Free Hemoglobin Levels Research Articles

Strategy for the hemocompatibility testing of microparticles.

Polymer-based microparticles are applied as non-thrombogenic or thrombogenic materials in a wide variety of intra- or extra-corporeal medical devices. As demanded by the regulatory agencies, the hemocompatibility of these blood contacting biomaterials has to be evaluated in vitro to ensure that the particle systems appropriately fulfill the envisioned function without causing undesired events such as thrombosis or inflammation. Currently described in vitro assays for hemocompatibility testing of particles comprise tests with different single cell types (e.g. erythrocytes or leukocytes), varying concentrations/dilutions of the used blood cells or whole blood, which are not standardized.Here, we report about an in vitro dynamic test system for studying the hemocompatibility of polymeric microparticles utilizing fresh human whole blood from apparently healthy subjects, collected and processed under standardized conditions. Spherical poly(ether imide) microparticles with an average diameter of 140±30 μm were utilized as model systems. Reported as candidate materials for the removal of uremic toxins, these microparticles are anticipated to facilitate optimal flow conditions in a dialyzer with minimal backflow and blood cell damage. Pristine (PEI) and potassium hydroxide (PEI-KOH) functionalized microparticles exhibited similarly nanoporous surfaces (PEI: ØExternal pore = 90±60 nm; PEI-KOH ØExternal pore = 150±130 nm) but varying water wettabilities (PEI: θadv = 112±10° PEI-KOH θadv = 60±2°). The nanoporosity of the microparticle surfaces allows the exchange of toxic solutes from blood towards the interconnective pores in the particle core, while an immigration of the substantially larger blood cells is inhibited.Sterilized PEI microparticles were incorporated -air-free -in a syringe-based test system and exposed to whole blood for 60 minutes under gentle agitation. Thereafter, thrombi formation on the particles surfaces were analyzed microscopically. In the collected whole blood the non-adherent/circulating single blood cells were quantified via a differentiated complete blood cell count and the activation of platelets (P-Selectin expression, secretion and release), platelet function (PFA100 closure time) as well as thrombin formation (thrombin-antithrombin-complex) was analyzed. Free hemoglobin (HGB) levels were quantified as a measure of hemolysis.Microscopic evaluation revealed thrombi formation and particle aggregates for all tested microparticles. Reduction of circulating blood cells differed significantly between the particle types. Particularly, platelet and monocyte counts decreased up to 50% compared to the control (syringe filled with whole blood but without microparticles). In accordance, platelet activation, thrombin levels and degrees of hemolysis were clearly elevated in the particle loaded test systems and allowed a differentiation between the particle types. Increased PFA100 closure times (as activating agent a combination of collagen/ADP was used) indicated a similarly reduced ability of platelets to adhere and form stable aggregates independent from the particle type tested. This observation is most probably a consequence of the strong thrombus formation in the test system, which is associated with a reduction of the circulating blood cells.The reported in vitro dynamic whole blood test system allowed the sensitive analysis of the hemocompatibility of polymer-based microparticles and was successfully validated for porous PEI microparticles with different water wettabilities. Beyond the qualitative and quantitative analysis of cell-material interactions, the test also allowed the functional evaluation of platelets in whole blood.

Catalytic iron in acute myocardial infarction complicated by cardiogenic shock — A biomarker substudy of the IABP-SHOCK II-trial

BackgroundCatalytic iron (CI) is unbound ferric iron with the potential to generate reactive oxygen species with further deleterious vascular effects. In acute coronary syndromes, high levels of CI are linked to all-cause mortality. The prognostic impact of CI and iron metabolism in cardiogenic shock (CS) is currently undetermined. Aims of this study were to investigate the prognostic impact of CI and to identify predictors of high CI levels in patients with CS complicating acute myocardial infarction. MethodsThe Intraaortic Balloon Pump in Cardiogenic Shock II (IABP-SHOCK II) trial randomized 600 patients with CS to either therapy with intraaortic balloon pump or control. In 185 of these patients, blood samples were systematically collected at baseline and day 3. CI levels were measured using a modified bleomycin detectable iron assay. Furthermore, levels of free hemoglobin, total serum iron, transferrin, total iron binding capacity, ferritin, hepcidin, and transferrin saturation were assessed. ResultsPatients with baseline CI levels in the highest quartile had a worse outcome in comparison to patients with lower CI (day 1: HR 1.91 [1.11–3.31], p=0.005; day 3: HR 2.15 [1.06–4.34], p=0.01). In multivariable Cox-regression analysis baseline CI remained an independent predictor of 30-day mortality (HR per 10LOG 2.08 [1.25–3.47], p=0.005). Predictors of CI levels on day 3 were baseline CI, bleeding events, and baseline troponin T. ConclusionsCI levels were associated with increased short-term mortality in CS complicating acute myocardial infarction. High levels of CI at day 3 were associated with bleeding and high troponin levels.

A mobile phone-based approach to detection of hemolysis

Preeclampsia and HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome are pregnancy-related complications with high rates of morbidity and mortality. HELLP syndrome, in particular, can be difficult to diagnose. Recent work suggests that elevated levels of free cell hemoglobin in blood plasma can, as early as the first trimester, potentially serve as a diagnostic biomarker for impending complications. We therefore developed a point-of-care mobile phone–based platform that can quickly characterize a patient's level of hemolysis by measuring the color of blood plasma. The custom hardware and software are designed to be easy to use. A sample of the whole blood (~10µL or less) is first collected into a clear capillary tube or microtube, which is then inserted into a low-cost 3D-printed sample holder attached to the phone. A 5–10min period of quiescence allows for gravitational sedimentation of the red blood cells, leaving a layer of yellowish plasma at the top of the tube. The phone camera then photographs the capillary tube and analyzes the color components of the cell-free plasma layer. The software converts these color values to a concentration of free hemoglobin, based on a built-in calibration curve, and reports the patient's hemolysis level: non-hemolyzed, slightly hemolyzed, mildly hemolyzed, frankly hemolyzed, or grossly hemolyzed.. The accuracy of the method is ~1mgdL−1. This phone-based point-of-care system provides the potentially life-saving advantage of a turnaround time of about 10min (versus 4+hours for conventional laboratory analytical methods) and a cost of approximately one dollar USD (assuming you have the phone and the software are already available).

Quality evaluation of red blood cell suspension and platelet concentrate separated and prepared from whole blood stored at 22℃ for 24 hours

Objective To evaluate the quality of red blood cell suspension and platelet concentrate separated and prepared from whole blood stored at 22℃ for 24 h. Methods From May 2014 to February 2015, a total of 60 voluntary blood donors who were recruited by Maoming Central Blood Station of Guangdong Province were included into this study, using simple random sampling method. The inclusion criterion for subjects was that the physical examination and related hematology tests results of all blood donors before blood donation met the relevant regulations in the Whole Blood and Blood Component Donor Selection Requirements (GB18467-2011). All the 60 donors were randomly divided into study group (n=30) and control group (n=30). A total of 60 bags of whole blood (400 mL) were collected from the blood donors in the two groups, using quintuple blood collection bags. In the study group, the whole blood was stored and transported at 22 ℃, and prepared into red blood cell suspension and platelet concentrate 24 h after blood collection. In the control group, the whole blood was also stored and transported at 22 ℃, and prepared into red blood cell suspension and platelet concentrate within 8 h after blood collection. The preparation of red blood cell suspension and platelet concentrate in both groups was conducted by the buffy coat method. Red blood cell suspension was stored at 4 ℃ until the end of the storage period (35 d after preparation), and its sterility test was using Bact/ALERT full-automatic microbial detection system. Moreover, red blood cell count, hematocrit (HCT), hemoglobin (Hb) level, free hemoglobin (FHb) level, hemolytic ratio at the end of the storage period, and K+ , Na+ and Cl- concentrations in red blood cell suspension were compared between the study group and control group. Also, platelet concentrate was stored at 22 ℃ until the end of the storage period (5 d after preparation), and its sterilty test was using Bact/ALERT full-automatic microbial detection system. Additionally, platelet content, red cell volume, FHb level, pH value at the end of the storage period, adhesion rate, aggregation rate, and K+ , Na+ and Cl- concentrations in platelet concentrate were compared between the study group and control group. Results ① In the study group and the control group, there was no bacterial growth found in red blood cell suspension 35 d after storage, and in platelet concentrate 5 d after storage. ② In the red blood cell suspension of both groups 35 d after storage, all test results of HCT, Hb level and hemolytic ratio at the end of the storage period met the relevant requirements in Quality Requirements for Whole Blood and Blood Components (GB18469-2012). There were no significant differences comparisons of red blood cell count, HCT, level of Hb, level of FHb, hemolytic ratio at the end of the storage period, and K+ , Na+ and Cl- concentrations in the red blood cell suspension of both groups (t=0.55, 0.51, 1.18, 0.48, 0.72, 2.86, 2.07, 2.40; P>0.05). ③ In the platelet concentrate of both groups 5 d after storage, all test results of platelet content, red cell volume and pH value at the end of the storage period met the relevant requirements in Quality Requirements for Whole Blood and Blood Components (GB18469-2012). There were no significant differences comparisons of platelet content, red cell volume, level of FHb, platelet adhesion rate, platelet aggregation rate, K+ , Na+ and Cl- concentrations, and pH value in the platelet concentrate of both groups (t=0.17, 0.16, 0.56, 2.43, 0.36, 2.50, 1.85, 1.75, 0.32; P>0.05). Conclusions The quality of the red blood cell suspension and platelet concentrate prepared by whole blood stored at 22 ℃ for 24 h met national standards. Therefore, the separation and preparation of red blood cell suspension and platelet concentrate using whole blood stored at 22 ℃ overnight is feasible. Key words: Blood storage; Blood component removal; Red blood cell; Platelet; Quality control

Human mannose-binding lectin inhibitor prevents Shiga toxin–induced renal injury

Hemolytic uremic syndrome caused by Shiga toxin-producing Escherichia coli (STEC HUS) is a worldwide endemic problem, and its pathophysiology is not fully elucidated. Here we tested whether the mannose-binding lectin (MBL2), an initiating factor of lectin complement pathway activation, plays a crucial role in STEC HUS. Using novel human MBL2-expressing mice (MBL2 KI) that lack murine Mbls (MBL2(+/+)Mbl1(-/-)Mbl2(-/-)), anovel STEC HUS model consisted of an intraperitoneal injection with Shiga toxin-2 (Stx-2) with or without anti-MBL2 antibody (3F8, intraperitoneal). Stx-2 induced weight loss, anemia, and thrombocytopenia and increased serum creatinine, free serum hemoglobin, and cystatin C levels, butasignificantly decreased glomerular filtration rate compared with control/sham mice. Immunohistochemical staining revealed renal C3d deposition and fibrin deposition inglomeruli in Stx-2-injected mice. Treatment with 3F8 completely inhibited serum MBL2 levels and significantly attenuated Stx-2 induced-renal injury, free serum hemoglobin levels, renal C3d, and fibrin deposition and preserved the glomerular filtration rate. Thus, MBL2 inhibition significantly protected against complement activation and renal injury induced by Stx-2. This novel mouse model can beused to study the role of complement, particularly lectin pathway-mediated complement activation, in Stx-2-induced renal injury.

(652) - LVAD Support Increases Plasma Iron Species: Implications for a Pathophysiologic Relationship between LVAD-Associated Hemolysis and Thrombosis

Emerging evidence suggests a relationship between left ventricular assist device (LVAD)-associated hemolysis and LVAD thrombosis. Plasma iron species such as free hemoglobin, hemin, and free iron (Fe2+, Fe3+), which are produced during hemolysis, generate oxidative stress and may promote thrombogenesis. As a preliminary study to guide in vitro mechanistic investigations into contributors to LVAD thrombosis, we examined plasma iron species in patients during LVAD support. Patients undergoing continuous-flow LVAD support were studied (n=23). Whole blood was obtained prior to LVAD implantation and after one week of LVAD support. Lactate dehydrogenase (LDH) was measured as a marker of hemolysis. Free hemoglobin, hemin, Fe2+, and Fe3+ were measured as plasma iron species. Paired Student’s t-tests were performed between pre-LVAD and post-LVAD samples. LVAD support increased LDH (pre-LVAD 245±20 units/L vs. post-LVAD 333±36 units/L, p=0.07), plasma free hemoglobin (pre-LVAD 46±6 mg/dL vs. post-LVAD 67±9 mg/dL, p=0.01), hemin (pre-LVAD 0.26±0.06 μg/dL vs. post-LVAD 4.8±2.3 μg/dL, p=0.06), Fe2+ (pre-LVAD 173±17 μg/dL vs. post-LVAD 220±27 μg/dL, p=0.02), and Fe3+ (pre-LVAD 29±7 μg/dL vs. post-LVAD 51±9 μg/dL, p=0.03). LVAD support significantly increased plasma levels of free hemoglobin, hemin, Fe2+, and Fe3+. Elevated plasma iron species during LVAD support may contribute to the pathophysiology of LVAD thrombosis. These data are guiding in vitro studies to determine whether the relationship between LVAD-associated hemolysis and thrombosis is causal or associative.

Extracorporeal treatment of hemolytic complications in multifield surgical clinic

to optimize the protocol of high-volume plasmapheresis for hemolytic complications in surgical practice. We studied 100 patients aged 7-73 years with hemolytic complications. In 83 patients with free hemoglobin level over 200 mg% 50-300% of plasma volume was removed. In 32 patients (15 of them with plasmapheresis and 17 without it) renal function was assessed comparatively. Plasmapheresis was used in case of significantly higher values of hemolysis (p<0.01). Cardiac patients make 95% of observations. They had intraoperative hemolysis due to plonged extracorporeal circulation (93) and injection of 16-18-day blood preserved with glugjcirum into pump oxygenator (2). Hemolytic complications were observed in 5 patients. Autoerythrocytes disintegration occurred in 95 cases. Efficacy of plasmapheresis to eliminate free hemoglobin was 95%. Severity of acute renal injury depended on hymolysis intensity. Renal function recovery was diagnosed in 2-3 days postoperatively. Plasmapheresis facilitated microalbuminuria and concentration function restoration.

Decreased Hemolysis of Red Cells Washed with Plasma-Lyte As Compared with 0.9% Sodium Chloride

Introduction -Washed red blood cell (RBC) and platelet transfusions have been a valuable therapeutic option for patients with repeated or severe allergic or febrile reactions. Recent data from randomized and observational clinical trials demonstrate that washed transfusions reduce inflammatory and immunologic complications, including recurrence of acute leukemia, death after coronary surgery, elevations of IL-6 and C-reactive protein after pediatric cardiac surgery, and transfusion related acute lung injury and transfusion associated cardiac overload. However, washing of RBCs may not be completely benign. Increased hemolysis may occur in vitro. Washing of RBCs stored for longer periods (>28 days) is associated with increased morbidity in pediatric cardiac surgery patients receiving massive transfusions of older RBCs. Current washing techniques and dilution of RBCs for transfusion employ 0.9% normal saline (NS) which is both hypertonic and acidic. We evaluated whether use of a more physiologic solution, Plasma-Lyte, causes less damage to RBCs than use of NS.Methods -We employed RBC units (n=15) collected for, but not suitable for clinical transfusion. Median storage age was 19 days (range 10-39 days). Each donor unit (11 AS-1, 3 AS-3 and 1 CPD-A1) was split in two using aseptic technique, and washed with either a liter of NS or Plasma-Lyte employing a standard clinical protocol on a Terumo 2991. Each half was resuspended in 20 ml of washing solution. Aliquots (12 ml) were removed prior to and immediately after washing, and after 24, 48 and 72 hours of storage at 1-6¡ C. These aliquots were immediately centrifuged (15 min at 4,300 x g) and the supernatant stored at -80¡ C. Hemoglobin and heme in the supernatant were quantitated using QuantiChrom kits. All comparisons were by Wilcoxon matched pairs non-parametric tests.Results -Supernatants immediately after washing had similar mean free hemoglobin (pre = 160 mg/dl ± 80 [SEM]; post = 66 ± 12 for NS and 58 ± 10 for Plasma-Lyte) and heme (pre = 92 µM ± 46; post = 36 ± 8 for NS and 33 ± 7 for Plasma-Lyte ). However, by 24 hours of storage after washing (the current limit for clinical use), supernatant heme and hemoglobin were significantly greater by about 50% in RBCs stored in NS (87 µM ± 22 and 151 mg/dl ± 36) as compared to Plasma-Lyte (59 µM ± 21 and 108 mg/dl ± 36; p = 0.014 and 0.02). These changes accelerated during subsequent storage (see figure for heme levels-differences in hemoglobin were comparable). Prolonged storage after NS washing predisposed to greater hemolysis than did Plasma-Lyte washing. Supernatant heme levels at 72 hours post washing increased from early (10-18 days; n=5) to late (31-39 days; n=5) storage by a mean of 61% in Plasma-Lyte washed RBCs (p=0.26) compared with 200% in NS washed RBCs (p=0.016)Conclusions -RBCs stored for 10-39 days and then washed with Plasma-Lyte experience strikingly less hemolysis. Whether such differences would occur in vivo is not known, and the clinical implications are yet to be determined. Circulating free heme and hemoglobin levels have been associated with thrombosis in animal models, transfused patients and patients with sickle cell disease. NS, as opposed to Plasma-Lyte, has been linked to increased renal injury and mortality in animal models and patients. We speculate that hemolysis may be a contributing cause. Our results indicate that RBC washing with Plasma-Lyte is a superior approach and potentially safer than NS. These findings also suggest the hypothesis for further investigation of whether NS is truly a suitable diluent for RBC transfusion, and whether NS is the safest choice when crystalloid is indicated for volume replacement or apheresis. Plasma-Lyte washing might also facilitate increasing the storage period of washed RBCs. [Display omitted] DisclosuresOff Label Use: See Dr. Blumberg's disclosure. Blumberg:Biomet: Consultancy; Terumo: Consultancy.

Free hemoglobin increases von Willebrand factor–mediated platelet adhesion in vitro: implications for circulatory devices

AbstractIntravascular hemolysis occurs in patients on extracorporeal membrane oxygenation. High levels of free acellular adult hemoglobin (free HbA) are associated with clotting in this mechanical device that can result in thrombotic complications. Adsorption of fibrinogen onto the surface of biomaterial correlates with platelet adhesion, which is mediated by von Willebrand factor (VWF). Because free Hb interacts with VWF, we studied the effect of hemoglobin (Hb) on platelet adhesion to fibrin(ogen) under conditions of different hydrodynamic forces. This effect was investigated using purified human HbA and fibrinogen, extracellular matrix, collagen, or purified plasma VWF as surface-coated substrates to examine flow-dependent platelet adhesion. Antibodies and VWF-deficient plasma were also used. Free Hb (≥50 mg/dL) effectively augmented platelet adhesion, and microthrombi formation on fibrin(ogen), extracellular matrix, and collagen at high shear stress. The effect of free Hb was effectively blocked by anti-glycoprotein Ibα (GPIbα) antibodies or depletion of VWF. Unexpectedly, free Hb also promoted firm platelet adhesion and stable microthrombi on VWF. Lastly, we determined that Hb interacts directly with the A1 domain. This study is the first to demonstrate that extracellular Hb directly affects the GPIbα-VWF interaction in thrombosis, and describes another mechanism by which hemolysis is connected to thrombotic events.

Abstract 19379: Red Blood Cell Washing Increases Kidney Injury in Cardiac Surgery Patients - The Role of Oxidised Lipids and Oxidative Stress

Introduction: Allogeneic red blood cell (RBC) transfusion in cardiac surgery patients increases the risk of pulmonary and renal morbidity. These associations are attributable to the ‘storage lesion’ - accumulation of metabolites in the RBC supernatant that includes pro-inflammatory lipids, cell-free hemoglobin and energy depletion. Hypothesis: Storage lesion components trigger different events that lead to organ injury. Reducing storage lesion by RBC washing or rejuvenation will reduce organ injury. Methods: Samples from 60 cardiac patients participating in a randomized controlled trial of RBC washing prior to transfusion versus standard care were analysed for inflammatory markers. In a complementary experiment, 4 groups of pigs received crystalloid infusion or 14-day old RBC that were left untreated, washed or rejuvenated (Rejuvesol, Citra Labs, IN). Inflammatory markers, free hemoglobin and iron were measured in blood. Leukocyte invasion was determined by immunohistochemistry. The results were verified and further analysed in an in vitro inflammatory model. Results: Transfusion of stored RBC (~22 days) into humans or pigs was associated with leukocyte activation, and attenuated by washing. That was attributed to oxidised lipids on transfused RBC derived microvesicles that activated monocytes and platelets, as revealed in depletion experiments. Unexpectedly, transfusion of washed RBC increased kidney injury, which correlated with higher free haemoglobin levels, oxidative stress (confirmed by HMOX-1 levels and protein carbonylation), and endothelial injury in washed RBC recipients. Oxidative stress triggered non-standard endothelial activation defined by expression of VLA5 and fibronectin retention. Either pathway was reduced when rejuvenated blood was transfused. Conclusion: Monocytes/platelets and endothelium are activated by oxidized lipids/MV and reactive free haem, respectively, and can be alleviated by RBC rejuvenation.

Influencia de un observador en la hemólisis producida durante las extracciones de muestras sanguíneas en atención primaria

ObjectiveTo check whether an intervention based on direct observation and complementary information to nurses helps reduce haemolysis when drawing blood specimens. Material and methodsRandom sampling study in primary care centres in the serrania de Málaga health management area, using a cross-sectional, longitudinal pre- and post-intervention design. The study period was from August 2012 to January 2015. The level of free haemoglobin was measured by direct spectrophotometry in the specimens extracted. It was then checked whether the intervention influenced the level of haemolysis, and if this was maintained over time. ResultsThe mean haemolysis measured pre-intervention was 17%, and after intervention it was 6.1%. A year later and under the same conditions, the frequency of haemolysis was measured again the samples analysed, and the percentage was 9% These results are low when compared to the level obtained pre-intervention, but are higher when compared to the levels obtained immediately after the intervention. The transport and analysis conditions were the same. ConclusionsAn intervention based on a direct and informative observation in the process of collecting blood samples contributes significantly to reduce the level of haemolysis. This effect is maintained in time. This intervention needs to be repeated to maintain its effectiveness. Audits and continuing education programs are useful for quality assurance procedures, and maintain the level of care needed for a good quality of care.

Arginase Level in Suspended Red Blood Cells Storaged for Different Time

To explore the effect of storage time on arginase level, and the possible source of arginase in suspended red blood cells (RBC). The arginase and myeloperoxidase (MPO) levels in suspended RBC and control plasma were detected by ELISA. The free hemoglobin level in suspended RBC and control plasma were detected by colorimetric method. The relationship between arginase level, MPO level and free hemoglobin level in suspended RBC was analyzed by the related methods. The arginase and free hemoglobin levels in suspended RBC were higher than those in control plasma. Otherwise, MPO level was not significantly different between suspended RBC and control plasma. All of them did not increase along with prolonging of storage time. There was not a significant correlation between arginase level and free hemoglobin level in suspended RBC of different storage time (r = 0.03), but arginase level positively correlated with MPO level in the suspended RBC of different storage time (r = 0.76). The arginase level in suspended RBC storaged for different time increases significantly, but not along with prolonging of storage time. The main possible source of arginase in the suspended RBC is the residual white blood cell, especially neutrophils.

Twelve Hours In Vitro Biocompatibility Testing of Membrane Oxygenators.

In vitro test systems for extracorporeal membrane oxygenation (mock loop) represent an interesting alternative to complex and expensive in vivo test systems to analyze the pathomechanisms leading to insufficient biocompatibility. Data on mock loop systems are limited, and operation times are constricted to a maximum duration of 6 hr. This study aims at a 12 hr operation time and frequent monitoring of markers for insufficient biocompatibility in two experimental settings. Porcine blood circulated in a mock loop without any modifications, or the circuit was operated with a CO2-enhanced gas (5% CO2/21% O2/74% N2) in combination with a nutrient solution (phosphate-adenine-glucose-guanosine-saline-mannitol). Coagulation parameters changed over time without differences between the two groups. In the unmodified test setting, a pH increase was detected after 1 hr, followed by significantly increased levels of free hemoglobin as a marker for hemolysis and elevated numbers of activated platelets, which correlate with detected von Willebrand factor, microparticles, and interleukin-β. Proinflammatory cytokine levels were significantly increased after 12 hr. In contrast, these parameters remained constant in the modified test setting providing proof of a stable operating in vitro mock loop system with an extended/prolonged operation time.

199 Red Cell Rejuvenation Targets Inflammatory Microparticles, free Haemoglobin and Redox Active Iron: Key Mediators of Transfusion Related Acute Lung Injury in Cardiac Surgery

<h3>Background</h3> Using porcine model of TRALI, we have previously demonstrated that red cell washing prior to transfusion fails to protect against organ injury after cardiac surgery. Red cell rejuvenation however, eliminated all metabolic, biochemical and histological features of TRALI. In current study we investigate the mechanisms that drive endothelial dysfunction and organ injury in response to stored RBC transfusion. <h3>Method</h3> Recipient swine of allogenic blood were randomised as: Sham (n = 6), transfusion of: 1-day old RBC (n = 8), 14-day old RBC (n = 6), 14-day old RBC washed (n = 8) and rejuvenated-washed 14-day stored RBC (n = 8). Microparticles (MP), free haemoglobin and iron levels were measured in plasma and serum by flow cytometry and colorimetric methods. Expression of heme oxygenase-1 was assessed by RT-PCR. Parallel serial analysis of human stored, washed and rejuvenated RBC packs was conducted to co-relate with porcine data. Data was analysed using and t-tests. The data are reported as mean ± SE. <h3>Results</h3> Removing inflammatory MP by washing suppressed leucocyte adhesion in lung but increased serum free haemoglobin and serum/pulmonary iron levels (see Table 1). That in turn activated endothelium through a non-standard pathway, which lead to higher in-vivo pulmonary vascular resistance index and reduced renal vasodilation (see table). Rejuvenation eliminated generation of free haemoglobin and iron, prevented endothelial dysfunction and functional and histopathological features of TRALI. <h3>Conclusions</h3> We identified two components of stored blood transfusion that contribute to organ injury. MP activate leukocytes and are removed by RBC washing. Free iron and haemoglobin is generated during washing and is eliminated by Rejuvenation.

Cisplatin combined with hyperthermia kills HepG2 cells in intraoperative blood salvage but preserves the function of erythrocytes

The safe use of intraoperative blood salvage (IBS) in cancer surgery remains controversial. Here, we investigated the killing effect of cisplatin combined with hyperthermia on human hepatocarcinoma (HepG2) cells and erythrocytes from IBS in vitro. HepG2 cells were mixed with concentrated erythrocytes and pretreated with cisplatin (50, 100, and 200 μg/ml) alone at 37 °C for 60 min and cisplatin (25, 50, 100, and 200 μg/ml) combined with hyperthermia at 42 °C for 60 min. After pretreatment, the cell viability, colony formation and DNA metabolism in HepG2 and the Na(+)-K(+)-ATPase activity, 2,3-diphosphoglycerate (2,3-DPG) concentration, free hemoglobin (Hb) level, osmotic fragility, membrane phosphatidylserine externalization, and blood gas variables in erythrocytes were determined. Pretreatment with cisplatin (50, 100, and 200 μg/ml) combined with hyperthermia (42 °C) for 60 min significantly decreased HepG2 cell viability, and completely inhibited colony formation and DNA metabolism when the HepG2 cell concentration was 5×10(4) ml(-1) in the erythrocyte (P<0.01). Erythrocytic Na(+)-K(+)-ATPase activity, 2,3-DPG level, phosphatidylserine externalization, and extra-erythrocytic free Hb were significantly altered by hyperthermia plus high concentrations of cisplatin (100 and 200 μg/ml) (P<0.05), but not by hyperthermia plus 50 μg/ml cisplatin (P>0.05). In conclusion, pretreatment with cisplatin (50 μg/ml) combined with hyperthermia (42 °C) for 60 min effectively eliminated HepG2 cells from IBS but did not significantly affect erythrocytes in vitro.

Plasma Free Hemoglobin Is an Independent Predictor of Mortality among Patients on Extracorporeal Membrane Oxygenation Support.

BackgroundHemolysis is common in all extracorporeal circuits as evident by the elevated plasma free hemoglobin (PFHb) level. We investigated whether increased hemolysis during extracorporeal membrane oxygenation (ECMO) is an independent mortality predictor.MethodsWe performed a retrospective observational study of consecutive subjects who received ECMO at a tertiary care facility from 2007-2013 to investigate independent predictors of in-hospital mortality. We examined variables related to patient demographics, comorbidities, markers of hemolysis, ECMO characteristics, transfusion requirements, and complications. 24-hour PFHb> 50 mg/dL was used as a marker of severe hemolysis.Results154 patients received ECMO for cardiac (n= 115) or pulmonary (n=39) indications. Patients’ mean age was 51 years and 75.3% were males. Compared to nonsurvivors, survivors had lower pre-ECMO lactic acid (p=0.026), lower 24-hour lactic acid (p=0.023), shorter ECMO duration (P=0.01), fewer RBC transfusions on ECMO (p=0.008) and lower level of PFHb 24-hours post ECMO implantation (p=0.029). 24-hour PFHb> 50 mg/dL occurred in 3.9 % versus 15.5% of survivors and nonsurvivors, respectively, p=0.002. A Cox proportional hazard analysis identified PFHb> 50 mg/dL 24-hours post ECMO as an independent predictor of mortality (OR= 3.4, 95% confidence interval: 1.3 – 8.8, p= 0.011).ConclusionPFHb> 50 mg/dL checked 24-hour post ECMO implantation is a useful tool to predict mortality. We propose the routine checking of PFHb 24-hours after ECMO initiation for early identification and treatment of the cause of hemolysis.

Effect of astaxanthin on oxidative stress of red blood cells and peroxidation damage of membrane

To explore the effect of astaxanthin (ASTA) on oxidative stress of intra- and extra- red blood cells during stored period and the protective function for cell membrane. The blood of volunteers was collected to prepare suspended red blood cells without leukocytes. Then the red blood cells were randomly divided into group A, group B, group C and group D. The ASTA was added into MAP preservation solution of group B, group C and group D, the final concentration of ASTA was 5, 10 and 20 µmol/L respectively. Group A was used as control group, in which only the dissolved liquid DMSO of ASTA was added. The red blood cells were stored in refrigerator at 2 °C-6 °C. On day 7, 14, 28 and day 42 of storage, the content of reactive oxygen species (ROS) in red blood cells was detected by fluorescence microplate reader. The content of malondialdehyde (MDA) was detected with TBA method. The content of hydrogen peroxide (H2O2) outside cell was detected with spectrophotometric method. The mean corpuscular volume(MCV) was detected with blood cell analyzer. The content of free hemoglobin(FHb) was detected with chemical colorimetry. The ROS, MDA, FHb and H2O2 levels in B, C and D groups were lower than those in control group during the stored period. On day 7 and 14 of storage, among group B, group C, group D and group A, the MCV showed no difference in comparison with control group. On day 28 and 42 of storage, the MCV in B, C and D groups was lower than that in control group. The ASTA can reduce the oxidative stress level of stored red blood cells inside and outside, relieve the peroxidation damage of cell membrane.

Blood Warming and Hemolysis: A Systematic Review With Meta-Analysis.

The use of fluid warmers during blood transfusion is recommended to avoid inducing hypothermia and its harmful effects. Fluid warmers offered by manufacturers can reach temperatures of 43°C. However, the recommendations of national regulatory organizations do not clearly indicate the maximum heating temperature in relation to the risk of hemolysis. To fill this gap, we conducted a systematic review of the literature with meta-analysis. To match clinical practice, this review was limited to fluid warmers that used contact heating; thus, studies that used radiofrequency or microwave heating were excluded. Twenty-four observational studies were included, 17 of which were the subject of a meta-analysis. A preliminary descriptive analysis indicated that multiple factors can influence the level of hemolysis during blood heating with a liquid warmer, including blood age, anticoagulant type, duration of exposure to heat, stirring the blood during heating, and various elements of the circuit through which blood flows (eg, type of infusion pump with pressure and flow, type of microfilter, and type of tubing). Moreover, the duration between sampling and hemolysis assay was a source of heterogeneity among studies, as were the initial free hemoglobin levels in the various experiments. In general, the increase generated by each of these factors other than temperature appears to have been limited except for blood age, which is an important parameter of hemolysis, the length of exposure to heat, and, in some studies, the type of infusion pump used. Regarding the meta-analysis, at temperatures at or less than 43°C and even up to 45-46°C, it appears that blood heating is safe and causes hemolysis only in clinically negligible proportions.

Non-invasive detection of free hemoglobin in red blood cell concentrates for quality assurance

AbstractThere is currently no non-invasive measurement method available on the market for the quality control of red cell concentrates (RCCs). As the level of free hemoglobin is an indicator for hemolysis resulting from destroyed or overaged red blood cells, it is an important parameter to assess the quality and usability of RCCs before transfusion. A new optical device has been developed and tested to enable the measurement of the free hemoglobin concentration non-invasively in RCCs.

Hemolysis of irradiated leukoreduced red blood cells during rapid warming: An in vitro experimental study.

BackgroundAlthough water chambers are often used as surrogate blood-warming devices to facilitate rapid warming of red blood cells (RBCs), these cells may be damaged if overheated. Moreover, filtered and irradiated RBCs may be damaged during the warming process, resulting in excessive hemolysis and extracellular potassium release.MethodsUsing hand-held syringes, each unit of irradiated and leukocyte-filtered RBCs was rapidly passed through a water chamber set to different temperatures (baseline before blood warming, 50℃, 60℃, and 70℃). The resulting plasma potassium and free hemoglobin levels were then measured.ResultsWarming RBCs to 60℃ and 70℃ induced significant increases in free hemoglobin (median [interquartile ranges] = 60.5 mg/dl [34.9–101.4] and 570.2 mg/dl [115.6–2289.7], respectively). Potassium levels after warming to 70℃ (31.4 ± 7.6 mEq/L) were significantly higher compared with baseline (29.7 ± 7.1 mEq/L; P = 0.029). Potassium levels were significantly correlated with storage duration after warming to 50℃ and 60℃ (r = 0.450 and P = 0.001; r = 0.351 and P = 0.015, respectively).ConclusionsRapid warming of irradiated leukoreduced RBCs to 50℃ may not further increase the extracellular release of hemoglobin or potassium. However, irradiated leukoreduced RBCs that have been in storage for long periods of time and contain higher levels of potassium should be infused with caution.