Patricia A. Oneal, Geraldine P. Schechter, Griffin P. Rodgers and Jeffery L. Miller
Many diseases share the clinical feature of red blood cell (RBC) hemolysis. Hemoglobinopathies and immune-mediated hemolysis are the most common causes (see discussions in Chapters 4 and 24, respectively). Very rare inherited or acquired diseases may also directly or indirectly result in increased red cell destruction.1 Understanding the mechanisms that lead to hemolysis assists with the diagnosis, prognosis, and consideration of the most appropriate therapy. In this post-genomic era, correlations between genotype and phenotype are being pursued in cases of inherited hemolytic syndromes. Genetics-based discoveries are being translated into new clinical tools in anticipation of mechanism-specific therapies.
Hemolytic anemia is defined as decreased levels of erythrocytes in circulating blood because of their accelerated destruction. All circulating erythrocytes are subject to physiologic stresses such as turbulence in blood flow, endothelial damage, and age-related catabolic changes. Normally, damaged RBCs are removed from the circulation by the reticuloendothelial system. In hemolytic syndromes, erythrocyte clearance by the reticuloendothelial system may be increased (extravascular hemolysis) or the cells may be lysed within the circulation (intravascular hemolysis). As a result, RBC survival is generally shortened to less than 100 days (normal survival is approximately 120 days). When sufficient numbers of erythrocytes are destroyed, oxygen delivery to tissues is impaired. Tissue hypoxia leads to increased release of erythropoietin, which signals the bone marrow to produce more RBCs.
A hallmark of hemolytic anemia is an elevated number of immature erythrocytes (reticulocytes) in the peripheral blood. In low-level hemolysis, erythrocyte production may adequately compensate for blood cell destruction and minimize the anemia. Alternatively, patients with acute hemolysis or with underlying defects in hematopoiesis may present with pronounced anemia without reticulocytosis. Hence, the evaluation of suspected hemolysis requires consideration of the hemolysis itself as well as the marrow’s ability to compensate. The diagnostic strategy usually begins with a search for common causes of hemolysis and proceeds toward rare etiologies. The extent of diagnostic studies should be guided by the magnitude of hemolysis and the available therapeutic options. With the information contained here, practicing clinicians should be able to develop a clinical approach, differential diagnosis, and therapeutic plan for patients with suspected hemolysis.
ETIOLOGY AND DIFFERENTIAL DIAGNOSIS
Grouping the various causes of the disease generates a differential diagnosis for hemolysis. As shown in Figure 3.1, hemolysis results from pathology intrinsic or extrinsic to the erythrocytes. Intrinsic hemolysis may be categorized further according to hemoglobin, membrane, or enzyme-based factors. Alternatively, the patient’s immune status or infectious agents can lead to hemolysis in the absence of intrinsic defects. Other chemical or physical features of the erythrocyte environment can also cause hemolysis. A more complete differential organized according to these categories is shown in Table 3.1.
FIGURE 3.1 Intrinsic and extrinsic causes of hemolysis.
Most intrinsic causes for hemolysis are inherited, while the extrinsic causes of hemolysis are typically acquired. In some cases, such as paroxysmal nocturnal hemoglobinuria (PNH) or glucose-6-phosphate dehydrogenase (G6PD) deficiency, both intrinsic and extrinsic factors may contribute to the hemolytic picture. Consideration of the primary site of hemolysis (intravascular versus extravascular) may also be helpful in determining the origin of erythrocyte destruction.6
To complete the differential diagnosis, diseases or events that may in part mimic a typical hemolytic episode should be considered. The laboratory evaluation may be normal with the exception of a single variable such as hemoglobin, absolute reticulocyte count (ARC), or unconjugated bilirubin. For instance, the compensatory reticulocytosis that occurs after an acute hemorrhagic event may be mistaken for evidence of hemolysis. In the absence of other clinical or laboratory abnormalities, artifactual reticulocytosis may be caused by a malfunction in the automated cell counter. While hypersplenism may be associated with increased red cell clearance and anemia, the abnormal RBC morphology seen in patients with asplenia is usually not associated with hemolysis. Finally, patients with chronic idiopathic unconjugated hyperbilirubinemia (Gilbert’s syndrome) are sometimes erroneously referred to a hematologist to rule out hemolysis.7
CLINICAL APPROACH TO PATIENTS WITH SUSPECTED HEMOLYSIS
As with most diseases, the approach to hemolysis involves a combination of clinical and laboratory investigations directed by the judgment and skills of the clinician (Fig. 3.2).
Low-level or chronic hemolysis should be suspected in all patients with unexplained anemia. A detailed history and physical examination should be the cornerstone of each patient’s evaluation.
History
Onset/duration (hereditary versus acquired)
History of fatigue
History of jaundice
Abdominal pain/cholelithiasis (chronic hemolysis)
Medications (may exacerbate enzyme deficiencies)
Travel (consider infection)
History of recent or current infection
Vascular/cardiac surgery
Blood loss or sequestration (increases reticulocytes in the absence of hemolysis)
Discolored urine (intravascular hemolysis)
Complete family history (jaundice, gallbladder disease, splenectomy, hereditary anemia, or other inherited diseases)
Physical
Pallor
Increased temperature
Rapid pulse
Jaundice (chronic hemolysis)
Mechanical click from heart valves
Splenomegaly
FIGURE 3.2 A clinical approach to hemolytic anemia. LDH, lactate dehydrogenase; PNH, paroxysmal nocturnal hemoglobinuria; RBC, red blood cell.
The laboratory evaluation is performed to confirm the suspected diagnosis, provide insight regarding the underlying mechanism, and gauge a therapeutic response. The complete blood count (CBC) usually confirms the diagnosis of anemia. Reticulocytosis increases the mean cell volume (MCV) and red cell distribution width (RDW). A critical test in the evaluation of all patients with suspected hemolysis is the reticulocyte count. An increased number of reticulocytes are present in hemolysis unless erythropoiesis is suppressed. Stressed erythropoiesis associated with acute hemolysis also causes the release of large polychromatic reticulocytes with a decreased area of central pallor into the circulation, called shift cells, which can be appreciated by evaluating the peripheral blood smear.8 Reticulocytes are also identified by their RNA content, so automated detection of RNA in the cells provides an accurate alternative to manual inspection. Normal values for reticulocytes in newborn infants range from 2.5% to 6.5% and fall to less than 2% by the second week of life. In adults, reticulocytes comprise 0.5% to 1.5% of circulating erythrocytes in the absence of anemia, consistent with the normal turnover of 1% of normal red cell mass per day in adults. Percentages above the normal range are usually detected in the setting of hemolysis due to increased erythropoiesis. However, in the setting of anemia an uncorrected reticulocyte percentage may also reflect the prolonged survival of stress reticulocytes and the lower total number of circulating RBC. Therefore, an ARC more accurately measures the compensatory response than does the uncorrected reticulocyte percentage.9
Absolute reticulocyte count (ARC) = Reticulocyte percentage/100 × RBC count/µL
The normal ARC ranges between 25,000 and 75,000/µL. In patients with hemolysis, the ARC is usually elevated to levels greater than 100,000/µL. If hemolysis is acute, a rise in reticulocytes may be delayed by 3 to 5 days.
While a bone marrow examination is generally not required to determine the etiology of uncomplicated hemolysis, the peripheral blood smear should never be overlooked. This simple test is rapid, inexpensive, and can provide important clues regarding the mechanism of hemolysis (Table 3.2).
ACUTE INTRAVASCULAR HEMOLYSIS
The clinical syndrome associated with acute intravascular hemolysis deserves special attention because of its potential catastrophic consequences. Its recognition can lead to rapid institution of specific therapies and prevention of acute renal failure and death. Diagnosis and treatment of Clostridium perfringens sepsis or thrombotic thrombocytopenia purpura may be triggered by a hemolysis workup. Intravascular hemolysis is almost exclusively caused by extrinsic mechanisms, which may have the potential to be rapidly modified or reversed (Table 3.1).
Examination of several key laboratory values may also be used to assess the severity of intravascular hemolysis. Lactate dehydrogenase is released from hemolyzed RBCs. Small amounts of hemoglobin released into the circulation are metabolized in the liver after binding and clearance by haptoglobin. With robust intravascular hemolysis, a rapid decrease of serum haptoglobin to undetectable levels occurs. Free hemoglobin not bound to haptoglobin can be oxidized to methemoglobin or bound to transport proteins such as hemopexin or albumin, which the liver will then remove from the circulation. Free hemoglobin at levels of 100 to 200 mg/dL can be detected by visual examination of plasma or serum. The capacity of renal tubular cells to reabsorb free hemoglobin is limited resulting in hemoglobinuria. Because renal tubular cells slough, iron staining can identify the tubular epithelium containing hemosiderin in the urine sediment. Cessation of hemolysis leads to a rapid recovery of the haptoglobin levels, but urine hemosiderin is detectable for longer periods (Fig. 3.3). Urine hemosiderin in the absence of urine hemoglobin provides clinical evidence for subacute or chronic intravascular hemolysis. In the absence of cirrhosis, reduced levels of haptoglobin (<28 mg/dL) provide 92% sensitivity and 98% specificity for predicting hemolysis.10
SPECIAL CONSIDERATION OF ENZYME AND MEMBRANE DEFECTS
Once the more obvious causes of hemolysis are ruled out, the clinician must consider those etiologies less frequently encountered in daily practice, including enzyme or membrane defects. The laboratory evaluation can be confusing because of the numerous etiologies and the diversity of tests available. Therefore, the extent of diagnostic testing is dictated by the magnitude of hemolysis and the impact of a specific diagnosis on therapy. PNH is diagnosed by flow cytometry because of the associated absence of glycosylphosphatidylinositol-anchored proteins (e.g., CD59) on the plasma membranes of hematopoietic cells (see Chapter 6). General evaluation of the erythroid cytoskeleton abnormalities can usually be assessed by peripheral blood smear evaluation. In the case of enzymopathies, specific functional assays are available from reference laboratories.
FIGURE 3.3 Indicators of acute intravascular hemolysis. (From Hillman RS, Finch CA. Red Cell Manual. 7th ed. Philadelphia, PA: F. A. Davis; 1996, with permission.)
ERYTHROID ENZYMOPATHIES
Enzyme deficiencies are most often associated with congenital nonspherocytic hemolytic anemia. Inheritances of G6PD and phosphoglycerate kinase (PGK) deficiencies are chromosome X-linked. Because the other red cell enzyme abnormalities exhibit an autosomal recessive mode of inheritance, they may be suspected in cases of unexplained hemolysis during infancy or childhood. While G6PD deficiency may be the most common enzyme deficiency in humans,11 the other enzymopathies associated with hemolysis are rarely diagnosed. Based on their low incidence, laboratory evaluation of suspected enzymopathies requires assays performed at specialized or research laboratories (e.g. Mayo Medical Laboratories, Rochester, MN), which measure the functional properties of each enzyme. In the setting of acute hemolysis, however, the magnitude of the functional deficit may be underestimated because of the generally higher levels of enzyme activity in reticulocytes and other “young” erythrocytes. As the clinical application of information contained in the human genome improves, genetic testing may become more practical for these enzymopathies. The success of clinical genotyping in this regard will depend upon the number of mutations identified, as well as the strength of correlation between genotype and phenotype. A genome-based profile of the known hemolysis-related enzymes is available on the Internet (http://fmp-8.cit.nih.gov/hembase/index.php).
Enzyme deficiencies most commonly associated with hemolysis are linked to the prevention of oxidative damage or the generation of energy (ATP) in RBCs. Glutathione reduction (hexose monophosphate shunt) is necessary for the prevention of oxidative damage from hydrogen peroxide to cellular proteins including hemoglobin. Glycolysis (Embden-Meyerhof pathway) provides the sole source of energy to the RBCs once they lose their mitochondria. Below is a brief synopsis of the enzymopathies associated with hemolysis (organized according to the involved metabolic pathway).
Enzymes Involved in Glutathione Metabolism
G6PD deficiency is the most common RBC enzyme disorder associated with hemolysis. As an X-linked disorder, it is far more common in males, but females do present with the disease due to mosaicism of the X chromosome as well as compound heterozygosity of inheritance. It has been estimated that this disorder affects 400 million people throughout the world, with the highest frequencies occurring in populations from the Mediterranean region, Africa, and China.11 Clinical classification is made according to the magnitude of the enzyme deficiency and the severity of hemolysis.
Severe enzyme deficiency (less than 10% of normal activity) with chronic or intermittent hemolysis (Mediterranean and Asian populations).
Moderate enzyme deficiency (10%–60% of normal) with intermittent hemolysis usually associated with infection or drugs. Approximately 10% to 15% of African American males are moderately deficient in G6PD activity.
Mild enzyme deficiency (greater than 60% activity) without significant hemolysis.
Importantly, the severity of hemolysis among all G6PD-deficient patients depends on two major variables: G6PD protein and oxidative stress. G6PD deficiency is defined at the genetic level by mutations that cause either reduced synthesis of functional G6PD (quantitative defect) or production of abnormal G6PD (qualitative defect). The Johns Hopkins University (http://omim.org/entry/305900)has catalogued the known 400 G6PD variants. The neonatal hyperbilirubinemia in G6PD-deficient infants is caused by increased bilirubin production from erythrocyte breakdown and inadequate clearance by an immature liver. Neonates with severe G6PD deficiency are at greatest risk of developing neonatal hyperbilirubinemia.
The second major factor in determining the level of hemolysis is the level of intracellular oxidative stress. G6PD acts to catalyze the conversion of glucose-6-phosphate to 6-phosphogluconate. That biochemical reaction is coupled to the production of NADPH and subsequent reduction of glutathione. Erythrocytes that are exposed to oxidants or oxidative stresses become depleted of reduced glutathione (GSH). Once GSH is depleted, oxidation of other red blood cell sulfhydryl-containing proteins (including hemoglobin) occurs. Oxidation of hemoglobin leads to the formation of sulfhemoglobin and hemoglobin precipitates called Heinz bodies. Heinz body inclusions are generated during acute, drug-induced hemolytic episodes. Patients with moderate G6PD deficiency are generally asymptomatic in the steady state. They present episodically with acute hemolytic anemia due to oxidative stress from infections such as acute viral hepatitis and pneumonia. Hemolysis is also associated with the ingestion of fava beans, which contain pyrimidine aglycones (divicine and isouramil). Favism is most commonly associated with the G6PD variant in Mediterranean populations. Certain drugs and chemicals (Table 3.3) that increase the risk of hemolysis in G6PD-deficient patients should be avoided.12 Hemolysis occurs 1 to 3 days after ingestion of these drugs or fava beans, with resolution usually within one week of cessation.
Testing for suspected G6PD deficiency can be performed by simple qualitative or quantitative fluorescence tests that measures the production of NADPH. During episodes of acute hemolysis, measured levels of enzyme activity may be increased due to the loss of older erythrocytes with the least activity. A more definitive diagnosis of G6PD deficiency requires genetic testing of the involved patient or family. Treatment of a G6PD-deficient individual depends on the degree of hemolysis. Potentially harmful foods and drugs should always be avoided by the patient, as well as nursing mothers of infants with G6PD deficiency. Patients with infection should be carefully monitored for early signs of increased hemolysis. Blood transfusion may be life-saving during acute hemolytic episodes. While controversial, splenectomy may be considered in cases of G6PD deficiency presenting with severe hemolysis that do not respond to other measures.
γ-Glutamylcysteine synthetase is the rate-limiting enzyme in glutathione biosynthesis. Hemolytic anemia is associated with low activity of this enzyme and normal glutathione synthetase levels. These rare patients present with a history of lifelong anemia, intermittent jaundice and spinocerebellar degeneration in adulthood.13
Glutathione peroxidase (GSH-Px) is primarily responsible for the elimination of hydrogen peroxide from erythrocytes. Production of this protein is dependent upon adequate nutritional levels of selenium.14Moderate deficiencies in GSH-Px activity may result in the formation of Heinz bodies and nonspherocytic hemolytic anemia in infants. Like G6PD deficiency, oxidizing agents should be avoided in these patients.
Glutathione reductase is the enzyme that reduces oxidized GSH in the presence of flavin adenine dinucleotide. Deficiency of glutathione reductase causes increased susceptibility to drug-induced hemolysis. Glutathione reductase activity increases with dietary supplementation of riboflavin, and a subset of these patients respond well to riboflavin dietary supplements. In some cases of glutathione reductase deficiency, the enzymatic activity cannot be restored by riboflavin supplementation due to a 2,246 base pair deletion found in the gene encoding for glutathione reductase.15
Glutathione synthetase deficiency is caused by autosomal recessive inheritance of mutations of the glutathione synthetase gene with subsequently low levels of glutathione in RBCs. The disease is marked by accumulation of the metabolite oxyproline in the urine.16 The patients present with the clinical triad of hemolysis, metabolic acidosis, and mental deterioration. Treatment includes vitamin C, vitamin E, bicarbonate, and avoidance of oxidative drugs.
Table 3.3 Common Drugs and Chemicals to Avoid in Patients with Glucose-6-Phosphate Dehydrogenase Deficiency
Dapsone
Methylene blue
Nitrofurantoin
Phenazopyridine (Pyridium)
Primaquine
Rasburicase
Toluidine blue
Enzymes Involved in Glycolysis
Pyruvate kinase (PK) deficiency is associated with over 150 genetic mutations.17 PK deficiency is the second most common enzymopathy associated with congenital nonspherocytic hemolytic anemia with a prevalence of approximately 1:20,000 in Caucasian populations. PK converts phosphoenolpyruvate to pyruvate, simultaneously generating adenosine triphosphate (ATP) from adenosine diphosphate (ADP). PK activity decreases during RBC aging, as the enzyme is gradually denatured. The eventual result is failure of glycolysis as PK activity falls below a critical level. Because glycolysis is the sole source of ATP synthesis in the mature RBC, ATP depletion and hemolysis follow glycolytic failure.
Consistent with several other inherited causes of hemolysis, PK deficiency is postulated to provide some protection from malarial infection.18 Selection for variations of this gene may involve other factors as well, because PK deficiency is less common in Africa and other malarial-endemic regions. Most patients are compound-heterozygotes for the two most common mutant forms of the enzyme. Approximately one-third of the cases present with jaundice during the newborn period, and one-third of those cases are severe enough to require transfusion. Death during the neonatal period may result from severe anemia. In individuals with milder forms of the enzymopathy, the anemia is less severe and the diagnosis may not be established until later in childhood. Unfortunately, poor correlation between PK activity and the severity of clinical hemolysis confounds the accuracy of prognosis.17 There is currently no reliable method to predict success of splenectomy for individual cases.
Glucose phosphate isomerase (GPI) deficiency is the third most common glycolytic enzyme deficiency associated with hemolytic anemia. GPI catalyzes the production of fructose-6-phosphate from glucose-6-phosphate. It is found in all ethnic groups, but is prevalent in individuals of European descent. Over two-dozen genetic variants have been identified to date with considerable variability in disease severity. In severe cases, anemia and hyperbilirubinemia are evident at birth. In addition to chronic hemolysis and hyperbilirubinemia, acute hemolytic crises can occur with viral and bacterial infections.19
Aldolase A deficiency has been found to cause moderately severe lifelong hemolytic anemia, sometimes requiring transfusions during acute hemolytic crises. Aldolase catalyzes the conversion of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. Abnormal expression of the aldolase variant causes hemolysis and myopathy.20 Other congenital anomalies include short stature, mental retardation, delayed puberty, and a distinct facial appearance.
2,3-Diphosphoglycerate mutase (DPGM) deficiencies of greater than 50% cause a compensated hemolytic anemia. DPGM converts 1,3-biphosphoglycerate to 2,3-diphosphoglycerate (2,3-DPG). Deficiencies of this enzyme may lead to a combination of hemolysis and polycythemia because of the resulting 2,3-DPG deficiency.21
Enolase is the enzyme that converts 2-phosphoglycerate to phosphoenolpyruvate. Case studies have shown a decrease in the enzyme activity in patients with a mild spherocytic hemolytic anemia.22
Hexokinase deficiency causes a rare congenital hemolytic anemia, predominantly in persons of northern European ancestry. Hexokinase acts at the initial enzymatic step in glycolysis, catalyzing the conversion of glucose to glucose-6-phosphate. Hexokinase activity in reticulocytes is considerably higher than in mature cells. Anemia is associated with a reduction of this enzyme activity to 25% that of normal erythrocytes.23
Phosphofructokinase deficiency (also called Tarui disease) results in a glycogen storage disorder characterized by hemolysis and myopathy. Phosphofructokinase is an allosteric enzyme that catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-diphosphate. Most affected individuals have exhibited exertional myopathy resulting in weakness, easy fatigability, muscle cramps on exercise, and myoglobinuria. Hemolysis is caused by decreased erythrocyte deformability from leakage of calcium ions.24
PGK deficiency results in a moderate-to-severe nonspherocytic hemolytic anemia. PGK converts 1,3 biphosphoglycerate to 3-phosphoglycerate. PGK deficiency is the only X-linked disorder involved in glycolysis. The disease phenotype is unusually pleomorphic and may include varying degrees of hemolytic anemia, mental retardation, and myopathy.25
Triosephosphate isomerase (TPI) deficiency is a rare disorder characterized by severe hemolytic anemia and increased susceptibility to infection. In addition, progressive neurological deterioration is a hallmark of the associated disease. Deficiencies usually become evident during infancy, with spasticity, motor retardation, hypotonia, weakness, and seizures.26
Other Enzymopathies Associated with Hemolysis
Pyrimidine 5′ nucleotidase (uridine 5′ monophosphate hydrolase) deficiency leads to accumulation of high concentrations of pyrimidine nucleotides within the erythrocytes that precipitate and cause basophilic stippling. Diagnosis is confirmed by a decrease in the nucleotide OD260:OD280 ratio and measurement of enzyme activity. Disease severity is variable, but the patients typically manifest lifelong hemolysis with the expected sequelae.27
Adenosine deaminase (ADA) is a purine catabolic enzyme that converts adenosine to inosine. ADA deficiency causes inherited severe combined immunodeficiency. In contrast, elevations in ADA cause hemolytic anemia. Studies show that ADA amplification in reticulocytes results from increased translation of ADA mRNA.28
Heme oxygenase-1 is an enzyme involved in the conversion of heme to bilirubin. Heme oxygenase-1 further provides protection against certain oxidative stresses. Two pediatric patients were described with severe growth retardation, asplenia, an abnormal coagulation/fibrinolysis system, and persistent hemolytic anemia. This enzyme deficiency may cause the unique findings of erythrocyte fragmentation and intravascular hemolysis in the absence of hyperbilirubinemia or decreased haptoglobin potentially due to a defect in the macrophage’s ability to catabolize heme.29
Lecithin cholesterol acyltransferase (LCAT) is an enzyme involved in lipoprotein metabolism. Deficiencies of LCAT result in erythroid membrane defects caused by excess unesterified cholesterol. The patients also develop renal disease with proteinuria, corneal opacifications, and their serum lipid profiles include decreased serum HDL levels (55–10% normal).30
ERYTHROID MEMBRANE DEFECTS
The RBC membrane comprises integral and peripheral proteins distributed in the context of a lipid bilayer. Integral membrane proteins interact to form a lattice-like structure (cytoskeleton) at the cytoplasmic surface of the lipid bilayer that is responsible for the strength and deformability of the red blood cell. Band 3, a protein that functions as an anion exchanger (AE1), is the major protein that physically links the lipid bilayer to the underlying membrane cytoskeleton. The cytoskeleton proteins include spectrin, ankyrin, actin, band 3, band 4.1, and band 4.2. Other red cell membrane proteins serve roles in maintaining osmotic equilibrium or have adhesive properties. The exact function of a number of erythroid membrane proteins remains vague.
Immune-mediated hemolysis results from antibodies directed toward erythrocyte membrane proteins. Approximately 24 proteins are largely responsible for transfusion-related alloimmunity (see Chapter 24). Nonimmune hemolysis may also be due to rare erythroid phenotypes involving those proteins. Nonimmune hemolysis due to the Rh-null phenotype is generally mild and well compensated with a reticulocyte count below 10%. The red cell morphology may be stomatocytic or spherocytic, and the RBC osmotic fragility is increased.31 Weak expression of the Kell blood group results in the so-called “McLeod phenotype.” This phenotype is X-linked and occurs with relatively high frequency in individuals who have chronic granulomatous disease (CGD). Neurodegeneration and acanthocytosis are the characteristic features of the McLeod phenotype. The associated hemolysis is mild with slightly elevated reticulocyte counts.32
The first indication that a patient may have a membrane abnormality as a cause for hemolysis usually comes from microscopic examination of the peripheral blood smear. As shown in Table 3.2, the presence of spherocytes, elliptocytes, stomatocytes, acanthocytes, or pyropoikilocytes may be the primary alert for an underlying membrane defect.
Hereditary spherocytosis (HS) is the most common hereditary anemia among people of northern European descent, occurring at a frequency of 1 in 5,000. It is commonly caused by mutations in the genes that encode the components of the erythroid cytoskeleton (α- or β-spectrin, ankyrin, band 3, protein 4.2). Autosomal recessive or dominant patterns of inheritance have been identified, and a positive family history is gathered in over half of the cases. Patients are usually diagnosed in childhood with the clinical triad of anemia, jaundice, and splenomegaly. Spherocytes are identified by their small size (low MCV) and absence of the central pallor seen in normal erythrocytes. Immune-based spherocytosis is ruled out by negative DAT testing.
In most cases, the clinical presentation and hematological parameters are sufficient to make the diagnosis.33 If the diagnosis is subtle or complicated, confirmation of the diagnosis using assays of membrane fragility, electrophoretic quantitation of membrane proteins, or genetic analyses may be considered. The osmotic fragility test for HS has been used to detect hemolysis by measuring the fraction of total hemoglobin released from red cells at progressively more dilute salt concentrations. Hemolysis occurs in circulating HS spherocytes at salt concentrations that do not affect normal RBC. A cryohemolysis test may also be used to detect increased hemolysis in HS erythrocytes. Red cells are suspended in a hypertonic solution, briefly heated to 37°C, and then cooled to 4°C for 10 minutes. A widely separated degree of hemolysis between spherocytes and normal cells is seen with the cryohemolysis test, and asymptomatic disease carriers may also be identified.34 Although interesting historically, membrane fragility tests lack sensitivity and specificity, and therefore should be ordered conservatively as they add little to the evaluation of a well-prepared blood smear.
Hereditary elliptocytosis (HE) is endemic in areas of Africa and Asia. The disease also results from mutations in the α-spectrin, β-spectrin, and band 4.1 genes. In the homozygous state, hemolysis may be lifelong and exacerbated by acute or chronic illnesses.35 In the heterozygous state, people with HE have no clinical syndrome, with slight reticulocytosis and the characteristic abnormalities of the red blood cell morphology providing the only clues toward diagnosis.
Hereditary pyropoikilocytosis is a severe type of HE resulting from a mutation in either protein 4.1 or α-spectrin. The usual presentation involves mild to moderate hemolytic anemia with evidence of marked poikilocytosis. The spectrin in these abnormal cells has an increased sensitivity to thermal denaturation, and the cells exhibit mechanical fragility. As a result, the red cell volume distribution is broad, and a striking number of fragmented cells and microspherocytes are observed on the peripheral smear.36
Hereditary stomatocytosis is identified by a pinched rather than circular area of central pallor in erythrocytes. While all the patients share the common clinical feature of stomatocytes, research in defining the underlying cause of the morphological change has resulted in more specific clinical descriptions including dehydrated hereditary stomatocytosis (xerocytosis), overhydrated hereditary stomatocytosis, and cryohydrocytosis. The common features include hemolysis and red cell cation leaks and variable severity of hemolysis.37 Most importantly, splenectomy is not helpful and may result in a high risk of thromboembolic disease.38
Acanthocytosis on the peripheral smear may be caused by abnormal lipids in hepatic cirrhosis, or by other abnormalities in lipids or band 3. Abetalipoproteinemia is a rare genetic disorder resulting in hypolipidemia, acanthocytosis, malabsorption of fat, retinitis pigmentosa, and ataxia. Infants with this autosomal recessive disorder are normal at birth but soon develop steatorrhea, abdominal distension, and growth failure. Retinitis pigmentosa and ataxia appear between ages 5 and 10 years and are progressive. Therefore, acanthocytosis seen on the peripheral smear of children in the absence of hemolysis or liver disease should alert the clinician to consider associated neurodegenerative conditions.39
TREATMENT OPTIONS FOR CONFIRMED HEMOLYSIS
Therapeutic strategies for hemolytic anemia are determined by the underlying cause of red cell destruction, the magnitude of the anemia, and cardiopulmonary status of the patient.
For extrinsic causes, the treatment plan usually becomes obvious at the time of diagnosis. Immune-mediated hemolysis may require immunoglobulin infusion, corticosteroids, or other immunosuppressive therapies. Transfusion therapy (packed RBCs) should be avoided unless absolutely necessary. However, RBC transfusion should not be withheld if a severely compromised cardiopulmonary status exists even when compatibility of the donor cells is incomplete. In those rare cases, the department of transfusion medicine involved should be asked to identify the most compatible product available, and the transfusion should be closely monitored. Infections are treated with antimicrobials. For thrombotic thrombocytopenic purpura, plasmapheresis is specifically indicated along with immunosuppressive therapy to reverse the depletion of ADAMTS-13 that results in excessive high molecular weight multimers of von Willebrand factor triggering disseminated platelet thrombi. Prevention of immune- or G6PD-mediated hemolysis involves discontinuation or avoidance of the associated medications. Monitoring urine hemoglobin and hemosiderin levels can determine the response to intravascular hemolysis therapy.
One acquired intrinsic red cell disorder, PNH, can be effectively treated by eculizumab, an anti-C5 antibody that can control the intravascular hemolysis by converting it to a milder extravascular hemolysis (Chapter 6). Eculizumab has also been shown to reduce the thrombosis risk that accompanies the intravascular hemolysis. Immunization with anti-meningococcal vaccination is required before eculizumab is prescribed.40,41 Eculizumab has also been reported to reverse thrombotic microangiopathy and renal failure in patients with atypical hemolytic uremic syndrome.42
Despite the considerable advances in defining these genetic diseases at the molecular level, equally specific treatment regimens for other intrinsic causes of hemolysis are lacking. In general, intrinsic causes of hemolysis are inherited, and they are present during infancy or childhood. In those cases, prognosis and treatment are complex, as the hemolytic picture may change over time. The first question to ask is whether treatment is necessary. Chronic hemolysis may only require a yearly clinical evaluation of the CBC, ARC, and blood smear to determine if the patient is able to maintain adequate levels of erythropoiesis. Parvovirus infections in these patients may result in acute worsening of their anemia due to a sudden decrease in their erythropoiesis. Folic acid should be given to all patients with chronic hemolysis (1 mg/day) because this vitamin is consumed with the accelerated production of erythrocytes. Whether folic acid supplementation is still necessary in the United States where the fortification of food folate has occurred since the mid-1990s has not been evaluated. Transfusion regimens should be tailored for individual patients, and iron overload should be anticipated. Even in the absence of transfusion, iron overload may result from ineffective erythropoiesis. The increased metabolism of heme also leads to a significant increase in pigmented gallstone formation.
Treatments such as splenectomy or bone marrow transplantation should be reserved for marked hemolysis producing life-threatening anemia. As stated above, splenectomy should be discouraged for patients with hereditary stomatocytosis syndromes.38 When severe hemolysis is due to other membrane defects, splenectomy may be beneficial and indicated. In children, splenectomy should be delayed until the age of 6 (if possible) due to the increased risk of sepsis. In general, splenectomy risks must be compared to those associated with lifelong transfusion. After a splenectomy is performed, special care must be taken to compensate for the loss of splenic function. The spleen is responsible for the clearance of encapsulated bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, or Neisseria meningitidis. The combined use of pneumococcal polysaccharide immunization and early empiric antibiotic therapy offers a high level of protection for post-splenectomy patients. It is estimated that sepsis is fatal in 40% to 50% of all splenectomized patients. Within that group, children with thalassemia and sickle cell syndromes have the highest risk of death.43 In all cases, patients must be informed that the asplenic state carries a significant risk of overwhelming and life-threatening infection.
CONCLUSION
A broad range of genetic and acquired diseases are manifested by hemolysis. The differential diagnosis is useful in developing diagnostic and therapeutic strategies and should be thought of in terms of intrinsic or extrinsic causes of erythrocyte damage. A careful search for the cause of hemolysis should be pursued because treatments are so different. When a common cause of hemolysis is not found, an underlying enzyme or membrane defect should be sought.
Clinical severity in all cases of hemolysis is determined by the rate of red cell destruction, and the host’s ability to compensate by producing fresh erythrocytes. Disease can vary from a subtle and clinically silent syndrome to hemolysis of sufficient intensity to dominate the clinical picture and even cause death if left untreated. Every therapeutic plan should be designed for both the severity of disease as well as the cause of hemolysis.
HELPFUL INTERNET SITES
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References