With respect to primary etiology, anemias due to increased destruction of red cells, which we know as hemolytic anemias (HAs), may be inherited or acquired (Table 10-1). From the clinical point of view they may be more acute or more chronic, they may vary from mild to very severe, and the site of hemolysis may be predominantly intravascular or extravascular. With respect to mechanisms, HAs may be due to intracorpuscular causes or to extracorpuscular causes. But before reviewing the individual types of HA, it is appropriate to consider what they have in common.
TABLE 10-1CLASSIFICATION OF HEMOLYTIC ANEMIAS* ||Download (.pdf) TABLE 10-1CLASSIFICATION OF HEMOLYTIC ANEMIAS*
| ||INTRACORPUSCULAR DEFECTS ||EXTRACORPUSCULAR FACTORS |
|Hereditary || |
|Familial (atypical) hemolytic uremic syndrome |
|Acquired || |
Paroxysmal nocturnal hemoglobinuria
Mechanical destruction (microangiopathic)
GENERAL CLINICAL AND LABORATORY FEATURES
The clinical presentation of a patient with anemia is greatly influenced in the first place by whether the onset is abrupt or gradual, and HAs are no exception. A patient with autoimmune HA or with favism may be a medical emergency, whereas a patient with mild hereditary spherocytosis or with cold agglutinin disease may be diagnosed after years. This is due in large measure to the remarkable ability of the body to adapt to anemia when it is slowly progressing (Chap. 2).
What differentiates HAs from other anemias is that the patient has signs and symptoms arising directly from hemolysis (Table 10-2). At the clinical level, the main sign is jaundice; in addition, the patient may report discoloration of the urine. In many cases of HA, the spleen is enlarged because it is a preferential site of hemolysis, and in some cases, the liver may be enlarged as well. In all severe congenital forms of HA, there also may be skeletal changes due to overactivity of the bone marrow (although they are never as severe as they are in thalassemia).
TABLE 10-2SOME COMMON FEATURES OF HEMOLYTIC DISORDERS ||Download (.pdf) TABLE 10-2SOME COMMON FEATURES OF HEMOLYTIC DISORDERS
|General examination ||Jaundice, pallor |
|Other physical findings ||Spleen may be enlarged; bossing of skull in severe congenital cases |
|Hemoglobin level ||From normal to severely reduced |
|MCV, MCH ||Usually increased |
|Reticulocytes ||Increased |
|Bilirubin ||Increased (mostly unconjugated) |
|LDH ||Increased (up to 10× normal with intravascular hemolysis) |
|Haptoglobin ||Reduced to absent (if hemolysis is part intravascular) |
The laboratory features of HA are related to hemolysis per se and the erythropoietic response of the bone marrow. Hemolysis regularly produces in the serum an increase in unconjugated bilirubin and aspartate transaminase (AST); urobilinogen will be increased in both urine and stool. If hemolysis is mainly intravascular, the telltale sign is hemoglobinuria (often associated with hemosiderinuria); in the serum, there is increased hemoglobin, lactate dehydrogenase (LDH) is increased, and haptoglobin is reduced. In contrast, the bilirubin level may be normal or only mildly elevated. The main sign of the erythropoietic response by the bone marrow is an increase in reticulocytes (Table 10-2), a test all too often neglected in the initial workup of a patient with anemia. Usually the increase will be reflected in both the percentage of reticulocytes (the more commonly quoted figure) and the absolute reticulocyte count (the more definitive parameter). The increased number of reticulocytes is associated with an increased mean corpuscular volume (MCV) in the blood count. On the blood smear, this is reflected in the presence of macrocytes; there is also polychromasia and sometimes one sees nucleated red cells. In most cases, a bone marrow aspirate is not necessary in the diagnostic workup; if it is done, it will show erythroid hyperplasia. In practice, once a HA is suspected, specific tests will usually be required for a definitive diagnosis of a specific type of HA.
The mature red cell is the product of a developmental pathway that brings the phenomenon of differentiation to an extreme. An orderly sequence of events produces synchronous changes whereby the gradual accumulation of a huge amount of hemoglobin in the cytoplasm (to a final level of 340 g/L, i.e., about 5 mM) goes hand in hand with the gradual loss of cellular organelles and of biosynthetic abilities. In the end, the erythroid cell undergoes a process that has features of apoptosis, including nuclear pyknosis and actual loss of the nucleus. However, the final result is more altruistic than suicidal; the cytoplasmic body, instead of disintegrating, is now able to provide oxygen to all cells in the human organism for some remaining 120 days of the red cell "life" span.
As a result of this unique process of differentiation and maturation, intermediary metabolism is drastically curtailed in mature red cells (Fig. 10-1); for instance, cytochrome-mediated oxidative phosphorylation has been lost with the loss of mitochondria (through a process of physiologic autophagy); therefore, there is no backup to anaerobic glycolysis for the production of adenosine triphosphate (ATP). Also the capacity of making protein has been lost with the loss of ribosomes. This places the cell's limited metabolic apparatus at risk because if any protein component deteriorates, it cannot be replaced, as it would be in most other cells, and in fact the activity of most enzymes gradually decreases as red cells age. Another consequence of the relative simplicity of red cells is that they have a very limited range of ways to manifest distress under hardship: in essence, any sort of metabolic failure will eventually lead either to structural damage to the membrane or to failure of the cation pump. In either case, the life span of the red cell is reduced, which is the definition of a hemolytic disorder. If the rate of red cell destruction exceeds the capacity of the bone marrow to produce more red cells, the hemolytic disorder will manifest as HA.
Red blood cell metabolism. The Embden-Meyerhof pathway (glycolysis) generates adenosine triphosphate (ATP) for energy and membrane maintenance. The generation of nicotinamide adenine dinucleotide phosphate (NADPH) maintains hemoglobin in a reduced state. The hexose monophosphate shunt generates nicotinamide adenine dinucleotide phosphate (NADPH) that is used to reduce glutathione, which protects the red cell against oxidant stress. Regulation of 2,3-bisphosphoglycerate levels is a critical determinant of oxygen affinity of hemoglobin. Enzyme deficiency states in order of prevalence: glucose-6-phosphate dehydrogenase (G6PD) > pyruvate kinase > glucose-6-phosphate isomerase > rare deficiencies of other enzymes in the pathway. The more common enzyme deficiencies are encircled. ADP, adenosine diphosphate.
Thus, the essential pathophysiologic process common to all HAs is an increased red cell turnover. The gold standard for proving that the life span of red cells is reduced (compared with the normal value of about 120 days) is a red cell survival study, which can be carried out by labeling the red cells with 51Cr and measuring residual radioactivity over several days or weeks; however, this classic test is now available in very few centers, and it is rarely necessary. If the hemolytic event is transient, it does not usually cause any long-term consequences, except for an increased requirement for erythropoietic factors, particularly folic acid. However, if hemolysis is recurrent or persistent, the increased bilirubin production favors the formation of gallstones. If a considerable proportion of hemolysis takes place in the spleen, as is often the case, splenomegaly may become increasingly a feature, and hypersplenism may develop, with consequent neutropenia and/or thrombocytopenia.
The increased red cell turnover also has metabolic consequences. In normal subjects, the iron from effete red cells is very efficiently recycled by the body; however, with chronic intravascular hemolysis, the persistent hemoglobinuria will cause considerable iron loss, needing replacement. With chronic extravascular hemolysis the opposite problem, iron overload, is more common, especially if the patient needs frequent blood transfusions. Chronic iron overload will cause secondary hemochromatosis: this will cause damage particularly to the liver, eventually leading to cirrhosis, and to the heart muscle, eventually causing heart failure.
Compensated hemolysis versus hemolytic anemia
Red cell destruction is a potent stimulus for erythropoiesis, which is mediated by erythropoietin (EPO) produced by the kidney. This mechanism is so effective that in many cases the increased output of red cells from the bone marrow can fully balance an increased destruction of red cells. In such cases, we say that hemolysis is compensated. The pathophysiology of compensated hemolysis is similar to what we have just described, except there is no anemia. This notion is important from the diagnostic point of view because a patient with a hemolytic condition, even an inherited one, may present without anemia. It is also important from the point of view of management because compensated hemolysis may become "decompensated"—i.e., anemia may suddenly appear—in certain circumstances, for instance pregnancy, folate deficiency, or renal failure, interfering with adequate EPO production. Another general feature of chronic HAs is seen when any intercurrent condition, for instance, an acute infection, depresses erythropoiesis. When this happens, in view of the increased rate of red cell turnover, the effect will be predictably much more marked than in a person who does not have hemolysis. The most dramatic example is infection by parvovirus B19, which may cause a rather precipitous fall in hemoglobin, an occurrence sometimes referred to as aplastic crisis.
INHERITED HEMOLYTIC ANEMIAS
There are three essential components in the red cell: (1) hemoglobin, (2) the membrane–cytoskeleton complex, and (3) the metabolic machinery necessary to keep (1) and (2) in working order. Diseases caused by abnormalities of hemoglobin, or hemoglobinopathies, are covered in Chap. 8. Here we will deal with diseases of the other two components.
Hemolytic anemias due to abnormalities of the membrane-cytoskeleton complex
The detailed architecture of the red cell membrane is complex, but its basic design is relatively simple (Fig. 10-2). The lipid bilayer incorporates phospholipids and cholesterol, and it is spanned by a number of proteins that have their hydrophobic transmembrane domains embedded in the membrane. Most of these proteins have hydrophilic domains extending toward both the outside and the inside of the cell. Other proteins are tethered to the membrane through a glycosylphosphatidylinositol (GPI) anchor, and they have only an extracellular domain. These proteins are arranged roughly perpendicular to or lying across the membrane; they include ion channels, receptors for complement components, receptors for other ligands, and some of unknown function. The most abundant of these proteins are glycophorins and the so-called band 3, an anion transporter. The extracellular domains of many of these proteins are heavily glycosylated, and they carry antigenic determinants that correspond to blood groups. Underneath the membrane and tangential to it is a network of other proteins that make up the cytoskeleton: the main cytoskeletal protein is spectrin, the basic unit of which is a dimer of α-spectrin and β-spectrin. The membrane is physically linked to the cytoskeleton by a third set of proteins (including ankyrin and the so-called band 4.1 and band 4.2), which thus make these two structures intimately connected to each other.
Diagram of red cell membrane-cytoskeleton. GPA, glycophorin A; GPC, glycophorin C; RhAG, Rh-associated glycoprotein (For explanation see text.) (From N Young et al: Clinical Hematology. Copyright Elsevier, 2006; with permission.)
The membrane–cytoskeleton complex is indeed so integrated that, not surprisingly, an abnormality of almost any of its components will be disturbing or disruptive, causing structural failure, which results ultimately in hemolysis. These abnormalities are almost invariably inherited mutations; thus, diseases of the membrane–cytoskeleton complex belong to the category of inherited HAs. Before the red cells lyse, they often exhibit more or less specific morphologic changes that alter the normal biconcave disk shape. Thus, the majority of the diseases in this group have been known for over a century as hereditary spherocytosis and hereditary elliptocytosis.
Hereditary spherocytosis (HS)
This is a relatively common type of HA, with an estimated frequency of at least 1 in 5000. Its identification is credited to Minkowksy and Chauffard, who at the end of the nineteenth century reported families in which HS was inherited as an autosomal dominant condition (Fig 10-3A). From this seminal work, HS came to be defined as an inherited form of HA associated with the presence of spherocytes in the peripheral blood. In addition, in vitro studies revealed that the red cells were abnormally susceptible to lysis in hypotonic media: indeed, the presence of osmotic fragility became the main diagnostic test for HS. Today we know that HS, thus defined, is genetically heterogeneous, i.e., it can arise from a variety of mutations in one of several genes (Table 10-3). Whereas classically the inheritance of HS is autosomal dominant (with the patients being heterozygous), some severe forms are instead autosomal recessive (with the patient being homozygous).
TABLE 10-3INHERITED DISEASES OF THE RED CELL MEMBRANE–CYTOSKELETON ||Download (.pdf) TABLE 10-3INHERITED DISEASES OF THE RED CELL MEMBRANE–CYTOSKELETON
|GENE ||CHROMOSOMAL LOCATION ||PROTEIN PRODUCED ||DISEASE(S) WITH CERTAIN MUTATIONS (INHERITANCE) ||COMMENTS |
|SPTA1 ||1q22-q23 ||α-Spectrin || |
Mutations of this gene account for about 65% of HE. More severe forms may be due to coexistence of an otherwise silent mutant allele
|SPTB ||14q23-q24.1 ||β-Spectrin || |
Mutations of this gene account for about 30% of HE, including some severe forms
|ANK1 ||8p11.2 ||Ankyrin ||HS (dominant) ||May account for majority of HS |
|SLC4A1 ||17q21 ||Band 3 (anion channel) ||HS (dominant) ||Mutations of this gene may account for about 25% of HS |
| || || || |
Southeast Asia ovalocytosis (dominant)
Polymorphic mutation (deletion of 9 amino acids); clinically asymptomatic; protective against Plasmodium falciparum
Certain specific missense mutations shift protein function from anion exchanger to cation conductance
|EPB41 ||1p33-p34.2 ||Band 4.1 ||HE (dominant) ||Mutations of this gene account for about 5% of HE: mostly with prominent morphology but no hemolysis in heterozygotes; severe hemolysis in homozygotes |
|EPB42 ||15q15-q21 ||Band 4.2 ||HS (recessive) ||Mutations of this gene account for about 3% of HS. |
|RHAG ||6p21.1-p11 ||Rhesus antigen ||Chronic nonspherocytic hemolytic anemia ||Very rare; associated with total loss of all Rh antigens |
Peripheral blood smear from patients with membrane-cytoskeleton abnormalities. A. Hereditary spherocytosis. B. Hereditary elliptocytosis, heterozygote. C. Elliptocytosis, with both alleles of the α-spectrin gene mutated.
Clinical presentation and diagnosis
The spectrum of clinical severity of HS is broad. Severe cases may present in infancy with severe anemia, whereas mild cases may present in young adults or even later in life. In women, HS is sometimes first diagnosed when anemia is investigated during pregnancy. The main clinical findings are jaundice, an enlarged spleen, and often gallstones; indeed, it is often the finding of gallstones in a young person that triggers diagnostic investigations.
The variability in clinical manifestations that is observed among patients with HS is largely due to the different underlying molecular lesions (Table 10-3). Not only are mutations of several genes involved, but individual mutations of the same gene can also give very different clinical manifestations. In milder cases, hemolysis is often compensated (discussed earlier), and this may cause variation in time, even in the same patient, because intercurrent conditions (e.g., infection) cause decompensation. The anemia is usually normocytic, with the characteristic morphology that gives the disease its name. A characteristic feature is an increase in mean corpuscular hemoglobin concentration (MCHC); this is almost the only condition in which an increased MCHC is seen.
When there is a family history (Fig. 10-3A), it is usually easy to suspect the diagnosis, but there may be no family history for at least two reasons. (1) The patient may have a de novo mutation, i.e., a mutation that has taken place in a germ cell of one of his or her parents or early after zygote formation. (2) The patient may have a recessive form of HS (Table 10-3). In most cases, the diagnosis can be made on the basis of red cell morphology and of a test for osmotic fragility, a modified version of which is called the "pink test." In some cases, a definitive diagnosis can be obtained only by molecular studies demonstrating a mutation in one of the genes underlying HS. This is usually carried out in laboratories with special expertise in this area.
TREATMENT: Hereditary Spherocytosis
We don't have a causal treatment for HS; i.e., no way has yet been found to correct the basic defect in the membrane–cytoskeleton structure. However, it has been apparent for a long time that the spleen plays a special role in HS through a dual mechanism. On one hand, like in many other HAs, the spleen itself is a major site of destruction; on the other hand, transit through the splenic circulation makes the defective red cells more spherocytic and therefore accelerates their demise, even though lysis may take place elsewhere. For these reasons, splenectomy has long been regarded as a prime, almost obligatory, therapeutic measure in HS. Therefore, current guidelines (not evidence-based) are as follows. (1) Avoid splenectomy in mild cases. (2) Delay splenectomy until at least 4 years of age, after the risk of severe sepsis has peaked. (3) Antipneumococcal vaccination before splenectomy is imperative, whereas penicillin prophylaxis postsplenectomy is controversial. (4) There is no doubt that HS patients often may require cholecystectomy, in which case the practice has been to also carry out a splenectomy at the same time. Today the decision regarding this combined surgery should not be regarded as automatic; cholecystectomy is usually done via the laparoscopic approach, and splenectomy should be carried out if clinically indicated.
Hereditary elliptocytosis (HE)
HE is at least as heterogeneous as HS, both from the genetic point of view (Table 10-3) and from the clinical point of view. Again, it is the shape of the red cells that gives the name to these conditions, but there is no direct correlation between the elliptocytic morphology and clinical severity. In fact, some mild or even asymptomatic cases may have nearly 100% elliptocytes, whereas in severe cases, it is all sorts of bizarre poikilocytes that predominate. Clinical features and recommended management are similar to those outlined above for HS. Although the spleen may not have the specific role it has in HS, in severe cases, splenectomy may be beneficial. The prevalence of HE causing clinical disease is similar to that of HS. However, an asymptomatic form, referred to as Southeast Asia ovalocytosis, has a frequency of up to 7% in certain populations, presumably as a result of malaria selection.
Disorders of cation transport
These rare conditions with autosomal dominant inheritance are characterized by increased intracellular sodium in red cells, with concomitant loss of potassium: indeed, they are sometimes discovered through the incidental finding, in a blood test, of high serum K+ (pseudohyperkalemia). In patients from some families, the cation transport disturbance is associated with gain of water: as a result, the red cells are overhydrated (low MCHC), and on a blood smear, the normally round-shaped central pallor is replaced by a linear-shaped central pallor, which has earned this disorder the name stomatocytosis. In patients from other families, the red cells are instead dehydrated (high MCHC), and their consequent rigidity has earned this disorder the name xerocytosis. In these disorders, one would suspect that the primary defect may be in a cation transporter. In most cases, this has not yet been demonstrated, but interestingly, certain missense mutations of the SLC4A1 gene encoding band 3 (Table 10-3) give stomatocytosis. Hemolysis can vary from relatively mild to quite severe. From the practical point of view, it is important to know that splenectomy is contraindicated, as it has been followed in a majority of cases by severe thromboembolic complications.
When there is an important defect in the membrane or in the cytoskeleton, hemolysis is a direct consequence of the fact that the very structure of the red cell is abnormal. Instead, when one of the enzymes is defective, the consequences will depend on the precise role of that enzyme in the metabolic machinery of the red cell, which, in first approximation, has two important functions: (1) to provide energy in the form of ATP and (2) to prevent oxidative damage to hemoglobin and to other proteins.
Abnormalities of the glycolytic pathway
Since red cells, in the course of their differentiation, have sacrificed not only their nucleus and their ribsomes but also their mitochondria, they rely exclusively on the anaerobic portion of the glycolytic pathway for producing energy in the form of ATP. Most of the ATP is required by the red cell for cation transport against a concentration gradient across the membrane. If this fails, due to a defect of any of the enzymes of the glycolytic pathway, the result will be hemolytic disease (Table 10-4).
TABLE 10-4RED CELL ENZYME ABNORMALITIES CAUSING HEMOLYSIS ||Download (.pdf) TABLE 10-4RED CELL ENZYME ABNORMALITIES CAUSING HEMOLYSIS
| ||ENZYME (ACRONYM) ||CHROMOSOMAL LOCATION ||PREVALENCE OF ENZYME DEFICIENCY (RANK) ||CLINICAL MANIFESTATIONS EXTRA-RED CELL ||COMMENTS |
|Glycolytic pathway ||Hexokinase (HK) ||10q22 ||Very rare || ||Other isoenzymes known |
| ||Glucose 6-phosphate isomerase (G6PI) ||19q31.1 ||Rare (4)* ||NM, CNS || |
| ||Phosphofructokinase (PFK) ||12q13 ||Very rare ||Myopathy || |
| ||Aldolase ||16q22-24 ||Very rare || || |
| ||Triose phosphate isomerase (TPI) ||12p13 ||Very rare ||CNS (severe), NM || |
| ||Glyceraldehyde 3-phosphate dehydrogenase (GAPD) ||12p13.31-p13.1 ||Very rare ||Myopathy || |
| ||Diphosphoglycerate mutase (DPGM) ||7q31-q34 ||Very rare || ||Erythrocytosis rather than hemolysis |
| ||Phosphoglycerate kinase (PGK) ||Xq13 ||Very rare ||CNS, NM ||May benefit from splenectomy |
| ||Pyruvate kinase (PK) ||1q21 ||Rare (2)* || ||May benefit from splenectomy |
|Redox ||Glucose 6-phosphate dehydrogenase (G6PD) ||Xq28 ||Common (1)* ||Very rarely granulocytes ||In almost all cases only AHA from exogenous trigger |
| ||Glutathione synthase ||20q11.2 ||Very rare ||CNS || |
| ||γ-Glutamylcysteine synthase ||6p12 ||Very rare ||CNS || |
| ||Cytochrome b5 reductase ||22q13.31-qter ||Rare ||CNS ||Methemoglobinemia rather than hemolysis |
|Nucleotide ||Adenylate kinase (AK) ||9q34.1 ||Very rare ||CNS || |
|Metabolism ||Pyrimidine 5′- nucleotidase (P5N) ||3q11-q12 ||Rare (3)* || ||May benefit from splenectomy |
Pyruvate kinase deficiency
Abnormalities of the glycolytic pathway are all inherited and all rare. Among them, deficiency of pyruvate kinase (PK) is the least rare, with an estimated prevalence of the order of 1:10,000. The clinical picture is that of an HA that often presents in a newborn with neonatal jaundice; the jaundice persists, and it is usually associated with a very high reticulocytosis. The anemia is of variable severity; sometimes it is so severe as to require regular blood transfusion treatment; sometimes it is mild, bordering on a nearly compensated hemolytic disorder. As a result, the diagnosis may be delayed, and in some cases, it is made in young adults, for instance, in a woman, during her first pregnancy, when the anemia may get worse. In part, the delay in diagnosis is due to the fact that the anemia is remarkably well tolerated because the metabolic block at the last step in glycolysis causes an increase in bisphosphoglycerate (or DPG), a major effector of the hemoglobin–oxygen dissociation curve; thus, the oxygen delivery to the tissues is enhanced.
TREATMENT: Pryuvate Kinase Deficiency
The management of PK deficiency is mainly supportive. In view of the marked increase in red cell turnover, oral folic acid supplements should be given constantly. Blood transfusion should be used as necessary, and iron chelation may have to be added if the blood transfusion requirement is high enough to cause iron overload. In these patients, who have more severe disease, splenectomy may be beneficial. There is a single case report of curative treatment of PK deficiency by bone marrow transplantation from an HLA-identical PK-normal sibling. This seems a viable option for severe cases when a sibling donor is available.
Other glycolytic enzyme abnormalities
All of these defects are rare to very rare (Table 10-4), and all cause HA with varying degrees of severity. It is not unusual for the presentation to be in the guise of severe neonatal jaundice, which may require exchange transfusion. If the anemia is less severe, it may present later in life, or it may even remain asymptomatic and be detected incidentally when a blood count is done for unrelated reasons. The spleen is often enlarged. When other systemic manifestations occur, they involve the central nervous system, sometimes entailing severe mental retardation (particularly in the case of triose phosphate isomerase deficiency), the neuromuscular system, or both. The diagnosis of HA is usually not difficult because of the triad of normo-macrocytic anemia, reticulocytosis, and hyperbilirubinemia. Enzymopathies should be considered in the differential diagnosis of any chronic Coombs-negative HA. In most cases of glycolytic enzymopathies, the morphologic abnormalities of red cells characteristically seen in membrane disorders are conspicuous by their absence. A definitive diagnosis can be made only by demonstrating the deficiency of an individual enzyme by quantitative assays carried out in only a few specialized laboratories. If a particular molecular abnormality is already known in a family, then of course one could test directly for that at the DNA level, bypassing the need for enzyme assays.
Abnormalities of redox metabolism
Glucose 6-phosphate dehydrogenase (G6PD) is a housekeeping enzyme critical in the redox metabolism of all aerobic cells (Fig. 10-1). In red cells, its role is even more critical because it is the only source of NADPH that directly and via glutathione (GSH) defends these cells against oxidative stress. G6PD deficiency is a prime example of an HA due to interaction between an intracorpuscular cause and an extracorpuscular cause because in the majority of cases, hemolysis is triggered by an exogenous agent. Although a decrease in G6PD activity is noted in most tissues of G6PD-deficient subjects, the decrease is less marked than in red cells, and it does not seem to have a clinical impact.
The G6PD gene is X-linked, and this has important implications. First, as males have only one G6PD gene (i.e., they are hemizygous for this gene), they must be either normal or G6PD-deficient. By contrast, females, having two G6PD genes, can be normal, deficient (homozygous), or intermediate (heterozygous). As a result of the phenomenon of X-chromosome inactivation, heterozygous females are genetic mosaics, with a highly variable ratio of G6PD-normal to G6PD-deficient cells and an equally variable degree of clinical expression: some heterozygotes can be just as affected as hemizygous males. The enzymatically active form of G6PD is either a dimer or a tetramer of a single protein subunit of 514 amino acids. G6PD-deficient subjects have been found invariably to have mutations in the coding region of the G6PD gene (Fig. 10-4). Almost all of some 150 different mutations known are single missense point mutations, entailing single amino acid replacements in the G6PD protein. In most cases, these mutations cause G6PD deficiency by decreasing the in vivo stability of the protein; thus, the physiologic decrease in G6PD activity that takes place with red cell aging is greatly accelerated. In some cases, an amino acid replacement can also affect the catalytic function of the enzyme.
Diagram of redox metabolism in the red cell. G6P, glucose 6-phosphate; G6PD, glucose 6-phosphate dehydrogenase; GSH, reduced glutathione; GSSG, oxidized glutathione; Hb, hemoglobin; MetHb, methemoglobin; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, reduced nicotinamide adenine dinucleotide phosphate, 6PG, 6-phosphogluconate.
Among these mutations, those underlying chronic nonspherocytic hemolytic anemia (CNSHA; see "Clinical Manifestations" below) are a discrete subset. This much more severe clinical phenotype can be ascribed in some cases to adverse qualitative changes (for instance, a decreased affinity for the substrate, glucose 6-phosphate) or simply to the fact that the enzyme deficit is more extreme because of a more severe instability of the enzyme. For instance, a cluster of mutations map at or near the dimer interface, and clearly they compromise severely the formation of the dimer.
G6PD deficiency is widely distributed in tropical and subtropical parts of the world (Africa, Southern Europe, the Middle East, Southeast Asia, and Oceania) (Fig. 10-5) and wherever people from those areas have migrated. A conservative estimate is that at least 400 million people have a G6PD deficiency gene. In several of these areas, the frequency of a G6PD deficiency gene may be as high as 20% or more. It would be quite extraordinary for a trait that causes significant pathology to spread widely and reach high frequencies in many populations without conferring some biologic advantage. Indeed, G6PD is one of the best characterized examples of genetic polymorphisms in the human species. Clinical field studies and in vitro experiments strongly support the view that G6PD deficiency has been selected by Plasmodium falciparum malaria, by virtue of the fact that it confers a relative resistance against this highly lethal infection. Whether this protective effect is exerted mainly in hemizygous males or in females heterozygous for G6PD deficiency is still not quite clear. Different G6PD variants underlie G6PD deficiency in different parts of the world. Some of the more widespread variants are G6PD Mediterranean on the shores of that sea, in the Middle East, and in India; G6PD A in Africa and in Southern Europe; G6PD Vianchan and G6PD Mahidol in Southeast Asia; G6PD Canton in China; and G6PD Union worldwide. The heterogeneity of polymorphic G6PD variants is proof of their independent origin, and it supports the notion that they have been selected by a common environmental agent, in keeping with the concept of convergent evolution (Fig. 10-5).
Epidemiology of G6PD deficiency throughout the world. The different shadings indicate increasingly high levels of prevalence, up to about 20%; the different colored symbols indicate individual genetic variants of G6PD, each one having a different mutation. (From L Luzzatto et al, in C Scriver et al [eds]: The Metabolic & Molecular Bases of Inherited Disease, 8th ed. New York, McGraw-Hill, 2001.)
The vast majority of people with G6PD deficiency remain clinically asymptomatic throughout their lifetimes; however, all of them have an increased risk of developing neonatal jaundice (NNJ) and a risk of developing acute hemolytic anemia (AHA) when challenged by a number of oxidative agents. NNJ related to G6PD deficiency is very rarely present at birth. The peak incidence of clinical onset is between day 2 and day 3, and in most cases the anemia is not severe. However, NNJ can be very severe in some G6PD-deficient babies, especially in association with prematurity, infection, or environmental factors (such as naphthalene–camphor balls used in babies' bedding and clothing), and the risk of severe NNJ is also increased by the coexistence of a monoallelic or biallelic mutation in the uridyl transferase gene (UGT1A1; the same mutations are associated with Gilbert syndrome). If inadequately managed, NNJ associated with G6PD deficiency can produce kernicterus and permanent neurologic damage.
AHA can develop as a result of three types of triggers: (1) fava beans, (2) infections, and (3) drugs (Table 10-5). Typically, a hemolytic attack starts with malaise, weakness, and abdominal or lumbar pain. After an interval of several hours to 2–3 days, the patient develops jaundice and often dark urine, due to hemoglobinuria. The onset can be extremely abrupt, especially with favism in children. The anemia is from moderate to extremely severe. It is usually normocytic and normochromic, and it is due partly to intravascular hemolysis. Hence, it is associated with hemoglobinemia, hemoglobinuria, high LDH, and low or absent plasma haptoglobin. The blood film shows anisocytosis, polychromasia, and spherocytes (Fig. 10-6). The most typical feature is the presence of bizarre poikilocytes, with red cells that appear to have unevenly distributed hemoglobin ("hemighosts") and red cells that appear to have had parts of them bitten away ("bite cells" or "blister cells"). A classical test, now rarely carried out, is supravital staining with methyl violet that, if done promptly, reveals the presence of Heinz bodies, consisting of precipitates of denatured hemoglobin and regarded as a signature of oxidative damage to red cells (except for the rare occurrence of an unstable hemoglobin). LDH is high and so is the unconjugated bilirubin, indicating that there is also extravascular hemolysis. The most serious threat from AHA in adults is the development of acute renal failure (this is exceedingly rare in children). Once the threat of acute anemia is over, and in the absence of comorbidity, full recovery from AHA associated with G6PD deficiency is the rule.
TABLE 10-5DRUGS THAT CARRY RISK OF CLINICAL HEMOLYSIS IN PERSONS WITH G6PD DEFICIENCY ||Download (.pdf) TABLE 10-5DRUGS THAT CARRY RISK OF CLINICAL HEMOLYSIS IN PERSONS WITH G6PD DEFICIENCY
Peripheral blood smear from a 5-year-old G6PD-deficient boy with acute favism.
A very small minority of subjects with G6PD deficiency have chronic nonspherocytic hemolytic anemia (CNSHA) of variable severity. The patient is always a male, usually with a history of NNJ, who may present with anemia or unexplained jaundice or because of gallstones later in life. The spleen may be enlarged. The severity of anemia ranges in different patients from borderline to transfusion-dependent. The anemia is usually normo-macrocytic, with reticulocytosis. Bilirubin and LDH are increased. Although hemolysis is, by definition, chronic in these patients, they are also vulnerable to acute oxidative damage, and therefore the same agents that can cause acute HA in people with the ordinary type of G6PD deficiency will cause severe exacerbations in people with the severe form of G6PD deficiency. In some cases of CNSHA, the deficiency of G6PD is so severe in granulocytes that it becomes rate-limiting for their oxidative burst, with consequent increased susceptibility to some bacterial infections.
The suspicion of G6PD deficiency can be confirmed by semiquantitative methods often referred to as screening tests, which are suitable for population studies and can correctly classify male subjects, in the steady state, as G6PD-normal or G6PD-deficient. However, in clinical practice, a diagnostic test is usually needed when the patient has had a hemolytic attack. This implies that the oldest, most G6PD-deficient red cells have been selectively destroyed, and young red cells, having higher G6PD activity, are being released into the circulation. Under these conditions, only a quantitative test can give a definitive result. In males, this test will identify normal hemizygotes and G6PD-deficient hemizygotes; among females, some heterozygotes will be missed, but those who are at most risk of hemolysis will be identified.
TREATMENT: G6PD Deficiency
The acute HA of G6PD deficiency is largely preventable by avoiding exposure to triggering factors of previously screened subjects. Of course, the practicability and cost-effectiveness of screening depends on the prevalence of G6PD deficiency in each community. Favism is entirely preventable in G6PD-deficient subjects by not eating fava beans. Drug-induced hemolysis can be prevented by testing for G6PD deficiency before prescribing; in most cases, one can use alternative drugs. When AHA develops and once its cause is recognized, in most cases no specific treatment is needed. However, if the anemia is severe, it may be a medical emergency, especially in children, requiring immediate action, including blood transfusion. This has been the case with an antimalarial drug combination containing dapsone (called Lapdap, introduced as recently as 2003) that has caused severe acute hemolytic episodes in children with malaria in several African countries; after a few years, it was taken off the market. If there is acute renal failure, hemodialysis may be necessary, but if there is no previous kidney disease, recovery is the rule. The management of NNJ associated with G6PD deficiency is no different from that of NNJ due to other causes.
In cases with CNSHA, if the anemia is not severe, regular folic acid supplements and regular hematologic surveillance will suffice. It will be important to avoid exposure to potentially hemolytic drugs, and blood transfusion may be indicated when exacerbations occur, mostly in concomitance with intercurrent infection. In rare patients, regular blood transfusions may be required, in which case appropriate iron chelation should be instituted. Unlike in hereditary spherocytosis, there is no evidence of selective red cell destruction in the spleen; however, in practice, splenectomy has proven beneficial in severe cases.
Other abnormalities of the redox system
As mentioned earlier, GSH is a key player in the defense against oxidative stress. Inherited defects of GSH metabolism are exceedingly rare, but each one of them can give rise to chronic HA (Table 10-4). A rare, peculiar, usually self-limited severe HA of the first month of life, called infantile poikilocytosis, may be associated with deficiency of glutathione peroxidase (GSHPx) due not to an inherited abnormality but to transient nutritional deficiency of selenium, an element essential for the activity of GSHPx.
Pyrimidine 5′-nucleotidase (P5N) deficiency
P5N is a key enzyme in the catabolism of nucleotides arising from the degradation of nucleic acids that takes place in the final stages of erythroid cell maturation. How exactly its deficiency causes HA is not well understood, but a highly distinctive feature of this condition is a morphologic abnormality of the red cells known as basophilic stippling. The condition is rare, but it probably ranks third in frequency among red cell enzyme defects (after G6PD deficiency and PK deficiency). The anemia is lifelong, of variable severity, and may benefit from splenectomy.
Familial (atypical) hemolytic uremic syndrome (aHUS)
This phrase is used to designate a group of rare disorders, mostly affecting children, characterized by microangiopathic HA with presence of fragmented erythrocytes in the peripheral blood smear, thrombocytopenia (usually mild), and acute renal failure. (The word atypical is part of the phrase because it is the HUS caused by infection with Escherichia coli producing the Shiga toxin that is regarded as typical). The genetic basis of aHUS has been elucidated only recently. Studies of more than 100 families have revealed that family members who have developed HUS have mutations in any one of several genes encoding complement regulatory proteins: complement factor H (CFH), CD46 or membrane cofactor protein (MCP), complement factor I (CFI), complement component C3, complement factor B (CFB), and thrombomodulin. Thus, whereas all other inherited HAs are due to intrinsic red cell abnormalities, this group is unique in that hemolysis results from an inherited defect external to red cells (Table 10-1). Because the regulation of the complement cascade has considerable redundancy, in the steady state, any of the above abnormalities can be tolerated. However, when an intercurrent infection or some other trigger activates complement through the alternative pathway, the deficiency of one of the complement regulators becomes critical. Endothelial cells get damaged, especially in the kidney, and at the same time and partly as a result of this, there will be brisk hemolysis (thus, the more common Shiga toxin–related HUS can be regarded as a phenocopy of aHUS). aHUS is a severe disease with up to 15% mortality in the acute phase and up to 50% of cases progressing to end-stage renal disease. aHUS often undergoes spontaneous remission, and the best tested form of treatment is plasma exchange, which supplies the deficient complement regulator. Because the basis of aHUS is an inherited abnormality, it is not surprising that given exposure to an appropriate trigger, the syndrome will tend to recur: when it does, the prognosis is always serious. In some cases, kidney (and liver) transplantation has been carried out, but the role of these procedures is controversial.
ACQUIRED HEMOLYTIC ANEMIA
Mechanical destruction of red cells
Although red cells are characterized by the remarkable deformability that enables them to squeeze through capillaries narrower than themselves for thousands of times in their lifetimes, there are at least two situations in which they succumb to shear, if not to wear and tear. The result is intravascular hemolysis, resulting in hemoglobinuria. One situation is acute and self-inflicted, march hemoglobinuria. Why sometimes a marathon runner may develop this complication, whereas on another occasion this does not happen, we do not know (perhaps her or his footwear needs attention). A similar syndrome may develop after prolonged barefoot ritual dancing. The other situation is chronic and iatrogenic (it has been called microangiopathic hemolytic anemia); it takes place in patients with prosthetic heart valves, especially when paraprosthetic regurgitation is present. If the hemolysis consequent to mechanical trauma to the red cells is mild, and provided the supply of iron is adequate, it may be largely compensated. If more than mild anemia develops, reintervention to correct regurgitation may be required.
A number of chemicals with oxidative potential, whether medicinal or not, can cause hemolysis even in people who are not G6PD-deficient (discussed earlier). Examples are hyperbaric oxygen (or 100% oxygen), nitrates, chlorates, methylene blue, dapsone, cisplatin, and numerous aromatic (cyclic) compounds. Other chemicals may be hemolytic through a nonoxidative, largely unknown mechanism; examples are arsine, stibine, copper, and lead. The HA caused by lead poisoning is characterized by basophilic stippling. It is in fact a phenocopy of that seen in P5N deficiency (discussed earlier), suggesting it is mediated at least in part by lead inhibiting this enzyme.
In these cases, hemolysis appears to be mediated by a direct chemical action on red cells. But drugs can cause hemolysis through at least two other mechanisms. (1) A drug can behave as a hapten and induce antibody production. In rare subjects, this happens, for instance, with penicillin. Upon a subsequent exposure, red cells are caught, as innocent bystanders, in the reaction between penicillin and antipenicillin antibodies. Hemolysis will subside as soon as penicillin administration is stopped. (2) A drug can trigger, perhaps through mimicry, the production of an antibody against a red cell antigen. The best known example is methyldopa, an antihypertensive agent no longer in use, which in a small fraction of patients stimulates the production of the Rhesus antibody anti-e. In patients who have this antigen, the anti-e is a true autoantibody, which would then cause an autoimmune HA (discussed later). Usually this would gradually subside when methyldopa was discontinued.
Severe intravascular hemolysis can be caused by the venom of certain snakes (cobras and vipers), and HA can also follow spider bites.
By far, the most frequent infectious cause of HA, in endemic areas, is malaria. In other parts of the world, the most frequent cause is probably Shiga toxin–producing Escherichia coli O157:H7, now recognized as the main etiologic agent of the hemolytic-uremic syndrome, more common in children than in adults. Life-threatening intravascular hemolysis, due to a toxin with lecithinase activity, occurs with Clostridium perfringens sepsis, particularly following open wounds or septic abortion or as a disastrous accident due to a contaminated blood unit. Occasionally, HA is seen, especially in children, with sepsis or endocarditis from a variety of organisms.
Autoimmune hemolytic anemia (AIHA)
Except for countries where malaria is endemic, AIHA is the most common form of acquired hemolytic anemia. In fact, not quite appropriately, the two phrases are sometimes used as synonymous.
AIHA is caused by an autoantibody directed against a red cell antigen, i.e., a molecule present on the surface of red cells. The autoantibody binds to the red cells. Once a red cell is coated by antibody, it will be destroyed by one or more mechanisms. In most cases the Fc portion of the antibody will be recognized by the Fc receptor of macrophages, and this will trigger erythrophagocytosis (Fig. 10-7). Thus, destruction of red cells will take place wherever macrophages are abundant, i.e., in the spleen, liver, and bone marrow. Because of the special anatomy of the spleen, it is particularly efficient in trapping antibody-coated red cells, and often this is the predominant site of red cell destruction. Although in severe cases even circulating monocytes can take part in this process, most of the phagocytosis-mediated red cell destruction takes place in the organs just mentioned, and it is therefore called extravascular hemolysis. In some cases, the nature of the antibody (usually an IgM antibody) is such that the antigen–antibody complex on the surface of red cells is able to activate complement (C). As a result, a large amount of membrane attack complex will form, and the red cells may be destroyed directly; this is known as intravascular hemolysis.
Mechanism of antibody-mediated immune destruction of red cells. (From N Young et al: Clinical Hematology. Philadelphia, Elsevier, 2006; with permission.)
The onset of AIHA is very often abrupt and can be dramatic. The hemoglobin level can drop, within days, to as low as 4 g/dL; the massive red cell removal will produce jaundice; and sometimes the spleen is enlarged. When this triad is present, the suspicion of AIHA must be high. When hemolysis is (in part) intravascular, the telltale sign will be hemoglobinuria, which the patient may report or for which the physician must inquire and test. The diagnostic test for AIHA is the antiglobulin test worked out in 1945 by R. R. A. Coombs and known since by his name. The beauty of this test is that it directly detects the pathogenetic mediator of the disease, i.e., the presence of antibody on the red cells themselves. When the test result is positive, it clinches the diagnosis, and when it is negative, the diagnosis is unlikely. However, the sensitivity of the Coombs test varies depending on the technology that is used, and in doubtful cases, a repeat in a specialized lab is advisable; the term "Coombs-negative AIHA" is a last resort. In some cases, the autoantibody has a defined identity: it may be specific for an antigen belonging to the Rhesus system (it is often anti-e). In many cases, it is regarded as "unspecific" because it reacts with virtually all types of red cells.
As in autoimmune diseases in general, the real cause of AIHA remains obscure. However, from the clinical point of view, an important feature is that AIHA can appear to be isolated, or it can develop as part of a more general autoimmune disease, particularly systemic lupus erythematosus, of which sometimes it may be the first manifestation. Therefore, when AIHA is diagnosed, a full screen for autoimmune disease is imperative. In some cases, AIHA can be associated, on first presentation or subsequently, with autoimmune thrombocytopenia (Evans's syndrome).
TREATMENT: Autoimmune Hemolytic Anemia
Severe acute AIHA can be a medical emergency. The immediate treatment almost invariably includes transfusion of red cells. This may pose a special problem because if the antibody involved is unspecific, all the blood units cross-matched will be incompatible. In these cases, it is often correct, paradoxically, to transfuse incompatible blood, the rationale being that the transfused red cells will be destroyed no less but no more than the patient's own red cells, but in the meantime the patient stays alive. Clearly, this rather unique situation requires good liaison and understanding between the clinical unit treating the patient and the blood transfusion/serology lab. Apart from emergency blood transfusion, the first-line treatment of AIHA is by using corticosteroids. In at least one-half of the cases, prednisone (1 mg/kg per day) will produce a remission promptly. Whereas some patients are then apparently cured, relapses are not uncommon. Although unfortunately most of the management of AIHA is not evidence-based, for patients who do not respond and for those who have relapsed (or who require more than 15 mg/d of prednisone to prevent relapse), it is highly recommended to consider a second-line treatment option, which might be either splenectomy or rituximab (anti-CD20). Splenectomy, although it does not cure the disease, can produce significant benefit by removing a major site of hemolysis, thus improving the anemia and/or reducing the need for other therapies (e.g., the dose of prednisone). Rituximab has emerged as a significant alternative to splenectomy because it can produce remissions in up to 80% of patients and it can be used repeatedly, even though progressive multifocal leukoencephalopathy is a dreaded if rare side effect. Azathioprine, cyclophosphamide, cyclosporine, and IV immunoglobulin have become third-line agents since the introduction of rituximab. In severe refractory cases, either auto- or allohematopoietic stem cell transplantation has been used, sometimes successfully.
Paroxysmal cold hemoglobinuria (PCH)
PCH is a rather rare form of AIHA occurring mostly in children, usually triggered by a viral infection, usually self-limited, and characterized by the involvement of the so-called Donath-Landsteiner antibody. In vitro this antibody has unique serologic features: it has anti-P specificity and it binds to red cells only at a low temperature (optimally at 4°C), but when the temperature is shifted to 37°C, lysis of red cells takes place in the presence of complement. Consequently, in vivo there is intravascular hemolysis, resulting in hemoglobinuria. Clinically, the differential diagnosis must include other causes of hemoglobinuria (Table 10-6), but the presence of the Donath-Landsteiner antibody will prove PCH. Active supportive treatment, including blood transfusion, is needed to control the anemia; subsequently, recovery is the rule.
TABLE 10-6DISEASES AND CLINICAL SITUATIONS WITH PREDOMINANTLY INTRAVASCULAR HEMOLYSIS ||Download (.pdf) TABLE 10-6DISEASES AND CLINICAL SITUATIONS WITH PREDOMINANTLY INTRAVASCULAR HEMOLYSIS
| ||ONSET OR TIME COURSE ||MAIN MECHANISM ||APPROPRIATE DIAGNOSTIC PROCEDURE ||COMMENTS |
|Mismatched blood transfusion ||Abrupt ||Nearly always ABO incompatibility ||Repeat cross-match || |
|Paroxysmal nocturnal hemoglobinuria (PNH) ||Chronic with acute exacerbations ||Complement (C)-mediated destruction of CD59(–) red cells ||Flow cytometry to display a CD59(–) red cell population ||Exacerbations due to C activation through any pathway |
|Paroxysmal cold hemoglobinuria (PCH) ||Acute ||Immune lysis of normal red cells ||Test for Donath-Landsteiner antibody ||Often triggered by viral infection |
|Septicemia ||Very acute ||Exotoxins produced by Clostridium perfringens ||Blood cultures ||Other organisms may be responsible |
|Microangiopathic ||Acute or chronic ||Red cell fragmentation ||Red cell morphology on blood smear ||Different causes ranging from endothelial damage to hemangioma to leaky prosthetic heart valve |
|March hemoglobinuria ||Abrupt ||Mechanical destruction ||Targeted history taking || |
|Favism ||Acute ||Destruction of older fraction of G6PD-deficient red cells ||G6PD assay ||Triggered by ingestion of large dish of fava beans, but trigger can be infection or drug instead |
Cold agglutinin disease (CAD)
This designation is used for a form of chronic AIHA that usually affects the elderly and has special clinical and pathologic features. First, the term cold refers to the fact that the autoantibody involved reacts with red cells poorly or not at all at 37°C, whereas it reacts strongly at lower temperatures.1 As a result, hemolysis is more prominent the more the body is exposed to the cold. The antibody is usually IgM with an anti-I specificity (the I antigen is present on the red cells of almost everybody), and it may have a very high titer (1:100,000 or more has been observed). Second, the antibody is produced by an expanded clone of B lymphocytes, and sometimes its concentration in the plasma is high enough to show up as a spike in plasma protein electrophoresis, i.e., as a monoclonal gammopathy. Third, since the antibody is IgM, CAD is related to Waldenström macroglobulinemia (WM) (Chap. 17), although in most cases the other clinical features of this disease are not present. Thus, CAD must be regarded as a form of WM, i.e., as a low-grade mature B cell lymphoma that manifests at an earlier stage precisely because the unique biologic properties of the IgM that it produces give the clinical picture of chronic HA.
In mild forms of CAD, avoidance of exposure to cold may be all that is needed to enable the patient to have a reasonably comfortable quality of life, but in more severe forms, the management of CAD is not easy. Blood transfusion is not very effective because donor red cells are I-positive and will be rapidly removed. Immunosuppressive/cytotoxic treatment with azathioprine or cyclophosphamide can reduce the antibody titer, but clinical efficacy is limited and, in view of the chronic nature of the disease, the side effects may prove, in the long run, unacceptable. Unlike in AIHA, prednisone and splenectomy are ineffective. Plasma exchange is in theory a rational approach, but it is laborious and must be carried out at frequent intervals if it is to be beneficial. Since the advent of rituximab, the picture has changed significantly for those 60% of patients with CAD who respond to this agent. Given the long clinical course of CAD, it remains to be seen with what periodicity rituximab will need to be administered.
Paroxysmal nocturnal hemoglobinuria (PNH)
PNH is an acquired chronic HA characterized by persistent intravascular hemolysis (Table 10-6) subject to recurrent exacerbations. In addition to hemolysis, there are often pancytopenia and a distinct tendency to venous thrombosis. This triad makes PNH a truly unique clinical condition. However, when not all of these three features are manifest on presentation, the diagnosis is often delayed, although it can be always made by appropriate laboratory investigations (discussed later).
PNH has about the same frequency in men and in women, and it is encountered in all populations throughout the world, but it is a rare disease. Its prevalence is estimated to be between 1 and 5 per million (it may be somewhat less rare in Southeast Asia and in the Far East). There is no evidence of inherited susceptibility. PNH has never been reported as a congenital disease, but it can present in small children or as late as in the seventies, although most patients are young adults.
The patient may seek medical attention because, one morning, she or he has "passed blood instead of urine" (Fig. 10-8). This distressing or frightening event may be regarded as the classical presentation; however, more frequently, this symptom is not noticed or is suppressed. Indeed, the patient often presents simply as a problem in the differential diagnosis of anemia, whether symptomatic or discovered incidentally. Sometimes the anemia is associated from the outset with neutropenia, thrombocytopenia, or both, thus signaling an element of bone marrow failure (discussed later). Some patients may present with recurrent attacks of severe abdominal pain, defying a specific diagnosis and eventually found to be related to thrombosis. When thrombosis affects the hepatic veins, it may produce acute hepatomegaly and ascites, i.e., a full-fledged Budd-Chiari syndrome, which in the absence of liver disease ought to raise the suspicion of PNH.
Consecutive urine samples from a patient with paroxysmal nocturnal hemoglobinuria. The variation in the severity of hemoglobinuria within hours is probably unique to this condition.
The natural history of PNH can extend over decades. Without treatment, the median survival time is estimated to be about 8–10 years. In the past, the most common cause of death was venous thrombosis, followed by infection secondary to severe neutropenia and hemorrhage secondary to severe thrombocytopenia. PNH may evolve into aplastic anemia (AA), and PNH may manifest itself in patients who previously had AA. Rarely (estimated 1–2% of all cases), PNH may terminate in acute myeloid leukemia. On the other hand, full spontaneous recovery from PNH has been well documented, albeit rarely.
Laboratory investigations and diagnosis
The most consistent blood finding is anemia, which may range from mild to moderate to very severe. The anemia is usually normo-macrocytic, with unremarkable red cell morphology; if the MCV is high, it is usually largely accounted for by reticulocytosis, which may be quite marked (up to 20%, or up to 400,000/μL). The anemia may become microcytic if the patient is allowed to become iron-deficient as a result of chronic urinary blood loss through hemoglobinuria. Unconjugated bilirubin is mildly or moderately elevated, LDH is typically markedly elevated (values in the thousands are common), and haptoglobin is usually undetectable. All these findings make the diagnosis of HA compelling. Hemoglobinuria, the telltale sign of intravascular hemolysis (Table 10-6), may be overt in a random urine sample. If it is not, it may be helpful to obtain serial urine samples, since hemoglobinuria can vary dramatically from day to day and even from hour to hour (Fig. 10-8). The bone marrow is usually cellular, with marked to massive erythroid hyperplasia, often with mild to moderate dyserythropoietic features (these do not justify confusing PNH with myelodysplastic syndrome). At some stage of the disease, the marrow may become hypocellular or even frankly aplastic (discussed later).
The definitive diagnosis of PNH must be based on the demonstration that a substantial proportion of the patient's red cells have an increased susceptibility to complement (C), due to the deficiency on their surface of proteins (particularly CD59 and CD55) that normally protect the red cells from activated C. The sucrose hemolysis test is unreliable, and the acidified serum (Ham) test is carried out in few labs. The gold standard today is flow cytometry, which can be carried out on granulocytes as well as on red cells. A bimodal distribution of cells, with a discrete population that is CD59-, CD55-, is diagnostic of PNH. Usually this population is at least 5% of the total in the case of red cells and at least 20% of the total in the case of granulocytes.
Hemolysis in PNH is due to an intrinsic abnormality of the red cell, which makes it exquisitely sensitive to activated C, whether it is activated through the alternative pathway or through an antigen–antibody reaction (Fig. 10-9). The former mechanism is mainly responsible for intravascular hemolysis in PNH. The latter mechanism explains why the hemolysis can be dramatically exacerbated in the course of a viral or bacterial infection. Hypersusceptibility to C is due to deficiency of several protective membrane proteins, of which CD59 is the most important because it hinders the insertion into the membrane of C9 polymers. The molecular basis for the deficiency of these proteins has been pinpointed not to a defect in any of the respective genes, but rather to the shortage of a unique glycolipid molecule, glycosylphosphatidyl-inositol (GPI), which, through a peptide bond, anchors these proteins to the surface membrane of cells. The shortage of GPI is due in turn to a mutation in an X-linked gene, called PIG-A, required for an early step in GPI biosynthesis. In virtually every patient, the PIG-A mutation is different. This is not surprising, since these mutations are not inherited; rather, each one takes place de novo in a hematopoietic stem cell (i.e., they are somatic mutations). As a result, the patient's marrow is a mosaic of mutant and nonmutant cells, and the peripheral blood always contains both PNH cells and normal (non-PNH) cells. Thrombosis is one of the most immediately life-threatening complications of PNH and yet one of the least understood in its pathogenesis. It could be that deficiency of CD59 on the PNH platelet causes inappropriate platelet activation; however, other mechanisms are possible.
The complement cascade and the fate of red cells. A. Normal red cells are protected from complement activation and subsequent hemolysis by CD55 and CD59. These two proteins, being GPI-linked, are missing from the surface of PNH red cells as a result of a somatic mutation of the X-linked PIG-A gene that encodes a protein required for an early step of the GPI molecule biosynthesis. B. In the steady state, PNH erythrocytes suffer from spontaneous (tick-over) complement activation, with consequent intravascular hemolysis through formation of the membrane attack complex (MAC); when extra complement is activated through the classical pathway, an exacerbation of hemolysis will result. C. On eculizumab, PNH erythrocytes are protected from hemolysis from the inhibition of C5 cleavage; however, upstream complement activation may lead to C3 opsonization and possible extravascular hemolysis. GPI, glycosylphosphatidylinositol; PNH, paroxysmal nocturnal hemoglobinuria reticuloendothelial system. (From L Luzzatto et al: Haematologica 95:523, 2010.)
Bone marrow failure (BMF) and relationship between PNH and aplastic anemia (AA)
It is not unusual that patients with firmly established PNH have a previous history of well-documented AA. On the other hand, sometimes a patient with PNH becomes less hemolytic and more pancytopenic and ultimately has the clinical picture of AA. Since AA is probably an organ-specific autoimmune disease in which T cells cause damage to hematopoietic stem cells, the same may be true of PNH, with the specific proviso that the damage spares PNH stem cells. Skewing of the T cell repertoire in patients with PNH lends some support to this notion. In addition, there is evidence in mouse models that PNH stem cells do not expand when the rest of the bone marrow is normal, and by using high-sensitivity flow cytometry technology, very rare PNH cells harboring PIG-A mutations can be demonstrated in normal people. In view of these facts, it seems that an element of BMF in PNH is the rule rather than the exception. An extreme view is that PNH is a form of AA in which BMF is masked by the massive expansion of the PNH clone that populates the patient's bone marrow. The mechanism whereby PNH stem cells escape the damage suffered by non-PNH stem cells is not yet known.
TREATMENT: Paroxysmal Nocturnal Hemoglobinuria
Unlike other acquired HAs, PNH may be a lifelong condition; standard care was formerly supportive treatment only, including transfusion of filtered red cells2 whenever necessary, which, for some patients, means quite frequently. Folic acid supplements (at least 3 mg/d) are mandatory, and the serum iron should be checked periodically and iron supplements administered as appropriate. Long-term glucocorticoids are not indicated because there is no evidence that they have any effect on chronic hemolysis; in fact, they are contraindicated because of their many dangerous side effects. A major advance in the management of PNH has been the development of a humanized monoclonal antibody, eculizumab, directed against the complement component C5. In an international, multicenter, placebo-controlled randomized trial of 87 patients (so far the only controlled therapeutic trial in PNH) who had been selected on grounds of having severe hemolysis making them transfusion-dependent, eculizumab proved effective and was licensed in 2007 (Fig. 10-10). By blocking the complement cascade downstream of C5, eculizumab abrogates complement-dependent intravascular hemolysis in all PNH patients, which in itself significantly improves their quality of life. One would expect that, as a result, the need for blood transfusion would be also abrogated, and this indeed is the case in about one-half of the patients, in many of whom there is also a rise in hemoglobin levels. In the remaining patients, the anemia remains sufficiently severe to require blood transfusion, apparently because of ongoing extravascular hemolysis of red cells opsonized by complement (C3) fragments. Based on its half-life, eculizumab must be administered intravenously every 14 days. The only form of treatment that currently can provide a definitive cure for PNH is allogeneic bone marrow transplantation (BMT). When an HLA-identical sibling is available, BMT should be offered to any young patient with severe PNH; the availability of eculizumab has probably decreased significantly the proportion of those who take up this option.
For patients with the PNH-AA syndrome, immunosuppressive treatment with antilymphocyte globulin (ALG or ATG) and cyclosporine A may be indicated. Although no formal trial has ever been conducted, this approach has helped particularly to relieve severe thrombocytopenia and/or neutropenia in patients in whom these were the main problem(s). By contrast, there is often little immediate effect on hemolysis. Any patient who has had venous thrombosis or who has a genetically determined thrombophilic state in addition to PNH should be on regular anticoagulant prophylaxis.
Therapeutic efficacy of an anti-C5 antibody on the anemia of paroxysmal nocturnal hemoglobinuria. (From P Hillmen et al: N Engl J Med 355:1233, 2006; with permission.)