Age of Blood in Sickle Cell Transfusion -The Effects of Phosphotidylserine Expression on Older Red Cell Units in Adults With Sickle Cell Disease
Phase: PHASE2
Conditions: Sickle Cell Disease
Sponsor: University of North Carolina, Chapel Hill

 
The Effects of Phosphatidylserine Expression  
on Older Red Cell Units in Adults with Sickle Cell Disease 
 
 
 
NCT number NCT03704922 
Document Date 10/10/2023 
 

1 
 
 
 
 
 
 
 
 
 
The Effects of Phosphatidylserine Expression  
on Older Red Cell Units in Adults with Sickle Cell Disease 
 
Protocol Version 11 –  Oct 10, 2023 
 
Study Sponsor: NHLBI 
 
Principle Investigator: Matthew S Karafin, MD, MS 
Clinical co-investigators: Jane Little, MD, Ross Fasano, MD  
 
 
Conducted at the University of North Carolina Sickle Cell Clinic,  
 And Emory University 
 
A randomized trial to determine if there is a mechanistic 
or clinically important difference between the PS-high vs. PS-low units 
on patient immune systems & clinical outcomes 
 
 
 

2 
 
Table of Contents 
 
X. Synopsis and Study Schema -  
 
 
 
1 
1. BACKGROUND AND SIGNIFICANCE -  
 
 
6 
2. OBJECTIVES -   
 
 
 
 
 
9 
3. STUDY POPULATION -  
 
 
 
 
11 
4. TRIAL ENROLLMENT -  
 
 
 
 
11 
5. INTERVENTIONS -  
 
 
 
 
 
13 
6. SCHEDULE OF MEASUREMENTS -  
 
 
14 
7. SPECIMEN COLLECTION PROCEDURES -  
 
18 
8. ADVERSE EVENT CRITERIA AND REPORTING -  
18 
9. SAFETY MONITORING AND STOPPING GUIDELINES - 22 
10. STATISTICAL CONSIDERATIONS -  
 
 
22 
11. DATA COLLECTION AND VALIDATION -  
 
25 
12. PROTECTION OF HUMAN SUBJECTS -   
 
25 
13. INVESTIGATOR RESPONSIBILITY -  
 
 
25 
14. MONITORING AND QUALITY CONTROL -  
 
26 
15. RECORDS AND REPORTS -  
 
 
 
26 
16. REFERENCES -  
 
 
 
 
 
28 
 
 

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Quick List of Abbreviations: 
   RBC – Red blood cell 
   SCD – Sickle cell disease 
   PS – Phosphatidylserine 
   PE – Phosphatidylethanolamine 
   NTBI – non transferrin bound iron 
   MFI – mean fluorescence intensity 
 
Concept Synopsis and Study Schema  
The desired impact of red blood cell (RBC) transfusion in patients with sickle cell 
disease (SCD) is to reduce the burden of patient sickled red cells with the goal of 
reducing stroke risk and reducing the burden of frequent vaso-occlusive pain events 
that is otherwise refractory to other treatment modalities. However, controversy exists 
over transfusions with blood banked for short vs. long storage intervals. Pre-clinical 
animal studies and observational human models suggest that increased 
phosphatidylserine exposure (PS), seen often from red cells units with longer storage 
intervals, may lead to downstream immune effects that have detrimental consequences, 
such as increased clinical infections. No randomized prospective human study has 
attempted to correlate age of transfused RBCs with PS-exposure, and then evaluate 
how this exposure modulates patient immune systems and subsequent clinical 
outcomes. 
 
Primary Hypothesis:  
There is a clinically significant difference between the effect of PS-high RBCs vs. PS-
low RBCs on patient macrophages.  
 
Study Schema: 
 
Trial design: Adults and adolescents (post puberty)  with SCD who are treated with 
outpatient red cell transfusions will be randomized in blocks of 5 (S. Piantadosi block 
randomization software, 2010) to receive ≥30 day or ≤10 day old units for 3 months (3 
consecutive transfusion events). The study statistician will provide the blood bank with 
the schema who will then be responsible for providing the units for these subjects. 
Participants and all other investigators will be blinded to treatment assignment.  
 
Two baseline venous blood samples will be obtained to be used as a baseline 
measurement of the patients underlying inflammatory status. The first will be drawn about 
1- week prior to the first randomized transfusion, and the second sample just prior to the 
first randomized transfusion. Subjects will be asked to hold their iron chelation 72 hours 
before the study transfusion.  Pre-transfusion, sterile samples will be extracted from red 
cell units. Patient venous blood samples will be also obtained 2 hours post transfusion, 
24 hours post-transfusion, and 2 weeks post-transfusion (Table 1). Participants will 
complete standardized diaries daily to document symptoms of infection, SCD pain, 
medications and emergency department (ED) or hospital use. Diaries will be collected 
and participants will be assessed with each transfusion encounter, up to 3 transfusion 
events, and 4 weeks after the last transfusion. To ensure compliance, coordinators will 

4 
 
contact subjects regularly to remind them to complete diaries. All data will be captured 
with standardized case report forms and entered into an electronic database. 
 
Subject Recruitment and Anticipated Numbers: We intend to recruit and enroll 40 
subjects. Participants will be recruited from the Adult SCD Clinic at UNC and 
Emory/Grady Health System. We expect that UNC will recruit between 5-10 subjects over 
the next 2 years (15 subjects have already been enrolled at Medical College of 
Wisconsin), and Emory will recruit between 10-20 subjects to reach the study goal of 40 
randomized subjects.  
 
Subject Inclusion/exclusion criteria:  
 
Inclusion criteria:  
1) Ages 16 to 60 years old 
2) Subject must have sickle cell anemia confirmed by hemoglobin analysis 
3) Must be receiving red cell transfusion therapy 
4) Must be outpatient at the time of study transfusions  
 
Exclusion criteria:  
1) History of severe reactions to transfusion therapy 
2) Receive red cell transfusions that are crossmatch incompatible 
3) Current participation in another therapeutic trial for sickle cell disease 
4) Current pregnancy  
5) History of HIV infection 
6) Having an uncontrolled inter-current illness, or psychiatric illness/social situations that 
would limit compliance with study requirements as determined by the principal 
investigator. 
 
Study procedures:  
 
Red cell unit preparation: Red cell units for transfusion will be crossmatch-compatible, 
sickle-negative, and phenotype-matched for D, C, E, and K red cell antigens per current 
hospital protocols. Red cell unit age will be documented by the blood bank at the time of 
randomization. Study transfusions will take place only in the outpatient infusion clinic.  
 
Pain and infection diary: The diary is adapted from a model widely used in SCD studies. 
Daily, participants will rate pain on a numeric ordinal pain rating 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.  
 
Physical exam: A basic, focused, physical exam will be performed on each subject by 
the PI or trained delegate prior or during each transfusion and prior to the completion of 
the study. The physical exam will assess a general review of systems, a review of the 
pre-transfusion vital signs, and a basic evaluation of patient general status, heart, lung, 

5 
 
and neurological organ systems. We will also document if the patient has an indwelling 
catheter. 
 
Blood measurements:  
Unit samples: Red cell characteristics: 2 ml blood from each red cell unit will be obtained 
from 6 sterile unit links pre-transfusion. RBC PE and PS surface exposure, and plasma 
microparticles will be quantified by flow cytometry using the LSR2-green, and all flow 
analyses will be recorded using FACSDiVa software. The plasma fraction from each unit 
sample will be diverted to EPR (Bruker Elexys X-band system, Billerica, MA, USA), a Bio-
Plex cytokine assay kit (Bio-Rad Laboratories), and a fluorescent immunoassay (DxFerr 
kit) to measure concentrations of free heme/hemoglobin, cytokines (IL-1, IL-6, IL-8, MCP-
1), and NTBI, respectively. A small sample will also be frozen for future metabolomic 
analysis. 
 
 
Patient Samples: 10-30 ml per lab draw of whole blood will be obtained for each blood 
draw as defined in Table 1, and will be stored per tube storage requirements. . Blood will 
be obtained by port or peripheral access as available. Patient white cells will be isolated 
with established Ficoll separation techniques.. White cells will be tested by flow cytometry 
as above for key markers of activation (macrophages: CD64, CD62L, CD80, CD86, Tim1, 
neutrophils: The plasma fraction from each sample will be diverted to EPR (Bruker Elexys 
X-band system, Billerica, MA, USA), a Bio-Plex cytokine assay kit (Bio-Rad Laboratories), 
and a fluorescent immunoassay (DxFerr kit) to measure concentrations of free 
heme/hemoglobin, cytokines (IL-1, IL-6, IL-8, MCP-1), and NTBI, respectively. Clinical 
measurements obtained from patients will also include: CBC (4ml EDTA), reticulocyte 
count (4ml EDTA), HbS% (3ml EDTA), haptoglobin (1ml red top), chemistry labs (ferritin 
(0.5ml red top), iron saturation (1ml red top), lactate dehydrogenase (1ml red top), high 
sensitivity CRP (HS-CRP, 1ml red top)), urinalysis (12 ml) and blood culture (8-
10ml/bottle). These clinical labs will be measured at intervals specifically defined in Table 
1. Individual human samples of plasma and RBC will also be frozen and sent to Angelo 
D’Alessandro at the University of Colorado Denver to measure lipid metabolites and 
metabolomic profiles pre vs. post transfusion as an additional exploratory goal of this trial. 
The laboratory of Dr. Steven Spitalnik and his representatives will be receiving 30-200 
individual samples of plasma and RBCs for this purpose. All samples will be de-identified.  
 
 
 
 
 
 
 
 
 
 
 
 

6 
 
1. BACKGROUND AND SIGNIFICANCE 
 
Sickle cell disease (SCD) is an autosomal recessive disorder that affects 1 of every 
400 African-American newborns in the United States.42,43 Abnormal hemoglobin, due to 
a mutation in the sixth codon of the β-globin gene, is the basis for SCD.44 Under hypoxic 
conditions, the abnormal hemoglobin polymerizes, causing red blood cells to become 
sickle-shaped and unable to transverse the microvasculature.44 Occlusion of small 
vessels results in tissue ischemia, infarcts and organ damage.44,45 Commonly affected 
organs include the brain, lungs and bone, which result in stroke, acute chest syndrome 
and pain, respectively.45 Red cell transfusion is the most critical therapy to treat and 
prevent complications of SCD.1 
 
Adults with SCD depend on red cell transfusion to prevent stroke and critical 
illness. Red cell transfusion corrects anemia, decreases sickle hemoglobin (HbS) 
percent, suppresses HbS synthesis, and reduces red cell hemolysis.1,44,45 These 
beneficial effects are used to successfully manage multiple complications from SCD, 
including severe anemia, stroke, acute chest syndrome and multi-organ failure.1,44-55 
When administered on a chronic basis, typically monthly, transfusion decreases rate of 
cerebral infarcts by up to 45%51 and vaso-occlusive events by up to 50%.53-57 Other than 
transfusion, hydroxyurea is the only medication available for SCD.1,58 Although 
hydroxyurea decreases rate of vaso-occlusion and prolongs life, the drug is not indicated 
for stroke prevention and is ineffective in 50% of patients.59-64 Since red cell transfusion 
remains the cornerstone of therapy for this population, provision of the safest units 
possible is critical. 
 
Changes occur in the physiology of red cell units with prolonged storage 
that may trigger a cascade of deleterious events in the transfusion recipient: 
macrophage activation, inflammation and release of toxic iron species (Figure 1). 
Red cells preserved with Adsol solution can be stored up to 42 days,65 a recommendation 
based on integrity, not red cell efficacy or safety. Significant oxidative and metabolic 
changes occur with storage: hemolysis with release of cell-free hemoglobin, heme, and 
non-transferrin bound iron (NTBI), as well as alterations to the lipid composition of the red 
cell membrane.6-15 Surface exposure of phosphatidylserine (PS), a phospholipid normally 
found on the inner leaflet of the red cell membrane but which becomes externally exposed 
with storage, may be the most important of these changes.23-27 Macrophages recognize 
PS as an “eat me” signal via specific receptors (Tim1, Tim4, and Stabilin-2) and rapidly 
phagocytize these red cells.66-71 In pre-clinical studies, phagocytosis of old red cells 
results in activation and secretion of pro-inflammatory cytokines, such as IL-1, IL-6, IL-8, 
CXCL-1 and monocyte chemoattractant protein-1 (MCP-1).19,28 Phagocytosis of a bolus 
of old red cells also overwhelm the macrophage’s metabolic capacity, leading them to 
release cell-free hemoglobin, heme and NTBI with downstream effects.21,22,29-31 NTBI has 
been associated with fatal proliferation of ferrophilic bacteria and the generation of toxic 
reactive oxygen species.19-22,37-39 Of note, these effects on macrophages are only seen 
after the transfusion of older intact red cells, not from the products of lysed red cells, which 
suggests that phagocytosis of red cells is required to generate this pathophysiology.29 

7 
 
Although some patients may be able to tolerate the aberrant physiology of older red cells, 
others may be at risk of poor outcomes. 
 
Adults with SCD may be uniquely susceptible to the deranged physiology of 
older units because they are prone to infections, inflamed and poorly equipped to 
handle excess red cells with high PS. Vaso-occlusion-induced tissue damage activates 
macrophages and leukocytes, which in turn leads to a chronic pro-inflammatory state.72-
76 Reperfusion injury further exacerbates inflammation with the generation of reactive 
oxygen species as well as additional endothelial and leukocyte activation.77,78 Activated 
macrophages, leukocytes and endothelium then promote more vaso-occlusion in what 
becomes a vicious cycle of ischemia, injury and inflammation.78 Intravascular hemolysis 
with release of cell-free hemoglobin and heme also contributes to this pathophysiology 
as cell-free hemoglobin is a sink for nitric oxide, a potent vasodilator and platelet 
antagonist,32-34,78 and heme activates endothelial cells via TLR4 receptors.35,36 Prior to 
penicillin prophylaxis, infection was the primary cause of death in patients with SCD due 
to splenic dysfunction.79-81 The cumulative result of SCD’s pathology is an adult who is 
prone to infections, chronically inflamed and susceptible to the pathology of PS-high, 
older red cells, and the consequent increased levels of cell-free hemoglobin, heme and 
NTBI in circulation. In fact, on sickle erythrocytes (not transfused cells) PS exposure is 
well-known to cause adhesion to the endothelium and thrombin generation, both of which 
worsen the pathology of vaso-occlusion.82,83 Since older red cell units with increased PS 
exposure can promote a similar physiology as SCD, they may exacerbate the illness of 
these vulnerable patients. 
 
No prospective study has evaluated the effects of an older unit with high PS 
exposure in an adult with SCD. There has only been one recently published prospective 
study in patients with SCD to examine the effects of age of blood.84 This was a 
randomized trial, conducted in Africa, of young children with either SCD or malaria 
transfused with > 25 day-old units versus < 10 day-old units to treat severe anemia (mean 
Hgb 3.7g/dl) with evidence of lactic acidosis. Although the trial found no difference 
between the two arms in the primary outcome measure, blood lactate levels, the results 
do not address the question of the current proposal: do PS-high, older units trigger a 
pathobiology in chronically transfused adults with SCD that leads to infection? Differences 
in unit physiology, patient inflammation, infection rates, and pain were not examined in 
the prior study, which is why the current study needs to be conducted. Other than that 
one prospective study, the only data about the effects of older units in SCD comes from 
three observational cohort studies, all of which showed an adverse outcome (increased 
red cell alloimmunization and decreased vascular dilatation) associated with older 
units.85-87  
 
The effects of the older units have been equally inconsistent in other patient 
populations, mostly trauma and surgery. While multiple observational studies have 
associated receipt of old units with poor outcomes, including infection and death,88-90 
recent randomized prospective studies did not show a difference in mortality.91,92 
Limitations of these other previous studies were a small difference in age between older 
and younger units, as well as the fact that no evaluation was performed of the unit’s 

8 
 
physiology. Not all older units show the same degree of pathology.66 Most pertinent to our 
study, however, is that the patients were not adults with SCD. Organ dysfunction, 
inflammation, hemolysis, along with a dependence on blood to prevent critical illnesses, 
creates a situation in adults with SCD that is different from other patient populations, 
including children with SCD, and makes studies in other populations difficult to apply. To 
address a critical gap in knowledge, we propose for the first time, a prospective, 
randomized study to elucidate the immunological and clinical effects of an older 
unit with high PS exposure on adults with SCD. 
 
Preliminary Studies 
 
In Milwaukee, 1/3 of units transfused to adults with SCD are >30 days old, 
but nation-wide some restrict the transfusion of older units. Four hundred adults 
receive care in the Adult Sickle Cell Clinic at UNC in Chapel Hill, 47 of who receive chronic 
outpatient transfusions for chronic pain or stroke prophylaxis. Transfusions are 
administered to adults regardless of storage age. In a 3-year retrospective review of 
transfusions administered to adults with SCD, 896 units were transfused over 407 simple 
transfusion encounters. Median storage age was 23 days (range: 2-42 days); 32% of 
units provided were ≥ 30 days-old (Karafin et al., 2015).41 To evaluate the opinions and 
policies of other hospital blood bank directors about the use of older red cell units for 
patients with SCD, we conducted a nation-wide survey (n=90). While only 23% of 
respondents had a storage age restriction policy for patients with SCD, 31% thought that 
older units were not as effective as younger units, and 65% believed that evidence-based 
policies were needed (Karafin et al., 2015).40 These data from our institution and across 
the US demonstrate equipoise among blood bank directors about the potential harm of 
older units for patients with SCD. At UNC, we have determined that the average unit age 
transfused is 18 days, with a standard deviation of 10 days (range: 5 days- 39 days). 
 
Transfusion of older units to adults with SCD stimulates expansion and 
activation of macrophages. Adults with SCD may be susceptible to the effects of an 
older unit’s physiology because they are prone to infection, chronically inflamed, and 
poorly equipped to handle excess iron. In a cohort of 40 steady-state adults with SCD, 
we specifically measured markers of inflammation and iron excess. High sensitivity C-
reactive protein, a marker of global inflammation, was found to be markedly elevated 
(median 5.6 mg/L, range 0.4-60 mg/L; reference range <1.0 mg/L), as was ferritin, a 
marker of iron stores (median 2,969 ng/ml, range 20-12,300 ng/ml; reference range 13-
400 ng/ml) (Karafin et al., 2015).93 
 
To characterize whether older units would exacerbate the adult’s pro-inflammatory 
physiology, we next examined activation of macrophages, neutrophils, B and T cells in 
the peripheral blood of 8 adults with SCD before and after transfusion and 8 adults with 
SCD who do not receive transfusion (ages 19-35). All transfused units were greater than 
14 days old (range, 14-36 days). In support of our hypothesis, we identified that absolute 
macrophage and neutrophil numbers were higher post-transfusion compared to pre 
transfusion in 5/8 (63%) and 6/8 (75%) subjects, respectively. There was also evidence 
of macrophage activation post-transfusion (Figure 2). Among transfused subjects, 

9 
 
macrophages increased in size in 7/8 (88%) subjects, and showed greater expression of 
CD64+ (12% increase) and CD62L (34% increase) as measured by mean fluorescence 
intensity (MFI) 24 hrs post-transfusion as compared to pre-transfusion values.  
 
 
Transfusion of older red cell units to adults with SCD increases rate of 
infection. To explore whether the pro-inflammatory state stimulated by older units was 
associated with infection risk, as has been shown in pre-clinical models, we examined 29 
adults with SCD who were treated with a chronic transfusion program. A random effects 
model, which accounts for a subject’s contribution to repeat encounters, was used to 
define the association between infection and storage age. This model revealed that a 
mean unit age > 25 days old was associated with a significant increase in ED or hospital 
admissions for infection (p=0.03). After receipt of older units, adults developed 
pneumonia (n=6), urinary tract/kidney infections (n=2), gynecologic infections (n=2), an 
ear infection (n=1), a wound infection (n=1), and sepsis (n=1). Eight (62%) of these 
infections contributed to a subsequent inpatient admission (Karafin et al., submitted for 
publication). 
 
Our preliminary data demonstrates the need for a prospective, randomized trial to 
characterize the effects of an older unit’s physiology on adults with SCD. Because little 
data exist, blood bank directors across the country have varied policies about the 
provision of older units to adults with SCD. At our institution, adults are frequently 
transfused with older units, which we have shown activate macrophages and, ultimately, 
promote infection. 
 
Summary: Finding ways to maximize the safety and efficacy of red cell transfusion 
therapy for patients with SCD is desperately needed. Our group has data suggesting that 
older units of stored red cells contribute to immune activation and the increased risk of 
infection. If found to be effective, restricting transfusions to PS-low, younger units of blood 
in this population has the potential to improve patient outcomes and significantly alter the 
field of transfusion medicine. 
 
2. OBJECTIVES 
 
2.1: Primary Aims 
 
Note: This randomized clinical study should be considered a feasibility study to determine 
the ability to design and run a protocol of this nature.  Each Aim was specifically selected 
Figure 2: CD64 macrophage activation at 
time 0 and 24 hours in adults with SCD not 
transfused (A), transfused (B) and a 
comparison between not transfused and 
transfused (C). Macrophages were identified by 
CD14 expression (rectangles on scatter plots in 
(A) and (B)), and further analyzed for activation 
by CD64 expression (histograms gray=time 0, 
red=24 hours). There is a trend suggesting 
transfused subjects have a greater increase in 
CD64+ macrophages 24 hrs post transfusion. 

10 
 
to maximize the possibility that novel information could be obtained from this trial. 
However, power calculations and associated precision estimates should be considered 
preliminary, as the data derived from this study will inform the calculations necessary to 
design a larger, more definitive study.  
 
Aim 1:  
To characterize the physiologic differences between >30 vs <10 day-old units. As part of 
the randomized study, we will measure PS exposure on donor red cells, as well as PE, 
microparticles, plasma free hemoglobin, heme, and NTBI. We hypothesize that >30 day-
old units will have significantly more biochemically old units (see section 10 for the 
definition) than <10 day-old units.  With a total sample size of 40 patients (20 in each 
group), we will have at least 80% power (α=0.05) to detect a difference of at least 47% 
between the two randomized groups for biochemically old red cell units (close to a 100% 
difference is expected). 
 
Aim 2:  
To quantify whether physiologic differences between >30 day-old units versus <10 day-
old units promote immune cell activation in transfused adults with SCD. We will perform 
flow cytometry to measure the activation of monocytes and neutrophils, as well as 
measure cell-free hemoglobin, heme and NTBI on blood samples before, 2, and 24 hours 
after transfusion. We hypothesize that those administered >30 day-old (biochemically old) 
units will have a greater increase in the number of CD62L + activated monocytes at 2 
hours post-transfusion compared to those administered <10 day-old units.  With a sample 
size of 40 patients, we will have 80% power (α=0.05) to detect a change in MFI ratio of 
1.35. 
 
Aim 3:  
To explore whether the physiology of >30 day-old units versus <10 day-old units is 
associated with adverse clinical outcomes in transfused adults with SCD. We will perform 
a systematic evaluation of hospital admissions, pain scores, medications, and clinical 
symptoms prior to each transfusion and at the end of the study period (Table 1). We 
hypothesize that reports of infection will be greater in subjects randomized to receive >30 
day-old  older units. 
 
2.2 Secondary Aims 
 
Aim 1:  
To explore whether: >30 day-old units will have significantly greater mean 1) 
phosphatidylethanolamine expression, 2) cell free hemoglobin concentration, 3) cell free 
heme expression, and/or 4) NTBI expression in comparison to <10 day-old units. 
 
 
 
 
 
 

11 
 
Aim 2:  
To explore whether:  
 
1) Those administered >30 day-old units will have a greater increase in the number of 
activated monocytes or neutrophils as defined by flow cytometry markers of activation at 
2 or 24 hours post-transfusion compared to those administered <10 day units. 
 
2) Those administered >30 day-old day units will have a greater increase in the 
concentration of NTBI, cell free hemoglobin, and free heme, compared to those 
administered <10 day-old units at 2 or 24 hours post transfusion. 
3) Recipient RBC PS, PE, and microparticle expression will increase more between 2 and 
24 hours after receiving >30 day-old units than after receiving <10 day units. 
 
Aim 3:  
To explore whether those administered >30 day-old units will have a more rapid increase 
in the concentration of HbS% and a more rapid decrease in hemoglobin over time 
compared to those administered <10 day-old units. 
 
Aim 4: 
To explore whether those administered >30 day-old units will have a significantly different 
RBC metabolomic profile 2 and 24 hours post transfusion than those provided <10 day-
old units.   
 
Aim 5: 
We will address aspects of study feasibility, such as subject drop-out, rates of missing 
data, and the confidence intervals/precision of our recorded variables, including PS, PE, 
microparticles, CD62L, and the other recorded clinical variables. 
 
3. STUDY POPULATION 
 
3.1 Enrollment Inclusion Criteria 
 
1)  Ages 16 to 60 years old 
2) Subject must have sickle cell anemia confirmed by hemoglobin analysis 
3)  Must be receiving chronic red cell transfusion therapy 
4) Must be outpatient at the time of transfusion 
 
3.2 Enrollment Exclusion Criteria 
 
1)  History of severe reactions to transfusion therapy 
2)  Receiving red cell units that are crossmatch incompatible 
3)  Current participation in another therapeutic trial for SCD 
4)  Current pregnancy (due to the rapidly changing blood volumes that would alter the 
number of units needed for successful randomization, and due to the increased 
likelihood of hospitalization during the study protocol)  
5)  History of HIV infection 

12 
 
6)  Having an uncontrolled inter-current illness, or psychiatric illness/social situations 
that would limit compliance with study requirements. 
 
4. TRIAL ENROLLMENT 
 
4.1 Screening/Recruitment 
 
Patients ≥ 16 years of age undergoing outpatient red cell transfusion therapy will be 
identified through either the adult outpatient sickle cell clinic at UNC, the Infusion Clinic 
at UNC and at Emory/Grady Health System. Emory/Grady Health System will only 
enroll subjects who are ≥ 18 years of age. Basic features of patient medical and surgical 
histories (i.e. age, gender, reason for receiving chronic transfusion therapy) will be 
recorded. 
 
Potentially eligible patients, as described in the previous paragraph, will be approached 
for study consent prior to their transfusion. 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 evaluated. If the subject is 
eligible for the study based on the inclusion/exclusion criteria (section 3.1 and 3.2), they 
will be enrolled in the study and will be randomized. The availability of suitable products 
(see sections 4.2 and 5.1) will determined when the study will start for that individual. 
 
If a subject is enrolled and receives a red cell transfusion prior to randomization, that 
subject will still be randomized, and will start the study at their next outpatient red cell 
transfusion appointment, pending the availability of the appropriately aged units. 
 
4.2 Stratification and Randomization 
 
Subjects will be randomized to receive RBCs stored either ≤ 10 days at time of 
transfusion or ≥ 30 days at the time of transfusion. Storage arm assignment applies to 
up to 3 consecutive outpatient transfusions, beginning at the time of randomization and 
continuing through the end of the 3rd randomized outpatient transfusion. Randomization 
assignment can occur at any time between the date of consent till 2-3 days prior to the 
first study transfusion. Only the transfusion service will have access to the 
randomization assignment as designated by the study biostatistician. 
 
Due to the pilot nature of this study, randomization will not be stratified by gender, age, 
ethnicity, or hemoglobinopathy type. 
 
For the first study transfusion, after the number of units requested for crossmatch is 
known, but no earlier than 2-3 calendar days before transfusion, a study staff member 
will contact the institution’s transfusion service to determine whether the transfusion 
service has enough suitable units stored ≤ 10 days and ≥ 30 days to meet the 
crossmatch request for this subject.  
 

13 
 
If there is not sufficient inventory to meet the randomization criteria, the subject’s study 
participation will be postponed, and unit availability will be assessed again prior to their 
next outpatient transfusion. 
 
Once the subject has received their first study transfusion, the remaining 2 outpatient 
study transfusion events will occur regardless of whether the transfusion service can 
meet the transfusion needs of the study.  
 
4.3 Masking of Treatment Allocation 
 
At enrollment, the subject’s eligibility status and ABO/Rh type will be entered into the  
data management system. If eligible, the subject will be randomized at the time of 
consent, but no later than 2-3 calendar days before the first outpatient study transfusion. 
Unit age availability will be assessed no earlier than 2-3 calendar days before the first 
outpatient study transfusion, and the study will commence if unit availability is 
satisfactory for that subject. The study start will be delayed until the next outpatient 
transfusion if blood unit availability is deemed insufficient for that first study transfusion. 
Only blood bank staff with the appropriate security level in the data management 
system will be able to access the treatment arm assignment. Access to case report 
forms containing information about the age of the RBC products sent for each subject 
will also be restricted at the site to the appropriate blood bank staff. Clinical staff 
overseeing the subject’s participation in the trial, collecting data, and reporting the data 
into  the data management system will not have access to the treatment arm 
assignment or information about the age 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. Medical 
infusion clinic personnel physically providing the RBC transfusions will verify product 
and patient identity according to hospital-specific procedures. These personnel will be 
instructed to not divulge the patients’ randomization assignments. Other infusion clinic 
staff (other than those actually infusing RBCs), will be instructed not to seek to identify 
the age of the products the patients are receiving. The subjects themselves will not be 
informed of their randomization assignment and will also be instructed not to seek to 
identify the age of the products they are receiving. However, as the key components of 
this study are objective, inadvertent unblinding of the randomization assignment will not 
compromise the validity of the study.  
 
5. INTERVENTIONS  
 
5.1 Preparation 
  
In Section 5 of this protocol, the term “study RBC units” includes all RBC units provided 
to a randomized subject, beginning at the time of the first study transfusion and 
continuing through the end of study. Transfusions will always be ordered based on 
patient need, following all institutional policies and practices. The decision to order (or 
not order) an RBC transfusion is not dictated in any way by the study protocol 

14 
 
5.1.1 Preparation criteria applying to both treatment arms 
 
The following criteria apply to all study RBC units in both treatment arms. Any study 
RBC unit which does not meet all these criteria will be considered a protocol violation.  
1. Institutional processing standards will be maintained for all units.  
2. All study RBC units should be pre-storage leukoreduced.  
3. All study RBC units should be stored in either AS1, AS3, or AS5.  
4. No study RBC unit should be washed before release from the transfusion service.  
5. No study RBC unit should be volume-reduced.  
6. No study RBC unit should include a frozen product.  
7. No study RBC unit should be deglycerolized.  
8. No study RBC unit should be irradiated. 
 
5.1.2 Preparation of RBC units for subjects randomized to receive units stored ≤ 
10 days 
  
All study RBC units released from the blood bank for subjects randomized to receive 
units stored ≤10 days should have been stored ≤10 days at the time of transfusion.  
 
If there are not enough suitable units stored ≤10 days at the time a study RBC 
transfusion is needed, the blood bank should release suitable units with the minimum 
available storage time. The transfusion itself should not be cancelled or postponed 
because there are not enough suitable units stored ≤ 10 days.  
 
Study units stored longer than 10 days will be considered protocol deviations for 
subjects in this treatment arm if used for an outpatient transfusion and after the first 
successful randomization. 
 
Subjects randomized to this treatment arm are likely to receive units with shorter 
storage times than the units they would receive if not in the study, but this is not 
guaranteed to be true for any particular subject. 
 
5.1.3 Preparation of RBC units for subjects randomized to receive units stored ≥ 
30 days 
  
All study RBC units released from the blood bank for subjects randomized to receive 
units stored ≥ 30 days should have been stored ≥ 30 days at the time of transfusion.  
 
If there are not enough suitable units stored ≥ 30 days at the time a study RBC 
transfusion is needed, the blood bank should release suitable units with the maximum 
available storage time. The transfusion should not be cancelled or postponed because 
there are not enough suitable units stored ≥ 30 days.  
Units stored less than 30 days will be considered protocol deviations for subjects in this 
treatment arm.  
 
 

15 
 
5.2 Administration 
 
RBC units will be administered according to local institutional policy and safety 
standards as ordered by the medical team for patient care needs.  
 
5.3 Concomitant Treatments 
  
Standard medical care according to the local institutional standard will be provided to 
the subject. 
 
6. SCHEDULE OF MEASUREMENTS 
 
6.1 Red Cell Unit Sample Collection:  
 
2 ml blood from each donor unit will be obtained from 6 sterile unit links/segments on 
the day of the 3 randomized outpatient transfusion events. PE and PS exposure, and 
microparticles, will be measured by flow cytometry using the LSR2-green, and all flow 
analyses will be recorded using FACSDiVa software. The plasma fraction from each red 
cell unit will be diverted to EPR (Bruker Elexys X-band system, Billerica, MA, USA), a 
Bio-Plex cytokine assay kit (Bio-Rad Laboratories), and a fluorescent immunoassay 
(DxFerr kit) to quantify concentrations of free heme/hemoglobin, cytokines (IL-1, IL-6, 
IL-8, MCP-1), and NTBI, respectively. 
 
6.2 Patient Sample Collection (Table 1): 
  
A. One - four weeks prior to the first transfusion: 
-  15ml whole blood for flow cytometry and EPR measurements  
 
B. Immediately prior to each red cell transfusion: 
 
- 15ml whole blood for flow cytometry and EPR measurements 
 
- 15ml to McLendon Labs 
- blood culture to McLendon Labs (8-10ml blood/culture bottle) 
- urine to McLendon Labs (12 ml urine) 
 
C. Two hours after each red cell transfusion (1 ½- 3 hours) 
 
-15ml whole blood for flow cytometry and EPR measurements 
 
-15ml to McLendon Labs 
 
D. 1 day after each red cell transfusion (about 21-27 hours after) 
 
-15ml whole blood for flow cytometry and EPR measurements 
 
-15ml to McLendon Labs 
 
E.  2 weeks after each red cell transfusion (+/- 2 days) 
- 10ml McLendon Labs  
 

16 
 
Patient Sample Processing: Blood will be obtained by port or peripheral access as 
available. Patient white cells will be isolated with established Ficoll separation 
techniques.. White cells will be tested by flow cytometry as above for key markers of 
activation (macrophages: CD62L, CD64, CD80, CD86, Tim1, neutrophils: The plasma 
fraction from each sample will be diverted to EPR (Bruker Elexys X-band system, 
Billerica, MA, USA), a Bio-Plex cytokine assay kit (Bio-Rad Laboratories), and a 
fluorescent 
immunoassay 
(DxFerr 
kit) 
to 
measure 
concentrations 
of 
free 
heme/hemoglobin, cytokines (IL-1, IL-6, IL-8, MCP-1), and NTBI, respectively. Clinical 
measurements obtained from patients will also include: CBC (4ml EDTA), reticulocyte 
count (4ml EDTA), HbS% (3ml EDTA), haptoglobin (1ml red top), chemistry labs (ferritin 
(0.5ml red top), iron saturation (1ml red top), lactate dehydrogenase (1ml red top)), 
urinalysis (12 ml) and blood culture (8-10ml/bottle). These clinical labs will be measured 
at intervals specifically defined in Table 1. Individual human samples of plasma and RBC 
will also be sent to Angelo D’Alessandro at the University of Colorado Denver to measure 
lipid metabolites and metabolomic profiles pre vs. post transfusion as an additional 
exploratory goal of this trial. The laboratory of Dr. Steven Spitalnik and his representatives 
will be receiving 30-200 individual samples of plasma and RBCs for this purpose. All 
samples will be de-identified.  
 
6.3 Patient Clinical Outcomes:  
 
A daily pain diary will be used and subjects will rate their pain on a ordinal numeric pain 
rating 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 any antibiotics taken if applicable. Subject ED and hospitalization events, and etiologies 
(see definitions in 6.7), will be recorded at regular intervals by the study coordinator during 
the study period using the available electronic medical record. Of note, diary 
completeness will be assessed at each face-to-face visit. 
 
6.4 Patient Physical Exam:  
 
Prior to each randomized transfusion, the PI or appropriately trained designee will 
perform a basic assessment of the patient. This exam will include a basic interval 
history (including review of any ED or hospital visits), a focused review of systems, and 
a focused physical exam.   
 
 
 
 
 
 
 
 
 
 

17 
 
Table 1: Schedule of Measurements 
Study 
Measure 
Daily 
1-4 weeks pre-
transfusion 
Pre-every 
transfusion 
 
2hr post 
every 
transfusion 
24 hr post 
every 
transfusion 
2 weeks 
post every 
transfusion 
End 
of 
Study 
Physical exam 
AE assessment 
Med 
assessment 
 
 
X 
 
 
 
X 
CBC w/ 
differential* 
 
 
X 
 
X 
X 
 
HbS%  
 
 
X 
 
X 
X 
 
Blood culture 
 
 
X 
 
 
 
 
Urinalysis w/ 
reflex culture 
 
 
X 
 
 
 
 
Haptoglobin  
 
 
X 
X 
X 
 
 
Plasma 
Hemoglobin 
 
 
X 
X 
X 
 
 
Basic 
Metabolic 
panel+ 
 
 
X 
 
 
 
 
Ferritin & Iron 
Panel# 
 
 
X 
X 
X 
 
 
Lactate 
dehydrogenase 
 
 
X 
X 
X 
 
 
HS-CRP 
 
 
X 
X 
X 
 
 
RBC Unit blood 
donor sample 
 
 
X 
 
 
 
 
Flow cytometry 
sample 
 
X 
X 
X 
X 
 
 
Study Diary 
X 
 
X 
 
 
X 
X 
*WBC, RBC, Hgb. Hct. Plt ct 
+Creatinine, Bicarb, Chloride, BUN, Potassium (K), Sodium (Na), Glucose, Calcium 
#Iron, Transferrin, TIBC, % Saturation 
 
Table 2: Lab Sample Type and Storage Conditions  
Blood 
Samples 
Sample 
required 
Tube type 
Storage 
Processed By 
Red cell units 
6 unit 
segments 
NA 
Room temp 
PI or designee 
Flow 
8ml 
Green top 
Room temp 
(cold after 30 
minutes) 
PI or designee 
Flow/EPR 
Labs 
10 ml 
Cytochex 
Room temp 
PI or designee 
Hematology 
Labs 
10 ml 
EDTA 
Room temp 
McLendon 
Labs 
Chemistry 
Labs 
5 ml 
Red top 
Room temp 
McLendon 
Labs 
Microbiology 
Blood Culture 
10 ml 
Culture vial 
Room temp 
McLendon 
Labs 
Microbiology 
Urine Culture 
12ml 
Sterile cup 
Room temp 
McLendon 
Labs 
 
 

18 
 
6.5 RBC Transfusions  
 
The following data will be collected for each RBC unit ordered for the subject while on 
study:  
• Unit ID number  
• Source of unit (apheresis or whole blood collection)  
• Blood group of unit (ABO and Rh)  
• Leukoreduction status 
• Irradiation status  
• Volume reduction/washed unit status 
• Storage medium (AS1, AS3, or AS5)  
• Antigens matched (D, Cc, Ee, Kk,or other) 
• Collection or expiration date (specifically recorded by the blood bank to maintain 
study coordinator blinding) 
• Start time and end time of transfusion  
 
6.6 Unblinding 
 
Investigators will remain blind to treatment assignment until the entire study is complete.  
Only the DSMB, blood bank staff, and the designated study statistician at Medical 
College of Wisconsin should be aware of the treatment that is assigned to each subject. 
Unintentional unblinding of study participants will not be a reason for study 
discontinuation. Adverse events or urgent clinical care may lead to study termination as 
documented in section 6.7.  
6.7 Ending of Study Participation 
 
1) The subject completes the 3 transfusions  
2) The subject decides to withdraw from the study 
3) The subject moves away, dies, or is lost to follow-up 
4) The patient no longer needs outpatient red cell transfusions as determined by the 
patient’s provider 
5) 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 transfusion per the opinion of the PI 
• PI decides to withdraw the subject due to noncompliance 
• Withdrawal by the PI or subject because of treatment side effects or 
complications 
 
 
 

19 
 
6.8 Key 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 does 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 3-8 week schedule as part of 
routine, non-acute care). 
 
7. SPECIMEN COLLECTION PROCEDURES  
 
Blood samples will be obtained per institutional protocol and assayed in local laboratory 
facilities as defined in Section 6.  
 
8. ADVERSE EVENT CRITERIA AND REPORTING  
 
This study will be using the descriptive terminology developed by the National Cancer 
Institute for use in reporting adverse events: Common Toxicology Criteria for Adverse 
Events (CTCAE) version 4.0, dated May 28, 2009. The CTCAE includes a grading 
(severity) scale for each adverse event term. Grades were developed using the 
following guidelines:  
 
Grade 1: Mild; asymptomatic or mild symptoms; clinical or diagnostic observations only; 
intervention not indicated.  
 
Grade 2: Moderate; minimal, local or noninvasive intervention indicated 
 
Grade 3: Severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; limiting self care 
ADL.  
 
Grade 4: Life-threatening consequences; urgent intervention indicated.  
 
Grade 5: Death related to AE. 
 

20 
 
In general adverse events will be reported as diseases or syndromes whenever 
possible, instead of reporting individual component symptoms, signs, laboratory 
abnormalities or sequelae.  
 
 
8.1 Definitions  
 
Adverse Event (AE) –For this study, it shall be defined as any untoward or unfavorable 
medical occurrence in a human subject requiring medical intervention (grade 2 or 
above) and outside of standard treatment for their underlying illness (i.e. sickle cell 
disease), including any symptom, or disease state, temporally associated with the 
subject’s participation in the research, whether or not considered related to the subject’s 
participation in the research  
 
Serious Adverse Event (SAE) – Any adverse event temporally associated with the 
subject’s participation in research that meets any of the following criteria:  
• results in death;  
• is life-threatening (places the subject at immediate risk of death from the event as 
it occurs);  
• requires inpatient hospitalization or prolongation of existing hospitalization (not 
including usual sickle cell disease pain crises);  
• results in a persistent or significant disability/incapacity;  
• any other adverse event that, based upon appropriate medical judgment, may 
jeopardize the subject’s health and may require medical or surgical intervention 
to prevent one of the other outcomes listed in this definition  
 
Unexpected Adverse Event – Any adverse event occurring in one or more subjects in 
a research protocol, the nature, severity, or frequency of which is not consistent with  
either:  
• the known or foreseeable risk of adverse events associated with the procedures 
involved in the research that are described in (a) the protocol–related documents, 
such as the IRB-approved research protocol or the current IRB-approved 
informed consent document, and (b) other relevant sources of information, such 
as product labeling and package inserts; or  
• the expected natural progression of any underlying disease, disorder, or 
condition of the subject(s) experiencing the adverse event and the subject’s 
predisposing risk factor profile for the adverse event.  
 
Unanticipated problem involving risks to subjects or others (UP): Any incident, 
experience, or outcome that meets all of the following criteria:  
• unexpected (in terms of nature, severity, or frequency) given (a) the research 
procedures that are described in the protocol-related documents, such as the 
IRB-approved research protocol and informed consent document; and (b) the 
characteristics of the subject population being studied;  
• related or possibly related to a subject’s participation in the research; and  

21 
 
• suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) related to the 
research than was previously known or recognized.  
 
Attribution – the determination of whether an adverse event is related to a transfusion 
procedure.  
 
Attribution categories:  
1. Not Related - Event clearly related to other factors (e.g., clinical state, other 
therapies; concomitant drugs)  
2. Possibly Related - Sequence of event is compatible with study drug, device, or 
procedure, but could have been produced by other factors  
3. Probably Related - Sequence of event is compatible with study drug, device, or 
procedure and cannot be explained by other factors without much doubt  
4. Definitely Related - Sequence of event is compatible with study drug, device, or 
procedure and beyond doubt cannot be explained by other factors  
 
8.2. Types of Adverse Events to be Collected 
 
Specific information about the following events will be collected for this study on the 
adverse event checklist case report form:  
 
1. Serious adverse events, regardless of attribution to red cell transfusion  
2. Specific adverse events, regardless of attribution to red cell transfusion  
• Myocardial infarction  
• Pulmonary embolism  
• Stroke  
• Renal failure or acute injury  
• Infection/sepsis  
• Ventricular tachycardia  
• Ventricular fibrillation  
• Transfusion-associated congestive heart failure/transfusion-associated 
circulatory overload (TACO)  
• Transfusion-related acute lung injury (TRALI)  
• Anaphylaxis  
• Graft vs. Host Disease (GVHD)  
• Grade 2 (medical intervention needed) or higher events of the following types:  
o Allergic reaction  
o Sinus bradycardia  
o Sinus tachycardia  
o Hypertension  
o Hypotension  
o Dyspnea  
o Hypoxia  

22 
 
o Wheezing  
o Fever  
o Hemolysis  
o Hyperbilirubinemia  
o Hemoglobinuria.  
 
3. Unexpected adverse events (all grades), attributed as possibly, probably or definitely 
related to red cell transfusion  
4. Unanticipated problems  
 
8.3. Timeline for Reporting of Adverse Events 
 
Study staff must fill out an Adverse Event Checklist form within 7 days of each 
randomized 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. 
 
8.4. Review of Events for Subjects Enrolled 
 
The Data Safety Monitoring Board (DSMB) is an independent board appointed by the 
PI. The principal role of the DSMB 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.  
 
The DSMB 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). The DSMB chair has expertise in benign hematology and 
transfusion medicine, will review the serious adverse event materials, and determine if 
the information is complete.  
 
The DSMB members will meet annually via teleconference. All adverse events will be 
reviewed during the call, and the group will vote if additional DSMB review is required, 
and make recommendations about the study. 
 
8.5. Reporting Events to Local Institutional Review Boards 
  
The investigator will notify the local Institutional Review Board (IRB) of all adverse 
events in accordance with institutional policy. 

23 
 
8.6. Annual DSMB Reports 
 
The DSMB will meet once a year, either in-person or via teleconference. The PI will 
provide the DSMB prior to the meeting with:  
• Accrual totals entered into Velos 
• Adverse event log reports for each study subject entered into the electronic data 
system 
• Serious adverse event reports for each study subject entered into the electronic 
data collection system  
• Study compliance issues if applicable 
• The DSMB will also be provided each subject’s study arm assignment by the 
primary biostatistician, Dr. Pippa Simpson. 
 
The DSMB will define: 
• All-cause mortality, with p-value to compare.  
• Number, type, and severity of serious adverse events, overall and by treatment 
arm. No formal stopping rule is set for this comparison.  
• Number of subjects in each treatment arm with at least one clinically documented 
infection leading to an ED or hospital admission with p-value. No formal stopping 
rule is set for this comparison. 
• Unexpected adverse events and unanticipated problems overall and by treatment 
arm. 
• Evaluate statistically significant difference in serious infection or pain admissions 
(Section 10) between the two arms.  
• Recommend stopping guidelines as needed 
 
9. SAFETY MONITORING AND STOPPING GUIDELINES 
  
Interim monitoring plans and stopping guidelines will be defined by the DSMB. This 
section describes proposed plans. The study is expected to take three years to 
complete. Stopping guidelines are proposed for two outcomes, serious infection and 
pain crisis requiring ED or hospital admission. For these two outcomes, yearly looks at 
the data will take place. Therefore, three looks at the data (including the final look) are 
anticipated. 
 
In designing the study, a serious infection difference of 20% between the 2 arms would 
be of clinical interest. As we do not expect to have adequate power in this study of 30 
subjects to detect this difference, with an alpha of 0.05 with 15 in each arm, we will be 
able to detect an infection rate difference of 47-56% between the two arms. Any 
statistically significant difference in serious infection or pain admissions between the two 
arms will be considered as evidence for stopping the clinical trial. 
 
 
 
 

24 
 
10. STATISTICAL CONSIDERATIONS 
 
10.1. Sample Size and Power 
 
Participants will be recruited from UNC and Emory for this proposal. There are about 50 
eligible patients at UNC and about 100 chronically transfused subjects at Emory. We 
anticipate no difficulties reaching our 40 patient study target. We anticipate that we will 
enroll at least 40 total individuals aged 16-60 years who are managed with chronic 
transfusions at UNC or Emory and will meet the study inclusion and exclusion criteria. 
This study will not attempt to stratify the study population for age or gender.  
 
10.2. Analysis Plan 
  
The analysis will be an intent to treat (ITT) with all those randomized being included in 
the study. For all aims, the distribution of, and relationship between, variables will be 
explored with summaries, plots, and tree analysis. All statistical estimates of population 
parameters will be tabulated along with corresponding confidence intervals (CIs). All 
hypothesis tests yielding large p-value (e.g., p > α) will be reported as being inconclusive. 
Dr. Pippa Simpson, the lead biostatistician, or her designee, will perform these analyses. 
Where necessary for parametric assumptions, appropriate transformations will be 
employed with justification for their use; otherwise, non-parametric tests will be used. P-
values will be reported without dichotomization wherever applicable. Data obtained from 
transfusion events where units of the appropriate age could not be obtained (study 
deviation) will be excluded from a subanalysis of the dataset, and the same distribution 
of, and relationship between, variables will be explored with similar summaries, plots, and 
tree analysis as in the ITT analysis.  All subjects, regardless of age and gender, will be 
analyzed together as one cohort. Statistical software used in this study will be Cytel, 
StatXact and LogXact, SAS version 9.4, and Salford systems CART (Classification and 
Regression Trees). To adjust for multiple comparisons, we will use the false discovery 
rate approach which is less conservative than the Bonferroni approach. Missing data: 
Logistic regression will be used to investigate the possible causes for missing data and 
to investigate if data are missing at random (MAR). Reasons for missing data will be 
documented in the study database. We do not anticipate any missing data or drop-outs, 
but we will monitor these events. Assuming data are MAR, multiple imputations will be 
used for items, and for repeated measures, a random effects models will be used; the 
structure for the variance covariance matrix will be explored under the constraints of 
sample size. A sensitivity analysis will also be performed to evaluate the 
robustness/fragility of the study’s 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. 
 
10.2.1 Primary Outcome Analysis  
 
Key variables of interest: 
PS – Measured as a percent of RBC positive 
PE – Measured as a percent of RBC positive 

25 
 
Microparticles – Measured by number positive per microliter 
CD62L – Measured as percent of monocytes positive 
Plasma free hemoglobin – Measured as mg/dL 
Non-transferrin bound iron – Measured as mg/dL 
Rate of clinical infections – Measured as number per days on study 
 
Aim 1:  
The primary outcome measure is the proportion of biochemically old red cell units. 
Previous studies and our own work have suggested that PS, PE, and microparticles 
increase in a sigmoidal fashion over time.7,10 For this study, the cut-off for a biologically 
old unit would be the point at which the sigmoidal curve enters its log phase, which for 
PE, PS, and microparticles will be the mean value at day 21, or 8%, 3%, and 3/μl 
respectively based on these preliminary studies. This cutoff is biologically meaningful 
and is supported temporally by the metabolic changes that occur with red cell 
storage.119 Consequently, biochemically old units will be defined as having either a 
surface PE, surface PS, or a microparticle concentrations ≥ the a priori-defined cutoff. 
We will compare the transfusions provided to the two groups (the proportion of 
biochemically old units when stored ≥30 days compared to when stored ≤10 days) using 
a Fisher exact test at an alpha of 0.05. Power: With a total sample size of 40 patients 
(20 in each group), we will have at least 80% power (α=0.05) to detect a difference of at 
least 47% between the two randomized groups for biochemically old red cell units (close 
to a 100% difference is expected).  
 
Exploratory: We will calculate the mean, minimum, and maximum levels of PS, PE, and 
microparticles for each study group, and we will compare the secondary outcome 
measures of plasma free hemoglobin, heme, and NTBI and also PS, PE, and 
microparticle concentrations using a two sided two sample t-test at an alpha of 0.05.We 
will also plan for auxiliary analyses that characterize the relationship between the PS 
level and the age (days) of the RBC units.  This analysis will focus on estimation of the 
mean PS, PE, and microparticle level as a function of age (days), visualization of the 
data values, estimation of correlation coefficients, and evaluation of how well age (days) 
predicts PS, PE, and microparticle levels. 
 
Aim 2:  
The primary outcome measure is the change in CD62L (activated) circulating 
monocyte/macrophage MFI at 2 hours post-transfusion compared to the subject pre-
transfusion. We will compare the two groups (≥30 day-old compared to ≤10 day-old unit 
exposure) using a two sample two-sided t-test of the log at an alpha of 0.05. Power: We 
will have at least 80% power to detect a change in the ratio of 1.35 post-transfusion if 
the variability is similar to that seen in our preliminary data.  
 
Exploratory: We will use a general(ized) linear model to include biochemically old units 
transfused (Aim 1 outcome), regardless of unit age, as a covariate in our analysis. Other 
co-variates in this model will include free heme, cell free hemoglobin, and NTBI. We will 
similarly compare other obtained activation measures as outcomes: activation markers 
for monocytes, neutrophils (as listed in section 6) and measured plasma cytokine 

26 
 
concentrations (section 6). We will also plan for auxiliary analyses of the outcome 
variables in which the explanatory variable is the age (days) of the RBC units  --to 
characterize the degree to which age (days) can accurately predict the clinical and 
physiologic outcome measures defined. Lastly, as an exploratory aim, we will evaluate 
the in vivo change in recipient red cell PE/PS positivity at 2 and 24 hours post 
transfusion.  
 
Aim 3:  
The primary outcome variable is the rate of post-transfusion infections in the patient.  
The primary estimand is the treatment effect defined as the difference between the 
regimen-specific proportions of patients in the target populations that would experience 
post-transfusion infections.  This population parameter will be estimated as a function of 
the proportions observed in the sample of patients studied.  The point estimate will be 
reported along with the corresponding 95% confidence interval estimate. The presence 
of an indwelling catheter will be used as a key co-variate in this analysis. Power: A 
difference of 20% will be of clinical interest. We do not expect to have adequate power 
for this pilot study but at an alpha of 0.05 with 20 subjects in each group we will be able 
to detect a difference of 47% between the proportions. 
 
Exploratory: We will explore the relationship of blood age and transfusion of 
biochemically old red cell units on the change in Hb and HbS% over time, daily pain 
scores (0-10 as an ordinal scale), opioid use and dose, ED and hospitalization rate, 
infection symptoms, new alloantibody formation, and antibiotic use during the 3-month 
study period. We will compare groups using a Fisher exact test. We will also plan for 
auxiliary analyses of the outcome variables in which the explanatory variable is the age 
(days) of the RBC units  --to characterize the degree to which age (days) can accurately 
predict the clinical and physiologic outcome measures defined.   
 
AIM 4: 
We will calculate the mean, median, minimum, and maximum levels of each metabolic 
compound analyzed by Dr. Alessandro in his laboratory at the University of Colorado. 
Analysis tools and methods will be determined by his research team, and will focus on 
changes in the patient and the unit pre and post transfusion. 
 
AIM 5:  
Feasibility Analysis: We will assess important variables necessary to develop a future 
larger clinical trial on this subject.  Areas that we will investigate during the course of 
this trial will include frequency and content of missing data, subject drop out rates, and 
estimates of outcome measure precision. For this study, we will evaluate the number of 
males, females, and adolescents that enroll in the study to inform future studies. We do 
not expect there to be differences in the key outcomes based on these variables, and 
plan to analyze all 40 subjects together as one cohort, but differences by age and 
gender will be evaluated and considered. 
 
 
 

27 
 
11. DATA COLLECTION AND VALIDATION  
 
Data will be collected and entered into a web-based data management system.  The 
lead coordinator will routinely validate that all data entry fields are entered. Validations 
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 – The principle investigator will serve as the trial coordinator for this 
study. The PI 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. 
 
Data Quality - The principal investigator will routinely check for data accuracy and data 
completeness. When data are found to be missing, the principal investigator or his 
designee will attempt to document in the electronic record why the values are missing. 
The principal investigator and his designee will maintain a data dictionary and will 
evaluate adherence to the codebook at regular intervals. 
 
12. PROTECTION OF HUMAN SUBJECTS 
  
This study will be conducted according to Good Clinical Practices (GCP), the rules and 
regulations of the Institutional Review Board at the University of North Carolina, and in 
accordance with state and federal agencies. 
 
13. INVESTIGATOR RESPONSIBILITY  
 
13.1 Institutional Review Board (IRB) / Approval 
  
No patient will be enrolled in the study until the IRB has approved the protocol and the 
Informed Consent Form. Documentation of approval will be maintained at all times. At 
study termination, a study summary will be submitted by the PI to the IRB. Copies of all 
submissions to and correspondence (approvals and disapprovals) from the IRB/EC will 
be maintained on file.  
 
13.2 Informed Consent  
 
If a patient is potentially eligible for the study as defined, the patient will be approached 
to obtain written informed consent. The background of the study and the potential 
benefits and risks will be explained. The patient or patient’s legally authorized 

28 
 
representative must sign the consent form that has been approved by the IRB prior to 
enrollment. Failure to obtain signed informed consent renders the patient ineligible for 
the study. Copies of the signed informed consent shall be kept in subject’s study files.  
 
13.3 Subject Data Protection 
  
Subjects will be identified in the electronic case report form (eCRF) by a subject 
identification number. All information and data concerning subjects or their participation 
in this study will be considered confidential. All data used in analysis and reports will be 
used without identifiable reference to the subject. At all times throughout the study, 
confidentiality shall be observed by all parties involved. All data shall be secured against 
unauthorized access. 
 
14. MONITORING AND QUALITY CONTROL  
 
14.1 Site Training 
  
Only trained personnel can perform study related procedures. All investigators and 
relevant staff such as study coordinators and blood bank staff members will be trained 
on the protocol. These individuals will be trained by the PI or lead coordinator. The 
study team will be responsible for ensuring that the hospital staff directly responsible for 
patient care (staff nurses and physicians) is adequately trained in the management of 
these study patients and blood bank procedures. 
 
14.2 Monitoring of the Study 
  
The PI or designee will monitor the investigation to ensure that it is conducted in 
accordance with the protocol. 
 
15. RECORDS AND REPORTS  
 
15.1 Case Report Forms 
  
Case Report Forms (CRFs) will be used to collect all subject data during the course of 
the study. 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.  
 
15.2 Source Documents 
  
Good Clinical 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:  

29 
 
• 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  
 
15.3 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 University 
of North Carolina protocol after study completion or termination. In addition, the 
Investigator will not discard or destroy any study-specific materials unless otherwise 
instructed. 
 
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