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• N

onsense, 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

HbAA2F

HbD

HbF

HbE

HbS

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

anemia2-15% HPFH

BTT  Sickle10-15% 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/dlMCV = 67.3 FlMCH = 21.3 pgRDW = 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 = 13.4MCV = 68.8MCH = 22.4

ImpressionS/O Delta beta thal trait

HbF

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 ?