Platelet disorders and VWD Flashcards
What is the VWD called which is associated with a short half life of VWF?
Type 1 Vicenza
**may be diagnosed by looking at post DDAVP VWF levels over 24 hours and noticing a quick decline.
What initial tests are recommended in VWD testing?
- FBC, PFA, APTT and PT
2. VWF:Ag, VWF:CB, VWF:RCo, Factor VIII level
How is VWF:Ag measured?
Immunoassay - our lab uses latex immunoturbidimetric method.
Other options = ELISA and ECLIA
What can interfere with an immunoturbidimetric assay?
- RF/heterophile antibodies - may give falsely high results
- Human anti-mouse antibodies may neutralise the antibody and give falsely low results
- Paraproteins - can give falsely high and low results.
How does your lab perform the VWF:RCo Assay
Ristocetin cofactor assay = high CV
Recombinant GP1b with mutation that allows VWF binding without unfolding.
Incubate rGP1b with patient plasma (PPP). Add beads coated in antibody against GP1b.
Immunoturbidimetric assay (VWF-dependent agglutination). Read off standard curve.
Describe the original ristocetin cofactor assay
Commercial platelets + patient plasma + ristocetin ==> measure agglutination on aggregometer and read off standard curve.
N.B. must first check that the platelets do not aggregate in the presence of ristocetin alone
Describe the collagen binding assay at your laboratory (VWF:CB)
The VWF:CB assay is sensitive to deficiencies in HMW VWF.
This is a manual ELISA test.
Collagen is on plates
Add patient plasma containing VWF. Wash off unbound material (VWF binds to collagen).
Add rabbit anti-VWF coupled to an enzyme peroxidase then wash off unbound antibody.
Add substrate. Colour change is proportional to VWF bound.
How is VWF multimeric analysis performed?
- Heat plasma to get rid of charge so separation is based only on size
- Run on gel electrophoresis
- Bands are stained (enzyme linked detection)
Describe the RIPA test for diagnosing type 2B VWD
Patient platelet rich plasma is mixed with low and high dose ristocetin
==> VWD type 2B is diagnosed if there is a response to low dose ristocetin as well as high dose.
- increased binding of VWF to GP1b
- measured on platelet aggregometer
How is the VWF-Factor VIII binding assay performed
Manual - ELISA
- Platelets coated with anti-VWF
- Patient plasma is added and VWF binds. Add calcium chloride to strip endogenous FVIII
- Add recombinant factor VIII in a known quantity
- Add enzyme linked antibodies against VWF and FVIII and then add substrates
- Calculate ratio
What are the typical Factor VIII levels in VWD?
Factor VIII is typically 1.5 times the VWF
Therefore will be low in most type 1
Factor VIII is <10u/dL in type 3 VWD
Evaluation of suspected HIT
Primarily clinical - use the 4T score. If low then the diagnosis can be excluded and no further testing is required.
1. Timing 4-14 days
2. Thrombocytopenia (>50% fall, nadir >20)
3. Thrombosis or skin necrosis at site of injection or systemic reaction after IV bolus
4. oTher causes excluded
High (6-8) or intermediate (5-5) requires further testing.
Risk factors for developing HIT
- UFH 10x higher than LMWH
- Surgical and trauma patients are at higher risk than medical and pregnant patients
- 1:1 ratio of PF4 to heparin (very high doses of heparin used in cardiothoracic surgery may be protective against HITT although the incidence of antibodies is high)
- Prophylactic heparin dose is more likely to cause HIT than the therapeutic dose
Name 6 different methods used to test for HIT antibodies
Immunoassays:
1. ELISA (PF4-heparin coats well, add patient plasma)
2. Particle gel immunoassay (PF4-heparin coats red particles, add with patient plasma to column)
Functional assays:
3. Serotonin release assay (SRA)
4. Heparin-induced platelet aggregation test (HIPA)
5. Rapid aggregation test
6. Whole blood impedance aggregometry
What is HIT?
Heparin induced thrombocytopenia is a life and limb threatening condition. Autoantibodies develop against the PF4-heparin complex and are platelet activating ==> platelet aggregation and thrombosis.
