Rodak's Hematology Clinical Principles and Applications

PART IV

Erythrocyte Disorders

CHAPTER 24

Intrinsic defects leading to increased erythrocyte destruction

Elaine M. Keohane

OUTLINE

Red Blood Cell Membrane Abnormalities

Red Blood Cell Membrane Structure and Function

Hereditary Red Blood Cell Membrane Abnormalities

Acquired Red Blood Cell Membrane Abnormalities

Red Blood Cell Enzymopathies

Glucose-6-Phosphate Dehydrogenase Deficiency

Pyruvate Kinase Deficiency

Other Enzymopathies

Objectives

After completion of this chapter, the reader will be able to:

  1. Describe the intrinsic cell properties that affect red blood cell (RBC) deformability.
  2. Explain how defects in vertical and horizontal membrane protein interactions can result in a hemolytic anemia.
  3. Compare and contrast the inheritance pattern, membrane proteins mutated, mechanism of hemolysis, typical RBC morphology, and clinical and laboratory findings of hereditary spherocytosis, hereditary elliptocytosis, and hereditary ovalocytosis.
  4. Compare and contrast the RBC morphology and laboratory findings of hereditary spherocytosis and immune-associated hemolytic anemias.
  5. Explain the principle, interpretation, and limitations of the osmotic fragility and eosin-5′-maleimide (EMA) binding tests in the diagnosis of hereditary spherocytosis.
  6. Describe the causes, pathophysiology, RBC morphology, and clinical and laboratory findings of hemolytic anemias characterized by stomatocytosis.
  7. Describe the causes and pathophysiology of hereditary and acquired conditions characterized by acanthocytosis.
  8. Describe the cause, pathophysiology, clinical manifestations, laboratory findings, and treatment for paroxysmal nocturnal hemoglobinuria.
  9. Compare and contrast the inheritance pattern, pathophysiology, clinical symptoms, and typical laboratory findings of glucose-6-phosphate dehydrogenase deficiency and pyruvate kinase deficiency.
  10. Given the history, symptoms, laboratory findings, and a representative microscopic field from a peripheral blood film for a patient with a suspected intrinsic hemolytic anemia, discuss possible causes of the anemia and indicate the data that support these conclusions.

CASE STUDY

After studying the material in this chapter, the reader should be able to respond to the following case study:

A 45-year-old man sought medical attention for the onset of chest pain. Physical examination revealed slight jaundice and splenomegaly. The medical history included gallstones, and there was a family history of anemia. A CBC yielded the following results:

 

Patient Results

Reference Interval

WBCs (×109/L)

13.4

3.6–10.6

RBCs (×1012/L)

4.20

4.60–6.00

HGB (g/dL)

11.9

14.0–18.0

HCT (%)

32.4

40–54

MCV (fL)

77.1

80–100

MCH (pg)

28.3

26–32

MCHC (g/dL)

36.7

32–36

RDW (%)

22.9

11.5–14.5

Platelets (×109/L)

290

150–450

The peripheral blood film revealed anisocytosis, polychromasia, and spherocytes (Figure 24-1).

  1. From the data given, what is a likely cause of the anemia?
  2. What additional laboratory tests would be of value in establishing the diagnosis, and what abnormalities in the results of these tests would be expected to confirm your impression?
  3. What is the cause of this type of anemia?

 
FIGURE 24-1 Peripheral blood film for the patient in the case study (×500).  Source:  (From Carr JH, Rodak BF: Clinical hematology atlas, ed 3, Philadelphia, 2009, Saunders.)

Intrinsic hemolytic anemias comprise a large group of disorders in which defects in the red blood cells (RBCs) themselves result in premature hemolysis and anemia. Intrinsic disorders can be divided into abnormalities of the RBC membrane, metabolic enzymes, or hemoglobin. Most of these defects are hereditary. This chapter covers defects in the RBC membrane and enzymes causing hemolytic anemia. Chapter 27 covers qualitative hemoglobin disorders, and Chapter 28 covers quantitative hemoglobin disorders.

Red blood cell membrane abnormalities

Red blood cell membrane structure and function

The RBC maintains a biconcave discoid shape that is essential for normal function and survival for 120 days in the peripheral circulation. The key to maintaining this shape is the plasma membrane, a lipid bilayer embedded with proteins and connected to an underlying protein cytoskeleton (Figure 9-2). The insoluble lipid portion serves as a barrier to separate the vastly different ion and metabolite concentrations of the interior of the RBC from its external environment, the blood plasma. The concentration of the constituents in the cytoplasm is tightly regulated by proteins embedded in the membrane that serve as pumps and channels for movement of ions and other material between the RBC’s interior and the blood plasma. Various membrane proteins also act as receptors, RBC antigens, enzymes, and support for the surface carbohydrates to form a protective glycocalyx with the surface glycolipids. The lipid bilayer remains intact because transmembrane proteins embedded in the membrane anchor it to a two-dimensional protein lattice (cytoskeleton) immediately beneath its surface.1 Together, the transmembrane proteins and underlying cytoskeleton provide structural integrity, cohesion, and mechanical stability to the cell.

In a normal life span of 120 days, RBCs must repeatedly maneuver through very narrow capillaries and squeeze through the splenic sieve (narrow slits or fenestrations in the endothelial cell lining of the splenic sinuses) as they move from the splenic cords to the sinuses. To accomplish this without premature lysis, the RBCs must have deformability, or the ability to repeatedly bend, stretch, distort, and then return to the normal discoid, biconcave shape.2 The cellular properties that enable RBC deformability include the RBC’s biconcave, discoid geometry; the elasticity (pliancy) of its membrane; and its cytoplasmic viscosity (Chapter 9).3,  4

The biconcave, discoid geometry of the RBC is dependent on vertical and horizontal interactions between the transmembrane and cytoskeletal proteins (listed in Tables 9-5 and 9-6).3Two transmembrane protein complexes, the ankyrin complex and protein 4.1 complex, provide vertical structural integrity to the cell by anchoring the lipid bilayer to the underlying spectrin cytoskeleton.3,  5,  6 In the ankyrin complex, ankyrin and protein 4.2 link transmembrane proteins, band 3 and RhAG (the Rh-associated glycoprotein) to the cytoskeleton.6-8 In the protein 4.1 complex, protein 4.1 links transmembrane proteins, glycophorin C, XK, Rh, and Duffy to the cytoskeleton, and adducin and dematin link with transmembrane proteins, band 3 and glucose transport (glut 1) (Figure 9-2).6,  9 These interactions are called vertical because they are perpendicular to the plane of the cytoskeleton. They prevent loss of membrane and the resultant decrease in the surface area–to–volume ratio of the RBC.5 Two major cytoskeletal proteins, α-spectrin and β-spectrin, interact laterally with each other to form antiparallel heterodimers, which link with other spectrin heterodimers to form tetramers. The spectrin heterodimers also form a spectrin–actin–protein 4.1 junctional complex with accessory proteins (tropomyosin, tropomodulin, dematin, and adducin), thus linking the spectrin tetramers in a two-dimensional lattice.10,  11 These proteins provide horizontal mechanical stability, which prevents the membrane from fragmenting in response to mechanical stress.3,  5

Factors that impact the elasticity of the cell are not as clear. Interactions between the spectrin dimers and their junctional complexes are flexible and allow for movement as the RBCs stretch and bend (Figure 9-4).11 In addition, the ability of spectrin repeats to unfold and refold is likely to be one of the determinants of membrane elasticity.11,  12

The cytoplasmic viscosity depends on the concentration of hemoglobin, as well as the maintenance of the proper cell volume by the normal functioning of various channels and pumps that allow the passage of ions, water, and macromolecules in and out of the cell.3

A defect in the RBCs that changes the membrane geometry, its elasticity, or the viscosity of the cytoplasm affects RBC deformability and can result in premature hemolysis and anemia.3The RBC membrane is discussed in more detail in Chapter 9; the reader is encouraged to review that chapter when studying defects in the membrane.

Hereditary red blood cell membrane abnormalities

Most defects in the RBC membrane that can cause hemolytic anemia are hereditary; however, acquired defects also exist. Hereditary RBC membrane defects have historically been classified by morphologic features. The major disorders are hereditary spherocytosis, characterized by spherocytes, and hereditary elliptocytosis, characterized by elliptical RBCs. Hereditary pyropoikilocytosis, a variant of hereditary elliptocytosis, is characterized by marked poikilocytosis and heat sensitivity. Other less common membrane disorders include hereditary ovalocytosis, overhydrated hereditary stomatocytosis, and dehydrated hereditary stomatocytosis (also called hereditary xerocytosis). Hereditary membrane defects can also be classified as those that affect membrane structure (and alter geometry and elasticity) and those that affect membrane transport (and alter cytoplasmic viscosity) (Box 24-1).11 The membrane structural defects can be further divided into those that affect vertical membrane protein interactions and those that affect horizontal membrane protein interactions.5,  11 The major hereditary membrane defects are described in Table 24-1.

TABLE 24-1

Characteristics of Major Hemolytic Anemias Caused by Hereditary Membrane Defects

RBC, Red blood cell.

BOX 24-1

Classification of Major Hereditary Membrane Defects Causing Hemolytic Anemia

Mutations that alter membrane structure

Hereditary spherocytosis

Hereditary elliptocytosis/pyropoikilocytosis

Hereditary ovalocytosis (Southeast Asian ovalocytosis)

Mutations that alter membrane transport proteins

Overhydrated hereditary stomatocytosis

Dehydrated hereditary stomatocytosis or hereditary xerocytosis

Mutations that alter membrane structure

Hereditary spherocytosis.

Hereditary spherocytosis (HS) is a heterogeneous group of hemolytic anemias caused by defects in proteins that disrupt the vertical interactions between transmembrane proteins and the underlying protein cytoskeleton. HS has worldwide distribution and affects 1 in 2000 to 3000 individuals of northern European ancestry.6,  11,  13 In 75% of families, it is inherited as an autosomal dominant trait and is expressed in heterozygotes who have one affected parent.6 Homozygotes are rare; such patients present with severe hemolytic anemia but have asymptomatic parents.13 In approximately 25% of cases, the inheritance is nondominant, with some autosomal recessive cases.11,  13

Pathophysiology.

HS results from gene mutations in which the defective proteins disrupt the vertical linkages between the lipid bilayer and the cytoskeletal network.5 Various mutations in five known genes can result in the HS phenotype (Table 24-1). Mutations can occur in genes for (1) cytoskeletal proteins, including ANK1, which codes for ankyrin (40% to 65% of cases in the United States and Europe; 5% to 10% of cases in Japan); SPTA1, which codes for α-spectrin (fewer than 5% of cases); SPTB, which codes for β-spectrin (15% to 30% of cases); and EPB42, which codes for protein 4.2 (fewer than 5% of cases in the United States and Europe; 45% to 50% of cases in Japan); and (2) transmembrane protein, SLC4A1, which codes for band 3 (20% to 35% of cases).6,  11 Less than 10% of cases involve de novo mutations, with most affecting the ankyrin gene.13 A mutation database for hereditary spherocytosis lists 130 different mutations (ANK1, 52; SCL4A1, 49; SPTA1, 2; SPTB, 19; and EPB42, 8).14 Because of the vertical interactions of the transmembrane proteins and cytoskeletal protein lattice, a primary mutation in one gene may have a secondary effect on another protein in the membrane. For example, primary mutations in ANK1 result in both ankyrin and spectrin deficiencies in the RBC membrane.1315,  16 In approximately 10% of patients, no mutation is identified.11

The defects in vertical membrane protein interactions cause RBCs to lose unsupported lipid membrane over time because of local disconnections of the lipid bilayer and underlying cytoskeleton. Essentially, small portions of the membrane form vesicles; the vesicles are released with little loss of cell volume.13 The RBCs acquire a decreased surface area–to–volume ratio, and the cells become spherical. These cells do not have the deformability of normal biconcave discoid RBCs, and their survival in the spleen is decreased.4,  13,  15 As the spherocytes attempt to move through the narrow, elliptical fenestrations of the endothelial cells lining the splenic sinusoids, they acquire further membrane loss or become trapped and are rapidly removed by the macrophages of the red pulp of the spleen.4,  13 In addition, as the RBCs are sequestered in the spleen, the membrane can acquire yet more damage, lose more lipid membrane, and become more spherical due to splenic conditioning.4,  13,  15 The conditioning may be enhanced by the acidic conditions in the spleen and the prolonged contact of the RBCs with macrophages.13,  15 Low levels of adenosine triphosphate (ATP) and glucose, and phagocyte-produced free radicals, which cause oxidative damage, may also play a role (Figure 24-2).13,  15

 
FIGURE 24-2 Pathophysiology of splenic trapping and destruction of spherocytes. ATP, Adenosine triphosphate; RBC, red blood cell.  Source:  (Modified from Becker PS, Lux SE: Disorders of the red cell membrane. In Nathan DG, Oski FA, editors: Hematology of infancy and childhood, ed 4, Philadelphia, 1993, Saunders, pp. 529-633.)

In HS, RBC membranes also have abnormal permeability to cations, particularly sodium and potassium, which is likely due to disruption of the integrity of the protein cytoskeleton.4,  17The cells become dehydrated, but the exact mechanism is not clear. Overactivity of the Na+-K+ ATPase may cause a reduction in intracellular cations, causing more water to diffuse out of the cell.4,  13 This results in an increase in viscosity and cellular dehydration.13 The abnormality is not related to defects in cation transport proteins, and dehydration occurs regardless of the type of primary mutation causing the HS.17

Clinical and laboratory findings.

Symptomatic HS has three key clinical manifestations: anemia, jaundice, and splenomegaly. Symptoms of HS may first appear in infancy, childhood, or adulthood, or even at an advanced age.16 There is wide variation in symptoms. Silent carriers are clinically asymptomatic with normal laboratory findings and usually are identified only if they are the parents of a child with recessively inherited HS.4,  13,  16 Approximately 20% to 30% of patients have mild HS and are asymptomatic because an increase in erythropoiesis compensates for the RBC loss.46 They usually have normal hemoglobin levels but may show subtle signs of HS, with a slight increase in serum bilirubin in the range of 1.0 to 2.0 mg/dL, an increased reticulocyte count of up to 6%, and a few spherocytes on the peripheral blood film.4,  6,  16 Mild HS may first become evident during pregnancy, during illnesses that cause splenomegaly (such as infectious mononucleosis), or in aging, when the rate of erythropoiesis starts to decline.13,  16 Approximately 60% of patients have moderate HS with incompletely compensated hemolytic anemia, hemoglobin levels greater than 8 g/dL, serum bilirubin over 2 mg/dL, reticulocyte counts in the range of 6% to 10%, and spherocytes on the peripheral blood film.4,  6,  16 Jaundice is seen at some time in about half of these patients, usually during viral infections. About 5% to 10% of patients have moderate to severe HS, with hemoglobin levels usually in the range of 6 to 8 g/dL, serum bilirubin between 2 to 3 mg/dL, and reticulocyte counts greater than 10%.4,  6,  16 About 3% to 5% of patients have severe HS, with hemoglobin levels below 6 g/dL, serum bilirubin over 3 mg/dL, and reticulocyte counts greater than 10%.4,  6,  15 Patients with severe HS are usually homozygous for HS mutations and require regular transfusions.13 Splenomegaly is found in about half of young children and in 75% to 95% of older children and adults with HS.4

The hallmark of HS is spherocytes on the peripheral blood film. When present in patients with childhood hemolytic anemia and a family history of similar abnormalities, the uniform spherocytes are highly suggestive of HS. Some of these are microspherocytes—small, round, dense RBCs that are filled with hemoglobin and lack a central pallor (Figure 24-3). Normal-appearing RBCs, along with polychromasia and varying degrees of anisocytosis and poikilocytosis, are present. In addition to spherocytes, occasionally other RBC morphologic variants may be observed in some types of mutations: acanthocytes in some β-spectrin mutations,18 pincered or mushroom-shaped cells in some cases of band 3 deficiency in patients without splenectomy,19 and ovalostomatocytes in homozygous EPB4.2 mutations.20 Note that spherocytes are not specific for HS and can be seen in other hereditary and acquired conditions.

 
FIGURE 24-3 Spherocytes (peripheral blood, ×1000).

Because of the spherocytosis, HS patients have an increase in the mean cell hemoglobin concentration (MCHC) to between 35 and 38 mg/dL and an increase in the red cell distribution width (RDW) to greater than 14%.4,  16,  21 The mean cell volume is variable, ranging from within the reference interval to slightly below.6,  13 Using laser-based automated analyzers, there is an increase in hyperchromic or hyperdense RBCs representing cells with an MCHC over 41 g/dL.6,  13 A cutoff of greater than 4% hyperchromic cells has been proposed to screen patients for HS.6,  22 Biochemical evidence of extravascular hemolysis may be present in moderate to severe forms of HS, and the extent is dependent on the severity of the hemolysis. This includes a decrease in serum haptoglobin level and an increase in levels of serum indirect bilirubin and lactate dehydrogenase (Chapter 23). The bone marrow shows erythroid hyperplasia due to the increased demand for RBCs to replace the circulating spherocytes that are prematurely destroyed, but bone marrow analysis is not required for diagnosis.

Additional tests.

In patients with a family history of HS, splenomegaly, an increased MCHC and reticulocyte count, and spherocytes on the peripheral blood film, no further special testing is needed for the diagnosis of HS.16 In cases in which HS is suspected, but the family history and mode of inheritance are not clear, or there are atypical clinical and/or laboratory findings, further special testing is needed to confirm a diagnosis.16 No one method will detect all cases of HS, so a combination of methods is needed for definitive diagnosis.

Osmotic fragility.

The osmotic fragility test demonstrates increased RBC fragility in blood specimens in which the RBCs have decreased surface area–to–volume ratios. Blood is added to a series of tubes with increasingly hypotonic sodium chloride (NaCl) solutions. In each tube, water enters and leaves the RBCs until equilibrium is achieved. In 0.85% NaCl, the amount of water entering the cell is equivalent to the water leaving the cell because the intracellular and extracellular osmolarity is the same. In a hypotonic solution, more water will enter the cell to dilute the intracellular contents until equilibrium is reached between the cytoplasm and the hypotonic extracellular solution. As this phenomenon occurs, the cells swell. As the RBCs are subjected to increasingly hypotonic solutions, even more water will enter the RBCs until the internal volume is too great and lysis occurs. Because spherocytes already have a decreased surface area–to–volume ratio, they lyse in less hypotonic solutions than normal-shaped, biconcave RBCs and thus have increased osmotic fragility.

In the procedure, a standard volume of fresh, heparinized blood is mixed with NaCl solutions ranging from 0.85% (isotonic saline) to 0.0% (distilled water) in 0.05% to 0.1% increments.23 After a 30-minute incubation at room temperature, the tubes are centrifuged and the absorbance of the supernatant is measured spectrophotometrically at 540 nm.23 The percent hemolysis is calculated for each tube as follows:23

Ax% is the absorbance in the tube being measured, A0.85% is the absorbance in the 0.85% NaCl tube (representing 0% hemolysis), and A0.0% is the absorbance in the 0.0% NaCl tube (representing 100% hemolysis). The % hemolysis for each % NaCl concentration is plotted, and an osmotic fragility curve is drawn (Figure 24-4). Normal biconcave RBCs show initial hemolysis at 0.45% NaCl, and 100% or complete hemolysis generally occurs between 0.35% and 0.30% NaCl. If the curve is shifted to the left, the patient’s RBCs have increased osmotic fragility, and in this case, initial hemolysis begins at an NaCl concentration greater than 0.5%. Conversely, if the curve is shifted to the right, the RBCs have decreased osmotic fragility. Decreased osmotic fragility is found in conditions characterized by numerous target cells, such as thalassemia.

 
FIGURE 24-4 Erythrocyte osmotic fragility curve. A, Curve in thalassemia showing two cell populations: one with increased fragility (lower left of curve) and one with decreased fragility (upper right of curve). B, Normal curve. C, Curve indicating increased fragility, as in hereditary spherocytosis.

The existence of a distinct subpopulation of the most fragile cells, those most conditioned by the spleen, is reflected by the presence of a “tail” on the osmotic fragility curve (Figure 24-4).13 After splenectomy is performed, the osmotic fragility improves, and this subpopulation of conditioned cells disappears.13

Incubating the blood at 37° C for 24 hours before performing the test (called the incubated osmotic fragility test) allows HS cells to become more spherical and is often needed to detect mild cases. Patients who have increased osmotic fragility only when their blood is incubated tend to have mild disease and a low number of spherocytes in the total RBC population. The osmotic fragility test is time-consuming, and it requires a fresh heparinized blood specimen collected without trauma (to avoid hemolysis) and accurately made NaCl solutions. Specimens are stable for 2 hours at room temperature or 6 hours if the specimen is refrigerated.23 A major drawback of the osmotic fragility test is its lack of sensitivity.16 In a 2011 study of 150 HS patients by Bianchi and colleagues, the sensitivity for the unincubated test was 68%, with only a modest increase in sensitivity to 81% with the incubated test.24 In nonsplenectomized HS patients with compensated anemia, those sensitivity figures dropped to only 53% and 64%, respectively.24 The osmotic fragility test is also nonspecific. A result indicating increased fragility does not differentiate between HS and spherocytosis caused by other conditions, such as burns, immune hemolytic anemias, and other acquired disorders.1316 These disadvantages have led some not to recommend this test for routine use.16

Eosin-5′-maleimide (EMA) binding test.

The eosin-5′-maleimide (EMA) binding test has been proposed as a more sensitive alternative for confirmation of HS.16 EMA is a fluorescent dye that binds to transmembrane proteins band 3, Rh, RhAg, and CD47 in the RBC membrane.25 When measured in a flow cytometer, specimens from HS patients show a lower mean fluorescence intensity (MFI) than RBCs from normal controls and from patients with spherocytes due to immune-mediated hemolysis.25 The result is reported in % decrease in MFI when the patient specimen is compared to normal controls.6 The sensitivity and specificity of the EMA-binding assay varies from 93% to 96% and 94% to 99%, respectively.6,  24,  26 Positive tests can also occur with congenital dyserythropoietic anemia type II, Southeast Asian ovalocytosis, and hereditary pyropoikilocytosis, but these are rare conditions.6,  24,  27 The EMA binding test offers advantages in that it is suitable for low-volume pediatric specimens, it can be performed within 3 hours, specimens are acceptable for analysis up to 7 days after collection, and gating can be used to eliminate the interference from transfused or fragmented RBCs.6 However, there is disagreement among laboratories on the % MFI decrease cutoff value for HS, and standardization is needed across laboratories.6,  27

Other tests.

In atypical HS cases, additional tests may be required to identify the defective proteins.16 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) can be used to identify membrane protein deficiencies by electrophoretic separation of the various proteins in solubilized RBC membranes with quantitation of the proteins by densitometry.13,  16 Membrane proteins can also be quantitated by radioimmunoassay.13 Variation in membrane surface area and cell water content can be determined by osmotic gradient ektacytometry.28 The ektacytometer is a laser-diffraction viscometer that records the laser diffraction pattern of a suspension of RBCs exposed to constant shear stress in solutions of varying osmolality from hypotonic to hypertonic. An RBC osmotic deformability index is calculated and plotted against the osmolality of the suspending solution to generate an osmotic gradient deformability profile.28 This method, however, is available only in specialized laboratories.6,  13

Several other tests have been used for diagnosis of HS, but they are cumbersome to perform; lack sensitivity and specificity, or both; and are not widely used in the United States.4,  13The acidified glycerol lysis test measures the amount of hemolysis after patient RBCs are incubated with a buffered glycerol solution at an acid pH. The test has a sensitivity of 95% but is not specific in that other conditions with spherocytes will yield a positive result, including autoimmune hemolytic anemias.24,  29 In the autohemolysis test, patient’s RBCs and serum are incubated for 48 hours, with and without glucose. Normal controls generally have less than 5.0% hemolysis at the end of the 48-hour incubation period, and they have less than 1.0% hemolysis if glucose is added. Glucose catabolism (anaerobic glycolysis) provides the ATP to drive the cation pumps to help maintain the osmotic balance in the RBCs. In HS, the hemolysis is 10% to 50%, which corrects considerably but not to the reference interval when glucose is added. The test has a sensitivity similar to that of the incubated osmotic fragility test.4,  13 The hypertonic cryohemolysis test is based on the fact that cells from HS patients are particularly sensitive to cooling at 0° C in hypertonic solutions.30 The percent hemolysis is calculated after the patient’s RBCs are incubated in buffered 0.7 mol/L sucrose, first at 37° C for 10 minutes and then at 0° C for 10 minutes. Normal cells show 3% to 15% hemolysis, whereas RBCs in HS have greater than 20% hemolysis.30 Increased hemolysis can also occur in Southeast Asian ovalocytosis, some types of hereditary elliptocytosis, and congenital dyserythropoietic anemia type II.16,  30

Molecular techniques for detection of genetic mutations are not usually required.13,  16 The polymerase chain reaction procedure followed by single-strand conformational polymorphism analysis can identify potential regions in HS genes that may contain a mutation.13,  16 Once a region has been identified, nucleic acid sequencing can be performed to identify the specific mutation.13 Genetic diagnosis is likely to become more important in the future.

Complications.

Although most patients with HS have a well-compensated hemolytic anemia and are rarely symptomatic, complications may occur that require medical intervention. Patients may experience various crises, classified as hemolytic, aplastic, and megaloblastic.13 Hemolytic crises are rare and usually associated with viral syndromes. In aplastic crises there is a dramatic decrease in hemoglobin level and reticulocyte count. The crisis usually occurs in conjunction with parvovirus B19 infection, which suppresses erythropoiesis, and patients can become rapidly and severely anemic, often requiring transfusion.16 This complication is more common in children, but it can occur in adults.13,  16 Patients with moderate and severe HS can also develop folic acid deficiency resulting from increased folate utilization to support the chronic erythroid hyperplasia in the bone marrow.13 This phenomenon is termed megaloblastic crisisand is particularly acute during pregnancy and during recovery from an aplastic crisis. Providing folic acid supplementation to patients with moderate and severe HS avoids this complication.16 About half of patients, even those with mild disease, also experience cholelithiasis (bilirubin stones in the gallbladder or bile ducts) due to the chronic hemolysis.13 Chronic ulceration or dermatitis of the legs is a rare complication.13

Treatment.

Mild HS usually requires no treatment.16 Splenectomy is reserved for moderate to severe cases, and laparoscopy is the recommended method.16 Splenectomy results in longer RBC survival in the peripheral blood and helps prevent gallstones by decreasing the amount of hemolysis and thus the amount of bilirubin produced.13,  16 The major drawbacks of splenectomy are the lifelong risk of overwhelming sepsis and death from encapsulated bacteria and an increased risk of cardiovascular disease with age.13,  16 Prior to splenectomy, patients receive vaccines for pneumococcus, meningococcus, and H. influenza type b, and postsplenectomy antibiotics may be recommended.13 Because infants and young children are especially susceptible to postsplenectomy sepsis, splenectomy is usually postponed until after the age of 6 years.16 In a nationwide sample of 1657 children (aged 5 to 12 years) with HS who underwent splenectomy, there were no cases of postoperative sepsis and no fatalities from any cause during hospitalization for the surgery.31 Partial splenectomy has been performed in young children with severe HS, but further evaluation of the risks and benefits of this procedure is needed.13,  16

After splenectomy, spherocytes are still apparent on the blood film, and all of the typical changes in RBC morphology seen after splenectomy also are observed, including Howell-Jolly bodies, target cells, and Pappenheimer bodies (Figure 24-5). Reticulocyte counts decrease to high-normal levels, and the anemia is usually corrected. Leukocytosis and thrombocytosis are present. Bilirubin levels decrease but may remain in the high-reference interval.

 
FIGURE 24-5 Red blood cell morphology in hereditary spherocytosis after splenectomy. Howell-Jolly bodies and spherocytes are seen (peripheral blood, ×1000).

Occasionally, a patient does not improve because of an accessory spleen missed during surgery or the accidental autotransplantation of splenic tissue during splenectomy. In these cases hemolysis may resume years later.13 The resumption of splenic function can be ascertained from liver or spleen scans.13 Patients with severe HS usually require regular transfusions. Transfusions are rarely needed in the less severe forms of HS.13,  15,  16

Differential diagnosis.

HS must be distinguished from immune-related hemolytic anemia with spherocytes. Family history and evaluation of family members, including parents, siblings, and children of the patient, help differentiate the hereditary disease from the acquired disorder. The immune disorders with spherocytes are usually characterized by a positive result on the direct antiglobulin test (DAT), whereas the results are negative in HS. The eosin-5′-maleimide (EMA) binding test shows decreased fluorescence typical of HS. Increased osmotic fragility is not diagnostic of HS, because the cells in acquired hemolytic anemia with spherocytes also show increased osmotic fragility. The typical clinical and laboratory findings in HS are summarized in Table 24-2.

TABLE 24-2

Typical Clinical and Laboratory Findings in Hereditary Spherocytosis (HS)13,  16

Clinical manifestations

Splenomegaly

Anemia*

Jaundice (can be intermittent)

Mode of inheritance

75% autosomal dominant

25% nondominant

Complete blood count results

↓Hemoglobin*

↑Mean cell hemoglobin concentration

↑Red cell distribution width

↑Reticulocyte count*

↑Hyperchromic (hyperdense) RBCs**

Peripheral blood film findings

Spherocytes

Polychromasia

Direct antiglobulin test result

Negative

Indicators of hemolysis

↓Serum haptoglobin

↑Serum lactate dehydrogenase

↑Serum indirect bilirubin

Selected additional tests for atypical cases

Not required for diagnosis of HS with the typical features listed above ↓ Fluorescence in eosin-5´-maleimide binding test by flow cytometry ↑ Osmotic fragility and incubated osmotic fragility tests 
SDS-PAGE analysis of membrane proteins

* Varies with severity of HS and ability of the bone marrow to compensate for the hemolysis.

** As measured on some automated blood cell analyzers.

† With some rare mutations, acanthocytes, pincered cells, stomatocytes, or ovalocytes may be seen in addition to spherocytes.

‡ A result within the reference interval does not rule out HS; similar results can be observed in conditions other than HS.

↑, Increased; ↓, decreased; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Hereditary elliptocytosis.

Hereditary elliptocytosis (HE) is a heterogeneous group of hemolytic anemias caused by defects in proteins that disrupt the horizontal or lateral interactions in the protein cytoskeleton. It reportedly exists in all of its forms in 1 in 2000 to 1 in 4000 individuals, but because the majority of cases are asymptomatic, the actual prevalence is not known.13,  32 The disease is more common in Africa and Mediterranean regions, where there is a high prevalence of malaria. The prevalence in West Africa of certain spectrin mutations associated with HE is between 0.6% and 1.6%.13,  33 The molecular basis for the association of spherocytosis and malaria is unknown. The inheritance pattern in HE is mainly autosomal dominant, with a small number of autosomal recessive cases.11,  13

Pathophysiology.

HE results from gene mutations in which the defective proteins disrupt the horizontal linkages in the protein cytoskeleton and weaken the mechanical stability of the membrane.4,  5 The HE phenotype can result from various mutations in at least three genes: SPTA1, which codes for α-spectrin (65% of cases); SPTB, which codes for β-spectrin (30% of cases); and EPB41,which codes for protein 4.1 (5% of cases) (Table 24-1).11,  32 A mutation database for hereditary elliptocytosis is available and lists 46 different mutations.14 The spectrin mutations disrupt spectrin dimer interactions and the EPB41 mutations result in weakened spectrin–actin–protein 4.1 junctional complexes.6,  11 RBCs are biconcave and discoid at first, but become elliptical over time after repeated exposure to the shear stresses in the peripheral circulation.13 The extent of the disruption of the spectrin dimer interactions seems to be associated with the severity of the clinical manifestations.11 In severe cases, the protein cytoskeleton is weakened to such a point that cell fragmentation occurs. As a result, there is membrane loss and a decrease in surface area–to–volume ratio that reduces the deformability of the RBCs. The damaged RBCs become trapped or acquire further damage in the spleen, which results in extravascular hemolysis and anemia. In general, patients who are heterozygous for a mutation are asymptomatic, and their RBCs have a normal life span; those who are homozygous for a mutation or compound heterozygous for two mutations can have moderate to severe anemia that can be life-threatening.6,  11

The RBCs in HE patients all show some degree of decreased thermal stability. Cases in which the RBCs show marked RBC fragmentation upon heating were previously classified as hereditary pyropoikilocytosis (HPP). HPP is now considered a severe form of HE that exists in either the homozygous or compound heterozygous state.11,  13

Patients with the Leach phenotype lack the Gerbich antigens and glycoprotein C (GPC).4 The phenotype is due to a mutation in the genes for GPC and results in the absence of the glycoprotein in the RBC membrane.34 The Gerbich antigens are normally expressed on the extracellular domains of GPC and thus are absent in this condition. Heterozygotes have normal RBC morphology, and homozygotes have mild elliptocytosis but no anemia.4 The reason for the elliptocyte morphology may be a defect in the interaction between GPC and protein 4.1 in the junctional complex.4

Clinical and laboratory findings.

The vast majority of patients with HE are asymptomatic, and only about 10% have moderate to severe anemia.11 Some may have a mild compensated hemolytic anemia as evidenced by a slight increase in the reticulocyte count and a decrease in haptoglobin level, or develop transient hemolysis in response to other conditions such as viral infections, pregnancy, hypersplenism, or vitamin B12 deficiency.13 Often an asymptomatic patient is diagnosed after a peripheral blood film is examined for another condition. Rarely, heterozygous parents with undiagnosed, asymptomatic HE have offspring who are homozygous or compound heterozygous for their mutation(s) and have moderate to very severe hemolysis. Some of these asymptomatic parents have normal RBC morphology and laboratory tests.13

The characteristic finding in HE is elliptical or cigar-shaped RBCs on the peripheral blood film in numbers that can vary from a few to 100%4 (Figure 24-6). The number of elliptocytes does not correlate with disease severity.4 Investigation of elliptocytosis begins by taking a thorough patient and family history and performing a physical examination, and examining the peripheral blood films of the parents.4 Other laboratory tests may be needed to rule out other conditions in which elliptocytes may be present, such as iron deficiency anemia, thalassemia, megaloblastic anemia, myelodysplastic syndrome, and primary myelofibrosis.4 In these cases, the elliptocytes usually comprise less than one third of the RBCs.35 An acquired defect in the gene for 4.1 may be found in myelodysplastic syndrome.36 In the homozygous or compound heterozygous states, the anemia is moderate to severe, the osmotic fragility is increased, and biochemical evidence of excessive hemolysis is present. The peripheral blood film in patients with the HPP phenotype shows extreme poikilocytosis with fragmentation, microspherocytosis, and elliptocytosis similar to that in patients with thermal burns (Figure 24-7A). The mean cell volume (MCV) is very low (50 to 65 fL) because of the RBC fragments.435 RBCs in the HPP phenotype show marked thermal sensitivity. After incubation of a blood sample at 41° C to 45° C, the RBCs fragment (Figure 24-7B).13 Normal RBCs do not fragment until reaching a temperature of 49° C. Thermal sensitivity is not specific for HPP, however; it also occurs in cases of HE with spectrin mutations.4,  13 RBCs with the HPP phenotype show a lower fluorescence than RBCs in HS when incubated with eosin-5′-maleimide and analyzed by flow cytometry (see earlier section on special tests for HS).37 Mutation screening using molecular tests or quantitation of membrane proteins by SDS-PAGE may be also be performed.

 
FIGURE 24-6 Red blood cell morphology in hereditary elliptocytosis (peripheral blood, ×1000).  Source:  (From Carr JH, Rodak BF: Clinical hematology atlas, ed 3, Philadelphia, 2009, Saunders.)

 
FIGURE 24-7 A, Morphology of red blood cells in hereditary pyropoikilocytosis before incubation. B, Morphology after 1 hour at 45° C (peripheral blood, ×500).

As in HS, patients with moderate or severe hemolytic anemia due to HE can develop cholelithiasis due to bilirubin gallstones, and hemolytic, aplastic, and megaloblastic crises can occur (see earlier section on HS complications).

Treatment.

Asymptomatic HE patients require no treatment. All HE patients who are significantly anemic and show signs of hemolysis respond well to splenectomy.4 Transfusions are occasionally needed for life-threatening anemia.

Hereditary ovalocytosis (southeast asian ovalocytosis).

Hereditary ovalocytosis or Southeast Asian ovalocytosis (SAO) is a condition caused by a mutation in the gene for band 3 that results in increased rigidity of the membrane and resistance to invasion by malaria.3,  4 It is common in the malaria belt of Southeast Asia, where its prevalence can reach 30%.13,  38 The inheritance pattern is autosomal dominant, and all patients identified are heterozygous.3,  4

Pathophysiology.

SAO is the result of one mutation, a deletion of 27 base pairs in SLC4A1, the gene that codes for band 3.39 Deletion occurs at the interface between the transmembrane and cytoplasmic domains.4,  40 The mutation causes an increase in membrane rigidity that may be due to tighter binding of band 3 to ankyrin or decreased lateral mobility of band 3 in the membrane.4,  13,  38RBC membranes also have decreased elasticity as measured by ektacytometry and micropipette aspiration.3,  38 The molecular mechanism responsible for the increased membrane rigidity that results from the band 3 mutation has not yet been elucidated.3,  4

Clinical and laboratory findings.

In patients with SAO, hemolysis is mild or absent. On a peripheral blood film, typical cells of SAO are oval RBCs with one to two transverse bars or ridges and usually comprise about 30% of the RBCs.3,  13 No treatment is required for this condition.

Mutations that alter membrane transport proteins

RBC volume is regulated by various membrane proteins that serve as passive transporters, active transporters, and ion channels. When RBCs lose the ability to regulate volume, the cells are prematurely hemolyzed. Cell volume is determined by the intracellular concentration of cations, particularly sodium.11 If the total cation content is increased, water enters the cell and increases the cell volume, forming a stomatocyte. If the total cation content is decreased, water leaves the cell, which decreases the cell volume and produces a dehydrated RBC, also called a xerocyte.

Hereditary stomatocytosis comprises a group of heterogeneous conditions in which the RBC membrane leaks monovalent cations. The two major categories are overhydrated hereditary stomatocytosis and dehydrated hereditary stomatocytosis or hereditary xerocytosis. Molecular characterization of these conditions is ongoing and should provide a better means of classification and clearer understanding of their pathophysiology.

Overhydrated hereditary stomatocytosis.

Overhydrated hereditary stomatocytosis (OHS) is a very rare hemolytic anemia due to a defect in membrane cation permeability that causes the RBCs to be overhydrated.3,  6 It is inherited in an autosomal dominant pattern.11,  13

Pathophysiology.

In OHS, the RBC membrane is excessively permeable to sodium and potassium at 37° C.41 There is an influx of sodium into the cell that exceeds the loss of potassium, which results in a net increase in the intracellular cation concentration. As a result, more water enters the cell, and the cell swells and becomes stomatocytic. Because of the water influx, the cytoplasm has decreased density and viscosity. The increase in cell volume without an increase in membrane surface area causes premature hemolysis in the spleen.3 The exact molecular defect is unknown, but mutations in the RHAG gene that codes for RhAG protein, along with a deficiency of the RhAG protein in the membrane, has been found in some patients with OHS.41,  42Most patients also have a secondary deficiency of stomatin, a transmembrane protein, but its gene is not mutated.6,  41,  42 Stomatin may participate in regulation of ion channels, but its role in OHS is unclear.41 The RhAG is a transmembrane protein and a component of the ankyrin complex.

Clinical and laboratory findings.

OHS can cause moderate to severe hemolytic anemia. The diagnostic features include 5% to 50% stomatocytes on the peripheral blood film (Figure 24-8), macrocytes (MCV of 110 to 150 fL), decreased MCHC (24 to 30 g/dL), reticulocytosis, reduced erythrocyte potassium concentration, elevated erythrocyte sodium concentration, and increased net cation content in the erythrocytes.3,  6 Because of the increased cell volume, the RBCs have increased osmotic fragility due to a decreased surface area–to–volume ratio.11 Splenectomy should be carefully considered in OHS because it is associated with an increased risk of thromboembolic complications.3

 
FIGURE 24-8 Red blood cell morphology in hereditary stomatocytosis (peripheral blood, ×1000).

Dehydrated hereditary stomatocytosis or hereditary xerocytosis.

Dehydrated hereditary stomatocytosis (DHS) or hereditary xerocytosis (HX) is an autosomal dominant hemolytic anemia due to a defect in membrane cation permeability that causes the RBCs to be dehydrated.3,  4 It is the most common form of stomatocytosis.4

Pathophysiology.

In DHS/HX, the RBC membrane is excessively permeable to potassium. The potassium leaks out of the cell, but this is not balanced by an increase in sodium. Because of the reduced intracellular cation concentration, water is lost from the cell.3,  4 It is due to mutations in the PIEZO1 gene that codes for the Piezo-type mechanosensitive ion channel component 1 protein in the RBC membrane.43,  44 Two mutations have been identified to date.44 The PIEZO1 protein combines with other proteins to form a pore in a channel to mediate cation transport.43 The mutations result in an increase in ion channel activity and an increase in cation transport.45

Clinical and laboratory findings.

Patients with DHS/HX generally have mild to moderate anemia, reticulocytosis, jaundice, and mild to moderate splenomegaly.4,  11 Fetal loss, hydrops fetalis, and neonatal hepatitis can also be features of DHS/HX.4 The RBCs are dehydrated, as evidenced by the elevated MCHC and decreased osmotic fragility.4,  11 The RBC morphology includes stomatocytes (usually fewer than 10%), target cells, burr cells, desiccated cells with spicules, and RBCs in which the hemoglobin appears to be puddled in discrete areas on the cell periphery.4,  11 Most patients with DHS/HX do not require treatment. Splenectomy does not improve the anemia and is contraindicated because it increases the risk of thromboembolic complications.3,  4

Other hereditary membrane defects with stomatocytes

Familial pseudohyperkalemia.

Familial pseudohyperkalemia (FP) is a rare disorder in which excessive potassium leaks out of the RBCs at room temperature in vitro but not at body temperature in vivo.6 CBC parameters are near the reference intervals, although some patients have a mild anemia.6,  29 Occasional stomatocytes may be observed on the peripheral blood film.6 The gene defect associated with this disorder has not yet been identified.

Cryohydrocytosis.

Cryohydrocytosis (CHC) is another rare disorder that manifests as a mild to moderate hemolytic anemia with leakage of sodium and potassium from the RBCs and stomatocytosis.6 The RBCs have marked cell swelling and hemolysis when stored at 4° C for 24 to 48 hours.29 Mutations in band 3 have been identified in some patients, which cause it to leak cations out of the RBCs, while other patients have a deficiency in membrane stomatin.4,  6,  29

Rh deficiency syndrome.

Rh deficiency syndrome comprises a group of rare hereditary conditions in which the expression of Rh membrane proteins is absent (Rh-null) or decreased (Rh-mod).4,  13 Cases of the syndrome can be genetically divided into the amorph type (caused by mutations in Rh proteins) and the regulatory type (caused by mutations in a protein that regulates Rh gene expression).4,  13 Patients with Rh deficiency syndrome present with mild to moderate hemolytic anemia. Stomatocytes and occasional spherocytes may be observed on the peripheral blood film. Symptomatic patients may be treated by splenectomy, which improves the anemia.

Other hereditary membrane defects with acanthocytes

Acanthocytes (spur cells) are small, dense RBCs with a few irregular projections that vary in width, length, and surface distribution (Figure 24-9). These are distinct from burr cells (echinocytes), which typically have small, uniform projections evenly distributed on the surface of the RBC (Chapter 19). The differentiation is easier to make on scanning electron micrographs and on wet preparations than on dried blood films.35,  36 A small number of burr cells (less than 3% of RBCs) can be present on the peripheral blood films of healthy individuals, while increased numbers of burr cells are observed in uremia and pyruvate kinase deficiency.13,  35 Acanthocytes, however, are not present on peripheral blood films of healthy individuals but can be found in hereditary neuroacanthocytosis (including abetalipoproteinemia), and acquired conditions such as spur cell anemia in severe liver disease (discussed below), myelodysplasia, malnutrition, and hypothyroidism.13

 
FIGURE 24-9 Two acanthocytes (arrows). Note that cells are dense with irregularly spaced projections of varying length (peripheral blood, ×1000).

Neuroacanthocytosis.

Neuroacanthocytosis is a term used to describe a group of rare inherited disorders characterized by neurologic impairment and acanthocytes on the peripheral blood film. Three disorders are provided as examples in this group: abetalipoproteinemia, McLeod syndrome, and chorea acanthocytosis.46

Abetalipoproteinemia (ABL) is a rare autosomal recessive disorder characterized by fat malabsorption, progressive ataxia, neuropathy, retinitis pigmentosa, and acanthocytosis.46 ABL can first manifest with steatorrhea and failure to thrive in young children (due to fat malabsorption) or as ataxia and neuropathy in young adults (due to decreased absorption of fat-soluble vitamin E).46ABL is caused by mutations in the MTP (microsomal triglyceride transfer protein) gene; MTP is needed to transfer and assemble lipids onto apolipoprotein B.46 The mutations result in an absence in the plasma of chylomicrons, very low-density lipoproteins (which transport triglycerides), and low-density lipoproteins (which transport cholesterol).13,  46Consequently, triglycerides and cholesterol are decreased, but sphingomyelin is increased in the plasma.13 Because RBC membrane lipids are in equilibrium with plasma lipids, the RBC membrane acquires increased sphingomyelin, which decreases the fluidity of the RBC membrane and results in the shape change. The shape defect is not present in developing nucleated RBCs or reticulocytes but progresses as RBCs age in the circulation.13 Other unknown mechanisms may also contribute to the formation of acanthocytes in ABL. Usually, 50% to 90% of the RBCs are acanthocytes.4,  13 Affected individuals have a mild hemolytic anemia, normal RBC indices, and normal to slightly elevated reticulocyte counts.13 Early treatment with high doses of vitamins A and E can reduce the neuropathy and retinopathy.13 Patients with other related disorders, such as hypobetalipoproteinemia due to mutations in the APOB gene, also may have acanthocytosis and neurologic disease.4,  46

The McLeod syndrome (MLS) is an X-linked disorder caused by mutations in the KX gene.47 KX codes for the Kx protein, a membrane precursor of the Kell blood group antigens.46 Men who lack Kx on their RBCs have reduced expression of Kell antigens, reduced RBC deformability, and shortened RBC survival.46 Patients with MLS have variable acanthocytosis (up to 85%), mild anemia, and late-onset (aged 40 to 60 years), slowly progressive chorea (movement disorders), peripheral neuropathy, myopathy, and neuropsychiatric manifestations.46,  47Some female heterozygote carriers may have acanthocytes, but neurologic symptoms are rare. Clinical manifestations in females depend on the proportion of RBCs with the normal X chromosome inactivated versus those with the mutant X chromosome inactivated.13,  47

Chorea acanthocytosis (ChAc) is a rare autosomal recessive disorder characterized by chorea, hyperkinesia, cognitive impairments, and neuropsychiatric symptoms.46 The mean age of onset of the neurologic symptoms is 35 years.46 In patients with ChAc, 5% to 50% of RBCs on the peripheral blood film are acanthocytes.46 ChAc is caused by mutations in VPS13A, a gene that codes for chorein, a protein with uncertain cellular functions.47Chorein may be involved in trafficking proteins to the cell membrane; consequently, a deficiency of chorein may lead to abnormal membrane protein structure and acanthocyte formation.46

Acquired red blood cell membrane abnormalities

Acquired stomatocytosis

Stomatocytosis occurs frequently as a drying artifact on Wright-stained peripheral blood films. A medical laboratory professional should examine many areas on several films before categorizing the result as stomatocytosis, because in true stomatocytosis such cells should be found in all areas of the blood film. In normal individuals, 3% to 5% of RBCs may be stomatocytes.4 In wet preparations in which RBCs are diluted in their own plasma and examined under phase microscopy, stomatocytes tend to be bowl shaped or uniconcave, rather than the normal biconcave shape. This technique can eliminate some of the artifactual stomatocytosis, but target cells also may appear bowl shaped in solution. Acute alcoholism and a wide variety of other conditions (such as malignancies and cardiovascular disease) and medications have been associated with acquired stomatocytosis.4,  13

Spur cell anemia

A small percentage of patients with severe liver disease develop a hemolytic anemia with acanthocytosis called spur cell anemia. The acanthocytes are due to a defect in the lipid component of the membrane. In severe liver disease, there is excess free cholesterol because of the presence of abnormal plasma lipoproteins. An equilibrium is sought between free cholesterol in the plasma and the RBC membrane cholesterol, and cholesterol preferentially accumulates in the outer leaflet of the membrane.4,  13 The spleen remodels the membrane into the acanthocyte shape.13 Acanthocytes have long, rigid projections and become entrapped and hemolyzed in the spleen, which results in a rapidly progressive anemia of moderate severity, splenomegaly, and jaundice.4,  13 Spur cell anemia in end-stage liver disease has a poor prognosis. The anemia may resolve, however, if the patient is able to undergo liver transplantation.4

Paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare chronic intravascular hemolytic anemia caused by an acquired clonal hematopoietic stem cell mutation that results in circulating blood cells that lack glycosylphosphatidylinositol (GPI)–anchored proteins, such as CD55 and CD59.48 The absence of CD55 and CD59 on the surface of the RBCs renders them susceptible to spontaneous lysis by complement. Because the mutation occurs in a hematopoietic stem cell, the defect is also found in platelets, granulocytes, monocytes, and lymphocytes.49 PNH is uncommon, with an annual incidence of two to five new cases per million persons in the United States.50

Pathophysiology of the hemolytic anemia.

The GPI anchor consists of a phosphatidylinositol (PI) molecule and a glycan core. The phosphatidylinositol is incorporated in the outer leaflet of the lipid bilayer. The glycan core consists of glucosamine, three mannose residues, and ethanolamine phosphate. At least 24 genes code for enzymes and proteins involved in the biosynthesis of the GPI anchor.50 GPI-anchored proteins attach to the ethanolamine in the glycan core by an amide bond at their C-termini (Figure 24-10).

 
FIGURE 24-10 Glycosylphosphatidylinositol (GPI) anchor for attachment of surface proteins to the cell membrane. Left: The structure of a GPI-anchored protein. The GPI anchor consists of phosphatidylinositol in the outer leaflet of the lipid bilayer, which is connected to a glycan core consisting of glucosamine (GlcN), three mannose (Man) residues, and ethanolamine phosphate (EtN). The protein is linked to the anchor at its C-terminus by an amide bond. The result is a surface protein with a fluid and mobile attachment to the cell surface. The GPI anchor and GPI-linked protein is extracellular. In paroxysmal nocturnal hemoglobinuria (PNH), a mutation occurs in the PIGA gene coding for phosphatidylinositol glycan class A (PIG-A), one of seven subunits of a glycosyl transferase enzyme needed to add N-acetylglucosamine to the inositol (Inos) component of the phosphatidylinositol molecule (see arrow for location of the PNH defect). The mutated PIG-A enzyme subunit inhibits or prevents the first step in the biosynthesis of the GPI anchor. Right: In contrast, a transmembrane protein has an extracellular domain, a short transmembrane domain, and an intracellular domain.  Source:  (Adapted from Ware RE, Rosse WF: Autoimmune hemolytic anemia. In Nathan DG, Orkin SH, editors: Nathan and Oski’s hematology of infancy and childhood, ed 5, Philadelphia, 1998, Saunders, p. 514.)

In PNH, a hematopoietic stem cell acquires a mutation in the PIGA gene that codes for phosphatidylinositol glycan class A (PIG-A), also known as phosphatidylinositol N-acetylglucosaminyltransferase subunit A.51 It is one of seven subunits of a glycosyl transferase enzyme needed to add N-acetylglucosamine to phosphatidylinositol. This is the first step in the biosynthesis of the GPI anchor in the endoplasmic reticulum membrane. The PIGA gene is located on the X chromosome, and over 180 different mutations have been identified.49Without a fully functional glycosyl transferase enzyme, the hematopoietic stem cell is unable to effectively synthesize the glycan core on phosphatidylinositol in the membrane; therefore, the cell is deficient in membrane GPI anchors. Without GPI anchors, all the progeny of the mutated stem cell are unable to express any of the approximately 16 currently known GPI-anchored proteins found on normal hematopoietic cells.49 The GPI-anchored proteins are complement regulators, enzymes, adhesion molecules, blood group antigens, or receptors.Relevant to the expression of the hemolysis in PNH, two GPI-anchored proteins are absent or deficient on the RBC membrane: decay-accelerating factor (DAF, or CD55) and membrane inhibitor of reactive lysis (MIRL, or CD59).50 CD55 and CD59 are complement-inhibiting proteins. CD55 inhibits the complement alternate pathway C3 and C5 convertases, and CD59 prevents the formation of the membrane attack complex.48 When CD55 and CD59 are absent from the RBC surface, the cell is unable to prevent the activation of complement, and spontaneous and chronic intravascular hemolysis occurs. Out of all the genes needed for GPI anchor synthesis, PIGA is the only one located on the X chromosome. Therefore, only one acquired mutation in a stem cell is needed for the PNH phenotype (males have only one X chromosome, and in females one of the X chromosomes is inactivated).48

The PIGA mutant clone coexists with normal hematopoietic stem cells and progenitors, which results in a population of RBCs that is GPI-deficient and a population that is normal.48Some patients have a greater expansion of the mutant clone, a higher percentage of circulating GPI-deficient RBCs, and a more severe chronic hemolytic anemia.48 On the other hand, other patients have minimal expansion of the mutant clone and have a lower percentage of circulating GPI-deficient RBCs. These patients may be asymptomatic and may not require any treatment. It is unclear why there is a greater expansion of the GPI-deficient clone and chronic hemolysis in some patients and not others.

Patients with PNH also display phenotypic mosaicism.48,  50 The mosaicism results when a single patient is able to harbor normal clones as well as mutant clones with different PIGAmutations. Those different mutations result in variable expression of CD55 and CD59 on the RBCs within an individual patient, giving rise to three RBC phenotypes: type I, type II, and type III.48,  49 Type I RBCs are phenotypically normal, express normal amounts of CD55 and CD59, and undergo little or no complement-mediated hemolysis. Type II RBCs are the result of a PIGA mutation that causes only a partial deficiency of CD55 and CD59, and these cells are relatively resistant to complement-mediated hemolysis. Type III RBCs are the result of a PIGAmutation that causes a complete deficiency of the GPI anchor, and therefore no CD55 and CD59 proteins are anchored to the RBC surface. Type III RBCs are highly sensitive to spontaneous lysis by complement. The most common RBC phenotype in PNH is a combination of type I and type III cells, while the second most common has all three types.48 When the severity of the hemolysis in PNH is being assessed, both the relative amount and the type of circulating RBCs are considered.

In addition to hemolysis, patients with PNH may have bone marrow dysfunction that contributes to the severity of the anemia. Many patients have a history of bone marrow failure caused by acquired aplastic anemia or myelodysplastic syndrome that precedes or coincides with the onset of PNH.52

Clinical manifestations.

The onset of PNH most frequently occurs in the third or fourth decade, but it can occur in childhood and advanced age.48-50 The major clinical manifestations and complications of PNH are those associated with hemolytic anemia, thrombosis, and bone marrow failure (Box 24-2).49 Anemia is mild to severe, depending on the predominant type of RBC, the degree of hemolysis, and the presence of bone marrow failure. Free hemoglobin released during intravascular hemolytic episodes rapidly scavenges and removes nitric oxide (NO). The decreased NO can manifest as esophageal spasms and dysphagia (difficulty swallowing), erectile dysfunction, abdominal pain, or platelet activation and thrombosis.49,  50 The most common thrombotic manifestation is hepatic vein thrombosis, which obstructs venous outflow from the liver (Budd-Chiari syndrome), a serious, often fatal complication.50

BOX 24-2

Major Clinical Manifestations and Complications of Paroxysmal Nocturnal Hemoglobinuria

Related to intravascular hemolysis

  • Anemia
  • Hemoglobinuria
  • Chronic renal failure
  • Cholelithiasis
  • Esophageal spasm, erectile dysfunction

Related to thrombosis

  • Venous thrombosis
  • Abdominal vein thrombosis: hepatic (Budd-Chiari syndrome), splenic, renal veins
  • Portal hypertension
  • Cerebral vein thrombosis
  • Retinal vein thrombosis and loss of vision
  • Deep vein thrombosis, pulmonary emboli
  • Arterial thrombosis (less common)
  • Stroke
  • Myocardial infarction

Related to bone marrow failure

  • Pancytopenia: fatigue, infections, bleeding
  • Myelodysplastic syndrome

Adapted from Bessler M, Hiken J: The pathophysiology of disease in patients with paroxysmal nocturnal hemoglobinuria, Hematology Am Soc Hematol Educ Program, pp. 104-110, 2008.

Laboratory findings.

Biochemical evidence of intravascular hemolysis includes hemoglobinemia, hemoglobinuria, decreased level of serum haptoglobin, increased levels of serum indirect bilirubin and lactate dehydrogenase, and hemosiderinuria (Chapter 23). Hemolysis can be exacerbated by conditions such as infections, strenuous exercise, and surgery.50 Hemoglobinuria is present in only 25% of patients at diagnosis, but it will occur in most patients during the course of the illness.52 Very few patients report periodic hemoglobinuria at night, a symptom for which the condition was originally named.50 Hemosiderinuria due to chronic intravascular hemolysis (Chapter 23) may be detected with Prussian blue staining of the urine sediment.

Reticulocyte counts are mildly to moderately increased, with less elevation than would be expected in other hemolytic anemias of comparable severity. The MCV may be slightly elevated due to the reticulocytosis. The direct antiglobulin test (DAT) is negative. If the patient does not receive transfusions, iron deficiency develops due to the loss of hemoglobin iron in the urine, and the RBCs become microcytic and hypochromic. Serum iron studies (serum iron, total iron-binding capacity, and serum ferritin) are performed to detect iron deficiency (Chapter 20). Folate deficiency often occurs if there is chronic erythroid hyperplasia and a greater need for folate, which leads to secondary macrocytosis. Pancytopenia may occur if there is concomitant bone marrow failure.

The bone marrow aspirate and biopsy specimen are examined for evidence of an underlying bone marrow failure syndrome, abnormal cells, and cytogenetic abnormalities.52 The bone marrow may be normocellular to hypercellular with erythroid hyperplasia in response to the hemolysis, or it may be hypocellular in concomitant bone marrow failure.50 A finding of dysplasia or certain chromosome abnormalities is helpful in diagnosis of a myelodysplastic syndrome (Chapter 34). An abnormal karyotype is found in 20% of patients with PNH.50

Confirmation of PNH requires demonstration of GPI-deficient cells in the peripheral blood. Flow cytometric analysis (Chapter 32) of RBCs with fluorescence-labeled anti-CD59 determines the proportion of types I, II, and III RBCs, and thus can provide an assessment of the severity of the hemolysis (Figure 24-11, and Figure 32-17).48,  50 Type I cells with normal expression of CD59 show the highest intensity level of fluorescence; type II cells with a partial deficiency of CD59 show moderate fluorescence; and type III cells with no CD59 are negative for fluorescence. Patients with a greater proportion of type III cells (complete deficiency of GPI-anchored proteins) are expected to have a high-grade hemolysis.48,  52 Patients with a high percentage of type II cells (partial deficiency of GPI-anchored proteins) and a low percentage of type III cells may have only modest hemolysis.48-52 A high-sensitivity two-parameter flow cytometry method using labeled anti-CD59 and anti-CD235a (anti-glycophorin A) was able to detect type III PNH RBCs with a sensitivity of 0.002% (1 in 50,000 normal cells).53However, flow cytometry methods to detect PNH RBCs have two major disadvantages. They underestimate the percentage of type III cells be cause RBCs lacking CD59 undergo rapid complement lysis in the circulation.52 In addition, these methods cannot accurately determine the percentage of PNH RBCs after recent transfusion.

 
FIGURE 24-11 Flow cytometric analysis of peripheral blood cells from a patient with paroxysmal nocturnal hemoglobinuria (PNH). A, Fluorescence intensity of erythrocytes from a healthy control participant after staining with anti-CD59. B, Fluorescence intensity of erythrocytes from an untransfused PNH patient after staining with anti-CD59. Type II cells are “blended” between the type I (normal) and type III cells. C, Fluorescence intensity of granulocytes from a healthy control participant stained with FLAER. D, Fluorescence intensity of granulocytes from the same PNH patient as in B after staining with FLAER. Note that the granulocytes are almost exclusively type III cells. A small population of type I granulocytes is present. FITC, Fluorescein isothiocyanate; FLAER, fluorescein-labeled proaerolysin variant.  Source:  (From Brodsky RA: Paroxysmal nocturnal hemoglobinuria. In Hoffman R, Benz EJ, Jr., Silberstein LE, et al, editors: Hematology: basic principles and practice, ed 6, Philadelphia, 2013, Saunders, an imprint of Elsevier. Figure 29-2, p. 375.)

Because of the inherent problems with flow cytometry methods to detect PNH RBCs, diagnosis of PNH is accomplished by detection of the absence of GPI-anchored proteins on WBCs using multiparameter flow cytometry. The absence of at least two GPI-anchored proteins in two WBC lineages (usually granulocytes and monocytes) is recommended for greater diagnostic accuracy.50,  53 Methods typically use fluorescent monoclonal antibodies to GPI-anchored proteins (such as CD59, CD55, CD24, CD16, CD66b, CD14), along with lineage-specific antibodies to non-GPI-anchored proteins (CD15 and CD33 or CD64) to identify granulocytes and monocytes, respectively.50,  53,  54 An alternative flow cytometric method uses a fluorescein-labeled proaerolysin variant (FLAER). FLAER binds directly to the glycan core of the GPI anchor with a high signal-to-noise ratio. The absence of binding on granulocytes and/or monocytes is indicative of GPI deficiency (Figure 24-11). Using multiparameter flow cytometry with FLAER in combination with monoclonal antibodies to GPI-anchored antigens and lineage-specific antigens increases sensitivity and specificity for detection of GPI-deficient granulocytes and monocytes.53,  54 A high-sensitivity four-color protocol using FLAER, CD24, CD15, and CD45 for granulocytes and FLAER, CD14, CD64, and CD45 for monocytes was able to detect GPI-deficient granulocytes and monocytes with a sensitivity of 0.01% (1 in 10,000) and 0.04% (1 in 2500), respectively.53 The results are not affected by recent transfusions, since donor neutrophils and monocytes have a short life span in stored blood; therefore, it can reliably be used to estimate the percentage of GPI-deficient granulocytes and monocytes in recently transfused patients.50 The high sensitivity is also important for posttherapy monitoring of PNH clones.

The sugar water test (sucrose hemolysis test) and the Ham test (acidified serum lysis test) have insufficient sensitivity for diagnosis of PNH and have been replaced by flow cytometric techniques.

Classification.

The International PNH Interest Group proposed three subcategories of PNH: classic PNH, PNH in the setting of another specified bone marrow disorder, and subclinical PNH.48,  52 In classic PNH, there is clinical and biochemical evidence of intravascular hemolysis, reticulocytosis, a cellular bone marrow with erythroid hyperplasia and normal morphology, and a normal karyotype. In addition, more than 50% of the circulating neutrophils are GPI-deficient.52 In PNH in the setting of another bone marrow disorder, patients have evidence of hemolysis but have a history of or concomitant aplastic anemia, myeloproliferative disorder, or other myelopathy. The number of GPI-deficient neutrophils is variable but is usually less than 30% of the total neutrophils. In subclinical PNH, patients have no clinical or biochemical evidence of hemolysis but have a small subpopulation of GPI-deficient neutrophils that comprise less than 1% of the total circulating neutrophils.52 This subcategory is found in association with bone marrow failure syndromes. Features of each subgroup are summarized inTable 24-3.

TABLE 24-3

Classification of Paroxysmal Nocturnal Hemoglobinuria (PNH)

* Subclassification proposed by the International PNH Interest Group.48,52

† Bone marrow failure syndromes include aplastic anemia, refractory anemia/myelodysplastic syndrome, and other myelopathy (e.g., myelofibrosis).

‡ Determined by high-sensitivity flow cytometric analysis.

Treatment.

In 2007, the U.S. Food and Drug Administration approved eculizumab (Soliris®) for the treatment of hemolysis in PNH.56 Eculizumab is a humanized monoclonal antibody that binds to complement C5, prevents its cleavage to C5a and C5b, and thus inhibits the formation of the membrane attack unit.56 Eculizumab is the treatment of choice for patients with classic PNH. It results in an improvement of the anemia and a decrease in transfusion requirements.56,  57 There is also a reduction in the lactate dehydrogenase level in the serum, reflecting a reduction in hemolysis.56,  57 In a study by Hillman and colleagues (2013) of 195 patients taking eculizumab for a duration of 30 to 66 months, 96.4% of patients did not have an episode of thrombosis, and in 93%, markers of their chronic kidney disease stabilized or even improved.57 Because of the inhibition of the complement system, patients taking eculizumab have an increased risk for infections with Neisseria meningitidis and need to be vaccinated prior to administration of the drug.56 Patients continue to have a mild to moderate anemia and reticulocytosis likely due to extravascular hemolysis of RBCs sensitized with C3 (eculizumab does not inhibit complement C3).48,  58 To address this issue, research is under way to identify therapies that target theearly events of complement activation, including monoclonal antibodies to C3, but a universal inhibition of C3 may increase the patient’s susceptibility to infections and immune complex disease.58 A promising new therapy under investigation is a novel recombinant fusion protein (TT30) designed to prevent the formation of C3 convertase only on the membranes of GPI-deficient RBCs.58

Eculizumab is not curative and does not address the bone marrow failure complications of PNH. Other treatments for PNH are mainly supportive. Iron therapy is given to help alleviate the iron deficiency caused by the urinary loss of hemoglobin, and folate supplementation is given to replace the folate consumed in accelerated erythropoiesis. Administration of androgens and glucocorticoids to ameliorate anemia is not universally accepted.48 Anticoagulants are used in the treatment of thrombotic complications. In suitable patients with severe intravascular hemolysis, hematopoietic stem cell transplantation with an HLA-matched sibling donor may be an option and can be a curative therapy, but with an overall survival of only 50% to 60%.48

PNH is a disease with significant morbidity and mortality. Prior to eculizumab, thrombosis was the major cause of death, and the median survival after diagnosis was approximately 10 years.48 Long-term studies of patients on eculizumab therapy are in progress, and early results show a decrease in the debilitating complications and an increase in survival of patients with PNH.57

Red blood cell enzymopathies

The major function of the RBCs is to transport oxygen to the tissues over their life span of 120 days. For the RBCs to do that effectively, they need functional enzymes to maintain glycolysis, preserve the shape and deformability of the cell membrane, keep hemoglobin iron in a reduced state, protect hemoglobin and other cellular proteins from oxidative denaturation, and degrade and salvage nucleotides. Deficiencies in RBC enzymes may impair these functions to varying degrees and decrease the life span of the cell. The most important metabolic pathways are the Embden-Meyerhof pathway (anaerobic glycolysis) and the hexose monophosphate (pentose) shunt59 (Figure 9-1). The most commonly encountered enzymopathies are deficiencies of glucose-6-phosphate dehydrogenase and pyruvate kinase. Other RBC enzymopathies are rare.59

Glucose-6-phosphate dehydrogenase deficiency

RBCs normally produce free oxygen radicals (O2) and hydrogen peroxide (H2O2) during metabolism and oxygen transport, but they have multiple mechanisms to detoxify these oxidants (Chapter 9). Occasionally RBCs are subjected to an increased level of oxidants (oxidant stress) due to exposure to certain oxidizing drugs, foods, chemicals, herbal supplements, and even through reactive oxygen molecules produced in the body during infections. If allowed to accumulate in the RBCs, these reactive oxygen species would oxidize and denature hemoglobin, membrane proteins and lipids, and ultimately cause premature hemolysis. Therefore, the RBCs’ capacity to detoxify oxidants, especially during oxidant stress, is critical to maintain their normal life span.

Glucose-6-phosphate dehydrogenase (G6PD) is one of the important intracellular enzymes needed to protect hemoglobin and other cellular proteins and lipids from oxidative denaturation. G6PD catalyzes the first step in a series of reactions that detoxify hydrogen peroxide formed from oxygen radicals (Figure 24-12). In the hexose monophosphate shunt (Chapter 9), G6PD generates reduced nicotinamide adenine dinucleotide phosphate (NADPH) by converting glucose-6-phosphate to 6-phosphogluconate. In the next step, glutathione reductase uses the NADPH to reduce oxidized glutathione (GSSG) to reduced glutathione (GSH) and NADP. In the final reaction, glutathione peroxidase uses the GSH generated in the previous step to detoxify hydrogen peroxide to water (H2O). GSSG is formed in the reaction and is rapidly transported out of the cell. During oxidant stress, RBCs with normal G6PD activity are able to readily detoxify hydrogen peroxide to prevent cellular damage and safeguard hemoglobin. G6PD is especially critical to the cell because it provides the only means of generating NADPH. Consequently, G6PD-deficient RBCs are particularly vulnerable to oxidative damage and subsequent hemolysis during oxidant stress.

 
FIGURE 24-12 Function of glucose-6-phosphate dehydrogenase (G6PD) in generating the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) and reduced glutathione. G6PD converts glucose-6-phosphate (from the Embden-Meyerhof pathway) to 6-phosphogluconate. In the reaction, oxidized NADP is reduced to NADPH. Glutathione reductase uses the NADPH to reduce oxidized glutathione (GSSG) to reduced glutathione (GSH). Glutathione peroxidase uses the GSH to detoxify hydrogen peroxide to water.

The G6PD gene is located on the X chromosome. It codes for the G6PD enzyme, which assembles into a dimer and tetramer in its functional configuration.59 With the X-linked inheritance pattern, men can be normal hemizygotes (have the normal allele) or deficient hemizygotes (have a mutant allele). Women can be normal homozygotes (both alleles normal), deficient homozygotes (both alleles have same mutation), compound heterozygotes (each allele has a different mutation), or heterozygotes (have one normal allele and one mutant allele). The G6PD enzyme activity in female heterozygotes lies between normal and deficient due to the random inactivation of one of the X chromosomes in each cell (lyonization).59-61 Therefore, the RBCs of female heterozygotes are a mosaic, with some cells having normal G6PD activity and some cells having deficient G6PD activity. Because the X inactivation is random, the proportion of normal to G6PD-deficient RBCs varies among different heterozygous women.61 Some heterozygous women experience acute hemolytic episodes after exposure to oxidants if they have a high proportion of G6PD-deficient RBCs.

G6PD deficiency is the most common RBC enzyme defect, with a prevalence of 5% of the global population, or approximately 400 million people worldwide.62 The prevalence of G6PD deficiency varies by geographic location: sub-Saharan Africa (7.5%), the Middle East (6.0%), Asia (4.7%), Europe (3.9%), and the Americas (3.4%).62 In the United States, the prevalence of G6PD deficiency in African American males is approximately 10%.59 G6PD deficiency has the highest prevalence in geographic areas in which malaria is endemic because of the selective pressure of malaria.63 Studies in Africa show that G6PD deficiency (A− variant) in hemizygous males confers protection against life-threatening Plasmodium falciparum malaria.64 This protective effect is not observed in heterozygous females because of their mosaicism of normal and G6PD-deficient RBCs.64 In a 2010 case-control study in Pakistan, G6PD deficiency (Mediterranean variant) conferred protection against Plasmodium vivax infection, also with a greater protective effect in hemizygous males.65 This protective effect may be due to parasite susceptibility to excess free oxygen radicals produced in G6PD-deficient RBCs.66 In addition, significant oxidative damage may occur to the RBCs early after parasite invasion so that these early-infected cells are more readily phagocytized with elimination of the parasite.67

A mutation database published in 2012 reported 186 known mutations in the G6PD gene, with over 85% of them being single missense mutations.68An amino acid substitution changes the structure of the enzyme and thus affects its function, stability, or both. In addition, more than 400 variant isoenzymes have been identified.61 The normal or wildtype G6PD variant is designated G6PD-B.59,  61 Some G6PD variants have significantly reduced enzyme activity, while others have mild or moderately reduced activity or normal activity. The different variants of G6PD have been divided into classes by the World Health Organization, based on clinical symptoms and amount of enzyme activity (Table 24-4).68,  69

TABLE 24-4

Classification of Glucose-6-Phosphate Dehydrogenase Variants by the World Health Organization

From Beutler E, Gaetani G, der Kaloustian V et al. World Health Organization (WHO) Working Group. Glucose-6-phosphate dehydrogenase deficiency. Bull World Health Organ 67:601-611, 1989; and Minucci A, Moradkhani K, Hwang MJ, et al. Glucose-6-phosphate dehydrogenase (G6PD) mutations database: Review of the “old” and update of the new mutations. Blood Cells Mol Dis 48:154-165, 2012.

Pathophysiology

G6PD-deficient RBCs cannot generate sufficient NADPH to reduce glutathione and thus cannot effectively detoxify the hydrogen peroxide produced upon exposure to oxidative stress. Oxidative damage to cellular proteins and lipids occurs, particularly affecting hemoglobin and the cell membrane. Oxidation converts hemoglobin to methemoglobin and forms sulfhydryl groups and disulfide bridges in hemoglobin polypeptides. This leads to decreased hemoglobin solubility and precipitation as Heinz bodies.59 Heinz bodies adhere to the inner RBC membrane, causing irreversible membrane damage (Figure 14-11). Because of the membrane damage and loss of deformability, RBCs with Heinz bodies are rapidly removed from the circulation by intravascular and extravascular hemolysis.61 Reticulocytes have approximately five times more G6PD activity than older RBCs, because enzyme activity decreases as the cells age. Therefore, during exposure to oxidants, the older RBCs with less G6PD are preferentially hemolyzed.63

Clinical manifestations

The vast majority of individuals with G6PD deficiency are asymptomatic throughout their lives. However, some patients have clinical manifestations. The clinical syndromes are acute hemolytic anemia, neonatal jaundice (hyperbilirubinemia), and chronic hereditary nonspherocytic hemolytic anemia (HNSHA).63

Acute hemolytic anemia.

Oxidative stress can precipitate a hemolytic episode, and the main triggers are certain oxidizing drugs or chemicals, infections, and ingestion of fava beans. Hemolysis secondary to drug exposure is the classic manifestation of G6PD deficiency. The actual discovery of G6PD deficiency in the 1950s was a direct consequence of investigations into the development of hemolysis in certain individuals after ingestion of the antimalarial agent primaquine.59Box 24-3 lists drugs that show strong evidence-based association with hemolysis in G6PD-deficient individuals or have been reported in well-documented case reports.70,  71 The degree of hemolysis can vary, depending on the dosage, coexisting infection, concomitant use of other drugs, or type of mutation.70 Exposure to naphthalene in mothballs and some herbal supplements have also been associated with hemolysis in some G6PD-deficient individuals.59

BOX 24-3

Drugs Causing Predictable Hemolysis in Glucose-6-Phosphate Dehydrogenase Deficiency

Drugs with strong evidence-based support for an association with drug-induced hemolysis:1

Dapsone

Methylthioninium chloride (methylene blue)

Nitrofurantoin

Phenazopyridine

Primaquine

Rasburicase

Tolonium chloride (toluidine blue)

Drugs with well-documented case reports for an association with drug-induced hemolysis:2

Cotrimoxazole

Quinolones

Sulfadiazine

1Youngster I, Arcavi L, Schechmaster R, et al: Medications and glucose-6-phosphate dehydrogenase deficiency: an evidence-based review, Drug Saf 33:713-726, 2010.

2Luzzatto L, Seneca E. G6PD deficiency: a classic example of pharmacogenetics with on-going clinical implications. Br J Haematol 164:469-480, 2014.

Individuals with classes II and III G6PD deficiency are clinically and hematologically normal until the offending drug is taken. Clinical hemolysis can begin abruptly within hours or occur gradually 1 to 3 days after the drug is taken.61,  63 Typical symptoms include chills, fever, headache, nausea, and back pain.59 A rapid drop in hemoglobin may occur, and the anemia can range from mild to very severe. Hemoglobinuria is a usual finding and indicates that the hemolysis is intravascular, although some extravascular hemolysis occurs. Reticulocytes increase within 4 to 6 days.61 Generally, with class III variants such as G6PD-A, the hemolytic episode is self-limiting because the newly formed reticulocytes have higher G6PD activity. With class II variants such as G6PD-Mediterranean, the RBCs are severely G6PD-deficient, so the hemolytic episode may be longer and the hemolysis is not self-limiting.61

Infection is probably the most common cause of hemolysis in individuals with G6PD deficiency. During the episode, the hemoglobin can drop 3 to 4 g/dL if reticulocyte production is suppressed by the infection.59 The hemolysis resolves after recovery from the infection.61 The mechanism of hemolysis induced by acute and subacute infection is poorly understood, but the generation of hydrogen peroxide by phagocytizing leukocytes may play a role.61 Diminished liver function may contribute further to the oxidant stress by allowing the accumulation of oxidizing metabolites. Infectious agents implicated in hemolytic episodes include bacteria, viruses, and rickettsia.

Favism is a rare, severe hemolytic episode that occurs in some G6PD-deficient individuals after ingestion of fava beans. Favism can initially manifest with a sudden onset of acute intravascular hemolysis within hours of ingesting fava beans, or hemolysis can occur gradually over a period of 24 to 48 hours.61 Hemoglobinuria is one of the first signs. Specific patient factors may affect the severity of the hemolysis, including the type of mutation, presence of underlying disorders, and the amount of fava beans ingested. Only a small percentage of G6PD-deficient individuals manifest favism, and most of these have the G6PD-Mediterranean variant.

Neonatal hyperbilirubinemia.

Neonatal hyperbilirubinemia is associated with G6PD deficiency. Jaundice generally appears 2 to 3 days after birth without concomitant anemia.63 The jaundice is mainly attributed to inefficient conjugation of indirect bilirubin by the liver rather than to excessive hemolysis.72 These neonates must be closely monitored because the hyperbilirubinemia can be severe and cause bilirubin encephalopathy (kernicterus) and permanent brain damage.61 Severe hyperbilirubinemia occurs more often in infants who, in addition to a mutated G6PD gene, are homozygous for a mutation in the promoter region of the bilirubin-uridine diphosphoglucuronate glucuronosyltransferase 1 (UGT1A1) gene, which impairs their ability to conjugate and excrete indirect bilirubin.61 In addition, the particular G6PD variant and environmental factors (such as drugs given to the mother or infant, the presence of infection, and gestational age) probably play an important role in the occurrence of neonatal hyperbilirubinemia, because a wide variation exists in frequency and severity in different populations with G6PD deficiency.

Chronic hereditary nonspherocytic hemolytic anemia.

A small percentage of G6PD-deficient patients have chronic HNSHA, as evidenced by persistent hyperbilirubinemia, decreased serum haptoglobin level, and increased serum lactate dehydrogenase level. Most of these patients are diagnosed at birth as having neonatal hyperbilirubinemia, and the hemolysis continues into adulthood. They usually do not have hemoglobinuria, which suggests that the ongoing hemolysis is extravascular as opposed to intravascular. The RBC morphology is unremarkable. These patients also are vulnerable to acute oxidative stress from the same agents as those affecting other G6PD-deficient individuals and may have acute episodes of hemoglobinuria. The severity of HNSHA is extremely variable, likely related to the type of mutation in the G6PD gene.

Laboratory findings

General tests for hemolytic anemia.

The anemia occurring during a hemolytic crisis may range from moderate to extremely severe and is usually normocytic and normochromic. The morphology of G6PD-deficient RBCs is normal except during a hemolytic episode. The degree of change in morphology during a hemolytic episode varies, depending on the severity of the hemolysis. In some patients, the change is not striking, but in other individuals with severe variants, marked anisocytosis, poikilocytosis, spherocytosis, and schistocytosis may occur.61 Bite cells (RBCs in which the margin appears indented and the hemoglobin is concentrated) may be observed in rare cases of drug-induced hemolysis but should not be considered a specific feature of G6PD deficiency.61,  73 Bite cells are absent in acute and chronic hemolytic states associated with common G6PD-deficient variants, and they can also be found in other conditions.61,  73 Heinz bodies cannot be detected with Wright staining. They can be visualized with supravital stains, such as crystal violet, as dark purple inclusions attached to the inner RBC membrane (Figure 14-11). The reticulocyte count is increased and may reach 30% of RBCs. Consistent with intravascular hemolysis, the serum haptoglobin level is severely decreased, the serum lactate dehydrogenase activity is elevated, and there is hemoglobinemia and hemoglobinuria. The indirect bilirubin level is also elevated. The white blood cell (WBC) count is moderately elevated, and the platelet count varies. Importantly, the direct antiglobulin test (DAT) is negative, indicating that an immune cause of the hemolysis is unlikely (Chapter 26). Table 24-5contains a summary of the clinical and laboratory findings in G6PD deficiency during an acute hemolytic episode.

TABLE 24-5

Typical Clinical and Laboratory Findings in Glucose-6-Phosphate Dehydrogenase Deficiency During Acute Hemolytic Episode

History

Recent infection, administration of drugs associated with hemolysis, or ingestion of fava beans

Clinical manifestations

Chills, fever, headache, nausea, back pain, abdominal pain

Jaundice

Dark urine

Complete blood count results

Hemoglobin (moderate to severe)

 

Reticulocyte count

Peripheral blood film findings

Polychromasia

 

RBC morphology varies from normal to marked anisocytosis, poikilocytosis, spherocytosis, or schistocytosis, depending on severity

Direct antiglobulin test result

Negative

Indicators of hemolysis

Serum haptoglobin (severe)

Serum lactate dehydrogenase

Serum indirect bilirubin

Hemoglobinemia

Hemoglobinuria

Selected additional tests

G6PD activity (mild to severe); may be falsely normal due to reticulocytosis, leukocytosis, thrombocytosis, and in individuals with mild deficiencies 
DNA-based mutation detection usually needed to identify heterozygous females 
Heinz bodies observed on supravital stain

↓, Decreased; ↑, increased; G6PD, glucose-6-phosphate dehydrogenase; RBC, red blood cell.

Tests for G6PD deficiency.

The two major categories of tests for G6PD deficiency are quantitative and qualitative biochemical assays for G6PD activity (phenotypic assays) and DNA-based molecular tests for mutation detection (genotypic assays). Quantitative spectrophotometric assays are the gold standard to determine G6PD activity, make a definitive diagnosis, and assess the severity of the deficiency.59,  74 The assays are based on the direct measurement of NADPH generated by the patient’s G6PD in the reaction shown in Figure 24-13. The assays require venous blood collected in heparin or ethylenediaminetetraacetic acid (EDTA) anticoagulant. A hemolysate is prepared and incubated with the substrate/cofactor (glucose-6-phosphate/NADP) reagent. The rate of NADPH formation is proportional to G6PD activity and is measured as an increase in absorbance at 340 nm using a spectrophotometer.59,  74 The activity is typically reported as a ratio of the units of G6PD activity per gram of hemoglobin (IU/g Hb), so a standard hemoglobin assay must be done on the same specimen used for the G6PD assay. Cutoff points to determine G6PD deficiency are usually set at less than 20% of normal activity (usually less than 4.0 IU/g Hb), but this varies by method, laboratory, and population screened.71

 
FIGURE 24-13 Principle of the glucose-6-phosphate dehydrogenase (G6PD) activity assay. G6PD (in patient’s hemolysate) converts glucose-6-phosphate to 6-phosphogluconate with the conversion of oxidized nicotinamide adenine dinucleotide phosphate (NADP) (not fluorescent) to the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) (fluorescent). In the quantitative assay, the rate of production of NADPH is proportional to G6PD activity and is measured as an increase in absorbance at 340 nm using a spectrophotometer. In the qualitative assay, the appearance of a fluorescent spot under ultraviolet light when the reaction mixture is spotted on filter paper indicates normal G6PD activity.

Qualitative tests are designed as rapid screening tools to distinguish normal from G6PD-deficient patients. G6PD deficiency is defined by various methods as less than 20% to 50% of normal G6PD activity.71,  74,  75 Similar to quantitative assays, these tests also incubate a lysate of heparin or EDTA-anticoagulated blood with a glucose-6-phosphate/NADP reagent to generate NADPH (Figure 24-13). The endpoint in qualitative tests, however, is visually observed, and the results are reported as “G6PD-deficient” or “normal.” Qualitative tests with deficient or intermediate results are reflexed to the quantitative assay for verification of the G6PD deficiency.

The fluorescent spot test is based on the principle that the NADPH generated in the reaction is fluorescent, while the NADP in the reagent is not fluorescent. Blood and glucose-6-phosphate/NADP reagent are incubated and spotted on filter paper in timed intervals. Specimens with normal G6PD activity appear as moderate to strong fluorescent spots under long-wave ultraviolet (UV) light; specimens with decreased or no activity do not fluoresce or display weak fluorescence compared to a normal control.

Dye-reduction qualitative assays use the same G6PD enzymatic reaction but have a second step in which the NADPH reduces a dye, giving a visually observed color change. An example is BinaxNOW® (Alere/Inverness Medical, Waltham, MA), which is a handheld device that uses the enzyme chromographic test (ECT) method.76 Hemolysate is applied to one section of a lateral flow test strip in the device. The specimen migrates to the reaction pad of the strip containing the glucose-6-phosphate/NADP substrate/cofactor and a nitrobluetetrazolium dye.76 If the specimen has normal G6PD activity, the NADPH generated reduces the dye to a formazan product that is visually observed as a brown-black color on the reaction pad.76 This method has a sensitivity of 98% in detecting deficient specimens with G6PD activity less than 4.0 U/g Hb.76 In another formazan-based test, the NADPH reduces a tetrazolium monosodium salt (WST-8) substrate, forming a formazan orange-colored product.75 The dye-reduction methods have an advantage, since they do not need a UV light for visualization. A disadvantage of the BinaxNOW® method, however, is its higher cost compared to other assays. Rapid, point-of-care screening methods that do not require a venipuncture specimen are in development.

Biochemical qualitative screening tests are reliable to identify hemizygous males and homozygous or compound heterozygous mutant females with severe deficiency (less than 20% of normal activity), but lack the sensitivity to detect mild and moderate deficiencies found in some class III G6PD deficiency and in heterozygous females. They also have subjective endpoints that may affect test reproducibility and accuracy.

Phenotypic assays for G6PD activity have several additional limitations. As covered earlier in the chapter, reticulocytes have higher G6PD activity compared to mature RBCs. Reticulocytosis typically occurs as a response to an acute hemolytic episode and will falsely increase the patient’s G6PD activity over baseline values.59,  74 Patients who are not G6PD deficient are expected to have high G6PD activity during hemolytic episodes. When a patient with reticulocytosis has an unexpectedly normal G6PD activity result, G6PD deficiency should be suspected. To avoid falsely elevated or falsely normal results, biochemical assays for G6PD activity should not be performed during acute hemolysis. The testing should be performed after the reticulocyte and total RBC counts have returned to baseline, which may take 2 weeks to 2 months after the hemolytic episode.71,  74

Another limitation of phenotypic assays is that testing cannot be done after recent transfusion because the mixture of donor and patient RBCs does not reflect the patient’s true G6PD activity. WBCs and platelets also contain G6PD activity. In cases of severe anemia, or in severe leukocytosis or thrombocytosis, the buffy coat should be removed from the specimen before preparing the hemolysate for testing.74

DNA-based mutation detection (genotyping) is available in larger hospital and reference laboratories. Typically, the DNA is extracted from WBCs isolated from whole blood. Since the vast majority of G6PD mutations involve single nucleotide substitutions, molecular testing is straightforward. Although there are approximately 186 known mutations, rapid PCR-based methods can be used that target specific mutations with high prevalence in a particular geographic area, racial group, or ethnic group.74 If targeted mutation testing is negative, whole gene DNA sequencing is required.74

DNA-based testing is best suited for prenatal testing, family studies, and identification of heterozygous females who typically have indeterminate or normal biochemical (phenotypic) tests owing to their mosaicism of normal and G6PD-deficient cells.59,  74 DNA-based methods can be done on patients who were recently transfused because donor WBCs have a short half-life in stored blood and will not interfere with the test. In addition, DNA-based tests are not affected by reticulocytosis and can be done during an acute hemolytic episode.

The disadvantage of DNA-based methods is the requirement for technical expertise and specialized equipment. In addition, knowing the genotype of heterozygous females does not predict the clinical phenotype in terms of the proportion of normal and G6PD-deficient cells.71

An additional application of tests for G6PD deficiency is screening asymptomatic patients prior to prescribing the drugs listed in Box 24-3. Screening is recommended for patients with a family history of G6PD deficiency or for those who are members of ethnic/racial groups with a high prevalence of G6PD variants.

Treatment

Treatment for G6PD-deficient patients with acute hemolysis begins with discontinuing drugs associated with hemolysis. Most hemolytic episodes, especially in individuals with G6PD-A, are self-limited. In patients with more severe types, such as G6PD-Mediterranean, RBC transfusions may be required. Screening is important in populations that have a high incidence of G6PD deficiency. The prevention of acute hemolytic anemia is difficult because multiple causes exist; however, some cases of acute hemolytic anemia are easily preventable, such as by avoidance of fava bean consumption in families in which this sensitivity exists. Neonates in high risk populations should be screened for hyperbilirubinemia associated with G6PD deficiency, and treated immediately to prevent kernicterus and permanent brain damage.

Favism is a relatively dangerous manifestation and potentially fatal in individuals who do not have access to appropriate medical facilities and a transfusion service. Prevention of drug-induced disease is possible by choosing alternate drugs when possible. In cases in which the offending drugs must be used, especially in individuals with G6PD-A, the dosage canbe lowered to decrease the hemolysis to a manageable level. Infection-induced hemolysis is more difficult to prevent but can be detected early in the course of the episode and treated if necessary. Most episodes resolve without treatment but may be severe enough to warrant RBC transfusion. In patients with hemoglobin levels greater than 9 g/dL with persistent hemoglobinuria, close monitoring is important. Neonates with moderate hyperbilirubinemia and jaundice secondary to G6PD deficiency can be treated with phototherapy, but those with severe hyperbilirubinemia may require exchange transfusion.59

Pyruvate kinase deficiency

Pyruvate kinase (PK) is a rate-limiting key enzyme of the glycolytic pathway of RBCs. It catalyzes the conversion of phosphoenolpyruvate to pyruvate, forming ATP (Figure 9-1). PK deficiency is an autosomal recessive disorder, with an estimated prevalence of 1 per 20,000 in the white population.77 It is the most common form of hereditary nonspherocytic hemolytic anemia and is found worldwide.59,  61 PK deficiency is due to a mutation in the PKLR gene that codes for PK in red blood cells and hepatocytes. Over 180 mutations (predominantly missense) have been reported.78 Symptomatic hemolytic anemia occurs in homozygotes or compound heterozygotes. Certain mutations are more common in the United States, parts of Europe, and Asia.59 There is a high prevalence of PK-deficient homozygotes with the same point mutations in two isolated, consanguineous communities in the United States: 27 Amish kindred in Pennsylvania (1436G> A) and 6 children born into polygamist families in a small town in the Midwest (1529G> A).79,  80

Pathophysiology

The mechanisms causing hemolysis and premature destruction of PK-deficient cells are not completely known. The metabolic consequence of PK deficiency is a depletion of cellular ATP and an increase in 2,3-bisphosphoglycerate (2,3-BPG).81,  82 The increase in 2,3-BPG shifts the hemoglobin-oxygen dissociation curve to the right and decreases the oxygen affinity of hemoglobin81 (Chapter 10). This promotes greater release of oxygen to the tissues and enables affected individuals to tolerate lower levels of hemoglobin.59,  81,  82 ATP depletion also affects the ability of the cell to maintain its shape and membrane integrity.

Clinical manifestations

Individuals with PK deficiency have a wide range of clinical presentations, varying from severe neonatal anemia and hyperbilirubinemia requiring exchange or multiple transfusions to a fully compensated hemolytic process in apparently healthy adults.81 Most patients, however, have manifestations of chronic hemolysis, including anemia, jaundice, splenomegaly, and increased incidence of gallstones (due to the production of excessive bilirubin).59 Rarely, folate deficiency (due to accelerated erythropoiesis), bone marrow aplasia (usually due to parvovirus B19 infection), and skin ulcers can occur.73,  82 Pregnancy carries the risk of fetal loss and exacerbation of the anemia in the mother.79 There is an increased risk of iron overload and organ damage that occurs with age, even in the absence of transfusions.79,  82,  83 The mechanism of dysregulation of iron homeostasis is not clear but may be related to a decrease in or lack of response to hepcidin, the major iron-regulating protein.79

Laboratory findings

The hemoglobin level is variable, depending on the extent of the hemolysis. Reticulocytosis is usually present, but not in proportion to the severity of the anemia, because the reticulocytes are preferentially destroyed in the spleen.59,  82 After splenectomy, the number of circulating reticulocytes can increase fivefold.82 In addition to showing anisocytosis, poikilocytosis, and polychromasia, the peripheral blood film reveals a variable number of burr cells, or echinocytes (in the range of 3% to 30%), which increase in number after splenectomy.82 The postsplenectomy peripheral blood film may also show Howell-Jolly bodies, Pappenheimer bodies, and target cells. The WBC and platelet counts are normal or slightly increased. Patients usually have the characteristic laboratory findings of chronic hemolysis, including an increased serum indirect bilirubin level, a decreased serum haptoglobin level, and increased urinary urobilinogen. The osmotic fragility is usually normal, and the direct antiglobulin test is negative.

Tests for PK deficiency include quantitative and qualitative biochemical assays for PK activity (phenotypic assays) and DNA-based molecular tests for mutation detection (genotypic assays). In the quantitative PK assay, a hemolysate is prepared from patient’s anticoagulated blood after careful removal of the WBCs. WBCs have a very high PK level, and contamination of the hemolysate with WBCs falsely increases the result (i.e., in a PK deficiency, the result could be falsely normal).82 The reagents include phosphoenolpyruvate, adenosine diphosphate (ADP), lactate dehydrogenase, and the reduced form of nicotinamide adenine dinucleotide (NADH). In the first step of the reaction, the patient’s PK converts phosphoenolpyruvate to pyruvic acid, and a phosphate is transferred to ADP, forming adenosine triphosphate (ATP). In the second step, lactate dehydrogenase converts the pyruvic acid to lactic acid, and the NADH is converted to its oxidized form, NAD (Figure 24-14). The rate of NAD formation is proportional to PK activity and is measured as a decrease in absorbance at 340 nm using a spectrophotometer. The activity is typically reported as a ratio of the units of PK activity per gram of hemoglobin (IU/g Hb). More complex techniques may be necessary when some variant forms of PK are suspected.82

 
FIGURE 24-14 Principle of the pyruvate kinase (PK) activity test. The reagent contains adenosine diphosphate (ADP), phosphoenolpyruvic acid, lactate dehydrogenase, and the reduced form of nicotinamide adenine dinucleotide (NADH). In the first step, PK (in patient’s hemolysate) converts phosphoenolpyruvic acid to pyruvic acid, and a phosphate is transferred to ADP, forming adenosine triphosphate (ATP). In the second step, lactate dehydrogenase converts pyruvic acid to lactic acid with the conversion of NADH (fluorescent) to the oxidized form of nicotinamide adenine dinucleotide (NAD) (not fluorescent). In the quantitative assay, the rate of production of NAD is proportional to PK activity and is measured as a decrease in absorbance at 340 nm using a spectrophotometer. In the qualitative assay, the disappearance of fluorescence under ultraviolet light when the reaction mixture is spotted on filter paper indicates normal PK activity.

Qualitative tests for PK deficiency are used for screening and are based on the same principle as that described earlier, except the hemolysate and reagents are incubated and spotted onto filter paper. The loss of fluorescence is visually evaluated to determine the oxidation of NADH to NAD (Figure 24-14).84

Mutation detection (genotypic testing) can be accomplished by sequencing the exons, flanking regions, and promoter region of the PKLR gene.82 Molecular diagnosis is superior in sensitivity and specificity, is applicable for use in prenatal testing, and enables correlation of certain mutations with disease severity.59,  79,  82

Treatment

No specific therapy is available for PK deficiency except supportive treatment and RBC transfusion as necessary. Splenectomy is beneficial in severe cases, and after this procedure the hemoglobin level usually increases enough to reduce or eliminate the need for transfusion.73 Splenectomy, however, results in a lifelong increased risk of sepsis by encapsulated bacteria. Hematopoietic stem cell transplant may be curative for children with severe hemolytic disease who have an unaffected HLA-identical sibling for a donor.79

Other enzymopathies

Pyrimidine 5′-nucleotidase type 1 (P5’NT-1) is an enzyme needed for the degradation and elimination of ribosomal ribonucleic acid (RNA) in reticulocytes. P5’NT-1 removes the phosphate from pyrimidine 5’ ribonucleoside monophosphate to form ribonucleoside and inorganic phosphate. These degradation products are then able to diffuse out of the cell.59P5’NT-1 deficiency is inherited in an autosomal recessive manner.59,  85 It is the third most common RBC enzyme deficiency that causes hereditary nonspherocytic hemolytic anemia (after G6PD and PK).59,  85 The NT5C3A gene codes for P5’NT-1, and over 20 different mutations have been reported.85

Patients who are homozygotes or compound heterozygotes for NT5C3A mutations develop chronic hemolytic anemia. The P5’NT-1-deficient RBCs accumulate pyrimidine ribonucleoside monophosphates, which precipitate and appear as very coarse basophilic stippling in the cell.59,  85 These RBCs ultimately undergo premature hemolysis. Most patients have a mild to moderate anemia with reticulocytosis, jaundice, and splenomegaly.85 Mental retardation has been reported in some patients.61,  85 Diagnostic tests include measurement of P5’NT-1 activity and the concentration of intracellular pyrimidine nucleotides in RBCs and DNA-based testing for mutations.85 Therapy consists of RBC transfusion as needed.

Other RBC enzymopathies are rarely encountered. In addition to PK deficiency, deficiencies of other enzymes of the RBC Embden-Meyerhof pathway that cause hereditary nonspherocytic hemolytic anemia have been described, including hexokinase, glucose-6-phosphate isomerase, 6-phosphofructokinase, fructose-bisphosphate aldolase, triosephosphate isomerase, and phosphoglycerate kinase.61 All of these deficiencies are autosomal recessive conditions, except for phosphoglycerate kinase deficiency, which is X-linked. Mutations in enolase are rare, and their association with hemolytic anemia is uncertain.61 Deficiencies in glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase are not associated with hemolytic anemia.61 Phosphoglycerate mutase deficiency results in a depletion of 2,3-bisphosphoglycerate. This causes a shift in the hemoglobin-oxygen dissociation curve to the left and an increased affinity of hemoglobin for oxygen. The resulting tissue hypoxia manifests as a mild erythrocytosis.61

Summary

  • The RBC membrane must have deformability for the RBC to maneuver through the microcirculation and the splenic sieve over its life span of 120 days. The cellular properties that enable deformability are the biconcave, discoid shape of the cell; the viscoelasticity of the membrane; and cytoplasmic viscosity.
  • Two transmembrane protein complexes, the ankyrin complex and protein 4.1 complex, provide vertical structural integrity to the cell by anchoring the lipid bilayer to the underlying spectrin skeleton. α-Spectrin, β-spectrin, and their accessory proteins form a two-dimensional lattice to provide horizontal mechanical stability to the membrane.
  • Hereditary spherocytosis (HS) is caused by mutations that disrupt the vertical membrane protein interactions, which results in loss of membrane, decrease in surface area–to–volume ratio, and formation of spherocytes that are destroyed in the spleen. Patients with HS have anemia, splenomegaly, jaundice, an increased MCHC, a negative DAT result, biochemical evidence of hemolysis, and spherocytes and polychromasia on the peripheral blood film. RBCs in HS show decreased fluorescence in the eosin-5′-maleimide binding test when measured by flow cytometry, and the osmotic fragility test usually shows increased fragility.
  • Hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) are caused by mutations that disrupt the horizontal interactions in the protein cytoskeleton, which results in loss of mechanical stability of the membrane. Elliptocytes are present on the peripheral blood film. Only 10% of HE patients have moderate or severe anemia. HPP is a severe thermal-sensitive form of HE in which extreme poikilocytosis along with schistocytes, microspherocytes, and elliptocytes are seen on the peripheral blood film.
  • Hereditary ovalocytosis, also called Southeast Asian ovalocytosis (SAO), is caused by a mutation in band 3 that increases membrane rigidity. The prevalence is high in Southeast Asia, and hemolysis is mild or absent; typical cells are oval with one to two transverse bars or ridges.
  • Hereditary stomatocytosis is a group of disorders characterized by an RBC membrane that leaks cations. In overhydrated hereditary stomatocytosis (OHS), the RBCs have decreased cytoplasmic viscosity and stomatocytes are observed on the peripheral blood film. In dehydrated hereditary stomatocytosis (DHS) or hereditary xerocytosis (HX), the RBCs have increased cytoplasmic viscosity, and the peripheral blood film shows burr cells, target cells, few stomatocytes, and cells with puddled hemoglobin at the periphery. Stomatocytosis may also occur in Rh deficiency syndrome and in a variety of acquired conditions.
  • Neuroacanthocytosis comprises a group of inherited disorders characterized by neurologic impairment and the presence of acanthocytes on the peripheral blood film. Major disorders in this group include abetalipoproteinemia, McLeod syndrome, and chorea acanthocytosis. Acquired acanthocytosis can occur in severe liver disease (spur cell anemia).
  • Paroxysmal nocturnal hemoglobinuria (PNH) is due to an acquired hematopoietic stem cell mutation that results in the lack of GPI-anchored proteins on blood cell surfaces. CD55 and CD59, complement-regulating proteins, are partially or completely deficient on RBCs, which makes the RBCs susceptible to spontaneous complement lysis. Flow cytometry is a sensitive method to detect the absence of GPI-anchored proteins on cell surfaces. The rate of hemolysis in classic PNH improves after treatment with eculizumab, a complement C5 inhibitor.
  • Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common RBC enzymopathy, but the vast majority of patients are asymptomatic. Patients with classes II and III G6PD variants may develop acute hemolytic anemia after infections or after ingestion of certain drugs or fava beans. A small percentage of patients have class I G6PD variant and chronic hereditary nonspherocytic hemolytic anemia (HNSHA).
  • Most patients with pyruvate kinase (PK) deficiency have symptoms of hemolysis. Burr cells are commonly observed on the peripheral blood film. PK deficiency is the most common cause of HNSHA.

Now that you have completed this chapter, go back and read again the case study at the beginning and respond to the questions presented.

Review questions

Answers can be found in the Appendix.

  1. In HS a characteristic abnormality in the CBC results is:
  2. Increased MCV
  3. Increased MCHC
  4. Decreased MCH
  5. Decreased platelet and WBC counts
  6. The altered shape of the spherocyte in HS is due to:
  7. An abnormal RBC membrane protein affecting vertical protein interactions
  8. Defective RNA synthesis
  9. An extrinsic factor in the plasma
  10. Abnormality in the globin composition of the hemoglobin molecule
  11. Which of the following results are consistent with HS?
  12. Increased osmotic fragility, negative DAT result
  13. Decreased osmotic fragility, positive DAT result
  14. Increased osmotic fragility, positive DAT result
  15. Decreased osmotic fragility, negative DAT result
  16. The RBCs in HE are abnormally shaped and have unstable cell membranes as a result of:
  17. Abnormal shear stresses in the circulation
  18. Defects in horizontal membrane protein interactions
  19. Mutations in ankyrin
  20. Lack of all Rh antigens in the RBC membrane
  21. The peripheral blood film for patients with mild HE is characterized by:
  22. Elliptical RBCs
  23. Oval RBCs with one or two transverse ridges
  24. Overhydrated RBCs with oval central pallor
  25. Densely stained RBCs with a few irregular projections
  26. Laboratory test results for patients with HPP include all of the following except:
  27. RBCs that show marked thermal sensitivity at 41° C to 45° C
  28. Marked poikilocytosis with elliptocytes, RBC fragments, and microspherocytes
  29. Low fluorescence when incubated with eosin-5′-maleimide
  30. Increased MCV and normal RDW
  31. Acanthocytes are found in association with:
  32. Abetalipoproteinemia
  33. G6PD deficiency
  34. Rh deficiency syndrome
  35. Vitamin B12deficiency
  36. The most common manifestation of G6PD deficiency is:
  37. Chronic hemolytic anemia caused by cell shape change
  38. Acute hemolytic anemia caused by drug exposure or infections
  39. Mild compensated hemolysis caused by ATP deficiency
  40. Chronic hemolytic anemia caused by intravascular RBC lysis
  41. A patient experiences an episode of acute intravascular hemolysis after taking primaquine for the first time. The physician suspects that the patient may have G6PD deficiency and orders an RBC G6PD assay 2 days after the hemolytic episode begins. How will this affect the test result?
  42. No effect
  43. False increase due to reticulocytosis
  44. False decrease due to hemoglobinemia
  45. Absence of enzyme activity
  46. The most common defect or deficiency in the anaerobic glycolytic pathway that causes chronic HNSHA is:
  47. Pyruvate kinase deficiency
  48. Lactate dehydrogenase deficiency
  49. Glucose-6-phosphate dehydrogenase deficiency
  50. Methemoglobin reductase deficiency
  51. Which of the following laboratory tests would be best to confirm PNH?
  52. Acidified serum test (Ham test)
  53. Osmotic fragility test
  54. Flow cytometry for detection of eosin-5′-maleimide binding on erythrocytes
  55. Flow cytometry for detection of CD55, CD59, and FLAER binding on neutrophils and monocytes

References

  1.   Mohandas N, Evans E. Mechanical properties of the red cell membrane in relation to molecular structure and genetic defectsAnnu Rev Biophys Biomol Struct; 1994; 23:787-818.
  2.   Mohandas N, Chasis J. A. Red blood cell deformability, membrane material properties and shaperegulation by transmembrane, skeletal and cytosolic proteins and lipids. Semin Hematol; 1993; 30:171-192.
  3.   Mohandas N, Gallagher P. G. Red cell membranepast, present, future. Blood; 2008; 112:3939-3948.
  4.   Gallagher P. G. The red blood cell membrane and its disordershereditary spherocytosis, elliptocytosis, and related diseases. In: Lichtman M. A, Kipps T. J, Seligsohn U, et al. Williams Hematology. 8th ed. New York : McGraw-Hill 2010.
  5.   Palek J, Jarolim P. Clinical expression and laboratory detection of red blood cell membrane protein mutationsSemin Hematol; 1993; 30:249-283.
  6.   Da Costa L, Galimand J, Fenneteau, et al. Hereditary spherocytosis, elliptocytosis, and other red cell membrane disordersBlood Rev; 2013; 27:167-178.
  7.   Bennett V. Proteins involved in membrane-cytoskeleton association in human erythrocytesspectrin, ankyrin, and band 3. Methods Enzymol; 1983; 96:313-324.
  8.   Nicolas V, Le Van Kim C, Gane P, et al. Rh-RhAG/ankyrin-R, a new interaction site between the membrane bilayer and the red cell skeleton, is impaired by Rhnull-associated mutationJ Biol Chem; 2003; 278:25526-25533.
  9.   Salomao M, Zhang X, Yang Y, et al. Protein 4.1R-dependent multiprotein complexnew insights into the structural organization of the red blood cell membrane. Proc Natl Acad Sci U S A; 2008; 105:8026-8031.
  10.   An X, Debnath G, Guo X, et al. Identification and functional characterization of protein 4.1R and actin-binding sites in erythrocyte beta-spectrinregulation of the interactions by phosphatidylinositol-4,5-bisphosphate. Biochemistry; 2005; 44:10681-10688.
  11.   An X, Mohandas N. Disorders of red cell membraneBr J Haematol; 2008; 141:367-375.
  12.   An X, Guo X, Zhang X, et al. Conformational stabilities of the structural repeats of erythroid spectrin and their functional implicationsJ Biol Chem; 2006; 281:10527-10532.
  13.   Gallagher P. G. Red blood cell membrane disorders. In: Hoffman R, Benz E. J, Jr Silberstein L. E, et al. Hematology: Basic Principles and Practice.6th ed. Philadelphia : Saunders, an imprint of Elsevier 2013.
  14.   National Human Genome Research Institute. Red Cell Membrane Disorder Mutations Database. Available at: http://research.nhgri.nih.gov/RBCmembrane/Accessed 08.03.14.
  15.   Perrotta S, Gallagher P. G, Mohandas N. Hereditary spherocytosisLancet; 2008; 372:1411-1426.
  16.   Bolton-Maggs P.H.B, Langer J. C, Iolascon A, et al. Guidelines for the diagnosis and management of hereditary spherocytosis—2011 updateBr J Haematol; 2011; 156:37-49.
  17.   De Franceschi L, Olivieri O, Miraglia del Giudice E, et al. Membrane cation and anion transport activities in erythrocytes of hereditary spherocytosiseffects of different membrane protein defects. Am J Hematol; 1997; 55:121-128.
  18.   Hassoun H, Vassiliadis J. N, Murray J, et al. Characterization of the underlying molecular defect in hereditary spherocytosis associated with spectrin deficiencyBlood; 1997; 90:398-406.
  19.   Dhermy D, Galand C, Bournier O, et al. Heterogenous band 3 deficiency in hereditary spherocytosis related to different band 3 gene defectsBr J Haematol; 1997; 98:32-40.
  20.   Bouhassira E. E, Schwartz R. S, Yawata Y, et al. An alanine-to-threonine substitution in protein 4.2 cDNA is associated with a Japanese form of hereditary hemolytic anemia (protein 4.2NIPPON)Blood; 1992; 79:1846-1854.
  21.   Michaels L. A, Cohen A. R, Zhao H. J, et al. Screening for hereditary spherocytosis by use of automated erythrocyte indexesJ Pediatr; 1997; 130:957-960.
  22.   Cynober T, Mohandas N, Tchernia G. Red cell abnormalities in hereditary spherocytosisrelevance to diagnosis and understanding of the variable expression of clinical severity. J Lab Clin Med; 1996; 128:259-269.
  23.   Hansen D. M. Hereditary anemias of increased destruction. In: Steine-Martin E. A, Lotspeich-Steininger C. A, Koepke J. A. Clinical Hematology: Principles, Procedures, Correlations.2nd ed. Philadelphia : Lippincott 1998; 255-256.
  24.   Bianchi P, Fermo E, Vercellati C, et al. Diagnostic power of laboratory tests for hereditary spherocytosisa comparison study in 150 patients groups according to molecular and clinical characteristics. Haematologica; 2012; 97:516-523.
  25.   King M. J, Smythe J, Mushens R. Eosin-5-maleimide binding to band 3 and Rh-related proteins forms the basis of a screening test for hereditary spherocytosisBr J Haematol; 2004; 124:106-113.
  26.   Kar R, Mishra P, Pati H. P. Evaluaiton of eosin-5-maleimide flow cytometric test in diagnosis of hereditary spherocytosisInt J Lab Hematol; 2008; 32:8-16.
  27.   Mackiewicz G, Bailly F, Favre B, et al. Flow cytometry test for hereditary spherocytosisHaematologica; 2012; 97:e47.
  28.   Clark M. R, Mohandas N, Shohet S. B. Osmotic gradient ektacytometrycomprehensive characterization of red cell volume and surface maintenance. Blood; 1983; 61:899-910.
  29.   King M. J, Zanella A. Hereditary red cell membrane disorders and laboratory diagnostic testingInt J Lab Hem; 2013; 35:237-243.
  30.   Streichman S, Gescheidt Y. Cryohemolysis for the detection of hereditary spherocytosiscorrelation studies with osmotic fragility and autohemolysis. Am J Hematol; 1998; 58:206-212.
  31.   Abdullah F, Zhang Y, Camp M, et al. Splenectomy in hereditary spherocytosisreview of 1,657 patients and application of the pediatric quality indicators. Pediatr Blood Cancer; 2009; 52:834-837.
  32.   Gallagher P. G. Hereditary elliptocytosisspectrin and protein 4.1R. Semin Hematol; 2004; 41:142-164.
  33.   Dhermy D, Schrevel J, Lecomte M. C. Spectrin-based skeleton in red blood cells and malariaCurr Opin Hematol; 2007; 14:198-202.
  34.   Winardi R, Reid M, Conboy J, et al. Molecular analysis of glycophorin C deficiency in human erythrocytesBlood; 1993; 81:2799-2803.
  35.   Gallagher P. G. Abnormalities of the erythrocyte membranePediatr Clin N Am; 2013; 60:1349-1362.
  36.   Ideguchi H, Yamada Y, Kondo S, et al. Abnormal erythrocyte band 4.1 protein in myelodysplastic syndrome with elliptocytosisBr J Haematol; 1993; 85:387-392.
  37.   King M. J, Telfer P, MacKinnon H, et al. Using the eosin-5-maleimide binding test in the differential diagnosis of hereditary spherocytosis and hereditary pyropoikilocytosisCytometry B Clin Cytom; 2008; 74B:244-250.
  38.   Mohandas N, Winardi R, Knowles D, et al. Molecular basis for membrane rigidity of hereditary ovalocytosis. A novel mechanism involving the cytoplasmic domain of band 3J Clin Invest; 1992; 89:686-692.
  39.   Liu S. C, Zhai S, Palek J, et al. Molecular defect of the band 3 protein in Southeast Asian ovalocytosisN Engl J Med; 1990; 323:1530-1538.
  40.   Schofield A.E, Tanner M.J.A, Pinder J. C, et al. Basis of unique red cell membrane properties in hereditary ovalocytosisJ Mol Biol; 1992; 223:949-958.
  41.   Bruce L. J. Hereditary stomatocytosis and cation leaky red cells—recent developmentsBlood Cells Mol Dis; 2009; 42:216-222.
  42.   Bruce L. J, Guizouam H, Burton N. M, et al. The monovalent cation leak in overhydrated stomatocytic red blood cells results from amino acid substitutions in the Rh-associated glycoprotein.Blood; 2009; 113:1350-1357.
  43.   Zarychanski R, Schulz V. P, Houston B. L, et al. Mutations in the mechanotransduction protein PIEZO1 are associated with hereditary xerocytosisBlood; 2012; 120:1908-1915.
  44.   Bae C, Gnanasambandam R, Nicolai C, et al. Xerocytosis is caused by mutations that alter the kinetics of the mechanosensitive channel PIEZO1Proc Natl Acad Sci U S A; 2013; 110:E1162-E1168.
  45.   Andolfo I, Alper S. L, De Franceschi L, et al. Multiple clinical forms of dehydrated hereditary stomatocytosis arise from mutations in PIEZO1Blood; 2013; 121:3925-3935.
  46.   Rampoldi L, Danek A, Monaco A. P. Clinical features and molecular bases of neuroacanthocytosisJ Mol Med; 2002; 80:475-491.
  47.   Walker R. H, Jung H. H, Dobson-Stone C, et al. Neurologic phenotypes associated with acanthocytosisNeurology; 2007; 68:92-98.
  48.   Parker C. J. Paroxysmal nocturnal hemoglobinuria. Available at: http://accessmedicine.mhmedical.com.libproxy2.umdnj.edu/content.aspx?bookid=358& Sectionid=39835858Accessed 09.03.14. In: Lichtman M. A, Kipps T. J, Seligsohn U, et al. Williams Hematology.8th ed. New York : McGraw-Hill 2010.
  49.   Besslar M, Hiken J. The pathophysiology of disease in patients with paroxysmal nocturnal hemoglobinuria. : Hematology Am Soc Hematol Educ Program 2008; 104-110.
  50.   Brodsky R. A, et al. Paroxysmal nocturnal hemoglobinuria. In: Hoffman R, Benz E.J, Jr Silberstein L.E, et al. Hematology: Basic Principles and Practice.6th ed. Philadelphia : Saunders, an imprint of Elsevier 2013.
  51.   Takeda J, Miyata T, Kawagoe K, et al. Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuriaCell; 1993; 73:703-711.
  52.   Parker C, Omine M, Richards S, et al. Diagnosis and management of paroxysmal nocturnal hemoglobinuriaBlood; 2005; 106:3699-3709.
  53.   Sutherland R. D, Keeney M, Illingworth A. Practical guidelines for the high-sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry.Cytometry Part B; 2012; 82B:195-208.
  54.   Preis M, Lowrey C.H. Laboratory tests for paroxysmal nocturnal hemoglobinuriaAm J Hematol; 2014; 89:339-341.
  55.   Sutherland D. R, Kuek N, Azcona- Olivera J. Use of FLAER-based WBC assay in the primary screening of PNH clonesAm J Clin Pathol; 2009; 132:564-572.
  56.   Dmytrijuk A, Robie-Suh K, Cohen M. H, et al. FDA reporteculizumab (Soliris) for the treatment of patients with paroxysmal nocturnal hemoglobinuria. Oncologist; 2008; 13:993-1000.
  57.   Hillmen P, Muus P, Röth A, et al. Long-term safety and efficacy of sustained eculizumab treatment in patients with paroxysmal nocturnal hemoglobinuriaBr J Haematol; 2013; 162:62-73.
  58.   Risitano A. M, Notaro R, Pascariello C, et al. The complement receptor 2/factor H fusion protein TT30 protects paroxysmal nocturnal hemoglobinuria erythrocytes from complement-mediated hemolysis and C3 fragment opsonizationBlood; 2012; 119:6307-6316.
  59.   Price E. A, Otis S, Schrier S. L. Red blood cell enzymopathies. In: Hoffman R, Benz E. J, Jr Silberstein L. E, et al. Hematology: Basic Principles and Practice.6th ed. Philadelphia : Saunders, an imprint of Elsevier 2013.
  60.   Cappellini M. D, Fiorelli G. Glucose-6-phosphate dehydrogenase deficiencyLancet; 2008; 371:64-74.
  61.   van Solinge W. W, van Wijk R. Disorders of red cells resulting from enzyme abnormalities. Available at: http://accessmedicine.mhmedical.com.libproxy2.umdnj.edu/content.aspx?bookid=358& Sectionid=39835864In: Lichtman M.A, Kipps T.J, Seligsohn U, Kaushansky K, Prchal J.T. Williams Hematology. 8th ed. New York : McGraw-Hill 2010 Accessed 31.03.14.
  62.   Nkhoma E. T, Poole C, Vannappagari V, et al. The global prevalence of glucose-6-phosphate dehydrogenase deficiencya systematic review and meta-analysis. Blood Cells Mol Dis; 2009; 42:267-278.
  63.   Luzzatto L. Glucose 6-phosphate dehydrogenase deficiencyfrom genotype to phenotype. Hematologica; 2006; 91:1303-1306.
  64.   Guindo A, Fairhurst R. M, Doumbo O. K, et al. X-linked G6PD deficiency protects hemizygous males but not heterozygous females against severe malariaPLoS; 2007; 4(3):e66.
  65.   Leslie T, Briceno M, Mayan I, et al. The impact of phenotypic and genotypic G6PD deficiency on risk of Plasmodium vivax infectiona case-control study amongst Afghan refugees in Pakistan. PLoS; 2010; 7(5):e1000283.
  66.   Clark I. A, Hunt N. H. Evidence for reactive oxygen intermediates causing hemolysis and parasite death in malariaInfect Immun; 1983; 39:1-6.
  67.   Cappadoro M, Giribaldi G, O’Brien E. Early phagocytosis of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes parasitized by Plasmodium falciparum may explain malaria protection in G6PD deficiencyBlood; 1998; 92:2527-2534.
  68.   Minucci A, Moradkhani K, Hwang M. J, et al. Glucose-6-phosphate dehydrogenase (G6PD) mutations databasereview of the “old” and update of the new mutations. Blood Cells Mol Dis; 2012; 48:154-165.
  69.   Beutler E, Gaetani G, der Kaloustian V, et al. World Health Organization (WHO) Working Group. Glucose-6-phosphate dehydrogenase deficiencyBull World Health Organ; 1989; 67:601-611.
  70.   Youngster I, Arcavi L, Schechmaster R, et al. Medications and glucose-6-phosphate dehydrogenase deficiencyan evidence-based review. Drug Saf; 2010; 33:713-726.
  71.   Luzzatto L, Seneca E. G6PD deficiencya classic example of pharmacogenetics with on-going clinical implications. Br J Haematol; 2014; 164:469-480.
  72.   Kaplan M, Muraca M, Vreman H. J, et al. Neonatal bilirubin production-conjugation imbalanceeffect of glucose-6-phosphate dehydrogenase deficiency and borderline prematurity.Arch Dis Child Fetal Neonatal Ed; 2005; 90:F123-F127.
  73.   Prchal J. T, Gregg X. T. Red cell enzymes. : Hematol Am Soc Hematol Educ Program 2005; 19-23.
  74.   Minucci A, Giardina B, Zuppi C, et al. Glucose-6-phosphate dehydrogenase laboratory assayhow, when, and why. IUBMB Life; 2009; 61:27-34.
  75.   Tantular I. S, Kawamoto F. An improved simple screening method for detection of glucose-6-phosphate dehydrogenase deficiencyTrop Med Int Health; 2003; 8:569-574.
  76.   Tinley K. E, Loughlin A. M, Jepson A, et al. Evaluation of a rapid qualitative enzyme chromatographic test for glucose-6-phosphate dehydrogenase deficiencyAm J Trop Med Hyg; 2010; 82:210-214.
  77.   Beutler E, Gelbart T. Estimating the prevalence of pyruvate kinase deficiency from the gene frequency in the general white populationBlood; 2000; 95:3585-3588.
  78.   Zanella A, Bianchi P, Fermo E, et al. Pyruvate kinase deficiencyHematologica; 2007; 92:721-722.
  79.   Rider N. L, Strauss K. A, Brown K, et al. Erythrocyte pyruvate kinase deficiency in an old-order Amish cohortlongitudinal risk and disease management. Am J Hematol; 2011; 86:827-834.
  80.   Christensen R. D, Yaish H. M, Johnson C. B, et al. Six children with pyruvate kinase deficiency in one small townMolecular characterization of the PK-LR gene. J Pediatr; 2011; 159:695-697.
  81.   Van Wijk R, van Solinge W. W. The energy-less red blood cell is losterythrocyte enzyme abnormalities of glycolysis. Blood; 2005; 106:4034-4042.
  82.   Zanella A, Fermo E, Bianchi P, et al. Red cell pyruvate kinase deficiencymolecular and clinical aspects. Br J Haematol; 2005; 130:11-25.
  83.   Andersen F. D, d’Amore F, Nielsen F. C, et al. Unexpectedly high but still asymptomatic iron overload in a patient with pyruvate kinase deficiencyHematol J; 2004; 5:543-545.
  84.   Tsang S. S, Feng C. S. A modified screening procedure to detect pyruvate kinase deficiencyAm J Clin Pathol; 1993; 99:128-131.
  85.   Zanella A, Bianchi P, Fermo E, et al. Hereditary pyrimidine 5’-nucleotidase deficiencyfrom genetics to clinical manifestations. Br J Haematol; 2006; 133:113-123.