Key points

Introduction

Primary abnormalities of the erythrocyte membrane lead to a variety of clinical syndromes, including hereditary spherocytosis (HS), hereditary elliptocytosis, and related disorders. Clinical and laboratory manifestations, as well as associated molecular defects, of these disorders vary widely. Abnormalities of erythrocyte shape on peripheral blood smear often provide clues to the underlying pathobiology and clinical diagnosis of the underlying disorder.

Introduction

Primary abnormalities of the erythrocyte membrane lead to a variety of clinical syndromes, including hereditary spherocytosis (HS), hereditary elliptocytosis, and related disorders. Clinical and laboratory manifestations, as well as associated molecular defects, of these disorders vary widely. Abnormalities of erythrocyte shape on peripheral blood smear often provide clues to the underlying pathobiology and clinical diagnosis of the underlying disorder.

Hereditary spherocytosis

HS syndromes are a group of disorders associated with a primary defect in erythrocyte membrane proteins. HS was first described based on the finding of spherocytes, characteristic erythrocytes lacking central pallor, on peripheral blood smear. HS occurs worldwide in all racial and ethnic groups. It is the most common inherited anemia in individuals of northern European ancestry, affecting approximately 1 person per 2500 individuals in the United States. Clinical, laboratory, and molecular heterogeneity characterize the HS syndromes.

The principal abnormality in HS erythrocytes is loss of membrane surface area relative to intracellular volume, which leads to spherically shaped erythrocytes with decreased deformability. The loss of surface area results from increased membrane fragility due to primary and secondary abnormalities in erythrocyte membrane proteins, particularly ankyrin, α- and β-spectrin, band 3, and protein 4.2 ( Fig. 1 ). Increased erythrocyte fragility leads to vesiculation and membrane loss. Splenic destruction of poorly deformable HS erythrocytes is the primary cause of hemolysis experienced by HS patients.

The erythrocyte membrane. A model of the major proteins of the erythrocyte membrane is shown: α- and β-spectrin, ankyrin, band 3 (the anion exchanger), 4.1 (protein 4.1) and 4.2 (protein 4.2), actin and glycophorin.

( From Perrotta S, Gallagher PG, Mohandas N. Hereditary spherocytosis. Lancet 2008;372:1412; with permission.)

HS is inherited as in autosomal-dominant manner in approximately two-thirds of patients, associated with mutations in the ankyrin, β-spectrin, or band 3 genes. In the remaining patients, inheritance is not dominant because of autosomal recessive inheritance or a de novo mutation. Autosomal recessive inheritance is associated with mutations of either the α-spectrin or protein 4.2 genes. Several de novo mutations have been reported in the HS genes.

Clinical manifestations and classification

Clinical manifestations of the spherocytosis syndromes vary widely. Typical HS is associated with pallor, jaundice, splenomegaly, anemia, reticulocytosis, spherocytes on peripheral blood smear, positive osmotic fragility or flow cytometric analysis of eosin-5-maleimide-labeled erythrocytes (EMA binding) (see below), and a positive family history. Mild, moderate, and severe forms of HS have been defined according to the severity of anemia and the degree of compensation for the hemolysis ( Table 1 ).

HS may present at any age, but typically it presents in childhood. Anemia is the most frequent finding at presentation (50%), followed by splenomegaly, jaundice, or a positive family history. Most HS patients have incompletely compensated hemolysis with mild-to-moderate anemia that is asymptomatic except for fatigue and pallor. Jaundice is visible at some time in over half of HS patients, usually in association with viral infection or other stress. The jaundice is typically acholuric (ie, unconjugated hyperbilirubinemia without detectable bilirubinuria). By late childhood, palpable splenomegaly is found in most HS patients. Approximately 25% of HS patients have compensated hemolysis (ie, erythrocyte production and destruction are balanced). These patients are not anemic and are usually asymptomatic. The remaining 5% to 10% of HS patients experience moderate-to-severe anemia. This category includes patients with both dominant and recessive HS. The most severely affected patients are transfusion-dependent and almost always have recessive HS. Chronically transfused patients are at risk for developing complications of recurrent transfusion and iron overload.

HS may present in the neonatal period. Some patients present with significant neonatal jaundice requiring phototherapy or even exchange transfusion. Others present with significant anemia presenting in the first few weeks of life and may require several transfusions in infancy. Most of these patients become transfusion-independent during the first year of life. A subset of patients presents with severe anemia in utero or immediately after birth and requires red blood cell transfusion. These patients frequently remain transfusion-dependent and suffer from severe HS.

Initial assessment/physical examination

Initial assessment of a patient with suspected HS includes a detailed family history and questions about a history of pallor, jaundice, anemia, gallstones, and splenectomy. Physical examination includes attention to pallor, scleral icterus, and splenomegaly.

After diagnosing a patient with HS, family members should be examined for the presence of HS.

Laboratory findings

Laboratory findings in HS are heterogeneous. Initial studies in a patient with suspected HS include:

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  Complete blood count/erythrocyte indices

  
  
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  Peripheral blood smear

  
  
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  Reticulocyte count

  
  
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  Bilirubin

  
  
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  Flow cytometric analysis of eosin-5-maleimide-labeled erythrocytes (EMA binding) or incubated osmotic fragility

Peripheral Blood Smear

Typical HS patients have blood smears with easily detectable spherocytes (ie, erythrocytes lacking central pallor), which are distinctive but not diagnostic ( Fig. 2 A ). Occasionally, only a few spherocytes are seen on peripheral smear, or, in contrast, numerous small, dense spherocytes and bizarre erythrocyte morphology with anisocytosis and poikilocytosis may be observed in severely affected patients (see Fig. 2 B). Specific findings have been identified in some patients with specific membrane protein abnormalities such as pincered erythrocytes (band 3) or spherocytic acanthocytes (β spectrin).

Peripheral blood smears in hereditary spherocytosis. ( A ) Typical hereditary spherocytosis. Characteristic spherocytes lacking central pallor are seen. ( B ) Severe, recessively inherited spherocytosis. Numerous small, dense spherocytes and bizarre erythrocyte morphology with anisocytosis and poikilocytosis associated with severe hemolysis are seen.

EMA Binding and Osmotic Fragility

The EMA (eosin-5-maleimide) binding test is a flow cytometry-based analysis of the relative amounts of fluorescence, reflecting the amount EMA binding to band 3 and Rh-related proteins in the erythrocyte membrane. The basis of the test is the reduction in band 3 and related membrane proteins, leading to a decrease in fluorescence intensity, typically to approximately 65% of normal ( Fig. 3 A ). Although defects of band 3 protein are only found in approximately 25% of typical HS patients, decreased fluorescence intensity is also observed in HS erythrocytes with defects in ankyrin and spectrin, thought to be due to transmission of long range effects of varying protein defects across the membrane, thereby influencing EMA binding to band 3. In laboratories with the ability to perform Fluorescence-activated cell sorting-based studies, EMA binding has high sensitivity and specificity and is simple and rapidly performed. EMA binding results are not influenced by shipping or storage for several days, and may be suitable for study of patients who have been recently transfused.

Testing in hereditary spherocytosis. ( A ). EMA binding. Histogram of fluorescence of EMA-labeled erythrocytes from a normal control and a patient with typical hereditary spherocytosis. Decreased fluorescence in observed from HS erythrocytes. ( B ) Osmotic fragility curves in hereditary spherocytosis. The shaded region is the normal range. Results representative of typical and severe spherocytosis are shown. A tail, representing fragile erythrocytes conditioned by the spleen, is common in spherocytosis patients prior to splenectomy.

The osmotic fragility (OF) test, which measures the in vitro lysis of erythrocytes suspended in solutions of decreasing osmolarity, is frequently utilized in the diagnosis of HS. Because of the loss of membrane surface area relative to intracellular volume, HS erythrocytes are unable to withstand the introduction of small amounts of free water that occur when they are placed in increasingly hypotonic saline solutions. As a consequence, HS erythrocytes hemolyze more readily than normal erythrocytes at any saline concentration. Hemolysis is determined by measuring the amount of hemoglobin released from red cells into the extracellular fluid. Approximately 25% of HS patients have a normal OF on freshly drawn blood, with the OF curve approximating the number of spherocytes on peripheral smear. After incubation at 37°C for 24 hours, HS erythrocytes lose membrane surface area more readily than normal, because their membranes are leaky and unstable, revealing the membrane defect in OF testing. When the spleen is present, a subpopulation of fragile erythrocytes that have been conditioned by the spleen form the tail of the OF curve that disappears after splenectomy (see Fig. 3 B).

Neither EMA binding nor OF testing detects all HS patients. Both tests struggle in the identification of cases of mild HS, with OF suffering from poorer sensitivity. Other conditions such as congenital dyserythropoietic anemia type 2, southeast Asian ovalocytosis, and hereditary pyropoikilocytosis may yield reduced fluorescence in EMA binding. OF testing is unreliable in patients who have small numbers of spherocytes, including those who have been recently transfused, and it is abnormal in other conditions where spherocytes are present. Although some laboratories in Europe combine EMA-binding studies with the acidified glycerol lysis test to improve diagnostic sensitivity, this test is not available in most clinical laboratories in North America.