Cell-free Hemoglobin Concentrations Research Articles

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

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

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

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

Potential of cell-free hemoglobin and haptoglobin as prognostic markers in patients with ARDS and treatment with veno-venous ECMO

BackgroundHemolysis is associated with increased mortality in patients with sepsis, ARDS, or therapy with extracorporeal membrane oxygenation (ECMO). To quantify a critical threshold of hemolysis in patients with ARDS and treatment with veno-venous ECMO, we aimed to identify cutoff values for cell-free hemoglobin (CFH) and haptoglobin (Hp) plasma concentrations associated with a significant increase in ICU mortality.MethodsPatients with ARDS admitted to a tertiary ARDS referral center between 01/2007 and 12/2018 and treatment with veno-venous ECMO were included. Cutoff values for mean CFH (mCFH) and mean Hp (mHp) plasma concentrations dividing the cohort into groups with significantly different ICU mortalities were calculated and patient characteristics were compared. A multiple logistic regression model with stepwise backward variable selection was included. In addition, cutoff values for vulnerable relative timespans for the respective CFH and Hp concentrations were calculated.ResultsA quantitative cutoff value of 11 mg/dl for mCFH separated the cohort (n = 442) regarding ICU mortality (mCFH ≤ 11 mg/dl: 38%, [95%-CI: 32.22–43.93] (n = 277) vs. mCFH > 11 mg/dl: 70%, [61.99–76.47] (n = 165), p < 0.001). Analogously, a mHp cutoff value ≤ 0.39 g/l was associated with a significant increase in ICU mortality (mHp ≤ 0.39 g/l: 68.7%, [60.91–75.61] (n = 163) vs. mHp > 0.39 g/l: 38.7%, [33.01–44.72] (n = 279), p < 0.001). The independent association of ICU mortality with CFH and Hp cutoff values was confirmed by logistic regression adjusting for confounders (CFH Grouping: OR 3.77, [2.51–5.72], p < 0.001; Hp Grouping: OR 0.29, [0.19–0.43], p < 0.001). A significant increase in ICU mortality was observed when CFH plasma concentration exceeded the limit of 11 mg/dl on 13.3% of therapy days (≤ 13.3% of days with CFH > 11 mg/dl: 33%; [26.81–40.54] (n = 192) vs. > 13.3% of days with CFH > 11 mg/dl: 62%; [56.05–68.36] (n = 250), p < 0.001). Analogously, a mortality increase was detected when Hp plasma concentration remained ≤ 0.39 g/l for > 18.2% of therapy days (≤ 18.2% days with Hp ≤ 0.39 g/l: 27%; [19.80–35.14] (n = 138) vs. > 18.2% days with Hp ≤ 0.39 g/l: 60%; [54.43–65.70] (n = 304), p < 0.001).ConclusionsModerate hemolysis with mCFH-levels as low as 11 mg/dl impacts mortality in patients with ARDS and therapy with veno-venous ECMO. Furthermore, a cumulative dose effect should be considered indicated by the relative therapy days with CFH-concentrations > 11 mg/dl. In addition, also Hp plasma concentrations need consideration when the injurious effect of elevated CFH is evaluated.

Haptoglobin 1 allele predicts higher serum haptoglobin concentration and lower multiorgan failure risk in sickle cell disease

AbstractHaptoglobin (HP) is an acute-phase protein and the main scavenger of cell-free hemoglobin. When HP is depleted, as observed in hemolytic conditions such as sickle cell disease (SCD), cell-free hemoglobin can lead to acute organ damage. The impact of the HP 1-1, 2-1, and 2-2 isoforms on HP and cell-free hemoglobin concentrations and SCD-related complications is unclear. In a longitudinal cohort of patients with SCD, the HP 1 allele was associated with higher HP and lower cell-free hemoglobin concentrations at a routine clinic visit as well as during hospitalization for a vaso-occlusive episode or acute chest syndrome. With a median follow-up of 6.8 years, acute chest syndrome occurred in 42% (n = 163) and multiorgan failure in 14% (n = 53) of 391 patients with SCD with a minimum follow-up of 6 months. The HP 1 allele was independently associated with lower risk of developing multiorgan failure during acute chest syndrome (additive model hazard ratio, 0.5; P < .001). Future studies assessing the regulation of HP concentrations and ability to bind cell-free hemoglobin according to the HP genotype may help to identify patients with SCD at high risk for multiorgan failure and to guide interventions, such as rapid initiation of exchange transfusion or HP replacement therapy.

Cell-Free Hemoglobin Concentration in Blood Prime Solution Is a Major Determinant of Cell-Free Hemoglobin Exposure during Cardiopulmonary Bypass Circulation in the Newborn

Exposure to circulating cell-free hemoglobin is a ubiquitous feature of open-heart surgery on cardiopulmonary bypass circulation. This study aims to determine the origins and dynamics of circulating cell-free hemoglobin and its major scavenger proteins haptoglobin and hemopexin during neonatal cardiopulmonary bypass. Forty neonates with an isolated critical congenital heart defect were included in a single-center prospective observational study. Blood samples were obtained preoperatively, hourly during bypass circulation, after bypass separation, at admission to the pediatric intensive care unit, and at postoperative days 1–3. Concentrations of cell-free hemoglobin, haptoglobin and hemopexin were determined using ELISA. Neonates were exposed to significantly elevated plasma concentrations of cell-free hemoglobin and a concomitant depletion of scavenger protein supplies during open-heart surgery. The main predictor of cell-free hemoglobin exposure was the concentration of cell-free hemoglobin in blood prime solution. Concentrations of haptoglobin and hemopexin in prime solution were important determinants for intra- and postoperative circulating scavenger protein resources.

Differential impacts of hemolysis on coagulation parameters of blood samples: A STROBE-compliant article.

This study aimed at investigating the impact of hemolysis on different coagulation parameters.A total of 216 venous blood samples without visible hemolysis were collected from adult patients at a tertiary referral center over six months. The plasma obtained was quantified for six coagulation parameters including prothrombin time, activated partial thromboplastin time, fibrinogen, D-dimer, antithrombin III, and protein C. The rest of the plasma from each blood sample was aliquoted into three tubes, each containing 1 mL of plasma with three different volumes of cell-free hemoglobin (i.e., 2, 4, 8 μL) from lysed RBCs to create simulated hemolyzed blood samples with hemoglobin concentration of approximately 0.1, 0.2, and 0.4 g/dL to mimic mild (1+), moderate (2+), and severe (3+) hemolysis, respectively, before repeating the coagulation tests to determine possible correlation between the simulated degree of hemolysis and the changes in test results of the coagulation parameters.Spearman correlation analysis showed significant decreases in the values of activated partial thromboplastin time, fibrinogen, D-dimer, and protein C values with an increasing degree of simulated hemolysis (all P < .01). Comparison of the percentage bias of biological variance showed significant positive associations of cell-free hemoglobin concentrations with the percentage bias of D-dimer and protein C. However, only the former was still within the range of biological variance under condition of simulated hemolysis. Besides, the presence of cell-free hemoglobin regardless of concentration had a notable impact on the percentage bias of activated partial thromboplastin time, whereas the influence was non-significant for prothrombin time, fibrinogen, and antithrombin III.The results showed different impacts of simulated hemolysis on six coagulation parameters, highlighting the dependence of clinical reliability on the coagulation parameter to be investigated in hemolytic blood samples.

Synergistic Effect of β-alanine and Aprotinin on Mesenteric Ischemia

BackgroundAcute mesenteric ischemia arises through sudden interruption of mesenteric blood flow, mostly due to an occlusion of the superior mesenteric artery and is associated with a high mortality of approximately 50% to 90%. In previous studies, the single application of β-alanine or aprotinin caused an ameliorated intestinal damage but without any systemic effects. MethodsTo analyze the combined effect of β-alanine and aprotinin on acute ischemia and reperfusion of the small intestine, a model with anesthetized rats was used. Ischemia and reperfusion were initiated by occluding and reopening the superior mesenteric artery. After 120 min of ischemia and 180 min of reperfusion, the intestine was analyzed for tissue damage, the activity of the saccharase, and accumulation of granulocytes. In addition, systemic and metabolic as well as inflammatory parameters were measured in blood at certain points in time. ResultsThe combination of β-alanine and aprotinin resulted in a clearly stabilized mean arterial blood pressure and blood glucose level during the reperfusion period. Furthermore, the combined administration resulted in significantly reduced tissue damage parameters, cytokine and cell-free hemoglobin concentrations in blood plasma. In addition, the damage to the small intestine was significantly attenuated, so that the animals ultimately survived the entire test period because of the administration of both substances. ConclusionsOverall, the simultaneous application of both substances leads to a synergistic protection without the occurrence of undesirable side effects. The combined usage of β-alanine and aprotinin can be seen as a promising approach to inhibit the onset of acute mesenteric ischemia.

Bedside Allogeneic Erythrocyte Washing with a Cell Saver to Remove Cytokines, Chemokines, and Cell-derived Microvesicles.

Removal of cytokines, chemokines, and microvesicles from the supernatant of allogeneic erythrocytes may help mitigate adverse transfusion reactions. Blood bank-based washing procedures present logistical difficulties; therefore, we tested the hypothesis that on-demand bedside washing of allogeneic erythrocyte units is capable of removing soluble factors and is feasible in a clinical setting. There were in vitro and prospective, observation cohort components to this a priori planned substudy evaluating bedside allogeneic erythrocyte washing, with a cell saver, during cardiac surgery. Laboratory data were collected from the first 75 washed units given to a subset of patients nested in the intervention arm of a parent clinical trial. Paired pre- and postwash samples from the blood unit bags were centrifuged. The supernatant was aspirated and frozen at -70°C, then batch-tested for cell-derived microvesicles, soluble CD40 ligand, chemokine ligand 5, and neutral lipids (all previously associated with transfusion reactions) and cell-free hemoglobin (possibly increased by washing). From the entire cohort randomized to the intervention arm of the trial, bedside washing was defined as feasible if at least 75% of prescribed units were washed per protocol. Paired data were available for 74 units. Washing reduced soluble CD40 ligand (median [interquartile range]; from 143 [1 to 338] ng/ml to zero), chemokine ligand 5 (from 1,314 [715 to 2,551] to 305 [179 to 488] ng/ml), and microvesicle numbers (from 6.90 [4.10 to 20.0] to 0.83 [0.33 to 2.80] × 106), while cell-free hemoglobin concentration increased from 72.6 (53.6 to 171.6) mg/dl to 210.5 (126.6 to 479.6) mg/dl (P < 0.0001 for each). There was no effect on neutral lipids. Bedside washing was determined as feasible for 80 of 81 patients (99%); overall, 293 of 314 (93%) units were washed per protocol. Bedside erythrocyte washing was clinically feasible and greatly reduced concentrations of soluble factors thought to be associated with transfusion-related adverse reactions, increasing concentrations of cell-free hemoglobin while maintaining acceptable (less than 0.8%) hemolysis.

Save it-don't waste it! Maximizing utilization of erythrocytes from previously stored whole blood.

Recent military and civilian experience suggests that fresh whole blood may be the preferred for treatment of hemorrhagic shock, but its use is limited by its 21-day shelf life. The red blood cell storage lesion and coagulation status of packed red blood cells (pRBCs) salvaged from expired whole blood are unknown. We hypothesized that pRBCs can be salvaged from previously stored whole blood. Cold stored, low-titer, O-positive, nonleukoreduced, whole blood units were obtained at 21 days of storage. Erythrocytes were separated by centrifugation, resuspended in AS-3, and stored for 21 additional days as salvaged pRBCs. The red blood cell storage lesion parameters of microvesicles, Band-3, free hemoglobin, annexin V, and erythrocyte osmotic fragility were measured and compared with pRBCs prepared at the time of donation and stored in AS-3 for 42 days (standard pRBCs). In additional experiments, murine pRBCs were prepared from expired whole blood units and compared with those stored under standard conditions. Mice underwent hemorrhage and resuscitation with standard and salvaged pRBC units, and serum cytokines and free hemoglobin were determined. There were no significant differences in microvesicle formation or cell-free hemoglobin concentration between salvaged and standard pRBCs. There was decreased Band-3 and increased phosphatidylserine in the salvaged units as well as greater osmotic fragility. Salvaged pRBCs maintained consistent clot firmness. After hemorrhage and resuscitation in a murine model, salvaged pRBCs did not demonstrate increased serum cytokine levels. Salvaged pRBCs from previously stored whole blood accumulate the red blood cell storage lesion in a similar fashion to standard pRBCs and maintain consistent coagulability when reconstituted with plasma. Salvaged pRBCs are not associated with an increased inflammatory response when used for resuscitation in a murine model. Salvaged pRBCs may be a viable product for utilization in the treatment of traumatic hemorrhagic shock.

Human Lung Microvascular Endothelial Cell Death in Response to Cell‐free Hemoglobin

During sepsis, circulating levels of cell‐free hemoglobin (CFH) are often elevated due to the lysis of red blood cells, and higher concentrations are associated with worse clinical outcomes. Sepsis is caused by a widespread inflammation response, which leads to the release of pro‐inflammatory molecules and cytokines into the circulation. These compounds can prime and activate the endothelium rendering it more susceptible to damage from other circulating molecules. Sepsis is a leading cause of acute respiratory distress syndrome (ARDS), which is characterized by vascular leak and fluid accumulation in the lungs. In our previous studies in murine models of sepsis, lung endothelial cell death was observed, which may contribute to the alveolar capillary barrier dysfunction that characterizes ARDS. Here we investigate whether CFH can initiate cell death in vitro and whether this is potentiated by exposure to pro‐inflammatory signals. Primary human lung microvascular endothelial cells (HLMVECs) were cultured to confluence and exposed to various clinically relevant concentrations of CFH diluted in complete media for up to 24 hours or pre‐treated for 4 hours with 5 ng/ml Cytomix (equal parts IL‐1β, TNF‐α, and INFγ), followed by treatment with 1.0 mg/ml CFH for 24 hours. Cell viability was determined using Draq7, a cell membrane impermeable nuclear stain. Cell death was measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, which detects DNA fragmentation from late apoptosis or necrosis. While there was a time‐ and concentration‐dependent trend, no significant differences between CFH treated and untreated cells were seen in the absence of cytomix, even at the highest concentration of 1.0 mg/ml CFH at 24 hours for the TUNEL assay (57 TUNEL+ cells per FOV in CFH‐treated vs. 17 in control, p=0.8245). However, there was a significant increase in Draq7 staining with 1.0 mg/ml CFH treatment after 24 hours (7.9% Draq7+ cells in CFH‐treated vs. 2.3% in control, p=0.0240). Exposure to cytomix augmented the decrease in viability from CFH as measured by Draq7 assay (32.4% Draq7+ cells in Cyto + CFH‐treated cells vs. 14.5% in CFH‐treated cells, p&lt;0.0001). These findings indicate that CFH alone has modest effects on lung endothelial cell viability. However, during sepsis when circulating cytokine levels are increased, the effect of CFH on endothelial cell death is enhanced, which may contribute to the microvascular leak and alveolar fluid accumulation that are characteristic of acute lung injury.Support or Funding InformationThis work is funded by NIH HL135849, HL103836, and HL094296.Figure 1

Handling of hemolyzed serum samples in clinical chemistry laboratories: the Nordic hemolysis project.

Background Some clinical chemistry measurement methods are vulnerable to interference if hemolyzed serum samples are used. The aims of this study were: (1) to obtain updated information about how hemolysis affects clinical chemistry test results on different instrument platforms used in Nordic laboratories, and (2) to obtain data on how test results from hemolyzed samples are reported in Nordic laboratories. Methods Four identical samples containing different degrees of hemolysis were prepared and distributed to 145 laboratories in the Nordic countries. The laboratories were asked to measure the concentration of cell-free hemoglobin (Hb), together with 15 clinical chemistry analytes. In addition, the laboratories completed a questionnaire about how hemolyzed samples are handled and reported. Results Automated detection of hemolysis in all routine patient samples was used by 63% of laboratories, and 88% had written procedures on how to handle hemolyzed samples. The different instrument platforms measured comparable mean Hb concentrations in the four samples. For most analytes, hemolysis caused a homogenous degree of interference regardless of the instrument platform used, except for alkaline phosphatase (ALP), bilirubin (total) and creatine kinase (CK). The recommended cut-off points for rejection of a result varied substantially between the manufacturers. The laboratories differed in how they reported test results, even when they used the same type of instrument. Conclusions Most of the analytes were homogeneously affected by hemolysis, regardless of the instrument used. There is large variation, however, between the laboratories on how they report test results from hemolyzed samples, even when they use the same type of instrument.

The Relevance of Hemolysis in Anesthesia and Intensive Care Medicine

Hemolysis leads to an increase of circulating intravascular cell-free hemoglobin. Increased plasma concentrations of cell-free hemoglobin are relevant in critically ill patients because cell-free hemoglobin causes vasoconstriction by depletion of endothelial nitric oxide, oxidative stress, and inflammation. Furthermore, cell-free hemoglobin contributes to tissue injuries such as renal failure and intestinal mucosa damage after cardiac surgery. High concentrations of cell-free hemoglobin are associated with an increased mortality in patients with sepsis. Currently, it is unclear if hemolysis associated with transfusion of packed red blood cells that have been stored for prolonged periods of time is relevant for the clinical outcome. However, humans possess plasma proteins haptoglobin and hemopexin which bind to plasma hemoglobin and cell-free heme, respectively. The haptoglobin-hemoglobin and hemopexin-heme complexes are then eliminated from the plasma by hepatic or splenic uptake.

Moderate glucose supply reduces hemolysis during systemic inflammation.

BackgroundSystemic inflammation alters energy metabolism. A sufficient glucose level, however, is most important for erythrocytes, since erythrocytes rely on glucose as sole source of energy. Damage to erythrocytes leads to hemolysis. Both disorders of glucose metabolism and hemolysis are associated with an increased risk of death. The objective of the study was to investigate the impact of intravenous glucose on hemolysis during systemic inflammation.Materials and methodsSystemic inflammation was accomplished in male Wistar rats by continuous lipopolysaccharide (LPS) infusion (1 mg LPS/kg and h, 300 min). Sham control group rats received Ringer’s solution. Glucose was supplied moderately (70 mg glucose/kg and h) or excessively (210 mg glucose/kg and h) during systemic inflammation. Vital parameters (eg, systemic blood pressure) as well as blood and plasma parameters (eg, concentrations of glucose, lactate and cell-free hemoglobin, and activity of lactate dehydrogenase) were measured hourly. Clot formation was analyzed by thromboelastometry.ResultsContinuous infusion of LPS led to a so-called post-aggression syndrome with disturbed electrolyte homeostasis (hypocalcemia, hyperkalemia, and hypernatremia), changes in hemodynamics (tachycardia and hypertension), and a catabolic metabolism (early hyperglycemia, late hypoglycemia, and lactate formation). It induced severe tissue injury (significant increases in plasma concentrations of transaminases and lactate dehydrogenase), alterations in blood coagulation (disturbed clot formation), and massive hemolysis. Both moderate and excessive glucose supply reduced LPS-induced increase in systemic blood pressure. Excessive but not moderate glucose supply increased blood glucose level and enhanced tissue injury. Glucose supply did not reduce LPS-induced alterations in coagulation, but significantly reduced hemolysis induced by LPS.ConclusionIntravenous glucose infusion can diminish LPS-related changes in hemodynamics, glucose metabolism, and, more interestingly, LPS-induced hemolysis. Since cell-free hemoglobin is known to be a predictor for patient’s survival, a reduction of hemolysis by 35% only by the addition of a small amount of glucose is another step to minimize mortality during systemic inflammation.

Practical recommendations for managing hemolyzed samples in clinical chemistry testing.

We suggest here a pragmatic approach for managing results of clinical chemistry testing in hemolyzed samples collected from adults/older children, attempting to balance the need to produce quality laboratory data with clinical urgency of releasing test results. Automatic measurement of the hemolysis index (H-index) in serum or plasma is highly advisable, whilst low-quality assessment of this test remains less good than a visual inspection. Regarding its practical use, when the H-index value does not generate an analytically significant bias, results can be released, whilst when the value is associated with analyte variation in a range between analytically and clinically significant bias (i.e. variation does not exceed the reference change value [RCV]), results of hemolysis-sensitive tests can be released in association with a comment describing the direction in which data are potentially altered, suggesting the need to collect another sample. When the H-index is associated with analyte variation exceeding clinically significant bias (i.e. variation exceeds the RCV), results of hemolysis-sensitive tests should be suppressed and replaced with a comment that biased results cannot be released because the sample is preanalytically compromised and advising the recollection of another sample. If H-index values reach an even higher critical cut-off (i.e. H-index corresponding to a cell-free hemoglobin concentration ≥10 g/L), all laboratory data may be unreliable and should hence be suppressed and replaced with a comment that all data cannot be released because the sample is grossly hemolyzed, also suggesting the recollection of another sample. Due to inaccuracy and imprecision, the use of corrective formulas for adjusting data of hemolysis-sensitive tests is discouraged.

Polluted water exacerbates Barbus callensis oocyte oxidative status

Abstract The deleterious effects of environmental pollutants on cellular components and tissues damage in fish have been studied extensively. However, there is no data about the oxidative status of fish oocytes once released into water. This study aimed to investigate the effects of polluted (Soummam River) and unpolluted (Agrioun River) fresh water on the oxidative biomarkers of Barbus callensis (=Lucibarbus callensis) (Val.) oocytes. The experimental design consisted of collecting fish oocytes from polluted and unpolluted rivers and then activating these oocytes separately in water collected from each site. Four groups were considered: oocytes from the Agrioun River activated in Agrioun fresh water (A-oocytes/A-fresh water); oocytes from the Agrioun River activated in Soummam fresh water (A-oocytes/S-fresh water); oocytes from the Soummam River activated in Agrioun fresh water (S-oocytes/A-fresh water); and oocytes from the Soummam River activated in Soummam fresh water (S-oocytes/S-fresh water). Oxidative stress biomarkers were evaluated by measuring total antioxidant status (TAS), catalase (CAT) activity, and cell-free hemoglobin (Hb) concentrations. The results showed that the oxidative status of fish oocytes was significantly affected by the quality of fresh water. Unpolluted fresh water improved the antioxidant activity of the fish oocytes. The results of this study suggest that once oocytes are released into polluted water, antioxidant protection is affected with subsequent cellular oxidative damage and potential reproduction impairment.

B-type natriuretic peptide and plasma hemoglobin levels following transfusion of shorter-storage versus longer-storage red blood cells: Results from the TOTAL randomized trial

Prior studies have suggested that transfusion of stored red blood cells (RBCs) with increased levels of cell-free hemoglobin might reduce the bioavailability of recipient nitric oxide (NO) and cause myocardial strain. Ugandan children (ages 6-60 months) with severe anemia and lactic acidosis were randomly assigned to receive RBCs stored 1-10 days versus 25-35 days. B-type natriuretic peptide (BNP), vital signs, renal function test results, and plasma hemoglobin were measured. Most children had either malaria or sickle cell disease and were thus at risk for reduced NO bioavailability. Seventy patients received RBCs stored 1-10 days, and 77 received RBCs stored 25-35 days. The median (interquartile range) cell-free hemoglobin was nearly 3 times higher in longer-storage RBCs (26.4 [15.5-43.4] μmol/L) than in shorter-storage RBCs (10.8 [7.8-18.6] μmol/L), P < .0001. Median (interquartile range) BNP 2 hours posttransfusion was 156 (59-650) pg/mL (shorter storage) versus 158 (59-425) pg/mL (longer storage), P = .76. BNP values 22 hours posttransfusion were 110 (46-337) pg/mL (shorter storage) versus 96 (49-310) pg/mL (longer storage), P = .76. Changes in BNP within individuals from pretransfusion to 2 hours (or 22 hours) posttransfusion were not significantly different between the study groups. BNP change following transfusion did not correlate with the concentration of cell-free hemoglobin in the RBC supernatant. Blood pressure, blood urea nitrogen, creatinine, and change in plasma hemoglobin were not significantly different in the 2 groups. In a randomized trial among children at risk for reduced NO bioavailability, we found that BNP, blood pressure, creatinine, and plasma hemoglobin were not higher in patients receiving RBCs stored for 25-35 versus 1-10 days.

Transfusion of Old RBCs Induces Neuroinflammation and Cognitive Impairment.

Transfusing RBCs stored for longer than 14 days (old RBC) in humans is common. This transfusion can injure organs, such as lungs and kidneys. We determined whether transfusion with old RBC injured brain. Prospective, controlled animal study. University research laboratory. Adult male Sprague-Dawley rats. Six-month-old Sprague-Dawley rats lost 20% total blood volume and then received RBC prepared from equal volume of the lost blood. RBC was stored for 1 day (fresh RBC) or 7 days (old RBC, storage lesions similar to those of human RBC stored for 28 d). Some rats received IV cell-free hemoglobin. These rats were not subjected to hemorrhage and RBC transfusion. Rats were subjected to Barnes maze and fear conditioning tests from 1 week after blood transfusion. Rats transfused with old RBC but not fresh RBC took a longer time to identify the target hole in the Barnes maze and had less context-related fear conditioning behavior than control rats. Old RBC significantly increased interleukin 6 and ionized calcium-binding adapter molecule 1 in the hippocampus at 24 hours after the transfusion. These effects were attenuated by sulforaphane and minocycline, an antibiotic with anti-inflammatory property. Old RBC solution had a higher concentration of cell-free hemoglobin. Sulforaphane increased haptoglobin, a chelator of cell-free hemoglobin. Rats that received cell-free hemoglobin had a pattern of neuroinflammation and impairment of learning and memory similar to that of rats that received old RBC. These results provide initial evidence to suggest that transfusion of old RBC induces neuroinflammation and impairment of learning and memory. These effects may be mediated by cell-free hemoglobin.

Genetic variants and cell-free hemoglobin processing in sickle cell nephropathy

Intravascular hemolysis and hemoglobinuria are associated with sickle cell nephropathy. ApoL1 is involved in cell-free hemoglobin scavenging through association with haptoglobin-related protein. APOL1 G1/G2 variants are the strongest genetic predictors of kidney disease in the general African-American population. A single report associated APOL1 G1/G2 with sickle cell nephropathy. In 221 patients with sickle cell disease at the University of Illinois at Chicago, we replicated the finding of an association of APOL1 G1/G2 with proteinuria, specifically with urine albumin concentration (β=1.1, P=0.003), observed an even stronger association with hemoglobinuria (OR=2.5, P=4.3×10(-6)), and also replicated the finding of an association with hemoglobinuria in 487 patients from the Walk-Treatment of Pulmonary Hypertension and Sickle cell Disease with Sildenafil Therapy study (OR=2.6, P=0.003). In 25 University of Illinois sickle cell disease patients, concentrations of urine kidney injury molecule-1 correlated with urine cell-free hemoglobin concentrations (r=0.59, P=0.002). Exposing human proximal tubular cells to increasing cell-free hemoglobin led to increasing concentrations of supernatant kidney injury molecule-1 (P=0.01), reduced viability (P=0.01) and induction of HMOX1 and SOD2. HMOX1 rs743811 associated with chronic kidney disease stage (OR=3.0, P=0.0001) in the University of Illinois cohort and end-stage renal disease (OR=10.0, P=0.0003) in the Walk-Treatment of Pulmonary Hypertension and Sickle cell Disease with Sildenafil Therapy cohort. Longer HMOX1 GT-tandem repeats (>25) were associated with lower estimated glomerular filtration rate in the University of Illinois cohort (P=0.01). Our findings point to an association of APOL1 G1/G2 with kidney disease in sickle cell disease, possibly through increased risk of hemoglobinuria, and associations of HMOX1 variants with kidney disease, possibly through reduced protection of the kidney from hemoglobin-mediated toxicity.

Cell-Free Hemoglobin, HMOX1 and APOL1 in Sickle Cell Nephropathy

We recently reported that hemoglobinuria is associated with chronic kidney disease (CKD) stage and progression in sickle cell disease (SCD) (Saraf et al. BJH 2014). To further investigate a potential role of cell-free hemoglobin in SCD nephropathy, we measured urinary concentrations of kidney injury molecule-1 (KIM-1), a biomarker of tubular injury, and nephrin, a marker of glomerular injury, in 32 University of Illinois at Chicago (UIC) patients. Urine KIM-1 concentration directly correlated with increasing urine cell-free hemoglobin concentration (P = 0.003) (Figure 1) but urine nephrin concentration did not. To determine biological responses of tubular cells to cell-free hemoglobin, we added lyophilized hemoglobin to cultured human kidney-2 (HK2) tubular cells. Supernatant KIM-1 concentrations increased progressively with increasing cell-free hemoglobin exposure (Figure 2) while total cell number and cell viability were stable. Using a fluorescein-labeled hemoglobin assay, we found that cell-free hemoglobin bound to HK2 cells in a competitive manner. To determine whether enzymes for metabolizing hemoglobin and protecting from reactive oxygen species are affected by exposure of HK2 cells to cell-free hemoglobin, we evaluated candidate gene expression using rt-PCR. The relative expression of heme oxygenase-1 (HMOX1) (Figure 3) progressively increased with increasing cell-free hemoglobin dose while expression of superoxide dismutase 1 and 2, catalase, glutathione reductase, and glutathione synthetase did not change.We then focused on variants of HMOX1 in a cohort of 247 UIC SCD patients. Genotyping was carried out using Affymetrix Axiom genome-wide Pan-African array in DNA isolated from peripheral blood mononuclear cells. Examination of 11 tag SNPs within +/-10 kb of HMOX1 identified a SNP in the promoter region of HMOX1 (rs743811, minor allele frequency 0.14) that had a significant association with CKD stage (β = -1.0, P = 0.00032) and non-significant associations of the same direction with end stage renal disease (ESRD) (β = -0.9, P = 0.2) and hemoglobinuria (β = -0.2, P = 0.6). Validation studies were conducted in 517 SCD patients from the Walk-Treatment of Pulmonary Hypertension and Sickle Cell Disease with Sildenafil Therapy (Walk-PHaSST) cohort. Genotyping was performed using the Illumina Human 610-Quad SNP array and imputed using the HapMap II reference panels for HMOX1 tag SNPs. HMOX1 rs743811 had a significant associated with ESRD (β = -1.6, P = 0.014) and non-significant associations of a similar direction with CKD stage (β = -0.2, P = 0.3) and hemoglobinuria (β = -0.3, P = 0.3).Homozygosity or compound heterozygosity for the G1 and G2 variants of APOL1, encoding apolipoprotein 1, have been implicated in CKD in African Americans with and without SCD (Ashley-Koch et al. BJH 2011), and we therefore examined their association with hemoglobinuria-associated CKD in the UIC cohort. The S342G and I384M substitutions are in almost complete linkage disequilibrium and are termed G1; the deletion of two amino acids, N388 and Y389, is termed G2. The G2 variant was further imputed using reference panels of the 1000 Genomes Project. Homozygosity or compound heterozygosity of the G1/G2 variant was associated with hemoglobinuria (β = 2.0, P = 0.00018), CKD stage (β = 1.0, P = 0.022), and ESRD (β = 1.9, P = 0.036). For validation studies in Walk-PHaSST, the G1 and G2 variants of APOL1 were imputed using the 1000 Genomes Project. Homozygosity or compound heterozygosity of G1/G2 in the Walk-PHaSST cohort was associated with hemoglobinuria (β = 0.7, P = 0.021), but associations of a similar direction with CKD stage (β = 0.2, P = 0.6) and ESRD (β = 1.1, P = 0.3) were not statistically significant.Our findings are consistent with the possibility that cell-free hemoglobin contributes to sickle cell nephropathy through tubular injury. A SNP in the promoter region of HMOX1 is associated with CKD stage in the UIC cohort and ESRD in the Walk-PHaSST cohort, raising the possibility that altered HMOX1 expression can have a role in SCD-associated CKD. Our results also point to a novel association of the G1/G2 variants of APOL1 with cell-free hemoglobin-mediated CKD in SCD subjects. Future studies to explore the potential roles of HMOX1 and APOL1 in cell-free hemoglobin-associated sickle cell nephropathy are warranted. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Reference range of hemolysis index in serum and lithium-heparin plasma measured with two analytical platforms in a population of unselected outpatients

BackgroundThe hemolysis index (HI) is now available in several laboratory analyzers, but doubts remain about the thresholds for suppressing test results, the degree of standardization among different instrumentations and the use of different reference ranges in different biological matrices. This study was hence planned to establish the reference ranges of HI in serum and lithium-heparin plasma in a population of unselected outpatients, using two analytical platforms. Materials and methodsWe analyzed the HI in serum and lithium-heparin samples collected from 135 unselected outpatients, and we also defined the relative reference ranges according to Clinical and Laboratory Standards Institute (CLSI) recommendations. Samples were collected in the morning by expert nurses, using straight needle venipuncture. The HI in serum and lithium-heparin plasma was assessed with Roche Cobas c501 and Siemens Dimension Vista 1500. ResultsThe median concentration of cell-free hemoglobin was significantly higher in serum than in lithium-heparin plasma when measured with Cobas c501, but not with Dimension Vista 1500. After categorizing values according to cell-free hemoglobin thresholds, the agreement between instruments was 0.75 (p<0.01) for serum and 0.95 (p<0.01) for lithium-heparin plasma. The upper limits calculated according to CLSI document C28-A3 were 0.22g/L for Roche Cobas c501 and 0.25g/L for Siemens Dimension Vista 1500 in serum, whereas they were 0.13g/L for Cobas c501 and 0.10g/L for Dimension Vista 1500 in lithium-heparin plasma. ConclusionsAccording to our data, different thresholds of cell-free hemoglobin should be used between serum and lithium-heparin plasma for monitoring phlebotomy practice.