4) Tema 2 Modul 1 (Red Cell Disorders)

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Theme 2

Clinical Aspects of Hematopoietic and Myeloproliferative Disorders

Module 1

Red Cell Disorders

Competency Based Curriculum

Medical Programme

FACULTY OF MEDICINE UNIVERSITY OF BRAWIJAYA

MALANG

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IRON DEFICIENCY ANEMIA

Definition and Overview Iron deficiency is defined as a decreased total iron body content. Iron

deficiency anemia occurs when iron deficiency is sufficiently severe to diminish erythropoiesis and cause the development of anemia. Iron deficiency is the most prevalent single deficiency state on a worldwide basis. It is important economically because it diminishes the capability of individuals who are affected to perform physical labor & it diminishes both growth and learning in children.

Competency Area Area of competence : 3rd of the Doctor Competencies Standard from Indonesian Medical Council.

Competency Component To apply the concepts and principles of etiology, pathogenesis and pathophysiology, staging of disease, clinical processes to diagnose iron deficiency anemia, and its management.

Clinical Competence 1. Student can describe the etiology, pathogenesis and pathophysiology of iron deficiency anemia.

2. Student can describe the stages in development of iron deficiency anemia.

3. Student can diagnose iron deficiency anemia based on clinical features and laboratory findings.

4. Student can describe the management of iron deficiency anemia.

Learning Methode Expert lecture, active learning/modul task & group discussion

Equipment Classroom, worksheet, computer, LCD and screen.

Time Expert lecture 50 minutes, active learning/modul task 100 minutes, and group discussion 50 minutes

Contributors 1. Department of Child Health 2. Department of Internal Medicine

Evaluation 1. Formative assessment at the end learning of some topics using true false questions

2. Module exam at the end learning of module using MCQs 3. Final exam at the end of semester using MCQs

Suggested Readings 1. Killip S, Bennet JM, Chambers MD. Iron Deficiency Anemia. Am Fam Physician 2007; 75:671-8.

2. United Nations Children’s Fund, United Nations University, World Health Organization. Iron Deficiency Anaemia : Assessment, Prevention and Control A Guide for Programme Managers. World Health Organization 2001.

3. Ciesla B. Iron Deficiency Anemia. In: Ciesla B, eds. Hematology in Practice. 1st ed. Philadelphia: F.A. Davis Company. 2007.

4. Andrews NC. Iron Deficiency and Related Disorders. In: Greer JP, Foerster J, Lukens JN, Rodgers GM et al, eds. Wintrobe’s Clinical Hematology. 11th ed.. Philadelphia. Lippincott Williams and Wilkins.2004.

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IRON DEFICIENCY ANEMIA

Department of Child Health Department of Internal Medicine

Faculty of Medicine University of Brawijaya INTRODUCTION Iron deficiency is defined as a decreased total iron body content. Iron deficiency anemia occurs when iron deficiency is sufficiently severe to diminish erythropoiesis and cause the development of anemia. Iron deficiency is the most prevalent single deficiency state on a worldwide basis. It is important economically because it diminishes the capability of individuals who are affected to perform physical labor, and it diminishes both growth and learning in children. ETIOLOGY Iron deficiency occurs as a late manifestation of prolonged negative iron balance, as a result of major blood loss, or because of failure to meet an increased physiologic need for iron. Factors leading to negative iron balance, increased requirements, or inadequate iron for erythropoiesis are listed in Table 1. In many instances, multiple etiologic factors are involved. The association of a marginal diet with some source of blood loss, such as that associated with menstruation, is a common combination. Another example is hookworm infection, which produces anemia primarily in those people whose diets are marginally adequate. Table 1. Etiologic Factors in Iron Deficiency Anemia Negative iron balance Decreased iron intake Inadequate diet Impaired absorption Increased iron loss Gastrointestinal blood loss : epistaxis, varices, gastritis, ulcer, tumor, Meckel’s diverticulum, parasitosis, milk-induced enteropathy of early childhood, vascular malformations, inflammatory bowel disease, diverticulosis, hemorrhoids Genitourinary blood loss : menorrhagia, chronic infections, cancer Pulmonary blood loss : pulmonary hemosiderosis, infection Other blood loss : trauma, excessive phlebotomy, large vascular malformations Increased requirements Infancy Pregnancy Lactation Inadequate presentation to erythroid precursors Atransferrinemia Antitransferrin receptor antibodies Abnormal iron balance Aceruloplasminemia Autosomal-dominant hemochromatosis due to mutations in ferroportin PATHOGENESIS AND PATHOPHYSIOLOGY Three pathogenetic factors are implicated in the anemia of iron deficiency. The first is impaired hemoglobin synthesis, a concequence of reduced iron supply. The second is a generalized defect in cellular proliferation. Third, there may be reduced erythrocyte survival, particularly when the anemia is severe. Iron is vital for all living organisms because it is essential for multiple metabolic processes, including oxygen transport, DNA synthesis, and electron transport. Iron equilibrium in the body is

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regulated carefully to ensure that sufficient iron is absorbed in order to compensate for body losses of iron. While body loss of iron quantitatively is as important as absorption in terms of maintaining iron equilibrium, it is a more passive process than absorption. Consistent errors in maintaining this equilibrium lead to either iron deficiency or iron overload. Iron balance is achieved largely by regulation of iron absorption in the proximal small intestine. Either diminished absorbable dietary iron or excessive loss of body iron can cause iron deficiency. Diminished absorption usually is due to an insufficient intake of dietary iron in an absorbable form. Hemorrhage is the most common cause of excessive loss of body iron, but it can occur with hemoglobinuria from intravascular hemolysis. Malabsorption of iron is relatively uncommon in the absence of small bowel disease or previous gastrointestinal surgery. Iron uptake in the proximal small bowel occurs by 3 separate pathways. These are the heme pathway and separate pathways for ferric and ferrous iron. One third of dietary iron is heme iron, but two thirds of body iron is derived from dietary myoglobin and hemoglobin. Heme iron is not chelated and precipitated by numerous constituents of the diet that renders nonheme iron nonabsorbable. Examples are phytates, phosphates, tannates, oxalates, and carbonates. Heme is maintained soluble and available for absorption by globin degradation products produced by pancreatic enzymes. Heme iron and nonheme iron are absorbed into the enterocyte noncompetitively. Heme enters the cell as an intact metalloporphyrin, presumably by a vesicular mechanism. Heme is degraded within the enterocyte by heme oxygenase with release of iron so that it traverses the basolateral cell membrane in competition with nonheme iron to bind transferrin in the plasma. Ferric iron utilizes a different pathway to enter cells than ferrous iron. This was shown by competitive inhibition studies, the use of blocking antibodies against divalent metal transporter-1 (DMT-1) and beta3-integrin, and transfection experiments using DMT-1 DNA. This indicated that ferric iron utilizes beta3-integrin and mobilferrin, while ferrous iron uses DMT-1 to enter cells. Which pathway transports most nonheme iron in humans is not known. Most nonheme dietary iron is ferric iron. Iron absorption in mice and rats may involve more ferrous iron because they excrete moderate quantities of ascorbate in intestinal secretions. Contrariwise, humans are a scorbutic species and are unable to synthesize ascorbate to reduce ferric iron. Other proteins are described that appear related to iron absorption. These are stimulators of iron transport (SFT), which are reported to increase the absorption of both ferric and ferrous iron, and hephaestin, which is postulated to be important in the transfer of iron from enterocytes into the plasma. The relationship and interactions between the newly described proteins is not known at this time and is being explored in a number of laboratories. The iron concentration within enterocytes varies directly with the body's requirement for iron. Absorptive cells in iron-deficient humans and animals contain little stainable iron, whereas this is increased significantly in subjects who are replete in iron. Untreated phenotypic hemochromatosis creates little stainable iron in the enterocyte, similar to iron deficiency. Iron within the enterocyte may operate by up-regulation of a receptor, saturation of an iron-binding protein, or both. In contrast to findings in iron deficiency, enhanced erythropoiesis, or hypoxia, endotoxin rapidly diminishes iron absorption without altering enterocyte iron concentration. This suggests that endotoxin and, perhaps, cytokines alter iron absorption by a different mechanism. Most iron delivered to nonintestinal cells is bound to transferrin. Transferrin iron is delivered into nonintestinal cells via 2 pathways, the classical transferrin receptor pathway (high affinity, low capacity) and the pathway independent of the transferrin receptor (low affinity, high capacity). Otherwise, the nonsaturability of transferrin binding to cells cannot be explained.

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In the classical transferrin pathway, the transferrin iron complex enters the cell within an endosome. Acidification of the endosome releases the iron from transferrin so that it can enter the cell. The apotransferrin is delivered by the endosome to the plasma for reutilization. The method by which the transferrin receptor–independent pathway delivers iron to the cell is not known. Nonintestinal cells also possess the mobilferrin integrin and DMT-1 pathways. Their function in the absence of an iron-saturated transferrin is uncertain; however, their presence in nonintestinal cells suggests they may participate in intracellular functions in addition to their capability to facilitate cellular uptake of iron. STAGES IN THE DEVELOPMENT OF IRON DEFICIENCY ANEMIA The first stage, also called prelatent iron deficiency or iron depletion, represents a reduction in iron stores without reduced serum iron levels. This stage is usually detected by a low serum ferritin measurement. Latent iron deficiency is said to exist when iron stores are exhausted, but the blood hemoglobin level remains higher than the lower limit of normal. In this second stage (latent iron deficiency or iron deficiency), certain biochemical abnormalities of iron-limited erythropoiesis may be detected, including reduced transferrin saturation, increased TIBC, increased free erythrocyte protoporphyrin, increased zinc protoporphyrin, and increased serum TFRC. Other findings include subnormal urinary iron excretion after deferoxamine injection and decreased tissue cytochrome oxidase levels. The mean corpuscular volume (MCV) usually remains within normal limits, but a few microcytes may be detected on a blood smear. Many patients report generalized fatique or malaise, even though they are not yet anemic. Finally, in the third stage (iron deficiency anemia), the blood hemoglobin concentration falls below the lower limit of normal, and iron deficiency anemia is apparent. Iron-containing enzymes, such as the cytochrome, also reach abnormally low levels during this period. Epithelial manifestations of iron deficiency oocur very late in iron deprivation. This progression forms the basis for the stages of iron deficiency outlined in Table 2. Table 2. Laboratory Findings for Identifying Iron Deficiency

Stage I Prelatent/Iron Depletion

Stage II Latent/Iron Deficiency

Stage III Iron Deficiency Anemia

Symptoms Fatique, malaise in some patients Pallor, pica, epithelial changes

Hemoglobin levels Normal Normal Decreased Mean corpuscular volume Normal Normal Decreased Reticulocyte Hb content Normal Decreased Decreased Serum iron Normal < 60 ug/dl < 40 µg/dl Total iron binding capacity 360-390 µg/dl > 390 µg/dl > 410 µd/dl Transferrin saturation Normal < 16% < 16% Serum ferritin < 20 µg/L < 12 µg/L < 12 µg/L Free erythrocyte protoporphyrin, zinc protoporphyrin

Normal Increased Increased

Bone marrow iron Decreased Absent Absent CLINICAL FEATURES History of Patients While iron deficiency anemia is a laboratory diagnosis, a carefully obtained history can lead to its recognition. The history can be useful in establishing the etiology of the anemia and, perhaps, in estimating its duration.

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1. History of diet A dietary history is important. Vegetarians are more likely to develop iron deficiency, unless their diet is supplemented with iron. National programs of dietary iron supplementation are initiated in many portions of the world where meat is sparse in the diet and iron deficiency anemia is prevalent. Unfortunately, affluent nations also supplement iron in foodstuffs and vitamins without recognizing the potential contribution of iron to free radical formation and the prevalence of genetic iron overloading disorders. Elderly patients, because of poor economic circumstances, may try to survive on a "tea and toast" diet because they do not wish to seek aid. They may also be hesitant to share this dietary information. Pica can be the etiology of iron deficiency among people who habitually eat either clay or laundry starch. Hippocrates recognized clay eating; however, physicians do not recognize it unless the patient and family are specifically queried. Both substances decrease the absorption of dietary iron. Clay eating occurs worldwide in all races, though it is more common in Asia Minor. Starch eating is a habit in females of African heritage, and it often is started in pregnancy as a treatment for morning sickness. 2. Hemorrhage Two thirds of body iron is present in circulating red blood cells as hemoglobin. Each gram of hemoglobin contains 3.47 mg of iron; thus, each mL of blood lost from the body (hemoglobin 15 g/dL) results in a loss of 0.5 mg of iron. Bleeding is the most common cause of iron deficiency in North America and Europe. Patients report a history of bleeding from most orifices (hematuria, hematemesis, hemoptysis) before they develop chronic iron deficiency anemia; however, gastrointestinal bleeding may go unrecognized, and excessive menstrual losses may be overlooked. Patients often do not understand the significance of a melanotic stool. Unless menstrual flow changes, patients do not seek medical attention. If they do, they report that their menses are normal in response to inquiry for self-evaluation. Because of the marked differences among women with regard to menstrual blood loss (10-250 mL per menses), query the patient about a specific history of clots, cramps, and the use of multiple tampons and pads. 3. Duration Iron deficiency in the absence of anemia is asymptomatic. One half of patients with moderate iron deficiency anemia develop pagophagia. Usually, they crave ice to suck or chew. Occasionally, patients are seen who prefer cold celery or other cold vegetables in lieu of ice. Leg cramps, which occur on climbing stairs, also are common in patients deficient in iron. Often, patients can provide a distinct point in time when these symptoms first occurred, providing an estimate of the duration of the iron deficiency. Signs and Symptoms There are many signs and symptoms that mark an individual as being iron deficient. Some of these symptoms are unique to iron deficiency and some are general signs and symptoms of anemia. Clinically, a patient with anemia may present with fatique, pallor (Figure 1a), vertigo, dyspnea, cold intolerance, and lethargy. Additionally, these patients may experience cardiac problems such as palpitations and angina. Symptoms unique to the iron deficiency anemia patient are pica (an abnormal craving for unusual substances such as dirt, ice, or clay), cheilitis (inflammation around the lips), and koilonychias (spooning of the nail beds( (Figure 1b).

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Additionally, evidence suggest that iron deficiency in infants may result in developmental delays and behavioral disturbances. In pregnant women, iron deficiency in the first two trimesters may lead to an increase in preterm delivery and an increase in delivering a low-birth-weight baby.

Figure 1. (a) Pallor, and (b) Koilonychia LABORATORY STUDIES From a clinical standpoint, if iron deficiency is suspected, testing for iron deficiency must analyze the patient’s red cell status and iron status. In terms of the complete blood cell count (CBC), hemoglobin levels will be below the normal reference range. The definition of anemia varies by sex and age. The most commonly used definitions of anemia come from the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) (Table 3). Table 3. Definition of Anemia by Hemoglobin Value

The mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) will be markedly lower than normal, the red cell distribution width (RDW) may be mildly elevated, and the peripheral blood smear will show small red cells, which are deficient in hemoglobin. Target cells and elliptocytes may occasionally be seen (Figure 2). The reticulocyte count will be low in comparison to the level of anemia, indicating a slightly ineffective erythropoiesis. Test to assess a patient’s iron status include serum iron (SI), transferrin or total iron binding capacity (TIBC), serum ferritin, and transferrin saturation. Serum iron is a measure of the total amount of iron in the serum with a normal value of 50 to 150 µg/L. The TIBC measures the availability of iron binding sites on the transferrin molecule. If an individual is iron deficient, there will be many binding sites available searching for iron and the TIBC value will be increased. This value is elevated in iron deficient patients (reference range 250 to 450 µg/L) but subject to fluctuations in patients who use oral contraceptives or have liver disease, chronic infections or nephrotic syndrome. The TIBC is less sensitive to iron deficiency and must be evaluated in terms of the patient’s other health issues. Transferrin saturation (% saturation) is derived as the

(a) (b)

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product of the serum iron concentration divided by the TIBC and multiplied by 100. The normal value is 20% to 50%. Serum ferritin is one of the most sensitive indicators of iron stores, with a normal value of 20 to 250 µg/L for men and 10 to 120 µg/L for women. Ferritin is an acute phase reactant, and conditions such as chronic inflammation or chronic infection may falsely elevate the serum ferritin level. In these cases, an accurate assessment of iron stores will be difficult.

A bone marrow aspirate can be diagnostic of iron deficiency. The absence of stainable iron in a bone marrow aspirate that contains spicules and a simultaneous control specimen containing stainable iron permit establishment of a diagnosis of iron deficiency without other laboratory tests. Other laboratory tests are useful to establish the etiology of iron deficiency anemia and to exclude or establish a diagnosis of 1 of the other microcytic anemias. a. Testing stool for the presence of hemoglobin is useful in establishing gastrointestinal bleeding as

the etiology of iron deficiency anemia. Usually, chemical testing that detects more than 20 mL of blood loss daily from the upper gastrointestinal tract is employed. More sensitive tests are available; however, they produce a high incidence of false-positive results in people who eat meat. Severe iron deficiency anemia can occur in patients with a persistent loss of less than 20 mL/d.

b. To detect blood loss, the patient can be placed on a strict vegetarian diet for 3-5 days and the stool can be tested for hemoglobin using a benzidine method, or red blood cells can be radiolabeled with radiochromium and retransfused. Stools are collected, and the radioactivity is quantified in a gamma-detector and compared to the radioactivity in a measured quantity of the patient's blood. An immunological method of detecting human species-specific hemoglobin in stool is under development and could increase specificity and sensitivity.

c. Hemoglobinuria and hemosiderinuria can be detected by laboratory testing as described under Causes. This documents iron deficiency to be due to renal loss of iron and incriminates intravascular hemolysis as the etiology.

d. Hemoglobin electrophoresis and measurement of hemoglobin A2 and fetal hemoglobin are useful in establishing either beta-thalassemia or hemoglobin C or D as the etiology of the microcytic anemia. Unfortunately, simple tests do not exist for alpha-thalassemia in most laboratories, and it is a diagnosis of exclusion.

MANAGEMENT Treatment 1. Medication The most economical and effective medication in the treatment of iron deficiency anemia is the oral administration of ferrous iron salts. Among the various iron salts, ferrous sulfate most commonly is used. Claims are made that other iron salts are absorbed better and have less morbidity. Generally, the toxicity is proportional to the amount of iron available for absorption. If the quantity of

Figure 2. Microcytic and hypochromic red cells

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iron in the test dose is decreased, the percentage of the test dose absorbed is increased, but the quantity of iron absorbed is diminished. Ferrous sulfate is the mainstay treatment for treating patients with iron deficiency anemia. They should be continued for about 2 months after correction of the anemia and its etiological cause in order to replenish body stores of iron. Ferrous sulfate is the most common and cheapest form of iron utilized. Tablets contain 50-60 mg of iron salt. Other ferrous salts are used and may cause less intestinal discomfort because they contain a smaller dose of iron (25-50 mg). Oral solutions of ferrous iron salts are available for use in pediatric populations. Doses of ferrous sulfate in adult patient is 325 mg (60 mg iron) orally with each meal three times daily, and in pediatric patient is 3-6 mg/kg/day orally divided in 1 to 3 dosis suggested, depending on severity of anemia Reserve parenteral iron for patients who are either unable to absorb oral iron or who have increasing anemia despite adequate doses of oral iron. It is expensive and has greater morbidity than oral preparations of iron. Reserve transfusion of packed RBC for patients with either significant acute bleeding or patients in danger of hypoxia and/or coronary insufficiency. 2. Dietetic On a worldwide basis, diet is the major cause of iron deficiency. To suggest that iron-deficient populations correct the problem by the addition of significant quantities of meat to their diet is unrealistic. The addition of nonheme iron to national diets is initiated in some areas of the world. Problems encountered in these enterprises include changes in taste and appearance of food after the addition of iron and the need to supplement foodstuffs that are consumed by most of the population in predictable quantities. In addition, many dietary staples, such as bread, contain iron chelators that markedly diminish the absorption of the iron supplement (phosphates, phytates, carbonates, oxalates, tannates). Persons on an iron-poor diet need to be identified and counseled on an individual basis. Educate older individuals on a tea and toast diet about the importance of improving their diet, and place them in contact with community agencies that will provide them with at least 1 nutritious meal daily. Patients who have dietary-related iron deficiency due to pica need to be identified and counseled to stop their consumption of clay and laundry starch. 3. Surgical Treatment Surgical treatment consists of stopping hemorrhage and correcting the underlying defect so that it does not recur. This may involve surgery for treatment of either neoplastic or nonneoplastic disease of the gastrointestinal tract, the genitourinary tract, the uterus, and the lungs. 4. Consultations Surgical consultation often is needed for the control of hemorrhage and treatment of the underlying disorder. In the investigation of a source of bleeding, consultation with certain medical specialties may be useful to identify the source of bleeding and to provide control. Gastroenterology consultation is the most frequently sought consult among the medical specialties. Endoscopy has become a highly effective tool in identifying and controlling gastrointestinal bleeding. If bleeding is brisk, angiographic techniques may be useful in identifying the bleeding site and controlling the hemorrhage. Radioactive technetium labeling of autologous erythrocytes also is used to identify the site of bleeding. Unfortunately, these radiographic techniques do not detect bleeding at rates less than 1 mL/min and may miss lesions with intermittent bleeding.

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5. Activity Restriction of activity is usually not required. Patients with moderately severe iron deficiency anemia and significant cardiopulmonary disease should limit their activities until the anemia is corrected with iron therapy. If these patients become hypoxic or develop evidence of coronary insufficiency, they should be hospitalized and placed at bed rest until improvement of their anemia cells can be accomplished by transfusion of packed red blood. Obviously, these decisions need to be made on an individual basis and differ somewhat depending upon the severity of the anemia and the comorbid conditions. March hemoglobinuria can produce iron deficiency, and its treatment requires modification of activity. Cessation of jogging or wearing sneakers while running usually diminishes the hemoglobinuria. Prevention Mineral supplementations These agents are used to provide adequate iron for hemoglobin synthesis and to replenish body stores of iron. Iron is administered prophylactically during pregnancy because of anticipated requirements of the fetus and losses that occur during delivery. Patient Education Physician education is needed to ensure a greater awareness of iron deficiency and the testing needed to establish the diagnosis properly. Physician education also is needed to investigate the etiology of the iron deficiency. Public health officials in geographic regions where iron deficiency is prevalent need to be aware of the significance of iron deficiency, its effect upon work performance, and the importance of providing iron during pregnancy and childhood. The addition of iron to basic foodstuffs is employed in these areas to diminish the problem. Table 4 provided recommendations to prevent and control iron deficiency in the United States.

Table 4. Recommendations to Prevent and Control Iron Deficiency in the United States For infants (0 to 12 months) and children (1 to 5 years) • Encourage breastfeeding or • Iron-fortified formula • Serve one serving of fruits, vegetables, juice by 6 months • Screen children for anemia every 6 months School-age children (5 to 12 years) and adolescent boys (12 to 18 years) • Screen only those with history of IDA or low iron intake groups Adolescent girls (12 to 18 years) and nonpregnant women of childbearing age • Encourage intake of iron-rich food and foods that increase iron absorption • Screen nonpregnant women every 5 to 10 years through childbearing years Pregnant women • Start oral doses of iron at first prenatal visit • Screen for anemia at first prenatal visit • If hemoglobin is _9 g/dL, provide further medical attention Postpartum women • Risk factors include continued anemia, excessive blood loss, and multiple births Males older than 18 years/postmenopausal women • No routine screening is recommended COMPLICATIONS • Iron deficiency anemia diminishes work performance by forcing muscles to depend, to a greater

extent than in healthy individuals, upon anaerobic metabolism. This is believed to be due to deficiency in iron-containing respiratory enzymes rather than anemia.

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• Severe anemia due to any cause may produce hypoxemia and enhance the occurrence of coronary insufficiency and myocardial ischemia. Likewise, it can worsen the pulmonary status of patients with chronic pulmonary disease.

• Defects in structure and function of epithelial tissues may be observed in iron deficiency. Fingernails may become brittle or longitudinally ridged with development of koilonychia (spoon-shaped nails). The tongue may show atrophy of the lingual papillae and develop a glossy appearance. Angular stomatitis may occur with fissures at the corners of the mouth. Dysphagia may occur with solid foods, with webbing of the mucosa at the junction of the hypopharynx and the esophagus (Plummer-Vinson syndrome); this has been associated with squamous cell carcinoma of the cricoid area. Atrophic gastritis occurs in iron deficiency with progressive loss of acid secretion, pepsin, and intrinsic factor and development of an antibody to gastric parietal cells. Small intestinal villi become blunted.

• Cold intolerance develops in one fifth of patients with chronic iron deficiency anemia and is manifested by vasomotor disturbances, neurologic pain, or numbness and tingling.

• Rarely, severe iron deficiency anemia is associated with papilledema, increased intracranial pressure, and the clinical picture of pseudotumor cerebri. These manifestations are corrected with iron therapy.

• Impaired immune function is reported in subjects who are iron deficient, and there are reports that these patients are prone to infection; however, evidence that this is directly due to iron deficiency is not convincing because of the presence of other factors.

• Children deficient in iron may exhibit behavioral disturbances. Neurologic development is impaired in infants and scholastic performance is reduced in children of school age. The IQ of school children deficient in iron is reported as significantly less than their nonanemic peers. Behavioral disturbances may manifest as an attention deficit disorder. Growth is impaired in infants with iron deficiency. All these manifestations improve following iron therapy.

PROGNOSIS Iron deficiency anemia is an easily treated disorder with an excellent outcome; however, it may be caused by an underlying condition with a poor prognosis, such as neoplasia. Similarly, the prognosis may be altered by a comorbid condition such as coronary artery disease. MODUL TASK: Discuss this case in your small group!

CASE: A 9 month-old girl, body weight 8450 grams History taking: pale 1 week; no bleed; no fever; no history of blood transfusion Physical examination: conjunctiva palpebra anemic, facies dysmorphic (-); lymph nodes, liver and spleen are not palpable Laboratory results: Hb 9,3 g/dl; white blood cells 7250/mm3; platelet 314.000mm3; MCV, MCH and MCHC low; serum iron (SI) low; total iron binding capacity (TIBC) high; Hb-electrophoresis normal

QUESTIONS: 1. What is THE MOST PROBABLE diagnosis of this patient? 2. What are THE ETIOLOGIC FACTORS of this disease? 3. What are THE MECHANISMS OF THIS DISEASE? 4. Describe in brief THE CLINICAL STAGES in the development of this disease! 5. How TO DIAGNOSE this disease based on the clinical features and laboratory findings? 6. How TO MANAGE this patient?

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SUGGESTED READINGS 1. Killip S, Bennet JM, Chambers MD. Iron Deficiency Anemia. Am Fam Physician 2007; 75:671-8. 2. Conrad ME. Iron Deficiency Anemia. e-Medicine Pediatrics 2006. URL: http://emedicine.

medscape. com/article/202333-overview 3. United Nations Children’s Fund, United Nations University, World Health Organization. Iron

Deficiency Anaemia : Assessment, Prevention and Control A Guide for Programme Managers. World Health Organization 2001.

4. Ciesla B. Iron Deficiency Anemia. In: Ciesla B, eds. Hematology in Practice. 1st ed. Philadelphia: F.A. Davis Company. 2007.

5. Andrews NC. Iron Deficiency and Related Disorders. In: Greer JP, Foerster J, Lukens JN, Rodgers GM et al, eds. Wintrobe’s Clinical Hematology. 11th ed.. Philadelphia. Lippincott Williams and Wilkins.2004.

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MEGALOBLASTIC ANEMIA

Definition and Overview Megaloblastic anemias are a heterogeneous group of anemia that share

common morphologic characteristics. Erythrocytes are larger and have higher nuclear-to-cytoplasmic ratios compared to normoblastic cells. The underlying defect accounting for the asynchronous maturation of the nucleus is defective DNA synthesis and in clinical practice, this is usually caused by deficiency of vitamin B12 (cobalamine) or folate.


Competency Component To apply the concepts and principles of etiology, pathophysiology, clinical processes to diagnose megaloblastic anemia caused by vitamin B12 and folate deficiency, and its management.

Clinical Competence 1. Student can describe the etiology of megaloblastic anemia caused by vitamin B12 and folate deficiency.

2. Student can describe the pathophysiology of megaloblastic anemia. 3. Student can diagnose megaloblastic anemia based on clinical

features and laboratory findings. 4. Student can describe the management of megaloblastic anemia

caused by vitamin B12 and folate deficiency.

Learning Methode Active learning with modul task, group discussion, expert lecture, and skill development (history taking).


Time Active learning with modul task 2x50 minutes; group discussion 1x50 minutes; expert lecture 1x50 minutes.

Contributors 1. Department of Child Health. 2. Department of Internal Medicine.

Evaluation Module exams at the end of module programmes, middle and final exams at the end of semester with multiple choice questions, and skill exam with OSCE.

Suggested Readings 1. Hoffbrand AV, Pettit JE, Moss PAH. Megaloblastic Anaemia and Other Macrocytic Anaemias. In: Essential Haematology. 4th ed. London: Blackwell Science. 2001.

2. Ciesla B. The Macrocytic Anemias. In: Ciesla B, eds. Hematology in Practice. 1st ed. Philadelphia: F.A. Davis Company. 2007.

3. Schik P. Megaloblastic Anemia. e-Medicine Pediatrics 2007. URL: http://emedicine. medscape. com/article/204066-overview

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MEGALOBLASTIC ANEMIA


Faculty of Medicine University of Brawijaya INTRODUCTION In macrocytic anemia the red cells are abnormally large (mean corpuscular volume or MCV > 95 fl). There are many several causes but they can be broadly subdivided into megaloblastic and non-megaloblastic, based on the appearance of developing erythroblasts in the bone marrow. Megaloblastic anemias are a heterogeneous group of anemia that share common morphologic characteristics. Erythrocytes are larger and have higher nuclear-to-cytoplasmic ratios compared to normoblastic cells. Neutrophils can be hypersegmented, and megakaryocytes are abnormal. On the molecular level in megaloblastic cells, the maturation of nuclei is delayed, while cytoplasmic development is normal. ETIOLOGY The underlying defect accounting for the asynchronous maturation of the nucleus is defective DNA synthesis and in clinical practice, this is usually caused by deficiency of vitamin B12 (cobalamine) or folate. Less commonly, abnormalities of metabolism of these vitamins or other lesions in DNA synthesis may cause an identical hematological appearance. Vitamin B12 Deficiency Vitamin B12 deficiency is usually caused by pernicious anemia (Table 1). Less commonly it may be caused by veganism in which the diet lacks B12 (usually in Hindu Indians), gastrectomy or small intestinal lesions. There is no syndrome of B12 deficiency as a result of increased utilization or loss of the vitamin, so the deficiency inevitably takes at least 2 years to develop, i.e. the time needed for body stores to deplete at the rate of 1-2 µg each day when there is no new B12 entering the body from the diet. Nitrous oxide, however, may rapidly inactivate body B12. Table 1. Causes of Vitamin B12 Deficiency Nutritional Especially vegans Malabsorption Gastric causes Pernicious anemia Congenital lack or abnormality of intrinsic factor Total or partial gastrectomy Intestinal causes Intestinal stagnant loop syndrome – jejunal diverticulosis, blind loop, stricture, etc Chronic tropical sprue Ileal resection and Crohn’s disease Congenital selective malabsorption with proteinuria (autosomal recessive megaloblastic anemia) Fish tapeworm Pernicious anemia is caused by autoimmune attack on the gastric mucosa leading to atrophy of the stomach. The wall of the stomach becomes thin, with a plasma cell and lymphoid infiltrate of the lamina propria. Intestinal metaplasia may occur. There is achlorhydria and secreation of intrinsic factor is absent or almost absent. More females than males are affected (1.6:1) with a peak

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occurrence at 60 years, and there may be associated autoimmune disease. The disease is found in all races but is most common in Nothern Europeans and tends to occur in families. There is also an increased incidence of carcinoma of the stomach (about 2-3% of all cases of pernicious anemia). Folate Deficiency This is most often a result of a poor dietary intake of folate alone or in combination with a condition of increased folate utilization or malabsorption (Table 2). Excess cell turnover of any sort, including pregnancy, is the main cause of an increased need for folate, since the folate molecul becomes degraded when DNA synthesis is increased. The mechanism by which anticonvulsants and barbiturates cause the deficiency is still controversial. Alcohol, sulfasalazine and other drugs may have multiple effects on folate metabolism. Table 2. Causes of Folate Deficiency Nutritional Especially old age, institutions, poverty, famine, special diets, goat’s milk anemia, etc Malabsorption Tropical sprue, gluten-induced enteropathy (adult or child) Possible contributory factor to folate deficiency in some patients with partial gastrectomy, extensive jejunal resectionor Crohn’s disease Excess utilization Physiological Pregnancy and lactation, prematurity Pathological Hematological diseases: hemolytic anemia, myelofibrosis Malignant diseases: carcinoma, lymphoma, myeloma Inflammatory diseases: Crohn’s disease, tuberculosis, rheumatoid arthritis, psoriasis, exfoliative dermatitis, malaria Excess urinary folate loss Active live disease, congestive heart failure Drugs Anticonvulsants, sulfasalazine Mixed Liver disease, alcoholism, intensive care PATHOPHYSIOLOGY The molecular basis for megaloblastosis is a failure in the synthesis and assembly of DNA. The most common causes of megaloblastosis are cobalamin and folate deficiencies. Cobalamin metabolism and folate metabolism are intricately related, and abnormalities in these pathways are believed to lead to the attenuated production of DNA. Methotrexate-induced megaloblastosis has been ascribed to a deficiency in deoxythymidine triphosphate (dTTP) that is consumed by the methyl folate trap. Evidence exists that megaloblastosis is caused by interference of folate metabolism by the inhibition of methionine synthesis. However, because of dietary folate deficiency, the size of the dTTP pool is normal or increased in persons with megaloblastosis. Impairment in the deoxyuridine monophosphate (dUMP) and deoxythymidine monophosphate (dTMP) pathway may be responsible for nutritional megaloblastosis. Despite this information, the biochemical basis for megaloblastosis is not fully understood. This is especially true of the cobalamin-related neuropathy that can occur independently of megaloblastic changes in hematopoietic cells. One hypothesis for the cause of cobalamin neuropathy is that a defect exists in the conversion of adenosyl-cobalamin-dependent conversion of methylmalonyl coenzyme A to succinyl coenzyme A.

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A hallmark of megaloblastic anemia is ineffective erythropoiesis, as evidenced by erythroid hyperplasia in the bone marrow, a decreased peripheral reticulocyte count, and an elevation in lactate dehydrogenase (LDH) and indirect bilirubin levels. The pathogenesis of these findings is the intramedullary destruction of fragile and abnormal megaloblastic erythroid precursors. An understanding of the source of cobalamin and folate is important to understand the pathogenesis of the development of megaloblastosis. Dietary intake is the source of cobalamin and folate because humans cannot synthesize these substances. Cobalamin must be bound to intrinsic factor (IF), and this complex is taken up in the terminal ileum. Once absorbed, cobalamin is bound to another protein, transcobalamin II (TCII), and is transported to storage sites. Abnormalities in any of these steps in cobalamin transport can lead to deficiencies in this substance. Considerable amounts of cobalamin are accumulated in storage sites; this explains why years elapse before cobalamin deficiency develops in patients who cannot take up dietary cobalamin. Although the processing and transport of ingested folate is complex, folate-induced megaloblastosis is rarely caused by abnormalities in transport but instead is most often caused by dietary insufficiency. Folate deficiency can be caused by malabsorption in patients with sprue. In contrast to cobalamin, very little folate is stored; this explains why folate deficiency can occur within months of cessation of folate ingestion. Megaloblastosis can also be caused by disorders in which cobalamin and folate uptake and metabolism are not affected. Myeloproliferative syndromes and viral infections (eg, HIV) can lead to megaloblastosis by disrupting DNA synthesis. Megaloblastosis can occur in patients who are on certain medications, including many cancer chemotherapy drugs. CLINICAL FEATURES The onset is usually insidious with gradually progressive symptoms and signs of anemia. The patients may be mildly jaundiced (lemon yellow tint) due to the excess breakdown of hemoglobin resulting from increased ineffective erythropoiesis in the bone marrow. Glossitis (a beefy-red, sore tongue), angular stomatitis (Figure 1) and mild symptoms of malabsorption with loss of weight may be present caused by the epithelial abnormality.

Figure 1. Megaloblastic anemia: glossitis, and angular cheilosis (stomatitis) Purpura as a result of thrombocytopenia and widespread melanin pigmentation (the cause for which is unclear) are less frequent presenting features (Table 3). Many asymptomatic patients are diagnosed when a blood count that has bee performed for another reason reveals macrocytosis. Vitamin B12 neuropathy (subacute combined degeneration of the cord) Severe B12 deficiency may cause a progressive neuropathy affecting the peripheral sensory nerves, and posterior and lateral columns. The neuropathy is symmetrical and affects the lower limbs more than the upper limbs. The patient notices tingling in the feet, difficulty in walking and may fall over in the dark. Rarely, optic atrophy or severe psychiatric symptoms are present. Anemia may be

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severe, mild or even absent, but the blood film and bone marrow appearances are always abnormal. The cause of the neuropathy is likely to be related to the accumulation of S-adenosyl homocysteine and reduced levels of S-adenosyl nethionine in nervous tissue resulting in defective methylation of myelin and other substrates. The evidence that folate deficiency in the adult can cause a neutropathy in conflicting although there are more substantial data suggesting it causes psychiatric changes. Table 3. Effects of Vitamin B12 or Folate Deficiency Megaloblastic anemia Macrocytosis of epithelial cell surfaces Neuropathy (for vitamin B12 only) Sterility Rarely, reversible melanin skin pigmentation Decreased osteoblast activity Neural tube defects in the fetus are related to folate or B12 deficiency Cardiovascular disease Neural tube defect Folate or B12 deficiency in the mother predisposes to neural tube defect (NTD) (anencephaly, spina bifida or encephalocele) in the fetus (Figure 2). The lower the maternal serum or red cell folate or serum B12 levels (even when these are in the normal range) the higher the incidence of NTDs. Moreover, supplementation of the diet with folic acid at the time of conception and in early pregnancy reduces the incidence of NTD by 75%. The exact mechanism is uncertain but is thought to be related to build-up of homocysteine and S-adenosyl homocysteine in the fetus which may impair methylation of various proteins and lipids. A common polymorphism in the enzyme 5,10-methylene tetrahydrofolate reductase (5,10-MTHFR)(677C→T) results in higher serum homocysteine and lower serum and red cell folate levels compared to controls. The incidence of the mutation is higher in the parents and fetus with NTD than in controls.

Cardiovascular disease Raised serum homocysteine levels are associated with an increased incidence of myocardial infarct, peripheral and cerebral vascular disease and venous thrombosis. Raised serum homocysteine levels are associated with low serum and red cell folate and low serum B12 or vitamin B6 levels. In addition homocysteine levels tend to be higher in men than in premenopausal women, in old age, in heavy smokers and those with excess alcohol consumption, with impaired renal function and with some drugs. Although folate deficiency has been associated with an increased incidence of cardiovascular disease, results of studies showing a reduction in the rate of myocardial infarction or stroke by the use of prophylactic folic acid have yet to be reported.

Figure 2. A baby with neural tube defect (spina bifida)

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Other tissue abnormalities Sterility is frequent in either sex with severe B12 or folate deficiency. Macrocytosis, excess apoptosis and other morphological abnormalities of cervical, buccal, bladder and other epithelia occurs. Widespread reversible melanin pigmentation may also occur. B12 deficiency is associated with reduced osteoblastic activity. Uncontrolled trials suggest folate deficiency may predispose to colon cancer. LABORATORY FINDINGS The CBC shows a pancytopenia (low white count, low red count, and low platelet count), although the platelet count may be only borderline low (see normal values on the front cover of this textbook). Pancytopenia in the CBC combined with macrocytosis should raise the index of suspicion toward a megaloblastic process because few other conditions (aplastic anemia, hypersplenism) show this pattern. Red cell inclusions such as basophilic stippling and Howell-Jolly bodies may be observed. Howell-Jolly bodies formed from megaloblastic erythropoiesis are larger and more fragmented in appearance than normal Howell-Jolly bodies. There is a low reticulocyte count (less than 1%) and the RDW is increased, owing to schistocytes, targets, and teardrop cells. The blood smear in megaloblastic anemia is extremely relevant in the diagnosis and shows macrocytes, macro-ovalocytes, hypersegmented multilobed neutrophils, and little polychromasia with respect to the anemia (Figure 3).

The presence of hypersegmented neutrophils (lobe count of more than five lobes) in combination with macrocytic anemia is a morphological marker for megaloblastic anemias. This qualitative white cell abnormality appears early in the disease and survives through treatment. It is usually the last morphology to disappear. The MCV initially is extremely high and may be in the range of 100 to 140 fL. A bone marrow examination is not necessary for the diagnosis of megaloblastic anemia, because the diagnosis of this disorder can be adequately made without this time-consuming, costly, and invasive procedure. Laboratory Diagnosis of Megaloblastic Anemias The megaloblastic anemias show striking similarities in their clinical and hematological presentations. Common features of the megaloblastic anemias include : • Pancytopenia • Increased MCV and MCHC • Hypersegmented neutrophils (five lobes or more in segmented neutrophils) • Increased bilirubin • Increased LDH • Hyperplasia in the bone marrow

Figure 3. Peripheral smear from a patient with megaloblastic anemia. Note the hypersegmented neutrophils and the macro-ovalocytes.

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• Decreased M:E ratio • Reticulocytopenia TREATMENT Most patients with megaloblastosis are treated with cobalamin and folate therapy to treat deficiencies in these substances (Table 4). Transfusion therapy should be restricted to patients with severe, uncompensated, and life-threatening anemia. Because megaloblastic anemias usually develop gradually, most patients have adjusted to low Hgb levels and do not require transfusions. Table 4. Treatment of Megaloblastic Anemia

Vitamin B12 deficiency Folate deficiency

Compound

Hydroxocobalami

Folic acid

Route Intramuscular * Oral

Dose 1000 µg 5 mg

Initial dose 6 x 1000 µg over 1-3 weeks Daily for 4 months

Maintenance 1000 µg every 3 months Depends on underlying disease; life-long therapy may be needed in chronic inherited hemolytic anemia, myelofibrosis, renal dialysis

Prophylactic Total gastrectomy, ileal resection Pregnancy, severe hemolytic anemia, dialysis, prematurity

* Some authors have recommended daily oral or sublingual therapy of vitamin B12 deficiency • Cobalamin (1000 mcg) should be given parenterally daily for 2 weeks, then weekly until the

hematocrit value is normal, and then monthly for life. This dose is large, but it may be required in some patients. Patients with neurological complications should receive cobalamin at 1000 mcg (more in some cases) every day for 2 weeks, then every 2 weeks for 6 months, and monthly for life.

- Oral cobalamin (1000 mcg) can be administered to patients with hemophilia (to avoid intramuscular injections) and to patients with severe malnutrition or those who have abnormalities in the terminal ileum. Doses and schedules differ in recent publications. However, oral dosages should be monitored for desired response, since absorption can be variable and may be insufficient in some patients.

- It may be practical to initially administer parenteral cobalamin to a patient with vitamin B-12 deficiency and then to continue treatment with oral cobalamin. Oral cobalamin is cost effective and better accepted by patients.

• Folate (1-5 mg) should be administered orally. If this is difficult, comparable doses can be administered parenterally.

• Therapeutic options when the etiology of megaloblastosis is uncertain include therapeutic doses of both cobalamin and folate after serum level measurements for cobalamin and folate levels, bone marrow, and other studies have been initiated. The Schilling test is not affected by previous therapy. Another option is to administer a trial of a physiological dose of folate. Cobalamin deficiency does not respond to daily folate doses of 100-400 mcg (physiological dose), but this dose results in complete response in patients with folate deficiency. Under no circumstances should therapeutic doses of folate (1-5 mg/d) be administered without cobalamin. The reason is that folate therapy corrects the anemia, but folate does not correct a cobalamin-induced neurological disorder and thus results in the progression of neuropsychiatric complications.

• Prophylactic folate therapy (1 mg/d) should be administered during pregnancy and the perinatal period to meet the increased demand for folate by the fetus and during lactation. Folate should also be given daily to patients with chronic hemolysis. Folate therapy is currently recommended for

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individuals with high levels of homocysteine who have a propensity for thromboembolic disease to prevent this complication. Multivitamins that contain folate have been recommended for elderly persons.

- Fortification of foods with folic acid has been recommended to prevent hyperhomocysteinemia-related thrombosis, folate deficiency–related neoplasia, and pregnancy-related fetal abnormalities.

- However, opponents to the fortification plan are concerned that folate-fortified foods given to patients with unrecognized cobalamin deficiencies will increase the frequency of cobalamin-induced neuropsychiatric disorders.

• Cobalamin therapy can be beneficial for patients with borderline cobalamin deficiency or in patients who present with only neuropsychiatric disorders. The role of minimal cobalamin deficiency in patients with borderline neuropsychiatric dysfunction has recently been recognized because of more sensitive tests and a greater awareness of this potential problem. One cause of borderline cobalamin deficiency is food-cobalamin malabsorption, described in the protein-bound absorption test discussion. Treatment with 50 mcg of oral cyanocobalamin daily can restore cobalamin stores in these patients.

• Blind loop syndrome should be treated with antibiotics. • Patients with TCII deficiency may require higher doses of cobalamin. • Tropical sprue should be treated with cobalamin and folate. • Acute megaloblastic anemias due to nitrous oxide exposure can be treated with folate (5 mg/d) and

cobalamin (1 mg IM). • Fish tapeworm infection, pancreatitis, Zollinger-Ellison syndrome, and inborn errors should be

treated with appropriate measures. MODULE TASK

CASE: A 3 year-old boy, body weight 11kgs History taking: pale 4 weeks; no bleed; no fever; feeding difficulties (patient don’t like meat and sea-food) Physical examination: conjunctiva palpebra anemic; stomatitis; glossitis; bleeding (-); lymph nodes, liver and spleen are not palpable; neuropathy on lower limbs bilateral Laboratory results: Hb 8,9 g/dl; white blood cells 5640/mm3; platelet 284.000mm3; MCV high, MCH and MCHC normal

QUESTIONS: 1. What are THE WORKING DIAGNOSIS and DIFFERENTIAL DIAGNOSIS of this patient? 2. Describe in brief THE CAUSES of this disease! 3. What are THE MECHANISMS of this disease? 4. How TO DIAGNOSE this disease based on the clinical features and laboratory findings? 5. How TO TREAT this patient? SUGGESTED READINGS 1. Hoffbrand AV, Pettit JE, Moss PAH. Megaloblastic Anaemia and Other Macrocytic Anaemias. In:

Essential Haematology. 4th ed. London: Blackwell Science. 2001. 2. Ciesla B. The Macrocytic Anemias. In: Ciesla B, eds. Hematology in Practice. 1st ed. Philadelphia:

F.A. Davis Company. 2007. 3. Schik P. Megaloblastic Anemia. e-Medicine Pediatrics 2007. URL: http://emedicine. medscape.

com/article/204066-overview

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THALASSEMIA

Definition and Overview Thalassemia is a hereditary anemia resulting from defects in hemoglobin

production. The thalassemias are characterized by their clinical severity (phenotype). Thalassemia major refers to disease requiring more than eight red blood cell (RBC) transfusions per year and thalassemia intermedia to disease that requires no or infrequent transfusions. it is important for physician (especially pediatricians, obstetricians and hematologist) to be aware of a possible diagnosis of thalassemia wherever they practice and for any patients they evaluate who have anemia.


Competency Component To apply the concepts and principles of pathophysiology, clinical and genetic classification, clinical processes to diagnose thalassemia, and its management.

Clinical Competence 1. Student can describe the pathophysiology of thalassemia. 2. Student can describe the clinical and genetic classification of

thalassemia. 3. Student can diagnose thalassemia based on clinical features and

laboratory findings. 4. Student can describe the management of thalassemia. 5. Student can describe the complications of thalassemia.

Learning Methode Active learning with modul task, group discussion, expert lecture, and skill development (history taking).


Time Active learning with modul task 2x50 minutes; group discussion 1x50 minutes; expert lecture 1x50 minutes.

Contributors 1. Department of Child Health. 2. Department of Internal Medicine.

Evaluation Module exams at the end of module programmes, middle and final exams at the end of semester with multiple choice questions, and skill exam with OSCE.

Suggested Readings 1. Cunningham MJ. Update on Thalassemia: Clinical Care and Complications. Pediatr Clin N Am 2008; 55: 447-60.

2. Rund D, Rachmilewitz E. β-Thalassemia. N Engl J Med 2005; 353(11): 1135-46.

3. Higgs DR, Thein SL, Wood WG. Thalassaemia: Classification, Genetics and Relationship to Other Inherited Disorders of Haemoglobin. In: Weatherall DJ, Clegg JB, eds. The Thalassaemias Syndromes. 4th ed. Oxford, England: Blackwell Science, 2001: 121-32.

4. Hoffbrand AV, Pettit JE, Moss PAH. Genetic Disorders of Haemoglobin. In:Essential Haematology. 4th ed. London: Blackwell Science. 2001.

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THALASSEMIA


Faculty of Medicine University of Brawijaya INTRODUCTION Thalassemia, originally named Cooley anemia, initially was described by Dr. Thomas Cooley and Dr. Pearl Lee in 1925 in North America as an inherited blood disease. Thalassemia is a hereditary anemia resulting from defects in hemoglobin production. The thalassemia syndromes are named according to the globin chain affected or the abnormal hemoglobin produced. Thus, α-globin gen mutations cause α-thalassemia and β-globin gene mutations give rise to β-thalassemia. In addition, the thalassemias are characterized by their clinical severity (phenotype). Thalassemia major refers to disease requiring more than eight red blood cell (RBC) transfusions per year and thalassemia intermedia to disease that requires no or infrequent transfusions. Thalassemia trait refers to carriers of mutations; such individuals have microcytosis and hypochromia but no or only mild anemia. EPIDEMIOLOGY Thalassemia is among the most common genetic disorders worldwide; 4.83 percent of the world’s population carry globin variants including 1.67 percent of the population who are heterozygous for α-thalassemia and β-thalassemia. α-thalassemia originated in Africa, the Middle East, China, India, and Southeast Asia, including Indonesia. β-thalassemia arose in the Mediterranean, Middle East, South and Southeast Asia, and Southern China. Immigration and emigration, however, have led to changing demographics, and patients who have thalassemia syndromes and heterozygous carriers now reside in all parts of the world. Thus, it is important for physician (especially pediatricians, obstetricians and hematologist) to be aware of a possible diagnosis of thalassemia wherever they practice and for any patients they evaluate who have anemia. PATHOPHYSIOLOGY The thalassemia syndromes were among the first genetic diseases to be understood at the molecular level. More than 200 β-globin and 30 α-globin mutations deletions have been identified; these mutations result in decreased or absent production of one globin chain (α or β) and a relative excess of the other. The resulting imbalance leads to unpaired globin chains, which precipitate and cause premature death (apoptosis) of the red cell precursors within the marrow, termed ineffective erythropoiesis. Of the damaged but viable RBCs that are released from the bone marrow, many are removed by the spleen or hemolyzed directly in the circulation due to the hemoglobin precipitants (Figure 1). Combined RBC destruction in the bone marrow, spleen, and eriphery causes anemia and, ultimately, an escalating cycle of pathology resulting in the clinical syndrome of severe thalassemia. Damaged erythrocytes enter the spleen and are trapped in this low pH and low oxygen environment; subsequent splenomegaly exacerbates the trapping of cells and worsens the anemia. Anemia and poor tissue oxygenation stimulate increased kidney erythropoietin production that further drives marrow erythropoiesis, resulting in increased ineffective marrow activity and the classic bony deformities associated with poorly managed thalassemia major and severe thalassemia intermedia. Anemia in the severe thalassemia phenotypes necessitates multiple RBC transfusions and, over time, without proper chelation, results in transfusion-associated iron overload. In addition, ineffective

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erythropoiesis enhances gastrointestinal iron absorption and can result in iron overload, even in untransfused patients who have thalassemia intermedia. It has long been recognized that the severity of ineffective erythropoiesis affects the degree of iron loading, but until the recent discovery of hepcidin and understanding, its role in iron metabolism the link was not understood. Hepcidin, an antimicrobial hormone, is recognized as playing a major role in iron deficiency and overload. Hepcidin initially was discovered due to its role in the etiology of anemia of chronic inflammation or chronic disease. Elevated levels, associated with increased inflammatory markers, maintain low levels of circulating bioavailable iron in two important ways: (1) by preventing iron absorption and transport from the gut and (2) by preventing release and recycling of iron from macrophages and the reticuloendothelial system. Conversely, inadequate hepcidin allows increased gastrointestinal absorption of iron and ultimately may lead to excess iron sufficient to result in organ toxicity.

Figure 1. Pathophysiology of thalassemia

Iron not bound to transferrin, also referred to as nontransferrin-bound iron, damages the endocrine organs, liver, and heart. Nontransferrinbound iron can result in myocyte damage leading to arrhythmias and congestive heart failure, the primary causes of death in patients who have thalassemia. Appropriate chelation therapy and close monitoring of cardiac siderosis can avoid this devastating complication (see the article by Kwiatkowski elsewhere in this issue for discussion of iron chelators). GENERAL CHARACTERISTICS Studies of the interactions between different thalassemia alleles and structural hemoglobin variants, later combined with in vitro analysis of the relative rates of synthesis of the different globins, allowed the thalassemia to be subdivided into two broad groups, α and β thalassemia. Although each can be further classified into different subgroups, all these disorders have one thing in common; there is always imbalanced globin synthesis. This is the hallmark of thalassemia, and it is the deleterious consequences of the globin that is produced in excess that are responsible for the ineffective erythropoiesis and shortened red-cell survival that characterizes all the severe forms of the disease.

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These principles are summarized in Figure 2. Since β-chain synthesis is only fully activated after birth, it follows that β thalassaemia is not expressed as a disease in intrauterine life, but only becomes manifest as γ-chain synthesis declines during the first year after birth. It is characterized by persistent γ-chain, that is HbF, production and an elevated level of HbA2 in heterozygotes. Since there is defective β-chain synthesis, it might be expected that there would be a relative increase in δ-chain production and therefore the increased level of HbA2 would be expected.However, the reasons for persistent γ-chain production in β thalassemia, and its marked variability from case to case, are much more difficult to explain, reflecting as it does both selective survival of cells with HbF and increased HbF production. It clearly distinguishes β thalassemia from almost every other genetic or acquired hematological disorder and is of prime importance in determining the severity of its phenotype. Unfortunately, however, persistent g-chain production is insufficient to compensate for the deficit of β-chains in the more severe forms of β thalassemia. Hence there is always an excess of α-chains, aggregates of which cause damage both to developing red-cell precursors and to mature red cells. Thus the central pathophysiological mechanism of b thalassemia is α-chain excess and the damage that it causes at every stage of erythropoiesis (Figure 2).

Since α-chains are shared by both fetal and adult hemoglobin, it is not surprising that the a thalassemias are manifest in both fetal and adult life. However, unlike the surfeit of α-chains that is produced in β thalassemia, excess γ and β-chains that result from defective α-chain production (Figure 1) are able to form soluble homotetramers, γ4 (or Hb Bart’s) and β4 (or HbH). These molecules are physiologically useless because of their very high oxygen affinity and, at least in the case of HbH, instability. Thus the clinical features of the more severe forms of a thalassemia are a reflection of the properties of hemoglobins Bart’s and H and their effects on erythropoiesis, and in particular on red-cell survival. In milder forms of a thalassemia it may be difficult to demonstrate HbH, for example. It appears that a critical level of β-chain excess is needed before viable β4 molecules are formed. CLASSIFICATION

Figure 2. The α and β-thalassamias. A simplified representation of the differences in the hemoglobin patterns between the two main forms of thalassemia. Shaded boxes indicate defective globin synthesis.

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As knowledge about the thalassemias has grown, different approaches to their classification have evolved and gradually become more sophisticated. The disease can now be described at several levels. First, there is a phenotypic classification based on its severity: this classification says nothing about the genetic constitution of a particular patient, but simply describes, in very general terms, a constellation of clinical features. Second, the thalassemias can be defined by the particular globin(s) that is (are) synthesized at a reduced rate. In effect, this constitutes a genetic classification in that, in most cases, it describes the gene (or genes) that must be affected by the thalassemia mutation. Finally, it is now often possible to subclassify many thalassemias according to the particular mutation that is responsible for defective globin synthesis. In clinical practice it is very useful to retain each of these classifications. Much of our approach to treatment is still determined by characterization of the disease at a clinical level. However, for an accurate assessment of the likely outcome it is becoming increasingly important to go to at least the next step, that is, a genetic classification by the particular globins involved. Indeed, in current day-to-day management of thalassemia it is often extremely helpful to be able to analyse the disorder at the molecular level, particularly if its prenatal detection is contemplated. Clinical Classification Based on clinical assessment, the thalassemias can be divided into hydrops fetalis which are four genes deletion α-thalassemia, thalassemia major which are severe and transfusion dependent, and thalassemia minor which can only be identified hematologically and usually represent the carrier states or traits (Table 1). Table 1. Clinical Classification of the Thalassemias Hydrops fetalis Four genes deletion α-thalassemia Thalassaemia major Transfusion dependent, homozygous β0-thalassemia or other combonations of β-thalassemia trait Thalassaemia intermedia Homozygous β-thalassemia Heterozygous β-thalassemia δβ-thalassemia and hereditary persistence of fetal Hb Hemoglobin H disease

Thalassemia minor β0-thalassemia trait β+-thalassemia trait Hereditary persistence of fetal Hb δβ-thalassemia trait α0-thalassemia trait α+-thalassemia trait

Although β-thalassemia major usually results either from the homozygous inheritance of a particular mutation or from the compound heterozygous state for two different mutations, it has become apparent that there are rare forms of moderately severe β-thalassemia that result from the action of a single mutant gene; that is, they are dominantly inherited. Another term,‘thalassemia intermedia’, though it has an old-fashioned ring about it, is still retained and is extremely useful in clinical practice. It describes conditions which, though not as severe as the major forms, are associated with a more severe degree of anemia than is found in the trait. In practice, this term encompasses a wide spectrum, ranging from disorders which are almost as serious as major forms to asymptomatic conditions which are only slightly more severe than the trait. Finally, some heterozygotes for thalassemia mutations are clinically and hematologically normal; they are sometimes designated ‘silent’ carriers. Genetic Classification The thalassemias are classified according to their genetic basis by describing the globin subunit which is synthesized at a reduced rate. A classification of the syndromes at this level is shown

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in Table 2. The genetic classification of the thalassemias divides them broadly into α, β, γ, δβ, δ and εγδβ varieties, depending on which globin or globins are underproduced. Newcomers to the field may be confused when they see that, as well as the thalassemias, Table 2 includes ‘hereditary persistence of fetal hemoglobin’. It seems reasonable to include this heterogeneous collection of conditions with the thalassemias since many of them are, in effect, forms of β or δβ thalassemia in which globin imbalance is almost entirely compensated by a genetically determined persistence of relatively high levels of fetal hemoglobin production. In each of the later chapters that deal with particular forms of thalassemia in detail, their classification is considered at greater length. But as a general introduction it may be helpful to outline the main features of the different genetic forms here. Table 2. Genetic Classification of the Thalassemias and Related Disorders α-thalassaemia α0 α+ Deletion (–α) Non-deletion (αT) β-thalassaemia β0 β+ Variants with unusually high level of HbF or A2 Normal HbA2 ‘Silent’ Dominant Unlinked to b-gene cluster δβ-thalassaemia (δβ)+ (δβ)0 (Aγδβ)0

γ-thalassaemia δ-thalassaemia δ0

δ+ εγδβ-thalassaemia Hereditary persistence of fetal haemoglobin Deletion (δβ)0 Non-deletion Linked to b-globin-gene cluster Gγβ+ Aγβ+ Unlinked to β-globin-gene cluster

α-THALASSEMIA SYNDROMES These are usually caused by gene deletions(Table 2). As there are normally four copies of the α-globin gene the clinical severity can be classified according to the number of genes that are missing or inactive. Loss of all four genes completely suppresses α-chain synthesis and since the α-chain is essential in fetal as well as in adult hemoglobin this is incompatible with life and leads to death in utero (hydrops fetalis). Three α-gene deletions leads to a moderately severe (Hb 7-11 g/dl) microcytic, hypochromic anemia with splenomegaly. This is known as HbH disease because HbH (β4) can be detected in red cells of these patients by electrophoresis or in reticulocyte preparations. In fetal life, Hb Barts (γ4) occurs. The α-thalassemia traits are caused by loss of one or two genes and are usually not associated with anemia, although the MCV and MCH are low and the red cell count is over 5.5 x 1012/L. Hemoglobin electrophoresis is normal and α/β-chain synthesis studies or DNA analyses are needed to be certain of the diagnosis. The normal α/β-synthesis ratio is 1:1 and this is reduced in the α-thalassemias and raised in β-thalassemias. Uncommon non-deletional forms of α-thalassemia are caused by point mutations producing dysfunction of the genes or rarely by mutations affecting termination of translation which give rise to an elongated but unstable chain, e.g. Hb Constant Spring.

β-THALASSEMIA SYNDROMES 1. β-THALASSEMIA MAJOR This condition occurs on average in one in four offspring if both parents are carriers of the β-thalassemia trait. Either no β-chain (β0) or small amounts (β+) are synthesized. Excess α-chains precipitate in erythroblasts and in mature red cells causing the severe ineffective erythropoiesis and

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hemolysis that are typical of this disease. The greater the α-chain excess, the more severe the anemia. production of γ-chains helps to ‘mop up’ excess α-chains and to ameliorate the condition. Over 200 different genetic defects have now been detected. Unlike α-thalassemia, the majority of genetic lesions are point mutations rather than gene deletions. These mutations may be within the gene complex itself or in promoter or enhancer regions. Certain mutations are particularly frequent in some communities and this may simplify antenatal diagnosis aimed at detecting the mutations in fetal DNA. Thalassemia major is often a result of inheritance of two different mutations, each affecting β-globin synthesis (compound heterozygotes). In some cases deletion of the β gene, δ and β genes or even δ, β, and γ genes occurs. In others, unequal crossing-over has produced δβ fusion genes (so called Lepore syndrome named after the first family in which this was diagnosed). Clinical Features 1. Severe anemia becomes apparent at 3-6 months after birth when the switch from γ- to β-chain

production should take place. 2. Enlargement of the liver and spleen occurs as a result of excessive red cell destruction,

extramedullary hematopoiesis and later because of iron overload. The large spleen (splenomegaly) increases blood requirements by increasing red cell destruction and pooling, and by causing expansion of the plasma volume.

3. Expansion of bones caused by intense marrow hyperplasia leads to a thalassemic facies (Figure 3), and to thinning of the cortex of many bones with a tendency to fractures and bossing of the the skull with a ‘hair-on-end’ appearance on X-ray (Figure 4).

4. The patient can be sustained by blood transfusions but iron overload caused by repeated transfusions is inevitable unless chelation therapy is given. Each 500 ml of transfused blood contains about 250 mg iron. To make matters worse,iron absorption from food is increased in β-thalassemia, probably secondary to ineffective erythropoiesis. Iton damages the liver, the endocrine organs (with failure of growth, delayed or absent puberty, diabetes mellitus, hypothyroidism, hypoparathyroidism) and the myocardium. In the absence of intensive iron chelation death occurs in the second or third decade, usually from congestive heart failure or cardiac arrhytmias. Skin pigmentation as result of excess melanin and hemosiderin gives a slatey grey appearance at an early stage of iron overload.

Figure 3. Thalassemic facies Figure 4. Hair-on-end appearance 5. Infections may occur for a variety of reasons. In infancy, without adequate transfusion, the anemic

child is prone to bacterial infections. Pneumococcal, Hemophilus and meningococcal infections are likely if splenectomy has been carried out and prophylactic penicillin is not taken. Yersinia enterocolitica occurs particularly in iron-loaded patients being treated with desferrioxamine; it may

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cause severe gastroenteritis. Transmition of viruses by blood transfusion may occur. Liver disease in thalassemia is most frequently a result of hepatitis C but hepatitis B is also common where the virus is endemic. Human immunodeficiency virus (HIV) has been transmitted to some patients by blood transfusion.

6. Osteoporosis may occur in well-transfused patients. It is more common in diabetic patients. Laboratory Findings 1. There is a severe hypochromic, microcytic anemia with raised reticulocyte percentage with

normoblasts, target cells and basophilic stippling in the blood film (Figure 5). 2. Hemoglobin electrophoresis reveals absence or almost complete absence of HbA with almost all

the circulating hemoglobin being HbF. The HbA2 percentage is normal, low or slightly raised. α/β-globin chain synthesis studies on reticulocytes show an increased α : β ratio with reduced or absent β-chain synthesis. DNA analysis can be used to identify the defect on each allele.

Figure 5. Thalassemia: the blood film shows marked hypochromic microcytic cells with target cells & poikilocytosis

Assessment of Iron Status The tests that may be performed to assess iron overload are listed in Table 3. Tests may also be carried out to determine the degree of organ damage caused by iron. The serum ferritin is the most widely used test. It is usual in thalassemia major to attempt to keep the level between 1000 and 1500 µg/L, when the body iron stores are about 5 to 10 times normal. However, the serum ferritin is raised in relation to iron status in viral hepatitis and other inflammatory disorders, and should therefore be interpreted in conjunction with other tests such as liver biopsy, urine excretion of iron in response to deferrioxamine, skin pigmentation and function of the heart, liver & endocrine & the clinical picture. Table 3. Assessment of Iron Overload Assessment of iron stores Serum ferritin Serum iron & percentage saturation of transferrin (TIBC) Bone marrow biopsy (Perls’ stain) for reticuloendothelial stores DNA test for mutation resulting in Cys282 Tyr in the HFE gene Liver biopsy (parenchymal & reticuloendothelial stores) Liver CT scan or MRI Cardiac MRI Desferrioxamine iron excretion test (chelatable iron) Repeated phlebotomy until iron deficiency occurs Assessment of tissue damage caused by iron overload Cardiac : clinical, chest x-ray, ECG, 24-h monitor, echocardiography, radionuclide (MUGA) scan to

check LV ejection fraction at rest & with stress Liver : liver function tests, liver biopsy, CT scan Endocrine : clinical examination (growth & sexual development), glucose tolerance test, pituitary

gonadotrophin release tests, thyroid, parathyroid, gonadal & adrenal function, growth hormone assays, radiology for bone age, isotopic bone density study

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2. β-THALASSEMIA MINOR (TRAIT) This is a common, usually symptomless, abnormality characterized like α-thalassemia trait by a hypochromic, microcytic blood picture (MCV and MCH very low) but high red cell count (>5.5 x 1012/L) and mild anemia (Hb levels 10-15 g/dl). It is usually more severe that α trait; a raised HbA2 (>3.5%) confirms the diagnosis. One of the most important indications for making the diagnosis is that it allows the possibility of prenatal counseling to patients with a partner who also has a significant hemoglobin disorder. If both carry β-thalassemia trait there is a 25% risk of a thalassemia major child. Table 4. Genetic Basis and Clinical Manifestations of Common β-Thalassemia Syndromes

THALASSEMIA INTERMEDIA Cases of thalassemia of moderate severity (Hb 7.0-10.0 g/dl) who do not need regular transfusions are called thalassemia intermedia. This is a clinical syndrome which may be caused by a variety of genetic defects. It may be caused by homozygous β-thalassemia with production of more HbF than usual or with mild defects in β-chain synthesis, or by β-thalassemia trait alone but of unusual severity (‘dominant’ β-thalassemia) or β-thalassemia trait in association with mild globin abnormalities such as Hb Lepore. The coexistence of α-thalassemia trait improves the hemoglobin level in homozygous β-thalassemia by reduction the degree of chain imbalance and thus of α-chain precipitation and ineffective erythropoiesis. Conversely, patients with β-thalassemia trait who also have excess (five or six) α genes tend to be more anemic than usual. The patient with thalassemia intermedia may show bone deformity, enlarged liver and spleen, extramedullary erythropoiesis and features of iron overload caused by increased iron absorption. HbH disease, three-gene deletion α-thalassemia is a type of thalassemia intermedia without iron overload or extramedullary hematopoiesis. MANAGEMENT 1. Regular blood transfusions are needed to maintain the Hb over 10 g/dl at all times (Figure 6). This

usually requires 2-3 units every 4-6 weeks. Fresh blood, filtered to remove white cells, gives the

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best red cell survival with fewest reactions. The patients should be genotyped at the start of the transfusion programme in case red cell antibodies against transfused red cells develop.

Figure 6. Management of Thalassemia and Treatment-Related Complications 2. Iron chelation therapy is used to treat iron overload. Unfortunately desferrioxamine is inactive

orally. It may be given by a separate infusion bag 1-2 g with each unit of blood transfused and by subcutaneous infusion 20-40 mg/kg over 8-12 hours, 5-7 days weekly. It is commenced in infants after 10-15 units of blood have been transfused. Iron-chelated by desferrioxamine is mainly excreted in the urine but up to one-third is also excreted in the stools. If patients comply with this intensive iron chelation regime, life expectancy for patients with thalassemia major and other chronic refractory anemias receiving regular blood transfusion improves considerably. Desferrioxamine is not without side-effects, especially in children with relatively low serum ferritin levels, including high tone deafness, retinal damage, bone abnormalities and growth retardation. Patients should have auditory and funduscopic examinations at regular intervals. Deferiprone (L1) is used alone or in combination with desferrioxamine. The two drugs have an additive or even synergistic action on iron excretion. Alone it is less effective than desferrioxamine. Compliance is usually better. Side-effects include an arthropathy, agranulocytosis or severe neutropenia, gastrointestinal disturbance and zinc deficiency.

3. Regular folic acid (e.g. 5 mg daily) is given if the diet is poor. 4. Vitamin C 200 mg daily increases excretion of iron produced by desferrioxamine. 5. Splenectomy may be needed to reduce blood requirements. This should be delayed until the

patient is over 6 years old because of the high risk of dangerous infections post-splenectomy.

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6. Endocrine therapy is given either as replacement because of end-organ failure or to stimulate the pituitary if puberty is delayed. Diabetic will require insulin therapy. Patients with osteoporosis may need additional therapy with increased calcium and vitamin D in their diet, together with administration of a bisphosphonate.

7. Immunization against hepatitis B should be carried out in all non-immune patients. Treatment for transfusion-transmitted hepatitis C with α-interferon and ribavirin is needed if viral genomes are detected in plasma.

8. Allogeneic bone marrow transplantation offers the prospect of permanent cure. The success rate (long-term thalassemia major-free survival) is over 80% in well-chelated younger patients without liver fibrosis or hepatomegaly. A human leucocyte antigen (HLA) matching sibling (or rarely other family member or matching unrelated donor) acts as donor. Failure is mainly a result of recurrence of thalassemia, death (e.g. from infection) or severe chronic graft-versus-host disease.

MODULE TASK 1. Describe in brief pathophysiology of thalassemia ! 2. Describe in brief clinical and genetic classification of thalassemia ! 3. Describe clinical features and laboratory findings to diagnose β-thalassemia major ! 4. Describe in brief assessment of iron overload ! 5. Describe in brief the management of thalassemia ! 6. Describe in brief the complications of thalassemia ! SUGGESTED READINGS 1. Cunningham MJ. Update on Thalassemia: Clinical Care and Complications. Pediatr Clin N Am

2008; 55: 447-60. 2. Rund D, Rachmilewitz E. β-Thalassemia. N Engl J Med 2005; 353(11): 1135-46. 3. Higgs DR, Thein SL, Wood WG. Thalassaemia: Classification, Genetics and Relationship to Other

Inherited Disorders of Haemoglobin. In: Weatherall DJ, Clegg JB, eds. The Thalassaemias Syndromes. 4th ed. Oxford, England: Blackwell Science, 2001: 121-32.

4. Hoffbrand AV, Pettit JE, Moss PAH. Genetic Disorders of Haemoglobin. In:Essential Haematology. 4th ed. London: Blackwell Science. 2001.

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IMMUNE HEMOLYTIC ANEMIA

Definition and Overview Immune hemolytic anemia (IHA) is the clinical condition in which IgG

and/or IgM antibodies bind to RBC surface antigens and initiate RBC destruction via the complement system and the reticuloendothelial system. IHA is classified as either autoimmune, alloimmune, or drug-induced based on the antigenic stimulus responsible for the immune response. Autoimmune hemolytic anemia (AIHA) is characterized by the production of antibodies directed against self RBCs. In contrast, alloimmune hemolytic anemia requires exposure to allogeneic RBCs, and the resulting alloantibodies show no reactivity toward autologous RBCs. Sources of allogeneic RBC exposure include pregnancy, blood product transfusion, and transplantation.


Competency Component To apply the concepts and principles of classification and pathogenesis, clinical processes to diagnose immune hemolytic anemia, and its management.

Clinical Competence 1. Student can describe the classification and pathogenesis of immune hemolytic anemia.

2. Student can diagnose immune hemolytic anemia based on clinical features and laboratory findings.

3. Student can describe the management of immune hemolytic anemia.

Learning Methode Expert lecture, active learning/modul task & group discussion


Time Expert lecture 50 minutes, active learning/modul task 100 minutes, and group discussion 50 minutes

Contributors 1. Department of Child Health 2. Department of Internal Medicine

Evaluation 1. Formative assessment at the end learning of some topics using true false questions

2. Module exam at the end learning of module using MCQs 3. Final exam at the end of semester using MCQs

Suggested Readings 1. Dhaliwal G, Cornett PA, Tierney LM. Hemolytic Anemia. Am Fam Physician 2004; 69: 2599-606.

2. Hoffman PC. Immune Hemolytic Anemia—Selected Topics. ASH Hematology 2006: 13-8.

3. Gehrs BC, Friedberg RC. Autoimmune Hemolytic Anemia. Am J Hematol 2002; 69: 258-71.

4. Hoffbrand AV, Pettit JE, Moss PAH. Haemolytic Anaemias. In: Essential Haematology. 4th ed. London: Blackwell Science. 2001.

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IMMUNE HEMOLYTIC ANEMIA


Faculty of Medicine University of Brawijaya INTRODUCTION Immune hemolytic anemia (IHA) is the clinical condition in which IgG and/or IgM antibodies bind to RBC surface antigens and initiate RBC destruction via the complement system and the reticuloendothelial system. IHA is classified as either autoimmune, alloimmune, or drug-induced based on the antigenic stimulus responsible for the immune response. Autoimmune hemolytic anemia (AIHA) is characterized by the production of antibodies directed against self RBCs. Since the autoantibodies usually are directed against high-incidence antigens, they often exhibit reactivity against allogeneic RBCs as well. AIHA is a fairly uncommon disorder, with estimates of the incidence at 1-3 cases per 100,000 per year. In contrast, alloimmune hemolytic anemia requires exposure to allogeneic RBCs, and the resulting alloantibodies show no reactivity toward autologous RBCs. Sources of allogeneic RBC exposure include pregnancy, blood product transfusion, and transplantation. The principal manifestations of RBC alloimmunization are hemolytic transfusion reactions and hemolytic disease of the newborn (HDN). The incidence of acute hemolytic transfusion reactions has been estimated to be 0.003-0.008%, while 0.05-0.07% of transfused patients develop a clinically recognized delayed hemolytic transfusion reaction. Drug-induced IHA is the final classification of IHA. Drug-induced antibodies can recognize either intrinsic RBC antigens or RBC-bound drug. Antibodies that react with intrinsic RBC antigens are serologically indistinguishable from autoantibodies. In contrast, antibodies that react against RBC-bound drug require the drug for hemolysis. CLASSIFICATION AND PATHOGENESIS The pathogenesis of IHA ultimately overlaps for these three classifications. The degree of hemolysis depends on characteristics of the bound antibody (e.g. quantity, specificity, thermal amplitude, ability to fix complement, ability to bind tissue macrophages) as well as the target antigen (density, expression, patient age). IgG antibodies are relatively poor activators of the classical complement pathway, but they (in particular IgG1 and IgG3 antibodies) are recognized rapidly by Fc receptors on various phagocytic cells. Therefore, IgG-sensitized RBCs generally are eliminated by phagocytes of the reticuloendothelial system. Since reticuloendothelial cells also have receptors for complement factors C3b and iC3b, these complement components, if present, can potentiate the extravascular hemolysis. On the other hand, IgM-sensitized RBCs generally are associated with a combination of intravascular and extravascular hemolysis. Intravascular hemolysis occurs because IgM antibodies, unlike IgG antibodies, readily activate the classical complement pathway and produce cytolysis. However, due to the presence of regulatory RBC proteins such as decay accelerating factor (DAF, CD55) and membrane inhibitor of reactive lysis (MIRL, CD59), overhelming complement activation usually is required to produce clinically evident intravascular hemolysis, e.g. as seen with ABO-incompatible blood transfusions. More commonly, IgM-sensitized RBCs undergo extravascular hemolysis. While reticuloendothelial cells do not have receptors for the Fc fragment of IgM antibodies with comparable activity to the receptors directed against the Fc fragment of IgG, they do have receptors for the abundant RBC-bound C3b and iC3b resulting from complement activation. Whereas

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the spleen is the principal site of IgG-associated extravascular hemolysis, Kupffer cells in the liver are the principal effectors of IgM-associated extravascular hemolysis. AUTOIMMUNE HEMOLYTIC ANEMIA Autoimmune hemolytic anemia (AIHA) are caused by antibody production by the body against its own red cells. They are characterized by a positive direct antiglobulin test (DAT) also known as the Coombs’ test (Figure 1) and divided intro warm, cold and mixed-types (Table 1) according to whether the antibody reacts more strongly with red cells at 370C or 40C. Warm hemolysis refers to IgG autoantibodies, which maximally bind red blood cells at body temperature (37°C [98.6°F]). In cold hemolysis, IgM autoantibodies (cold agglutinins) bind red blood cells at lower temperatures (0° to 4°C [32° to 39.2°F]).

WARM AUTOIMMUNE HEMOLYTIC ANEMIA When warm autoantibodies attach to red blood cell surface antigens, these IgG-coated red blood cells are partially ingested by the macrophages of the spleen, leaving microspherocytes, the characteristic cells of AIHA. These spherocytes, which have decreased deformability compared with normal red blood cells, are trapped in the splenic sinusoids and removed from circulation. Table 1. Classification of Autoimmune Hemolytic Anemia Warm autoimmune hemolytic anemia Idiopathic Secondary Systemic lupus erythematosus, other autoimmune disease Chronic lymphocytic leukemia, lymphomas Drugs, e.g. methyldopa, fludarabine Cold autoimmune hemolytic anemia Idiopathic Secondary Infections – Mycoplasma pneumonia, infectious mononucleosis Lymphoma Paroxysmal cold hemoglobinuria rare, sometimes associated with infections, e.g. syphilis Mixed-type autoimmune hemolytic anemia Idiopathic Secondary Lymphoproliferative disorders Autoimmune disorders

Figure 1. Direct antiglobulin test (DAT), demonstrating the presence of autoantibodies (shown here) or complement on the surface of the red blood cell. (RBCs = red blood cells)

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Clinical Features The disease may occur at any age in either sex and present as a hemolytic anemia of varying severity. The spleen in often enlarged. The disease tend to remit and relapse. It may occur alone or in association with other diseases or arise in some patients as a result of methyldopa therapy (Table 1). When associated with immune thrombocytopenic purpura (ITP), which is a similar condition affecting platelets, it is known as Evan’s syndrome. When secondary to systemic lupus erythematosus, the cells typically are coated with immunoglobulin and complement. Laboratory Findings The hematological and biochemical findings are typical of an extravascular hemolytic anemia with spherocytosis prominent in the peripheral blood (Figure 2). The DAT is positive as a result of IgG, IgG and complement or IgA on the cells and, in some cases, the autoantibody shows specificity within the rhesus system. The antibodies both on the cell surface and free in serum are best detected at 370C.

Treatment î Remove the underlying cause (e.g. methyldopa, fludarabine) î Corticosteroids. Prednisolone is the usual first-line treatment; 60 mg daily is a typical starting dose

in adults and should then be tapered down. Those with predominantly IgG on red cells do best whereas those with complement often respond poorly, both to corticosteroids or splenectomy.

î Splenectomy may be of value in those who fail to respond well or fail to maintain a satisfactory hemoglobin level on an acceptably small steroid dosage.

î Immunosupression may be tried after other measures have failed but is not always of great value. Azathioprine, cyclophosphamide, chlorambucil, cyclosporine and mycophenolate mofetil have been tried.

î Folic acid is given to severe cases. î Blood transfusion may be needed if anemia is severe and causing symptoms. The blood should

be the least incompatible and if the specificity of the autoantibody is known, donor blood is chosen which lacks the relevant antigen(s). The patients also readily make alloantibodies against donor red cells.

î High-dose immunoglobulin has been used but with less success than in ITP. COLD AUTOIMMUNE HEMOLYTIC ANEMIA

Figure 2. Blood film in warm autoimmune hemolytic anemia.

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In these syndrome the autoantibody, whether monoclonal (as in the idiopathic cold hemagglutinin syndrome or associated with lymphoproliferative disorders) or polyclonal (as following infection, e.g. infectious mononucleosis or mycoplasma pneumonia) attaches to red cells mainly in the peripheral circulation where the blood temperature is cooled. The antibody is usually IgM and binds to red cells best at 40C. IgM antibodies are highly efficient at fixing complement and both intravascular and extravascular hemolysis can occur. Complement alone is usually detected on the red cells, the antibody having eluted off the cells in warmer parts of the circulation. Interestingly, in nearly all these cold AIHA syndromes (CAS), the antibody is directed against the ‘I’ antigen on the red cell surface. In infectious mononucleosis it is anti-i. Clinical Features The patient may have a chronic hemolytic anemia aggravated by the cold and often associated with intravascular hemolysis. Mild jaundice and splenomegaly may be present. The patient may develop acrocyanosis (purplish skin discoloration) at the tip of the nose, ears, fingers, and toes caused by the agglutination of red cells in small vessles. Laboratory Findings Laboratory findings are similar to those of warm AIHA, except that spherocytosis is less marked, red cells agglutinate in the cold (Figure 3) and the DAT reveals complement (C3d) only on the red cell surface.

Treatment î Treatment consists of keeping the patient warm and treating the underlying cause, if present. î Alkylating agents such as chlorambucil may be helpful in the chronic varieties. î Splenectomy does not usually help unless massive splenomegaly is present, and steroids are not

helpful. Underlying lymphoma should be excluded in ‘idiopathic’ cases. MIXED-TYPE AUTOIMMUNE HEMOLYTIC ANEMIA Some patients with warm AIHA also possess a cold agglutinin. While the majority of these cold agglutinin are not clinically significant, occasionally they have a sufficient thermal amplitude (greater than 300C) or high titer (greater than 1:1,000 at0-40C) to indicate cold agglutinin syndrome (CAS). Similar to the separate entities, mixed-type AIHA can be either idiopathic or secondary to lymphoproliferative disorders or SLE. Patients usually have a chronic course interrupted by severe exacerbations, which can result in a hemoglobin level below 5.0 g/dl. These exacerbation do not

Figure 3. Blood film in cold autoimmune hemolytic anemia.

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appear to be associated with cold exposure, and they do not result in acrocyanosis or Raynaud’s phenomenon. Laboratory Findings The laboratory work-up shows that the DAT is positive for both IgG and C3. As with the separate diseases, the mixed-type AIHA produces difficulties with the antibody screen and the cross-match. The RBC eluate typically indicates pancreative warm IgG autoantibody. The cold autoantibody usually exhibits specificity against the ‘I’ antigen, but reactivity against i has been reported. Donor units have to be released as cross-match least-incompatible due to the autoantibodies. Treatment Mixed-type AIHA appears to respond to treatment in a similar manner as warm AIHA. Patients generally respomnd to steroids, and immunosuppressive agents and splenectomy have been employed successfully as well. Associated diseases, if present, also need to be treated to optimize recovery. ALLOIMMUNE HEMOLYTIC ANEMIA The most severe alloimmune hemolysis is an acute transfusion reaction caused by ABO-incompatible red blood cells. For example, transfusion of A red cells into an O recipient (who has circulating anti-A IgM antibodies) leads to complement fixation and a brisk intravascular hemolysis. Within minutes, the patient may develop fever, chills, dyspnea, hypotension, and shock. Delayed hemolytic transfusion reactions occur three to 10 days after a transfusion and usually are caused by low titer antibodies to minor red blood cell antigens. On exposure to antigenic blood cells, these antibodies are generated rapidly and cause an extravascular hemolysis. Compared with the acute transfusion reaction, the onset and progression are more gradual. DRUG-INDUCED HEMOLYTIC ANEMIA Drug-induced immune hemolysis is classified according to three mechanisms of action: drug-absorption (hapten-induced), immune complex, or autoantibody (Table 2). These IgG- and IgM-mediated disorders produce a positive DAT and are clinically and serologically indistinct from AIHA. Table 2. Selected Drugs that Causes Immune Hemolytic Anemia

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Hemolysis resulting from high-dose penicillin therapy is an example of the drug-absorption mechanism, in which a medication attached to the red blood membrane stimulates IgG antibody production. When large amounts of drug coat the cell surface, the antibody binds the cell membrane and causes extravascular hemolysis. Quinine-induced hemolysis is the prototype of the immune complex mechanism, in which the drug induces IgM antibody production. The drug-antibody complex binds to the red blood cell membrane and initiates complement activation, resulting in intravascular hemolysis. Alpha-methyldopa is the classic example of antierythrocyte antibody induction. Although the exact mechanism is unknown, the drug (perhaps by altering a red blood cell membrane protein and rendering it antigenic13) induces the production of antierythrocyte IgG antibodies and causes an extravascular hemolysis. TREATMENT OF REFRACTORY CASES OF AIHA The standard therapeutic approaches to treatment of AIHA include corticosteroids, splenectomy and immunosuppressive drugs. In the past several years, certain newer therapies have become available, and have shown evidence of success. These are primarily used in patients who are not candidates for or fail to respond to splenectomy, those who relapse after splenectomy, and those who cannot maintain stable hemoglobin levels without unacceptably high doses of corticosteroids. 1. Intravenous immune globulin (IVIG) Flores et al reviewed the cases of 73 patients treated with IVIG, and found responses in 29 (40%).37 Children were more likely to respond, as were patients with initial hepatomegaly and lower initial hemoglobin levels. 2. Danazol Danazol, which has been used more in refractory cases of immune thrombocytopenia, has also been used in AIHA. Ahn reported good to excellent results in the majority of patients treated.38 In another series of 17 patients treated with the combination of prednisone and danazol, excellent responses were noted in 80% who received the combination as first-line therapy; treatment was less effective in patients who had relapsed and in those with Evans’ syndrome. 3. Newer immunosuppressives Howard et al reported on the use of mycophenolate mofetil in 4 patients with refractory AIHA.40 Patients were treated with 500 mg per day initially, then 1000 mg per day. All 4 had a complete or good response. 4. Monoclonal antibodies There has been considerable interest in the past several years in the use of the monoclonal antibodies widely used in the treatment of B-cell lymphoid neoplasms, namely rituximab (Rituxan®), and to a lesser extent alemtuzumab (Campath-1H®). Zecca et al first reported on a child with pure red cell aplasia and AIHA treated successfully with rituximab and IVIG.41 Another report, in 5 children with AIHA, described excellent responses, but with a resultant not-unexpected prolonged B-cell deficiency. Shanafelt et al reported on 5 patients, of whom 2 had a complete response. In an additional 4 patients with Evans’ syndrome, complete responses occurred in either the immune thrombocytopenia or the AIHA, but not both. Trape et al noted the benefits of rituximab for residual AIHA in 5 patients following chemotherapy of a lymphoproliferative disorder. Mantadakis et al offered a case report of a patient with refractory Evans’ syndrome who responded for 7 months to rituximab, and then responded a second time after relapse. Ramanathan et al noted 2 patients with refractory disease who demonstrated prolonged remissions with rituximab. Not all reports have been favorable, however: Zaja

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et al noted no response to rituximab in 2 patients with AIHA, though a patient with cold agglutinin disease responded well. Gupta et al reported on the combined use of rituximab, cyclophosphamide and dexamethasone in 8 patients with refractory AIHA in the setting of chronic lymphocytic leukemia. The results were excellent, including in relapsed patients, with 5 patients converting to negative DAT status. There has been only limited experience with alemtuzumab in AIHA, with one report noting responses in 3 of 4 patients treated. The role of monoclonal antibodies in the therapy of autoimmune cytopenias has been reviewed in detail recently. It is reasonable to conclude that monoclonal antibody therapy, specifically rituximab, is a safe and effective therapy for AIHA. It is likely that as our experience with the drug evolves, it will be used at an earlier point in therapy, before more toxic immunosuppressives, rather than only in refractory cases. COMPLICATIONS OF AIHA 1. Thromboembolism In an early review of AIHA, the most common cause of death was pulmonary embolism (4 of 47 patients). All of these patients had had a splenectomy, and all were receiving corticosteroid therapy. In a more recent review by Pullarkat et al, 8 of 30 patients (27%) suffered from an episode of venous thromboembolism. A total of 9 had a detectable lupus anticoagulant and 17 had anticardiolipin antibodies detected. Among the 8 with thrombosis, 5 had a lupus anticoagulant and 4 had anticardiolipin antibodies. The authors attributed the thrombosis to disruption and loss of red cell membranes, resulting in exposure of phosphatidyl serine, and a subsequent surface for formation of tenase and prothrombinase complexes. Other factors implicated in the thrombotic tendency in patients with AIHA included cytokine-induced expression of monocyte or endothelial tissue factors. The authors postulated that the detection of a lupus anticoagulant identifies patients with AIHA at particularly high risk for venous thromboembolism and suggested that serious consideration be given to prophylactic anticoagulation in such patients. However, they point out that thrombosis also occurred in 15% of AIHA patients who did not have a lupus anticoagulant, so other factors are likely at work. Kokori et al, in a review of AIHA in patients with systemic lupus erythematosus, found the risk of thrombosis to be increased more than 4-fold, particularly in the presence of IgG anticardiolipin antibody. The association of serologic indications of lupus, namely a false-positive test for syphilis, has been noted in the past in patients with AIHA by Conley and Savarese. Hendrick has reviewed this issue recently and concluded that patients with AIHA are indeed at high risk for thromboembolism. In an audit of 23 patients with warm antibody AIHA and 5 with cold agglutinin hemolysis, venous thromboembolism was noted in 6, of which 4 cases were fatal. These patients did not have detectable antiphospholipid antibodies. In an analysis of 36 hemolytic episodes, venous thromboembolism occurred in 5 of 15 without anticoagulant prophylaxis, but in only 1 of 21 in which prophylaxis was used. Although it is premature to recommend anticoagulant prophylaxis in general for patients with hemolytic episodes from AIHA, consideration might be given to those at particularly high risk, such as those with evidence of coexisting antiphospholipid antibodies. 2. Lymphoproliferative disorders Patients with lymphoproliferative disorders are well known to have a higher risk for development of AIHA; this is particularly true of chronic lymphocytic leukemia. Interestingly, there may also be an increased risk for future development of lymphoproliferative disorders in patients with AIHA. Sallah et al reported on 107 patients with AIHA, of 16 American Society of Hematology whom 67 had idiopathic AIHA, and 40 had an associated immune disorder (e.g., rheumatoid arthritis, temporal arteritis, Crohn’s disease, lupus, thyroiditis, Sjögren’s syndrome). Nineteen of the 107 (18%) subsequently developed a malignant lymphoproliferative disorder, at a median of 26 months after

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onset of the AIHA.36 Risk factors for development of such a disorder were age, the presence of an underlying autoimmune disease, and a coexistent serum gammopathy. None of the patients had underlying HIV infection. The authors postulate that the development of a malignant lymphoid disorder is likely a multistep process, with an earlier proliferative phase involving chronic antigenic stimulation prior to a mutation leading to malignant change. MODULE TASK 1. Describe in brief classification and pathogenesis of immune hemolytic anemia ! 2. Describe in brief classification, clinical features and laboratory findings of :

a. Warm AIHA b. Cold AIHA

3. Describe in brief the treatment of warm AIHA and cold AIHA ! 4. Describe in brief the treatment of refractory cases of AIHA ! 5. Describe the complications of AIHA ! SUGGESTED READINGS 1. Dhaliwal G, Cornett PA, Tierney LM. Hemolytic Anemia. Am Fam Physician 2004; 69: 2599-606. 2. Hoffman PC. Immune Hemolytic Anemia—Selected Topics. ASH Hematology 2006: 13-8. 3. Gehrs BC, Friedberg RC. Autoimmune Hemolytic Anemia. Am J Hematol 2002; 69: 258-71. 4. Hoffbrand AV, Pettit JE, Moss PAH. Haemolytic Anaemias. In: Essential Haematology. 4th ed.

London: Blackwell Science. 2001.

4) Tema 2 Modul 1 (Red Cell Disorders)

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