Lecture 3 & 4: Sickle Cell Anaemia

Sickle Cell Disease: Frequency of Disease

• UK 10-15,000 affected in all ages

• Worldwide 300,000 affected individuals born each year

• In high incidence areas affects 2-3% of all births

Sickle Cell Disease: Life Expectancy

• UK: 40-60 years

  • Improving year-on-year

• Developing nations: 50-90% childhood mortality

Sickle Cell Disease: Economic Costs

• Developed Countries (US) $27,779 per patient per year

  • Gene therapy £1 million

• Developing – folic acid (£15)

Balanced Polymorphism

• A (genetic) disease that causes a very serious illness, where the gene should have died out but hasn’t due to it providing heterozygotes with a genetic advantage

Sickle Cell Disease: Genetic Basis

• A single base change (A to T) causes glutamic acid on the 6^th position of the beta-globin chain to be replaced with valine.

  • Charged amino acid replaced with a neutral amino acid

    mutation provides protection from malaria

• Example of polymorphism

  • heterozygous - HbAS

  • homozygous recessive HbSS

  • homozygous dominant - Hb AB

Heterozygous State of Sickle Cell (HbAS) + Malaria

• Advantageous: provides protection against malaria due to the interaction of sickle Hb and actin RBC cytoskeleton

  • Mutation has arisen on multiple occasions within malaria-affected areas.

  • highly advantageous in very high-frequency malarial areas (up to 40%).

• Prevents parasite from altering the red cell cytoskeleton (and making it ‘sticky’ – an essential part of parasite life);

  • in _______ parasites cannot complete their life cycle as effectively.

Homozygous State of Sickle Cell (HbSS) + Malaria

• Associated with severe disease

  • absence of modern health care - few affected individuals survive past childhood

• No effective protection - malaria is a very severe disease in those with sickle cell disease as they are often already vulnerable to chronic ill health

  • Anaemia and poor nutritional status

Biochemistry of SCD: Disorder of Protein-Protein Interaction

• Replacement of charged glutamic acid on the 6^th position of beta-globin chain with neutral valine causes a structural change and charge change.

• When haemoglobin is deoxygenated, the valine becomes exposed on the Hb surface.

  • Can interact with other Hb molecules

Significance of Change from GLU to VAL in B-globin chain

• Charge of GLU prevents the chains from interacting,

  • normal haemoglobin: GLU molecules are charged and oppose interactions between the adjacent Hb molecules

• Neutral VAL promotes interactions

  • sickle haemoglobin: VAL molecules have no charge and so permit hydrophobic interactions between the adjacent Hb molecules

Biochemistry of SCD: Polymerisation

• Deoxygenated sickle haemoglobin may polymerise and form long fibrous linked chains

• These chains distort the red cell, preventing normal flexing and producing a characteristic long-form cell with sharp ends (long sharp cell)

  • difficulties in flowing through capillaries and exchanging oxygen

Polymerisation of Hb in Healthy RBCs

• The molecules of normal haemoglobin are separate within the erythrocyte and form a gel that can be shaped by the flexible cytoskeleton to form the typical flexible disk shape

Are RBCs Sickled all The Time?

• No, Sickling reverses when red cells return to high oxygen, so initially the red cell shape is not permanently altered

  • reversibility of sickling is important to understanding the clinical presentation, damage to red cells, and treatment  

• Long chains only form following the loss of oxygen

  • Sickling only occurs in deoxygenated tissues

• Most of the time not heavily deoxygenated

  • If sickled all the time, would not be able to through lungs = death

If all RBCs Form A Sickle Shape When Oxygen Is This A Problem?

• Sickling process takes time to occur, so in health, most cells can return to the lung before major sickling occurs

  • Affected individuals do not have sickling at all times - dependent on crystalisation

Crystallisation

• Not an immediate process - 2 parts

  • Slow nucleation occurs, where cells begin to stick together – unlikely for cells to sickle – happens too slowly – blood reoxygenated

  • Followed by rapid chain formation – gives rise to the sickled shape

• The time taken for sickling of red cells is critical to the clinical illness of patients

How are Foetuses Protected Against Sickle Cell Disease

• in the developing baby the beta haemoglobin chain is replaced by HbF to form foetal haemoglobin - no sickle haemoglobin present until after delivery.

• Hbf not lost until 3-6 months after birth – B chains not initially produced  - no sickle Hb – gives babies protection

  • has a role in treatment and disease severity

Variation in Sickle Cell Phenotype

• Not all patients with sickle cell disease are equally affected - some have much milder disease

  • All have the same sickle cell disease mutation but have co-inherited factors that change disease severity

Hereditary Persistence of HbF In Sickle Cell Disease (HPFH)

• Hereditary persistence of foetal haemoglobin (HPFH) in affected individuals the foetal haemoglobin remains in adult cells at a high concentration (up to 20%)

• Has two effects:

  • fewer sickle haemoglobin molecules, but

  • HbF does not form polymers and interferes with the formation of long chains of haemoglobin - sickling is less severe

    • Interfere with crystallisation proteins

Cell Biology of Sickle Cell Disease

• A disease of

  • Chronic red cell damage

  • Acute crisis

Sickle Cell Disease - Chronic Damage

• Damaged to Hb in response to repeated cycles of polymerisation and depolymerisation    

• Eventually, the polymerisation does not reverse, and haemoglobin may become denatured

  • Cell may become ‘irreversibly sickled’ – can’t enter lungs

• The repeated cycles of shape-change then reversal damage the cells causing permanent partial shape change and damaging and degrading membrane pumps so that cells become dehydrated

  • Unable to transport oxygen or sit in gel   

Sickle Cell Disease- Anaemia

• Red cell survival in blood is greatly reduced

  • often from 120 days to as little as 8 days.      

• Production is increased to compensate but cannot replace cells rapidly enough, so patients are chronically anaemic.

• Typical haemoglobin levels are 70-80g/l compared with normal around 130-140g/l.;

  • low level is compensated for by other systems

Spleen

• Responsible for removing damaged cells and circulating pathogens in the blood

  • Sickle cells enter and are destroyed – shortening their lifespan

Blood Appearance in SCD (General)

• Red blood cell damage:

  • Repeated cycles of sickling and recovery damages red blood cells.

  • Damaged cells are prematurely destroyed.

  • This leads to anaemia.

• Most cells are not typically sickle-shaped, but few have a normal shape

Sickle Cell - Blood Appearance: True Sickle Cells

• Permanent, irreversible sickling

  • Pointy ends

Sickle Cell - Blood Appearance: Boat Cells

• Permanent partial sickle change

  • elongated but not spikey

Sickle Cell - Blood Appearance: Dense Irregular Cells

• Dehydrated cells

  • Hb is squashed due to repeated cycles of polymerisation/depolymerisation

Sickle Cell - Blood Appearance: Target Cells

• Sickle Hb forms a central pool

Disruption to the Equilibrium Between RBC and Sickle Shape

• Gives rise to acute crisis

• Don’t readily form sickle shapes in capillaries but equilibrium can be shifted and can occur more readily and rapidly - blocks blood flow

  • Hypoxia, fever, and dehydration favour sickle-shape

• Reason for Hospitalisation

Why does Dehydration Favour Sickle Shape and Assit Acute Crisis

• Affects peripheral circulation - slows down blood flow

• This give a longer time for the sickling process to occur

  • In some physiological conditions, the sickle cells may form in small vessels

Acute Crisis

• Sickle cells block the small vessels causing a failure of blood supply and tissue damage

  • Cells can pass round bends and stick to other cells and form meshes

• Can cause the activation of white cells in response to blockages – release cytokines

• Process affects tissue beyond the site of blockage as blood supply is lost – tissue death

Clinical Effects of Acute Crisis

• Damage to many organs

  • Bone pain and damage – oxygen supplied to bones via small vessels – blockages occur here

  • Chest crisis – pain in ribs – less likely to take deep breaths – hypoxic conditions - sickle

  • Stroke – loss of blood to the cerebrum  

  • Sepsis – due to loss of blood supply to tissue – infection more likely as tissue dies

    • WBC protection lost

Clinical Effects of Sickle Cell: Chronic Anaemia

• Red cell damage and premature destruction

• Adaption through increased heart output to increase O₂ transport - symptoms less apparent when young

  • RBC don’t survive as long

• When older it is problematic - chronic problems to heart and lungs e.g. acute chest syndrome, pulmonary hypertension and renal diseases

  • this along with effects of repeated crisis limits life expectancy

Clinical Effects of Sickle Cell: Bone Pain

• Blockage of smell vessels supplying the bones causes sudden onset of very severe pain in children and adults.

• This can sometimes be managed at home but may need admission and strong painkillers.      

• Chronic pain can be debilitating and areas of dead bone can become a focus for infection e.g. necrosis and ulceration

Dactylitis

• Severe swelling and pain      

• Can impair bone growth and dead areas of bone can be prone to infection

  • Long term treatment - antibiotics

Clinical Effects of Sickle Cell: Skeletal Deformity

• Occurs in response to blockage of small vessels that supply bones in children can damage the ‘growth plate’ preventing their normal growth and resulting in long-term problems

  • Can result in shorter fingers

Clinical Effects of Sickle Cell: Stroke

• Rare but severe

• the blockage of small vessels in brain leads to death of the brain tissue they supply

• People at risk must be treated much more intensively to reduce the chance of further ____ and permanent disability

  • Risk due to abnormal circulation

Clinical Effects of Sickle Cell: Spleen and Infection

• Removes damaged cells but is full of tiny capillaries – resulting in damage      

• Repeated sickling destroys the tissue leading to fibrosis and calcification. 

• Organ becomes non-functional, increasing the risk of sudden and severe infection with particular types of bacteria (encapsulated bacteria)

  • Organ becomes smaller and shrinks as a result of damage

Clinical Effects of Sickle Cell: Chest Crisis

• Severe sickling can cause the blockage of small vessels in the lungs – results in fluid leaking into tissues  - results in inflammation and damage

  • Poor gas exchange  - increases chance of sickling      

• Lung tissue and heart enlarge to compensate  

• Reduces oxygen further and the pain can prevent individuals from taking full breaths   

• Leads to a cycle of further hypoxia and sickling.

• a medical emergency and is one of the major causes of death in sickle cell disease.  

  • In the long term, can result in chronic lung disease.

General Aim of Treatment (For Crisis)

•  To slow the sickling process by reversing any causes and improving oxygenation and circulation through fluids and removing pain

• Tissue damage can then be limited allowing the body to heal itself

Treatment for Mild/Simple Cases of Crisis:

• Pain relief: for comfort and to allow better movement breathing and self-care

  • Individuals in pain don’t drink/ inflate lungs well – further problems

  • E.g. paracetamol, ibroberaphen, morphine

• Hydration: adequate fluids will preserve blood flow and red cell hydration

  • given a drip in hospitals or drink 3L of water at home

• Treat causes: reverse causes to prevent ongoing sickling e.g. high altitude or infection – give antibiotics, treat pain

• Assess: who is seriously ill and may need hospital care

• Return to normal life: as soon as possible

Treatment for Severe Cases of Crisis

• Use simple measures to treat the bone pain and crisis and

  • Transfusion

  • Exchange transfusion

  • Experimental therapies of clinical trial

Transfusion

• Used to treat severe cases of crisis if Hb levels are very low

  • Raising Hb increases oxygenation – reducing likelihood of sickling

  • Can’t have too many transfusions – can develop antibodies – difficult to cross-match

Exchange Transfusions

• Treatment for those with severe chest crisis

  • Used if life-threatening

• It aims to replace most of the circulating sickle cell blood with normal blood – if the % of sickle blood is lowered to less than 30% sickle damage is prevented

  •  Replacement of sickle blood with normal blood → take one unit and replace with a new unit

Long Term Treatments: Preventative Measure

• Used to reduce the frequency of crises and maintain health:

  • Education (primary approach) – safety!

  •   Vaccination – protection for spleen  

  • Antibiotics -prevents pneumonia and meningitis  

  • Folic acid – promotes RBC production

Long Term Treatment: Modifying Red Cell Physiology

• Raise HbF

• the drug hydroxycarbamide is recognised to increase intracellular HbF and can reduce the frequency severity of crises

  • Reduces instances of sickling

Long Term Treatment: Chronic Transfusion

• Used in severe disease

• Transfusion done monthly to replace all sickle blood with normal blood, or bone marrow transplant for very selected case

Problem with Bone Marrow Transplantation

• Dangerous

• Long term problems

• Mortality risk

Experimental Therapies

• Gene therapy - replace HbSS/ increase HbF

• Anti-sickling molecules - prevent HbSS protein-protein interactions → prevent crisis

• Anti-adhesion molecules - stop crisis by making Hb less sticky and block vessels

• Modify vascular tone - relax capillaries and improve blood flow

  • nitrous oxide improves vascular tone

Treatment of SCD in Developing Countries

• Most cases are in developing countries

• Low-cost interventions are critical!

  • Screening – to identify cases early

  • Education – to discuss what can be done

  • Folic acid – cheap and supports blood cell production

  •  Vaccination, antibiotics – reduce crises

  • Malaria treatment – as needed – biggest treatment of homozygous

• Hydroxycarbamide (£250 per year)

Factors Affecting Choice of Diagnostic Test

• Speed – sometimes you need a fast result e.g. surgery 

• Accuracy – most often accuracy is more important than speed      

• Expertise – simplicity makes tests easier to provide      

• Throughput – high capacity may need test that can be automated      

• Cost – the cost of the health service must always be considered

Types of Diagnostic Test

• Cell biological approach: Look for sickle cells      

• Physiological approach: Detect sickle haemoglobin polymerisation      

• Biochemical analysis: Detect the abnormal charge properties of haemoglobin 

• Gene analysis: Detect the typical mutation

Blood Smear

• Looks for sickled cells themselves – distinct characteristics can be seen on a blood film

  • Cheap and (fairly rapid)

• ROLE: may spot an unexpected diagnoses

Problem of Using Blood Smear as A Diagnostic Test

• Expertise required – may be overlooked

• Relatively low sensitivity in healthy patients such cells can be rare – can’t see characteristics cells

• Not good for complex diagnoses where there are multiple other problems affecting cell appearances

• Doesn’t consider Hb or genes

Haemaglobin Solubilty Test

• Detects SHb – forms polymers      

• Use detergent to lyse red cells then add a reducing agent  

  • normal haemoglobin forms a clear suspension of monomers - relatively transparent. 

  • Sickle haemoglobin forms polymers - solution is cloudy and cannot be seen through

• ROLE: Great in emergencies e.g. Pre-operative

Advantages of Using Haemoglobin Solubility Test

• Cheap,

• Quick,

• very sensitive,

• Little training is required

Problem of Using Haemgolbin Solubity Test as Diagnostic Tool

• Too broad

• Test doens’t distinguish between homozygous and heterozygous → not good for complex diagnoses

Test Based on The Altered Charge of Sickle Cell

• Different charge of sickle haemoglobin means it will move at a different rate than normal haemoglobin when placed in an electric field.

•   Shown using flatbed electrophoresis   

  • relatively slow with lower capacity    

• Hb lysed and placed at the end of the paper

  • Normal Hb – glutamic acid – charged

    • More charge – moves faster

  • Sickled Hb – valine (neutral) moves slowest

• ROLE: Used for non-urgen diagnosis

Use of Chromoatographic Machines

• Analysis done using a high-pressure column chromatography detecting different haemoglobin forms as they emerge under high pressure from a charged column.

• Differentiates betwen Homozygous and heterozygous

  • one band healthyHeterozygous – 2 bands (middle)

  • One band further right – disease – moves slowly in chromatographic column

• Machines are fast

Advantages of Using Chromatographic Machienes

• Accurate and intermediate cost

• Insufficiently rapid for very urgent samples, and requires expertise to interpret

• Great for complex cases and can be scaled up for large sample numbers – differentiates between homozygous and heterozygous

Genetic Testing

• Used to detect the abnormal sickle gene - if detected, diagnosis is proven

• Accurate and useful in very complex cases where results of other tests are unclear

• Generally retained for particular circumstances where diagnostic material may be limited, and accuracy is essential – particularly pre-natal testing

Problems of Using Genetic Testing As A Diagnositic Tool

• Cost

• Time

• Expertise