Hemoglobin Electrophoresis
Dr Nisha S AhmadChief of Lab ServicesMetropolis Health Services
Agenda
• Brief overview of hemoglobin• The globin genes• The Thalassemias• Structural hemoglobinopathies• Testing
Hemoglobin
• 4 Heme groups • 4 polypeptide chains
B
B
A
A
heme
Hemoglobin structure
A
A
A
A A
A
B G D
B DG
HbA HbF HbA2
95-98% ~1% <3.5%
Hemoglobins in normal adults
The Globin Genes
Hemoglobin Type
Name Components
Adult A 22
A2 22
Fetal F 22
Embryonic Portland 22
Gower 1 22
Gower 2 22
Abnormal H 4
Bart’s 4
Hemoglobin
Types Quantity Alpha Thal
HbA (α2 β2) >95% HbH
HbA2 (α2 δ2) <3.5% -
HbF (α2 γ2) <2.0% Hb Barts
An inherited mutation of the globin genes leading to a quantitative or qualitative abnormality of globin synthesis
Hemoglobinopathy
Thalassemia - Defined
• A family of genetic anemias characterized by a reduced rate of production of 1 or more globin subunits of hemoglobin (Hb)
• Symptoms are caused by the deleterious effects of the normally produced subunits that are now in excess
Pathophysiology
• Excess alpha chains precipitate and form inclusion bodies that associate with the RBC cell membrane
• Cause membrane damage and shortened cell survival
• Large scale destruction of precursor cells in bone marrow
• Decreased B production causes increased δ production and an elevated A2 (α2δ2)
Types of B-globin mutations
• B0 – No B-globin chains are produced
• B+ - some beta chains produced Decreased
Alpha thalassemia
AA / AA Normal
AA / A - Mild microcytosis (Silent Carrier)
AA / - -A - / A -
Mild microcytosis (Trait or Carrier)(cis vs trans)
A - / - - Hemoglobin H disease-clinically variable
- - / - - Hydrops Fetalis (Alpha Thal Major)
α- and β-thalassemia
Alpha Thalassemia• Deletions of alpha- globin gene (s)• Symptoms can begin in fetal life• Complicated inheritance – 4 alpha genes
Beta Thalassemia• Nonsense, splice and frameshift mutations in beta-globin
gene• Symptoms begin in infancy/childhood• Simple AR inheritance; genotype-phenotype correlation
Structural variant - Defined
• Abnormal globin protein that is produced at a normal rate, with varying consequences
• Oxygen affinity, stability and function
Normal
Normal Thalassemia
Normal Structural Variant
Laboratory Investigation
CBC-MCV,MCH,RDW Tests Hemoglobin electrophoresis
• Cellulose acetate: Alkaline pH• Citrate agar: Acid pH• Capillary Electrophoresis
HPLCIEFDNA
Preanalytical
• EDTA sample• Age• H/O Transfusion• Area of Residence E/D/C
SCREENING
ANEMIA PROFILE
ANTENATAL PROFILE
PRE MARITAL NEONATAL PROFILE
HbA1c
Manual System
Cellulose Acetate
• pH-8.6• In a alkaline solution ,Hb molecules have a
net negative charge and move towards the anode
Cellulose Acetate Hb Electrophoresis
- A2 F A +
C/E/O S/D/G
BTT
NORMAL
Cellulose Acetate Hb Electrophoresis
- A2/C S F A+
Normal
Hb AS
Hb SS
Cellulose Acetate Hb Electrophoresis
- A2/C/E S /D F A+
Normal
Hb AS
HB AD
Citrate Agar Electrophoresis
• pH-6.4• Citrate agar electrophoresis at an acid Ph
provides ready separation of hemoglobins that migrate together on cellulose acetate
• S from D and G• C from E and O
Citrate Agar Hb Electrophoresis
+ C S A F _
Normal
Citrate Agar Hb Electrophoresis
+ C S A F _
Normal
Sickle trait
HPLC
° Separation based on interaction between Stationary Phase & Mobile Phase
° Stationary Phase is Analytical Cartridge; Mobile Phase is Buffer
° Compounds are separated to target analytes according to physical properties: - size, shape, charge, hydrophobicity & affinity for
other molecules° Bound analytes elute off the stationary phase by
manipulating the mobile phase
Contd…
• Desorption is then brought about by increasing the salt concentration or by altering the pH of the mobile phase
• Charged particles (matrix) bind reversibly to sample molecules (proteins, etc.)
HPLC Automated system;precalibrated column and gradient
Stationary Phase: Cation Exchange Cartridge
Carboxyl groups attached to a resin base
Direction of flow Detector
Hemoglobin Introduction
Positively charged hemoglobin fragments in the hemolysate attach to the carboxyl groups at varying strengths.
Starting Gradient: Low Ionic Strength Buffer
• The gradient starts with a low % of Buffer B (high % Buffer A)
• At this gradient, hemoglobin fragments with an ionic strength lower than the buffer gradient, such as A and F, are displaced from the cartridge and pass into the detector
Ending Gradient: High Ionic Strength Buffer
• As the % of the High Ionic Strength Buffer B increases, the more hemoglobin fragments will be displaced
• Once the gradient is 100% Buffer B all remaining hemoglobin fragments, including any variant hemoglobins such as S, D and C, will be removed
CHROMATOGRAMS
Output
Time
Peak
RT
Area
Normal HPLC Graph
Hb O
Hb C
Hb H
Hb J
Hb Köln
Hb Q
A2
A2 Suggests
<3.5% Normal
3.5-8.0% BTT /(Megaloblastic anemia)
11-15% Hb Lepore
20-40% HbE
30-48% HbD
Hb Lepore
HbE
HbD
Hb EE/DD
HbFF Suggests
<2% Normal
<5%HPFH/Pregnancy/ Aplastic
anemia
2-15% HPFH
BTT
Sickle
10-15% Delta Beta Thalassemia
>50% Beta Thal Major
HPFH (homozygous)
Delta Beta Thalassemia (homo)
Hb F
1.Delta beta Thal Trait
2.HPFH+IDA
1.Iron Studies2.DNA Analysis
HPLC GRAPH OF CHILD
Hb F= 100%
Hb A2= 0%
HbA0 =0%
CBC:
Hb = 6.8 g/dl
MCV = 67.3 Fl
MCH = 21.3 pg
RDW = 24.7%
HPLC GRAPH MOTHER
Hb F = 7.0%HbA2 = 2.6%HbA0 = 90.4%
CBC:Hb = 11.9MCV = 64.7MCH = 21.1
Impression:
S/o Delta beta thal trait
HPLC GRAPH FATHER
HbF = 8.0%
HbA2= 2.8%
HbA0 = 89.2%
CBC: Hb = 13.4
MCV = 68.8
MCH = 22.4
Impression
S/O Delta beta thal trait
Low A2
• Iron Deficiency Anemia• Alpha Thalassemia trait• HbH Disease• Delta Thalassemia• Delta –Beta Thalassemia
Peak before 1 min
• Sample Integrity• Icteric sample• HbH
HbHAge-8 yrs
Hb-7.8g/dl
MCV-61 fl
MCH-17.8pg
HbH
Extended Retic stain
HbH inclusions
Capillary Electrophoresis
Advantages• HbH • HbE vs HbD
Disadvantages• Preanalyticals (Cap piercing model)• Interpretation
Limitations….
• Silent carriers• Alpha Thalassemia• DNA analysis-gold standard
DNA analysis
• Silent carriers• Prenatal period• 5 common mutations• 23 Mutations
Conclusion…..
• Clinical Suspicion• CBC• PBS• Reticulocyte count• Specific tests• Electrophoresis• HPLC• Capillary Electrophoresis• DNA analysis-Pre natal/gold standard
Questions ?