autoimmune hemolytic anemias

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1 DCTH REVIEW Non immune hemolytic anemias: diagnostic and clinical aspects Alberto Zanella, Paola Bianchi UOC Oncoematologia, Fondazione IRCCS Ca Granda, Ospedale Maggiore Policlinico, Milano, Italy SUMMARY Non-immune hemolytic anemias are a heterogeneous group of rare diseases whose identification is the final step of a laboratory and clinical diagnostic process primarily aimed at defining whether the hemolysis is acute or chronic, extra or intravascular, congenital or acquired, associated or not with extra hematological signs. Autoimmune hemolytic anemias (AIHA), the acquired forms most commonly encountered in clinical practice, are identified by the direct antiglobulin test (DAT), although this test is known to be negative in 8-11% of cases (the so called DAT-negative AIHA). In case of suspected congenital hemolysis, the blood smear analysis remains by far the most important step of the diagnostic procedure, allowing the identification of typical red cell membranopathies; in less typical cases, further investigations are needed, i.e. red cell osmotic fragility tests and ektacytometry that respectively exploit the surface area-tovolume ratio and measure red cell deformability, or method that directly target specific red cell membrane defects as SDS-PAGE, EMA-binding test and molecular analysis. In the absence of definite red cell morphologic abnormalities, an erythrocyte enzymopathy should be suspected and the most common enzyme activities determined, starting with enzymes of the pentosephosphate shunt in the case of acute hemolysis, and from those of glycolysis or nucleotide metabolism if the hemolysis is chronic. The most common hereditary defects of red cell membrane and metabolism are briefly reviewed. Key words: Non-immune hemolytic anemia, Inherited red cell membrane disorders, Inherited red cells enzymopathy, Congenital hemolysis. Correspondence: Alberto Zanella zanella@policlinico.mi.it Non-immune hemolytic anemias are an heterogeneous group of rare diseases, congenital or acquired. The identification of these disorders is the final step of a diagnostic workout based not only on laboratory investigations, but also on patient s and family medical history taking and clinical examination, aimed at defining whether the hemolysis is acute or chronic, extra or intravascular, congenital or acquired, associated or not with extra hematological signs. As the congenital nature of hemolysis may sometimes be difficult to establish, the first step is the exclusion of all the acquired causes of hemolysis, in particular autoimmune hemolytic anemias (AIHA) which can be ruled out by the direct antiglobulin test (DAT), although this test is known to be negative in 8-11% of cases (the so called DAT-negative AIHA) (1). In case of suspected congenital hemolysis, the blood smear analysis remains

2 28 A. Zanella, P. Bianchi by far the most important step of the diagnostic procedure. In the presence of typical RBC morphologic abnormalities (like spherocytes, elliptocytes, ovalocytes, stomatocytes), or marked anysocytosis and poikilocytosis with normal hemoglobin screening, red cell membrane defects or congenital dyserythropoietic anemias may be suspected and in the more typical cases with positive family history a prompt diagnosis may be made. In less typical cases, further investigations are needed, i.e. red cell osmotic fragility tests and ektacytometry that respectively exploit the surface area-to-volume ratio and measure red cell deformability, or method that directly target the specific underlying defect as SDS-PAGE, EMA-binding test and molecular analysis (2). In the absence of definite red cell morphologic abnormalities, an erythrocyte enzymopathy should be suspected and the most common enzyme activities determined, starting with enzymes of the pentosephosphate shunt in the case of acute hemolysis (oxidative hemolysis), and from those of glycolysis or nucleotide metabolism if the hemolysis is chronic. Hereditary red cell membrane disorders are the most frequent cause of chronic hemolysis in Caucasians. HEREDITARY RED BLOOD CELL MEMBRANE DEFECTS The most common hereditary red cell membrane disorders are hereditary spherocytosis (HS), ellyptocytosis (HE) and its most severe expression hereditary pyropoikilocytosis (HPP), and hereditary stomatocytosis characterized by a stoma-like slit in some red cells (3). Hereditary spherocytosis Hereditary spherocytosis has been reported worldwide but the highest frequency is found among the Northern European populations (estimated 1: 2000 births). Approximately 75% of cases display an autosomal dominant pattern of inheritance, the remaining comprise recessive forms and de novo mutations. The molecular defect is highly heterogeneous, involving the genes encoding for spectrin, ankyrin, band 3 and protein 4.2. The deficiency or dysfunction of any of these proteins, which are involved in the attachment of the cytoskeleton to the membrane integral domain, hastens red cell vesiculation, results in a loss of surface area and leads to spheroid, osmotically fragile cells that are selectively trapped in the spleen (4). Typical HS patients present with anemia, jaundice, splenomegaly, and reticulocytosis. The disease severity of HS is classified on the basis of the degree of anemia as asymptomatic state, mild, moderate, and severe sometimes requiring exchange transfusion at birth and/or repeated blood transfusions. It is worth mentioning that many of the HS newborns have a normal hemoglobin level at birth but develop a transient and sometimes severe anemia during the first few weeks of life, due to the inability of the infants to mount an appropriate reticulocyte response to compensate for the filtering function of the spleen (5). Most of the patients have mild HS, and up to 20-30% have a purely compensated hemolysis: in this latter category, HS could be evoked when finding gallstones, persistent jaundice, clinical splenomegaly, or a transient anemia in a hemolytic crisis during infection or pregnancy. The clinical picture for a family with HS

3 Non immune hemolytic anemias: diagnostic and clinical aspects 29 is often fairly homogeneous. Variable clinical severity in isolated HS families has been mainly linked to co-inheritance of low expression polymorphism in trans to a HS(Sp) allele, namely, recessive HS and the a-spectrin polymorphism SpaLEPRA allele (Low Expression PRAgue) (6), which has a frequency of 3.6% in the normal population. A preliminary laboratory diagnosis includes examination of red cell morphology and full blood count results. The laboratory hallmark of HS, albeit non specific, is the presence of spherocytes at blood smear examination; however, spherocytes are very few in 10-20% of patients and can easily be undetected by non expert operators (7). The laboratory diagnosis of HS therefore most commonly relies on indirect tests that exploit the surface area-to-volume ratio, typically reduced in spherocytes i.e. NaCl osmotic fragility tests, Glycerol Lysis and Acidified Glycerol Lysis tests (8), and Pink test. These methods do not differentiate HS from secondary spherocytosis. More recently, a direct flow cytometric EMA-binding test has been proposed (9), that shows high sensitivity and specificity. By the analysis of a series of 150 HS patients it has been observed that the association of EMA-binding and AGLT allowed to identify all the examined patients and therefore may represent an effective diagnostic tool for HS also in mild/compensated cases (10). In severe/atypical cases, the diagnostic workout may be more complex requiring SDS-PAGE analysis of RBC membrane proteins. SDS-PAGE analysis is also required in the differential diagnosis of CDAII that displays clinical and laboratory features similar to HS (7). Recently, the use of ektacytometry and Laser-assisted Optical Rotational Cell Analyzer (LoRRca) have been proposed as a laboratory method to detect red cell membrane abnormalities and shown to be particularly useful in the diagnosis of hereditary stomatocytosis (11). Finally, the recent advent of Next Generation Sequencing (NGS) will make more feasible molecular analysis in these disorders, in particular atypical cases. ICSH Guidelines for the laboratory diagnosis of non immune hereditary red cell membrane disorders have been very recently published (12). In HS anemia is almost constantly corrected by splenectomy (which has on the contrary a limited effect in CDAII) that should however be performed only in selected cases, mainly due to the risk of sepsis. Erythropoietin may be useful to treat anemia in the neonatal period. Hereditary elliptocytosis HE is ubiquitously distributed with an estimated frequency of 1:1000-1:4000; it is a clinically and genetically heterogeneous disorder: common HE cases are asymptomatic or present mild hemolysis. In neonatal/infantile poikilocytic HE the onset may be severe but hemolysis decline with time and the phenotype after the age of 4 months-2 years becomes that of mild HE. The most severe expression of HE, named hereditary pyropoikilocytosis (HPP) because morphology mimics the thermal injury of erythrocytes, is characterized by severe, transfusion-dependent hemolytic anemia with onset in infancy; hydrops foetalis may occur in rare cases. HE is caused by abnormalities of proteins involved in the horizontal skeletal network including the spectrin dimer-dimer interaction or the

4 30 A. Zanella, P. Bianchi spectrin-actin-protein 4.1 junctional complex (3). The genes involved in HE are: alpha-spectrin (SPTA1, 1q21), beta-spectrin (SPTB, 14q23-q24.2), 4.1R (EPB41, 1p33-p32) or glycophorin C (GYPC, 2q14.3). Heterozygous mutations usually result in common HE. Patients with HPP are either compound heterozygotes or homozygotes for a missence mutation of alpha/beta spectrin. HPP is also due to presence of one alpha spectrin mutation in trans to a low-expression alpha-spectrin allele (alpha LELY) (13). The laboratory hallmark is the presence of elliptical-shaped red cells on a peripheral blood smear; marked poikilocytosis and red cells fragmentation occur in neonatal/infantile poikilocytic HE and in pyropoichilocytosis. Jaundice, splenomegaly and gallstones are common symptoms in HE. Osmotic fragility is not informative in common HE, usually increased in HPP. SDS-PAGE analysis of red cell membrane proteins may reveal quantitative/qualitative abnormalities of cytoskeletal proteins. Hereditary stomatocytosis The hereditary stomatocytoses (HSt) are a group of hemolytic conditions in which the primary lesion is a leak to the monovalent cations sodium (Na+) and potassium (K+) (14), resulting in an altered hydration status shown by a significant change in mean cell volume (MCV) Four subtypes have been identified: overhydrated HSt and dehydrated HSt, Cryohydrocytosis (CHC), and Familial Pseudohyperkalemia (FP); only FP is an asymptomatic trait whereas the other three conditions vary in both clinical severity and in qualitative features.(15). All subtypes are inherited as dominants, but new mutations are not uncommon. Overhydrated hereditary stomatocytosis Overhydrated HSt (also known as hydrocytosis) is the prototypical example, characterized by a moderate to severe hemolytic anemia with a mild macrocytosis and often numerous typical stomatocytes. The cell membrane is extremely leaky to the cations. The intracellular Na+ and K+ levels are very abnormal, presumably due to an increased pore size of the mutated Rh-associated glycoprotein that facilitates a more rapid rate of cation leak (16). Dehydrated hereditary stomatocytosis (xerocytosis) Dehydrated hereditary stomatocytosis (xerocytosis) is an autosomal dominant disorder characterised by decreased intracellular potassium content and loss of cell water, increased cytoplasmic viscosity and a typically increased mean cell haemoglobin concentration. Anemia is usually well-compensated, with only a mild to moderately enlarged spleen. Recently dehydrated HSt has been associated to several mutations of PIEZO1 protein gene (FAM38A) (17). This mechanosensitive channel protein probably regulates the response of ion fluxes accompanying mechanical stress when circulating through narrow passages in capillaries. The mutated PIEZO1 proteins can cause a gain-of-function (or increased permeability) (18), and this may explain why a healthy athlete with a firm diagnosis of DHSt presented with hemolysis after every intense training session (19). Cryohydrocytosis Cryohydrocytosis, a name coined to describe red cells which become wet at refrigerator temperatures, comes in three forms. The key abnormality is that the cells leak Na+ and K+ profusely at

5 Non immune hemolytic anemias: diagnostic and clinical aspects 31 refrigerator temperatures (4 C - 8 C). Hemolysis is variable. The low-temperature leaks cause pseudohyperkalemia, artefactually high plasma K+ readings due to loss of this cation from the abnormal red cells when they are cooled down from body to room temperatures after venesection. The mildest form of cryohydrocytosis presents with pseudohyperkalemia only; the next most severe presents with a mild hemolytic state, normocytic red cells, some stomatocytes and normal MCHC. The most severe form caused by mutations in GLUT1 presents with marked hemolysis, marked pseudohyperkalemia, and a pediatric neurological syndrome which is due to deficiency in glucose transport across the blood-brain barrier ( GLUT1 deficiency ) (20). It is worth mentioning that the laboratory diagnosis of hereditary stomatocytosis is often difficult, with the exception of very typical cases, due to the marked heterogeneity of morphological and clinical features. These disorders are often misdiagnosed as hereditary spherocytosis and sometimes splenectomized. Since splenectomy in HSt is known to be associated with an increased risk of venous thromboembolic complications, it is very important to avoid misdiagnosis. Ektacytometry has been proved to be very usfull in this sense (12). DEFECTS OF RED CELL METABOLISM Three main pathways are active in RBCs: the glycolysis, involved in the conversion of glucose into pyruvate and lactate with production of ATP, NADH and 2,3 DPG; the nucleotide methabolism that acts in the final part of glycolysis, and the hexose-monophosphate shunt that generates NADPH to keep glutathione in the reduced state and to protect red cells from oxidative damage. Metabolic energy is used to maintain red cell shape, to keep the iron of hemoglobin in the divalent form, to pump ions against electrochemical gradients and to keep sulphydryl groups of red cell enzymes, hemoglobin, and membranes in the active, reduced forms. Several red cell enzyme, when qualitatively or quantitatively defective, may be associated with hemolytic anemia (21). Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common erythrocyte enzyme defect usually associated with acute hemolysis occuring during oxidative stress, with the exception of class I variants which lead to chronic hemolysis. Other defects of hexose-monophosphate shunt which may cause acute oxidative hemolysis involve a-glutamylcystein synthetase, glutathione synthetase and glutathione reductase. Chronic hemolytic anemia is usually associated to defects of enzymes of glycolysis [in particular pyruvate kinase (PK), glucosephosphate isomerase, hexokinase, phosphofructokinase (PFK) triosephosphate isomerase (TPI)], or nucleotide metabolism, as pyrimidine 5 -nucleotidase (P5 N). When the gene involved are ubiquitous and expressed not only in the hemopoietic tissue, the deficiency may lead to non-hematological signs such as myopathy in PFK deficiency, neuromuscular dysfunction and susceptibility to infections in TPI deficiency, or neurological abnormalities and mental retardation in the defects of phosphoglycerate kinase and aldolase (22). The diagnosis relies on the determination of red cell enzyme activity by

6 32 A. Zanella, P. Bianchi quantitative assays or screening tests. Except for the basophilic stippling of erythrocytes which is characteristic for pyrimidine 5 nucleotidase deficiency, red cell morphology is unusally unremarkable. Molecular testing for RBC enzyme defects is helpful to confirm the diagnosis. Pyruvate kinase deficiency PK deficiency, inherited in an autosomal recessive form, is the most common enzymopathy of the glycolytic pathway The prevalence of this disease, as assessed by gene frequency studies, has been estimated to be 1: in the general white population. More than 220 different mutations have been described in PKLR gene, the majority involving missense mutations, a few deletions, or mutations in the promoter region. The most commonly reported are missense mutations, including 1529G A in the United States and Europe, 1456C T in Southern Europe, and 1468C T in Asia. Most patients are compound heterozygotes (21). The clinical features of PKD are highly variable, reflecting the genetic heterogeneity between affected individuals. Prenatally, hydrops fetalis has been reported in rare cases. After birth, infants with indirect hyperbilirubinemia often require phototherapy and/or exchange transfusion. PKD should always be considered in newborns with severe hyperbilirubinemia of unknown etiology. Infants and young children can be transfusion dependent for years until (or sometimes even after) splenectomy. Although the anemia tends to stabilize in adulthood, and some patients have a symptomless compensated haemolysis with only a minor apparent anaemia; exacerbations occur with acute infections, stress, and pregnancy. Since 2,3 diphosphoglycerate is elevated in individuals with PKD, the anemia may be better tolerated than in other conditions, because the oxygen dissociation curve is shifted to favor unloading of oxygen in the tissues (22). Patients with PKD have a variable laboratory phenotype ranging from mild anemia to transfusion dependent hemolysis. In a prior report of 61 patients, the median pre-splenectomy hemoglobin was 9.8 g/dl with a range of g/dl (23). The reticulocyte count is increased but is not proportional to the severity of hemolysis. Since the reticulocytes are preferentially sequestered in the spleen, splenectomy paradoxically causes a rise in both hemoglobin and reticulocytes. Although multiple reports describe a genotype-phenotype relationship (23), the clinical manifestations of PKD vary depending on many other factors including the patient s genetic background, presence of red cell abnormalities, ineffective erythropoiesis to variable degree, and differences in splenic function. These findings are most readily noted in the variability among members of the same family with identical genotypes. Since the haematological features of PK deficiency are common to other hereditary non-spherocytic haemolytic diseases and red cell morphology is commonly unremarkable (although some spur cells or acanthocytes may be observed particularly after splenectomy), the diagnosis ultimately depends on the demonstration of low enzyme activity and the confirmation at DNA level. No curative therapy for PK deficiency is available to date, and the treatment is therefore based on supportive measures (24). Red cell transfusions may be required in severely anaemic cases,

7 Non immune hemolytic anemias: diagnostic and clinical aspects 33 particularly in the first years of life; the haemoglobin then tends to stabilise in many cases at about 6-8 g/dl, and transfusions are no more necessary unless the anaemia is exacerbated by infections, pregnancy or other conditions. Because the delivery of oxygen to tissues is highly efficient due to the high 2,3-DPG content, the decision to transfuse a PK deficient patient should be based on the clinical conditions rather than on the haemoglobin levels. Splenectomy does not arrest haemolysis, and should be reserved for severely affected, young patients who need regular blood transfusions, and to patients who do not tolerate anaemia. It usually results in an increase of 1-3 g/ dl in haemoglobin, reducing or even eliminating transfusion requirement. Indications and risks of splenectomy, particularly the risk of overwhelming sepsis, are the same reported for red cell membrane disorders. Iron chelation may be required since iron overload is common in PKD, in both chronically transfused and transfusion independent individuals. In non transfused patients, additional factors for iron loading include splenectomy, a degree of ineffective erythropoiesis, and co-inheritance of hereditary hemochromatosis mutations (25). Bone marrow transplantation has been successfully performed in one case (26). A gene therapy strategy has not been attempted in human PKD: studies in mice have shown prolonged expression of PK after introduction of a viral vector expressing human liver-type PK cdna (27). Additionally, other investigators have shown increased expression of PK in transgenic mice using a gene-addition strategy, although this did not fully rescue the mice from their phenotype with continued evidence of ineffective erythropoiesis (28). No specific drug therapies are available for treatment of PKD. Historic attempts to develop pharmacologic treatments, such as riboflavin and sulphydryl compounds, have been unsuccessful (24). A pharmacologic activator of red cell pyruvate kinase is presently in early phase clinical trials: the drug can both activate pyruvate kinase and enhance glycolytic flux in mice, and increases ATP and decreases 2,3-DPG in healthy individuals (29, 30). To date, no human data are available from patients with the disorder. REFERENCES 1. Zanella A, Barcellini W. Treatment of autoimmune hemolytic anemias. Haematologica. 2014; 99: Bolton-Maggs P, Langer JC, Iolascon A, Tittensor P, King M-J. Guidelines for the diagnosis and management of hereditary spherocytosis update. Br J Haematol. 2012; 156: Mohandas N, Gallagher PG. Red cell membrane: past, present, and future. Blood. 2008; 112: Perrotta S, Gallagher PG, Mohandas N. Hereditary spherocytosis. Lancet 2008; 372: Saada V, Cynober T, Brossard Y, et al. Incidence of hereditary spherocytosis in a population of jaundiced neonates. Pediatric Hematol Oncol. 2006; 23: Dhermy D, Steen-Johnsen J, Bournier O, et al. Coinheritance of two g-spectrin gene defects in a recessive spherocytosis family. Clin Lab Haem. 2000; 22: Mariani M, Barcellini W, Vercellati C, et al. Clinical and hematologic features of 300 patients affected by hereditary spherocytosis grouped according to the type of the membrane protein defect. Haematologica. 2008; 93: Zanella A, Izzo C, Rebulla P, et al. Acidified glygerol lysis test: a screening test for spherocytosis. Brit J Haematol. 1980; 45:

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