Impact of Leukoreduction Timing on Plasma Citrullinated Histone H3 (H3cit) Dynamics and Red Blood Cell Storage Lesions

Packed red blood cells (PRBCs) are the most commonly transfused blood component and play a critical role in treating anemia, trauma, and surgical patients. However, during refrigerated storage, PRBCs undergo a sequence of biochemical, metabolic, and structural changes collectively referred to as the "storage lesion." These changes include depletion of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG), glucose utilization, lactate production, pH changes, potassium loss, oxidative stress, increased free hemoglobin release, and morphological alterations such as decreased deformability and microparticle formation. Such changes can impair erythrocyte function and survival following transfusion, particularly in critically ill patients and those requiring massive transfusion Studies have demonstrated that markers of NETs increase during RBC storage and are significantly higher in non-leukoreduced units compared to leukoreduced units(5) Specifically, in canine models, day 42 non-leukoreduced units contained approximately 1000-fold more cell-free DNA than leukoreduced units, with significantly higher Cit-H3 levels Leukoreduction, the removal of white blood cells from blood products, is performed to reduce transfusion reactions and immunomodulation. Pre-storage leukoreduction has been shown to prevent cytokine accumulation during storage and reduce the risk of febrile nonhemolytic transfusion reactions and TRALI The timing of leukoreduction is critical, as pre-storage filtration prevents the accumulation of leukocyte-derived bioactive substances that occur during storage, whereas post-storage filtration does not Importantly, universal leukoreduction has been associated with an 83% decrease in TRALI rates, highlighting the clinical significance of leukocyte removal Neutrophil extracellular traps (NETs) are web-like structures composed of DNA, histones, and neutrophil granular proteins extruded from activated neutrophils. NETs have been implicated in cytotoxicity, thrombosis, and autoimmunity, and they play a role in the pathogenesis of transfusion-related acute lung injury (TRALI) Citrullinated histone H3 (Cit-H3) is a specific marker of NETosis, as NET formation requires histone citrullination by peptidylarginine deiminase 4 (PAD4) Given the established role of NETs in transfusion-related complications and the potential for Cit-H3 to serve as a quality marker, this study aims to evaluate Cit-H3 levels in stored RBC units and assess its correlation with storage duration and leukoreduction timing. The findings could provide insights into optimizing storage conditions and improving transfusion outcomes through better identification and mitigation of storage lesions

Inclusion criteria

• • Standard whole blood units collected from healthy voluntary donors who meet the national blood transfusion service fitness criteria (age 18-60 years, body weight ≥50 kg).

• Collection Volume & Container: Standard unit volume (450 ± 45 mL) collected into closed bag systems with additive/anticoagulant solution (CPDA-1).
• Thermal Storage Chain: Blood units maintained continuously within standard blood bank temperature limits (2 C to 6C )from collection until the end of the storage period.

Baseline Hematological Parameters: Normal baseline hematological profile immediately post-collection (Hemoglobin ≥12.5g dl)

Exclusion criteria

• Donor Clinical Factors: Donors with a history of acute or chronic inflammatory conditions, active infections, autoimmune diseases, or those who ingested anti-inflammatory/antiplatelet medications (e.g., NSAIDs, aspirin) within 14 days prior to donation.
• Technical Collection Complications: Phlebotomy draw time exceeding 12 minutes, difficult venipuncture, or units displaying macro-clots or excessive lipemia post-collection.
• Storage Excursions & Bag Damage: Any blood unit subjected to cold-chain temperature violations outside 2-6C, physical damage, or system breaches/leaks.

Transfusion-Transmitted Infections (TTI): Units testing reactive for screening markers, including HBsAg, Anti-HCV, Anti-HIV-1/2, or Syphilis

• Donor Clinical Factors: Donors with a history of acute or chronic inflammatory conditions, active infections, autoimmune diseases, or those who ingested anti-inflammatory/antiplatelet medications (e.g., NSAIDs, aspirin) within 14 days prior to donation.