9. Haemolytic anaemias and Haemoglobinopathies

Question 1

What are haemoglobinopathies?

Answer 1

Haemoglobinopathies are inherited disorders where expression of one or more of the globin chains of haemoglobin is abnormal.

Question 2

What are the two types of haemoglobinopathies?

Answer 2

1. Abnormalities in the globin chains which alters the stability and/ or function e.g sickle cell disease.
   This can be as a result of mutations in the genes for α or β globin chains which alter structure/function/stability of the haemoglobin tetramer.
2. Reduced or absent expression of normal globin chains e.g Thalassemias.
   Globin gene mutations reduce expression of specific individual globin proteins resulting in an imbalance in the composition of the haemoglobin tetramer. This leads to a reduced level of haemoglobin rather than the presence of an abnormal haemoglobin.

Question 3

Describe the structure of haemoglobin

Answer 3

• Haemoglobin is a tetramer made up of 4 globin polypeptide chains.
• There are 2 alpha chains and 2 non alpha chains (e.g beta, gamma or delta).
• Each globin group is then bound to an oxygen binding group.

Question 4

Give the major haemoglobin types in adults

Answer 4

• HbA - 2 alpha, 2 beta
• HbA2 - 2 alpha, 2 delta
• HbF < 1 % - 2 alpha, 2 gamma

Question 5

Why are there different types of haemoglobins?

Answer 5

• Different Haemoglobins expressed during development as a adaptive response to variations in oxygen requirements
• Several embryonic forms expressed early in development
• Fetal haemoglobin (HbF) main form just before birth.

Question 6

When does HbA develop in an individual and becomes dominant?

Answer 6

HbA commences before birth and steadily increases to become dominant after birth

Question 7

How many alpha globin genes are there in humans?

Answer 7

There are 4 alpha globin genes. 2 on the maternal chromosome and 2 on the paternal chromosome

Question 8

Which chromosome can alpha globin genes be found on?

Answer 8

Chromosome 16

Question 9

How many beta globin genes are there in humans?

Answer 9

There are 2 beta globin genes, 1 on each chromosme.

Question 10

Which chromosome can beta globin genes be found on?

Answer 10

Chromosome 11

Question 11

What is the function of locus control regions on chromosome 11 and 16?

Answer 11

They control and regulate the expression of the globin genes.

Question 12

Explain why the expression of globin genes is kept under tight control and the consequences when this goes wrong

Answer 12

Normal expression of globin genes is under tight control to ensure a 1:1 ratio of α to non-α globin chain proteins.

However defects in this regulation of expression of globin genes can result in abnormalities in both the relative and absolute amounts of the globin chain proteins.

It can then result in:
β thalassaemia (β globin gene expression affected)
α thalassaemia (α globin gene expression affected)

Question 13

In which population groups is Thalassemia most prevalent?

Answer 13

It’s traditionally more prevalent in South Asian, Mediterranean, Middle east (β thalassaemia) and Far East (α thalassaemia) populations.
Important in clinical practice to be aware of the ethnicity of your individual patients and patient population (e.g. for prenatal counselling)

Question 14

What is the scale of aplha thalassaemia conditions?

Answer 14

• 1 α GLOBIN GENE DELETED: silent carrier state
• 2 α GLOBIN GENE DELETED: alpha thalassaemia trait
• 3 α GLOBIN GENE DELETED: haemoglobin H disease,
• 4 α α GLOBIN GENE DELETED: Hydrops fetalis

Question 15

Describe the severity of silent carrier state α-Thalassaemia

Answer 15

Asymptomatic

Question 16

Describe the severity of alpha thalassaemia trait

Answer 16

• minimal or no anaemia
• Either both genes on one chromosome 16 or one gene on each chromosome 16 deleted.
• Microcytosis and hypochromia in RBCs
• Resembles β-thalassemia minor

Question 17

Describe the severity of haemoglobin H disease

Answer 17

• moderately severe
• Tetramers of β-globin (called HbH) form resulting in microcytic, hypochromic anaemia with target cells and Heinz bodies.
• Resembles β-thalassemia intermedia

Question 18

Describe the severity of Hydrops fetalis

Answer 18

• Severe, usually results in intrauterine death
• All 4 α genes deleted.
• Excess γ-globin forms tetramers in foetus (called Hb Bart) that is unable to deliver oxygen to tissues.

Question 19

Is β-Thalassaemia a result of mutation or deletion?

Answer 19

Disease is often caused by gene mutation rather than deletion - β-globin gene on chr11

Question 20

Differentiate between β0 and β+

Answer 20

β0 refers to a total absence of production of β-globin gene, whilst β+ refers to a reduction of globin production

Question 21

Describe the severity of β-Thalassaemia minor or β thalassemia trait

Answer 21

• Usually asymptomatic with a mild anaemia (very microcytic and hypochromic rbcs)
• Resembles α-Thalassemia trait
• Heterozygous with 1 normal and one abnormal gene
(βo/β or β+/β)
• Despite the reduction in red blood cell size, the total level of haemoglobin in blood tends to remain normal because the bone marrow responds by producing more red blood cells. Anaemia only really occurs in such patients in times of increased demand such as pregnancy or persistent infections

Question 22

Describe the severity of β-Thalassaemia intermedia

Answer 22

• Severe anaemia, but not enough to require regular blood transfusions
• Resembles Haemoglobin H (HbH) disease

Question 23

Describe the severity of β-Thalassaemia major

Answer 23

• Severe transfusion-dependent anaemia.
• Becomes manifest 6-9 months after birth as synthesis of haemoglobin A (α2β2) cannot replace Haemoglobin F (α2γ2) due to the lack of β globin.
• Homozygous (βo/βo or β+/β+)

Question 24

What is the inheritance pattern of Haemoglobinopathies?

Answer 24

Inherited disorders typically autosomal recessive

Question 25

What will the peripheral blood smear of a patient with severe Thalassemia show?

Answer 25

• Hypochromic & microcytic red cells (due to low haemoglobin)
• Anisopoikilocytosis with frequent target cells and circulating nucleated red blood cells and Heinz bodies.

Question 26

What effect can an excess in the unaffected globin chain in Thalassemia have on a RBC

Answer 26

Relative excess of the unaffected globin chain also contributes to the defective nature of the red cell (e.g.
insoluble aggregates of α chains form in β thalassaemia)

Question 27

What effect can premature haemoglobin aggregates have on erythropoiesis and on RBC?

Answer 27

Haemoglobin aggregates get oxidised and result in:
• Premature death of erythroid precursors within bone marrow leading to ineffective erythropoiesis
• Excessive destruction of mature red cells in spleen leading to shortened red blood cell survival - Thus, in addition to defective Hb production, thalassaemia is also a form of haemolytic anaemia as red cells are destroyed

Question 28

List the consequences of Thalassaemia

Answer 28

1. EXTRMEDULLARY HAEMOPOIESIS - occurs in an attempt to compensate but results in splenomegaly, hepatomegaly and expansion of haemopoiesis into the bone cortex ..this impairs growth and causes classical skeletal abnormalities
2. STIMULATION OF EPO due to reduced oxygen delivery which further contributes to the drive to make more defective red cells
3. IRON OVERLOAD - occurs due to:
   • Excessive absorption of dietary iron due to ineffective haematopoiesis
   • Repeated blood transfusions required to treat the anaemia
4. REDUCED LIFE EXPECTANCY

Question 29

What is Thalassaemia?

Answer 29

Thalassaemias are a group of inherited disorders which result from decreased or absent α or β globin chain production (α- and βthalassaemia respectively) resulting in an imbalance in the composition of the α2b2 tetramer.

Question 30

What is β Thalassaemia?

Answer 30

β-thalassaemia results from mutation in one or both of the β globin genes leading to a reduction in the amount or total absence of the β globin polypeptide chain

Question 31

What is α Thalassaemia?

Answer 31

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

Question 32

Give the treatments for Thalassaemia

Answer 32

• Red cell transfusion from childhood
• Iron chelation (delays iron overload)
This is the removal of excess iron from the body using special drugs.
• Folic acid (this s to help support healthy erythropoiesis)
• Immunisation
This is because Thalassaemia patients are more at risk of infection than a healthy individual
• Holistic care - cardiology, endocrine, psychological, ophthalmology input to pick up and manage complications
• Stem cell transplantation in some -This is to replace the defective red cell production
• Pre-conception counselling for at risk couples and antenatal screening

Question 33

What is sickle cell disease?

Answer 33

• Sickle cell disease is an autosomal recessive disease resulting from the mutation of beta-globin genes.
• The GAG codon is changed to GTG resulting in charged glutamic acid being substituted by uncharged valine at position 6.
• This produces a mutant haemoglobin molecule that contains the mutated β-globin protein.
• This is referred to as Haemoglobin S (HbS).

Question 34

Describe the inheritance of sickle cell disease

Answer 34

• Heterozygous HbS carrier state causes a mild asymptomatic anaemia = Sickle cell trait
• HbSS = homozygous sickle cell anaemia, is most common cause of severe sickling syndrome = Sickle cell disease
• HbS can also be co-inherited with another abnormal Hb e.g. HbC (HbSC ) or ß-thal (HbS ß-thal) to cause a sickling disorder

Question 35

What is the advantage of having sickle cell trait?

Answer 35

The HbS variant is found mainly in people of
Black African descent but is also common in Arab, Mediterranean, and South Asian populations. Heterozygous individuals for HbS have some resistance to malaria due to changes in the red blood cell making it difficult for the Falciparum parasite to grow

Question 36

In sickle cell disease, describe the effect of varying oxygen concentrations of on RBC

Answer 36

• Anaemia usually mild and well tolerated as HbS readily gives up oxygen in comparison to HbA
• Problems come in low oxygen state as In a low oxygen state, the deoxygenated HbS forms polymers s that can
result in the deformation the red blood cell membrane leading to the cell taking on a sickle shape.
• However when the oxygen levels are normal, HbS is fine and will cause the RBC to return back to its normal concave structure.
• However, After repeated episodes of sickling,
damage occurs to the red cell membrane causing it to lose elasticity. Such damaged cells fail to return to a normal shape when normal oxygen tension is restored.. • Irreversibly sickled red are cells less deformable and can cause occlusion in small blood vessels – ‘sticky’

Question 37

What are the consequences of sickle cell formation?

Answer 37

• Vaso-occlusive episodes:
due to occlusion of small capillaries from sickle cells getting trapped. This leads to recurrent acute pain and syndromes such as stroke or acute chest syndrome as
well as chronic kidney disease and joint damage from avascular necrosis.
• Anaemia:
due to sickle cells undergoing haemolysis resulting in
a shortened erythrocyte lifespan from ~120 days to ~20-30 days.
• Jaundice and gallstones:
due to increased bilirubin resulting from chronic haemolysis.
• Splenic atrophy:
due to splenic infarction with an associated susceptibility to infection by encapsulated bacteria such as Streptococcus pneumoniae and Streptococcus meningitidis

Question 38

What are the 3 crises in sickle cell?

Answer 38

1. Vaso-occlusive crisis
2. Aplastic crisis
   This is most often triggered by the parvovirus and it causes a complete stop to RBC production for a few days.
   This causes reticulocyte counts to drop significantly, the rapid turnover of RBCs results in a drop in haemoglobin.
3. Haemolytic crisis
   These are acute accelerated drops in haemoglobin levels. The blood cells break down at a faster rate. This is particularly prevalent in those with a G6PDH deficiency.

You can then get end organ damage as a result of acute thromboses or O2 deprivation.

Question 39

What are other complications of sickle cell cell disease?

Answer 39

• Retinopathy
• Splenic atrophy
• Avascular necrosis (e.g. in femoral head)
• Acute chest syndrome
• Stroke
• Osteomyelitis
• Skin ulcers
• Kidney infarcts
• Priaprism

There’s a reduced life expectancy of around 67 years usually due to either stroke, multi organ failure and acute chest syndrome

Question 40

What are the treatments of sickle cell?

Answer 40

Haematopoietic stem cell transplantation is the only cure for sickle cell disease but this is rarely performed due to difficulty in finding a donor with a sufficient genetic match. Treatment therefore is typically concentrated on reducing symptoms with regular medical care to prevent complications.

Question 41

What is aplastic crisis?

Answer 41

An aplastic crisis is when the body does not make enough new red blood cells to replace the ones that are already in the blood

Question 42

. What is haemolytic anaemia?

Answer 42

This refers to anaemia as a result of an abnormal increase in the breakdown (haemolysis) of RBCs.

Question 43

. Give the 2 sites abnormal breakdown of RBCs can take place in

Answer 43

• Blood vessels (intravascular haemolysis)

* Spleen & wider RES (extravascular haemolysis)

Question 44

How does the bone marrow try to help in haemolytic anaemia?

Answer 44

• Bone marrow can compensate by increasing production but only up to a point.
• If haemolysis exceeds capacity of marrow to compensate (~ 6 fold increase max) then the rate of destruction exceeds rate of production and anaemia develops

Question 45

What is the normal lifespan of RBC?

Answer 45

~120 days

Question 46

What are the key laboratory findings that’d be present in a sample of a patient with haemolytic anaemia

Answer 46

• Raised reticulocytes (as the marrow tries to
compensate)
• Raised bilirubin (breakdown of Haem)
• Raised LDH (red cells rich in this enzyme)

Question 47

What are the two forms of haemolytic anaemia?

Answer 47

• Inherited (defective gene)

* Acquired (damage to cells)

Question 48

What are the 4 examples of inherited haemolytic anaemia?

Answer 48

• Glycolysis defect - pyruvate kinase deficiency limits ATP production
• Pentose-P Pathway - G6PDH deficiency leads to oxidative damage
• Membrane protein e.g hereditary spherocytosis
• Haemoglobin defect e.g Sickle cell

Question 49

What are the 4 examples of acquired haemolytic anaemia?

Answer 49

• Mechanical damage - microangiopathic anaemia
• Antibody damage - autoimmune haemolytic anaemia
• oxidant damage - exposure to chemicals or oxidants
• heat damage - e.g severe burns
• enxymatic damage - e.g snake venom

Question 50

What are the consequences of haemolytic anaemia

Answer 50

• Severity of anaemia is typically worse than chronic disease if Hb is very low or there is a sudden fall in Hb
• Accumulation of bilirubin leading to jaundice and associated risk of complications such as pigment gallstones.
• Overworking of red pulp leading to splenomegaly
• Massive sudden haemolysis e.g. from incompatible blood transfusion can cause cardiac arrest due to lack of oxygen delivery to tissues & hyperkalaemia due to release of intracellular contents

Question 51

Describe the appearance of pigmented gallstones

Answer 51

• Small
• Irregular
• Dark

Question 52

Describe the symptoms of jaundice

Answer 52

High bilirubin causes yellowish discoloration of skin and sclera of eye.
Urine dark in colour due to conjugated bilirubin

Question 53

Function of red pulp in the spleen

Answer 53

A. Removal of old, damaged and dead red blood cells along with antigens and microorganisms -

B. Phagocytosis of opsonised bacteria by macrophages

C. Sequestration of platelets.

D. Storage of red blood cells in case of hypovolaemia, these can then be released following an injury resulting in blood loss

E. Prenatally, it is haematopoietic until about the fifth month of gestation when bone marrow becomes the main site for haematopoiesis.

Question 54

What is Hereditary Spherocytosis?

Answer 54

Hereditary spherocytosis is an inherited autosomal dominant disease resulting in abnormalities in erythrocyte membrane proteins which impede the ability of the cell to change shape. Many cells take on
spherical shape. Cells less flexible and more easily damaged

Question 55

Mutations in the genes coding for which 4 proteins have been shown to cause the Hereditary Spherocytosis?

Answer 55

• Spectrin (an actin crosslinking and molecular scaffold protein that links the plasma membrane to the actin cytoskeleton).
• Ankyrin (links integral membrane proteins to the underlying spectrin-actin cytoskeleton).
• Band 3 (facilitates chloride and bicarbonate exchange across the plasma membrane and is also involved in a physical linkage of the plasma membrane to the underlying cytoskeleton (via binding with ankyrin and protein 4.2)).
• Protein 4.2 (an ATP-binding protein which may regulate the association of protein 3 with Ankyrin).

Question 56

How does mutations in the 4 proteins lead to hereditary spherocytosis?

Answer 56

• the common role of the proteins is in facilitating “vertical interactions” between the cytoskeleton and the lipid bilayer of the plasma membrane.
• mutation of the genes coding for these proteins results in a local disconnection of the cytoskeleton and membrane.
• This is followed by vesiculation of the unsupported membrane components leading to progressive reduction in membrane surface area and production of a “spherocyte”/ spherical shape to the red blood cell.
• Red cell membranes are normally very flexible but become increasingly less deformable as surface area is lost

Question 57

What are the effects of red cells becoming spherical shaped?

Answer 57

• The poor deformability of spherocytes means that they become trapped and damaged as they pass through the spleen (extravascular haemolysis) resulting in a reduction in the lifespan of erythrocytes and haemolytic anaemia.
• The deficit in cell deformability of spherocytes only appears to be a problem in the spleen rather than small capillaries since the red cells return to a nearly normal lifespan following splenectomy.
• In addition to anaemia, symptoms include jaundice and splenomegaly.
• Howell-Jolly bodies may be seen within red blood cells.

Question 58

What is the treatment for hereditary spherocytosis?

Answer 58

Patients with mild symptoms do not usually require treatment but for those with severe symptoms partial or full splenectomy improves the anaemia.
The spherocytes still remain but are no longer lysed by the spleen.

Question 59

What is Hereditary Eliptocytosis?

Answer 59

• Many cells elliptical rather than biconcave disc shape
• Spectrin defect most common
• Also defects in band 4.1, Band 3 and glycophorin C proteins

Question 60

What is Hereditary Pyropoikilocytosis?

Answer 60

• Spectrin defect
• Severe form of hereditary elliptocytosis
• Abnormal sensitivity of red cells to heat
• Similar morphology to that seen in thermal burns

Question 61

What are Microangiopathic haemolytic anaemias?

Answer 61

Microangiopathic haemolytic anaemias result from mechanical damage e.g.
• Shear stress as cells pass through a defective heart
valve (e.g. in aortic valve stenosis) - Cells get damaged
when forced through narrowed opening under high pressure
• Cells snagging on fibrin strands in small vessels where increased activation of clotting cascade has occurred
- e.g. in Disseminated Intravascular coagulation - a condition where bleeding and clotting occur at the same time in the patient e.g. in malignancy, obstetric complications, trauma, and sepsis
- e.g in Thrombotic thrombocytopenic purpura - a syndrome where small thrombi form within the microvasculature

Question 62

What are schistocytes?

Answer 62

The red blood cell fragments resulting from mechanical damage are called schistocytes and the presence of schistocytes in a blood sample is a good indicator that some form of pathology is present.

Question 63

What are autoimmune haemolytic anaemias caused by?

Answer 63

• Caused by autoantibodies binding to red cell membrane proteins
• Can result from infections (e.g. chest infection in children) and lymphoproliferative disorders such as leukaemia or lymphoma and reactions to drugs such as cephalosporins

Question 64

What is are the classification of autoimmune haemolytic anaemias?

Answer 64

Classified as either “warm” (IgG) or “cold” (IgM) based
on temperature antibodies react best at under laboratory conditions
• Spleen recognises antibody bound cells as abnormal
and removes them
• Red cell lifespan reduced resulting in anaemia

Question 65

Describe what happens in warm autoimmune haemolytic anaemia

Answer 65

• In warm autoimmune haemolytic anaemia, IgG antibodies recognise epitopes on the red cell membrane.
• This leads to macrophages in the spleen recognising these antibody-coated red cells and either disposing of the whole cell by phagocytosis or “nibbling” a bit off.
• In the latter case, since some membrane is lost, the red cell tends to “round up” forming a spherocyte.
• Splenomegaly often occurs in these patients as the spleen is doing extra work.

Question 66

Describe what happens in cold autoimmune haemolytic anaemia

Answer 66

• In cold autoimmune haemolytic anaemia IgM autoantibodies recognise red cell epitopes
• As the IgM autoantibodies bind best at cooler temperatures, they tend to bind to the red cells in more distal parts of the body e.g fingertips, especially if cold weather.
• The IgM autoantibodies span several red cells creating large agglutinates which can block small capillaries, creating ischaemic conditions in peripheral body parts causing numb fingertips, earlobes etc. and pallor, blue discoloration or in extreme cases gangrene.
• When the red cells circulate back to warmer, more central parts of the body the IgM falls off and the agglutination disappears.
• It is the complement binding to the red cells in cold Immune haemolytic anaemia which causes most damage both by directly creating holes in the membrane and also by causing macrophages in the spleen to recognise and destroy the cells.

Question 67

What is Pyruvate kinase deficiency?

Answer 67

• Pyruvate kinase deficiency is an inherited metabolic disorder (typically autosomal recessive but there is also a dominant form) due to a mutations in the PKLR gene.
• There are four pyruvate kinase isoenzymes, two of which are encoded by PKLR (isoenzymes L and R
expressed in liver and erythrocytes, respectively).
• Mutations in the PKLR gene therefore cause a deficiency in pyruvate kinase in erythrocytes

Question 68

How does pyruvate kinase deficiency lead to inherited haemolytic anaemia?

Answer 68

• This enzyme catalyses the final step in glycolysis which produces ATP
• Since red blood cells lack mitochondria, pyruvate kinase deficiency inhibits their only metabolic pathway which can supply ATP for cellular processes.
• The sodium potassium ATPase pump activity is inhibited from insufficient ATP and the red cells lose
potassium to plasma.
• Water moves down its concentration gradient out of cells causing them to shrink resulting in cellular death and haemolytic anaemia

Question 69

What is the treatment for haemolytic anaemia resulting from pyruvate kinase deficiency?

Answer 69

Most affected individuals with pyruvate kinase deficiency have only a mild deficiency in enzyme activity and do not require treatment. Individuals with a more severe deficiency may require regular blood transfusion.

Question 70

What is G6PDH deficiency?

Answer 70

Glucose-6-phosphate dehydrogenase (G6PDH) deficiency is an X-linked recessive inborn error of metabolism.

Question 71

Why does G6PDH deficiency lead to haemolytic anaemia?

Answer 71

• G6PDH is the rate limiting enzyme of the pentose phosphate pathway which maintains NADPH levels.
• NADPH is required to protect against oxidative stress by maintaining the level of reduced glutathione.
• Since the pentose phosphate pathway is the only source of reduced glutathione in red blood cells, these are particularly affected by defects in the glucose-6-phosphate dehydrogenase enzyme.
• Patients with G6PDH deficiency are therefore at risk of
haemolytic anaemia in states of oxidative stress such as infection or exposure to certain chemicals or medications.
• Damaged red cells are phagocytosed in the spleen and metabolism of the excessive haemoglobin to bilirubin can lead to jaundice.

Question 72

What is the direct Coombs test used for?

Answer 72

used to 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 haemolysis is immune-related.

Question 73

What are the possible Causes of splenomegaly?

Answer 73

• Immune response work hypertrophy
• RBC destruction work hypertrophy
• Congestive causes such as in splenic vein thrombosis or portal hypertension
• Myeloproliferative causes e.g. chronic myeloid metaplasia
• Infiltrative causes e.g. sarcoidosis and some neoplasms
• Neoplastic causes e.g. chronic lymphocytic leukaemia