Complement-targeting therapies in hemolytic diseases.

Micafungin and other echinocandins are commonly used for the prophylaxis and empirical treatment of invasive fungal infections in neutropenic patients due to their broad-spectrum activity and favorable safety profile. Drug-induced hemolytic anemia (DIHA) is a rare condition, and micafungin-related cases are exceptionally uncommon. We describe a fatal case of intravascular hemolysis triggered by re-administration of micafungin in a patient receiving chemotherapy for peripheral T cell lymphoma. The patient experienced convulsions and shock within minutes of infusion of the drug, followed by severe hemolysis and disseminated intravascular coagulation. Subsequently, hemolytic mechanisms were evaluated using drug-adsorption and immune complex models. Additionally, we had sera from eight patients treated with micafungin between January 2019 and December 2022, including our index case, tested for anti-micafungin antibodies. Immune complex-mediated hemolysis was confirmed in the index case, supported by positive indirect antiglobulin tests and in vitro hemolysis. Anti-micafungin antibodies were only detected in this single patient, indicating an extremely rare immunologic reaction. Although rare, micafungin-induced DIHA can be life-threatening. Clinicians should remain vigilant, particularly when resuming or continuing administration of micafungin.

Background: Bites from spiders of the genus Loxosceles, popularly known as the brown recluse spider, can cause a syndrome called loxoscelism, which is classified into cutaneous and viscerocutaneous forms. The cutaneous form, which is more common, causes the development of a necrotizing lesion with gravitational spread at the bite site. The viscerocutaneous form, which is rarer, includes systemic symptoms such as intravascular hemolysis, thrombocytopenia, and acute kidney injury, which can lead to death. However, clinical reports suggest a potential cardiotoxic role of Loxosceles venom, although this has been little studied. It should be noted that phospholipase-D is the main toxin present in the venom and has been implicated as the factor responsible for the development of cardiac dysfunction. Given this context, the present study sought to deepen the understanding of the effects of L. intermedia spider venom on the hearts of guinea pigs, using a detailed approach involving cardiac biomarkers, electrocardiograms, and histopathological analysis through optical and electron microscopy. Materials, Methods & Results: Eighteen male guinea pigs (Cavia porcellus) weighing 600 g were used. Of these, 16 animals received intradermal injections of Loxosceles intermedia venom at doses ranging from 11.6 to 350 μg diluted in 0.5 mL of 0.9% saline, and 2 animals received only 0.5 mL of 0.9% saline, serving as controls. Cardiac function was assessed by serial electrocardiograms and by measuring serum cardiac biomarkers, such as creatine kinase (CK) and its MB fraction (CK-MB), lactate dehydrogenase (LDH), troponin I (TnI), N-terminal fragment of B-type natriuretic peptide (NT-ProBNP) and D-dimer at 24 and 72 h post-injection. After 72 h, the animals were euthanized for histopathological and ultrastructural analysis of cardiac tissue using optical and transmission electron microscopy. Survival time varied inversely with the venom dose. Electrocardiograms recorded before envenomation showed no abnormalities. However, after Loxosceles envenomation, ECG changes were observed, including cardiac arrhythmias such as junctional beats and supraventricular extrasystoles, paired ventricular extrasystoles associated with isolated junctional beats, isolated junctional beats and atrioventricular block, junctional arrhythmia, and isolated atrial extrasystole. Elevated CK, CK-MB, LDH, and TnI levels were observed. Histopathological changes included diffuse cardiac congestion and focal hemorrhage, while ultrastructural analysis revealed autophagic vacuoles and mitochondrial damage in cardiac fibers. Discussion: This research was the 1st to conduct a clinical study on systemic loxoscelism and its cardiovascular impacts. The data obtained demonstrate that loxoscelism envenomation can cause significant cardiac and biochemical alterations, with rapidly manifesting effects, such as atrial and ventricular premature beats. Atrial premature beats result from early atrial ectopic beats, and ventricular premature beats present as beats originating early in the ventricle, with a post-extrasystolic pause. In envenomations, ventricular premature beats are associated with damage to cardiac muscle fibers due to myotoxic activity and impaired perfusion of cardiac tissue. Junctional beats, which are early ectopic beats originating in the atrioventricular junction, were also observed. Therefore, these results indicate that loxoscelism venom can directly interfere with cardiac electrophysiology, causing conduction disturbances and arrhythmias. Furthermore, the venom was capable of generating histological lesions consistent with myocardial damage that may also indirectly contribute to impaired cardiac function through mechanisms such as hypoxia and hemorrhages. Keywords: Loxosceles intermedia, loxoscelism, cardiotoxicity, cardiac biomarkers, electrocardiogram.

This study investigated the pathophysiological effects of cell-free hemoglobin (Hb) generated by mechanical hemolysis during venovenous extracorporeal circulation (VVECC). We hypothesized that Hb scavenger protein constructs that bind Hb, heme and iron, could attenuate end-organ injury caused by intravascular hemolysis during VVECC. Scavenger constructs consisted of an apohemoglobin-haptoglobin (apoHb-Hp) complex designed to bind Hb and heme, as well as a separate preparation of haptoglobin, albumin, hemopexin, transferrin, termed the protein cocktail. To test the hypothesis, Golden Syrian hamsters were instrumented with dorsal window chambers and catheters, and VVECC was maintained for a total of 2 h, with a maximum flow rate equivalent to 50% of the animal's cardiac output. VVECC circuits were primed with either two binding materials, the apoHb-Hp and the protein cocktail, or a control solution of 5% human serum albumin (HSA). Microvascular Hb oxygen saturation in arterioles (saO2) and venules (svO2) were studied. All groups displayed a significant decrease in saO2 and svO2 at maximum VVECC when compared to baseline, while statistically significant changes between treatment groups showed no consistent trend. The protein cocktail bound 24% of cell-free Hb, while the apoHb-Hp bound 66% of cell-free Hb. Additionally, markers of renal damage and inflammation such as plasma creatinine, urinary NGAL, 4-HNE, and KIM-1 were significantly reduced in both Hb scavenger groups as compared to the HSA control. Results from this study suggest that Hb, heme and iron scavenging solutions used to prime VVECC circuits are indicated to support organ function.

Circuit-induced hemolysis is relatively common in extracorporeal membrane oxygenation (ECMO) patients. Intravascular release of cell-free hemoglobin can lead to complications and requires timely recognition. Validation of plasma free hemoglobin (PFH) measurement using a direct spectrophotometric method is presented. We evaluated a method modified from Kahn et al. (Ann Clin Lab Sci 1981;11:126-31) on a stand-alone spectrophotometer (Cary 60) and compared its performance to the semiquantitative H-index on an Abbott Alinity c, including precision, linearity, recovery, reference interval verification, interference, and stability. Method comparison was performed relative to the H-index and the same method on a different spectrophotometer (Beckman DU 720). Lipemia interference was performed on the Cary 60, Cary 3500, and Beckman DU 720. Surrogate biomarkers for hemolysis detection were also investigated in ECMO patients. The PFH method on the Cary 60 demonstrated imprecision ranging from 1% (96.0 mg/dL) to 4% (3.0 mg/dL), linearity to 100 mg/dL, and recovery >80% for values >2 mg/dL hemoglobin-spiked plasma. Dilution expanded the reportable range to the maximum dilution tested (1000 mg/dL). Lipemia interfered with PFH measurement by the direct method, but the same method on the Cary 3500 was resistant to lipemia. Bilirubin did not cause significant interference. Direct and H-index methods were comparable with a mean difference of 5.03 mg/dL (95% CI -1.38, 11.44). Lactate dehydrogenase was the most reliable surrogate biomarker for hemolysis. with AUC of 0.921 (0.894, 0.949) at >50 mg/dL. PFH measurement by a direct spectrophotometric method is more precise and sensitive compared to the H-index; however, PFH measurement is susceptible to lipemia unless performed on a high-end spectrophotometer.

Background: Intravascular hemolysis is a common complication in patients undergoing extracorporeal membrane oxygenation (ECMO), with plasma free hemoglobin (pfHb) serving as a biomarker for detection. Without standardized protocols, laboratories face challenges in interpreting and reporting results. Hemolysis indices may enhance reporting accuracy. Methods: This retrospective observational study at University Hospital Center Zagreb included 61 lithium heparin plasma samples from ECMO patients. pfHb was measured using the Harboe method (fHb) and estimated from hemolysis indices on Abbott Alinity c analyzer (efHb). Total and conjugated bilirubin, hemolysis, icterus, and lipemia indices (HIL) were recorded. Method comparison used Passing-Bablok regression and Bland-Altman analysis. An algorithm for pfHb reporting accounting for HIL interferences was developed. Results: Significant differences were observed between methods, with Harboe yielding higher median fHb (261 mg/L) versus efHb (58 mg/L). Regression analysis showed constant negative bias of -91 mg/L (95% CI: -143 to -16) for efHb relative to fHb. Bland-Altman analysis demonstrated wide limits of agreement. Correlation between fHb and efHb was moderate (Spearman's rho = 0.618, p < 0.001). The delta between methods increased with higher bilirubin concentrations. An algorithm integrating HIL indices with the Harboe method was developed to guide result validation and reporting. Conclusions: Accurate hemolysis assessment in ECMO patients requires careful interpretation, appropriate method selection, and laboratory-clinician collaboration. The proposed algorithm improves the clinical utility of pfHb testing by accounting for analytical interferences and supporting informed decision-making.

Red blood cells (RBCs) play a crucial role in delivering oxygen to tissues with their distinctive shape. However, the mechanisms underlying cellular deformation and rupture due to stress, which lead to diseases such as acute renal failure, pulmonary hypertension, anemia, and gallstone formation, remain poorly understood. Here, we investigate the mechanism of membrane protrusion and present a highly effective method to restore the morphology and function of damaged RBCs. We propose ultrasound-triggered nanodroplets loaded with oxygen and glucose to increase the local concentrations of these substances around RBCs and facilitate the rapid release of their payloads to repair fatigued RBCs under ultrasound. We identify a critical membrane protrusion length threshold of one-third the cell's diameter, beyond which the skeleton structure fractures and prevents repair. Our findings demonstrate how nanodroplets can efficiently deliver oxygen and glucose to expose membrane connection and trigger cytoskeleton reorganization to repair cellular structure. In an in vitro extracorporeal membrane oxygenation (ECMO) model, our method reduces the percentage of abnormal RBCs to 5% and decreases free hemoglobin concentration by 50%. This work offers new insights into RBC rejuvenation and strongly supports the potential of ultrasound-triggered nanodroplets for revitalizing fatigued RBCs, contributing to long-term health and well-being.

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, acquired clonal hemopoietic stem cell disorder characterized by complement mediated chronic intravascular haemolysis, bone marrow failure and life-threatening thrombosis. PNH results from a somatic mutation in the PIG-A gene on the X chromosome leading to deficiency of glycosylphosphatidylinositol (GPI) anchored complement regulatory protein predominantly CD55 &amp; CD 59. The absence of CD55 &amp; CD59 causes unregulated complement activation &amp; ultimately formation of membrane attack complex (MAC) causes cell lysis. The discovery of complement inhibitors has dramatically improved survival and quality of life for patients with PNH. This review summarizes the pathophysiology, clinical manifestations, diagnostic approach, and evolving treatment strategies for PNH.

Sickle cell disease (SCD) is a genetic inherited hemoglobinopathy arising from homozygosity or compound heterozygosity for a single base pair mutation in hemoglobin β-globin gene (HBB) and the severity is affected by allelic combinations, haplotypes and gene products. Numerous SCD mouse models exist to study mechanism and therapeutic intervention, and each display some phenotypic features of human disease. Berkeley SCD mice demonstrate clinically relevant pulmonary hypertension (mean pulmonary artery pressure = 25-35 mmHg) when housed under sub-chronic (3-months) exposure to a moderately decreased oxygen level, approximately 15%. This model accelerates red blood cell sickling and hemolysis, which perpetuates precapillary pulmonary vascular disease and right ventricular dysfunction. Iron restriction in SCD is reported to attenuate the frequency and severity of vaso-occlusive crisis through reducing HbS in RBCs. Vamifeport is an oral clinical stage ferroportin inhibitor shown to improve microcirculatory blood flow in Townes SS mice. We hypothesized that vamifeport treatment may attenuate right ventricular dysfunction and pulmonary vascular remodeling in Berkeley SCD mice that express a pulmonary hypertension phenotype. Further, we hypothesized that lung and right ventricle metabolism and protein expressions would show an antioxidant and iron regulatory response that favored the attenuation of cardiopulmonary dysfunction. Indeed, attenuation of red cell sickling, less extra- and intravascular hemolysis and normalization of cardiopulmonary dysfunction was observed after vamifeport treatment. We suggest that induction of mild iron deficiency anemia may attenuate deadly sequelae of SCD, including cardiopulmonary dysfunction.

Sickle cell disease (SCD) is the most common monogenic-hemolytic disorder affecting people of African ancestry. Adenosine diphosphate (ADP) released following intravascular hemolysis activates platelets by stimulating purinergic receptors to promote thrombosis. Despite brisk intravascular hemolysis, which releases high levels of ADP into plasma, and evidence of platelet and hemostatic activation, it remains elusive why only a subset of SCD patients develop lung thrombosis. Using real-time in vivo lung microscopy, we report a surprising finding that humanized SCD mice are protected from ADP-induced lung thrombosis, which is secondary to the degradation of ADP by CD39 present in circulating extracellular vesicles released by the lung endothelium. ADP-induced platelet aggregation is also impaired in the blood of SCD patients with elevated levels of CD39+ extracellular vesicles. CD39 polymorphism rs3176891A→G is associated with the incidence of lung thrombosis in SCD patients but not healthy humans of African ancestry. Remarkably, CD39+ extracellular vesicles are fewer and ADP-induced platelet aggregation is higher in the blood of SCD patients with rs3176891G allele. This study identifies a novel extracellular vesicle-dependent mechanism preventing lung thrombosis in SCD and reveals how CD39 polymorphisms may impair this protection to increase the risk for lung thrombosis in a subset of SCD patients.

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disease characterized primarily by intravascular hemolysis, thrombosis, and bone marrow failure. Complement inhibitors are commonly used in clinical treatment and show limited efficacy, highlighting the urgent need to identify new therapeutic targets and explore alternative treatment strategies to provide theoretical guidance for clinical practice. Methods: We established a PNH cell model and constructed an miRNA-mRNA regulatory network to identify key miRNAs and core target genes. Single-cell sequencing data were analyzed to further clarify the critical genes. Finally, integrated drug database analysis identified potential therapeutic agents for PNH, which were validated by molecular docking and molecular dynamics simulations. Results: Using CRISPR/RNP technology, we successfully constructed a PIGA-knockout (PIGA-KO) THP-1 cell model. Differential expression analysis identified 1979 differentially expressed mRNAs (DEmRNAs) and 97 differentially expressed miRNAs (DEmiRNAs). The multiMiR package in R was used to predict the target genes of DEmiRNAs, from which those experimentally validated through dual-luciferase reporter assays were selected. After integration with the DEmRNAs, an miRNA-mRNA regulatory network was constructed, comprising 26 miRNAs and 38 mRNAs. Subsequent miRNA pathway enrichment analysis identified hsa-miR-23a-3p as a key miRNA, with CXCL12, CXCL8, HES1, and TRAF5 serving as core target genes. The integration of single-cell sequencing datasets (PRJNA1061334 and GSE157344) was performed, followed by cell communication and enrichment analysis. This approach, combined with clinical relevance, identified the neutrophil cluster as the key cluster. Intersection analysis of neutrophil cluster differential analysis results with key modules from hdWGCNA further clarified the critical genes. Drug prediction using EpiMed, CMap, and DGIdb identified Leflunomide, Dipyridamole, and Pentoxifylline as potential therapeutic agents. Molecular docking and molecular dynamics simulations showed stable binding of these potential drugs to the critical molecules, indicating a viable molecular interaction foundation. Conclusions: Leflunomide, Dipyridamole, and Pentoxifylline may serve as promising therapeutic agents for PNH, and the hsa-miR-23a-3p/CXCL8 regulatory axis could play a pivotal role in the pathogenesis and progression of PNH.

Antioxidant and antibacterial effects of human plasma-based scavenging protein cocktail on burn wound healing in vitro.

Acute intravascular hemolysis and acute hepatic injury caused by consuming Paxillus orientalis.

Hemolysis and Renal Safety of Pulsed-Field Versus Thermal Ablation for Atrial Fibrillation: A Systematic Review and Meta-Analysis.

Low-titer O whole blood (LTOWB) is increasingly used to improve trauma outcomes, but maintaining supplies remains challenging, particularly in austere environments. This study evaluated EnzAO1 (FpGalNAcDeAc) and EnzAO2 (FpGalNase) to generate enzyme-converted O type whole blood (ECO-WB) from A type swine whole blood (WB). WB from 16 A type and 4 O type swine were collected in Citrate Phosphate Dextrose Adenine-1 (CPDA-1). Type A WB was randomized to enzymatic conversion at either room temperature or 4°C. Each unit was divided into 45 mL aliquots and treated with no enzyme or one of four enzyme concentrations (1.75-17.5 μg/mL). Samples were analyzed at 15, 30, 60, and 120 min. A-antigen reduction was measured by flow cytometry. Blood counts, blood gases, rotational thromboelastometry, osmotic fragility, and free hemoglobin were used to assess blood post-conversion. Enzymatic treatment converted A type swine WB to ECO-WB in a concentration- and time-dependent manner. At the highest enzyme concentration (17.5 μg/mL), A antigen expression was reduced to 0.6% ± 0.2% by 15 min, comparable to the signal observed in type O controls (0.45% ± 0.45%). No significant differences in antigen conversion were observed between temperature conditions. ECO-WB demonstrated stable hematologic profiles compared to controls. This study demonstrates enzymatic conversion of type A swine WB into ECO-WB, reducing A antigen expression comparable to type O WB without compromising blood quality. The process is compatible with refrigerated and room temperature storage, supporting its application in diverse clinical and operational settings.

IntroductionVacuum-assisted venous drainage (VAVD) has been proposed as a better alternative option than conventional gravitational venous drainage (GVD) in cardiac surgery. However, the literature reports conflicting results between both methods in terms of post-cardiac surgery complications. Therefore, we aimed to perform a meta-analysis to compare clinical outcomes between VAVD and GVD in patients undergoing cardiac surgery.MethodsPubMed, Scopus, and Web of Science databases were searched for any randomized control trials or cohort studies that compared clinical outcomes between VAVD and GVD in patients undergoing cardiac surgery.ResultsSixteen studies with 8426 patients were included in our study. The pooled effect estimate of the postoperative results showed a statistically significant association between VAVD and decreased blood loss/chest tube drainage (MD = -88.7, 95% CI = -154.71 to -22.69, p-value = 0.008), amount of packed red blood cells (pRBC) transfusion (MD = -0.25, 95% CI = -0.27 to -0.22, p < 0.00,001), re-exploration (RR = 0.6, 95% CI = 0.35 to 1, p = 0.05), and re-operation (RR = 0.47, 95% CI = 0.23 to 0.99, p-value = 0.05). However, our study revealed no significant difference between both groups in terms of postoperative mortality, hospital/ICU stay, other blood product transfusions, change of free hemoglobin at 24h, and other clinical outcomes.ConclusionOur study revealed that VAVD is at least equivalent and may provide some benefits compared to GVD in patients undergoing cardiac surgery. While, VAVD requires specific expertise and training in order to optimize its outcomes, its ability to reduce blood loss and blood transfusion, support its use as a valuable alternative for GVD in high-risk groups.

Autologous transfusion of filtered, unwashed blood is a technique to mitigate the effects of blood loss during mechanical thrombectomy, but the degree of hemolysis resulting from this approach and guidance for blood return in this setting is not well understood. To assess hemolysis in aspirated and filtered porcine blood using a commercially available syringe-based aspiration thrombectomy blood return device. A large-bore, syringe-based aspiration thrombectomy blood return device was tested to assess extent of hemolysis in blood from a porcine model, following the manufacturer's instructions for use (IFU). The aspiration sequence consisted of three stages: blood was aspirated via the inferior vena cava (IVC) from a single animal using a 60-cc syringe through a 24 F catheter (stage 1), filtered through a blood return filter (stage 2), and processed through a transfusion filter (stage 3). Blood was sampled from each stage using a new 3-cc syringe and tested for hemolysis. This was repeated for five separate sequences using the same 60-cc syringe for a total of 15 samples. Plasma free hemoglobin (PFH) was measured as a hemolysis marker. Systemic samples were taken throughout to establish a control PFH value. Samples were compared to control and evaluated against the Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) Registry threshold of 20 mg/dL PFH for hemolysis. Using the blood return device, PFH levels were elevated relative to control (6.0 ± 1.8 mg/dL) in all samples, and 33.3% (5/15) of samples exceeded the 20 mg/dL INTERMACS threshold. A significant increase in mean PFH occurred across sequences (p < 0.001), while there was a statistically nonsignificant increase in mean PFH (p = 0.059) across stages. Porcine blood processed through a syringe-based aspiration thrombectomy blood return device exhibited threshold-exceeding hemolysis, which increased across sequences and remained post-filtration. This study highlights the need to further assess hemolysis when returning aspirated blood to patients during thrombectomy.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired disorder of complement dysregulation, predisposing patients to complications of intravascular hemolysis, thrombophilia, and marrow failure, with a high risk of mortality without treatment. Allogeneic stem cell transplantation is the only current cure but is typically reserved for marrow failure-predominant disease or when targeted therapies are not available. Terminal complement inhibition with eculizumab has significantly altered management and outcomes for patients with PNH, and the last several years have seen the development and approval of many new complement inhibitors with different molecular targets. Newer inhibitors may also provide options for extended time between doses, for self-administration, and for management of iatrogenic extravascular hemolysis, which can occur secondary to C5 inhibition. This essay reviews the various therapeutic options potentially available to PNH patients, the pros and cons of each treatment, considerations regarding the monitoring of side effects, and the possible complications, as well as breakthrough hemolysis and an approach to shared decision-making.

The purpose of this study was to compare selected iron markers (serum iron, total iron-binding capacity (TIBC), transferrin and ferritin concentration, transferrin saturation, and free haemoglobin) in children from marginalised Roma communities (MRCs) with children from the majority population and explore their associations with diet composition. We obtained cross-sectional data (questionnaires, blood samples from children) from 119 mother-child dyads from MRCs and the majority population. Group differences were tested using Chi-square and Mann-Whitney U tests. Associations of belonging to MRCs and diet with iron markers (transferrin, ferritin, TIBC, serum iron, transferrin saturation) were examined using bootstrapped linear regression models, and mediation analyses assessed whether eating habits mediated group differences. Statistically significant differences between children from MRCs, and the majority were found in serum transferrin, ferritin, and TIBC levels. The more frequent consumption of sweetened drinks, sweets, and salty snacks is associated with lower levels of transferrin, and more frequent consumption of dairy products is associated with higher levels of total iron-binding capacity. Current breastfeeding was found to be negatively associated with ferritin. Consumption of sweets and salty snacks partially mediates the differences in transferrin between children from MRCs and the majority. Our findings suggest that the observed low ferritin levels, elevated TIBC, and reduced transferrin saturation in Roma children are likely indicative of early-stage iron deficiency, potentially driven by underlying malnutrition. This study underscores the significant disparities in iron metabolism between children from MRCs and those from the majority population, primarily driven by social determinants of health, including diet composition.

Introduction: Cholestasis is a common complication of Extracorporeal Membrane Oxygenation (ECMO) secondary to patient physiology and circuit-induced factors. In our institution’s Neonatal Intensive Care Unit (NICU), we noted cases of severe cholestasis, with peak conjugated bilirubin levels much higher than previously reported in the literature. The objective of our study was to identify the contributing factors to the development of severe cholestasis in neonatal ECMO cases. Methods: Using our institutional ECMO database, all neonates who received ECMO at our institution were identified. A retrospective chart review was completed for a sample of 30 neonates. Univariate, multivariate, and logistic regression models were utilized. Results: Twenty percent of the patients in our study developed severe cholestasis (peak conjugated bilirubin > 10.0 mg/dL). Comparing the group of neonates that developed severe cholestasis to those who did not, we found that severe cholestasis was associated with the use of the Getinge Pediatric Quadrox-iD oxygenator. Mean plasma free hemoglobin levels were significantly higher in cases using pediatric oxygenators vs. adult (204.6 mg/dL vs. 110.4 mg/dL, p = 0.01). Longer ECMO courses and percent time within the ACT goal were also associated with severe cholestasis. Conclusion: Our study describes a cohort of neonatal ECMO cases complicated by severe cholestasis that was mediated by hemolysis due to circuit factors. In particular, circuit factors (the use of a pediatric oxygenator), longer duration of ECMO, and anticoagulation management were all significant factors. Future studies are needed to further elucidate the impact of these circuit factors and how they interplay with neonatal physiology.