Cell-free Plasma Hemoglobin Research Articles

Accuracy of the HemoCue® to measure cell-free plasma hemoglobin and detect clinically significant hemolysis.

Monitoring cell-free plasma hemoglobin (PHb) during extracorporeal therapies allows early intervention of significant hemolysis, but timely measurements are often challenging. We thus present an analysis of a rapid benchtop device's ability to detect clinically significant hemolysis (PHb ≥50mg/dL). PHb was measured in 419 plasma samples from 88 pediatric patients undergoing cardiopulmonary bypass via both the benchtop device (HemoCue® Plasma/Low Hb system) and the clinical laboratory at the Children's Hospital of Pittsburgh (reference standards). Values of PHb ≥50mg/dL as measured by the reference standard was defined as the binary outcome of clinically significant hemolysis. Analyses included Pearson correlations, logistic regression, receiver operating characteristic curves, and Bland-Altman. Because the manufacturer specifications identify the measurement range of the HemoCue® system as 30-3000mg/dL, a secondary analysis was completed using PHb ≥30mg/dL. Using reference measurements, 66/88 subjects had at least one PHb level that fell within the range of detection (≥30mg/dL) of the benchtop device and 46/88 had significant hemolysis as defined by PHb ≥50mg/dL. PHb levels ≥30mg/dL largely correlated with measurements made with the benchtop device (r = 0.82, p < .001). The device was able to predict PHb values ≥30mg/dL (AUROC 0.9582) and ≥50mg/dL (AUROC 0.9637). The Bland-Altman demonstrated a mean difference of 7.0mg/dL with <5% outside the 95% limits of agreement. The HemoCue® system is an effective surrogate for more robust laboratory testing to identify clinically significant hemolysis during cardiopulmonary bypass.

Acetaminophen for Prevention and Treatment of Organ Dysfunction in Critically Ill Patients With Sepsis

Acetaminophen (paracetamol) has many pharmacological effects that might be beneficial in sepsis, including inhibition of cell-free hemoglobin-induced oxidation of lipids and other substrates. To determine whether acetaminophen increases days alive and free of organ dysfunction in sepsis compared with placebo. Phase 2b randomized, double-blind, clinical trial conducted from October 2021 to April 2023 with 90-day follow-up. Adults with sepsis and respiratory or circulatory organ dysfunction were enrolled in the emergency department or intensive care unit of 40 US academic hospitals within 36 hours of presentation. Patients were randomized to 1 g of acetaminophen intravenously every 6 hours or placebo for 5 days. The primary end point was days alive and free of organ support (mechanical ventilation, vasopressors, and kidney replacement therapy) to day 28. Treatment effect modification was evaluated for acetaminophen by prerandomization plasma cell-free hemoglobin level higher than 10 mg/dL. Of 447 patients enrolled (mean age, 64 [SD, 15] years, 51% female, mean Sequential Organ Failure Assessment [SOFA] score, 5.4 [SD, 2.5]), 227 were randomized to acetaminophen and 220 to placebo. Acetaminophen was safe with no difference in liver enzymes, hypotension, or fluid balance between treatment arms. Days alive and free of organ support to day 28 were not meaningfully different for acetaminophen (20.2 days; 95% CI, 18.8 to 21.6) vs placebo (19.6 days; 95% CI, 18.2 to 21.0; P = .56; difference, 0.6; 95% CI, -1.4 to 2.6). Among 15 secondary outcomes, total, respiratory, and coagulation SOFA scores were significantly lower on days 2 through 4 in the acetaminophen arm as was the rate of development of acute respiratory distress syndrome within 7 days (2.2% vs 8.5% acetaminophen vs placebo; P = .01; difference, -6.3; 95% CI, -10.8 to -1.8). There was no significant interaction between cell-free hemoglobin levels and acetaminophen. Intravenous acetaminophen was safe but did not significantly improve days alive and free of organ support in critically ill sepsis patients. ClinicalTrials.gov Identifier: NCT04291508.

Hemoglobin-Mediated Oxidation of Low-Density Lipoprotein (oxLDL) Contributes to Lung Microvascular Endothelial Dysfunction Via Lectin-Like Oxidized LDL Receptor 1 (LOX-1)

Introduction: Lung microvascular endothelial barrier disruption is a critical feature of acute lung injury during several pathologies, including sepsis. Plasma cell-free hemoglobin (CFH) is elevated in approximately 80% of patients with sepsis and is independently associated with development of acute respiratory distress syndrome (ARDS) and mortality. However, the precise signaling mechanisms involved in CFH-mediated lung microvascular barrier dysfunction are incompletely understood. One potential mechanism by which CFH might disrupt the microvascular endothelial barrier is through its known ability to oxidize lipids and lipoproteins, including low-density lipoprotein (LDL). Oxidized LDL (oxLDL) is a known endothelial barrier disruptor, primarily signaling through scavenger receptors such as lectin-like oxidized LDL receptor 1 (LOX-1). We hypothesized that CFH-mediated oxidation of LDL exacerbates lung microvascular endothelial barrier dysfunction via LOX-1. Methods: For experimental studies, LDL was oxidized by combining LDL with CFH in a test tube overnight at 37°C and oxidation of LDL was quantified by TBARS assay. In primary human lung microvascular endothelial cells (HLMVEC), barrier dysfunction was assessed with expert permeability testing (XPerT) assay. HLMVEC were stimulated with vehicle control (PBS), LDL (0.05 mg/mL), CFH (0.5 mg/mL), or LDL oxidized by CFH (LDL+CFH) with or without one-hour pre-treatment of hemoglobin scavenger haptoglobin (1:1) or LOX-1 inhibitor BI-0115 (Boehringer Ingelheim, 10 μM). Cleaved caspase-3 (CST 9661, 1:500) was assessed with immunofluorescence microscopy. To test whether CFH and oxLDL are associated in clinical sepsis, circulating levels of CFH and oxLDL were measured in 60 sepsis patients via ELISA. Results: Oxidation of LDL by CFH (p=0.0006 by TBARS) exacerbated HLMVEC barrier dysfunction (p&lt;0.0001 by XPerT) but was partially prevented by scavenging CFH with haptoglobin (p&lt;0.0001 by XPerT) or blocking LOX-1 receptor (p&lt;0.0001 by XPerT). Oxidation of LDL by CFH stimulation of HLMVEC also increased caspase-3 activation (p&lt;0.0001 by immunofluorescence of cleaved caspase-3). In sepsis patients, circulating oxLDL levels correlated with CFH (r=0.4, p=0.002). Conclusions: Oxidation of LDL by CFH contributes to lung microvascular endothelial injury via LOX-1 receptor and activation of caspase-3; targeting this pathway may hold therapeutic potential to treat pathologies in which CFH is elevated, including sepsis-induced acute lung injury/ARDS. Funding: Parker B. Francis Fellowship (JEM); NIH K99HL166865 (JEM), R01HL158906 (LBW), R01HL164937 (LBW), R21GM144915 (LBW, JAB), R35HL150783 (JAB), RF1AG075341 (JAB). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Metabolic Reprogramming of Human Macrophages by Sickle Cell Hemoglobin

BACKGROUND: Sickle cell disease is caused by a single nucleotide mutation in the β-globin gene, resulting in the substitution of valine for glutamic acid, polymerization of hemoglobin and sickling of red blood cells (RBCs) under low oxygen conditions causing chronic hemolysis, and organs damage. SCA is characterized by the presence of cell-free plasma hemoglobin. Organ damage is associated with chronic inflammation and pro-inflammatory macrophages phenotype. Typically, anti-inflammatory response is elicited by the phagocytosis of senescent RBCs and cell-free hemoglobin. We have demonstrated that treatment of human macrophages with sickle cell hemoglobin (HbS) leads to lysosome accumulation, autophagy activation, and pro-inflammatory phenotype. The propensity of mutant HbS to polymerize under low pH may promote lysosomal HbS polymer accumulation, hindering degradation, and inducing macrophage differentiation towards a pro-inflammatory state. The differentiation of macrophages into specific phenotypes is associated with significant metabolic reprogramming. HYPOTHESIS: We hypothesize that the uptake of HbS by human macrophages induces their metabolic reprogramming via mitochondrial remodeling. METHODS: Human THP-1 cells were differentiated into macrophages with PMA and treated with either purified HbS or normal hemoglobin (HbA). Mitochondria were detected with MitoTrackerTM Green FM dye, and mitochondrial ROS (mtROS) level was detected by Mito SOX Red Kit. Transmission electron microscopy imaging was performed at Talos 200X transmission electron microscope. EPView (Olympus) software was used for the structure analysis. Total RNA was isolated by Trizol and sequenced on an Illumina® NextSeq 500. Comparisons between treatments were conducted using Gene Set Enrichment Analysis (GSEA). RESULTS: Immunofluorescent staining demonstrated accumulation of mitochondria after treatment with HbS compared to untreated (ctrl) or HbA-treated macrophages. Electron microscopy revealed an increase in the number and size of mitochondria. In 25 random microphotographs of cytoplasm, we detected 51 mitochondria in control, 79 in HbA treated and, 96 in HbS treated macrophages. The mitochondria volume and cristae size were significantly increased in macrophages treated with HbS. There was no increase in mtROS production in HbS treatment compared to ctrl. As expected, HbA treatment reduced mtROS production. The mitochondrial gene set (242 genes) was the second most enriched GSEA set in HbS-treated compared to HbA-treated macrophages. The larger mitochondrial size may be associated with a reduction of fission or an increase in fusion. We did not find differences in the expression of mitochondrial fission and fusion genes between HbS and HbA-treated macrophages. The increase in the mitochondrial size and cristae size may also be associated with increased oxidative phosphorylation. We found significant enrichment of genes responsible for oxidative phosphorylation in the HbS-treated macrophages. We did not find significant differences in the expression of glycolysis genes. CONCLUSION: Treatment of human macrophages with HbS significantly increases the number and volume of mitochondria, size of the cristae, and oxidative phosphorylation without upregulation of mtROS production and reduction of glycolysis. This work was supported by NIH Research Grants SC1HL150685, P50HL118006, and R01HL125005. We thank Drs. Castle Raley and Keith Crandall, George Washington University SPH Genomics Core, for sequencing and guidance in data analysis; Dr. Christine Brantner, GW Nanofabrication and Imaging Center, for TEM imaging. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Factors that determine cell-free hemoglobin concentration in blood prime during cardiac surgery with cardiopulmonary bypass: a prospective observational study

INTRODUCTION: Plasma cell-free hemoglobin (Hbf) has a negative effect on the results of cardiac surgery. OBJECTIVE: Analysis of the dynamics of the Hbf during cardiosurgical operations with CPB. Identification of the main factors for increasing the Hbf. MATERIALS AND METHODS: A prospective, single-center, cohort study included 50 patients who underwent cardiac surgery with CPB. Hbf was monitored before surgery, during CPB (10 minutes after the start, then every 30 minutes) and every 6 hours for two days. RESULTS: An increase in Hbf levels was found in 90 % of surgical procedure. In 8 % of cases, hemolysis was mild (0.1 &lt; Hbf ≤ 0.4 g/l), in 48 % — moderate (0.4 &lt; Hbf &lt; 1 g/l), severe (Hbf ≥ 1 g/l) was after 34 % of operations. After weaning from CPB Hbf rapidly decreases and after 12 hours drop to the preoperative level. Aortic cross-clamping is a risk factor with an odds ratio of 1.038 (p = 0.036). A positive correlation was established between the Hbf level and the duration of CPB (r = 0.76; p &lt; 0.001). In ROC-analysis, a good predictive ability for development of severe hemolysis was demonstrated by the time of CPB: AUC 0.74 (p = 0.014) with a threshold value of 76 minutes. During CABG, severe hemolysis developed significantly less frequently than during valve corrections — 12.5 % versus 44.1 %, p = 0.026. Hbf content was significantly lower with an initial hemoglobin concentration &lt; 140 g/l than in patients with an initial hemoglobin level ≥ 140 g/l — 0.7 (0.5; 0.95) g/l versus 0.9 (0.7; 1.28) g/l, p = 0.036. CONCLUSIONS: Hemolysis is typical for the cardiac surgeries with CPB. The time of aoral clamping and CPB, the type of surgcry, the hemoglobin content before surgcry have an impact on the severity of hemolysis.

Caspase-1 As a Therapeutic Target in Intravascular Hemolysis-Induced Microvascular Inflammation and Occlusive Processes

Hemolytic anemias, such as sickle cell disease (SCD), are a group of acute and acquired conditions characterized by the accelerated destruction of red blood cells (RBC). Intravascular hemolysis (IVH), or RBC lysis within the bloodstream, incurs the release of damage-associated molecular patterns (DAMPs), trigging cell activation and immune responses. Caspase-1 (Casp-1), a protease enzyme, plays a critical role during inflammatory processes by initiating programed cell death and promoting the cleavage of pro-interleukin (IL)-1β into its active form, IL-1β, through inflammasome assembly. Here, we aimed to elucidate the complex mechanisms involved in IVH-induced inflammation, leukocyte-endothelium interactions, and microvascular dynamics. Acute IVH was induced in C57BL/6 mice (HEM) by the injection of sterile water (i.v., 150 µl), and control mice received i.v. saline (CON) (n=3-8 per group). Intravital microscopy was performed at 15 min post IVH induction on the cremaster muscle of mice to quantify venular leukocyte (WBC) rolling and adhesion, and non-invasive laser Doppler flowmetry (LDF) measured blood perfusion in the murine pelvis microvasculature. Blood samples were collected at 1-hour post-IVH for ELISA and flow cytometry analyses. Immortalized human endothelial cells (HUVEC; n=7 per group) were stimulated with heme or S100A8 for 3 hours. HEM mice demonstrated significantly increased plasma heme (P&amp;lt;0.0001) and cell-free hemoglobin (P&amp;lt;0.001), in association with significantly decreased plasma haptoglobin (P&amp;lt;0.05), compared to CON mice, thereby confirming the occurrence of IVH. Furthermore, HEM mice exhibited significantly increased plasma concentrations of the S100A8 alarmin (P&amp;lt;0.001) and IL1-β (P&amp;lt;0.001), as well as augmentations (P&amp;lt;0.001) in plasma TNF-ɑ and IL-6 levels. Flow cytometry was used to measure active Casp-1 in neutrophils and monocytes of mice using the FAM-FLICA probe. FLICA staining was significantly higher in the neutrophils (P&amp;lt;0.05) and monocytes (P&amp;lt;0.05) of HEM mice, compared to CON mice, implying Casp-1 activation in these cell subsets. WBC-endothelium interactions are a hallmark of vaso-occlusion in SCD. HEM mice exhibited elevated venular WBC rolling and adhesion to the endothelium, compared to CON mice (P&amp;lt;0.001), as previously shown (Almeida et al., 2015; PMID: 26019278). In addition, IVH significantly reduced the velocity of microvascular blood flow in mice (P&amp;lt;0.001), and impaired perfusion (P&amp;lt;0.001). To address the role of IVH-induced Casp-1 activation in microvascular dynamics, mice received i.p. administrations of the Casp-1 inhibitor, YVAD, at 1 hour prior to induction of IVH, or not, and were subjected to intravital microscopy or LDF. Interestingly, the inhibition of Casp-1 significantly abrogated IVH-induced WBC rolling (P&amp;lt;0.01) and adhesion to vessel walls (P&amp;lt;0.05) in HEM mice and significantly improved microvascular blood perfusion and flow velocity (P&amp;lt;0.05), suggesting a role for Casp-1 activation in the WBC-endothelium and occlusive interactions observed (See Figure). We then evaluated how heme and S100A8 may induce endothelial cell activation, as these DAMPs were found elevated in the plasma of HEM mice. As expected, compared to unstimulated HUVEC, heme induced endothelial cell activation in vitro, as shown by higher expressions of ICAM-1 (P&amp;lt;0.05), VCAM-1 (P&amp;lt;0.01) and E-selectin (P&amp;lt;0.01), and increased Casp-1 activity (P&amp;lt;0.01). In turn, S100A8 alone significantly elevated ICAM-1 (P&amp;lt;0.05) and E-selectin (P&amp;lt;0.01) expressions on HUVEC, as well as IL-1β release (P&amp;lt;0.001), however no significant increase in Casp-1 activity was observed. HUVEC, when pre-treated with YVAD, displayed a significant reduction in S100A8-induced E-selectin expression (P&amp;lt;0.05), but neither ICAM-1 nor VCAM-1 expression were significantly decreased. In contrast, no changes in the expressions of these adhesion molecules were observed in heme-stimulated cells after YVAD treatment. Taken together, these findings suggest that targeting caspase-1 activation and its associated pathways may represent a potential therapeutic intervention to mitigate the adverse inflammatory and microvascular effects of IVH, a disease mechanism that occurs in severe hemolytic anemias such as SCD.

Abstract 12733: The Effect of Mavacamten Treatment on Hemolysis Biomarkers in Patients With Obstructive Hypertrophic Cardiomyopathy in the EXPLORER-HCM Study

Introduction: In patients with obstructive hypertrophic cardiomyopathy (HCM), blood flow changes from left ventricular outflow tract (LVOT) obstruction can lead to hemolysis and release of red blood cell proteins into circulation. Increased cell-free plasma hemoglobin (Hb) and reduced haptoglobin (Hp) are biomarkers of intravascular hemolysis. Mavacamten, a cardiac myosin inhibitor, reduced LVOT obstruction in the phase 3 EXPLORER-HCM study; however, its effect on hemolysis biomarkers is unknown. Aim: To assess the effect of 30 weeks of mavacamten treatment versus placebo on levels of intravascular hemolysis biomarkers in patients with obstructive HCM. Methods: Hemolysis biomarkers were measured as part of exploratory proteomic analyses from randomized EXPLORER-HCM (NCT03470545) patients at baseline and Week 30 (mavacamten, n = 115; placebo, n = 120) using a proteomic platform (SomaScan v4.1). Results: At Week 30, mavacamten treatment showed decreases in cell-free plasma Hb (Log 2 fold change = -1.57, FDR = 1.7х10 -4 ) and increases in cell-free plasma Hp (Log 2 fold change = 1.83, FDR = 1.2х10 -4 ) vs placebo (Figure). Hb and Hp levels were negatively correlated at baseline and at Week 30. In general, patients can be grouped into high- or low- hemolysis biomarker profile (high Hb with low Hp or low Hb with high Hp, respectively) at baseline and Week 30. Patients who received mavacamten and changed from high- to low hemolysis biomarker profile at Week 30 trended towards greater LVOT reductions under resting and Valsalva conditions. No changes were detected for either group in whole blood Hb or hematocrit. Conclusions: Reduced cell-free plasma Hb and increased cell-free plasma Hp levels, suggest a reduction in intravascular hemolysis by mavacamten in obstructive HCM. These results are consistent with improved hemodynamic effects (less outflow obstruction) and indicate that mavacamten may reduce shear forces on red blood cells in the heart.

Plasma Nitric Oxide Consumption Is Elevated and Associated With Adverse Outcomes in Critically Ill Patients.

Impaired nitric oxide (NO) bioavailability may contribute to microvascular dysfunction in sepsis. Excessive plasma NO consumption has been attributed to scavenging by circulating cell-free hemoglobin. This may be a mechanism for NO deficiency in sepsis and critical illness. We hypothesized that plasma NO consumption is high in critically ill patients, particularly those with sepsis, acute respiratory distress syndrome (ARDS), shock, and in hospital nonsurvivors. We further hypothesized that plasma NO consumption is correlated with plasma cell-free hemoglobin concentration. Retrospective cohort study. Adult ICUs of an academic medical center. Three hundred sixty-two critically ill patients and 46 healthy control subjects. None. Plasma NO consumption was measured using reductive chemiluminescence and cell-free hemoglobin was measured with a colorimetric assay. Mean (95% CI) plasma NO consumption (µM) was higher in critically ill patients versus healthy control subjects (3.9 [3.7-4.1] vs 2.1 [1.8-2.5]), septic versus nonseptic patients (4.1 [3.8-4.3] vs 3.6 [3.3-3.8]), ARDS versus non-ARDS patients (4.4 [4.0-4.9] vs 3.7 [3.6-3.9]), shock vs nonshock patients (4.4 [4.0-4.8] vs 3.6 [3.4-3.8]), and hospital nonsurvivors versus survivors (5.3 [4.4-6.4] vs 3.7 [3.6-3.9]). These relationships remained significant in multivariable analyses. Plasma cell-free hemoglobin was weakly correlated with plasma NO consumption. Plasma NO consumption is elevated in critically ill patients and independently associated with sepsis, ARDS, shock, and hospital death. These data suggest that excessive intravascular NO scavenging characterizes sepsis and adverse outcomes of critical illness.

Cell-free hemoglobin-mediated oxidation of low-density lipoprotein (oxLDL) contributes to lung microvascular endothelial dysfunction during sepsis

Introduction: Disruption of the microvascular endothelial barrier is a critical pathological feature of sepsis-induced acute lung injury. Plasma cell-free hemoglobin (CFH) is elevated in approximately 80% of patients with sepsis and is independently associated with development of acute respiratory distress syndrome (ARDS) and mortality. Oxidized CFH (ferric, 3+) can oxidize low-density lipoprotein (oxLDL), which signals through its major endothelial receptor, the lectin-like oxidized LDL receptor 1 (LOX-1), to cause endothelial dysfunction through several pro-inflammatory pathways including activation of mitogen-activated protein kinases (MAPKs). However, little is known regarding whether LOX-1 receptor signaling leads to microvascular endothelial hyperpermeability or acute lung injury, especially in the context of sepsis. We hypothesized that oxidation of LDL by CFH contributes to lung microvascular endothelial barrier dysfunction and worse outcomes during sepsis through LOX-1 and downstream p38 MAPK. Methods: To test whether generation of oxLDL by CFH is associated with endothelial injury in clinical sepsis, circulating levels of CFH, oxLDL, and vascular injury marker sVE-cadherin were measured in 24 sepsis patients via ELISA and tested for association with development of ARDS. LDL was oxidized by combining LDL with CFH in a test tube overnight at 37°C and oxLDL was quantified by TBARS assay. In primary human lung microvascular endothelial cells (HLMVEC) transendothelial electrical resistance (TER), a measure of barrier dysfunction, was assessed by Electric Cell-substrate Impedance Sensing (ECIS). LOX-1 receptor was blocked using BI-0115 (Boehringer Ingelheim, 20 μM) and p38 MAPK was inhibited using NiPp (100 μM). Results: In sepsis patients, plasma oxLDL levels correlated with CFH (r=0.686, p=0.016) and sVE-cadherin (r=0.603, p=0.012), and tended to be higher in those who developed ARDS (38 U/L [IQR 27, 45] vs. 27 U/L [IQR 19, 35], p=0.1). Oxidation of LDL by CFH exacerbated HLMVEC barrier dysfunction compared to control, LDL, or CFH. Barrier dysfunction induced by CFH or oxLDL was attenuated by blocking the LOX-1 receptor or p38 MAPK. Conclusions: Increased plasma CFH and oxLDL are associated with vascular injury during clinical sepsis; one mechanism by which CFH may cause vascular hyperpermeability and sepsis-mediated lung injury is through oxidation of LDL which can drive signaling through the endothelial LOX-1 receptor and activation of p38 MAPK. NIH R35HL150783, R21GM144915, R01HL158906, R01HL164937, T32HL094296; Parker B. Francis Fellowship This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cell‐free Hemoglobin‐Oxidized LDL Axis Contributes to Microvascular Endothelial Barrier Dysfunction and Poor Outcomes During Sepsis

IntroductionSepsis, a dysregulated host response to infection with high morbidity and mortality, is characterized by a systemic inflammatory response and widespread vascular hyperpermeability leading to edema, organ dysfunction, and death with no specific treatments. Disruption of the microvascular endothelial barrier is a critical pathological feature of sepsis‐induced organ dysfunction driven by circulating inflammatory mediators, oxidants, and proteolytic enzymes. Cell‐free hemoglobin (CFH) is one such factor, released from damaged red blood cells during sepsis due to their increased deformability and fragility. Though CFH is not detectable in healthy subjects, plasma CFH is elevated in patients with sepsis and independently associated with mortality. CFH contributes to endothelial dysfunction, but the signaling mechanisms are incompletely understood. CFH can oxidize circulating low‐density lipoproteins (oxLDL), which could potentiate endothelial barrier disruption. We hypothesized that elevated levels of CFH during sepsis are associated with increased oxLDL and contribute to microvascular endothelial barrier dysfunction through the oxLDL receptor, lectin‐like oxidized LDL receptor 1 (LOX‐1).MethodsCirculating levels of CFH and oxLDL were measured in sepsis patients. In human lung microvascular endothelial cells (HLMVECs), transendothelial electrical resistance (TER), a measure of barrier dysfunction, was assessed by Electric Cell‐substrate Impedance Sensing (ECIS).Results (Figure)In sepsis patients, plasma oxLDL levels correlated with CFH (r=0.686; p=0.016, n=12) and were higher in patients who died in hospital (33.24 U/L [IQR 28.08, 48.28] vs. 27.19 U/L [IQR 16.24, 38.24]; p=0.045, n=25). HLMVEC barrier dysfunction induced by CFH (p&lt;0.0001, n=6‐7) or oxLDL (p=0.002, n=4‐6) was attenuated by blocking the LOX‐1 receptor with small molecule inhibitor BI‐0115 (Boehringer Ingelheim, 20 µM; p=0.018 vs CFH, n=5; p=0.0002 vs oxLDL, n=6).ConclusionIncreased plasma CFH and oxLDL are associated with HLMVEC barrier dysfunction and poor clinical outcomes during sepsis; one mechanism by which CFH may cause vascular hyperpermeability during sepsis is through oxidation of LDL which can drive signaling through the LOX‐1 receptor.

Association Between Hyperoxemia and Increased Cell-Free Plasma Hemoglobin During Cardiopulmonary Bypass in Infants and Children.

To determine potential risk factors for severe hemolysis during pediatric cardiopulmonary bypass and examine whether supraphysiologic levels of oxygen and cardiopulmonary bypass duration are associated with hemolysis. Prospective observational study. Cardiac ICU in a university-affiliated children's hospital. Greater than 1 month to less than 18 years old patients undergoing cardiopulmonary bypass for cardiac surgery. None. Plasma samples from 100 patients to assess cell-free plasma hemoglobin levels were obtained at start cardiopulmonary bypass, at the end of cardiopulmonary bypass, and 2 and 24 hours after reperfusion. Arterial blood gas samples were obtained before and every 30 minutes during cardiopulmonary bypass. Patient demographics and laboratory data were collected from the electronic medical record. Plasma hemoglobin levels peaked at the end of cardiopulmonary bypass and haptoglobin levels continued to fall throughout all time points. There were 44 patients with severe hemolysis (change in cell-free plasma hemoglobin > 50 mg/dL). Younger age (odds ratio/sd 0.45 [95% CI, 0.25-0.81]) and higher mean Pao2 × cardiopulmonary bypass duration (31.11 [1.46-664.64]) were identified as risk factors for severe hemolysis in multivariable analysis. Severe hemolysis was associated with longer hospital and ICU lengths of stay as well as acute kidney injury. We observed younger age and the exposure to both oxygen and duration of cardiopulmonary bypass as risk factors for hemolysis. Oxygen delivery through the cardiopulmonary bypass circuit is an easily modifiable risk factor. Its role in the production of reactive oxygen species that could alter the erythrocyte membrane deserves further examination in larger prospective studies.

Identifying adolescent and young adult patients with sickle cell disease at highest risk of death.

Identifying adolescent and young adult patients with sickle cell disease at highest risk of death.

Bioactive Oxylipins in Infants and Children With Congenital Heart Disease Undergoing Pediatric Cardiopulmonary Bypass.

To determine the production of 9-hydroxyoctadecadienoic acid and 13-hydroxyoctadecadienoic acid during cardiopulmonary bypass in infants and children undergoing cardiac surgery, evaluate their relationship with increase in cell-free plasma hemoglobin, provide evidence of bioactivity through markers of inflammation and vasoactivity (WBC count, milrinone use, vasoactive-inotropic score), and examine their association with overall clinical burden (ICU/hospital length of stay and mechanical ventilation duration). Prospective observational study. Twelve-bed cardiac ICU in a university-affiliated children's hospital. Children were prospectively enrolled during their preoperative clinic appointments with the following criteria: greater than 1 month to less than 18 years old, procedures requiring cardiopulmonary bypass INTERVENTIONS:: None. Plasma was collected at the start and end of cardiopulmonary bypass in 34 patients. 9-hydroxyoctadecadienoic acid, 13-hydroxyoctadecadienoic acid, plasma hemoglobin, and WBC increased. 9:13-hydroxyoctadecadienoic acid at the start of cardiopulmonary bypass was associated with vasoactive-inotropic score at 2-24 hours postcardiopulmonary bypass (R = 0.25; p < 0.01), milrinone use (R = 0.17; p < 0.05), and WBC (R = 0.12; p < 0.05). 9:13-hydroxyoctadecadienoic acid at the end of cardiopulmonary bypass was associated with vasoactive-inotropic score at 2-24 hours (R = 0.17; p < 0.05), 24-48 hours postcardiopulmonary bypass (R = 0.12; p < 0.05), and milrinone use (R = 0.19; p < 0.05). 9:13-hydroxyoctadecadienoic acid at the start and end of cardiopulmonary bypass were associated with the changes in plasma hemoglobin (R = 0.21 and R = 0.23; p < 0.01). The changes in plasma hemoglobin was associated with milrinone use (R = 0.36; p < 0.001) and vasoactive-inotropic score less than 2 hours (R = 0.22; p < 0.01), 2-24 hours (R = 0.24; p < 0.01), and 24-48 hours (R = 0.48; p < 0.001) postcardiopulmonary bypass. Cardiopulmonary bypass duration, 9:13-hydroxyoctadecadienoic acid at start of cardiopulmonary bypass, and plasma hemoglobin may be risk factors for high vasoactive-inotropic score. Cardiopulmonary bypass duration, changes in plasma hemoglobin, 9:13-hydroxyoctadecadienoic acid, and vasoactive-inotropic score correlate with ICU and hospital length of stay and/mechanical ventilation days. In low-risk pediatric patients undergoing cardiopulmonary bypass, 9:13-hydroxyoctadecadienoic acid was associated with changes in plasma hemoglobin, vasoactive-inotropic score, and WBC count, and may be a risk factor for high vasoactive-inotropic score, indicating possible inflammatory and vasoactive effects. Further studies are warranted to delineate the role of hydroxyoctadecadienoic acids and plasma hemoglobin in cardiopulmonary bypass-related dysfunction and to explore hydroxyoctadecadienoic acid production as a potential therapeutic target.

Anti-Inflammatory Effects of Hydroxyurea in a Murine Model of Chronic Intravascular Hemolysis

Intravascular hemolysis is associated with a number of disorders, including sickle cell anemia (SCA). In addition to diminishing red blood cell (RBC) counts, clinical manifestations associated with intravascular hemolysis include leg ulcers, pulmonary hypertension and priapism. Recent studies indicate that intravascular hemolysis propagates the inflammatory processes associated with many hemolytic diseases. This study aimed to establish a murine model of chronic hemolysis (CH) that elevates circulating cell-free heme to concentrations similar to those seen in SCA and observe the effects of CH on inflammatory parameters. We also evaluated the effects of concomitant administration of hydroxyurea (HU), a drug commonly used as therapy for SCA, on hemolytic inflammation. For induction of CH, C57BL/6 mice received twice weekly low concentration doses (10 mg/Kg/day) of phenylhydrazine (PHZ, i.p.) for four weeks (wks). Control mice received saline (CON) and some mice also received HU (50 mg/Kg/day, 5 times/wk for 4 wks, i.p.). Plasma cell-free hemoglobin (Hb) and heme were elevated to 0.4±0.1 g/L and 51.4±8.1 µM, respectively (N=4, P&lt;0.05 compared to CON), after four wks of PHZ; concentrations similar to those observed in humans and mice with SCD (data not shown). Four wks of CH induced significant leukocytosis and reduced RBC counts (WBC; 6.1±0.8, 20.2±3.2 x103/µL: RBC; 6.6±0.4, 4.8±0.3 x106/µL for CON and CH, respect., P&lt;0.01, N=4-5), and also significantly increased spleen weight in mice (0.33±0.01, 1.00±0.09 % of body weight for CON and CH, respect., P&lt;0.0001, N=4-5). CH mice displayed substantial depletion of the Hb- and heme-binding proteins, haptoglobin (HP) and hemopexin (HPX) (HP; 129.9±5.2, 1.01±0.2 µg/ml: HPX; 1.98±0.22, 0.44±0.13 mg/ml for CON and CH, respect., P&lt;0.0001, N=5), and increased plasma hemoxygenase-1 (HO-1) (17.4±1.7, 37.8±3.3 ng/ml, P&lt;0.0001, N=5). With regard to inflammatory markers, four wks of CH significantly increased circulating levels of cytokines IL-1β and IL-10 (IL-1β; 3.77±0.53, 6.06±1.14 pg/mL: IL-10, 6.75±0.28, 8.46±0.87 pg/mL for CON and CH, respect., P&lt;0. 05, N=4-5), but not TNF-α (0.70±0.17, 0.76±0.24 pg/mL for CON and CH, respect., N=4-5). Western blotting demonstrated that CH elevated hepatic protein expressions of CD163 (Hb-HP complex receptor), HO-1 and LRP1 (CD91, Heme-HPX complex membrane receptor), while depleting hepatic HPX (CD163; 0.3±0.02, 1.8±0.4 AU: LRP1; 0.4±.0.1, 1.2±0.4 AU: HO-1; 0.93±0.03, 2.66±1.01 AU: HPX; 0.7±0.2, 0.14±0.02 AU relative to GAPDH, for CON and CH, respect., N=3-8, P&lt;0.05 for CD163, HO-1, HPX). Concomitant treatment of mice with 50 mg/Kg/day HU throughout hemolysis induction (CHHU) did not significantly alter hematological data, spleen weight or plasma markers of hemolysis, compared to CH (WBC, 20.6±1.91 x103/µL: RBC, 4.6±0.1 x106/µL: spleen weight; 1.01±0.06 % body weight: HP; 0.71±0.04 pg/mL: HPX; 0.34±0.64 mg/mL, respect., P&gt;0.05 compared to CH, N=5), although a slight decrease in plasma HO-1 was observed (30.7 ± 2.2 ng/ml for CHHU, P&gt;0.05). Notably, HU therapy significantly decreased plasma levels of cytokines IL-1β and IL-10, in mice undergoing CH (IL-1β; 2.22±0.24 pg/mL: IL-10; 5.81±0.29 pg/mL, N=5, P&lt;0.01 compared to CH) and partially abolished the effects of CH on hepatic protein levels of HO-1 (1.39±0.23 AU, N=3) and HPX (0.4±0.08 AU, N= 7, P&lt;0.05 compared to CH). In summary, using repeated low dose PHZ administrations, we successfully induced moderate and continuous hemolysis in mice, similar to that observed in SCA. This CH was associated with the depletion of hemoglobin and heme scavenger proteins, and with the production of pro- and anti-inflammatory cytokines, in addition to higher expression of scavenger receptors in the livers of these mice. Concomitant therapy of mice with HU, frequently used to induce fetal hemoglobin (HbF) in SCA patients, did not alter hematological data or markers of hemolysis, indicating that this drug does not inhibit hemolysis in our CH model. However, HU had significant anti-inflammatory effects, reducing markers of inflammation and elevating hepatic HPX protein expression. As such, HU has anti-inflammatory effects during long term use that are independent of its HbF-inducing properties, providing further evidence of the mechanism of action HU in SCA and further supporting its use in other hemolytic disorders. Disclosures No relevant conflicts of interest to declare.

Cell-Free Hemoglobin Is Associated With Increased Vascular Resistance and Reduced Peripheral Perfusion in Severe Malaria.

In severe falciparum malaria, unlike sepsis, hypotension on admission is uncommon. We hypothesized that low nitric oxide bioavailability due to the presence of cell-free hemoglobin (CFH) increases vascular tone in severe malaria. Patients with severe malaria (n = 119), uncomplicated malaria (n = 91), or suspected bacterial sepsis (n = 56), as well as healthy participants (n = 50), were recruited. The systemic vascular resistance index (SVRI) was estimated from the echocardiographic cardiac index and the mean arterial pressure. SVRI and hematocrit levels were lower and plasma CFH and asymmetric dimethylarginine levels were higher in patients with malaria, compared with healthy participants. In multivariate linear regression models for mean arterial pressure or SVRI in patients with severe malaria, hematocrit and CFH but not asymmetric dimethylarginine were significant predictors. The SVRI was lower in patients with suspected bacterial sepsis than in those with severe malaria, after adjustment for hematocrit and age. Plasma CFH levels correlated positively with the core-peripheral temperature gradient and plasma lactate levels and inversely with the perfusion index. Impaired peripheral perfusion, as reflected by a low perfusion index or a high core-peripheral temperature gradient, predicted mortality in patients with severe malaria. CFH is associated with mean arterial pressure, SVRI, and peripheral perfusion in patients with severe malaria. This may be mediated through the nitric oxide scavenging potency of CFH, increasing basal vascular tone and impairing tissue perfusion.

Haptoglobin therapy has differential effects depending on severity of canine septic shock and cell-free hemoglobin level.

During sepsis, higher plasma cell-free hemoglobin (CFH) levels portend worse outcomes. In sepsis models, plasma proteins that bind CFH improve survival. In our canine antibiotic-treated Staphylococcus aureus pneumonia model, with and without red blood cell (RBC) exchange transfusion, commercial human haptoglobin (Hp) concentrates bound and compartmentalized CFH intravascularly, increased CFH clearance, and lowered iron levels, improving shock, lung injury, and survival. We now investigate in our model how very high CFH levels and treatment time affect Hp's beneficial effects. Two separate canine pneumonia sepsis Hp studies were undertaken: one with exchange transfusion of RBCs after prolonged storage to raise CFH to very high levels and another with rapidly lethal sepsis alone to shorten time to treat. All animals received continuous standard intensive care unit supportive care for 96 hours. Older RBCs markedly elevated plasma CFH levels and, when combined with Hp therapy, created supraphysiologic CFH-Hp complexes that did not increase CFH or iron clearance or improve lung injury and survival. In a rapidly lethal bacterial challenge model without RBC transfusion, Hp binding did not increase clearance of complexes or iron or show benefits seen previously in the less lethal model. High-level CFH-Hp complexes may impair clearance mechanisms and eliminate Hp's beneficial effect during sepsis. Rapidly lethal sepsis narrows the therapeutic window for CFH and iron clearance, also decreasing Hp's beneficial effects. In designing clinical trials, dosing and kinetics may be critical factors if Hp infusion is used to treat sepsis.

Impact of different standard red blood cell storage temperatures on human and canine RBC hemolysis and chromium survival.

Storage temperature is a critical factor for maintaining red-blood cell (RBC) viability, especially during prolonged cold storage. The target range of 1 to 6°C was established decades ago and may no longer be optimal for current blood-banking practices. Human and canine RBCs were collected under standard conditions and stored in precision-controlled refrigerators at 2°C, 4°C, or 6°C. During 42-day storage, human and canine RBCs showed progressive increases in supernatant non-transferrin-bound iron, cell-free hemoglobin, base deficit, and lactate levels that were overall greater at 6°C and 4°C than at 2°C. Animals transfused with 7-day-old RBCs had similar plasma cell-free hemoglobin and non-transferrin-bound iron levels at 1 to 72 hours for all three temperature conditions by chromium-51 recovery analysis. However, animals transfused with 35-day-old RBCs stored at higher temperatures developed plasma elevations in non-transferrin-bound iron and cell-free hemoglobin at 24 and 72 hours. Despite apparent impaired 35-day storage at 4°C and 6°C compared to 2°C, posttransfusion chromium-51 recovery at 24 hours was superior at higher temperatures. This finding was confounded by a preparation artifact related to an interaction between temperature and storage duration that leads to removal of fragile cells with repeated washing of the radiolabeled RBC test sample and renders the test sample unrepresentative of the stored unit. RBCs stored at the lower bounds of the temperature range are less metabolically active and produce less anaerobic acidosis and hemolysis, leading to a more suitable transfusion product. The higher refrigeration temperatures are not optimal during extended RBC storage and may confound chromium viability studies.

High cut-off membrane for in-vivo dialysis of free plasma hemoglobin in a patient with massive hemolysis

BackgroundThe possibility of clearing Cell-free Plasma Hemoglobin (CPH) from human plasma may appear attractive, especially when considering the noxious effects that CPH has on the immune function and the renal damage caused by its filtration. The existence of the so-called High Cut-Off (HCO) filters, possessing pores as big as 60 kDa, could potentially allow the clearance of the αβ dimers (31.3 kDa), the form in which the α2β2 hemoglobin tetramers (62.6 kDa) physiologically dissociate in plasma. We present herein the first reported case in which such an attempt was made.Case presentationThe patient was a 51-year-old man with hemolytic crisis due to glucose-6-phosphate dehydrogenase deficiency, further complicated by pigment-induced nephropathy. He underwent a 48-h CVVHD session, in which a HCO filter was used. The Sieving Coefficient (SC) for CPH was initially 0.08 and decreased to 0.02 after 24 h. This unexpected low SC was due to the initial high concentration of CPH (4.24 g/L). At such concentrations, the α2β2 tetramer poorly dissociates into the αβ dimer; but increases exponentially at concentrations lower than 1 g/L.ConclusionsClearance of CPH through a HCO filter is technically feasible but its performance markedly relies on the initial concentration of CPH. Critically ill patients with smoldering hemolysis, as it happens during septic shock or ECMO treatment, may benefit the most from the use of this membrane in order to clear CPH.

Cell-Free Plasma Hemoglobin and Male Gender Are Risk Factors for Acute Kidney Injury in Low Risk Children Undergoing Cardiopulmonary Bypass.

To determine the relationship between the production of cell-free plasma hemoglobin and acute kidney injury in infants and children undergoing cardiopulmonary bypass for cardiac surgery. Prospective observational study. Twelve-bed cardiac ICU in a university-affiliated children's hospital. Children were prospectively enrolled during their preoperative outpatient appointment with the following criteria: greater than 1 month to less than 18 years old, procedures requiring cardiopulmonary bypass, no preexisting renal dysfunction. None. Plasma and urine were collected at baseline (in a subset), the beginning and end of cardiopulmonary bypass, and 2 hours and 24 hours after cardiopulmonary bypass in 60 subjects. Levels of plasma hemoglobin increased during cardiopulmonary bypass and were associated (p < 0.01) with cardiopulmonary bypass duration (R = 0.22), depletion of haptoglobin at end and 24 hours after cardiopulmonary bypass (R = 0.12 and 0.15, respectively), lactate dehydrogenase levels at end cardiopulmonary bypass (R = 0.27), and change in creatinine (R = 0.12). Forty-three percent of patients developed acute kidney injury. There was an association between plasma hemoglobin level and change in creatinine that varied by age (overall [R = 0.12; p < 0.01]; in age > 2 yr [R = 0.22; p < 0.01]; and in < 2 yr [R = 0.03; p = 0.42]). Change in plasma hemoglobin and male gender were found to be risk factors for acute kidney injury (odds ratio, 1.02 and 3.78, respectively; p < 0.05). Generation of plasma hemoglobin during cardiopulmonary bypass and male gender are associated with subsequent renal dysfunction in low-risk pediatric patients, especially in those older than 2 years. Further studies are needed to determine whether specific subgroups of pediatric patients undergoing cardiopulmonary bypass would benefit from potential treatments for hemolysis and plasma hemoglobin-associated renal dysfunction.

Cell-Free Hemoglobin-mediated Increases in Vascular Permeability. A Novel Mechanism of Primary Graft Dysfunction and a New Therapeutic Target.

Cell-free hemoglobin (CFH) is a potent oxidant associated with poor clinical outcomes in a variety of clinical settings. Recent studies suggest that acetaminophen (APAP), a specific hemoprotein reductant, can abrogate CFH-mediated oxidative injury and organ dysfunction. Preoperative plasma CFH levels are independently associated with primary graft dysfunction (PGD) after lung transplant ( 1 ). Our objectives were to determine whether CFH would increase lung vascular permeability in the isolated perfused human lung and whether APAP would limit these effects. Human lungs declined for transplant were inflated and perfused with Dulbecco's modified Eagle medium/5% albumin at a pulmonary artery pressure of 8-12 mm Hg. After steady state was achieved, CFH (100 mg/dl) was added to the perfusate ± APAP (15 μg/ml). Lung permeability was measured by continuous monitoring of lung weight gain and by extravasation of Evans blue dye-labeled albumin from the vasculature into bronchoalveolar lavage. To test the mechanism of increased permeability, human pulmonary microvascular endothelial cells were exposed to CFH (0.5 mg/ml) ± APAP (160 μM) for 24 hours and permeability was assessed by electrical cell-substrate impedance sensing. In the isolated perfused human lung, CFH increased lung permeability over 2 hours compared with control lungs (12% vs. 2% weight gain from baseline, P = 0.03). Increased vascular permeability was confirmed by a 4.8-fold increase in Evans blue dye-labeled albumin in the airspace compared with control lungs. Pretreatment with APAP prevented lung weight gain (P = 0.06 vs. CFH). In human pulmonary microvascular endothelial cells, CFH increased monolayer permeability (P = 0.03 vs. control), and this was attenuated by APAP (P = 0.045 vs. CFH). Circulating CFH increases vascular permeability in the isolated perfused human lung and paracellular permeability in lung microvascular endothelial cells. These effects may explain the association of plasma CFH levels with PGD. The hemoprotein reductant APAP attenuates the effects of CFH and merits further exploration as a potential therapy for PGD prevention.