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Hemoglobin
Sarah Walter, M.D.
Electrophoresis
Normal Hemoglobin
Structure
Hemoglobin
A is a tetramer
composed of 4 subunits:
2 and 2
Each
Normal Hemoglobin
Structure
Hemoglobin tetramer
Normal Hemoglobin
Structure
O
O
Fe
Porphyrin ring
O2 binding site
Hemoglobin Function
The
Hemoglobin Function
Normal Hemoglobin
Function
Normal Hemoglobin
Function
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
A2
2 2
2.5%
A1C
3%
2 (-Nglucose)
2 2
Gower-1
2 2
0*
Gower-2
2 2
0*
92%
<1%
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
Abnormal Hemoglobin
Reasons
disorder:
to suspect a hemoglobin
Laboratory Methods to
evaluate Hemoglobin
Red
cell morphologies:
Laboratory Methods to
evaluate Hemoglobin
Red
cell morphologies:
Laboratory Methods to
evaluate Hemoglobin
Red
cell morphologies:
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.
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.
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.
Alpha
Beta
Hemoglobinopathies
Case 1
Labs:
Hgb: 10.2
Hct: 30.9
MCV: 96.4
B12: 338
Iron: 122
Ferritin: 304.5
IBC: 226
Case 1
HbF: 1.3%
HbA2: 4.1%
Case 1
Case 1
Hemoglobin
S/C disease:
Case 1
Hemoglobin
S/C disease:
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
Case 2
Case 2
Case 3
Case 3
Case 3
Case 3
Sickle
cell anemia:
Case 3
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
5-10
20-30
70-80 2-3
Case 4
Case 4
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
THE END!!!