A Randomized Trial to Determine if RBCs From Donors With Iron Deficient Erythropoiesis Have Decreased Post-transfusion RBC Recovery and Whether Iron Repletion Improves Recovery
Phase: NA
Conditions: Iron Deficiency
Sponsor: Columbia University

 
 
    
Red blood cells from iron-deficient 
donors: recovery and storage quality 
Donor Iron Deficiency Study: Protocol version 2.5 
CUMC IRB-AAAQ8875 
 
1 
 
 
Donor Iron Deficiency Study 
 
 
Protocol Version 2.5 – December 6, 2019 
 
Study Sponsor: National Heart, Lung, and Blood Institute 
 
Grant: NIH R01-HL133049 
 
 
A randomized trial to determine if RBCs from donors with iron 
deficient erythropoiesis have decreased post-transfusion RBC 
recovery and whether iron repletion improves recovery 
ClinicalTrials.gov #NCT02889133

 
 
    
Red blood cells from iron-deficient 
donors: recovery and storage quality 
Donor Iron Deficiency Study: Protocol version 2.5 
CUMC IRB-AAAQ8875 
 
2 
 
Concept Synopsis and Study Schema 
Each year, ~5 million regular donors meet almost half the transfusion 
needs of the United States by voluntarily donating their blood. 
Despite fulfilling all requirements for blood donation, almost two-
thirds of the women and half of the men who are regular blood 
donors are iron deficient. In order to manage the inventory, red blood 
cell (RBC) units destined for transfusion can be stored for up to 42 
days prior to transfusion based on FDA guidelines. This guideline is 
based on a post-transfusion recovery study in which one must prove 
to the FDA that, on average, using healthy volunteers, more than 
75% of transfused red cells remain in circulation for 24 hours at 
outdate. This research will determine if RBC units from iron-
deficient volunteer blood donors fail to meet U.S. FDA standards 
for 24-hour post-transfusion recovery. RBCs from iron-deficient 
donors may be specifically damaged by refrigerated storage. In 
studies with a mouse model of iron deficiency (with hemoglobin 
levels similar to those that are acceptable for human blood donation), 
we found a mean 10.6% further decrease in the 24-hour post-
transfusion recovery of refrigerator-stored red cells from iron-deficient 
animals, as compared to the post-transfusion recovery from iron-
replete littermates. A comparable decrement in post-transfusion 
recovery of refrigerator-stored red cells from iron-deficient human 
volunteer blood donors would result in many of these units failing to 
meet established quality standards for clinical use. This study will 
also test whether giving iron deficient donors their iron back will 
improve the quality of their red cells during refrigerator storage.  
 
Research Question(s)/Hypothesis(es): 
 
Primary 
1.  The 24-hour post-transfusion RBC recovery from units 
obtained from iron-deficient donors will not meet FDA standards 
for clinical use. 
 
2.  The 24-hour post-transfusion RBC recovery from units 
obtained after intravenous iron repletion will improve 
significantly and will meet FDA standards for clinical use. 
 
Secondary  
1.  Iron therapy will improve neurocognition and emotional well-
being in donors with iron deficient erythropoiesis. 
 
2.  Metabolite levels in the transfusate will be associated with 
24-hour post-transfusion RBC recovery and iron status. 
 

 
 
    
Red blood cells from iron-deficient 
donors: recovery and storage quality 
Donor Iron Deficiency Study: Protocol version 2.5 
CUMC IRB-AAAQ8875 
 
3 
Study Schema: 
This is a randomized, controlled, double-blind clinical trial. 60 healthy 
regular donors who meet donation standards, while exhibiting iron-
deficient erythropoiesis by laboratory test criteria, will donate a single 
standard RBC unit that will be leukoreduced and stored under 
standard conditions for 42 days. The FDA gold standard measure for 
the quality of RBCs destined for transfusion is the 51Cr-radiolabeled 
24-hour RBC recovery study. Thus, a small aliquot of the transfused 
RBCs will be radiolabeled and injected into the volunteers on day 41 
or 42 of storage. RBC recovery will be calculated from samples 
obtained at 5min, 7.5min, 10min, 12.5min, 15min, 30-minutes, 1-hour 
and 24 hours after autologous infusion as per the standardized 
protocol.4 For secondary outcomes, study surveys and 
neurocognitive assessments to assess symptoms of anemia and iron 
deficiency will also be performed prior to donation and prior to 
infusion. 
In a prospective, randomized, double-blind manner, these 
donors will then receive either intravenous saline or low-molecular 
weight iron dextran (or ferric carboxymaltose if iron dextran is 
unavailable due to supply) within 1-day to 4-weeks after the first post-
transfusion recovery study. After five months, they will donate a 
second RBC unit, similarly stored for 42 days, and autologous 51-
chromium 24-hour post-transfusion RBC recoveries will again be 
determined. The primary outcome will be the group mean difference 
in 24-hour post-transfusion recovery difference from after treatment 
to baseline, between the groups receiving intravenous saline and 
iron. 
 
 
 

 
 
    
Red blood cells from iron-deficient 
donors: recovery and storage quality 
Donor Iron Deficiency Study: Protocol version 2.5 
CUMC IRB-AAAQ8875 
 
4 
 
Figure 1. Study Schema. 60 frequent blood donors with iron-deficient erythropoiesis 
will donate one unit of RBCs and a 51Cr-labeled post-transfusion RBC recovery study 
will be performed. Each volunteer will then be randomized to placebo (intravenous 
saline) or iron repletion (intravenous low molecular weight iron-dextran or ferric 
carboxymaltose) within 4-weeks of completing the post-transfusion RBC recovery 
study. 5 months ± 4 weeks later (target 5 months) they will donate another RBC unit. 
42 days after the second blood donation, another 51Cr-labeled post-transfusion RBC 
recovery study will be performed and the result compared to the first 51Cr-labeled 
post-transfusion RBC recovery study.  
Donate first 
unit of blood 
1 
unit 
51-Cr 24-hour  
Post-Transfusion 
Recovery Study 
 
42 days 
1 
unit 
51-Cr 24-hour  
Post-Transfusion  
Recovery Study 
42 days 
5 months 
1 
unit 
42 days 
IV iron 
IV saline 
5 months 
51-Cr 24-hour  
Post-Transfusion 
Recovery Study 

 
 
    
Red blood cells from iron-deficient 
donors: recovery and storage quality 
Donor Iron Deficiency Study: Protocol version 2.5 
CUMC IRB-AAAQ8875 
 
5 
TABLE OF CONTENTS 
 
1. Background and Significance………..…………………………...  7 
2. Objectives 
2.1.   Primary Hypothesis………………………………………… 11 
2.2.   Secondary Hypothesis…………………………………..… 11 
3. Study Population 
3.1.   Enrollment Inclusion criteria………………………………. 11 
3.2.   Enrollment Exclusion criteria……………………………… 12 
4. Trial Enrollment 
4.1.   Screening / Recruitment…………………………………… 12 
4.2.   Randomization…………………………..……………….…. 13 
4.3.   Blinding……………………………..…………………….…. 14 
5. Interventions 
5.1.   Blood donation........………………………………………... 15 
5.2.   Chromium-51 post-transfusion RBC recovery ...…….…. 15 
5.3.   Total Dose Iron Infusion……………………...……………. 16 
5.4.   Cognitive and Emotional Status Assessment…………… 18 
5.4.1. Cognition……………………………………………….   18 
 
 
 
5.4.3. Hearing…………………………………………………. 20 
 
 
 
 
 
6. Schedule of Measurements 
6.1.   Summary Timeline…………………………………………. 21 
6.2.   Assessment Procedures 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
6.2.5. Type and Screen Testing……………………………. 23 
7. Specimen Collection Procedures……………………………..… 23 
8. Compensation…………………………………………………….. 24 
9. Adverse Event Criteria and Reporting  
9.1.   Definitions………………………………………………….... 24 
9.2.   Types of Adverse Events Reported………………………. 26 
9.3.   Reporting Timelines…………………………………………  28 
10. Interim Reporting 
     10.1 Semi-Annual DSMB Reports………………………………. 29 
11. Statistical Considerations 
11.1. Analysis Plan……………………………………………… 30 
11.2. Sample Size Estimate……………………………………. 30 
11.3. Interim Analyses………………………………………….. 31 
5.4.2. Emotion………………………………………………… 20
5.4.4. Restless Leg Syndrome Severity……………………. 21
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
6.2.2. Hepcidin……………………………………………….. 23
6.2.1. Zinc Protoporphyrin, Serum Iron, Total Iron Binding
 
Capacity, Transferrin Saturation, Ferritin, C-Reactive
 
Protein, Soluble Transferrin Receptor………………… 23
6.2.3. Complete Blood Counts……………………………… 23
 
 
 
 
6.2.4. Urine Pregnancy Test………………………………... 23

 
 
    
Red blood cells from iron-deficient 
donors: recovery and storage quality 
Donor Iron Deficiency Study: Protocol version 2.5 
CUMC IRB-AAAQ8875 
 
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12.  Data Collection and Validation…………………………………. 31 
13.  Protection of Human Subjects………………………………….. 32 
14. Investigator Responsibility 
14.1. Institutional Review Board (IRB) Approval…………….. 32 
14.2. Informed Consent………………………………………… 32 
 
 
 
 
 
15.  Records and Reports 
15.1. Case Report Forms…………………………………….. 
33 
15.2. Source Documents……………………………………... 
33 
15.3. Record Retention……………………………………….. 
33 
16.  References……………………………………………………… 
34 
17.  Appendix 1 (Recruitment letter)………………………………. 
39 
18.  Appendix 2 (SF-36 Health and well-being survey)………….. 
40 
19.  Appendix 3 (Multidimensional Assessment of Fatigue/MAS). 41 
20.  Appendix 4 (Beck Depression Inventory-II/BDI-II)….……….. 42 
21.  Appendix 5 (Beck Anxiety Inventory/BAI)…………………….. 43 
22.  Appendix 6 (Discharge instructions)………………………….. 
44 
23.  Appendix 7 (Case Report Forms)….………………………….. 45 
 
 
14.3. Subject Data Protection………………………………… 
33

 
 
    
Red blood cells from iron-deficient 
donors: recovery and storage quality 
Donor Iron Deficiency Study: Protocol version 2.5 
CUMC IRB-AAAQ8875 
 
7 
1.  Background and Significance: 
General Introduction. Iron deficiency is common among regular blood 
donors, but the recovery and quality of RBC units from iron-deficient 
donors has not been rigorously examined. Evidence from both animal 
and human studies indicate that when the iron supply for 
erythropoiesis is inadequate, the RBCs produced have multiple 
metabolic defects that impair their ability to tolerate refrigerated 
storage. Our studies in a mouse model demonstrated decreased 
post-transfusion recovery of refrigerator-stored RBCs obtained from 
iron-deficient donors. The planned studies will identify human donors 
at greatest risk of providing RBCs with poor post-transfusion recovery 
by using a combination of a decreased serum ferritin concentration 
and increased RBC zinc protoporphyrin, as described below. To 
evaluate unequivocally the role of iron deficiency in poor post-
transfusion RBC recovery, intravenous iron will be used for iron 
repletion.  
 
Serum ferritin and RBC zinc protoporphyrin detect iron-deficient 
erythropoiesis in volunteer blood donors. Iron deficiency, a decrease 
in the amount of body iron, is detected clinically by measuring 
indicators of iron storage (e.g., serum ferritin), and of iron supply 
(e.g., RBC zinc protoporphyrin). In the absence of complicating 
factors, as iron stores decrease, serum ferritin levels decline; a 
serum ferritin level less than 12 µg/L is virtually diagnostic of the 
absence of marrow iron stores. RBC zinc protoporphyrin monitors the 
supply of iron available for RBC production. In the developing RBC, 
the insertion of iron into protoporphyrin IX is the final step in 
producing heme for incorporation into hemoglobin. If iron is 
unavailable, divalent zinc is incorporated instead, producing zinc 
protoporphyrin, which binds to hemoglobin and persists for the life of 
the RBC as a biochemical indicator of an inadequate supply of iron 
for RBC production.1,3  
Four successive stages of iron deficiency can be distinguished (Fig. 
2). Reduced iron stores (not shown): As iron stores decrease, 
serum ferritin levels decline proportionally. Iron depletion: Iron 
depletion develops when iron stores are absent, but iron delivery to 
the erythroid marrow for producing hemoglobin and other functional 
iron compounds is maintained by the combination of iron recycling 
from senescent RBCs and gastrointestinal iron absorption. With 
absent iron stores, the serum ferritin falls to <15 µg/L. Because the 
iron supply for RBC production is maintained, RBC zinc 

 
 
    
Red blood cells from iron-deficient 
donors: recovery and storage quality 
Donor Iron Deficiency Study: Protocol version 2.5 
CUMC IRB-AAAQ8875 
 
8 
protoporphyrin levels remain in the reference range. Iron-deficient 
erythropoiesis: With further reductions in body iron, the lack of iron 
limits production of hemoglobin and other iron-requiring compounds, 
resulting in iron-deficient erythropoiesis. Nonetheless, the effect on 
the circulating hemoglobin concentration is insufficient to be detected 
by the standards used to screen blood donors, which currently only 
includes a hemoglobin test. As newly formed RBCs replace 
senescent RBCs, RBC zinc protoporphyrin progressively increases, 
providing an index reflecting the severity and duration of the 
inadequate supply of iron for erythropoiesis. Further decreases in the 
nominal serum ferritin levels have no physiological meaning. Thus, 
the combination of a serum ferritin <15 µg/L and an increased 
RBC zinc protoporphyrin (i.e., >80 µmol/mol heme), is highly 
specific for iron-deficient erythropoiesis.1,3 
Iron-deficiency anemia: Further diminution in body iron produces 
frank iron-deficiency anemia, which would result in donor deferral.  
 
Iron deficiency is common in volunteer blood donors. In the United 
States in 2011, of the donors who provided the ~15.7 million units of 
RBCs that were collected, 69% were repeat donors.5 In addition, in 
Canada, ~90% of RBC units collected for transfusion are provided by 
repeat donors.6 Although iron deficiency is surprisingly prevalent in 
first-time donors,7,8 its 
prevalence is even 
higher in the particularly 
altruistic frequent 
donors, especially 
among women of 
childbearing age.9,10 In 
the REDS-II Donor Iron 
Status Evaluation (RISE) 
study,11 up to 49% and 
66% of male and female 
frequent donors, 
respectively, manifested 
either iron depletion (i.e., 
absent iron stores) or 
iron-deficient 
erythropoiesis. Similar 
frequencies of iron 
deficiency were also 
reported in Canadian,6 
Austrian,12 Danish,13 and 
Dutch14 populations.  
 
RBCs from donors with 
iron-deficient 
 
Figure 2. RBC zinc protoporphyrin progressively 
increases with increasing severity of iron 
deficiency. Iron deficiency progresses from reduced 
iron stores to iron depletion, in which there is normal 
erythropoiesis with normal zinc protoporphyrin 
levels, but decreased ferritin due to absent iron 
stores. The next stage is iron-deficient 
erythropoiesis, in which increased RBC levels of 
zinc protoporphyrin and very low serum ferritin levels 
reflect both absent iron stores and the lack of 
sufficient iron for normal hemoglobin production. As 
iron deficiency progresses further, frank iron 
deficiency anemia develops with insufficient iron for 
maintaining adequate hemoglobin levels. Our target 
population is human donors with iron-deficient 
erythropoiesis. Adapted from Hastka et al.1 and 
Brittenham3. 
240$
200$
160$
120$
80$
40$
$$0$
Threshold 
for anemia 
Normal 
Iron  
Depletion 
Iron  
Deficient 
Erythropoiesis 
Iron  
Deficiency 
Anemia 
Erythrocyte zinc protoporphyrin progressively increases 
with increasing severity of iron deficiency 
Erythrocyte 
Zinc Protoporphyrin 
(µmol/mol heme) 
Iron stores 
Liver/Bone Marrow 
Circulating 
red blood  
cells 

 
 
    
Red blood cells from iron-deficient 
donors: recovery and storage quality 
Donor Iron Deficiency Study: Protocol version 2.5 
CUMC IRB-AAAQ8875 
 
9 
erythropoiesis have impaired tolerance for refrigerated storage and 
decreased post-transfusion RBC recovery. RBCs from individuals 
with iron deficiency anemia have decreased levels of endogenous 
antioxidants,15,16 have evidence of oxidative damage,17,18 and are 
more sensitive to oxidative stress15,18 and low pH;16 the latter 
decreases progressively during RBC storage.19 Furthermore, 
refrigerated storage induces oxidative stress in donor RBCs and 
inhibits their oxidative stress defense mechanisms.17,20-24 Oxidative 
damage per se also impairs RBC deformability25 and impaired 
deformability was seen in humans,18 rats,18 and rabbits with iron 
deficiency anemia26 and in stored RBCs from healthy human 
donors.27 Rigid RBCs are less able to pass through previously 
negotiable microcirculatory beds and are more prone to undergo 
extravascular hemolysis in the spleen.28 Indeed, circulatory RBC 
lifespan is decreased in humans with iron deficiency anemia16,29-31 
and in relevant animal models.26,32 In humans, decreased circulatory 
lifespan is most likely due to extravascular hemolysis in the spleen29-
31 and is corrected by iron repletion.16,30 
Remarkably, in several older studies,16,29,33 RBCs obtained from 
donors with iron deficiency anemia were transfused into healthy 
recipients, without prior refrigerated storage. In each study, the 
transfused iron-deficient RBCs had a decreased circulatory 
lifespan/recovery, most likely due to splenic clearance. In addition, 
when RBCs obtained from healthy donors were transfused into 
recipients with iron deficiency anemia, the transfused RBCs had a 
normal lifespan, suggesting that the iron deficiency-induced defect 
was intrinsic to the RBC and not due to enhanced clearance 
mechanisms.33,34 
 
Iron deficiency decreased the 24-hour post-transfusion RBC recovery 
in a mouse model. Replicating the human studies described just 
above from the 1940-70’s is no longer ethically permissible. Although 
the RBCs transfused in these studies were from donors with iron 
deficiency anemia, not from donors with iron-deficient erythropoiesis, 
deliberate allogeneic human transfusion studies using such donors 
and healthy volunteer recipients are no longer feasible. Instead, we 
used a mouse model to obtain proof-of-principle preliminary data. 
Three mouse donor cohorts were prepared: iron replete, iron-

 
 
    
Red blood cells from iron-deficient 
donors: recovery and storage 
quality 
Donor Iron Deficiency Study: Protocol version 2.5 
CUMC IRB-AAAQ8875 
 
10 
deficient erythropoiesis, and iron deficiency anemia (Fig. 3). Similar 
to our prior studies,35 refrigerator-stored, transfused RBCs from iron-
replete donors had a normal 24-hour post-transfusion recovery 
(mean 77.1%). In addition, as expected from other publications,16,29,33 
the 24-hour post-transfusion recovery was poor using RBCs from 
donors with severe iron-deficiency anemia (mean 46.7%; p<0.001). 
In contrast, results using donors with iron-deficient erythropoiesis 
were subnormal (mean 66.5%; p<0.05) and would be less than the 
FDA-mandated minimum (in this mouse model). Taken together, 
these data support our hypothesis that, after refrigerated 
storage, RBCs from donors with iron-deficient erythropoiesis 
without anemia are suboptimal. 
Figure 3. RBCs from iron-deficient donor mice have 
reduced 24-hour recovery and increased zinc 
protoporphyrin levels. Weanling male C57BL/6 mice were 
placed on a defined iron control diet of 45ppm iron (TD.110593, 
Harlan Laboratories) or an iron-deficient diet of 0-4ppm iron 
(TD.110592). A third group was fed the iron-deficient diet and 
was also phlebotomized weekly (50-100 μL per bleed) to 
generate a total of three groups of mice with varying iron status: 
(1) iron replete, (2) iron-deficient erythropoiesis (IDE) with mild 
iron deficiency, and (3) severe iron deficiency anemia (IDA).  At 
~10 weeks of age, blood was collected from each cohort, 
leukoreduced, packed, and refrigerator stored in CPDA-1. After 
12 days of storage, 24-hour post-transfusion recovery was 
determined in recipient transgenic mice expressing enhanced 
Green Fluorescent Protein under the direction of the human 
ubiqutin C promoter using flow cytometric detection of 
transfused RBCs (left panel). The experiment was repeated 
three times in its entirety (N=24 per group total). Mean 
hemoglobin levels prior to blood donation are in parenthesis. 
The dotted red line represents 75% RBC recovery. Statistical 
significance determined by one-way ANOVA with Dunnett’s 
Replete (16.1 g/dL)
IDE (11.2 g/dL)
IDA (7.7 g/dL)
0
50
100
24-hour post-transfusion recovery
77.1
66.5%
46.7%
Mean = 
P<0.05
P<0.001
Zinc Protoporphyrin 
(µmol/mol heme)
Replete
IDE
IDA
0
100
200
300

 
 
    
Red blood cells from iron-deficient 
donors: recovery and storage 
quality 
Donor Iron Deficiency Study: Protocol version 2.5 
CUMC IRB-AAAQ8875 
 
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2. Objectives 
 
2.1 Primary Hypothesis 
1) The 24-hour post-transfusion RBC recovery of units 
obtained from donors exhibiting iron-deficient 
erythropoiesis will not meet FDA standards for clinical 
use. 
2) The 24-hour post-transfusion RBC recovery of units 
obtained after intravenous iron repletion will improve 
significantly and will meet FDA standards for clinical use. 
2.2 Secondary Hypothesis 
1) The extent of iron-deficient erythropoiesis, as 
measured by RBC zinc protoporphyrin levels, will predict 
the 24-hour post-transfusion RBC recovery. 
2) There will be a good correlation (R2>0.5) between RBC 
zinc protoporphyrin levels and other markers of iron 
status: ferritin, hepcidin, soluble transferrin receptor, 
reticulocyte hemoglobin. 
3) Iron repletion will improve fatigue score36 and self-
reported health and wellbeing score.37 
4) Iron repletion will decrease RBC zinc protoporphyrin 
and soluble transferrin receptor levels. 
5) Iron repletion will increase hepcidin, ferritin, and 
hemoglobin levels. 
6) Iron repletion will improve the following neurocognitive 
scores: 
 a) Cognitive Function Composite Score 
b) Executive Function  
c) Attention  
d) Episodic Memory 
e) Language 
f) Processing Speed 
g) Working Memory 
7) Iron repletion will improve the following emotional well-
being scores: 
 a) quality of life 
 b) fatigue 
 c) depression 
 d) anxiety 
8) Metabolite levels in the RBC unit will correlate with iron 
status and 24-hour post-transfusion RBC recovery. 
3. Study Population 
 
3.1 Enrollment Inclusion Criteria: 
1) 18-75 years old;  
2) healthy (by self report);  

 
 
    
Red blood cells from iron-deficient 
donors: recovery and storage 
quality 
Donor Iron Deficiency Study: Protocol version 2.5 
CUMC IRB-AAAQ8875 
 
12 
3) body weight >110 lbs;  
4) female hematocrit >38%, male hematocrit >39%;  
5) frequent blood donor (men ≥2 and female ≥1 RBC unit 
donations in past year);  
6) ferritin <15 ng/mL;  
7) zinc protoporphyrin >60 µmol/mol heme. 
 
3.2 Enrollment Exclusion Criteria: 
 
 
1) ineligible for donation based on the New York Blood 
Center donor autologous questionnaire;  
2) C-reactive protein >10 mg/L;  
3) sickle cell trait (by self report);  
4) systolic blood pressure >180 or <90 mm Hg, diastolic 
blood pressure >100 or <50 mm Hg;  
5) heart rate <50 or >100;  
6) temperature >99.5°F prior to donation (attempts will be 
made to reschedule donation if possible);  
7) temperature >100.4°F or subjective feeling of illness 
prior to 51Cr 24-hour RBC recovery study (to avoid the 
subject having a concurrent illness that may affect post-
transfusion recovery); (attempts will be made to 
reschedule donation and 51Cr 24-hour RBC recovery 
study); 
8) positive results on standard blood donor infectious 
disease testing;  
9) pregnancy;  
10) taking, or planning to take, iron supplements and not 
willing to stop for duration of study;  
11) history of severe asthma requiring hospitalization, 
allergic eczema (atopic dermatitis), or other atopic allergy 
causing anaphylaxis.  
4. Trial Enrollment 
 
4.1 Screening/Recruitment 
The New York Blood Center study staff will send a recruitment 
letter/email (see Appendix 1) to potential subjects who are 18-
75 years old and frequent blood donors donating in the 
Manhattan catchment area. We will define frequent blood 
donors as (1): men who have donated the equivalent of at 
least 2, and women who have donated the equivalent of at 
least 1, RBC units in the past year.  
 
Volunteers responding to the recruitment email will be 
screened for participation in the study by phone or email (see 
Appendix 7 for all screening forms) and then invited for a 

 
 
    
Red blood cells from iron-deficient 
donors: recovery and storage 
quality 
Donor Iron Deficiency Study: Protocol version 2.5 
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screening meeting (P&S 14-434) to confirm eligibility and 
provide informed consent.  
Screening day (P&S 14-434): All volunteers will be given the 
health questionnaire they would receive upon donation at the 
New York Blood Center (see Appendix 7). All volunteers who 
decide to proceed with screening and sign consent will have 
four tubes of blood drawn to determine baseline laboratory 
values and to perform a blood type and antibody screen. If the 
baseline hematocrit is >38% (female) or >39% (male), ferritin 
< 15ng/mL, zinc protoporphyrin level is > 60 umol/mol heme, 
and C-reactive protein is <10mg/L (see inclusion/exclusion 
criteria above) then the volunteer will be scheduled for an 
autologous whole blood donation at the New York Blood 
Center, and scheduled for an autologous 51-Chromium red 
cell recovery study 6 weeks after donation. At that time, 
neurocognitive assessments and emotional wellbeing will be 
performed as well (see Appendix 2-5 for surveys and section 
5.4). Re-screening: Those subjects who meet the ferritin 
<15ng/mL and the zinc protoporphyrin > 60umol/mol heme 
criteria, but do not qualify due to a low hematocrit may be re-
screened between 2 weeks to 3 months later. 
4.2 Randomization 
Randomization will be performed using a computerized 
system with equal allocation (1:1) to iron repletion or placebo. 
Randomization will be stratified by gender; randomly permuted 
block sizes of 4, 6, or 8 will be used. The moment of 
randomization will be recorded and will occur only after 
successful completion of the first post-transfusion RBC 
recovery study (i.e., between Day #43-71; target is the day 
after the post-transfusion recovery study; performed on 
Harkness Pavillion 10). Volunteers will be admitted to the 
Columbia General Clinical Research Center (GCRC) 
(supported by the Columbia Clinical and Translational Science 
Award (CTSA)). One peripheral intravenous line will be 
inserted and blood (2 tubes, 10mL) will be drawn for 
determining the 24-hour post-transfusion recovery. The 
subject will be randomized at this point. The randomization 
scheme will be created by a statistician unaffiliated with the 
study team and provided to the research pharmacy and DSMB 
prior to study initiation. The CUMC research pharmacy will be 
responsible for randomizing each subject to the appropriate 
treatment arm and will prepare either the iron treatment or 
saline placebo and provide it to the study team in a blinded 

 
 
    
Red blood cells from iron-deficient 
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quality 
Donor Iron Deficiency Study: Protocol version 2.5 
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14 
fashion. If a subject arrives on the day of randomization and 
has a fever (T>100.4F) or feels ill, then the 24-hour post-
transfusion recovery sample will be drawn, but randomization 
may be held until the subject feels better and reschedules. 
4.3 Blinding 
The randomized group will only be known to the Columbia 
University Medical Center Research Pharmacy and the 
statistician who generated the randomization scheme. The 
study will be double-blind. As described in section 5.3, both a 
test dose of the iron treatment/placebo and the 
treatment/placebo will be blinded. The test dose will be 
administered prior to giving the full dose to ensure safety of 
treatment. A research pharmacist will provide the placebo (IV 
saline) or treatment (IV low molecular weight iron-dextran or 
ferric carboxymaltose) and test dose of iron/placebo in a tinted 
infusion bag and tubing specifically designed to maintain 
blinding in clinical research studies (Medipak). A research 
nurse unaffiliated with the study team, from the Outpatient or 
Inpatient Center at the Columbia General Clinical Research 
Center (Harkness Pavillion 10th Floor), will be responsible for 
the test infusion and total dose iron infusion. With this design, 
volunteers and study investigators will be blinded to whether 
volunteers receive the active intervention or placebo. Subjects 
in both groups will receive similar discharge instructions as if 
they had received low molecular weight iron-dextran (see 
Appendix 6 for discharge instructions). Only the research 
pharmacist and the statistician on the DSMB will not be 
blinded. Scheduling and logistic communications with 
volunteers will be made by the study coordinator, who will also 
be blinded to the treatment group. The research pharmacy will 
provide a standard 1-gram dose of low molecular weight iron-
dextran, ferric carboxymaltose, or saline in special bags 
designed to conceal group assignment.  
Situations may arise in which breaking the blind earlier would 
be in the best interest of the volunteer. In any situation in 
which a physician or the subject asks to be un-blinded to study 
treatment, a sealed copy of the randomization scheme for 
each subject will be kept in the investigator’s office and will be 
broken upon request. Furthermore, the research pharmacy 
can be reached on an emergency basis to provide this 
information. Finally, any un-blinding that occurs will be 
reported to the DSMB and ultimately reported in the resulting 
publication. 
 

 
 
    
Red blood cells from iron-deficient 
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Donor Iron Deficiency Study: Protocol version 2.5 
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15 
5. Interventions 
 
5.1 Blood Donation: 
The blood donation will be performed at the New York Blood 
Center at 310 East 67th Street, New York, NY per standard 
protocol for autologous RBC donations. A urine pregnancy test 
will be performed on all female participants < age 55 on the 
day of donation. A positive pregnancy test will result in 
exclusion from the study (see Appendix 7 for donation day 
eligibility form). At the New York Blood Center, subjects will be 
screened per the standard protocol for autologous RBC 
donations. This will include health questionnaire, finger stick 
hemoglobin determination, temperature, heart rate and blood 
pressure measurement. Following donation, the blood will be 
leukoreduced, packed, and stored in AS-3 solution. The unit 
will be transferred to the CUMC-NYPH blood bank for storage 
following standard hospital procedures.  
 
5.2 51Chromium 24-hour post-transfusion RBC recovery 
study 
Volunteers will be admitted to the Columbia General Clinical 
Research Center (GCRC) (supported by the Columbia Clinical 
and Translational Science Award (CTSA) on Harkness 
Pavillion 10). A urine pregnancy test will be performed on all 
female participants < age 55 years on the day of infusion. A 
positive pregnancy test will result in exclusion from the study 
(see Appendix 7 for infusion eligibility form). Two intravenous 
lines will be placed in contralateral arms. Four tubes of blood 
will be drawn to determine laboratory values and to perform a 
repeat blood type and antibody screen. If the subject chooses 
to come in for an optional blood draw within 30 days of this 
study visit, only three tubes will be drawn as the type and 
screen will not be necessary. 
A 30 mL aliquot of the autologous blood unit donated 6 
weeks prior will be removed using sterile technique by a 
licensed radiopharmacist into a syringe. This syringe will be 
labeled with the volunteer's identifying information per hospital 
policy and radiolabeled in the Kreitchman PET Center. The 
radiolabeling will be performed using 20 microcurie of sodium 
chromate (51Cr) based on the methods of Moroff et al4 and the 
recommendations from the International Committee for 
Standardization in Hematology.38 The 51Cr labeled red cells 
will be washed with saline and then infused intravenously 

 
 
    
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(over 1 minute through one IV line) into the volunteer. For 
calculating counts per minute per ml of RBC at time zero (T0), 
samples (two tubes, 10 mL) from the contralateral arm will 
also be obtained after 5, 7.5, 10, 12.5, 15 minutes, 30 minutes, 
and 1-hour after infusion. Thus, in total, the volunteers will be 
infused approximately 30mL of packed radiolabeled RBCs and 
will have 16 tubes or 80 mL of blood drawn overall (8 time 
points x 2 tubes x 5mL per tube = 80 mL). All blood draws will 
be performed from the peripheral intravenous line to avoid 
multiple needle sticks. Finally, because 30mL of blood will be 
infused and only 80mL of blood removed, this will leave the 
volunteer with a net loss of only 50mL of blood on the day of 
infusion. Following the 1-hour timed blood draw, the subject 
will be discharged. 
 
5.3 Low Molecular Weight Iron Dextran (LMWID) or Ferric 
Carboxymaltose (INJECTAFER) Infusion 
Volunteers will be admitted to the Columbia General Clinical 
Research Center (GCRC) (supported by the Columbia Clinical 
and Translational Science Award (CTSA) on Harkness 
Pavillion 10). One peripheral intravenous line will be inserted 
and blood (2 tubes, 10mL) will be drawn for determining the 
24-hour post-transfusion recovery. The allowable time frame 
for this blood draw will be from 20-28 hours of infusion the 
prior day. The subject will be randomized from this point to 4-
weeks later by the research pharmacy. A research pharmacist 
will provide the placebo (IV saline) or treatment (IV low 
molecular weight iron-dextran or ferric carboxymaltose) and 
test dose (25 mg LMWID, 12.5 mL of a 2 mg/mL solution of 
iron dextran or 0.5 mL of 50 mg/mL solution of ferric 
carboxymaltose, diluted in normal saline) in tinted infusion 
bags and tubing specifically designed to maintain blinding in 
clinical research studies (Medipak). If low molecular weight 
iron dextran is unavailable due to shortages, the research 
pharmacist will provide ferric carboxymaltose instead. A 
research nurse unaffiliated with the study team, from the 
Outpatient or Inpatient Center at the Columbia General 
Clinical Research Center (Harkness Pavillion 10th Floor), will 
be responsible for the test infusion and total dose iron infusion. 
The research pharmacy will provide a standard 1-gram dose 
of low molecular weight iron-dextran, ferric carboxymaltose, or 
saline in special bags designed to conceal group assignment. 
In addition, the clinical pharmacy will provide Solumedrol 125 

 
 
    
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mg IV both before and after infusion and Tylenol 650mg PO 
and Benadryl 25mg PO before the infusion. The Solumedrol 
has been shown to reduce the risk of myalgias and arthralgias 
after LMWID infusions.39 Premedication with Tylenol and 
Benadryl have also been shown to be effective in reducing 
adverse events,40 and this is the current standard of care at 
Columbia University Irving Medical Center for adults receiving 
LMWID. After the intravenous test dose (25 mg of LMWID or 
ferric carboxymaltose, 12.5 mL of the complete dose, infused 
over 20 minutes), patients will be observed for any side effects 
for 40 minutes (one hour from start of infusion); if no adverse 
effects are seen, then the entire dose diluted in 500 mL normal 
saline (i.e., 2 mg/mL of LMWID) will be infused over a period 
of 2 to 6 hours as tolerated (target 2 hours). If ferric 
carboxymaltose is used, the same test dose and the full 1 
gram dose will be administered in 250 mL of normal saline 
over the same time frame. The same premedication will be 
provided to minimize differences in treatment effects. Adverse 
events will be identified by observation, direct inquiry, and 
physical examination of each volunteer. Vital signs will be 
measured before, during (after 15 minutes and then hourly), 
and after each infusion. Resuscitation equipment and 
personnel trained in the detection and treatment of 
anaphylactic-type reactions will be readily available during 
drug administration. 
 
Recent studies support the convenience, safety, and 
efficacy of a single infusion of 1g of LMWID as therapy for iron 
deficiency in adults.41 The calculation for iron repletion using 
LMWID (from INFeD package insert) in adults is as follows: 
Dose (mL) = 0.0442 (Desired Hb - Observed Hb) x LBW + 
(0.26 x LBW) 
 
Based on:  
Desired Hb = the target Hb in g/dl. 
Observed Hb = the patient’s current hemoglobin in g/dl. 
 
LBW = Lean body weight in kg. A patient’s lean body weight 
(or actual body weight if less than lean body weight) should be 
utilized when determining dosage. 
 
For males: LBW = 50 kg + 2.3 kg for each inch of patient’s 
height over 5 feet 

 
 
    
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For females: LBW = 45.5 kg + 2.3 kg for each inch of patient’s 
height over 5 feet 
To calculate a patient’s weight in kg when lbs are known: 
patient’s weight in pounds = weight in kilograms/2.2 
 
Using this formula, the minimum dose that would be required 
to raise the hemoglobin by 2 g/dL would be ~800mg (minimum 
donation requirement is 110lbs = 50 kg) in a female and 
almost 900mg in a male. Thus, given that all subjects will be 
healthy volunteers with ferritin < 15 ng/mL with evidence of 
iron-deficient erythropoiesis, and will be dosed following a 
whole blood donation for study purposes, 1 gram of LMWID 
was chosen for simplicity, safety, consistency, and design 
considerations. Based on similar considerations, 1 gram of 
ferric carboxymaltose would provide equivalent total dose iron 
repletion with a similar safety profile.42  
 
5.4 Cognitive and Emotional Status Assessment 
 
As outlined in the Schedule of Measurements section below, 
cognitive and emotional functioning will be assessed with a 
thorough battery of neuropsychological tests and 
questionnaires assessing overall wellbeing. Tests will be 
administered in person by a psychometrician who has 
received training on test administration.   
 
Neuropsychological tests will be administered to subjects on 
an iPad or Tablet.  Subjects will be supervised at all times 
during this portion of the evaluation and will be encouraged to 
complete the tests within one session (i.e. without 
interruption). Total time for test administration is approximately 
30 minutes and the time of start and completion of the test 
battery will be recorded.  When the neuropsychological battery 
has been completed, the subject will be provided with a 
clipboard containing printed copies of four questionnaires (see 
appendices 4-7). The subject will be asked to complete the 
surveys on his/her own without interruption. The time of start 
and completion of the surveys will be recorded. 
 
Neuropsychological tests included were selected based on the 
following criteria: All (1) meet high psychometric standards, 
(i.e. are reliable, valid, and well standardized); (2) have 
appropriate normative data; (3) maintain the lowest potential 

 
 
    
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for patient burden, e.g. duration; (4) can be administered 
serially without significant practice effects. 
5.4.1 Cognition  
 
Subjects will be administered subtests from the NIH 
Toolbox Cognition Battery (NIHTB-CB), a computerized 
battery of neuropsychological tests. The NIH Toolbox 
provides a standard set of royalty-free, comprehensive 
assessment tools that can be used by researchers and 
clinicians in a variety of settings, with a particular 
emphasis on measuring outcomes in longitudinal 
epidemiologic studies and prevention or intervention 
trials. The battery has been normed and validated 
across the lifespan in subjects age 3-85 and its use 
ensures that assessment methods and results can be 
used for comparisons across existing and future 
studies. By providing a “common currency” for the study 
of neurological research, the NIH Toolbox enables 
economies of scale and enhances efficiency. The NIH 
Toolbox is capable of monitoring neurological and 
behavioral function over time, and measuring key 
constructs across developmental stages.  
 
Scores from the NIH Toolbox Cognition Battery will 
produce a Cognitive Function Composite Score.  
Individual measure scores reflecting Executive 
Function, Attention, Episodic Memory, Language, 
Vocabulary, Processing Speed and Working Memory 
will also be obtained. 
 
NIH Toolbox Cognition Battery (NIHTB-CB)   
Vocabulary 
NIH Toolbox Picture Vocabulary Test 
 
Memory  
NIH Toolbox Auditory Verbal Learning Test (Rey) 
NIH Toolbox Picture Sequence Memory Test 
 
Attention/Executive Functioning 
NIH Toolbox Flanker Inhibitory Control and Attention 
Test 
NIH Toolbox Dimensional Change Card Sort Test 
(DCCS) 

 
 
    
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Processing Speed 
NIH Toolbox Pattern Comparison Processing Speed 
Test 
  
Working Memory 
NIH Toolbox List Sorting Working Memory Test 
  
5.4.2 Emotion  
 
Subjects will complete four paper and pencil self-administered 
questionnaires to evaluate quality of life, fatigue, depression, 
and anxiety.   
 
Short Form 36 Health Survey (SF-36) 
36-item, patient-reported survey assessing overall health 
status and quality of life (Appendix 4).43 
 
Multidimensional Assessment of Fatigue (MAS) 
16 item scale used to measure fatigue according to four 
dimensions:  degree/severity, distress that it causes, timing of 
fatigue, and its impact on ADLs (Appendix 5).44 
 
Beck Depression Inventory-II (BDI-II) 
21 item self-report multiple choice inventory used to assess for 
depression (Appendix 6).45 
 
Beck Anxiety Inventory (BAI) 
21-question multiple-choice self-report inventory that is used 
for measuring how the subject has been feeling in the last 
week, focusing primarily on somatic symptoms (Appendix 7).46  
 
5.4.3 Hearing 
 
Words-In-Noise (WIN) Test 
Measures a person’s ability to recognize single words amid 
varying levels of background noise, measuring difficulty a 
person might have hearing in a noisy environment. A recorded 
voice tells the participant to listen and repeat words. 
Background noise gets louder, reducing the signal-to-noise 
ratio.  
Hearing Handicap Inventory Screening 
10 questions to assess hearing impairment. 

 
 
    
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5.4.4 Restless Leg Syndrome (RLS) 
 
Restless Leg Syndrome Rating Scale 
10 questions to assess restless leg symptoms. 
 
 
6. Schedule of Measurements 
 
 
6.1 Summary Timeline 
The schedule of measurements is summarized in Table 1. 
Following successful screening and signed informed consent, 
subjects will donate a unit of autologous red blood cells 
(Donation1). This will be followed 40-42 days later by the 
baseline 51-Chromium post-transfusion recovery study. 
Following the 24-hour post-chromium infusion blood draw, the 
subjects will be randomized to iron infusion or placebo (Iron 
infusion1). Five months ± 4 weeks later, subjects will donate 
the second unit of autologous blood (Donation2). The second 
51-Chromium post-transfusion recovery study will be 
performed 40-42 days after this donation. Subjects will have to 
return 24-hours after chromium infusion to determine the 24-
hour post-transfusion recovery. Surveys and neurocognitive 
assessments will be conducted prior to Donation1, PTR1, 
Donation2, and PTR2. 
Because an active type and screen must be obtained 
before the blood bank issues the stored RBC unit and the 
radioactive 51-Cr-labeling can occur, we will provide subjects 
with the option of coming in up to 30 days prior to the 
scheduled PTR study to obtain a type and screen sample (1 
tube of blood by standard venipuncture). This will provide for a 
shorter day for the PTR study because there will no longer be 
the need to wait for the type and screen testing and the study 
can begin once the subject arrives. If a subject chooses not to 
provide this optional blood draw, a blood sample will be taken 
when the IV lines are placed for the PTR study and the PTR 
study will proceed after the blood bank issues the blood and it 
is radiolabeled per hospital guidelines.  
 
 
 
 
 

 
 
    
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Table 1: Schedule of Measurements 
Day of 
study 
Urine 
pregnancy 
test 
CBC with 
reticulocy
te count 
Iron 
paramet
ers*  
C-
reactive 
protein 
Type 
and 
Screen 
51-Cr 
PTR** 
Iron/Placebo 
infusion 
Surveys/ 
Neuro-
cognitive 
Metabolo
mics**** 
Screening 
 
X 
X 
X 
X 
 
 
 
 
Donation1  
X 
X 
X 
X 
 
 
 
X 
X 
Blood draw 
(optional)*** 
 
 
 
 
X 
 
 
 
 
PTR1 (40-
42 days 
after 
Donation1) 
X 
X 
X 
X 
X 
X 
 
X 
 
Iron 
infusion1  
(1 day after 
PTR1) 
X 
 
 
 
 
 
X 
 
 
Donation2 
(5months ± 
4 weeks 
after iron 
infusion1) 
X 
X 
X 
X 
 
 
 
X 
X 
Blood Draw 
(optional)*** 
 
 
 
 
X 
 
 
 
 
PTR2 (40-
42 days 
after 
Donation2) 
X 
X 
X 
X 
X 
X 
 
X 
 
Final blood 
draw (1 day 
after PTR2) 
 
 
 
 
 
X – 
collectio
n of 24 
hour 
(+/- 4hr) 
time 
point for 
final 
measur
ement 
 
 
 
*Iron parameters = Zinc Protoporphyrin (ZPP), iron, total iron binding capacity (TIBC), transferrin 
saturation, ferritin, soluble transferrin receptor, hepcidin. 
**PTR = post-transfusion recovery study with associated blood draws for hemoglobin and 
Chromium-51 determination taken pre-infusion and 5, 7.5, 10, 12.5, 15, 30, 60 minutes and 24-
hours post-infusion (+/- 4 hours). 
***The option to come in up to 30 days prior to the PTR study will be provided to obtain a type and 
screen sample. This will provide an active type and screen specimen so that the PTR study can 
begin without delay. Alternatively, the type and screen blood sample will be drawn when the IV is 
paced for the PTR study and then the study will begin once the type and screen is tested and the 
blood is issued and labeled. 
****Metabolomics will be performed by mass spectrometry on blood obtained from the unit at the 
time of radiolabeling by metabolomics facilities at BloodWorks NW and University of Colorado.  
 
 
 
 
 
 

 
 
    
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6.2 Assessment Procedures 
  
 
6.2.1 Zinc Protoporphyrin, Serum Iron, Total Iron Binding 
Capacity, Transferrin Saturation, Ferritin, C-Reactive 
Protein, Soluble Transferrin Receptor 
Samples for zinc protoporphyrin and soluble transferrin 
receptor will be sent to ARUP Laboratories for testing. The 
other parameters will be measured in the Columbia University 
Medical Center Automated Core Laboratory using clinically-
validated instruments. 
 
6.2.2 Hepcidin 
This will be measured using an ELISA kit (Bachem) following 
the manufacturer’s instructions. 
 
6.2.3 Complete Blood Counts 
Blood counts will be performed in the Columbia University 
Medical Center Automated Core Laboratory using clinically-
validated instruments. 
 
6.2.4 Urine Pregnancy Test 
Point of care urine pregnancy testing will be performed using 
QuickVue One-Step hCG Urine tests (Quidel Corp) following 
the manufacturer’s instructions. 
 
6.2.5 Type and Screen Testing 
All type and screen testing will be performed by licensed 
medical technologists in the Columbia University Medical 
Center – New York Presbyterian Hospital blood bank following 
standard procedures. 
 
6.2.6 Metabolomics Testing 
Comprehensive metabolomics testing will be performed for 
research use only at the metabolomics core facilities at the 
University of Denver and Bloodworks NW using coded 
specimens. CUIMC investigators will not share identifying 
information with the collaborating investigators.  
 
7. Specimen Collection Procedures 
All blood samples will be obtained by peripheral venipuncture or from 
an indwelling peripheral intravenous line following institutional 
protocols. 
 

 
 
    
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8. Compensation 
Subjects will be provided monetary compensation in cash for 
participation per visit. $20 will be provided for the screening. $10 will 
be provided for re-screening for those who meet criteria to be invited 
to be screened again. $80 will be provided for each donation and 
PTR study ($320 total). $80 will be provided for the iron/placebo 
infusion and for the final blood draw at the end of the study ($160 
total). Finally, $40 will be provided for each of the optional blood 
draws. Thus, the maximum total compensation for subjects is $590 
and the minimum compensation is $500 for completing the study. 
Subjects who dropout of the study prior to completion will keep any 
compensation received up to the dropout. 
 
9. Adverse Event Criteria and Reporting 
 
9.1 Definitions 
Adverse Event (AE) – Any untoward or unfavorable medical 
occurrence in a human subject, including any abnormal sign 
(for example, abnormal physical exam or laboratory finding), 
symptom, or disease, temporally associated with the subject’s 
participation in the research, whether or not considered related 
to the subject’s participation in the research. 
 
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; limiting age-appropriate instrumental ADL. 
 
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. 
 
Serious Adverse Event (SAE) – Any adverse event 
temporally associated with the subject’s participation in 

 
 
    
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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; 
results in a persistent or significant disability/incapacity; results 
in a congenital anomaly/birth defect; or 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. 
 
Note that seriousness and severity are separate concepts. 
The term “severe” refers to the intensity of a specific event; a 
severe event may be of minor medical significance (e.g., a 
severe leg cramp). The term “serious” is based on outcome or 
action criteria that are usually associated with events that pose 
a threat to the patient’s life or functioning. An event that is mild 
in severity is serious if it leads to one of the outcomes defined 
above. 
 
Grade 4 and 5 events will always be considered Serious 
Adverse Events. Many Grade 3 events and some Grade 1 and 
2 events may meet the definition of a Serious Adverse Event. 
 
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, 

 
 
    
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severity, or frequency) given (a) the research procedures that 
are described in the protocol-related 
 
Attribution – the determination of whether an adverse event 
is related to a medical treatment 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 
 
9.2 Types of Adverse Events Reported 
 
Strict definitions and monitoring protocols of adverse effects 
(AEs) and serious AEs (SAEs) will be developed with the Data 
Safety Monitoring Board (DSMB). An SAE defined as being 
one or more of the following: (i) life threatening, (ii) results in 
hospitalization, or (iii) causes irreversible, persistent, or 
significant disability/incapacity; these will be reported to 
regulatory agencies within 7 days of their occurrence. Any AE 
or SAE temporally related with study procedures will be 
reported by the site investigators or coordinators. An alert will 
be sent to the DSMB, and the Institutional Review Board and 
Sponsor (NIH-NHLBI). Safety outcomes will be assessed at 
each study visit and with a follow-up phone call the day after 
each visit using a checklist of known adverse events and an 
open-ended question for volunteers to describe other adverse 
events. Participants will be encouraged to call the coordinator 
throughout the study if they experience 1) a new symptom, 2) 
change in severity of an existing symptom, 3) see a doctor/ER, 
4) start a new medication. The most common serious acute 
adverse reaction of blood donation and insertions of 
intravenous lines is a vasovagal reaction, which may lead to 
loss of consciousness. Witnessed or un-witnessed vasovagal 
reactions will be considered as AEs, unless resulting in 
hospitalization (e.g., due to loss of consciousness causing 

 
 
    
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head trauma), in which case the event will be considered an 
SAE. Permanent or persistent peripheral arm nerve damage 
(in a phlebotomized arm) causing disability will be considered 
an SAE. Bruising and pain at the site of needle insertion that 
results in volunteer complaint at the follow-up phone call a day 
after phlebotomy will be considered an AE. Anaphylactoid 
reactions to iron infusions are usually evident within a few 
minutes, and close observation is necessary to ensure 
recognition. If at any time during the intravenous 
administration, any signs of a hypersensitivity reaction or 
intolerance are detected, administration will be stopped 
immediately. The reaction will be considered an SAE if it 
meets one of the criteria listed above. Furthermore, subjects 
will be called one day and one week after infusion to inquire 
about delayed side effects, such as myalgias, arthralgias, and 
gastrointestinal problems. Finally, iron deficiency is associated 
with fatigue, restless leg syndrome, decreased physical 
endurance and work capacity, and impaired concentration, 
attention, and other cognitive functions. Thus, subjects 
randomized to the placebo group may have AEs due to 
continued iron deficiency. These will be assessed using the 
validated Multidimensional Assessment of Fatigue, Beck 
Depression Inventory-II, Beck Anxiety Inventory, and the 
overall well-being SF-36 Health Survey incorporating both 
physical and mental summary measures. Each subject will 
take these short surveys as outlined in Table 1.  
 
Medical follow-up of any Serious Adverse Event will be 
provided by the medical staff of Columbia University Medical 
Center in the New York Presbyterian Hospital or, if needed, in 
the out-patient clinic at appropriate intervals until resolution of 
the condition related to the Serious Adverse Event. An 
Adverse Event that does not meet the criteria of a Serious 
Adverse Event will be reported promptly to Drs. Spitalnik and 
Hod, appropriate management will be provided by the medical 
staff at Columbia University Medical Center, and the 
occurrence of any adverse events reviewed periodically by Dr. 
Spitalnik, Dr. Hod, and their Co-Investigators. A Data Safety 
Monitoring Board will be established and will review all 
reported events at least annually and all serious adverse 
events within one month of occurrence. A report summarizing 
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recommendations will be submitted to the Institutional Review 
Board. 
 
9.3 Reporting timelines 
 
Table 2 below details the SAE and UP safety reporting 
requirements and timelines. Reporting timelines for all non-
serious AEs will follow the Data and Safety Monitoring Plan for 
the study. 
 
Table 2: SAE and UP event reporting timelines 
What Event is 
Reported 
When is Event 
Reported 
By Whom is 
Event 
Reported 
To Whom is Event 
Reported 
Fatal or life-
threatening 
unexpected, 
suspected serious 
adverse reactions 
Within 7 calendar 
days of initial receipt 
of information 
Investigator 
Local/internal IRBs 
and Data Safety 
Monitoring Board 
(DSMB) 
Sponsor  
NHLBI 
Non-fatal, non-life-
threatening 
unexpected, 
suspected serious 
adverse reactions 
Within 15 calendar 
days of initial receipt 
of information 
Investigator 
Local/internal IRBs and 
Data Safety Monitoring 
Board (DSMB) 
Sponsor  
NHLBI 
Unanticipated adverse 
effects 
Within 10 working 
days of investigator 
first learning of effect 
Investigator 
Local/internal IRBs and 
Data Safety Monitoring 
Board (DSMB) 
Sponsor  
Local/internal IRBs and 
Data Safety Monitoring 
Board (DSMB) 
Unanticipated Problem 
that is not an SAE 
Within 14 days of the 
investigator 
becoming aware of 
the problem 
Investigator 
Local/internal 
IRBs/Institutional 
Officials, 
NHLBI and/or DCC 
All Unanticipated 
Problems1 
Within 30 days of the 
IRB’s receipt of the 
report of the UP from 
the investigator. 
IRB 
OHRP 
Investigator 
External IRBs 

 
 
    
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1. Per OHRP guidance: only when a particular AE or series of AEs is 
determined to meet the criteria for an UP should a report of the AE(s) be 
submitted to the IRB at each institution under the HHS regulations at 45 
CFR part 46. Typically, such reports to the IRBs are submitted by 
investigators. 
 
 
10. Interim Reporting 
 
This section describes scheduled reports that will be sent to the 
DSMB. Reporting requirements for events that will be monitored 
continuously (i.e. all fatal events and all serious adverse events 
possibly, probably, or definitely related to red cell transfusion) are 
described above. 
 
 
10.1 Semi-Annual DSMB Reports 
The DSMB will meet twice a year, either in-person or via 
teleconference. Reports will include:  
• Baseline characteristics overall and by group  
• Primary study endpoint overall and by group, (p-values to 
compare to early stopping boundary will only be provided after 
20, 40, and 60 volunteers have been completed)  
• Number, type, and severity of serious adverse events, overall 
and by treatment arm, with p-value for comparison of number 
of serious adverse events per group. This p-value will be 
compared to the p-value boundary from an alpha-spending 
approach approximating O’Brien-Fleming boundaries. No 
formal stopping rule is set for this comparison.  
• Number, type, and severity of adverse events attributed as 
possibly, probably, definitely related to donation, iron or red 
cell infusion, overall and by treatment arm, with p-value for 
comparison of number of such adverse events per group. This 
p-value will be compared to the p-value boundary from an 
alpha-spending approach approximating O’Brien-Fleming 
boundaries. No formal stopping rule is set for this comparison.  
• Proportion of subjects in each treatment arm with at least one 
serious adverse event possibly, probably, or definitely related 
to a donation, red cell or iron infusion, with p-value. This p-
value will be compared to the p-value boundary from an alpha-
spending approach approximating O’Brien-Fleming 
boundaries. No formal stopping rule is set for this comparison.  
• Unexpected adverse events, and unanticipated problems 
overall and by treatment arm  
• Site status  

 
 
    
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• Accrual  
• Site/study compliance issues 
• Data entry completeness and data QA 
 
 
11. Statistical Considerations 
 
 
11.1 Analysis Plan 
The primary null hypothesis will be tested in an intent-to-treat 
analysis using a t-test, or nonparametric equivalent, of the between-
group difference in means of the within-subject change in the post-
transfusion RBC recovery from the initial study (under iron-deficient 
erythropoiesis conditions) and the subsequent study performed after 
randomization to iron repletion or placebo. A similar approach will 
be used to test the secondary objectives (i.e., the between-group 
difference in means of the within-subject change in fatigue score,36 
self-reported health and wellbeing score,37 zinc protoporphyrin, 
hepcidin, soluble transferrin receptor, serum ferritin and hemoglobin 
concentration). 
 
Furthermore, because we expect variable responses to iron 
repletion in the experimental group, and some crossover in the 
placebo group, we will also perform a secondary analysis to explore 
the effect of iron status on post-transfusion RBC recovery. We will 
use multiple regression to assess whether RBC zinc protoporphyrin 
level increases the R2 of a model of post-transfusion recovery 
predicted by treatment group membership. We will examine pre-
specified demographic variables (gender [male/female], race 
[white/not white], age [<50/≥50 years]) to see if we can identify one 
or more that may be effect modifiers. These factors will be 
considered for inclusion in an adjusted model. The specific criteria 
for inclusion are: (i) difference by treatment group significant at α = 
0.10 two-sided, and (ii) related to outcome at level α = 0.10 two-
sided. If any of the pre-specified covariates meet the criteria for 
inclusion, they will be incorporated in an adjusted model, and that 
model will become the primary analysis. Otherwise, the simple 
model will be primary. With 60 subjects, we will have 80% power to 
detect a partial correlation coefficient increase of 0.33 for the unique 
contribution of RBC zinc protoporphyrin level.47    
 
 
11.2 Sample Size Estimate 
Based on preliminary data from our prior 51Cr RBC recovery 
studies (IRB protocol #AAAI-0835), the standard deviation of 
the measure in our single site is 5.0%. Furthermore, the 
expected mean difference in post-transfusion RBC recovery 

 
 
    
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between iron replete mice and mice with iron-deficient 
erythropoiesis is 10.6%. If the difference were this large in 
humans, we would require <6 subjects (alpha = 0.05, two-
sided, power = 0.80). However, we expect the difference to be 
less conspicuous in humans than in inbred mice. Thus, we 
power the study to detect a clinically relevant difference in 
post-transfusion recovery of 4%. Under this assumption, the 
calculated sample size required for each arm is 26 (alpha = 
0.05, two-sided, power = 0.80). Furthermore, to allow for up to 
a 15% dropout rate (i.e., 4/26 subjects), we plan to randomize 
30 subjects per arm for a total sample size of 60 subjects. Of 
note, randomization will occur after successful completion of 
the first post-transfusion recovery study; thus, we predict up to 
70 individuals will need to be recruited. 
 
 
11.3 Interim Analyses 
Interim analyses will be performed twice (after every 20 
subjects have completed study participation) in addition to the 
final analysis. The DSMB will conduct the analyses using a 
two-sided asymmetric Lan-DeMets alpha-spending approach 
with an O’Brien-Fleming two-sided symmetric stopping 
boundary and overall alpha = 0.05. The DSMB criteria for 
early stopping will include: (i) the Z-score at interim analysis 
lies outside of the group sequential boundaries as calculated 
(Table 3); (ii) major safety violations; and (3) convincing 
evidence of futility in the context of AEs. Interim boundaries 
together with terminal criteria (z-scores and associated p-
values) calculated using the WinLD version 2 program are 
provided in Table 3. 
 
Table 3. Lan-DeMets Group Sequential Boundaries Calculations 
Volunteers 
completed 
Lower 
boundary 
Upper 
boundary 
Nominal 
upper 
alpha 
Cumulative 
alpha 
20 
-3.7103 
3.7103 
0.00010 
0.00021 
40 
-2.5114 
2.5114 
0.00601 
0.01210 
60 
-1.9930 
1.9930 
0.02313 
0.05000 
 
 
12. Data Collection and Validation 
Data will be collected and entered into an encrypted web-based data 
management system (DMS). Reports of outstanding edits, generated 
upon completion of data entry, will enable continuous cleaning of the 
data. 
 

 
 
    
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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 office in a cabinet. 
 
Data Management – The principal investigators will monitor timely 
entry of data into the study database. Access to all source 
documentation maintained by the Investigator, including 
correspondence and source data, will be available for monitoring and 
audit purposes. 
 
Data archives – at all times, appropriate backup copies of the 
database and related software files will be maintained and the 
information will be appropriately protected from illegitimate access. 
 
13. Protection of human subjects 
All aspects of this proposed research will be conducted according to 
the defined protocol, relevant FDA regulations, ICH-GCP Guidelines, 
and HIPAA for protection of human subjects under local IRB 
oversight. 
 
14. Investigator Responsibility 
 
14.1 Institutional Review Board (IRB) Approval 
No patient will be enrolled in the study until the Columbia 
University Medical Center and New York Blood Center IRB 
has approved the protocol and the Informed Consent Form. 
Copies of all submissions to and correspondence (approvals 
and disapprovals) from the IRB must be maintained on file at 
the study site. 
 
14.2 Informed Consent  
If a subject is potentially eligible for the study and responds to 
the letter sent by the New York Blood Center, the subject will 
be contacted to be screened and obtain written informed 
consent. The background of the study and the potential 
benefits and risks will be explained. The subject or legally 
authorized representative must sign the consent form that has 
been approved by the IRB prior to enrollment. Failure to obtain 
signed informed consent renders the subject ineligible for the 
study. Copies of the signed informed consent shall be kept in 
the study files.  

 
 
    
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14.3 Subject Data Protection  
Subjects will be identified in the electronic case report form 
(eCRF) by a subject identification number. All information and 
data sent to NHLBI, 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. All subjects enrolled in 
this study will be informed and must agree to the use and 
disclosure of their study information by the institution and 
investigators to NHLBI, their agents and representatives, or 
other review boards. 
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. The 
Investigator will allow regulatory bodies to review the study 
files, subject CRFs, medical records and other study-related 
documents.  
 
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:  
• Laboratory data before enrollment sufficient to verify 
protocol entry criteria 
• Dated and signed notes for specific results of 
procedures and exams  
 
15.3 Record Retention  
NHLBI and the investigators must establish and maintain 
records and reports. The Investigator must maintain the 
signed Informed Consent Forms, CRFs, study documentation 
(listed above) and source documents for at least 3 years and 3 
months after study completion or termination. In addition, the 

 
 
    
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Investigator must not discard or destroy any study-specific 
materials unless otherwise instructed by NHLBI. 
 
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