Red Cells 1: Red cell physiology and congenital anaemias

Question 1

What are 4 general causes of Anaemia?

*remember that Anaemia is not a diagnosis but a result of something else going on*

Answer 1

• Blood loss
• Increased destruction of RBCs
• Lack of production of RBCs
• Defective production of RBCs

Question 2

What is the name given to an immature red cell?

Answer 2

Reticulocyte

Question 3

Where do you find reticulocytes?

Answer 3

You find reticulocytes in the bone marrow but also in the peripheral blood if the bone marrow is stressed or you have to compensate for RBCs loss for some reason

Question 4

Describe the Red Blood Cell development process

Answer 4

Stem cells in the bone marrow can be influenced by things like growth factors, hormones (i.e erythropoietin) which push it down the erythroid lineage

Once a cell commits to becoming a RBC, it starts developing in the bone marrow. They start to produce Hb (protein via ribosome synthesis). As they mature they have more Hb, their cytoplasm matures and then the cells lose their nucleus to form a reticulocyte.

Question 5

What substances are required for red cell production?

Answer 5

• Metals - Iron, copper
• Vitamins - B12, folic acid, thiamine etc
• Amino acids - to build proteins (Hb) inside blood cells
• Hormones - erthropoitein, GM-CSF, androgens (testosterone), thyroxine

Question 6

Which form of red cell should you never see in the blood?

Answer 6

You should never see any nucleated red cells in the blood and if you do this is tells us the patient’s bone marrow is under stress / loss of blood / very unwell

Question 7

Where does red cell breakdown occur? (naturally)

Answer 7

In the reticuloendothelial system - macrophages in the spleen, liver, lymph nodes or lungs etc are all called together

At the end of the life span (120 days approx) the reticuloendothelial system recognises the abnormal red cells and takes them out of the circulation. A lot of it is recycled for use in the body.

Question 8

How are old red cells recycled/reincorporated in the body?

Answer 8

• Globin from Haemoglobin is reused as protein
• Haem:
  
  • Iron - recycled into haemoglobin
  • Haem group is broken down into bilirubin

Question 9

What is the link between bilirubin levels and red cell breakdown?

Answer 9

• Bilirubin is produced from red cell breakdown (from haem).
• This bilirubin is bound to albumin which carries it back to the liver. When it is bound to albumin it is called unconjugated bilirubin
• It only becomes conjugated bilirubin within the liver.
• So increased RBC breakdown results in increased levels of unconjugated bilirubin in the blood.
• If you were to have increased conjugated bilirubin levels then you are looking at a liver problem.

Question 10

What is the key characteristic of a mature red cell on a blood film?

Answer 10

Pale centre as the haemoglobin is pushed to the extremities of the cell due to biconcave shape

Question 11

Genetic defects can affect which 3 components of the red cell?

Answer 11

• Red cell membrane i.e hereditary spherocytosis
• Metabolic pathways (enzymes) i.e G6PD
• Haemoglobin i.e sickle cell disease and thalassaemias

Most reduce red cell survival and result in haemolysis - red cell breakdown (before 120 days)

Question 12

Skeletal proteins in the red cell membrane are crucial in keeping the biconcave RBC structure in tact. Name some of these proteins.

Answer 12

• Band 3
• Ankyrin
• Alpha spectrin
• Beta spectrin

A problem in these leads to red cell destruction

Question 13

What is Hereditary Spherocytosis?

Answer 13

A common autosomal dominant disorder (50/50 chance) affecting the red cell membrane = Hereditary/congenital haemolytic anaemia

• Caused by mutations in genes relating to red cell membrane structural proteins that result in loss of skeletal integrity
• There are lots of different mutations in different proteins but they have the same outcome - hence why it varies between families
• Spherical shape on the blood film - see image - round, darker cells with no pale centre = RBC with loss of skeletal integrity

Question 14

How does someone present with Hereditary Spherocytosis?

Answer 14

Very variable clinical severity that depends on which structural protein/s is/are affected. It can be picked up early in life if severe phenotype or later in adult life.

• Anaemia - the reticuloendothelial system detects the abnormal spherical RBCs and removes them
• Jaundice (neonatal - severe) - increase in unconjugated bilirubin due to RBC breakdown
• Splenomegaly - often picked up in adulthood, ongoing haemolysis over the years, the r.s has worked overtime to remove red cells and spleen enlarges because of that
• Pigment gallstones - increased bilirubin in the gallbladder can crystalise and form these - can happen in young or older patients

Question 15

How do you treat Hereditary Spherocytosis in mild and severe cases?

Answer 15

Mild:

• Folic acid - you have increased folate requirement with increased red cell turnover

More severe:

• Blood transfusion
• Splenectomy - if very severe phenotype and patient is suffering from persistent anaemia or requiring regular blood transfusions

Question 16

Which metabolic pathway…

1. Produces energy for the RBC?
2. Protects the red cell from oxidative damage?

Answer 16

1. Glycolytic pathway
2. Pentose Phosphate shunt

Question 17

Which enzyme links the glycolytic and pentose phosphate pathways?

Answer 17

Glucose - 6 - phosphate dehydrogenase (G6PD)

Question 18

1. What is the most common red cell metabolism disorder/genetic enzyme deficiency?
2. And what is a rare one that you should still know about?

Answer 18

1. G6PD deficiency - cells are less able to protect themselves from oxidative damage which results in the breakdown of red cells
2. Pyruvate kinase deficiency - results in build up of metabolites in the glycolytic pathway – particularly 2,3 DPG – which again results in RBC haemolysis

Question 19

How does the G6PD enzyme protect haemoglobin from oxidative damage?

Answer 19

• It produces NADPH which is vital for the reduction of glutathione enzyme
• Reduced glutathione scavanges and detoxifies free radicals therefore preventing oxidative damage

Question 20

What are the genetics behind G6PD deficiency?

Answer 20

• GP6D has many genetic variants
• It is persistant as it confers protection against malaria - it comes into populations whose ethnicity originates from malarial areas
• It is X-linked:
  
  • Affect males - 1 abnormal X and normal Y
  • Female carriers - X abnormal but also X normal so this protects them

Question 21

Blood film of G6PD deficiency

Answer 21

Oxidative damage causes blister cells / bite cells

Question 22

How does G6PD deficiency present?

Answer 22

Spectrum of severity as some people have:

• No enzymes - chronic background haemolysis
• Reduced enzyme levels - only have haemolysis when exposed to things that cause oxidative damage anyways that then require G6PD to mop up

Presentation:

• Variable degrees of anaemia
• Neonatal jaundice or jaundice in adulthood during episodes of haemolysis
• Splenomegaly due to chronic background haemolysis
• Pigment gallstones - chronically high unconjugated bilirubin

Question 23

What sorts of things can trigger haemolysis in G6PD deficiency?

Answer 23

• Drugs - antimalarials
• Components of the diet - fava beans
• Intercurrent illness - (A disease that intervenes during the course of another disease) Infection causes bone marrow to become stressed so when you are ill you become slightly anaemic transiently. If your RBCs don’t last that long anyway due to a cell membrane or enzyme deficiency then any intercurrent illness will exaggerate that anaemia

Question 24

Extravascular vs intravascular haemolysis

Answer 24

Intravascular haemolysis - is not normal - RBCs burst within your circulation resulting in free Hb which is toxic to the kidneys. You also get free iron in the circulation which produces free radicals as a result.

Extravascular haemolysis - happens in the reticuloendothelial system where RBCs are being removed - this is a normal process of RBC breakdown that keeps our bilirubin levels at a normal level but this can increase in some forms of haemolysis. If RBC is detected as abnormal shape for example in hereditary spherocytosis the liver/spleen/macrophages will remove those faster than normal

Question 25

What is the normal structure of haemoglobin?

Answer 25

* 2 polypeptide alpha chains * 2 polypeptide beta chains * Each chain has an associated haem group (4 haem molecules in total) Haem is composed of a porphyrin ring and iron

Question 26

What is the 'Bohr effect' in relation to Hb?

Answer 26

As CO2 levels rise (hypercapnia) in the body you get carbonic acid forming =\> Acidosis =\> pH levels fall in cells =\> O2 is given up to the tissues more easily. This ^ happens whilst exercising - helps O2 get to your tissues. Some variant Hb holds onto O2 more tightly (higher affinity) such as foetal haemoglobin - doesn't give up O2 to tissues and so the foetus makes more RBCs to compensate

Question 27

Normal adult haemoglobin

Answer 27

**Normal adult Hb = haem molecule and 2 alpha and 2 beta chains. There are:** * **4 alpha genes (on Chr16)** * **2 beta genes (on Chr11)** So in adults haemoglobin is 97% of HbA (alpha and beta) and 3% HbA2 (alpha and delta) or HbF (alpha and gamma). * At the alpha gene locus there are _only_ alpha genes so if you lose alpha genes due to an inherited abnormality there are no other genes that can make an equivalent alpha chain and this is incompatible with life. * Beta genes are different as they have delta and gamma genes nearby that can make delta chains and gamma chains (HbF) so therefore they can compensate for the loss = compatible with life

Question 28

Foetal haemoglobin

Answer 28

When you’re born you have almost 97% of HbF which is made up of alpha and gamma chains. In the first year of life what gradually happens is that the gamma genes are switched off and the beta genes are switched on which marks the shift from HbF to HbA. However, as adults we still have the ability to use these gamma genes if needs be

Question 29

What are haemoglobinopathies?

Answer 29

Inherited abnormalities of haemoglobin synthesis. This can be caused by: * Reduced or absent globin chain production * Mutations leading to structurally abnormal globin chains

Question 30

Reduced or absent globin chains results in what Haemoglobinopathy?

Answer 30

**Thalassaemias** * You can have alpha or beta thalassaemias * Very rarely get delta or gamma thalassaemias (this doesn't affect adults much) Basically thalassaemias cause a **chain imbalance** which results in chronic haemolysis and anaemia

Question 31

What is the most common condition resulting from mutations that cause structurally abnormal globin chains?

Answer 31

Sickle Cell disease (haemoglobinopathy)

Question 32

Nearly all haemoglobinopathies have which type of inheritance?

Answer 32

Autosomal Recessive Inheritance - you have asymptomatic carriers (silent in populations) 1 in 4 chance of having affected 1 in 2 chance of being a carrier or 'trait' - this is the reason for persistence

Question 33

What happens to the haemoglobin structure in Sickle Cell disease?

Answer 33

Sickle haemoglobin (HbS) is composed of haem molecule and: * 2 normal alpha chains * 2 abnormal beta chains - point mutations in both (beta sickle chains) These mutations cause crystals to form inside the RBCs which causes it to change shape and become more rigid sickle cell

Question 34

What is a consequence of sickle cells disease in terms of damage caused by the sickle cells?

Answer 34

Due to their shape, sickle cells can cause damage as they try to move through the microvasculature. These crystallised cells damage their membranes causing leakage. The cells become dehydrated and they haemolyse. As a result of haemolysis and just the mere presence of these abnormal cells in the circulation you get things like: * Damage to the endothelium * Triggering of an inflammatory response * Activation of coagulation due to Hb in the circulation * Narrowing of vessels as a result All of this results in vaso-occlusion - plugs which obstruct capillaries and restrict blood flow to an organ.

Question 35

Presentation of Sickle cell disease

Answer 35

SCD is a multi-system disorder that presents with multi-system disease. Damage is caused by vaso-occlusion, endothelial damage and inflammation. The commonest presentation = relatively well patient between times with background sickling who then gets intercurrent illness / infection and sickling increases. They then present with: * **Bone pain** - infarction of the bone causing severe pain requiring opiates * **Chest crisis** - sickling in the lungs causing worsening hypoxia which in turn causes more sickling and results in this crisis * **Stroke** - sickling in the brain * **Increased infection risk** - SCD causes patients to autoinfarct their spleen over their lifetime - lots of sickling crises in the spleen - it becomes non-functional * **Chronic haemolysis** - increased risk of gallstones or aplastic crisis (where a virus slows down RBC production) * **Sequestration crises** - in spleen or liver - pooling of blood

Question 36

Acute management of sickle cell painful crisis?

Answer 36

* Severe pain - often requires opiates * Hydration * O2 - to prevent hypoxia and further sickling * Consider antibiotics for infection * Blood transfusion

Question 37

Life long management of sickle cell disease

Answer 37

* Vaccination - due to damage to spleen - to avoid infection * Folic acid - because of increased haemolysis - folate requirement is increased * Penicillin (and malarial) prophylaxis * Regular transfusion of HbA from donor to try and reduce the % of HbS in the circulation - this is not given to every patient due to risk of alloimmunisation and iron overload * Hydroxycarbamide - stops sickling * Severe patients - bone marrow transplantation / gene therapy

Question 38

Beta Thalassaemia Intermedia

Answer 38

A non-transfusion dependent thalassaemia Range of mutations / genotypes that result in an anaemia where you only need 'intermittent' transfusion at times of intercurrent illness / growth spurts / pregnancy (more demand on haematopoeisis)

Question 39

Beta thalassaemia minor

Answer 39

This is common in the population. It is the 'trait' or carrier state where you may have lost one beta chain or 1 or 2 alpha chains and your'e still making alpha and beta chains but there's an imbalance It gives a mild anaemia and small red cells (hypochromic microcytic red cells)

Question 40

Beta Thalassaemia major

Answer 40

* **Severe, transfusion dependent condition** * **No beta chains** * Presents as anaemia in babies at 3-6 months old as this is when gamma chains are switched off and the beta chains are supposed to be switched on but aren't because they don't have them (beta thalassaemia) * If this is not picked up and treated then the bone marrow will try hard to make as much blood as it can with the delta and gamma chains available to it and even the liver and spleen can take over to try help too * They are not successful and as a result you get bony deformities, splenomegaly and growth retardation as a result * Untreated or inadequately transfused you die before 10 years - however this is not the case now as women are screened during pregnancy for thalassaemias

Question 41

Treatment of beta thalassaemia major

Answer 41

* **Chronic transfusion support every 4-6 weeks throughout their life.** * If they are transfused then they will grow normally, however, transfusion results in _iron overload_ (bag of blood = bag of iron). * Need to be treated with _iron chelators_ to remove excess iron from the body - otherwise iron will build up in organs. With good adherence to chelation, the life expectancy = near normal * Bone marrow transplantation = curative * New gene therapies too

Question 42

Rare defect in Haem synthesis

Answer 42

Hereditary sideroblastic anaemia - ALA synthase mutations

Question 43

What is Homozygous alpha zero thalassaemia (α0/α0 )?

Answer 43

* Where you lose all your alpha chains which is incompatible with life * Causes severe thalassemia syndrome known as hydrops fetalis. Increased risk of severe maternal complications and infants with this syndrome usually die in utero, or soon after birth.