Personalizing Preterm Neonatal Transfusions With Fetal Hemoglobin-Enriched Cord Blood (ANH-Prestige)

Advancing Neonatal Health: Personalizing Preterm Neonatal Transfusions With Fetal Hemoglobin-Enriched Cord Blood

Long-term morbidities among very low birth weight infants remain a significant challenge. Oxidative stress is a key factor in the pathogenesis of 'free radical (FR) diseases of prematurity,' including retinopathy of prematurity, bronchopulmonary dysplasia, necrotizing enterocolitis, and intraventricular hemorrhage. Red blood cell (RBC) transfusions are recognized as a contributing factor to FR-related diseases. RBCs contain adult hemoglobin (HbA), which has a lower affinity for oxygen. This characteristic increases oxygen delivery and tissue uptake, leading to a potentially harmful state of hyperoxia and over-generation of FRs. The strategy employs a multidisciplinary approach to evaluate the impact of cord blood transfusions in anemic newborns. Results will be assessed in relation to short- and long-term neonatal outcomes to determine the effectiveness of this new preventive strategy. Improving the current data are critical for setting action priorities for and monitoring progress

Study Overview

• Status: Not yet recruiting
• Conditions: Anemia; Prematurity Complications
• Intervention / Treatment: Combination product: Cord Blood Red Blood Cell Transfusion (CB-RBC); Other: Adult Donor Red Blood Cell Transfusion (A-RBC)

Detailed Description

Specific AIM 1 To assess whether CB-RBC transfusions can offer a protective advantage of CB-RBC transfusions in preterm newborns by preserving HbF, reducing the risk of oxidative stress-related complications, and potentially lowering the incidence of 'free radical diseases of prematurity' .

Biomarkers of oxidative stress will be correlated with short term outcome of the newborns (diagnosis at discharge). Moreover with the aim to evaluate the social, economic, organizational implications of this intervention the investigators will perform an Health Impact Assessment (HIA).

The goal is to provide policymakers, healthcare providers, and stakeholders with evidence-based insights on the effectiveness, cost-effectiveness, safety, and potential impact of CB-RBC transfusions.

Specific AIM 2 To evaluate pathways involved in inflammatory and hypoxic-hyperoxic processes through microRNAs expression. OS cascade comprises the activation of microglia, infiltration of macrophages and release of pro-inflammatory mediators such as cytokines, chemokines, nitric oxide, free radicals. MicroRNAs, (miRs), play an important role in the regulation of OS modulated genes, with the potentiality to induce protective protein synthesis such as neuroglobin and sirtuin. Specifically, miRNA 107 e 17-5p, involved in angiogenesis and arterial remodeling are upregulated; miRNA 223-3p associated with chronic kidney disease is downregulated.

Specific AIM 3 To assess the protective role CB-RBC transfusions in newborns, by performing neurodevelopmental outcome of enrolled babies through clinical and neurobehavioral evaluations at 3-6-9-12-month of age.

Experimental Design Aim 1 This study is designed as a multicenter, randomized, controlled, double-blind trial.

Preterm neonates born between 24+0 and 31+6 weeks of gestational age who require at least one red blood cell transfusion during hospitalization will be randomized in a 1:1 ratio to receive either standard A-RBCs (control group) or CB-RBCs (intervention group). Randomization will be stratified by gestational age and study center and conducted using a centralized computer generated system. Treating physicians, caregivers, and outcome assessors including clinical, laboratory, and imaging personnel will be blinded to treatment allocation.

--Interventions: All transfusions will follow uniform clinical guidelines and thresholds for transfusion, aligned with current Italian neonatal transfusion standards. The experimental intervention involves transfusion with CB-RBC concentrates prepared from cord blood units donated to public cord blood banks. These units are leukodepleted using BioR Flex filters, fractionated via Compomat G5 cell separators, suspended in SAG-M additive solution, and stored in DEHP-free pediatric bags. All units will be gamma-irradiated before administration and transfused within 24 hours of irradiation. Safety screening will include infectious disease testing (HIV, HBV, HCV, syphilis, bacterial and fungal cultures), identical to the procedures for adult donor blood.

--Sample Size: Based on an expected 50% incidence of the composite endpoint in the control group and a hypothesized reduction to 30% in the CB-RBC group (absolute risk reduction = 20%), a one-sided Chi-square test with alpha = 0.05 and 80% power requires 74 infants per group. To account for an interim analysis at 50% of enrollment and for up to 10% attrition rate, a total of 200 infants (100 per group) will be enrolled.

--Data Collection and Quality Assurance: Data will be captured using the REDCap electronic data capture system, with predefined validation rules and role-specific access rights. Clinical and perinatal data, transfusion details, and outcomes will be recorded prospectively.

Experimental Design Aim 2

--Biological Sampling and Time Points:

Peripheral blood samples will be collected from each enrolled neonate at three defined time points:

• B1 - Before the first transfusion (baseline);
• B2 - 48 hours (±6h) after the transfusion;

• B3 - 14 days after the transfusion. At each time point, 1 ml of whole blood will be obtained and processed for molecular and biochemical analyses. Samples will be aliquoted and stored at -80°C until batch analysis. Pre-analytical procedures, storage, and shipping conditions will follow standardized SOPs across centers. All laboratory staff performing analyses will be blinded to treatment allocation, ensuring unbiased biomarker evaluation.

  • Oxidative Stress and Antioxidant Biomarkers:

The oxidative stress (OS) profile will be assessed using a panel of protein and lipid biomarkers, including:

• Protein oxidation: Advanced Oxidation Protein Products (AOPP);
• Lipid peroxidation: Isoprostanes (IsoPs), malondialdehyde (MDA);
• Non-enzymatic antioxidants: Vitamin E, glutathione (GSH), and ascorbic acid (AA);
• Enzymatic antioxidants: Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx).

Lipid mediators related to inflammation and oxidative balance, such as Resolvin D1, will also be quantified, alongside neuroglobin and sirtuin-1, which are involved in cellular protection mechanisms.

• MicroRNA Profiling:

Total RNA will be extracted from 400 μl of serum using the miRVana PARIS kit (Invitrogen). Reverse transcription will be performed using the TaqMan Advanced miRNA cDNA Synthesis Kit, followed by quantification using TaqMan miRNA assays on a 7900HT Fast PCR system.

MicroRNAs of interest include miR-107, miR-17-5p, and miR-223-3p, selected for their involvement in oxidative stress, angiogenesis, and vascular remodeling. Expression will be normalized using hsa-miR-16-5p as the endogenous control.

The 2^deltadeltaCt method will be used to quantify expression changes, and miRNAs with Ct > 35 will be excluded from analysis. Total RNA will be isolated from 400μl of serum using the miRVana PARIS kit (Invitrogen, AM1556). Samples will be stored at -80°C. Reverse transcription will be performed using the TaqMan Advanced miRNA cDNA Synthesis Kit (Applied Biosystems, A28007). Candidate miRNAs will be quantified using TaqMan Advanced miRNA assays (Applied Biosystems) on a 7900HT Fast PCR System, with hsa-miR-16-5p (Assay ID: 477860_mir) as an endogenous control. Expression levels will be analyzed using the 2^deltadeltaCt method; miRNAs with Ct > 35 will be considered not expressed and excluded.

--Comparative Analyses: Between-group comparisons (CB-RBC vs A-RBC) will be conducted at each time point and across time points (B1-B3), allowing evaluation of both absolute differences and temporal changes in biomarker levels. Analyses will be adjusted for baseline clinical variables, transfusion volume, and number of transfusions received.

This aim will allow to link the transfusion source to molecular signatures of oxidative stress and inflammation, providing mechanistic insight into the observed clinical outcomes

Experimental Design Aim 3 --Follow-up Time Points--

• Neurodevelopmental assessments will be conducted at:

  • 1 month corrected age;
  • 3 months corrected age;
  • 6 months corrected age;
  • 12 months corrected age. These time points will allow for early detection of neurological delays and tracking of developmental trajectories over the first year of life.
• Neurodevelopmental Assessment Tools:

Standardized and validated instruments will be used to ensure comparability and reliability across study sites:

• The Bayley Scales of Infant and Toddler Development - Third Edition (BSID-III) will be used at 12 months to assess cognitive, motor, and language development;
• Structured neurological examinations will be performed at each follow-up visit to detect signs of abnormal motor tone, reflexes, or neurosensory deficits;

• Information on growth, oxygen dependence, feeding difficulties, and the presence of multi-organ sequelae will be collected longitudinally.

  --Neuroimaging: Magnetic Resonance Imaging (MRI) of the brain will be conducted according to routine clinical practice and scored retrospectively using the Woodward scoring system, which quantifies white and gray matter injury, ventricular dilation, and global structural anomalies. MRI reviewers will be blinded to treatment allocation and clinical outcomes to ensure unbiased evaluation.

• Analysis Plan The primary neurodevelopmental outcome will be the proportion of infants with BSID-III scores below 85 (i.e., more than one standard deviation below the normative mean) in at least one domain (cognitive, motor, or language) at 12 months.

Group comparisons will be performed using Chi-square tests, and multivariable logistic regression models will be used to adjust for potential confounders, including gestational age, IVH grade, BPD, and birth weight.

Secondary analyses will explore continuous BSID-III scores, composite neurodevelopmental indices, and MRI findings.

Correlations between biological markers (from Aim 2) and neurodevelopmental outcomes will be examined to identify predictive biomarkers and potential mechanistic pathways

• Study Type: Interventional
• Enrollment (Estimated): 200
• Phase: Not Applicable

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Child
• Accepts Healthy Volunteers: No

Description

Inclusion Criteria:

• Preterm neonates born between 24+0 and 31+6 weeks of gestational age;
• Requirement for at least one red blood cell transfusion during hospitalization, according to current Italian transfusion thresholds;
• Written informed consent obtained from parents or legal guardians prior to any study procedure.

Exclusion Criteria:

• Gestational age > 32+0 weeks;
• Pregnancy complicated by maternal-fetal alloimmunization (e.g., hemolytic disease of the newborn);
• Pregnancy complicated by fetal hydrops;
• Major congenital anomalies or genetic syndromes;
• Previous red blood cell transfusions (prior to enrollment);
• Perinatal hemorrhage at delivery;
• Documented congenital infections (TORCH).

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Other
• Allocation: Randomized
• Interventional Model: Factorial Assignment
• Masking: Quadruple

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: Cord Blood Red Blood Cell Transfusion (CB-RBC); The experimental intervention consists of transfusion of cord blood red blood cell concentrates (CB-RBC), prepared from cord blood units donated to public cord blood banks. The units are processed as follows: Leukodepletion using BioR Flex filters;; Fractionation using Compomat G5 cell separators;; Suspension in SAG-M additive solution;; Storage in DEHP-free pediatric blood bags. All units are irradiated with gamma rays prior to administration and transfused within 24 hours after irradiation. Safety is ensured through screening for infectious diseases (HIV, HBV, HCV, syphilis, bacterial and fungal cultures). The dose, volume, and frequency of transfusion follow the same guidelines as adult red blood cell concentrates (A-RBC), according to current Italian neonatal standards. Transfusion is performed by the clinical staff of the Neonatology Unit according to the standard protocol.	Combination product: Cord Blood Red Blood Cell Transfusion (CB-RBC); Transfusion of leukodepleted, gamma-irradiated cord blood-derived red blood cell concentrates (CB-RBC), prepared from donated public cord blood bank units, characterized by high fetal hemoglobin (HbF) content and administered according to standard neonatal transfusion thresholds
Active Comparator: Adult Donor Red Blood Cell Transfusion (A-RBC); The control arm receives transfusions of adult donor red blood cell concentrates (A-RBC), leukodepleted and irradiated according to the same thresholds and doses as the experimental arm, representing the Italian standard of care for anemic preterm neonates and ensuring that the only difference between the two arms is the source of the transfusion product - and therefore the HbF content - with respect to which CB-RBC is expected to demonstrate superiority	Other: Adult Donor Red Blood Cell Transfusion (A-RBC); Transfusion of leukodepleted, gamma-irradiated adult-donor derived red blood cell concentrates (A-RBC) administered according to standard neonatal transfusion thresholds.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Incidence of free radical-related morbidities in preterm neonates receiving CB-RBC transfusions	Composite incidence of: Retinopathy of prematurity (ROP) Bronchopulmonary dysplasia (BPD) Necrotizing enterocolitis (NEC) Intraventricular hemorrhage (IVH) diagnosed according to standard neonatal criteria. Metric / Unit Number (%) of infants with at least one morbidity	From birth until hospital discharge or 36 weeks postmenstrual age

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Severity of retinopathy of prematurity	Severity of ROP classified according to the International Classification of Retinopathy of Prematurity. Metric / Unit ROP stage (Stage 0-5)	Until hospital discharge or 44 weeks postmenstrual age
Severity of bronchopulmonary dysplasia	Severity of BPD classified according to Jensen criteria. Metric / Unit Mild / Moderate / Severe BPD	At 36 weeks postmenstrual age
Bayley Scales of Infant Development score (Bayley-III)	Neurodevelopmental assessment performed using Bayley Scales of Infant and Toddler Development. Metric / Unit Composite cognitive, language, behavioral and motor scores Cognitive, language, and motor composite scores range from 40 to 160, with higher scores indicating better neurodevelopmental performance.	At 12 months corrected age
Safety of CB-RBC transfusions	Assessment of adverse events associated with CB-RBC transfusions. Metric / Unit Number (percent) of participants with transfusion-related adverse events	From the first CB-RBC transfusion to hospital discharge (up to 6 months)
Transfusion-associated complications	Occurrence of transfusion-associated complications including transfusion reactions, infections, or circulatory overload. Metric / Unit Number (percent) of complications	From enrollment until hospital discharge (up to 6 months)
Expression levels of oxidative stress-related microRNAs	Evaluation of microRNAs involved in oxidative stress response, inflammation, and neurodevelopment using molecular profiling techniques. Metric / Unit Relative expression levels (fold change)	Baseline and after first transfusion assessment during hospitalization (up to 6 months)
Changes in neuroprotective and antioxidant protein expression	Assessment of antioxidant and neuroprotective proteins following CB-RBC transfusion. Metric / Unit Protein concentration (ng/mL or pg/mL)	Baseline and within 72 hours after transfusion

Collaborators and Investigators

• Sponsor: University of Parma

Study record dates

Study Major Dates

• Study Start (Estimated): November 1, 2026
• Primary Completion (Estimated): January 1, 2028
• Study Completion (Estimated): November 1, 2029

Study Registration Dates

• First Submitted: May 15, 2026
• First Submitted That Met QC Criteria: June 3, 2026
• First Posted (Actual): June 9, 2026

Study Record Updates

• Last Update Posted (Actual): June 9, 2026
• Last Update Submitted That Met QC Criteria: June 3, 2026
• Last Verified: May 1, 2026

More Information

Terms related to this study

• Keywords: transfusion; preterm newborns; Cord Blood red blood cells
• Additional Relevant MeSH Terms: Hematologic Diseases; Hemic and Lymphatic Diseases; Anemia
• Other Study ID Numbers: AOUPR; RF-2024-12380404 - ER (Other Grant/Funding Number: Italian Ministry of Health)

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: YES
• IPD Plan Description: De-identified individual participant data underlying the reported results will be made available upon reasonable request to qualified researchers, following publication of the primary results and subject to institutional and ethical approval
• IPD Sharing Time Frame: At the end of the sudy
• IPD Sharing Access Criteria: Access to de-identified individual participant data will be provided to qualified researchers upon reasonable request, subject to approval by the study investigators and institutional ethics requirements. Data sharing will be permitted only for scientifically sound research purposes and after execution of a data access agreement
• IPD Sharing Supporting Information Type: STUDY_PROTOCOL; SAP

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No