Preparation, characterization and antioxidant and anticancerous potential of Quercetin loaded ß-glucan particles derived from mushroom and yeast.

ß-glucans are polysaccharides found in the cell walls of various fungi, bacteria and cereals. ß-glucan have been found to show various kinds of anti-inflammatory, antimicrobial, antidiabetic antioxidant and anticancerous activities. In the present study, we have isolated ß-glucan from the baker's yeast Saccharomyces cerevisiae and white button mushroom Agaricus bisporus and tested their antioxidant potential and anticancerous activity against prostate cancer cell line PC3. Particles were characterized with zeta sizer and further with FTIR that confirmed that the isolated particles are ß-glucan and alginate sealing made slow and sustained release of the Quercetin from the ß-glucan particles. Morphological analysis of the hollow and Quercetin loaded ß-glucan was performed with the SEM analysis and stability was analyzed with TGA and DSC analysis that showed the higher stability of the alginate sealed particles. Assessments of the antioxidant potential showed that Quercetin loaded particles were having higher antioxidant activity than hollow ß-glucan particles. Cell viability of the PC3 cells was examined with MTT assay and it was found that Quercetin loaded alginate sealed Agaricus bisporus derived ß-glucan particles were having lowest IC50. Further ROS generation was found to increase in a dose dependent manner. Apoptosis detection was carried out with Propidium iodide and AO/EtBr staining dye which showed significant death in the cells treated with higher concentration of the particles. Study showed that particles derived from both of the sources were having efficient anticancer activity and showing a dose dependent increase in cell death in PC3 cells upon treatment.

Serum microRNAs as new biomarkers for detecting subclinical hemolysis in the nonacute phase of G6PD deficiency.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common enzymopathies worldwide. Patients with G6PD deficiency are usually asymptomatic throughout their life but can develop acute hemolysis after exposure to free radicals or certain medications. Several studies have shown that serum miRNAs can be used as prognostic biomarkers in various types of hemolytic anemias. However, the impact of G6PD deficiency on circulating miRNA profiles is largely unknown. The present study aimed to assess the use of serum miRNAs as biomarkers for detecting hemolysis in the nonacute phase of G6PD deficiency. Patients with severe or moderate G6PD Viangchan (871G > A) deficiency and normal G6PD patients were enrolled in the present study. The biochemical hemolysis indices were normal in the three groups, while the levels of serum miR-451a, miR-16, and miR-155 were significantly increased in patients with severe G6PD deficiency. In addition, 3D analysis of a set of three miRNAs (miR-451a, miR-16, and miR-155) was able to differentiate G6PD-deficient individuals from healthy individuals, suggesting that these three miRNAs may serve as potential biomarkers for patients in the nonhemolytic phase of G6PD deficiency. In conclusion, miRNAs can be utilized as additional biomarkers to detect hemolysis in the nonacute phase of G6PD deficiency.

Relationships among hemolysis indicators and neuron-specific-enolase in patients undergoing veno-arterial extracorporeal membrane oxygenation.

Neuron-specific-enolase is used as a marker of neurological prognosis after cardiopulmonary resuscitation. It is also present in red blood cells and platelets. It is not known whether hemolysis increases the values of neuron-specific-enolase enough to clinically affect its interpretation in critically ill patients who are to be introduced to veno-arterial extracorporeal oxygenation. In this study, we examined the relationships among neuron-specific-enolase and hemolysis indicators such as free hemoglobin and lactate dehydrogenase after the introduction of veno-arterial extracorporeal oxygenation. Of the 91 patients who underwent veno-arterial extracorporeal membrane oxygenation in our hospital from January 1, 2018, to February 24, 2021, 68 patients survived for more than 24 h. Of these, 14 patients who were categorized into the better cerebral performance categories (1-3) and 19 patients who were categorized into the poor neurological prognosis category (4) were included. After the introduction of veno-arterial extracorporeal membrane oxygenation, neuron-specific-enolase was markedly higher in the poor neurological prognosis group than in the good neurological prognosis group (41.6 vs. 92.0, p = 0.04). A significant positive correlation was revealed between neuron-specific-enolase and free hemoglobin in the good neurological prognosis group (rs = 0.643, p = 0.0131). A similar relationship was observed for lactate dehydrogenase and neuron-specific-enolase in both the conscious (rs = 0.737, p = 0.00263) and non-conscious groups (rs = 0.544, p = 0.0176). When neuron-specific-enolase is used as a marker for neuroprognostic evaluation, an abnormally high value is likely to indicate the lack of consciousness, whereas a lower elevation should be interpreted with caution, taking into account the effects of hemolysis.

Naphthalene Ball Ingestion Leading to Intravascular Hemolysis and Acute Kidney Injury.

Naphthalene is an aromatic hydrocarbon found in mothballs, deodorizers, or insecticides. Naphthalene poisoning is not commonly seen in the pediatric age group due to its pungent odor and taste, water insolubility, and poor absorption from the gastrointestinal tract (GIT). This case report describes a five-year-old boy who experienced accidental naphthalene mothball ingestion resulting in intravascular hemolysis and acute kidney injury (AKI). Naphthalene exposure can cause severe complications, especially in children. The clinical presentation included fever, abdominal pain, vomiting, decreased urine output, and hematuria. The laboratory findings revealed hemolytic anemia, elevated serum creatinine, and proteinuria. The child received supportive treatment including intravenous fluids, packed red blood cell transfusions, and hemodialysis for AKI. Early diagnosis and intervention are crucial for a favorable outcome. This case highlights the importance of considering naphthalene poisoning in the differential diagnosis of children with hemolysis and AKI.

Association of Serum Metabolites with Serum Indices and Preanalytical Factors of Biobanked Serum Samples.

Background: Serum indices (hemolysis, icterus, and lipemia; HIL) are known to impact clinical chemistry assay results. This study aimed to investigate the impact of HIL indices on serum metabolite profiles and the association of serum metabolite levels with pre-analytical factors of serum samples. Methods: A cohort of serum samples (n = 12,196) from the Korean Genome and Epidemiology Study (KoGES) was analyzed for HIL indices and the pre-analytical variables (SPRECs) which were generated in the process of serum collection. We further performed targeted metabolomics on a subset comprising hemolyzed (n = 60), icteric (n = 60), lipemic (n = 60) groups, and a common control group of non-HIL samples (n = 60) using the Absolute IDQ p180 kit. Results: We found 22 clinical chemistry analytes significantly associated with hemolysis, 25 with icterus, and 24 with lipemia (p < 0.0001). Serum metabolites (n = 27) were associated with all of hemolysis, icterus, and lipemia (p < 0.05). The PC ae C36 2 had exhibited a significant association with pre-analytical factors corresponding to the third (pre-centrifugation delay between processing) and sixth (post-centrifugation) elements of the SPREC. Conclusions: This study showed the association of the serum index and pre-analytical factors with serum metabolite profiles. In addition, the association of pre-analytical factors with serum metabolite concentrations would corroborate the utility of SPRECs for the quality control of biobanked serum samples.

Biological and genetic determinants of glycolysis: Phosphofructokinase isoforms boost energy status of stored red blood cells and transfusion outcomes.

Mature red blood cells (RBCs) lack mitochondria and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo or storage in blood banks. Here, we leveraged 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study to identify associations between end-of-storage levels of glycolytic metabolites and donor age, sex, and ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (detected in mature RBCs); hexokinase 1 (HK1); and ADP-ribosyl cyclase 1 and 2 (CD38/BST1). Gene-metabolite associations were validated in fresh and stored RBCs from 525 Diversity Outbred mice and via multi-omics characterization of 1,929 samples from 643 human RBC units during storage. ATP and hypoxanthine (HYPX) levels-and the genetic traits linked to them-were associated with hemolysis in vitro and in vivo, both in healthy autologous transfusion recipients and in 5,816 critically ill patients receiving heterologous transfusions, suggesting their potential as markers to improve transfusion outcomes.

Exploring the Possible Link Between Diabetic Ketoacidosis, Glucose-6-Phosphate Dehydrogenase Deficiency, and Methemoglobinemia.

One of the most widespread enzymopathies affecting human beings is glucose-6-phosphate dehydrogenase (G6PD) deficiency, which is brought on by inherited mutations in the X-linked gene. Red blood cells (RBCs) with a G6PD deficiency are more sensitive to oxidative assault and consequently to hemolysis. There are more than 200 known G6PD mutations, of which around half are polymorphic and thus prevalent in a variety of populations. We present a case of diabetic ketoacidosis (DKA), with severe hemolytic anemia and methemoglobinemia. The patient was admitted to the intensive care unit, treated for DKA, and received a blood transfusion. In addition, the patient presented with high methemoglobin levels and features of severe hemolytic anemia from the onset, which made the diagnostic consideration of G6PD highly likely. Accordingly, the patient was treated with several doses of ascorbic acid instead of methylene blue. In a nutshell, a patient with DKA who has hemolytic anemia has to have it properly evaluated and controlled. The link between methemoglobinemia, G6PD deficiency, and DKA should be recognized by medical professionals, particularly when oxygen saturation gaps are found.

The impact of the Impeller's hub design on the performance and blood damage in a microaxial mechanical circulatory support device - A numerical study.

This study uses CFD methods to investigate the effects of the impeller's geometry on the hemodynamic characteristics, pump performance, and blood damage parameters, in a percutaneous microaxial Mechanical Circulatory Support (MCS) device. The numerical simulations employ the steady state Reynolds-Averaged Navier-Stokes approximation using the SST k-ω turbulent model. Three different impeller models are examined with different hub conversion angles (α = 0○, 3○ and 5○). The analysis includes 23 cases for different pressure heads (Δp = 60-80 mmHg) and angular velocities (ω = 30-52 kRPM). The obtained flow rate is compared between the cases to assess the effect of the impeller's design and working conditions on the pump performance. The comparative risk of shear-induced platelet activation is estimated using the statistical median of the stress-accumulation values calculated along streamlines. The risk of hemolysis is estimated using the average exposure time to shear stress above a threshold (τ > 425 Pa). The results reveal that the shape of the impeller's hub has a great impact on the flow patterns, performance, and risk of blood damage, as well as the angular velocity. The highest flow rate (Q = 3.7 L/min) and efficiency (η = 11.3 %) were achieved using a straight hub (α = 0○). Similarly, for the same condition of flow and pressure, the straight hub impeller has the lowest blood damage risk parameters. This study shed light on the effect of pump design on the performance and risk of blood damage, indicating the roles of the hub shape and angular velocity as dominant parameters.

Continuous training in young athletes decreases hepcidin secretion and is positively correlated with serum 25(OH)D and ferritin.

Background: Iron deficiency is known to impair muscle function and reduce athletic performance, while vitamin D has been reported to induce iron deficiency. However, the mechanism underlying exercise-induced changes in iron metabolism and the involvement of vitamins in this mechanism are unclear. The present study examined changes in biological iron metabolism induced by continuous training and the effects of vitamin D on these changes. Methods: Diet, physical characteristics, and blood test data were collected from 23 female high school students in a dance club on the last day of each of a 2-month continuous training period and a 2-week complete rest periods. Results: Serum hepcidin-25 levels were significantly lower during the training period than the rest period (p = 0.013), as were the red blood cell count, hemoglobin, and hematocrit (all p < 0.001). Serum erythropoietin was significantly higher (p = 0.001) during the training period. Significant positive correlations were observed between 25(OH)D levels and serum iron, serum ferritin, and transferrin saturation during the training period. Multiple regression analysis with serum 25(OH)D level as the dependent variable and serum ferritin and iron levels as independent variables during the training period revealed a significant association with serum ferritin. Conclusion: Continuous training may promote hemolysis and erythropoiesis, contributing to the suppression of hepcidin expression. The relationship between serum 25(OH)D and iron in vivo may be closely related to metabolic changes induced by the exercise load.

Resolution of extravascular hemolysis with oral iptacopan monotherapy in a patient with treatment experienced paroxysmal nocturnal hemoglobinuria (PNH).

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematologic disorder characterized by a loss of glycosyl-phosphatidyl-inositol-linked (GPI) proteins on various hematopoietic cells. Some GPI proteins are involved in the regulation of the complement system, and their absence renders erythrocytes susceptible to complement-mediated lysis. Current standard of care in PNH is to block the complement system at the level of C5 using ravulizumab or eculizumab; however, some patients with PNH may develop extravascular hemolysis (EVH) during treatment with C5 inhibitors. The proximal complement inhibitor iptacopan has recently been shown to be efficacious in patients with PNH. This article reports on a 43-year-old female patient with PNH who was successfully treated with iptacopan. The patient had received ravulizumab for several years and developed a clinically relevant EVH. After obtaining informed consent, the patient received oral iptacopan 200 mg twice daily and ravulizumab was discontinued. Over the next few weeks hemoglobin levels and reticulocyte counts normalized. The patient reported mild flushes with erythema, chills, and mild muscle pain, all of which resolved during follow-up. No breakthrough hemolysis occurred, and no severe adverse events were recorded.

Hepatolithiasis and Choledocholithiasis Secondary to Warm Autoimmune Hemolytic Anemia in the Setting of COVID-19 and Lupus.

Two major etiologies of hyperbilirubinemia include hemolysis and cholestasis. Although rare, the former can give rise to the latter through the formation of pigment gallstones and subsequent biliary tree obstruction. We report a case of a 57-year-old woman with systemic lupus erythematosus who presented with dyspnea and right upper quadrant abdominal pain. She was found to have hepatolithiasis and choledocholithiasis secondary to warm autoimmune hemolytic anemia in the setting of COVID-19. In patients with symptomatic anemia secondary to acute hemolysis and concomitant right upper quadrant abdominal pain, elevated hepatocellular laboratory results should prompt a high clinical suspicion for biliary tree pigment stones.

Hemolysis during open heart surgery in patients with hereditary spherocytosis - systematic review of the literature and case study.

BACKGROUND: Due to the distinctive nature of cardiac surgery, patients suffering from hereditary spherocytosis (HS) are potentially at a high risk of perioperative complications resulting from hemolysis. Despite being the most prevalent cause of hereditary chronic hemolysis, the standards of surgical management are based solely on expert opinion. OBJECTIVE: We analyze the risk of hemolysis in HS patients after cardiac surgery based on a systematic review of the literature. We also describe a case of a patient with hereditary spherocytosis who underwent aortic valve repair. METHODS: This systematic review was registered in the PROSPERO international prospective register of systematic reviews (CRD42023417666) and included records from Embase, MEDLINE, Web of Science, and Google Scholar databases. The case study investigates a 38-year-old patient who underwent surgery for an aortic valve defect in mid-2022. RESULTS: Of the 787 search results, 21 studies describing 23 cases of HS undergoing cardiac surgery were included in the final analysis. Hemolysis was diagnosed in five patients (one coronary artery bypass graft surgery, two aortic valve bioprosthesis, one ventricular septal defect closure, and one mitral valve plasty). None of the patients died in the perioperative period. Also, no significant clinical hemolysis was observed in our patient during the perioperative period. CONCLUSIONS: The literature data show that hemolysis is not common in patients with HS undergoing various cardiac surgery techniques. The typical management of a patient with mild/moderate HS does not appear to increase the risk of significant clinical hemolysis. Commonly accepted beliefs about factors inducing hemolysis during cardiac surgery may not be fully justified and require further investigation.

Testosterone supplementation increases red blood cell susceptibility to oxidative stress, decreases membrane deformability, and decreases survival after cold storage and transfusion.

BACKGROUND: Blood collection from donors on testosterone therapy (TT) is restricted to red blood cell (RBC) concentrates to avoid patient exposure to supraphysiological testosterone (T). The objective of this study was to identify TT-related changes in RBC characteristics relevant to transfusion effectiveness in patients. STUDY DESIGN: This was a two-part study with cohorts of patients and blood donors on TT. In part 1, we conducted longitudinal evaluation of RBCs collected before and at three time points after initiation of T. RBC assays included storage and oxidative hemolysis, membrane deformability (elongation index), and oximetry. In part 2, we evaluated the fate of transfused RBCs from TT donors in immunodeficient mice and by retrospective analyses of NIH's vein-to-vein databases. RESULTS: TT increased oxidative hemolysis (1.45-fold change) and decreased RBC membrane deformability. Plasma free testosterone was positively correlated with oxidative hemolysis (r = .552) and negatively correlated with the elongation index (r = -.472). Stored and gamma-irradiated RBCs from TT donors had lower posttransfusion recovery in mice compared to controls (41.6 ± 12 vs. 55.3 ± 20.5%). Recipients of RBCs from male donors taking T had 25% lower hemoglobin increments compared to recipients of RBCs from non-TT male donors, and had increased incidence (OR, 1.80) of requiring additional RBC transfusions within 48 h of the index transfusion event. CONCLUSIONS: TT is associated with altered RBC characteristics and transfusion effectiveness. These results suggest that clinical utilization of TT RBCs may be less effective in recipients who benefit from longer RBC survival, such as chronically transfused patients.

In vitro Effect of Cannabidiol on Red Blood Cells: Implication in Long-Lasting Pathology Treatment.

BACKGROUND: Cannabidiol (CBD) is the principal non-hallucinogenic compound of Cannabis plants with high clinical interest because CBD has been described as having anti-inflammatory, analgesic and anticonvulsant properties. CBD is considered a multitarget compound as it can interact with a wide range of targets, explaining their multiplicity of effects. Some clinical studies have indicated certain side effects of CBD, including somnolence, anemia and diarrhea, while the elevation of transaminases is considered as an exclusion criterion from the trial. Since the red blood cells (RBCs) are a source of transaminase, we assayed in vitro effect on RBCs stability. METHODS: We performed in vitro experiments with RBCs obtained from human peripheral blood with normal hematological parameters exposed to CBD in the range of therapeutic uses. We evaluated RBCs morphological changes, membrane fragility and hemoglobin release as a reflection of hemolysis. RESULTS: CBD induced an increase in the hemoglobin release (3.27 µg/106 RBC), without altered RBC osmotic fragility. When RBCs suspensions were incubated with CBD the initial number of elements (RBCs + vesicles) was increased up to 65% after 20 min and returned to basal level after 40 min of incubation. In the first 20 min, the accounts of elements were enriched in the smaller vesicles that disappeared after the remaining 20 minutes. CONCLUSION: These results suggest that CBD affects the indemnity of erythrocytes in vitro, inducing the formation of hemolytic vesicles that can provide the basis for the development of anemia, transaminase elevation and underlying tissular iron overload in patients chronically treated with CBD.

Empirical and Computational Evaluation of Hemolysis in a Microfluidic Extracorporeal Membrane Oxygenator Prototype.

Microfluidic devices promise to overcome the limitations of conventional hemodialysis and oxygenation technologies by incorporating novel membranes with ultra-high permeability into portable devices with low blood volume. However, the characteristically small dimensions of these devices contribute to both non-physiologic shear that could damage blood components and laminar flow that inhibits transport. While many studies have been performed to empirically and computationally study hemolysis in medical devices, such as valves and blood pumps, little is known about blood damage in microfluidic devices. In this study, four variants of a representative microfluidic membrane-based oxygenator and two controls (positive and negative) are introduced, and computational models are used to predict hemolysis. The simulations were performed in ANSYS Fluent for nine shear stress-based parameter sets for the power law hemolysis model. We found that three of the nine tested parameters overpredict (5 to 10×) hemolysis compared to empirical experiments. However, three parameter sets demonstrated higher predictive accuracy for hemolysis values in devices characterized by low shear conditions, while another three parameter sets exhibited better performance for devices operating under higher shear conditions. Empirical testing of the devices in a recirculating loop revealed levels of hemolysis significantly lower (<2 ppm) than the hemolysis ranges observed in conventional oxygenators (>10 ppm). Evaluating the model's ability to predict hemolysis across diverse shearing conditions, both through empirical experiments and computational validation, will provide valuable insights for future micro ECMO device development by directly relating geometric and shear stress with hemolysis levels. We propose that, with an informed selection of hemolysis parameters based on the shear ranges of the test device, computational modeling can complement empirical testing in the development of novel high-flow blood-contacting microfluidic devices, allowing for a more efficient iterative design process. Furthermore, the low device-induced hemolysis measured in our study at physiologically relevant flow rates is promising for the future development of microfluidic oxygenators and dialyzers.

Puzzling undetectable haptoglobin in a transfused pregnant patient.

BACKGROUND-AIM: Pregnancy induces physiological changes that can affect serologic and immunologic markers, potentially resulting in lower or undetectable haptoglobin values compared to non-pregnant counterparts. Such variations may lead to inaccurate diagnosis of hemolysis. METHODS: We report a case of a patient in second trimester of pregnancy receiving induction chemotherapy due to B-cell acute lymphocytic leukemia with undetectable haptoglobin levels in a routine laboratory sample collected less than 12 h posttransfusion of red cell unit. Despite undetectable haptoglobin, lactate dehydrogenase (LD) was within reference intervals (RI). The patient was evaluated for acute hemolytic transfusion reaction (AHTR) and followed up. Haptoglobin levels showed an upward trend during follow-up visits, reaching 15 mg/dL, and within RI in the third trimester. RESULTS: The patient did not meet the Center for Disease Control (CDC) criteria for AHTR. Alternative explanations for the observed laboratory findings were explored. Undetectable haptoglobin levels were attributed to various factors, including recent RBC transfusion, pregnancy-related physiological changes, and potential hyperhydration treatment plan due to chemotherapy. CONCLUSION: This case underscores the importance of cautious interpretation of laboratory results in pregnant patients, necessitating trimester-specific reference intervals for haptoglobin. A multidisciplinary approach to patient care is crucial for accurate diagnosis and management.

Purinergic Receptor Antagonists Inhibit Hemolysis Induced by Clostridium perfringens Alpha Toxin.

Clostridium perfringens alpha toxin (CPA), which causes yellow lamb disease in sheep and gas gangrene and food poisoning in humans, is produced by all types of C. perfringens and is the major virulence determinant of C. perfringens type A. CPA induces hemolysis in many species, including humans, murines, sheep and rabbits, through its enzymatic activity, which dissolves the cell membrane. Recent studies have shown that some pore-forming toxins cause hemolysis, which is achieved by the activation of purinergic receptors (P2). However, the relationship between P2 receptors and non-pore-forming toxin hemolysis has not been investigated. In the present study, we examined the function of P2 receptors in CPA toxin hemolysis and found that CPA-induced hemolysis was dependent on P2 receptor activation, and this was also true for Staphylococcus aureus ß-Hemolysin, another non-pore-forming toxin. Furthermore, we use selective P2 receptor antagonists to demonstrate that P2X1 and P2X7 play important roles in the hemolysis of human and murine erythrocytes. In addition, we found that redox metabolism was mainly involved in CPA-induced hemolysis using metabolomic analysis. We further demonstrate that CPA activates P2 receptors and then activates NADPH oxidase through the PI3K/Akt and MEK1/ERK1 pathways, followed by the production of active oxygen to induce hemolysis. These findings contribute to our understanding of the pathological effects of CPA, clarify the relationship between P2 activation and non-pore-forming toxin-induced hemolysis, and provide new insights into CPA-induced hemolysis.

Retrospective Evaluation of Clinical and Clinicopathologic Findings, Case Management, and Outcome for Dogs and Cats Exposed to Micrurus fulvius (Eastern Coral Snake): 92 Cases (2021-2022).

This retrospective, observational study describes the clinical findings, case management trends, and outcomes of 83 dogs and nine cats exposed to eastern coral snakes in a university teaching hospital setting. The medical records of dogs and cats that received antivenom following coral snake exposure were reviewed. Data collected included signalment, time to antivenom administration, physical and laboratory characteristics at presentation, clinical course during hospitalization, length of hospitalization, and survival to discharge. The mean time from presentation to coral snake antivenom administration was 2.26 ± 1.46 h. Excluding cases where the owner declined in-hospital care, the mean hospitalization time for dogs and cats was 50.8 h and 34 h, respectively. The mean number of antivenom vials was 1.29 (1-4). Gastrointestinal signs (vomiting and ptyalism) occurred in 42.2% (35/83) of dogs and 33.3% (3/9) of cats. Peripheral neurologic system deficits (ataxia, paresis to plegia, absent reflexes, and hypoventilation) were noted in 19.6% (18/92) of dogs and cats. Hemolysis was also common in 37.9% (25/66) of dogs but was not observed in cats. Mechanical ventilation (MV) was indicated in 12% (10/83) of dogs but no cats. Acute kidney injury (AKI), while rare, was a common cause of euthanasia at 20% (2/5) and was the most common complication during MV at 44.4% (4/9). Pigmenturia/hemolysis occurred in 88.9% (8/9) of MV cases and in all cases with AKI. Despite delays in antivenom administration by several hours, dogs and cats with coral snake exposure have low mortality rates (6% of dogs (5/83) and 0% of cats). Gastrointestinal signs were common but were not predictive of progression to neurological signs. Thus, differentiating between coral snake exposure and envenomation before the onset of neurological signs remains challenging.

Quantitative analysis of heme and hemoglobin for the detection of intravascular hemolysis.

BACKGROUND: Intravascular hemolysis is associated with massive release of hemoglobin and consequently labile heme into the blood, resulting in prothrombotic and proinflammatory events in patients. Though heme is well-known to participate in these adverse effects, it is not monitored. Instead, haptoglobin and hemoglobin serve as clinical biomarkers. The quantification of labile heme together with hemoglobin, however, should be considered in clinical diagnosis as well, to obtain a complete picture of the hemolytic state in patients. So far, quantification techniques for labile heme were not yet systematically analyzed and compared for their clinical application potential, especially in the presence of hemoglobin. RESULTS: Two commercial assays (Heme Assay Kit®, Hemin Assay Kit®) and five common approaches (pyridine hemochromogen assay, apo-horseradish peroxidase-based assay, UV/Vis spectroscopy, HPLC, mass spectrometry) were analyzed concerning their linearity, accuracy, and precision, as well as their ability to distinguish between hemoglobin-bound heme and labile heme. Further, techniques for the quantification of hemoglobin (Harboe method, SLS method, Hemastix®) were included to study their selectivity for hemoglobin and potential interference by the presence of labile heme. Both, indirect and direct approaches were suitable for the determination of a wide concentration of heme (∼0.02-45 µM) and hemoglobin (∼0.002-17 µM). A clear distinction between hemoglobin-bound heme and labile heme with one method was not possible. Thus, a novel combined approach is presented and applied to human and porcine plasma samples for the determination of hemoglobin and labile heme. SIGNIFICANCE: Our results demonstrate the need to develop improved techniques to differentiate labile and protein-bound heme for early detection of intravascular hemolysis. Here, we present a novel strategy by combining two spectroscopic methods, which is most reliable as an easy-to-use tool for the determination of hemoglobin and heme levels in plasma samples for the diagnosis of intravascular hemolysis and in basic biomedical research.