Cell Identification
VP
BS
-14
Participants
Identification No. % Evaluation
Spherocyte 885 97.3 Educational
Erythrocyte, normal 19 2.1 Educational
The arrows point to spherocytes, correctly identified by 97.3% of the participants. Spherocytes are
erythrocytes that are hyperchromic and lack central pallor due to their spherical shape. This contrasts
with normal erythrocytes, which have a biconcave shape and visible central pallor on smear preparations.
Spherocytes are often smaller than normal erythrocytes and may be very small (microspherocytes,
defined as <4 µm in diameter). Spherocytes form as a consequence of membrane loss, resulting in a
decreased ratio of cell surface membrane to cytoplasmic volume. Increased spherocytes are most
commonly seen in cases of immune hemolytic anemia and hereditary spherocytosis.
4
VP
BS
-15
Participants
Identification No. % Evaluation
Polychromatophilic RBC 887 97.7 Educational
Macrocyte oval/round 18 2.0 Educational
The arrowed cell is a polychromatophilic erythrocyte, correctly identified by 97.7% of the participants.
Polychromatophilic red cells are non-nucleated cells that are larger than normal erythrocytes, lack central
pallor, and have characteristic gray-blue cytoplasm. These cells correspond to reticulocytes, which can be
identified using supravital stains, and represent the final stage of red cell maturation. Normal blood
smears are expected to contain occasional polychromatophilic erythrocytes. Increased numbers indicate
that the bone marrow is actively working to increase red cell production, usually in response to bleeding or
hemolysis.
5
VP
BS
-16
Participants
Identification No. % Evaluation
Eosinophil 905 99.5 Educational
The arrowed cell is an eosinophil, correctly identified by 99.5% of the participants. Eosinophils are
leukocytes with characteristic bright, orange-red, refractile cytoplasmic granules of uniform size. The
granules typically do not overlie the nucleus. The nucleus usually contains two round to oval lobes of
equal size connected by a very thin filament.
6
VP
BS
-17
Participants
Identification No. % Evaluation
nRBC, normal/abnormal morphology 908 99.8 Educational
The arrowed cells are nucleated erythrocytes, correctly identified by 99.8% of the participants. Nucleated
erythrocytes seen in blood smears are typically at the orthochromic normoblast stage of maturation, which
is characterized by a round nucleus with markedly dense chromatin. Some nuclear irregularity may be
seen and does not necessarily indicate dyserythropoiesis, as the nucleus may develop an abnormal
shape as it migrates from the bone marrow into the blood. As seen in these examples, the cytoplasm of
circulating nucleated red cells typically displays polychromasia, and the cells are somewhat larger than
mature erythrocytes, reflecting their more immature maturation stage.
7
V
PB
S-1
8
Participants
Identification No. % Evaluation
Platelet, normal 907 99.7 Educational
The arrows point to normal platelets, correctly identified by 99.7% of the participants. Platelets are blue-
gray fragments of megakaryocytic cytoplasm that typically measure 1.5 to 3 µm in diameter and contain
fine, purple-red granules. Large platelets measure approximately 4 to 7 µm in diameter. The term “giant
platelet” is used when the platelet is larger than the size of an average red cell, assuming a normal MCV.
All of the platelets in this field demonstrate normal size and cytoplasmic granulation. No large or giant
platelets are seen.
8
VPBS-B 2012: Hereditary Spherocytosis
1- Education All material is © 2012 College of American Pathologists, all rights reserved.
Case History
The patient is a full-term, large for gestational age, baby boy with jaundice. There is a strong and extended
family history of spherocytosis on the father's side. Laboratory data include: WBC = 10.8 x 109/L; HGB =
7.6 g/dL; MCV = 90.8 fL; RDW = 23.3%; PLT = 849 x 109/L; Reticulocyte = 20.3%; Reticulocyte
Absolute = 537.8 K/UL; and elevated MCHC.
OVERVIEW OF HEMOLYTIC ANEMIA
Anemia is defined as a decrease in the number of red blood cells or decrease in blood hemoglobin
concentration that may result from a variety of causes such as red cell loss (eg, bleeding), decreased
production by the bone marrow, or increased destruction (eg, hemolytic). Case VPBS-B represents a patient
with hemolytic anemia secondary to hereditary spherocytosis (HS). Hemolytic anemia develops when the
survival of the red cells in the circulation is decreased from the normal life span of 110 to 120 days due to
their destruction within the circulation (intravascular hemolysis), by premature phagocytosis and
destruction by the spleen and reticuloendothelial system (extravascular hemolysis), or a combination of
both processes. Patients may have ongoing hemolysis without development of anemia (compensated
hemolysis) due to the ability of the bone marrow to increase the proliferation and differentiation of red cell
precursors by six- to eight-fold. However, when red cell life span is markedly shortened, usually to 15 to 20
days or less, the bone marrow is no longer able to adequately compensate for the red cell destruction. This
will lead to development of anemia as fewer red cells are produced than are destroyed. In addition, anemia
may develop in patients who have a longer red cell life span in situations where there is an acute
impairment of bone marrow function such as due to infection or drug exposure, leading to decreased red
cell production. An increase in red cell destruction due to activation of splenic function (usually due to viral
infection) may also lead to acute development of anemia in a patient with a hemolytic process that is
usually compensated.
CLASSIFICATION OF HEMOLYTIC ANEMIA
Hemolytic anemias may be subclassified in multiple ways, taking into account the various pathophysiologic
mechanisms underlying the anemia. It is often useful to think of the cause of red cell destruction when
investigating the etiology of hemolysis (Table 1). In this paradigm, anemia may be separated into either an
intrinsic or intracorpuscular defect of the red cell or hemolysis due to an extrinsic or extracorpuscular
process. An extracorpuscular defect implies that if the red cells were removed and transfused into another
patient they would have a normal life span, as the hemolysis is due to a process occurring within the
patient that is independent of the red cell (eg, thrombi that disrupt red cell integrity, hypersplenism).
Extracorpuscular hemolysis is usually an acquired disorder, and hemolysis will be decreased by treatment of
the underlying cause. In contrast, intracorpuscular hemolysis includes both acquired and inherited disorders
that directly affect red cell structure or essential functions. Affected red cells will have a shortened life
span even after being transfused into an unaffected patient due to the inherent abnormalities of the red
cell.
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Many intrinsic hemolytic states are due to an inherited red blood cell defect, including hemoglobinopathies,
red cell membrane defects and enzymatic defects (Table 1). Thus, a good clinical history can provide a
great deal of insight into the underlying pathophysiology of the hemolysis, particularly if a family history is
identified, suggesting an inherited disorder. Examination of the peripheral blood smear is also an essential
component in evaluating hemolysis. Specific morphologic features, including identification of spherocytes,
poikilocytes, elliptocytes, stomatocytes, sickle cells, intraerythrocytic parasites, target cells, acanthocytes,
or prominent basophilic stippling can all provide clues as to the possible cause of hemolysis (Table 2). It is
important to utilize a well prepared blood smear that is free of artifacts to ensure optimal identification of
the specific red cell morphologic features.
Table 1 – Pathophysiologic Causes of Hemolysis
Intracorpuscular (intrinsic) causes of hemolysis:
Inherited defects
1. Red cell membrane defects a. Hereditary spherocytosis b. Pyropoikilocytosis
2. Enzymatic defects a. Glycolytic pathway defects – pyruvate kinase deficiency, etc.
3. Hemoglobinopathies a. Qualitative defects – sickle cell disease, hemoglobin C disease, hemoglobin E disease,
etc. b. Quantitative defects – thalassemias
Acquired defects
1. Paroxysmal nocturnal hemoglobinuria (PNH)
Extracorpuscular (extrinsic) causes of hemolysis:
1. Immune hemolytic anemias 2. Infections – malaria, etc. 3. Physical agents – burns, chemicals, toxins, etc. 4. Microangiopathic processes – disseminated intravascular coagulations (DIC), thrombotic
thrombocytopenia purpura (TTP), hemolytic uremic syndrome (HUS), etc. 5. Splenic sequestration/hypersplenism
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Table 2 – Morphologic Features Associated with Specific Causes of Hemolysis
Spherocytes
Hereditary spherocytosis
Immune based hemolysis
Severe thermal injury or burn
Spider, bee, or snake venom
Clostridium septicemia
Poikilocytes
Microangiopathic and macroangiopathic anemias
Hereditary pyropoikilocytosis
Sickle Cells
Sickle cell anemia and other HbS hemoglobinopathies
Basophilic Stippling
Thalassemias
Lead poisoning
Pyrimidine 5’nucleosidase enzyme deficiency
Target Cells
Hemoglobinopathies (HbS, HbC, etc.)
Thalassemias
Acanthocytes
Uremia
Pyruvate kinase deficiency
Intracellular Parasites
Malaria
Babesiosis
Hb = hemoglobin
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Another method of classification of hemolytic anemias is based on clinical presentation, such as separating
hemolytic anemias into inherited or hereditary causes versus those that are acquired (Table 1). Acquired
causes include most extrinsic processes as well as an uncommon acquired intrinsic defect in red cell
membrane proteins called paroxysmal nocturnal hemoglobinuria (PNH). This approach to classification is
often helpful in determining therapeutic approaches to hemolytic anemia but may also be useful in guiding
the workup of a hemolytic process.
LABORATORY FINDINGS INDICATIVE OF HEMOLYSIS
The laboratory approach to establishing a diagnosis of hemolysis depends on demonstrating sequelae of
increased red cell destruction. Often times, patients will not come to the attention of a physician until
anemia develops, and many patients with low level hemolysis will not be recognized until something
exacerbates the hemolysis or impairs the marrow’s ability to compensate for the shortened red cell life
span. This is typically manifested by increases in serum lactate dehydrogenase (LDH) and unconjugated
(indirect) bilirubin as well as decreased serum haptoglobin levels. Other tests that may be useful in
documenting hemolysis, primarily intravascular hemolysis, include detection of hemoglobinemia,
hemoglobinuria, and hemosiderinuria resulting from increased red cell breakdown.
Once hemolysis is identified, the first step in working up the cause of hemolysis is often performance of a
Coombs’ test (direct antiglobulin test) to identify anemias that arise due to immune-based hemolysis
(Coombs’ test positive) versus those that are not immune based (Coombs’ test negative). Because
hemolysis causes a decrease in red cell life span, the bone marrow will compensate by increasing
erythropoiesis. This will lead to erythroid hyperplasia in the marrow as well as early release of immature red
cells into the circulation. These immature red cells may be identified in the peripheral blood as reticulocytes
or nucleated red blood cells. Reticulocytes may be identified by supravital staining and are able to be
detected by many CBC analyzers as well as by manual methods. In peripheral blood smears, reticulocytes
are often macrocytic and demonstrate distinct blue to blue-gray coloration (polychromasia) that reflects the
presence of RNA, the Golgi complex and mitochondria within the immature red cell cytoplasm (Figure 1).
Enumeration of the number of reticulocytes as a percentage of all red cells yields the reticulocyte count.
Reticulocyte counts performed by manual methods using supravital stains have limited accuracy. In
contrast, CBC analyzers provide a very accurate reticulocyte count due to the higher number of red cells
sampled. The presence of nucleated red blood cells may also reflect premature release of erythrocytes into
the circulation in an attempt to compensate for anemia. Most often these will contain a condensed or
pyknotic nucleus (orthochromic normoblasts), but in cases of severe hemolysis earlier forms
(polychromatophilic or basophilic normoblasts) may be seen in the peripheral blood smear (Figure 2).
VPBS-B 2012: Hereditary Spherocytosis
5- Education All material is © 2012 College of American Pathologists, all rights reserved.
Figure 1. Polychromatophilic red cells. The three arrowed red cells show the characteristic larger size and blue-gray or basophilic color compared to the other erythrocytes in the image.
Figure 2. Nucleated red cells (orthochromic normoblasts). The two arrowed cells are circulating nucleated red cells that have small nuclei with condensed chromatin and pink to blue-gray cytoplasm.
SPHEROCYTES
The patient presented in case VPBS-B has a large number of spherocytes on the peripheral smear. A
spherocyte is defined as an abnormal red cell that is spherical, resulting in a greater density of hemoglobin
in the center of the red cell compared to a normal biconcave-shaped red cell that lacks central pallor.
Usually spherocytes are slightly smaller than normal red blood cells and occasionally they may be quite
small (microspherocytes) (Figure 3).
Figure 3. Spherocytes. The two arrowed red cells are spherocytes that appear smaller than the other red cells in the image and appear round with dense and homogenous cytoplasm that lacks central pallor.
Spherocytes occur due to loss of membrane from the red cell resulting in a decrease in the cell surface to
cytoplasmic ratio. To accommodate the loss in surface membrane in the setting of a constant cytoplasmic
volume the red cell is forced to assume a spherical shape. Spherocytes have a shortened life span in the
VPBS-B 2012: Hereditary Spherocytosis
6- Education All material is © 2012 College of American Pathologists, all rights reserved.
circulation because they lose the ability to deform as they pass through the spleen and small vessels of the
circulation and thus are lysed.
Spherocytes may be seen in a variety of different disease states (Table 3), and identification of spherocytes
in a blood smear mandates Coombs’ testing to rule out the possibility of an autoimmune or other antibody-
mediated hemolysis. HS is the most common cause of spherocytosis. Other causes of increased
spherocytes include septicemia with Clostridia species, severe burns or thermal injuries, as well as exposure
to venoms or severe hypophosphatemia. An appropriate clinical history should help to exclude those
differential diagnostic possibilities.
Table 3 –Conditions Associated with Increased Spherocytes
Common
Hereditary spherocytosis
Immune (warm antibody) based hemolysis
ABO incompatibility in neonates
Uncommon
Transfusion reaction hemolysis
Severe burns or thermal injury
Spider, bee, or snake venom
Clostridium sepsis
Acute red cell oxidation injury (glucose-6-phosphate dehydrogenase deficiency)
Severe hypophosphatemia
HEREDITARY SPHEROCYTOSIS
Overview of HS:
Hereditary spherocytosis (HS) is the most prevalent inherited cause of hemolytic anemia in patients of
Northern European ancestry and is usually the underlying etiology for increased spherocytes in the setting
of hemolysis when immune-based destruction is excluded. HS infrequently occurs in other ethnic groups. In
the United States, HS is seen in a frequency of approximately 1 out of 3,000 - 5,000. HS is typically
inherited as an autosomal dominant disorder, but other inheritance patterns may occur.
Red Cell Membrane Defects in HS:
Hereditary spherocytosis is a disorder of the red cell membrane. The normal red cell membrane is composed
of a lipid bilayer with an underlying protein cytoskeleton that acts to maintain the biconcave disk shape.
VPBS-B 2012: Hereditary Spherocytosis
7- Education All material is © 2012 College of American Pathologists, all rights reserved.
The biconcave shape allows for cellular deformability so that the red cell can pass undamaged through
small vessels and the splenic sinusoids as well as react to changes in pH, oxygen tension, and osmotic
gradients while in the circulation. The lipid bilayer is composed of phospholipids, cholesterol, and
glycolipids. These lipids arrange around the protein cytoskeleton, which contains a large number of proteins
that chemically interact to form a meshwork that allows for both vertical and horizontal interactions (Figure
4). The major proteins found in the red cell membrane skeleton include spectrin, actin, ankyrin, protein
4.1R, protein 4.2, and protein band 3 (also termed protein AE1). Spectrin is a principle component of the
membrane, comprising approximately 30% of the proteins in the red cell membrane. Spectrin is composed
of alpha and beta chains that interact to form long helical structures that support the biconcave shape of
the red cell and allow lateral or horizontal movement of the other cytoskeletal proteins. Spectrin binds
directly to ankyrin. In turn, ankyrin binds to protein band 3, and protein band 3 will bind to protein 4.2,
allowing for direct connection of the protein cytoskeleton to the lipid bilayer and vertical movement of the
protein cytoskeleton.
Figure 4. Schematic diagram of the red cell membrane. The figure shows a schematic representation of the interactions between the proteins of the red cell cytoskeleton and the lipid membrane demonstrating vertical and horizontal interactions.
Red cell membrane disorders result from alterations in either the binding qualities or the quantity of the
individual proteins within this red cell protein meshwork (Table 4). This leads to disruption of the typical
interactions between proteins and results in an uncoupling of the protein meshwork from the lipid bilayer
and normal binding of the integral membrane proteins to each other. This protein uncoupling results in
instability of the lipid bilayer so that it tends to form small vesicles or blebs that are subsequently removed
by the spleen. This cumulative membrane loss causes loss of the normal biconcave disc structure and
formation of spherocytes, as the decreased membrane is stretched to cover the constant volume of red cell
Horizontal Interactions
Band 3 (AE1)
Ve
rtic
al I
nte
rac
tio
ns
VPBS-B 2012: Hereditary Spherocytosis
8- Education All material is © 2012 College of American Pathologists, all rights reserved.
cytoplasm. Thus, the spleen plays a critical role in the development of hemolysis as it is responsible for
removal of the membrane microvesicles that lead to the formation of spherocytes as well as the
subsequent final destruction of the inflexible spherocytes.
Table 4 – Red Cell Membrane Protein Defects in Hereditary Spherocytosis
Protein Deficiencies
Ankyrin
Spectrin
Combined spectrin and ankyrin deficiencies
Band 3
Protein 4.2
Protein Dysfunction
β Spectrin to protein 4.1 binding
Band 3 binding to the lipid bilayer or ankyrin
Hereditary spherocytosis can be caused by abnormalities in several of the different components of the red
cell skeletal membrane including deficiencies in spectrin, ankyrin, band 3, and protein 4.2. Initially, spectrin
deficiency was thought to be the major underlying cause of HS; however, it has been found that in many
cases spectrin deficiency is due to qualitative or quantitative deficiencies of the other proteins which help
to integrate spectrin into the cell membrane leading to secondary loss of spectrin from the cell rather than
an inherited deficiency of spectrin production. For example, hereditary ankyrin defects (one of the most
common defects observed in HS) are often associated with a decreased amount of red cell spectrin due to
the lack of tethering of spectrin to the red cell protein meshwork, leading to loss of the protein. Studies of
red cell membrane protein synthesis and function in patients with hereditary spherocytosis have
demonstrated a variety of protein abnormalities including spectrin deficiency alone, combined spectrin and
ankyrin deficiency, band 3 deficiency, protein 4.2 deficiency, and some cases that have no obvious
biochemical abnormality but have protein dysfunction in that the proteins do not bind to each other or the
lipid membrane appropriately (Table 4). Each of these disease subsets is associated with specific mutations
that have specific ethnic associations, genetic findings and degrees of associated hemolysis (Table 5).
Although there are a wide variety of different proteins and molecular defects that underlie HS, the common
pathophysiologic defect appears to be the weakening of the protein-to-protein interactions and the resultant
lack of linkage of the lipid bilayer to the proteins of the cellular cytoskeleton in all cases.
VPBS-B 2012: Hereditary Spherocytosis
9- Education All material is © 2012 College of American Pathologists, all rights reserved.
Table 5 – Genetic Heterogeneity of Hereditary Spherocytosis
Genetic defect Hemolysis Inheritance pattern Frequency
Spectrin α-chain Severe Autosomal recessive Rare
Spectrin β-chain Mild to moderate Autosomal dominant Common, often associated with ankyrin deficiency ~20% of cases
Ankyrin deficiency Mild to severe Autosomal dominant Common ~60% of cases
Band 3 deficiency Mild to moderate Autosomal dominant Common ~20% of cases
Protein 4.1 deficiency Mild Autosomal dominant Rare, most common in North Africa
Protein 4.2 deficiency Moderate to severe (not responsive to splenectomy)
Autosomal recessive Rare, most common in Japan, rare in European population
Clinical Findings in HS:
Clinically, patients with HS often present with anemia, jaundice, and splenomegaly. However, the clinical
manifestations are highly variable and range from patients who have no anemia due to a relatively longer
red cell life span with bone marrow compensation to those who have severe hemolytic anemia due to a
very short red cell life span. The onset of symptoms is highly variable and many patients are not identified
until later in life when an infection or other process exacerbates the hemolysis or impairs the bone
marrow’s ability to compensate for more rapid red cell turnover (aplastic episode). The anemia seen in HS is
usually mild to moderate, but may be exacerbated with fatigue, cold exposure, pregnancy, or infection.
Often times, increased anemia is associated with increased jaundice due to increased red cell destruction
and hyperbilirubinemia. Some patients develop pigment (calcium bilirubinate) gallstones due to chronic
hemolysis.
Hereditary spherocytosis is clinically subclassified based on the severity of disease (Table 6). Most cases
are classified as moderate HS, which is a chronic hemolysis with characteristic spherocytes on the blood
smear, a negative Coombs’ test and a family history suggesting an autosomal dominant pattern of
inheritance. Mild disease is seen in 20 to 30% of patients. These patients will have no significant anemia
due to full compensation of hemolysis by the bone marrow. Usually splenomegaly is mild or absent and
patients are asymptomatic unless they have a hemolytic or aplastic episode that is triggered by an
infection. Moderate HS accounts for 60 to 75% of all cases. These patients will have mild to moderate
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anemia, mild to moderate splenomegaly and intermittent jaundice. They will have increased reticulocyte
counts and bilirubin levels. Patients with moderate HS may require occasional transfusions. Moderately
severe to severe HS occurs in approximately 5% of cases and is characterized by significant hemolytic
anemia that may require multiple transfusions. Most cases of severe HS present during infancy and early
childhood and are more likely to be associated with an unusual nondominant pattern of inheritance.
Table 6 – Clinical Classification of Hereditary Spherocytosis
Mild HS Moderate HS
Moderately Severe HSa Severe HSb
Percent of cases 20-30% 60-75% 5% <10%
Hemoglobin (g/dL)
11-15 8-12 6-8 <6
Reticulocytes (%)
3-8 8 10 10
Bilirubin (mg/dL) 1-2 2 2-3 3
Peripheral smear Mild spherocytosis
Spherocytosis Spherocytosis Spherocytosis and poikilocytosis
Osmotic fragility fresh blood
Normal or slightly increased
Moderately increased
Moderately to severely increased
Severely increased
a Values in untransfused patients
b Patients with severe spherocytosis are always transfusion-dependent.
Laboratory Findings in HS:
The laboratory findings in most patients with HS include anemia, reticulocytosis, and an increased mean
corpuscular hemoglobin concentration (MCHC) with identification of spherocytes on the peripheral blood
smear (Table 7). There are usually normal numbers of white cells and platelets unless there is a
superimposed infection. Most patients will have increased bilirubin and LDH levels with decreased
haptoglobin. The anemia is typically mild to moderate and the MCV, despite the increased number of
spherocytes, may be normal due to the increased number of reticulocytes. There is varying amounts of
polychromasia and anisocytosis. The increase in MCHC is thought to be due to mild cellular dehydration
secondary to nonspecific loss of potassium through the membrane. The number of spherocytes may vary
considerably, and patients with severe hereditary spherocytosis also may have many poikilocytes.
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Table 7 – Laboratory Features of Hereditary Spherocytosis
Blood
RBC
Variable normochromic, normocytic, or microcytic anemia (9-15 g/dL)
Variably increased reticulocytes
MCHC increased
Spherocytes present
WBC
Normal
Platelets
Normal
Bone Marrow
Variable cellularity (normocellular to hypercellular)
Variable erythroid hyperplasia
Laboratory
Evidence of hemolysis
Increased LDH
Elevated indirect bilirubin
Decreased haptoglobin
Specific Testing
Increased osmotic fragility
Decreased fluorescence intensity for eosin-maleimide by flow cytometry
LDH = lactate dehydrogenase; MCHC = mean corpuscular hemoglobin concentration; RBC = red blood cell; WBC = white blood cell
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Additional testing for HS usually involves determination of red cell osmotic fragility. Spherocytes have
increased osmotic fragility due to the decrease in red cell membrane surface area relative to the volume of
cytoplasm. When red cells are placed in solutions that have differing salt concentrations, the ability of the
red cell to absorb water from solution is dependent on the ability to expand the red cell membrane. Thus,
the typical biconcave red cell is able to absorb more water from progressively more dilute salt
concentrations before lysis occurs compared to spherocytes. For spherocytes, the lower membrane to cell
surface ratio allows for less water to be absorbed from a hypotonic solution before lysis occurs. Osmotic
fragility testing is a good screening tool for hereditary spherocytosis but is not specific. Any patient with
increased numbers of spherocytes, including those with an immune-based hemolytic anemia, will show
increased osmotic fragility. Conversely, 10 to 20% of patients with hereditary spherocytosis, primarily
those with mild hereditary spherocytosis, will have normal to only slightly increased osmotic fragility.
A more specific test for diagnosis of hereditary spherocytosis can be done using flow cytometry.
Immunophenotypic detection of the levels of the band 3 (also referred to as AE1) protein and enumeration
by flow cytometry is a strong indicator of HS. The band 3 protein will bind the fluorescent dye eosin-
maleimide (EMA) at a protein site in one of the extramembrane domains. As band 3 is an essential
component of the red blood cell cytoskeleton, it will be decreased if there is a hereditary defect that impairs
the normal assembly of the protein framework. The level of EMA dye binding can be evaluated by flow
cytometry using the green fluorescent channel and gating on the red cell population. This test will detect
most cases of HS, as a defect in any of the cytoskeleton proteins (including spectrin and ankyrin) will lead
to a relative decrease in the levels of band 3 and a decreased fluorescent signal.
Use of clinical history, peripheral smear analysis to identify spherocytes, and in some cases the use of the
osmotic fragility and/or eosin-maleimide flow cytometry test are sufficient to allow a diagnosis of hereditary
spherocytosis in virtually all cases. In rare cases, additional testing by analysis of specific levels of each of
the red cell protein cytoskeletal components may be necessary; however, this is not a widely available test
and is usually not indicated.
Complications of HS:
Patients with long-standing HS are at risk for development of bilirubin gallstones. Patients also may have
episodes of worsening anemia that are due to physical stresses or infection that increase the rate of
hemolysis. This may be due to increased splenic activity (particularly in the case of viral infections) or due
to circulatory conditions that are more detrimental to the red cell, such as hypoxemia or changes in pH
(hemolytic crisis). Another complication that is usually due to infection with parvovirus B19 is an aplastic or
hypoplastic crisis in which the bone marrow’s ability to replace the hemolyzed red cells is markedly
impaired due to diminished erythropoiesis. Parvovirus infects red cell precursors and inhibits their growth,
resulting in a profound decrease in hemoglobin concentration and marked reticulocytopenia. Parvovirus
infection may be the initiating cause that brings a patient with HS to clinical attention due to the inability of
the bone marrow to compensate for hemolysis, even in mild to moderate cases of HS. Exaggeration of
anemia may also be seen with vitamin deficiencies, particularly deficiencies of folate due to pregnancy or
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liver disease. These deficiencies act to inhibit compensatory increases in erythropoiesis within the bone
marrow, leading to an inability to produce sufficient numbers of red cells to compensate for hemolysis.
Clinical Management of HS:
As HS symptoms are dependent on the degree of hemolysis due to the alterations in red cell cytoskeletal
protein stability, each patient will have a unique clinical presentation. Most patients will have mild to
moderate hemolysis and a relatively benign clinical course. When hemolysis exceeds the ability of the bone
marrow to compensate, splenectomy may be performed and can greatly improve symptoms of anemia and
decrease the frequency of episodes of hemolysis associated with infection and other stresses. Red cell
transfusions are sometimes needed to treat intermittent increases in the severity of anemia during
hemolytic or aplastic crises.
REFERENCES:
1. Bolton-Maggs PHB, Stevens RF, Dodd NJ, et al. Guidelines for the diagnosis and management of
hereditary spherocytosis. Brit J Haematol. 2004;126:455-474.
2. Coetzer TL, Zail S. Introduction to Hemolytic Anemias. Intracorpuscular Defects: I. Hereditary Defects
of the Red Cell Membrane. In: Harmening DM, ed. Clinical Hematology and Fundamentals of
Hemostasis. 5th ed. Philadelphia, PA: F.A. Davis Company; 2009:176-195.
3. Gallagher PG, Glader B. Hereditary Spherocytosis, Hereditary Elliptocytosis, and Other Disorders
Associated with Abnormalities of the Erythrocyte Membrane. In: Greer JP, Forester J, Rodgers GM, et
al, eds. Wintrobe’s Clinical Hematology. 12th ed. Philadelphia, PA: Lippincott, Williams & Wilkins;
2009:911-930.
4. Glassy EF, ed. Color Atlas of Hematology: An Illustrated Field Guide Based on Proficiency Testing.
Northfield, IL: College of American Pathologists; 1998:100-103.
5. Grace RF, Lux SE. Disorders of the Red Cell Membrane. In: Orkin SH, Nathan DG, Ginsburg D, Look AT,
Fisher DE, Lux SE, eds. Nathan and Oski’s Hematology of Infancy and Childhood. 7th ed. Philadelphia,
PA: Saunders Elsevier; 2009:659-837.
6. Perkins, SL. Hereditary Erythrocyte Membrane Defects. In: Kjeldsberg CR, Perkins SL, eds. Practical
Diagnosis of Hematologic Disorders. 5th ed. Chicago, IL: ASCP Press; 2010:93-103.
Kyle T. Bradley, MD and Sherrie L. Perkins, MD Hematology and Clinical Microscopy Committee
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14- Education All material is © 2012 College of American Pathologists, all rights reserved.
AUTHOR’S BIO:
Kyle T. Bradley, MD, MS, FCAP is an assistant professor in the Department of Pathology and Laboratory
Medicine at Emory University Hospital in Atlanta, GA. He is board certified in anatomic pathology, clinical
pathology, and hematology by the American Board of Pathology. His primary responsibilities are in clinical
service and resident/fellow teaching in the areas of hematopathology and surgical pathology. Dr. Bradley
has authored a number of original articles, abstracts, and educational activities in the fields of
hematopathology and anatomic pathology and is a member of the College of American Pathologists (CAP)
Hematology and Clinical Microscopy Resource Committee.
Sherrie L. Perkins MD, PhD, FCAP is a professor of pathology at the University of Utah Health Sciences
Center and the chief medical officer for ARUP Laboratories in Salt Lake City, UT. She is the director of
hematopathology for ARUP Laboratories and has responsibilities in teaching, resident training, clinical
service, and research. Dr. Perkins has written more than 170 peer-reviewed papers and 70 book chapters
in the areas of hematology and hematopathology. Dr. Perkins is currently a member of the College of
American Pathologists (CAP) Hematology and Clinical Microscopy Resource Committee.