Hemoglobin Electrophoresis and

HPLC Study

Normal Hemoglobin Structure

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

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

The hemoglobin dissociation curve

Other Hemoglobins in normal adults

Hemoglobin

Structure

2 2

92%

A2

2 2

2.5%

A1C

2 (-N-glucose)

3%

2 2

<1%

Gower-1

2 2

0*

Gower-2

2 2

0*

Portland

2 2

0*

* Indicates early embryonic form not seen in adults

Fetal Hb

Hemoglobin Abnormalities /Hemoglobinopathies

Three main categories:
1. Quantitative: Production of one or more globin chains is reduced
or absent (Thalassemia).

2. Structural or qualitative: Amino acid sequence is altered

because of incorrect DNA code (4 groups):
Affecting function: HbS
Unstable Hb: Torino, Zurich, Hasharon (Hb precipitates as Heinz bodies,
jaundice, splenomegaly, hemolytic anemia)
Decreased affinity: Cyanosis, anemia: Kansas, Providence, Seattle, Beth
Israel
Increased affinity: Polycythemia, family erythrocytosis, electrophoresis is
not useful: J.Capetown, Chesapeake, Malmo, Bethesda
Hb M: methhemoglobin: Cyanosis, develops 6 months after birth: Boston,
iwate, Saskatoon, Milwaukee.

3. Hereditary persistence of Fetal Hemoglobin (HPFH):

Complete or partial failure of globin to switch to globin.

 Over 1000 hemoglobin variants are current known.

Worldwide, estimated over 150 million people carry a Hb
variant, which make hemoglobinopathy the commonest
inherited disease.

Laboratory Methods to evaluate

Hemoglobin
Red cell morphologies:
HbS: Sickle cells
HbC: Target cells, crystals
Thalassemias: Microcystosis, target cells,
basophilic stippling

Methods of hemoglobin identification

Hb electrophoresis

Alkaline
Acid
Capillary
Etc.

HPLC (High performance liquid chromatography)

Gene sequencing or amino acid analysis

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.

Capillary Electrophoresis (SEBIA)

CAPILLARYS 2 System
Automate electrophoresis testing in free
solution
Hemoglobins are seperated by their
electrophoretic mobility in alkaline buffer
Hbs detected at 415nm
Amino acid substitutions in hemoglobin variants alter
net charge and mobility

Capillary Electrophoresis (SEBIA)

+
-

Normal CZE

 Electrophoresis:
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

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

PEAK Names

Retention
times (min)

Peak names

Retention
times (min)

F Window

0.98-1.20

A2 Window

3.30-3.90

P2 Window

1.24-1.40

D Window

3.90-4.30

P3 Window

1.40-1.90

S Window

4.30-4.90

A0 Window

1.90-3.10

C Window

4.90-5.30

INTERPRETATION OF CZE/HPLC RESULTS

Presence of Hb Variant?
Yes, Location? Percentage*? Number of
Variants?
A2 percentage*
F percentage
CBC Indices and RBC morphology*
Age, Clinical Course*
Family history/Ethnic origin
Other Lab tests
Iron/feritin/TIBC

*Changed By Thalassemia

Increased Hb F
>2-80% Babies
90-100% Homozygous Hereditary Persistent Fetal
Hemoglobin, 0, 0
15-40% Heterozygous HPFH
10-25% SS with Hydroxyurea Treatment
3-10% Homozygous Hemoglobinopathies, Anemia,
Leukemia, or Other Malignancy
<5% -Thal, Lepore
Marked elevation in JMML (up to 70%)
Increased in congenital bone marrow failure syndrome

INTERPRETATION OF RESULTS
Hemoglobin A2
Increased

4.0-7.0% -Thalassemia Minor, S + Thal

3.5-4.5% HB AS, AC, SC, SS, CC
6.5-14% Hb Lepore
Also elevated in megaloblastic anemia, hyperthyroidism

Decreased
1.3-1.7% Iron Deficiency, sideroblastic, aplastic anemia
1.5-2.3% chain variant (A2), chain variant

Most common Hemoglobin abnormalities

Thalassemias: Alpha,Beta
Common Hemoglobin Point Mutations
Beta Chain Variants
S (6 Glu Val)
C (6 Glu Lys)
E (26 Glu Lys)
D Los Angeles (22 Glu Gln)
O Arab (121 Glu Lys)
Alpha Chain Variants
G Philadelphia ( 68 AsnLys)
Delta Chain Variants
A2 (16 Gly Arg)

Hereditary Persistence of Fetal Hemoglobin F (HPFH)

Case 1
2 yo male

Case 1

Diagnosis: beta thalassemia minor

Thalassemia
Mutation or Deletion Causing Reduction or
Absence of 1 or More Chains
CBC/smear: microcytosis, target cells,
basophilic stippling
Thalassemia -4 Genes ( / )
Thalassemia -2 Genes ( / )
0- No Production
+- Reduced Production

Delta Thalassemia -2 Genes

Combos i.e. Thalassemia

Case 2
16 months old male

Case 2

Hb S trait

Hb S
Most Common Hemoglobin Disease, 6 Glu Val
Trait (Hb AS):
Most Common Hb Variant in USA, 9% in American Blacks
Asymptomatic, + solubility test
Protective Against Malaria, 30% Africans at Malaria Area

Disease (Hb SS):

Severe Symptoms, Sickling in Vivo

Crises: Bone Pain, Hemolysis, Stroke, etc
Hydroxy Urea Treatment Induce HbF Production
Similar Symptoms: Hb SC, Hb SD, etc

Case 3
12 yo Female

Case 3

Hb C trait

Hb C
2nd most common Hb variant in USA, 2-3% of US
Blacks, 6 Glu Lys
Trait (Hb AC):
Clinically and hematologically Normal
Moderate Target Cells seen

Disease (Hb CC):

Mild Hemolytic Anemia, Splenomegaly

Rod Shaped Crystals in RBCs
Normochromic Normocytic Anemia
Prominent Target Cells

Case 4
14 months old female

Case 4

Hb E trait

Hb E
Found in SE Asia, 26 Glu Lys, mildly unstable
Most Common Hemoglobinopathy Worldwide
Trait (Hb AE)
Asymtomatic, No CBC Abnormalities

Disease (Hb EE)

Mild Anemia, Target Cells, Microcytosis
May have mild splenomegaly
+Beta Thal= Severe Anemia, hemolysis, need transfusion

Case 5
1 yo Male

Case 5

Heterozygous Hb A2

Hb A2
Delta Chain Variant, 16 Gly Arg
Most Common in Blacks (2%)
Asymptomatic and Normal CBC
Little Consequence:
Low A2 in Heterozygous A2
Absent A2 in Homozygous A2
Add A2 to A2 when Interpret -Thal

Case 6
9 months old female

Case 6

Z6
Z4
Z1

Hb G-Philadelphia

+alpha-Thal

Case 6

Hb G-Philadelphia
68 AsnLys
Most Common Chain Variant, in Blacks,
Italians, Chinese
Heterozygous: Normal CBC
Associated -Thal: MCV, Hb G (30%, 45%)

Case 7
10 yo Male HbA1c=50%

Case 7

Case 7
Chain Variant Elute with HbA1C on HPLC

Case 8
11-month old African American female with
Sickle Cell disease presented in ER with acute
chest syndrome
Hb 6.5 g/dL with marked polychromasia, many
sickle cells, moderate schistocytes and target
cells
Patient received two RBC transfusions using
the same donor unit

Pre-transfusion

Post-transfusion

Donors blood

Case 8
Acquired alpha Hb variant

Thank You!