Blood Cell Identification: 2011-C Mailing: Iron Deficiency Anemia (IDA) CASE HISTORY BACKGROUND

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1 Blood Cell Identification: 2011-C Mailing: Iron Deficiency Anemia (IDA) Please Note: To view the Figures and Images contained within this education activity in color, access the electronic version of the reading. CASE HISTORY The patient is a 38-year-old female with complaints of diarrhea. Laboratory data include: WBC = 3.1 x 10 9 /L; RBC = 2.1 x /L; HGB = 5.6 g/dl; HCT = 18.9%; MCV = 60 fl; MCH = 17.8 pg; MCHC = 29.5 g/dl; RDW = 19.0; PLT = 364 x 10 9 /L. This is a case of iron deficiency anemia due to blood loss and decreased iron absorption. In this education activity, the etiology, diagnosis, differential diagnosis, and laboratory features of iron deficiency anemia will be discussed. BACKGROUND Iron, a chemical element with the symbol Fe, is the most common element on earth, forming much of the earth's outer and inner core, and the fourth most common element in the earth's crust. It is not surprising then, that this element is incorporated into virtually all living organisms from the smallest microbes to plants and complex mammals. Iron not only facilitates oxygen flow in mammals but also is integrated into small internal magnets that facilitate migration of birds, turtles, salmon, and other animals. Studies have shown that primitive organisms could use iron-based biochemistry to harness energy in the earth's early anaerobic environment. As oxygen became plentiful in the earth's atmosphere, evolution from anaerobic to aerobic organisms occurred to utilize the vast sources of oxidizing energy in living organisms. Oxygen, however, is toxic to biological molecules and can produce free radicals inside living cells, which can damage essential cellular components such as proteins, lipids, and nucleic acids. Iron played a vital role in this process as 90% of the cells molecular oxygen uptake is in cytochrome oxidase, a molecule with a center of iron and copper. Cytochrome oxidase binds molecular oxygen tightly between its copper and iron atoms, protecting the cell from toxic free oxygen. Iron, however, so extensively available on an ancient and anaerobic earth in its ferrous (Fe 2+ ) form in living organisms now became oxidized into its ferric form (Fe 3+ ), which is not chemically available to living organisms. These highly insoluble ferric oxides precipitated out of the oceans and are visible throughout the earth in banded iron formations, which are a source of iron ore and produced the red soils and rocks of southwestern United States. Eventually, little biochemically available iron remained on earth. Living organisms now walk a tightrope between too little iron and iron toxicity. To assimilate this trace element, ancient organisms evolved siderophores, ferritins, and transferrins to acquire, store, and recycle iron atoms. The normal human body content of iron is 3 to 4 grams, distributed in hemoglobin within circulating red blood cells, in iron containing proteins myoglobin, cytochromes, catalases, bound to transferrin in the plasma and stored in ferritin or hemosiderin. Iron is stored in the liver, spleen, and bone marrow. Adult men 1-Education

2 Blood Cell Identification: 2011-C Mailing: Iron Deficiency Anemia (IDA) have iron stores of 10 mg/kg; adult women 5.5 +/- 3.4 mg/kg. Seven percent of women have deficient stores of 3.9 +/- 3.2 mg/kg and up to 20 percent of menstruating women in the United States have no iron stores. CASE DISCUSSION Further evaluation of this 38-year-old mother of three included serum iron of 38 µg/dl (decreased), transferrin (total iron binding capacity, TIBC) of 420 mg/dl (increased), and plasma ferritin of 8 ng/ml (decreased). Upper gastrointestinal endoscopy showed no lesions of the stomach or esophagus, but did reveal mucosal atrophy of the duodenum. Biopsy of this area showed villous atrophy and lymphocytic infiltration of the epithelium. These findings are characteristic histopathologic features of celiac sprue, a condition in which reaction to the gluten found in wheat, barley, and rye, damages the lining of the small intestine resulting in malabsorption. Lower gastrointestinal endoscopy was negative for colitis, polyps, and colon cancer. On history review, the patient indicated she had been experiencing extremely heavy menstrual periods (menorrhagia). The patient was diagnosed with iron deficiency anemia due to menstrual blood loss, possibly compounded by iron malabsorption due to celiac sprue. She was begun on oral iron therapy and a gluten free diet. She responded with reticulocytosis and an elevation in her hemoglobin level and hematocrit. She subsequently underwent endometrial ablation which cured her menorrhagia. IRON DEFICIENCY ANEMIA Iron deficiency anemia is present in 1-2% of adults in the United States. Iron deficiency without anemia, however, is more common, affecting 11% of women and 4% of men. In affluent countries, the major cause of iron deficiency is blood loss. Obvious examples include severe trauma, active bleeding from the gastrointestinal tract with blood loss in vomitus (hematemesis) or stool (melena), as well as blood loss in sputum (hemoptysis), urine (hematuria), and with menstruation (menorrhagia). Less obvious, or occult bleeding, necessitates more extensive investigation to determine the source. In men, it is most often from the gastrointestinal tract, from gastric and duodenal ulcers or tumors, especially colon cancer. Repeated voluntary blood donations, extensive blood drawing during hospitalization, and surgical blood loss greater than that transfused are additional causes. In women, excessive menstrual blood loss, blood loss during delivery of an infant, and iron loss to the fetus during pregnancy and to the neonate during lactation are factors that may all contribute to iron deficiency. Diets deficient in iron can lead to iron deficiency and iron deficiency anemia. Meat and fish are rich in iron. Vegetables contain little iron, except for black beans, soy beans, and cornflower. Some iron in foods, such as spinach, is unavailable to the body due to other components in the food. Grain cereals, including 2-Education

3 Blood Cell Identification: 2011-C Mailing: Iron Deficiency Anemia (IDA) unpolished rice, contain little iron and contain complexes that inhibit the absorption of iron. Therefore, individuals on vegetarian diets lacking legumes or those who do not have access to meat and fish are at risk of iron deficiency and iron deficiency anemia. The major site for iron absorption is in the duodenum. Reduced gastric acid due to chronic atrophic gastritis and Helicobacter pylori gastritis, as well as alterations in the duodenal mucosa in celiac disease may lead to iron deficiency due to inadequate absorption. Celiac disease is not uncommonly found to be the cause of lack of response to oral iron therapy in patients with iron deficiency anemia. In gastric bypass surgery for morbid obesity, the duodenum is also bypassed, resulting in loss of absorptive area combined with reduced gastric acid for iron absorption. Other causes of iron deficiency include intravascular hemolysis due to malfunctioning cardiac valves, paroxysmal nocturnal hemoglobinuria, and response to erythropoietin therapy given in patients with renal failure without concomitant iron therapy. Clinical symptoms in iron deficiency anemia include weakness, fatigue, headache, irritability, and exercise intolerance. An appetite for substances not normally considered food (pica), such as clay, paper, or ice (pagophagia) may be noted. Restless leg syndrome has been associated with iron deficiency, but the association is not conclusive. Older individuals may present with exacerbation of an underlying disease such as increased angina with underlying coronary artery disease, increased shortness of breath with underlying congestive heart failure, or increasing confusion in dementia. LABORATORY FINDINGS Classic laboratory findings in iron deficiency anemia are a low hemoglobin and decreased mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC), increased red cell distribution width (RDW), low serum iron, low serum ferritin, elevated total iron binding capacity (TIBC), also measured as transferrin, and low transferrin saturation. The peripheral blood smear demonstrates hypochromic, microcytic red blood cells, thin elliptocytes, anisopoikilocytosis, and decreased reticulocytes (Figure 1 on the following page), while the bone marrow lacks iron stores. Initiation of oral or parenteral (intravenous) iron therapy should result in a brisk reticulocytosis and elevation in hemoglobin concentration and hemoglobin level. As a patient is recovering from iron deficiency, red blood cells may have a dimorphic appearance with hypochromic microcytes remaining from the period of iron deficiency and normochromic, normocytic red cells that are formed after initiation of iron therapy. 3-Education

4 Blood Cell Identification: 2011-C Mailing: Iron Deficiency Anemia (IDA) Figure 1. Iron Deficiency Peripheral blood smear in a patient with iron deficiency. Note the hypochromic, microcytes shown by the arrows. The central pallor in these red cells is significantly more than one third the cell diameter (hypochromic), while the red cell size is smaller than the small lymphocyte in this image. In addition, a thin elliptocyte is present (arrowhead). In developed countries, however, this presentation is not common. Many patients in the United States with iron deficiency anemia will have early iron deficiency with normal red cell indices and a relatively normal peripheral blood smear. Only later will microcytosis, hypochromasia, anisocytosis, and poikilocytosis, including thin elliptocytes, be seen on the blood smear. Iron-deficient red cells have abnormally stiff plasma membranes, which contribute to the formation of elongated and elliptical hypochromic red cells. The white blood cell count in iron deficiency anemia is normal or slightly decreased. Granulocytes may be decreased and a few hypersegmented neutrophils may be observed. Greater numbers of the latter, though, should raise the suspicion for concurrent folate or vitamin B12 deficiency. Platelets may be increased; in severe anemia, they may be decreased. Although bone marrow iron stores determined with Prussian blue stain of a bone marrow sample is the gold standard, this invasive procedure is usually not needed in a straight forward case of iron deficiency anemia. Expected response to a trial of oral iron therapy, as noted above, can be used. In addition, decreased serum ferritin level can often supplant bone marrow sampling. Serum ferritin is in equilibrium with the tissue and is a good, sensitive indicator of iron stores in most patients. It is an acute phase reactant and may be elevated in inflammatory conditions, autoimmune diseases, malignancy, and liver disease. In these conditions, a ferritin level within the normal range may inaccurately mask underlying iron depletion (a false positive result for iron stores). Theoretically, raising the lower end of the reference range in these patients may overcome this limitation; although standards by which to do this are not well established. A low serum ferritin is generally not seen in conditions other than iron deficiency. 4-Education

5 Blood Cell Identification: 2011-C Mailing: Iron Deficiency Anemia (IDA) DIFFERENTIAL DIAGNOSIS The differential diagnosis of iron deficiency anemia includes thalassemia and sideroblastic anemia, also microcytic hypochromia anemias, as well as anemia of chronic disease, an entity that most often results in normochromic, normocytic anemia or in some cases, hypochromic (mildly), microcytic (mildly) anemia. It is especially important to exclude thalassemia and sideroblastic anemia, as these patients usually have excess iron stores, and iron therapy would be contraindicated. Overlap in some of the diagnostic laboratory tests may make this a difficult task, especially in early cases of iron deficiency, as is common in developed countries. Table 1 below lists common laboratory tests used to evaluate and distinguish these microcytic anemias. Table 1. Laboratory Evaluation of Microcytic Anemias Anemia Iron deficiency anemia Hypochromic, microcytic Anemia of chronic disease Normochromic, normocytic or mildly hypochromic, microcytic Thalassemia Hypochromic, microcytic Sideroblastic anemia Hypochromic, microcytic (inherited); may be normo- /macrocytic (acquired) RBC number Decreased Decreased Normal or increased Decreased RDW Increased Normal Normal or increased Normal or increased Peripheral blood smear Hypochromic microcytes; thin elliptocytes; decreased polychromasia; platelets may be normal or increased Normochromic, normocytic or mildly hypochromic, microcytic; other findings are variable and nonspecific Hypochromic microcytes; target cells; increased polychromasia; +/- coarse basophilic stippling in β- thalassemia Hypochromic microcytes (inherited) or normocytic/macrocytic (acquired); variable anisopoikilocytosis; some cases show dimorphic red cell population or siderocytes Serum Fe Decreased Decreased Normal or increased Increased Ferritin Decreased Normal or increased Normal or increased Increased Transferrin (TIBC) Increased Decreased Decreased or normal Decreased or normal Bone marrow iron Absent Increased Increased Increased Other useful information Dietary, surgical, blood loss history Identification of underlying disorder Family history; hemoglobin analysis (such as HPLC, capillary or gel electrophoresis) Family, medication, and nutritional history; if considering myelodysplastic syndrome, bone marrow evaluation with cytogenetics Distinguishing between iron deficiency and anemia of chronic disease is the most common clinical problem. Anemia of chronic disease is seen with a variety of chronic medical illnesses, including infections, chronic immune activation (eg, systemic lupus erythematosus, rheumatoid arthritis), malignancies, and a number of other disorders. The underlying medical illness causes release of cytokines and acute phase reactant proteins that contribute to the anemia of chronic disease. Hepcidin, an acute phase reactant protein, inhibits iron release from macrophages via its effect on the iron export protein ferroportin and decreases iron absorption in the small intestine. These alterations result in inadequate iron availability for developing erythroid precursors. Similar to iron deficiency, serum iron is low, but in contrast to iron deficiency, iron transport proteins, as measured by TIBC, are decreased and bone marrow iron stores are increased. 5-Education

6 Blood Cell Identification: 2011-C Mailing: Iron Deficiency Anemia (IDA) Sideroblastic anemias are a group of hereditary and acquired disorders that exhibit abnormal iron metabolism and heme synthesis within the red blood cell. Inherited sideroblastic anemias are most often hypochromic and microcytic, while acquired sideroblastic anemia may be normocytic or macrocytic. Causes of acquired sideroblastic anemia include: myelodysplastic syndromes (clonal myeloid neoplasms), as well as alcoholism, medications (eg, isoniazid, chloramphenicol), copper deficiency, and lead poisoning. In sideroblastic anemias, iron becomes sequestered in the red blood cell mitochondria and is not available for heme syntheses. In some cases, red cells with iron granules, a finding referred to as Pappenheimer bodies, can be seen on the peripheral blood smear (Figure 2 below). Characteristic ring sideroblasts in the red cell precursors show a ring-like accumulation of siderotic granules in mitochondria surrounding the nucleus (Figure 3 on the following page). Serum iron, serum ferritin and bone marrow iron stores are all increased. Figure 2. Sideroblastic Anemia This image shows a peripheral blood smear from a patient with inherited sideroblastic anemia. This blood smear shows numerous hypochromic microcytes, many of which contain blue-purple staining iron granules referred to as Pappenheimer bodies. Red cells with iron granules can also be referred to as siderocytes. 6-Education

7 Blood Cell Identification: 2011-C Mailing: Iron Deficiency Anemia (IDA) Figure 3. Ring Sideroblasts This image shows a Prussion blue iron stain performed on bone marrow aspirate material in a patient with the myelodysplastic syndrome refractory anemia with ring sideroblasts. The iron sequestered in mitochondria stains blue within the late erythroid precursors, and in some cells forms a ring around the nucleus (arrows). Thalassemias are hereditary disorders of globin synthesis resulting in decreased numbers of hemoglobin tetramers within red blood cells. Normal hemoglobin A tetramers are comprised of two α and two β globin chains. Thalassemias result from decreased production of the normal α-globin (α-thalassemia) or β-globin (βthalassemia) subunit of the hemoglobin tetramer and result in variable clinical features depending on the specific abnormality, number of affected genes, and degree of anemia. The peripheral blood smear will show hypochromic, microcytes, and target cells (Figure 4 on the following page). In contrast to iron deficiency anemia, increased polychromasia is seen on the blood smear, an indication of bone marrow response to anemia. In addition, β-thalassemia may show basophilic stippling and/or circulating nucleated red cells. Thalassemias occur predominantly in persons of Mediterranean, African, and Asian ancestry. Family history and hemoglobin studies, such as high pressure liquid chromatography (HPLC) and gel or capillary electrophoresis, may be useful. Homozygous hemoglobin E, a hemoglobinopathy characterized by a mutation in the β-globin subunit of hemoglobin, shows similar clinical and blood smear findings to α- thalassemia. 7-Education

8 Blood Cell Identification: 2011-C Mailing: Iron Deficiency Anemia (IDA) Figure 4. Thalassemia This blood smear is from a patient with α-thalassemia. The red cell population is hypochromic, microcytic, and occasional target cells are seen (arrows). In contrast to iron deficiency, the RBC numbers may be normal or increased in thalassemia. 8-Education

9 Blood Cell Identification: 2011-C Mailing: Iron Deficiency Anemia (IDA) References 1. Glassy EF, ed. Color Atlas of Hematology: An Illustrated Field Guide Based on Proficiency Testing. Northfield, IL: College of American Pathologists; Gulati G, Care J. Blood cells: An Atlas of Morphology with Clinical Relevance. Chicago, IL: ASCP Press; Mielczarek EV, McGrayne SB. Iron, Nature's Universal Element. Why People Need Iron and Animals Make Magnets. New Brunswick NJ; London: Rutgers University Press; Looker AC, Dallman PR, Carroll MD, et al. Prevalence of iron deficiency in the United States. JAMA. 1997;277: Cook JD, Flowers CH, Skikne BS. The quantitative assessment of body iron. Blood. 2003;101: Cook JD, Finch CA, Smith NJ. Evaluation of the iron status of a population. Blood. 1976;48: Schrier SL. Causes and diagnosis of anemia due to iron deficiency. UpToDate. 2010;1-33. Available at: Accessed August 5, Kjeldsberg CR, Perkins SL. Practical Diagnosis of Hematologic Disorders, Vol. 1 Benign Disorders. 5th ed. Chicago, IL: ASCP Press; 2010; Elghetany MT, Banki K. In: Henry's Clinical Diagnosis and Management by Laboratory Methods. 21st ed. Philadelphia, PA: Saunders Elsevier; 2007: Education

10 Blood Cell Identification: 2011-C Mailing: Iron Deficiency Anemia (IDA) Education Activity Authors Martha R. Clarke, MD, FCAP: Martha R. Clarke, MD, is Chair, Department of Pathology and Laboratory Medical Director at St. Clair Hospital and Medical Director of Pathstar*, a private surgical pathology laboratory. Dr. Clarke is AP/CP, Hematology and Cytopathology boarded by the American Board of Pathology. She has authored over 60 papers, abstracts and educational activities in diverse areas of expertise. Dr. Clarke is currently a member of the Hematology and Clinical Microscopy Resource Committee. Joan Etzell, MD, FCAP: Joan Etzell, MD, is a Professor of Clinical Laboratory Medicine and the Director of the Clinical Hematology Laboratory at the University of California, San Francisco (UCSF). She is AP/CP and Hematology Board certified by the American Board of Pathology. Dr. Etzell is actively involved in the education of medical technologists, medical students, residents, and fellows in hematology / hematopathology. She serves as the Hematopathology Fellowship Director and Associate Residency Program Director in Laboratory Medicine in UCSF. Dr. Etzell has authored over 50 papers, book chapters, educational activities and abstracts in the areas of hematology and hematopathology. Dr. Etzell currently serves as the Vice-Chair of the Hematology and Clinical Microscopy Resource Committee for the College of American Pathologists (CAP). 10-Education

11 Please Note: To view the Figures and Images contained within this education activity in color, access the electronic version of the reading. CASE HISTORY This peripheral blood smear is from a 1-month-old female with a history of prematurity, hypothermia, and respiratory distress. She tested positive for respiratory syncytial virus (RSV). Laboratory data include: WBC = x 10 9 /L; RBC = 2.57 x /L; HGB = 6.3 g/dl; HCT = 20%; MCV = 77 fl; and PLT = 329 x10 9 /L. CASE DISCUSSION This case illustrates a case of an infant with hereditary pyropoikilocytosis (HPP). The morphologic findings seen in the blood smear include multiple bizarre red cell forms with fragmentation (poikilocytes), microspherocytes, and scattered elliptocytes. These clinical and morphologic features suggest a diagnosis of hereditary elliptocytosis, and more specifically, a diagnosis of hereditary pyropoikilocytosis. Hereditary elliptocytosis (HE) is a heterogenous group of inherited erythrocyte membrane disorders that are characterized by the presence of elongated, oval, or elliptical red blood cells (RBCs) on a peripheral blood smear. HE is divided into three groups based on clinical and morphologic features: (1) Common HE, which includes hereditary pyropoikilocytosis (HPP) (2) Spherocytic HE (3) Southeast Asian ovalocytosis (SAO), also known as stomatocytic ovalocytosis This group of red cell membrane disorders displays a wide spectrum of clinical presentations, ranging from asymptomatic elliptocytosis, which is primarily a cosmetic disorder, to patients with severe and lifethreatening hemolytic anemia. The severity of the disease and attendant hemolysis depends on the underlying defect in red cell membrane proteins and the inheritance pattern. Severe hemolysis is usually associated with homozygosity or compound heterozygosity for more than one membrane protein mutation, as discussed below. In the United States, the prevalence of HE is 3 5 persons per 10,000, and HE is more common in individuals of African or Mediterranean origin, where it may help to confer resistance of the red cell to infection by malaria. Before we delve into further detail regarding hereditary pyropoikilocytosis, let us briefly discuss the three groups of elliptocytic disorders (Figure 1 and Table 1 on page 3). COMMON HEREDITARY ELLIPTOCYTOSIS Most individuals (~90%) with common HE are asymptomatic and do not have anemia. The peripheral blood smear is remarkable for a striking number of elliptocytes, well above 25% of erythrocytes, in contrast to normal individuals who can have up to 5% elliptocytes. A minority of patients (~10-20%) can have mild hemolysis, while certain subgroups, particularly those of African descent, can have moderate hemolytic 1-Education

12 anemia in the neonatal period, morphologically indistinguishable from HPP, which later resolves. Peripheral blood smears from these infants exhibit fragmented and budding red blood cells, and other poikilocytes. Over time, hemolysis usually abates, poikilocytes and fragmented red cells disappear, and a more typical clinical course of HE with minimal or no hemolysis, and no anemia emerges. The most common molecular abnormality seen in common HE is a defect in α-spectrin, although abnormalities in β-spectrin and protein 4.1 have also been described. Hereditary pyropoikilocytosis (HPP), as in this case presentation, is a subset of common HE. In its classical form, it is characterized by persistent severe hemolytic anemia, bizarre red cell forms secondary to fragmentation (poikilocytes) and increased membrane fragility. Many of the cells are very small, and the patients may have very low mean corpuscular volumes (MCV) and will also have increased osmotic fragility. While normal erythrocytes hemolyze at 49 C, red cells in an HPP individual have a lower thermal threshold for fragmentation, and will hemolyze at C. These patients present in infancy with moderate to severe hemolysis that is life-long. HPP is also associated with α-spectrin abnormalities, but unlike common HE, these are usually homozygous or double heterozygous mutations, leading to the much more severe phenotype. SPHEROCYTIC HEREDITARY ELLIPTOCYTOSIS Spherocytic HE is characterized by the presence of two distinct populations of red cells, elliptocytes and spherocytes, but a distinct lack of poikilocytes. This subtype of HE is more common in Caucasians, particularly of European descent. The peripheral blood smears show variable numbers of spherocytes as well as elliptocytes, which are plumper than in common HE (Figure 1 on the following page). Affected individuals typically have mild to moderate red cell hemolysis and are often anemic. Splenectomy will usually improve the anemia. The molecular basis of this disorder is still largely unknown. SOUTH ASIAN OVALOCYTOSIS Southeast Asian ovalocytosis (SAO) is an asymptomatic red blood cell membrane disorder commonly seen in malaria endemic areas in Melanesia, Malaysia, Philippines, Indonesia, and southern Thailand. The disease is inherited in an autosomal dominant manner with a heterozygous mutation in band 3 protein. The mutation results in a very rigid, but mechanically stable, red cell membrane. Peripheral blood film findings include ovalocytes that contain one or two transverse ridges or a single longitudinal slit. Some of these cells may have the appearance of either stomatocytes or shoe buckles with two areas of central pallor bisected by a hemoglobin bridge. Affected individuals experience no or minimal red cell hemolysis, despite the increased red cell membrane rigidity. 2-Education

13 Figure 1. Comparison of Variants of Hereditary Elliptocytosis (HE) Table 1. Comparison of Variants of Hereditary Elliptocytosis (HE) Population affected Inheritance Mutation in red cell membrane Blood smear findings Clinical features Manifestation Common HE Spherocytic HE Southeast Asian Ovalocytosis (aka Most CHE HPP stomatocytic HE) African descent Autosomal dominant α-spectrin β-spectrin protein 4.1 Elliptocytes Most patients non-anemic African and Mediterranean descent Caucasians Individuals from Far East, esp. Malaysia Autosomal recessive Autosomal dominant Autosomal dominant Homozygous α-spectrin Unknown Band 3 Microcytosis, striking micropoikilocytosis, fragmentation Severe hemolytic anemia Hemolysis at C (normal RBCs fragment at 49 C) Elliptocytes and spherocytes (lack poikilocytes) Mild to moderate hemolytic anemia Resemble stomatocytic ovalocytes Absent to mild hemolysis Overall, the most commonly defined genetic abnormality in the elliptocytic disorders is a defect in one of the spectrin protein subunits, which leads to impaired association of spectrin dimers into tetramers and spectrin oligomers. Other abnormalities, such as protein 4.1 deficiency, are much rarer. Before we proceed with a more in-depth discussion of the pathophysiology underlying HE & HPP, it is first important to review the structure of a normal red blood cell and normal hemoglobin membrane. 3-Education

14 NORMAL RED BLOOD CELL AND HEMOGLOBIN STRUCTURE Figure 2. Normal Red Blood Cell Erythrocyte (normocytic) Erythrocyte (normochromic) The normal human red blood cell, as shown in the diagrams above (Figure 2) has a concave shape with excess surface area as compared with cell volume. The excess surface area allows for the cell to easily change its shape as it transits through the spleen. Significant deformation of the cell is required to move through the small slit-like spaces in basement membranes separating splenic cords from sinuses. A second requirement of the red cell is that the membrane must be highly elastic so that it does not break apart from the normal fluid stresses of circulation. The structure of the red blood cell membrane is key to maintaining both the membrane elasticity and stability necessary to prevent cellular fragmentation or breakage. It is composed of a lipid bilayer containing several unique proteins that transverse the membrane or interact to form a membrane cytoskeleton (Figure 3 on the following page). The positions and configurations of the proteins in the membrane cytoskeleton determine the function and include both vertical and horizontal interactions that impart maximal flexibility to red cell structure. Vertically positioned proteins, including the transmembrane proteins band 3 (also known as the anion exchanger or transmembrane protein AE1) and glycophorin C, contribute to vertical interactions. These include stabilizing the membrane lipid layer to prevent surface RBC membrane loss during circulation and transit though the spleen. An inherited defect in one of these vertical proteins, band 3, for example, will lead to increased formation of spherocytes due to more rapid loss of membrane volume, such as seen in hereditary spherocytosis. 4-Education

15 Figure 3. Normal Red Cell Membrane Vertical Interactions Horizontal Interactions Schematic model of red cell membrane exhibiting the lipid bilayer with transmembrane proteins (including band 3 and glycophorin C) and skeletal membrane proteins (including α-spectrin, ß-spectrin, and protein 4.1). Hereditary elliptocytosis and hereditary pyropoikilocytosis, on the other hand, are caused by defective horizontal interactions. Horizontal interactions involve the proteins found at the inner surface of the RBC membrane, including spectrin, actin, ankyrin, protein 4.1, and protein 4.2. The spectrin proteins form long, linear arrays that create a meshwork that are anchored to the red cell lipid membrane by interactions with ankyrin and protein 4.1. These skeletal proteins are essential in preserving membrane stability and counteracting membrane fragmentation as the RBCs enter capillary beds and transit through the circulation. The abnormalities in red cell shapes seen in the red cell membrane disorders are due to either a quantitative decrease (or absence) in the amounts of proteins or in production of proteins that do not interact and selfassociate normally. The defect that underlies most cases of HE and HPP is a failure of spectrin heterodimers to self-associate into spectrin heterotetramers that form the meshwork of proteins that create the cytoskeletal structure. This is thought to arise from several different mutations that affect the structural integrity of the horizontal red cell membrane cytoskeleton proteins. Thus, abnormal interactions between the different proteins can arise due to mutations in α-spectrin, β-spectrin, band 3, and protein 4.1 genes (Figure 3 above). When the interactions between these proteins are impaired, the horizontal stability of the cytoskeleton is weakened as these proteins are essential for formation of a functional red cell cytoskeleton. In addition, other mutations may impact the structural integrity of the red cell cytoskeleton. For example, the necessary spectrin tetramer association is impaired by mutations in the alpha or beta-spectrin dimerdimer association regions. Finally, formation of the spectrin-actin-4.1 complex and its interaction with the glycophorin protein to stabilize the membrane in a vertical fashion can be compromised by a defect in protein Education

16 PATHOPHYSIOLOGY OF HEMOLYSIS IN HE/HPP The red cell precursors in all of the red cell membrane defects are originally normally shaped as they enter into the circulation. The abnormalities in shape, such as elliptocytosis, are thought to arise due to the disconnection of the red cell membrane proteins from the lipid bilayer. This causes destabilization of the red cell cytoskeleton structure. Typically red cells will lose their typical biconcave disk shape as the red cell matures and ages by multiple episodes of deformation as they pass through capillaries, requiring deformation to an elliptical shape. When the normal protein interactions are disrupted, the cell will remain in an elliptical shape and form new protein interactions that cause the cell to retain its elliptical shape, rather than returning to a biconcave disk after passing through a capillary. This disruption in membrane stability may lead to increase in membrane fragility and susceptibility to red cell fragmentation and hemolysis. In typical HE, hemolysis may be minimal, however in HPP the membrane instability is greater, leading to increased fragmentation and variable degrees of hemolysis. DIAGNOSIS OF HE/HPP MEMBRANE DISORDERS Diagnosis of HE or HPP is usually based on morphologic evaluation of the blood smear and clinical information. Blood Smear Findings Unaffected (normal) individuals may have up to ~5% elliptical forms in their peripheral blood smears. Patients with hereditary elliptocytosis, however, generally show >15-20% elliptocytes, and may have as many as 100% of the red cells demonstrating elliptocyte morphology. Elliptocytes are defined as red cells that have an oval shape with a long axis that is 2 to 3 times the length of the shorter axis. Increased elliptocytes may also be seen in patients with iron deficiency anemia, megaloblastic anemia and myelodysplasia. Thus it is important to evaluate other red cell and leukocyte morphologic features (such as microcytosis and hypochromia) as well as iron studies and other laboratory tests to exclude these disorders. Hereditary pyropoikilocytosis is so named because the red blood cell morphology is similar to that seen in patients suffering from thermal/burn injury. There is a marked increase in red cell fragmentation due to the marked red cell membrane instability. The red cell fragments include bite or helmet cells, keratocytes or horn cells, triangular cells, microspherocytes, and microcytes (Figures 4 and 5 on the following page). 6-Education

17 Figure 4. Figure 5. Classic morphologic features of HPP. Numerous fragmented cells and extensive polychromatophilic cells. Rare spherocytes are also seen. Classic morphologic features of HPP. Red cell fragments, helmet cells, and elliptocytes are present. Dependent on the degree and type of mutation present, cases of HPP may have more prominent fragmented red cells or may have more prominent spherocyte formation. As a result of the increase in red cell destruction, the bone marrow tries to offset the hemolysis by producing elevated numbers of red cell precursors. These manifest as nucleated red blood cells and increased polychromatophilic cells in the peripheral smear. As mentioned above, this severe red cell fragmentation manifests at temperatures of C (as opposed to normal red blood cells fragmenting at 49 C). HPP is generally distinguished from hemolytic forms of common HE by the abundance of spherocytes and fragmented cells and variable numbers of intact elliptocytes in the blood smear. Hematologic evaluation of parents and siblings can also be helpful in understanding the inheritance pattern and nature of the defect. Useful additional laboratory data to aid in detecting hemolysis include elevated lactate dehydrogenase (LDH), elevated indirect bilirubin, and low levels of haptoglobin. Reticulocyte counts will be high as a compensatory mechanism for increased red cell destruction. Interestingly, the blood smear and some laboratory abnormalities may also bear a close resemblance to those seen in microangiopathic hemolytic anemia (MAHA), although its pathophysiology is quite separate and distinct. While red cells in HPP fragment because of a congenital deficiency in red cell cytoskeletal membrane proteins, the red cells in MAHA fragment as they undergo rips and tears by passing through fibrin strands that have formed in the microcirculation. The blood smear findings of MAHA and HPP are so similar that knowledge of the clinical history may be the only way to reliably distinguish the two entities. Table 2 on the following page shows a comparison table of the two hemolytic disorders. 7-Education

18 Table 2. Comparison of HE/HPP and MAHA Hereditary Elliptocytosis / Hereditary Pyropoikilocytosis (HE/HPP) Microangiopathic Hemolytic Anemia (MAHA) Cause Congenital/inherited disorder Acquired disorder; secondary to prior insult Etiology Peripheral Blood Smear Findings Hemolysis Inciting Event Mutation in red cell cytoskeletal membrane proteins (such as spectrin, etc.) Red cell fragmentation/poikilocytosis; spherocytes; polychromatophilia Predominantly extravascular hemolysis with splenic sequestration Membrane instability due to homozygous spectrin deficiency. Manifests at temperatures C (normal RBC fragments at 49 C) Disseminated intravascular coagulation (DIC); thrombotic thrombocytopenic purpura (TTP); hemolytic uremic syndrome (HUS) Red cell fragmentation/poikilocytosis; polychromatophilia; thrombocytopenia may be seen Primarily intravascular hemolysis Shearing of RBCs through intravascular fibrin strands Other Tests for Red Cell Membrane Disorders Although nonspecific, osmotic fragility testing can show increases in osmotic fragility in both HPP and homozygous common HE, but is usually normal in less severe subtypes of HE. Increases in osmotic fragility may be seen with a variety of other red cell membrane defects, most notably hereditary spherocytosis, but may also be positive in cases of autoimmune hemolytic anemia or other conditions where increased spherocytes are present. Heating the red cells to C and observing fragmentation may suggest HPP and spectrin deficiency, however, depending on the temperature threshold associated with that patient s specific mutation, this testing may yield many false positive and/or false negative results. Osmotic gradient ektacytometry and eosin-5-maleimide binding testing (which binds to the band 3 protein) performed by flow cytometric analysis are sometimes abnormal in HPP, reflecting the abnormal composition of the red cell cytoskeletal proteins. Other testing performed at specialized laboratories includes denaturing gel electrophoresis to detect cytoskeletal protein deficiencies; non-denaturing acrylamide gel electrophoresis of spectrin to quantitate abnormally high ratio of spectrin dimers to tetramers or tryptic peptide mapping to identify abnormal proteins. In addition, PCR-based DNA analysis of specific cell cytoskeletal protein genes may be performed to detect and identify mutations. However, in most cases these highly specialized tests are not necessary to make the diagnosis of HPP. Identification of the characteristic red cell morphologic features in the proper clinical context and exclusion of other causes of red cell fragmentation and hemolysis is sufficient. 8-Education

19 CLINICAL IMPLICATIONS As aforementioned, most patients with typical HE are not anemic, but others may display transient episodes of mild to moderate hemolytic anemia. Individuals with HPP, however, first experience hemolytic anemia in the neonatal period, and this may be severe. These patients usually develop splenomegaly secondary to extravascular hemolysis and splenic sequestration, and may require multiple transfusions and eventual splenectomy in severe cases. With lifelong chronic hemolysis, patients may suffer from pigmented gallstones and other sequelae of hyperbilirubinemia. Increased red cell production by the marrow may lead to erythroid hyperplasia in the marrow and increased nutritional requirements for iron, vitamin B12, and folate to support formation of red cells to replace those that are removed prematurely from the circulation by hemolysis. Severe hemolysis may lead to expansion of the bone marrow spaces and frontal bossing. As in other red cell disorders, viral infections may lead to a severe episode of anemia due to decreased red cell production by the bone marrow to compensate for hemolysis or increased splenic activity that further shortens the red cell life span. Treatment of HPP is based on the severity of clinical symptoms and ranges from no treatment to splenectomy and transfusion support. 9-Education

20 References 1. McPherson RA, Pincus MR, eds. In: Henry s Clinical Diagnosis and Management by Laboratory Methods. 21st ed. Philadelphia, PA: Saunders Elsevier; Glassy EF, ed. Color Atlas of Hematology: An Illustrated Field Guide Based on Proficiency Testing. Northfield, IL: College of American Pathologists; Tse WT, Lux SE. Red blood cell membrane disorders. Br J Haematol. 1999;104: Iarocci TA, Wagner GM, Mohandas N, et al. Hereditary poikilocytic anemia associated with the coinheritance of two alpha spectrin abnormalities. Blood. 1988;71: Mentzer WC. Hereditary elliptocytosis: Clinical features and diagnosis. In: UpToDate. Landaw SA, Hoppin AG, eds. UpToDate. Waltham, MA; Mentzer WC. Hereditary elliptocytosis: Genetics and pathogenesis. In: UpToDate. Landaw SA, Hoppin AG, eds. UpToDate. Waltham, MA; Gaetani M, Mootien S, Harper S, et al. Structural and functional effects of hereditary hemolytic anemia-associated point mutations in the alpha spectrin tetramer site. Blood. 2008;111: Tolpinrud W, Maksimova YD, Forget BG, Gallagher PG. Nonsense mutations of the a-spectric gene in hereditary pyropoikilocytosis. Haematologica. 2008;93: Education

21 Education Activity Authors Maria E. Vergara-Lluri, MD: Ria Vergara-Lluri, MD, is in her fifth year of postgraduate training in anatomic pathology and clinical pathology, and is completing her final year of residency training at the University of California, Los Angeles (UCLA) Medical Center in Los Angeles, California. Dr. Vergara-Lluri served as cochief resident for the anatomic pathology department at UCSF ( ), acting as leader, liaison, and advocate for resident education and training. She is the junior member of the Hematology and Clinical Microscopy Resource Committee for the College of American Pathologists (CAP). Sherrie L. Perkins, MD, PhD, FCAP: Sherrie L. Perkins, MD, PhD, 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 over 140 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. Joan Etzell, MD, FCAP: Joan Etzell, MD, is a Professor of Clinical Laboratory Medicine and the Director of the Clinical Hematology Laboratory at the University of California, San Francisco (UCSF). She is AP/CP and Hematology Board certified by the American Board of Pathology. Dr. Etzell is actively involved in the education of medical technologists, medical students, residents, and fellows in hematology / hematopathology. She serves as the Hematopathology Fellowship Director and Associate Residency Program Director in Laboratory Medicine in UCSF. Dr. Etzell has authored over 50 papers, book chapters, educational activities and abstracts in the areas of hematology and hematopathology. Dr. Etzell currently serves as the Vice-Chair of the Hematology and Clinical Microscopy Resource Committee for the College of American Pathologists (CAP). 11-Education