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α-Hemolysin (HlyA) is an extracellular protein toxin secreted by uropathogenic strains of Escherichia coli that inserts into membranes of eukaryotic cells. The main goal of this work was to investigate the involvement of tryptophan (W) residues in the hemolytic activity of HlyA. We investigated the hemolytic activity of six single-point mutant proteins, in which one of the four Ws was replaced by cysteine (C) or leucine (L). We also analyzed the photoinactivation of HlyA with pterin (Ptr), an endogenous photosensitizer, as a method of unspecific oxidation of W and tyrosine (Y) residues. HlyA photoinactivation was analyzed by ultraviolet-visible spectrophotometry, hemolytic activity measurement, fluorescence spectroscopy, and electrophoretic analysis. The results indicate that Ws are important in the hemolytic process. Specifically, the chemical structure of the amino acid at position 578 is important for the acylation of HlyA at residue K563. Furthermore, the exposure of HlyA to ultraviolet radiation, with energy similar to that experienced under sun exposure, in the presence of Ptr induces the inactivation of the toxin, causing chemical changes in, at least, W and Y, the rate of damage to W residues being faster than that observed for Y residues. This work not only deepens our understanding of the structure-function relationship of the toxin but also introduces the possibility of using photoinactivation of HlyA for potential applications such as obtaining innocuous molecules for vaccine production and the elimination of the toxin from contaminated surfaces and drinking water.

Hemolytic toxicity, as one of the key toxicity endpoints for small molecules, can cause lysis of the erythrocyte membrane and subsequent release of hemoglobin into blood plasma, leading to multiple acute and chronic adverse effects. Hence, it is necessary to assess the hemolytic toxicity of small molecules in an early stage of drug discovery and development process, and it is more significant to quantitatively predict the hemolytic toxicity of small molecules before costly and time-consuming experiments. Nevertheless, this endpoint has never been quantitatively predicted due to the lack of an appropriate dataset. In this work, we manually collected a quantitative hemolytic toxicity dataset containing 805 small molecules with experimental values of HD50 (50% hemolytic dose) from a variety of literature, built the first machine learning-based regression model to quantitatively predict the hemolytic toxicity of small molecules, and developed a pragmatic software for automatic prediction. Based on this model, we further implemented an automatic recursive fragmentation module to predict the hemolytic fragments with high fragment efficiency for the given compound(s), which may be of particular interest to experimental medicinal chemists. Therefore, we anticipate that this quantitative model may help medicinal chemists boost the development of promising lead compounds with low hemolytic toxicity or fuel the discovery of highly hemolytic chemical probes to delve into the in-depth mechanism of the hemolytic process.

Hemolytic anemia (HA) is not a common presentation in infectiousendocarditis (IE) and has only been reported in very few cases. Althoughdiseased valves may cause shearing stress that fragments RBCs, similar to thatassociated with mechanical heart valves, an autoimmune hemolytic process hasalso been implicated. We reported a case of patient who had HA secondary toacute IE due to MRSA. The anemia completely resolved after treating the (IE) .The cause was most likely mechanical shearing (schistocytes or fragmentedRBCs present on peripheral smear) by the large vegetation on native valve ;autoimmune hemolysis was considered unlikely in this case due to consistently negative Coombs tests and failure to respond to corticosteroids.

Paullinia cupana var. sorbilis (Mart.) Ducke, popularly known as guarana, is one of the most promising plants in Brazilian flora and has attracted considerable interest from the scientific community owing to its numerous therapeutic activities and less side effects. Hence, using nanotechnology is a viable alternative to primarily improve the physicochemical characteristics and bioavailability of guarana. The objective of the present study was to develop, characterize, and evaluate the stability of liposomes containing guarana powder and to evaluate their antioxidant and hemolytic activity in vitro. Three different concentrations of guarana powder and two methods of liposome preparation were tested. Liposomes were developed and characterized, and their stability was analyzed by evaluating physicochemical parameters. Hemolytic activity of guarana liposomal formulation (G-Lip) was compared with that of guarana in its free form (FG) and of liposome without guarana (W-Lip). Red blood cells from rats were exposed to these different formulations dissolved in phosphate buffer solution (PBS; pH 7.4). The best stability was achieved for the formulation containing 1 mg mL−1 guarana powder produced by the reverse phase evaporation method. FG showed dose-dependent antioxidant activity, which was maintained in G-Lip. W-Lip showed high hemolytic activity in PBS at pH 7.4 possibly because of the presence of polysorbate 80, and on addition of guarana to these structures, the hemolytic process was reversed. The same protective effect was observed for FG. It is believed that the complex structure of guarana, primarily the presence of polyphenols, exerts a powerful antioxidant action, helping to protect erythrocytes.

During active multiple sclerosis (MS), red blood cells (RBCs) harvested from patients reportedly display increased osmotic fragility and increased cellular volume (macrocytosis). The cause of these abnormalities remains unknown. We have previously proposed thatClostridium perfringensepsilon toxin (ETX) may be a blood-borne trigger for newly forming MS lesions based on its tropism for blood-brain barrier vasculature and CNS myelin. Recently, Gao et al. have reported that ETX binds to and damages human RBCs, leading to hemolysis. Moreover, the authors suggest that purinergic nucleotide (P2) receptor activation amplifies the hemolytic process. Here, we confirm that ETX indeed causes human-specific RBC lysis. However, our data suggest that the hemolytic process is mediated by metal-catalyzed oxidation of the swell-induced, nucleotide-sensitive ICln chloride channel. We use spectrophotometry, flow cytometry and Western blotting to show that ETX targets human RBCs and T lymphocytes via their shared expression of Myelin and Lymphocyte protein (MAL); a protein shown to be both necessary and sufficient for ETX binding and toxicity. ETX likely triggers T cells to release redox-active heavy metals, Cu+and Fe3+, via the lysosomal exocytosis pathway, while RBCs likely release these heavy metals via ETX pore formation within the RBC membrane. Extracellular Cu+and Fe3+may then amplify hemolysis by oxidizing a previously identified heavy metal-binding site within the ICln channel pore, thus deregulating its normal conductance. Elucidating the precise mechanism of ETX-mediated hemolysis may shed light on the underlying etiology of MS, as it would explain why MS RBC abnormalities occur during active disease.IMPORTANCEDuring active MS, numerous reports suggest that circulating RBCs are larger than normal and fragment more easily. The exact trigger(s) for these RBC abnormalities and for newly forming MS lesions remains unidentified. We have proposed that ETX, secreted by the gut bacteriumClostridium perfringens, may be an environmental trigger for newly forming MS lesions. Indeed, ETX has been shown to breakdown the BBB, enter the brain and damage the myelin sheath. Because ETX is typically spread through the circulatory system, we wished to determine how the toxin affects human blood. Provocatively, there has been a recent report that ETX produces cellular abnormalities in human RBCs, reminiscent of what has been described during active MS. In our study, we sought to elucidate the precise mechanism for how ETX causes RBC damage. In addition to triggering BBB breakdown and CNS demyelination, ETX might also explain why RBCs appear abnormal during MS attacks.

[Figure][1] These images were captured from the peripheral blood smear of a 56-year-old patient with Child-Pugh C alcoholic cirrhosis, ongoing alcohol abuse, and chronic macrocytic anemia with baseline hemoglobin 7 to 8 g/dL. Laboratory work suggested a hemolytic process, with a mixed

The hemolytic activity of N,N′-substituted piperazines and their influence on the hemolytic process initiated by radiation (red LED, 653 nm) in the presence of radachlorin (RADA-FARMA, Russia) photosensitizer and on complement-dependent hemolysis were studied. All compounds did not cause human RBC lysis in the studied concentration range (0.15 – 3.0 mM) although dose-dependent inhibition of photo-induced hemolysis that manifested as an increase of the 50% lysis time was observed. The piperazine derivatives did not substantially influence RBC lysis by the activated complement. Therefore, the antihemolytic effect of the N,N’-substituted piperazines was due to their antioxidant activity.

The classical complement pathway and, to some extent, the terminal pathway, are involved in the immune pathogenesis of autoimmune hemolytic anemia (AIHA). In primary cold agglutinin disease (CAD), secondary cold agglutinin syndrome and paroxysmal cold hemoglobinuria, the hemolytic process is entirely complement dependent. Complement activation also plays an important pathogenetic role in some warm-antibody AIHAs, especially when immunoglobulin M is involved. This review describes the complement-mediated hemolysis in AIHA with a major focus on CAD, in which activation of the classical pathway is essential and particularly relevant for complement-directed therapy. Several complement inhibitors are candidate therapeutic agents in CAD and other AIHAs, and some of these drugs seem very promising. The relevant in vitro findings, early clinical data and future perspectives are reviewed.

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause hemolytic uremic syndrome (HUS). This study investigated whether Stx2 induces hemolysis and whether complement is involved in the hemolytic process. RBCs and/or RBC-derived microvesicles from patients with STEC-HUS (n = 25) were investigated for the presence of C3 and C9 by flow cytometry. Patients exhibited increased C3 deposition on RBCs compared with controls (p < 0.001), as well as high levels of C3- and C9-bearing RBC-derived microvesicles during the acute phase, which decreased after recovery. Stx2 bound to P1k and P2k phenotype RBCs, expressing high levels of the Pk Ag (globotriaosylceramide), the known Stx receptor. Stx2 induced the release of hemoglobin and lactate dehydrogenase in whole blood, indicating hemolysis. Stx2-induced hemolysis was not demonstrated in the absence of plasma and was inhibited by heat inactivation, as well as by the terminal complement pathway Ab eculizumab, the purinergic P2 receptor antagonist suramin, and EDTA. In the presence of whole blood or plasma/serum, Stx2 induced the release of RBC-derived microvesicles coated with C5b-9, a process that was inhibited by EDTA, in the absence of factor B, and by purinergic P2 receptor antagonists. Thus, complement-coated RBC-derived microvesicles are elevated in HUS patients and induced in vitro by incubation of RBCs with Stx2, which also induced hemolysis. The role of complement in Stx2-mediated hemolysis was demonstrated by its occurrence only in the presence of plasma and its abrogation by heat inactivation, EDTA, and eculizumab. Complement activation on RBCs could play a role in the hemolytic process occurring during STEC-HUS.

Uropathogenic Escherichia coli often produce the virulence factor α-hemolysin (HlyA), and the more severe the infection, the likelier it is to isolate HlyA-producing E. coli from patients. HlyA forms pores upon receptor-independent insertion of the toxin into biological membranes and it has been substantiated that HlyA-induced hemolysis is amplified by toxin-induced ATP release and activation of P2X receptors. Thus, hemolysis inflicted by HlyA is a protracted process involving signal transduction. It consists of early, marked cell shrinkage followed by swelling and eventually lysis. The initially shrinkage is a consequence of a substantial Ca(2+)-influx and activation of Ca(2+)-sensitive K(+) and Cl(-) channels (KCa3.1/TMEM16A). The shrinkage is followed by gradual cell swelling, which ultimately lyses the cells. These findings clearly show that the HlyA pore provides a substantial volume challenge for the cells, and the fate of the given cell is co-determined by intrinsic erythrocytal volume regulation. We therefore speculated that other mechanisms involved in erythrocyte volume regulation may influence the hemolytic process inflicted by HlyA. Strikingly, HlyA-induced hemolysis is markedly reduced in erythrocytes isolated from NKCC1-deficient (NKCC1(-/-)) mice compared to controls. The NKCC1 inhibitors furosemide and bumetanide concentration-dependently inhibit HlyA-induced lysis of human and murine erythrocytes. However, in high concentrations bumetanide further reduced hemolysis in erythrocytes from NKCC1(-/-) mice and, thus, also exhibit indirect effects on hemolysis. The effect of loop diuretics on the hemolysis is not unique to HlyA but is similarly seen in LtxA- and α-toxin-induced hemolysis. Bumetanide clearly potentiates HlyA-induced volume reduction and delays the following erythrocyte swelling. This allows increased phagocytosis of damaged erythrocytes by THP-1 cell as a result of prolonged cell shrinkage. These data suggest that erythrocyte susceptibility to cytolysins is modified by NKCC1 and signifies intrinsic volume regulators as important determinants of cellular outcome of pore-forming toxins.

Introduction: Hemolytic anemia is a common cause of anemia in our country. It may results from a cellular defect that changes the shape of RBCs from biconcave to spherical as in spherocytosis or elliptical as in elliptocytosis. It may results from RBCs enzyme deficiency as in cases with pyruvate kinase deficiency or glucose-6-phosphate dehydrogenase enzyme deficiency (G6PD deficiency). Other causes of hemolytic anemia include hemoglobinopathy‚ autoimmune antibodies against RBCs‚ hypersplenism and others.G6PD deficiency is the most important disease of the hexose monophosphate pathway and is responsible for two clinical syndromes‚ an episodic hemolytic anemia induced by infections‚ certain drugs or fava beans and a spontaneous chronic non spherocytic hemolytic anemia.G6PD deficiency is an inherited disorder caused by a genetic defect in the RBCs enzyme G6PD‚which generates NADPH and protects RBCs from oxidative injury. G6PD deficiency is the most common enzymatic disorder of RBCs. Although G6PD is a critical enzyme in the redox metabolism of all aerobic cells‚ yet its role in the RBCs is more critical because it is the only source of NADPH which directly and via GSH defends these cells against oxidative stress. G6PD deficiency is an example of hemolytic anemia due to interaction between an intra-corpuscular cause and an extra corpuscular cause‚ because in the majority of cases hemolysis is triggered by an exogenous agent. The severity of hemolytic anemia varies among individuals with G6PD deficiency. Education of patients and their parents regarding safe and unsafe medications and foods is crucial to prevent future episodes of hemolysis. In many cases all members of the patient’s family should avoid such precipitating foods. G6PD deficiency is a sex-linked disorder. As result males who inherit a G6PD mutation are hemizygous for the defect all their RBCs are affected. Females who inherit a heterozygous G6PD mutation usually do not have severe hemolytic anemia‚ since half of their RBCs express the abnormal allele. The majority of females who inherit an abnormality in G6PD are unaffected carriers. However, the cells that express the abnormal allele are vulnerable to hemolysis as the enzyme deficient RBCs in males. The presence of anemia will vary depending on the severity of the deficiency in the affected cells and whether there is skewed x-inactivation (lyonization) that results in a greater expression in a large percentage of RBCs. G6PD deficiency is a common genetic disorder‚ affecting nearly 400 million individuals worldwide. Whilst it is known that a number of drugs‚ foods and chemicals can trigger hemolysis in G6PD deficient individuals‚ the association between herbal and dietary supplements and hemolysis is less clear. Amongest nutrients blamed as triggers for hemolysis in G6PD deficient individuals are fava beans and related ligaments. Oxidant drugs and infections also predispose to hemolytic attacks. Provided that the blood of a G6P deficient person is normal‚ acute hemolysis results from the action of an exogenous factor on the RBCs deficient in glucose-6-phosphate dedydrogenase (G6PD). The function of G6PD in RBCs is to provide the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) necessary for the conversion of oxidized glutathione to the reduced state (GSH) as protection against the oxidation of RBCs. So that reduced glutathione (GSH) acts as an anti-oxidant that inactivates oxidant compounds such as hydrogen peroxide‚ that are normally generated within the RBCs. If GSH or any enzyme needed for maintaining glutathione in the reduced form (GSH) is deficient‚ the SH group in the RBCs membrane is oxidized and the hemoglobin of the RBCs is denatured and may precipitate in the RBC inclusions called Heinz bodies. An acute hemolytic process (crisis) results from damage to the RBC membrane by precipitated hemoglobin induced by the exogenous oxidant agent. The damaged RBCs are rapidly removed from the circulation causing acute drop in Hb level and the acute hemolytic crisis. This episodic acute hemolytic anemia may be induced by infections‚ certain drugs and fava beans. Glucose-6-phosphate dedydrogenase (G6PD) deficiency is inherited in an x-linked manner‚ so that the synthesis of RBCs G6PD is determined by a gene on x-chromosome. Thus most females do not usually have evident clinical hemolysis after exposure to oxidant agents‚ unless new gene mutation has occurred. In Egypt ingestion of fava beans produces an acute severe hemolytic crisis known as favism. Fava beans contain divicine‚ isouramil and convicine which ultimately lead to production of hydrogen peroxide and other reactive oxygen species production. Therefore, seasonal increased prevalence of G6PD deficiency crisis occurs in the green beans growth season. This is usually manifested by drop in Hb‚ hemoglobinuria‚ hyperbilirubinemia and reticulocytosis. These patients usually have to be rescued by packed RBCs transfusion. The list of drugs to be avoided in G6PD deficient individuals (whether causing predictable or possible hemolysis) must be given to all cases that present in crisis.The aim of the study: To find out how much the staff in the Emergency unit are sticking to the agreed upon unit’s protocol as well as to compare the unit’s protocol with European Guidelines.Subjects: Children from 1 day to 18 years of age with G6PD acute hemolytic crisis attending Assiut University Children Hospital over one year 2015-2016.Inclusion criteria: All cases of G6PD deficiency acute hemolytic crisis.Tools of study: The investigations stated in the unit’s protocol included CBCs‚ urine dipsticks‚ blood urea and creatinine‚ serum bilirubin (direct and indirect) as well as blood grouping and cross matching. After treatment of the crisis (as stated in the unit’s protocol) a list of drugs and agents to be avoided was given to the patients. The patients were advised to return to follow up after four weeks for the measurement of G6PD enzyme level in the outpatient department in Assiut university children Hospital.Patients and methods: This study was performed in the Emergency Unit in Assiut University Children Hospital for one year 2015-2016 (Nov‚ 2015 to Nov‚ 2016). The study included fifty cases of acute G6PD hemolytic crisis admitted to the emergency unit: they were aged nine months to four years. They were 45 males and 5 females. The investigations stated in the unit’s protocol included CBCs‚ urine dipsticks‚ blood urea and creatinine‚ serum bilirubin (direct and indirect) as well as blood grouping and cross matching. After treatment of the crisis (as stated in the unit’s protocol) a list of drugs and agents to be avoided was given to the patients. The patients were advised to return to follow up after four weeks for the measurement of G6PD enzyme level in the outpatient department in Assiut university children Hospital.Results: The study included fifty cases of acute G6PD hemolytic crisis admitted to the emergency unit: they were aged nine months to four years. They were 45 males and 5 females.

Background Hemolytic anemias are defined by the premature destruction of red blood cells. These anemias have many causes that are mostly due to chronic diseases, but, occasionally, cases of acute life-threatening hemolysis can occur. Objective The objectives of this article were to review the pathophysiology of hemolytic anemias, to discuss the general emergency department (ED) evaluation, and to discuss the assessment and treatment of important and “cannot miss” hemolytic diseases. Discussion Because hemolytic anemias are rarely seen, the emergency physician may consider a patient's anemia as due to blood loss rather than hemolysis, and the workup and treatment may not be appropriate. The primary goal for the emergency provider is to resuscitate, but he or she also must recognize that a hemolytic process is present. Appropriate laboratory work and specialist consultation should be obtained. While focused treatment is rarely necessary in the ED, the avoidance of certain treatments, such as early platelet transfusion in thrombotic thrombocytopenic purpura, may be necessary. Conclusions Hemolytic anemias are rare, but should still be considered in the ED differential diagnosis of low hemoglobin. Emergency physicians should first resuscitate, but should also be able to identify the presence of hemolysis and obtain the appropriate laboratory tests. Occasionally, specific treatments are needed but should be discussed in conjunction with a specialist.

The objective of this study was to investigate the in vitro effect of Vipera lebetina venom on human erythrocytes’ osmotic fragility with respect to different venom concentrations, time of incubation with the venom, initial volume of red blood cells, and presence of antivenom. In vitro osmotic fragility of human erythrocytes was determined using Dacie and Lewis method. Osmotic fragility is increased significantly after incubation with the venom at different concentrations. The maximal effect was detected at the venom concentration of 400 μg. Time of incubation with the venom was an important factor in hemolytic process while the initial cell volume has no significant effect. Incubation of envenomed erythrocytes for 120 min with antivenom did not reverse the changes in osmotic fragility induced by the venom. The study suggests that the venom could alter the susceptibility of erythrocytes to hemolysis when subjected to osmotic stress and the degree of hemolysis depends on venom concentration and time of intubation with the venom.

In the paper, we present an improved method for evaluation of a compound ability to destabilize erythrocyte plasma membrane. The proposed method is based on the continuous monitoring of the light scattered by erythrocytes exposed to osmotic pressure differences. The kinetics of hemolysis depends on the plasma membrane mechanics and the extent of the osmotic stress. Generally, the osmotic pressure difference of approximately 150 mOsm is taken for measurements, as a result of the equal volume mixing with the physiological salt solutions. In this approach the hemolytic process completion is not established which may result in poor quality and reproducibility of the experimental data. In consequence, inaccurate parameters of the kinetic are determined due to the low quality fitting to the, widely used, single exponential model. In the paper we propose a new experimental protocol allowing to determine the extended set of parameters for kinetics of hemolysis. Namely, the method of the minimal osmotic pressure difference determination is proposed which ensures the completeness of the hemolytic process. This step allows improving the quality and exactness of the calculated parameters. The developed methodology was tested on two qualitatively different, biologically relevant, experiments; evaluation of the peptide effect on the plasma membrane properties and differentiating between human and rabbit erythrocytes.

Although recently published case reports suggest the significance of Jr(a) alloimmunization in the obstetric setting, the involved mechanism still remains unclear. Here we report a case of severe fetal and neonatal anemia associated with anti-Jr(a) alloimmunization, which was successfully managed using Doppler assessment of peak systolic velocity of the fetal middle cerebral artery (MCA-PSV). A Japanese woman with anti-Jr(a) (titer 1024) was referred to our department at 20 weeks' gestation. As fetal MCA-PSV exceeded 1.5 multiple of median, labor was induced and a female neonate of 1998 g was delivered vaginally at 33 weeks and 5 days of gestation. The infant's hematocrit and hemoglobin levels were 25.4% and 82 g/L, respectively, but her total bilirubin level (15 µmol/L; 0.9 mg/dL) and reticulocyte counts (4.5%) were low. During the course, the infant showed no apparent signs of hemolysis. Jr(a) alloimmunization should be recognized as a possible cause of fetal anemia with no direct hemolytic process.

Tolaasin, a pore-forming peptide toxin produced by Pseudomonas tolaasii, causes brown blotch disease on cultivated mushrooms. Hemolysis using red blood cells was measured to evaluate the cytotoxicity of tolaasin. To investigate the mechanism of tolaasin-induced cell disruption, we studied the effect of temperature on the hemolytic process. At 4 degrees Celsius, poor binding of the tolaasin molecules to the erythrocyte membrane was observed and most of the tolaasin molecules stayed in the solution. However, once tolaasin bound to erythrocytes at 37 degrees Celsius and the temperature was decreased, complete hemolysis was observed even at 4 degrees Celsius. These results indicate that tolaasin binding to cell membrane is temperature-sensitive while tolaasin-induced membrane disruption is less sensitive to temperature change. The effect of erythrocyte concentration was measured to understand the membrane binding and pore-forming properties of tolaasin. The percentage of hemolysis measured by both hemoglobin release and cell lysis decreased as erythrocyte concentration increased in the presence of a fixed amount of tolaasin. The result shows that hemolysis is dependent on the amount of tolaasin and multiple binding of tolaasin is required for the hemolysis of a single cell. In analysis of dose-dependence, the hemolysis was proportional to the tenth power of the amount of tolaasin, implying that tolaasin-induced hemolysis can be explained by a multi-hit model.

The understanding of the oxidative stress mechanisms helps to explain many of the processes of cellular lesion and death, especially those related to the hemolytic diseases. Sickle cell anemia, thalassemias and G6-PD deficiency are among the more frequent genetic anomalies accompanied by oxidative stress. In the sickle cells, one of the factors that predisposes to the hemolytic process is the oxidative degradation of the hemoglobin S due to its deoxigenation leading to hemichrome formation and precipitation as Heinz bodies. The oxidative stress contributes to the sickle process and shortening of the erythrocyte survival. Here we analyzed the oxidative process in erythrocytes of patients with two different genotypes for HbS (AS and SS). Units of blood from donors of the Center of Hematology and Hemotherapy of Paraná (HEMEPAR), from normal individuals (AA) and from heterozygote individuals (AS), and venous blood collected from patients with sickle cell anemia (SS) were analyzed. In order to evaluate the protective action of the vitamins C and E in oxidative stress, erythrocytes were treated with antioxidant substances, vitamin C and vitamin E, and then treated with the oxidant tert-butilhydroperoxide (TBHP). The oxidative action induced by TBHP was observed in erythrocytes AAASSS, by the increase in the content of Heinz bodies, methemoglobin, hemolysis, GSH depletion and lowering activities of the enzymes G6-PD and GR. The protective actions of the vitamins C and E for the oxidative stress induced by TBHP were observed for the erythrocytes in the lowering Heinz bodies, methemoglobin, hemolysis, and partial recovery of GSH more efficiently in AS and SS erythrocytes. Recovery was not observed in the levels of the activities of the enzymes G6-PD and GR, under the vitamins actions. The results obtained confirmed the higher susceptibility of the sickle erythrocyte to oxidation which necessitates precaution in the transfusion adequacy of AS erythrocytes. On the other hand, the protective effect of the vitamins C and E over the oxidative stress observed in erythrocytes AS and SS open perspectives for their use for treatment of patients with sickle cell anemias, as well as in the preservation of transfusional erythrocyte bag units.

None of experimental models used to study the toxic effect of unconjugated bilirubin brain accumulation, reproduce the conditions in which the hyperbilirubinemia is a consequence of a hemolytic process, i.e. when important amounts of bilirubin and iron are released. The aim was to develop an animal model to determine the role of bilirubin and iron, in the encephalopathy secondary to a hemolytic disease. Male Wistar rats 7 days old (n = 30) were treated with phenylhydrazine as hemolytic at 75 mg/kg body weight intraperitoneally for 2 days and euthanized 24 h after the last dose. Hemoglobin, hematocrit, serum and brain bilirubin, serum iron and lipoperoxidation products, as well as neuronal damage and iron positive staining were evaluated and compared among treated and untreated (n = 10) animals. The animals with induced hemolysis showed significant reduction in hemoglobin and hematocrit, increased concentration of total and conjugated bilirubin, as well as of serum iron and lipid peroxidation products. The neuronal damage in treated animals included the presence of altered neurons spread out among normal cells, as well as of iron-staining positive cells. With the use of appropriated pharmacological procedures, the characteristics of the model can be useful to dissect the participation of both bilirubin and iron, on the bilirubin encephalopathy secondary to hemolysis.

Interactions between maltose functionalized hyperbranched poly(ethylene imine)s (95% maltose decoration denoted as Mal-PEI A; 33% maltose decoration denoted as Mal-PEI B) and red blood cells (RBCs) and between red blood cell membranes were investigated. We monitored the degree of hemolysis, the change in cell shape, the influence of polymers on the fluidity of the cell membrane and some cell membrane enzymes to determine their possible cytotoxic impact on them. To observe the extent of hemolysis, the RBCs were incubated with different concentrations of Mal-PEIs. The first significant lysis of RBCs was observed after 6h of incubation. Prolongation of the incubation time increased the number of ruptured cells. Moreover, we observed that Mal-PEI B was more hemolytic than Mal-PEI A in buffer solution. In contrast, an incubation of RBCs with Mal-PEIs in human plasma significantly decreased the hemolytic process and showed higher hemolytic property of Mal-PEI A compared to Mal-PEI B. Also several changes in the shape of the RBCs occurred after incubation with Mal-PEIs. Some of the erythrocytes shrank (echinocytes), but their morphology generally remained unchanged during the incubation. As shown by fluorescence experiments, both polymers induced the increase of fluidity of RBCs membranes. In summary, both types of hyperbranched poly(ethylene imine)s were practically non-hemolytic even at high polymer concentrations. Mal-PEI B was slightly more noxious than the Mal-PEI A in a buffer solution, while in blood plasma, the situation was opposite. Decrease of Na+/K+ ATPase and total ATPase enzymes activity was related with molecule size and number of maltose groups on the surface of molecule. The low hemolytic properties only observed at higher concentration (100μM and 400μM) indicated that Mal-PEIs are promising macromolecules in the area of drug delivery systems.

RBC transfusions in a patient with a history of autoimmune hemolytic anemia (AIHA) can represent both a laboratory and a clinical challenge. The development of high-titer low-avidity antibodies and antibodies to high-frequency antigens may further impair the ability to identify compatible donor RBCs. Not infrequently, incompatible RBCs must be used and the desire to increase oxygen carrying capacity conflicts with the desire to avoid exacerbating the autoimmune hemolytic process with RBC transfusions. A 66-year-old Caucasian female with coronary artery disease and a history of refractory AIHA had recently developed anemia and required multiple RBC transfusions. The patient had maintained adequate RBC counts with erythropoietin and prednisone therapy for the previous 16 months. With the recent worsening of her hemolytic anemia, she had developed angina that was treated with RBC transfusions in an outpatient setting. However, her angina increased as her RBC counts decreased, leading to hospital admission for further management of her hemolytic anemia and angina. She subsequently required multiple incompatbile RBC transfusions despite increased prednisone therapy and did not improve until after coronary artery stent placement and high dose IVIG therapy. This case demonstrates the usefulness of early patient phenotyping in a case of accelerating hemolytic anemia to aid in donor RBC selection, the value of communicating with clinicians and the patient regarding the use of least-incompatible RBCs, and the importance of optimizing the patient’s clinical condition to avoid ischemia. In addition, it demonstrates the value of repeated attempts with IVIG treatment despite previous refractoriness to this treatment.

Hemoglobin F (HbF) is an effective inhibitor of HbS polymerization. Hydroxyurea (HU) is used to increase HbF synthesis and improve the clinical course of sickle cell disease (SCD) patients. We studied a series of laboratory parameters concerning HbF production and reticulocyte response, and compared data between two groups: 1) 13 SCD patients treated with HU, and 2) 33 untreated SCD patients. Higher values of Hb concentration, mean cell volume (MCV), mean cell hemoglobin (MCH), mean reticulocyte volume (MRV), HbF concentration, percentage of F‐cells, and amount of HbF/F‐cells were observed in the treated group of patients. There was no correlation between Hb and HbF elevations. The reticulocyte count, immature reticulocyte count, mean fluorescence index (MFI), and neutrophil count were significantly lower in treated patients. Taken together, these findings suggest that a decreased hemolytic process occurred in patients undergoing HU treatment. There was a significant correlation between MCV and HbF, between MRV and HbF, and between MRV and F‐cell in patients taking HU. These data indicate that macroreticulocytes correspond to F‐reticulocytes, and that an increase in MRV in SCD patients using HU may be an indirect signal of F‐cell production. The concentration of HbF/F‐cells was higher in patients treated with HU, but this increase apparently was independent of F‐cell production. Reticulocyte (RTC) parameters, as assessed by hematological analyzers, may be useful for following erythropoietic changes in patients receiving HU, and can indirectly indicate HbF and F‐cell production induced by HU therapy. J. Clin. Lab. Anal. 17:66–72, 2003. © 2003 Wiley‐Liss, Inc.

Increased hemolysis in the presence of severe neonatal hyperbilirubinemia appears to augment the risk of bilirubin neurotoxicity. The mechanism of this intensifying effect is uncertain. In direct antiglobulin titer (DAT) positive, isoimmune hemolytic disease, the bilirubin threshold at which neurotoxicity occurs appears to be lower than in DAT-negative hyperbilirubinemia. In other hemolytic conditions, the hemolysis may simply facilitate the development of extremely high serum bilirubin levels. Whether the hemolytic process per se exerts an independent effect or whether a very rapid rise in serum bilirubin might lead to greater penetration of the blood–brain barrier is unclear. In this review, we survey the synergistic role of hemolysis associated with severe hyperbilirubinemia in the potentiation of bilirubin-induced neurotoxicity and suggest methods of identifying at-risk babies with increased hemolysis to allow for their increased surveillance.

BACKGROUND Maternal serum anti-D is actively transported across the placenta into the fetal blood where it adheres to D-positive erythrocytes. The anti-D coated red cells attach to Fc-receptors on fetal reticuloendothelial cells and are subsequently phagocytosed and hemolysed. It is not known if anti-D is consumed or recirculated in this process. MAIN QUESTION: Is anti-D in the fetus consumed in the hemolytic process in the erythroblastotic fetus and can the consumption be modulated by high-dose immunoglobulins (i.v.IG) given to the mother? METHODS Fetal/maternal serum anti-D concentration ratios were calculated for simultaneously taken blood samples from three groups of Rh(D) immunized pregnant women; six women with fetuses who were of Rh(D) negative phenotype, 19 women with fetuses who were Rh(D) positive and received no treatment and, seven women who were treated with i.v.IG because they bore anemic, Rh(D) positive fetuses. RESULTS Fetuses with a Rh(D) negative phenotype expressed an increase in fetal/maternal anti-D concentration ratios from 10 to 55% between 25 and 31 gestational weeks, while Rh(D) positive fetuses without i.v.IG treatment had stable values at the 10% level between 24 and 35 gestational weeks. During i.v.IG-treatment of the mothers, Rh(D) positive fetuses showed an increase in ratio from 10 to 30% between 26 and 34 gestational weeks. There was a statistically significant (p

Human erythrocytes, preincubated with different concentrations of calcium chloride (0.17–1.67 mM) showed hemolysis after addition of bilirubin (72 μM). Hemolysis was observed only when cells were incubated first with calcium followed by bilirubin and not vice versa. This hemolysis was found to be dependent upon several factors such as concentration of bilirubin, time of incubation of erythrocytes with calcium and time of incubation of bilirubin with the calcium-loaded erythrocytes. Inclusion of EDTA in the incubation medium reduced the percentage hemolysis to a significant extent. Involvement of activated oxygen species in hemolytic process seems to be unlikely as inclusion of sodium azide and catalase did not prevent hemolysis. A comparison of other bivalent cations such as Ba2+, Mg2+, Mn2+ and Cu2+ with Ca2+ for their ability to hemolyse cells in presence of bilirubin shows that Ba2+ and Mg2+ are ineffective, whereas both Mn2+ and Cu2+ induce hemolysis both in the absence as well as in the presence of bilirubin. However, their mechanism of hemolysis is different from that of calcium-induced hemolysis. Formation of calcium-induced hydrophobic aggregates of phospholipid molecules in erythrocyte membrane may open the new binding sites for bilirubin on these membranes which may perturb the membrane conformation.

Amphotericin B (AmB) is a very effective antifungal and antiparasitic drug with a narrow therapeutic window. To improve its efficacy/toxicity balance, new controlled release formulations have been developed based on different encapsulation systems, aggregation states and particle sizes modifications. The kinetics of the hemolytic process was studied not only to characterize the toxicity of different formulations but also as an indicator of drug release. Pharmacokinetic studies in beagle dogs were carried out with those formulations that exhibited the least hemolytic toxicity: liposomal formulation (AmBisome®), poly-aggregated AmB and encapsulated particulate AmB formulation. A novel poly-aggregated AmB formulation proved to be comparable in terms of low hemolytic activity with the marketed gold standard formulation: AmBisome®. Its pharmacokinetic profile, characterized by a smaller area under the curve and larger volume of distribution, was markedly different from AmBisome®, resulting in a cost-effective alternative for the treatment of leishmaniasis which can enhance the AmB passive target by the uptake by the cells of the reticulo-endothelial system. Effects of different variables such as type of formulation, dose, microencapsulation, anesthesia and dog's healthy state on AmB pharmacokinetics were studied.

Transesophageal echocardiography (TEE) has become increasingly important in the assessment of mitral valve prosthetic dysfunction. We report on three patients who developed medically refractory hemolytic anemia after St. Jude mitral valve replacement secondary to perivalvular mitral prosthetic regurgitation. Two of the three cases had corrective surgical intervention that resolved the hemolytic process. Careful transesophageal echocardiographic evaluations of the perivalvular area proved diagnostic and resulted in appropriate management decisions.

The biochemical effect of N-acylethanolamines on trout erythrocytes from Salmo irideus was studied by following their susceptibility to hemolysis and measuring steady-state fluorescence anisotropy of the fluorescent probe, 1,6diphenyl-1,3,5-hexatriene (DPH) on isolated membranes. The hemolytic process was strongly affected by different ethanolamine derivatives. However, only slight changes in fluorescence anisotropy were detected in these models, therefore, it appears that acylethanolamine derivatives do not produce significant variations in the physico-chemical characteristics of the membrane. The localization of these molecules in particular microdomains is hypothesized, thus locally modifying physiological membrane activities.

SLS複合体のpIは5.4, 分子量は32,000であり, 非還元状態でも溶血活性を示した. SLSおよびSLOの溶血作用機序はすでに報告されているが, K+およびHbの遊離態度より機序の差異を確認した. また, 溶血保護物質を用いた実験からは dextran 4以上の分子径でHbの遊離が阻止された. このことよりSLSの惹起する溶血では, 膜のイオン透過性変化過程を経て, 膜内への水の流入が開始する段階では膜に孔が形成されている可能性が示唆された. また, 孔径は種々分子径の異なる物質による溶血保護効果の成績より, 約3.5nmと測定された. これまでSLSによる溶血では膜上に孔様構造は認められないと報告されてきたが, 溶血保護効果の成績からは孔の形成が強く示唆された. さらに, SLSの示すHEは3.49, SLOは9.00と大きな差異が認められることより, HEは溶血機序解析の指標となりうることが明らかになった.

Publisher Summary There are two major biochemical pathways in the red cell: the Embden–Myerhof anaerobic pathway (energy potential of the cell) and the hexose monophosphate shunt (reduction potential of the cell). In this chapter enzyme reactions in the red cell are presented as a flow diagram. Enzyme defects have also been highlighted in this chapter. For example pyruvate kinase (PK) is an enzyme active in the penultimate conversion in the Embden–Meyerhof pathway. Although deficiency is rare, it is the most common enzyme abnormality in the Embden–Meyerhof pathway. In diagnosis of haemolytic anemia it is evident that premature destruction of red cells may result from corpuscular, that is, abnormalities of membrane, enzymes, or hemoglobin; or from extracorpuscular abnormalities, that is, immune or nonimmune mechanisms. An essential feature of hemolytic anemia is a reduction in the normal red cell survival of 120 days. The approach to the diagnosis of hemolytic anemia should include consideration of the clinical features suggesting hemolytic disease, laboratory demonstration of the presence of a hemolytic process, and determination of the precise cause of the hemolytic anemia by special hematologic investigations. Causes of hemolytic anemia due to corpuscular defects have also been listed in this chapter. Hereditary spherocytosis, elliptocytosis, stomatocytosis, acanthocytosis, xerocytosis and pyropoikilocytosis are diagnosed on the basis of their characteristic morphologic abnormalities. Enzyme defects and many hemoglobinopathies have nonspecific morphologic abnormalities related to secondary effects on red cell membrane proteins and pumps.

Publisher Summary During neonatal period anemia is caused by hemorrhage, hemolysis, or failure of red blood cell production. The condition of hemorrhage arises when blood loss occurs during the prenatal, intranatal, or postnatal period. Prenatal blood loss may be transplacental, intraplacental, or retroplacental or may be due to a twin-to-twin transfusion. Hemolytic anemia in the newborn is usually associated with an abnormally low haemoglobin level, an increase in the reticulocyte count and with unconjugated hyperbilirubinemia. The hemolytic process is often first detected as a result of investigation for jaundice during the first week of life. The causes of hemolytic anemia in the newborn are listed in this chapter. The failure of red blood cell production can be congenital or acquired. Viral interference (e.g., CMV, HIV) with fetal hematopoiesis may cause anemia, leukopenia and thrombocytopenia in the newborn. HIV disease may be associated with a number of hematologic abnormalities. Other kinds of anemia such as anemia of prematurity and physiologic anemia are also described in this chapter. Lastly, the chapter outlines the diagnostic approach to anemia in new born and also highlights the treatment procedure in respective cases.

Vitamin B 12 deficiency is an uncommon disorder in infancy. Most cases are because of maternal deficiency resulting from insufficient storage and/or reduced intake and are generally seen in exclusively breast-fed infants. Accentuation of the hemolytic process has never been described in association with Varicella Zoster Virus (VZV) infections.We describe a 9-months-old breast-fed infant with megaloblastic anemia secondary to maternal vitamin B 12 deficiency. He presented severe pancytopenia and regression of motor functions and developed hemolytic crisis during a VZV infection.Nutritional cobalamin deficiency should be considered in anemic infants with a history of prolonged exclusive breastfeeding and delayed developmental milestones. VZV infection can trigger a hemolytic process in infants with severe megaloblastic anemia secondary to B12 deficiency. A normal mean corpuscular volume does not rule out megaloblastic anemia, when the condition is combined with severe hemolysis.

Hemolytic and subhemolytic blood damage by mechanical heart valve prostheses have been observed in both clinical and in vitro investigations. A direct comparison between these studies is not possible. Nevertheless the transfer of some in vitro results to the behaviour of the valve in situ may be performed considering the similarity principle. This requires the use of dimensionless similarity numbers such as the plasma's hemoglobin concentration (PHb) or others, instead of dimensioned parameters. To evaluate the in vitro hemolysis of valve prosthesis a test chamber filled with human banked blood was used. An artificial ventricle ensuring an oscillatory flow through the valve was also used. The rise of PHb was evaluated in terms of a similarity number, called the lysis number. This number describes the probability of destroying a single red blood cell participating once in the hemolytic process under consideration. The lysis number, a Björk-Shiley valve (TAD 29), was found to be in the order of 2 × 10−4. From this, the survival time of erythrocytes in patients with an artificial heart valve was estimated. It was found to be in the order of 20 d of T50 Cr in agreement with clinical results

Background and objectives: Atypical hemolytic uremic syndrome (aHUS) is associated with a congenital or acquired dysregulation of the complement alternative pathway that leads to continuous complement activation on host cells causing inflammation and damage. Eculizumab, a humanized mAb against complement protein C5, inhibits activation of the terminal complement pathway. Design, setting, participants, & measurements: We report an adolescent with relapsing unclassified aHUS. On admission, a high plasma creatinine level indicated a poor prognosis, and hemodialysis had to be started. Plasma exchanges were initially effective against the microangiopathic hemolytic activity and allowed a temporary improvement of renal function with termination of hemodialysis after 7 wk. Subsequently, plasma exchanges (three times per week) failed to prevent ongoing aHUS activity and progressive renal failure. After 12 wk, aHUS treatment was switched to eculizumab. Results: Eculizumab was effective in terminating the microangiopathic hemolytic process in two aHUS relapses; however, after normalization of complement activity, aHUS recurred and ultimately led to anuric end-stage renal failure. Conclusions: In this patient, complement inhibition by eculizumab temporarily terminated the microangiopathic hemolytic activity. Nevertheless, renal damage as a result of preceding and subsequent aHUS activity resulted in end-stage renal failure; therefore, therapeutic success may depend on early administration of eculizumab. The optimal duration of treatment may be variable and remains to be determined.

Anemia is a common finding in post-liver transplant patients. Causes for the anemia include nutritional deficiencies, red cell aplasia as well as immune-mediated hemolysis. One of the immunologic causes of hemolytic anemia is drug-induced hemolysis. Tacrolimus is a common immunosuppressant used in post-liver transplant patients to prevent graft rejection. There have been reports of tacrolimus-associated hemolytic anemia secondary to hemolytic uremic syndrome as well as autoimmune hemolysis. There are also case-reports of severe hemolytic anemia related to cold agglutinin production in post-liver transplant patients. We described in this paper three cases of severe cold agglutinin hemolytic anemia in three pediatric liver transplant patients. Steroid therapy, plasmapheresis and withdrawal of tacrolimus led to resolution of the severe hemolytic process in each case. Whether the immune-mediated hemolysis is related to tacrolimus is not clear and needs to be characterized further.

Cephalosporin-induced hemolytic anemia is an acquired form of hemolytic anemia caused by interaction of drug with the immune system. Drug adsorption, drug-dependent antibody, and autoimmune induction are the three mechanisms of hemolysis. Cefotetan-induced hemolytic anemia (CIHA) has been described to occur through all three mechanisms. We report four cases of CIHA that occurred after appropriate perioperative use of cefotetan. All of our patients developed an acute and severe hemolytic episode that caused significant symptoms and led to hospitalization within 1-2 weeks after exposure to cefotetan. The hemolytic process was self-limited, and all our patients responded to supportive measures and blood transfusion. This report adds to our knowledge of CIHA, a rare complication of cefotetan use. Our cases suggest that cefotetan-induced acute severe hemolysis is caused by membrane modification (nonimmunologic protein adsorption) in addition to immune complex formation. Prompt diagnosis and aggressive supportive measures are essential in minimizing morbidity and mortality from hemolysis. Physicians should warn their patients about this complication. Given that hemolysis occurs when the subject is no longer under direct clinical supervision, patient awareness on how to recognize signs and symptoms of hemolysis is paramount to reducing the likelihood of this potentially lethal side effect. Finally, physicians might consider restricting cefotetan use.

Autoimmune hemolytic anemia in pregnancy is a rare cause of hemolytic disease of the newborn. This report describes a neonate with a mild hemolytic process and a positive direct antiglobulin test (DAT) presenting as the first manifestations of a maternal warm-reactive autoantibody. A full-term male neonate, blood group O, had a strongly positive DAT and laboratory evidence suggestive of a mild hemolytic process. The neonate’s mother was also group O and had a negative antibody screen. Umbilical cord blood testing revealed a panreactive eluate though the antibody was not detected in cord serum. The neonate’s mother was also found to have a positive DAT. A panagglutinin was identified in an eluate of her red cells, although the autoantibody could not be detected in her serum by a variety of sensitive techniques. There was no clinical or laboratory evidence of maternal hemolysis. Immunohematology 1997;13:6–8.

Publisher Summary Hemolytic anemia is a reduction in the normal red cell survival of 120 days. This chapter highlights the causes, diagnosis, clinical features, and treatments for hemolytic anemia. Premature destruction of red cells may result from corpuscular abnormalities of membrane, enzymes, hemoglobin, and extracorpuscular abnormalities. The causes of hemolytic anemia due to corpuscular and extracorpuscular defects are membrane defects, enzyme defects, hemoglobin defects, and congenital dyserythropoietic defects. Similarly, the causes of hemolytic anemia due to extracorpuscular defects majorly are immune and nonimmune. The approach to the diagnosis of hemolytic anemia should include consideration of the clinical features suggesting hemolytic disease, laboratory demonstration of the presence of a hemolytic process, and determination of the precise cause of the hemolytic anemia by special hematologic investigations. Two major laboratory findings of hemolytic anemia consist of: reduced red cell survival and evidence of accelerated hemoglobin catabolism, and evidence of increased erythropoiesis. Once the presence of a hemolytic process has been established, the root cause of the hemolytic anemia can be determined. Various treatments have been proposed for the treatment of hemolytic anemia, such as transfusion, splenectomy, and folic acid supplementation.

Karakus, sema/0000-0001-7615-4581; Haznedaroglu, Ibrahim C./0000-0001-8028-9462; Haznedaroglu, Ibrahim C./0000-0001-8028-9462 WOS: 000178860600009 PubMed: 12412737 A 41-year-old woman, Philadelphia positive chronic myeloid leukemia patient, had progressive decline in hemoglobin levels. She had been receiving interferon alpha treatment for five years. Autoimmune hemolytic anemia was diagnosed. Subsequent bone marrow examination revealed translocation t(7;14). She was placed on prednisone treatment. The patient responded to prednisone as treatment for the hemolytic process. The result of a direct Coombs' test remained positive. The patient died shortly after the diagnosis of autoimmune hemolytic anemia. Autoimmune hemolytic anemia should be considered in the evaluation of chronic myeloid leukemia patients with a sudden decrease in hemoglobin levels.

Hemolytic disease of the newborn occurs as a result of sensitization of the mother's immune system to red-cell antigens of the fetus. This sensitization results from the transplacental passage of fetal red cells possessing an antigen that is not present on maternal red cells. The IgG antibodies produced by the mother in response to the foreign antigen cross the placenta into the fetal circulation, bind to the red cells there, and destroy them. As a consequence of the hemolytic process, there is both extramedullary hematopoiesis and reticuloendothelial clearance of sensitized fetal cells, leading to hepatic and splenic enlargement. As the . . .

Intravascular mechanical fragmentation of erythrocytes is an uncommon occurrence in native valve infective endocarditis. We report a case of fragmentation hemolysis in a patient with tricuspid valve endocarditis due to Staphylococcus aureus. She received transfusion of multiple units of packed red blood cells and ultimately required surgical removal of the affected valve to control the hemolytic process. We believe this to be only the fifth such reported case and the first in which surgical therapy was necessary to control the hemolytic process.

Membrane fractions from homogenized adult Schistosoma mansoni are known to lyse host red blood cells (RBC's), Which serve as an important nutrient source for the parasite, In order to learn more about the homolytic process, we investigated the effects of pH and temperature on the steps involved in the hemolytic process. For maximum schistosome induced hemolysis to occur the worm lytic agent must be in the contact with RBCs in a low pH (pH 5.1), high temperature (37°C) environment for a short time (30 min), after which hemolysis occurs at both pH 7.5 and 5.1 At pH 7.5 the hemolytic process is relatively temperature independent and highly concentration dependent. Dose-response experiments suggest that a multi-hit process of hemolysis is probably involved. Temperature and dextran experiments suggest that a pore is formed in the RBC membrane at pH 7.5 At pH 5.1 hemolysis is temperature dependent and not very concentration dependent. Dose-response data suggest that a single-hit process of hemolysis is utilized at low pH. The hemolytic process at pH 7.5, the pH of the host blood, and pH 5.1, the approximate pH of the worm gut, appears to be very different. © 1995 Wiley-Liss, Inc.

We report on a Japanese girl with Upshaw-Schulman syndrome, a congenital disorder characterized by recurrent thrombocytopenia, microangiopathic hemolytic anemia, proteinuria and hematuria that can be transiently improved by the transfusion of plasma or various plasma components. Unusually Large von Willebrand Factor (ULvWF) multimers were found during both relapse and remission phases. Serial plasma levels of 6-keto-prostaglandin F1 alpha (PGF1 alpha), the stable metabolite of prostacyclin (PGI2), were low at relapse. When the patient was treated with continuous PGI2 infusion, the microangiopathic hemolytic process gradually subsided within 10 days. These results suggest that PGI2 may be partly involved in the pathogenesis of this congenital disorder.

Publisher Summary Simple aliphatic, aromatic, and heterocyclic thiols are poisonous to animals. Such thiols and the disulfides derived from them are selectively toxic to erythrocytes and cause hemolytic anemia in vivo. Only a fraction of the thiols and disulfides present in food and elsewhere in the environment have been tested for hemolytic activity in vivo; however, with the large number of compounds involved, such testing is not practicable. An in vitro screening test is required for identifying substances that are likely to pose the highest risk. Studies on the mechanism of toxicity of thiols and disulfides make this a possibility. The hemolytic process is initiated by the one-electron oxidation of the thiolate anion by oxyhemoglobin. The thiyl radical so produced is converted to the disulfide through intermediate formation of the disulfide radical anion. By thiol–disulfide exchange with glutathione, which is present at high concentration in the erythrocyte, the original thiol is reformed so that a redox cycle is established, with formation of active oxygen species. Such a mechanism explains the fact that the toxic effects of thiols and disulfides are identical and that these compounds are specifically toxic to erythrocytes. The electronic and steric properties of thiols that control their reactivity toward oxyhemoglobin are discussed in this chapter.

A case of drug-induced immune hemolytic anemia is described. A 2 year old boy exhibited sudden anemia and hemoglobinuria after administration of minocycline (MINO). The specific immunoglobulin G antibody against MINO was demonstrated in the patient's serum by western blotting. This is a rare example where anti-minocycline immune complex-mediated hemolysis was responsible for an intravascular hemolytic process.

Synthetic 2-hydroxyethylmethacrylate copolymer particles (HEMA) can be opsonized in the coelomic fluid of Eisenia foetida earthworms. The incomplete coelomic fluid (i.e., the coelomic fluid after incubation with HEMA particles) exerts a lower level of hemolytic activity compared to complete coelomic fluid. The decreased hemolysis can be compensated by the addition of isolated opsonins. On the other hand, isolated opsonins do not possess direct hemolytic capacity. It can be suggested that at least one of the isolated opsonins is involved in the hemolytic process. These results support the hypothesized cooperation of humoral and cellular mechanisms in earthworm defence.

PURPOSE We have developed and applied simple techniques for the detection of irreversibly and reversibly sickled cells (ISCs and RSCs) in blood samples. Both ISCs and RSCs are found in the venous and arterial circulation, and sickling and unsickling occurs continuously in the tissues and arterial blood, respectively. PATIENTS AND METHODS Using these techniques, we have studied the number of RSCs and ISCs in 100 children with sickle cell disease (SS) and 20 with sickle cell C disease (SC). RESULTS Both RSC and ISC numbers were significantly higher in blood samples of children with SS than those with SC disease. The number of RSCs and ISCs bears a direct relationship to the severity of the hemolytic process but has a negative relationship to the frequency of vaso-occlusive crises. CONCLUSIONS The manifestations of sickle cell disease appear not to be the result of large numbers of circulating sickled cells randomly lodging in vessels but more likely are the result of local vascular stasis with subsequent sickling and vaso-occlusion.

Abstract 3214 Background: We have identified a kindred in Manitoba and Saskatchewan, Canada, affected by non-immune hemolytic anemia. Red cell morphology, an elevated MCHC and decreased osmotic fragility are consistent with hereditary xerocytosis, a rare hemolytic disorder, for which the causative genetic mutation is unknown. Objectives: To describe the clinical phenotype and inheritance of an uncharacterized chronic hemolytic disorder in a large kindred. Methods: With assistance from each consenting family member, a pedigree was constructed. A focused history was taken and the presence of splenomegaly was assessed by physical examination. Laboratory analysis included a CBC, reticulocyte count, osmotic fragility and peripheral blood film. Biochemical measurements of LDH, ALT, bilirubin, ferritin, haptoglobin, plasma hemoglobin and methemoglobin were performed. Glycolytic enzymes were evaluated in a subset of patients to rule out other rare causes of hemolysis. Results: The family pedigree captured the genetic relations of 342 individuals spanning 5 generations. Consent to participate in the detailed family study was obtained from 137 family members. The average age of the study population was 29 years (range 8 months to 76 years). Laboratory specimens were collected from 26 unrelated spouses and 111 related individuals. Males represented 48% of the studied population. The distribution of reticulocyte counts was distinctly bimodal with no overlap between the two populations, allowing classification of individuals as phenotypically affected or non-affected. The mean percent reticulocyte count of non-affected subjects (related family members and unrelated spouses) was 1.1% (± 0.4, range 0.5–2.3%). Affected subjects had a mean percent reticulocyte count of 9.7% (± 2.6, range 5.3–14.6%). Using this classification, the hemolytic process segregated in an autosomal dominant fashion with complete penetrance. A history of anemia (46 vs. 8%), jaundice (45 vs. 4%), red or brown urine (45 vs. 1%), and either gallstones or cholecystectomy (41 vs. 4%) was more prevalent in affected than unaffected individuals. Episodes of anemia tended to be associated with illness or stress. There was no association between the hemolytic phenotype and neuromuscular, cardiovascular, pulmonary, renal, hepatic, or endocrine disorders. Despite a mean percent reticulocyte count of 9.7% in affected individuals, the mean hemoglobin concentration was not statistically different between affected and unaffected individuals (13.5 ± 1.2 g/dL vs. 13.8 ± 1.4 g/dL, p=0.26). The MCV (96.7 ± 5.5 fL vs. 87.3 ± 5.2 fL, p900 μg/L. Osmotic fragility performed on 4 affected individuals was decreased. Glycolytic enzymes, screens for unstable hemoglobins and hemoglobinopathies were normal in those tested. Conclusions: In this family study, elevated percent reticulocyte counts were used to characterize the presence of a well compensated, autosomal dominant hemolytic process associated with an elevated MCHC and decreased osmotic fragility. Clinically this condition is associated with gallstones and progressive iron loading. Features are consistent with hereditary xerocytosis. Molecular analysis is currently underway to locate the causative gene and identify the underlying mutation. Disclosures: No relevant conflicts of interest to declare.

A docile substrain of lymphocytic choriomeningitis virus (LCMV) causes a persistent infection in adult C3HeB mice and induces a severe autoimmune hemolytic anemia (AIHA) which is maximal around three weeks post infection (PI). Evaluations of serum immunoglobulin levels of these mice demonstrated grossly elevated IgG2a levels along with increased IgG1 and IgG2b levels, suggesting that these animals also develop polyclonal B cell activation (PBA). Interestingly, LCMV-infected B10.BR mice did not demonstrate a marked hypogammaglobulinemia nor did they experience a severe hemolytic anemia. Although evaluations of the hematocrits indicated that these animals endure a mild anemia 21 days PI, a below normal reticulocyte count until day 18 PI suggests that there was a prolonged suppression in hematopoiesis. It is clear from RBC survival studies that there is not an accelerated rate of RBC elimination, as seen in infected C3H mice, demonstrating that the anemia in B10.BR mice is not due to a hemolytic process. These results imply a correlation between the development of PBA and AIHA, suggesting a cause and effect relationship.

The kinetics of hemolysis of erythrocytes in glycerol-containing media was studied spectrophotometrically. The hemolytic process starts by a rapid process, obeying a first order rate law, which is followed by a slow change in absorbance. The kinetics of hemolysis may be described by (a) the maximum absorption, Emax, due to cellular expansion, (b) the rate constant, k, of the fast process and (c) the final absorption at its end, Einf and the ratio Einf/Emax. At pH 6.85 in normal human cells, k = 0.72min−1 while in hereditary spherocytosis cells, k = 1.06min−1, iron deficiency k = 0.52 and β-thalassemia minor k = 0.36min−1. The percentages of Einf/Emax were 35.3 in control cells, while they were 9.8, 50.0 and 88.3 in spherocytosis, iron deficiency and thalassemia, respectively. Thus these kinetic parameters may help to distinguish and understand the above mentioned erythocyte disorders. At physiological pH (7.4–7.2), no hemolysis was detected in the medium used. When the pH decreased, hemolysis occurred, its rate increasing gradually until pH 6.3. On further acidosis, the hemolytic rate slowed down again. Addition of DIDS to the whole blood prior to the test inhibits hemolysis. Similar effect of DIDS was noted in washed cells; this effect was partially reversed by albumin. These results suggest that a process involving band 3 affects the rate and degree of glycerol-induced hemolysis of normal red blood cells.

Severe autoimmune hemolytic anemia was found in an 89-year-old man who presented with fatigue and chest pain. A search for an underlying condition revealed a tumor in the cecum. Cecostomy and right hemicolectomy were performed and were followed by a prompt resolution of the hemolytic process. The tumor was a moderately differentiated adenocarcinoma. The immediate cessation of hemolysis upon removal of the malignant tissue may indicate a linkage between, rather than a coincidental presence of, the two diseases.