Hemoglobulin Electropheresis

2. Normal Hemoglobin Structure: Hb is a tetramer composed of 4 subunits: 2a and 2ß Each subunit has a porphyrin ring which holds an iron molecule. This is the binding site of O2

3. Structure of Normal Hemoglobin

4. Hemoglobin Function The normal hemoglobin molecule is well suited for its function Allows for O2 to be picked up at high O2 tension in the lung and delivered to the tissues at low O2 tension The oxygen binding is cooperative: As each O2 binds to hemoglobin, the molecule undergoes a conformational change increasing the O2 affinity for the remaining subunits. This creates the sigmoidal oxygen dissociation curve

5. Other Hemoglobins in normal adults HbA2 Decreased in iron deficiency, alpha-thalassemia Elevated in megaloblastic anemia, Beta-thalessemia HbF Sickle cell anemia, Beta thalessemia major Normal levels in Beta-thalassemia minor Normal or mildly elevated in congenital hemolytic anemia

6. Hemoglobin Abnormalities There are 3 main categories of inherited Hemoglobin abnormalities: Structural or qualitative: The amino acid sequence is altered because of incorrect DNA code (Hemoglobinopathy) Quantitative: Production of one or more globin chains is reduced or absent (Thalassemia) Hereditary persistence of Fetal Hemoglobin (HPFH): Complete or partial failure of ? globin to switch to ß globin.

7. Abnormal Hemoglobin Reasons to suspect a hemoglobin disorder: Patient presents with suspicious history or physical exam Laboratory tests: Microcytic hypochromic RBCs, hemolytic anemia Screening test abnormality (primarily in neonates)

9. HbC crystals with Target cells

10. Hemoglobin Electrophoresis (Identifying Thalassemia and hemoglobinopathy): Alkaline (Cellulose Acetate) pH 8.6: All Hemoglobin molecules have a negative charge, and migrate towards the anode proportional to their net negative charge. Amino acid substitutions in hemoglobin variants alter net charge and mobility. Acid (Citrate agar) pH 6.2: Hemoglobin molecules separate based on charge differences and their ability to combine with the agar. Used to differentiate Hemoglobin variants that migrate together on the cellulose gel (i.e. HbS from HbD and HbG, HbC from HbE).

12. Hemoglobins in normal adults

13. Hemoglobin Electrophoresis Patterns

14. Cellulose Acetate Hb Electrophoresis - A2/C S F A + Normal

15. - A2/C S F A + Normal Hb SS Cellulose Acetate Hb Electrophoresis

16. - A2/C S F A+ Normal Hb SS Hb AS Cellulose Acetate Hb Electrophoresis

17. - A2/C S F A+ Normal Hb SS Hb AS Hb SC Cellulose Acetate Hb Electrophoresis

18. - A2/C S F A+ Normal Hb SS Hb AS Hb SC Hb CC Cellulose Acetate Hb Electrophoresis

19. - A2/C S F A+ Normal Hb SS Hb AS Hb SC Hb CC HB AD Cellulose Acetate Hb Electrophoresis

20. Laboratory Methods to evaluate Hemoglobin High-Performance Liquid Chromatography (HPLC): Weak cation exchange column. The ionic strength of the eluting solution is gradually increased and causes the various Hemoglobin molecules to have a particular retention time. Amino acid substitutions will alter the retention time relative to HbA. There is some analogy between retention time and pattern on alkaline electrophoresis.

21. Most common Hemoglobin abnormalities Thalassemias Alpha Beta Hemoglobinopathies HbS trait; disease HbC trait; disease HbE Hereditary Persistence of Hemoglobin F (HPHF)

22. Case 1 A 45 year old German man who is asymptomatic is seen for microcytosis. Peripheral smear shows microcytosis, hypochromia, target cells, basophilic stippling, polychromasia Labs: Hgb: 11.8 Hct: 37.5 MCV: 65.9 Iron: 119 Ferritin: 506 IBC: 275 Fe Sat: 43%

23. Case 1

24. Case 1

25. Case 1 Beta Thalassemia Minor: The thalassemia seen most commonly is caucasians (primarily Mediterranean descent) Beta thalassemia minor is loss of one of two genes for Beta globin on chromosome 11 Patients generally asymptomatic May have mild microcytic anemia (MCV: 60-70; Hgb: 10-13) with a normal or slightly increased RBC count The peripheral smear will show target cells and basophilic stippling See increased HbA2 in the range of 5-9% with normal HbF Thalassemia found most commonly in caucasians See mild microcytosis

26. Case 1 Beta Thalassemia Minor: Primary indication is a slightly elevated HbA2 detected by HPLC (usually around 4-7%, up to 10%) typically without elevation of HbF Diagnosis may be obscured in concomitant iron deficiency present because Beta-thalassemia causes an increase in HbA2 while iron deficiency causes a decrease in HbA2. Both create a microcytosis. May see a anemia that partially responds to iron therapy Always want to look at iron studies when interpreting hemoglobin electrophoresis; usually wait to diagnose until nutritional deficiencies have first been corrected.

27. Case 1 Beta Thalassemia Major: Homozygous double gene deletion with no Beta globin production Presents with lethal anemia, jaundice, splenomegaly, growth retardation, bone malformations, death Severe hypochromic, microcytic anemia with very bizarre cells HbA2 is not increased HgF is at nearly 100% Abundant intra-erythrocyte precipitation of alpha monomers that are insoluble

28. Case 2 47 year old African American female presents to the ER with drug intoxication and marked anemia. She is unable to provide any adequate history to the clinicians. Labs: Hgb: 5.9 Hct: 17.8 MCV: 97.1 RDW: 20.9 Iron: 83 Ferritin: 394.3 IBC: 144 Fe Sat: 58%

29. Case 2

30. Case 2

31. Case 2 Sickle cell anemia: In sickle cell trait, usually see HbS concentrations of 35 to 45% of total Hemoglobin because the HbS has a slower rate of synthesis than HbA If HbS is less than 33%, start thinking about S-alpha-thalassemia If HbS is greater than 50%, worry about S-Beta-thalassemia or Sickle cell disease with transfusion

32. Case 2 Sickle cell anemia: This patient was transfused with two units of RBCs before the HPLC was performed. It is important to know the appropriate ratios of HbS: HbA expected. If the patient does not fit, always look at the transfusion history. If concerned about overlying Beta-thalassemia, repeat HPLC after four months of most recent transfusion

33. Case 2

34. Case 3 31 year old healthy female, pregnant with moderate target cells detected on routine peripheral smear Labs: Hgb: 15.0 Hct: 42.5 MCV: 87.8 MCH: 31.0 RDW: 12.6

35. Case 3

36. Case 3

37. Case 3 Hemoglobin C trait: Hemoglobin C trait (Heterozygotes) are clinically and hematologically well Moderate target cells seen on peripheral smear HbA and HbC in a 60:40 ratio on HPLC 2% of African Americans have HbC trait Homozygotes have mild hemolytic disease, cholelithiasis and occasional aplastic crisis. See reduced MCV with increased MCHC Intracellular HbC crystals, block-like structures may be seen and are pathognomonic of HbC.