Cell-free Plasma Hemoglobin Research Articles

Cell-free hemoglobin mediated oxidative stress is associated with acute kidney injury and renal replacement therapy in severe falciparum malaria: an observational study

BackgroundIntravascular hemolysis is an intrinsic feature of severe malaria pathophysiology but the pathogenic role of cell-free hemoglobin-mediated oxidative stress in severe malaria associated acute kidney injury (AKI) is unknown.MethodsAs part of a prospective observational study, enrolment plasma cell-free hemoglobin (CFH), lipid peroxidation markers (F2-isoprostanes (F2-IsoPs) and isofurans (IsoFs)), red cell deformability, and serum creatinine were quantified in Bangladeshi patients with severe falciparum malaria (n = 107), uncomplicated malaria (n = 80) and sepsis (n = 28). The relationships between these indices and kidney function and clinical outcomes were examined.ResultsAKI was diagnosed at enrolment in 58% (62/107) of consecutive patients with severe malaria, defined by an increase in creatinine ≥1.5 times expected baseline. Severe malaria patients with AKI had significantly higher plasma cell-free hemoglobin (geometric mean CFH: 8.8 μM; 95% CI, 6.2–12.3 μM), F2-isoprostane (56.7 pg/ml; 95% CI, 45.3–71.0 pg/ml) and isofuran (109.2 pg/ml; 95% CI, 85.1–140.1 pg/ml) concentrations on enrolment compared to those without AKI (CFH: 5.1 μM; 95% CI, 4.0–6.6 μM; P = 0.018; F2-IsoPs: 27.8 pg/ml; 95% CI, 23.7–32.7 pg/ml; P < 0.001; IsoFs: 41.7 pg/ml; 95% CI, 30.2–57.6 pg/ml; P < 0.001). Cell-free hemoglobin correlated with markers of hemolysis, parasite burden (P. falciparum histidine rich protein 2 (PfHRP2)), and F2-IsoPs. Plasma F2-IsoPs and IsoFs inversely correlated with pH, positively correlated with creatinine, PfHRP2 and fractional excretion of sodium, and were higher in patients later requiring hemodialysis. Plasma F2-IsoP concentrations also inversely correlated with red cell deformability and were higher in fatal cases. Mixed effects modeling including an interaction term for CFH and time showed that F2-IsoPs, IsoFs, PfHRP2, CFH, and red cell rigidity were independently associated with increasing creatinine over 72 h. Multivariable logistic regression showed that admission F2-IsoPs, IsoFs and red cell deformability were associated with the need for subsequent hemodialysis.ConclusionsCell-free hemoglobin and lipid peroxidation are associated with acute kidney injury and disease severity in falciparum malaria, suggesting a pathophysiological role in renal tubular injury. Evaluation of adjunctive therapies targeting cell-free hemoglobin-mediated oxidative stress is warranted.

Sickle Cell Trait Increases Red Blood Cell Storage Hemolysis and Post-Transfusion Clearance in Mice

BackgroundTransfusion of blood at the limits of approved storage time is associated with lower red blood cell (RBC) post-transfusion recovery and hemolysis, which increases plasma cell-free hemoglobin and iron, proposed to induce endothelial dysfunction and impair host defense. There is noted variability among donors in the intrinsic rate of storage changes and RBC post-transfusion recovery, yet genetic determinants that modulate this process are unclear. MethodsWe explore RBC storage stability and post-transfusion recovery in murine models of allogeneic and xenogeneic transfusion using blood from humanized transgenic sickle cell hemizygous mice (Hbatm1PazHbbtm1TowTg(HBA-HBBs)41Paz/J) and human donors with a common genetic mutation sickle cell trait (HbAS). FindingsHuman and transgenic HbAS RBCs demonstrate accelerated storage time-dependent hemolysis and reduced post-transfusion recovery in mice. The rapid post-transfusion clearance of stored HbAS RBC is unrelated to macrophage-mediated uptake or intravascular hemolysis, but by enhanced sequestration in the spleen, kidney and liver. HbAS RBCs are intrinsically different from HbAA RBCs, with reduced membrane deformability as cells age in cold storage, leading to accelerated clearance of transfused HbAS RBCs by entrapment in organ microcirculation. InterpretationThe common genetic variant HbAS enhances RBC storage dysfunction and raises provocative questions about the use of HbAS RBCs at the limits of approved storage.

Percutaneous Mitral Valve Repair in Mitral Regurgitation Reduces Cell-Free Hemoglobin and Improves Endothelial Function.

Background and ObjectiveEndothelial dysfunction is predictive for cardiovascular events and may be caused by decreased bioavailability of nitric oxide (NO). NO is scavenged by cell-free hemoglobin with reduction of bioavailable NO up to 70% subsequently deteriorating vascular function. While patients with mitral regurgitation (MR) suffer from an impaired prognosis, mechanisms relating to coexistent vascular dysfunctions have not been described yet. Therapy of MR using a percutaneous mitral valve repair (PMVR) approach has been shown to lead to significant clinical benefits. We here sought to investigate the role of endothelial function in MR and the potential impact of PMVR.Methods and ResultsTwenty-seven patients with moderate-to-severe MR treated with the MitraClip® device were enrolled in an open-label single-center observational study. Patients underwent clinical assessment, conventional echocardiography, and determination of endothelial function by measuring flow-mediated dilation (FMD) of the brachial artery using high-resolution ultrasound at baseline and at 3-month follow-up. Patients with MR demonstrated decompartmentalized hemoglobin and reduced endothelial function (cell-free plasma hemoglobin in heme 28.9±3.8 μM, FMD 3.9±0.9%). Three months post-procedure, PMVR improved ejection fraction (from 41±3% to 46±3%, p = 0.03) and NYHA functional class (from 3.0±0.1 to 1.9±1.7, p<0.001). PMVR was associated with a decrease in cell free plasma hemoglobin (22.3±2.4 μM, p = 0.02) and improved endothelial functions (FMD 4.8±1.0%, p<0.0001).ConclusionWe demonstrate here that plasma from patients with MR contains significant amounts of cell-free hemoglobin, which is accompanied by endothelial dysfunction. PMVR therapy is associated with an improved hemoglobin decompartmentalization and vascular function.

Cell-free plasma hemoglobin removal by dialyzers with various permeability profiles.

The release of hemoglobin from mechanically stressed erythrocytes into plasma is a general side effect of extracorporeal therapies, such as extracorporeal membrane oxygenation or hemodialysis. In many reported cases dialysis patients showed elevated cell-free plasma hemoglobin (CPH) levels which are associated with pathophysiological effects. In this in vitro study, the CPH clearance capacity of various filters with different permeability profiles was measured. Simulated dialysis treatments were conducted and clearance was calculated from variations in CPH concentrations over time by measuring plasma absorbance at 405 nm. Conventional high-flux filters exhibited no detectable clearance of CPH. High-flux filters with extended permeability exhibited clearances between 5.8 ± 1.2 and 12.7 ± 1.7 ml/min when tested with plasma and between 5.8 ± 1.2 and 11.3 ± 1.6 ml/min when tested with whole blood. septeX high-cutoff filters had clearances between 13.8 ± 1.8 and 15.5 ± 1.7 ml/min when tested with plasma and of 22.6 ± 2.9 ml/min when tested with whole blood. This study demonstrated that filters with extended permeability and the septeX filter enable CPH removal when used as in chronic and acute settings.

Haptoglobin: an emerging candidate for phenotypic modulation of sickle cell anemia?

Sickle cell anemia (SCA) is characterized by a single homozygous mutation (A→T) in the sixth codon of the -globin gene that results in hemoglobin S (Hb S), in which a glutamic acid residue is substituted by valine in the sixth position of the -globin chain (HBB; glu(E)6val(A); GAG-GTG; rs334).1 This change leads to a wide variety of symptoms, including chronic intravascular hemolysis, increased cell-free plasma hemoglobin (Hb) and heme levels and vascular alterations.2 SCA has been characterized as a chronic inflammatory state with abnormal endothelial activation as a result of various associated factors. The mechanisms that induce the production of inflammatory mediators and the effects of these molecules on the inflammatory response are little understood in this disease.3 Sickled red blood cells are stiff and therefore have a predisposition to hemolysis; one third of the cells are destroyed in the intravascular space leading to increased cell-free plasma Hb and heme levels.4 The pathophysiological effects associated with free Hb/heme are acute hemodynamic instability and acute or chronic vascular injury.5 The toxicity and inflammatory nature of free Hb are a result of the greater nitric oxide consumption it promotes and the consequent accumulation of hydroxyl radicals and reactive oxygen species in the blood vessels. The organism’s first defense mechanism against the harmful effects of free Hb involves haptoglobin (Hp), whose primary function is to bind to free Hb in the plasma, thereby preventing the excretion of iron by the kidneys and protecting blood vessels from its oxidative effects.6,7 In addition to being an antioxidant, Hp is a positive acute-phase glycoprotein present in plasma with immunomodulatory properties.6,7

Elevation of Plasma Cell-Free Hemoglobin in Pulmonary Arterial Hypertension

BACKGROUND:Cell-free hemoglobin (CFH) is a potent nitric oxide scavenger associated with poor outcomes in several diseases. Pulmonary arterial hypertension (PAH) is characterized by reduced nitric oxide availability. We hypothesized that CFH would be elevated in PAH and would associate with hemodynamics and clinical outcomes.METHODS:We measured CFH in 200 consecutively evaluated patients with PAH, 16 unaffected bone morphogenetic receptor protein type 2 (BMPR2) mutation carriers, 19 healthy subjects, and 29 patients with pulmonary venous hypertension (PVH). CFH values were tested for association with hemodynamics, time to hospitalization, and death.RESULTS:CFH was elevated in patients with PAH and BMPR2 carriers compared with healthy subjects and patients with PVH (P≤ .01 all comparisons). There were no differences in CFH across PAH subtypes. CFH modestly correlated with mean pulmonary artery pressure (ρ = 0.16,P= .03) and pulmonary vascular resistance (ρ = 0.21,P= .01) and inversely with cardiac index (ρ = −0.18,P= .02) in patients with PAH. CFH was not associated with hemodynamic response to nitric oxide or death. Patients with the highest CFH levels had increased risk of PAH-related hospitalization when adjusted for age, sex, and PAH cause (hazard ratio, 1.69; 95% CI,1.08-2.66;P= .02).CONCLUSIONS:CFH is elevated in patients with PAH and BMPR2 carriers compared with healthy subjects and patients with PVH. Elevated CFH levels are independently associated with an increased risk of hospitalization. Further study is required to understand the mechanism of CFH elevation and the potential pathologic contribution of CFH in PAH.

Vasculopathy and pulmonary hypertension in sickle cell disease.

Sickle cell disease (SCD) is an autosomal recessive disorder in the gene encoding the β-chain of hemoglobin. Deoxygenation causes the mutant hemoglobin S to polymerize, resulting in rigid, adherent red blood cells that are entrapped in the microcirculation and hemolyze. Cardinal features include severe painful crises and episodic acute lung injury, called acute chest syndrome. This population, with age, develops chronic organ injury, such as chronic kidney disease and pulmonary hypertension. A major risk factor for developing chronic organ injury is hemolytic anemia, which releases red blood cell contents into the circulation. Cell free plasma hemoglobin, heme, and arginase 1 disrupt endothelial function, drive oxidative and inflammatory stress, and have recently been referred to as erythrocyte damage-associated molecular pattern molecules (eDAMPs). Studies suggest that in addition to effects of cell free plasma hemoglobin on scavenging nitric oxide (NO) and generating reactive oxygen species (ROS), heme released from plasma hemoglobin can bind to the toll-like receptor 4 to activate the innate immune system. Persistent intravascular hemolysis over decades leads to chronic vasculopathy, with ∼10% of patients developing pulmonary hypertension. Progressive obstruction of small pulmonary arterioles, increase in pulmonary vascular resistance, decreased cardiac output, and eventual right heart failure causes death in many patients with this complication. This review provides an overview of the pathobiology of hemolysis-mediated endothelial dysfunction and eDAMPs and a summary of our present understanding of diagnosis and management of pulmonary hypertension in sickle cell disease, including a review of recent American Thoracic Society (ATS) consensus guidelines for risk stratification and management.

Hemodialysis induced release in plasma cell free hemoglobin as a source of catalytic iron in patients with end stage renal disease

Hemodialysis induced release in plasma cell free hemoglobin as a source of catalytic iron in patients with end stage renal disease

Transfusion of older stored blood worsens outcomes in canines depending on the presence and severity of pneumonia

In experimental pneumonia we found that transfused older blood increased mortality and lung injury that was associated with increased in vivo hemolysis and elevated plasma cell-free hemoglobin (CFH), non-transferrin-bound iron (NTBI), and plasma labile iron (PLI) levels. In this study, we additionally analyze identically treated animals that received lower or higher bacterial doses. Two-year-old purpose-bred beagles (n = 48) challenged intrabronchially with Staphylococcus aureus (0 [n = 8], 1.0 × 10(9) [n = 8], 1.25 × 10(9) [n = 24], and ≥1.5 × 10(9) [n = 8] colony-forming units/kg) were exchange transfused with either 7- or 42-day-old canine universal donor blood (80 mL/kg in four divided doses). The greater increases in CFH with older blood over days after exchange proved relatively independent of bacterial dose. The lesser increases in CFH observed with fresher blood were bacterial dose dependent potentially related to bacterial hemolysins. Without bacterial challenge, levels of CFH, NTBI, and PLI were significantly higher with older versus fresher blood transfusion but there was no significant measurable injury. With higher-dose bacterial challenge, the elevated NTBI and PLI levels declined more rapidly and to a greater extent after transfusion with older versus fresher blood, and older blood was associated with significantly worse shock, lung injury, and mortality. The augmented in vivo hemolysis of transfused older red blood cells (RBCs) appears to result in excess plasma CFH and iron release, which requires the presence of established infection to worsen outcome. These data suggest that transfused older RBCs increase the risks from infection in septic subjects.

Influence Of Inhaled Nitric Oxide On Stored Red Blood Cell autotransfusion In Overweight Volunteers With Endothelial Dysfunction: Preliminary Results

IntroductionTransfusion of stored packed erythrocytes is associated with excessive intra and extravascular hemolysis. High plasma cell-free hemoglobin levels rapidly scavenge endogenous nitric oxide (NO) leading in many species to systemic and pulmonary vasoconstriction, and a pro-inflammatory, pro-thrombotic state. MethodsWe conducted a crossover randomized interventional study of autologous stored blood transfusion. We enrolled fourteen overweight adults (BMI 28 to 40 Kg/m2) who also had evidence of impaired endothelial function.Endothelial dysfunction was determined by a reduced Reactive Hyperemia Index following 5 minutes of forearm tourniquet ischemia (RHI<1.8).Over 8 months each volunteer returned to the blood bank on 3 occasions to donate a unit of blood. In a randomized scheme,autologous blood was leukoreduced and stored in AS-3, and re-infused after a) 3-days of storage (3-d), b) 40-days of storage (40-d), or c) 40-days of storage and breathing 80 parts per million (ppm) NO (40-d+NO).Venous blood was sampled before and at 10min, 1h, 2h, 4h and 24h after autotransfusion.Plasma hemoglobin measurement Plasma hemoglobin was measured with a QuantiChrom Hemoglobin Assay Kit (BioAssaySystems, Hayward, CA). These samples were collected using 16 G catheters avoiding negative aspirating pressures to minimize ex vivo hemolysis (1).Plasma NO consumption The ability of plasma hemoglobin to scavengeNO was measured with an NO consumption assay with a NO chemiluminescenceanalyzer (Sievers, Boulder, CO) (2) ResultsEleven volunteers completed the entire study. Volunteer characteristics at baseline were (mean ± SD): age: 41± 13 years; sex: 9 males, 5 females; BMI 33.2 ± 4.9 Kg/m2; reactive hyperemia index 1.59±0.18, and plasma levels of C-Reactive Protein 4.75 ±3.44 md/L.After autotransfusion the plasma hemoglobin and serum iron concentrations were increased from 1 to 4 hours after transfusing 40-days stored blood. No increase was detected after transfusing 3-days stored blood (p<0.01 value differs from b and c) (Figures 1a and 2). [Display omitted] [Display omitted] The plasma NO consumption assay showed an increased level in the 40-d group (p<0.05 value differs from both b and c) (Figure 1b). Plasma concentrations of potassium, lactate dehydrogenase, haptoglobin, cytokines, and systemic blood pressure did not differ between a, b and c and all remained within the normal range. ConclusionsTransfusion of autologous packed erythrocytes stored for 40 days in AS3 is associated with increased hemolysis and increased plasma NO consumption,the latter is prevented by breathing 80 ppm NO.

The antioxidant tempol decreases acute pulmonary thromboembolism-induced hemolysis and nitric oxide consumption

Acute pulmonary thromboembolism (APT) is a critical condition associated with acute pulmonary hypertension. Recent studies suggest that oxidative stress and hemolysis contribute to APT-induced pulmonary hypertension, possibly as a result of increased nitric oxide (NO) consumption. We hypothesized that the antioxidant tempol could attenuate APT-induced hemolysis, and therefore attenuate APT-induced increases in plasma NO consumption. APT was induced in anesthetized sheep with autologous blood clots. The hemodynamic effects of tempol infused at 1.0mg/kg/min 30 min after APT were determined. Hemodynamic measurements were carried out every 15 min. To assess oxidative stress, serum 8-isoprostanes levels were measured by ELISA. Plasma cell-free hemoglobin concentrations and NO consumption by plasma samples were determined. An in vitro oxidative AAPH-induced hemolysis assay was used to further validate the in vivo effects of tempol. APT caused pulmonary hypertension, and increased pulmonary vascular resistance in proportion with the increases in 8-isoprostanes, plasma cell-free hemoglobin concentrations, and NO consumption by plasma (all P<0.05). Tempol attenuated the hemodynamic alterations by approximately 15-20% and blunted APT-induced increases in 8-isoprostanes, in cell-free hemoglobin concentrations, and the increases in NO consumption by plasma (P<0.05). Tempol dose-dependently attenuated AAPH-induced in vitro hemolysis (P<0.05). Our findings are consistent with the idea that antioxidant properties of tempol decrease APT-induced hemolysis and nitric oxide consumption, thus attenuating APT-induced pulmonary hypertension.

Direct sGC activation bypasses NO scavenging reactions of intravascular free oxy-hemoglobin and limits vasoconstriction.

Hemoglobin-based oxygen carriers (HBOC) provide a potential alternative to red blood cell (RBC) transfusion. Their clinical application has been limited by adverse effects, in large part thought to be mediated by the intravascular scavenging of the vasodilator nitric oxide (NO) by cell-free plasma oxy-hemoglobin. Free hemoglobin may also cause endothelial dysfunction and platelet activation in hemolytic diseases and after transfusion of aged stored RBCs. The new soluble guanylate cyclase (sGC) stimulator Bay 41-8543 and sGC activator Bay 60-2770 directly modulate sGC, independent of NO bioavailability, providing a potential therapeutic mechanism to bypass hemoglobin-mediated NO inactivation. Infusions of human hemoglobin solutions and the HBOC Oxyglobin into rats produced a severe hypertensive response, even at low plasma heme concentrations approaching 10 μM. These reactions were only observed for ferrous oxy-hemoglobin and not analogs that do not rapidly scavenge NO. Infusions of L-NG-Nitroarginine methyl ester (L-NAME), a competitive NO synthase inhibitor, after hemoglobin infusion did not produce additive vasoconstriction, suggesting that vasoconstriction is related to scavenging of vascular NO. Open-chest hemodynamic studies confirmed that hypertension occurred secondary to direct effects on increasing vascular resistance, with limited negative cardiac inotropic effects. Intravascular hemoglobin reduced the vasodilatory potency of sodium nitroprusside (SNP) and sildenafil, but had no effect on vasodilatation by direct NO-independent activation of sGC by BAY 41-8543 and BAY 60-2770. These data suggest that both sGC stimulators and sGC activators could be used to restore cyclic guanosine monophosphate-dependent vasodilation in conditions where cell-free plasma hemoglobin is sufficient to inhibit endogenous NO signaling.

Elevated Plasma Hemoglobin Levels Increase Nitric Oxide Consumption in Experimental and Clinical Acute Pulmonary Thromboembolism*

We examined whether experimental lung embolization with autologous blood clots or with the infusion of microspheres increase cell-free hemoglobin levels and nitric oxide consumption by plasma samples from anesthetized lambs. These parameters were also measured in patients with acute pulmonary thromboembolism at baseline conditions and after thrombolysis, and in healthy controls. Controlled animal and clinical studies. University research laboratory and university hospital. Sheep and humans. Anesthetized lambs were embolized with intravenous injections of autologous blood clots or repeated injections of 300 μm microspheres. Control animals received saline. Blood samples were drawn from patients with acute pulmonary thromboembolism at baseline conditions and after thrombolytic therapy with streptokinase or alteplase. Hemodynamic measurements were performed and plasma cell-free hemoglobin concentrations were measured. A nitric oxide consumption assay was used to measure nitric oxide consumption by plasma samples. Embolization with blood clots or microspheres increased mean pulmonary artery pressure from ~15 to ~40 mm Hg in lambs. Both plasma hemoglobin concentrations and nitric oxide consumption increased in proportion to the hemodynamic alterations and correlated significantly. Patients with acute pulmonary thromboembolism had higher plasma hemoglobin concentrations and nitric oxide consumption than healthy controls. Thrombolysis with streptokinase or alteplase further increased both parameters, which peaked 1-3 days after thrombolysis. Our results show consistent evidence indicating a new mechanism involving increased hemoglobin decompartmentalization and augmented nitric oxide consumption, possibly contributing to the hemodynamic derangement of acute pulmonary thromboembolism.

Association Between Cell-Free Hemoglobin, Acetaminophen, and Mortality in Patients With Sepsis

To determine the association of circulating cell-free hemoglobin with poor clinical outcomes in patients with sepsis and to characterize the potential protective effects of acetaminophen, an inhibitor of hemoprotein-mediated oxidation. Retrospective observational study. A total of 391 critically ill patients with sepsis in multiple ICUs in an academic tertiary care hospital. None. Nonsurvivors had significantly higher plasma cell-free hemoglobin concentrations (median 20mg/dL, interquartile range 10-40) measured on enrollment compared to survivors (10mg/dL, interquartile range 10-30, p = 0.002). After controlling for potential confounders, patients with higher cell-free hemoglobin concentrations were significantly more likely to die in the hospital (odds ratio 1.078, 95% confidence interval 1.012-1.149, p = 0.02). In addition, receiving acetaminophen in the setting of increased cell-free hemoglobin was independently associated with a protective effect against death (odds ratio 0.48, 95% confidence interval 0.25-0.91, p = 0.026) and lower plasma concentrations of the lipid peroxidation product F2-isoprostanes (18.5 pg/mL, interquartile range 9-22.2) compared to no acetaminophen (42 pg/mL, interquartile range 29.7-86, p = 0.009). In critically ill patients with sepsis, elevated concentrations of circulating cell-free hemoglobin are independently associated with an increased risk of death. Acetaminophen may exert a protective effect by reducing cell-free hemoglobin-induced oxidative injury.

Mortality increases after massive exchange transfusion with older stored blood in canines with experimental pneumonia

AbstractTwo-year-old purpose-bred beagles (n = 24) infected with Staphylococcus aureus pneumonia were randomized in a blinded fashion for exchange transfusion with either 7- or 42-day-old canine universal donor blood (80 mL/kg in 4 divided doses). Older blood increased mortality (P = .0005), the arterial alveolar oxygen gradient (24-48 hours after infection; P ≤ .01), systemic and pulmonary pressures during transfusion (4-16 hours) and pulmonary pressures for ∼ 10 hours afterward (all P ≤ .02). Further, older blood caused more severe lung damage, evidenced by increased necrosis, hemorrhage, and thrombosis (P = .03) noted at the infection site postmortem. Plasma cell–free hemoglobin and nitric oxide (NO) consumption capability were elevated and haptoglobin levels were decreased with older blood during and for 32 hours after transfusion (all P ≤ .03). The low haptoglobin (r = 0.61; P = .003) and high NO consumption levels at 24 hours (r = −0.76; P < .0001) were associated with poor survival. Plasma nontransferrin-bound and labile iron were significantly elevated only during transfusion (both P = .03) and not associated with survival (P = NS). These data from canines indicate that older blood after transfusion has a propensity to hemolyze in vivo, releases vasoconstrictive cell-free hemoglobin over days, worsens pulmonary hypertension, gas exchange, and ischemic vascular damage in the infected lung, and thereby increases the risk of death from transfusion.

Association of Plasma Cell-Free Hemoglobin with Physiological Measures of Nitric Oxide Responsiveness and Estimated Pulmonary Artery Systolic Pressure: Requirement for Rigorously Optimized Blood Processing Variables

AbstractAbstract 3253 Background:Accurate plasma hemoglobin (PHb) measurements are needed to investigate a model proposing a link between intravascular hemolysis and decreased nitric oxide (NO) bioavailability in sickle cell disease (SCD). Artifactual hemolysis hypothetically can produce excessive noise that might obscure the signal and lead to misinterpretation of data. We systematically studied possible blood processing variables that could cause artifactual hemolysis to obscure in vivo levels of free hemoglobin in adults with SCD. Methods:We systematically compared the effects on PHb measured by ELISA of anticoagulant type, Vacutainer materials and processing time on the same blood specimens from healthy controls and adults with SCD divided into aliquots that were then processed using a variety of in vitro conditions. We analyzed the results of PHb measurements from archived specimens from 467 SCD patients. We evaluated in greater detail plasma specimens collected and processed at bedside from 44 patients with SCD under optimal conditions: drawn from indwelling wide-bore brachial artery catheters by minimal negative pressure into heparinized syringes, gently transferred to tubes for centrifugation within 5 minutes, evaluating the associations of PHb levels from these optimally processed samples with relevant in vivo physiological measurements. Results:Analysis of specimens from healthy control subjects (n=10) and adults with SCD (n=10) for each experiment, split into aliquots to test each variable under defined conditions yielded significant evidence indicating that: (1) arterial catheter syringe specimens are superior to conventional Vacutainer venipuncture specimens; (2) heparin is superior to EDTA anticoagulant; (3) glass tubes are superior to plastic tubes; (4) processing in 2 hours or less is superior to longer processing times. Comparison of 467 archived EDTA plasma specimens processed under comparably variable conditions provided results consistent with the bench top results, confirming the importance of these processing variables on research specimens. Conventional venipuncture EDTA Vacutainer tubes with conventional processing yielded median PHb levels four-fold that of arterial catheter heparin syringe specimens processed at bedside, suggesting that on average 75% of the PHb in conventional specimens may represent background noise and only 25% of the PHb level may represent in vivo free hemoglobin.Blood specimens were collected and processed by the optimal conditioned defined by the preceding experiments from 44 adults with sickle cell anemia. This set of specimens yielded a median PHb level of 6.07μM (CI95=5.27–7.03μM). PHb correlated significantly with blunting of vasodilation in vivo after brachial artery infusion of each of three graded doses of the NO donor sodium nitroprusside (SNP), an indicator of vascular NO responsiveness; at doses of 0.8μg/min(r=−0.46 p=0.0022), 1.6μg/min (r=−0.41, p=0.0082), and 3.2μg/min (r=−0.46, p=0.0024). Quartile analysis revealed a significant difference in forearm blood flow response across all doses of SNP among the highest, middle, and lowest PHb quartiles (p=0.0386). PHb also correlated significantly with tricuspid regurgitant velocity (R=0.37, p=0.0147), suggesting a significant association with Doppler echocardiography estimates of pulmonary artery pressure. A limited number of patients from this specimen pool (n=7) subsequently underwent clinically indicated right heart catheterization. There was a trend in this small subset correlating PHb with mean pulmonary artery pressure (r=0.61, p=0.1482). Conclusions:Plasma hemoglobin levels collected and processed under optimal conditions show statistically significant associations with in vivo physiological measures of nitric oxide responsiveness and echocardiographically estimated pulmonary artery pressure, with supportive trends from right heart catheterization in a limited subgroup of patients with SCD. This supports a role for plasma hemoglobin as a biomarker of nitric oxide resistance and pulmonary hypertension in SCD. Conventional phlebotomy into Vacutainer tubes introduces a high background level of artifactual ex vivo hemolysis in SCD specimens that is capable of obscuring these associations, especially if processing is delayed 2 hours or more. Disclosures:No relevant conflicts of interest to declare.

The relationship between the severity of hemolysis, clinical manifestations and risk of death in 415 patients with sickle cell anemia in the US and Europe

The intensity of hemolytic anemia has been proposed as an independent risk factor for the development of certain clinical complications of sickle cell disease, such as pulmonary hypertension, hypoxemia and cutaneous leg ulceration. A composite variable derived from several individual markers of hemolysis could facilitate studies of the underlying mechanisms of hemolysis. In this study, we assessed the association of hemolysis with outcomes in sickle cell anemia. A hemolytic component was calculated by principal component analysis from reticulocyte count, serum lactate dehydrogenase, aspartate aminotransferase and total bilirubin concentrations in 415 hemoglobin SS patients. Association of this component with direct markers of hemolysis and clinical outcomes was assessed. As primary validation, both plasma red blood cell microparticles and cell-free hemoglobin concentration were higher in the highest hemolytic component quartile compared to the lowest quartile (P≤0.0001 for both analyses). The hemolytic component was lower with hydroxyurea therapy, higher hemoglobin F, and alpha-thalassemia (P≤0.0005); it was higher with higher systemic pulse pressure, lower oxygen saturation, and greater values for tricuspid regurgitation velocity, left ventricular diastolic dimension and left ventricular mass (all P<0.0001). Two-year follow-up analysis showed that a high hemolytic component was associated with an increased risk of death (hazard ratio, HR 3.44; 95% confidence interval, CI: 1.2-9.5; P=0.02). The hemolytic component reflects direct markers of intravascular hemolysis in patients with sickle cell disease and allows for adjusted analysis of associations between hemolytic severity and clinical outcomes. These results confirm associations between hemolytic rate and pulse pressure, oxygen saturation, increases in Doppler-estimated pulmonary systolic pressures and mortality (Clinicaltrials.gov identifier: NCT00492531).

Hemolysis and cell-free hemoglobin drive an intrinsic mechanism for human disease

Blood transfusion represents the first and most prescribed cell-based therapy; however, clinical safety and efficacy trials are lacking. Clinical cohort studies have suggested that massive transfusion and/or transfusion of aged stored blood may contribute to multiorgan dysfunction in susceptible patients. In this issue of the JCI, Baek and colleagues report that aged stored blood hemolyzes after massive transfusion in a guinea pig model. Hemolysis led to vascular and kidney injury that was mediated by cell-free plasma hemoglobin and prevented by coinfusion of the specific hemoglobin scavenger protein, haptoglobin. These studies support an expanding body of research indicating that intravascular hemolysis is a pathological mechanism in several human diseases, including multiorgan dysfunction after either massive red blood cell transfusion or hemoglobin-based blood substitute therapy, the hemoglobinopathies, malaria, and other acquired and genetic hemolytic conditions.

Impact of Severe Pain on Outcomes in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

Abstract 5273 Introduction:Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder that renders affected red blood cells sensitive to complement-mediated lysis resulting in the release of hemoglobin. Excessive and persistent complement activation leads to chronic red cell hemolysis and direct activation of platelets and macrophages, causing inflammation, hemostatic activation, thromboembolism, ischemia, and, ultimately organ damage. Cell-free plasma hemoglobin in patients with PNH has been shown to deplete nitric oxide (NO), leading to smooth muscle constriction. The combination of thrombosis and vasoconstriction can lead to symptoms of end organ damage such as abdominal pain, chest pain and dyspnea. The relation of pain to severe morbidities and mortality has not been previously examined in PNH patients. Aim/Methods:To determine whether severe pain in PNH patients is predictive of mortality, thrombosis or life-disabling morbidities, we retrospectively analyzed medical charts of 301 PNH patients from a national data registry in South Korea over the last 41 years. Patients with severe pain, defined as patients receiving opioids and/or NSAIDs for pain management, were compared to patients with no history of pain medication. We compared life-threatening morbidities and mortality between patients using pain medication (n=83) and patients with no pain medication (n=214). Results:Twenty-eight percent (83/297) of patients reported the use of opioid or NSAIDs. There was no significant difference between patients with severe pain and patients with no pain medication with respect to age at diagnosis (38 vs 40 years, P=0.412), granulocyte clone size (54% vs 48%, P=0.223) and LDH fold above normal at diagnosis (6.2 vs 5.3 fold, P=0.279). Thrombosis was recorded in 43% of patients with severe pain in contrast to 8% in patients with no pain medication (P<0.001). Patients with severe pain had an 8.4-fold greater risk for thrombosis compared to patients with no pain management (OR=8.4, 95% CI [4.15–17.08]; P<0.001). Twenty-five of the 83 patients with pain medication (30%) died during the follow up period of observation, a significantly higher rate of mortality compared to patients with no pain medication (8%; P<0.001). Patients with severe pain are roughly 5 times at greater risk for early mortality compared to patients with no pain medication (OR=4.8, 95% CI [2.44–9.38]; P<0.001). Conclusion:These data demonstrated that PNH patients with severe pain experience an increased risk of thrombosis and early mortality. Almost 1 in 3 PNH patients experienced severe pain requiring pain medication during their disease. Patients with severe pain had an increased risk of thrombosis, which may support the idea that ischemia or microthrombi of small vessels, in addition to smooth muscle dystonia due to NO depletion, drives the severe pain in these patients. Our data also suggests that symptoms of consistent pain, including abdominal pain, chest pain and flank pain, may represent a continued progression and manifestation of complement activation and progressive organ damage that leads to early mortality and poor quality of life. Along with thrombosis and renal impairment as the major risk factors affecting survival, severe pain should be considered as a predictive factor of mortality. These results suggest that early signs of pain in PNH should be managed at the root cause (uncontrolled hemolysis), and that pain is a risk factor of poor outcomes in patients with PNH. Disclosures:Lee:Novartis: Honoraria; Alexion: Honoraria. Chung:Novartis: Research Funding.

Transfusion of Stored Autologous Blood Alters Reactive Hyperemia and Circulating Nitrite Levels in Healthy Volunteers

Abstract 2327 Background:Transfusion of human blood stored for over 2 weeks is associated with increased mortality and morbidity. During storage, packed red blood cells (PRBC) progressively release hemoglobin, which avidly binds nitric oxide (NO). We hypothesized that the NO mediated hyperemic response following ischemia would be reduced after transfusion of PRBC stored for 40 days. Methods and results:We conducted a cross-over randomized interventional study, enrolling ten healthy adults. Nine volunteers completed the study. Each volunteer received one unit of 40-day and one of 3-day stored autologous leukoreduced PRBC, on different study days according to a randomization scheme. Blood withdrawal and reactive hyperemia index (RHI) measurements were performed before and 10 minutes, 1 hour, 2 hours, and 4 hours after transfusion. The RHI was lower (13% mean post-transfusion difference) after transfusion of 40-day as compared to 3-day stored PRBC (p=0.01). Plasma cell-free hemoglobin and bilirubin levels were higher after transfusion of 40-day than after 3-day stored PRBC (p=0.02 and 0.001, respectively). Plasma levels of potassium, LDH, haptoglobin, cytokines, as well as blood pressure, did not differ between the two transfusions and remained within the normal range. Plasma nitrite concentrations increased after transfusion of 40-day stored PRBC, but not after transfusion of 3-day stored PRBC (p=0.01). Conclusions:Transfusion of autologous PRBC stored for 40 days is associated with increased hemolysis and reduced RHI, possibly due to enhanced NO scavenging by cell-free oxyhemoglobin. Disclosures:Bloch:Massachusetts General Hospital: Patents & Royalties, The Massachusetts General Hospital has filed patents related to the use of small molecule inhibitors of BMP signaling to modulate iron metabolism, and PBY, RTP and KDB may be eligible to receive royalties. Yu:Massachusetts General Hospital: Patents & Royalties. Zapol:Dr. Warren Zapol receives royalties from patents on inhaled nitric oxide licensed by Massachusetts General Hospital to Linde Corp, Munich, Germany, and Ikaria Corp, Clinton, New Jersey. Dr. Zapol has applied for patents on inhaled nitric oxide and blood t: Patents & Royalties.