1. A Brief Overview of Hemoglobin Electrophoresis
Sarah Walter, M.D.

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

3. Normal Hemoglobin Structure
Hemoglobin tetramer

4. Normal Hemoglobin StructureFe
Porphyrin ring
O2 binding site
The oxygen atom binds to the Fe atom
perpendicular to the porphyrin ring

5. Hemoglobin Function
The function of the Hemoglobin molecule is to pick up oxygen in the lung and deliver it to the tissues utilizing none of the oxygen along the way.

6. 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

7. Normal Hemoglobin Function
The hemoglobin dissociation curve

8. Normal Hemoglobin Function
Many variables influence the dissociation curve:
pH:
An increase in pH (dec. CO2) shifts the curve to the left (increased O2) affinity
A decrease in pH (inc. CO2) shifts the curve to the right (decreased O2 ) affinity
Temperature:
Increased temp with increased metabolic demands causes decreased O2 affinity (right shift) and increased O2 delivery
2,3 DPG:
Lowers O2 affinity by preferentially binding to Beta chain of deoxyhemoglobin, stabilizing it and reduces the intracellular pH
As hemoglobin concentration decreases, 2,3 DPG increases, allowing more O2 to be unloaded

9. Other Hemoglobins in normal adults
Structure % A
α2 β2
92% A2
α2 δ2
2.5%
A1C
α2 (β-N-glucose) 3% F
α2 γ2
<1%
Gower-1
ζ2 ε2 0*
Gower-2
α2 ε2
Portland
ζ2 γ2
* Indicates early embryonic form not seen in adults

10. Other Hemoglobins in normal adults
HbA2:
Decreased in iron deficiency, alpha-thalassemia
Elevated in megaloblastic anemia, hyperthyroidism, Beta-thalessemia
HbF:
Elevated in HPFH, Sickle cell anemia (preferential survival of RBCs because HgF inhibits sickling), Beta thalessemia major
Normal levels in Beta-thalassemia minor
Normal or mildly elevated in congenital hemolytic anemia
Marked elevation in juvenile CML (up to 70%)

11. 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 γ globin to switch to β globin.

12. 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)

13. Laboratory Methods to evaluate Hemoglobin
Red cell morphologies:
HbS: Sickle cells

14. Sickle cells on peripheral smear

15. Laboratory Methods to evaluate Hemoglobin
Red cell morphologies:
HbS: Sickle cells
HbC: Target cells, crystals after splenectomy

16. HbC crystals with Target cells

17. Laboratory Methods to evaluate Hemoglobin
Red cell morphologies:
HbS: Sickle cells
HbC: Target cells, crystals after splenectomy
Thalassemias: Microcystosis, target cells, basophilic stippling

18. Alpha Thalassemia with basophilic stippling
The basophilic stippling is due to precipitated globins that formed in excess (I.e. HbH, Beta tetramer)

19. Laboratory Methods to evaluate Hemoglobin
Electrophoresis:
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).

20. Hemoglobin Electrophoresis Patterns

21. 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.

22. Normal HPLC pattern

23. Laboratory Methods to evaluate Hemoglobin
Solubility test (Sickledex):
Test to identify HbS. HbS is relatively insoluble compared to other Hemoglobins.
Add reducing agent
HbS will precipitate forming and opaque solution compared with the clear pink solution seen in HbS is not present.

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

25. Case 1
47 year old female presents with a history of peptic ulcer disease, H. Pylori an anemia.
Labs:
Hgb: 10.2
Hct: 30.9
MCV: 96.4
B12: 338
Iron: 122
Ferritin: 304.5
IBC: 226

26. Case 1
HbF: 1.3%
HbA2: 4.1%
Sickledex test POSITIVE

27. Case 1

28. Case 1 Hemoglobin S/C disease:
Second most common hemoglobin variant in Africans; 1 in 1000 births of African Americans
Relatively benign condition; Milder disease than Sickle cell disease. Patients have normal growth and development
Do not see the classic sickle cells
Peripheral smear reveals anisocytosis, target cells, poikilocytosis, polychromasia

29. Case 1 Hemoglobin S/C disease:
Most patients have moderate splenomegaly with many having autosplenectomy, usually older age than with Sickle cell disease
May have veno-occlusive disease, but less common and less severe than in sickle cell disease
May have aseptic necrosis of bone with osteomyelitis
~50% HbS: 50% HbC; rarely is HbF >2%

30. Case 2
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%

31. Case 2
HbF: 1.6%* HbA2: 5.1%

32. Case 2
Cellulose acetate gel performed
HbS
HbS

33. Case 2 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

34. Case 2 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.

35. Case 2 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

36. Case 3
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: 17.8MCV: 97.1
RDW: 20.9
Iron: 83
Ferritin: 394.3
IBC: 144
Fe Sat: 58%

37. Case 3 HbF: 1.0%; HbA: 38.7%; HbA2: 4.4%; HbS: 56.1%
Sickledex is POSITIVE; Peripheral smear with 2+ sickle cells

38. Case 3

39. Case 3 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

40. Case 3 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

41. Case 3 Expected ratios HbA HbS HbA2 HbF Hb AS 55-60 40-45 2-3 <1
Hb SS
90-95
5-10
Hb S-α-thal 75 25
Hb S- β thal major
Inc.
Hb S- β thal minor
5-30
60-90
Hb S HPFH
70-80
20-30
Hb SC 50

42. Case 4
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

43. Case 4
HbF: 0.6%; HbA2: 2.9%; HbA: 56.3%

44. Case 4

45. Case 4 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.

46. THE END!!!