MLT 06210

Haemoglobinopathies
SESSION 4

Aljawadi

BMLS
 Learning Objectives

 By the end of this session, students

are expected to be able to:
 Define Haemoglobinopathy.

 List the types Haemoglobinopathies

 To describe thalassaemia

 Describe the etiology, clinical feature, complication and

laboratory diagnosis of HbS, HbC, HbDpunjab and HbE

 Describe principle and procedure of Hb electrophoresis

 Definition of Haemoglobinopathy

 Haemoglobinopathy refers to a disease state

involving the haemoglobin (Hb) molecules.

 Disorders of globin synthesis rather than haem

synthesis

 These are the disorders which results due to

impairment of the globin production during
haemoglobin synthesis
 Haemoglobinopathies

Qualitative Disorders
•Abnormal haemoglobins are formed when the sequence of globin
chain amino acids is altered.

•There is usually only a single amino acid substitution in one of the

globin (polypeptide) chains.

Quantitative Disorders
•Thalassaemias result from a lack of production of particular globin
chains to maintain adequate Hb levels.

•Hereditary persistence of fetal haemoglobin (HPFH) results from a

failure to switch from fetal to adult Hb.
Cont..

 All Haemoglobinopathies result from a genetic

mutation in one or more genes that affect
haemoglobin synthesis.
 The genes that are mutated can code for either the
proteins that make up the haemoglobin molecule
(globin chains) or the proteins involved in
synthesizing or regulating synthesis of the globin
chains.
 Regardless of the mutation encountered, all
Haemoglobinopathies affect haemoglobin synthesis
in one of two ways:
Quantitative haemoglobinopathies

 The disorders of haemoglobin resulted due to decreased

in production of the Alpha and Beta haemoglobin chain

 It is also known as THALASSAEMIA

 Thalassaemia are classified based on:-

 Specific or type of haemoglobin chain affected ( α and β)

 Severity of the disorder in developing of hemolytic anaemia ( Major,

intermedia and Minor)
 Specific or type of globin chain affected

 Normally we inherit four genes which code for synthesis

of alpha haemoglobin chain (αα/αα)
 And also two genes which code for synthesis of Beta
haemoglobin chain (β/β)
 Deletion of one, two, three or all four α genes affects the
rate of production of α haemoglobin chain.
 While any mutation or deletion in β will affect the rate of
production of β haemoglobin chains
Cont…

 Decrease in production of the alpha haemoglobin

chain is known as alpha thalassemia can occur due
to:-
 Deletion of one gene (-α/αα)
 Deletion of two genes (-α/-α) or (--/αα)
 Deletion of three genes (--/-α)
 Deletion of four genes (--/--)
 Decrease in synthesis of Beta haemoglobin chains is
known as Beta thalassemia, occur due to mutation or
by deletion of beta gene
 Severity of the disorder in developing of
hemolytic anaemia

 Thalassemia Major
 Alpha thalassemia major (HbBarts hydrops syndrome):
causes a severe fatal hemolysis of fetus red blood cells. Infants
are stillborn premature or dies soon after birth
 Beta thalassemia major: causes severe hemolytic anaemia
of infants red blood cells, HbF is not replaced by HbA. Infants
needs a regular blood transfusion (blood transfusion
dependent)
Cont….

 Thalassemia intermedia

 Beta thalassemia intermedia: causes a moderate to severe

hemolytic anaemia. Patient require a regular blood transfusion

 Alpha thalassemia intermedia: (HBH) occurs due to

deletion of three of the four alpha gene. Symptoms are the
same as beta thalassemia intermedia
Cont..

 Thalassemia minor:

 Both alpha and beta thalassemia minor are known as

thalassemia trait (carrier)

 Both causes mild anemia or sometimes asymptomatic

 Qualitative Haemoglobin Disorders

 Whereas thalassaemia syndromes are caused by

abnormalities in the rate of production of α and β
haemoglobin chains
 Disorders due to abnormal haemoglobins are caused
by structural changes in the chains
 Abnormal structure of haemoglobin chain affect the
oxygen carrying capacity of haemoglobin
 Also abnormal haemoglobin chain structure makes
red blood cells to be prone to hemolysis
 Common types of abnormal haemoglobin

 Hb S Haemolytic anaemia sickling

 Hb C Mild haemolytic anaemia

 Hb Dpunjab No anaemia

 Hb E Mild microcytic anaemia

 The etiology, clinical features, and
laboratory diagnosis of HbS

 HbS (ß2) is formed when valine replaces glutamic

acid in the ß globin chain (6th amino acid position).

 In the deoxygenated state, HbS has poor solubility

forming polymers (long fibres) in red cells.

 Polymerization leads to changes in the red cell

membrane and metabolism causing the cells to
become rigid and distorted with a sickle shape.
Cont…

 The sickle cells adhere to vascular endothelium,

blocking small blood vessels causing organ damage
or failure such as kidney and other complications.

 They become trapped in the spleen and are

haemolysed easily.
Cont..

 HbAS: When the abnormal ßs gene is inherited from

one parent and normal ß gene from the other parent
(heterozygous state or HbAS), the disorder is
referred to as sickle cell trait (ßs/ß).

 This is mainly an asymptomatic condition with 50%

or more of the person’s Hb being normal HbA.
Under exceptional (hypoxic) circumstances, a sickle
cell crisis can arise in persons with sickle cell trait.
 Clinical features/complications

 Haemolytic anaemia,
 Jaundice,
 Fever,
 Painful swelling of the hands and feet,
 Skeletal changes due to increased erythroid
hyperplasia,
 Painful infarct,
 Pulmonary complications,
 Leg ulcers, etc.
 Laboratory diagnosis

 Hb estimation which is usually ranges between 6.0 –

8.0g/dl (60-80 g/L)
 Sickling test is positive
 FBP shows marked poikilocytosis with sickle cells,
nucleated red cells and target cells. Macrocyte may
present due to folate deficiency, also there is
polychromasia due to high reticulocytes in peripheral
blood.
 Hb solubility test becomes Positive (shows opacity)
 Hb electrophoresis shows Hb S
Sickle cells
 The etiology, clinical features, and
laboratory diagnosis of HbC

 Hb C is defined by the structural formula α2ß26 glu-

Lys, in which lysine substitutes glutamic acid in the
sixth position of the beta chain.
 HbC is inherited in the same manner as HbS but
manifest as the milder disease. Similar to HbS, HbC
polymerizes under low oxygen tension, but the
structure of the polymers differs.
 HbS polymers are long and thin whereas the
polymers in HbC are short and do not alter RBCs
shape to the extent that HbS does.
 Clinical features

 The condition can cause symptoms similar to but

less severe than sickle cell anaemia.

 Splenomegally is common than in HbS, eye disease

is common.

 In some patient HbSC disease causes only a mild

haemolytic anaemia. Crisis can arise during
pregnancy.
 Laboratory diagnosis

 In HbC disease the blood film shows anisocytosis,

and poikilocytosis with small dense cells, many
target cells, and occasionally sickle cells and cells
containing HbC crystals.
 HbC yields a negative result on the haemoglobin
solubility test, and definitive diagnosis is made using
electrophoresis or HPLC. No HbA is present in
HbCC disease.
 HbS and HbC
COOH 146 2 2 6GluVal = Hb S
lys
val
glu

6 glu

pro

thr
lys
2 2 6GluLys = Hb C
leu
 chain
his

1 val

NH2
 The etiology, clinical features, and
laboratory diagnosis of Hb Dpunjab

 Formed when Glutamine replaces Glutamic acid at

position 121 of the β –globin chain (β121)

 HbD Punjab is important clinically because of its

interaction with HbS.

 In HbSD Punjab disease there is moderate to severe

haemolytic anaemia and the blood film is similar to
that seen in sickle cell anaemia.
 Cont…

 Sickle cell test are positive (solubility test result

similar to that seen with sickle cell trait).

 On alkaline electrophoresis, HbD Punjab (and other

forms of HbD) has the same mobility as HbS.
 Clinical features

 HbD and HbC are asymptomatic in the heterozygous

state.

 HbD disease (Hb DD) is marked by mild haemolytic

anaemia and chronic non-progressive splenomegaly.
No treatment is required.
 The etiology, clinical feature, and
laboratory diagnosis of HbE

 Formed when Lysine replaces Glutamic acid at

position 26 of the β-globin chain
 Homozygous inheritance of Hb E causes only mild
anemia.
 The blood film shows microcytosis and hypochromia
with target cells.
 Haemoglobin E can be detected with alkaline and
acid haemoglobin electrophoresis methods.
 Hb electrophoresis Procedure

 Haemoglobin electrophoresis is a blood test

that can detect different types of haemoglobin.
 It uses the principles of gel electrophoresis to
separate out the various types of haemoglobin in an
acid or alkaline medium within electric field.
 The test can detect abnormal levels of HbS, the form
associated with sickle-cell disease, as well as other
abnormal haemoglobin-related blood disorders, such
as haemoglobin C
 Purpose of Haemoglobin electrophoresis

 Haemoglobin electrophoresis is used to separate and

identify the different haemoglobins by their
migration within an electric field.
 Haemoglobin variants separate at different rates due
to differences in their surface electrical charge as
determined by their amino acid structure.
 Haemoglobin electrophoresis at pH 8.4–8.6 using
cellulose acetate membrane is simple, reliable, and
rapid. It is satisfactory for the detection of most
common clinically important haemoglobin variants.
 Principle of Electrophoresis:

 Haemoglobin types are separated based on electric

charge in an electrical field because they are amphoteric
like other proteins.
 In an alkaline buffer haemoglobins become negatively
charged ions and migrate toward the anode (positive
terminal) while in acid buffer particles migrate toward
the cathode.
 Due to differences in charge, size and weight each
haemoglobin migrates to a different spot on the
electrophoresis medium and when the voltage is stopped,
they can be visualized with densitometry and
quantitated.
 Cont…

 The speed of each haemoglobin migration depends

largely on the degree of ionization of the protein at
the pH of the buffer system, the voltage strength, size
& shape of molecule, temperature, and buffer
characteristics.
 Haemoglobin A migrates faster and closer to the
anode, followed by F, then S if present, and then A2
or C if present compared to the origin of the sample
placement on the electrophoresis medium.
Migration of different haemoglobin
 Key Points

 Haemoglobinopathy refers to a disease state involving

the haemoglobin (Hb) molecules. All
Haemoglobinopathies result from a genetic mutation in
one or more genes that involve haemoglobin synthesis.
They can be qualitative or quantitative.

The variants that are most common are,

 Hb S - haemolytic anaemia sickling
 Hb C - mild haemolytic anaemia
 Hb Dpunjab - no anaemia
 Hb E mild microcytic anaemia
 Key points cont…

 Haemoglobin electrophoresis is used to separate and

identify the different haemoglobins by their
migration within an electric field.
 Migration of the haemoglobin molecules within the
electrical field is determined by
 Degree of ionization of the protein at the pH of the buffer
system,
 The voltage strength,
 Size & shape of molecule,
 Temperature
 Buffer characteristics
END