Blood Cell Identification: 2011-C Mailing: Iron Deficiency ... · The differential diagnosis of...

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

1-Education All material is © 2011 College of American Pathologists, all rights reserved

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

109/L; RBC = 2.1 x 1012/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 109/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 (Fe2+) form in living organisms now became oxidized into its ferric form

(Fe3+), 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



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



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.



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.



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

Iron deficiency anemia Anemia of chronic disease

Thalassemia

Sideroblastic anemia

Anemia Hypochromic, microcytic

Normochromic, normocytic or mildly hypochromic, microcytic

Hypochromic, microcytic

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.



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.



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 (β-

thalassemia) 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.



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.



References

1. Glassy EF, ed. Color Atlas of Hematology: An Illustrated Field Guide Based on Proficiency Testing.

Northfield, IL: College of American Pathologists; 1998.

2. Gulati G, Care J. Blood cells: An Atlas of Morphology with Clinical Relevance. Chicago, IL: ASCP

Press; 2007.

3. Mielczarek EV, McGrayne SB. Iron, Nature's Universal Element. Why People Need Iron and Animals

Make Magnets. New Brunswick NJ; London: Rutgers University Press; 2000.

4. Looker AC, Dallman PR, Carroll MD, et al. Prevalence of iron deficiency in the United States. JAMA.

1997;277:973.

5. Cook JD, Flowers CH, Skikne BS. The quantitative assessment of body iron. Blood.

2003;101:3349.

6. Cook JD, Finch CA, Smith NJ. Evaluation of the iron status of a population. Blood. 1976;48:449.

7. Schrier SL. Causes and diagnosis of anemia due to iron deficiency. UpToDate. 2010;1-33. Available

at: http://www.uptodate.com/contents/causes-and-diagnosis-of-anemia-due-to-iron-deficiency.

Accessed August 5, 2011.

8. Kjeldsberg CR, Perkins SL. Practical Diagnosis of Hematologic Disorders, Vol. 1 Benign Disorders.

5th ed. Chicago, IL: ASCP Press; 2010;17-29.

9. Elghetany MT, Banki K. In: Henry's Clinical Diagnosis and Management by Laboratory Methods.

21st ed. Philadelphia, PA: Saunders Elsevier; 2007: 504-507.

http://www.uptodate.com/contents/causes-and-diagnosis-of-anemia-due-to-iron-deficiency�



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).

Blood Cell Identification: 2011-C Mailing: Hereditary Pyropoikilocytosis (HPP)


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 = 14.82 x 109/L; RBC = 2.57 x 1012/L; HGB = 6.3 g/dL; HCT = 20%; MCV = 77 fL; and PLT =

329 x109/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 life-

threatening 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



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 45-46°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.



Figure 1. Comparison of Variants of Hereditary Elliptocytosis (HE)

Table 1. Comparison of Variants of Hereditary Elliptocytosis (HE)

Common HE Spherocytic HE Southeast Asian Ovalocytosis (aka stomatocytic HE) Most CHE HPP

Population affected

African descent African and Mediterranean descent

Caucasians Individuals from Far East, esp. Malaysia

Inheritance Autosomal dominant

Autosomal recessive Autosomal dominant Autosomal dominant

Mutation in red cell membrane

α-spectrin β-spectrin protein 4.1

Homozygous α-spectrin Unknown Band 3

Blood smear findings

Elliptocytes Microcytosis, striking micropoikilocytosis, fragmentation

Elliptocytes and spherocytes (lack poikilocytes)

Resemble stomatocytic ovalocytes

Clinical features Most patients non-anemic

Severe hemolytic anemia Mild to moderate hemolytic anemia

Absent to mild hemolysis

Manifestation Hemolysis at 45-46 °C (normal RBCs fragment at 49 °C)

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.



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.



Figure 3. Normal Red Cell Membrane

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 self-

associate 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 dimer-

dimer 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 4.1.

Horizontal Interactions

Ver

tica

l Int

erac

tions



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).



Figure 4. Figure 5.

Classic morphologic features of HPP. Numerous Classic morphologic features of HPP. Red cell fragments, fragmented cells and extensive polychromatophilic helmet cells, and elliptocytes are present. cells. Rare spherocytes are also seen.

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 45–46°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.



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 Mutation in red cell cytoskeletal membrane proteins (such as spectrin, etc.)

Disseminated intravascular coagulation (DIC); thrombotic thrombocytopenic purpura (TTP); hemolytic uremic syndrome (HUS)

Peripheral Blood Smear Findings

Red cell fragmentation/poikilocytosis; spherocytes; polychromatophilia

Red cell fragmentation/poikilocytosis; polychromatophilia; thrombocytopenia may be seen

Hemolysis Predominantly extravascular hemolysis with splenic sequestration

Primarily intravascular hemolysis

Inciting Event

Membrane instability due to homozygous spectrin deficiency. Manifests at temperatures 45–46°C (normal RBC fragments at 49°C)

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 45-48°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.



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.



References

1. McPherson RA, Pincus MR, eds. In: Henry’s Clinical Diagnosis and Management by Laboratory

Methods. 21st ed. Philadelphia, PA: Saunders Elsevier; 2007.

2. Glassy EF, ed. Color Atlas of Hematology: An Illustrated Field Guide Based on Proficiency Testing.

Northfield, IL: College of American Pathologists; 1998.

3. Tse WT, Lux SE. Red blood cell membrane disorders. Br J Haematol. 1999;104:2-13.

4. Iarocci TA, Wagner GM, Mohandas N, et al. Hereditary poikilocytic anemia associated with the co-

inheritance of two alpha spectrin abnormalities. Blood. 1988;71:1390.

5. Mentzer WC. Hereditary elliptocytosis: Clinical features and diagnosis. In: UpToDate. Landaw SA,

Hoppin AG, eds. UpToDate. Waltham, MA; 2011.

6. Mentzer WC. Hereditary elliptocytosis: Genetics and pathogenesis. In: UpToDate. Landaw SA,

Hoppin AG, eds. UpToDate. Waltham, MA; 2011.

7. 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;111:5712-

5720.

8. Tolpinrud W, Maksimova YD, Forget BG, Gallagher PG. Nonsense mutations of the a-spectric gene

in hereditary pyropoikilocytosis. Haematologica. 2008;93:1752-1754.



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 co-

chief resident for the anatomic pathology department at UCSF (2010-2011), 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).

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