L13 - Coagulation and Haemophilia Flashcards
Haemostasis
• A complex process involving:
- Platelets
- Plasma proteins
- Endothelial cells
- Smooth muscle cells
•Keeps the blood fluid while ensuring that blood loss is minimised at a site of vessel injury
Old theories of blood coagulation
- Cooling
- Blood clotted when it cooled
- Rest
- Motion inhibited clotting, rest encouraged it
- Vital Force
- Blood was alive in blood vessels and died outside them
- Air
- Exposure to air (either oxygen or nitrogen) caused the blood to clot
Prothrombin Time
- Plasma (100 µL)
- Normal (control) or patient plasma
- Thromboplastin - 200 µL (Tissue factor, phospholipids, calcium (0.025 M))
- Reactions proceed through extrinsic/common pathways
- Generates fibrin (clot)
- Record clotting time (1 decimal place)
- Clotting factor deficiencies (VII, X, V, II, I)
- Monitor oral anticoagulant therapy (warfarin)
Partial Thromboplastin Time
- Plasma (100 µL)
- Partial Thromboplastin (Phospholipid -100 µL)
- Activator (100 µL) -negatively charged e.g. kaolin
- Incubate for 3 minutes
- Calcium chloride (0.025 M) (100 µL)
- Reaction proceeds through intrinsic/common pathways
- Generates fibrin clot -record clotting time (1 d.p.)
- Clotting factor deficiencies (I, II, V, VIII, IX, X, XI, XII)
- Monitor anticoagulant therapy (UF heparin)
Normal PT, Prolonged aPTT
- Extrinsic and common pathways are normal
* Deficiency in intrinsic pathway (Factor VIII, IX, XI, or XII)
Prolonged PT, Normal aPTT
- Intrinsic and common pathways are normal
* Deficiency in extrinsic pathway (Factor VII)
Prolonged PT, Prolonged aPTT
- Deficiency in common pathway (Factor X, V, Prothrombin, Fibrinogen)
- Deficiency in multiple coagulation factors
Pattern of inheritance of haemophilia
- Males were affected by the disease
* Females were vary rarely affected, but they could pass the disease onto their offspring (carriers)
Those affected from haemophilia suffered from bleeding into:
- Muscle/soft tissue
- Brain
•Joints were involved (? Arthritis)
Haemophilia A
deficiency of Factor VIII
Haemophilia B
deficiency of Factor IX
Incidence of haemophilia in males
1:5000 –1:25 000 male births
Haemophilia as an X-linked, recessive disease
- Females that carry the mutation are called ‘carriers’
- Carriers have a 50% chance of passing the mutation onto their sons
- Male haemophiliacs will pass the mutation to their daughters, while their sons are unaffected
- 1980’s -Both Factor VIII and IX genes were cloned and found to reside on the long arm of the X-chromosome
Haemophilia in females (rare cases):
- Offspring of a male haemophiliac and a female carrier
- Occurrence of two factor VIII gene mutations
- Extreme Lyonisation (or X-inactivation)
Lab diagnosis of haemophilia
•Mixing studies
- Mix an equal volume of patient plasma with normal plasma
- Perform an aPTT
- If the patient has a factor deficiency, the missing factor is replaced by the same factor that is present in the normal plasma
- The aPTT is corrected back to normal (i.e. normalized)
•Factor assays to determine the concentration of FVIII and FIX present in the patient plasma
Haemophilia Clinical Manifestations in newborns
- Bleeding at the umbilical stump
- Bleeding after circumcision
- Bleeding with heel pricks/immunisation
- Rarely, newborns will present with severe bleeds
- Bleeding in infants often does not occur until they become mobile
Development of haemophilia joint disease
- An acute bleed into the joint increases pressure within the synovial cavity
- Recurrent bleeding causes chronic synovitis and damage to the cartilage and bone, → haemarthrosis
- Haemarthrosisoccurs in three stages
Stages of haemoarthosis
- Acute haemarthrosis
• Bleed into the joint - Chronic synovitis
• Iron (from lysed RBCs) promotes the production of inflammatory cytokines
• Synovium becomes vascular and inflammatory cells are recruited
- Degenerative arthritis • Exposure of blood and cytokines to the cartilage results in its destruction
Pseudotumours
- A clinical manifestation of haemophilia
* Large encapsulated haematomas in large muscle groups and some bones
Intracranial haemorrhage
- Can occur in the neonatal period (3-5% of haemophiliacs)
* Childhood and adult ICH usually occurs after trauma
Treatment of haemophilia
- Early “treatments”
- Russell’s Viper Venom
- Blood transfusion
- Fresh Frozen Plasma (FFP)
- Cryoprecipitate
- Factor VIII and IX concentrates
- Haemophilia Treatment Centres –early 1970’s
Early “treatments”
• Administration of lime, O2, use of thyroid gland, bone marrow, H2O2, gelatin etc.
Russell’s Viper Venom
• Used in the 1930’s for “local” applications
Blood transfusion
- First documented case for the treatment of haemophilia was in 1840
- Not accepted as a routine treatment until the 1940’s
Fresh Frozen Plasma (FFP)
- Used for treatment in the 1950s/1960s
- Recipient required hospitalisation
- Required large volumes
Cryoprecipitate
- Prof Judith Pool discovers that, if FFP is allowed to slowly thaw in the cold, a precipitate forms. This can be collected by centrifugation and the remaining plasma recombined with the remaining blood
- The precipitate, called cryoprecipitate, contains FVIII and fibrinogen and can be stored frozen
- Allowed injection of concentrated FVII
Factor VIII and IX concentrates
- Late 1960’s –methods to purify fVIII and fIX from plasma were described
- Allowed the production of fVIII and fIX concentrates
- Large volumes of plasma were pooled from multiple donors to make one batch of concentrate
- Early 1970’s –factor concentrates were available for home use
Blood related discoveries in the late 1970s/1980s
- 1984 –HIV was identified
- 1985 –HIV testing in blood facilities implemented
- 1989 –Hepatitis C was isolated
- 1990 –Hepatitis C testing of blood donors began
- Was estimated that 50% of haemophiliacs succumbed to viral infection
Recombinant factor treatments 1980s/1990s
- 1982 –factor IX was cloned
- 1984 –factor VIII was cloned (These important discoveries allowed the production of recombinant fVIII/fIX protein)
- 1992 –recombinant fVIII was licensed for the treatment of haemophilia A
- 1997 –recombinant fIX was licensed for the treatment of haemophilia B
On-demand treatment regimes
- Affected individuals are treated with replacement factor only when a bleeding episode begins
- Does not prevent bleeding episodes from starting
Prophylactic treatment regimes
- Affected individuals are treated with replacement factor 1-4 times a week
- Achieves a basal level of factor that maintains haemostasis (~30%)
- A significant amount of factor is used
Life expectancy of haemophilia by the 1980’s
- Before the advent of cryoprecipitate, males rarely lived to reproductive age. The average life expectancy was 11 years.
- by 1980s this was increased to 50-60 years
- Use of factor concentrate had a significant effect
Life expectancy of haemophilia today
- Normal life expectancy
- Use of recombinant factor and prophylactic treatment
- Death from other causes (i.e. cardiovascular disease etc)
Future treatment of haemophilia
Gene therapy:
- Recent studies have investigated the use of gene transfer to insert a normal VIII/IX gene into affected individuals
- Factor IX gene is put into a vector which targets the production of factor IX protein to the liver
- Maintains FIX at 1-6% of normal in individuals with severe haemophilia B
- Decreases the amount of factor IX concentrate used
- Decreases the number of bleeding episodes
Royal family haemophilia
- It was hypothesised that those affected by Haemophilia in the Royal Family had Haemophilia A
- In 2009, it was reported that the remains of all Russian Royal Family members were found
- They found that there was no mutation in the factor VIII gene (F8), but there was one in IX gene
- Therefore, the Royal Family had haemophilia B
