Pre-Bypass Ultrafiltration reduces Cytokine Burden of Blood Prime in Pediatric Cardiac Surgery

Pre-Bypass Ultrafiltration reduces Cytokine Burden of Blood Prime in Pediatric Cardiac Surgery | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Pre-Bypass Ultrafiltration reduces Cytokine Burden of Blood Prime in Pediatric Cardiac Surgery Aylin Poertecene, Svea Kleiner, Leonie Trachte, Sebastian Tiedge, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6193879/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Allogeneic red blood cells (RBCs) are commonly used for cardiopulmonary bypass (CPB) circuit priming in congenital heart surgery. While convection-based pre-bypass ultrafiltration (PBUF) corrects acid-base, electrolyte, and metabolite imbalances, its efficacy in removing RBC cytokines/chemokines remains unclear. In a prospective observational study, 22 children (median age: 4.1 months) undergoing congenital heart surgery were enrolled. PBUF of RBC-primed CPB circuits was conducted using bicarbonate-buffered hemofiltration solution. Cytokines/chemokines were quantified in RBC supernatants, CPB priming (before and after PBUF), preoperative patient plasma, and PBUF effluent using Luminex-based multiplex technology. 30 of 50 cytokines were detected in > 50% of RBC supernatants. RBC priming significantly elevated concentrations of 25 cytokines, with 20 further rising after PBUF. At CPB onset, eight mediators (MIF, IL-15, CCL11/Eotaxin, CCL2/MCP-1, VEGF, IL-5, VCAM-1, ICAM-1) exceeded patient plasma concentrations. PBUF filtered cytokines with different efficiencies (0.6–97%). Despite poor filtration or increased concentrations, total mediator load of 42 cytokines decreased significantly (33.3–69.1% of pre-processing levels) after PBUF. In conclusion, PBUF effectively removed multiple cytokines/chemokines released from RBC. Beyond filtration, decrease of total mediator load may be attributed to adsorption to circuit components or rebinding to RBCs. Improved washing techniques may further optimize mediator levels in RBC-primed CPB circuits. Biological sciences/Immunology/Cytokines Health sciences/Diseases/Cardiovascular diseases/Congenital heart defects cytokines cardiopulmonary bypass ultrafiltration red blood cell priming children Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Congenital heart disease (CHD) is the most common congenital malformation 1 . Most children require cardiac surgery with cardiopulmonary bypass (CPB) at an early age 1 . Neonatal and infant congenital heart surgery is further complicated by the significant size mismatch between the CPB system and the patient's blood volume 2 . Therefore, to prevent excessive hemodilution and severe anemia, whole blood or stored allogeneic red blood cells (RBCs) are often added to the CPB circuit during priming prior to cannulation to maintain a hematocrit above 24% during CBP, as recommended by the Network for the Advancement of Patient Blood Management, Haemostasis and Thrombosis 3 . This practice results in transfusion of stored allogeneic RBCs during initiation of CPB. RBC transfusion is associated with immunosuppression, infection, electrolyte imbalance, acute kidney injury, and lung injury 4 . These effects are partly related to storage-dependent changes in RBCs, which undergo morphological and functional changes 5 . Recently, packed RBCs have also been shown to be a major reservoir of cytokines, chemokines, and growth factors, suggesting a potential role in inflammatory processes 5 , 6 . In addition, stored RBCs develop unphysiologic acid-base, electrolyte, and metabolite values 7 . To optimize unprocessed RBCs for CPB circuit priming, the American Society of Extra Corporeal Technology recommends pre-bypass ultrafiltration (PBUF) 2 . At our institution, using a bicarbonate-buffered solution for PBUF, improved electrolyte, lactate, and acid-base levels of the priming solution 8 . However, the ability of PBUF to remove pro-inflammatory mediators from packed RBCs used for CPB priming has not been evaluated. Therefore, we conducted a single-center, prospective observational study to assess the inflammatory mediator burden of allogeneic RBC-primed CPB circuits in neonatal and infant congenital heart surgery, as well as the efficacy of PBUF in reducing cytokine/chemokine levels delivered to the patient. Results Study population 22 children with various congenital heart defects with a median (range) age of 4.1 months (10 days to 34 months) and Risk Adjustment for Congenital Heart Surgery score of 3 (2–6) were included in the study (Supplementary Table S1). Two children were treated in the intensive care unit prior to surgery. PBUF was conducted during each surgery at the perfusionists' discretion until pH, metabolic parameters and electrolytes of the CPB circuit prime reached normal levels. This required a median (range) duration of 10 minutes (4–35) and a median volume of 400 mL (250–770) of hemofiltration solution. Mediator burden in RBC supernatants Quantification of cytokines was performed on 22 RBC units obtained immediately before their application to the CPB prime (Table 1). Of the 50 cytokines assayed, 46 were detected in at least one RBC supernatant showing a high inter-sample variability, with 30 of the 50 mediators detected in at least 50% of the RBC supernatants. Notably, 16 of the 50 mediators were present in more than 90% of RBC supernatant samples (Figure 2). Only 6 mediators, namely Interleukin (IL)-1b, IL-12p70, IL-2, Monocyte chemotactic protein 3 (CCL7/MCP-3), Nerve growth factor b (b-NGF) and Granulocyte-macrophage colony-stimulating factor (GM-CSF), were detectable in 10% or less of RBC supernatants. The median duration of RBC storage before use was 12.5 days (range 7–28 days). Five of 50 cytokines, namely Stem cell growth factor b (SCGFb), CCL11/Eotaxin, Interferon-gamma-inducible protein 10 (CXCL10/IP-10), (Supplementary Figure S1), IL-16 and Cutaneous T-Cell attracting chemokine (CCL27/CTACK), (data not shown) showed a positive correlation between cytokine concentrations in RBC supernatants and storage duration. However, among these, CCL27/CTACK and IL-16 exhibited only a moderate correlation and showed extremely low levels that seem unlikely to be clinically relevant. Levels of CXCL10/IP-10 and SCGFb, which showed an association indicating potential storage-dependent changes, were several orders of magnitude lower in RBC supernatant compared to patient baseline levels (Supplementary Figure S1). CCL11/Eotaxin, on the other hand, showed significantly higher concentrations in RBC supernatants compared to patient baseline (Supplementary Figure S1). Cytokine concentration in RBC-free priming Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), soluble IL-2 receptor alpha (IL-2Ra), Regulated upon activation normal T cell expressed and presumably secreted (CCL5/RANTES), SCGFb, Stem cell factor (SCF) and Macrophage colony-stimulating factor (M-CSF) were identified at low levels in control samples of asanguine priming solution before the addition of RBC; all other cytokines were not detected at quantifiable levels (Supplementary Table S2). Therefore, it must be assumed that negligible traces of a few cytokines are present in the other components involved in the priming process. Mediator concentration during priming and PBUF After priming of the CPB circuit, circulation and homogeneous mixing of all prime components, all cytokines identified in RBC supernatants remained detectable. Most mediators were present at either comparable or higher concentrations than in RBC supernatants (Table 1). Of the 50 mediators analyzed, the concentrations of 25 (IL-15, IL-9, Tumor necrosis factor beta [TNF-b], TRAIL, IL-17, Interferon-gamma [IFN-g], IL-2Ra, IL-4, Leukemia inhibitory factor [LIF], Growth-regulated alpha protein [CXCL1/GRO-a], CCL5/RANTES, CCL11/Eotaxin, Stromal cell-derived factor 1 alpha [CXCL12/SDF-1a], Macrophage inflammatory protein 1b [CCL4/MIP-1b], Monocyte chemotactic protein 1 [CCL2/MCP-1], CXCL8/IL-8, Vascular Endothelial Growth Factor [VEGF], IL-5, Granulocyte-Colony Stimulating Factor [G-CSF], SCF, IL-7, Macrophage colony-stimulating factor [M-CSF], IL-3, b-NGF, Intracellular adhesion molecule 1 [ICAM-1]) increased significantly, while 4 cytokines (Macrophage migration inhibitory factor [MIF], IL-18, Hepatocyte growth factor [HGF] and soluble Vascular cell adhesion molecule 1 [VCAM-1]) decreased significantly in concentration. For the remaining cytokines, no change in concentration was observed (Figure 3 and Table 1, Supplementary Figure S2). Irrespective of the changes in concentration, many of the mediators were detected in a larger number of priming samples than in the RBC samples. After complete assembly, the priming solution was circulated in the CPB circuit and PBUF was performed. After PBUF, 20 out of 50 cytokines further increased significantly in concentration (IL-9, TNF-b, IL-1a, IL-17, IFN-g, IL-2Ra, IL-13, IL-4, CCL11/Eotaxin, CCL4/MIP-1b, CCL2/MCP-1, CXCL8/IL-8, G-CSF, basic fibroblast growth factor [FGF-b], SCF, IL-7, M-CSF, b-NGF, VCAM-1, ICAM-1), while the others remained stable, none decreased (Table 1 and Figure 3, Supplementary Figure S2). Some of the cytokines (IL-1a, IFN- g, IL-1 RA, IL-13, CCL27/CTACK, FGF-b, IL-7, b-NGF) were also detected in a relevant higher number of samples than before PBUF. Only IL-12p70 was undetectable in both, pre- and post-PBUF samples (Table 1). Priming vs. patient mediator concentration To establish a threshold for the potential clinical relevance of mediator burden in RBC priming, we compared the cytokine levels detected in the priming after PBUF with the median preoperative plasma levels of the patients in our study cohort for each of the cytokines. After PBUF, the concentration of 8 out of 50 cytokines (MIF, IL-15, CCL11/Eotaxin, CCL2/MCP-1, VEGF, IL-5, VCAM-1, and ICAM-1) in the CPB circuit significantly exceeded the median preoperative baseline levels of the patients (Table 1, Figure 3, Supplementary Figure S2). Levels of all other mediators showed a non-significant trend to be elevated above the patient baseline (IL-3, IL-4, M-CSF [p-value <0.1]), ranged below the median patient baseline, or were undetectable (Table 1). Mediator removal by PBUF The majority of the 50 cytokines analyzed were also detectable in the PBUF effluent at the end of PBUF (Supplementary Table S2). Among the 8 cytokines whose concentrations in the priming solution after PBUF exceeded baseline serum levels in patients, 7 showed a positive correlation between their spot concentrations in the PBUF effluent and their respective levels in the CPB prime. The slope of the linear regression line varied for each cytokine, reflecting differences in filtration properties (Figure 4). The removal effect, calculated as ratio of respective cytokine concentrations in the priming and PBUF effluent, ranged from 0.67 ± 1.03% for VCAM-1 to 97.73 ± 83.92% for VEGF (Table 2, Supplementary Table S2). However, there was variability in the removal effect, indicating better filtration of smaller molecules such as VEGF (43 kDa) or IL-15 (18k Da) and restricted filtration of larger molecules such as M-CSF (107 kDa), ICAM-1 (58 kDa in monomeric form), and VCAM-1 (81-110 kDa). Absolute mediator content during priming and PBUF To further estimate the total mediator load, the mediator concentration was multiplied by i) the volume of applied RBCs and ii) the respective filling volume of the CPB circuit at specific stages of the priming process, accounting for the particular volume used for PBUF. After priming the CPB circuit, the total mediator load for 48 cytokines increased significantly compared to the mediator load from the applied packed RBC volume (Supplementary Figure S3 and Table S4). CCL7/MCP-3 and IL-12(p70), which were below the lower detection limit in RBCs, were not detectable. After further processing of the prime comprising PBUF, the load of 42 out of 50 mediators decreased significantly (Supplementary Figure S3 and Table S4), whereas the contents of 5 mediators did not decrease significantly (IL-2, b-NGF, GM-CSF) or remained stable (IL-13, FGF-b). These 5 mediators were consistently detected at low concentrations in all samples (Table 1). Only IL-1b and CCL7/MCP-3 levels increased significantly, but this was negligible, as it was detected in only 1 or 2 samples above the detection limit. IL-12(p70) was undetectable. The final mediator load ranged from 33.3 % (CXCL9/MIG) to 69.1 % (IL-16) of pre-PBUF baseline levels. Notably, mediators that exceeded the median preoperative baseline concentrations in our patient cohort, as well as those identified to be poorly filtered (Table 2), were also significantly reduced. Discussion We conducted a single-center observational study to assess the inflammatory mediator burden of allogeneic RBC-primed CPB circuits during neonatal and infant congenital heart surgery, as well as the efficacy of pre-bypass ultrafiltration (PBUF) in reducing cytokine and chemokine levels. To the best of our knowledge, this is the first comprehensive analysis of a broad range of pro- and anti-inflammatory factors, chemokines, growth factors and endothelial markers during the priming process. Our findings confirm that packed RBC, once thought to be immunologically inert, are sources of a wide variety of cytokines. After RBC priming of the CPB circuit, circulation and homogeneous mixing of all components, RBCs continue to release mediators, resulting in higher cytokine concentrations and loads in CPB prime compared to packed RBC supernatants. Subsequent PBUF was highly effective and successfully removed a significant portion of mediators through the effluent. Notably, our study is the first to demonstrate that the entire priming process further reduced the overall inflammatory mediator load. First, most of the cytokines we found in packed RBC were detectable at low levels in the supernatants of RBC units, with a large variability most likely due to donor specifications. This observation is consistent with recent studies, including those by Karsten et al., which highlighted RBCs as dynamic cytokine reservoir 5 , 9 . Their investigation identified 46 mediators in RBC lysates and 46 cytokines in RBC-conditioned media 5 , 9 . Our analysis supplements their findings, detecting 46 out of 50 mediators in RBC units used for CPB priming, with 30 present in more than half of the stored RBC unit supernatants. However, we observed slight differences in the detectable cytokines. Four mediators (IL-1β, IL-12 (p70), CCL7/MCP-3, and GM-CSF) previously reported in RBC-conditioned media were undetectable in our RBC supernatant samples 5 , 9 , while three cytokines (CCL7/MCP-3, β-NGF, and SCGF-ß) were previously not been described 5 , 9 . These discrepancies may be due to high inter-individual and donor-dependent cytokine variability in RBC units and limited sample sizes in both studies. Additionally, while Karsten et al. used freshly isolated RBCs for their experiments, we investigated packed RBC after routine handling and storage. Remarkably, of the 50 mediators analyzed, only five showed a correlation between mediator burden and prolonged storage time. This is in contrast to the literature, which describes changes in immunological profiles of leukocyte-depleted RBCs depending on storage time or manufacturing methods 10 , 11 . A possible explanation for these discrepancies is that our study did not track mediator concentrations in packed RBCs across the complete storage period. Second, RBCs continue to release mediators after administration into the CPB circuit, leading to significantly higher cytokine levels in CPB prime compared to initial RBC supernatants. This suggests an ongoing release mechanism, likely amplified by mechanical stimulation from CPB circuit components 12 . It is reasonable to assume, that shear stress and surface contact with circuit components induce some degree of hemolysis, resulting in the release of intracellular mediators from RBC 5 , 12 , 13 . However, the literature indicates that a fraction of cytokines, such as CXCL8/IL-8, exist in a bound form within the RBC cytoplasm and might therefore not be detectable in our assays after hemolysis 14 . Additional active release of monomeric cytokines by intact RBCs is likely to cause a measurable increase in mediator levels throughout the priming preparation 5 . To that effect, versatile binding and adaptive release of mediators on RBCs through the Duffy antigen receptor for chemokines (DARC) and the erythrocyte glycocalyx have been previously described 5 . To date, two major triggers for CCL2/MCP-1 release from DARC have been identified: the exposure to unfractionated heparin and the coagulation process 15 . Both conditions are frequently encountered when donor blood is used for CPB priming. Third, PBUF of the CPB prime was effective, successfully removing a significant portion of the mediator load, as indicated by cytokines detection in the PBUF effluent. The concomitant increase in the concentration of 20 of 50 inflammatory mediators after PBUF is likely due to several factors. As a convection-based process, PBUF cannot achieve an absolute reduction in mediator concentrations 16 . The effectiveness of filtration is further constrained by the filter membrane properties and the specific characteristics of each cytokine 17 , 18 . According to the manufacturer 19 , a comparable larger hemofilter with an identical polyarylsulfone membrane used in our study exhibits a sieving coefficient of 0.6 for 10 kDa molecules. Since most chemokines have a molecular mass of approximately 15 kDa 20 , they are expected to be effectively filtered, whereas larger molecules are known to be less removed 17 . Notably, some mediators exhibited unexpected filtration properties relative to their molecular weight. For instance, SCF (30 kDa) was largely undetectable in PBUF effluent, while IL-5 (15 kDa) appeared only in a subset of effluent samples (5 out of 21). This suggests that probably additional factors such as hydrophobicity or molecular structure may influence cytokine removal by PBUF 17 , 18 . Removal efficiency by PBUF varied from 0.6–97% across different cytokines, which is consistent with findings from other studies, such as Bierer et al., who documented extensive removal of 20 out of 39 mediators via ultrafiltration 18 . The low mediator concentrations in CPB prime preclude drawing generalized conclusions about the removal capacity for some of the cytokines that were undetectable in PBUF effluent. However, a difference was found for CCL5/RANTES, which was well filtered in our system, but was not removed in the study by Bierer et al. 18 . This difference may be attributed to the use of different filtration membranes: polysulfone in their study versus polyarylsulfone in ours. Both materials likely have different filtration characteristics for specific cytokines, as previously published for comparison of polyamide and polysulfone membranes 21 . Overall, we observed a significant reduction in the total load of 42 different mediators during the entire priming processing. The final mediator load ranging from 33.3% (CXCL9/MIG) to 69.1% (IL-16) of pre-processing baseline levels. This reduction is primarily attributed to the PBUF described above; however, even mediators considered to be poorly filtered showed a significant decrease in load, suggesting additional mechanisms, not specifically addressed in our study, to be involved. Beyond filtration, some polymers used in hemofiltration have been reported to adsorb various inflammatory cytokines 22 , thereby lowering inflammatory mediator levels. Cytokine adsorption to CPB components, especially to the inner heparin-coated surfaces 23 , or binding to circulating albumin 24 may also contribute. Furthermore, re-binding of mediators to RBCs through DARC or glycocalyx could also be a relevant mechanism 5 . At our institution, sanguineous CPB prime is used when indicated to maintain recommended intraoperative hematocrit levels in neonates and infants 3 . To assess the potential clinical relevance of cytokines levels detected in CPB prime after PBUF, we used the median baseline concentration of patients measured in preoperative plasma samples as a threshold. At the time of CPB cannulation, the concentration of 8 out of 50 cytokines exceeded those in patient plasma. Among these, the vast majority constitutes pro-inflammatory chemotactic cytokines that affect monocyte and macrophage migration and differentiation, including MIF 25 , CCL2/MCP-1 26 and neutrophil migration such as CXCL1/GROα 27 . For instance, MIF is a pleiotropic protein with multiple biological functions playing a critical role in a variety of infectious and autoimmune diseases, as well as in kidney injury 28 . Erythrocytes have been shown to be the largest reservoir of MIF in the blood 5 , 9 . We also detected T helper type 2-associated eosinophil chemoattractants like IL-5 29 and CCL11/Eotaxin 30 as well as pleiotropic pro-inflammatory mediators such as IL-15 31 . The cell adhesion molecules ICAM-1 and VCAM-1 belong to the immunoglobulin superfamily and primarily play roles in leucocyte migration, endothelial adherence and lymphocyte activation 32 . The clinical impact of cytokine exposure from RBC-primed CPB within the complex inflammatory environment of neonatal and infant congenital heart surgery is challenging to assess. Two studies have already shown that PBUF could mitigate an intra- and postoperative rise in inflammatory markers such as Procalcitonin, TNF- α, IL-1β, IL-6 and CXCL8/IL-8 in children undergoing CPB heart surgery 33 , 34 . In these studies, PBUF was associated with reduced inotropic support, shorter ventilation times and intensive care stays. Furthermore, Cholette et al. showed that the immunomodulatory effects of RBC transfusions in pediatric cardiac surgery could be mitigated by removing supernatants through washing procedures 35 . Our current institutional approach to PBUF focuses on normalizing pH, glucose and electrolytes. A single filtration cycle is effectively counteracting the ongoing mediator release from RBCs and reduces the mediator content, but falls short in reducing some cytokine concentrations below patient's baseline. However, there are potential strategies to improve the efficacy of PBUF: increasing the number of filtration cycles, pre-treating RBCs using cell saver devices 35 or incorporating techniques such as countercurrent dialysis 36 , might further reduce mediator levels. Our study faces several limitations. First, the results obtained from RBC supernatants and CPB prime were not corrected for potential residual leukocyte and platelet contamination following leukocyte depletion of RBCs. Second, the proportion of positive samples for each cytokine and sample type, as well as the median mediator concentration for the cytokine-positive samples are provided. This approach accounts for the large inter-individual variability and documents the likelihood of a specific mediator being present and its expected concentration if detected. Consequently, the overall median, encompassing both positive and negative samples, is generally lower than the median reported here for most mediators. Third, due to the overall low concentration for some cytokines in the prime, the removal effect of PBUF could only be assessed for mediators that were reliably detectable in the prime. Fourth, since we did not conduct repetitive analyzes of RBC supernatants during storage, our results on mediator accumulation reflect only a correlation between storage time and cytokine content. The described effects could also be attributed to inter-individual donor-specific differences in packed RBCs. Finally, we chose the median preoperative baseline concentration of patients for specific cytokines as an arbitrary threshold for the potential clinical relevance of mediator load in RBC priming. However, this approach does not fully exclude the possibility that lower concentrations or levels of mediators may have an effect on the immune system in children undergoing cardiac surgery. In conclusion, multiple cytokines and chemokines were present in RBCs and were significantly released after priming the CPB circuit. PBUF effectively removed inflammatory mediators via the effluent, but with the current approach of a single filtration cycle, their concentrations remained largely stable. This limitation is likely due to the fact that the convective elimination of mediators through the hemofilter is combined with simultaneous volume reduction and the filtration characteristics of each cytokine. Beyond filtration, the decrease in total mediator content may be due to adsorption to circuit components or re-binding to RBCs. Improved washing techniques may further optimize mediator levels in RBC-primed CPB circuits. Methods Study design This prospective observational study was performed at Hannover Medical School between October 2019 and January 2021. The study was approved by the local ethics committee of the Hannover Medical School (No. 8591_BO_S_2019). All procedures involving human participants were performed in accordance with the ethical standards of the institutional and national research committee and with the Helsinki Declaration of 1964 and its later amendments or comparable ethical standards. Written informed consent was obtained for each child from their legal guardians. Trial registration: DRKS, DRKS00027572. Registered 07 February 2025 - Retrospectively registered, https://www.drks.de/DRKS00027572 . Study population Children with a body weight of less than 10 kg scheduled for congenital heart surgery with RBC priming of the CPB system were eligible for enrollment. Exclusion criteria were known immunodeficiency, suspected preoperative infection, and preoperative mechanical ventilation. Written informed consent was obtained for each child from their legal guardians. Cardiopulmonary bypass General anesthesia was induced intravenously with etomidate, sufentanil, esketamine and atracurium and maintained at the discretion of the anesthesiologist. For all operations, CPB system (Stöckert S5, Munich, Germany) and setup (Terumo FX05 Oxygenator, Eschborn, Germany; Terumo 3/16"x1/4" tubing set, Eschborn, Germany) (Fig. 1 ) were standardized. Heparin-coated tubing was used in all systems. CPB circuits were uniformly primed with 10 mL/kg of 20% human albumin (max. 100 mL), 1 mL of 10% calcium, 3 mL/kg of 20% mannitol, bicarbonate-buffered hemofiltration solution (Duosol, B. Braun, Melsungen, Germany), and heparin (150 IE/kg). After replacing the pre-bypass filter, 125 mL of CPB-buffered RBCs, stored in saline adenine-glucose-mannitol solution, was added to the circuit. Subsequently, circuit prime blood gas and electrolyte levels were analyzed to identify metabolic abnormalities. PBUF was performed at the perfusionists' discretion using a hollow-fiber polyarylsulfone membrane hemofilter (Maquet BC 20 Plus, Rastatt, Germany) 19 , with a maximum pressure of 300 mmHg, until pH status and electrolyte levels were normalized. Before cannulation, circuit volume was further reduced to the CPB circuit priming volume of 240 mL through continued PBUF. Study Protocol Blood samples were collected from stored RBC units, from the CPB prime 1 minute after RBCs were added and distributed throughout the circuit, and from CPB prime following PBUF immediately before cannulation. Additional control samples were collected from asanguine primes from three CPB circuits before RBC addition. All circuit samples were collected from the venous line (Fig. 1 ). For each time point, 0.5-1.0 ml sample volume was collected in EDTA tubes (Sarstedt, Nümbrecht, Germany) and immediately cooled on ice until further processing. PBUF samples were collected from the hemofilter outflow line (Fig. 1 ) at the end of PBUF in uncoated sterile sample tubes, simultaneously with the second CPB prime sample. Baseline patient samples were collected after induction of anesthesia and central venous line placement. All samples were centrifuged (2000 rpm, 8 min, 4°C), and supernatants were frozen at -80°C for later analysis. Mediator concentrations in plasma/ PBUF effluent/ RBC supernatant were quantified using Luminex-based multiplex technology and Bio-Plex assays (Bio-Plex Pro Human Cytokine Screening Panel, 48-Plex and Bio-Plex Pro Human Cytokine VCAM-1 and ICAM-1 Set, Fa Bio-Rad, Hercules, Ca, USA) as previously described according to manufacturer’s instructions 37 . All samples were diluted 1:1 with sample diluent provided with the kits. Standards were reconstituted and prepared according to the manufacturer’s instructions. Standard curves and concentrations were determined using the Bio-Plex Manager 6.1 software. Six baseline samples were measured in a separate analysis and were not included in further analyses. Laboratory data, patient clinical data, and CPB-related data were documented throughout the entire study. The following 50 mediators were analyzed: Cutaneous T-Cell attracting chemokine (CTACK, CCL27), CCL11/Eotaxin, basic fibroblast growth factor (FGF-β), Granulocyte-Colony Stimulating Factor (G-CSF), Granulocyte-macrophage colony-stimulating factor (GM-CSF), Growth-regulated alpha protein (GRO-α, CXCL1), Hepatocyte growth factor (HGF), Intracellular adhesion molecule 1 (ICAM-1), Interferon-gamma (IFN-g) a2, Interleukin (IL) 1a, 1b, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12(p40), 12(p70), 13, 15, 16, 17, 18, Interleukin receptor antagonist 1 (IL-1RA), soluble IL-2 receptor alpha (IL-2Rα), Interferon-gamma-inducible protein 10 (IP-10, CXCL10), Leukemia inhibitory factor (LIF), Monocyte chemotactic protein (MCP) 1 (CCL2) and 3 (CCL7), Macrophage colony-stimulating factor (M-CSF), Macrophage migration inhibitory factor (MIF), Monokine induced by interferon gamma (MIG, CXCL9), Macrophage inflammatory protein (MIP) 1α (CCL3) and 1β (CCL4), Nerve growth factor β (β-NGF), Platelet-derived growth factor-BB (PDGF-bb), Regulated upon activation normal T cell expressed and presumably secreted (RANTES, CCL5), Stem cell factor (SCF), Stem cell growth factor β (SCGFβ), Stromal cell-derived factor 1a (SDF-1α, CXCL12), Tumor necrosis factor (TNF) α and β, Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), Vascular cell adhesion molecule 1 (VCAM-1), and vascular endothelial growth factor (VEGF). Statistical analysis: Statistical analysis was performed in R (version 2022.07.2). All analyte concentration data showed non-normal distributions and are presented as median and interquartile range. Median concentrations were calculated from all samples that were above the lower limit of quantification. To enable comparative statistical analyses including mediator-negative samples, the lowest measurable concentration for each respective cytokine was used in statistical hypothesis testing for mediator-negative samples. The correlation between cytokine concentration and RBC storage time was evaluated using the Kendall´s rank correlation. Comparisons of concentrations between two time points were performed using the Wilcoxon rank-sum test (RBC vs. CPB prime pre-PBUF vs. patient baseline, RBC vs. CPB prime after PBUF vs. patient baseline) or the Wilcoxon Signed-Rank Test (CPB pre-PBUF vs. after PBUF) from the stats package. A p-value of < 0.05 was considered statistically significant. The removal effect 17 , 38 was calculated as follows: $$\:Removal\:Effect\:=\frac{1}{n}\:\sum\:_{i=1}^{n}\left(\frac{Cytokine\:Concentration\:in\:Ultrafiltration\:{Effluent}_{i}}{Cytokine\:Concentration\:in\:CPB\:{Prime}_{i}}\right)$$ For the calculation of cytokine removal, only those sample pairs were included in which the concentration in the prime was at least twice the lower limit of quantification. The absolute cytokine load was estimated from the CPB volume and the measured concentration during prime preparation before and after PBUF. Abbreviations CPB Cardiopulmonary bypass CTACK Cutaneous T-Cell attracting chemokine FGF-β Basic fibroblast growth factor GM-CSF Granulocyte-macrophage colony-stimulating factor G-CSF Granulocyte colony-stimulating factor GRO-α Growth-regulated alpha protein ICAM Intracellular adhesion molecule IL Interleukin IP-10 Interferon-gamma-inducible protein MCP Monocyte chemotactic protein M-CSF Macrophage colony-stimulating factor MIF Macrophage migration inhibitory factor MIP Macrophage inflammatory protein NGF Nerve growth factor PBUF Pre-bypass ultrafiltration RANTES Regulated upon activation normal T cell expressed and presumably secreted RBC Red blood cell SCF Stem cell factor SCGF Stem cell growth factor TNF Tumor necrosis factor TRAIL Tumor necrosis factor-related apoptosis-inducing ligand VCAM Vascular cell adhesion molecule VEGF Vascular endothelial growth factor Declarations Authors' contributions AP carried out data acquisition, analysis, interpretation and manuscript writing; SK performed data analysis, interpretation and manuscript writing; LT carried out data acquisition, analysis, and interpretation; ST, JO, AH and ND were involved in data acquisition and critical interpretation, NR and AK carried out data analysis and critical interpretation, CF performed experiments, data analysis and interpretation, PB was involved in data interpretation and critical review, MB designed the study and was involved in data acquisition, analysis, interpretation and manuscript writing. All authors read and approved the final manuscript. Data availability statement The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Additional information Competing Interests Statement The authors declare no competing interests. Funding This work was supported by a research grant from Hannover Medical School. Dr. Svea Kleiner and Dr. Martin Boehne were supported in part by a research grant from the German Society of Pediatric Cardiology (grant number: Forschungsförderung 2021). Dr. Svea Kleiner is participating in “YoungAcademy—PRACTIS” (PRogram of hAnnover medical school forClinician scienTISts), which is a clinician-scientist program funded by the German Research Foundation (DFG; ME-3696/3-1). Leonie Trachte was supported by the KlinStrucMed program of Hannover Medical School, funded by Else-Kröner-Fresenius Foundation. Acknowledgements The authors would like to thank Dr. Christoph Kammeyer for proofreading of the manuscript. References Dolk, H., Loane, M. & Garne, E. European Surveillance of Congenital Anomalies Working, G. Congenital heart defects in Europe: prevalence and perinatal mortality, 2000 to 2005. Circulation 123 , 841–849. https://doi.org:10.1161/CIRCULATIONAHA.110.958405 (2011). Oldeen, M. E., Angona, R. E., Hodge, A. & Klein, T. American Society of ExtraCorporeal Technology: Development of Standards and Guidelines for Pediatric and Congenital Perfusion Practice (2019). J. Extra Corpor. Technol. 52 , 319–326. https://doi.org:10.1182/ject-2000045 (2020). Faraoni, D., Meier, J., New, H. V., Van der Linden, P. J. & Hunt, B. J. Patient Blood Management for Neonates and Children Undergoing Cardiac Surgery: 2019 NATA Guidelines. J. Cardiothorac. Vasc. Anesth. 33 , 3249–3263. https://doi.org:10.1053/j.jvca.2019.03.036 (2019). Guzzetta, N. A. Benefits and risks of red blood cell transfusion in pediatric patients undergoing cardiac surgery. Paediatr Anaesth. 21 , 504–511. https://doi.org:10.1111/j.1460-9592.2010.03464.x (2011). Karsten, E. & Herbert, B. R. The emerging role of red blood cells in cytokine signalling and modulating immune cells. Blood Rev. 41 , 100644. https://doi.org:10.1016/j.blre.2019.100644 (2020). Karsten, E., Hill, C. J. & Herbert, B. R. Red blood cells: The primary reservoir of macrophage migration inhibitory factor in whole blood. Cytokine 102 , 34–40. https://doi.org:10.1016/j.cyto.2017.12.005 (2018). Sumpelmann, R., Schurholz, T., Thorns, E. & Hausdorfer, J. Acid-base, electrolyte and metabolite concentrations in packed red blood cells for major transfusion in infants. Paediatr Anaesth. 11 , 169–173. https://doi.org:10.1046/j.1460-9592.2001.00637.x (2001). Osthaus, W. A., Sievers, J., Breymann, T. & Suempelmann, R. Bicarbonate buffered ultrafiltration leads to a physiologic priming solution in pediatric cardiac surgery. Interact. Cardiovasc. Thorac. Surg. 7 , 969–972. https://doi.org:10.1510/icvts.2008.179333 (2008). Karsten, E., Breen, E. & Herbert, B. R. Red blood cells are dynamic reservoirs of cytokines. Sci. Rep. 8 , 3101. https://doi.org:10.1038/s41598-018-21387-w (2018). Bal, S. H. et al. Effect of storage period of red blood cell suspensions on helper T-cell subpopulations. Blood Transfus. 16 , 262–272. https://doi.org:10.2450/2017.0238-16 (2018). Almizraq, R. J. et al. Blood manufacturing methods affect red blood cell product characteristics and immunomodulatory activity. Blood Adv. 2 , 2296–2306. https://doi.org:10.1182/bloodadvances.2018021931 (2018). Freitas Leal, J. et al. The impact of circulation in a heart-lung machine on function and survival characteristics of red blood cells. Artif. Organs . 44 , 892–899. https://doi.org:10.1111/aor.13682 (2020). Kohne, I. Haemolysis induced by mechanical circulatory support devices: unsolved problems. Perfusion 35 , 474–483. https://doi.org:10.1177/0267659120931307 (2020). Fernando, H., Chin, C., Rosgen, J. & Rajarathnam, K. Dimer dissociation is essential for interleukin-8 (IL-8) binding to CXCR1 receptor. J. Biol. Chem. 279 , 36175–36178. https://doi.org:10.1074/jbc.C400283200 (2004). Schnabel, R. B. et al. Duffy antigen receptor for chemokines (Darc) polymorphism regulates circulating concentrations of monocyte chemoattractant protein-1 and other inflammatory mediators. Blood 115 , 5289–5299. https://doi.org:10.1182/blood-2009-05-221382 (2010). Bierer, J., Stanzel, R., Henderson, M., Sett, S. & Horne, D. Ultrafiltration in Pediatric Cardiac Surgery Review. World J. Pediatr. Congenit Heart Surg. 10 , 778–788. https://doi.org:10.1177/2150135119870176 (2019). Clar, A., Bowers, M. C. & Larson, D. F. Derivation of sieving coefficients to determine the efficacy of the hemoconcentrator in removal of four inflammatory mediators produced during cardiopulmonary bypass. ASAIO J. 43 , 163–170 (1997). Bierer, J. et al. Novel inflammatory mediator profile observed during pediatric heart surgery with cardiopulmonary bypass and continuous ultrafiltration. J. translational Med. 21 , 439. https://doi.org:10.1186/s12967-023-04255-8 (2023). Maquet Getinge Group. Hemoconcentrators reduced hemodilution BC 20 Plus, BC 60 Plus and BC 140 Plus. MCP_BR_10033_EN_10032 10004/12018. Miller, M. C. & Mayo, K. H. Chemokines from a Structural Perspective. Int. J. Mol. Sci. 18 https://doi.org:10.3390/ijms18102088 (2017). Berdat, P. A. et al. Elimination of proinflammatory cytokines in pediatric cardiac surgery: analysis of ultrafiltration method and filter type. J. Thorac. Cardiovasc. Surg. 127 , 1688–1696. https://doi.org:10.1016/j.jtcvs.2004.01.030 (2004). Kishikawa, T., Fujieda, H. & Sakaguchi, H. Comprehensive analysis of cytokine adsorption properties of polymethyl methacrylate (PMMA) membrane material. J. Artif. Organs . 25 , 343–349. https://doi.org:10.1007/s10047-022-01323-6 (2022). Fujita, M. et al. Adsorption of inflammatory cytokines using a heparin-coated extracorporeal circuit. Artif. Organs . 26 , 1020–1025. https://doi.org:10.1046/j.1525-1594.2002.07017.x (2002). Pfensig, C. et al. A New Application for Albumin Dialysis in Extracorporeal Organ Support: Characterization of a Putative Interaction Between Human Albumin and Proinflammatory Cytokines IL-6 and TNFalpha. Artif. Organs . 40 , 397–402. https://doi.org:10.1111/aor.12557 (2016). Cheng, Q. et al. Macrophage migration inhibitory factor increases leukocyte-endothelial interactions in human endothelial cells via promotion of expression of adhesion molecules. J. Immunol. 185 , 1238–1247. https://doi.org:10.4049/jimmunol.0904104 (2010). Singh, S., Anshita, D. & Ravichandiran, V. MCP-1: Function, regulation, and involvement in disease. Int. Immunopharmacol. 101 , 107598. https://doi.org:10.1016/j.intimp.2021.107598 (2021). Ahuja, S. K. & Murphy, P. M. The CXC chemokines growth-regulated oncogene (GRO) alpha, GRObeta, GROgamma, neutrophil-activating peptide-2, and epithelial cell-derived neutrophil-activating peptide-78 are potent agonists for the type B, but not the type A, human interleukin-8 receptor. J. Biol. Chem. 271 , 20545–20550. https://doi.org:10.1074/jbc.271.34.20545 (1996). Boor, P. MIF in kidney diseases: A story of Dr. Jekyll and Mr. Hyde. Pathologe 40 , 25–30. https://doi.org:10.1007/s00292-018-0548-1 (2019). Zhu, Y. et al. Cutting edge: IL-5 primes Th2 cytokine-producing capacity in eosinophils through a STAT5-dependent mechanism. J. Immunol. 173 , 2918–2922. https://doi.org:10.4049/jimmunol.173.5.2918 (2004). Conroy, D. M. et al. The role of the eosinophil-selective chemokine, eotaxin, in allergic and non-allergic airways inflammation. Mem. Inst. Oswaldo Cruz . 92 Suppl 2 , 183–191. https://doi.org:10.1590/s0074-02761997000800024 (1997). Perera, P. Y., Lichy, J. H., Waldmann, T. A. & Perera, L. P. The role of interleukin-15 in inflammation and immune responses to infection: implications for its therapeutic use. Microbes Infect. 14 , 247–261. https://doi.org:10.1016/j.micinf.2011.10.006 (2012). Singh, V., Kaur, R., Kumari, P., Pasricha, C. & Singh, R. ICAM-1 and VCAM-1: Gatekeepers in various inflammatory and cardiovascular disorders. Clin. Chim. Acta . 548 , 117487. https://doi.org:10.1016/j.cca.2023.117487 (2023). Gholampour Dehaki, M., Niknam, S., Azarfarin, R., Bakhshandeh, H. & Mahdavi, M. Zero-Balance Ultrafiltration of Priming Blood Attenuates Procalcitonin and Improves the Respiratory Function in Infants After Cardiopulmonary Bypass: A Randomized Controlled Trial. Artif. Organs . 43 , 167–172. https://doi.org:10.1111/aor.13325 (2019). Shimpo, H. et al. Ultrafiltration of the priming blood before cardiopulmonary bypass attenuates inflammatory response and improves postoperative clinical course in pediatric patients. Shock 16 (Suppl 1), 51–54. https://doi.org:10.1097/00024382-200116001-00010 (2001). Cholette, J. M. et al. Longer RBC storage duration is associated with increased postoperative infections in pediatric cardiac surgery. Pediatr. Crit. Care Med. 16 , 227–235. https://doi.org:10.1097/PCC.0000000000000320 (2015). Mitzner, S. et al. P1110in Vitro Cytokine Removal - Comparism of Conventional Hdf and Hdx (Middle-Cut-Off-Dialyzer, Baxter Theranova). Nephrol. Dialysis Transplantation . 35 https://doi.org:10.1093/ndt/gfaa142.P1110 (2020). Ledwoch, N. et al. Identification of distinct secretory patterns and their regulatory networks of ischemia versus reperfusion phases in clinical heart transplantation. Cytokine 149 , 155744. https://doi.org:10.1016/j.cyto.2021.155744 (2022). Guan, Y., Wan, C., Wang, S., Sun, P. & Long, C. Balanced ultrafiltration: inflammatory mediator removal capacity. Artif. Organs . 36 , 894–900. https://doi.org:10.1111/j.1525-1594.2012.01471.x (2012). Tables Table 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table12.docx SupplementaryInformationPBUFCytokines.pdf Cite Share Download PDF Status: Published Journal Publication published 25 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 09 May, 2025 Reviews received at journal 04 May, 2025 Reviews received at journal 30 Apr, 2025 Reviewers agreed at journal 27 Apr, 2025 Reviewers agreed at journal 24 Apr, 2025 Reviewers agreed at journal 23 Apr, 2025 Reviewers invited by journal 10 Apr, 2025 Editor assigned by journal 02 Apr, 2025 Submission checks completed at journal 31 Mar, 2025 First submitted to journal 31 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6193879","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":441302221,"identity":"52a66d08-e4b3-4856-831a-0faf788dc0d9","order_by":0,"name":"Aylin Poertecene","email":"","orcid":"","institution":"Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Aylin","middleName":"","lastName":"Poertecene","suffix":""},{"id":441302222,"identity":"deecdf35-5e81-4e61-bd16-25cce5e53aee","order_by":1,"name":"Svea Kleiner","email":"","orcid":"","institution":"Hannover Medical 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09:23:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6193879/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6193879/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-15746-7","type":"published","date":"2025-08-25T15:58:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80660800,"identity":"71085964-9dbd-4c77-ba56-9d5417dcb8ed","added_by":"auto","created_at":"2025-04-15 16:26:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":142908,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCPB setup\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStandardized cardiopulmonary bypass (CPB) circuit setup for all 22 patients. Prime samples were collected at the venous line during the priming process. Pre-bypass ultrafiltration (PBUF) effluent samples were directly taken from the hemofilter outlet at the end of the priming process.\u003c/p\u003e","description":"","filename":"Figure1101.png","url":"https://assets-eu.researchsquare.com/files/rs-6193879/v1/644ec00a7b2e4b264f4b2ae0.png"},{"id":80661950,"identity":"30720629-101b-4913-86da-04d8f9201ad5","added_by":"auto","created_at":"2025-04-15 16:42:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":97119,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytokines in packed red blood cells (RBCs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProportion of mediator-positive units of red blood cell (RBC) concentrates from n=22 samples used for pediatric cardiopulmonary bypass circuit priming for 50 measured cytokines.\u003c/p\u003e","description":"","filename":"Figure1102.png","url":"https://assets-eu.researchsquare.com/files/rs-6193879/v1/46ed5d861ef814fa54709008.png"},{"id":80661192,"identity":"c3db20e0-a014-4d1b-8044-80153ae88b2f","added_by":"auto","created_at":"2025-04-15 16:34:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":648082,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLongitudinal course of cytokine concentrations during priming\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea-h) Longitudinal course of 8 cytokines out of 50 during prime preparation. Figures show mediator concentrations in red blood cell (RBC) supernatants and prime samples before pre-bypass ultrafiltration (PBUF) and after PBUF. After PBUF, the concentrations of eight cytokines (MIF, IL-15, CCL11/Eotaxin, CCL2/MCP-1, VEGF, IL-5, VCAM-1 and ICAM-1) exceeded the median patient baseline concentration, while the remaining cytokines were at or below patient baseline. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001; see Supplementary Figure S2 for figures of the remaining 42 cytokines. ICAM, Intracellular adhesion molecule; IL, Interleukin; MCP, Monocyte chemotactic protein, MIF, Macrophage migration inhibitory factor; VCAM, Vascular cell adhesion molecule; VEGF, Vascular endothelial growth factor\u003c/p\u003e","description":"","filename":"Figure1103.png","url":"https://assets-eu.researchsquare.com/files/rs-6193879/v1/9d628adb84af621939070917.png"},{"id":80659773,"identity":"c24fa260-8a89-4cb8-b48d-779189db0baa","added_by":"auto","created_at":"2025-04-15 16:18:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":102915,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytokine concentrations in pre-bypass ultrafiltration effluent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea-b) Figure illustrates the spot concentrations of cytokines in the pre-bypass ultrafiltration (PBUF) effluent during the terminal phase of PBUF in relation to the concomitant mediator concentrations of CPB priming in a) low concentration range and b) high concentration range. The selection of these eight cytokines presented was based on their levels in the prime after PBUF, which exceeded the patient's baseline.\u003c/p\u003e\n\u003cp\u003ec-f) The figure depicts the density plots of the overall distribution of cytokine concentrations in CPB priming (red) and PBUFeffluent (blue) for four exemplary cytokines. The height of the curve at a given point along the x-axis indicates the relative likelihood of the concentration of IL-15, IL-5, MIF and VCAM-1 in the respective sample type. Strong overlap indicates convergence of cytokine concentrations in the PBUFeffluent and therefore more efficient cytokine removal.\u003c/p\u003e","description":"","filename":"Figure1104.png","url":"https://assets-eu.researchsquare.com/files/rs-6193879/v1/85fa5d510f7aebe87485e301.png"},{"id":90344946,"identity":"5940f8fc-5456-4fad-a56b-bc27ec86fa75","added_by":"auto","created_at":"2025-09-01 16:08:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1864110,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6193879/v1/a78c4d74-0637-45b4-894d-d2fd831f248b.pdf"},{"id":80659807,"identity":"00c80328-bec2-48d4-b7a4-aa26f83f8b9e","added_by":"auto","created_at":"2025-04-15 16:19:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":47389,"visible":true,"origin":"","legend":"","description":"","filename":"Table12.docx","url":"https://assets-eu.researchsquare.com/files/rs-6193879/v1/c3c072c701769d580492e093.docx"},{"id":80659784,"identity":"14121da2-f8b0-4778-b3d9-957addd526e6","added_by":"auto","created_at":"2025-04-15 16:18:55","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1439659,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationPBUFCytokines.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6193879/v1/75b33f20af460ea1aafd83b3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pre-Bypass Ultrafiltration reduces Cytokine Burden of Blood Prime in Pediatric Cardiac Surgery","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCongenital heart disease (CHD) is the most common congenital malformation \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Most children require cardiac surgery with cardiopulmonary bypass (CPB) at an early age \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Neonatal and infant congenital heart surgery is further complicated by the significant size mismatch between the CPB system and the patient's blood volume \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Therefore, to prevent excessive hemodilution and severe anemia, whole blood or stored allogeneic red blood cells (RBCs) are often added to the CPB circuit during priming prior to cannulation to maintain a hematocrit above 24% during CBP, as recommended by the Network for the Advancement of Patient Blood Management, Haemostasis and Thrombosis \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. This practice results in transfusion of stored allogeneic RBCs during initiation of CPB.\u003c/p\u003e \u003cp\u003eRBC transfusion is associated with immunosuppression, infection, electrolyte imbalance, acute kidney injury, and lung injury \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. These effects are partly related to storage-dependent changes in RBCs, which undergo morphological and functional changes \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Recently, packed RBCs have also been shown to be a major reservoir of cytokines, chemokines, and growth factors, suggesting a potential role in inflammatory processes \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In addition, stored RBCs develop unphysiologic acid-base, electrolyte, and metabolite values \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. To optimize unprocessed RBCs for CPB circuit priming, the American Society of Extra Corporeal Technology recommends pre-bypass ultrafiltration (PBUF) \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. At our institution, using a bicarbonate-buffered solution for PBUF, improved electrolyte, lactate, and acid-base levels of the priming solution \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. However, the ability of PBUF to remove pro-inflammatory mediators from packed RBCs used for CPB priming has not been evaluated.\u003c/p\u003e \u003cp\u003eTherefore, we conducted a single-center, prospective observational study to assess the inflammatory mediator burden of allogeneic RBC-primed CPB circuits in neonatal and infant congenital heart surgery, as well as the efficacy of PBUF in reducing cytokine/chemokine levels delivered to the patient.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eStudy population\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e22 children with various congenital heart defects with a median (range) age of 4.1 months (10 days to 34 months) and Risk Adjustment for Congenital Heart Surgery score of 3 (2\u0026ndash;6) were included in the study (Supplementary Table S1). Two children were treated in the intensive care unit prior to surgery.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePBUF was conducted during each surgery at the perfusionists\u0026apos; discretion until pH, metabolic parameters and electrolytes of the CPB circuit prime reached normal levels. This required a median (range) duration of 10 minutes (4\u0026ndash;35) and a median volume of 400 mL (250\u0026ndash;770) of hemofiltration solution. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMediator burden in RBC supernatants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantification of cytokines was performed on 22 RBC units obtained immediately before their application to the CPB prime (Table 1). Of the 50 cytokines assayed, 46 were detected in at least one RBC supernatant showing a high inter-sample variability, with 30 of the 50 mediators detected in at least 50% of the RBC supernatants. Notably, 16 of the 50 mediators were present in more than 90% of RBC supernatant samples (Figure 2). Only 6 mediators, namely Interleukin (IL)-1b, IL-12p70, IL-2, Monocyte chemotactic protein 3 (CCL7/MCP-3), Nerve growth factor b (b-NGF) and Granulocyte-macrophage colony-stimulating factor (GM-CSF), were detectable in 10% or less of RBC supernatants.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe median duration of RBC storage before use was 12.5 days (range 7\u0026ndash;28 days). Five of 50 cytokines, namely Stem cell growth factor b (SCGFb), CCL11/Eotaxin, Interferon-gamma-inducible protein 10 (CXCL10/IP-10), (Supplementary Figure S1), IL-16 and Cutaneous T-Cell attracting chemokine (CCL27/CTACK), (data not shown) showed a positive correlation between cytokine concentrations in RBC supernatants and storage duration. However, among these, CCL27/CTACK and IL-16 exhibited only a moderate correlation and showed extremely low levels that seem unlikely to be clinically relevant. Levels of CXCL10/IP-10 and SCGFb, which showed an association indicating potential storage-dependent changes, were several orders of magnitude lower in RBC supernatant compared to patient baseline levels (Supplementary Figure S1). CCL11/Eotaxin, on the other hand, showed significantly higher concentrations in RBC supernatants compared to patient baseline (Supplementary Figure S1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytokine concentration in RBC-free priming\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTumor necrosis factor-related apoptosis-inducing ligand (TRAIL), soluble IL-2 receptor alpha (IL-2Ra), Regulated upon activation normal T cell expressed and presumably secreted (CCL5/RANTES), SCGFb, Stem cell factor (SCF) and Macrophage colony-stimulating factor (M-CSF) were identified at low levels in control samples of asanguine priming solution before the addition of RBC; all other cytokines were not detected at quantifiable levels (Supplementary Table S2). Therefore, it must be assumed that negligible traces of a few cytokines are present in the other components involved in the priming process.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMediator concentration during priming and PBUF\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter priming of the CPB circuit, circulation and homogeneous mixing of all prime components, all cytokines identified in RBC supernatants remained detectable. Most mediators were present at either comparable or higher concentrations than in RBC supernatants (Table 1). Of the 50 mediators analyzed, the concentrations of 25 (IL-15, IL-9, Tumor necrosis factor beta [TNF-b], TRAIL, IL-17, Interferon-gamma [IFN-g], IL-2Ra, IL-4, Leukemia inhibitory factor [LIF], Growth-regulated alpha protein [CXCL1/GRO-a], CCL5/RANTES, CCL11/Eotaxin, Stromal cell-derived factor 1 alpha [CXCL12/SDF-1a], Macrophage inflammatory protein 1b\u0026nbsp;[CCL4/MIP-1b],\u0026nbsp;Monocyte chemotactic protein 1 [CCL2/MCP-1], CXCL8/IL-8, Vascular Endothelial Growth Factor [VEGF], IL-5, Granulocyte-Colony Stimulating Factor [G-CSF], SCF, IL-7, Macrophage colony-stimulating factor [M-CSF], IL-3, b-NGF, Intracellular adhesion molecule 1 [ICAM-1]) increased significantly, while 4 cytokines (Macrophage migration inhibitory factor [MIF], IL-18, Hepatocyte growth factor [HGF] and soluble Vascular cell adhesion molecule 1 [VCAM-1]) decreased significantly in concentration. For the remaining cytokines, no change in concentration was observed (Figure 3 and Table 1, Supplementary Figure S2). Irrespective of the changes in concentration, many of the mediators were detected in a larger number of priming samples than in the RBC samples.\u003c/p\u003e\n\u003cp\u003eAfter complete assembly, the priming solution was circulated in the CPB circuit and PBUF was performed. After PBUF, 20 out of 50 cytokines further increased significantly in concentration (IL-9, TNF-b, IL-1a, IL-17, IFN-g, IL-2Ra, IL-13, IL-4, CCL11/Eotaxin, CCL4/MIP-1b, CCL2/MCP-1, CXCL8/IL-8, G-CSF, basic fibroblast growth factor [FGF-b], SCF, IL-7, M-CSF, b-NGF, VCAM-1, ICAM-1), while the others remained stable, none decreased (Table 1 and Figure 3, Supplementary Figure S2). Some of the cytokines (IL-1a, IFN- g, IL-1 RA, IL-13, CCL27/CTACK, FGF-b, IL-7, b-NGF) were also detected in a relevant higher number of samples than before PBUF. Only IL-12p70 was undetectable in both, pre- and post-PBUF samples (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePriming vs. patient mediator concentration\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo establish a threshold for the potential clinical relevance of mediator burden in RBC priming, we compared the cytokine levels detected in the priming after PBUF with the median preoperative plasma levels of the patients in our study cohort for each of the cytokines. After PBUF, the concentration of 8 out of 50 cytokines (MIF, IL-15, CCL11/Eotaxin, CCL2/MCP-1, VEGF, IL-5, VCAM-1, and ICAM-1) in the CPB circuit significantly exceeded the median preoperative baseline levels of the patients (Table 1, Figure 3, Supplementary Figure S2). Levels of all other mediators showed a non-significant trend to be elevated above the patient baseline (IL-3, IL-4, M-CSF [p-value \u0026lt;0.1]), ranged below the median patient baseline, or were undetectable (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMediator removal by PBUF\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe majority of the 50 cytokines analyzed were also detectable in the PBUF effluent at the end of PBUF (Supplementary Table S2). Among the 8 cytokines whose concentrations in the priming solution after PBUF exceeded baseline serum levels in patients, 7 showed a positive correlation between their spot concentrations in the PBUF effluent and their respective levels in the CPB prime. The slope of the linear regression line varied for each cytokine, reflecting differences in filtration properties (Figure 4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe removal effect, calculated as ratio of respective cytokine concentrations in the priming and PBUF effluent, ranged from 0.67 \u0026plusmn; 1.03% for VCAM-1 to 97.73 \u0026plusmn; 83.92% for VEGF (Table 2, Supplementary Table S2). However, there was variability in the removal effect, indicating better filtration of smaller molecules such as VEGF (43 kDa) or IL-15 (18k Da) and restricted filtration of larger molecules such as M-CSF (107 kDa), ICAM-1 (58 kDa in monomeric form), and VCAM-1 (81-110 kDa).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbsolute mediator content during priming and PBUF\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further estimate the total mediator load, the mediator concentration was multiplied by i) the volume of applied RBCs and ii) the respective filling volume of the CPB circuit at specific stages of the priming process, accounting for the particular volume used for PBUF.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter priming the CPB circuit, the total mediator load for 48 cytokines increased significantly compared to the mediator load from the applied packed RBC volume (Supplementary Figure S3 and Table S4). CCL7/MCP-3 and IL-12(p70), which were below the lower detection limit in RBCs, were not detectable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter further processing of the prime comprising PBUF, the load of 42 out of 50 mediators decreased significantly (Supplementary Figure S3 and Table S4), whereas the contents of 5 mediators did not decrease significantly (IL-2,\u0026nbsp;b-NGF, GM-CSF) or remained stable (IL-13,\u0026nbsp;FGF-b). These 5 mediators were consistently detected at low concentrations in all samples (Table 1). Only IL-1b\u0026nbsp;and\u0026nbsp;CCL7/MCP-3 levels increased significantly, but this was negligible, as it was detected in only 1 or 2 samples above the detection limit. IL-12(p70) was undetectable.\u003c/p\u003e\n\u003cp\u003eThe final mediator load ranged from 33.3 % (CXCL9/MIG) to 69.1 % (IL-16) of pre-PBUF baseline levels. Notably, mediators that exceeded the median preoperative baseline concentrations in our patient cohort, as well as those identified to be poorly filtered (Table 2), were also significantly reduced.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe conducted a single-center observational study to assess the inflammatory mediator burden of allogeneic RBC-primed CPB circuits during neonatal and infant congenital heart surgery, as well as the efficacy of pre-bypass ultrafiltration (PBUF) in reducing cytokine and chemokine levels. To the best of our knowledge, this is the first comprehensive analysis of a broad range of pro- and anti-inflammatory factors, chemokines, growth factors and endothelial markers during the priming process. Our findings confirm that packed RBC, once thought to be immunologically inert, are sources of a wide variety of cytokines. After RBC priming of the CPB circuit, circulation and homogeneous mixing of all components, RBCs continue to release mediators, resulting in higher cytokine concentrations and loads in CPB prime compared to packed RBC supernatants. Subsequent PBUF was highly effective and successfully removed a significant portion of mediators through the effluent. Notably, our study is the first to demonstrate that the entire priming process further reduced the overall inflammatory mediator load.\u003c/p\u003e \u003cp\u003eFirst, most of the cytokines we found in packed RBC were detectable at low levels in the supernatants of RBC units, with a large variability most likely due to donor specifications. This observation is consistent with recent studies, including those by Karsten et al., which highlighted RBCs as dynamic cytokine reservoir \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Their investigation identified 46 mediators in RBC lysates and 46 cytokines in RBC-conditioned media \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Our analysis supplements their findings, detecting 46 out of 50 mediators in RBC units used for CPB priming, with 30 present in more than half of the stored RBC unit supernatants. However, we observed slight differences in the detectable cytokines. Four mediators (IL-1β, IL-12 (p70), CCL7/MCP-3, and GM-CSF) previously reported in RBC-conditioned media were undetectable in our RBC supernatant samples \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, while three cytokines (CCL7/MCP-3, β-NGF, and SCGF-ß) were previously not been described \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. These discrepancies may be due to high inter-individual and donor-dependent cytokine variability in RBC units and limited sample sizes in both studies. Additionally, while Karsten et al. used freshly isolated RBCs for their experiments, we investigated packed RBC after routine handling and storage. Remarkably, of the 50 mediators analyzed, only five showed a correlation between mediator burden and prolonged storage time. This is in contrast to the literature, which describes changes in immunological profiles of leukocyte-depleted RBCs depending on storage time or manufacturing methods \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. A possible explanation for these discrepancies is that our study did not track mediator concentrations in packed RBCs across the complete storage period.\u003c/p\u003e \u003cp\u003eSecond, RBCs continue to release mediators after administration into the CPB circuit, leading to significantly higher cytokine levels in CPB prime compared to initial RBC supernatants. This suggests an ongoing release mechanism, likely amplified by mechanical stimulation from CPB circuit components \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. It is reasonable to assume, that shear stress and surface contact with circuit components induce some degree of hemolysis, resulting in the release of intracellular mediators from RBC \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, the literature indicates that a fraction of cytokines, such as CXCL8/IL-8, exist in a bound form within the RBC cytoplasm and might therefore not be detectable in our assays after hemolysis \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Additional active release of monomeric cytokines by intact RBCs is likely to cause a measurable increase in mediator levels throughout the priming preparation \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. To that effect, versatile binding and adaptive release of mediators on RBCs through the Duffy antigen receptor for chemokines (DARC) and the erythrocyte glycocalyx have been previously described \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. To date, two major triggers for CCL2/MCP-1 release from DARC have been identified: the exposure to unfractionated heparin and the coagulation process \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Both conditions are frequently encountered when donor blood is used for CPB priming.\u003c/p\u003e \u003cp\u003eThird, PBUF of the CPB prime was effective, successfully removing a significant portion of the mediator load, as indicated by cytokines detection in the PBUF effluent. The concomitant increase in the concentration of 20 of 50 inflammatory mediators after PBUF is likely due to several factors. As a convection-based process, PBUF cannot achieve an absolute reduction in mediator concentrations \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The effectiveness of filtration is further constrained by the filter membrane properties and the specific characteristics of each cytokine \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. According to the manufacturer \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, a comparable larger hemofilter with an identical polyarylsulfone membrane used in our study exhibits a sieving coefficient of 0.6 for 10 kDa molecules. Since most chemokines have a molecular mass of approximately 15 kDa \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, they are expected to be effectively filtered, whereas larger molecules are known to be less removed \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Notably, some mediators exhibited unexpected filtration properties relative to their molecular weight. For instance, SCF (30 kDa) was largely undetectable in PBUF effluent, while IL-5 (15 kDa) appeared only in a subset of effluent samples (5 out of 21). This suggests that probably additional factors such as hydrophobicity or molecular structure may influence cytokine removal by PBUF \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRemoval efficiency by PBUF varied from 0.6–97% across different cytokines, which is consistent with findings from other studies, such as Bierer et al., who documented extensive removal of 20 out of 39 mediators via ultrafiltration \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The low mediator concentrations in CPB prime preclude drawing generalized conclusions about the removal capacity for some of the cytokines that were undetectable in PBUF effluent. However, a difference was found for CCL5/RANTES, which was well filtered in our system, but was not removed in the study by Bierer et al. \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. This difference may be attributed to the use of different filtration membranes: polysulfone in their study versus polyarylsulfone in ours. Both materials likely have different filtration characteristics for specific cytokines, as previously published for comparison of polyamide and polysulfone membranes \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOverall, we observed a significant reduction in the total load of 42 different mediators during the entire priming processing. The final mediator load ranging from 33.3% (CXCL9/MIG) to 69.1% (IL-16) of pre-processing baseline levels. This reduction is primarily attributed to the PBUF described above; however, even mediators considered to be poorly filtered showed a significant decrease in load, suggesting additional mechanisms, not specifically addressed in our study, to be involved. Beyond filtration, some polymers used in hemofiltration have been reported to adsorb various inflammatory cytokines \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, thereby lowering inflammatory mediator levels. Cytokine adsorption to CPB components, especially to the inner heparin-coated surfaces \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, or binding to circulating albumin \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e may also contribute. Furthermore, re-binding of mediators to RBCs through DARC or glycocalyx could also be a relevant mechanism \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAt our institution, sanguineous CPB prime is used when indicated to maintain recommended intraoperative hematocrit levels in neonates and infants \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. To assess the potential clinical relevance of cytokines levels detected in CPB prime after PBUF, we used the median baseline concentration of patients measured in preoperative plasma samples as a threshold. At the time of CPB cannulation, the concentration of 8 out of 50 cytokines exceeded those in patient plasma. Among these, the vast majority constitutes pro-inflammatory chemotactic cytokines that affect monocyte and macrophage migration and differentiation, including MIF \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, CCL2/MCP-1 \u003csup\u003e26\u003c/sup\u003e and neutrophil migration such as CXCL1/GROα \u003csup\u003e27\u003c/sup\u003e. For instance, MIF is a pleiotropic protein with multiple biological functions playing a critical role in a variety of infectious and autoimmune diseases, as well as in kidney injury \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Erythrocytes have been shown to be the largest reservoir of MIF in the blood \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. We also detected T helper type 2-associated eosinophil chemoattractants like IL-5 \u003csup\u003e29\u003c/sup\u003e and CCL11/Eotaxin \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e as well as pleiotropic pro-inflammatory mediators such as IL-15 \u003csup\u003e31\u003c/sup\u003e. The cell adhesion molecules ICAM-1 and VCAM-1 belong to the immunoglobulin superfamily and primarily play roles in leucocyte migration, endothelial adherence and lymphocyte activation \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe clinical impact of cytokine exposure from RBC-primed CPB within the complex inflammatory environment of neonatal and infant congenital heart surgery is challenging to assess. Two studies have already shown that PBUF could mitigate an intra- and postoperative rise in inflammatory markers such as Procalcitonin, TNF- α, IL-1β, IL-6 and CXCL8/IL-8 in children undergoing CPB heart surgery \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In these studies, PBUF was associated with reduced inotropic support, shorter ventilation times and intensive care stays. Furthermore, Cholette et al. showed that the immunomodulatory effects of RBC transfusions in pediatric cardiac surgery could be mitigated by removing supernatants through washing procedures \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur current institutional approach to PBUF focuses on normalizing pH, glucose and electrolytes. A single filtration cycle is effectively counteracting the ongoing mediator release from RBCs and reduces the mediator content, but falls short in reducing some cytokine concentrations below patient's baseline. However, there are potential strategies to improve the efficacy of PBUF: increasing the number of filtration cycles, pre-treating RBCs using cell saver devices \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e or incorporating techniques such as countercurrent dialysis \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, might further reduce mediator levels.\u003c/p\u003e \u003cp\u003eOur study faces several limitations. First, the results obtained from RBC supernatants and CPB prime were not corrected for potential residual leukocyte and platelet contamination following leukocyte depletion of RBCs. Second, the proportion of positive samples for each cytokine and sample type, as well as the median mediator concentration for the cytokine-positive samples are provided. This approach accounts for the large inter-individual variability and documents the likelihood of a specific mediator being present and its expected concentration if detected. Consequently, the overall median, encompassing both positive and negative samples, is generally lower than the median reported here for most mediators. Third, due to the overall low concentration for some cytokines in the prime, the removal effect of PBUF could only be assessed for mediators that were reliably detectable in the prime. Fourth, since we did not conduct repetitive analyzes of RBC supernatants during storage, our results on mediator accumulation reflect only a correlation between storage time and cytokine content. The described effects could also be attributed to inter-individual donor-specific differences in packed RBCs. Finally, we chose the median preoperative baseline concentration of patients for specific cytokines as an arbitrary threshold for the potential clinical relevance of mediator load in RBC priming. However, this approach does not fully exclude the possibility that lower concentrations or levels of mediators may have an effect on the immune system in children undergoing cardiac surgery.\u003c/p\u003e \u003cp\u003eIn conclusion, multiple cytokines and chemokines were present in RBCs and were significantly released after priming the CPB circuit. PBUF effectively removed inflammatory mediators via the effluent, but with the current approach of a single filtration cycle, their concentrations remained largely stable. This limitation is likely due to the fact that the convective elimination of mediators through the hemofilter is combined with simultaneous volume reduction and the filtration characteristics of each cytokine. Beyond filtration, the decrease in total mediator content may be due to adsorption to circuit components or re-binding to RBCs. Improved washing techniques may further optimize mediator levels in RBC-primed CPB circuits.\u003c/p\u003e "},{"header":"Methods","content":"\u003ch2\u003eStudy design\u003c/h2\u003e\u003cp\u003eThis prospective observational study was performed at Hannover Medical School between October 2019 and January 2021. The study was approved by the local ethics committee of the Hannover Medical School (No. 8591_BO_S_2019). All procedures involving human participants were performed in accordance with the ethical standards of the institutional and national research committee and with the Helsinki Declaration of 1964 and its later amendments or comparable ethical standards. Written informed consent was obtained for each child from their legal guardians. Trial registration: DRKS, DRKS00027572. Registered 07 February 2025 - Retrospectively registered, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.drks.de/DRKS00027572\u003c/span\u003e\u003cspan address=\"https://www.drks.de/DRKS00027572\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e\u003ch2\u003eStudy population\u003c/h2\u003e\u003cp\u003eChildren with a body weight of less than 10 kg scheduled for congenital heart surgery with RBC priming of the CPB system were eligible for enrollment. Exclusion criteria were known immunodeficiency, suspected preoperative infection, and preoperative mechanical ventilation. Written informed consent was obtained for each child from their legal guardians.\u003c/p\u003e\u003ch2\u003eCardiopulmonary bypass\u003c/h2\u003e\u003cp\u003eGeneral anesthesia was induced intravenously with etomidate, sufentanil, esketamine and atracurium and maintained at the discretion of the anesthesiologist. For all operations, CPB system (Stöckert S5, Munich, Germany) and setup (Terumo FX05 Oxygenator, Eschborn, Germany; Terumo 3/16\"x1/4\" tubing set, Eschborn, Germany) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were standardized. Heparin-coated tubing was used in all systems. CPB circuits were uniformly primed with 10 mL/kg of 20% human albumin (max. 100 mL), 1 mL of 10% calcium, 3 mL/kg of 20% mannitol, bicarbonate-buffered hemofiltration solution (Duosol, B. Braun, Melsungen, Germany), and heparin (150 IE/kg). After replacing the pre-bypass filter, 125 mL of CPB-buffered RBCs, stored in saline adenine-glucose-mannitol solution, was added to the circuit. Subsequently, circuit prime blood gas and electrolyte levels were analyzed to identify metabolic abnormalities. PBUF was performed at the perfusionists' discretion using a hollow-fiber polyarylsulfone membrane hemofilter (Maquet BC 20 Plus, Rastatt, Germany) \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, with a maximum pressure of 300 mmHg, until pH status and electrolyte levels were normalized. Before cannulation, circuit volume was further reduced to the CPB circuit priming volume of 240 mL through continued PBUF.\u003c/p\u003e\u003ch2\u003eStudy Protocol\u003c/h2\u003e\u003cp\u003eBlood samples were collected from stored RBC units, from the CPB prime 1 minute after RBCs were added and distributed throughout the circuit, and from CPB prime following PBUF immediately before cannulation. Additional control samples were collected from asanguine primes from three CPB circuits before RBC addition. All circuit samples were collected from the venous line (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For each time point, 0.5-1.0 ml sample volume was collected in EDTA tubes (Sarstedt, Nümbrecht, Germany) and immediately cooled on ice until further processing. PBUF samples were collected from the hemofilter outflow line (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e) at the end of PBUF in uncoated sterile sample tubes, simultaneously with the second CPB prime sample. Baseline patient samples were collected after induction of anesthesia and central venous line placement. All samples were centrifuged (2000 rpm, 8 min, 4°C), and supernatants were frozen at -80°C for later analysis.\u003c/p\u003e\u003cp\u003eMediator concentrations in plasma/ PBUF effluent/ RBC supernatant were quantified using Luminex-based multiplex technology and Bio-Plex assays (Bio-Plex Pro Human Cytokine Screening Panel, 48-Plex and Bio-Plex Pro Human Cytokine VCAM-1 and ICAM-1 Set, Fa Bio-Rad, Hercules, Ca, USA) as previously described according to manufacturer’s instructions \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. All samples were diluted 1:1 with sample diluent provided with the kits. Standards were reconstituted and prepared according to the manufacturer’s instructions. Standard curves and concentrations were determined using the Bio-Plex Manager 6.1 software. Six baseline samples were measured in a separate analysis and were not included in further analyses. Laboratory data, patient clinical data, and CPB-related data were documented throughout the entire study.\u003c/p\u003e\u003cp\u003eThe following 50 mediators were analyzed: Cutaneous T-Cell attracting chemokine (CTACK, CCL27), CCL11/Eotaxin, basic fibroblast growth factor (FGF-β), Granulocyte-Colony Stimulating Factor (G-CSF), Granulocyte-macrophage colony-stimulating factor (GM-CSF), Growth-regulated alpha protein (GRO-α, CXCL1), Hepatocyte growth factor (HGF), Intracellular adhesion molecule 1 (ICAM-1), Interferon-gamma (IFN-g) a2, Interleukin (IL) 1a, 1b, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12(p40), 12(p70), 13, 15, 16, 17, 18, Interleukin receptor antagonist 1 (IL-1RA), soluble IL-2 receptor alpha (IL-2Rα), Interferon-gamma-inducible protein 10 (IP-10, CXCL10), Leukemia inhibitory factor (LIF), Monocyte chemotactic protein (MCP) 1 (CCL2) and 3 (CCL7), Macrophage colony-stimulating factor (M-CSF), Macrophage migration inhibitory factor (MIF), Monokine induced by interferon gamma (MIG, CXCL9), Macrophage inflammatory protein (MIP) 1α (CCL3) and 1β (CCL4), Nerve growth factor β (β-NGF), Platelet-derived growth factor-BB (PDGF-bb), Regulated upon activation normal T cell expressed and presumably secreted (RANTES, CCL5), Stem cell factor (SCF), Stem cell growth factor β (SCGFβ), Stromal cell-derived factor 1a (SDF-1α, CXCL12), Tumor necrosis factor (TNF) α and β, Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), Vascular cell adhesion molecule 1 (VCAM-1), and vascular endothelial growth factor (VEGF).\u003c/p\u003e\u003ch2\u003eStatistical analysis:\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed in R (version 2022.07.2). All analyte concentration data showed non-normal distributions and are presented as median and interquartile range. Median concentrations were calculated from all samples that were above the lower limit of quantification. To enable comparative statistical analyses including mediator-negative samples, the lowest measurable concentration for each respective cytokine was used in statistical hypothesis testing for mediator-negative samples. The correlation between cytokine concentration and RBC storage time was evaluated using the Kendall´s rank correlation. Comparisons of concentrations between two time points were performed using the Wilcoxon rank-sum test (RBC vs. CPB prime pre-PBUF vs. patient baseline, RBC vs. CPB prime after PBUF vs. patient baseline) or the Wilcoxon Signed-Rank Test (CPB pre-PBUF vs. after PBUF) from the stats package. A p-value of \u0026lt; 0.05 was considered statistically significant. The removal effect \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e was calculated as follows:\u003c/p\u003e\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Removal\\:Effect\\:=\\frac{1}{n}\\:\\sum\\:_{i=1}^{n}\\left(\\frac{Cytokine\\:Concentration\\:in\\:Ultrafiltration\\:{Effluent}_{i}}{Cytokine\\:Concentration\\:in\\:CPB\\:{Prime}_{i}}\\right)$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003eFor the calculation of cytokine removal, only those sample pairs were included in which the concentration in the prime was at least twice the lower limit of quantification. The absolute cytokine load was estimated from the CPB volume and the measured concentration during prime preparation before and after PBUF.\u003c/p\u003e"},{"header":"Abbreviations","content":" \u003cp\u003eCPB Cardiopulmonary bypass\u003c/p\u003e \u003cp\u003eCTACK Cutaneous T-Cell attracting chemokine\u003c/p\u003e \u003cp\u003eFGF-β Basic fibroblast growth factor\u003c/p\u003e \u003cp\u003eGM-CSF Granulocyte-macrophage colony-stimulating factor\u003c/p\u003e \u003cp\u003eG-CSF Granulocyte colony-stimulating factor\u003c/p\u003e \u003cp\u003eGRO-α Growth-regulated alpha protein\u003c/p\u003e \u003cp\u003eICAM Intracellular adhesion molecule\u003c/p\u003e \u003cp\u003eIL Interleukin\u003c/p\u003e \u003cp\u003eIP-10 Interferon-gamma-inducible protein\u003c/p\u003e \u003cp\u003eMCP Monocyte chemotactic protein\u003c/p\u003e \u003cp\u003eM-CSF Macrophage colony-stimulating factor\u003c/p\u003e \u003cp\u003eMIF Macrophage migration inhibitory factor\u003c/p\u003e \u003cp\u003eMIP Macrophage inflammatory protein\u003c/p\u003e \u003cp\u003eNGF Nerve growth factor\u003c/p\u003e \u003cp\u003ePBUF Pre-bypass ultrafiltration\u003c/p\u003e \u003cp\u003eRANTES Regulated upon activation normal T cell expressed and presumably secreted\u003c/p\u003e \u003cp\u003eRBC Red blood cell\u003c/p\u003e \u003cp\u003eSCF Stem cell factor\u003c/p\u003e \u003cp\u003eSCGF Stem cell growth factor\u003c/p\u003e \u003cp\u003eTNF Tumor necrosis factor\u003c/p\u003e \u003cp\u003eTRAIL Tumor necrosis factor-related apoptosis-inducing ligand\u003c/p\u003e \u003cp\u003eVCAM Vascular cell adhesion molecule\u003c/p\u003e \u003cp\u003eVEGF Vascular endothelial growth factor\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAP carried out data acquisition, analysis, interpretation and manuscript writing; SK performed data analysis, interpretation and manuscript writing; LT carried out data acquisition, analysis, and interpretation; ST, JO, AH and ND were involved in data acquisition and critical interpretation, NR and AK carried out data analysis and critical interpretation, CF performed experiments, data analysis and interpretation, PB was involved in data interpretation and critical review, MB designed the study and was involved in data acquisition, analysis, interpretation and manuscript writing. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a research grant from Hannover Medical School. Dr. Svea Kleiner and Dr. Martin Boehne were supported in part by a research grant from the German Society of Pediatric Cardiology (grant number: Forschungsf\u0026ouml;rderung 2021). Dr. Svea Kleiner is participating in \u0026ldquo;YoungAcademy\u0026mdash;PRACTIS\u0026rdquo; (PRogram of hAnnover medical school forClinician scienTISts), which is a clinician-scientist program funded by the German Research Foundation (DFG; ME-3696/3-1). Leonie Trachte was supported by the KlinStrucMed program of Hannover Medical School, funded by Else-Kr\u0026ouml;ner-Fresenius Foundation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Dr. Christoph Kammeyer for proofreading of the manuscript.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDolk, H., Loane, M. \u0026amp; Garne, E. European Surveillance of Congenital Anomalies Working, G. 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Organs\u003c/em\u003e. \u003cb\u003e36\u003c/b\u003e, 894\u0026ndash;900. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1111/j.1525-1594.2012.01471.x\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1111/j.1525-1594.2012.01471.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2012).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cytokines, cardiopulmonary bypass, ultrafiltration, red blood cell, priming, children","lastPublishedDoi":"10.21203/rs.3.rs-6193879/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6193879/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAllogeneic red blood cells (RBCs) are commonly used for cardiopulmonary bypass (CPB) circuit priming in congenital heart surgery. While convection-based pre-bypass ultrafiltration (PBUF) corrects acid-base, electrolyte, and metabolite imbalances, its efficacy in removing RBC cytokines/chemokines remains unclear.\u003c/p\u003e \u003cp\u003eIn a prospective observational study, 22 children (median age: 4.1 months) undergoing congenital heart surgery were enrolled. PBUF of RBC-primed CPB circuits was conducted using bicarbonate-buffered hemofiltration solution. Cytokines/chemokines were quantified in RBC supernatants, CPB priming (before and after PBUF), preoperative patient plasma, and PBUF effluent using Luminex-based multiplex technology. 30 of 50 cytokines were detected in \u0026gt;\u0026thinsp;50% of RBC supernatants. RBC priming significantly elevated concentrations of 25 cytokines, with 20 further rising after PBUF. At CPB onset, eight mediators (MIF, IL-15, CCL11/Eotaxin, CCL2/MCP-1, VEGF, IL-5, VCAM-1, ICAM-1) exceeded patient plasma concentrations. PBUF filtered cytokines with different efficiencies (0.6\u0026ndash;97%). Despite poor filtration or increased concentrations, total mediator load of 42 cytokines decreased significantly (33.3\u0026ndash;69.1% of pre-processing levels) after PBUF. In conclusion, PBUF effectively removed multiple cytokines/chemokines released from RBC. Beyond filtration, decrease of total mediator load may be attributed to adsorption to circuit components or rebinding to RBCs. Improved washing techniques may further optimize mediator levels in RBC-primed CPB circuits.\u003c/p\u003e","manuscriptTitle":"Pre-Bypass Ultrafiltration reduces Cytokine Burden of Blood Prime in Pediatric Cardiac Surgery","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-15 16:18:47","doi":"10.21203/rs.3.rs-6193879/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-09T17:51:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-04T22:19:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-30T14:38:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"92303250365833440989640049360806008090","date":"2025-04-27T11:18:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223449106771444487669085891014082971324","date":"2025-04-24T16:34:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"302420710442973675480213080563822086407","date":"2025-04-23T19:43:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-10T17:18:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-02T15:46:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-31T15:32:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-31T15:31:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bfbb7802-8c60-4c5f-8a7c-ec10427cb9b6","owner":[],"postedDate":"April 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46990752,"name":"Biological sciences/Immunology/Cytokines"},{"id":46990753,"name":"Health sciences/Diseases/Cardiovascular diseases/Congenital heart defects"}],"tags":[],"updatedAt":"2025-09-01T16:04:00+00:00","versionOfRecord":{"articleIdentity":"rs-6193879","link":"https://doi.org/10.1038/s41598-025-15746-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-08-25 15:58:23","publishedOnDateReadable":"August 25th, 2025"},"versionCreatedAt":"2025-04-15 16:18:47","video":"","vorDoi":"10.1038/s41598-025-15746-7","vorDoiUrl":"https://doi.org/10.1038/s41598-025-15746-7","workflowStages":[]},"version":"v1","identity":"rs-6193879","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6193879","identity":"rs-6193879","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}