A Brief Overview of

Hemoglobin
Sarah Walter, M.D.
Electrophoresis

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

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
Hemoglobi Structure
n
A
2 2

A2

2 2

2.5%

A1C

3%

2 (-Nglucose)
2 2

Gower-1

2 2

0*

Gower-2

2 2

0*

92%

<1%

* Indicates early embryonic

adults
Portland
2 2 form not seen in 0*

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 globin
to switch to globin.

Abnormal Hemoglobin
Reasons

disorder:

to suspect a hemoglobin

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: 1013) 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 HbA 2
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 Labs:

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

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
S-alpha-thalassemia
If HbS is greater than 50%, worry about SBeta-thalassemia 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 Betathalassemia, repeat HPLC after four months of
most recent transfusion

Case 3
Expected ratios
Hb AS
Hb SS
Hb S--thal
Hb S- thal
major
Hb S- thal
minor
Hb S HPFH

HbA

HbS

HbA2 HbF

5560
0
75
0

40-45 2-3

<1

90-95 2-3
25
2-3
90-95 Inc.

5-10
<1
5-10

5-30 60-90 Inc.

5-10

20-30

70-80 2-3

Case 4

31 year old healthy Labs:

female, pregnant
Hgb: 15.0
with moderate
Hct: 42.5
target cells
MCV: 87.8
detected on routine
MCH: 31.0
peripheral smear
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!!!