken from this tube).  
• 
Draw 10 mL of whole blood for CBCD, reticulocyte count, and hemoglobin 
electrophoresis.   
• 
Draw 4 mL of whole blood for cell free hemoglobin 
• 
Process and store samples for NTBI and metabolomics studies. 
• 
Review of daily diary 
 
72hrs - post infusion 
• 
Draw 6 mL of whole blood l for chromium measurements taken at 72 hrs (+/- 3 hours) 
post infusion (samples for Omics will be taken from this tube).  
• 
Draw 10 mL of whole blood mL for CBCD, reticulocyte count, and hemoglobin 
electrophoresis.   
• 
Process and store samples for NTBI and metabolomics studies. 
• 
Review of daily diary 
 
1 Week – post infusion 
• 
Draw 6 mL of whole blood  for chromium measurements taken at 1 week (+/- 1 day) 
post infusion (samples for Omics will be taken from this tube).  
• 
Draw 10 mL of whole blood for CBCD, reticulocyte count, and hemoglobin 
electrophoresis.   
• 
Draw 4 mL of whole blood for cell free hemoglobin 
• 
Draw 15 mL of whole blood for Hapto, Iron panel, BILI TD, HSCRP, LDH, Ferritin, Iron 
Saturation, and MPO at  1 week (+/- 1 day) post infusion. 
• 
Process and store samples for NTBI and metabolomics studies. 
• 
Review of daily diary 
 
2 Week – post infusion 
• 
Draw 6 mL of whole blood  for chromium measurements taken at 2 weeks (+/- 2 days) 
post infusion (samples for Omics will be taken from this tube).  

 
 
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• 
Draw 10 of whole blood for mL for CBCD, reticulocyte count, and hemoglobin 
electrophoresis.   
• 
Draw 4 mL of whole blood for cell free hemoglobin 
• 
Draw 15 mL of whole blood for Hapto, Iron panel, BILI TD, HSCRP, LDH, Ferritin, Iron 
Saturation, and MPO at 2 weeks (+/- 2 days), 3 weeks (+/- 2 days) post infusion. 
• 
Process and store samples for NTBI and metabolomics studies. 
• 
Review of daily diary 
 
3 Week – post infusion 
• 
Draw 6 mL of whole blood for chromium measurements taken at 3 weeks (+/- 2 days) 
post infusion (samples for Omics will be taken from this tube).  
• 
Draw 10 mL of whole blood for CBCD, reticulocyte count, and hemoglobin 
electrophoresis.   
• 
Draw 4 mL of whole blood for cell free hemoglobin 
• 
Draw 15 mL of whole blood for Hapto, Iron panel , BILI TD,  HSCRP, LDH, Ferritin, Iron 
Saturation, and MPO at 3 weeks (+/- 2 days) post infusion. 
• 
Process and store samples for NTBI and metabolomics studies. 
• 
Review of daily diary 
 
4 Week - post infusion 
• 
Physical Exam  
• 
Draw 6 mL of whole blood  for chromium measurements taken at 4 weeks (+/- 2 days) 
post infusion (samples for Omics will be taken from this tube).  
• 
Draw 10 mL of whole blood for CBCD, reticulocyte count, and hemoglobin 
electrophoresis.   
• 
Draw 4 mL of whole blood for cell free hemoglobin 
• 
Draw 15 mL of whole blood for Hapto, Iron panel, BILI TD, HSCRP, LDH, Ferritin, Iron 
Saturation, and MPO at 4 weeks (+/- 2 days) post infusion. 
• 
Process and store samples for NTBI and metabolomics studies. 
• 
Review of daily diary 
 
Follow-Up Visit 
 
After day 28, the subject will be given a (washout period) break of about 4 months (120 days). 
The patient will continue their transfusion treatements as prescribed by their physician. After this 
break, the subject will be contacted to repeat the study blood draws and visits. 
 
Specimen Processing 
 
NTBI: 0.2mL from SST tube will be obtained from each required time point, snap frozen, and 
stored at -80C until mailing. Samples will be sent yearly to the research laboratory of Dr. Eldad 
Hod (Columbia University) 
 
Metabolomics:  Unit samples: 0.5 ml (max, Min 0.1 ml) transfusate (no separation); 0.1 ml of 
packed RBC (pellet) and 0.1 ml of supernatants after gentle centrifugation at 2,500g at 4C for 
10 Min. All samples will be snap frozen and stored at -80C until mailing. Patient samples: 20 ul 
of plasma and 50 ul of RBCs after gentle centrifugation at 2,500g at 4C for 10 Min will snap 
frozen and stored at -80 until mailing. Samples will be sent yearly to the research laboratory of 
Dr. Angelo D’Alessandro (University of Colorado) 
 

 
 
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4.9  Discontinuation of Study Treatment, Withdrawal, and Compliance 
Discontinuation from the study does not mean discontinuation from any clinically guided medical 
care. Subjects will be monitored for AEs/SAEs during the 4 week infusion testing period so long 
as an infusion took place.  
 
In the absence of delays due to adverse events, study activities may continue until:  
 
• 
Disease progression or instability as determined by the principal investigator.  
• 
General or specific changes in the subject’s condition renders the subject 
unacceptable for further treatment in the investigator’s judgment.  
• 
Intercurrent illness that prevents further participation. 
• 
Subject decides to withdraw from the study.  
• 
The subject has significant noncompliance with the protocol (defined as greater than 
3 missed appointments).   
• 
Unacceptable adverse event(s)  
• 
Study stopping rules are met. 
 
Subjects who sign the informed consent form, and are enrolled and receive the study 
intervention, but subsequently withdraw, or are withdrawn or discontinued from the study, will 
not be replaced. 
 
Consent Withdrawal 
 
A subject may decide to withdraw from the study at any time. UNC will follow its IRB of record’s 
SOPs regarding consent withdrawal.  
 
If a subject intends on withdrawing consent, staff should confirm which of the following options 
the subject chooses and document the discussion: 
 
-  Full consent withdrawal with no study follow-up. 
 
-  Selective consent withdrawal from interventional portion of the study, but agree to continued 
follow-up of associated clinical outcome information (i.e. study diary and/or blood draws). 
 
Investigator-initiated Withdrawal 
 
The investigator will withdraw a subject whenever continued participation is no longer in the 
subject’s best interests. Reasons for withdrawing a subject include, but are not limited to, 
disease progression or instability, the occurrence of an severe adverse event or a concurrent 
illness, a subject’s request to end participation, a subject’s noncompliance or simply significant 
uncertainty on the part of the investigator that continued participation is prudent. The reason for 
study withdrawal and the date the subject was removed from the study must be documented. 
 
4.10  Lost to Follow-up 
The following actions must be taken if a participant fails to return to the clinic for a required 
study visit and/or is unable to be reached for follow-up: 
 

 
 
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• 
The investigator or designee must make every effort to regain contact and/or reschedule a 
missed visit with the participant. 
• 
A participant is deemed lost to follow-up if his/her status cannot be obtained after all of the 
following occurs at two consecutive scheduled protocol calendar timepoints: 
o Three telephone calls (at least one day apart) from the study team are unanswered  
AND  
o A letter to the participant’s last known mailing address goes unanswered  
AND 
o These contact attempts must be documented in the participant’s study file.  
• 
Update the EDC when a participant is officially considered lost to follow-up. 
• 
If a subject is considered lost to follow-up, but subsequently contacts the participating site 
study team, the subject should be considered in follow-up again. 
 
4.11  Accrual Suspension and Closure 
The UNC PI facilitates the suspension and closing of accrual in the following manner: 
 
• 
EDC tracks accrual throughout the study. 
• 
If the study must be suspended, EDC and Clinical trial management software (ie Velos) is 
updated to a ‘suspended’ status. 
• 
When the accrual number is reached, PI notifies staff of study closure. 
 
4.12  End of Study Definition 
A participant is considered to have completed the study if he or she has completed all phases of 
the study including the last visit or the last scheduled procedure shown in the calendar of events 
or has been discontinued. 
 
4.13  Study Discontinuation and Closure 
This study may be temporarily suspended or prematurely terminated if there is sufficient 
reasonable cause (as determined by the study principal investigator, DSMC, sponsor, and/or 
IRB). Written notification, documenting the reason for study suspension or termination, will be 
provided by the suspending or terminating party to study participants, investigator, funding 
agency, and regulatory authorities. If the study is prematurely terminated or suspended, the 
principal investigator (PI) will promptly inform the MCW Institutional Review Board (IRB) and 
sponsor and will provide the reason(s) for the termination or suspension. Study participants will 
be contacted, as applicable, and be informed of changes. 
5 STUDY PLAN 
5.1 RBC Unit Administration 
Treatment will be administered on an outpatient basis. The study RBC unit will be administered 
per hospital protocol by the UNC RN staff per normal administration protocols. Subject vital 
signs will be taken at regular intervals per hospital protocol. The study staff will aseptically 
remove 50 ml (~4 tablespoons) of the same crossmatch-compatible RBCs into a syringe,  and 
members of the Nuclear Medicine Department will label that sample with a radioisotope 
(51Chromium) per standard protocols. A portion of that tagged aliquot will be transferred into a 

 
 
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syringe to be infused into the subject. A second portion from that sample, pre chromium 
labeling, will be saved for future “omics” studies. The UNC Nuclear Medicine department study 
staff will be specifically responsible for the preparation and infusion of the chromium-labeled 
aliquot to the patient.  The RN staff will be responsible for the transfusion of the non-labeled 
compatible red cell unit and lab blood draws. 
 
5.2 Monitoring Subject Compliance  
Pain and infection diary: The diary is adapted from previously published SCD studies.87 Pain 
and infection symptoms will also be documented daily for 4 weeks along with the type, duration, 
and dose of antibiotics, if applicable. Daily, participants will rate pain on a numeric pain rating 
ordinal scale (0-10), indicate whether the pain was consistent with a “crisis,” indicate whether 
they utilized a healthcare facility, and record the amount of opioids used. Infection symptoms 
will also be documented daily along with the type, duration, and dose of antibiotics, if applicable. 
5.3 Follow-up Period 
Patients will be followed for up to 28 days following their last study-related blood transfusion. 
Patients will not be followed after completion of the study protocol. 
Patients removed from the study treatment for unacceptable SAEs will be followed until 
resolution or stabilization of the adverse event. SAEs will be followed until completion.  
6 DOSING DELAYS/DOSE MODIFICATIONS 
6.1 Monitoring and Toxicity Management 
Each patient receiving G6PD-deficient and G6PD-normal RBCs will be evaluated for safety. The 
safety parameters include all laboratory tests and hematological abnormalities, physical findings 
and spontaneous reports of adverse events reported to the investigator by patients, and are 
outside laboratory values and clinical events considered usual for SCD. 
The chromium agent used for red cell labeling has no known or previously reported risks of 
adverse events.  Consequently, general tracking of adverse events will be conducted as noted 
in the following sections.  
Each patient will be assessed periodically for any toxicity development. Toxicity will be 
assessed according to the CTCAE v 4.0. 
We will specifically monitor for evidence of red cell hemolysis as a result of the red cell 
transfusion. Evidence of hemolysis will be obtain from clinical symptoms (fever, chills) and 
laboratory values obtained per study protocol (LDH, haptoglobin, and hemoglobin values).  
Acute toxicity will be managed according to the symptoms and determined etiology. Most 
adverse events from transfusion can be managed symptomatically by standard over-the-counter 
medications, such as Tylenol for fevers, or Benadryl for allergic symptoms. Further 
management will depend upon the judgment of the clinician and may include ED admission or 
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Patients will also be monitored for exacerbations of the subjects underlying sickle cell disease: 
Vaso-occlusive pain crises, acute chest crises, stroke, serious infection, and priapism. This will 
be monitored by review of the patients electronic medical record, subject verbal report, and/or 
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7 ADVERSE EVENTS: DEFINITIONS AND REPORTING 
REQUIREMENTS 
7.1 Definitions 
1) Vaso-occlusive pain crisis: an episode of acute pain with no cause other than a vaso-
occlusive event lasting at least 24 hours that requires the administration of oral or parenteral 
opioids in a medical facility. 
 
2) Acute Chest Syndrome: an acute illness requiring medical facility attention characterized by 
fever and/or respiratory symptoms, accompanied by a new pulmonary infiltrate on a chest X-ray. 
 
3) Priapism: an acute illness requiring medical facility attention characterized by an erect penis  
that does not return to its flaccid state, despite the absence of both physical and psychological 
stimulation, within four hours. 
 
4) Acute infection: an acute illness requiring medical facility intervention (i.e prescription of 
antibiotics or hospital admission) characterized by any subjective symptom. 
 
5) Chronic transfusion: receiving red cell units on a 4-8 week schedule as part of routine, non-
acute care). 
 
6) Acute transfusion: receiving red cell units on an unscheduled basis to resolve an acute issue. 
 
7.1.1 Adverse Event (AE) and Serious Adverse Events (SAE) 
The investigator and his or her team will follow UNC policies related to adverse event reporting. 
This information may be found on the Human Research Protection Program website. 
Serious AE (SAE) means any untoward medical occurrence that at any dose: 
• 
Death. Results in death. 
• 
Life threatening. Is life threatening (refers to an AE in which the patient was at risk of 
death at the time of the event. It does not refer to an event which hypothetically might 
have caused death if it were more severe). 
• 
Hospitalization. Requires inpatient hospitalization or prolongation of an existing 
hospitalization (see clarification in the paragraph below on planned hospitalizations). 
• 
Disability/incapacity. Results in persistent or significant disability or incapacity. 
(Disability is defined as a substantial disruption of a person’s ability to conduct normal 
life functions). 
• 
Medically important event. This refers to an AE that may not result in death, be 
immediately life threatening, or require hospitalization, but may be considered serious 
when, based on appropriate medical judgment, may jeopardize the patient, require 
medical or surgical intervention to prevent one of the outcomes listed above, or involves 
suspected transmission via a medicinal product of an infectious agent. Examples of such 

 
 
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medical events include allergic bronchospasm requiring intensive treatment in an 
emergency room or at home, blood dyscrasias or convulsions that do not result in 
inpatient hospitalization, or the development of drug dependency or drug abuse; any 
organism, virus, or infectious particle (e.g., prion protein transmitting transmissible 
spongiform encephalopathy), pathogenic or nonpathogenic, is considered an infectious 
agent.  
Clarification should be made between a serious AE (SAE) and an AE that is considered severe 
in intensity (Grade 3 or 4), because the terms serious and severe are NOT synonymous. The 
general term severe is often used to describe the intensity (severity) of a specific event; the 
event itself, however, may be of relatively minor medical significance (such as a Grade 3 
headache). This is NOT the same as serious, which is based on patient/event outcome or action 
criteria described above, and is usually associated with events that pose a threat to a patient’s 
life or ability to function. A severe AE (Grade 3 or 4) does not necessarily need to be considered 
serious. For example, a white blood cell count of 1000/mm3 to less than 2000 is considered 
Grade 3 (severe) but may not be considered serious. Seriousness (not intensity) serves as a 
guide for defining regulatory reporting obligations. 
7.1.2 Unanticipated Problem Involving Risk to Subject or Other  
The investigator and his or her team will follow UNC  policies related to unanticipated problems 
involving risks to subjects or others. This information may be found on the Human Research 
Protection Program website.  
7.1.3 AE Attribution and Grading 
Adverse Event Grading 
Grade 
Description 
0 
No AE (or within normal limits). 
1 
Mild; asymptomatic or mild symptoms; clinical or diagnostic 
observations only; intervention not indicated. 
2 
Moderate; minimal, local or noninvasive intervention (e.g., 
packing cautery) indicated; limiting age-appropriate 
instrumental activities of daily living (ADL). 
3 
Severe or medically significant but not immediately life-
threatening; hospitalization or prolongation of hospitalization 
indicated; disabling; limiting self-care ADL. 
4 
Life-threatening consequences; urgent intervention indicated. 
5 
Death related to AE. 
 
Adverse Event Attribution 

 
 
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Attribution is an assessment of the relationship between the AE and the medical intervention. 
 
Relationship 
Attribution 
Description 
Unrelated to investigational 
agent/intervention 
Unrelated 
The AE is clearly NOT related to the 
intervention 
Unlikely 
The AE is doubtfully related to the 
intervention 
Related to investigational 
agent/intervention 
Possible 
The AE may be related to the 
intervention 
Probable 
The AE is likely related to the 
intervention 
Definite 
The AE is clearly related to the 
intervention 
 
Relationship Assessment:  In-Depth Definitions 
For all collected AEs, the clinician who examines and evaluates the subject will determine the 
adverse event’s causality based on temporal relationship and his/her clinical judgment. The 
degree of certainty about causality will be graded using the categories below: 
Definitely Related: There is clear evidence to suggest a causal relationship, and other possible 
contributing factors can be ruled out. The clinical event, including an abnormal laboratory test 
result, occurs in a plausible time relationship to drug administration and cannot be explained by 
concurrent disease or other drugs or chemicals. The response to withdrawal of the drug 
(dechallenge) should be clinically plausible. The event must be pharmacologically or 
phenomenologically definitive, with use of a satisfactory rechallenge procedure if necessary.  
Probably Related: There is evidence to suggest a causal relationship, and the influence of 
other factors is unlikely. The clinical event, including an abnormal laboratory test result, occurs 
within a reasonable time sequence to administration of the drug, is unlikely to be attributed to 
concurrent disease or other drugs or chemicals, and follows a clinically reasonable response on 
withdrawal (dechallenge). Rechallenge information is not required to fulfill this definition.  
Possibly Related: There is some evidence to suggest a causal relationship (e.g., the event 
occurred within a reasonable time after administration of the trial medication). However, the 
influence of other factors may have contributed to the event (e.g., the subject’s clinical condition, 
other concomitant events). Although an adverse drug event may rate only as “possibly related” 
soon after discovery, it can be flagged as requiring more information and later be upgraded to 
“probably related” or “definitely related,” as appropriate. 
Unlikely: A clinical event, including an abnormal laboratory test result, whose temporal 
relationship to drug administration makes a causal relationship improbable (e.g., the event did 
not occur within a reasonable time after administration of the trial medication) and in which other 
drugs or chemicals or underlying disease provides plausible explanations (e.g., the subject’s 
clinical condition, other concomitant treatments).  
Unrelated: The AE is completely independent of study drug administration, and/or evidence 
exists that the event is definitely related to another etiology. There must be an alternative, 
definitive etiology documented by the clinician.  

 
 
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7.2 Known AEs List 
Below are know AE’s related to blood cell transfusion. Many of these risks are common to sickle 
cell disease and are known to occur. Reporting of these instances will be based on frequency and 
severity under the determination of trained study staff. 
 
• 
Myocardial infarction  
• 
Pulmonary embolism  
• 
Stroke  
• 
Renal failure  
• 
Sepsis  
• 
Ventricular tachycardia  
• 
Ventricular fibrillation 
• 
Red cell hemolysis (immune and non-immune)  
• 
Transfusion-associated congestive heart failure/transfusion-associated circulatory 
overload (TACO)  
• 
Transfusion-related acute lung injury (TRALI)  
• 
Anaphylaxis  
• 
Graft vs. Host Disease (GVHD)  
• 
Grade 2 or higher events of the following types:  
o Allergic reaction  
o Sinus bradycardia  
o Sinus tachycardia  
o Hypertension  
o Hypotension  
o Dyspnea  
o Hypoxia  
o Wheezing  
o Cough  
o Fever  
o Chills  
o Hemolysis  
o Hyperkalemia  
o Hypocalcemia  
o Hyperbilirubinemia  
o Hemoglobinuria.  
 
7.2.1 Other Risks 
7.2.1.1 Loss of confidentiality. 
• 
While participating in this project all study records kept electronically will be on encrypted 
drives and all paper copies will be kept in locked cabinets in a room requiring badge 
access for entry but there is always the potential some of the subjects information could 
be stolen. 
 
Risks of Transfusion/Apheresis catheter  
• 
Injury to local structures 
• 
Phlebitis at insertion site 
• 
Air embolism 
• 
Hetamtoma 
• 
Arrhythmia 

 
 
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• 
Catheter malposition  
• 
Infection 
• 
thrombosis 
 
7.3 Time Period and Grade of AE Capture 
The study staff will start recording AE’s from the time of the pre-transfusion type-and-cross 
blood sample until the end of the subject’s last visit on week 4. This process will occur twice due 
to the cross-over design of the study. 
7.4 Monitoring and Recording an Adverse Event  
Definition. Any clinically relevant deterioration in laboratory assessments in the opinion of the 
study investigator or other clinical finding determined relevant by the study investigator is 
considered an AE.  
Reporting source. AEs may be spontaneously reported by the patient and/or in response to an 
open question from study personnel or revealed by observation, physical examination or other 
diagnostic procedures.  
Prior to the trial. Planned hospital admissions or surgical procedures for an illness or disease 
that existed before the patient was enrolled in the trial are not to be considered AEs unless the 
condition deteriorated in an unexpected manner during the trial (e.g., surgery was performed 
earlier or later than planned).  
Pretreatment events following signed informed consent. For serious pretreatment events, 
the investigator must determine both the intensity of the event and the relationship of the event 
to study procedures.  
Treatment events. For serious AEs, the investigator must determine both the intensity of the 
event and the relationship of the event to study drug administration.  
Not serious AEs. For non-serious AEs, the investigator must determine both the intensity of the 
event and the relationship of the event to study drug administration. 
Follow-up of Adverse Events 
All adverse events will be followed with appropriate medical management 30 days following the 
last dose of the study transfusion or until they are resolved, if they are related to the study 
treatment. 
7.4.1 Procedure for Reporting Drug Exposure during Pregnancy and Birth Events 
If a woman becomes pregnant, or suspects that she is pregnant, while participating in this study, 
she must inform the investigator immediately and permanently discontinue study participation. 
The investigator must notify the DSMC by email. The pregnancy must be followed for the final 
pregnancy outcome. 
7.4.2 Subject Complaints 

 
 
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If a complaint is received by anyone on the study staff, it will be discussed with the study staff 
and will be addressed on a case-by-case basis. The PI will be notified of any complaints. 
Complaints will be reported to the IRB if indicated.  
If the subject has questions about his or her rights as a study subject, wants to report any 
problems or complaints, obtain information about the study or offer input, the subject can call 
the UNC Hospital research subject advocate or the study PI at 984-974-1583. This information 
is provided to the subject in their consent.  
7.4.3 Routine Reporting Procedures for AEs 
Study staff must report the Adverse Event form within 7 days of each study transfusion. The 
Adverse Event Checklist will indicate whether or not one or more of the events listed in Section 
8.2 occurred during the transfusion. The PI will be also be notified of the identified event within 24 
hours of learning of the event by phone or email. 
 
Since this is an investigator-initiated study, the principal investigator, also referred to as the 
sponsor-investigator, is responsible for reporting serious adverse events (SAEs) to any 
regulatory agency and to the sponsor-investigator’s IRB. Regardless of expectedness or 
causality, all SAEs (including serious pretreatment events) must also be reported to the DSMB 
as soon as possible, but no later than five calendar days after the sponsor-investigator’s 
observation or awareness of the event.  
 
Signs or symptoms reported as adverse events will be graded and recorded by the investigator, 
according to the CTCAE. When possible, signs and symptoms indicating a common underlying 
pathology should be noted as one comprehensive event.  
 
The investigator will assess all adverse events and determine reporting requirements to the 
UNC Safety Monitoring Committee (SMC) and MCW’s Institutional Review Board. The 
investigator will report SAEs to any required regulatory agency and to the sponsor-investigator’s 
IRB. 
 
All adverse events, whether or not unexpected, and whether or not considered to be associated 
with the use of the study drug, will be entered into the studies AE log. 
 
Reporting to the Safety Monitoring Committee 
 
Regardless of expectedness or causality, all SAEs (including serious pretreatment events) must 
also be reported to the SMC as soon as possible, but no later than five calendar days of the 
sponsor-investigator’s observation or awareness of the event.  
Report Method:  The investigator will use email to report SAEs to the SMC. The SAE report 
must include event term(s), serious criteria and the sponsor-investigator’s or sub-investigator’s 
determination of both the intensity of the event(s) and the relationship of the event(s) to study 
drug administration. Intensity for each SAE, including any lab abnormalities, will be determined 
by using the NCI CTCAE as a guideline whenever possible.  
Reporting to MCW Committee Institutional Review Board 
The principal investigator must report events to the MCW IRB within five business days of 
his/her awareness of the event.  

 
 
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Event Type 
Report Recipients  
PI/Study Chair/ 
Coordinating 
Center 
Institutional 
Review  
Board 
DSM
C 
 
 
FDA 
CTO  
Regulator
y 
Office  
Othe
r 
Serious Adverse Event 
ASAP 
5 days (or annual 
CPR)1 
5 
days 
7 or 15 
days2 
ASAP 
 
Unanticipated Problems Involving Risks to 
Subjects of Others 
ASAP 
5 days (or annual 
CPR)1 
5 
days 
7 or 15 
days2 
ASAP 
 
Evidence of Causal Relationship between Drug 
and AE 
ASAP 
5 days (or annual 
CPR)1 
5 
days 
7 or 15 
days2 
ASAP 
 
 
 
 
 
 
 
 
Contacts 
Role 
Name 
Entity/Department 
Institution 
Telephone 
Email 
Investigator 
Matthew Karafin 
 
UNC 
984-974-1583 
Matthew.karafin@unc.edu 
Research 
Coordinator 
David Wichlan 
 
UNC 
919-966-6876 
david_wichlan@med.unc.ed
Footnotes 
1 Consult UNC IRB Policies (contact your regulatory representative) 
2  FDA guidelines:  Suspected adverse reaction, Unexpected and Serious = 7 Days; If not = 15 days 

 
 
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8 INTERVENTION INFORMATION 
8.1 Agent #1: Chromium-labeled red cells 
8.1.1 Product Description: Chromium-labeled red cells is an FDA approved cross-match compatible 
red cell transfusion product.   
Contraindications: Pregnancy 
Side Effects:  None known. 
Solution Preparation  
1. Prepare the Cr-51 dose. 
 
2. With a 20 gauge needle, withdraw 50 millliter of the unit and immediately transfer syringe contents to the 
ACD vial using a 25 gauge needle as an airway.  Mix gently. 
 
3. From the ACD vial, withdraw 4-6 milliliter of whole blood and place in a lavender top tube labelled 
background.  
 
4. Label the red blood cells by adding 200 microcurie of Cr-51 to the ACD vial and mix gently.  Incubate at 
room temperature for 30 minutes, mixing gently every 10 minutes. 
 
5. After the 30-minute incubation of Cr-51 with blood, add 50 milligram of ascorbic acid to the ACD vial, mix 
gently and let stand for 5 minutes. 
 
6. Withdraw all of the Cr-51 labelled RBC into a 60 milliliter syringe and inject the patient.  Make note of the 
location of injection. 
 
8.1.2 Investigational Agent Administration 
The 51Cr labeled red cells will be  infused intravenously (over 1 minute through one IV line) into the subject. 
 
8.1.3 Storage Requirements 
The 51Cr labeled red cells will be  stored at 1-6° C per standard blood bank storage conditions for red cells. 
8.1.5 Route of Administration  
The 51Cr labeled red cells will be  infused intravenously (over 1 minute through one IV line) into the subject 
via a midline catheter that is placed by interventional radiology prior to the infusion. 
 
8.1.6 Nursing Implications 
The infusion will be provided by a trained assigned staff member of the Nuclear Medicine Department. 
8.1.7 Handling 
The 51Cr labeled red cells will be  stored at 1-6° C per standard blood bank storage conditions for red cells. 
The prepared red cell product is intended to be infused as soon as the labeling step is complete. 

 
 
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8.1.8 Agent Ordering 
The UNC Nuclear Medicine Department will order and create the compound as necessary for this single site 
study.    
9 STATISTICAL CONSIDERATIONS 
9.1 Study Measures 
9.1.1 Outcome Variables 
 
For Aims 1 and 2, the outcome measures of interest will be obtained from blood samples on 15 occasions 
within each of Period1 and Period2:  At baseline prior to the infusion treatment. 
Post-infusion at 5, 7.5, 10, 12.5, 15, 30 minutes,  and at 1, 24, 48, 72 hours,  and at 1, 2, 3, 4 weeks. 
 
For Aim 1, for purposes of the main analysis the measurements of  51CR-labeled PTR of RBCs prior to 
infusion and at 24 hours post-infusion are of interest.  The units of measurement will be % recovery.   
 
For Aim 2a and b, the outcome measures are Hemoglobin A and the following other clinically-relevant 
measures. We will evaluate their longitudinal tragectories over 4 weeks:  
• 
complete blood count with differential (CBCD: RBCs, WBCs, PLTsHCT, PCV, Hgb, MPV, throbocytes)  
• 
reticulocyte count per RBCs (%),  
• 
Hgb A, Hgb A2, Hgb F (% of total by electrophoresis)   
• 
cell-free hemoglobin (mg/dL),  
• 
haptoglobin (mg/dL),  
• 
Iron (mcg/dL), TIBC (mcg/dL), transferrin saturation (%), ferritin (mcg/L),  
• 
bilirubin values (total, direct, indirect) (mg/dL),  
• 
high-sensitivity C-reactive protein (HSCRP) (mg/L),  
• 
lactate dehydrogenase (LDH) (U/L),  
• 
myeloperoxidase (MPO) (pmol/L),  
• 
labile plasma iron (LPI) (μmol/L) 
• 
non-transferrin bound iron (NTBI) (μmol/L) 
 
Note: normal and valid values of these measurements will be determined by the clinical laboratories 
performing these assays. 
 
For Aim 2c, the outcomes of interest are the metabolomic profile estimates obtained for each blood sample. 
The unit of measure will be (μmol/L)   
 
For Aim 2d, pain scores, occurrence of infection, and antibiotic use will be obtained via a daily 4-week diary 
for evaluation of pain on an ordinal scale (0,1,2,… , 10),  occurrence of infection symptoms,  and use of 
antibiotics (type, duration, dose).   
 
9.1.2 Baseline Characteristics of the Participants 
At enrollment, the following will be recorded: age, gender, history of past red cell transfusions, red cell 
phenotype, medical history information, and surgical history information.   
9.2 Study Design 
For this Phase II, single-blind, controlled trial, the experimental design is a two-treatment, two-period, two-
sequence cross-over design: CO(2,2,2). The two treatment regimens are  
 
Regimen A  
transfusion of one G6PD-deficient donor units stored 15-25 days 

 
 
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Regimen B  
transfusion of one G6PD-normal  donor  units stored 15-25 days   
The two sequences of treatment are AB and BA. The two 4-week periods will be separated by a 4-month 
washout interval.  We believe the 4-month washout interval will be adequate to prevent carryover effects from 
period1 into period2 because RBCs normally only survive 120 days (4 months) at their maximum normal 
survival. 
 
9.3 Randomization, Concealment and Blinding 
The two sequences (AB and BA) will be assigned to the N=16 enrollees as determined by RBC unit 
availability at the time of the first exchange transfusion.  The two 4-week periods will be separated by a 4-
month washout interval. 
 
In crossover studies, reliably concealed unit assignment is the basis for the important assumption that 
‘sequence effects are zero’. Per the CONSORT Statement, allocation concealment will be used to prevent 
selection bias by concealing the allocations from the study personnel who are responsible for enrolling and 
assigning participants  --until the moment of assignment.  
 
After consent, one RBC unit will be requested for the study transfusion.  The study staff member will then 
contact UNC transfusion service to confirm that the transfusion service has a units that meet the crossmatch 
requirements (ABO, sickle negative, antigen matched) for this subject. The patient will then be enrolled in the 
study and assigned to a treatment sequence (AB or BA) depending upon RBC unit availability. The subject 
will be contacted regarding the date of their study transfusion once a suitable unit (G6PD deficient or G6PD 
normal) becomes available, and reaches the appropriate storage age, per protocol.   
 
9.4 Replacement Policy 
  
Replacement of enrolled participants who discontinue the study is not recommended because it be a source 
of selection bias. It is preferable to choose a target enrollment that is sufficiently large to be able to cope with 
dropout. 
9.5 Accrual Estimates 
 
The Adult Sickle Cell Disease clinic at UNC sees about 450 patients per year, of which only 50 would be 
eligible due to being on chronic transfusion.  Given known estimates of alloimmunization, 75% of these 
patients would be eligible to approach for consent (N=37). Due to the complexity of the study, target 
enrollment will aim for 4-8 research subjects per year, with a 3-4 year timeframe for study completion. 
Additional sites may be considered at the discretion of the principal investigator to meet enrollment goals.  
9.6 Statistical Analysis Plans  
Overview.   The aim-specific analysis plans include (1) detailed steps for the major estimators and inferential 
analyses, (2) sensitivity analyses performed to assess the robustness of the main results to reasonable 
perturbations/modifications of the a priori assumptions, choices, and methods used, (3) a role for outcome-
dependent exploratory analyses for hypothesis generation, and (4) necessary descriptive graphical and 
tabular methods used to characterize the sample of cows, visualize the data, and examine relationships 
among variables. Assuming the reasons for missing data values were appropriately documented in/with the 
database,  then best practices for dealing with incomplete data will depend on the documented causes of 
missing values.  
 
The main analyses will focus on the magnitude and direction of point- and interval-estimates of the 
population parameters of interest;  e.g., the treatment effect and the regimen-specific means and variances.  
To indicate precision, all statistical estimates of population parameters will be tabulated along with 
corresponding confidence intervals (CI) and standard errors (SE). The CI will be interpreted as a set of 
plausible values of the population parameter that are most compatible with the observed data.  The point- and 
interval-estimates will be treated as the most important results of the study because it is most appropriate to 

 
 
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ask quantitative research questions and obtain quantitative answers. P-values for hypothesis tests will be of 
less interest because they only address binary questions such as “Is the treatment effect exactly zero in the 
target population of cows?”  Following the recent recommendations of the American Statistical Association as 
explained by Wasserstein, et al. (2019), and Wasserstein, et al. (2016),  p-values will be reported to four 
decimal places and will not be dichotomized. This approach acknowledges that no p-value can reveal the 
plausibility, presence, truth, or importance of an association or effect. Smaller p-values indicate a larger 
degree of evidence against the (null) hypothesis tested and/or assumptions made, whereas larger p-values 
indicate that the test is inconclusive due to scarcity of information.  
 
Uncertainty about the optimal choice of methods and assumptions is best handled by relegating 
competing approaches to an important role in the domain of sensitivity analyses. The various sensitivity 
analyses will be used to evaluate the robustness/sensitivity of the study’s main results to reasonable 
perturbations/modifications of the statistical methods and assumptions used.  Results of the sensitivity 
analyses will be used to guide our level of trust in the main results. 
 
[ Reference: 2019 ICH E9-R1 addendum, “Estimands and Sensitivity Analysis in Clinical Trials”. 
 
www.gmp-compliance.org/guidemgr/files/E9-R1_Step4_Guideline_2019_1203.pdf  ] 
 
Exploratory analyses will be distinguished from confirmatory analyses. The purpose of exploratory 
analyses will be to generate new hypotheses or to refine hypotheses; p-values will not be used in exploratory 
analyses.  
 
Statistical computations will be performed using software from Cytel (StatXact and LogXact), SAS 
Institute (SAS version 9.2), and Salford systems (CART). 
 
Analysis Plan for Aim 1.  The longitudinal analysis of the effects of treatment on 24-hour 51CR-labeled PTR of 
RBCs will rely on a generalized linear mixed-effects model which expresses mean response as a function of 
treatment_regimen,  treatment_period, and occasion. The baseline PTR value will be treated as being one of 
the outcomes. The fitted model will be used to obtain parameter estimates that characterize the 24-hour 
trajectory of the mean response for each of the two regimens.  Graphical figures will be used to display the 
resulting point- and interval-estimates.   
 
Hypothesis testing.  An F-test procedure will be used to test the null hypothesis “the treatment effect is 
exactly zero in the target population”.   
 
Missing data. For this main analysis, all enrollees with at least one post-exchange transfusion 
evaluation will be included. We anticipate that the main analyses will rely on an assumption that missing data 
values, if any, are attributable to ignorable causes (i.e., the mechanisms satisfy the “missing at random” 
(MAR) criteria.)   
 
Sensitivity analyses.   After the main analysis has been conducted,  sensitivity analyses will be used to 
evaluate the robustness/fragility of the main results to reasonable perturbations of the statistical methods and 
assumptions used. Results of the sensitivity analyses will only be used to guide trust in the main results.  The 
sensitivity analyses will include, for example: diagnostics for goodness of fit and influential observations, and 
analysis of residuals for examination of distributional assumptions; evaluation of the impact of using 
alternative statistical procedures (for coping with missing values); investigation of the impact of including / 
excluding questionable data values.  As part of the sensitivity analyses, investigation of missing values will 
include use of logistic regression methods to investigate apparent causes for missing data, and multiple 
imputations methods.  The impact of using alternative assumptions regarding the variance covariance 
structure will also be included in the sensitivity analyses. The impact of using alternative distributional 
assumptions and transformations of scales will also be considered.  
 
Auxiliary analyses.  Longitudinal analyses of the PTR data spanning all 15 occasions per period will 
also be performed to characterize the trajectory of the mean response for each regimen over 4 months.  
 
 
Analysis Plan for Aim 2a. The longitudinal analysis of the effects of treatment on Hemoglobin A will rely on the 
same analysis strategy as described for Aim 1 for Estimation, Hypothesis testing, handling Missing data, and 
Sensitivity analyses.  
 
Analysis Plan for Aim 2b.  For each of the 26 outcome variables, a longitudinal analysis of the effects of 
treatment on mean response will rely on an analysis strategy similar to that described in Aim 1 for Estimation, 
handling Missing data, and Sensitivity analyses.   
Regardless of scale, the strategy for obtaining the main 
results is the same for each regimen and for the difference between the regimens, point estimates of central 

 
 
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tendency and variance will be obtained along with 95% confidence intervals to indicate precision. 
 
 
Analysis Plan for Aim 2c.  For the purpose of hypothesis generation, exploratory statistical methods 
(reference: Tukey, 1977) and exploratory predictive modeling methods will be used to investigate associations 
between treatment_regimen and longitudinal metabolomic profiles.   
 
Analysis Plan for Aim 2d.  For each of the outcome variables [ pain on an ordinal scale {0,1,2,… , 10},  
occurrence of infection symptoms, and use of antibiotics (type, duration, dose) ], a longitudinal analysis of the 
effects of treatment on mean response will rely on an analysis strategy similar to that described in Aim 1 for 
Estimation, handling Missing data, and Sensitivity analyses.   
 
9.7 Interim Analyses 
No interim analyses will be performed.  
9.8 Analysis of Safety Data 
Descriptive and tabular analyses of the safety data will be performed based on information from all patients 
having received at least one transfusion. The study will use the NCI CTCAE v4.0. 
9.9 Sample Size Rationale 
Given that the experimental design is a 2-treatment 2-period 2-sequence crossover,  the number of patients 
enrolled should be an even number;  thus, we will enroll N=16 participants and assign them depending upon 
RBC unit availability for the first exchange transfusion. The primary considerations in choosing N = 16 
enrollees as the target sample size included:  
o the anticipated frequency of missing data,  
o anticipated levels of precision of estimators,  
o anticipated levels of power of hypothesis tests,  
o costs and time requirements.   
Our assessment of anticipated levels of precision and power focused on Aim 1.  Those considerations were 
informed by a previous study79 of 24-hour posttransfusion RBC recovery (PTR %), in which the 
point-estimates and interval-estimates of the population standard deviation (σ) of PTR were as follows: 
 
Previous study79 
 
    Estimates of the mean               Estimates of σ 
 
n 
 estimate 
95%CI 
  estimate 
95%CI 
Normal donors 
27  
85.3% 
[84.1, 86.5]   
3.2% 
[2.5,   4.4]% 
G6PD-deficient 
10  
78.5% 
[73.2, 83.7]   
8.4% 
[5.8, 15.3]% 
 
Precision of estimators of the means in Aim 1.  The above table suggests conjectures about the magnitudes 
of standard errors (SE) of the means that can be anticipated for the proposed crossover study:   
  
Proposed crossover study (n=16)               SE of the mean = SD/(161/2) 
 
Expected  Plausible Range 
A:  units from normal donors 
0.8% 
[0.6, 1.1]% 
B:  units from G6PD-deficient donors 
2.1% 
[1.5, 3.8]% 
A vs B:  mean difference (if ρ=0.5)1 
1.8% 
[1.3, 3.4]% 
A vs B:  mean difference (if ρ=0.0)2 
2.2% 
[1.6, 4.0]% 
1  The SEs in the 3rd row correspond to SD = 7.3% and [5.0, 13.6]%. 
2  The SEs in the 4th row correspond to SD = 9.0% and [6.3,15.9]%. 
This table of SEs suggests that precision will be adequate for estimation of regimen-specific mean PTR% in 
the proposed crossover study of n=16 patients.  For example, the width of the 95%CI for the regimen-A mean 
is expected to be  ± 1.6%  (i.e., 1.96 standard errors). The width of the 95%CI for the regimen-B mean is 

 
 
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expected to be  ± 4.2%, approximately.  The third row in this table conservatively assumes that the correlation 
between a pair of PTR values is “moderate” (ρ=0.5). The fourth row’s assumption (ρ=0) is ultra-conservative.  
 
Power of a test procedure in Aim 1.  We consider a test of the null hypothesis that “The PTR(%) mean 
difference between regimens A and B is exactly zero in the target population of patients.”  If we assume that  
   (1) the mean absolute PTR difference is ∆=10% in the target population of current and future SCD patients, 
   (2) the SD of the differences in the target population is σdiff =7.3%  (or plausibly 5.0% ≤ σdiff ≤ 13.6%)    
   (3) the correlation between paired PTR measures (for A and B) is ρ = 0.50 in the target population,  and  
   (4) the sample size is n = 16 participants with complete data,   
then the chance of drawing a sample of patients that would yield a p-value smaller than α = 0.05 is 99%;  this 
power level drops to 78% if σdiff = 13.6%.   If ∆=5%  and  σdiff = 7.3%  in the target population, then the power 
level would be 72%;  this power level drops to 28% if σdiff = 13.6%.    
 
While the planned analysis strategy for Aim 1 will rely on a longitudinal linear mixed-effects model that 
accounts for period effects, the above estimates of anticipated precision and power are based on the 
assumptions of a simplistic model for paired 24hr PTR (%) outcomes. The resulting estimates are thus 
approximate but provide reasonable guidance about precision and power for the proposed study. 
    
In summary, our sample size analysis suggests that a target enrollment of N=16 participants is adequate to 
provide satisfactory levels of precision for the estimators of mean PTR(%) and is expected to easily provide a 
high level of power for a test of difference when the treatment effect is near 10% or larger  --even if a few 
participants do not have complete data.  The estimates suggest that a smaller sample size would not provide 
adequate precision for key estimators (i.e., the confidence intervals would be too wide).  
10 PLANS TO ENSURE DATA QUALITY 
Database System for Data Capture 
Data will be collected and entered into a web-based data management system (RedCAP). The lead coordinator 
will routinely verify that all data entry fields are entered. Verifications are question-by-question checks that give 
immediate feedback to help catch data entry errors, form completion errors, and out-of-range values. Reports 
of outstanding edits, generated upon completion of data entry, will enable continuous cleaning of data.  
 
Confidentiality 
Each subject is assigned a unique number to assure confidentiality. Any publication or presentation will refer to 
subjects by this number and not by name. The medical records department, affiliated with the institution where 
the subject receives medical care, maintains all original inpatient and outpatient chart documents. Subject 
research files will be kept in a locked room or locked cabinet.  
 
Data Management Plans 
The Principal Investigator will serve as the trial coordinator for this study. The PI or designee will monitor timely 
entry of data into the study database. Access to all source documentation maintained by the lead coordinator, 
including correspondence and source data, will be available for monitoring and audit purposes. 
 
 
 
 
 
 
 
 
 

 
 
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11 DATA AND SAFETY MONITORING PLAN (DSMP) 
Disposition of Participants 
 
1) The subject completes the 2 chromium infusions and associated lab draws  
2) The subject decides to withdraw from the study 
3) The subject moves away, dies, or is lost to follow-up 
4) Subjects may be removed from the study, or treatment stopped by the Investigator for any of the following 
reasons: 
• 
Occurrence of CTCAE Criteria of grade 3 or higher that are deemed attributable to the chromium 
infusion per the opinion of the PI 
• 
PI decides to withdraw the subject due to noncompliance 
• 
Withdrawal by the PI or subject because of transfusion side effects or complications 
 
 
Data and Safety Management Overview 
The UNC Safety Monitoring Committee (SMC) and the UNC Institutional Review Board (IRB) will approve 
protocol-specific DSM plans. A local, investigator-initiated trial will be required to be continuously monitored by 
the principal investigator of the study with annual safety and progress reports submitted to the SMC.  
11.1 Study Team 
The study team minimally consists of the principal investigator, the clinical research coordinator, regulatory 
specialist and the study biostatistician. While subjects are on treatment, the principal investigator will meet 
regularly with the research coordinator and the study biostatistician to review study status. This review will 
include but not be limited to reportable SAEs and update of the ongoing study summary that describes study 
progress in terms of the study schema. The appropriateness of further subject enrollment and the specific 
intervention for a next subject enrollment is addressed.   
11.2 Quality Assurance  
The UNC Clinical Trials Office provides ongoing quality assurance audits.  
11.3 SMC 
UNC places the highest priority on ensuring the safety of patients participating in clinical trials. Every clinical 
trial conducted at UNC includes a plan for safety and data monitoring. 
The SMC is an independent board appointed by the PI composed of three members. The principal role of the 
SMC is to regularly monitor the data from the clinical trial, review and assess the performance of its operations, 
and make recommendations, as appropriate, to the PI and IRB. 
 
The PI will be notified within 24 hours of learning of an event when a serious adverse event possibly, probably 
or definitely related to red cell transfusion or an event resulting in death (regardless of attribution) is reported. 
The PI will review the event as soon as the materials are available. The PI may request additional information 
regarding the event and may request the subject’s treatment arm assignment. Following the review, the PI will 
sign a log summarizing the event.  
 

 
 
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The SMC chairperson will receive information on all serious adverse events possibly, probably, or definitely 
related to red cell transfusion and all events resulting in death (regardless of attribution) within 7 days of the PI 
becoming aware of the event. The SMC chair will have expertise in benign hematology and transfusion 
medicine, will review the serious adverse event materials, and determine if the information is complete.  
 
The SMC members will meet annually via teleconference. All adverse events will be reviewed during the call, 
and the group will determine if additional SMC review is required, and make recommendations about the study. 
 
This study will be reviewed by  a Safety Monitoring Committee (SMC). A summary of the SMC activities are 
as follows:  
 
Review the clinical trial for data integrity and safety.  
 
Review all unexpected grade 3, and all grade 4, and 5 adverse events, as well as any others requiring 
expedited reporting as defined in this protocol. (Grades 4 and 5 events must be reported to the SMC 
within five calendar days of study staff’s knowledge.) 
 
Submit a summary of any recommendations related to study conduct.  
 
Terminate the study if deemed unsafe for patients.  
A copy of the UNC Data and Safety Monitoring Plan and membership roster will be maintained in the study 
research file and updated as membership changes. The committee will review reports from the study principal 
investigator annually (or more frequently if needed) and provide recommendations on trial continuation, 
suspension or termination as necessary.  
For this pilot study, the site-PI will appoint three members to a Safety Monitoring Committee (SMC). These will 
be investigators from institutions with expertise in transfusion medicine, SCD, and biostatistics. They will be 
charged with monitoring the accruing data to confirm that the patients in the trial are being cared for safely.  
 
The SMC will meet at least once per year, and will be responsible for:  
1) reviewing and analyzing the progress of the study  
2) approving amendments to the trial protocol 
3) monitoring the safety of the study treatments and diagnostic procedures 
4) ensuring data quality 
5) reviewing interim analyses and recommending early stopping or continuation of the trial 
6) reviewing recruitment and event rates.  
 
The data safety monitoring plan will ensure that the site is in compliance with Federal regulations, Good Clinical 
Practice and Good Manufacturing Practice Guidelines, as applicable. 
 
The SMC will meet at least once per year, either in-person or via teleconference. Prior to the 
meeting, the site-PI will provide the SMC with: 
• Accrual totals 
• Adverse event log reports for each study subject 
• Serious adverse event reports for each study subject 
• Study compliance issues 
• The SMC will also be provided each subject’s study arm assignment by the primary site biostatistician. 
 
The SMC will determine: 
• All-cause mortality 
• Number, type, and severity of serious adverse events 
• Number of subjects in each treatment arm with at least one clinically documented pain crisis leading to an ED 
or hospital admission 

 
 
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• Unexpected adverse events and unanticipated problems 
• Serious infection or pain admissions 
 
For the sessions, results will be overall and by RBC unit group. 
 
Any available SMC letters will be submitted to the IRB of record as required. 
12 REGULATORY COMPLIANCE, ETHICS AND STUDY MANAGEMENT 
12.1 Ethical Standard 
This study will be conducted in accordance with the ethical principles that have their origin in the Declaration 
of Helsinki as stated in 21 CFR §312.120(c)(4); consistent with GCP and all applicable regulatory 
requirements. 
12.2 Regulatory Compliance 
This study will be conducted in compliance with: 
 
The protocol 
 
Federal regulations, as applicable, including: 21 CFR 50 (Protection of Human Subjects/Informed 
Consent); 21 CFR 56 (Institutional Review Boards) and §312 (Investigational New Drug Application; 
and 45 CFR 46 Subparts A (Common Rule), B (Pregnant Women, Human Fetuses and Neonates), C 
(Prisoners), and D (Children), GCP/ICH guidelines, and all applicable regulatory requirements. The 
IRB must comply with the regulations in 21 CFR §56 and applicable regulatory requirements. 
12.3 Prestudy Documentation  
Prior to implementing this protocol at UNC, the protocol, informed consent form, HIPAA authorization and any 
other information pertaining to participants must be approved by the UNC IRB.  
13.4  Institutional Review Board 
The protocol, the proposed informed consent form and all forms of participant information related to the study 
(e.g., advertisements used to recruit participants) will be reviewed and approved by the MCW Institutional 
Review Board. Prior to obtaining MCW approval. The initial protocol and all protocol amendments must be 
approved by the IRB prior to implementation.  
Informed Consent Process 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in the study and 
continues throughout the individual’s study participation. Extensive discussion of risks and possible benefits of 
this therapy will be provided to the subjects and their families. Consent forms describing in detail the study 
interventions/products, study procedures and risks are given to the subject and written documentation of 
informed consent is required prior to starting intervention/administering study product. 
Consent forms will be IRB-approved and the subject (and legally authorized representative, if necessary) will 
be asked to read and review the document. Upon reviewing the document, the investigator will explain the 
research study to the subject and answer any questions that may arise. In accordance with 46 CR 46.111, the 
subject will sign and date the informed consent document prior to any procedures being done specifically for 
the study.  

 
 
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A witness should only sign when required, per UNC IRB policy. If a witness signs the document when not 
required, the study staff should document in the legal medical record (or note to file) the relationship to the 
patient and why a witness signed. (i.e., “Although not required, the subject’s spouse was present during the 
consenting process and signed as the witness.” Or “Although not required, hospital staff was present for 
consenting process and signed as a witness.”) 
The subjects will have the opportunity to discuss the study with their surrogates or think about it prior to 
agreeing to participate. The subjects may withdraw consent at any time throughout the course of the trial.  
A copy of the informed consent document will be given to the subjects for their records. The rights and welfare 
of the subjects will be protected by emphasizing to them that the quality of their medical care will not be 
adversely affected if they decline to participate in this study. If there are changes to the consent form, all 
revisions will be reviewed with study subject at the next appropriate opportunity. Patients who require 
reconsenting will be defined in the IRB approved amendment submission. The process for obtaining informed 
consent will again be performed. Study subjects will not be reconsented for continuing reviews. The UNC 
research staff will follow the UNC IRB’s policy for subjects who demonstrate limited English proficiency or 
limited literacy.  
After the subject’s visit in which the consent is signed, it is documented in the clinic chart that the consent has 
been signed and that all questions have been answered to the subject’s satisfaction after adequate time for 
review of the consent. It is also documented that a copy of the consent is given to the subject. The original 
consent is kept with the subject’s study file, and a copy of the consent is sent to the OCRICC office, which will 
then submit to HIM a copy of the signed consent to be scanned into EPIC, the legal medical record. 
13.5  Subject Confidentiality and Access to Source Documents/Data 
Subject confidentiality is strictly held in trust by the sponsor/sponsor-investigator, participating investigators, 
and any staff. This confidentiality includes the clinical information relating to participating subjects, as well as 
any genetic or biological testing. 
The study protocol, documentation, data and all other information generated will be held in strict confidence. 
No information concerning the study or the data will be released to any unauthorized third party without prior 
written approval of the principal investigator. 
The conditions for maintaining confidentiality of the subjects’ records are required for the life of the data.  
One risk of taking part in a research study is that more people will handle the personal health information 
collected for this study. The study team will make every effort to protect the information and keep it 
confidential, but it is possible that an unauthorized person might see it. Depending on the kind of information 
being collected, it might be used in a way that could embarrass the subject or affect his/her ability to get 
insurance. 
While data are being collected and after all data have been collected but are still in the process of being 
analyzed, the subject’s data/PHI are stored in the locked UNC Clinical Research Office. Databases in which 
the study subject information is stored and accessed are password protected, allowing for limited access by 
authorized personnel only. Data/PHI kept in the case report forms contain the study identifiers, subject initials, 
date of birth and date of service.  
Personal identifiers, such as name and medical record number, will be removed from accompanying lab 
reports and test results. Any data/PHI that are not stored for the purposes of the study are shredded in the 
Clinical Trials Office. 
After all study queries and analyses are completed, the data/PHI will not be destroyed but will be archived  in 
a secure long-term storage site in order to keep an accurate record of screened and enrolled subjects for the 
sponsor and potential audit purposes only specific for this study.  

 
 
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The Investigator will maintain the signed Informed Consent Forms, CRFs, study documentation and source 
documents for at least 10 years after study completion or termination per UNC Institutional policy. In addition, 
the Investigator will not discard or destroy any study-specific materials unless otherwise instructed. 
 
The principal investigator will allow access to all source data and documents for the purposes of monitoring, 
audits, IRB review and regulatory inspections.  
13.6  Protection of Human Subjects 
13.6.1  Protection from Unnecessary Harm 
The PI and study personnel are responsible for protecting all subjects involved in human experimentation. 
This is accomplished through the IRB mechanism and the informed consent process. The IRB reviews all 
proposed studies involving human experimentation and ensures that the subject’s rights and welfare are 
protected and that the potential benefits and/or the importance of the knowledge to be gained outweigh the 
risks to the individual. The IRB also reviews the informed consent document associated with each study in 
order to ensure that the consent document accurately and clearly communicates the nature of the research to 
be done and its associated risks and benefits. 
13.6.2  Protection of Privacy 
As noted, patients will be informed of the extent to which their confidential health information generated from 
this study may be used for research purposes. Following this discussion, they will be asked to sign informed 
consent documents. The original signed document will become part of the patient’s medical records, and each 
patient will receive a copy of the signed document.  
13.7  Changes in the Protocol 
Once the protocol has been approved by the UNC IRB, any changes to the protocol must be documented in 
the form of an amendment. The amendment must be signed by the investigator and approved by IRB prior to 
implementation.  
If it becomes necessary to alter the protocol to eliminate an immediate hazard to patients, an amendment may 
be implemented prior to IRB approval. In this circumstance, however, the investigator must then notify the IRB 
in writing within five working days after implementation.  
The IRB may provide, if applicable regulatory authority(ies) permit, expedited review and approval/favorable 
opinion for minor change(s) in ongoing studies that have the approval /favorable opinion of the IRB. The 
investigator will submit all protocol modifications to the sponsor and the regulatory authority(ies) in 
accordance with the governing regulations. 
Changes to the protocol may require approval from the sponsor. 
Any departures from the protocol must be fully documented in the source documents. 
13.8  Investigator Compliance  
The investigator will conduct the study in compliance with the protocol given approval/favorable opinion by the 
IRB and the appropriate regulatory authority(ies).  
Onsite Audits 
Auditing is essential to ensure that research conducted at the UNC is of the highest quality and meets UNC 
and regulatory agency standards.  

 
 
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Regulatory authorities, the IRB may request access to all source documents, data capture records and other 
study documentation for on-site audit or inspection. Direct access to these documents must be guaranteed by 
the investigator, who must provide support at all times for these activities. 
13 DATA HANDLING AND RECORD KEEPING 
Case Report Forms  
 
Case Report Forms (CRFs) will be used to collect all subject data during the course of the study. Data from 
these forms will be entered into the study EDC. The Principal Investigator or predetermined designated 
individual shall be responsible for completion of the CRFs. All protocol deviations shall be documented and a 
justification for any missed assessments shall be provided on the protocol deviation log. Completed CRFs will 
be verified at regular intervals throughout the study by the principal investigator.  
 
 
Record Retention  
 
The Investigator will maintain the signed Informed Consent Forms, CRFs, study documentation (listed above) 
and source documents for at least 10 years per after study completion or termination per UNC Institutional 
policy. In addition, the Investigator will not discard or destroy any study-specific materials unless otherwise 
instructed. 
 
13.1 Overview 
Every effort is made to uphold the integrity of the project, the research, the institution and the researchers 
involved. Data collection guidelines and methodologies are carefully developed before the research begins. 
Investigators focus on the following to ensure data integrity:  well-trained data collectors/recorders to ensure 
consistency and quality, well-designed data collection protocols and ongoing monitoring. In this way, study 
rigor and validity are maintained. Data is protected from physical damage as well as from tampering, loss or 
theft. This project’s data management is a multidisciplinary activity that includes investigators, research 
coordinators and nurses, data mangers, support personnel, biostatisticians and database programmers. 
Quality control will be applied to each stage of data handling to ensure that all data are reliable and have been 
processed correctly.  
14.2  Data Management Responsibilities 
14.2.1  Principal Investigator 
The principal investigator oversees the management of patient records/case report forms and ensures that a) 
complete and accurate data will be obtained and provided to the sponsor; b) patient records are maintained to 
include history, prescribed medication and investigational product(s), measurements, exams, evaluations and 
adverse events; c) corrections are applied to clinical research data according to principles of good research 
practice (i.e., single-line delete, date and initial). He or she will ensure that there is correlation between the 
case report forms and the source documents.  
14.2.2  Research Coordinator  
A research coordinator creates, collects and organizes clinical trial documentation. He or she ensures that 
source documentation and data abstraction and entry are being done at protocol specified time points. 
 
14.2.3  Research Nurse/Medical Staff 
The research nurse and medical staff document protocol-required care or assessment of the subject’s 
outcomes, adverse events and compliance to study procedures. 
14.2.4  Biostatistician 

 
 
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The biostatistician may assist in CRF development (content and design), dataset specifications (annotation of 
CRFs and record layout) and validation. The study biostatisican will all perform all statistical computations 
associated with Aims 1 and 2a-d and be involved in manuscript preparation from this data. 
14.4  Source Documents 
Good Manufacturing Practice Guidelines require that investigators maintain information in the subject's medical 
records, laboratory reports, clinic charts, etc. that corroborate data recorded on the CRFs. In order to comply 
with these requirements, the following information should be maintained:  
• 
Medical history/physical condition of the subject before enrollment sufficient to verify protocol entry 
criteria  
• 
Dated and signed notes for specific results of procedures and exams  
• 
Good Manufacturing Practices require the nuclear pharmacist (or designee) to record date, time, and 
responsible person completing all critical steps in the manufacturing processing, including but not limited 
to the receipt, preparation, processing/manufacturing, labeling, distribution, and infusion of the 
radiolabeled red blood cell product.  This should include records of accountability and chain of identity / 
chain of custody for the radiolabeled product.   
All source documents will be written following ALCOA standards:  
ALCOA Attribute 
Definition 
Attributable 
Clear who has documented the data. 
Legible 
Readable and signatures identifiable. 
Contemporaneous 
Documented in the correct time frame along with the flow of events. 
If a clinical observation cannot be entered when made, chronology 
should be recorded. Acceptable amount of delay should be defined 
and justified. 
Original  
Original, if not original should be exact copy; the first record made 
by the appropriate person. The investigator should have the original 
source document. 
Accurate 
Accurate, consistent and real representation of facts. 
Enduring 
Long-lasting and durable. 
Available and 
accessible 
Easily available for review by treating physicians and during 
audits/inspections. The documents should be retrievable in 
reasonable time. 
Complete 
Complete until that point in time. 
Consistent 
Demonstrate the required attributes consistently. 
Credible 
Based on real and reliable facts. 
Corroborated 
Data should be backed up by evidence. 
 
14.5  Case Report Forms  
The principal investigator and/or his/her designee will prepare and maintain adequate and accurate participant 
case histories with observations and data pertinent to the study. Study-specific case report forms (CRFs) will 
document safety and treatment outcomes for safety monitoring and data analysis. All study data will be 
entered into the EDC via standardized CRFs, in accordance with the study calendar, using single data entry 
with a secure access account. The clinical research coordinator will complete the CRFs as soon as possible 
upon completion of the study visit; the investigator will review and approve the completed CRFs.  
The information collected on CRFs shall be identical to that appearing in original source documents. Source 
documents will be found in the patient’s medical records maintained by UNC personnel. All source 
documentation should be kept in separate research folders for each patient. 

 
 
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In accordance with federal regulations, the investigator is responsible for the accuracy and authenticity of all 
clinical and laboratory data entered in CRFs. The principal investigator will approve all completed CRFs to 
attest that the information contained on the CRFs is true and accurate.  
All source documentation and data will be available for review/monitoring by the SMC and regulatory 
agencies. 
14.6  Study Record Retention 
Source documents will be maintained for 10 years after data analysis has been completed and the study in 
closed. After which all materials will be destroyed. 
The principal investigator is required to maintain adequate records of the disposition of the drug, including 
dates, quantity and use by subjects, as well as written records of the disposition of the drug when the study 
ends.  
The principal investigator is required to prepare and maintain adequate and accurate case histories that 
record all observations and other data pertinent to the investigation on each individual administered the 
investigational drug or employed as a control in the investigation. Case histories include the case report forms 
and supporting data including, for example, signed and dated consent forms and medical records including, 
for example, progress notes of the physician, the individual's hospital chart(s), and the nurses' notes. The 
case history for each individual shall document that informed consent was obtained prior to participation in the 
study. 
Study documentation includes all CRFs, data correction forms or queries, source documents, and regulatory 
documents (e.g., protocol and amendments, IRB correspondence and approval, signed patient consent 
forms). 
Source documents include all recordings of observations or notations of clinical activities and all reports and 
records necessary for the evaluation and reconstruction of the clinical research study. 
14.7  Publishing Data 
The UNC PI will possess the data and be responsible for publishing. The University of Columbia will have 
access to the data and may also utilize the data for publishing with the approval of the UNC’s PI. 
 
 

 
 
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