Increase In Plasma Hemoglobin Research Articles

Assessment of Donkey (Equus asinus africanus) Whole Blood Stored in CPDA-1 and CPD/SAG-M Blood Bags.

Simple SummaryThe development of conservative solutions was essential to store blood for different periods, but there is no blood bag available for animals. Currently, the solution containing citrate, phosphate dextrose, and adenine (CPDA-1) and the solution containing citrate, phosphate, and dextrose plus mannitol and sodium chloride (CPD/SAG-M) are the most used in human blood conservation. In this study, we propose to evaluate whether the CPDA-1 and CPD/SAG-M blood bags designed for humans are efficient for the conservation of donkey whole blood for 42 days. During storage, both blood bags resulted in mild alterations in the stored blood, but the two bags were efficient and very similar in preserving donkey blood for up to 42 days. Both types of human-designed blood bags can be used for donkey transfusion medicine.Hemotherapy using whole blood and its components is being increasingly used in veterinary therapy. Since it is important to store animal blood while maintaining acceptable hematological, blood gas, and biochemical characteristics, increasing our knowledge of available technologies for strategic blood storage is imperative. Thus, we aimed to assess the hematological, blood gas, and biochemical changes in donkey whole blood using blood bags with two different types of storage agents. Eight adult healthy male donkeys were used; 900 mL of blood was collected from each, with 450 mL stored in citrate-phosphate-dextrose and adenine bags (CPDA-1) and 450 mL stored in bags containing citrate-phosphate-dextrose, adenine, mannitol, and sodium chloride (CPD/SAG-M). Both bags were kept refrigerated between 1 and 6 °C for 42 days. Blood samples were removed from the bags eight times (T): T0 (immediately after blood collection), T1, T3, T7, T14, T21, T35, and T42 (1, 3, 7, 14, 21, 35 and 42 days after storage). Hematological, blood gas, biochemical, and microbiological parameters were assessed. The CPDA-1 bags had a higher packed cell volume when compared to CPD/ SAG-M. The red blood cell count reduced by around 19% in both the bags due to hemolysis, which was confirmed by an increase in plasma hemoglobin. The white blood cell count; pH; concentrations of glucose, sodium, bicarbonate, and 2,3 diphosphoglycerate were reduced in both bags. Meanwhile, pO2, pCO2, lactate dehydrogenase, and levels of potassium increased in the CPDA-1 and CPD/SAG-M bags. Blood bags were efficient for the storage of donkey blood for up to 42 days.

Application of plasma glucosylsphingosine detection in the follow-up of patients with Gaucher disease

Objective: Explore the application of plasma glucosylsphingosine level in the follow-up treatment of patients with Gaucher disease. Methods: Two groups of patients with Gaucher disease were enrolled, who regularly received imiglucerase treatment in Xinhua Hospital, Shanghai Jiao Tong University School of Medicine between January 2017 and July 2020. Group 1 was 6 initially treated patients, including 1 case of chitotriosidase deficiency, aged 10-43 years old, 4 females and 2 males. The blood routine test, chitotriosidase activity and plasma glucosylsphingosine level were measured during pre-and post-treatment. Group 2 were 6 cases of Gaucher disease including 3 cases of chitotriosidase deficiency, who received long-term specific treatment in the same hospital, aged 17 to 32 years, 2 females and 4 males. The plasma glucosylsphingosine level was detected in the follow-up treatment during January 2017 to July 2020. Results: Patients in group 1 had a significant increase in plasma platelets after 12 months of treatment (P<0.05), and also a significant increase in plasma hemoglobin after 30 months of treatment (P<0.05). The chitotriosidase activity of 5 patients in group 1 significantly decreased after 18 months of treatment (P<0.05), the median value of the chitotriosidase activity decreased by 7 278 nmol·ml(-1)·h(-1) at 30 months of treatment. While only 3 months after treatment, the plasma glucosylsphingosine levels of 6 patients in group 1 decreased significantly (P<0.05), the median value of the glucosylsphingosine levels decreased by 259.7 μg/L at 30 months of treatment. The plasma glucosylsphingosine levels in group 1 patients were positively correlated with chitotriosidase activity, with spearman of 0.863, P<0.001. In group 2, 6 patients with Gaucher disease that had been treated for a long period of time, showed normal peripheral blood routine tests, normal liver and spleen volume. However, the plasma glucosphingosine levels in group 2 patients decreased significantly during 2017-2020 (P<0.05). Compare to the initial values, the median value of the last glucosphingosine levels in group 2 patients had been reduced by 23.4 μg/L. Conclusion: The detection of plasma glucosylsphingosine levels in patients with Gaucher disease could be used for short-term and long-term follow-up of treatment.

Interference in the anti-Xa heparin activity assay due to hemolysis and icterus during pediatric extracorporeal life support.

Chromogenic anti-Xa assays for unfractionated heparin monitoring (heparin activity) are susceptible to interference from hemolysis and icterus. The purpose of this study was to better understand the effect of hemolysis and icterus on anti-Xa heparin activity and to predict the magnitude of the error. Increasing levels of hemoglobin and unconjugated bilirubin were added to pooled normal plasma or buffer containing known levels of heparin. Increased plasma hemoglobin or bilirubin produced falsely increased residual factor Xa activity as measured by the absorbance change (OD/min) in the Stago heparin activity assay. This increased absorbance change slope resulted in falsely lower estimates of heparin activity. The falsely lower heparin activity measurement occurred even when heparin was not present, indicating it was not due to heparin neutralization. In a sample containing 0.62±0.06 U/mL heparin and 228 mg/dL hemoglobin, the measured heparin activity was 0.41±0.03 U/mL, underestimating heparin activity by 0.21±0.07 U/mL. Interference occurred if plasma hemoglobin was above 70 mg/dL or bilirubin was above 16 mg/dL, which happened in 16%-26% of samples from pediatric patients on extracorporeal life support (ECLS). In conclusion, hemolysis and icterus were common in ECLS patients, leading to underestimates of unfractionated heparin activity and potentially higher doses of heparin than intended. The magnitude of the heparin activity measurement error could be predicted based on plasma hemoglobin and bilirubin levels until these levels exceeded the technical limits of the assay, ~230 mg/dL hemoglobin and 55 mg/dL bilirubin.

Pulmonary and Systemic Vascular Resistances After Cardiopulmonary Bypass: Role of Hemolysis

Prolonged cardiopulmonary bypass (CPB) is associated with hemolysis, resulting in increased plasma oxyhemoglobin and vascular nitric oxide depletion. The authors hypothesized that hemolysis associated with CPB would reduce nitric oxide bioavailability, resulting in high pulmonary and systemic vascular resistances that after CPB would normalize gradually over time, due to clearance of plasma oxyhemoglobin. The authors also investigated whether prolonged CPB (≥140 min) produced increased levels of hemolysis and greater pulmonary and systemic vasoconstriction. Prospective cohort study. Single-center university hospital. The study comprised 50 patients undergoing elective cardiac surgery requiring CPB. Plasma hemoglobin and plasma nitric oxide consumption were measured before surgery and after CPB. Pulmonary and systemic hemodynamics were measured after CPB. The effects of short (<140 min) and prolonged (≥140 min) CPB on these parameters were considered. Pulmonary and systemic vascular resistances and plasma hemoglobin and nitric oxide consumption were highest at 15 minutes after CPB and then decreased over time. Pulmonary and systemic vascular resistances and plasma hemoglobin and plasma nitric oxide consumption were higher in patients requiring prolonged CPB. The reduction in plasma nitric oxide consumption from 15 minutes to 4 hours after CPB was correlated independently with the reductions in pulmonary and systemic vascular resistances. Prolonged CPB was associated with increased plasma hemoglobin and plasma nitric oxide consumption and pulmonary and systemic vascular resistances. The reduction in plasma nitric oxide consumption at 4 hours after CPB was an independent predictor of the concomitant reductions in pulmonary and systemic vascular resistances.

Leukocyte expression of heme oxygenase-1 [hmox1] varies inversely with severity of tricuspid regurgitation in acute pulmonary embolism

ObjectivePulmonary embolism (PE) can cause intracardiac hemolysis and increased plasma hemoglobin and arginase-1, which can worsen pulmonary vasoconstriction. We test the hypothesis that patients with PE that causes tricuspid regurgitation (TR), indicative of higher pulmonary arterial pressures, have decreased leukocyte expression of hmox-1 compared with patients with PE and no TR and patients without PE. DesignProspective, noninterventional study. PatientsNormotensive patients with suspected PE (n=87) who underwent CT pulmonary angiography and transthoracic Doppler-echocardiography. MeasurementsSignificant TR was defined as a jet velocity >2.7m/s. Leukocyte expression of hmox-1, haptoglobin, haptoglobin related gene, the haptoglobin receptor, CD163 and cox-2 genes were assessed by quantitative rtPCR, and the hmox-1 promoter was examined for the −413 A→T SNP and GT repeat polymorphisms. ResultsOf the 44 (50%) with PE+, 22 had TR+, and their mean pulmonary vascular occlusion (39±32%) did not differ significantly from patients who were TR− (28±26%, P=0.15). Patients with PE+ and TR+ had significantly lower expression of hmox-1 and haptoglobin genes than patients without PE+ and no TR. Expression of hmox-1 varied inversely with TR velocity (r2=0.45, P<0.001) for PE+ (n=22) but not patients without PE. Hmox-1 expression did not vary significantly with genotype. Cox-2 did not differ between groups and had no correlation with TR. ConclusionsSeverity of TR varied inversely with hmox-1 expression, suggesting that hmox-1 expression affects pulmonary vascular reactivity after PE.

Assessment of changes in plasma hemoglobin and potassium levels in red cell units during processing and storage

Red cell units undergo changes during storage and processing. The study was planned to assess plasma potassium, plasma hemoglobin, percentage hemolysis during storage and to determine the effects of outdoor blood collection and processing on those parameters. Blood collection in three types of blood storage bags was done – single CPDA bag (40 outdoor and 40 in-house collection), triple CPD + SAGM bag (40 in-house collection) and quadruple CPD + SAGM bag with integral leukoreduction filter (40 in-house collection). All bags were sampled on day 0 (day of collection), day 1 (after processing), day 7, day 14 and day 28 for measurement of percentage hemolysis and potassium levels in the plasma of bag contents. There was significant increase in percentage hemolysis, plasma hemoglobin and plasma potassium level in all the groups during storage (p < 0.001). No significant difference was found between any parameter analyzed for outdoor and in-house collected single CPDA red cell units. There was significant lower percentage hemolysis (p < 0.001) and potassium (day 7 to day 14 – p < 0.05 and day 14 to day 28 – p < 0.001) in red cell units from day 7 onward until day 28 of storage in the leukoreduced quadruple bag as compared to the triple bag. The in-house single CPDA red cell units showed significantly more hemolysis (p < 0.001) as compared to the triple bags with SAGM additive solution after 28 days of storage. There is gradual increase in plasma hemoglobin and plasma potassium levels during the storage of red blood cells. Blood collection can be safely undertaken in outdoor blood donation camps even in hot summer months in monitored blood transport boxes. SAGM additive solution decreases the red cell hemolysis and allows extended storage of red cells. Prestorage leukoreduction decreases the red cell hemolysis and improves the quality of blood.

Hyperglycaemic Environment: Contribution to the Anaemia Associated with Diabetes Mellitus in Rats Experimentally Induced with Alloxan.

Background. Diabetes mellitus characterized by hyperglycaemia presents with various complications amongst which anaemia is common particularly in those with overt nephropathy or renal impairment. The present study has examined the contribution of the hyperglycaemic environment in diabetic rats to the anaemia associated with diabetes mellitus. Method. Sixty male albino rats weighing 175–250 g were selected for this study and divided equally into control and test groups. Hyperglycaemia was induced with 170 kgbwt−1 alloxan intraperitoneally in the test group while control group received sterile normal saline. Blood samples obtained from the control and test rats were assayed for packed cell volume (PCV), haemoglobin (Hb), red blood cell count (RBC), reticulocyte count, glucose, plasma haemoglobin, potassium, and bilirubin. Result. Significant reduction (P < 0.01) in PCV (24.40 ± 3.87 versus 40.45 ± 3.93) and haemoglobin (7.81 ± 1.45 versus 13.39 ± 0.40) with significant increase (P < 0.01) in reticulocyte count (12.4 ± 1.87 versus 3.69 ± 0.47), plasma haemoglobin (67.50 ± 10.85 versus 34.20 ± 3.83), and potassium (7.04 ± 0.75 versus 4.52 ± 0.63) was obtained in the test while plasma bilirubin showed nonsignificant increase (0.41 ± 0.04 versus 0.24 ± 0.06). Conclusion. The increased plasma haemoglobin and potassium levels indicate an intravascular haemolytic event while the nonsignificant increased bilirubin showed extravascular haemolysis. These play contributory roles in the anaemia associated with diabetes mellitus.

NANO-COPPER AS A NEW GROWTH PROMOTER IN THE DIET OF GROWING NEW ZEALAND WHITE RABBITS

Seventy two rabbits, 35 days old and weighing 622g average body weight, were used for the present study. The rabbits were randomly assigned into 4 equal treatments (n=18). Animals in treatment 1 served as a control group and were given basal diet without supplementation, while rabbits in treatments 2, 3 and 4 were given nano-copper in the diet at levels 25, 50 and 75 mg/kg diet, respectively. The experiment lasted for 8 weeks. The results revealed that, rabbits fed diets supplemented with 50 or 75 mg Cu/ kg diet as Nano-Cu recorded significantly higher (P<0.01) final body weight and performance index also, recorded better feed conversion ratio during all growth periods than the control group. The group of rabbits fed diet supplemented with 50 mg Nano-Cu/kg diet significantly achieved the best relative growth rate percentage. Dietary supplementation with 50 mg Nano-Cu /kg diet significantly recorded higher carcass, fore part, trunk and hind part percentages comparing with the control group, it also significantly reduced abdominal fat by 28.4%. The Nano- Cu supplementation significantly (P<0.05) increased activity of superoxide dismutase enzyme compared with the control group. Copper content in rabbits liver significantly increased by increasing dietary Nano-Cu. Dietary Nano-Cu supplementation significantly (P<0.05) increased the population of total bacterial count and lactolacillus counts and decreased the population of ureolytic bacteria, Escherichia coli and clostridium spp. Supplementation of Nano-Cu to rabbit diets significantly increased plasma hemoglobin, red blood cells count and lymphocytes percentage. Economical efficiency improved for rabbits fed either 50 or 75 mg Nano-Cu supplemental diet. Conclusively, Nano-Cu is considered a new substitution for high dose of inorganic copper in growing NZW rabbits feeding and can be fed up to 50 mg/kg diet without any deleterious effects on production and meat safety of rabbits.

Repeated effect of hyperbaria on rat blood system

The reactions of the blood system in male Wistar rats to repeated effects of nitrogenoxygen hyperbaria (pressure 0.5 MPa, density of gas medium 6 g/l, pO2 = 0.02–0.03 MPa) were studied. Rats were placed into a pressure chamber for 5 h-in 24, 72, and 120 h after first exposure (ambient air pressure control). The red blood cell parameters were analyzed and the general condition of animals was estimated. It was established that after first exposure the stress reaction developed in rats with all objective stress markers (corticosteroid and adrenal ascorbic acid levels). On repeated exposure after 24 h, almost complete normalization of all stress markers occurred except for the increased plasma hemoglobin level as a result of impaired erythrocyte membrane permeability. After repeated exposures in 72 and 120 h, the stress markers recurred. After exposure in 120 h, the general condition of animals sharply deteriorated: they did not move in the “open” field, could not hold themselves on a horizontal bar and were mainly lying. The immobilization stress control showed that after the initial stress reaction rats recovered completely with no changes in the blood system and general condition of animals occurred on repeated exposures. We believe that the hyperbaria-induced stress is associated with breathing difficulty under pressure. Thus, the repeated effect of hyperbaria is a harmful factor which causes no habituation.

Autologous transfusion of stored red blood cells increases pulmonary artery pressure.

Transfusion of erythrocytes stored for prolonged periods is associated with increased mortality. Erythrocytes undergo hemolysis during storage and after transfusion. Plasma hemoglobin scavenges endogenous nitric oxide leading to systemic and pulmonary vasoconstriction. We hypothesized that transfusion of autologous blood stored for 40 days would increase the pulmonary artery pressure in volunteers with endothelial dysfunction (impaired endothelial production of nitric oxide). We also tested whether breathing nitric oxide before and during transfusion could prevent the increase of pulmonary artery pressure. Fourteen obese adults with endothelial dysfunction were enrolled in a randomized crossover study of transfusing autologous, leukoreduced blood stored for either 3 or 40 days. Volunteers were transfused with 3-day blood, 40-day blood, and 40-day blood while breathing 80 ppm nitric oxide. The age of volunteers was 41 ± 4 years (mean ± SEM), and their body mass index was 33.4 ± 1.3 kg/m(2). Plasma hemoglobin concentrations increased after transfusion with 40-day and 40-day plus nitric oxide blood but not after transfusing 3-day blood. Mean pulmonary artery pressure, estimated by transthoracic echocardiography, increased after transfusing 40-day blood (18 ± 2 to 23 ± 2 mm Hg; P < 0.05) but did not change after transfusing 3-day blood (17 ± 2 to 18 ± 2 mm Hg; P = 0.5). Breathing nitric oxide decreased pulmonary artery pressure in volunteers transfused with 40-day blood (17 ± 2 to 12 ± 1 mm Hg; P < 0.05). Transfusion of autologous leukoreduced blood stored for 40 days was associated with increased plasma hemoglobin levels and increased pulmonary artery pressure. Breathing nitric oxide prevents the increase of pulmonary artery pressure produced by transfusing stored blood. Clinical trial registered with www.clinicaltrials.gov (NCT 01529502).

Can haptoglobin attenuate hemoglobin‐induced vascular damage?

Hemolytic diseases and infections can lead to intravascular hemolysis, which causes increased plasma hemoglobin (Hb) concentration. Over time, this “cell free Hb” is toxic to the blood vessels, exerting oxidative damage, nitric oxide (NO) scavenging, or inflammatory responses within the vasculature. The exposure of increased Hb on the lung tissue can lead to pulmonary hypertension (PH), which is high blood pressure in the pulmonary arteries due to changes in arterial wall. Haptaglobin is an acute phase protein responsible for binding and clearance of Hb. We hypothesized that haptoglobin (Hp) can attenuate PH secondarily to vascular hemoglobin exposure. To test our hypothesis, Hb was chronically administered to rats at physiologically relevant concentrations as observed in the sickle cell disease (SCD) (0.2 μmole/day) for 5 weeks. To further mimic the SCD process, rats were placed in hypobaric hypoxic chambers (~17,000 ft). Animals were divided into 4 groups; (1) Control (sham), (2) Hb infusion, (3) Hp injections (biweekly), and (4) Hb: Hp (chronically infused with Hb treated biweekly with Hp). At the end of the 5 weeks, we evaluated pulmonary blood pressure, cardiac output, and pulmonary vascular wall thickness. Additionally, lung analysis for indicators of endothelial nitric oxide synthase (eNOS) and intercellular adhesion molecule 1 (ICAM‐1) expression were performed. Preliminary data demonstrates that groups infused with haptoglobin had lower pulmonary blood pressure, decreased vessel wall thickness, and decreased ICAM‐1 expression. We observed no difference in eNOS expression between groups. This suggests that Hb induced inflammation may be attenuated by Hp.

Inhaled nitric oxide attenuates the adverse effects of transfusing stored red blood cells in mice with endothelial dysfunction after hemorrhagic shock

BackgroundTransfusion of red blood cells (RBCs) stored for prolonged periods is associated in some retrospective studies with adverse clinic outcomes in patients. We investigated whether transfusion with stored RBCs would increase tissue injury, inflammation, oxidative stress and mortality (adverse effects of transfusing stored RBCs) in a murine model of hemorrhagic shock. We tested whether the adverse effects associated with transfusing stored RBCs were exacerbated by endothelial dysfunction and ameliorated by inhaling nitric oxide.MethodsWe studied mice fed a high‐fat diet (HFD‐fed, to induce endothelial dysfunction) or a standard diet for 4–6 weeks. Mice were subjected to 90 min of hemorrhagic shock, followed by resuscitation with leukoreduced syngeneic RBCs stored for less than 24 h (fresh RBCs) or stored for 2 weeks (stored RBCs).ResultsIn standard diet‐fed mice at 2 h after resuscitation, transfusion with stored RBCs increased tissue injury more than transfusion with fresh RBCs. The adverse effects of transfusing stored RBCs were more marked in HFD‐fed mice and associated with increased lactate levels and short‐term mortality. Compared with fresh RBCs, resuscitation with stored RBCs was associated with a reduction in P50, increased plasma hemoglobin levels, and increased indices of inflammation and oxidative stress, effects that were exacerbated in HFD‐fed mice. Inhaled nitric oxide reduced tissue injury, lactate levels, and indices of inflammation and oxidative stress, as well as improved short‐term survival in HFD‐fed mice resuscitated with stored RBCs.ConclusionsResuscitation with stored RBCs adversely impacts outcome in mice with hemorrhagic shock, an effect which is exacerbated in mice with endothelial dysfunction. Inhaled nitric oxide reduces tissue injury and improves short‐term survival in HFD‐fed mice resuscitated with stored RBCs.

Linear accelerator: A reproducible, efficacious and cost effective alternative for blood irradiation

The dedicated devices for blood irradiation are available only at a few centers in developing countries thus the irradiation remains a service with limited availability due to prohibitive cost. To implement a blood irradiation program at our center using linear accelerator. The study is performed detailing the specific operational and quality assurance measures employed in providing a blood component-irradiation service at tertiary care hospital. X-rays generated from linear accelerator were used to irradiate the blood components. To facilitate and standardize the blood component irradiation, a blood irradiator box was designed and fabricated in acrylic. Using Elekta Precise Linear Accelerator, a dose of 25 Gy was delivered at the centre of the irradiation box. Standardization was done using five units of blood obtained from healthy voluntary blood donors. Each unit was divided to two parts. One aliquot was subjected to irradiation. Biochemical and hematological parameters were analyzed on various days of storage. Cost incurred was analyzed. Progressive increase in plasma hemoglobin, potassium and lactate dehydrogenase was noted in the irradiated units but all the parameters were within the acceptable range indicating the suitability of the product for transfusion. The irradiation process was completed in less than 30 min. Validation of the radiation dose done using TLD showed less than ± 3% variation. This study shows that that the blood component irradiation is within the scope of most of the hospitals in developing countries even in the absence of dedicated blood irradiators at affordable cost.

Simultaneous determination of phagocytosis of Plasmodium falciparum-parasitized and non-parasitized red blood cells by flow cytometry

BackgroundSevere falciparum malaria anaemia (SMA) is a frequent cause of mortality in children and pregnant women. The most important determinant of SMA appears to be the loss of non-parasitized red blood cells (np-RBCs) in excess of loss of parasitized (p-) RBCs at schizogony. Based on data from acute SMA where excretion of haemoglobin in urine and increased plasma haemoglobin represented respectively less than 1% and 0.5% of total Hb loss, phagocytosis appears to be the predominant mechanism of removal of np- and p-RBC.Estimates indicate that np-RBCs are cleared in approximately 10-fold excess compared to p-RBCs. An even larger removal of np-RBCs has been described in vivax malaria anaemia. Estimates were based on two single studies both performed on neurosyphilitic patients who underwent malaria therapy. As the share of np-RBC removal is likely to vary between wide limits, it is important to assess the contribution of both np- and p-RBC populations to overall RBC loss, and disclose the mechanism of such variability. As available methods do not discriminate between the removal of np- vs p-RBCs, the purpose of this study was to set up a system allowing the simultaneous determination of phagocytosis of p- and np-RBC in the same sample.Methods and ResultsPhagocytosis of p- and np-RBCs was quantified in the same sample using double-labelled target cells and the human phagocytic cell-line THP-1, pre-activated by TNF and IFNγ to enhance their phagocytic activity. Target RBCs were double-labelled with fluorescent carboxyfluorescein-succinimidyl ester (CF-SE) and the DNA label ethidium bromide (EB). EB, a DNA label, allowed to discriminate p-RBCs that contain parasitic DNA from the np-RBCs devoid of DNA. FACS analysis of THP-1 cells fed with double-labelled RBCs showed that p- and np-RBCs were phagocytosed in different proportions in relation to parasitaemia.ConclusionsThe assay allowed the analysis of phagocytosis rapidly and with low subjective error, and the differentiation between phagocytosed p- and np-RBCs in the same sample. The presented method may help to analyse the factors or conditions that modulate the share of np-RBC removal in vitro and in vivo and lead to a better understanding of the pathogenesis of SMA.

Inhaled Nitric Oxide Attenuates the Adverse Effects of Transfusing Stored Syngeneic Erythrocytes in Mice with Endothelial Dysfunction after Hemorrhagic Shock

The authors investigated whether transfusion with stored erythrocytes would increase tissue injury, inflammation, oxidative stress, and mortality (adverse effects of transfusing stored erythrocytes) in a murine model of hemorrhagic shock. They tested whether the adverse effects associated with transfusing stored erythrocytes were exacerbated by endothelial dysfunction and ameliorated by inhaling nitric oxide. The authors studied mice fed a high-fat diet (HFD-fed; to induce endothelial dysfunction) or a standard diet for 4-6 weeks. Mice were subjected to 90 min of hemorrhagic shock, followed by resuscitation with leukoreduced syngeneic erythrocytes stored less than 24 h (fresh erythrocytes) or stored for 2 weeks (stored erythrocytes). In standard-diet-fed mice at 2 h after resuscitation, transfusion with stored erythrocytes increased tissue injury more than transfusion with fresh erythrocytes. The adverse effects of transfusing stored erythrocytes were more marked in HFD-fed mice and associated with increased lactate levels and short-term mortality. Compared with fresh erythrocytes, resuscitation with stored erythrocytes was associated with a reduction in P50, increased plasma hemoglobin levels, and increased indices of inflammation and oxidative stress, effects that were exacerbated in HFD-fed mice. Inhaled nitric oxide reduced tissue injury, lactate levels, and indices of inflammation and oxidative stress and improved short-term survival in HFD-fed mice resuscitated with stored erythrocytes. Resuscitation with stored erythrocytes adversely impacts outcome in mice with hemorrhagic shock, an effect that is exacerbated in mice with endothelial dysfunction. Inhaled nitric oxide reduces tissue injury and improves short-term survival in HFD-fed mice resuscitated with stored erythrocytes.

Pulmonary Hypertension in Lambs Transfused with Stored Blood Is Prevented by Breathing Nitric Oxide

During extended storage, erythrocytes undergo functional changes. These changes reduce the viability of erythrocytes leading to release of oxyhemoglobin, a potent scavenger of nitric oxide. We hypothesized that transfusion of ovine packed erythrocytes (PRBC) stored for prolonged periods would induce pulmonary vasoconstriction in lambs, and that reduced vascular nitric oxide concentrations would increase this vasoconstrictor effect. We developed a model of autologous stored blood transfusion in lambs (n = 36). Leukoreduced blood was stored for either 2 days (fresh PRBC) or 40 days (stored PRBC). Fresh or stored PRBC were transfused into donors instrumented for awake hemodynamic measurements. Hemodynamic effects of PRBC transfusion were also studied after infusion of N-nitro-L-arginine methyl-ester (25 mg/kg) or during inhalation of nitric oxide (80 ppm). Cell-free hemoglobin levels were higher in the supernatant of stored PRBC than in supernatant of fresh PRBC (Mean ± SD, 148 ± 20 vs. 41 ± 13 mg/dl, respectively, P < 0.001). Pulmonary artery pressure during transfusion of stored PRBC transiently increased from 13 ± 1 to 18 ± 1 mmHg (P < 0.001) and was associated with increased plasma hemoglobin concentrations. N-nitro-L-arginine methyl-ester potentiated the increase in pulmonary arterial pressure induced by transfusing stored PRBC, whereas inhalation of nitric oxide prevented the vasoconstrictor response. Our results suggest that patients with reduced vascular nitric oxide levels because of endothelial dysfunction may be more susceptible to adverse effects of transfusing blood stored for prolonged periods. These patients might benefit from transfusion of fresh PRBC, when available, or inhaled nitric oxide supplementation to prevent the pulmonary hypertension associated with transfusion of stored PRBC.

Pulmonary vascular reserve during experimental pulmonary embolism: Effects of a soluble guanylate cyclase stimulator, BAY 41-8543*

Pulmonary embolism causes pulmonary hypertension by mechanical obstruction and vasoconstriction. Therapeutic potential of pharmacologic dilation of unblocked vessels has received limited attention. We tested pulmonary vasodilator reserve using a soluble guanylate cyclase stimulator, BAY 41-8543. Controlled animal study. Medical center research laboratory. Male Sprague-Dawley rats. Pulmonary embolism was induced by infusing 25-μm plastic microspheres in the right jugular vein, producing mild or moderate pulmonary hypertension. Control animals with no pulmonary embolism received suspension medium for microspheres. Mild pulmonary embolism increased right ventricular peak systolic pressure (from 28 to 38 mm Hg) and decreased cardiac output (from 46 to 34 mL/min) with no change in mean arterial pressure. Infusion of BAY 41-8543 (50-200 μg/kg) decreased right ventricular peak systolic pressure. Five hrs moderate pulmonary embolism increased right ventricular peak systolic pressure (from 28 to 47 mm Hg) and decreased cardiac output (from 48 to 27 mL/min), causing right ventricular peak systolic pressure/cardiac output to increase from 0.6 control with no pulmonary embolism to 1.8 mm Hg/mL/min in 5-hr moderate pulmonary embolism + solvent for BAY 41-8543. Treatment of 5-hr moderate pulmonary embolism with BAY 41-8543 (50 μg/kg) caused a 2.2-fold increase in cardiac output (59 mL/min) with a 46% reduction in right ventricular peak systolic pressure (38 mm Hg), suggesting significant pulmonary vasodilation. Moderate pulmonary embolism decreased arterial sO2 (from 83% to 71%) and increased lactate (from 0.5 to 2.3 mmol/L). Treatment with BAY 41-8543 normalized sO2 and lactate. Hemolysis occurred during moderate experimental pulmonary embolism (60-fold increase in plasma hemoglobin). Treatment with BAY 41-8543 reduced free plasma hemoglobin content by 80%. In the setting of moderate impervious pulmonary embolism, treatment with a guanylate cyclase stimulator normalized pulmonary hemodynamics, reduced hemolysis, and improved oxygenation. These data support the hypothesis that pharmacologic dilation of nonobstructed pulmonary vasculature can effectively treat acute pulmonary hypertension from pulmonary embolism.

Clinical and Genetic Variability of Red Blood Cell Hemolysis in Sickle Cell Anemia

Abstract 1077Intravascular hemolysis is an important pathological mechanism underlying some complications of sickle cell disease and other hemolytic anemias. Hemolysis contributes to endothelial dysfunction, pulmonary and systemic vasculopathy, and platelet and hemostatic activation via nitric oxide catabolism by plasma hemoglobin and arginine catabolism by red blood cell arginase. Little is known about the molecular mechanisms of hemolysis and how the propensity of erythrocytes to hemolyze is modulated. Hemoglobin F concentration and the presence of ∝ thalassemia affect the level of hemolysis but it is likely that other genes and their products are also important. We hypothesize that genetic variation, much of which is outside the β-globin gene-like cluster, underlies the susceptibility of erythrocytes to hemolyze in response to diverse disease stressors.We first characterized hemolysis by creating a principal component analysis (PCA) of age-adjusted values for LDH, AST, reticulocyte count and total bilirubin, but not hemoglobin concentration, to develop a hemolytic component that reflects shared variability among markers. The development of such a component helps to resolve the problem of dealing with correlated predictors in multivariate analyses and confounding variables such as site, and it permits for adjustment for the degree of anemia. To validate the PCA, we measured the plasma hemoglobin levels and red cell microparticle levels in the first and fourth quartile of PCA intensity of hemolysis in 118 HbS-only patients without detectable HbA, from the Walk-PHASST cohort We observed a highly significant increase in plasma hemoglobin (p<0.0001) and red cell microparticles (p=0.0004) based on PCA quartile. Despite the small sample size of this validating cohort we reproduced significant associations between high hemolytic rate and the subphentypes of low arterial oxygen saturation, high pulse pressure, leg ulcers, TRV, high NT-proBNP levels, and low 6-minute walk test distance. More patients with ∝ thalassemia and more females were present in the lower hemolytic index quartile (p=0.006).The hemolytic index and its individual components were then used as phenotypes in genome-wide association studies (GWAS) in the CSSCD (Cooperative Study of Sickle Cell Disease) and walk-PHaSST cohorts to discover novel genes that might be associated with hemolysis. As further validation of our approach using PCA stratification, patients in the quartile with the lowest hemolytic index from the CSSCD also had a much higher prevalence of ∝ thalassemia than patients within the highest quartile of hemolytic index (p=2.2E-16). We first examined 1117 cases from the CSSCD and found 303 SNPs, 265 with a MAF >0.05, that reached a threshold of significance of p<5E-4. For replication, we examined these SNPs in the Walk-PHASST cohort. Eight SNPs replicated with the same effects in a GWAS in 449 subjects from Walk-PHAAST and p-value<0.01. Of the 8 SNPs that replicated, 4 SNPs were in olfactory receptor (OR) genes on chromosome (chr) 11p; OR51L2 (rs7948471, rs7938426. rs1391617), and OR51L1 (rs2445284). Several of these SNPs were also associated with HbF in previous GWAS analyses. Polymorphisms in the OR gene cluster upstream of HBG might modulate HbF levels by altering chromatin structure within the HBB globin gene-like cluster. One SNP in an intron of NPRL3 (rs7203566) on chr16p is ∼34 kb upstream from a SNP causing ∝ thalassemia. In CSSCD cases there was an association of SNPs in NPRL3 with reticulocytes (p=5.1E-0006) and LDH (p=0.0003). In silico analysis did not predict any function for this SNP.Genetic studies to discover new biologic modifiers of hemolysis will help to identify critical molecular determinants of hemolysis for functional studies, to develop new disease severity biomarkers, and to suggest candidate therapies for some common human diseases with intravascular hemolysis. We anticipate that our studies will identify genetic variants enriched in the African-American population primarily determined by the evolved human response to endemic malaria infection. These studies are expected to broadly impact many human diseases and blood banking by providing genomic markers of susceptibility to hemolytic anemia, red cell storage stability and transfusion risk, and insights into novel strategies to reduce anemia and to enhance red blood cell storage and post-transfusion erythrocyte recovery. Disclosures:Gladwin:Patents filed related to treating hemolysis.: Patents & Royalties.

Arginase depletes plasma l-arginine and decreases pulmonary vascular reserve during experimental pulmonary embolism

The experiments test if experimental PE causes red blood cell hemolysis, arginase release and depletion of l-arginine and determine if arginase inhibition preserves l-arginine and improves pulmonary hemodynamics during PE. Experimental PE was induced in male Sprague–Dawley rats by infusing 25 μm microspheres (1.8 million/100 g body wt) in the jugular vein, producing moderate pulmonary hypertension. Pulmonary vascular resistance was estimated from the quotient of the right ventricular peak systolic pressure/cardiac output. Arterial plasma hemoglobin (ELISA), arginase activity (colorimetric assay) and l-arginine (high performance liquid chromatography) were determined. Arginase activity was inhibited by infusion of N-omega-hydroxy-nor-l-arginine (nor-NOHA, 400 mg/kg body wt, i.v.). Values are means ± s.e. Five hours of PE caused red blood cell hemolysis (15-fold increase in plasma hemoglobin) and release of arginase activity (2.7-fold increase). Plasma l-arginine concentration decreased significantly from 250 ± 20.6 to 118 ± 6.0 μmol/L (Control vs. PE) and estimated pulmonary vascular resistance increased 3-fold. Treatment with nor-NOHA prevented the depletion of plasma l-arginine (229 ± 15 μmol/L) and reduced the rise in pulmonary vascular resistance by 40%. In conclusion, experimental PE causes hemolysis, release of arginase activity, depletion of plasma l-arginine and increased estimated pulmonary vascular resistance. Inhibition of arginase activity preserves plasma l-arginine levels and improves estimated resistance, suggesting that the release of arginase during hemolysis contributes to the rise in estimated pulmonary resistance during experimental PE.

Gender differences in exercise – induced intravascular haemolysis during race training in thoroughbred horses

Exercise-induced intravascular haemolysis and “sport anemia” are widely reported in human sports medicine. It has been recognized also in horses, however, the clinical importance and the onset of this condition seem different than in human. In this study we investigated the episodes of intravascular haemolysis, indicated by the increase in plasma haemoglobin and the decrease in serum haptoglobin levels, after routine training sessions in race horses. Heart rate and changes in haematological parameters confirmed, that the exertion was relatively high. Intravascular haemolysis did not appear in stallions but was detected in mares after two training sessions. It has been determined that serum haptoglobin levels were higher in mares than in stallions before and after all training sessions. It is postulated that intravascular haemolysis induced by training is of limited clinical importance because it occurred only in mares which are better adapted due to higher haptoglobin level at rest, and it had no cumulative effect. Therefore gender differences should be taken into consideration in experiments with athletic horses.