1. MLAB 1415: Hematology Keri Brophy-Martinez Chapter 11: Thalassemia Part Two

2. Alpha Thalassemia 2

3. Predominant cause of alpha thalassemias is large number of gene deletions in the alpha-globin gene. Four types in alpha thalassemia: ◦ Silent Carrier State ◦ Alpha Thalassemia Trait (Alpha Thalassemia Minor) ◦ Hemoglobin H Disease ◦ Bart's Hydrops Fetalis Syndrome 3

4. 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 4

5. Laboratory Findings: Hemoglobin H Disease RBCs are microcytic, hypochromic with marked poikilocytosis. Numerous target cells. 5

6. FIGURE 11-4 b. Peripheral blood from patient in Figure 11-4a after incubation with brilliant cresyl blue. Notice the cells that have dimples and look like golf balls. These are the cells with precipitated HbH.

7. Bart’s Hydrops Fetalis Syndrome or α- Thalassemia major 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. Pregnancies dangerous to mother. Increased risk of toxemia and severe postpartum hemorrhage. 7

8. Bart’s Hydrops Fetalis Syndrome 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. 8

9. Laboratory Diagnosis of Thalassemia 9

10. 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. 10

11. 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. 11

12. 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. 12

13. Reticulocyte Count Usually elevated. Degree of elevation depends upon severity of thalassemia. 13

14. Osmotic Fragility Have decreased osmotic fragility. Is not very useful fact for diagnosing thalassemia. Is an inexpensive way of screening for carrier states. 14

15. 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. 15

16. 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. 16

17. Hemoglobin Electrophoresis Important role in diagnosing and differentiating various forms of thalassemias. Can differentiate among Hb A, Hb A 2, 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. 17

18. Hemoglobin Quantitation Elevation of Hb A 2 excellent way to detect heterozygote carrier of beta thalassemia. Variations in gene expression in thalassemias results in different amounts of Hb A 2 being produced. Can also quantitate levels of Hb F. 18

19. 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. 19

20. Differential Diagnosis of Microcytic, Hypochromic Anemias RDW Serum Iron TIBC Serum Ferritin Iron Deficiency IncDecIncDec Alpha Thal NormNormNormNorm Beta Thal NormNormNormNorm Hgb E Disease NormNormNormNorm Anemia of Chronic Disease NormDecDecInc Sideroblastic Anemia IncIncNormInc Lead Poisoning NormNormNormNorm 20

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