MLAB 1415: Hematology Keri Brophy-Martinez Chapter 11: Thalassemia

Introduction to Thalassemia

Thalassemia Diverse group of congenital disorders which manifest as anemia of varying degrees. Result of quantitative defective production of one or more globin portion(s) of hemoglobin molecule. Distribution is worldwide. Defect results from abnormal rate of synthesis in one or more of the globin chains. 

Thalassemia Results in overall decrease in amount of hemoglobin produced and may induce hemolysis. Two major types of thalassemia: Alpha (α) - Caused by defect in rate of synthesis of alpha chains. Beta (β) - Caused by defect in rate of synthesis in beta chains. May contribute protection against malaria.

Genetics of Thalassemia May be either homozygous defect or heterozygous defect. Adult hemoglobin composed two alpha and two beta chains. Genetic defects usually falls into one of below categories Gene deletion Promoter deletion Nonsence mutation Mutated termination Splice site mutation

Genetics of Thalassemia Alpha thalassemia usually caused by gene deletion Beta thalassemia usually caused by mutation.

Review of Hgb Structure Normal globin genes Alpha, beta, delta, gamma Form hgb A (97%), hgb A2, hgb F Epsilon, zeta: in utero Gamma: 3rd trimester until birth Ratio of β-chain to α-chain is 1:0 Thalassemia causes an excess of one of these chains

Pathophysiology α-chain excess β-chain excess unstable Precipitates within the cell, binding to the cell membrane causes damage Macrophages destroy the damaged RBCs in the bone marrow, leads to ineffective erythropoiesis Spleen also removes damaged RBCs, leads to chronic extravascular hemolysis β-chain excess Unstable Combines to form hgb molecules with 4 β-chains ( hemoglobin H) Infants: excess gamma chains combine with hgb molecules (hemoglobin Bart’s) High oxygen affinity, poor transporter of oxygen

General Clinical Findings Anemia Hypoxia Splenomegaly Gallstones Skeletal abnormalites Iron toxicity Fractures

Comparison of Hemoglobinopathies and Thalassemias Disease RBC count Indices RBC Morph Abnormal Hb Ancestry Retic Count Hemoglobinopathy Normocytic Normochromic Target cells, sickle cells (HbS), Crystals (HbC) HbS,HbC, HbE etc African Mediterranean Middle Eastern Asian Thalassemia Microcytic Hypochromic Target cells, basophilic stippling HbH Hb Bart’s Thalassemia: globin chains structurally normal Hemoglobinopathies: globin chain is abnormal

Beta Thalassemia

Classical Syndromes of Beta Thalassemia Beta thalassemia minima/ Silent carrier state – the mildest form of beta thalassemia. Beta thalassemia minor - heterozygous disorder resulting in mild hypochromic, microcytic hemolytic anemia. Beta thalassemia intermedia - Severity lies between the minor and major. Beta thalassemia major - homozygous disorder resulting in severe transfusion- dependent hemolytic anemia.

Beta thalassemia minima/ Silent Carrier State for β Thalassemia Are various heterogenous beta mutations that produce only small decrease in production of beta chains. Patients have nearly normal beta/alpha chain ratio and no hematologic abnormalities. Have normal levels of Hb A2.

Beta Thalassemia Minor Caused by heterogenous mutations that affect beta globin synthesis.  Usually presents as mild, asymptomatic hemolytic anemia unless patient in under stress such as pregnancy, infection, or folic acid deficiency. Have one normal beta gene and one mutated beta gene. Hemoglobin level in 9-14 g/dL range with normal or slightly elevated RBC count. 

Beta Thalassemia Minor Anemia usually hypochromic and microcytic with slight aniso and poik, including target cells and elliptocytes;  May see basophilic stippling. Rarely see hepatomegaly or splenomegaly. Have high Hb A2 levels (3.5-8.0%) and normal to slightly elevated Hb F levels. Are different variations of this form depending upon which gene has mutated. Normally require no treatment.  Make sure are not diagnosed with iron deficiency anemia.

Beta Thalassemia Intermedia Patients able to maintain minimum hemoglobin (7 g/dL or greater) without transfusions.  Expression of disorder falls between thalassemia minor and thalassemia major.  May be either heterozygous for mutations causing mild decrease in beta chain production, or may be homozygous causing a more serious reduction in beta chain production. See increase in both Hb A2 production and Hb F production. Peripheral blood smear picture similar to thalassemia minor.

Beta Thalassemia Intermedia Have varying symptoms of anemia, jaundice, splenomegaly and hepatomegaly. Have significant increase in bilirubin levels.  Anemia usually becomes worse with infections, pregnancy, or folic acid deficiencies. May become transfusion dependent as adults. Tend to develop iron overloads as result of increased gastrointestinal absorption. Usually survive into adulthood.

Beta Thalassemia Major/ Cooley’s anemia Characterized by severe microcytic, hypochromic anemia.  Detected early in childhood: Infants fail to thrive.  Have pallor, variable degree of jaundice, abdominal enlargement, and hepatosplenomegaly. Hemoglobin level between very low Severe anemia causes marked bone changes due to expansion of marrow space for increased erythropoiesis. See characteristic changes in skull, long bones, and hand bones. 

Beta Thalassemia Major Have protrusion upper teeth and Mongoloid facial features.  Physical growth and development delayed. Peripheral blood shows markedly hypochromic, microcytic erythrocytes with extreme poikilocytosis, such as target cells, teardrop cells and elliptocytes.  See marked basophilic stippling and numerous NRBCs.  MCV in range of 50 to 60 fL. Low retic count seen (2-8%). Most of hemoglobin present is Hb F with slight increase in Hb A2.

Beta Thalassemia Major Regular transfusions usually begin around one year of age and continue throughout life.  Excessive number of transfusions results in tranfusional hemosiderosis; Without iron chelation, patient develops cardiac disease. Danger in continuous tranfusion therapy: Development of iron overload. Development of alloimmunization (developing antibodies to transfused RBCs). Risk of transfusion-transmitted diseases. Bone marrow transplants may be future treatment, along with genetic engineering and new drug therapies.

Comparison of Beta Thalassemias GENOTYPE HGB A HGB A2 HGB F NORMAL Normal MINIMA MINOR Dec Normal to Inc INTERMEDIA Usually Inc MAJOR

Other Thalassemias Caused by Defects in the Beta-Cluster Genes 1. Delta Beta Thalassemia 2. Hemoglobin Lepore 3. Hereditary Persistence of Fetal Hemoglobin (HPFH)

Delta Beta Thalassemia Group of disorders due either to a gene deletion that removes or inactivates only delta and beta genes so that only alpha and gamma chains produced. Similar to beta thalassemia minor. Growth and development nearly normal.  Splenomegaly modest.  Peripheral blood picture resembles beta thalassemia.

Hemoglobin Lepore Rare class of delta beta thalassemia. Caused by gene crossovers between delta locus on one chromosome and beta locus on second chromosome.

Hereditary Persistence of Fetal Hemoglobin (HPFH) Rare condition characterized by  continued synthesis of Hemoglobin F in adult life.  Do not have usual clinical symptoms of thalassemia. Little significance except when combined with other forms of thalassemia or hemoglobinopathies. If combined with sickle cell anemia, produces milder form of disease due to presence of Hb F.

Hereditary Persistence of Fetal Hemoglobin (HPFH) Hb F more resistant to denaturation than Hb A. Can be demonstrated on blood smears using Kleihauer Betke stain.  Cells containing Hb F stain. Classified into two groups according to distribution of Hb F among red cells: Pancellular HPFH - Hemoglobin F uniformly distributed throughout red cells. Heterocellular HPFH - Hemoglobin F found in only small number of cells.

Beta Thalassemia with Hgb S Inherit gene for Hb S from one parent and gene for Hb A with beta thalassemia from second parent. Great variety in clinical severity.   Usually depend upon severity of thalassemia inherited.  Production of Hb A ranges from none produced to varying amounts.  If no Hb A produced, see true sickle cell symptoms.  If some Hb A produced, have lessening of sickle cell anemia symptoms.

Beta Thalassemia with Hgb C Shows great variability in clinical and hematologic symptoms. Symptoms directly related to which type thalassemia inherited. Usually asymptomatic anemia

Beta Thalassemia with Hgb E Is unusual because results in more severe disorder than homozygous E disease. Very severe anemia developing in childhood.  Transfusion therapy required.

Alpha Thalassemia

Alpha Thalassemia Predominant cause of alpha thalassemias is large number of gene deletions in the alpha-globin gene. Four clinical syndromes present in alpha thalassemia: Silent Carrier State Alpha Thalassemia Trait  (Alpha Thalassemia Minor) Hemoglobin H Disease Bart's Hydrops Fetalis Syndrome

Silent Carrier State Deletion of one alpha gene, leaving three functional alpha genes. The 3 remaining genes direct synthesis of adequate numbers of α-chains for normal hgb synthesis. No hematologic abnormalities present. Alpha/Beta chain ratio nearly normal. No reliable way to diagnose silent carriers by hematologic methods;  Must be done by globin gene analysis mapping. May see borderline low MCV  (78-80fL).

Alpha Thalassemia Trait (Alpha Thalassemia Minor) Also called Alpha Thalassemia Minor. Occurs when two of the four alpha genes are missing. The unaffected globin chains can compensate for the missing genes. Exhibits mild microcytic, hypochromic anemia. MCV between 70-75 fL. May be confused with iron deficiency anemia. Although some Bart's hemoglobin (γ4) present at birth, no Bart's hemoglobin present in adults.

Hemoglobin H Disease Second most severe form alpha thalassemia. Only one alpha gene out of four is functional Results in accumulation of excess unpaired gamma or beta chains. The excess chains pair up to form tetrads Beta: hemoglobin H (adults) Gamma: hemoglobin Bart’s (infants) Unstable Precipitates within RBCs triggers hemolysis High affinity for oxygen which reduces oxygen delivery to the tissues

Hemoglobin H Disease Live normal life; however, infections, pregnancy, exposure to oxidative drugs may trigger hemolytic crisis. RBCs are microcytic, hypochromic with marked poikilocytosis.  Numerous target cells. Hb H vulnerable to oxidation.  Gradually precipitate in vivo to form Heinz-like bodies of denatured hemoglobin.  Cells been described has having "golf ball" appearance, especially when stained with brilliant cresyl blue.

Bart’s Hydrops Fetalis Syndrome or α-Thalassemia major Most severe form.  Incompatible with life.  Have no functioning alpha chain genes Baby born with hydrops fetalis, which is edema and ascites caused by accumulation serous fluid in fetal tissues as result of severe anemia.  Also see hepatosplenomegaly and cardiomegaly. Predominant hemoglobin is Hemoglobin Bart, along with Hemoglobin Portland and traces of Hemoglobin H. Hemoglobin Bart's has high oxygen affinity so cannot carry oxygen to tissues.  Fetus dies in utero or shortly after birth. At birth, see severe hypochromic, microcytic anemia with numerous NRBCs. Pregnancies dangerous to mother.  Increased risk of toxemia and severe postpartum hemorrhage.

Comparison of Alpha Thalassemias Genotype Hb A Hb Bart Hb H Normal 97-98% Silent Carrier 96-98% 0-2% Alpha Thalassemia Trait 85-95% 5-10% Hemoglobin H Disease Dec 25-40% 2-40% Hydrops Fetalis 80% (with 20% Hgb Portland) 0-20%

Laboratory Diagnosis of Thalassemia

Laboratory Diagnosis of Thalassemia Need to start with patient's individual history and family history.  Ethnic background important. Perform physical examination: Pallor indicating anemia. Jaundice indicating hemolysis. Splenomegaly due to pooling of abnormal cells. Skeletal deformity, especially in beta thalassemia major.

CBC with Differential See decrease in hemoglobin, hematocrit, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH).  See normal to slightly decreased Mean Corpuscular Hemoglobin Concentration (MCHC).  Will see microcytic, hypochromic pattern. Have normal or elevated RBC count with a normal red cell volume distribution (RDW). Decrease in MCV very noticeable when compared to decrease in Hb and Hct.

CBC with Differential Elevated RBC count with markedly decreased MCV differentiates thalassemia from iron deficiency anemia. On differential, see microcytic, hypochromic RBCs (except in carrier states).  See mild to moderate poikilocytosis.  In more severe cases, see marked number of target cells and elliptocytes.  Will see polychromasia, basophilic stippling, and NRBCs.

Reticulocyte Count Usually elevated.  Degree of elevation depends upon severity of thalassemia.

Osmotic Fragility Have decreased osmotic fragility. Is not very useful fact for diagnosing thalassemia.  Is an inexpensive way of screening for carrier states.

Brilliant Cresyl Blue Stain Incubation with brilliant cresyl blue stain causes Hemoglobin H to precipitate.  Results in characteristic appearance of multiple discrete inclusions -golf ball appearance of RBCs.   Inclusions smaller than Heinz bodies and are evenly distributed throughout cell.

Acid Elution Stain Based on Kleihauer-Betke procedure.  Acid pH will dissolve Hemoglobin A from red cells.  Hemoglobin F is resistant to denaturation and remains in cell.  Stain slide with eosin.  Normal adult cells appear as "ghost" cells while cells with Hb F stain varying shades of pink.

Hemoglobin Electrophoresis Important role in diagnosing and differentiating various forms of thalassemias. Can differentiate among Hb A, Hb A2, and Hb F, as well as detect presence of abnormal hemoglobins such as Hemoglobin Lepore, hemoglobin Bart's, or Hemoglobin Constant Spring. Also aids in detecting combinations of thalassemia and hemoglobinopathies.

Hemoglobin Quantitation Elevation of Hb A2 excellent way to detect heterozygote carrier of beta thalassemia.  Variations in gene expression in thalassemias results in different amounts of Hb A2 being produced. Can also quantitate levels of Hb F.

Routine Chemistry Tests Indirect bilirubin elevated in thalassemia major and intermedia. Assessment of iron status, total iron binding capacity, and ferritin level important in differentiating thalassemia from iron deficiency anemia.

Other Special Procedures Globin Chain Testing - determines ratio of globin chains being produced. DNA Analysis - Determine specific defect at molecular DNA level.

Differential Diagnosis of Microcytic, Hypochromic Anemias RDW Serum Iron TIBC Serum Ferritin FEP Iron Deficiency Inc Dec Alpha Thal Norm Beta Thal Hgb E Disease Anemia of Chronic Disease Sideroblastic Anemia Lead Poisoning