HPLC study for hemoglobin

Introductory lecture
REASONS FOR HEMOGLOBIN ID AND
QUANTIFICATION

Newborn Screening
Prenatal Screening
Followup Newborn Screening
Diagnosing Causes of Microcytosis
Anemia, Polycythemia, Chronic Hemolysis
Hemoglobinopathy Blood Replacement
Unexplained A1c results

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
Fetal Hb
Other Hemoglobins in normal adults
Hemoglobin Structure %
A
2

2
92%
A
2

2

2
2.5%
A
1C

2
(-N-glucose) 3%
F
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
Over 1000 hemoglobin variants are current known.
Worldwide, estimated over 150 million people carry a hb
variant, which make hemoglobinopathy the commonest
inherited disease.
Hemoglobinopathies
1. Decreased production: Thalassemias
2. Structurally abnormal (4 groups):
Affecting function: HbS
Unstable Hb: Torino, Zurich, Hashron (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.


Method of hemoglobin identification
Hb electrophoresis
HPLC (High performance liquid chromatography)
Gene sequencing

HPLC:
Cellulose acetate pH 8.5
Cellulose Acetate pH 8.6
Hemoglobin Electrophoresis Patterns
C-Harlem

Citrate Agar pH 6.0
Hemoglobin Electrophoresis Patterns
C-Harlem
Claus Santa Obese And Fat
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
COMMON HEMOGLOBIN POINT MUTATIONS

Alpha Chain Variants
G Philadelphia ( 68 AsnLys)
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)
Delta Chain Variants
A2 (16 Gly Arg)

INTERPRETATION OF HPLC RESULTS

Hemoglobin Retention Time
Variant Hemoglobin Percentage*
A2 percentage*
Number of Variants*
CBC Indices*
Age
Clinical Course*
*Changed By Thalassemia
Alla Joutovsky,1 Joan Hadzi-Nesic,1 and Michael A. Nardi2*
BIO-RAD Variant Windows
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 the results
#Abnorml
peaks (%)
A% A2% Variants Examples
1 (25-40) 50-60 3.5-4.5 -chain AS, AC
2(25,1.0) 70-80 1.5-2.2* -chain

AG-phil
2 (50,45) 0 3.5-4.5 2 -chains

SC
3
(12,20,14)
40-50 2.0* 1-, 1-,
1-
chains

ASG-Phil
Normal Hb HPLC Result
Marked elevation in juvenile CML (up to 70%)
Increased in congenital bone marrow failure syndrome

HEMOGLOBIN S
S Trait (HbAS)
Common in Blacks; Other Populations
Asymptomatic, Blood Sickles in Vitro
Protective Against Malaria
S Disease (HbSS)
Severe symptoms, Sickling in Vivo
Hydroxy Urea Treatment- induce F
Crises- Bone pain, Hemolysis, stroke, etc
Similar symptoms, Other Double heterozygotes (SC)

HEMOGLOBIN E
Found in SE Asia
Most common Hemoglobinopathy Worldwide
Complicated by Iron Def, Thalassemia, A2 elution
Trait (HbAE)
Asymptomatic, No CBC Abnormalities
Disease (HbEE)
Mild anemia, Target cells, RBC survival
Osmotic Fragility
+beta Thal=severe, as homozygous b-tha
+alpha Thal= HbE

INTERPRETATION OF RESULTS
Hemoglobin A2
Increased
4.0-7.0% b-Thalassemia, SB+ 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


Very useful website: http://globin.cse.psu.edu/