The αO2-PRBC.Thal Study on the Use of Hemanext One® RBC Storage System for Blood Transfusion Support on Transfusion-dependent Thalassemia
Conditions: Transfusion-dependent Beta-Thalassemia, Blood Transfusion
Sponsor: University Research Institute for the Study of Genetic & Malignant Disorders in Childhood

  
1  
ClinicalTrials.gov Study Document 
 
 
 
Official Study Title: The O2-PRBC.Thal study on the use of Hemanext One 
RBC Storage System for blood transfusion support in transfusion-dependent 
thalassemia 
 
 
 
NCT Number: Pending 
 
 
 
Document Type: Study Protocol and Statistical Analysis Plan Document 
 
 
 
Date: NOV 2025 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

  
2  
Study/Protocol Title: The αO2-PRBC.Thal study on the use of Hemanext One® RBC 
Storage System for blood transfusion support on transfusion-dependent thalassemia   
  
  
Introduction   
Patients with transfusion dependence thalassemia (TDT) are regularly transfused every two to 
four weeks. Their long-term survival and quality of health is dependent on the quality of the 
transfused blood. TDT patients experience significant problems and organ dysfunction due to 
chronic anemia, chronic iron overload as well as long life use of different medications, like 
iron chelators. TDT patients usually receive 2-4 units of red blood cells (RBC) (each unit: 
250300ml), per month.   
  
RBCs storage processing aims to preserve the RBCs’ properties. Long-term storage leads to 
donor-dependent biochemical and morphological changes in RBCs collectively termed as 
“storage lesions”. Transfusion of stored RBCs for more than 1 to 2 weeks is associated with 
increased post-transfusion mortality rates [1-4]. Therefore, there is a need for developing 
optimized processing and storage conditions. Considering the primary functional role of RBCs, 
which is oxygen transport and exchange, focusing on these processes might be a potential way 
for improving storage quality [1,5].  
  
RBCs’ “storage lesions’’ have recently been correlated with metabolic reprogramming [6]. 
Dysregulation of RBCs’ homeostasis disrupts structural lipids and proteins, resulting in 
progressive pathological alterations in RBC morphology, and downregulates RBCs’ survival 
post-transfusion [6]. “Storage lesions” represent a wide spectrum of RBCs’ deformities being 
associated with oxidative damage, manifesting as post-translational changes in proteins (e.g., 
hemoglobin, peroxiredoxin 2, anion exchanger 1, catalase, pyruvate kinase, glucose 6phosphate 
dehydrogenase) and depletion of redox metabolites (e.g., glutathione) [7-13]. Glycolytic 
enzymes, especially glyceraldehyde 3-phosphate dehydrogenase (GADPH), have been shown 
in vitro to mediate this process; oxygen is considered to modulate the competitive binding of 
glycolytic enzymes and deoxyhemoglobin with N-terminal cytosolic domains [14]. 
Specifically, GADPH, is linked to the depletion of 2,3-diphosphoglycerate (2,3-DPG) during 
the first 2 weeks of storage [3,9,15-17]. Oxygen saturation levels are considered to modulate 
the competitive binding of GADPH and deoxyhemoglobin with N-terminal cytosolic domains 
[14]. Under pro-oxidant conditions, hemoglobin turns into its oxygenated substance decreasing 
its binding affinity, while GADPH, migrates to binding sited of the nearby structural proteins. 
Ectopic localization of GADPH to the membrane and binding with structural proteins, has been 
associated with loss of activity, blocking glycolysis, and causing various morphologic and 
functionality defects on the binding proteins [14].  
  
Studies have shown that RBCs stored under hypoxic conditions preserved faster O2 kinetics 
compared with the standard storage protocols, with this difference being reported in the first 
35 days of storage; that is, the longest permissible shelf-life of blood in the United Kingdom 
and many European countries [1]. In clinical practice, the median storage duration of transfused 
units is 21 days. The effects on O2 after RBCs’ storage in hypoxic conditions are similar to the 
ones of biochemical rejuvenation following standard storage – however RBCs’ storage in 
hypoxic conditions presents advantages in terms of easier clinical use, as it can be conducted 
on units in bulk and before dispatch to hospitals [1].  

  
3  
The Hemanext ONE RBC Processing and Storage system limits oxygen, the fuel for 
oxidative damage, thus, providing a higher quality RBCs. It has the potential to benefit all 
patients requiring transfusion for chronic conditions such as thalassemia, sickle cell disease 
(SCD), and myelodysplastic syndromes (MDS), as well as those in need of critical transfusions 
during post-traumatic surgery and other medical procedures [18-26]. Hemanext ONE creates 
hypoxic RBCs, RBCs that have been processed to reduce oxygen and carbon dioxide content 
of RBCs and to maintain this level throughout storage up to 42 days. Hypoxic RBCs have 
demonstrated positive impacts on multiple in vitro metrics of RBC quality in preclinical 
studies. Clinical studies are underway to determine the impact of hypoxic RBCs on patient 
outcomes and estimate potential cost savings from expected improvements in care and 
reductions in transfusion volumes.  
Hemanext ONE has been granted marketing authorization for commercial distribution via the 
De Novo process by the U.S. Food & Drug Administration. In Europe, Hemanext ONE is CE 
marked which allows the medical device to be placed in the market in the European Economic 
Area (EEA).  
Hemanext ONE has not been studied in chronically transfused patients like thalassemia.  
It is imperative to establish the safety and the efficacy, especially in regard to required quantity 
of blood transfusion prepared with the Hemanext ONE processing, before its general 
application to sensitive groups of patients like TDT. In this respect, there are many issues to be 
investigated, like long-term biochemical results and changes in the primary physiological 
function of RBCs in thalassemic recipients.   
  
The aim of our study is to investigate whether RBCs’ storage under hypoxic conditions is at 
least non inferior to standard conditions in providing transfusion support to TDT patients 
without increasing the transfusion burden, the degree of hemolysis and of metabolic 
disturbances, and the rate of iron and erythropoiesis, while improving our patients’ quality of 
life (QOL).  
  
  
Objectives  
• 
To validate the feasibility of applying the Hemanext ONE processing and storage of 
blood units in the routine care of transfusion dependent thalassemic (TDT) patients  
• 
To evaluate the safety of Hemanext ONE in TDT  
• 
To evaluate the changes in transfusion burden when using the Hemanext ONE system  
• 
To evaluate changes in surrogate markers of metabolism, hemolysis, iron overload and 
erythropoiesis, when using the Hemanext ONE system  
• 
Τo assess QOL changes  
 
  
Hypothesis  
The hypothesis is that using the hemanext one process is able to provide transfusion support 
to TDT patients without increasing the transfusion burden, the degree of hemolysis and 
metabolic disturbances, and the rate of iron and erythropoiesis.  
 
Primary Endpoint  
Changes from baseline in RBC transfusion burden. Significant changes are defined as a 
difference of >20% from baseline, corresponding to at least 1 unit/ 24weeks.  
 

  
4  
Secondary Endpoints  
• 
Changes from baseline in pre-transfusion hemoglobin  
• 
Changes on mean total Hb mass 
• 
Changes from baseline in metabolic markers  
• 
Changes from baseline in other markers of hemolysis and erythropoiesis  
• 
Changes in QOL  
• 
Overall changes from baseline in markers of iron overload   
• 
Type, frequency, severity, seriousness of AEs and relationship of AEs to Hemanext. AEs 
of interest include hemolytic reactions and    
 
  
METHODOLOGY  
  
Patients   
TDT patients followed at the Thalassemia Unit either of the First Dept of Pediatrics NKUA or 
at the LAIKON General Hospital and fulfilling the following criteria will be invited to 
participate in the study.   
  
Inclusion Criteria  
Subjects must satisfy the following criteria to be enrolled in the study:  
1. Subject is ≥ 18 years of age at the time of signing the informed consent form 
(ICF).   
2. Subject must understand and voluntarily sign an ICF prior to any study-related 
assessments/procedures being conducted.  
3. Subject has documented diagnosis of β- transfusion dependent thalassemia, 
defined as ect: ≥ 6 RBC units/24 weeks and no transfusion-free period for ≥ 42 
days during the 24 weeks prior enrollment.  
4. Transfusion history (including units or cc) for at least 6 months prior to 
enrollment needs to be available.  
5. Subject is on chelation therapy and a fairly stable dose for at least 6 months 
prior to enrollment.   
6. Chronic therapies (including hydroxyurea) are allowed as long as the 
medication dose has been stable for at least 6 months prior to enrollment.  
7. Having been exposed to PRBC transfusions prepared with the Hemanext One 
processing system 
  
Exclusion Criteria  
The presence of any of the following will exclude a subject from enrollment:  
1. Subject has currently or has a history of any significant medical condition, 
laboratory abnormality, or psychiatric illness.  
2. Subject has had positive Coombs (antiglobulin) test anytime during the 6 
months prior to enrollment.  
3. Subject is scheduled to undergo or has recently (in the last 6 months) undergone 
splenectomy.  
4. Use of luspatercept during the period of 6 months prior to enrollment and during 
the trial is not allowed.  

  
5  
5. Participation in an interventional clinical trial or use of experimental 
medications during the period of 6 months prior to enrollment and during the 
trial is not allowed.  
  
  
Methods  
The trial will consist of two phases. 
¥ Baseline phase (retrospective or prospective)  
The baseline phase consists of 12 weeks, evaluated retrospectively or prospectively, just prior 
or after receiving Hemanext transfusion. During this phase, the patients are expected to have 
received packed red blood cells (PRBC) processed using standard method. The phase will be 
used to establish their baseline transfusion and hematologic characteristics. Baseline values 
will be derived from this phase.  
 
¥ Treatment Phase 
The treatment phase will last for at least 3 months, during which patients will continue 
receiving PRBCs prepared using the Hemanext methodology. This phase will continue for as 
long as the patient receives Hemanext-prepared PRBC transfusions and may be extended until 
the data cut-off date. The data cut-off date is defined as the time point when data have been 
collected for at least 3 months from all patients included in the study. Each patient must sign 
the Informed Consent Form before any data collection begins. 
 
HEMANEXT ONE process and store CP2D/AS-3 Red Blood Cells, Leukocytes Reduced (LR 
RBC) that have been prepared within the standard 8-hour hold time. Processing must be 
initiated within 8 hours of collection and completed within 12 hours of collection. The Red 
Blood Cells must be processed at room temperature (20-26°C). The HEMANEXT ONE system 
limits O2 and CO2 levels in the storage environment. Red Blood Cells Leukocytes Reduced, 
O2/ CO2 Reduced may be stored for up to 42 days at 1-6°C. HEMANEXT ONE is used for 
volumes no greater than 350 mL of LR RBC.  
  
 
Transfusion Burden  
As this is the primary end point, the transfusion burden should be calculated based on cc/kg. 
Number of blood units could also be registered and if available hematocrit of the PRBC’s. 
Baseline values will be based on the baseline phase.  
  
 
Transfusion Efficacy 
To evaluate further the efficacy of the transfusion, the mean total Hb mass will be calculated 
based on Nadler’s formula for blood volume, as follows:  
 
 
"#$ %&'() = (0.3669 × ℎ(34ℎ5(%)!  +  0.03219 ×  :#;< =(34ℎ5(>4) +  0.6041)
×  %(&@ A: (4/C)  
 
"#$ D(%&'() = (0.3561 × ℎ(34ℎ5(%)!  +  0.03308 ×  :#;< =(34ℎ5(>4) +  0.1833)
×  %(&@ A: (4/C)  
 

  
6  
Mean Hemoglobin calculated as the sum of the values of the  mean hemoglobin between two 
measurements multiply by the × 
(#$%& ()*+)), - .)$&/0).),*)
(*2*$3 2(&)04$*52, 6)052#)
 
 
Analyses  
Different analysis will be evaluated as per standard follow up protocols for these patients. 
Samples for further exploratory analyses will be collected and stored   
 
                    
Samples collection and timepoints  
Visit Week:  
     Baseline Period  
 
Treatment Phase   
Open-Label 
Extension 
Period 
1  
  
  
  
  
  13  14  
  
  
  
  
  
  
  
38  Till LPLV 
Study Day:  1  
  
  
  
  
  85  92  
  
  
  
  
  
  
  260 
 
Transfusion 
Day: 
  
  
  
  
  
    
  
  
  
  
  
  
  
  
  
 
Informed consent   
  
  
  
  
  
  
X 
  
  
  
  
  
  
  
  
 
Demographics2  
X  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Eligibility  
X  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical and 
surgical history  
X  
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
X 
 
Transfused PRBC 
cc/kg – units 
X X X X X X 
X 
X 
X X X X X X X 
X 
X 
Medications  
X  
 
 
 
 
 
X  
 
 
 
 
 
 
X 
X 
Vital signs  
X X X X X X 
X 
X X X X X X X X 
X 
 
Hematology, 
including 
reticulocytes  
X 
 
 
 
As per standard 
evaluation 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
As per standard 
evaluation 
X 
 
X 
Serum chemistry  X 
 
X 
 
X 
As per 
standard 
practice 
Liver function 
tests  
X 
 
X 
 
X 
As per 
standard 
practice 
Lipid panel  
 
X 
 
X  
X 
As per 
standard 
practice 
Iron panel  
X 
 
 
 
 
 
 
X  
 
 
 
 
 
 
X Q 3months 
QOL  
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
X At last visit 

  
7  
Blood sample for 
exploratory 
disease 
parameters 
 
  
  
  
  
  
  
X  
  
  
    
  
  
  
X  At last visit 
Adverse events 
and serious 
adverse events  
X   
 
 
 
X (continuous)  
 
LV: last visit with Hemanext  
LPLV: Last patient last visit during treatment phase  
 
Hematology includes complete blood count with differential, HCT, Hb, RBC count, absolute 
reticulocyte count, percent reticulocyte count, MCV, MCH, MCHC, RDW, WBC count with 
differential, and platelet count.  
Serum chemistry includes sodium, potassium, chloride, calcium, magnesium, phosphorus, 
carbon dioxide or bicarbonate, albumin, total protein, glucose, BUN or urea, creatinine, and 
uric acid.   
Liver function tests include LDH, ALP, ALT, AST, and bilirubin (total, direct, and indirect). 
Lipid panel includes total cholesterol, LDL-C, HDL-C, and triglycerides; samples will be 
collected after an overnight fast. Iron panel may include iron, serum ferritin, total iron binding 
capacity, or transferrin saturation. 
 
  
Statistical Method:  
For continuous parameters, summary statistics will include the number of subjects, mean, 
standard deviation, quartiles, minimum and maximum, and the 95% confidence interval of the 
mean (if appropriate). For categorical parameters, summary statistics will include counts, 
percentages, and the 95% confidence interval of the percentages (if appropriate) in each 
category.  
 
Based on analysis on random patients from our unit, the standard deviation (SD) of transfusion 
burden (annual consumption in cc/kg/year) was estimated at 22.8. The non-inferiority limit was 
set at 15%. For a power of 90%, 25 participants are required. Assuming a drop-out rate of 10%, 
the target enrollment will be at least 28 patients. 
 
An interim analysis will be performed when half of the expected enrolled patients (the 15th 
patient) have reached week 12 of the treatment phase.  
  
  
 
 
 
 
 
  
  
  

  
8  
References  
  
1. Rabcuka J, Blonski S, Meli A, et al. Metabolic reprogramming under hypoxic storage 
preserves faster oxygen unloading from stored red blood cells. Blood Adv. 
2022;6(18):5415-5428. doi: 10.1182/bloodadvances.2022007774  
2. Bennett-Guerrero E, Veldman TH, Doctor A, et al.. Evolution of adverse changes in 
stored RBCs. Proc Natl Acad Sci USA.2007;104(43): 17063-17068.   
3. Hess JR. Measures of stored red blood cell quality. Vox Sang. 2014;107(1):1-9.   
4. Roussel C, Dussiot M, Marin M, et al.. Spherocytic shift of red blood cells during 
storage provides a quantitative whole cell-based marker of the storage lesion. 
Transfusion. 2017;57(4):1007-1018.   
5. Peter Klinken S. Red blood cells. Int J Biochem Cell Biol. 2002 Dec;34(12):1513-8. 
doi: 10.1016/s1357-2725(02)00087-0. PMID: 12379271.  
6. Reisz JA, Wither MJ et al. Oxidative modifications of glyceraldehyde 3-phosphate 
dehydrogenase regulate metabolic reprogramming of stored red blood cells. 
2016;128(12): e32-42.  
7. Rinalducci, Sara, Cristina Marrocco, and Lello Zolla. "Thiol‐based regulation of 
glyceraldehyde‐3‐phosphate dehydrogenase in blood bank–stored red blood cells: a 
strategy to counteract oxidative stress." Transfusion 55, no. 3 (2015): 499-506.  
8. Dandekar, Thomas, Astrid Fieselmann, Saman Majeed, and Zeeshan Ahmed. "Software 
applications toward quantitative metabolic flux analysis and modeling." Briefings in 
bioinformatics 15, no. 1 (2014): 91-107.  
9. D'Alessandro, Angelo, Monika Dzieciatkowska, Ryan C. Hill, and Kirk C. Hansen. 
"Supernatant protein biomarkers of red blood cell storage hemolysis as determined 
through an absolute quantification proteomics technology." Transfusion56, no. 6 
(2016): 1329-1339.  
10. Chouchani, Edward T., Andrew M. James, Ian M. Fearnley, Kathryn S. Lilley, and 
Michael P. Murphy. "Proteomic approaches to the characterization of protein thiol 
modification." Current opinion in chemical biology 15, no. 1 (2011): 120-128.  
11. Clasquin, Michelle F., Eugene Melamud, and Joshua D. Rabinowitz. "LC‐MS data 
processing with MAVEN: a metabolomic analysis and visualization engine." Current 
protocols in bioinformatics 37, no. 1 (2012): 14-11.  
12. Nemkov, Travis, Angelo D’Alessandro, and Kirk C. Hansen. "Three-minute method for 
amino acid analysis by UHPLC and high-resolution quadrupole orbitrap mass 
spectrometry." Amino acids 47 (2015): 2345-2357.  
13. Dzieciatkowska, Monika, Ryan Hill, and Kirk C. Hansen. "GeLC-MS/MS analysis of 
complex protein mixtures." Shotgun Proteomics: Methods and Protocols (2014): 5366.  
14. Julie A. Reisz, Matthew J. Wither, Monika Dzieciatkowska, Travis Nemkov, Aaron 
Issaian, Tatsuro Yoshida, Andrew J. Dunham, Ryan C. Hill, Kirk C. Hansen, Angelo 
D'Alessandro, Oxidative modifications of glyceraldehyde 3-phosphate dehydrogenase 
regulate metabolic reprogramming of stored red blood cells, Blood, Volume 128, Issue 
12, 2016, ISSN 0006-4971, https://doi.org/10.1182/blood-2016-05-714816.  
15. van der Meer PF, Cancelas JA, Cardigan R, et al.; BEST Collaborative . Evaluation of 
overnight hold of whole blood at room temperature before component processing: effect 
of red blood cell (RBC) additive solutions on in vitro RBC measures. Transfusion. 
2011;51(suppl 1):15S-24S.   

  
9  
16. Wilsher C, Garwood M, Sutherland J, Turner C, Cardigan R. The effect of storing whole 
blood at 22 degrees C for up to 24 hours with and without rapid cooling on the quality 
of red cell concentrates and fresh-frozen plasma. Transfusion.2008;48(11):2338-2347.  
17. Dern RJ, Brewer GJ, Wiorkowski JJ. Studies on the preservation of human blood. II. 
The relationship of erythrocyte adenosine triphosphate levels and other in vitro 
measures to red cell storageability. J Lab Clin Med. 1967;69(6):968-978.   
18. HEMANEXT ONE® (Blood container set used to process and store CP2D/AS-3 Red 
Blood Cells, Leukocytes Reduced, and O2/CO2 Reduced) [US Instructions for Use]. 
Lexington, MA: Hemanext Inc.  
19. Farmakis D, Porter J, Taher A, et al. 2021 Thalassemia International Federation 
Guidelines for the management of transfusion-dependent thalassemia. 2022;6:8.  
20. Chou S, Alsawas M, Fasano R, et al. American Society of Hematology 2020 guidelines 
for sickle cell disease: transfusion support. Blood Adv. 2020;4:2.  
21. Germing U, Oliva E, Hiwase D, and Almeida A. Treatment of anemia in 
transfusiondependent and non-transfusion-dependent lower-risk MDS: current and 
emerging strategies. 2019;3(6). doi: 10.1097/HS9.0000000000000314  
22. American College of Surgeons. ACS TQIP massive transfusion in trauma guidelines. 
ACS TQIP. 2014; https://www.facs.org/media/zcjdtrd1/transfusion_guildelines.pdf.  
23. Yoshida T, Blair A, D’Alessandro A, et al. Enhancing uniformity and overall quality of 
red cell concentrate with anaerobic storage. Blood Transfus. 2017;15(2):172-81.  
24. Yoshida T, McMahon E, Croxon H, et al. The oxygen saturation of red blood cell 
concentrates: The basis for a novel index of red cell oxidative stress. Transfusion. 
2022;62(1):183-193. doi: 10.1111/trf.16715.  
25. Reikvam H, Hetland G, Ezligini F, et al. Safety of hypoxic red blood cell administration 
in patients with transfusion-dependent hematological malignancies: An interim 
analysis. Transfus Apher Sci. 2023; doi: 10.1016/j.transci.2023.103755.