Haematology (Thrombophilia)

Question 1

How is haemostasis maintained?

Answer 1

A balance of pro and anti coagulant mechanisms

Question 2

What is thrombophilia?

Answer 2

An increased tendency of the blood to clot

Question 3

Thrombophilia leads to an increased risk of what?

Answer 3

Venous thrombosis

Question 4

Give some examples of inherited causes of thrombophilia

Answer 4

Protein S or Protein C deficiency

Antithrombin deficiency

Question 5

Give some examples of acquired causes of thrombophilia

Answer 5

• Persistent antiphospholipid syndrome
• Hormone therapy
• Contraceptive use

Question 6

What factors are usually elevated in thrombophilia?

Answer 6

Factor VIII, IX and XI

Question 7

Give some examples of thrombotic diseases

Answer 7

Deep vein thrombosis (DVT)

Pulmonary embolism

Question 8

What 2 types of defects result in Protein C, Protein S and antithrombin deficiencies?

Answer 8

Type I - affects concentration of the protein

Type II - affects the function

Question 9

Give an example of a type I antithrombin defect

Answer 9

Heterozygous Type I antithrombin deficiency:

• 50% reduction in antithrombin
• 10 fold higher risk of venous thromboembolism

Question 10

What lab assays for antithrombin can be used to distinguish between type I and type II patients?

Answer 10

• Laurell Rocket electrophoresis
• Radial immunodiffusion
• ELISA
• Latex bead-based assays

Question 11

What is the principle of the Laurell Rocket immunoelectrophoresis assay?

Answer 11

1) Gel plate prepared containing antibodies against target antigen
2) Plasma is added to wells and current in applied
3) Proteins move across plate and the antigen is captured and precipitated by the antibody
4) Distance travelled by antigen is proportional to the quantity of antigen in plasma
5) Can be visualised by staining the plate after dying, peak height plotted against percentage of protein present

Question 12

Describe the functional activity assay for antithrombin

Answer 12

1) Plasma incubated with excess of heparin and FXa
2) Excess heparin and FXa result in all the antithrombin forming AT-heparin-FXa complexes
3) Residual FXa is reacted with a chromogenic substrate

4) FXa cleaves the chromogenic substrate to release a coloured product
- colour intensity is inversely proportional to the antithrombin concentration

Question 13

Why do assays look at the activity of Protein C rather than the concentration?

Answer 13

Protein C levels are so low in the plasma

Question 14

Describe the assay use for Protein C

Answer 14

• Protac added - activates Protein C
• Addition of all the elements required for clotting - elongation of clotting time directly proportional to levels of Protein C
• Alternatively, a chromogenic substrate may be used

Question 15

What 3 assays can be used to measure Protein S?

Answer 15

1) Free protein S latex immunoassay
2) Protein S ELISA
3) Protein S functional assay

Question 16

Why is measuring total Protein S alone worthless?

Answer 16

Because 60% of Protein S in plasma is bound to complement C4b-binding protein

Question 17

Describe the free protein S latex immunoassay

Answer 17

1) Latex beads coated with C4b-binding protein are added to plasma
2) Only free Protein S not already bound to C4b binds to the beads
3) Addition of second set of beads with an antibody for protein S results in further binding and clumping of beads, which can be measured optically
4) Turbidometric changes (changes in light intensity of light passed through sample) are directly proportional to amount of free Protein S

Question 18

How does ELISA work for Protein S?

Answer 18

1) Microtitre plate coated with either polyclonal antibody against Protein S or monoclonal antibody that binds only yo free Protein S
- Protein S binds to antibody
- Addition of secondary antibody/enzyme conjugate that binds to protein S
- Addition of substrate that becomes coloured product

Question 19

How does the Protein S functional assay work?

Answer 19

Work similarly to Protein C assay

1) Test plasma diluted in PS-deficient plasma to ensure excess of clotting factors
2) Activation of coagulation factor and clotting factors added
3) Any increase in clotting time direct result of action of Protein S

Question 20

Give an example of a mutation that results in an increased risk of thrombosis

Answer 20

The Factor V Leiden mutation

Question 21

How id Factor Va cleaved in wild-type individuals and what is the result?

Answer 21

• Factor Va cleaved by activated Protein C and cofactor Protein S
• Cleavage at positions Arg306, Arg506 and Arg679 to inactivate enzyme to reduce coagulation

Question 22

What occurs in a mutated individual?

Answer 22

• Mutation at position 506 in the Factor V Leiden gene results in substitution of Gln for Arg
• Fva no longer cleaved, increasing clotting tendency

Question 23

In patients with Factor V Leiden mutations, what is the risk of clotting in heterozygous and homogyzous individuals?

Answer 23

Heterogygous - 2.5 fold increase in thrombosis risk

Homozygous - 80 fold increase

Question 24

What are some other causes of thombophilia?

Answer 24

• Mutations in genes for fibrinogen
• Defects in fibrinolysis
• High levels of clotting factors e.g FVIII, FIX and FXI
• Antiphospholipid syndrome

Question 25

What is antiphospholipid syndrome?

Answer 25

An autoimmune disorder characterised by one or more episodes of thrombosis or pregnancy morbidity (loss of foetus)

Question 26

What causes APS?

Answer 26

The persistent presence of antiphospholipid antibody (APA)

- a heterogeneous groups of Abs targeting phospholipid binding proteins such as prothrombin or beta-2-glycoprotein

Question 27

Is are APAs coincident, consequent or causative of thrombotic disease?

Answer 27

It is unclear which

Question 28

Name some other thrombosis related disorders

Answer 28

1) Arterial thrombosis - blood vessel is narrowed due to atheromas
2) Thrombotic thrombocytopenic purpura (TTP) - highly active Von Willebrand Factor (VWF)
3) Haemolytic uraemic syndrome - follows infections with E.coli 0157-H7 which produced Shiga toxin