A finite life span is a distinct characteristic of red cells. Hence, a logical, time-honored classification of anemias is in three groups: (1) decreased production of red cells, (2) increased destruction of red cells, and (3) acute blood loss. Decreased production is covered in Chaps. 93, 94, and 98; acute blood loss in Chap. 97; increased destruction is covered in this chapter.
All patients who are anemic as a result of either increased destruction of red cells or acute blood loss have one important element in common: the anemia results from overconsumption of red cells from the peripheral blood, whereas the supply of cells from the bone marrow is normal (indeed, it is usually increased). On the other hand, these two groups differ in that the consequences of physical loss of red cells from the bloodstream or from the body itself, as in acute hemorrhage, is fundamentally different from destruction of red cells within the body, as in hemolytic anemias (HAs).
With respect to primary etiology, HAs may be inherited or acquired; from a clinical point of view, they may be more acute or more chronic, and they may vary from mild to very severe; 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 (Table 96-1). But before reviewing the individual types of HA, it is appropriate to consider what general features they have in common, in terms of clinical aspects and of pathophysiology.
TABLE 96-1Classification of Hemolytic Anemiasa ||Download (.pdf) TABLE 96-1 Classification of Hemolytic Anemiasa
| ||Intracorpuscular Defects ||Extracorpuscular Factors |
Familial (atypical) hemolytic-uremic syndrome
Paroxysmal nocturnal hemoglobinuria (PNH)
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 (HS) or with cold agglutinin disease (CAD) 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. 59).
What differentiates HAs from other anemias is that the patient has signs and symptoms arising directly from hemolysis (Table 96-2). 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 may be also skeletal changes due to overactivity of the bone marrow: they are never as severe as in thalassemia major because there is less ineffective erythropoiesis, or none at all.
TABLE 96-2Features Common to Most Patients with a Hemolytic Disorder ||Download (.pdf) TABLE 96-2 Features Common to Most Patients with a Hemolytic Disorder
|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 ||Usually Increased |
|Bilirubin ||Almost always increased (mostly unconjugated) |
|LDH ||Increased (up to 10× normal with intravascular hemolysis) |
|Haptoglobin ||Reduced to absent if hemolysis is at least in part intravascular) |
The laboratory features of HA are related to (i) hemolysis per se, (ii) the erythropoietic response of the bone marrow. In most cases hemolysis is largely extravascular, and it produces an increase in unconjugated bilirubin and aspartate aminotransferase (AST) in the serum; 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 free hemoglobin, lactate dehydrogenase (LDH) is increased, and haptoglobin is reduced. In contrast, the serum 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 (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 in 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 an 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 eventually extrusion 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. 96-1); 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, which in the red cell is the only provider 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. At the same time, during their long time in circulation, various red cell components inevitably accumulate damage; in senescent red cells, the membrane protein band 3 molecules (see below and Fig. 96-1), having bound hemichromes on their intracellular domains, tend to cluster. Now they bind anti–band 3 IgG antibodies (present in most people) and C3 complement fragments; thus they become opsonized and are eventually removed by phagocytosis in the reticuloendothelial system.
Red blood cell (RBC) metabolism. The Embden-Meyerhof pathway (glycolysis) generates ATP required for cation transport and for membrane maintenance. The generation of NADH maintains hemoglobin iron in a reduced state. The hexose monophosphate shunt generates NADPH that is used to reduce glutathione, which protects the red cell against oxidant stress; the 6-phosphogluconate, after decarboxylation, can be recycled via pentose sugars to glycolysis. Regulation of the 2,3-bisphosphoglycerate level 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.
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.
Thus, the essential pathophysiologic process common to all HAs is an increased red cell turnover; and in many HAs, this is due at least in part to an acceleration of the senescence process described above. The gold standard for proving that the life span of red cells is reduced (compared to 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 the fall in radioactivity over several days or weeks (this classic test can now be replaced by a methodology using the non-radioactive isotope 15N). 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; however, if erythropoiesis is massively increased, the hepcidin-mediated regulation of iron absorption may be disturbed, to the extent that iron overload may set in even without blood transfusion. In the long run, in the absence of iron-chelation therapy 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; and 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 in pregnancy, folate deficiency, or renal failure interfering with adequate EPO production. Another general feature of chronic HAs is seen when any intercurrent condition, such as 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 hemoglobin and the membrane-cytoskeleton complex in working order. Diseases caused by inherited abnormalities of hemoglobin, or hemoglobinopathies, are covered in Chap. 94. 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. 96-2). The lipid bilayer incorporates phospholipids and cholesterol, and it is spanned by a number of proteins that have their hydrophobic transmembrane domain(s) embedded in the membrane; most of these proteins also extend to both the outside (extracellular domains) and the inside of the cell (cytoplasmic domains). Other proteins are tethered to the membrane through a glycosylphosphatidylinositol (GPI) anchor; these have only an extracellular domain, and they include ion channels, receptors for complement components, and receptors for other ligands. The most abundant red cell membrane 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.
The red cell membrane. In this figure, one sees, within the lipid bilayer, several membrane proteins, of which band 3 (anion exchanger 1 [AE1]) is the most abundant; the α-β spectrin dimers that associate to form most of the cytoskeleton; and several proteins (e.g., ankyrin) that connect the membrane to the cytoskeleton. In addition, as examples of glycosylphosphatidylinositol (GPI)-linked proteins, one sees acetylcholinesterase (AChE) and the two complement-regulatory proteins CD59 and CD55. The (non-realistic) shapes of the protein moieties of the GPI-linked proteins are meant to indicate that each one of them is different from others and that, unlike with the other membrane proteins shown, the entire polypeptide chain is extracellular. Branched lines symbolize carbohydrate moiety of proteins. The molecules are obviously not drawn to the same scale. Additional explanations can be found in the text. (From N Young et al: Clinical Hematology. Copyright Elsevier, 2006; with permission.)
The membrane-cytoskeleton complex has essentially three functions: It is an envelope for the red cell cytoplasm, it maintains the normal red cells shape, it provides highly specific cross-membrane transport of electrolytes and of metabolites such as glucose. In the membrane-cytoskeleton complex the individual components are so intimately integrated with each other that an abnormality of almost any of them will be disturbing or disruptive, causing structural or functional 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 (HS) and hereditary elliptocytosis (HE; as well as more rare ones like stomatocytosis, xerocytosis, etc). Now that their molecular basis has been elucidated, it has emerged (see Table 96-3) that, although we are dealing with monogenic disorders, there is no one-to-one correlation between a certain gene and a certain disorder. Rather, what has been regarded as a single disorder (e.g., HS) can arise through mutation of one of several genes; conversely, what have been regarded as different disorders can arise through different mutations of the very same gene (Fig. 96-3).
TABLE 96-3Inherited Diseases of the Red Cell Membrane-Cytoskeleton Complex ||Download (.pdf) TABLE 96-3 Inherited Diseases of the Red Cell Membrane-Cytoskeleton Complex
|Gene ||Chromosomal Location ||Protein Produced ||Disease(s) with Certain Mutations (Inheritance) ||Comments |
|SPTA1 ||1q22-q23 ||α-Spectrin ||HS (recessive) ||Rare |
| || || ||HE (dominant) ||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 ||HS (dominant) ||Rare |
| || || ||HE (dominant) ||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; also known as AE (anion exchanger) or AE1 ||HS (dominant) ||Mutations of this gene may account for about 25% of HS. |
| || || ||Southeast Asia ovalocytosis (dominant) ||Polymorphic mutation (deletion of 9 amino acids); in heterozygotes clinically asymptomatic and protective against Plasmodium falciparum. |
| || || ||Stomatocytosis ||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 little/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-associated glycoprotein ||Chronic nonspherocytic hemolytic anemia (recessive) || |
Very rare; associated with total loss of all Rh antigens.
One specific mutation in this gene causes overhydrated stomatocytosis.
|PIEZO1 ||16q23-q24 ||PIEZO1 (mechanosensitive cation channel) ||Dehydrated hereditary stomatocytosis (dominant) ||Also known as xerocytosis with pseudohyperkalemia. Patients may present with perinatal edema. |
|KCNN4 ||19q13.31 || |
Intermediate conductance calcium-activated potassium channel protein 4
|Dehydrated hereditary stomatocytosis (dominant) ||Clinical presentation similar to that of PIEZO1 mutants. |
|SLC2A1 ||1p34.2, ||GLUT1 glucose transporter ||Over-hydrated hereditary stomatocytosis ||Associated with serious neurological manifestations. |
Hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary stomatocytosis (HSt) are three morphologically distinct forms of congenital hemolytic anemia. It has emerged that each one can arise from mutation of one of several genes and that different mutations of the same gene can give one or another form. (See also Table 96-3.)
This is a relatively common type of genetically determined 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 who had the presence of numerous spherocytes in the peripheral blood (Fig. 96-4A). 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 96-3). It has been also recognized that the inheritance of HS is not always autosomal dominant (with the patient being heterozygous); indeed, some of the most severe forms are instead autosomal recessive (with the patient being homozygous).
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. The main clinical findings are jaundice, an enlarged spleen, and often gallstones; indeed, it may be 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 96-3). Not only are mutations of several genes involved: even different mutations of the same gene can give very different clinical manifestations. In milder cases, hemolysis is often compensated (see above), but changes in clinical expression may be seen even in the same patient because intercurrent conditions (e.g., pregnancy, infection) may cause decompensation. The anemia is usually normocytic, with the characteristic morphology that gives the disease its name. An increased mean corpuscular hemoglobin concentration (MCHC >34) on an ordinary blood count report should raise the suspicion of HS, because HS is almost the only condition in which this abnormality occurs. 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 that may take place elsewhere.
When there is a family history, it is usually easy to make a diagnosis based on features of HA and typical red cell morphology. However, there may be no family history for at least two reasons. First, the patient may have a de novo mutation, i.e., a mutation that has taken place in a germ cell of one of his parents or early after zygote formation. Second, the patient may have a recessive form of HS (Table 96-3). In such cases, more extensive laboratory investigations are required, including osmotic fragility, the acid glycerol lysis test, the eosin-5′-maleimide (EMA)–binding test, and SDS-gel electrophoresis of membrane proteins; these tests are usually carried out in laboratories with special expertise in this area. Sometimes a definitive diagnosis can be obtained only by molecular studies demonstrating a mutation in one of the genes underlying HS (Table 96-3).
TREATMENT Hereditary Spherocytosis
We do not have a causal treatment for HS; i.e., no way has yet been found to correct the basic defect in the membrane-cytoskeleton structure. Given the special role of the spleen in HS (see above), it has long been thought that an almost obligatory therapeutic measure was splenectomy. Because this operation may have more than trivial consequences, today we have more articulate recommendations, based on disease severity, as follows. In mild cases, avoid splenectomy; in moderate cases, delay splenectomy until puberty; in severe cases, proceed with splenectomy at the age of 4–6. It is also helpful, whenever possible, to know about the outcome of splenectomy in the patient’s affected relatives. Antipneumococcal vaccination before splenectomy is imperative, whereas penicillin prophylaxis after splenectomy is controversial. Along with splenectomy, cholecystectomy should not be carried out automatically; it should be carried out, usually by the laparoscopic approach, when clinically indicated.
HE is at least as heterogeneous as HS, both from the genetic point of view (Table 96-3, Fig. 96-3) and from the clinical point of view. Again, it is the shape of the red cells (Fig. 96-4B) that gives the name to the condition, 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 (or ovalocytes); whereas in severe cases all kinds of bizarre poikilocytes may 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, one particular in-frame deletion of nine amino acids in the SLC4A1 gene encoding band 3, which underlies the so-called Southeast Asia ovalocytosis (SAO), has a frequency of up to 7% in certain populations, presumably as a result of malaria selection; it is asymptomatic in heterozygotes and probably lethal in homozygotes.
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 a 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 (Fig. 96-3). In patients from other families, instead, the red cells are dehydrated (high MCHC), and their consequent rigidity has earned this disorder the name xerocytosis. One would surmise that in these disorders the primary defect may be in a cation transporter; indeed, xerocytosis results from mutations in PIEZO1. In other patients with stomatocytosis, mutations are found in other genes also related to solute transport (Table 96-3), including SLC4A1 (encoding band 3), the Rhesus gene RHAG, and the glucose transporter gene SLC2A1 responsible for a special form called cryohydrocytosis, an unusual variant in which the red cells swell and burst when they are cooled. In vivo hemolysis can vary from relatively mild to quite severe. From the practical point of view, it is important to know that in stomatocytosis splenectomy is strongly contraindicated because it has been followed in a significant proportion of cases by severe thromboembolic complications.
When there is an important defect in a component of the membrane cytoskeleton complex, hemolysis is a direct consequence of the fact that the very structure of the red cell is compromised. 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. This machinery has two main functions: (1) to provide energy in the form of ATP and (2) to prevent oxidative damage to hemoglobin and to other proteins by providing sufficient reductive potential; the key molecule for this is NADPH.
ABNORMALITIES OF THE GLYCOLYTIC PATHWAY
Because red cells, in the course of their differentiation, have sacrificed not only their nucleus and their ribosomes but also their mitochondria, they rely exclusively on the anaerobic portion of the glycolytic pathway for producing ATP, most of which 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 (Table 96-4), the result will be hemolytic disease.
TABLE 96-4Red Cell Enzyme Abnormalities Causing Hemolysis ||Download (.pdf) TABLE 96-4 Red Cell Enzyme Abnormalities Causing Hemolysis
| ||Enzyme (Acronym) ||Gene Symbol; Chromosomal Location ||Prevalence of Enzyme Deficiency (Rank) ||Clinical Manifestations Extra-Red Cell ||Comments |
|Glycolytic Pathway || || || || || |
| ||Hexokinase (HK) ||HK1; 10q22 ||Very rare || ||May benefit from splenectomy; BMTc |
| ||Glucose 6-phosphate isomerase (G6PI) ||GPI; 19q31.1 ||Rare (4)a ||NM, CNS ||May benefit from splenectomy |
| ||Phosphofructokinase (PFK)b ||PFKM; 12q13 ||Very rare ||Myopathy; myoglobinuria || |
| ||Aldolase ||ALDOA; 16q22-24 ||Very rare ||Myopathy || |
| ||Triose phosphate isomerase (TPI) ||TPI1; 12p13.31 ||Very rare ||CNS (severe), NM || |
| ||Glyceraldehyde 3-phosphate dehydrogenase (GAPD) ||GAPDH; 12p13.31- ||Very rare ||Myopathy || |
| ||Bisphosphoglycerate mutase (DPGM) ||BPGM; 7q33 ||Very rare || ||Erythrocytosis rather than hemolysis |
| ||Phosphoglycerate kinase (PGK) ||PGK1; Xq21.1 ||Very rare ||CNS, NM ||May benefit from splenectomy; BMTc |
| ||Pyruvate kinase (PK) ||PKLR; 1q22 ||Rare (2)a || ||May benefit from splenectomy; BMTc |
|Redox || || || || || |
| ||Glucose 6-phosphate dehydrogenase (G6PD) ||G6PD; Xq28 ||Common (1)a ||Very rarely granulocytes ||In almost all cases, only AHA from exogenous trigger |
| ||Glutathione synthase ||GSS; 20q11.22 ||Very rare ||CNS || |
| ||Glutathione reductase ||GSR: 8p12 ||Very rare ||Cataracts ||AHA from exogenous trigger (favism) |
| ||γ-Glutamylcysteine synthase ||GCLC; 6p12.1 ||Very rare ||CNS ||Mutations affect catalytic subunit |
| ||Cytochrome b5 reductase ||CYB5R3: 22q13.2 ||Rare ||CNS ||Methemoglobinemia rather than hemolysis |
|Nucleotide Metabolism || || || || || |
| ||Adenylate kinase (AK) ||AK1; 9q34.11 ||Very rare ||CNS ||May benefit from splenectomy |
| ||Pyrimidine 5’ nucleotidase (P5N) ||NTSC3A; 7p14.3 ||Rare (3)a || ||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 in most populations of the order of 1:10,000. However, recently, a polymorphic PK mutation (E277K) was found in some African populations, with heterozygote frequencies of 1–7%, suggesting that this may be another malaria-related polymorphism. The clinical picture of homozygous (or biallelic) PK deficiency is that of an HA that often presents in the newborn with neonatal jaundice; the jaundice persists, and it is often associated with reticulocytosis. The anemia is of variable severity; sometimes it is so severe as to require regular blood transfusion treatment, whereas sometimes it is mild, bordering on a nearly compensated hemolytic disorder. As a result, the diagnosis may be delayed: in some cases it is made, for instance, in a young woman during her first pregnancy, when the anemia may get worse. The delay in diagnosis may be caused in part by the fact that the anemia is often remarkably well tolerated, because the metabolic block at the last step in glycolysis causes an increase in 2,3-bisphosphoglycerate (or DPG; Fig. 96-1), a major effector of the hemoglobin-oxygen dissociation curve; thus, the oxygen delivery to the tissues is enhanced, a remarkable compensatory feat.
TREATMENT Pyruvate 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 be required even in some patients who, though not receiving blood transfusion, may be developing iron overload (see “General Pathophysiology” above). In patients who have more severe disease splenectomy may be beneficial, as the anemia improves (paradoxically, reticulocytes often increase considerably). There is a single case report of curative treatment of PK deficiency by bone marrow transplantation (BMT) from an HLA-identical PK-normal sibling. This seems a viable option for severe cases when a sibling donor is available. Prenatal diagnosis has been carried out in a mother who had already had an affected child. A clinical trial of a small molecule that is a specific PK ligand and may increase the stability and/or catalytic efficiency of mutant PK is currently on-going. Rescue of inherited PK deficiency through lentiviral-mediated human PK gene transfer has been successful in mice.
Other Glycolytic Enzyme Abnormalities
All of these defects are rare to very rare (Table 96-4), and most of them 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 can involve the central nervous system (sometimes entailing severe mental retardation, particularly in the case of triose phosphate isomerase deficiency), the neuromuscular system, or both (see Table 96-4). This is not altogether surprising, if we consider that these are housekeeping genes, i.e., expressed in all tissues. The diagnosis of HA is usually not difficult, thanks to the triad of normomacrocytic anemia, reticulocytosis, and hyperbilirubinemia. Enzymopathies should be considered in the differential diagnosis of any chronic Coombs-negative HA. Unlike with membrane disorders, in most cases of glycolytic enzymopathies morphologic abnormalities are conspicuous by their absence. A definitive diagnosis can be made only by demonstrating the deficiency of an individual enzyme by quantitative assays; these are carried out in only a few specialized laboratories. If a particular molecular abnormality is already known in the family, then one could test directly for that defect at the DNA level, thus bypassing the need for enzyme assays. Of course the time may be getting nearer when a patient will present with her or his exome already sequenced, and we will need to concentrate on which genes to look up within the file. The principles for the management of these conditions are similar as for PK deficiency. In isolated cases of glycolytic enzyme abnormalities BMT has been carried out successfully: although unfortunately non-hematologic manifestations, if any, are not reversed.
ABNORMALITIES OF REDOX METABOLISM
Glucose 6-Phosphate Dehydrogenase (G6PD) Deficiency
G6PD is a housekeeping enzyme critical in the redox metabolism of all aerobic cells (Fig. 96-1). In red cells, its role is even more critical because it is the only source of NADPH, which directly and via glutathione (GSH) defends these cells against oxidative stress (Fig. 96-5). G6PD deficiency-related HA 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 present in most tissues of G6PD-deficient subjects, in other cells the decrease is much less pronounced than in red cells, and it does not seem to impact on clinical expression.
The role of G6PD in protecting red cells from oxidative damage. A. In G6PD-normal red cells, G6PD and 6-phosphogluconate dehydrogenase – two of the enzymes of the pentose phosphate pathway—provide ample supply of NADPH, which in turn regenerates GSH when this is oxidized by reactive oxygen species (e.g., O2– and H2O2). O2– is one of the most reactive oxygen species that can be generated from the metabolism of pro-oxidant compounds such as primaquine; rasburicase, on the other hand, produces directly hydrogen peroxide in equimolar amount to uric acid degraded. B. In G6PD-deficient red cells, where the enzyme activity is reduced, NADPH production is limited, and it may not be sufficient to cope with the excess of reactive oxygen species generated in the presence of pro-oxidant compounds. This diagram also explains why a defect in glutathione reductase has very similar consequences to G6PD deficiency.
The G6PD gene is X-linked, and this has important implications. First, because 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, who have two G6PD genes, can be either normal or deficient (homozygous) or intermediate (heterozygous). Second, 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. 96-5). Almost all of the nearly 200 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.
Among these mutations, those underlying chronic nonspherocytic hemolytic anemia (CNSHA; see 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. 96-6) 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 because it confers a relative resistance against this highly lethal infection. As in other cases of balanced polymorphism, it is heterozygotes, therefore females, who are protected. 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, in the Middle East, 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. 96-6).
Epidemiology of glucose 6-phosphate dehydrogenase (G6PD) deficiency throughout the world. Color shades on the map indicate the median allele frequency of G6PD deficiency in malaria endemic and malaria-eliminating countries, according to a geostatistical model. Each colored circle illustrates the geographic distribution of one polymorphic G6PD allele present in more than one population. Dark grey circles indicate “local” polymorphic variants that have been detected only in one population. (From L Luzzatto et al: Hematology/Oncology Clinics of North America, 30:373, 2016.)
The vast majority of people with G6PD deficiency remain clinically asymptomatic throughout their lifetime; however, all of them have an increased risk of developing neonatal jaundice (NNJ) and a risk of developing acute HA (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, and/or environmental factors (such as naphthalene-camphor balls, which may be 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’s 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 96-5). 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. The onset can be extremely abrupt, especially with favism in children. The anemia is moderate to extremely severe, usually normocytic and normochromic, and 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; in addition, the most typical feature of G6PD deficiency 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”) (Fig. 96-7). A classical test, now rarely carried out, is supravital staining with methyl violet, which, if done promptly, reveals the presence of Heinz bodies (consisting of precipitates of denatured hemoglobin and hemichromes), which are regarded as a signature of oxidative damage to red cells (they are also seen with unstable hemoglobins). Not only LDH is high; also unconjugated bilirubin is high, 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 96-5Drugs That Carry Risk of Clinical Hemolysis in Persons with Glucose 6-Phosphate Dehydrogenase Deficiency ||Download (.pdf) TABLE 96-5 Drugs That Carry Risk of Clinical Hemolysis in Persons with Glucose 6-Phosphate Dehydrogenase Deficiency
Peripheral blood smear from a glucose 6-phosphate dehydrogenase (G6PD)-deficient boy experiencing hemolysis. Note the red cells that are misshapen and called “bite” cells. (From MA Lichtman et al: Lichtman’s Atlas of Hematology: http://www.accessmedicine.com. Copyright © The McGraw-Hill Companies, Inc. All rights reserved.)
It was primaquine (PQ)-induced AHA that led to the discovery of G6PD deficiency, but this drug has not been very prominent subsequently because it is not necessary for the treatment of life-threatening P. falciparum malaria. Today there is a revival of interest in PQ because it is the only effective agent for eliminating the gametocytes of P. falciparum (thus preventing further transmission) and for eliminating the hypnozoites of Plasmodium vivax (thus preventing endogenous relapse). In countries aiming to eliminate malaria, there may be a call for mass administration of PQ; this ought to be associated with G6PD testing. At the other end of the historic spectrum, the latest addition to the list of potentially hemolytic drugs (Table 96-5) is rasburicase; again G6PD testing ought to be made mandatory before giving this drug because fatal cases have been reported in newborns with kidney injury and in adults with tumor lysis syndrome.
Although drug-induced AHA has been prominent in the study of G6PD deficiency, the commonest clinical manifestations are in fact NNJ and favism, both of which are of public health importance in many populations. Contrary to beliefs that are still widespread, fava bean pollen inhalation does not cause favism, and other beans are safe.
A very small minority of subjects with G6PD deficiency have CNSHA of variable severity. The patient is nearly always a male, usually with a history of NNJ, who may present with anemia, unexplained jaundice, or 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 normomacrocytic, 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 AHA in people with the ordinary type of G6PD deficiency will cause severe exacerbations in people with CNSHA associated with 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 semi-quantitative 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. Of course, G6PD deficiency also can be diagnosed by DNA testing. Currently easy-to-use “point of care” tests for G6PD deficiency are becoming available, geared especially to the prospect of mass administration of PQ or other anti-malarials.
TREATMENT G6PD Deficiency
The AHA 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 depend on the prevalence of G6PD deficiency in each individual 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 many cases one can use alternative drugs. When AHA develops and once its cause is recognized, no specific treatment is needed in most cases. 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 in 2003) that has caused severe acute hemolytic episodes in children with malaria in several African countries; after a few years, the drug 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 HS, 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 previously, GSH is a key player in the defense against oxidative stress. Inherited defects of GSH metabolism are exceedingly rare, but each one can give rise to chronic HA (Table 96-4). A rare, peculiar, severe but usually self-limited HA occurring in 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 term 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 in this phrase should be consigned to history: it was introduced originally to distinguish this condition from the hemolytic-uremic syndrome [HUS] caused by infection with Escherichia coli producing the Shiga toxin, regarded as typical.) The genetic basis of atypical HUS (aHUS) has been elucidated. Studies of >100 families have revealed that those family members who developed HUS had 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), thrombomodulin, and others. 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 96-1). 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 briskly activates complement the deficiency of one of the complement regulators becomes critical. Endothelial cells get damaged, especially in the kidney; at the same time, and partly as a result of this, there will be brisk hemolysis (thus, the more common Shiga toxin–related HUS (Chap. 161) 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 (ESRD). Not infrequently, aHUS undergoes spontaneous remission; but because its basis is an inherited abnormality, it is not surprising that, given renewed exposure to a trigger, the syndrome will tend to recur; when it does, the prognosis is always serious. The traditional treatment has been plasma exchange, which will supply the deficient complement regulator. This has changed since the introduction of the anti-C5 complement inhibitor eculizumab (see “Paroxysmal Nocturnal Hemoglobinuria”) was found to greatly ameliorate the microangiopathic picture, with improvement in platelet counts and in renal function, thus abrogating the need for plasma exchange, which is not always effective and not free of complications. Since the basis of aHUS is genetic, and even after complete remission relapses are always possible, there is a rationale for continuing eculizumab indefinitely, especially in order to prevent ESRD. Patients who relapsed after discontinuing eculizumab have responded again. Discontinuation of eculizumab might be reasonable especially in patients heterozygous for a MCP mutation. However, there is no evidence base at the moment for balancing the pros and cons of lifetime eculizumab (a very expensive drug).
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 lifetime, 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 (Table 96-6). 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 or intense playing of bongo drums. 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 on mechanical trauma to the red cells is mild, and if the supply of iron is adequate, the loss may be largely compensated; if more than mild anemia develops, reintervention to correct regurgitation may be required.
TABLE 96-6Diseases and Clinical Situations in Which Hemolysis Is Largely Intravascular ||Download (.pdf) TABLE 96-6 Diseases and Clinical Situations in Which Hemolysis Is Largely Intravascular
| ||Onset/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 beansa |
By far the most frequent infectious cause of HA, in endemic areas, is malaria (Chap. 219). In other parts of the world, the most frequent direct cause is probably Shiga toxin–producing E. coli O157:H7, now recognized as the main etiologic agent of HUS, which is more common in children than in adults (Chap. 156). Life-threatening intravascular hemolysis, due to a toxin with lecithinase activity, occurs with Clostridium perfringens sepsis, particularly following open wounds, septic abortion, or as a disastrous accident due to a contaminated blood unit. Rarely, and if at all in children, HA is seen with sepsis or endocarditis from a variety of organisms. In addition, bacterial and viral infections can cause HA by indirect mechanisms (see previous section on G6PD deficiency and Table 96-6).
These can arise through at least two distinct mechanisms. First, when an antibody directed against a certain molecule (e.g., a drug) reacts with that molecule, red cells may get caught in the reaction (the so-called innocent bystander mechanism: see section below on Hemolytic Anemia from Toxic Agents and Drugs), whereby they are damaged or destroyed. Second, and more frequently, a true auto-antibody is directed against a red cell antigen, i.e., a molecule present on the surface of red cells.
AUTOIMMUNE HEMOLYTIC ANEMIA (AIHA)
This latter mechanism is common to a group of rare disorders (AIHA), with an estimated incidence in the United States of about 2/105/year. AIHA can be serious, since even with appropriate management the mortality is of the order of 5–10%.
The onset is often abrupt and can be dramatic. The hemoglobin level may 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 about which we must enquire or test for.
There are few situations in hematology where one laboratory test is so informative as the direct antiglobulin test developed in 1945 by R. R. A. Coombs, and known since then by this name. The currently recommended version of this test uses in the first instance a “broad spectrum” reagent: i.e., one that will detect not only immunoglobulins (Ig) but also complement (C) components (usually C3 fragments) bound to the surface of the patient’s red cells. If the test is positive (and barring special circumstances such as previous blood transfusion), it is practically diagnostic of AIHA; and one can then determine, by using specific reagents, whether Ig or C or both are implicated. The sensitivity of the Coombs test varies depending on the techniques that are used: in general, the test is positive if there are an average of at least 400 molecules of Ig and/or C on each red cell; but with more advanced techniques the sensitivity can be pushed to as low as 40 molecules per red cell: therefore liaison with a specialized laboratory is desirable. In the past the diagnosis of “Coombs-negative AIHA” was regarded as a last resort, but it is important to know that a patient with this label may have severe AIHA, because if the antibody is powerful (high affinity/avidity), few molecules may be sufficient to opsonize red cells. Based on the Coombs test findings as well as on the thermal characteristics and the antigenic specificities of the auto-antibodies (Table 96-7), AIHA has been classified into subtypes.
TABLE 96-7Classification of Acquired Immune Hemolytic Anemias ||Download (.pdf) TABLE 96-7 Classification of Acquired Immune Hemolytic Anemias
|Clinical Setting ||Type of Antibody |
|Cold, Mostly IgM, Optimal Temperature 4°C–30°C ||Warm, Mostly IgG, Optimal Temperature 37°C; or Mixed |
|Primary ||CAD ||AIHA (idiopathic) |
Secondary to viral infection
|Secondary to other infection ||Mycoplasma infection: paroxysmal cold hemoglobinuria || |
Secondary to/associated with other disease
Chronic inflammatory disorders (e.g., IBD)
After allogeneic HSCT
After immune check-point modulating drugs
|Secondary to drugs: drug-induced immune hemolytic anemia ||Small minority (e.g., with lenalidomide) ||Majority: currently most common culprit drugs are cefotetan, ceftriaxone, piperacillin |
| ||Drug-dependent: antibody destroys red cells only when drug present (e.g., rarely penicillin) |
| ||Drug-independent: antibody can destroy red cells even when drug no longer present (e.g., methyldopa) |
This is the more common type of AIHA. As the name suggests, the auto-antibody reacts best at 37°C: it will often react with most red cells, but it is usually Rhesus-specific (sometimes specifically anti-e). Warm antibody AIHA may be seen in isolation (and it is then called idiopathic) or as part of a systemic auto-immune disorder such as systemic lupus erythematosus (SLE: sometimes AIHA may be the first manifestation that leads to a diagnosis of SLE). Like all auto-immune diseases, AIHA must arise from a dysregulation of immunity. It is therefore not surprising that it is increasingly being recognized in chronic lymphocytic leukemia (CLL), whether treated or untreated; after BMT; and after solid organ transplantation entailing immuno-suppressive treatment. Recently, warm antibody AIHA has also occurred as a side effect of the use of immune checkpoint inhibitors, such as nivolumab, in patients with various types of cancer.
Once a red cell is coated by an autoantibody 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. Thus, destruction of red cells will take place wherever macrophages are abundant, i.e., in the spleen, liver, and bone marrow (extravascular hemolysis see Fig. 96-8). Because of the special anatomy of the spleen, this organ is particularly efficient in trapping antibody-coated red cells, and often this is the predominant site of red cell destruction. In some cases, the nature of the antibody is such (usually an IgM antibody) 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 (MAC) will form, and the red cells may be destroyed directly (intravascular hemolysis).
Mechanism of antibody-mediated immune destruction of red blood cells (RBCs). The three bottom images illustrate three different modalities of extravascular hemolysis. ADCC, antibody-dependent cell-mediated cytotoxicity. (From N Young et al: Clinical Hematology. Philadelphia, Elsevier, 2006; with permission.)
The hematological picture of AIHA includes in most cases reticulocytosis, as the bone marrow responds to anemia: but in some cases reticulocytes may not be increased because they themselves are attacked by the auto-antibody, and this may signify the disease is more severe. In some cases AIHA can be associated, on first presentation or subsequently, with autoimmune thrombocytopenia (Evans’ syndrome): this too usually signals severe disease. Evans’ syndrome may be a manifestation of common variable immune deficiency, and in children it may suggest one of several primary immune deficiency syndromes.
TREATMENT Warm Antibody 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 many or all of the blood units cross-matched may be incompatible. In these cases, it is often correct, if paradoxical, to transfuse ABO-matched but 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, and in the meantime the patient stays alive. A situation like this requires close liaison and understanding between the clinical unit treating the patient and the blood transfusion/serology lab. Whenever the anemia is not immediately life threatening, blood transfusion should be withheld (because compatibility problems may increase with each unit of blood transfused), and medical treatment started immediately with prednisone (1 mg/kg per day), which will produce a remission promptly in at least one-half of patients. Rituximab (anti-CD20), previously regarded as second-line treatment, is increasingly being used at a relatively low dose (100 mg/wk × 4), together with prednisone as part of first-line treatment. It is especially encouraging that this approach seems to reduce the rate of relapse, a common occurrence in AIHA. For patients who do relapse or are refractory to medical treatment, one may have to consider splenectomy: this procedure does not cure the disease, but it 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); of course splenectomy is not free of risk, as it entails increased risk of sepsis and of thrombosis. Since the introduction of rituximab, azathioprine, cyclophosphamide, cyclosporine, and intravenous immunoglobulin have become second- or third-line agents. In very rare severe refractory cases, one may have to consider myelo-immuno-ablative chemotherapy followed by rescue with either autologous or allogeneic hematopoietic stem cell transplantation.
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 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 96-6), but the presence of the Donath-Landsteiner antibody will prove PCH. Active supportive treatment, including blood transfusion, may be needed to control the anemia; subsequently, recovery is the rule.
This designation is used for a form of AIHA that usually affects the elderly and has special clinical and pathologic features. First, CAD is characteristically a chronic condition—in contrast to the abrupt onset of warm antibody AIHA. Second, 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. As a result, hemolysis is more prominent the more the body is exposed to the cold. The antibody is usually IgM; usually it has 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). Third, the antibody is produced by an expanded B lymohocyte clone (a low-grade mature B cell lymphoma): and sometimes the antibody concentration in the serum is high enough to show up as a spike in plasma protein electrophoresis, i.e., as a monoclonal gammopathy. Indeed, since we are dealing with a clonal disease and the antibody is IgM, CAD must be regarded as a form of Waldenström macroglobulinemia (see Chap. 107). The unique biologic properties of the IgM produced in a patient with CAD give the clinical picture of HA, often well before other clinical features of WM become manifest.
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. Plasma exchange will remove antibody and is, therefore, in theory, a rational approach, but it is laborious and must be carried out at frequent intervals if it is to be beneficial. The management of CAD has changed significantly with the advent of the anti-CD20 antibody rituximab: up to 60% of patients respond. If remission is followed by relapse a new course of rituxumab may be again effective, and remissions may be more durable with a rituximab-fludarabine combination. Therefore, even in the absence of a formal trial, rituximab has become de facto first-line treatment: especially since previously used immunosuppressive/cytotoxic agents such as azathioprine or cyclophosphamide, although they can reduce the antibody titer, have limited clinical efficacy and, in view of the chronic nature of CAD, their side effects may prove unacceptable. Unlike in AIHA, prednisone and splenectomy are ineffective. In terms of supportive treatment blood transfusion may be helpful—in spite of the fact that red cells from the donor, being I-positive, will survive no longer than those of the patient: both the blood bag and the patient’s extremities must be kept warm during transfusion.
Hemolytic Anemia from Toxic Agents and Drugs
A number of chemicals with oxidative potential, whether medicinal or not, can cause hemolysis even in people who are not G6PD-deficient (for which see above). Examples are hyperbaric oxygen (or 100% oxygen), nitrates, chlorates, methylene blue, dapsone, cisplatin, and numerous aromatic (cyclic) compounds. Other chemicals may be hemolytic through nonoxidative, largely unknown mechanisms; examples include 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 (see above), 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 anti-penicillin 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 stimulated the production of the Rhesus antibody anti-e. In patients who have this antigen, the anti-e is a true autoantibody, which then causes true AIHA (see above). Usually this will gradually subside once methyldopa is discontinued.
Severe intravascular hemolysis can be caused by the venom of certain snakes (cobras and vipers), and HA can also follow spider bites.
Paroxysmal Nocturnal Hemoglobinuria (PNH)
PNH is an acquired chronic HA characterized by persistent intravascular hemolysis with occasional or frequent recurrent exacerbations. In addition to (i) hemolysis, there may be (ii) pancytopenia and (iii) 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 always be made by appropriate laboratory investigations (see below).
PNH is encountered in all populations throughout the world, but it is a rare disease, with an estimated prevalence of ~5 per million (it may be somewhat less rare in Southeast Asia and in the Far East). PNH has about the same frequency in men and women. PNH is not inherited, and it 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.
When seeking medical attention, the patient may report that one morning, she or he “passed blood instead of urine” (Fig. 96-9). This distressing or frightening event may be regarded as the classic presentation; however, more frequently, this symptom is not noticed or not reported. 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 (see below). Some patients may present with recurrent attacks of severe abdominal pain eventually found to be related to thrombosis in abdominal veins, or attributable to NO depletion associated with intravascular hemolysis. When thrombosis affects the hepatic vein 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 (PNH). The variation in the severity of hemoglobinuria within hours is probably unique to this condition.
The natural history of PNH can extend over decades. In the past, with supportive treatment only, the median survival was estimated to be about 10–20 years; with the most common cause of death being venous thrombosis, followed by infection secondary to severe neutropenia and hemorrhage secondary to severe thrombocytopenia. 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 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 iron 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 of these findings make the diagnosis of HA compelling. Hemoglobinuria may be overt in a random urine sample; if it is not, it may be helpful to obtain serial urine samples (Fig. 96-9) because hemoglobinuria can vary dramatically from day to day and even from hour to hour. The bone marrow is usually cellular, with marked to massive erythroid hyperplasia, often with mild to moderate dyserythropoietic features (these overlap with those seen in myelodysplastic syndromes, but PNH remains a separate entity). At some stage of the disease, the marrow may become hypocellular or even frankly aplastic (see below).
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; in contrast, the acidified serum (Ham) test is highly reliable but is carried out only in a few labs. The gold standard today is flow cytometry, which can be carried out on granulocytes as well as on red cells, and has a very high sensitivity. In PNH, characteristically, one sees a bimodal distribution of cells, with a discrete population that is CD59 and CD55 negative. Although very small populations of CD59(-) cells are of interest in terms of pathophysiology (particularly of aplastic anemia [AA]), no patient should be diagnosed with PNH unless their proportion is substantial: in first approximation at least 5% of the total red cells and at least 20% of the total granulocytes.
Hemolysis in PNH is mainly intravascular and is due to an intrinsic abnormality of the red cell, which makes it exquisitely sensitive to activated C, whether C is activated through the alternative pathway or through an antigen-antibody reaction (classic pathway). The former mechanism is mainly responsible for chronic hemolysis in PNH; the latter explains why the hemolysis can be dramatically exacerbated in the course of a viral or bacterial infection. Hypersusceptibility to C is due to deficiency in the red cell membrane of several protective proteins (Fig. 96-10), among which CD59 is the most important because it is able to hinder 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, GPI (Fig. 96-2), which, through a peptide bond, anchors these proteins to the surface membrane of cells. The shortage of GPI is due in turn to a somatic mutation in an X-linked gene, called PIGA, required for an early step in GPI biosynthesis. As a result, the patient’s marrow is a mosaic of mutant and nonmutant cells, and the peripheral blood always contains both GPI-negative (PNH) cells and GPI-positive (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. (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 AA, sometimes well-documented; indeed, BMF preceding overt PNH is probably the rule rather than the exception. On the other hand, sometimes a patient with PNH becomes less hemolytic and more pancytopenic and ultimately has the clinical picture of AA. The relationship between PNH and AA manifested in the clinical course of patients may reflect a close link in pathogenesis. AA is thought to be an organ-specific autoimmune disease, in which T cells cause damage to hematopoietic stem cells via an as yet unidentified molecular target. The same may be true of PNH, and in this condition the target might be the GPI molecule itself. This would explain why GPI-negative (PNH) stem cells are spared; PIGA mutations can be demonstrated in normal people. Thus, PNH results from the combined action of two factors: failure of normal hematopoiesis and massive expansion of a PNH clone. There is evidence from mouse models that PNH stem cells do not expand on their own, and there is evidence from human patients that expansion is associated with negative selection against GPI-positive cells by GPI-specific T cells. Thus, PNH is a prime example of a clonal disease that is not malignant.
TREATMENT Paroxysmal Nocturnal Hemoglobinuria
Until 10 years ago there were essentially two treatment options for PNH: either allogeneic BMT, providing a definitive cure at the cost of non-negligible risks; or continued supportive treatment for what, unlike other acquired HAs, may be a lifelong condition. A major advance has been the introduction in 2007 of a humanized monoclonal antibody, eculizumab, which binds to the complement component C5 near the site that, when cleaved, will trigger the distal part of the complement cascade leading to formation of the MAC. With C5 blocked, the patient is relieved of intravascular hemolysis and of its attendant consequences, including hemoglobinuria. In the majority of those patients who needed regular blood transfusion, the transfusion requirement is either abolished or significantly reduced. For many PNH patients, eculizumab has meant a real improvement in the quality of life, as well as a decrease in complications, particularly thrombosis. At the same time, it is important to know that in patients on eculizumab the PNH red cells, now protected from being lysed through the MAC, do still bind C3 fragments and thus become opsonized. Therefore hemolysis continues, but it is now extravascular. The extent to which this happens depends in part on a genetic polymorphism of the complement receptor CR1. Those patients who, on eculizumab, are still receiving blood transfusion are at risk of iron overload. Based on its half-life, eculizumab must be administered intravenously every 14 days: a trial of a long-lived anti-C5 antibody is currently under way, and other complement inhibitors are under experimentation.
Eculizumab is very expensive and therefore not accessible to patients in many parts of the world. Therefore, the management of PNH by supportive treatment is still very important. Folic acid supplements (at least 3 mg/d) are mandatory; the serum iron should be checked periodically, and iron supplements should be administered as appropriate. Transfusion of filtered red cells should be used whenever necessary, which, for some patients, means quite frequently. 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 their side effects are considerable. A short course of prednisone may be useful when an inflammatory process exacerbates 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. With thrombotic complications that do not resolve otherwise, thrombolytic treatment with tissue plasminogen activator may be indicated.
Where eculizumab is available the proportion of PNH patients receiving BMT has decreased significantly. However, when an HLA-identical sibling is available, BMT should be taken into consideration for any young patient with severe PNH; and for patients with the so-called PNH-AA syndrome, since eculizumab has no effect on BMF. For these patients immunosuppressive treatment with antithymocyte globulin and cyclosporine A may be an alternative, and it may be compatible with concurrent administration of eculizumab.
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