ACP medicine, 3rd Edition

Hematology

Hemoglobinopathies and Hemolytic Anemias

Stanley L. Schrier M.D.1

1Professor of Medicine (Active Emeritus), Division of Hematology, Stanford University School of Medicine

The author has served as a consultant for Tularek Corp. and Receptron, Inc., during the past 12 months.

April 2004

Alteration of the erythrocyte membrane usually signals the reticuloendothelial macrophages to remove the damaged red blood cell (RBC) from the circulation. In extraordinary circumstances, however, the damage to the membrane is so great that the erythrocyte undergoes hemolysis, and its intracellular contents, including hemoglobin, are liberated into the plasma. This chapter describes structural and functional features of normal erythrocytes and diseases involving membrane architecture, RBC proteins, and extracorpuscular factors that can lead to shortened RBC survival.

Development, Structure, and Physiology of the Erythrocyte

Erythroid precursor cells undergo four or five cell divisions in the bone marrow and then extrude their nuclei and become reticulocytes. As these enucleate cells mature, hemoglobin synthesis decreases. The cells lose most of their transferrin receptors and enter the peripheral blood; they survive in the circulation for about 4 months.

As they move through the circulation, erythrocytes must withstand severe mechanical and metabolic stresses, deform to traverse capillaries with diameters half their own, resist high shearing forces while moving across the cardiac valves, survive repeated episodes of stasis-induced acidemia and substrate depletion, and avoid removal by the macrophages of the reticuloendothelial system. They must also maintain an internal environment that protects hemoglobin from oxidative attack and sustain the optimum concentration of 2,3-bisphosphoglycerate (2,3-BPG) needed for hemoglobin function.

HEMOGLOBIN

The normal adult RBC contains three forms of hemoglobin (Hb): HbA (96%), HbA2 (2% to 3%), and HbF (< 2%). Normal HbA (α2β2) is composed of two α chains, coded by four genes on chromosome 16, and two β chains, coded on chromosome 11. HbA2 is composed of two α chains and two δ chains (α2δ2), and fetal hemoglobin (HbF) is composed of two α chains and two γ chains (α2γ2). The genes for the β, δ, and γ chains are closely linked to one another on chromosome 11. The extraordinarily high concentration of hemoglobin in the RBC—33 to 35 g/dl (the mean corpuscular hemoglobin concentration, or MCHC)—produces a viscous solution intracellularly.

NONHEMOGLOBIN CYTOSOL

Erythrocytes principally utilize glucose to maintain the reducing power that protects the cell against oxidative attack, to generate the 2,3-BPG required to modulate the function of hemoglobin, and to control the salt and thus the water content of the RBC by the actions of adenosine triphosphate (ATP) and the transport adenosine triphosphatases (ATPases) [see Table 1]. The water and the hemoglobin content of the RBC determine the mean corpuscular volume (MCV) and the MCHC.

Table 1 Erythrocyte Metabolism

Pathway

Product

Functions of Metabolic Products

Glycolysis by Embden-Meyerhof pathway

ATP

Serves as a substrate for all kinase reactions, for the ATPase-linked sodium-potassium pump, for the ATPase-linked calcium efflux pump, and for other ATPases of the RBC membrane, including aminophospholipid translocase
Maintains deformable state of RBC membrane

2,3-DPG

Interacts with deoxyhemoglobin, shifting equilibrium to favor unloading of O2 from oxyhemoglobin
Acts as an intracellular anion that cannot cross the RBC membrane

NADH

Acts as a substrate for a methemoglobin reductase, enabling it to reduce methemoglobin (Fe3+) to hemoglobin (Fe2+)

Pentose phosphate pathway (hexose monophosphate shunt)

NADPH

Serves as a substrate for another methemoglobin reductase in methemoglobin reduction (a fail-safe mechanism)
Serves as a coenzyme for glutathione reductase in reduction of oxidized glutathione; reduced glutathione (GSH) protects RBC against oxidative denaturation

ATP—adenosine triphosphate  2,3-DPG—2,3-diphosphoglycerate  NADH—reduced nicotinamide-adenine dinucleotide  NADPH-reduced nicotinamide-adenine dinucleotide phosphate

PLASMA MEMBRANE

The RBC normally has a discoid shape with a diameter of 7 to 8 µm, an MCV of 85 to 90 femtoliters (fl) (1 fl = 10-15 L), and a surface area of 140 µm2 [see Figure 1]. Its unique shape enables it to squeeze through capillaries as narrow as 3 µm in diameter.

 

Figure 1. Changes in Erythrocyte Shape

The normal erythrocyte, or discocyte, undergoes shape changes in response to conditions created by treatment with certain agents. Most changes are reversible if inducing agents are removed before the permanent loss of membrane material.

Lipids (phospholipids and cholesterol) account for 50% of the weight of the surface membrane. The phospholipids are distributed asymmetrically in the membrane bilayer, with positively charged ones in the outer half and relatively negatively charged ones predominantly in the inner half. This asymmetry permits the selective intercalation of small charged molecules into either the outer or inner half of the bilayer, producing echinocytes or stomatocytes [see Figure 1].

The RBC membrane proteins include integral and peripheral proteins. Integral proteins interact with and span the hydrophobic phospholipid bilayer [see Figure 2]. The major integral proteins of the erythrocyte membrane are the glycophorins (which contain most of the membrane sialic acid and carry the MNSs blood group antigens) and band 3, which is the anion and bicarbonate transporter.

 

Figure 2. Red Cell Membrane Proteins

Band 3, the anion transport channel (orange), and the other integral proteins glycophorin A (not shown), glycophorin B (not shown), and glycophorin C (green) span the red cell membrane. Branching external carbohydrate side chains are attached to these proteins. The hydrophilic, polar heads of the phospholipid molecules that make up the bilayer are oriented toward the cell surface, whereas the hydrophobic fatty acid side chains are directed toward the interior of the bilayer. Cholesterol is intercalated between the fatty acid chains. Band 3 binds hemoglobin and glyceraldehyde-3-phosphate dehydrogenase on its cytosol surface. Spectrin (yellow), actin (red), tropomyosin (blue), and band 4.1 (light green) form a latticework on the inner membrane surface. The spectrin heterodimers associate to form heterotetramers. The lower figure depicts the hexagonal cytoskeletal lattice on the inner membrane surface. Band 2.1 (ankyrin) links the integral protein band 3 to the peripheral cytoskeleton through the β chain of spectrin. Additional linkage is provided by glycophorin C and band 4.1.

The peripheral proteins are all found at the cytosol face of the membrane. The interaction of these peripheral proteins, which include spectrin and actin, results in the tough but resilient cytoskeleton of the erythrocyte. The peripheral cytoskeleton, in turn, is connected to the integral proteins [see Figure 2].1,2

The membrane carbohydrates contribute to the external negative charge of the membrane and function partly as blood group antigens. Some of these glycolipids associate with phosphatidylinositol to form a glycolipid anchor, called the glycosylphosphatidylinositol (GPI) anchor. These GPI anchors provide the membrane-anchoring site for several classes of proteins that have important biologic functions at membrane surfaces, including several that serve to control complement action [see Paroxysmal Nocturnal Hemoglobinuria, below].3

CONTROL OF HYDRATION AND VOLUME

Control of RBC volume has considerable pathophysiologic importance because the water and cation contents of RBCs determine intracellular viscosity and the ratio of surface area to volume. The Na+ and K+ content is determined by passive diffusion and by active transport, primarily through Na+, K+-ATPase. The major intracellular anion is Cl-, which enters the RBC with high permeability through band 3. The K+-Cl- cotransporter drives the K+-Cl- gradient and is activated by RBC swelling and low intracellular pH, causing a net loss of K+ and Cl-. The Ca2+-ATPase actively pumps Ca2+ out of the RBC, making the free cytosolic Ca2+ content less than 0.1 µM—four orders of magnitude lower than the plasma concentration of 1 mM. The Gardos channel, which is a Ca2+-activated K+ efflux channel, plays an important role in volume regulation. Water enters and exits through a water channel called CHIP 28 (28 kd channel-forming integral membrane protein) or aquaporin. Other important intracellular anions are 2,3-BPG and hemoglobin, neither of which penetrates the cell membrane. When the concentration of free cytosolic Ca2+ rises to levels even as low as 0.3 µM, the channel is activated and results in a net loss of K+. If such a loss is not corrected, the affected RBC becomes dehydrated.4

SHAPE CHANGES

ATP depletion, calcium ion accumulation, or treatment with lysolecithin or with anionic amphipathic compounds transforms the normal erythrocyte, or discocyte, into an echinocyte—a crenated spiculated cell sometimes called a burr cell [see Figure 1]. Calcium, acting either alone or in concert with the calcium-binding protein calmodulin, can effect the echinocytic shape change. If the echinocytic process persists, fragmentation or budding of the tips of the echinocyte leads to loss of membrane components, particularly of band 3 and phospholipids. This results in loss of surface area, a reduction in the ratio of surface area to volume, and the formation of poorly deformable spheroechinocytes.

PRINCIPLES OF BLOOD FLOW

The major determinants of blood flow are the hematocrit; the plasma concentration of proteins such as fibrinogen and immunoglobulins, which influence the degree of rouleau formation or aggregation; RBC deformability; the caliber of blood vessels; and the shear rate (the ratio of flow rate to tube radius). At the low shear rates that exist in postcapillary venules, the RBCs tend to clump in asymmetrical masses, with a consequent increase in blood viscosity and resistance to flow.

CELL AGING AND DEATH

In the bone marrow, the developing reticulocyte progressively loses its residual RNA over a 4-day period after nuclear extrusion. At the conclusion of this stage, the reticulocyte can no longer engage in protein synthesis. The active K+-Cl- cotransporter functions to reduce cell volume. With the membrane protein assembly complete, the resulting mature cell enters the circulation and survives for a period of 100 to 120 days.5 Erythrocyte death is an age-dependent phenomenon and may be related to mechanical and chemical stresses the cell encounters in the circulation. As the erythrocyte ages, it loses water and its surface area diminishes. The ratio of surface area to volume decreases and the mean corpuscular hemoglobin concentration increases, impairing cell deformability. In addition, decreased enzymatic activity lowers the cell's ability to withstand metabolic stress. Aging may be manifested by changes at the erythrocyte's surface, such as a decrease in the density or type of surface charge or the appearance of a senescence neoantigen, perhaps oxidatively clustered band 3 [see Figure 2], that binds specific immunoglobulins and complement components.6 By such changes, the age-worn erythrocyte signals its incapacity to the reticuloendothelial system, triggering removal by macrophages.

Under physiologic conditions, slightly less than 1% of the RBCs are destroyed each day and are replaced by a virtually identical number of new cells. For a 70 kg (154 lb) man with a blood volume of about 5 L, about 50 ml of whole blood, containing approximately 22 ml of packed erythrocytes, is destroyed and replaced each day. Inasmuch as one third of each erythrocyte is hemoglobin, the replacement of these cells requires the synthesis of about 7 g of hemoglobin each day. Normal adult bone marrow can readily increase its erythroid output fivefold. After extensive and prolonged anemic stress, erythroid production can be raised by as much as seven or eight times. The supply of iron, however, places an important limit on RBC replacement: three fourths of the iron used in the synthesis of cells in a day comes from cells that were destroyed on the previous day.

General Features of Hemolytic Anemias

The severity of anemia is determined both by the rate of RBC destruction and by the marrow's capacity to increase erythroid production. When a person has a healthy marrow, erythrocyte survival time can be reduced from 120 days to 20 days without inducing anemia or jaundice; however, a substantial reticulocytosis will be present in such cases.

Most forms of hemolysis are extravascular; the damaged cell signals its changed status to the reticuloendothelial system via its membrane and is removed. In unusual circumstances in which damage to the erythrocyte is devastating—as in some forms of complement-mediated lysis—or in circumstances in which the reticuloendothelial system cannot cope with the burden of damaged cells, intravascular lysis develops and leads to hemoglobinemia.

Hemoglobin released to the plasma is degraded to αβ dimers, which bind to haptoglobin. The hemoglobin-haptoglobin complexes are removed by the reticuloendothelial system. When the haptoglobin-binding capacity is exceeded, αβ dimers pass into the glomerular filtrate. Some of the αβ dimers are excreted into the urine directly, producing hemoglobinuria, whereas others are taken up by renal tubule cells. Iron-containing renal tubule cells may be excreted for several days after an episode of intravascular hemolysis. Hemosiderinuria can be identified with Prussian blue stain. Free plasma hemoglobin can dissociate into globin and hemin. Hemin may bind to hemopexin and may reach the renal tubule cells in that form, or it may bind to plasma albumin, producing methemalbuminemia.

Intravascular hemolysis may produce severe anemia acutely. In addition, erythrocytic membrane particles released into the plasma may act as potent stimuli for disseminated intravascular coagulation. Acute severe hemolysis is also a cause of acute renal failure [see 10:VI Acute Renal Failure]. When a patient compensating for a marked increase in hemolysis has an infection that sharply impairs marrow erythroid activity,7 the hemoglobin level may fall dramatically—a condition called aplastic crisis. With chronic hemolysis, pigment stones often develop in the gallbladder.

Causes of hemolysis may be classified as either extracorpuscular or intracorpuscular. The intracorpuscular causes, which are essentially erythrocyte defects, comprise membrane abnormalities, metabolic disturbances, and disorders of hemoglobin structure or biosynthesis. Extracorpuscular causes represent abnormal elements within the vascular bed that attack and destroy normal erythrocytes. Because erythrocytes with intracorpuscular defects that cause hemolysis are intrinsically abnormal, when they are transfused into normal recipients, their survival time is characteristically short. Of the intracorpuscular defects, only one disorder, paroxysmal nocturnal hemoglobinuria, is not hereditary.

Erythrocyte Membrane Defects

DISORDERS OF SALT AND WATER METABOLISM

Hydrocytosis (Hereditary Stomatocytosis)

Hydrocytosis is a hereditary disorder that usually presents early in life as partly compensated hemolytic anemia; occasionally, the spleen is palpable. The MCV is usually elevated. The peripheral smear shows stomatocytes [see Figures 3a, 3b, and 3c]. Passive flux of both Na+ and K+ increases greatly. The Na+,K+-ATPase is overwhelmed; the cation concentration and thus the water content of the RBC increase, accounting for the increase in MCV and the decrease in the ratio of surface area to volume. Stomatocytes appear to adhere more avidly than normal RBCs, a finding that may account for the reported increase in thromboembolic events.8 Perhaps more importantly, the number of RBCs with phosphatidylserine exposed on the outer membrane surface is increased. Phosphatidylserine—a relatively negatively charged phospholipid that is normally found predominantly in the inner membrane layer—provides a nidus for thrombin formation and thus may also contribute to the tendency to thrombosis.9 Splenectomy may lead to improvement in the anemia. Other therapies may eventually prove useful; vaso-occlusive events were controlled in one patient by long-term RBC transfusion and in another by therapy with pentoxifylline.8

 

Figure 3a. Stomatocytes

Stomatocytes are identified by slitlike areas of central pallor; the smear also shows microspherocytes, which are a more advanced stage of stomatocytosis. On scanning electron microscopy or examination of wet preparations, the microspherocytes are shown to be stomatocytes. Microspherocytes are seen in hereditary spherocytosis and in autoimmune hemolytic anemia, as well as in other conditions characterized by relatively selective loss of membrane material or increase in cell volume.

 

Figure 3b. Supravital Stain of Erythrocytes

Supravital stain of erythrocytes shows single and multiple blue-staining Heinz bodies within counterstained erythrocytes. Phase microscopy can also be used to demonstrate Heinz bodies.

 

Figure 3c. Elliptocytes

Elliptocytes are visualized in a smear from a patient with hereditary elliptocytosis.

Xerocytosis

Xerocytosis, another hereditary hemolytic disorder, is characterized by a membrane defect that leads to loss of cations, particularly K+. Dehydration of erythrocytes occurs because the K+ leak exceeds the Na+ influx, possibly as a result of an overactive K+-Cl- cotransporter. Patients present with variably compensated hemolysis. Splenomegaly is not a prominent feature. The peripheral smear is variable, showing target cells, stomatocytes, echinocytes, or so-called hemoglobin puddling (i.e., hemoglobin collected around the circumference of the cell). MCHC is increased. Because these rigid cells are removed in many parts of the reticuloendothelial system, splenectomy is of little benefit.10In rare instances, xerocytosis can cause nonimmune hydrops fetalis.11

PROTEIN ABNORMALITIES

Hereditary Elliptocytosis

There are perhaps 250 to 500 cases of hereditary elliptocytosis per million population.10 Three morphologic variants are seen: (1) common hereditary elliptocytosis, (2) spherocytic hereditary elliptocytosis, and (3) stomatocytic hereditary elliptocytosis.12 Most patients with common hereditary elliptocytosis are heterozygous for this autosomal dominant disorder and have only elliptical RBCs or, at worst, compensated hemolysis. Homozygotes for the disorder may have severe uncompensated hemolytic anemia.

Under applied shear stress, erythrocytes assume an elliptical shape; when the stress is removed, the cell normally recoils to its discoid shape. It has been hypothesized that membrane defects in hereditary elliptocytosis interfere with normal recoil. The membrane defect appears to be a lesion in the membrane cytoskeleton; RBC membranes from patients with hereditary elliptocytosis are almost invariably mechanically fragile.

The diagnosis is made in patients with extravascular intracorpuscular hemolysis who have elliptocytes on the peripheral smear. Elliptocytosis can also be seen in severe iron deficiency, myeloproliferative and myelodysplastic disorders, and, occasionally, cobalamin and folate deficiencies.12 Results of the osmotic fragility test are usually normal. Splenectomy has been useful in patients with severe common hereditary elliptocytosis.

Hereditary Propoikilocytosis

The syndrome of hereditary (autosomal recessive) pyropoi kilocytosis, a variant of hereditary elliptocytosis, causes severe hemolysis in young children. It is caused by an abnormal α or β spectrin mutation. The blood smear shows extreme microcytosis and extraordinary variation in the size and shape of erythrocytes [see Figures 3a, 3b, and 3c]. Splenectomy may reduce the rate of hemolysis.

Hereditary Spherocytosis

Hereditary spherocytosis is usually inherited as an autosomal dominant trait and affects about 220 per million people worldwide. A rare autosomal recessive variant of hereditary spherocytosis has been described.

Because of a loss of surface membrane, RBCs assume a micro spherocytic shape and thus cannot deform sufficiently to pass through the splenic vasculature; splenic trapping of RBCs, hemolysis, and a compensatory increase in RBC production result. The underlying membrane defects lead to budding of membrane vesicles under conditions of metabolic depletion. These membrane vesicles are enriched in phospholipids from the bilayer, as well as in associated transmembrane proteins [see Figure 2]. The underlying molecular lesions appear to consist of deficiencies of spectrin, spectrin-ankyrin, band 3, and band 4.2 (palladin).12,13

About 25% of patients with hereditary spherocytosis have completely compensated hemolysis without anemia; their disorder is diagnosed only when a concomitant condition, such as infection or pregnancy, increases the rate of hemolysis or reduces the marrow's compensatory capacity. In other patients, mild anemia, pigmented gallstones, leg ulcers, and splenic rupture may develop. Aplastic crises may be precipitated by ordinary respiratory tract infections, especially by parvovirus infection.7 It is important to remember that this disease can become apparent during the first year of life, when increased splenic maturation resulting in RBC removal combined with a sluggish erythropoietic response can result in anemia severe enough to require RBC transfusion.14

This diagnosis is suggested by a predominance of microspherocytes on the peripheral smear [see Figure 3b], an MCHC of 35 g/dl or greater, reticulocytosis, mild jaundice, splenomegaly, and a positive family history, although at least half of newly diagnosed patients have no family history. Confirmation of the diagnosis is made by a 24-hour incubated osmotic fragility test. A negative Coombs test and a family history positive for hereditary spherocytosis rule against a diagnosis of acquired autoimmune hemolytic anemia. Splenectomy eradicates clinical manifestations of the disorder, including aplastic crises.

PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

Paroxysmal nocturnal hemoglobinuria (PNH) is a somatic clonal disorder of hematopoietic stem cells. PNH involves the PIG-A gene, which maps to the short arm of the X chromosome.15 The mutation results in a deficiency of the membrane-anchoring protein phosphatidylinositol glycan class A; the resulting mature hematopoietic cells are usually chimeric. Normal human erythrocytes, and probably platelets and neutrophils, modulate complement attack by at least three GPI membrane-bound proteins: DAF (CD55), C8-binding protein (C8BP), and MIRL (CD59). In the absence of the GPI anchor, all of the proteins that use this membrane anchor will be variably deficient in the blood cells of persons with PNH.16 Because the defective synthesis of GPI affects all hematopoietic cells, patients with PNH may have variable degrees of anemia, neutropenia, or thrombocytopenia, or they may have complete bone marrow failure.17

Diagnosis

Classically, acute episodes of intravascular hemolysis are superimposed on a background of chronic hemolysis. The patient typically notes hemoglobinuria on voiding after sleep.18,19 Recurrent venous occlusions lead to pulmonary embolism and hepatic and mesenteric vein thrombosis, possibly resulting from release of procoagulant microparticles derived from platelets.20 A literature review found that thrombotic events accounted for 22% of deaths in patients with PNH.21 Occasionally, PNH patients with thrombosis are mistakenly thought to have psychosomatic disorders because they complain of recurrent severe pain in the abdomen and back that has no obvious cause. In these cases, the associated anemia and hemolysis may be very mild, and episodes of hemolysis do not necessarily correlate with bouts of pain.

A diagnosis of PNH should be considered in any patient with chronic or episodic hemolysis. The diagnosis should also be considered for patients with recurrent venous thromboembolism, particularly if the thrombus occurs in a site such as the inferior vena cava or the portal mesenteric system or if it produces Budd-Chiari syndrome. Evidence of intravascular hemolysis, such as hemoglobinemia; reduced serum haptoglobin; increased serum methemalbumin; hemoglobinuria; or hemosiderinuria, suggests the diagnosis. The combination of marrow hypoplasia and hemolysis is an important clue. PNH may occur in association with aplastic anemia. Erythrocyte morphology is usually normal. Diagnosis is made by specific tests based on fluorescence-activated cell sorter analysis using antibodies that quantitatively assess DAF (CD55) and particularly MIRL (CD59) on the erythrocyte or on the leukocyte surface.22

Treatment

In PNH, the anemia is occasionally so severe (hemoglobin level < 8 g/dl) that the patient needs transfusions regularly19; therefore, the choice of transfusion component is critical. It is believed that infusion of blood products containing complement may enhance hemolysis. Infusion of donor white blood cells (WBCs), which are ordinarily present in a unit of packed RBCs, into an HLA-immunized recipient may provide the antigen-antibody reaction that activates complement by the classical pathway. In such a case, the use of special leukocyte-poor units may be helpful [see 5:X Transfusion Therapy].

A trial of prednisone (e.g., 60 mg a day with rapid tapering, or 20 to 60 mg every other day) may reduce transfusion requirements and may be helpful in alleviating the anemia. Splenectomy is of very questionable benefit. Surgery is risky in patients with PNH because stasis and trauma accentuate hemolysis and venous occlusion. If surgery is to be performed, prophylactic anticoagulation with warfarin in the perioperative period should be considered.

Patients with PNH are frequently iron deficient. The simple administration of iron to correct this defect, however, often aggravates hemolysis because iron therapy produces a cohort of new cells, many of which are susceptible to complement-mediated lysis. Transfusion before iron therapy may help circumvent this problem because it will decrease the erythropoietic stimulus to the marrow.

Thrombocytopenia resulting from poor platelet production may necessitate platelet transfusions [see 5:X Transfusion Therapy].18 Budd-Chiari syndrome and inferior vena cava thrombosis must be diagnosed and treated quickly with heparin, followed by long-term administration of warfarin. If heparinization is ineffective, thrombolytic therapy (e.g., streptokinase) may be used.23 Children and adolescents with PNH that is complicated by aplastic anemia should be considered for allogeneic bone marrow transplantation.19,24 In case reports, the anemia associated with PNH responded to erythropoietin,25 and four patients with severe neutropenia and thrombocytopenia responded to combinations of granulocyte-colony-stimulating factor (G-CSF) and cyclosporine.26

Prognosis

A study of 80 patients with PNH indicated that median survival was 10 years.18 The causes of PNH-related death were thrombocytopenia, PNH hemolysis, thromboses, or PNH-associated aplastic anemia [see 5:III Anemia: Production Defects]. Of interest is that 15% of patients experienced spontaneous remission.18 In rare instances, prolonged and severe iron loss may occur as a result of chronic hemosiderinuria, producing iron deficiency; some patients develop transfusion-associated hemochromatosis.19

Acute myeloid leukemia may develop during the course of PNH. In one series, this occurred in three of 80 patients; in another series, of 220 patients, the incidence of myelodysplastic syndromes was 5% and the incidence of acute leukemia was 1%.19

Abnormalities of Erythrocyte Metabolism

DEFECTIVE REDUCING POWER

The reducing power of the erythrocyte is provided by reduced glutathione (GSH) and the reduced coenzymes nicotin amide adenine dinucleotide (NADH) and nicotinamide-adenine dinucleotide phosphate (NADPH) [see Table 1]. When erythrocytic stores of these materials are inadequate, hemoglobin and membrane-associated proteins can be oxidized, leading to the production of Heinz bodies, which consist predominantly of oxidative degradation products of hemoglobin [see Figure 3b]. Erythrocytes containing Heinz bodies are rigid and are therefore selectively removed by the reticuloendothelial system.

Defective Glutathione Synthesis

Deficiencies of certain enzymes involved in GSH synthesis lead to oxidative attacks on erythrocytes and to hemolysis. Several reports have described families whose members show almost negligible GSH synthesis and have hemolysis associated with the production of Heinz bodies. Glutathione peroxidase deficiency apparently contributes to hemolysis in newborn infants.

GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY

Glucose-6-phosphate dehydrogenase (G6PD) is the first enzyme in the pentose phosphate pathway, or hexose monophosphate shunt. It catalyzes the conversion of NADP+ to NADPH, a powerful reducing agent. NADPH is a cofactor for glutathione reductase and thus plays a role in protecting the cell against oxidative attack. RBCs deficient in G6PD are therefore susceptible to oxidation and hemolysis.27,28

G6PD deficiency is one of the most common disorders in the world; approximately 10% of male blacks in the United States are affected, as are large numbers of black Africans and some inhabitants of the Mediterranean littoral. This disorder confers some selective advantage against endemic malaria. For example, in a study in Ghana on pregnant women (who are highly susceptible to falciparum malaria and its consequences), the prevalence of infection was 66% in normal women, 58% in G6PD heterozygotes, and 50% in homozygotes.29

The gene for G6PD is on the X chromosome at band q28; males carry only one gene for this enzyme, so those males that are affected by the disorder are hemizygous. Females are affected much less frequently because they would have to carry two defective G6PD genes to show clinical disease of the same severity as that in males. However, expression of a defective G6PD gene is not completely masked in heterozygous women; in fact, such women exhibit highly variable G6PD enzyme activity. According to the X-inactivation, or Lyon-Beutler, hypothesis,28 females heterozygous for G6PD have two cell lines: one that contains an active X chromosome with a gene for normal G6PD and another that contains an active X chromosome with a gene for deficient G6PD. Chance partly determines the relative proportions of the two cell lines, which in turn control the clinical severity of the defect.

Classification

There are three clinical classes of G6PD deficiency: class I, which is the uncommon chronic congenital nonspherocytic hemolytic anemia; class II, in which the enzyme deficiency is severe but hemolysis tends to be episodic; and class III, the most common variant, in which the enzyme deficiency is moderate and hemolysis is caused by oxidant attack. The severity of the hemolysis and the anemia is directly related to the magnitude of the enzyme deficiency, which is determined by the half-life of the enzyme. The normal G6PD half-life is 62 days; in class III G6PD deficiency, the enzyme has a half-life of 13 days; and in class II deficiency, G6PD has a half-life of several hours. The cloning and sequencing of the G6PD gene have clarified the classification of G6PD deficiency; before the sequencing of the G6PD gene, more than 300 variants of G6PD deficiency had been described.28

Etiology

Hemolysis occurs in persons with class III G6PD deficiency after exposure to a drug or substance that produces an oxidant stress. Ingestion of, or exposure to, fava beans may cause a devastating intravascular hemolysis (known as favism) in G6PD-deficient patients, but it usually occurs only in those with the Mediterranean variant of class II deficiency. Fava beans contain isouramil and divicine, two strong reducing agents whose actions eventuate in the oxidation of membrane proteins. This produces a rigid cell in which hemoglobin is confined to one part of the cytosol; the other part of the cytosol appears as a clear ghost (i.e., the classic bite, hemiblister, or cross-bonded cell) [seeFigure 4]. These membrane defects cause extravascular and intravascular hemolysis.27 Severe infections, diabetic ketoacidosis, and renal failure also reportedly trigger hemolysis.

 

Figure 4. Bite Cells

Bite, hemiblister, or cross-bonded cells are indicative of oxidative attack leading to oxidative hemolysis.

Diagnosis

Hemolytic anemia characterized by the appearance of bite cells and Heinz bodies after administration of certain drugs suggests the possibility of G6PD deficiency [see Table 2]. Dapsone, which is capable of inducing oxidant-type hemolysis, has increasingly come into use as prophylaxis for Pneumocystis carinii pneumonia in patients infected with HIV [see 7:XXXIII HIV and AIDS]. Therefore, it is important to screen potential users of dapsone for G6PD deficiency with the standard enzymatic tests. Other agents with oxidative potential, such as amyl nitrite (“poppers”), can cause hemolysis.30

Table 2 Drugs That Produce Hemolysis in G6PD-Deficient Patients

Class

Example

Antimalarials

Primaquine
Chloroquine

Sulfonamides

Sulfamethoxazole
Sulfapyridine

Sulfones

Dapsone

Analgesics

Acetanilid
Phenacetin
Acetylsalicylic acid (10 g/day)

Nitrofurans

Nitrofurantoin
Furazolidone

Water-soluble vitamin K derivatives

Menadiol

Other disorders to be considered in the differential diagnosis of oxidative hemolysis include unstable hemoglobinopathy, hemoglobin M disease, and deficiencies of other enzymes essential to glutathione metabolism. A G6PD screening test or direct enzyme assay usually resolves the question. Patients with A-type G6PD (class III) deficiency and brisk reticulocytosis, however, may have a near-normal G6PD level because young RBCs have relatively high G6PD levels. In such cases, it is best to repeat the tests when the reticulocyte count returns to normal. Information on genetic testing for G6PD deficiency can be found on the Internet at http://www.geneclinics.org.

Treatment

Avoidance of drugs that may produce hemolysis is critical in management. Acute favism requires circulatory support, maintenance of good renal blood flow, and transfusions with erythrocytes that are not G6PD deficient. The physician must also be alert to the possible onset of disseminated intravascular coagulation.

DEFECTS IN GLYCOLYSIS

The series of reactions constituting the glycolytic pathway generates several products, such as ATP, that have various essential functions in erythrocyte metabolism [see Table 1]. Defects involve the major glycolytic pathway (Embden-Meyerhof pathway) and generally interfere with ATP production.

Pyruvate kinase (PK) catalyzes the formation of pyruvate, a reaction associated with ATP synthesis. After G6PD deficiency, PK deficiency (autosomal recessive) is the second most common here ditary enzymopathy. Hemolysis, mild jaundice, and, occasionally, palpable splenomegaly are the presenting problems. The peripheral smear usually reveals normal RBCs, but in a few cases, the RBCs show extreme spiculation. Aplastic crises may occur.7

Congenital nonspherocytic hemolysis raises the possibility of PK deficiency. An enzyme assay establishes the diagnosis. Splenectomy should be considered for patients who require transfusions.

Glucose-6-phosphate isomerase deficiency is the third most common enzymopathy that leads to hemolysis. Other enzymopathies are quite rare. Screening tests and specific assays are available for deficiencies of such enzymes as hexokinase, phosphofructokinase, triose phosphate isomerase, phosphoglycerate kinase, and aldolase.

DEFECTS IN NUCLEOTIDE METABOLISM

In hemolytic anemia associated with pyrimidine 5′-nucleotidase deficiency, coarse basophilic stippling persists in mature erythrocytes, presumably because the enzyme deficiency prevents degradation of reticulocyte RNA. This accumulation results in expansion of the total RBC nucleotide pool to a level five times normal. Pyrimidine nucleotides accumulate, and adenine nucleotides are decreased. Glycolysis is impaired by an undetermined mechanism.

Disorders Involving Hemoglobin

CLASSIFICATION OF THE HEMOGLOBINOPATHIES

The clinically important hemoglobinopathies are classified into five categories on the basis of the underlying defect. The defects are as follows:

  1. Hemoglobin tends to gel or crystallize (e.g., sickle cell anemia or hemoglobin C disease).
  2. Hemoglobin is unstable (e.g., the congenital Heinz body anemias).
  3. Hemoglobin has abnormal oxygen-binding properties (e.g., the disorder caused by hemoglobin Chesapeake).
  4. Hemoglobin is readily oxidized to methemoglobin (e.g., methemoglobinemia).
  5. Hemoglobin chains are synthesized at unequal rates (e.g., the thalassemias).

SICKLE CELL DISEASE

Sickle Cell Anemia

Definition

Sickle cell anemia is an autosomal recessive disease caused by the substitution of the amino acid valine for glutamine at the sixth position of the β-hemoglobin chain, which results in the production of HbS.

Epidemiology

From 8% to 10% of African Americans and a lesser percentage of persons with eastern Mediterranean, Indian, or Saudi Arabian ancestry have the sickle (HbS) gene. Disease develops in persons who are homozygous for the sickle gene (HbSS), in whom 70% to 98% of hemoglobin is of the S type. About 0.2% of African Americans have sickle cell anemia. The fact that the sickle gene occurs in populations living in regions endemic for falciparum malaria suggests that sickle heterozygosity confers a protective advantage against malaria.31

Restriction endonuclease analyses indicate that the sickle gene mutation probably arose spontaneously in at least five geographic locations. These variations are called Senegal, Benin, Central African Republic (or Bantu), Saudi-Asian, Cameroon, and Indian (which may be the same as the Saudi-Asian variant). These variants are important clinically because some variants are associated with higher output of γ-globin chains (and thus higher HbF levels); others are associated more often with the gene for α-thalassemia-2 [see The Thalassemias, below]. Either of these associations may alleviate some aspects of the sickling process.31

Pathophysiology

Two major clinical features characterize sickle cell anemia: (1) chronic hemolysis and (2) acute, episodic vaso-occlusive crises that cause organ failure and account for most of the morbidity and mortality associated with the disease.

HbS liganded to oxygen or carbon monoxide shows near-normal solubility. When the molecule gives up its oxygen and changes to the deoxy S form, however, its solubility decreases. In an environment with reduced oxygen, HbS polymerizes into long tubelike fibers that induce erythrocytic sickling.32

The deoxyhemoglobin S polymer is in equilibrium with surrounding soluble molecules of deoxyhemoglobin S. An increase in the concentration of HbS, a decrease in pH, or an increase in the concentration of 2,3-BPG tends to stabilize the deoxy S form and enhances gelation.32 In addition, sickled erythrocytes retain the K+-Cl- cotransport function and have sufficient intracellular calcium to activate the Gardos efflux channel33 [see Control of Hydration and Volume, above]. These two mechanisms act together to produce a population of very dense sickled erythrocytes with MCHCs ranging up to 50 g/dl.33 HbF inhibits polymerization,33 so patients with high HbF values, such as those with the Saudi-Asian variant of sickle cell anemia, have milder disease.31 When hypoxemia and the MCHC reach a critical level, polymerization occurs after a variable delay33; this delay represents the period during which the deoxyhemoglobin S tetramers are slowly associating to form a nucleus. When the nucleus reaches a critical size, rapid, almost explosive gelation occurs. Free deoxyhemoglobin S tetramers rapidly attach to the nucleus to produce the long tubelike fibers that align to form parallel tubelike structures that distort the cell and produce the sickle shape [see Figures 5a, 5b, and 5c].

 

Figure 5a. Elongated Sickle Cells

Sickle cell anemia is characterized by markedly distorted sickle cells, including elongated forms.

 

Figure 5b. Target Cells

Target cells are seen in a variety of conditions, including hypochromia caused by iron deficiency, hemoglobinopathies such as HbC variants and the thalassemias, and liver disease.

 

Figure 5c. Cooley Anemia

Cooley anemia, or β-thalassemia major, is indicated by profound hypochromia, targeting, variation in size and shape of erythrocytes, and the presence of nucleated red cells.

Most cells in the venous circulation are not sickled. However, sickling will occur if the time to polymerization is shortened to less than 1 second or if RBCs become trapped in the microcirculation. Some RBCs contain polymerized sickle hemoglobin even in the arterial circulation. Another manifestation of membrane damage in sickle cells is the irreversibly sickled cell, which retains its sickle shape even when reoxygenated.34 Some of these poorly deformable RBCs are directly derived from a subpopulation of reticulocytes that are low in HbF30 and are removed predominantly in the reticuloendothelial system. The rapid removal of these young cells, as well as older, dense, rigid cells that cannot traverse the monocyte-macrophage system, results in chronic extravascular hemolysis.

Because of the extreme sensitivity of sickling to the local environment, attention has been focused on cellular factors. The extreme hyperosmolality of the renal medulla (1,200 mOsm) dehydrates RBCs and raises the MCHC. Consequently, sickling sufficient to abolish the renal medullary concentrating ability may occur even in patients who have only the sickle trait.

Sickle Crisis and Ischemic Infarction

Sickle crisis is a potentially life-threatening vaso-occlusive complication of sickle disease. The initiating event in the sickle crisis is not known, nor is it clear why some patients have severe crises and others do not.

Clusters of increasingly rigid sickle cells will occlude the microvasculature in the followng circumstances: (1) the pH falls, deoxygenation increases, or the MCHC rises; (2) nitric oxide production decreases or nitric oxide is trapped and removed by free hemoglobin in plasma35; (3) microvascular disease is present; or (4) capillary transit time is prolonged. Thrombosis may also play a role in sickle occlusion. There is some disorganization of the membrane phospholipid bilayer, with phosphatidylserine moving to the outer leaflet, possibly enhancing the thromboembolic manifestations of sickle disease.36 In sickle cell anemia, there also appears to be an increase in circulating endothelial cells, which abnormally express tissue factor and may provide an additional basis for thromboembolism.37

Blockage leads to ischemic infarction, the release of inflammatory cytokines, and an amplifying sequence of stasis-induced occlusion, which may progress to sickle crisis. Portal circulations in which oxygen tension is low, such as those in the liver or the kidney, are at particular risk for occlusion.

Risk factors predisposing to painful crises include a hemoglobin level greater than 8.5 g/dl, pregnancy, cold weather, and a high reticulocyte count. Nocturnal hypoxemia is an important risk factor in children.38 Conversely, the low hematocrit in sickle cell anemia reduces blood viscosity and is protective. Sickle cell patients also characteristically have a high plasma fibrinogen level, which enhances the aggregation of already rigid erythrocytes and increases viscosity, particularly at the low shear rates encountered in the microcirculation.39 Sickled RBCs also have a greater tendency to adhere to endothelial cells than do normal RBCs.40 The role of leukocytes in this adhesion process is becoming clearer. Adminstration of G-CSF has led to sickle crises and even death.41,42 Granulocyte-macrophage CSF (GM-CSF) has caused similar crises. The severity of sickle disease appears to parallel the level of the WBC count, and WBC cell-adhesion molecules seem to be critical to sickle vaso-occlusion.43,44

Diagnosis of Sickle Cell Disease

In the past, the diagnosis of sickle cell anemia was usually made on the basis of clinical manifestations occurring in childhood; the affected child was seen to have limitation in exercise tolerance, shortness of breath, tachycardia, frequent severe infections, and episodes of very painful dactylitis. Currently, many cases are identified on screening tests, which may be prompted by the diagnosis in a family member or performed as a routine neonatal procedure; in California and many other states, every fetal cord blood sample is examined by high-performance liquid chromatography (HPLC). Rarely, the disorder is diagnosed in adult life, occasionally during a first pregnancy, when prenatal screening reveals anemia. The general symptoms are limited exercise tolerance, exertional dyspnea, painful crises, bouts of jaundice, and even biliary colic.

The clinical appearance of the patient and a blood smear showing sickled cells, holly leaf cells, and erythrocytes with Howell-Jolly bodies are fairly suggestive of sickle cell anemia. Howell-Jolly bodies represent cytoplasmic remnants of nuclear chromatin that are normally removed by the spleen. Platelet and WBC counts are usually high. Unless an aplastic crisis is in progress, causing a virtual absence of normoblasts, the marrow shows erythroid hyperplasia. Diagnosis is confirmed by performing a sickle preparation: a drop of blood is incubated with fresh 2% sodium metabisulfite, and the proportion of sickle cells is measured immediately and then 1 hour later. Commercial testing sets such as Sickledex rely on the relative insolubility of HbS in 1.0 M phosphate buffers to make the diagnosis. The most definitive tests for sickle cell anemia, however, are hemoglobin electrophoresis or HPLC, which indicate the relative percentages of HbS and HbF. All of these tests are also useful in screening family members for sickle cell trait. Patients who are heterozygous for both the HbS gene and the β-thalassemia gene may appear to be homozygous for HbS. Other varieties of sickling hemoglobin are observed very infrequently. DNA-based methods can also be used to pinpoint the specific genetic abnormality and to identify the subpopulations from which the patient descended31; further description and information on diagnostic testing is available online at http://www.geneclinics.org. Persons with sickle cell anemia and α-thalassemia have higher hemoglobin levels, lower reticulocyte counts, a lower MCHC, a lower MCV, and less-dense RBCs than persons who have sickle cell anemia alone. Such patients may have increased life expectancy and perhaps a different pattern of manifestations of veno-occlusive complications.45 The combination of G6PD deficiency and sickle cell anemia has neither beneficial nor harmful effects.46,47

Management of Sickle Cell Disease

Sickle crisis

Standard conservative management of sickle crisis centers on rest, hydration, and analgesia. In demonstrably acidotic patients, mild alkalinization should be induced by administration of a bicarbonate solution, which is prepared by addition of an ampule of sodium bicarbonate to 1 L of either 5% dextrose in water or half-normal saline. The bicarbonate solution should be infused at a rate of 5 to 7 ml/kg/hr for the first 4 hours and at 4 ml/kg/hr for the next 20 hours. The role of supplemental oxygen in patients with normal arterial oxygen tension (PaO2) and no cardiopulmonary problems is untested.

Pain management

Pain [see 11:XIV Pain] is the major concern for 10% to 20% of patients with sickle cell anemia. Avascular necrosis of bone marrow produces excruciating pain that can last as long as 8 to 10 days. The need for pain relief sometimes results in habituation or addiction. Because there are few objective ways to monitor the sickle crisis, the physician may not know whether a demand for narcotics is a manifestation of drug-seeking behavior.

The patient who has sickle cell anemia should be provided with oral analgesics for use at home in an attempt to abort the pain crisis at its onset. Nonsteroidal anti-inflammatory drugs (NSAIDs), such as naproxen (500 mg) and ketorolac (10 mg), can be used initially. If NSAIDs alone are not sufficient, they can be followed by a narcotic-analgesic combination, such as hydrocodone and acetaminophen or oxycodone and aspirin. Adjuvants such as oral diphenhydramine (50 mg) or lorazepam (1 to 2 mg) may calm the patient and perhaps antagonize the actions of released histamine.48 If these measures, perhaps repeated every 6 hours, do not control the pain, the patient usually requires parenteral treatment. Care from the patient's regular physicians is far preferable to reliance on unfamiliar providers in emergency departments.48 The patient needs rapid evaluation for possible infection, acute chest syndrome, bone infarction, and other complications, and the pain should be treated either with 10 mg of intravenous morphine along with 50 mg of intramuscular diphenhydramine every 2 hours or with 4 mg of intramuscular hydromorphone along with 50 mg of intramuscular diphenhydramine every 2 hours. If there is no pain relief or inadequate pain relief 30 minutes after the first dose, 50% of the initial dose of opiates can be administered; the respiratory rate should be monitored closely, particularly if it approaches 10 respirations a minute. Some units have used patient-controlled analgesia with good results. It is important to continue to administer parenteral analgesia at regular intervals and to provide increased doses for breakthrough pain. The patient will probably need a laxative and may need an antiemetic, such as prochlorperazine (10 mg p.o. or I.M.). If the patient responds, home therapy with oral controlled-release morphine, such as MS Contin, is usually effective. If pain continues for more than 8 to 12 hours, the patient will probably need to be hospitalized to receive extended therapy with increased doses of analgesia and parenteral fluids, along with observation.48

Alteration of sickle cell pathophysiology

A clearer understanding of the kinetics of sickling suggests some future prospects for the therapy of sickle cell anemia. Decreasing the MCHC should diminish gelation. An approach that attempts to block the Ca2+-dependent K+ efflux (Gardos channel) [see Control of Hydration and Volume, above] has been tested in a sickle mouse model and shows promise in preventing RBC dehydration.49,50

Therapies to interfere with sickling are being actively pursued. The presence of 20% to 30% HbF in sickle RBCs markedly delays gelation, so a mechanism that would switch on the genes that control fetal hemoglobin synthesis and thus lessen the severity of sickle disease appears feasible.51,52 Hydroxyurea produces an increase in F reticulocyte and HbF levels. In a phase III trial, patients treated with hydroxyurea (starting dosage, 15 mg/kg/day) had fewer painful crises, admissions for crisis, and episodes of acute chest syndrome, as well as required fewer transfusions, than patients given a placebo.53 There was no effect on stroke; however, after 8 years of follow-up, mortality was reduced by 40%.54 The beneficial effect of hydroxyurea accrued after about 8 weeks of therapy and was accompanied by an increase in MCV and an increase in the proportion of F cells; in addition, there was a decrease in neutrophils and a decrease in sickle RBC adhesion to endothelial cells.55 Trials are also being conducted with butyrate, which can increase γ-chain production, thereby increasing HbF levels and interfering with gelation.56,57 Demethylating agents such as 5-azacytidine and decitabine can also increase HbF to therapeutically useful levels. Because sickle cells adhere abnormally to the endothelium, attempts have been made to block adhesion; thus far, these efforts have not proved useful.

Inflammatory cytokines appear to play an important role in the sickle crisis, as evidenced by the fact that a predictor of success in hydroxyurea therapy is a decrease in the WBC count.54,55 Other investigators are studying the possible vasodilatory role of nitric oxide.

Sibling-donor allogeneic bone marrow transplantation can result in cure or can lead to a substitution of sickle trait for sickle cell anemia. Bone marrow transplantation resulted in apparent cure in 15 of 22 carefully selected patients; there were two deaths (9%), and the remaining five patients had complications such as graft failure. Of the 22 patients, 12 had a history of stroke, five had a history of recurrent episodes of acute chest syndrome, and five had recurrent painful crises.58

Long-term transfusion therapy

Long-term transfusion therapy has been found to prevent stroke.59 Some investigators have shown that preventive transfusions reduce or eliminate pain crisis, episodes of acute chest syndrome, bacterial infection, and hospitalization.60,61 Other authors, however, warn against the dangers of iron overload,62,63 transfusion hepatitis, problems with venous access, and RBC alloimmunization.64 Further studies may clarify the role of long-term transfusion therapy.

Complications and Their Management

Skeletal problems

Aseptic necrosis (osteonecrosis) of the femoral head occurs in about 10% of patients, particularly those who also have α-thalassemia. Arthroplasty has been relatively ineffective, partly because of the presence of adjacent hard bone, which interferes with the placement of the prosthesis, and because of the increased risk of infection.65

Cardiopulmonary problems

Cardiac complications associated with anemia are the result of a large increase in cardiac output. Such complications include chamber enlargement, cardiomegaly, left ventricular hypertrophy, and flow murmurs.66 Acute myocardial infarction has occurred in relatively young adults who do not have coronary disease.67 The incidence of pulmonary hypertension is unknown, but its presence markedly shortens survival.68

Acute pulmonary complications are a major cause of morbidity and mortality; such complications include local infection, vascular occlusions in the pulmonary vessels (both in situ thrombosis and embolism), and pulmonary fat embolism from ischemic marrow fat necrosis.69 A large study of acute chest syndrome found that adult patients were afebrile but had shortness of breath, chills, and pain in the chest and in at least one extremity.70 Infarctions of the thoracic vertebrae contribute substantially to the pain.64 Physical examination frequently shows no abnormal chest findings. In one study, the PaO2 was found to be low, averaging 71 mm Hg but falling below 60 mm Hg in 25% of patients. In this study, the death rate in adults was 4.3%; death was preceded by a lower hemoglobin value, a higher WBC count, and multilobe involvement. Autopsy of 16 cases showed that nine patients had pulmonary embolism and fat emboli and possibly 20% had bacterial infections. In patients with acute chest syndrome and pulmonary infection, the most common infecting organism was Chlamydia pneumoniae(30%), followed by Mycoplasma pneumoniae (21%), respiratory syncytial virus (10%), Staphylococcus aureus (4%), and Streptococcus pneumoniae (3%).71

Usually, therapy for acute chest syndrome should include incentive spirometry,64 antimicrobial therapy for patients with evidence of infection, the cautious use of analgesia, aggressive fluid replacement, and consideration of bronchoalveolar lavage to identify microbial infection or the fat-laden macrophages of fat emboli. Meticulous monitoring is required; repeat measurements of oxygenation should be made, and transfusions should be performed when clinically necessary. One of the most important benefits of hydroxyurea therapy is its ability to reduce the frequency of acute chest syndrome.53,72 Children may also need supplementary penicillin prophylaxis.73

Hepatobiliary disease

Cholelithiasis occurs in 30% to 70% of patients, some of whom exhibit signs and symptoms of cholecystitis.74 There are conflicting data regarding frequency of cholecystitis or obstruction of the common bile duct.74,75 If cholecystectomy is to be done, one should wait until the painful crisis is over. Transfusions should be given to raise the hemoglobin to 10 g/dl before surgery, if necessary, and the procedure should be done laparoscopically.74

Hepatic complications include congestive hepatopathy secondary to heart failure and viral hepatitis from frequent transfusions. Sickling in the liver can also produce hepatopathy. Often, serum bilirubin levels exceed 30 mg/dl in patients with intrahepatic cholestasis, and coagulation abnormalities may lead to hemorrhagic complications and death.

Renal and urologic complications

Water loss as a result of an inability to concentrate urine may enhance the sickling process. The extremely hypertonic milieu of the renal medulla induces severe sickling and destruction of the vasa recta. Hematuria and papillary necrosis ensue. These complications are also observed in patients with sickle trait and in those who have sickle cell-hemoglobin C disease. The defect in renal concentrating ability appears to depend on the amount of HbS polymer contained in cells and is thus less severe in patients who also have α-thalassemia variants.76

Complications include renal tubular acidosis, hyperkalemia, and proteinuria. Treatment with enalapril reduces proteinuria, suggesting the presence of a component of glomerular capillary hypertension.77 Renal failure, in association with worsening anemia, contributes to the death of about one fifth of patients older than 40 years who have homozygous sickle disease.

Priapism is an extraordinarily painful complication of sickle cell anemia and may result in impotence.78 A United Kingdom study reported a good response in 13 of 18 patients treated for priapism with the alpha-adrenergic agonist etilefrine; however, this agent is not available in the United States.79

Neurologic disorders

Neurologic complications of sickle cell disease include stroke, subarachnoid hemorrhage, and isolated functional losses that suggest a focal occlusion. The pathogenesis of occlusion of the large cerebral arteries is probably different from that of the microvascular occlusive events that occur in hypoxic capillary beds. The most likely underlying causes are damage to the vascular endothelium, followed by extensive intimal proliferation and then thrombosis of the damaged vascular bed.45 In a multi-institutional study of 4,082 patients, the prevalence of cerebrovascular accidents (CVAs) was 4% to 5%; the incidence was 0.61 per 100 patient-years.80 Of the CVAs, 54% were infarcts, 34% were hemorrhagic in nature, 11% were transient ischemic attacks (TIAs), and 1% had both infarctive and hemorrhagic features. Of the patients who survived, the recurrence rate of CVA was 14%. Mortality was 11%. Virtually all patients who died had hemorrhagic CVAs.

In a prospective study in which transcranial Doppler ultrasonography was used to pinpoint children at risk for stroke, treatment with standard care or transfusion therapy (to reduce the HbS concentration to < 30%) resulted in only one CVA, compared with 10 CVAs and one intracerebral hematoma in the 65 control subjects (P < 0.002). The trial was terminated early.59 The success of this trial raises many serious questions about the necessity of ultrasonographic devices for successful management; the optimum duration of transfusion therapy; the inevitable consequences of transfusional hemochromatosis [see β-Thalassemia major (Cooley anemia), below] and the necessity for ethnically matched blood to minimize allotransfusion reaction; the willingness of patients and families to accept transfusion therapy; and the role of allogeneic bone marrow transplantation as a potential alternative.59,81 The risk of recurrent cerebrovascular events is increased in patients receiving long-term transfusion therapy who have multiple cerebral collateral vessels as a result of moyamoya disease (hazard ratio, 2.40).82

Ocular complications

The major ocular problems associated with sickle cell anemia are retinopathy, vitreous hemorrhage, and neovascularization. Annual ophthalmologic evaluations are recommended. The efficacy of laser photocoagulation in treating sickle-induced ocular changes is currently being investigated.

Dermatologic complications

Poorly healing leg ulcers can be an important cause of morbidity in patients with sickle cell anemia. The degree of anemia does not seem to correlate with the presence or severity of these ulcers, but incompetence of venous valves and the resulting venous insufficiency have been associated with ulceration.83 Standard management includes debridement, control of local infection, use of wet-dry dressings, and possibly RBC transfusion. Local treatment with GM-CSF enhances healing, perhaps by stimulating the local growth of macrophages.84 GM-CSF can be either injected perilesionally or added topically to the wound, but the more successful application method involves the subcutaneous injection of 100 µg of GM-CSF in each of four sites circumferentially around the ulcer at a distance of 5 mm from its edge (resulting in a total dose of 400 µg in the wound). In some circumstances, one treatment sufficed, whereas in others, weekly treatments for 4 to 12 weeks were necessary. This therapy has not been approved by the Food and Drug Administration.

Aplastic crisis

Aplastic crisis rapidly lowers hemoglobin and hematocrit levels and produces reticulocytopenia, as it does in any chronic hemolytic state. Parvovirus infection has been found to cause aplastic crisis,7 as has bone marrow necrosis.85

Susceptibility to infections

Patients with sickle cell anemia are hyposplenic and exhibit complement system abnormalities. Deficient serum opsonizing activity forSalmonella organisms may confer an increased susceptibility to those infections, including osteomyelitis.

Anesthesia complications

The hypoxemia and vascular stasis that may occur during general anesthesia enhance sickling and may lead to a sickle crisis in the postoperative period. In an analysis of almost 4,000 patients, 12 deaths were associated with 1,079 procedures, and there were more complications after regional anesthesia than after general anesthesia.86 A simple transfusion program to raise the hemoglobin level to 10 g/dl was as effective as more aggressive preoperative programs in reducing the rate of complications.87

Pregnancy and contraception

The dangers of pregnancy for women with sickle disease include pulmonary problems and an increased incidence of urinary tract infection, hematuria, preeclampsia, and maternal death. Presumably, pelvic hypoxemia and the vascular overload associated with pregnancy lead to enhanced sickling, with its attendant complications. Vaso-occlusion in the placenta may account for fetal death and low birth weight.

Experienced clinicians differ in their approach to the pregnant patient with sickle disease. Some advocate only meticulous conservative care, whereas others recommend prophylactic transfusions. A controlled study has indicated that there is no advantage to the use of prophylactic transfusions.88

Chorionic villus sampling (which can provide DNA for analysis in the first trimester of pregnancy), DNA amplification techniques, and probes that identify the specific nucleotide change of sickle cell anemia can give a relatively safe and very reliable prenatal diagnosis.89

Oral contraceptives may pose a special hazard to women with sickle cell anemia, because they have been associated with a slight increase in the incidence of stroke, venous thromboembolism, and myocardial infarction. However, the emerging evidence that daily use of oral contraceptives containing less than 50 mg of synthetic estrogens is relatively safe suggests that patients with sickle disease can take such medication with reasonable confidence. The use of the Norplant implantable contraceptive device is another alternative for some patients. In any event, pregnancy or abortion in sickle disease carries significant risk.88

Genetic Counseling

A key element to be considered in the provision of genetic counseling to patients with sickle trait or sickle disease is the significant morbidity in affected children and adults. Couples with sickle disease or sickle trait may want to have children despite the associated fetal and maternal risks. There are about 4,000 to 5,000 such pregnancies in the United States each year.89 In one study, 286 of 445 pregnancies (64%) in mothers with sickle cell anemia proceeded to delivery; 21% of the infants were small and thus would be expected to require additional care, which the mother might have difficulty providing. In this study, there was one maternal death caused by sickle cell disease90 [see Genetic Counseling and Prenatal Diagnosis, below].

Prognosis in Sickle Cell Disease

Whereas it was once assumed that most patients with sickle cell anemia would die by 20 years of age, the median age of death is now 42 years for men and 48 years for women.51 This life expectancy is 25 to 30 years less than that of the general African-American population. Of the identified causes of death, only 18% involved organ failure—predominantly renal disease, heart failure, or the consequences of chronic strokes. Thirty-three percent of patients died during acute pain crises; these crises were frequently associated with the acute chest syndrome and were less often associated with stroke. The presence of α-thalassemia had no measurable effect. Predictors of poor outcome were a white cell count greater than 15,000/µl; a low HbF level; and organ involvement manifested by renal disease, acute chest syndrome, and neurologic events. Taking hydroxyurea had a significant impact on prognosis, with a 40% decrease in mortality and a reduction in painful crises.91

SICKLE VARIANTS

Sickle Trait

Heterozygosity for the sickle cell gene results in sickle trait (HbAS). The RBCs of persons with sickle trait have an HbS concentration of less than 50%; frequently, the level is as low as 30%.

Generally, persons with sickle trait lead normal, healthy lives. A few complications occur: hyposthenuria; renal hematuria; and, during pregnancy, bacteriuria and pyelonephritis. Splenic infarction occurs under conditions of hypoxia; it also occurs at high altitudes, predominantly in nonblack persons who have sickle cell anemia.

Sickle trait has been identified as a major risk factor for sudden death during basic training in the military92; death has resulted from unexplained cardiac arrest, heatstroke, heat stress, or rhabdomyolysis. Increasing age has been correlated with an increased risk of sudden death. However, these events have occurred under extreme conditions: very strenuous physical activity, usually in untrained persons, occasionally at high altitudes or in extreme heat. Usually, persons with sickle trait who are accustomed to physical activity do not have an increased risk of sudden death. For example, the incidence of sudden death in African-American football players with sickle trait is not higher than in other players.93

Therapeutic options for renal hematuria include the administration of diuretics, parenteral bicarbonate, transfusions, or ε-aminocaproic acid.

Sickle Cell-β-Thalassemia

When combined with sickle trait, a defect in the β-thalassemia gene produces a disease very similar to sickle cell anemia. The β-thalassemia gene reduces the rate of synthesis of the βA chain, resulting in a predominance of βS in patients with sickle trait. Depending on whether the patient has a β0 or a β+ thalassemia, the RBCs contain varying amounts of HbS, HbA, HbA2, and HbF. Patients with β0 thalassemia have no HbA, but only HbS, HbF, and HbA2; thus, disease is severe in these patients. Diagnosis is based on an elevation in the level of HbA2, HbF, or both on hemoglobin electrophoresis, as well as a positive family history of thalassemia and the sickle gene. In a study of 55 Greek patients, treatment with hydroxyurea resulted in distinct clinical improvement.94,95 Further description and information on diagnostic testing is available online at http://www.geneclinics.org.

Sickle Cell-Hemoglobin C Disease

In sickle cell-hemoglobin C (HbSC) disease, almost equal amounts of HbS and HbC are formed. Between 1% and 2% of hemoglobin is HbF, and small amounts of HbA2 are also present; however, HbA is absent. The increased sickling seen in these patients results from the pathologic effect of HbC [see Hemoglobin C Disease, below].96 As many as 30% to 50% of patients with this disorder are not anemic and have only modest reticulocytosis. Patients may not be identified until the disorder manifests itself in the form of a vaso-occlusive crisis during surgery, pregnancy, or a medical emergency.97 Splenomegaly, proliferative retinopathy, aseptic necrosis of long bones, and the acute chest syndrome96 also occur. The peripheral smear [see Figures 5a, 5b, and 5c] shows irreversibly sickled cells in addition to target cells, stomatocytes, and erythrocytes with eccentric hemoglobin depositions, probably representing HbC aggregates or crystals. Diagnosis is confirmed by hemoglobin electrophoresis or HPLC.97 Further information on diagnostic testing is available online athttp://www.geneclinics.org.

Management is the same as that for sickle cell anemia. In a study of six patients with HbSC disease, treatment with hydroxy urea at a dosage of 1,000 mg/day resulted in an increase in MCV, a decrease in so-called stress reticulocytes, an increase in hemoglobin, and probably a reduction in cell density. Although not definitive, this small study suggests that hydroxyurea benefits patients with HbSC disease.98 Life expectancy for patients with HbSC disease is almost 20 years greater than that for patients with HbSS disease.51

OTHER HEMOGLOBINOPATHIES

Hemoglobin C Disease

The HbC molecule is α2β26glulys; the gene mutation probably originated at a single site in Burkina Faso, in West Africa.97 The presence of this hemoglobin produces almost no illness in the heterozygous state but causes mild compensated hemolysis and palpable splenomegaly in the homozygous state.

The relative insolubility of HbC is responsible for the pathologic changes associated with its presence. HbC probably interacts with the K+-Cl-cotransporter, which keeps it active, whereas the K+-Cl- cotransporter normally shuts off in RBCs after the reticulocyte stage. The result is a loss of K+, cellular dehydration with elevated MCHC, and then aggregation and crystallization of the poorly soluble HbC.97 The relative insolubility of HbC causes erythrocytes to become rigid and thereby subject to fragmentation and to loss of membrane material, resulting in the microspherocytes seen on a peripheral blood smear [see Figures 3a, 3b, and 3c].

Target cells, an important morphologic finding, constitute about 80% of the erythrocytes. HbC crystals are in the oxyhemoglobin state and dissolve when the RBCs are deoxygenated, probably accounting for the absence of vaso-occlusive episodes.

Diagnosis of hemoglobin C disease is based on blood-smear findings and the absence of evidence of either iron deficiency or thalassemia; the diagnosis is confirmed by hemoglobin electrophoresis. No therapy is required.

Hemoglobin E Disease

In hemoglobin E disease, lysine is substituted for glutamic acid at position 26 of the β-globin chain, resulting in an oxidatively unstable molecule. Hemoglobin E trait is found predominantly in Southeast Asia. It came to clinical attention in the United States as a result of the influx of Southeast Asians, in whom the incidence of this trait is about 10%.

Patients heterozygous for HbE have normal hemoglobin values, microcytosis, and no splenomegaly. Electrophoresis reveals that 70% of the hemoglobin is HbA, 25% is HbE, and the remainder is HbA2 or HbF. Inexperienced laboratories may mistake HbE for HbA2; the clue to this error is that HbA2 never accounts for more than 8% of the total hemoglobin. A laboratory report of an HbA2 level of 25% should prompt a review of the data.

Patients homozygous for HbE have mild anemia, with a hemoglobin level of about 12 to 13 g/dl, a low mean corpuscular volume, and an elevated RBC count but no reticulocytosis; they exhibit microcytes and target cells. Electrophoresis shows only HbE. Chronic hemolysis does not occur. Oxidant drugs such as dapsone should be avoided in both heterozygotes and homozygotes.

A serious clinical problem occurs when a patient is doubly heterozygous for HbE and β-thalassemia trait. Such patients present with β-thalassemia intermedia, characterized by severe anemia and splenomegaly (see below). These patients occasionally require transfusions of blood and even allogeneic bone marrow transplantation.99 Further information on diagnostic testing is available online athttp://www.geneclinics.org.

Unstable Hemoglobinopathies

Many individual variants make up the unstable hemoglobinopathies. The hemoglobin instabilities stem from amino acid substitutions that deprive the molecule of its heme group, alter the heme pocket, loosen the link between its α and β chains, or weaken the subunit structure [see 5:I Approach to Hematologic Disorders]. The result is disruption and precipitation of hemoglobin, particularly when it is subjected to oxidant attack. Precipitated hemoglobin forms Heinz bodies, which are observed even in persons heterozygous for the unstable hemoglobin variant. Because of the deleterious effects of Heinz bodies on the erythrocyte and its membrane, significant hemolysis can occur even in the heterozygous state.

Diagnosis of an unstable hemoglobinopathy is suggested by the presence of a partly compensated chronic nonspherocytic hemolysis. Heinz bodies are observed in the erythrocytes of patients who have undergone splenectomy. Erythrocytes from patients who have not undergone splenectomy demonstrate Heinz bodies on incubation with brilliant cresyl blue dye. The differential diagnosis of a hemoglobinopathy includes G6PD deficiency; this disorder can usually be ruled out by direct assay for the enzyme.

Management includes avoidance of oxidant drugs. Splenectomy may be considered when hemolysis is severe and inadequately compensated.

Hemoglobin with Abnormal Oxygen Affinity

The presence of hemoglobin with increased oxygen affinity should be considered in the differential diagnosis of unexplained erythrocytosis, particularly if there is a familial association [see 5:V The Polycythemias]. Hemoglobin electrophoresis may reveal the disorder, but in suspected cases, measurement of the oxyhemoglobin dissociation curve [see 5:I Approach to Hematologic Disorders] is preferable as a basis for diagnosis. Hemoglobin Chesapeake and hemoglobin Rainier are examples of forms with particularly increased oxygen affinity.

The rare instances of hemoglobin with low oxygen affinity, such as hemoglobin Kansas, represent mutations. Patients with low-oxygen-affinity hemoglobinopathy are sometimes cyanotic because of enhanced oxygen unloading.

Methemoglobinemia

Methemoglobin is an oxidation product of hemoglobin in which iron is in the ferric form; thus, the molecule cannot bind oxygen reversibly. Ordinarily, 1% of hemoglobin is in the ferric state. Between 0.5% and 3% of deoxyhemoglobin is normally spontaneously oxidized to methemoglobin every day. The normal reducing power of erythrocytes [see Table 1] maintains the balance between oxidation and reduction. The enzyme system that reduces 95% of methemoglobin to hemoglobin involves two proteins, NADH-cytochrome b5 reductase and cytochrome b5, and also requires NADH. As the name suggests, NADH-cytochrome b5 reductase uses NADH to reduce cytochrome b5. Reduced cytochrome b5 then reduces methemoglobin.100,101 Novel mutations in the affected gene have been described.102

Most often, methemoglobinemia is acquired by ingestion of or exposure to oxidants that oxidize Fe2+ so fast that the reducing systems are overwhelmed [see Mechanism of Oxidative Attack, below].

There are two congenital forms of methemoglobinemia. In the hereditary enzymopenic form of methemoglobinemia, patients are homozygous or doubly heterozygous for a deficiency of NADH-cytochrome b5 reductase.103 These patients appear blue even when only about 10% of their hemoglobin is in the form of methemoglobin, but they are not sick and easily tolerate methemoglobin levels of 25% or more. In contrast, the presence of about 5 g/dl of reduced, deoxygenated hemoglobin produces cyanosis. Patients with this form of methemoglobinemia do not exhibit hemolysis and generally do not require treatment. Assay of NADH-cytochrome b5 reductase, done by a special laboratory, can establish the diagnosis. If desired, methylene blue at a dosage of 100 to 300 mg/day orally can be used, but it may produce urinary discomfort.100 Methylene blue transfers electrons from NADPH to methemoglobin.

The other hereditary form of methemoglobinemia is caused by HbM, of which there are five rare variants. Each of these variants contains an amino acid substitution in the heme pocket, which allows stable bonds to be formed between the heme iron and the amino acid side chains. These bonds keep hemoglobin in the Fe3+ form—a form that is unable to bind oxygen and is inaccessible to the reducing enzymes. The disorder is seen only in heterozygotes; about 30% of hemoglobin is abnormal, as detected by electrophoresis. Cyanosis is noted at birth. Hemolysis is minimal, and therapy is not needed.

THE THALASSEMIAS

The thalassemias have a worldwide distribution; in many regions, they are responsible for major medical, social, and economic perturbations. Throughout the world, the regions in which the thalassemias occur are contiguous with regions endemic for malaria, indicating that the heterozygous forms of thalassemia provide protection against malaria.104 The techniques of molecular biology have helped elucidate the pathophysiology of these syndromes,104 which in turn has enabled investigators to make unambiguous antenatal diagnoses. Using these data, expectant parents can make thoughtful, informed choices regarding the outcome of pregnancies in which the fetus is severely affected.

Molecular Genetics

Thalassemias result from gene deletion, abnormalities in transcription and translation [see 3:VII Genetics for the Clinician], and instability of the mRNA directing globin synthesis or of the globin itself. The genes controlling the synthesis of the α and non-α chains of hemoglobin are located on chromosomes 11 and 16 [see Figure 6].

 

Figure 6. Hemoglobin Gene Loci

The genes encoding the α and non-α chains that come together to form the hemoglobin tetramer lie on chromosomes 16 and 11, respectively. The α genes are present at duplicated loci. Six distinct species of normal hemoglobin have been described. Three of these hemoglobins are synthesized only during embryonic stages of development (Hb Gower 1, Hb Portland, and Hb Gower 2). HbF predominates during fetal development, and a small amount continues to be synthesized in adult life. HbA and HbA2 constitute the major forms of adult hemoglobin. Different hemoglobin genes are activated at various stages of development. In the embryo, ξ chains combine with ε chains to yield Hb Gower 1 and with γ chains to form Hb Portland; α and ε chains are linked to form Hb Gower 2. There are two varieties of γ chains that are derived from separate loci and that differ in a single amino acid; Gγ contains glycine at position 136, whereas Aγ contains alanine at this position. The genes coding for the two other non-α chains, β and δ, which are required for the synthesis of adult hemoglobins, are switched on late in fetal development. The factors regulating this precisely coordinated sequence of changes in hemoglobin production are poorly understood; some evidence suggests that DNA segments intervening between the various hemoglobin genes may control the relative rates of synthesis of the adjacent gene products.

Pathophysiology

In a healthy person, the synthesis of α and β chains is meticulously coordinated to produce adult HbA (α2β2). In contrast, patients with thalassemia usually demonstrate imbalanced synthesis of normal globin chains. Occasionally, however, thalassemia-like syndromes can result from diminished production of a structurally abnormal chain.105 Because one of the globin chains is present in reduced amounts, the unpaired chain accumulates in the developing erythroid precursor cell, and toxicity results [see Figure 6]. Consequently, erythroid cells die in the marrow, giving rise to a classic form of ineffective erythropoiesis [see 5:III Anemia: Production Defects]; affected erythrocytes undergo hemolysis in the peripheral blood.

The β-thalassemias are characterized by diminished production of β-globin chains, causing unmatched α-globin chains to accumulate and aggregate. These aggregates of α chains precipitate, causing decreased ATP synthesis, potassium leak, and reduced amounts of surface sialic acid; the affected erythrocytes are misshapen and relatively rigid. The membrane Ca2+ barrier is breached, allowing Ca2+ to enter. These α-globin aggregates also appear to keep the K+-Cl- cotransporter functioning; as a result, in severe forms of β-thalassemia, dehydration of varying degree is seen.106 The RBC membranes are unstable and fragment easily; there is evidence of oxidation of RBC membrane proteins 4.1 and spectrin [see Figure 2]; and phosphatidylserine migrates to the outer membrane layer, perhaps forming a nidus for thromboembolic events.107,108 These destructive alterations of the membrane, which can be detected by macrophages, may in part be caused by local oxidation.109,110 Abnormal accumulations of α chains probably account for the accelerated apoptosis and ineffective erythropoiesis seen in marrow erythroid precursors.110 The overall decrease in hemoglobin synthesis per cell accounts for the observed hypochromia and target cell formation.

Patients with α-thalassemia demonstrate accumulations of excess β chains that, if present in sufficient amounts, form β4 tetramers (HbH) [see Figure 6]. Such tetramers have high oxygen affinity and are unstable, aggregating in the presence of oxidative stresses such as infection. β-Globin aggregates also become attached to the erythrocyte's membrane skeleton, but they produce lesions different from those produced by α-globin aggregates. In the severe α-thalassemias, ineffective erythropoiesis is less prominent85; rather, destruction of peripheral RBCs is the critical characteristic. RBCs in severe α-thalassemia are rigid, but in contrast to those in severe β-thalassemia, the membranes in severe α- thalassemia are more stable than normal.107,108 Also in contrast to β-thalassemia, the RBCs in severe α-thalassemia are uniformly overhydrated.106

Both α-thalassemia and β-thalassemia are characterized by variable degrees of anemia. This variation is attributable to varying degrees of ineffective erythropoiesis and hemolysis.110 When the anemia is severe, the associated hypoxia induces a vigorous compensatory erythropoiesis, leading to expansion of the marrow cavity, osteopenia, and enlargement of reticuloendothelial organs; tumors may arise at sites of extramedullary erythropoietic activity. Destruction of erythroblasts and erythrocytes may predispose to cholelithiasis and obstructive jaundice. Patients with the more severe forms of thalassemia require regular transfusions, which may eventually generate clinically significant iron overload [see 5:II Red Blood Cell Function and Disorders of Iron Metabolism].

Diagnosis and Treatment of Thalassemia

The HbF and HbA2 measurements that aid in the diagnosis of the β-thalassemias are readily available from clinical laboratories using hemoglobin electrophoresis and, more recently, HPLC. In contrast, the tests required to diagnose the α-thalassemias are quite sophisticated and in the past were performed only in institutions specifically engaged in thalassemia research. Currently, specialized laboratories can detect the number and position of deleted α-globin genes. Further description and information on diagnostic testing is available online athttp://www.geneclinics.org.

The clinical diagnostic tools used to assess patients suspected of having α-thalassemia include clinical history, smear evaluation, calculation of indices, brilliant cresyl blue staining, and family studies. In practice, α-thalassemia trait is diagnosed on the basis of a finding of microcytosis in an iron-replete patient who has normal HbA2 and HbF levels.

The diagnosis of either α- or β-thalassemia should be suspected when the MCV is less than 75 fl and the RBC count is greater than 5 million cells/µl. A patient with these two findings has an 85% chance of having a thalassemia syndrome.111 In one study, diagnosis of thalassemia was not considered in about half of the patients with the disease.

β-Thalassemia

The deficient synthesis of β-globin characteristic of β-thalassemia leads to accumulation of unmatched α chains. A diagnostically significant development in β-thalassemia is the partial compensatory increase of the δ and γ chains that yields elevated levels of HbA2 (α2δ2) and HbF (α2γ2), respectively [see Figures 5a, 5b, and 5c]. The β-thalassemia variants produce three clinical syndromes: β-thalassemia major, β-thalassemia minor, and thalassemia intermedia.

β-Thalassemia major (Cooley anemia)

β-Thalassemia major is usually a homozygous or doubly heterozygous condition; both parents of an affected individual carry a β-thalassemia trait. In β0-thalassemia, the most severe variant, no β chains are synthesized; only HbF and HbA2 are found. β+-Thalassemia is somewhat less severe. It is characterized by small amounts of β chains and small quantities of HbA in addition to HbF and HbA2. δβ-Thalassemia is yet milder; it is caused by deletion of the δ-globin and β-globin genes. This mutation prohibits production of HbA2 and HbA, permitting synthesis of fetal hemoglobin alone.

β-Thalassemia major is characterized by severe anemia that appears in the first year of life. Patients also have jaundice, hepatosplenomegaly, expansion of the erythroid marrow with secondary body changes (including retarded growth), and an increased susceptibility to infection.

Diagnosis is not difficult; no other condition closely resembles Cooley anemia. The peripheral smear shows nucleated RBCs, distorted hypochromic erythrocytes, and basophilic stippling, which represents aggregates of ribosomal RNA [see Figures 5a, 5b, and 5c]. Supravital staining reveals accumulations of excess unmatched α chains.

Management consists of aggressive transfusion therapy. The strategy involves transfusing to a hemoglobin level of about 12 g/dl, then allowing the hemoglobin level to fall to about 9 g/dl just before the next transfusion; this prevents such complications as heart failure, fluid overload, and skeletal deformity. Splenectomy is usually necessary to enhance survival of the patient's own RBCs as well as transfused RBCs.112 Vaccination with pneumococcal vaccine is indicated because of the risk of pneumococcal sepsis after splenectomy.

Long-term transfusions eventually generate iron overload, which if untreated leads to death from cardiac hemochromatosis during adolescence. Iron overload should be managed prophylactically by infusion of subcutaneous deferoxamine, an iron chelator, before iron buildup occurs. Subcutaneous deferoxamine at a dosage of 50 mg/kg/day can effect iron losses of 50 to 200 mg/day but only if infused continuously over 8 to 12 hours for 5 days each week.113 Such therapy not only prevents left ventricular dysfunction but also reverses already established abnormalities.114 The beneficial effects of iron chelation have improved the prognosis for persons with Cooley anemia114: it is no longer inevitable that patients die in their 20s of arrhythmia and left ventricular failure. With current deferoxamine therapy, 61% of patients born before 1976 have had no cardiovascular disease. Compliant patients whose ferritin levels are mostly below 2,500 ng/ml have a survival rate of 91% after 15 years.115 However, compliance with deferoxamine is a problem, and the cost of the drug, together with the cost of the pump and tubing that are required for administration, takes it out of reach of most patients in developing countries. Use of the oral iron chelator deferiprone remains a very contentious issue with regard to its safety and efficacy.116 A new orally active tridentate iron chelator (ICL670) looked very promising in early trials, but it is currently available only on fixed protocols.117 Bone marrow transplantation has been performed with HLA-matched sibling donors. More than 1,000 patients have now undergone allogeneic bone marrow transplantation from sibling donors who either were normal or had β-thalassemia trait.118 Some patients with hemoglobin E β-thalassemia have a phenotype fully as severe as β-thalassemia major and require the same therapy, including allogeneic bone marrow transplantation.99 Experience with cord blood transplantation is more limited.119 Depending on the condition of the patient at the time of transplantation, the rate of transplantation-related mortality was 5% to 19%; the cure rate was 54% to 90%.120,121 Other approaches to the treatment of severe β-thalassemia are still experimental.56 However, two small clinical trials have shown hydroxyurea to be of benefit. This treatment probably works by increasing the production of γ chains, which combine with and remove the excess α chains, and by causing an increase in the production of HbF, a useful hemoglobin.122,123

β-Thalassemia minor (β-thalassemia trait)

Patients with β-thalassemia minor are usually heterozygous for a β-globin mutation and have either mild or no anemia. The peripheral smear shows distinct hypochromia and microcytosis with basophilic stippling. Splenomegaly is occasionally found.

The HbA2 level is elevated above 5% in 90% of patients, and the HbF level is raised above 2% in 50% of patients. This increase in fetal hemoglobin occurs in varying proportions per RBC (a phenomenon known as heterocellular distribution), as shown by the Kleihauer-Betke stain. Patients with higher HbF levels have less severe anemia. Heterozygotes for δβ-thalassemia produce increased amounts of HbF but only normal amounts of HbA2.

Iron deficiency anemia should be excluded from the differential diagnosis of β-thalassemia trait [see 5:II Red Blood Cell Function and Disorders of Iron Metabolism]. Generally, it is easy to distinguish the two disorders. Both are associated with hypochromia and microcytosis, but iron deficiency produces hypo proliferation of RBCs, whereas β-thalassemia minor causes only a minimal reduction in their number. At a hemoglobin level of 9 g/dl, an iron-deficient patient has an RBC count of about 3 million cells/µl, whereas a patient with β-thalassemia trait has an RBC count of about 5 million cells/µl. If the diagnosis remains in doubt, measurement of the serum iron and iron-binding capacity or of the serum ferritin level can be used to distinguish these disorders. It is important to remember, however, that a patient with thalassemia trait may also be iron deficient as a consequence of vaginal bleeding, gastrointestinal bleeding, or both.

Thalassemia intermedia

As the term implies, thalassemia intermedia is characterized by clinical manifestations of moderate severity. Patients with this syndrome have distinct anemia, with hemoglobin levels as low as 6 to 7 g/dl; they exhibit variable degrees of hepatosplenomegaly, but they usually do not require regular transfusions. During infections or other erythropoietic insults, however, transfusions may be needed transiently. In two small clinical trials, isobutyramide was found to be of benefit.124,125

β-Thalassemia-like variants

The hemoglobinopathy associated with hemoglobin Lepore represents another β-thalassemia variant. Patients who are homozygous for this disorder present with Cooley anemia or thalassemia intermedia, and their RBCs contain only hemoglobin Lepore and HbF.105

Hereditary persistence of fetal hemoglobin

The RBCs of patients heterozygous for hereditary persistence of fetal hemoglobin (HPFH) contain about 50% of HbF, whereas homozygotes have 100% of HbF. It was once believed that patients with HPFH were well and had minimal or no anemia, but some clinical variants of HPFH associated with distinct anemia have been described.

α-Thalassemia

The α-globin gene and the β-globin gene differ in two major respects. First, there are no fetal, neonatal, or adult substitutes for the α-globin genes; second, there are only two β-globin genes but four α-globin genes—two α-globin genes on each chromosome 16 [see Figure 6]. The normal α-globin genotype is designated αα/αα. Patients who carry the α-thalassemia-1 variant exhibit a deletion of two α-chain genes from the same chromosome and thus have the —/ or α0 haplotype; this deletion is common among Asian patients. Patients who have the α-thalassemia-2 variant have lost one α gene on one chromosome and show the -α/ or α+ haplotype. Although this mutation is particularly frequent among blacks, it is also observed in Asian and Mediterranean populations. Five clinically distinct syndromes have been recognized among patients who carry different genotypes for the α-globin genes: hemoglobin Barts or hydrops fetalis (—/—); hemoglobin H disease (—/-α); heterozygous α-thalassemia-1 (—/αα); homozygous α-thalassemia-2 (-α/-α); and the silent carrier syndrome (-α/αα).

Hemoglobin Barts (hydrops fetalis)

Children with hemoglobin Barts syndrome are homozygous for α-thalassemia-1 (—/—) and therefore produce no α chains. The unmatched γ chains form γ4 tetramers (hemoglobin Barts). All infants with this condition are born hydropic, and most die unless rescued by intrauterine stem cell transplantation. The parents are usually heterozygous for α-thalassemia-1 (—/αα).

Hemoglobin H disease

The clinical picture of hemoglobin H disease is that of variable hemolytic anemia occurring in patients of Asian, Middle Eastern, or Mediterranean origin. HbH, which precipitates on staining with brilliant cresyl blue, can usually be detected in the patient's freshly drawn RBCs. The molecular mechanisms may involve deletion of three α genes, as would be the case if the patient were doubly heterozygous for α-thalassemia-1 (—/) and α-thalassemia-2 (-α/), yielding a —/-α genotype. Splenomegaly is common. Patients usually do not require regular transfusions, but transient RBC support may be necessary when the patient has infection or experiences other oxidative stresses that lead to the precipitation of the unstable HbH and enhanced hemolysis. During pregnancy, anemia may become clinically severe and require RBC transfusion. Partners of pregnant patients with HbH disease should be screened because if the partner carries an α-thalassemia trait, the fetus may have homozygous α-thalassemia hydrops fetalis. Occasionally there is associated growth retardation and even iron accumulation in the absence of RBC transfusions.126

Heterozygous α-thalassemia

Heterozygous α-thalassemia-1 (—/αα), a common genotype among Asians, causes mild or no anemia; rather, it engenders distinctly hypochromic, microcytic RBCs. Patients homozygous for α-thalassemia-2 (-α/-α), a common genotype among blacks, lack two α genes; the clinical manifestations of patients with this genotype resemble those of patients heterozygous for α-thalassemia-1. The heterozygous state for α-thalassemia-2 (-α/αα) is clinically undetectable and thus represents the silent carrier syndrome.

α-Thalassemia-like syndrome

Hemoglobin constant spring (hemoglobin CS) is a structurally abnormal hemoglobin common in some Asian populations. The α-globin gene contains a mutation in the termination codon, resulting in the synthesis of an α-globin that contains an additional 31 amino acids. Patients heterozygous for this defect have a clinical picture similar to that of a patient homozygous for α-thalassemia-2. Patients homozygous for hemoglobin CS tend to have slightly more severe clinical manifestations than patients heterozygous for α-thalassemia-1. In patients who are doubly heterozygous for α-thalassemia-1 and alpha CS (—/αCS α) and who have HbH/HbCS, disease is slightly more severe than in those with hemoglobin H disease.110

Genetic Counseling and Prenatal Diagnosis

Parents who have had a stillborn hydropic infant or a child with Cooley anemia are justifiably reluctant to repeat the experience. Adults from thalassemia families who know themselves to be heterozygous for thalassemia are often eager to receive genetic counseling when starting their own families. Genetic counseling entails screening prospective parents on the basis of routine diagnostic tests and family studies. In addition, advances in molecular genetics can now provide accurate, unambiguous prenatal diagnoses of the thalassemias. In the first trimester, chorionic villus sampling combined with the use of polymorphic DNA markers and synthetic oligonucleotide probes can provide the definitive diagnosis in about 80% of cases of β-thalassemia [see 3:VII Genetics for the Clinician].127,128 Indeed, the incidence of births of infants with thalassemia major has fallen in several parts of the world. Different ethnic groups respond differently to genetic counseling.

Extracorpuscular Defects

Erythrocytes can be damaged through trauma or by antibodies, drugs, abnormally functioning organs, and toxins. These causes of an extracorpuscular defect should be considered whenever hemolysis develops in a patient who has no personal or family history of anemia.

MECHANICAL INJURY: MICROANGIOPATHIC HEMOLYSIS

Microangiopathic hemolysis is characterized by the appearance of bizarre, fragmented erythrocytes (e.g., schistocytes, or helmet cells) on a peripheral smear and by signs of intravascular and extravascular hemolysis.

Pathophysiology

The normal erythrocyte can withstand considerable elongation and twisting, but it disintegrates when subjected to strong stretching or shearing forces. Stresses of this magnitude have been observed to occur in jets produced by deformed aortic valves, by arteriovenous shunts, by ventricular septal defects, or by the older valvular prostheses.

Localized intravascular coagulation, in which fibrin strands bridge the arteriolar lumen, is thought to occur in arterioles supplying inflamed or neoplastic tissues. Fibrin strands lop off fragments of RBCs, whose membranes promptly reseal. Some of the erythrocyte contents leak out, however, producing varying degrees of intravascular hemolysis. The distorted RBCs are then removed by the reticuloendothelial system.

Diagnosis

Hemolysis in conjunction with typical blood smear findings is diagnostic of microangiopathic hemolysis [see Figures 5a, 5b, and 5c]. If the angiopathy is extensive, thrombocytopenia and disseminated intravascular coagulation develop. Causes include hemodynamic jets, vasculitis,129 giant hemangiomas, thrombotic thrombocytopenic purpura, metastatic cancer,130 certain infections (especially men ingococcemia, rickettsial diseases, and hantavirus infection), hemolytic-uremic syndrome, disseminated intravascular coagulation, drugs (cocaine, cyclosporine, mitomycin, and tacrolimus), and even subclavian catheters.130,131 Quinine has been identified as a fairly common cause of drug-induced thrombotic thrombocytopenic purpura (TTP) and hemolytic-uremic syndrome.132 A single case of microangiopathic hemolysis has been described in an infant with cutaneous anthrax.133

Treatment

In treating microangiopathic hemolysis, clinicians must focus primary attention on the underlying disease. Patients may become iron deficient and require iron therapy. Supplementation of depleted folate stores may stimulate erythropoiesis. In rare cases, anemia caused by an old prosthetic aortic valve may be severe enough to warrant valve replacement. Plasmapheresis provides effective therapy for TTP [see5:X Transfusion Therapy and 5:XIII Hemorrhagic Disorders].

March hemoglobinuria

March hemoglobinuria, a disorder that somewhat resembles microangiopathic hemolysis, usually occurs in young persons after prolonged marching or running or playing on bongo drums. The severe and repetitive trauma to the feet or hands is thought to destroy RBCs circulating in the vessels of the soles and palms. The patient notices red urine that clears in 1 day or less after the activity. Transient hemoglobinemia and hemoglobinuria without anemia, smear abnormalities, or reticulocytosis confirm the diagnosis. The use of padded shoes and the avoidance of paved surfaces may prevent recurrences in persons who continue running.

IMMUNE HEMOLYSIS

General Mechanisms

A classic, well-delineated example of immune (not autoimmune) hemolysis involves fetomaternal incompatibility at Rh locus D, in which the D-negative mother, after contact with D-positive erythrocytes, may produce an IgG anti-D antibody; the antibody crosses the placenta and attacks and destroys fetal erythrocytes. The fetus becomes jaundiced and has spherocytic erythrocytes.

The fetal RBCs, now coated with maternal IgG anti-D antibody, attach to fetal macrophages and monocytes that contain receptors for the Fc portion of these IgG molecules. Macrophagic digestion of portions of the erythrocytic membrane leads to the loss of considerable surface area. The resulting rigid spherocyte returns to the circulation and becomes trapped in the fetal reticuloendothelial system, particularly in the spleen. Hemolysis results. The IgG antibody is maximally active at 37° C; it generally cannot extensively activate the complement pathway, and it cannot agglutinate attacked RBCs suspended in saline.

The direct Coombs antiglobulin test [see Figure 7] is used clinically to detect IgG coating of RBCs. This test is negative in the mother, because her erythrocytes lack D antigen and thus are not coated with anti-D antibody. The indirect Coombs test [see Figure 7], which detects the presence of free serum antibody that reacts with RBCs, is positive for the mother's serum because she has circulating anti-D antibody. In the fetus, in contrast, the direct Coombs test is strongly positive because the fetus's RBCs, which express D antigen, are coated with maternal anti-D antibody. The results of the fetus's indirect Coombs test may be positive or negative, depending on the amount of anti-D antibody that has been transferred by the mother, the avidity of the anti-D antibody for fetal D-positive RBCs, and the availability of D antigen sites on fetal RBCs.

 

Figure 7. Coombs Test

The Coombs test detects the presence of human antibodies or complement components on erythrocytes or the presence of antibodies in serum. The test is useful in diagnosing Rh hemolytic disease of the newborn, autoimmune hemolysis, or potential hemolytic transfusion reactions. The figure illustrates Rh hemolytic disease of the newborn.

In the direct Coombs test (a), fetal erythrocytes (RBCs) are shown with D antigen attached to their surfaces. Maternal anti-D antibody binds to the fetal erythrocytes at the D antigen sites in utero. Coombs antiserum, which contains antibody to human IgG, binds to the anti-D antibody on a sample of washed fetal erythrocytes, causing them to clump (a positive reaction). Washed maternal erythrocytes, having no D antigen, will have no attached anti-D antibody and are therefore not clumped by Coombs serum (a negative reaction).

In the indirect Coombs test (b), maternal or fetal serum is added to the red cells of another person or to panels of erythrocytes of known antigenic specificity; Coombs antiserum is then added. Clumping in this case occurs only if the test serum, such as the maternal serum, contains anti-D antibody and if the red cells chosen have the D antigen.

The direct Coombs test is used to detect immunoglobulin molecules already attached to erythrocytes, such as those found on the fetal erythrocytes in Rh hemolytic disease of the newborn or in autoimmune hemolytic anemia. Therefore, the test is done on the patient's thoroughly washed erythrocytes. The indirect Coombs test is used for determining whether specific antibodies are present in a serum sample, and it is performed on the patient's serum.

These antibodies are described as warm (maximum activity at 37° C [usually IgG1 or IgG3]) or cold (maximum activity at 5° C [usually IgM]). Antibodies have also been classified as complete (i.e., capable of agglutinating saline-suspended RBCs) and incomplete (i.e., incapable of agglutinating saline-suspended RBCs); their detection requires the use of techniques such as the direct Coombs antiglobulin test [see Figure 7] or enzyme treatment of RBCs.134 Warm autoantibodies are usually incomplete, whereas cold agglutinins, which are for the most part IgM, are usually complete.135

Autoimmune Hemolytic Anemia

Autoimmune hemolytic anemia is generally an acute disorder characterized by extravascular hemolysis. Intravascular hemolysis in this condition is rare and indicates that an extremely rapid rate of erythrocyte destruction is occurring or that the extravascular removal mechanisms have been overwhelmed.

Pathophysiology

In autoimmune hemolytic anemia, for reasons that are unclear, autoantibodies form and are directed against central components of the erythrocyte (e.g., Rh antigen, Kell antigen,134 glycophorin A).136 Alternatively, the patient's RBCs are sensitized with both an IgG antibody and a complement component, usually C3d. In other circumstances, however, it appears that complement is fixed to the RBC surface by an IgM antibody that is subsequently washed away. Occasionally, the RBCs exhibit only complement components, and no IgG can be detected by the Coombs test. Complement fixation in such cases may be explained by the continued presence of IgG at a level below that detectable in the usual direct antiglobulin test; alternatively, a complement-fixing IgG or IgM antibody had been attached to the cell but was eluted in the testing procedure.135

The severity of hemolysis correlates with the number and class of IgG and, in rare cases, IgA molecules attached to the RBC surface. Antibody-coated RBCs attach to the macrophages' receptors (FcRI, FcRII, or FcRIII) by the antibody's Fc portion. The firm binding of RBCs to these macrophage receptors is then followed either by removal of a portion of the RBC membrane, which results in the production of a spherocyte, or by phagocytosis of the entire RBC.134 Relatively low levels of IgG1 attachment to RBCs produce a positive result on direct Coombs antiglobulin testing without evidence of hemolysis (approximately 1,000 molecules per RBC), whereas much higher levels of IgG1 autoantibody per RBC are associated with frank hemolysis.134 The combined presence of IgG and complement components may enhance the severity of hemolysis.

Erythrocytes sensitized to IgG alone are usually removed in the spleen, whereas RBCs sensitized to IgG and complement or to complement alone are generally destroyed in the liver, because hepatic Kupffer cells carry receptors specific for complement component C3b.

Differential diagnosis

Both an idiopathic variety of autoimmune hemolytic anemia and a variety that occurs secondary to other disorders have been described. Such primary disorders include systemic lupus erythematosus, non-Hodgkin lymphoma (especially chronic lymphocytic leukemia), Hodgkin disease, cancer, myeloma, dermoid cyst, HIV infection, angioimmunoblastic lymphadenopathy with dysproteinemia, hepatitis C,137 and chronic ulcerative colitis.

Diagnosis

Patient presentations vary markedly, from asymptomatic to severe. A person may be found to have a positive Coombs test result when undergoing blood bank or blood donation screening. Such persons can usually be shown to have complement or the combination of complement and IgG (usually IgG1 or IgG4) on their RBCs, but they are generally not undergoing hemolysis. By contrast, an acute hemolytic episode can lower the hematocrit from 45% to 15% in 2 days. With this extreme presentation, severe fatigue and cardiorespiratory symptoms will develop, together with jaundice, lymphadenopathy, and hepatosplenomegaly.

In severe cases, the blood smear shows macrocytosis, polychromatophilia, variable spherocytosis, and autoagglutination of RBCs. The platelet count is also occasionally depressed (Evans syndrome), and there may be leukopenia. One third of patients may have reticulocytopenia at presentation.138 The direct Coombs test will be positive. Any or all of these findings may be absent in mild disease.

Whether complement, IgG, or both are present on RBCs should be determined by the use of Coombs reagents that are specifically directed against IgG, IgA, or complement components. Occasionally, an autoimmune hemolytic anemia is suspected, but the direct Coombs test is repeatedly negative; in such cases, the level of autoantibody may be below the level of detectability for very active autoantibodies, such as subclass IgG3 autoantibodies, or the autoantibody may be IgA or IgM.134

Patients with evidence of hemolytic anemia should be screened for autoimmune diseases (e.g., systemic lupus erythematosus) and other forms of hemolysis, such as paroxysmal nocturnal hemoglobinuria, cold agglutinin disease, and paroxysmal cold hemoglobinuria.

Treatment

Treatment of clinically affected patients is directed at decreasing autoantibody production and reducing the macrophagic attack on the RBCs. Initial therapy usually consists of 60 to 100 mg of prednisone a day, given in divided doses. This approach usually produces a slow decrease in antibody coating of RBCs and is thought to interfere with phagocytic attack on coated erythrocytes. A good response to corticosteroid use—indicated by a rise in the reticulocyte count and an improvement in hemoglobin and hematocrit—may be apparent within 1 or 2 days. Supplementation with 1 mg of folic acid a day is recommended.

After the initial response to therapy, which is usually satisfactory, the hemoglobin level and reticulocyte count may return to normal. The Coombs test is then repeated to determine whether the response has become weaker; if so, the prednisone dosage is tapered cautiously. Approximately 20% of patients remain well indefinitely, but the majority suffer from a chronic, treacherous disease that can produce sudden relapses with abrupt anemia. The prednisone should be titrated in accordance with the hemoglobin level, the reticulocyte count (elevation indicates continued hemolysis), and the direct Coombs titer; alternate-day therapy should be considered to minimize steroid side effects. If patients do not respond to standard prednisone therapy, high-dose dexamethasone (e.g., 40 mg/day orally for 4 consecutive days in 28-day cycles139) may be effective.

If the corticosteroid dose required for long-term therapy produces significant morbidity, one can proceed empirically either to splenectomy or to the use of immunosuppressive agents. Measurements of splenic sequestration of chromium-51 (51Cr)-labeled erythrocytes do not reliably indicate the benefits of splenectomy. Splenectomy rarely results in extended remission but is valuable as a prednisone-sparing measure. After splenectomy, low-dose prednisone (5 to 10 mg/day) may stabilize the hemoglobin concentration.

The immunosuppressive agent azathioprine or cyclophosphamide can be used as an alternative to splenectomy. There is no reliable evidence to support the use of one of these agents over the other. For patients with very aggressive disease, cyclosporine has been used successfully.140 Azathioprine should be started at a dosage of 100 to 200 mg a day; the peripheral blood count should be monitored with a view toward preventing reticulocytopenia or neutropenia. Cyclophosphamide is started at a dosage of 100 to 200 mg a day, with monitoring of blood counts and urine; however, because cyclophosphamide can cause therapy-related acute myeloid leukemia or myelodysplastic syndrome, its use should be limited [see 12:XVII Chronic Myelogenous Leukemia and Other Myeloproliferative Disorders].

Azathioprine or cyclophosphamide doses have to be adjusted to reduce the white cell count to about 3,000/mm3. Improvement usually comes in 3 to 4 weeks. When a response occurs, the prednisone dose can be reduced and the hemoglobin level, reticulocyte count, and Coombs titer monitored to determine the minimally required therapy. For patients with very refractory disease, therapy with intravenous cyclophosphamide at doses used for allogeneic bone marrow transplantation has been tried. This approach is clearly myelotoxic, and its usefulness awaits further confirmation. High-dose intravenous IgG has been used to treat autoimmune hemolytic anemia. In one report, only one third of patients had a transient response, and doses larger than those used in idiopathic thrombocytopenic purpura (i.e., 1g/kg/day for 5 days) were required.141,142

There are anecdotal reports that rituximab is useful in treating refractory and relapsing cases.143,144 However, a case of automimmune hemolytic anemia occurring after rituximab therapy for lymphoproliferative disorder has been reported.145

Patients with symptomatic anemia require an RBC transfusion, but often the blood bank reports an incompatibility. Many blood banks regularly perform a direct Coombs antiglobulin test on the recipient's RBCs. A patient who has free antibodies in the serum will exhibit very extensive and broad reactivity against donor panels of RBCs and will usually produce an incompatible major cross-match when tested with the antiglobulin reagent. If transfusions are needed to support cardiorespiratory and central nervous system functions, immediate consultation with the transfusion medicine service is recommended.135 No patient should be allowed to die because the blood bank does not have a perfectly compatible unit of RBCs. If transfusion is clinically indicated, the physician should administer the best units of blood that are available, because it has been shown that these patients can tolerate even imperfectly matched RBCs.146

Drug-Related Immune Hemolysis

Drug-initiated immune hemolysis is often indistinguishable from autoimmune hemolytic anemia. There are two variants: the hapten type and the hemolysis that results from alteration of a membrane antigen.147

Hapten type

Drugs such as the penicillins and the cephalosporins bind firmly to the erythrocyte membrane. In rare circumstances in which massive dosages of the drug (e.g., more than 10 million units of penicillin a day) are required, the protein-bound drug may act as a hapten and elicit an immune response. An IgG antibody that appears to be directed against the drug-RBC complex is produced148,149; this leads to a positive result on direct Coombs testing with the anti-IgG reagent and a negative result with the anti-C3d reagent. When the offending drug is stopped, hemolysis ends in a few days. In contrast, the drug may be bound loosely to produce a neoantigen that generates the immune response.147 In this circumstance, the result of direct Coombs testing with the anti-C3d reagent is usually positive, and the result of testing with the anti-IgG reagent may be negative.

If the patient's serum is tested against normal cells (i.e., the indirect Coombs test is used), no reaction occurs unless the offending drug and a source of complement are first added to the normal RBCs. Stopping or switching the drug is effective in eliminating the hemolysis because the antibody is usually very specific.

Alteration of a membrane antigen

Some drugs may alter a membrane antigen, thereby stimulating the production of IgG antibodies that cross-react with the native antigen. Methyldopa is the classic example of a drug that causes autoimmune hemolytic anemia. Other examples are levodopa, mefenamic acid, and procainamide. Drug administration leads to a positive direct Coombs test with anti-IgG reagents in 15% to 20% of treated patients, but hemolysis occurs in fewer than 1%. The eluted antibody is seen to be a classic IgG autoantibody directed against Rh components. The mechanism of hemolysis is identical to that of autoimmune hemolytic anemia.150

The NSAID diclofenac sodium has been reported to cause a devastating acute hemolytic anemia, with evidence of intravascular and extravascular hemolysis accompanied occasionally by shock, organ failure, and even disseminated intravascular coagulation.151 Patients develop both RBC autoantibodies and drug-dependent antibodies. It is thought that diclofenac sodium binds to the surface of RBCs, forming neoantigens that lead to the generation of true autoantibodies, as well as drug-dependent antibodies. The direct Coombs test is positive with both the IgG and the C3d reagents. Additional antibody reactivity occurs with the addition of diclofenac sodium metabolites obtained from the urine of patients treated with the drug. Therapy consists of recognizing the cause, stopping the diclofenac sodium, and supporting the patient for several days until the process stops.151

Delayed Hemolysis of Transfused Erythrocytes

Blood is usually typed only for ABO and Rh-D antigens, but other antigens are also present on RBCs. Thus, a patient who receives extensive transfusions over 1 to 2 weeks may develop an antibody response to one or more of these other antigens. Kell, Duffy, Kidd, and Rh antigens other than D are the usual offenders. When the patient with antibodies receives RBCs expressing these antigens, an acute self-limited hemolysis, usually extravascular, may ensue. Clues are a history of transfusion, spherocytosis on peripheral smear, a positive direct Coombs test, and the recent appearance of an antibody in the patient's serum (positive indirect Coombs test). Usually, no therapy is required, but further transfusions should be cross-matched with the patient's serum [see 5:X Transfusion Therapy]. Similar problems arise with transplantation of bone marrow and other tissue.152

Cold Agglutinin Disease

Cold agglutinin disease has several variants. One rare variant affects young adults and usually occurs after infection with Mycoplasma pneumoniae or infectious mononucleosis, although several cases have also been reported in association with chronic falciparum malaria. A more common variant affects persons about 60 years of age and may present as idiopathic cold agglutinin disease, as a prodrome to a lymphoproliferative or an immunoproliferative disorder, or in association with an already established lymphoproliferative disorder.153

Pathophysiology

Serologically, cold agglutinin disease is characterized by the presence of high titers of IgM agglutinins (> 1:1,000 and usually > 1:10,000) in serum. These antibodies are maximally active at 4° C, are capable of activating the complement sequence, and are directed against the polysaccharide antigens. Presumably, IgM reacts with erythrocytes circulating in the cooled blood of the nose, ears, and shins, where it fixes complement and then dissociates from the RBCs when they reach warmer areas of the body.

In the postinfectious variety of this disorder, IgM cold agglutinin is oligoclonal and short-lived. Conversely, the IgM is monoclonal in chronic idiopathic cold agglutinin disease or in cases associated with Waldenström macroglobulinemia, chronic lymphocytic leukemia, or other lymphomas. IgM predominantly contains λ light chains in patients with chronic idiopathic cold agglutinin disease or Waldenström macroglobulinemia; in patients with lymphoma, however, the IgM mainly contains κ light chains. Occasionally, the IgM cold agglutinin is detectable as an M protein spike on serum protein electrophoresis [see 12:XV Chronic Lymphoid Leukemias and Plasma Cell Disorders].

In the post-Mycoplasma variant, the mycoplasmas appear to bind to the RBC surface at the Ii antigen site. This receptor-ligand interaction results in the presentation of the I antigen in an immunogenic form.154 Listeria monocytogenes contains the I antigen,153 further supporting the idea that some infectious agents stimulate the naturally occurring cold agglutinins, as well as cause the postinfectious cold agglutinin disease.

Diagnosis

The clinical syndrome of cold agglutinin disease is quite variable. Patients occasionally show only low titers of cold agglutinins and have no other symptoms or have a history of recent pneumonia. In patients with warm-and-cold autoimmune hemolytic anemia, the associated hemolysis tends to be severe and chronic. The RBCs of these patients are coated with IgG and complement components, whereas their serum contains a relatively low titer of cold agglutinin that acts at 30° C and perhaps even at temperatures up to 37° C.

The diagnosis is suggested by hemolytic anemia with acral signs and symptoms. It may be difficult to draw blood, and the RBCs may visibly agglutinate in a cold syringe and on the blood smear. Automated blood cell counters may count the agglutinated RBCs as single cells and thus report absurdly high values for the MCV and MCHC. Usually, the laboratory detects a broadly active cold agglutinin. The direct Coombs test is positive with anticomplement reagents but infrequently positive with anti-IgG.

Findings that support the diagnosis of idiopathic cold agglutinin disease include a high IgM cold agglutinin titer with broad thermal reactivity134 and I specificity (reacting with erythrocytes from adults but not with cord erythrocytes), pure κ light-chain composition, occasionally an absolute serum IgM elevation, and an M protein pattern on serum protein electrophoresis. Investigation should be directed at discovering a possible lymphoma or other underlying disorder in these patients. Conversely, post-Mycoplasma and post-infectious mononucleosis cold agglutinins are polyclonal. The post-infectious mononucleosis antibody is usually directed against i antigens (cord RBCs).

Treatment

The post-Mycoplasma or the post-infectious mononucleosis variant is usually mild and self-limited and requires no specific treatment. Patients with the idiopathic variety who have acral symptoms must change their way of life, either by moving to a warmer climate or by keeping their ears, nose, hands, and feet covered during cold weather. In severely anemic patients, transfusions with packed RBCs may be required; in such patients, careful cross-matching and warming of the blood is necessary to minimize cold agglutination.

Splenectomy and corticosteroids are generally of no benefit in controlling hemolysis associated with cold agglutinin disease. Presumably, complement-coated cells are removed to a substantial degree by hepatic rather than splenic macrophages, and the cells that produce IgM are relatively insensitive to the effects of corticosteroids. Occasionally, however, high doses of cortico steroids (e.g., 100 mg of prednisone a day) have resulted in a reduction in the hemolytic rate in patients with relatively low titers of cold agglutinins. In the relatively rare variant caused by IgG cold agglutinins, corticosteroids and splenectomy may be of benefit. Use of penicillamine or other reducing agents containing sulfhydryl groups produces no benefit. Good responses are occasionally obtained by the use of chlorambucil at a dosage of 4 to 6 mg/day. Exchange transfusion and plasmapheresis appear to be logical therapies for acute disease, but further clinical studies are needed to evaluate these techniques. Interferon alfa, at a dosage of 3 million U/m2 three times weekly, was reported to produce an impressive drop in cold agglutinin titer, with a decrease in serum IgM monoclonal protein and in acral symptoms over a 1-month period.134 Treatment with rituximab in the doses used to treat non-Hodgkin lymphoma has been beneficial.155,156

Paroxysmal Cold Hemoglobinuria

Patients with the rare disorder of paroxysmal cold hemoglobinuria have cold-induced signs and symptoms of intravascular hemolysis. The hemolysis is associated with the presence of an IgG serum antibody that is directed against the RBC's P system. The IgG antibody is best demonstrated by the Donath-Landsteiner test; the serum is mixed either with the patient's own blood cells or with blood cells from a normal person. The mixture is chilled to 4° C. If the IgG antibody associated with this disorder is present, hemolysis occurs after warming to 37° C. In the past, paroxysmal cold hemoglobinuria was usually seen as a complication of syphilis, but it has recently been observed in association with viral infections and non-Hodgkin lymphoma.157

HYPERSPLENISM

Hypersplenic disorders constitute a diverse group of clinical conditions sharing the common features of splenomegaly and hemolysis. Splenic enlargement and hemolysis occur in many disorders, including hepatic cirrhosis with congestive splenome galy, Gaucher disease, lymphoma, connective tissue disorders, Felty syndrome, sarcoidosis, tuberculosis, and other infectious diseases.

Pathophysiology

The spleen's unique structure accounts for several of the pathophysiologic features of hypersplenism. Splenic arterioles have a few direct branches leading to the sinusoids, but most of the terminal arterioles open into the splenic cords. Blood cells pass from the cords to the pulp through slits in the sinus walls; the slits have dimensions of about 1 by 3 µm.158 Blood cells must squeeze through the longitudinal spaces, which are lined with reticular fibers, and between adventitial cells that are located outside the sinus. Macrophages and endothelial cells line the inside of the sinus. Repeated intimate contact occurs between blood cells and these macrophages.

Blood flow in the spleen is slow. The erythrocyte's pH and oxygen tension level fall, glucose is consumed, and the cell's metabolism is impaired. The hematocrit may increase, further elevating viscosity and resistance to flow. As a consequence, the blood cells are exposed to metabolic and mechanical stresses in the presence of macrophages and other leukocytes that can recognize cell membrane damage. As erythrocytes age, phagocytes remove defective surface areas, transforming the biconcave erythrocytes into rigid spherocytes or RBC fragments; these particles are later trapped and removed by the reticuloendothelial system. A big spleen has a greater than normal blood flow and exposes an unusually large proportion of blood cells to its culling activities. Thus, the problem in hypersplenism is essentially a quantitative one. A vicious circle may evolve in patients undergoing hemolysis, because hemolysis itself may cause splenomegaly.

Diagnosis

If the spleen is not palpable but the clinical situation is strongly suggestive of splenomegaly, ultrasonography or CT scanning may prove useful. Because blood cells other than erythrocytes are affected by a large spleen, the patient may be pancytopenic. Unless the underlying disease specifically involves the bone marrow, the marrow of patients with hypersplenism is generally hyperplastic because of rapid regeneration of all affected cell lines. The peripheral blood smear is not diagnostic of hypersplenism.

Treatment

If hypersplenism is producing clinically significant complications and if therapy for the patient's primary disease does not shrink the spleen, splenectomy may be necessary. Anemia, however, is not necessarily attributable to hypersplenism, irrespective of the size of the spleen. Hemodilution is another possible mechanism. Patients with massive splenomegaly who have very low hematocrit and hemoglobin values may have a normal RBC mass as assessed with the 51Cr technique. Massive splenomegaly often is associated with an increase in plasma volume that results in extraordinary hemodilution. Moreover, greatly enlarged spleens may contain a pool of erythrocytes that constitutes as much as 25% of the total RBC mass—in contrast to normal spleens, which have no such RBC pool. In patients with splenomegaly who have a true decrease in RBC mass, the underlying disease may act to reduce RBC production by suppressing erythropoietin production rather than by accelerating destruction. Therefore, it is prudent to determine RBC mass before making the diagnosis of hypersplenism.

DRUGS AND TOXINS AS CAUSES OF HEMOLYSIS

Drugs Causing Oxidative Attack

Pathogenesis

Dapsone, sulfasalazine, phenacetin, sodium perchlorate, nitroglycerin, phenazopyridine, primaquine,100 para quat, and vitamin K analogues can insert themselves into the oxygen-binding cleft of hemoglobin. By this action, such agents can generate oxidizing free radicals, such as superoxide, hydroxyl free radical, and peroxide. If the erythrocyte's protective reducing mechanisms are overwhelmed [see Table 1], hemoglobin is oxidized to form Heinz bodies and methemoglobin. Sulfhemoglobin is also produced by oxidative attack. The molecule contains a sulfur atom in the porphyrin ring, which gives it a blue-green color. The source of the sulfur atom is not clear, but the presence of sulfur in the heme ring makes it a poor oxygen transporter.159 The RBC membrane may also suffer from oxidative attack. Damaged cells are removed in the reticuloendothelial system. Hemolysis is usually, but not invariably, extravascular, and Heinz bodies can be seen on a specially stained blood smear. The smear may also show the bite, hemiblister, or cross-bonded cells typical of oxidative attack on erythrocytes [see Figure 4]. Severe oxidative damage apparently causes hemoglobin to puddle at one side of the RBC, leaving a plasma membrane-enclosed hemighost in the remainder. Such hemighosts can be detected in the peripheral blood. Severe oxidative destruction is associated with increased methemoglobin levels and a decrease in RBC levels of GSH. The methemoglobin level is elevated. As little as 1.5 g/dl of methemoglobin or 0.5 g/dl of sulfhemoglobin can produce the physical finding of cyanosis. By contrast, 5 g/dl of reduced deoxyhemoglobin is required to produce comparable cyanosis.100

Nitrites can oxidize hemoglobin to methemoglobin. Consequently, the recreational use of butyl and isobutyl nitrites as stimulants, psychedelics, and aphrodisiacs has led to clinical problems. When inhaled in usual amounts, these agents may produce a mild to modest increase in methemoglobin, raising its concentration from the normal level of 1% to 2% to as much as 20%. More extensive inhalation or ingestion of these agents has induced severe methemoglobinemia, characterized by methemoglobin levels approaching 62%. Because methemoglobin does not carry oxygen, these high levels are accompanied by manifestations of tissue hypoxia such as headache, shortness of breath, lethargy, and stupor. Physical examination shows tachycardia, postural hypotension, and cyanosis; the venous blood is purple-brown.160 If untreated, it is likely to be fatal.

Diagnosis

Diagnosis is based on a history of exposure to an oxidant drug or other toxin, together with characteristic peripheral blood smear findings and elevated methemoglobin measurements.

Treatment

Treatment should restore normal methemoglobin levels. Management starts with the identification and withdrawal of the offending agent. Patients who have severe met hemoglobinemia should be treated immediately with 1 to 2 mg/kg of methylene blue; the agent is infused intravenously in a 1 g/dl solution over a 5-minute period. In the presence of the RBC enzyme NADPH-methemoglobin reductase and adequate amounts of the electron donor NADPH [see Table 1], methylene blue is rapidly reduced to leukomethylene blue. This product in turn quickly reduces methemoglobin to hemoglobin. Cyanosis is thereby reversed, and the patient should turn pink immediately after the infusion. Several hours later, however, the patient may again become cyanotic, presumably because nitrates released from tissues reenter the peripheral blood at that time. Readministration of methylene blue at a dosage of 1 mg/kg intravenously over a 5-minute period should restore normal hemoglobin levels.

Successful methylene blue therapy requires adequate supplies of NADPH. Patients who have abnormalities of the pentose phosphate pathway, such as G6PD deficiency, will not respond to this approach and should receive emergency exchange transfusions.160 Patients with very high levels of methemoglobin (at least 60%) or those whose smears contain many hemighosts should undergo exchange transfusion, perhaps with hemodialysis.100,160

Lead-Induced Hemolysis

Lead exposure results in hypertensive encephalopathy, neuropathy, and hemolytic anemia characterized by coarse basophilic stippling in RBCs. The mechanism of lead-induced hemolysis is complex because the metal has several actions: it blocks heme synthesis, thus causing a buildup of RBC protoporphyrin; it produces a deficiency of pyrimidine 5′-nucleotidase161; and it attacks erythrocyte membrane phospholipids, producing potassium leak and interfering with Na+,K+-ATPase activity.

Diagnosis

Screening for lead poisoning entails measuring the free erythrocyte protoporphyrin level (sometimes called the zinc protoporphyrin level), which is elevated because lead blocks the last step in heme synthesis. The diagnosis is confirmed by measuring blood and urine lead levels.

Treatment

After the exposure to lead is stopped, use of a chelating agent such as edetate calcium disodium (CaNa2EDTA) may be considered. Treatment is started with 0.5 to 1 g of intravenous CaNa2EDTA, given over a period of 6 to 8 hours; the compound is given daily for 5 days.

After this initial course, 0.5 g of CaNa2EDTA is given as an intravenous bolus or intramuscular injection every 2 days for 2 weeks, during which time the urine lead levels are monitored. Alternatively, the initial 5-day course of CaNa2EDTA can be followed with oral penicillamine: 1 g a day is given for the first 7 days; the drug is withheld for the next 7 days; and during the final 7 days of the regimen, the dosage of 1 g a day is resumed and the urine lead level is measured at the end of the final day. Another study recommends giving 500 mg of penicillamine a day and continuing this dosage for 60 days after the patient has become asymptomatic.162

VENOMS AND PHYSICAL AGENTS AS CAUSES OF HEMOLYSIS

Agents Causing Enzymatic Attack

Classic examples of attacking enzymes are the snake-venom or clostridial lecithinases (e.g., phospholipase C). Such enzymes attack the phospholipids of the membrane bilayer and produce RBC fragmentation, spherocytosis, and intravascular and extravascular hemolysis. Disseminated intravascular coagulation and shock may occur. Prompt recognition and management of the primary disorder is critical, as is supportive therapy.

Venom from the brown spider, Loxosceles intermedia, releases sphingomyelinases and metalloproteinases that cleave the RBC membrane glycophorins. This in turn facilitates complement activation and lysis of affected RBCs.163

Physical Causes of Hemolysis

Freshwater drowning and accidental intravenous administration of sterile water can cause intravascular hemolysis by osmotic lysis. In such cases, RBCs swell and become spheroidal. Saltwater drowning can induce hemolysis by desiccating RBCs. Burns cause temperature-mediated denaturation of erythrocyte membrane polypeptides, resulting in hemolysis.

Infectious Diseases Causing Hemolysis

Malaria is the most important infectious cause of hemolysis. The resulting severe anemia causes the death of large numbers of pregnant women and 2- to 5-year-old children in sub-Saharan Africa. Plasmodium species, particularly P. falciparum, directly parasitize and destroy RBCs, but the anemia is a complex blend of impaired RBC production, hemolysis of parasitized and nonparasitized130 RBCs, and ineffective erythropoiesis.164 The diagnosis is made by pathognomonic findings on the blood smear; treatment is directed against the malarial parasite, with support of the circulation with RBC transfusions if required [see 7:XXXIV Protozoan Infections].

Other infectious causes of hemolysis

Infection with M. pneumoniae and infectious mononucleosis can cause cold agglutinin hemolysis. Infection with H. influenzae type b can cause hemolysis. The major virulence factor of H. influenzae, polyribose ribosyl phosphate (PRRP), allows the organism to escape phagocytosis. When PRRP is released into the circulation, it binds to RBCs. The binding of anti-PRRP antibodies then leads to complement-dependent hemolysis.165 Patients infected with HIV or cytomegalovirus may have autoimmune hemolytic anemia (see above).166

Clostridial sepsis can be devastating; the appearance of free plasma hemoglobin or hemoglobinuria should suggest this often fatal infection.Clostridium species are capable of sudden, explosive growth; they can release many enzymes, including phospholipases and proteases, that digest RBCs, producing intravascular hemolysis.

Some infections can cause splenomegaly and hypersplenic hemolysis. Meningococcemia or overwhelming gram-negative septicemia often produces disseminated intravascular coagulation and microangiopathic hemolysis.

Babesiosis is caused by a parasite that invades RBCs and that is transmitted from its rodent reservoir by the same ixodid tick that carries Lyme disease and human granulocytic ehrlichiosis. This disease is being more frequently diagnosed, particularly in New England. Immunocompromised persons, such as those with HIV, are more likely to have chronic and severe infections. The diagnosis has been made on peripheral blood smears, but polymerase chain reaction methods are more sensitive.167

HEMOLYSIS ASSOCIATED WITH LIVER DISEASE

Anemia in patients with liver disease is often the result of a production defect rather than hemolysis, but cirrhotic patients may have congestive splenomegaly with hypersplenic hemolysis. Macrocytes (with or without B12 or folate deficiency) and target cells (caused by cholesterol elevation) are also common findings in such cases.

Spur cell anemia

Severe liver disease, including alcoholic cirrhosis, may result in the formation of irregularly spiculated RBCs known as spur cells (acanthocytes).168 Spur cells have alterations in their membranes (a decreased ratio of phospholipids to cholesterol169) that shorten their survival, resulting in hemolytic anemia.

OTHER CAUSES OF HEMOLYSIS

Copper Accumulation

In rare instances, Wilson disease, a metabolic disorder associated with excessive copper deposition, is first detected during a coincident episode of dramatic, acute hemolysis. The release of free copper into the serum and its subsequent entry into RBCs are thought to be the underlying hemolytic mechanism. In addition to affecting hexokinase levels, the intracellular copper appears to cause formation of oxygen radicals that react with and oxidize membrane components. Although no successful therapeutic intervention has been reported, penicillamine can be given at a dosage of 2 to 4 g once a day orally to reduce the free copper level. The administration of 1,000 to 2,000 IU of vitamin E (α-tocopherol) a day for several days may also be helpful if oxidative attack is an important factor.

Cardiopulmonary Bypass

Free plasma hemoglobin increases after cardiopulmonary bypass. The increase is thought to be caused by activation of the complement pathway, leading to deposition of the C5b-C9 attack complex on the RBC surface [see 6:IV Disorders of the Complement System].170

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Editors: Dale, David C.; Federman, Daniel D.