A Brief Overview of
Hemoglobin Electrophoresis
Sarah Walter, M.D.
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
Normal Hemoglobin Structure
Hemoglobin tetramer
Normal Hemoglobin Structure
O
O
Fe
Porphyrin ring
O2 binding site
The oxygen atom binds to the Fe atom
perpendicular to the porphyrin ring
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.
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
Normal Hemoglobin Function
The hemoglobin dissociation curve
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
Other Hemoglobins in normal
adults
Hemoglobin 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
0*
Portland
ζ2 γ2
0*
* Indicates early embryonic form not seen in adults
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%)
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.
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)
Laboratory Methods to evaluate
Hemoglobin

Red cell morphologies:
– HbS: Sickle cells
Sickle cells on peripheral smear
Laboratory Methods to evaluate
Hemoglobin

Red cell morphologies:
– HbS: Sickle cells
– HbC: Target cells, crystals after splenectomy
HbC crystals with Target cells
Laboratory Methods to evaluate
Hemoglobin

Red cell morphologies:
– HbS: Sickle cells
– HbC: Target cells, crystals after splenectomy
– Thalassemias: Microcystosis, target cells,
basophilic stippling
Alpha Thalassemia with
basophilic stippling
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).
Hemoglobin Electrophoresis
Patterns
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.
Normal HPLC pattern
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.
Most common Hemoglobin
abnormalities

Thalassemias
– Alpha
– Beta

Hemoglobinopathies
– HbS trait; disease
– HbC trait; disease
– HbE
– Hereditary Persistence of Hemoglobin F
(HPHF)
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
Case 1
Sickledex test POSITIVE
HbF: 1.3%
HbA2: 4.1%
Case 1
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
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%
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%
Case 2
HbF: 1.6%*
HbA2: 5.1%
Case 2
Cellulose acetate gel performed
HbS
HbS
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
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.
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
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%
Case 3
HbF: 1.0%; HbA: 38.7%; HbA2: 4.4%; HbS: 56.1%
Sickledex is POSITIVE; Peripheral smear with 2+ sickle cells
Case 3
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 Salpha-thalassemia
 If HbS is greater than 50%, worry about S-Betathalassemia or Sickle cell disease with transfusion
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
Case 3
Expected ratios
HbA
HbS
HbA2 HbF
Hb AS
55-60 40-45 2-3
<1
Hb SS
0
90-95 2-3
5-10
Hb S-α-thal
75
25
<1
Hb S- β thal major
0
90-95 Inc.
5-10
Hb S- β thal minor
5-30
60-90 Inc.
5-10
Hb S HPFH
0
70-80 2-3
20-30
Hb SC
0
50
<1
2-3
2-3
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
Case 4
HbF: 0.6%; HbA2: 2.9%; HbA: 56.3%
Case 4
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.
THE END!!!