Participants Identification No. % Evaluation. Spherocyte Educational Erythrocyte, normal Educational

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1 Cell Identification VPBS-14 Participants Identification No. % Evaluation Spherocyte Educational Erythrocyte, normal 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

2 VPBS-15 Participants Identification No. % Evaluation Polychromatophilic RBC Educational Macrocyte oval/round 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

3 VPBS-16 Participants Identification No. % Evaluation Eosinophil 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

4 VPBS-17 Participants Identification No. % Evaluation nrbc, normal/abnormal morphology 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

5 VPBS-18 Participants Identification No. % Evaluation Platelet, normal Educational The arrows point to normal platelets, correctly identified by 99.7% of the participants. Platelets are bluegray 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

6 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 10 9 /L; HGB = 7.6 g/dl; MCV = 90.8 fl; RDW = 23.3%; PLT = 849 x 10 9 /L; Reticulocyte = 20.3%; Reticulocyte Absolute = 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. 1- Education

7 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 2- Education

8 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 3- Education

9 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). 4- Education

10 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 5- Education

11 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 antibodymediated 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. 6- Education

12 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. Band 3 (AE1) Vertical Interactions Horizontal Interactions 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 7- Education

13 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. 8- Education

14 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 9- Education

15 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 HS a Severe HS b Percent of cases 20-30% 60-75% 5% <10% Hemoglobin (g/dl) Reticulocytes (%) < Bilirubin (mg/dl) 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. 10- Education

16 Table 7 Laboratory Features of Hereditary Spherocytosis Blood RBC WBC Variable normochromic, normocytic, or microcytic anemia (9-15 g/dl) Variably increased reticulocytes MCHC increased Spherocytes present 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 11- Education

17 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 eosinmaleimide (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 12- Education

18 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: 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: 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: Glassy EF, ed. Color Atlas of Hematology: An Illustrated Field Guide Based on Proficiency Testing. Northfield, IL: College of American Pathologists; 1998: 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: Perkins, SL. Hereditary Erythrocyte Membrane Defects. In: Kjeldsberg CR, Perkins SL, eds. Practical Diagnosis of Hematologic Disorders. 5th ed. Chicago, IL: ASCP Press; 2010: Kyle T. Bradley, MD and Sherrie L. Perkins, MD Hematology and Clinical Microscopy Committee 13- Education

19 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. 14- Education