Plasma Free Hb Research Articles

A Novel Hemoglobin-Binding Agent Reduces Plasma Free Hemoglobin and Partially Improves Vascular Function In Murine Hemolytic Anemia

AbstractAbstract 267Humans with sickle cell disease (SCD) and hereditary spherocytosis (HS) have been shown to have increased plasma free hemoglobin, vascular dysfunction, and pulmonary hypertension. Increased plasma free hemoglobin (Hb) levels are associated with multiple physiologic findings, including hemoglobinuria, increased blood pressure, platelet activation, and increased mortality. Previously, we showed that SCD and HS mice have impaired systemic vasodilation and mild pulmonary hypertension. Furthermore, there is evidence of increased plasma free hemoglobin and plasma oxidizing potential in mice with SCD and HS, and plasma oxidizing potential correlates with plasma free hemoglobin levels. We hypothesize that increased plasma free Hb impairs systemic vasodilation and increases pulmonary hypertension in SCD and HS mice. To test this hypothesis, we developed a peptide (hE-Hb-B10) that specifically binds human hemoglobin and removes it from the plasma via the heparan sulfate proteoglycan (HSPG)-associated lipoprotein pathway, rather than the haptoglobin-Hb-CD163 receptor pathway. SCD, HS, and control mice were treated with daily intraperitoneal (ip) injections of PBS or peptide hE-Hb-B10 dissolved in PBS (10-20 mg/mouse/day). At the end of three weeks of treatment, anesthetized mice were phlebotomized, as well as utilized for either ex vivo vasodilation studies using the facialis artery (a distal branch of the carotid artery) or assessment of right ventricular systolic pressure (RVsP) as a surrogate measure of pulmonary hypertension. In addition, plasma samples from the mice were analyzed for plasma free hemoglobin (PFH), lactate dehydrogenase (LDH, a marker of hemolysis), and oxidizing potential. PFH, LDH and oxidizing potential were significantly increased in the plasma of PBS-treated SCD and HS mice as compared with levels in control mice. Treatment with hE-Hb-B10 significantly reduced PFH levels in both SCD and HS mice compared to PFH levels in PBS-treated SCD and HS mice. In contrast, hE-Hb-B10 treatment did not reduce oxidizing potential in SCD and HS mice, suggesting that factors other than PFH contribute to the increased oxidizing potential of plasma in SCD and HS mice. hE-Hb-B10 treatment did not alter hematocrit, reticulocyte count, or plasma LDH levels in SCD and HS mice, suggesting that the overall hemolytic rate was unaltered by peptide treatment. Plasma from control mice had low levels of PFH, LDH and oxidizing potential; these levels did not change with hE-Hb-B10 treatment. Finally, facialis artery vasodilation in response to acetylcholine was significantly improved in hE-Hb-B10 treated as compared to PBS-treated SCD and HS mice. However, pulmonary hypertension, as measured by RVsP, in SCD mice was not altered by treatment with hE-Hb-B10, while hE-Hb-B10-treated HS mice showed mild improvement. These data demonstrate that hE-Hb-B10 effectively reduces plasma free hemoglobin in SCD and HS mice. Although reducing PFH improves vascular function in a systemic vascular bed (facialis artery), we found minimal beneficial effects in the pulmonary vascular bed (as measured by RVsP). Our findings confirm that the mechanisms impairing vascular function in SCD and HS are complex, and suggest that targeting only one mechanism (i.e., plasma free Hb) may be insufficient to achieve notable improvements in vascular function in these complex disease states. Disclosures:No relevant conflicts of interest to declare.

Development of a Disposable Magnetically Levitated Centrifugal Blood Pump (MedTech Dispo) Intended for Bridge‐to‐Bridge Applications—Two‐Week In Vivo Evaluation

Last year, we reported in vitro pump performance, low hemolytic characteristics, and initial in vivo evaluation of a disposable, magnetically levitated centrifugal blood pump, MedTech Dispo. As the first phase of the two-stage in vivo studies, in this study we have carried out a 2-week in vivo evaluation in calves. Male Holstein calves with body weight of 62.4–92.2 kg were used. Under general anesthesia, a left heart bypass with a MedTech Dispo pump was instituted between the left atrium and the descending aorta via left thoracotomy. Blood-contacting surface of the pump was coated with a 2-methacryloyloxyethyl phosphorylcholine polymer. Post-operatively, with activated clotting time controlled at 180–220 s using heparin and bypass flow rate maintained at 50 mL/kg/min, plasma-free hemoglobin (Hb), coagulation, and major organ functions were analyzed for evaluation of biocompatibility. The animals were electively sacrificed at the completion of the 2-week study to evaluate presence of thrombus inside the pump,together with an examination of major organs. To date, we have done 13 MedTech Dispo implantations, of which three went successfully for a 2-week duration. In these three cases, the pump produced a fairly constant flow of 50 mL/Kg/min. Neurological disorders and any symptoms of thromboembolism were not seen. Levels of plasma-free Hb were maintained very low. Major organ functions remained within normal ranges. Autopsy results revealed no thrombus formation inside the pump. In the last six cases, calves suffered from severe pneumonia and they were excluded from the analysis. The MedTech Dispo pump demonstrated sufficient pump performance and biocompatibility to meet requirements for 1-week circulatory support. The second phase (2-month in vivo study) is under way to prove the safety and efficacy of MedTech Dispo for 1-month applications.

A first‐in‐man study of the reitan catheter pump for circulatory support in patients undergoing high‐risk percutaneous coronary intervention

To investigate the safety of a novel percutaneous circulatory support device during high-risk percutaneous coronary intervention (PCI). The Reitan catheter pump (RCP) consists of a catheter-mounted pump-head with a foldable propeller and surrounding cage. Positioned in the descending aorta the pump creates a pressure gradient, reducing afterload and enhancing organ perfusion. Ten consecutive patients requiring circulatory support underwent PCI; mean age 71 +/- 9; LVEF 34% +/- 11%; jeopardy score 8 +/- 2.3. The RCP was inserted via the femoral artery. Hemostasis was achieved using Perclose sutures. PCI was performed via the radial artery. Outcomes included in-hospital death, MI, stroke, and vascular injury. Hemoglobin (Hb), free plasma Hb (fHb), platelets, and creatinine (cre) were measured pre PCI and post RCP removal. The pump was inserted and operated successfully in 9/10 cases (median 79 min). Propeller rotation at 10,444 +/- 1,424 rpm maintained an aortic gradient of 9.8 +/- 2 mm Hg. Although fHb increased, there was no significant hemolysis (4.7 +/- 2.4 mg/dl pre vs. 11.9 +/- 10.5 post, P = 0.04, reference 20 mg/dl). Platelets were unchanged (pre 257 +/- 74 x 10(9) vs. 245 +/- 63, P = NS). Renal function improved (cre pre 110 +/- 27 micromol/l vs. 99 +/- 28, P = 0.004). The RCP was not used in one patient following femoral introducer sheath related aortic dissection. All PCI procedures were successful with no deaths or strokes, one MI, and no vascular complications following pump removal. The RCP can be used safely in high-risk PCI patients. This device may be an alternative to other percutaneous systems when substantial cardiac support is needed.

Quality control of blood irradiation with a teletherapy unit: damage to stored red blood cells after cobalt‐60 gamma irradiation

Previous publications have documented the damage caused to red blood cells (RBCs) irradiated with X-rays produced by a linear accelerator and with gamma rays derived from a 137Cs source. The biologic effects on RBCs of gamma rays from a 60Co source, however, have not been characterized. This study investigated the effect of 3000 and 4000 cGy on the in vitro properties of RBCs preserved with preservative solution and irradiated with a cobalt teletherapy unit. A thermal device equipped with a data acquisition system was used to maintain and monitor the blood temperature during irradiation. The device was rotated at 2 r.p.m. in the irradiation beam by means of an automated system. The spatial distribution of the absorbed dose over the irradiated volume was obtained with phantom and thermoluminescent dosimeters (TLDs). Levels of Hb, K+, and Cl(-) were assessed by spectrophotometric techniques over a period of 45 days. The change in the topology of the RBC membrane was investigated by flow cytometry. Irradiation caused significant changes in the extracellular levels of K+ and Hb and in the organizational structure of the phospholipid bilayer of the RBC membrane. Blood temperature ranged from 2 to 4 degrees C during irradiation. Rotation at 2 r.p.m. distributed the dose homogeneously (92%-104%) and did not damage the RBCs. The method used to store the blood bags during irradiation guaranteed that all damage caused to the cells was exclusively due to the action of radiation at the doses applied. It was demonstrated that prolonged storage of 60Co-irradiated RBCs results in loss of membrane phospholipids asymmetry, exposing phosphatidylserine (PS) on the cells' surface with a time and dose dependence, which can reduce the in vivo recovery of these cells. A time- and dose-dependence effect on the extracellular K+ and plasma-free Hb levels was also observed. The magnitude of all these effects, however, seems not to be clinically important and can support the storage of irradiated RBC units for at last 28 days.

Insight into Risk Factors for Pulmonary Hypertension in Thalassemia: Abnormal Platelet, Hemolysis and Vascular Markers.

Pulmonary hypertension (PAH), determined on echocardiogram by a tricuspid jet velocity (TJV)≥2.5m/sec in the absence of diastolic dysfunction is a common complication in patients with beta-thalassemia intermedia (TI) and some patients with thalassemia major (TM). The pathogenesis is thought to be multifactorial, primarily due to increased hemolysis, hypoxia, endothelial proliferation and thrombosis. Identifying biomarkers of these risk factors could increase understanding of the pathogenesis and facilitate screening and risk stratification in these patients. A cohort of 44 patients (mean age 29±11 years; TM-29; TI-15) was screened by echocardiogram for presence of PAH and evaluated by the following lab measures: Markers of platelet activation (P- selectin, soluble CD40 [sCD40L]); thrombin generation (thrombin-anti-thrombin [TAT]; prothrombin fragment 1+2 [F1.2]); increased ventricular pressure strain (brain natriuretic peptide [BNP]) indicating; and plasma free Hb, indirect bilirubin, LDH and NOx, for hemolytic activity. 14 patients had PAH, indicated by increase in TJV (range 2.5–3.1, mean 2.63±0.1 m/sec) and 30 patients had no PAH. 8/14 (57%) with PAH had prior splenectomy versus 6/30 (20%). 4/14 (28%) with PAH and 5/30 (16%) without PAH were non-transfused TI patients. 10/14 PAH patients had a mild increase in TJV (2.5–2.7m/sec) while 4/14 had a moderate increase in TJV (2.7–3.1m/sec). These 4 patients were splenectomized, 3 non transfused TI and one started transfusions only in adulthood. Results demonstrated an increase in P selectin which correlated with the severity of TJV: 52.4±14ng/ml in mild PAH, 60±18 ng/ml in moderate cases and 38±15 ng/ml in the PAH(−) group (R= 0.5 p<0.008). A similar trend in sCD40L was noted: 6.4±4 ng/ml in mild PAH, 7.8±2.7 ng/ml in moderate severity (R= 0.9, p<0.05) and 4.0 ± 0.7 ng/ml in patients without increase in TJV. Mean TAT and F1.2 were not helpful in distinguishing PAH(+) and PAH (−) patients. There was a trend of increased hemolysis as indicated by higher bilirubin levels and plasma free Hb: Bilirubin was 2.2±1.2, 3.2±2.1 and 1.8±0.9 mg/dL in patients with mild, moderate elevated and normal TJV, respectively. (R=0.3 p<0.04). Plasma free Hb was elevated in 35/44 patients but to a higher extent in 4 patients with a higher TJV; mean 120±180 vs 70±110mg/ml in the remaining 40 patients (nl<15). LDH was comparable in all groups. Reduced NOx levels were noted: 6.7±4.3 in mild cases, 4.7±1.9 in moderate cases and 10±16 in patients without indication of PAH. BNP levels were normal in all patients. Elevated adhesion molecules were mostly seen in patients with higher liver iron concentration suggesting further contribution of iron overload to the inflammatory stress and endothelial disruption. sVCAM-1, sICAM-1 were 890±700 ng/ml, 220±160 ng/ml, respectively in patients with PAH, and 454±200 ng/ml, 151±120 ng/ml respectively in those with no detected PAH. (p=0.05 for sVCAM, ns for sICAM). These preliminary studies demonstrate an association of platelet activation, hemolysis, vascular inflammation and PAH, providing insight into the pathogenesis. The identified biomarkers might serve for monitoring of increasing pulmonary pressure and need for intervention, in particular in non transfused TI patients.

Vascular Dysfunction in Murine Models of Hemolytic Anemia.

Pulmonary hypertension affects approximately 30% of young adults with sickle cell disease (SCD) and is a risk factor for early death. There is increasing evidence that intravascular hemolysis contributes to the pathophysiology of pulmonary hypertension in SCD as well as other hemolytic disorders. In this study, we compare measures of vascular dysfunction in Berkeley sickle cell mice (SCD mice) with a murine model of hereditary spherocytosis (sph/sph mice) that exhibit severe hemolytic anemia due to alpha spectrin deficiency, but without HbS-induced RBC sickling. We assessed right ventricular systolic pressure in vivo as a measure of pulmonary arterial function and endothelial-dependent vasodilation of facialis arteries ex vivo as a measure of systemic arterial function. Right ventricular systolic pressures and right ventricle to body weight ratios were increased to similar levels in both SCD and sph/sph mice as compared to control mice (p <0.025), consistent with pulmonary hypertension in both SCD and sph/sph mice. Acetylcholine-induced vasodilation of facialis arteries (a branch of the carotid artery) was severely impaired in both SCD and sph/sph mice compared to control mice (p<0.02). We also found increased plasma levels of soluble VCAM-1, E-selectin, and P-selectin in SCD and sph/sph mice compared to control mice (p<0.005 for all groups), providing further evidence for increased endothelial injury in both murine models. Interestingly, sVCAM-1 and E-selectin were more elevated in SCD mice compared to sph/sph mice (p<0.0001), suggesting hemolysis combined with HbS induces further endothelial injury. We hypothesized that plasma free hemoglobin released by intravascular hemolysis initiates endothelial impairment and injury due to scavenging of nitric oxide (NO) and generation of oxidative damage. Plasma methemoglobin levels were determined by helium electron paramagnetic resonance (EPR) spectroscopy before and after the addition of the NO donor molecule PROLI/NO. The difference between these two signals represents the total NO scavenging capacity of plasma. We found that both the plasma free Hb and NO scavenging capacity in plasma from SCD and sph/sph mice was much greater than that of healthy control mice (p<0.05). In addition, we developed an oxygen electrode assay to assess the oxidizing potential of plasma. The initiation of lipid peroxidation by oxidants present in the plasma results in the consumption of oxygen. We found that plasma from SCD and sph/sph mice has a significantly higher oxidizing potential than plasma from control mice (p<0.05). We obtained similar results (increased plasma free Hb, NO scavenging capacity, and oxidizing potential) comparing plasma from individuals with SCD to plasma from healthy control individuals. Consistent with the higher levels of hemolysis in the sph/sph mice, plasma free Hb, NO scavenging and oxidizing capacity were greater in sph/sph mice compared SCD mice (p<0.05). These results indicate that both pulmonary and systemic vascular function is impaired in these two different murine models of hemolysis, and suggest that nitric oxide-based vasoregulatory mechanisms are particularly affected. These data, combined with previous pathologic reports, suggest that while hemolysis importantly contributes to aberrant vasoregulation and initiating pathways of oxidative damage, sickle hemoglobin may uniquely provoke additional vascular and organ injury.

The % Tetrahydrobiopterin (BH4) and Serine 1177 Phosphorylation Is Reduced in Sickle Transgenic Mice.

Tetrahydrobiopterin (BH4) is an essential co-factor of nitric oxide synthase (NOS), the enzyme that generates nitric oxide (NO) from arginine and oxygen. NO plays an essential role in maintaining vascular tone and endothelial function, reducing levels of adhesion molecules and platelet aggregation, and reducing inflammation; all of which are critical in sickle cell disease (SCD). In addition to BH4, arginine, calmodulin, intracellular Ca++ levels, hsp90, phosphorylation at serine 1177 and threonine 495, and monomer/dimer equilibrium have all been shown to modulate NO and superoxide production by eNOS. Arginine levels are decreased in plasma in SCD. We have demonstrated that plasma arginine is reduced in sickle transgenic mice and that arginine supplementation partially restores both red cell and vascular function. Channon et al have demonstrated that, in atherosclerosis and diabetes, enhanced superoxide production is associated with BH4 depletion; superoxide can interfere with both NO production and, by reacting with NO, with bioavailability. We hypothesized that a similar depletion of BH4 occurs in SCD and that treatment protective of tissue BH4 levels may interrupt the cycle of polymerization, vaso-occlusion and inflammation that is the origin of pathology in SCD. We report here depletion of BH4 in tissue and plasma of several lines of sickle transgenic mice and the effect of hypoxia on % BH4 and serine 1177 phosphorylation in NY1DD mice, a mild sickle transgenic mouse that becomes more severe when exposed to hypoxia. Only BH4 is active as a NOS cofactor, the oxidized forms BH2 and biopterin (B) are inactive. Results are presented as %BH4 (BH4/(BH4+BH2+B)). We find that %BH4 is the same in C57BL and NY1DD mice under room air in plasma and brain. After exposure to 4 days of hypoxia (8% O2), % BH4 is reduced from 75.7%±3.8% to 53.7%±3.8% in plasma, p<0.004 and from 82.0%±3.7% to 68.7%±3.7% in brain, p<0.006. At the same time total biopterin (BT= BH4+BH2+B) increased in plasma, but remained constant in brain. We also measured eNOS phosphorylated at serine 1177 in brain in NY1DD mice. We find that the % phosphorylation decreases from 45.0%±7.9% to 28.3%±7.3%, p<0.007 after 4 days of hypoxia, but that at the same time total eNOS and total nNOS nearly doubled. BERK mice express exclusively HbS and, under normoxia, we found decreased %BH4 in brain in BERK mice (58%±3.3% vs 76.7%±3.0% for C57, p<0.003) with constant BT. In red cells of BERK mice, the %BH4 is about the same as C57BL (81.7% vs 76.7%, respectively), but BT doubled from 124 to 257, p>0.01. In conclusion, increased total expression of eNOS and nNOS and decreased %BH4 with decreased % phosphorylation at serine 1177 of eNOS suggest that NO production in brain in NY1DD mice after hypoxia may be severely compromised. Percent BH4 is also reduced in brain of BERK mice under normoxia, which suggests that BH4 is reduced in more severe mice under room air. Plasma and red cell %BH4 and BT do not have a simple relation to tissue levels, but do respond to pathology and may be the only readily measurable variable in human studies. The multiple pathways for disregulation of NOS in SCD involve not only NO scavenging by plasma free Hb and arginine deficiency, but also reduced bioavailability of BH4.

High Blood flow Rates with Adjustment of Needle Diameter do not Increase Hemolysis during Hemodialysis Treatment

Higher blood flow in dialysis therapy is often avoided due to concerns about shear-induced blood damage despite the lack of reliable data. This study investigated the influence of higher blood flow rates on plasma free hemoglobin (Hb) concentration after hemodialysis (HD) treatment. Thirty-two chronic HD patients were treated once with a blood flow rate of 250 mL/min using a 17G needle, and once with a blood flow rate of 500 mL/min using a 14G needle. Arterial and venous pressure and blood pressure (BP) were recorded before and after treatment. Blood samples were taken before and after treatment for analysis of plasma free Hb, pH, HCO3, base excess, hematocrit value, urea, sodium, potassium and calcium. HD treatment at blood flow rates of 500 mL/min did not increase plasma free Hb compared to treatments at blood flow rates of 250 mL/min. Frequency of intradialytic BP drops was not different either. By adaptation of the needle size, negative arterial pressure could be kept at a similar level. Urea reduction rates were significantly higher during treatments with higher blood flow rates. Higher blood flow rates can be applied without an increased hemolysis risk provided that needle sizes are adapted accordingly.

Tricuspid Regurgitant Jet Velocity Is Significantly Associated with Hemolysis in the Evaluation of Pulmonary Hypertension in Children and Young Adults with Sickle Cell Disease.

Pulmonary hypertension (PHT) has emerged as a frequent cause of increased morbidity and mortality in adults with sickle cell disease (SCD). However, the incidence, prevalence and etiology of PHT in children with SCD are currently unknown. An elevated tricuspid regurgitant jet velocity (TRJV) ≥ 2.5 m/sec on Doppler echocardiogram (ECHO) in adults may predict PHT usually diagnosed by traditional cardiac catheterization. We hypothesized that routinely measuring TRJV in children and young adults with SCD was feasible and that TRJV correlated with degree of baseline hemolysis.Methods Using a standard protocol, we prospectively measured steady state TRJV in a convenience, cross-sectional sample of 43 patients (mean age 14.2±2.8 years, range 10 to 20) with hemoglobin (Hb) SS, SC or S-β0 thalassemia at our institution as part of a PHT screening initiative beginning December 2005. Patients on chronic transfusions were excluded. The relationship between TRJV and same day laboratory studies and clinical data obtained from patient charts was examined.Results TRJV was not measurable in 5 of 43 (12%) patients, due presumably to normal pulmonary artery systolic pressures. Neither right ventricular hypertrophy nor decreased septal wall motion, both suggestive of PHT, was present when TRJV could not be determined. In the remaining 38 studies in which TRJV could be quantified (mean 2.34 m/sec±0.44), TRJV was ≥ 2.5 m/sec in 13 patients. Using Pearson's correlation coefficient, we found a significant correlation between TRJV and LDH (r=0.54, p=0.01), with higher TRJV associated with higher LDH. There were also significant, though more modest, positive correlations between TRJV and WBC (r=0.37, p=0.05) and reticulocyte count (r=0.40, p=0.05) and a significant negative correlation between TRJV and Hb (r= -0.46, p=0.01). Using t-test for independent samples, we found a significant difference in mean LDH (458 IU/L±192 vs. 338 IU/L±144, p=0.037), Hb (8.7 g/dL±1.3 vs. 10.2 g/dL±1.6, p=0.008) and reticulocyte count (17.3%±10.3 vs. 10.7%±6.9, p=0.027) between patients with TRJV ≥ 2.5 and <2.5 m/sec. A difference approaching significance in total WBC (11.4 x103/μL±5.3 vs. 8.3 x103/μL ±3.2, p=0.075) was also observed between the two groups. We found neither a significant difference in mean values between the two groups nor significant relationships with TRJV when we examined platelet count, plasma free Hb, percent fetal Hb or total bilirubin. Using Fisher's Exact Test, we did not demonstrate in our small cohort a difference in the proportion of patients with TRJV ≥ 2.5 or < 2.5 m/sec who had a history of hydroxyurea use, acute chest syndrome, frequent pain, asthma, splenectomy, gallstones, priapism, exchange transfusion, heart disease or tonsilloadenoidectomy.Conclusions We conclude that TRJV by ECHO is quantifiable in most children and young adults being evaluated for PHT and that a higher LDH and reticulocyte count and a lower Hb at baseline are observed more frequently with elevated TRJV. Larger cohort studies are needed to test the predictive value of one or more of these markers of hemolysis. Although long term outcomes associated with elevated TRJV, as an indication of PHT, in children with SCD remains unclear, decreasing hemolysis in this population may represent an early therapeutic target in the prevention of future clinically significant PHT.

Erythrocyte Glutathione Depletion Is Associated with Severity of Anemia and Pulmonary Hypertension in Patients with Sickle Cell Disease.

Background: Glutathione (GSH) is the most abundant intracellular non-protein thiol and is an anti-oxidant present in high millimolar concentration in erythrocytes. Additionally, GSH plays an important role in signal transduction, gene expression, apoptosis, protein glutathionylation, nitric oxide metabolism, and is the principal thiol redox buffer in erythrocytes. We hypothesized that, in sickle cell disease (SCD), GSH deficiency contributes to oxidative stress and pre-disposes the sickle erythrocyte to hemolysis.Methods: A sensitive liquid chromatography coupled to tandem mass spectrometric technique was used to examine the availability of GSH and its precursors (glutamate, cysteine, glycine and glutamine) in plasma and within the erythrocytes of 25 patients with SCD and 7 ethnically-matched normal volunteers. Given the association of hemolysis with pulmonary artery hypertension (PAH) in this population, tricuspid regurgitant jet velocity (TRV) by Doppler echocardiography was also determined in all patients.Results: Despite an increase in the amino acid precursors of GSH in the plasma of SCD patients compared with controls, total plasma GSH was significantly decreased (2.8 ± 1.5 vs. 4.1 ± 2.1 mM, p<0.05, SCD vs. controls). Additionally, a striking 61% depletion of GSH was found within the erythrocytes of SCD patients compared with normal volunteers (308.1 ± 112 vs. 790.8 ± 292 mM, p<0.0001). Erythrocyte glutamine concentrations were also decreased in SCD patients compared with controls (404.6 ± 252 vs. 1061 ± 277 mM, p<0.0001), while cysteine, glycine and glutamate levels trend higher. This indicates a potential loss of GSH synthesis capacity in SCD patients. The degree of erythrocyte GSH depletion correlated strongly with TRV (r = −0.64, p<0.0001), which is of significant clinical interest given the increased mortality risk of PAH in patients with SCD. While erythrocyte glutamine concentration also correlated inversely with TRV (r = −0.59, p < 0.001), erythrocyte glutamate and cysteine levels as well as plasma GSH and its precursors were not associated with TRV. Erythrocyte GSH depletion strongly associated with hematocrit (r = 0.60, p<0.001), suggesting a link to chronic anemia. Plasma arginase concentration strongly correlated with plasma-free Hb (r=0.83, p<0.0001) and inversely correlated with erythrocyte GSH (r=−0.41, p=0.02), linking lower erythrocyte GSH levels to increased red cell derived plasma arginase and therefore potentially an increased risk of mortality in SCD & PAH as we have previously reported (Morris et al, JAMA 2005).Conclusions: Lower levels of erythrocyte GSH may contribute to the development of PAH possibly by exacerbating oxidative stress and triggering hemolysis, resulting in release of erythrocyte arginase and dysregulation of arginine and NO metabolism. The role of GSH metabolism in the pathophysiology of SCD and its link to PAH warrants further investigation.

EFFECTS OF PLASMA FREE HEMOGLOBIN (Hb) ON THE RENIN ANGIOTENSIN SYSTEM (RAS)

A growing body of evidence suggests that the presence of plasma free Hb, even in extremely low concentrations, may be associated with adverse clinical signs and symptoms. Hb is linked to vasoconstrictive, pro-oxidative and pro-inflammatory events. While Hb’s oxidative and inflammatory effects are well established, the complete pathomechanism of Hb-induced vasoconstriction has not yet been fully elucidated. The hemodynamic disturbances mediated by Hb are of rapid onset, occurring within minutes, and sustained, lasting hours. Recently, we discovered that Hb, in its ferryl form, possesses angiotensin converting enzyme (ACE)-like activity, being able to convert, within minutes, angiotensin (Ang)-I to Ang-II and other active Ang fragments. This present study evaluated further the effect of Hb on RAS, focusing on the impact of ferrous- and ferryl-Hb on angiotensinogen (AGT) and ACE. The possible renin-like activity of Hb toward AGT was evaluated by reversed phase C-18 HPLC. The effect of Hb on ACE was investigated by monitoring changes in activity of purified and human lung microvascular endothelial ACE. Results indicate that both forms of Hb had no impact on AGT. However, incubation of purified ACE with ferryl-Hb increased its enzymatic activity. Hb also activated lung microvascular ACE. These events occurred within hours and could contribute to the sustained vasoconstrictive episodes associated with the presence of free Hb. Based on this study, it can be concluded that Hb has an impact on various elements of RAS, which could be an important factor in the not yet fully understood Hb-mediated vasoconstriction.

Hyperhemolysis during the evolution of uncomplicated acute painful episodes in patients with sickle cell anemia

Some patients with sickle cell anemia (SS) experience significant decrease in their hemoglobin (Hb) level and significant increase in reticulocyte count during the progression of uncomplicated acute painful episodes. These changes have been attributed to, but not proven, hyperhemolysis. This study provides evidence of hyperhemolysis in this group of patients. Hospitalized patients who met the above criteria were identified and followed prospectively, and the measures of red blood cell (RBC) survival were determined. The latter included 1) laboratory measures of hemolysis: Hb level, reticulocyte count, nucleated RBC count, RBC distribution width (RDW), total bilirubin, lactate dehydrogenase (LDH), and aspartate transaminase (AST); 2) determination of RBC survival in vivo by the (51)Cr method in selected patients; and 3) the determination of the RBC Hb:reticulocyte Hb ratio. Baseline values of these measures were also obtained and compared to the crisis data. During the evolution of the painful episode in the selected patients, there was a significant increase in total bilirubin, LDH, AST, nucleated RBC count, and RDW that are indicative of hyperhemolysis. There was a significant decrease in RBC Hb:reticulocyte Hb and (51)Cr RBC survival, which is diagnostic of hyperhemolysis. Together, the data indicate that hyperhemolysis occurs during uncomplicated acute painful episodes in some patients with SS. The presence of free plasma Hb, consequent to hyperhemolysis, reduces nitric oxide bioavailability, promotes endothelial dysfunction, and contributes to the development of pulmonary hypertension and vasoocclusion.

Hydroxyurea Treated Sickle Disorders Demonstrate Decreased Plasma Hb, Red Cell Vesicles and Thrombolysis.

Free plasma Hb has been recently implicated in the genesis of pulmonary hypertension in sickle cell disorders (SCD) through a putative NO scavenging mechanism. We have recently reported that free plasma Hb is correlated with increased plasma red cell derived vesicles (RBVes) in sickle cell and thalassemia disorders (Westerman, Blood 2004; 104:1671). Since high levels of HbF have been associated with decreased elevation of RBVes in children with SCD (Setty Blood 2000; 96:1119–1124), we hypothesized that patients treated with hydroxyurea (HU) might show a similar normalisation of RBVes. Given our finding of an association between RBVes and free plasma Hb, such treatment might also be predicted to correct increments in free plasma Hb. In this study we have examined whether sickle cell patients treated with HU show evidence of a decrease in free plasma Hb and RBVes compared to untreated SCD patients. RBVes, free plasma Hb, and markers of activation of thrombolysis were quantitated. 35 adult SCD patients, 27 in steady state, untransfused and not receiving HU and 7 patients on long term HU at a dose of 10–30 mg/kg/day. These parameters were also measured in 27 healthy control subjects without SCD. RBVes were significantly higher in untreated SCD (15.59±3.07 x 103/μL of blood) than in control subjects (2.13 ± 0.5)(p=0.001). In HU-treated SCD patients, vesicles were significantly lower (3.97±0.83)(p=0.0371). than in untreated patients and approached levels found in healthy controls. Free plasma Hb was significaly increased in untreated SCD (16.74±3.87) compared with healthy controls (1.77±0.2)(p=0.001); these levels were also corrected in HU treated SCD patients (13.42±4.48). The proportion of RBVes expressing phosphatadyl serine was also lower in HU-treated SCD (37.25± 9.79) than in untreated SCD (52.52±4.74). This was paralleled by a decrease in red cells expressing PS in HU treated patients (11.83± 3.95) compared with untreated patients (42.71±8.45)(p=0.011). This decrease in both RBVes and RBCs expressing PS in HU-treated patients might in principle decrease activation of coagulation. Indeed, plasma Ddimers, which are increased in untreated SCD (480 ±60.14) compared with healthy controls (117.43±38)(p=0.018). are normalised in HU treated patients (190±28.69). These data provide evidence of a link between RBVes and plasma Hb and suggest that PS expression on RBVes and red cells plays a role in the prothrombotic state seen in SCD. As HU treatment appears to correct plasma Hb, long term treatment may be associated with a decreased scavenging of NO and hence the risk of pulmonary hypertension. The correction of the prothrombotic tendency may also be beneficial in this regard. Prospective trials are indicated to confirm whether HU treated patients have evidence of decreased risk of pulmonary hypertension.

Nitric Oxide Scavenging, Abnormal Vasoregulation and Oxidative Damage in sph/sph Mice with Severe Hereditary Spherocytosis: Possible Consequences of Red Blood Cell Hemolysis.

The membrane skeleton, a multiprotein complex located just beneath the plasma membrane, provides the red blood cell (RBC) with the mechanical strength and deformability required to withstand high shear forces generated in the microcapillaries. Spectrin, a tetramer composed of a- and b- subunits, is the backbone of the erythroid membrane skeleton. Previously, we have shown that sph/sph mice have severe hereditary spherocytosis (HS) due to a spontaneous single-base deletion in the murine erythroid a-spectrin gene, Spna1. HS mice have severe hemolytic anemia, compensatory reticulocytosis, altered RBC morphology and increased fragility. Vascular dysfunction in sph/sph mice likely plays an important role in the mechanism by which these mice develop a high incidence of cardiac thrombosis and stroke between 6 and 12 weeks of age. We hypothesize that serum free hemoglobin released from intravascular hemolysis of sph/sph RBCs and xanthine oxidase, released from ischemic tissues, impairs endothelial cell function by scavenging nitric oxide (NO) and increasing oxidative damage. To test this hypothesis, we used helium electroparamagnetic resonance (EPR), to quantify plasma free Hb and NO scavenging capacity in the plasma of the mice; immunohistochemistry to determine tissue and vascular levels of xanthine oxidase and 3-nitrotyrosine; and, facialis arteries to measure changes in acetylcholine, endothelium and eNOS-dependent vasodilation. By EPR we found that the plasma free Hb and NO scavenging capacity in the plasma of sph/sph mice is much greater than that of the normal +/+ mice. Immunohistochemistry (IHC) for XO and NTyr revealed XO staining was decreased in livers of sph/sph mice as compared to livers from normal +/+ mice. XO staining was increased in local patches on the endothelium of lungs isolated from sph/sph mice compared to lungs from +/+ mice. NTyr, a marker of peroxynitrite formation was also increased in a focal manner in lungs of sph/sph mice compared to lungs of +/+ mice. Acetylcholine-induced and eNOS-dependent vasodilation in sph/sph mice was significantly impaired compared to vasodilation in normal +/+ mice. Taken together these data suggest the hemoglobin removal system in sph/sph mice is saturated, leading to increased free Hb and nitric oxide scavenging. IHC studies reveal XO is released from liver in sph/sph mice and once released binds the endothelium of lung, quite distal from the original site of injury. Such changes likely contribute to marked increases in NTyr staining and impaired endothelium and eNOS-dependent vasodilation in facialis arteries isolated from sph/sph mice. Taken together, these data indicate that sph/sph mice with severe HS have increased plasma free Hb and NO scavenging capacity as well as increased release of xanthine oxidase and subsequent binding to vascular endothelial cells to locations that are distal the original site of injury. Such plasma and vascular changes in hemoglobin and oxidative enzymes likely play a critical role in the mechanisms contributing to aberrant vasoregulation and initiating the pathways of oxidative damage found in sph/sph mice.

Therapeutic Inhibition of Endothelial Cell Tissue Factor Expression In Vivo by Nitric Oxide and Arginine in Sickle Transgenic Mice.

Sickle cell anemia is accompanied by biochemical activation of the coagulation system and by clinical thromboses. Therefore, we have focused on the expression of tissue factor (TF), the trigger of the coagulation system, in this disease. Previous studies from our laboratories described abnormal TF expression by monocytes and by circulating endothelial cells in human sickle blood. More recently, we reported (Blood 104:840, 2004) that severe-phenotype (BERK) sickle mice abnormally express TF on pulmonary vein endothelium; mild-phenotype (NY1DD) sickle mice are normal in this regard but assume the abnormal phenotype after exposure to hypoxia (3 hr at 8% O2) followed by reoxygenation for 18 hr at ambient air (H/R). Monitoring the % of pulmonary veins positive for TF on triple stained tissue sections (for nuclei, for murine TF, for murine CD31 to identify endothelial cells), we have now evaluated the role of NO in TF expression, employing these two models. In the NY1DD H/R model, NO inhalation (34 ppm) administered during the entire reoxygenation period led to 68±18% reduction (P<.001) of TF expression. Consistent with this, dietary supplementation with arginine (substrate for nitric oxide synthase [NOS]) prior to H/R led to 93±25% reduction (P<.001) of the H/R induced TF response. Conversely, dietary supplementation with L-NAME (inhibitor of NOS) converted the NY1DD mouse to a TF positive phenotype (P=.047) without even requiring H/R. Interestingly, inhalation of NO was not protective in the NY1DD H/R model if given only during the hypoxia period itself or only during the last 3 hours of the reoxygenation period, but it was protective if given only during the first 3 hours of the reoxgygenation period (68±24% reduction; P=.0008). Thus, the timing of clinical intervention is probably critical. In the BERK model at ambient air, treatment with dietary arginine for 2 weeks diminished TF expression by 60±33% (P=.04), an effect that was countervailed by concurrent administration of L-NAME. Data will soon be available on eNOS over-expressing sickle mice. Also in BERK mice, L-NAME largely prevented the TF inhibiting therapeutic benefit of lovastatin, suggesting that lovastatin's beneficial effect on TF is mediated via NO biology. These results suggest that NO biology determines endothelial TF expression in vivo in sickle mice. We note that NO is reported to suppress endothelial TF expression in vitro. We find that sickle mice have elevated plasma free Hb levels (increased by 40% in NY1DD mice and 57% in BERK mice), a risk factor for diminished NO bioavailability. On the other hand, H/R in NY1DD mice is not associated with a further increase in free plasma Hb. So it seems that the NO biology of sickle mice is not simply explained by plasma Hb. NO biology seems to exert a major role in regulating endothelial TF expression in the sickle mouse. Long-term NO modulating therapy, such as dietary arginine or lovastatin, may be beneficial in terms of the coagulopathy and thrombosis risk of sickle patients and should be tested with this in mind.

EVAHEART: an implantable centrifugal blood pump for long-term circulatory support.

We developed "EVAHEART": a compact centrifugal blood pump system as an implantable left ventricular assist device for long-term circulatory support. The 55 x 64 mm pump is made from pure titanium, and weighs 370 g. The entire blood-contacting surface is covered with an anti-thrombogenic coating of diamond like carbon (DLC) or 2-methacryloyloxyethyl phosphorylcholine (MPC) to improve blood compatibility. Flows exceeding 12 L/min against 100 mmHg pressure at 2600 rpm was measured. A low-temperature mechanical seal with recirculating cooling system is used to seal the shaft. EVAHEART demonstrated an acceptably low hemolysis rate with normalized index of hemolysis of 0.005 +/- 0.002 g/100L. We evaluated the pump in long-term in-vivo experiments with seven calves. Via left thoracotomy, we conducted left ventricular apex-descending aorta bypass, placing the pump in the left thoracic cavity. Pump flow rates was maintained at 5-9 L/min, pump power consumption remained stable at 9-10 W in all cases, plasma free Hb levels were less than 15 mg/dl, and the seal system showed good seal capability throughout the experiments. The calves were sacrificed on schedule on postoperative day 200, 222, 142, 90, 151, 155, and 133. No thrombi formed on the blood contacting surface with either the DLC or MPC coating, and no major organ thromboembolisms occurred except for a few small renal infarcts. EVAHEART centrifugal blood pump demonstrated excellent performance in long-term in-vivo experiments.

Haptoglobin polymorphism and body iron stores.

In humans the iron status is influenced by environmental and genetic factors. Among them, the genetic polymorphism of the hemoglobin (Hb)-binding plasma protein haptoglobin (Hp) has been shown to affect iron turnover. The best known biological function of Hp is capture of free Hb in plasma to allow hepatic recycling of heme iron and to prevent kidney damage during hemolysis. In healthy males, but not in females, the Hp 2-2 phenotype is associated with higher serum iron, higher transferrin saturation, and higher ferritin than Hp 1-1 and 2-1. Moreover, serum ferritin correlates with monocyte L-ferritin content, which is also highest in Hp 2-2 subjects due to endocytosis of multimeric Hb-Hp 2-2 complexes by the recently identified Hb scavenger receptor CD163 in macrophages. This iron delocalization pathway, occurring selectively in Hp 2-2 subjects, has important biological and clinical consequences. The Hp polymorphism is related to the prevalence and the outcome of various pathological conditions with altered iron metabolism such as hemochromatosis, infections, and atherosclerotic vascular disease.

Effects of different concentrations of anticoagulant on the in vitro characteristics of autologous whole blood.

Routinely, 450 mL of blood is collected into 63 mL of CPDA-1, for a final anticoagulant:blood ratio of approximately 1:7 in a whole-blood autologous unit. If less than 300 mL of blood is to be collected, the AABB standards suggest that there should be a proportionate decrease in anticoagulant. Data from an autologous blood program showed a range in volume from 92 mL to 667 mL per bag, which reflects an anticoagulant:blood ratio of 2:1 to 1:10. To determine the effects of these ratios on the in vitro function of RBCs at various anticoagulant ratios, blood was collected into different amounts of anticoagulant, and various measurements were made during storage. The number of RBCs and the MCV remained constant over time, regardless of the anticoagulant dilution used. Plasma free Hb increased with time with all dilutions. At a 1:2 ratio, it rose from 734 mg per L on Day 1 to 1805 mg per L on Day 35, and at 1:8, it was 355 mg per L for Day 1 and 854 mg per L on Day 35. Plasma sodium decreased and the potassium increased over time with all dilutions. From Day 1 to Day 35, there was a nine-fold increase in potassium at both the 1:2 and 1:8 dilutions (2.4 to 22.9 mmol/L, 3.2 to 29.6 mmol/L, respectively). The LDH increased over time and the pH decreased in all of the dilutions. Osmotic fragility remained constant at the 1:8 dilution but decreased at all of the other dilutions with storage, with 44-percent fragility on Day 35 at the 1:2 ratio. The WBC and platelet counts decreased consistently over time. Overall, 1 percent of the autologous units were below the cutoff volume of 300 mL at which an adjustment of the anticoagulant volume is required. Plasma Hb and plasma potassium concentrations are considerably higher in low-volume units, which indicates that deviation from standard collection procedures is deleterious to RBCs.

HeartMate III: pump design for a centrifugal LVAD with a magnetically levitated rotor.

A long-term, compact left ventricular assist device (LVAD), the HeartMate III, has been designed and fabricated, featuring a centrifugal pump with a magnetically levitated rotor. The pump has been optimized by in vitro testing to achieve a design point of 7 L/min against 135 mm Hg at high hydrodynamic efficiency (30%) and to be capable of up to 10 L/min under such a load. Furthermore, the pump has demonstrated no mechanical failures, low hemolysis (4-10 mg/dl plasma free Hb), and low thrombogenicity during six (40, 27, 59, 42, 27, and 49-day) in vivo bovine studies.

Usefulness of cord-blood harvesting for autologous transfusion in surgical newborns with antenatal diagnosis of congenital anomalies

Background/Purpose: The risks of homologous transfusion and the effectiveness of predeposit autologous transfusion have been described. The authors examined the clinical usefulness of cord-blood harvesting for autologous transfusion in newborns who had congenital anomalies antenatally diagnosed that would require surgical intervention at or near the time of delivery. Methods: Of 112 cases of antenatal diagnosis of congenital anomalies, 50 mothers gave informed consent and enrolled in this study. Cord-blood was withdrawn immediately after clamping of the umbilical cord and was used for autologous transfusion in newborns within the first 3 days postpartum. Results: A mean of 72 ± 54 mL of cord-blood was harvested (27 ± 18 mL/kg). While preserving cord-blood for 3 days at 4°C, no signs of clot formation or hemolysis were observed. The harvested cord-blood included plasma-free Hb ranging from 1 to 68 (13 ± 18) mg/dL and thrombin-antithrombin III complex ranging from 2 to 273 (18 ± 50) ng/mL. Bacteriologic examination of the stored cord-blood showed negative cultures, except for samples from 3 newborns after vaginal delivery. A mean of 46 ± 34 mL of cord-blood was used in 26 patients for autologous transfusion. No significant complications related to cord-blood transfusion were recognized clinically. Conclusions: Autologous cord-blood transfusion has the potential to be a useful alternative to homologous transfusion in newborns requiring surgery. Adequate collection and storage techniques for cord-blood must be developed. J Pediatr Surg 36:851-854. Copyright © 2001 by W.B. Saunders Company.