Haemolytic anaemias and haemoglobinopathies

Dr Mandeep Marwah
Lecture 5.2
Welcome everyone

• Learning objectives:

1. Classify haemolytic anaemias into acquired and inherited types,

Explain the underlying causes for each

2. Explain the haematological abnormalities of patients with haemoglobinopathies

3. Explain the important clinical features and complications of these disorders.
 Haemolytic anaemias
Inherited
(Defective gene)
• Glycolysis defect (Pyruvate kinase
deficiency limit ATP production)
• Pentose- P Pathways (G6PDH deficiency
leads to oxidative damage)
• Membrane protein
(e.g. hereditary spherocytosis)
• Haemoglobin defect
(Sickle cell anaemia)
Acquired
(Damage to cells)
• Microangiopathic haemolytic anaemia (MAHA)
• Antibody damage (Autoimmune haemolytic
anaemia)
• Oxidant damage (Exposure to chemicals
and oxidants)
• Heat damage (e.g. severe burns)
• Enzymatic damage (e.g. snake venom)
How is haemolytic anaemia diagnosed?

Two main principles:

• Confirm that it is haemolysis, acute reaction or chronic disease

• Determine aetiology
• Def: Haemolytic anaemia results from the abnormal breakdown (haemolysis) of red blood cells

• Origin: in blood vessels (intravascular haemolysis)

in the spleen (extravascular haemolysis) Inherited or

acquired
• Erythrocytes normally have a lifespan of ~120 days
• In haemolysis the average lifespan of the erythrocyte reduces to around 20-30 days
• The bone marrow only has a capacity to increase red cell production by around 5 to 6
fold
• Increased production by the bone marrow is unable to compensate and anaemia will
occur symptoms from relatively harmless to life-threatening

• Symptoms: same like any anaemia (e.g. fatigue, shortness of breath) and from
haemolysis increase bilirubin jaundice gall bladder stones
Glycolysis defect

Pyruvate kinase deficiency (limit ATP production) is an inherited metabolic disorder (typically autosomal
recessive but there is also a dominant form) due to:
• mutations in the PKLR gene

• Four pyruvate kinase isoenzymes, two of which are encoded by PKLR (isoenzymes L and R
expressed in liver and erythrocytes, respectively)

• Since red blood cells lack mitochondria, pyruvate kinase deficiency inhibits their only
metabolic pathway which can supply ATP for cellular processes.
• Patients with severe deficiency may require regular blood transfusion.
Pentose- P Pathways

G6PDH deficiency leads to oxidative damage

• Glucose-6-phosphate dehydrogenase (G6PDH) is an X- linked recessive inborn error of metabolism,
risk of haemolytic anaemia

• G6PDH is the rate limiting enzyme of the pentose phosphate pathway which supplies reducing
energy by maintaining NADPH levels.

• NADPH drives numerous anabolic reactions and is required to protect against oxidative stress
by maintaining the level of reduced glutathione
• The pentose phosphate pathway is the only source of reduced glutathione in red blood cells,
• Hereditary spherocytosis is an inherited autosomal dominant
disease
• resulting in abnormalities in erythrocyte membrane proteins
• Impede the ability of the cell to change shape
• Causes: Mutations in the genes coding for 4 different proteins necessary to maintain RBC
normal shape

Spectrin Ankirin Band 3 Protein 4.2

Local disconnection of the cytoskeleton and membrane

reduction in membrane surface area and production of a “spherocyte” shape
to the red blood cell lysed by spleen.
Treatment: splenectomy (partial or total)
Mechanism of spherocytosis
• Characteristic symptoms of HS are the destruction of red blood cells in the
spleen and their removal from the blood stream (haemolytic anaemia), a yellow
tone to the skin (jaundice), and an enlarged spleen (splenomegaly) and gall
stone development.
Haemolytic inherited anaemias
Case scenario:
• A 24 year old Caucasian man was referred to hospital by her GP.
Spleen
• Symptoms:
enlarged
- tiredness and abdominal pain after eating a meal
• Signs: R U Q LUQ
He had a left upper quadrant abdominal mass
Yellowish discolouration of the eyes
Spleen was found to be enlarged RLQ
(by 6cm below the left costal margin) LLQ
- His liver was not palpably enlarged.
A sample of blood sent to the haematology laboratory gave the
following data:
Measurement Patient Reference Range

Haemoglobin 100 g/L 115-165 g/L

Haematocrit 0.31 L/L 0.37 – 0.47 L/L

White blood cell 6.2 x10(9) /L 4.0 - 11.0 x10(9) /L

count
Platelet count 130 x10 (9) /L 140 - 400x10 (9) /L

Mean cell volume 82 fL 80 - 99 fL

Mean cell 30 pg 27.0 – 32.0 pg

haemoglobin
Reticulocytes 150 x10 (9) /L 20 – 80 x109 /L
Question
RBC lack mitochondria therefore _____ ______
deficiency inhibits their only metabolic pathway which
can supply ATP for cellular processes.
Haemoglobinopathies
Normal Haemoglobin

The haemoglobin molecule consists of a

tetramer of:
• four globin polypeptide chains
• two alpha chains and two non-alpha chains
(β, δ or γ)
• held together by noncovalent interactions
with each globin chain complexed with an
oxygen binding haem group
The globin chain compositions of the 3 main normal types of
haemoglobin present in adults
Proportion of total
Haemoglobin type Globin chains haemoglobin in
adults

A 2α/2β >95%

A2 2 α /2δ 2 - 3.5%

F 2 α /2γ < 1%
Haemoglobinopaties

Haemoglobinopathies are inherited disorders where expression of one or more of the

globin chains of haemoglobin is abnormal
There are two main categories:
1. Abnormal haemoglobin variants results:

– from mutations in the genes for α or β globin chains that alter the stability and/or
function of haemoglobin (e. g. Sickle cell disease)

2. Thalassaemias: result from reduced or absent expression of normal α or β-globin

chains. This leads to a reduced level of haemoglobin rather than the presence of an
abnormal haemoglobin
Sickle cell disease:

Most common Hb variant of clinical significance is

haemoglobin S (HbS)

• The HbS variant has an uncharged valine instead of a

charged glutamic acid at position 6 of β-globin

• More prone to polymerise at low oxygen tension.

Leads to formation of long twisted haemoglobin

polymers causing deformation the red blood cell
membrane leading to the sickle shape

• After repeated episodes of sickling, damage occurs to the

red cell membrane causing it to lose elasticity
• Damaged cells fail to return to a normal shape when
normal oxygen tension is restored
Two HbS types variant

• Hb S variant is found mainly in people of:

– Black African descent

– Arab
– Mediterranean and
– South Asia population

• Two HbS types:

1. Heterozygous individuals for HbS have some resistance to malaria

2. Homozygous individual develop sickle cell disease

– Combinations with other haemoglobinopathies produce sickling syndromes of variable
severity such as: sickle-β-thalassaemia, HbS/C or HbS/E.
Symptoms of Sickle Cell Disease

• Severe pain is a first-hand symptom of this

disease
• There are four patterns of the acute sickle cell
disease crisis:

1. Bone crisis

2. Acute chest syndrome

3. Joint crisis

4. Impaired organs

– (e.g. lungs, eyes, kidneys, genitals, liver,

spleen, heart attacks can even occur)
Sickle cell crisis treatment
1. Opioid pain medication such as morphine

2. Antibiotics for infection

3. Anti inflammatory medicines such as ibuprofen

4. Oxygen

5. Intravenous or oral fluids

6. An exchange transfusion:
– conducted with a special machine
– with the help of which the abnormal sickle red blood cells
are removed and replaced with normal ones
7. Hydroxyurea can decrease the frequency and severity of crisis

8. Haematopoietic stem cell transplantation is the only cure (rarely

performed – hard to find donor with sufficient genetic match)
Thalassaemias

Thalassaemias (blood disease from sea)

• result from reduced or absent expression of normal α or β- globin chains.

• This leads to a reduced level of haemoglobin rather than the presence of an abnormal haemoglobin
β Thalassaemia

• β-thalassaemia results from mutation in one or both of the β globin genes

• Leads to a reduction in the amount or total absence of the β globin polypeptide
chain
• In heterozygous individuals where only one of the two β globin genes are
mutated:
– the rate of β globin production is reduced resulting in microcytosis
• Hb level normal (reduced only in pregnancy or infections)
β-thalassaemia major

• In Homozygous individuals both β globin genes are mutated, is a life-

threatening condition called β- thalassaemia major.
• Synthesis of the β globin polypeptide chain is totally absent in such individuals
• Dependent on blood transfusions from the first few months of life onwards in
order to survive since the synthesis of haemoglobin A cannot replace
Haemoglobin F due to the lack of β globin.
α Thalassaemia

• α-thalassaemia results from deletion or loss of function of one or more of the four α globin genes

• The severity of the condition depends on how many genes are malfunctional ranging from:
– mild microcytosis when one or two genes are affected
– to death in utero where malfunction of all 4 genes leads to a complete absence of the α globin
chain
• Haemoglobin H disease: Lack of function in 3 of the 4 α globin genes: severe microcytosis, anaemia,
haemolysis and splenomegaly
Severity of thalassemia

Different combinations of α and β globin mutations in the same individuals lead to a wide range of
thalassaemia phenotypes (differences in the severity of disease):

1. Patients can either be transfusion dependent (thalassaemia major),

2. require transfusion intermittently (thalassaemia intermedia) or

3. require no transfusion (thalassaemia minor).

• NB: In view of the potential severity of disease, it is important that couples at risk are offered
screening and counselling
Question
Thalassaemias have a _____ level of haemoglobin
Acquired anaemia
Acquired haemolytic anaemia

• Microangiopathic haemolytic anaemia (MAHA)

• Antibody damage (Autoimmune haemolytic anaemia)

• Oxidant damage (Exposure to chemicals and oxidants)

• Heat damage (e.g. severe burns)

• Enzymatic damage (e.g. snake venom)

1. Microangiopathic haemolytic anaemias (MAHA):

• Group of acquired haemolytic anaemias where red cells are damaged by physical trauma.

• Often trauma results from red cells getting snagged as they try to pass through small vessels laden
with fibrin strands because of increased activation of coagulation

• Disseminated intravascular coagulation: a condition where bleeding and clotting occur at the same
time in the patient (e.g. in malignancy, obstetric complications, trauma, sepsis, haemolytic uremic
syndrome (HUS)in children) can cause this
• Thrombotic thrombocytopenic purpura - (microangiopathic haemolytic anaemia) a syndrome where
small thrombi form within the microvasculature.
• Red blood cells can also be damaged by the shear stress
produced by a defective heart valve such as in aortic valve
stenosis

The red blood cell fragments resulting from mechanical

damage are called schistocytes (blue arrows in diagram)
2. Antibody damage
(Autoimmune haemolytic anaemia)

• Immune haemolytic anaemias can result from:

– Infections
– lymphoproliferative disorders such as leukaemia or lymphoma
– Reactions to drugs such as cephalosporins (class of antibiotic)

• 2 types:
• 1. In warm autoimmune haemolytic anaemia IgG antibodies recognise epitopes on the
red cell membrane. This leads to macrophages in the spleen recognising these red
blood cell, destroy RBC or membrane spherocytes splenomegaly
2. In cold autoimmune haemolytic anaemia IgM autoantibodies recognise red cell
epitopes and there is also complement fixed to the patient’s red cells leading to
membrane instability and lysis:

– numb fingertips, earlobes etc. (peripheral body parts)

– pallor (paleness),
– blue discoloration or
– in extreme cases gangrene

Cold agglutinin autoimmune anaemia

Cold autoimmune haemolytic anaemia
The direct Coombs test
• Detect antibodies or complement bound to the surface of red blood cells.
• The patient's red cells are mixed with anti-human globulin antibody
• If the red cells are coated with antibodies the anti-human globulin will attach to those
antibodies making the red cells clump together suggesting the patient's haemolytic
Autoimmune haemolytic anaemia

Warm Ab Cold Ab Mixed Ab

lg 9 0 % lg
1

ReactsG,against M. -a g a in s t
Reacts
protein A g o n polysaccharide Ag
the RB,C
surface
Question
The direct Coombs test was done on a patient.
If the red cells clump together, this suggests the
patient is _____
Other causes of haemolysis

• Oxidant damage (Exposure to chemicals and oxidants) e.g. lead poisoning

• Heat damage (e.g. severe burns)
• Enzymatic damage (e.g. snake venom) and
• even “foot strike haemolysis" in runners
Summary

• Classify range of haemolytic anaemias into acquired and inherited types

• Explain the underlying causes for each

• Explain the haematological abnormalities of patients with haemoglobinopathies

• Explain the important clinical features of these disorders.