Hemolytic anemias recent advances in diagnosis and treatment

THE HEMOLYTIC ANEMIAS OF UNKNOWN CAUSE CAN BE SEPARATED INTO TWO MAIN GROUPS: (1) those produced by a defect in cell structure, which is usually hereditary, and (2) those due to a hemolysin of immune-body type.The hemolytic anemias associated with hypersensitivity to drugs and disease processes such as leukemia are less well understood and need further investigation. Splenectomy is the only effective treatment in congenital hemolytic jaundice and in acquired hemolytic anemia; the operation should be carried out promptly in most cases. Transfusion may be used in all varieties of hemolytic disease and is the only effective form of therapy in sickle-cell anemia and paroxysmal nocturnal hemoglobinuria.     

Hemolytic anemia in the mouse. Report of a new mutation and clarification of its genetics

A new mutation causing spherocytic, hemolytic anemia has been discovered in the house mouse. It is inherited as a single autosomal recessive gene, allelic with both sph and ha, which, in turn, were shown to be allelic with each other. A revised nomenclature for the three apparent alleles is proposed: sph (formerly sph), sphha (formerly ha), and sph2Bc (the new mutation). Like the other murine hemolytic anemias, sph2Bc involves a defect in the red blood cell membrane protein, spectrin.     

Recognition of sickle cell anemia in skeletal remains of children

The present study discusses in detail the osteological changes associated with sickle cell anemia in children and their importance in differential diagnosis. Posterior calcaneal and specific articular surface disruptive metacarpal lesions are diagnostic for sickle cell anemia. Calvarial thickening, tibial and femoral cortical bone thickening, and bowing are of more limited utility in differential diagnosis. Granular osteoporosis, pelvic demineralization and rib broadening are nonspecific. Localized calvarial “ballooning,” previously not described, may have diagnostic significance. Bone marrow hyperplastic response (porotic hyperostosis) in sickle cell anemia produces minimal radiologic changes contrasted with that observed in thalassemia and blood loss/hemolytic phenomenon. Two other issues, the osteological criteria for discriminating among the anemias and the purported relationship between porotic hyperostosis and iron deficiency anemia, are also discussed. There is sufficient information to properly diagnose the four major groups of anemias, and further, to establish that iron deficiency is only indirectly associated with porotic hyperostosis. The hyperproliferative bone marrow response (manifest as porotic hyperostosis) to blood loss or hemolysis exhausts iron stores, resulting in secondary iron deficiency. Am J Phys Anthropol 104:213–226, 1997. © 1997 Wiley-Liss, Inc.     

The role of oxidative stress in hemolytic anemia

The oxidative status of cells is determined by the balance between pro-oxidants and antioxidants. Pro-oxidants, referred to as reactive oxygen species (ROS), are classified into radicals and nonradicals. The radicals are highly reactive due to their tendency to accept or donate an electron and attain stability. When cells experience oxidative stress, ROS, which are generated in excess, may oxidize proteins, lipids and DNA - leading to cell death and organ damage. Oxidative stress is believed to aggravate the symptoms of many diseases, including hemolytic anemias. Oxidative stress was found in the beta-hemoglobinopathies (sickle cell anemia and thalassemia), glucose-6-phosphate dehydrogenase deficiency, hereditary spherocytosis, congenital dyserythropoietic anaemias and Paroxysmal Nocturnal Hemoglobinuria. Although oxidative stress is not the primary etiology of these diseases, oxidative damage to their erythroid cells plays a crucial role in hemolysis due to ineffective erythropoiesis in the bone marrow and short survival of red blood cells (RBC) in the circulation. Moreover, platelets and polymorphonuclear (PMN) white cells are also exposed to oxidative stress. As a result some patients develop thromboembolic phenomena and recurrent bacterial infections in addition to the chronic anemia. In this review we describe the role of oxidative stress and the potential therapeutic potential of anti-oxidants in various hemolytic anemias.     

Challenges for red blood cell biomarker discovery through proteomics

Red blood cells are rather unique body cells, since they have lost all organelles when mature, which results in lack of potential to replace proteins that have lost their function. They maintain only a few pathways for obtaining energy and reducing power for the key functions they need to fulfill. This makes RBCs highly sensitive to any aberration. If so, these RBCs are quickly removed from circulation, but if the RBC levels reduce extremely fast, this results in hemolytic anemia. Several causes of HA exist, and proteome analysis is the most straightforward way to obtain deeper insight into RBC functioning under the stress of disease. This should result in discovery of biomarkers, typical for each source of anemia. In this review, several challenges to generate in-depth RBC proteomes are described, like to obtain pure RBCs, to overcome the wide dynamic range in protein expression, and to establish which of the identified/quantified proteins are active in RBCs. The final challenge is to acquire and validate suited biomarkers unique for the changes that occur for each of the clinical questions; in red blood cell aging (also important for transfusion medicine), for thalassemias or sickle cell disease. Biomarkers for other hemolytic anemias that are caused by dysfunction of RBC membrane proteins (the RBC membrane defects) or RBC cytosolic proteins (the enzymopathies) are sometimes even harder to discover, in particular for the patients with RBC rare diseases with unknown cause. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Updates and advances in pyruvate kinase deficiency

Mutations in the PKLR gene lead to pyruvate kinase (PK) deficiency, causing chronic hemolytic anemia secondary to reduced red cell energy, which is crucial for maintenance of the red cell membrane and function. Heterogeneous clinical manifestations can result in significant morbidity and reduced health-related quality of life. Treatment options have historically been limited to supportive care, including red cell transfusions and splenectomy. Current disease-modifying treatment considerations include an oral allosteric PK activator, mitapivat, which was recently approved for adults with PK deficiency, and gene therapy, which is currently undergoing clinical trials. Studies evaluating the role of PK activators in other congenital hemolytic anemias are ongoing. The long-term effect of treatment with disease-modifying therapy in PK deficiency will require continued evaluation.     

Zinc deficiency states and carbonic anhydrase isozyme in experimental hemolytic and bleeding anemia of rabbits

Zinc deficiency states were produced in rabbit erythrocytes by experimentally induced bleeding anemia and hemolytic anemia. Parallel decreases in total zinc levels and the contents for major zinc protein, carbonic anhydrase I and II isozymes were observed in the erythrocytes. During the process of the anemias the zinc status in the erythrocytes varied remarkably and the relative increase of zinc ions other than that derived from carbonic anhydrase was observed, suggesting that the former zinc ions play an important role in forming a zinc pool in the erythrocytes under the anemic conditions.     

Human parvovirus B19: general considerations and impact on patients with sickle-cell disease and thalassemia and on blood transfusions

Human parvovirus B19 (B19V) is a small (22-24 nm) nonenveloped DNA virus belonging to the genus Erythrovirus (family Parvoviridae). Although it generally causes self-limiting conditions in healthy people, B19V infection may have a different outcome in patients with inherited hemolytic anemias. In such high-risk individuals, the high-titer replication may result in bone marrow suppression, triggering a life-threatening drop of hemoglobin values (profound anemia, aplastic crisis). To date there is no consensus concerning a B19V screening program either for the blood donations used in the hemotherapy or for high-risk patients. Moreover, questions such as the molecular mechanisms by which B19V produces latency and persistent replication, the primary site (sites) of B19V infection and B19V immunopathology are far from being known. This review summarizes general aspects of B19V molecular characteristics, pathogenesis and diagnostic approaches with a focus on the role of this pathogen in blood transfusions and in patients with some hemoglobinopathies (sickle-cell disease, thalassemia).     

The causes of porotic hyperostosis and cribra orbitalia: A reappraisal of the iron‐deficiency‐anemia hypothesis

Porosities in the outer table of the cranial vault (porotic hyperostosis) and orbital roof (cribra orbitalia) are among the most frequent pathological lesions seen in ancient human skeletal collections. Since the 1950s, chronic iron‐deficiency anemia has been widely accepted as the probable cause of both conditions. Based on this proposed etiology, bioarchaeologists use the prevalence of these conditions to infer living conditions conducive to dietary iron deficiency, iron malabsorption, and iron loss from both diarrheal disease and intestinal parasites in earlier human populations. This iron‐deficiency‐anemia hypothesis is inconsistent with recent hematological research that shows iron deficiency per se cannot sustain the massive red blood cell production that causes the marrow expansion responsible for these lesions. Several lines of evidence suggest that the accelerated loss and compensatory over‐production of red blood cells seen in hemolytic and megaloblastic anemias is the most likely proximate cause of porotic hyperostosis. Although cranial vault and orbital roof porosities are sometimes conflated under the term porotic hyperostosis, paleopathological and clinical evidence suggests they often have different etiologies. Reconsidering the etiology of these skeletal conditions has important implications for current interpretations of malnutrition and infectious disease in earlier human populations. Am J Phys Anthropol 2009. © 2009 Wiley‐Liss, Inc.     

Erythrocyte zincprotoporphyrin (ZPP) in subjects with heterozygote beta-thalassemia, acquired non-microcytic anemias and iron deficiency

The authors examine the utility of zinc protoporphyrin level in blood (ZPP) as diagnostic test for some anemias with different etiology. Our results show that the observed ZPP rise both in sideropenic anemia and lead poisoning, is related to TIBC increase. This relation demonstrates that both in these anemias a close correlation exists with disorders of iron metabolism. Furthermore such a correlation is not seen in thalassemic trait and in acquired non microcotic anemias.     

A genome-wide association study of total bilirubin and cholelithiasis risk in sickle cell anemia

Serum bilirubin levels have been associated with polymorphisms in the UGT1A1 promoter in normal populations and in patients with hemolytic anemias, including sickle cell anemia. When hemolysis occurs circulating heme increases, leading to elevated bilirubin levels and an increased incidence of cholelithiasis. We performed the first genome-wide association study (GWAS) of bilirubin levels and cholelithiasis risk in a discovery cohort of 1,117 sickle cell anemia patients. We found 15 single nucleotide polymorphisms (SNPs) associated with total bilirubin levels at the genome-wide significance level (p value <5 × 10(-8)). SNPs in UGT1A1, UGT1A3, UGT1A6, UGT1A8 and UGT1A10, different isoforms within the UGT1A locus, were identified (most significant rs887829, p = 9.08 × 10(-25)). All of these associations were validated in 4 independent sets of sickle cell anemia patients. We tested the association of the 15 SNPs with cholelithiasis in the discovery cohort and found a significant association (most significant p value 1.15 × 10(-4)). These results confirm that the UGT1A region is the major regulator of bilirubin metabolism in African Americans with sickle cell anemia, similar to what is observed in other ethnicities.     

Sequences in the U5-gag-pol region influence early and late pathogenic effects of Friend and Moloney murine leukemia viruses.

Friend replication-competent murine leukemia virus (F-MuLV), clone 57, induces a severe early hemolytic anemia and a later erythroleukemia after inoculation of newborn IRW or ICFW mice, whereas Moloney MuLV (M-MuLV) induces only lymphoid leukemia. We have shown previously that the attenuated hemolytic and erythroleukemogenic abilities of an F-MuLV variant, clone B3, were due mostly to changes in the env gene and long terminal repeat, respectively. For the present study, we derived two constructs exchanging env fragments of F-MuLV 57 and M-MuLV and compared them with two constructs described by Chatis et al. (J. Virol. 52:248-254, 1984) exchanging the U3 region of the long terminal repeat of the same parental viruses. When comparing the hemolytic effect of these constructs with those of the parent, we found that the U5-gag-pol region of F-MuLV was required for development of severe early hemolytic anemia and that, unlike the env of F-MuLV B3, the env of M-MuLV was fully competent in inducing severe early hemolytic anemia when associated with the F-MuLV U5-gag-pol and U3 regions. As expected, induction of erythroleukemia depended on the presence of the F-MuLV U3 region; however, the presence of both the U3 and U5-gag-pol regions of F-MuLV appeared to be synergistic and was associated with a more rapid appearance of erythroleukemia.     

Genetic modifiers in hemoglobinopathies

Hereditary anemias show considerable variation in their clinical presentation. In some cases, the causes of these variations are easily apparent. In thalassemia (or in HbE/thalassemia), genetic variation is primarily caused by the severity of the thalassemia mutation. However, not uncommonly, there is variation unexplained by the globin gene mutations themselves, which may be caused by genetic modifiers. In sickle cell disease, the primary mutation is the same in all patients. Therefore, variations in disease severity generally are due to genetic modifiers. In most genetic diseases involving beta globin, the most clearcut influence on phenotype results from elevated fetal hemoglobin levels. In addition, alpha globin gene number can influence disease phenotype. In thalassemia major or intermedia, reduction in the number of alpha globin genes can ameliorate the disease phenotype; conversely, excess alpha globin genes can convert beta thalassemia trait to a clinical picture of thalassemia intermedia. In sickle cell disease, the number of alpha globin genes has both ameliorating and exacerbating effects, depending on which disease manifestation is being examined. Unlinked genetic factors have substantial effects on the phenotype of hereditary anemias, both on the anemia and other disease manifestations. Recently, studies using genome-wide techniques, particularly studying QTLs causing elevated HbF, or affecting HbE/thalassemia, have revealed other genetic elements whose mechanisms are under study. The elucidation of genetic modifiers will hopefully lead to more rational and effective management of these diseases.     

Periodic auto-immune hemolytic anemia: An induced dynamical disease

Experimentally induced auto-immune hemolytic anemia (AIHA) in rabbits is characterized either by constant depressed erythrocyte numbers, or by oscillatory erythrocyte numbers about a depressed level (periodic auto-immune hemolytic anemia). Here the experimetallys observed characteristics of AIHA are satisfactorily accounted for by a simple model for erythropoiesis, assuming only the peripheral erythrocyte destruction rate is elevated with all other parameters normal. The onset of periodic AIHA is identified with the occurrence of a Hopf bifurcation in the model dynamics for certain values of the erythrocyte destruction rate.     

Serum hemolytic activity in dogs infected with Babesia gibsoni

The hemolytic activity in serum of Babesia gibsoni-infected dogs was examined. When the activity was assayed in a reaction system consisting of similar concentrations of the serum and canine red blood cells to those in blood, significant hemolysis was observed. The activity of the serum of B. gibsoni-infected dogs, either naturally or experimentally, was always higher than that of uninfected animals. Moreover, in the experimental infection with B. gibsoni, the change in serum hemolytic activity was parallel to those of anemia and parasitemia, whereas it was inversely parallel to that of the hematocrit value. The present study revealed the presence of a hemolytic factor(s) in the serum of B. gibsoni-infected dogs, suggesting that the progressive anemia was due to hemolysis by the factor(s).     

Effects of added dietary taurine on erythrocyte lipids and oxidative stress in rabbits fed a high cholesterol diet

Lipid peroxidation leads to damage of polyunsaturated fatty acids of membrane phospholipids. The contribution of oxidative stress to hypercholesterolemia-induced hemolytic anemia and the effects of addition of taurine on erythrocyte lipid composition, oxidative stress, and hematological data were studied in rabbits fed on a high cholesterol (HC) diet (1%, w/w) for 2 months. The effects of taurine on erythrocyte hemolysis and H2O2-induced lipid peroxidation were investigated in normal rabbit erythrocytes in vitro. The HC diet resulted in increases in plasma lipids and lipid peroxide levels as well as increases in cholesterol levels and the cholesterol:phospholipid ratio in the erythrocytes. This diet caused a hemolytic anemia, but lipid peroxide levels remained unchanged in the erythrocytes of the rabbits. Taurine (2.5%, w/w) added to the food has an ameliorating effect on plasma lipids and lipid peroxide levels in rabbits fed on a HC diet. This treatment also caused decreases in elevated erythrocyte cholesterol levels and cholesterol:phospholipid ratio due to the HC diet, but it did not prevent the hemolytic anemia and did not change erythrocyte lipid peroxide levels. In addition, in an in vitro study, taurine did not protect erythrocytes against H2O2-induced hemolysis or lipid peroxidation. These results show that the HC diet causes hemolytic anemia without any changes in erythrocyte lipid peroxidation, and taurine treatment was not effective against hemolytic anemia caused by the HC diet.     

The activity of the red blood cell Ca pump is decreased in hemolytic anemia of the beagle dog

A mild hereditary nonspherocytic anemia in Beagle dogs was studied. Compared to RBCs from normal dogs, RBCs from hemolytic Beagles were larger on average, contained more potassium, and exhibited an approximately 50% decrease in rate of loss of ATP induced by Ca and the ionophore, A23187. Under certain conditions, this rate of ATP loss can be taken as a measure of the Ca pump ATPase activity of intact RBCs. From RBC fractionation studies it appeared that the defective Ca pump ATPase was acquired during the relatively short life-span of the hemolytic RBC. Significant loss of Ca pump ATPase may be causally related to the hemolytic anemia. The mechanism(s) by which Ca pump ATPase activity is lost in this hemolytic anemia remain(s) to be determined.     

Hereditary erythrocyte pyruvate-kinase (PK) deficiency and chronic hemolytic anemia: Clinical,genetic and molecular studies in six new Spanish patients

Summary Clinical, familial and biochemical studies from six unrelated Spanish patients with hereditary hemolytic anemia and erythrocyte pyruvate-kinase (PK) deficiency are described. A remarkable molecular heterogeneity of mutant PK variants involving kinetic properties, molecular stability or electrophoretic mobility is demonstrated. In two patients whose PK variants showed abnormal electrophoretic pattern, and strongly aberrant kinetic properties, a chronic hemolytic anemia assciated with several other clinical manifestations of chronic hemolysis occurred. In patients whose PK variants showed less abnormal kinetic and electrophoretic characteristics, there was only moderate or mild hemolytic anemia. One patient's PK variant with no obvious kinetic or electrophoretic alterations, showed a markedly decreased heat stability with severe diminution of antigenic concentration of the enzyme. This patient presented a spectacular clinical and hematological improvement after splenectomy.The purpose of the present study is to describe six new PK variants of Spanish origin. In addition, an attempt is made to find relationships between molecular abnormalities of mutant PK variants and the severity of hemolytic anemia, in these patients. The possible role of some kinetic alterations, such as fructose diphosphate (FDP) activation or ATP inhibition of PK variants, in the clinical manifestations of chronic hemolysis is also suggested.     

An Imported Case of Severe Falciparum Malaria with Prolonged Hemolytic Anemia Clinically Mimicking a Coinfection with Babesiosis

While imported falciparum malaria has been increasingly reported in recent years in Korea, clinicians have difficulties in making a clinical diagnosis as well as in having accessibility to effective anti-malarial agents. Here we describe an unusual case of imported falciparum malaria with severe hemolytic anemia lasting over 2 weeks, clinically mimicking a coinfection with babesiosis. A 48-year old Korean man was diagnosed with severe falciparum malaria in France after traveling to the Republic of Benin, West Africa. He received a 1-day course of intravenous artesunate and a 7-day course of Malarone (atovaquone/proguanil) with supportive hemodialysis. Coming back to Korea 5 days after discharge, he was readmitted due to recurrent fever, and further treated with Malarone for 3 days. Both the peripheral blood smears and PCR test were positive for Plasmodium falciparum. However, he had prolonged severe hemolytic anemia (Hb 5.6 g/dl). Therefore, 10 days after the hospitalization, Babesia was considered to be potentially coinfected. A 7-day course of Malarone and azithromycin was empirically started. He became afebrile within 3 days of this babesiosis treatment, and hemolytic anemia profiles began to improve at the completion of the treatment. He has remained stable since his discharge. Unexpectedly, the PCR assays failed to detect DNA of Babesia spp. from blood. In addition, during the retrospective review of the case, the artesunate-induced delayed hemolytic anemia was considered as an alternative cause of the unexplained hemolytic anemia.     

An automated method of differential red blood cell classification with application to the diagnosis of anemia.

A method of automated red cell analysis suitable for the rapid classification of large numbers of red cells from individual blood specimens has been developed, and preliminarily tested on normal bloods and clinically proven cases of anemias and red cell disorders. According to this method digital image processing techniques provide several features relating to shape and internal central pallor configurations of red cells. These features are used with a fully automated decision logic to rapidly provide a quantitative "red cell differential" analysis, a report of the percentage subpopulations of recognized categories of red cells. For each subpopulation, measurements of mean cell area, mean cell hemoglobin content and mean cell hemoglobin density are provided. The nine types of red cell disorders studied with this method were: (a) iron deficiency anemia, (b) the anemia of chronic disease, (c) beta-thalassemia trait, (d) sickle cell anemia, (e) hemoglobin C disease, (f) intravascular hemolysis, (g) hereditary elliptocytosis, (h) hereditary spherocytosis, and (i) megaloblastic anemia due to folic acid deficiency. Preliminary indications are that the red cell differential is useful in distinguishing between these conditions.