A Brief Overview of Hemoglobin Electrophoresis

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A Brief Overview of Hemoglobin Electrophoresis

• Sarah Walter, M.D.

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Normal Hemoglobin Structure

• Hemoglobin A 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 oxygen

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Normal Hemoglobin Structure
Hemoglobin tetramer
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Normal Hemoglobin Structure
O
O
Fe
Porphyrin ring
O2 binding site
The oxygen atom binds to the Fe
atom perpendicular to the porphyrin ring
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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.

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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

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Normal Hemoglobin Function

• The hemoglobin dissociation curve

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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

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Other Hemoglobins in normal adults
Indicates early embryonic form not seen in
adults
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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)

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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.

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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)

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Laboratory Methods to evaluate Hemoglobin

• Red cell morphologies
• HbS Sickle cells

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Sickle cells on peripheral smear
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Laboratory Methods to evaluate Hemoglobin

• Red cell morphologies
• HbS Sickle cells
• HbC Target cells, crystals after splenectomy

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HbC crystals with Target cells
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Laboratory Methods to evaluate Hemoglobin

• Red cell morphologies
• HbS Sickle cells
• HbC Target cells, crystals after splenectomy
• Thalassemias Microcystosis, target cells,
  basophilic stippling

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Alpha Thalassemia with basophilic stippling
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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).

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Hemoglobin Electrophoresis Patterns
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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.

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Normal HPLC pattern
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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.

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Most common Hemoglobin abnormalities

• Thalassemias
• Alpha
• Beta
• Hemoglobinopathies
• HbS trait disease
• HbC trait disease
• HbE
• Hereditary Persistence of Hemoglobin F (HPHF)

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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

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Case 1
HbF 1.3 HbA2 4.1
Sickledex test POSITIVE
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Case 1
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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

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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 gt2

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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

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Case 2
HbF 1.6 HbA2 5.1
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Case 2
Cellulose acetate gel performed
HbS
HbS
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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

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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.

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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

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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

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Case 3
HbF 1.0 HbA 38.7 HbA2 4.4 HbS 56.1
Sickledex is POSITIVE Peripheral smear with 2
sickle cells
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Case 3
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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

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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

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Case 3
Expected ratios
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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

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Case 4
HbF 0.6 HbA2 2.9 HbA 56.3
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Case 4
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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 6040 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.

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THE END!!!