Hb electrophoresis- Types, Procedure and Analysis

Hb Electrophoresis
Dr. Reenaz Shaik
MD Pathology

CONTENTS
 Definition and types of electrophoresis
 Difference between zone and moving boundary
electrophoresis
 Hb Electrophoresis: Reagents, Materials,
Methods and Procedure
 Applications
 Normal and Abnormal Haemoglobins
 Understanding the bands
 Approach
 References
CONTENTS

 Electrophoresis is the separation of charged
compounds based on their electrical charge.
 An electrophoretic system consists of two electrodes
of opposite charge (anode, cathode), connected by a
conducting medium called an electrolyte.
 The separation effect on the ionic particles results
from differences in their velocity (v), which is the
product of the particle's mobility (m) and the field
strength (E).
DEFINITION
V=mE

USES
Biological molecules such as
Amino acids
Peptides
Proteins
Nucleotides
Nucleic acid
Inorganic material can be separated
using this technique.

Classification of electrophoresis
1. ZONE ELECTROPHORESIS:
 Paper electrophoresis
 Gel electrophoresis
 Thin layer electrophoresis
 Cellulose acetate electrophoresis
2. MOVING BOUNDARY ELECTROPHORESIS:
 Capillary electrophoresis
 Isotachophoresis
 Isoelectric Focussing
 Immune electrophoresis
TYPES

DIFFERENC
ES
 Zone electrophoresis: Migration of
charged molecules in a solution with the
supporting medium. The separated
components are formed like discrete zones
in the supporting medium.
 Moving boundary electrophoresis:
Migration of charged molecules in a free
moving solution, without the presence of a
supporting medium.

HB
ELECTROPHOR
ESIS
 Screening test to identify different variant
and abnormal haemoglobins.
 It helps in diagnosis of diseases caused by
abnormal haemoglobins like thalassemia,
sickle cell anemia, polycythemia rubra
vera.
 Helps in monitoring treatment and screen
for genetic disorders.

PRINCIPLE
 It uses the principle of Gel electrophoresis.
 Different haemoglobin have different
charges and according to those charges
and the amount of haemoglobin, different
chains move at different speed in gel are
separated.

HEMOGLOBIN STRUCTURE
 Electrophoresis
buffer(Tris/EDTA/borate(TEB), pH 8.5)
 Wetting agents (e.g., Zip zone Prep
solution)
 Fixative stain/solution(Ponceau S 5 g)
 Haemolysing reagents(0.5% Triton in 100
mg potassium cyanide)
 De-staining solution (3% acetic acid)
REAGENT

 Specimen : Blood
Packed red cells are preferred ; if whole
blood used paraprotein or high concentration
of polyclonal Ig may produce a band.
MATERIAL

 Cellulose acetate (CA) electrophoresis
 Alkaline electrophoresis
 Citrate Agar electrophoresis
 Alkaline and Citrate Agar electrophoresis
are commonly used methods
METHODS

STEPS
FIRST STEP: Cellulose acetate at alkaline
pH (8.4) done as an initial procedure. •
Separation is largely determined by electrical
charge. • At this pH, Hb is negatively
charged and moves toward the positively
charged anode.
SECOND STEP: Citrate agar or agarose gel
at acid pH (6)

 Sample Collection(blood)
 Centrifuge sample at 1200 g for 5 mins.
 Prepare the electrophoresis tank with TEB
buffer
 Soak the cellulose acetate into buffer for 5
mins
 Fill the sample well plate with 5 μl of each
diluted sample and cover with glass slide to
prevent evaporation
 Load a second sample well plate with Zip
Zone Prep solution
PROCEDURE

 Then Apply them to a blotter
 Blot the cellulose acetate strip twice
between two layers of clean blotting paper
 Do not allow the cellulose acetate to dry
 Load the applicator by depressing the tips
into the sample wells twice
 Place the cellulose acetate plates across
the bridges
 After 25 mins of electrophoresis
immediately transfer the cellulose acetate
to ponceau S and fix and stain for 5 mins
PROCEDURE
Cont..

 Remove excess stain by washing for 5 min in the
first acetic acid reservoir
 Label the membrane and store in protective
envelope
PROCEDURE
Cont..

 Hematoma
 Bleeding
 Infection at the puncture site
 Fainting or feeling lightheaded
 Swelling(also called phlebitis)
RISKS

Hb electrophoresis- Types, Procedure and Analysis

ABNORMAL
Hb’s RESULT
Abnormal Hb Manifesting Disease
High Hb A2 and Hb F Mild form of Thalassemia
High Hb F Hereditary Persistence of Foetal
Haemoglobin
High Hb S Sickle Cell Disease
High Hb C Anemia and enlarged spleen
High Hb E Anemia and small RBC’s
Hb DD, Hb SD Sickle disorder
Hb CC, Hb SC, Hb SS Mild, Moderate, Severe SCD
Hb H • Moderate to severe anemia,
jaundice, splenomegaly
• Blood: microcytosis,
hypochromia, target cells,
polychromasia
Hb Lepore- Boston Mild Thalassemia minor
Hb Koln Mild congenital hemolytic
anemia (AD, maybe mistaken for
hereditary spherocytosis) •
Hypochromia, macrocytosis •
Broad smudge in the S position

 Reasons you may not be able to have the
test or why the results may not be helpful
include:
 Having a blood transfusion in the last 3
months
 Having Iron deficiency anemia. This can
cause falsely low results for
haemoglobin A2
WHAT
AFFECTS
THE TEST?

 Evaluation of unexplained haemolytic
anemia
 Microcytic anemia unrelated to iron
deficiency, chronic disease, or lead toxicity
 A peripheral smear with abnormal red cell
features
 Positive family history of
haemoglobinopathy
 Positive neonatal screen results
 Positive results on sickle cell or solubility
test
APPLICATIONS

 There are different forms of normal
(e.g. HbA, Hb F, Hb A2)
 Abnormal (e.g. Hb S, Hb C, Hb D, Hb
G) hemoglobins.
HAEMOGLOBINS

NORMALHB VARIANTS
Hb
STRUCTUR
E

Proportions of different hemoglobins
% OF
Hb’s

HEMOGLOBINOPATH
YAPROACH
This list shows some of the commoner tests
used to investigate the hemoglobinopathies.
 Blood count
 Haemoglobin electrophoresis: Cellulose
acetate pH 8.4, Citrate agar pH 6
 Solubility tests
 Quantitation: Hb A2, Hb F, Hb Barts
 Tests for unstable hemoglobins
 Gene analysis

REFERENCES
 Chapter 49: Disorders of Hemoglobin Structure : Sickle Cell Anemia and
Related Abnormalities & chapter Part VI: The Erythrocyte-WILLIAMS
HEMATOLOGY.
 Dacie 12th edition, Investigation of variant Hemoglobins &Thalassemias
ch-14.
 Kawthalkar Clinical PATHOLOGY
 Alain J. Marengo-Rowe, MD, Structure-function relations of human
hemoglobins, Proc (Bayl Univ Med Cent). 2006 Jul; 19(3): 239–245.
 http://hemepathreview.com/Heme-Review/HemoglobinElectrophoresis.pdf
REFERENCE
S

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