Haematology (Thrombophilia) Flashcards
How is haemostasis maintained?
A balance of pro and anti coagulant mechanisms
What is thrombophilia?
An increased tendency of the blood to clot
Thrombophilia leads to an increased risk of what?
Venous thrombosis
Give some examples of inherited causes of thrombophilia
Protein S or Protein C deficiency
Antithrombin deficiency
Give some examples of acquired causes of thrombophilia
- Persistent antiphospholipid syndrome
- Hormone therapy
- Contraceptive use
What factors are usually elevated in thrombophilia?
Factor VIII, IX and XI
Give some examples of thrombotic diseases
Deep vein thrombosis (DVT)
Pulmonary embolism
What 2 types of defects result in Protein C, Protein S and antithrombin deficiencies?
Type I - affects concentration of the protein
Type II - affects the function
Give an example of a type I antithrombin defect
Heterozygous Type I antithrombin deficiency:
- 50% reduction in antithrombin
- 10 fold higher risk of venous thromboembolism
What lab assays for antithrombin can be used to distinguish between type I and type II patients?
- Laurell Rocket electrophoresis
- Radial immunodiffusion
- ELISA
- Latex bead-based assays
What is the principle of the Laurell Rocket immunoelectrophoresis assay?
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
Describe the functional activity assay for antithrombin
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
Why do assays look at the activity of Protein C rather than the concentration?
Protein C levels are so low in the plasma
Describe the assay use for Protein C
- 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
What 3 assays can be used to measure Protein S?
1) Free protein S latex immunoassay
2) Protein S ELISA
3) Protein S functional assay
Why is measuring total Protein S alone worthless?
Because 60% of Protein S in plasma is bound to complement C4b-binding protein
Describe the free protein S latex immunoassay
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
How does ELISA work for Protein S?
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
How does the Protein S functional assay work?
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
Give an example of a mutation that results in an increased risk of thrombosis
The Factor V Leiden mutation
How id Factor Va cleaved in wild-type individuals and what is the result?
- Factor Va cleaved by activated Protein C and cofactor Protein S
- Cleavage at positions Arg306, Arg506 and Arg679 to inactivate enzyme to reduce coagulation
What occurs in a mutated individual?
- Mutation at position 506 in the Factor V Leiden gene results in substitution of Gln for Arg
- Fva no longer cleaved, increasing clotting tendency
In patients with Factor V Leiden mutations, what is the risk of clotting in heterozygous and homogyzous individuals?
Heterogygous - 2.5 fold increase in thrombosis risk
Homozygous - 80 fold increase
What are some other causes of thombophilia?
- Mutations in genes for fibrinogen
- Defects in fibrinolysis
- High levels of clotting factors e.g FVIII, FIX and FXI
- Antiphospholipid syndrome
What is antiphospholipid syndrome?
An autoimmune disorder characterised by one or more episodes of thrombosis or pregnancy morbidity (loss of foetus)
What causes APS?
The persistent presence of antiphospholipid antibody (APA)
- a heterogeneous groups of Abs targeting phospholipid binding proteins such as prothrombin or beta-2-glycoprotein
Is are APAs coincident, consequent or causative of thrombotic disease?
It is unclear which
Name some other thrombosis related disorders
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
