Chronic Microcytic Anemia and Jaundice in a 36-Year-Old Male of Burmese Descent

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1 Chronic Microcytic Anemia and Jaundice in a 36-Year-Old Male of Burmese Descent Submitted Accepted Brit S. Shackley, MD, Thomas A. Drake, MD, Anthony W. Butch, PhD, DABCC, MT(ASCP) (Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, University of California at Los Angeles, Los Angeles, CA) DOI: /LM73OLT7NPYEHHWR Clinical History Patient: A 36-year-old male of Burmese descent. Chief Complaint: Anemia and jaundice. History of Present Illness: At the time of presentation, the patient was a recent immigrant from Burma who wished to establish care with an American physician. He had been chronically jaundiced and anemic since childhood, with multiple episodes of severe anemia requiring transfusion. He reported receiving more than 30 units of blood prior to age 17. Questions 1. What are the most significant clinical and laboratory findings? 2. How do you explain these findings? 3. What is the differential diagnosis for these findings? 4. What laboratory tests are routinely used to distinguish between the possible causes for this patient s condition? 5. How would you confirm the diagnosis? 6. What is this patient s most likely diagnosis? 7. What is the genetic basis of this disease? 8. Why is this patient s presentation more severe than the rest of his family? 9. What is the most appropriate treatment for this patient s disease? 10. Is genetic counseling recommended for this disease? Past Medical History: The patient contracted hepatitis C secondary to blood transfusions he received in Burma. Folic acid was his only medication. He has no history of tobacco or alcohol abuse. Family History: The patient is married with 1 child and works as an accountant. His mother and 1 sister both have thalassemia, although they are not as severely affected as he is. His father and 3 brothers have no known hematologic disorders. Possible Answers 1. The most significant clinical findings are the history of jaundice and severe anemia since childhood, which required multiple blood transfusions. The most significant laboratory findings are a hemoglobin (Hb) concentration of 8.3 g/dl, a decreased mean cell volume (MCV) and mean cell hemoglobin (MCH), along with an elevated red blood cell distribution width (RDW) (Table 1). The presence of nucleated red blood cells (RBCs) and an increased reticulocyte count are also significant, Physical Examination: Upon presentation, the patient was a well-nourished, welldeveloped male appearing jaundiced. Nontender splenomegaly was noted extending to the level of the umbilicus. There were no other abnormal findings on physical examination. The following vital signs were recorded: blood pressure, 130/75 mm Hg; pulse, 72; and respiration rate, 16. Principal Laboratory Findings: Table 1 and Images 1-2. Additional Diagnostic Tests: Hemoglobin (Hb) analysis was performed by high performance liquid chromatography (HPLC). The results are shown in Figure 1. as are the increased bilirubin and ferritin concentrations. The peripheral blood smear is notable for marked microcytosis, hypochromia, polychromasia, poikilocytosis, and anisocytosis, with numerous target cells, ovalocytes, teardrop forms, and schistocytes (red cell fragments) (Image 1). Basophilic stippling is also present on the peripheral smear (Image 2). 2. The low Hb, hematocrit, MCV, and MCH indicate a microcytic hypochromic anemia. This is further confirmed by peripheral blood smear review. The increased RDW (standard deviation of individual MCV measurements) indicates significant variation in RBC size and confirms the anisocytosis noted on the peripheral blood smear. The basophilic stippling is most likely caused by aggregates of ribosomes, mitochondria, and siderosomes, and is found in several clinical conditions including unstable Hbs. Nucleated RBCs and immature reticulocytes indicate hyperplastic erythropoiesis. The elevated bilirubin is consistent with intravascular hemolysis, and the extremely elevated ferritin indicates iron overload. 3. Anemias can be separated into macrocytic or microcytic based on the MCV being either increased (>100 fl) or decreased (<80 fl), respectively. Macrocytic anemias are normochromic and are most commonly associated with vitamin B12 and/or folate deficiency. Microcytic hypochromic anemias are caused by a quantitative defect in Hb synthesis. The differential diagnosis includes iron deficiency anemia, anemia of chronic disease, thalassemia, and sideroblastic anemia LABMEDICINE Volume 41 Number 2 February 2010 labmedicine.com

2 4. The RBC count is very helpful for identifying the etiology for this patient s microcytic hypochromic anemia. In iron deficiency, anemia of chronic disease, and sideroblastic anemia, the RBC count is decreased proportionally to the decrease in Hb. 1 Only thalassemia has an increased or high normal RBC count that is out of proportion to the Hb concentration (the RBC count is typically one-third the Hb concentration). In anemia of chronic disease, the MCV is usually only mildly decreased and the MCV is typically normal or increased in acquired sideroblastic anemia. 2 The MCV is significantly decreased in iron deficiency and thalassemia, and a MCV <72 fl favors the diagnosis of thalassemia. 3 An MCV to RBC ratio of <13 is also found in thalassemia, whereas a ratio of >15 favors the diagnosis of iron deficiency anemia. 4,5 A high RDW is characteristic of iron deficiency anemia and is also found with Hb S-β-thalassemia. 6 In β-thalassemia the RDW is typically normal but can be elevated, especially as the patient becomes anemic. 6 An elevated reticulocyte count favors thalassemia since the reticulocyte count is decreased in iron deficiency anemia due to a lack of available iron for hematopoiesis. 7 Biochemical markers of iron status are also helpful in differentiating between the various etiologies of microcytic hypochromic anemia. 8 In iron deficiency anemia, serum iron and ferritin are decreased, and transferrin receptors are increased. Although the iron concentration is also decreased in anemia of chronic disease, ferritin is normal or increased, and transferrin receptors are normal. In thalassemia, iron is normal, ferritin is normal or increased, and transferrin receptors are normal. In sideroblastic anemia, the iron and ferritin are both increased. 5. The diagnosis of thalassemia is not based on a single test. Rather, it requires a careful review of clinical and laboratory findings. Thalassemias resulting in reduced production of either α- or β-globin chains are classified as either α- or β-thalassemia, respectively. Mutations or deletions in 1 α-globin chain gene are clinically silent, and when 2 genes are affected, a mild anemia can be present. Deletion of 3 α-globin genes is classified clinically as thalassemia intermedia (Hb H disease) and can sometimes be difficult to differentiate Table 1_Principal Laboratory Findings Analyte Patient s Result Reference Interval Hematology WBC count /µl RBC count /µl Hemoglobin (Hb) g/dl Hematocrit % MCV fl MCH pg MCHC g/dl RDW % Platelet count /µl Nucleated RBCs % Reticulocyte count, auto % Absolute reticulocyte # /µl Immature reticulocyte fraction % Chemistry Sodium mmol/l Potassium mmol/l Chloride mmol/l Total CO mmol/l Glucose mg/dl Urea nitrogen mg/dl Total protein g/dl Albumin g/dl Bilirubin, total mg/dl Alkaline phosphatase U/L AST U/L ALT U/L Calcium mg/dl Creatinine mg/dl Uric acid mg/dl Ferritin ng/ml WBC, white blood cell; RBC, red blood cell; MCV, mean cell volume; MCH, mean cell Hb; MCHC, mean cell Hb concentration; RDW, red blood cell distribution width; CO 2, carbon dioxide; AST, aspartate aminotransferase; ALT, alanine aminotransferase. Image 1_Peripheral blood smear showing marked microcytosis, hypochromia, polychromasis, poikilocytosis, and anisocytosis. Image 2_Peripheral blood smear showing basophilic stippling (arrows). labmedicine.com February 2010 Volume 41 Number 2 LABMEDICINE 79

3 clinically and biochemically from β-thalassemia. 9 Only about one-quarter of patients with Hb H disease present with clinical symptoms of jaundice and anemia, and most Hb H patients are not dependent on blood transfusions. The deletion of all 4 α-globin genes is not compatible with life. Mild forms of β-thalassemia may not exhibit any clinical signs or symptoms. Intermediate to severe β-thalassemia is usually diagnosed during infancy or early childhood and presents with pallor, irritability, growth retardation, abdominal swelling due to hepatosplenomegaly, and jaundice. Laboratory tests reveal a severe anemia with markedly hypochromic, microcytic RBCs, with all of the classical findings of severe hemolytic anemia. The diagnosis of thalassemia is confirmed by fractionating Hb using electrophoretic or high performance liquid chromatography (HPLC) separation techniques. Hemoglobin H can be detected by both methods. 8,10 A diagnosis of β-thalassemia is confirmed by indentifying an increase in Hb A 2. Hemoglobin F is also elevated in approximately one-half of patients with β-thalassemia. 9 Hemoglobin fractionation of this patient s blood by HPLC revealed the presence of an extremely high level of the Hb A 2 fraction (90.2%) and elevated Hb F (9.0%) (Figure 1). No Hb H was detected. The very high level of the Hb A 2 fraction by HPLC was due to the presence of a co-migrating abnormal Hb with a similar retention time. Hemoglobin variants such as Hb E, Hb Osu Christianbourg, Hb G Copenhagen, and Hb Lepore co-elute with Hb A 2 by this method, making it impossible to identify an increase in Hb A 2. 1,11 Further testing by citrate agar electrophoresis at acid ph identified the presence of Hb E, which appears as a distinct band that does not co-migrate with Hb A Most likely diagnosis: Hb E-β 0 -thalassemia. The high normal RBC count with decreased Hb is most consistent with thalassemia being the etiology for the patient s microcytic hypochromic anemia. The MCV to RBC count ratio of 8.8 (48.7/5.52) also favors thalassemia and not iron deficiency, as does the elevated bilirubin and ferritin. The only α-thalassemia presenting with severe anemia and a low MVC is Hb H disease, and this was ruled out by the HPLC and gel electrophoresis studies. The absence of Hb A by HPLC rules out Hb E/β + -thalassemia, a condition in which Hb A usually represents approximately 30% of the total Hb. 9 Homozygous Hb E without concurrent thalassemia would have a similar appearance on HPLC fractionation and Hb electrophoresis but is inconsistent with the clinical picture, as this condition alone causes only mild, if any, anemia and microcytosis. Although family history, severity of disease, and laboratory studies all support the diagnosis, molecular studies are necessary as definitive proof for coinheritance of Hb E and β-thalassemia since Hb A 2 quantitation is not possible Hemoglobin E is second only to Hb S as the most common Hb variant worldwide. Hb E occurs when glutamic acid is replaced by lysine at codon 26 of the β-globin gene. 9 The mutation produces an alternative mrna splice site resulting in a structurally abnormal β-globin chain. Hb E is most common in Southeast Asia, particularly in individuals of Thai and Burmese descent. However, with increasing immigration from these areas, Hb E has become the third most common Hb variant in the United States. 7,14 The incidence of Hb E approaches 60% in many regions of Asia, including Thailand, Laos, and Cambodia. The high prevalence is likely Figure 1_Hb separation by HPLC (Bio-Rad Variant Beta-thal short program) revealed a Hb A2 fraction of 90.2% (retention time of 3.66), a Hb F of 9.0% (retention time of 1.13). due to its protective qualities against Plasmodium falciparum infection. 15,16 Hb E heterozygotes (1 β-globin chain gene affected) and homozygotes (both β-globin chain genes affected) are usually asymptomatic, and Hb E accounts for approximately 30% and 90% of the total Hb, respectively. 9 Mild elevations in Hb F are seen in Hb E homozygotes. β-thalassemia is a genetically heterogeneous group (>70 genetic mutations) characterized by reduced (β + -thalassemia) or lack of (β o -thalassemia) β-globin chain synthesis. There are more than 40 different genetic mutations resulting in β 0 - thalassemia, 17 and most are single nucleotide changes producing nonsense or frameshift mutations. 18,19 The reduction in β-globin chains leads to increased production of Hb A 2. Mutations in 1 β-globin gene (β + or β o heterozygotes) are classified clinically as β-thalassemia minor and are rarely associated with symptoms and do not require treatment. Patients with both β-globin genes affected (β ο and some β + homozygotes) are classified clinically as β-thalassemia major and have severe anemia requiring lifelong monitoring and transfusion therapy. Some patients homozygous for β + -thalassemia are classified as β-thalassemia intermedia and have a clinical presentation less severe than thalassemia major. 18 The combination of Hb E with β-thalassemia results in a compound heterozygote in which both β-globin chains are affected. The clinical picture of Hb E-β + -thalassemia is usually asymptomatic to mildly symptomatic with mild anemia. 20 Hemoglobin E-β 0 -thalassemia is the most severe manifestation of the Hb E variant, and the majority of patients present with either thalassemia intermedia or major. 21 The interaction of Hb E with β-thalassemia is complex and poorly understood. Ineffective erythropoiesis, shortened RBC survival, and apoptosis are hallmarks of the disease. 22 The much less common situation of homozygosity for Hb E co-inherited with the genotypes associated with Hb H disease can result in a comparable clinical and laboratory picture, whereas co-inheritance of 1 or 2 α-globin gene deletions behaves similarly to homozygosity for Hb E alone. This patient is atypical for Hb 80 LABMEDICINE Volume 41 Number 2 February 2010 labmedicine.com

4 E-β 0 -thalassemia because of the relatively low level of Hb F (11% instead of the expected 30% 60%). This finding along with a particularly low MCV suggests the patient may also harbor a concurrent 1 or 2 α-globin chain mutation. A recent study of Thai patients with Hb E-β 0 -thalassemia, who were also genotyped for α-globin mutations, found that among the subset of patients similar to ours who had Hb levels >7.5 g/ dl, MCV <60 fl, and Hb F <30%, approximately 70% had co-inherited 1 or 2 α-globin chain mutations This patient s presentation is more severe than that of his parents because the severity of the disease is a consequence of the co-inheritance of both a β o -thalassemia allele from 1 parent and the Hb E allele from the other. Either 1 alone is associated with mild or absent disease, as was observed. The sister, who was not clinically affected to the same degree, had a 1 in 4 chance of similarly inheriting the Hb E-β 0 -thalassemia genotype but most likely did not given her clinical condition. In the event she had, patients with Hb E-β-thalassemia from the same family with identical mutations may show large differences in clinical severity. The cause for this variability in clinical symptoms is unknown, and it is difficult to predict the clinical phenotype from the β-globin gene mutations. 24 A study of 378 patients with Hb E-β 0 -thalassemia from Thailand found Hb concentrations ranging from 3 13 g/dl, with a mean concentration of 7.7 g/dl. 25 Approximately half of the patients presented with thalassemia major and required regular blood transfusions, whereas the other half were clinically similar to thalassemia intermedia. 25,26 9. In Hb E-β o -thalassemia the marked erythropoiesis results in hepatosplenomegaly, extramedullary hematopoietic masses, restricted growth, and bone abnormalities. 26 The most common cause of death in Hb E-β o -thalassemia is due to cardiopulmonary disease. Splenectomy has been used in severe cases to increase Hb concentrations, but the increased risk of thromboembolism makes this treatment less attractive. 27 Iron overload is common in both nontransfused and transfused patients and chelation therapy must be considered in order to avoid the toxic effects of iron on various organs. In patients with Hb E-β-thalassemia requiring therapy, agents directed at elevating Hb F levels, such as hydroxyurea, are often effective. In 1 multicenter trial involving 42 patients, hydroxyurea resulted in a mean Hb increase of 1.5 g/dl due to increased Hb F production in approximately one-third of the patients with Hb E-β o -thalassemia. 28 Several of the patients receiving transfusions became transfusion independent with hydroxyurea treatment. Although hydroxyurea is beneficial in Hb E-β-thalassemia, the response is unpredictable and transfusion therapy is often required. 29 Other agents such as decitabine and short-chain fatty acids are currently being investigated for a more reliable clinical response Carrier screening is recommended in high-risk populations such as those of African American, Southeast Asian, and Mediterranean ancestry. The appropriate screening test for high-risk individuals is Hb electrophoresis. 31 Pre-natal diagnosis can be done by obtaining fetal DNA by way of amniocentesis or chorionic villus sampling. Fetal DNA is usually tested by polymerase chain reaction-based techniques for β-thalassemia mutations. The number of specific mutations tested for can be limited based on the ethnic background of the patient. For a given ethnic group, most cases are caused by 3 to 6 mutations, greatly reducing the work required to make the diagnosis. 12 If the diagnosis is not known before birth, newborn screening using HPLC is recommended. In the case of this patient with Hb E-β 0 -thalassemia, the results of the newborn screening would show only Hb F and Hb E. It is important to identify these patients in order to appropriately screen and initiate transfusion therapy to avoid the complications of severe anemia LM Keywords: Beta-thalassemia, hemoglobin E, microcytic anemia, hemoglobinopathy 1. Clarke GM, Trefor HN. Laboratory investigation of hemoglobinopathies and thalassemias: Review and update. Clin Chem. 2000;48: Meredith JL, Rosenthal NS. Differential diagnosis of microcytic anemias. 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Differential diagnosis of Hb EE and Hb E-β by protein and DNA analyses. Acta Haematol. 2000;103: Thein SL. Genetic insights into the clinical diversity of beta thalassaemia. Br J Haematol. 2004;124: McClatchey KD, ed. Clinical Laboratory Medicine. Baltimore, MA: Williams & Wilkins; 1994: Nguyen CK, Le TT, Duong BT, et al. Beta-thalassemia/haemoglobin E disease in Vietnam. J Trop Pediatr. 1990;36: Vichinsky E. Hemoglobin E syndromes. Hematol. 2007;1: Pootrakul P, Sirankapracha P, Hemsorach S, et al. A correlation of erythrokinetics, ineffective erythropoiesis, and erythroid precursor apoptosis in Thai patients with thalassemia. Blood. 2000;96: labmedicine.com February 2010 Volume 41 Number 2 LABMEDICINE 81

5 23. Sripichai O, Munkongdee T, Kumkhaek C, et al. Coinheritance of the different copy numbers of alpha-globin gene modifies severity of betathalassemia/hb E disease. Ann Hematol. 2008;87: Nuntakarn L, Fucharoen S, Fucharoen G, et al. Molecular hematological and clinical aspects of thalassemia major and thalassemia intermedia associated with Hb E-beta-thalassemia in Northeast Thailand. Blood Cells, Molecules, and Diseases. 2009;42: Fucharoen S, Ketvichit P, Pootrakul P, et al. Clinical manifestation of betathalassemia/hemoglobin E disease. J Pediatr Hematol Oncol. 2000;22: Fucharoen S, Winichagoon P. Clinical and hematologic aspects of hemoglobin E beta-thalassemia. Curr Opin Hematol. 2000;7: Phrommintikul A, Sukonthasarn A, Kanjanavanit R, et al. Splenectomy: A strong risk factor for pulmonary hypertension in patients with thalassaemia. Heart. 2006;92: Singer ST, Kuypers FA, Olivieri NF, et al. Fetal hemoglobin augmentation in E/beta(0) thalassemia: Clinical and hematological outcome. Br J Haematol. 2005;131: Panigrahi I, Agarwal S, Gupta T, et al. Hemoglobin E-beta thalassemia: Factors affecting phenotype. Indian Pediatr. 2005;42: Perrine SP, Castaneda SA, Boosalis MS, et al. Induction of fetal globin in beta-thalassemia: Cellular obstacles and molecular progress. Ann N Y Acad Sci. 2005;1054: Orion-Group. Coexisting iron deficiency anemia and thalassemia trait. The Orion Medical Journals. May Available at: journals/journals/vol21_may2005/259.htm. Accessed December 23, Rappaport VJ. Prenatal diagnosis and genetic screening integration into prenatal care. Obstet Gynecol Clin North Am. 2008;35: Rojnuckarin P, Settapiboon R, Vanichsetakul P, et al. Severe β 0 Thalassemia/ hemoglobin E disease caused by de novo 22-base pair duplication in the paternal allele of β globin gene. Am J Hematol. 2007;82: Cunningham MJ. Update on thalassemia: Clinical care and complications. Pediatr Clin North Am. 2008;55: LABMEDICINE Volume 41 Number 2 February 2010 labmedicine.com