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This review presents modern domestic and foreign published data on the effect of the SARS-CoV-2 virus on red blood cells in COVID-19 and on the effect of the resulting erythrocyte degradation products on the body. The mechanisms of detoxification of these products and the possibility of considering them as biomarkers of this severe disease, as well as therapeutic targets for combating it, are considered in detail. [ABSTRACT FROM AUTHOR]

Neuron-specific enolase (NSE) is one of the biomarkers used as an indicator of brain disorder, but since it is also found in blood cell components, there is a concern that a spurious increase in NSE may occur after cardiovascular surgery, where cardiopulmonary bypass (CPB) causes hemolysis. In the present study, we investigated the relationship between the degree of hemolysis and NSE after cardiovascular surgery and the usefulness of immediate postoperative NSE values in the diagnosis of brain disorder. A retrospective study of 198 patients who underwent surgery with CPB in the period from May 2019 to May 2021 was conducted. Postoperative NSE levels and Free hemoglobin (F-Hb) levels were compared in both groups. In addition, to verify the relationship between hemolysis and NSE, we examined the correlation between F-Hb levels and NSE levels. We also examined whether different surgical procedures could produce an association between hemolysis and NSE. Among 198 patients, 20 had postoperative stroke (Group S) and 178 had no postoperative stroke (Group U). There was no significant difference in postoperative NSE levels and F-Hb levels between Group S and Group U (p = 0.264, p = 0.064 respectively). F-Hb and NSE were weakly correlated (r = 0.29. p < 0.01). In conclusion, NSE level immediately after cardiac surgery with CPB is modified by hemolysis rather than brain injury, therefore it would be unreliable as a biomarker of brain disorder. [ABSTRACT FROM AUTHOR]

Gemtuzumab ozogamicin (GO) is a CD33 monoclonal antibody‐drug conjugate currently in use to treat myeloid malignancies. A unique adverse effect of this medication is destruction of CD33 positive macrophages resulting in reduced clearance of free hemoglobin leading to grossly red plasma. This build‐up of free hemoglobin can potentially lead to end organ damage and prevent performance of clinically necessary laboratory evaluation. We present a case of a pediatric patient who developed this adverse effect and was successfully treated with therapeutic plasma exchange (TPE). We also present results from a systematic review of the medical literature and share data from a query of the United States Food and Drug Administration (FDA) Adverse Event Reporting system for GO‐related hemoglobin scavenging impairment. Among reported cases, patients undergoing TPE and those receiving steroids had improved outcomes. Practitioners should be aware of this rare drug side‐effect and the potential utility of TPE for these patients. [ABSTRACT FROM AUTHOR]

We present a case of mechanical hemolysis as a complication of extracorporeal membrane oxygenation (ECMO) occurring in a COVID-19 patient as a result of pump head thrombosis. After emergency extracorporeal circuit replacement, hemoadsorption was initiated to address the negative hemolysis effects and plasma free hemoglobin rise in the setting of rapid clinical deterioration and impaired renal function. During therapy hemolysis severity reduced, the lactate dehydrogenase (LDH) levels decreased, while the P/F ratio increased two-fold. The patient was discharged from hospital on day 54 without the need for either oxygen therapy or dialysis. In the discussion section we addressed frequent issues of choosing therapy for ECMO complications.Conclusion. The timely, properly chosen, and clinically relevant use of hemoadsorption combined with advanced high-technology therapeutic procedures can have a positive impact on the patient's outcome.

Abstract Blood transfusions are mainly given to intensive care patients; therefore, additional complications that could arise from storage lesions in preserved blood should be avoided. It has been shown that human stored red blood cells are subject to changes that are considered to be a number of interdependent processes involving metabolic disarrangement and oxidative stress. The aim of our study was to determine alterations in selected hematological and biochemical parameters and to assess whether and when oxidative stress is a significant phenomenon in stored dog CPDA-1 whole blood. Ten ½ unit bags of whole blood donated from dogs and preserved with CPDA-1 (anticoagulant containing citrate, phosphate, dextrose and adenine) were stored for 5 weeks. Each week, a 9 ml sample was drawn aseptically to measure hematological parameters, selected metabolites, free hemoglobin content, osmotic fragility, antioxidant enzyme activity, total antioxidant capacity, malondialdehyde concentration and protein carbonyl content. The results revealed an MCV decrease in the first week of storage and then a gradual increase; osmotic fragility decreased at that time and remained low throughout the study period. Leukodepletion became significant in the fourth week of storage. The free hemoglobin concentration continuously increased, with the greatest changes observed in the last two weeks of storage. The total antioxidant capacity changed in a reverse manner. Superoxide dismutase and glutathione peroxidase activities decreased from week 0 to week 3, and catalase activity tended to decrease over time. The highest malondialdehyde concentrations in blood supernatant were measured in the first week of storage, and the carbonyl concentration increased after 35 days. Hematological changes and oxidative stress are already present in the first week of storage, resulting in depletion of the antioxidant system and subsequent accumulation of oxidation products as well as erythrocyte hemolysis, which are most pronounced at the end of the storage period.

Background: Ex situliver machine perfusion at subnormothermic/normothermic temperature isincreasingly applied in the field of transplantation to store and evaluateorgans on the machine prior transplantation. Currently, various perfusionconcepts are in clinical and preclinical applications. Over the last 6 years ina multidisciplinary team, a novel blood based perfusion technology wasdeveloped to keep a liver alive and metabolically active outside of the bodyfor at least one week. Methods: Within thismanuscript, we present and compare three scenarios (Group 1, 2 and 3) we werefacing during our research and development (R&D) process, mainly linked tothe measurement of free hemoglobin and lactate in the blood based perfusate. Apartfrom their proven value in liver viability assessment (ex situ), these twoparameters are also helpful in R&D of a long‐term liver perfusion machine and moreover supportive in the biomedical engineering process. Results: Group 1 ("good" liver on the perfusion machine) represents the best liver clearance capacity for lactate and free hemoglobin wehave observed. In contrast to Group 2 ("poor" liver on the perfusion machine), that has shown the worst clearance capacity for free hemoglobin. Astonishingly,also for Group 2, lactate is cleared till the first day of perfusion andafterwards, rising lactate values are detected due to the poor quality of theliver. These two perfusate parametersclearly highlight the impact of the organ quality/viability on the perfusion process. Whereas Group 3 is a perfusion utilizing a blood loop only (without a liver). Conclusion: Knowing the feasible ranges (upper‐ and lower bound) and the courseover time of free hemoglobin and lactate is helpful to evaluate the quality ofthe organ perfusion itself and the maturity of the developed perfusion device. Freehemoglobin in the perfusate is linked to the rate of hemolysis that indicates how optimizing (gentle blood handling, minimizing hemolysis) the perfusion machine actually is. Generally, a reduced lactate clearancecapacity can be an indication for technical problems linked to the blood supplyof the liver and therefore helps to monitor the perfusion experiments.Moreover, the possibility is given to compare, evaluate and optimize developed liverperfusion systems based on the given ranges for these two parameters. Otherresearch groups can compare/quantify their perfusate (blood) parameters withthe ones in this manuscript. The presented data, findings and recommendations willfinally support other researchers in developing their own perfusion machine ormodifying commercially availableperfusion devices according to their needs. [ABSTRACT FROM AUTHOR]

Artificial cardiovascular devices, such as vascular stents, artificial valves, and artificial hearts, can rebuild human cardiovascular functionalities via rebuilding the blood flow passing through these devices. To evaluate the red blood cells (RBCs) damage induced by a non-physiological blood flow in these devices, many hemolysis models have been proposed, of which the most popular one is a power function model. However, it was found that the newly obtained experimental data often did not match the existing power function model. In addition, the experimental period was usually short and the summarized power function model cannot reflect the RBCs damage after long-term exposure to shear stress. To address this issue, in this study a shear device was established on a torque rheometer; the changes of plasma free hemoglobin (FHB) of sheep blood under the shear stress from 10 to 70 Pa and exposure time from 5 to 30 min were recorded and compared. The results showed that as the shear stress and exposure time increased, FHB also increased, but the increase rate gradually decreased. As a result, after undergoing high shear stress or a long period of exposure time, FHB eventually became stable. Obviously, the existing power function model cannot describe this FHB change. In the current study, we used a sigmoidal logistic function model to describe the FHB increment upon the increase of shear stress and long exposure time. The results showed that the proposed model can provide better predictions of hemolysis, particularly in these cases under long exposure time. [ABSTRACT FROM AUTHOR]

The measurement of free hemoglobin (free Hb) in blood is crucial for assessing the risk of organ damage in patients with hemolytic diseases. However, the colorimetric method, commonly used in clinical practice, does not distinguish between free Hb and the hemoglobin-haptoglobin complex (Hb-Hp) in the blood, instead reflecting the total Hb level. Although size-exclusion high-performance liquid chromatography (SEC-HPLC) can specifically measure free Hb, its clinical use is limited by long assay times. Here, we developed a novel assay method for the rapid quantification of free Hb in serum, distinguishing it from Hb-Hp, using a latex agglutination immunoturbidimetric assay (LATIA). This method could be used to measure free Hb in sera in the range of 1–100 μg/mL in approximately 15 min using an automatic biochemistry analyzer. Using Hb-spiked serum samples from healthy adults, there was a high correlation with Hb levels determined using the newly developed method and SEC-HPLC, indicating a high specificity for free Hb. This novel assay can be used to monitor levels of free Hb in patients with various hemolytic diseases and to design therapeutic strategies based on measured values. However, further studies are required to assess its clinical performance. [Display omitted] • A latex agglutination immunoturbidimetric assay was developed for free Hb measurement. • The method distinguished free Hb from the hemoglobin-haptoglobin complex. • Hb levels determined using the LATIA method and SEC-HPLC were highly correlated. [ABSTRACT FROM AUTHOR]

Changes in classification criteria and active introduction of biomarkers of acute kidney injury (KDIGO, 2012) are changing approaches to diagnosis and treatment of postoperative renal dysfunction including cardiac surgery patients operated with cardiopulmonary bypass (CPB). The objective: to compare the detection rate of AKI after surgery with CPB with the use of biomarkers and kidney disease improving global outcomes criteria, as well as to evaluate the cause and localization of structural changes of the nephron.Subjects and Methods. A monocenter observational study among elective cardiac surgery patients (n = 97) was conducted. Inclusion criteria: age over 18 years, duration of surgery (coronary bypass surgery, prosthetic heart valves) from 90 to 180 minutes, no signs of end stage kidney disease. AKI was diagnosed based on changes in serum creatinine and biomarkers (NGAL, IgG, albumin in urine). The studied parameters were recorded 15 minutes after the start and end of anesthesia, as well as 24 and 48 hours after surgery. Retrospectively, the group was divided into three subgroups: 1) patients without AKI after surgery; 2) patients in whom signs of AKI were detected after 24 hours but regressed by the 48th hour; 3) patients in whom AKI persisted during all 48 hours of follow-up.Results. 24 hours after surgery, AKI based on KDIGO criteria was recorded in 56.3% of patients. Using biomarkers, signs of tubular damage (NGAL) at the end of anesthesia were detected in 95.9% of patients; after 24 hours, they were registered in 73.2% of cases. In a subgroup where AKI persisted for more than 24 hours, glomeruli were damaged in addition to tubules which was manifested not only by selective but also by non-selective proteinuria. The duration of CPB, hemodilution (Hb < 90 g/l), the release of free hemoglobin in the blood (> 1.5 mg/l) at low (< 1 g/l) values of haptoglobin were significantly associated with AKI development.Conclusion. The KDIGO criteria do not allow detecting a subclinical form of renal dysfunction which may occur in about 40% of patients after surgery with CPB. AKI can be caused by damage to both the tubular part of the nephron and glomeruli in cases of prolonged CPB with the development of hemolysis, the release of free hemoglobin in the blood, and persisting anemia at the end of the surgery. The NGAL assessment makes it possible to detect subclinical kidney injury in the absence of elevated serum creatinine levels.

Cell-free hemoglobin (CFH), a pro-oxidant and cytotoxic compound that is released in hemolysis, has been associated with nephrotoxicity. Lung transplantation (LuTx) is a clinical condition with a high incidence of acute kidney injury (AKI). In this study, we investigated the plasma levels of CFH and haptoglobin, a CFH-binding serum protein, in prospectively enrolled LuTx patients (n = 20) with and without AKI. LuTx patients with postoperative AKI had higher CFH plasma levels at the end of surgery compared with no-AKI patients, and CFH correlated with serum creatinine at 48 h. Moreover, CFH levels inversely correlated with haptoglobin levels, which were significantly reduced at the end of surgery in LuTx patients with AKI. Because multiple other factors can contribute to AKI development in the complex clinical setting of LuTx, we next investigated the role of exogenous CFH administration in a mouse model of mild bilateral renal ischemia reperfusion injury (IRI). Exogenous administration of CFH after reperfusion caused overt AKI with creatinine increase, tubular injury, and enhanced markers of renal inflammation compared with vehicle-treated animals. In conclusion, CFH is a possible factor contributing to postoperative AKI after LuTx and promotes AKI in an experimental model of mild transient renal ischemia. Targeting CFH might be a therapeutic option to prevent AKI after LuTx. [ABSTRACT FROM AUTHOR]

Blood transfusions are mainly given to intensive care patients; therefore, additional complications that could arise from storage lesions in preserved blood should be avoided. It has been shown that human stored red blood cells are subject to changes that are considered to be a number of interdependent processes involving metabolic disarrangement and oxidative stress. The aim of our study was to determine alterations in selected hematological and biochemical parameters and to assess whether and when oxidative stress is a significant phenomenon in stored dog CPDA-1 whole blood. Ten ½ unit bags of whole blood donated from dogs and preserved with CPDA-1 (anticoagulant containing citrate, phosphate, dextrose and adenine) were stored for 5 weeks. Each week, a 9 ml sample was drawn aseptically to measure hematological parameters, selected metabolites, free hemoglobin content, osmotic fragility, antioxidant enzyme activity, total antioxidant capacity, malondialdehyde concentration and protein carbonyl content. The results revealed an MCV decrease in the first week of storage and then a gradual increase; osmotic fragility decreased at that time and remained low throughout the study period. Leukodepletion became significant in the fourth week of storage. The free hemoglobin concentration continuously increased, with the greatest changes observed in the last two weeks of storage. The total antioxidant capacity changed in a reverse manner. Superoxide dismutase and glutathione peroxidase activities decreased from week 0 to week 3, and catalase activity tended to decrease over time. The highest malondialdehyde concentrations in blood supernatant were measured in the first week of storage, and the carbonyl concentration increased after 35 days. Hematological changes and oxidative stress are already present in the first week of storage, resulting in depletion of the antioxidant system and subsequent accumulation of oxidation products as well as erythrocyte hemolysis, which are most pronounced at the end of the storage period. [ABSTRACT FROM AUTHOR]

Removing free hemoglobin generated during extracorporeal circulation remains a challenge. Currently, there is no adsorbent with specificity and good biosafety for removing hemoglobin. In this study, a new chitosan/sodium alginate/carbon nitride (CS/SA/C 3 N 4) hydrogel adsorbent was prepared by blending SA with C 3 N 4 to drop into CS/CaCl 2 solution. The physicochemical properties of CS/SA/C 3 N 4 hydrogel were evaluated using some techniques, including scanning electron microscope, Zeta potential measurement, and thermogravimetric analysis. Hemoglobin adsorption in vitro, stability, hemocompatibility, cell compatibility, inflammatory reaction and blood extracorporeal circulation in vivo were also evaluated. The findings revealed that the CS/SA/C 3 N 4 -0.4 % hydrogel exhibited an impressive adsorption capacity of 142.35 mg/g for hemoglobin. The kinetic data of hemoglobin adsorption were well-described by pseudo second-order model, while the isothermal model data conformed to the Langmuir model. The hardness and modulus of CS/SA/C 3 N 4 -0.4 % was 11.7 KPa and 94.66 KPa respectively, which indicated robust resistance to breakage. CS/SA/C 3 N 4 demonstrated excellent hemocompatibility, biocompatibility and anti-inflammatory properties. In addition, the results of in vivo rabbit extracorporeal blood circulation experiment demonstrated that CS/SA/C 3 N 4 could adsorb free hemoglobin from blood while maintaining high biosafety standard. Consequently, CS/SA/C 3 N 4 hydrogel emerges as a promising candidate for use as a hemoglobin adsorbent in extracorporeal blood circulation system. [ABSTRACT FROM AUTHOR]

Introduction Maintaining blood supply is a challenge in blood banks. Red blood cells (RBCs) stored at 4°C experience issues of biochemical changes due to metabolism of cells, leading to changes collectively referred to as “storage lesions.” Oxidation of the red cell membrane, leading to lysis, contributes to these storage lesions. Methods Blood bags with CPD-SAGM stored at 4°C for 28 days were withdrawn aseptically on days 1, 14, and 28. Hematology analyzer was used to investigate RBC indices. Hemoglobin oxidation was studied through spectrophotometric scan of spectral change. RBC lysis was studied with the help of Drabkin's assay, and morphological changes were observed by light and scan electron microscopy. Results RBCs show progressive changes in morphology echinocytes and spherocytes on day 28. There was 0.85% RBC lysis, an approximately 20% decrease in percentage oxyhemoglobin, and a 14% increase in methemoglobin formation, which shows hemoglobin oxidation on day 28. Conclusions Oxidative damage to RBC, with an increase in storage time was observed in the present study. The observed morphological changes to RBC during the course of increased time shows that there is progressive damage to RBC membrane and a decrease in hemoglobin concentration; percentage RBC lysis is probably due to free hemoglobin and iron.

Purpose: Unraveling the pathophysiology of COVID-19 disease is of crucial importance for designing treatment. The purpose of this study is to investigate the effects of the disease on erythrocytes (RBCs) and to correlate the findings with disease severity. Materials and Methods: Hospitalized patients (n = 36) with COVID-19 and control group of healthy volunteers (n = 18) were included in the study. Demographic data, clinical, laboratory and chest Computed Tomography (CT) findings at time of admission were recorded. Laboratory measurements included: Hemoglobin (H b), indirect billirubin, LDH, D-Dimers, and plasma free hemoglobin (plasma free-Hb). On RBCs were performed: osmotic fragility (MCF), Free-Hb after mechanical stress (Free-Hb-MECH), intracellular RBC concentration of calcium ions (iCa2+), intracellular ROS (iROS), G6PD, intracellular active caspase-3 (RBC-caspase-3), IgG immunoglobulins (RBC-IgGs), which are bound on RBCs' senescent neo-antigen proteins and RBC surface phosphatidylserine (RBC-PS). Results: The percentage of males was 50 and 66% and the mean age was 65.16 ± 14.24 and 66.33 ± 13.48 years among patients and controls respectively (mean ± SD, p = 0.78). Upon admission patients' PO2/FiO2 ratio was 305.92 ± 76.75 and distribution of infiltration extend on chest CT was: 0–25% (N = 19), 25–50%: (N = 7), and 50–75% (N = 9). Elevated hemolysis markers (LDH and plasma free-Hb) were observed in patients compared to the control group. Patients' RBCs were more sensitive to mechanical stress, and exhibited significantly elevated apoptotic markers (iCa2+, RBC-PS). Plasma free Hb levels correlated with the extend of pulmonary infiltrates on chest CT in COVID-19 patients. Surprisingly, patients' RBC-iROS were decreased, a finding possibly related with the increased G6PDH levels in this group, suggesting a possible compensatory mechanism against the virus. This compensatory mechanism seemed to be attenuated as pulmonary infiltrates on chest CT deteriorated. Furthermore, RBC-IgGs correlated with the severity of pulmonary CT imaging features as well as the abnormality of lung function, which are both associated with increased disease severity. Lastly, patients' D-Dimers correlated with RBC surface phosphatidylserine, implying a possible contribution of the red blood cells in the thrombotic diathesis associated with the SARS-CoV-2 disease. Conclusion: This pilot study suggests that SARS-CoV-2 infection has an effect on red blood cells and there seems to be an association between RBC markers and disease severity in these patients. SARS-CoV-2 infection has significant effects on red blood cells that seems to be associated with disease severity in these patients. (A) Non-Intensive care unit hospitalized patients due to severe COVID-19 disease were included in this study (n = 36). (B) The control group included healthy subjects without renal or hepatic impairment, no diagnosis of anemia or myeloid malignancy, and no blood transfusion over the past 3 months (n = 18). (C) Demographic data, clinical, laboratory and chest Computed Tomography (CT) findings at time of admission were recorded. Hematological tests, standard biochemical analysis of serum components were performed both in the patient and in the control group and coagulation analysis were evaluated in the patient group. A peripheral blood smear examination was also performed in all patients and control samples. (D) Plasma free hemoglobin and red blood cell (RBC) osmotic fragility and free hemoglobin (Free-Hb) after mechanical stress (MECH) by mechanical hemolysis were assesed in both patients and controls. Intracellular (i) calcium (iCa2 +) levels, RBCs phosphatidylserine exposure (PS), RBC membrane-bound immunoglobulins G (IgGs), and activate RBC caspase-3 levels as cell clearance and apoptotic markers were measured by flow cytometry. A spectrophotometric assay was used to quantify the activity of G6PD enzyme. Intracellular RBC reactive oxidative species (iROS) were assesed in both patients and controls. (E) Elevated hemolysis markers (Free-Hb) and Lactate Dehydrogenase (LDH) were observed in patients compared to the control group. Patients' RBCs were more vulnerable to mechanical stress, and exhibited significantly elevated apoptotic markers (iCa2 + and RBC-PS+). Erythrocyte iROS levels were significantly lower in the patients' group compared to healthy controls. This finding possibly correlates with the increased G6PDH activity levels in this group, suggesting a compensatory mechanism against the virus. (F) Patients' D-Dimers correlated with RBC-PS +, implying a possible contribution of the erythrocytes in the thrombotic diathesis associated with the SARS-CoV-2 disease. (G) Free-Hb levels correlated with the extend of pulmonary infiltrates on chest CT in COVID-19 patients. RBC-IgGs+ correlated with the severity of pulmonary CT imaging features as well as the abnormality of lung function, expressed by the PO2/FiO 2 ratio. This finding suggests that hypoxia may lead to RBC senescence. [ABSTRACT FROM AUTHOR]

• Hemolysis due to CPB leads to an increase of serum free Hemoglobin(fHb) level. • Increase of serum fHb level had an independent association with postoperative AKI. • Hemolysis due to CPB leads to a decrease of serum Haptoglobin(Hp) level. • Decrease of serum Hp level had an independent association with postoperative AKI. • It might be possible to prevent postoperative AKI by attenuating the increase of fHb. To observe the perioperative free hemoglobin and haptoglobin levels and to assess their associations with the risk of postoperative acute kidney injury (pAKI) in adult patients undergoing valvular and aortic surgery requiring cardiopulmonary bypass (CPB). A single-center, prospective, observational study. Public teaching hospital. The study comprised 74 adult patients without chronic renal failure who underwent cardiovascular surgery requiring CPB from 2014 to 2020. Perioperative free hemoglobin and haptoglobin levels during the study period were obtained from study participants. The primary outcome was pAKI defined by the Kidney Disease: Improving Global Outcomes criteria. Of the 74 patients in this study, pAKI occurred in 25 patients (33.8%). The free hemoglobin level began to increase after the initiation of CPB and reached a peak level at 30 minutes after weaning from CPB. It returned to the baseline level on postoperative day one. Haptoglobin levels were the highest after anesthesia induction and decreased continuously until postoperative day one. In the multivariate analysis, maximum free hemoglobin and minimum haptoglobin were associated independently with increased risk of pAKI (adjusted odds ratio 1.33 [95% confidence interval 1.12-1.58; p = 0.001] and 0.95 [95% confidence interval 0.91-1.00; p = 0.03], respectively). The free hemoglobin level began to have an independent association with pAKI at one hour after commencement of CPB, and the independent association disappeared at postoperative day one. This study found that the perioperative increase of the free hemoglobin level and the decrease of the haptoglobin level had independent associations with the risk of pAKI. [ABSTRACT FROM AUTHOR]

Cell-free hemoglobin (CFH), a pro-oxidant and cytotoxic compound that is released in hemolysis, has been associated with nephrotoxicity. Lung transplantation (LuTx) is a clinical condition with a high incidence of acute kidney injury (AKI). In this study, we investigated the plasma levels of CFH and haptoglobin, a CFH-binding serum protein, in prospectively enrolled LuTx patients (n = 20) with and without AKI. LuTx patients with postoperative AKI had higher CFH plasma levels at the end of surgery compared with no-AKI patients, and CFH correlated with serum creatinine at 48 h. Moreover, CFH levels inversely correlated with haptoglobin levels, which were significantly reduced at the end of surgery in LuTx patients with AKI. Because multiple other factors can contribute to AKI development in the complex clinical setting of LuTx, we next investigated the role of exogenous CFH administration in a mouse model of mild bilateral renal ischemia reperfusion injury (IRI). Exogenous administration of CFH after reperfusion caused overt AKI with creatinine increase, tubular injury, and enhanced markers of renal inflammation compared with vehicle-treated animals. In conclusion, CFH is a possible factor contributing to postoperative AKI after LuTx and promotes AKI in an experimental model of mild transient renal ischemia. Targeting CFH might be a therapeutic option to prevent AKI after LuTx.

Methods based on paper-based analytical devices (PAD) and smartphone photographic colorimetric detection have become representative instrument-independent point-of-care testing (POCT) platforms due to their low cost and simplicity. However, the detection of target components from whole blood sample still presents challenges in terms of field preparation of small amounts of blood sample and detection sensitivity. This paper presents a rapid online processing method for whole blood samples on PAD based on plasma separation membrane (PSM), and combined with electrokinetic stacking and selective chromatic reaction. Real-time smartphone-based colorimetric detection of free hemoglobin (FHb) and human serum albumin (HSA) was successfully demonstrated. With the proposed method, both detections for low and high concentration analytes could be implemented. The limits of detection of 16.6 mg L-1 for FHb and 0.67 g L-1 for HSA were obtained, respectively, with RSD below 8 %. The reliability of the method was verified by the recovery test and desktop spectrophotometric method. The detection results for real blood samples were in agreement with that by clinical methods. The PAD method is inexpensive, simple and fast, and detection of a whole blood sample of 5 μL can be finished in 5 min. This work shows that POCT of biomarkers from whole blood with PAD is possible without using any desktop facilities. [Display omitted] • Disposable paper chip integrated with membrane and electrodes was used for whole blood sample. • Online plasma preparation was achieved in 1 min with only 5 μL whole blood sample without using any desktop facilities. • Real time colorimetric detection by smartphone camera was successfully demonstrated in 5 min for FHb and HSA. [ABSTRACT FROM AUTHOR]

Glucose-6-phosphate dehydrogenase is an enzyme that protects the erythrocytes against oxidative damage. G6PD deficiency is a common disorder in Gulf Countries that may present with hemolytic anemia and acute renal failure after exposure to oxidative triggers, such as drug, infection or some foodstuffs. In this study, a severe and complicated case of a G6PD deficient patient was presented. A 65-year old G6PD deficient patient presented with acute renal failure and intravascular hemolysis after eating fava beans. Therapeutic plasma exchange was used in the patient for two consecutive days. Clinical and laboratory findings were improved with plasma exchange. Although supportive therapy is enough for mild cases, therapeutic plasma exchange can be life-saving in complicated cases.

Objectives to evaluate the reference intervals for free hemoglobin concentration with the help of hemoglobin-cyanide method and a biochemical automatic analyzer. Material and methods.The reference group consisted of 120 healthy men and 120 healthy women from the Astrakhan region aged from 20 to 60 years. The fulfilment of standard procedures during the pre-analytical pre-laboratory, pre-analytical laboratory, analytical and post-analytical stages of the study ensured the quality control for free hemoglobin evaluation. Blood samples were taken with cubital vein puncture after applying a tourniquet (for not more than 1 minute), patient in lying position, using two-component blood sampling systems disposable polypropylene tubes with a blood coagulation activator (Sarstedt, Germany). To obtain blood serum, blood tubes were centrifuged at 2500 rpm during 10 minutes. The free hemoglobin in blood serum was detected by the hemoglobin-cyanide method using a ILAB 300 PLUS biochemical automatic analyzer (Laboratory Instrumentation, USA). The data was processed using the software package Statistica 6.0 for Windows (StatSoft Inc, США). Results.We used the classical approach and followed the strict inclusion and exclusion criteria, recommended by CLSI C 28-A3 guidelines. Gender and age differences in the content of free hemoglobin in blood serum in men and women of the Astrakhan region were not found. The reference interval of free hemoglobin in men and women of the Astrakhan region was evaluated as 0.020.84 g/l.

Abstract Spectroscopic methods for quantitative detection of molecules of the heme synthesis and heme metabolism are presented. This can be used to diagnose disease patterns such as iron deficiency, porphyria or free hemoglobin. This article combines an overview of clinical conditions associated with disorders of heme biosynthesis and presents selected laboratory findings in the development of portable diagnostic devices. An iron deficiency measurement device has already been validated as a portable device in clinical trials, two other methods have been tested according to EMA guidelines and are available for use in clinical validation studies. The presented rapid, low‐cost and low‐effort methods can support diagnosis in everyday clinical practice and initiate more targeted therapies, even for rare disease conditions.

Spectroscopic methods for quantitative detection of molecules of the heme synthesis and heme metabolism are presented. This can be used to diagnose disease patterns such as iron deficiency, porphyria or free hemoglobin. This article combines an overview of clinical conditions associated with disorders of heme biosynthesis and presents selected laboratory findings in the development of portable diagnostic devices. An iron deficiency measurement device has already been validated as a portable device in clinical trials, two other methods have been tested according to EMA guidelines and are available for use in clinical validation studies. The presented rapid, low‐cost and low‐effort methods can support diagnosis in everyday clinical practice and initiate more targeted therapies, even for rare disease conditions. [ABSTRACT FROM AUTHOR]

Severe intravascular hemolysis leads to the simultaneous presence of free heme pigments (oxyhemoglobin, methemoglobin, and methemalbumin) and bilirubin in human plasma. Standard spectrophotometric methods used to assess in vivo hemolysis inadequately address this complex analytical situation. Thus, we propose a novel quantification algorithm to ensure the highest analytical specificity. A corresponding second‐derivative fitting algorithm was validated according to the guideline of bioanalytical method validation from the European Medicines Agency using plasma specimens (n = 1759) spiked with different concentrations of oxyhemoglobin and methemoglobin. The results were compared to standard spectrophotometric quantification methods described by Harboe, Noe, and Fairbanks. Based on the second‐derivative method, simultaneous quantification of oxyhemoglobin and methemoglobin/methemalbumin in samples with total bilirubin concentrations ≤4.9 mg/dL (83.8 μmol/L) provided robust results (inaccuracy ≤20%, imprecision ≤16%). Analyzing UV/VIS spectra of plasma from patients with confirmed severe intravascular hemolysis evidenced an underestimation of up to 33% for the combined free heme pigment content. The employed second‐derivative algorithm allows for automated and highly specific quantification of the free heme pigment content in diluted human plasma, which cannot be realized with standard spectrophotometric evaluation methods. An Excel‐based tool readily applicable to clinical datasets accompanies this manuscript. [ABSTRACT FROM AUTHOR]

Abstract Background The possibility of clearing Cell-free Plasma Hemoglobin (CPH) from human plasma may appear attractive, especially when considering the noxious effects that CPH has on the immune function and the renal damage caused by its filtration. The existence of the so-called High Cut-Off (HCO) filters, possessing pores as big as 60 kDa, could potentially allow the clearance of the αβ dimers (31.3 kDa), the form in which the α2β2 hemoglobin tetramers (62.6 kDa) physiologically dissociate in plasma. We present herein the first reported case in which such an attempt was made. Case presentation The patient was a 51-year-old man with hemolytic crisis due to glucose-6-phosphate dehydrogenase deficiency, further complicated by pigment-induced nephropathy. He underwent a 48-h CVVHD session, in which a HCO filter was used. The Sieving Coefficient (SC) for CPH was initially 0.08 and decreased to 0.02 after 24 h. This unexpected low SC was due to the initial high concentration of CPH (4.24 g/L). At such concentrations, the α2β2 tetramer poorly dissociates into the αβ dimer; but increases exponentially at concentrations lower than 1 g/L. Conclusions Clearance of CPH through a HCO filter is technically feasible but its performance markedly relies on the initial concentration of CPH. Critically ill patients with smoldering hemolysis, as it happens during septic shock or ECMO treatment, may benefit the most from the use of this membrane in order to clear CPH.

Background: The aim of this study is to establish the contribution of blood cells subtypes on hemolysis. Methods: Separated blood cell subtype suspensions prepared with blood from 10 volunteers were serially diluted to obtain different concentrations of cell suspensions. The cells were fully lysed and cell hemolysates were added (1:20) to aliquots of serum pool. Thus, seven serum pools with different concentrations of interferent were obtained for each blood cell subtype. Biochemical parameters and serum indices were measured by an autoanalyzer. As cell lysis markers, free hemoglobin was measured by spectrophotometry while myeloperoxidase and b-thromboglobulin were measured by enzyme immunoassay. The percent changes in analyte levels of the serum pools were evaulated by Wilcoxon Signed Rank Test and compared with clinical thresholds defined for each test. Results: The clinical thresholds were exceeded in lactate dehydrogenase, potassium, aspartate aminotransferase, creatine kinase, magnesium, total protein, total cholesterol, inorganic phosphate, glucose for red blood cells (RBC); lactate dehydrogenase, aspartate aminotransferase, total protein, inorganic phosphate and glucose for platelets (PLT). Free hemoglobin was significantly correlated with RBC (r= 0.999; p= 0.001), while myeloperoxidase and b thromboglobulin showed no significant correlation to white blood cells (WBC) and PLT, respectively. Conclusions: The effect of RBC hemolysis in serum on the routine biochemical tests are clearly established, yet, additional studies are required in order to verify this kind of effects of PLT and WBC hemolysis.

Introduction  Maintaining blood supply is a challenge in blood banks. Red blood cells (RBCs) stored at 4°C experience issues of biochemical changes due to metabolism of cells, leading to changes collectively referred to as "storage lesions." Oxidation of the red cell membrane, leading to lysis, contributes to these storage lesions. Methods  Blood bags with CPD-SAGM stored at 4°C for 28 days were withdrawn aseptically on days 1, 14, and 28. Hematology analyzer was used to investigate RBC indices. Hemoglobin oxidation was studied through spectrophotometric scan of spectral change. RBC lysis was studied with the help of Drabkin's assay, and morphological changes were observed by light and scan electron microscopy. Results  RBCs show progressive changes in morphology echinocytes and spherocytes on day 28. There was 0.85% RBC lysis, an approximately 20% decrease in percentage oxyhemoglobin, and a 14% increase in methemoglobin formation, which shows hemoglobin oxidation on day 28. Conclusions  Oxidative damage to RBC, with an increase in storage time was observed in the present study. The observed morphological changes to RBC during the course of increased time shows that there is progressive damage to RBC membrane and a decrease in hemoglobin concentration; percentage RBC lysis is probably due to free hemoglobin and iron. [ABSTRACT FROM AUTHOR]

Sickle cell disease (SCD) causes exercise intolerance likely due to impaired skeletal muscle function and low nitric oxide (NO) bioavailability. Dietary nitrate improves hemodynamic and metabolic control during exercise in humans and animals. The purpose of this investigation was to assess the impact of nitrate supplementation on exercise capacity as measured by the running speed to exercise duration relationship [critical speed (CS)]in mice with SCD. We tested the hypothesis that nitrate supplementation via beetroot juice (BR) would attenuate the exercise intolerance observed in mice with SCD. Ten wild-type (WT) and 18 Berkley sickle-cell mice (BERK) received water (WT: n = 10, BERK: n = 10) or nitrate-rich BR (BERK+BR: n = 8, nitrate dose 1 mmol/kg/day) for 5 days. Following the supplementation period, all mice performed 3-5 constant-speed treadmill tests that resulted in exhaustion within 1.5 to 20 min. Time to exhaustion vs. treadmill speed was fit to a hyperbolic model to determine CS. CS was significantly lower in BERK vs. WT and BERK+BR with no significant difference between WT and BERK+BR (WT: 36.6 ± 1.6, BERK: 23.8 ± 1.5, BERK+BR: 31.1 ± 2.1 m/min, P < 0.05). Exercise tolerance, measured via CS, was significantly lower in BERK mice relative to WT. However, BERK mice receiving 5 days of nitrate supplementation exhibited no difference in exercise tolerance when compared with WT. These results support the potential utility of a dietary nitrate intervention to improve functionality in SCD patients. [ABSTRACT FROM AUTHOR]

Purpose: This study aimed to quantify the hemolytic responses of elite female football (soccer) players during a typical weekly training session. Methods: Ten elite female football players (7 field players [FPs] and 3 goalkeepers [GKs]) were recruited from the Australian National Women's Premier League and asked to provide a venous blood sample 30 min before and at the immediate conclusion of a typical weekly training session. During this training session, the players' movement patterns were monitored via a 5-Hz global positioning system. The blood samples collected during the training session were analyzed for iron status via serum fer- ritin (SF) analysis, and the hemolytic response to training, via serum free hemoglobin (Hb) and haptoglobin (Hp) measurement. Results: 50% of the participants screened were found to have a compromised iron stores (SF<35 (µ/L). Furthermore, the posttraining serum free Hb levels were significantly elevated (P = .011), and the serum Hp levels were significantly decreased (P- .005), with no significant differences recorded between the FPs and GKs. However, the overall distance covered and the movement speed were significantly greater in the FPs. Conclusions: The increases in free Hb and decreases in Hp levels provide evidence that a typical team-sport training session may result in significant hemolysis. This hemolysis may primarily be a result of running-based movements in FPs and/or the plyometric movements in GKs, such as diving and tackling. [ABSTRACT FROM AUTHOR]

Incidental azygos vein cannulation has been reported in a few cases of neonatal extracorporeal membrane oxygenation (ECMO). This complication is described in the literature mainly in pathological conditions wherein increased central venous pressure dilates the superior vena cava (SVC), i.e., right congenital diaphragmatic hernia (CDH) or pulmonary hypertension. Azygos vein cannulation should always be suspected in cases of impaired venous return and circuit failure. Although rare, it hinders proper venous aspiration of the ECMO circuit and generally requires repositioning or replacement of the venous cannula or conversion to central cannulation. In this report, we describe a newborn with severe right CDH who required ECMO assistance, wherein incidental cannulation of the azygos vein resulted in successful functioning of the circuit because of the concomitant presence of isolated interruption of the inferior vena cava and azygos continuation. To the best of our knowledge, this is the first report of successful neonatal ECMO despite azygos vein cannulation in a patient with such rare physiology.

Background: The authors report the relevance of using a point of care test (Helge ® ) for free hemoglobin determination and concordance of the values the with Cobas ® 8000 and spectrophotometer methods. Results: The within-run of the point of care test was <3%. Good correlations among the three methods were observed and an acceptable concordance for hemolysis index values from 50 mg/dl. An excellent agreement between the Cobas 8000 and the spectrophotometer was found. Conclusion: Automated methods represent methods of choice for free hemoglobin determination. An advantage of the Helge system is that it can be applied to samples experiencing a delay in evaluation due to the long distance between the collection site and the central laboratory. Another advantage is its use at the bedside, in the monitoring of extracorporeal membrane oxygenation patients.

Introduction: We have been developing a closed-circuit cardiopulmonary bypass (CPB) system ("Dihead CPB") for application during coronary artery bypass grafting (CABG) and valve surgery. To strive for minimal hemolysis during Dihead CPB, we compared the hemolysis caused by three different suction systems and performed a clinical study with the newly applied suction system. Materials & Methods: We evaluated the hemolysis caused by roller-pump suction, the SmartSuction® Harmony® and wall suction systems with respect to suction speed and compared the systems by means of in vitro studies. A clinical study was also performed on 15 volunteers to assess hemolysis and the adequacy of the newly applied suction system with Dihead CPB. Results: Pressure inside the suction cannula was −22.5 ± 0.1 mmHg at a maximum flow of 1.5 L/min for roller-pump suction and −43.4 ± 0.1 mmHg at −150 mmHg of the set vacuum pressure of wall suction. With the SmartSuction, the pressure inside the cannula varied from −76.3 ± 1.0 to −130.3 ± 1.5 mmHg, depending on suctioning conditions. Suction speed (to suction 50 ml of blood) was fastest with the SmartSuction (69.7 ± 3.58 s) whereas, with roller suction, it was 117.3 ± 8.47 s and with wall suction 96.9 ± 7.10 s. The SmartSuction had the highest hemolysis rate (2.00 ± 0.33%) vs. 0.61 ± 0.10% for roller suction and 0.41 ± 0.11% for wall suction (p<0.001). The clinical study with wall suction showed no significant increase in plasma free hemoglobin during or after CPB compared with before surgery. Conclusions: Wall suction had less hemolysis than roller suction or the SmartSuction in the in vitro study and the clinical study with wall suction showed efficient suction speed and acceptable hemolysis, suggesting that Dihead CPB with wall suction is feasible for CABG. [ABSTRACT FROM AUTHOR]

Free hemoglobin (FHB) concentration is considered a prospect quality indicator for erythrocyte suspensions (ES) under storage. Storage lesions alter the optical properties of ES and can be monitored by diffuse reflectance spectroscopy. Due to storage lesions, erythrocytes lyse and release hemoglobin into the extracellular medium. The purpose of the study is to model and assess the quality of ES units in a blood bank with diffuse reflectance measurements together with hematological variables reflecting absorption and scattering characteristics of ES. FHB concentrations were modeled based on the increased scattering in the extracellular medium. A semiempirical model was used for relating optical properties of ES to the diffuse reflectance measurements. The attenuation in the blood bag was computed and its influence was discarded via normalization, in accordance with Monte Carlo simulations. In the experiments, 40 ES units were measured multiple times during prolonged storage of 70 days. A generalized linear model was used for modeling the training set, and, in the validation, the highest correlation coefficient between predicted and actual FHB concentrations was 0.89. Predicting the actual value was accurate at a maximum level of R² = 0.80. The error rate of the model in diagnosing the true quality was about 10%. [ABSTRACT FROM AUTHOR]

Objective: Implementation of a laboratory test of free hemoglobin in plasma into routine laboratory practice using a calculation method based on hemolysis index (HI). The test should be available 24/7 and quality control of this method should be ensured. Design: Original Article. Settings: Institute for Clinical and Experimental Medicine, Vídeňská 1958/9, 140 21 Praha 4 Materials and methods: The method was calibrated using in-house calibration standards. The patients' samples were compared to a spectrophotometric method according to Fairbanks [1] and to the HI-based method (measured on the automated analyzer Cobas 6000, Roche) using the Passing-Bablok regression. The external quality assessment (EQA) samples (INSTAND e.V.) and the in-house internal quality control (IQC) samples, prepared just as the calibration standards, were measured in the same way. Yields relative to target (or theoretical) hemoglobin plasma concentrations were calculated for the both types of samples. Results: The linear regression equation for calculation of free hemoglobin in plasma [free Hb (mg/l) = 11.41*HI+21.1] was obtained by calibration. This formula was utilized to compare patients' samples to the Fairbanks procedure [1] using Passing-Bablok regression (intercept 45.9 mg/L; slope 1.28). Recovery of the Fairbanks method [1] relative to the target values of the EQA samples was 100.8 %. Recovery of the HI-based method was only 71.1%. The HI-based method correlated with theoretical concentration of hemoglobin; the spectrophotometric method by Fairbanks [1] did not. Conclusion: The free hemoglobin calculation method utilizing HI values is an easy-to-use, low-cost and fast method that can be available to clinical departments 24/7. The results are sufficient for timely identification of intravascular hemolysis. There are discrepant results of compared methods in patient samples, but we do not consider the bias as significant because of higher limit for organs damage (up 400 mg/l), which is caused by mechanical hemolysis. However, the EQA samples correlate only with the Fairbanks method [1], due to different matrix properties of these samples, and therefore cannot be used for the HI-based method. Ensuring traceability between spectrophotometrically measured EQA samples and the HIbased method by means of patients' samples has been the only option for trueness verification of the implemented method so far. [ABSTRACT FROM AUTHOR]

Though extracorporeal membrane oxygenation (ECMO) provides life-saving support, this intervention exposes patients to certain risks. Circulating free hemoglobin (fHb) resulting from mechanically induced hemolysis and insufficient haptoglobin/hemopexin may promote thrombosis within the ECMO circuit. Thrombi in the circuit can result in thromboembolic complications in these patients. Prevention of thrombus formation and propagation in the ECMO circuit may improve clinical outcome. fHb released during hemolysis has been shown to have multiple adverse effects, including thrombosis, but the mechanism by which fHb contributes to thrombosis in an ECMO circuit remains elusive. It is well established that (1) high shear stress generated in the circuit may cause hemolysis, and (2) plasma fibrinogen is adsorbed onto the inner tubing of the ECMO circuit over time. Plasma von Willebrand factor (pVWF) mediates platelet deposition at sites of vascular injury under high shear stress by sensing alterations in the hemodynamic environment. This biophysical property of pVWF that enables hemostasis may also contribute to the pathogenesis of ECMO-induced thrombosis. pVWF contains binding sites for both adsorbed fibrin(ogen) and fHb. High concentrations of fHb increase pVWF-mediated platelet adhesion and thrombus formation on a surface-adsorbed fibrin(ogen) under high shear stress. The molecular mechanism(s) by which fHb drives the conformation of pVWF into a prothrombotic state is currently unknown. Reduction of thrombotic risks during ECMO intervention warrants further investigations into the interaction between pVWF and fHb.

Glucose-6-phosphate dehydrogenase is an enzyme that protects the erythrocytes against oxidative damage. G6PD deficiency is a common disorder in Gulf Countries that may present with hemolytic anemia and acute renal failure after exposure to oxidative triggers, such as drug, infection or some foodstuffs. In this study, a severe and complicated case of a G6PD deficient patient was presented. A 65-year old G6PD deficient patient presented with acute renal failure and intravascular hemolysis after eating fava beans. Therapeutic plasma exchange was used in the patient for two consecutive days. Clinical and laboratory findings were improved with plasma exchange. Although supportive therapy is enough for mild cases, therapeutic plasma exchange can be life-saving in complicated cases. [ABSTRACT FROM AUTHOR]

The ability to characterize the mechanical properties of erythrocytes is important in clinical and research contexts: to diagnose and monitor hematologic disorders, as well as to optimize the design of cardiovascular implants and blood circulating devices with respect to blood damage. However, investigation of red blood cell (RBC) properties generally involves preparatory and processing steps. Even though these impose mechanical stresses on cells, little is known about their impact on the final measurement results. In this study, we investigated the effect of centrifuging, vortexing, pipetting, and high pressures on several markers of mechanical blood damage and RBC membrane properties. Using human venous blood, we analyzed erythrocyte damage by measuring free hemoglobin, phosphatidylserine exposure by flow cytometry, RBC deformability by ektacytometry and the parameters of a complete blood count. We observed increased levels of free hemoglobin for all tested procedures. The release of hemoglobin into plasma depended significantly on the level of stress. Elevated pressures and centrifuging also altered mean cell volume (MCV) and mean corpuscular hemoglobin (MCH), suggesting changes in erythrocyte population, and membrane properties. Our results show that the effects of blood handling can significantly influence erythrocyte damage metrics. Careful quantification of this influence as well as other unwanted secondary effects should thus be included in experimental protocols and accounted for in clinical laboratories.

Abstract. Forty four patients with persistent allergic rhinitis and sensitization to the house dust mites were observed. All the patients have been examined in conformity with international diagnostic standards. The first group of observation included fifteen patients who received intranasal therapy by flutikasone propionate nasal spray at a dose of 200 mkg once a day for two weeks. The second group of observation included fourteen patients who received 10 inhalations of Affinoleukin® (a lyophilized complex of low molecular weight proteins from human leukocyte membranes with glycine), as aerosol, at a single dose of 2 units. Fifteen non-treated patients comprised a group of comparison. Eosinophil counts and concentrations of free hemoglobin (by immunometrical method in co-agglutination reaction) were made in nasal secretions twice (before treatment and two weeks later). It was found that the increase of free hemoglobin levels, as well as increase in eosinophil count in nasal secretions represents a symptom typical of exacerbation in persistent allergic rhinitis and reflects severity of disease. The therapy led to significant clinical improvement, decrease in free hemoglobin levels and eosinophil counts in nasal secretions, corresponding to clinical effect of both therapeutic modes of topical pharmacotherapy, resp., in 73% and 79% of the patients with persisting allergic rhinitis. Decrease in free hemoglobin level has allowed for the first time to reveal the immunotherapeutic efficiency of treatment with Affinoleukin® aerosol during exacerbations of persisting allergic rhinitis.

The ability to characterize themechanical properties of erythrocytes is important in clinical and research contexts: to diagnose and monitor hematologic disorders, as well as to optimize the design of cardiovascular implants and blood circulating devices with respect to blood damage. However, investigation of red blood cell (RBC) properties generally involves preparatory and processing steps. Even though these impose mechanical stresses on cells, little is known about their impact on the final measurement results. In this study, we investigated the effect of centrifuging, vortexing, pipetting, and high pressures on several markers of mechanical blood damage and RBC membrane properties. Using human venous blood, we analyzed erythrocyte damage by measuring free hemoglobin, phosphatidylserine exposure by flow cytometry, RBC deformability by ektacytometry and the parameters of a complete blood count. We observed increased levels of free hemoglobin for all tested procedures. The release of hemoglobin into plasma depended significantly on the level of stress. Elevated pressures and centrifuging also altered mean cell volume (MCV) and mean corpuscular hemoglobin (MCH), suggesting changes in erythrocyte population, and membrane properties. Our results show that the effects of blood handling can significantly influence erythrocyte damage metrics. Careful quantification of this influence as well as other unwanted secondary effects should thus be included in experimental protocols and accounted for in clinical laboratories. [ABSTRACT FROM AUTHOR]

Objective The hemolytic product free-hemoglobin (fHb) reduces nitric oxide (NO) bioavailability. The present study aims to establish whether administration of different blood transfusions result in increased circulating fHb levels and NO consumption with effects on arterial NO-dependent blood flow in patients undergoing CABG surgery. Methods Ninety-five consecutive patients undergoing elective CABG surgery were prospectively divided in four groups based on blood transfusion requirements during surgery: stored blood cells (SBC, n. 21), intraoperative autologous salvaged blood (ASB, n. 25), SBC and ASB (n.22), no transfusion (control, n. 27). Blood samples were collected before and after intervention to analyse plasma levels of fHb and NO consumption. Endothelium-dependent relaxation was assessed in left internal mammary artery (LIMA) rings harvested before chest closure. Peripheral artery tonometry was assessed after intervention. Results Transfusions with SBC increased plasma fHb ( p < 0.05). Transfusions of ASB resulted in higher plasma fHb compared to SBC ( p < 0.01). fHb concentrations directly correlated with NO consumption ( r = 0.65, p < 0.001). Maximal endothelium-dependent relaxation in LIMA was significantly attenuated in SBC and ASB patients compared to control (15.2 ± 3.1% vs 21.1 ± 2.5% vs 43 ± 5.0% respectively; p < 0.01). Significant correlations were identified between the aortic pressure wave velocity, plasma fHb concentration and NO consumption ( p < 0.01). Conclusions Intraoperative blood transfusions and particularly autologous salvaged blood impair endothelium-dependent relaxation through NO scavenging by fHb. These findings obtained in vitro and in vivo provide new insights into the adverse relation between blood transfusions and patient outcome. [ABSTRACT FROM AUTHOR]

[Display omitted] • Disposable PAD was proposed for one drop blood samples analysis without cross-contamination and infection problems. • Colorimetric detection of Hb were enhanced by online electrokinetic stacking and chromatic reaction. • POCT of both THb and FHb from the same sample is possible with the same PAD. Total hemoglobin (THb) and free hemoglobin (FHb) are biomarkers of anemia and hemolysis, playing a critical role in human health status. This study reports a paper-based device by field amplified sample stacking (FASS) using colorimetric method to quantify both THb and FHb. The detection of THb was based on the color intensity of the Hb stacking band, a limit of detection (LOD) of 11 g∙L−1 and a linear working range of 16 ∼ 140 g∙L−1 were achieved. With selective chromatic reaction of the stacking band with o-tolidine, the sensitivity of Hb detection was further enhanced by 2000 times, a LOD of 25.8 mg∙L−1 and a linear range of 30 ∼ 1000 mg∙L−1 was obtained for FHb. This paper-based device was successfully used for detection of high concentration THb and low concentration FHb from the same blood sample, and both meeting the requirements of clinical diagnosis. In addition, the feasibility of on-site plasma preparation and online integration with the device was also preliminarily investigated with a blood separation membrane. This paper-based device could be used as a new platform for developing fast and low-cost Hb point-of-care testing devices. [ABSTRACT FROM AUTHOR]

Elevated soluble (s) CD163 and free hemoglobin (Hb) levels predict fatty liver progression; however, the molecular mechanisms underlying Hb metabolism and liver injury remain undefined. We investigated the effects of endoplasmic reticular (ER) stress on red blood cell (RBC) rheology and free Hb recycling pathways. ER stress was induced in Sprague-Dawley rats by an intraperitoneal injection of tunicamycin (TM) (50, 100, and 200 μg/100 g body weight (BW)) or an intravenous injection of Hb (5 mg/100 g BW). A TM injection increased sCD163 levels, attenuated free Hb uptake, and maintained RBC aggregability. An Hb injection increased serum LVV-hemorphin-7 and total bilirubin levels, but this effect was suppressed by TM. A Western blot analysis showed that ER stress suppressed Hb degradation in the liver through downregulation of globin degradation proteins cathepsin D and glyoxalase-1, as well as heme degradation protein heme oxyganase-1 and keap-1 expression. An ER stress activator also increased the translocation of nuclear factor (NF)-κB (p65) and nuclear factor-erythroid 2-related factor 2 (Nrf2) to nuclei. In conclusion, ER stress triggers ineffective Hb metabolism via altering globin and heme iron degradation pathways. Inability to recycle and metabolize free Hb may underlie the association between iron dysfunction and liver injury.