Thrombophilia Flashcards
How is the DRVVT performed?
- Pooled normal plasma is mixed with diluted PL at 37°C.
- Diluted RVV and then Calcium chloride are added and the clotting time is measured.
- The test is then repeated using patient plasma and the ratio of test: normal plasma is calculated.
- Excessive phospholipid in then added and the test repeated. If the DRVVT decreases, then this indicates that the prolongation in DRVVT is phospholipid dependent
Why is the phospholipid diluted in the DRRVT?
By diluting the phospholipid you make the test dependent on phospholipid. As the lupus anticoagulant binds to phospholipid, the more dilute the phospholipid, the more sensitive the test is to detect the lupus anticoagulant.
What are the ISTH recommendations for LAC testing?
- Fresh, citrated blood, doubled centrifuged
- Two tests that employ different principles should be used.
- Currently the ISTH recommends that that DRVVT be used as the initial test.
- The second test should be a sensitive activated partial thromboplastin time (low phospholipids and silica as the activator recommended i.e. to SCT)
- LAC positive if one of the two tests positive
- DRVVT should not be performed if the thrombin time of the plasma is significantly prolonged or if the patient is on anticoagulation ((warfarin when INR >3).
What constitutes a positive PL correction in LA testing?
> 10%
What are the causes of a positive DRVVT test result?
· Lupus anticoagulant
· Abnormalities of FII, FV, FX or fibrinogen
· Anticoagulation
What is the silica clotting time?
- APTT test in which Silica is the activator
- Contains a low dose of phospholipid
- Can also do the test with high phospholipid as confirmation
What is the Kaolin clotting time?
- Essentially an Activated Partial Thromboplastin Time test but without any added phospholipid.
- The test relies on residual cell membrane fragments and plasma lipids to provide a Phospholipid surface for coagulation reactions.
Dilutions of Normal:Patient plasma are prepared. Kaolin is added and then calcium to initiate coagulation. The KCT is the time from adding calcium to clot formation.
There is no phospholipid correction step for the KCT.
What is the biggest issue with the kaolin clotting time?
- The KCT relies on small quantities of phospholipid in the patient’s plasma and so is particularly sensitive to platelet contamination of the plasma sample, which greatly reduces the sensitivity of the test.
- A KCT of <60 s suggests contamination of the normal control plasma with platelet fragments and invalidates the result.
In which patients is it reasonable to do thrombophilia screening?
- Patients presenting with VTE under the age of 50 years
- History of recurrent thromboembolism
- Thrombosis occurring in an unusual site
- Warfarin-induced skin necrosis and thrombosis
- Following use of oestrogen-containing contraception or hormone replacement therapy (HRT).
- Strong family history
In what circumstances should thrombophilia screening be avoided?
- Provoked VTE
- VTE during pregnancy/ post-partum
- Unprovoked VTE (patients generally need long term treatment and recurrence risk is the same in patients with and without inherited thrombophilia).
- In relatives of VTE patients considering hormonal manipulation (first degree relatives have a predictable increased risk of thrombosis with oestrogen use, even when thrombophilia testing is negative).
- Recurrent arterial thromboses
- In patients undergoing investigation for infertility
How can antithrombin deficiency be diagnosed
Chromogenic assay
o Heparin activates antithrombin which leads to rapid inactivation of thrombin, FIXa and FXa.
o Patient plasma is incubated with heparin and either thrombin (bovine) or FXa
o Chromogenic substance that acts on thrombin or Xa added.
o Amount of residual thrombin/ Xa is measured spectrometrically. The antithrombin level in the patient sample is derived using a standard curve (inversely proportional)
Antigen assay (ELSIA, latex agglutination)
Genotyping/ PCR
How is protein C deficiency diagnosed?
1) Chromogenic assay using protac: snake venom which activates protein C
- After patients plasma is incubated with protac, a chromogenic substance is added and the amount of dye produced is measured.
- The amount of dye produced is inversely proportional to the amount of APC (more APC –> less Xa generated, less dye produced)
- Levels of protein C read of a standard curve generated using serial dilutions of a standard/ control plasma
2) Clot based assay: modified APTT using Protac to activate protein C
- Dilutions of normal and test plasma are mixed with PC deficient plasma.
- The clotting time of normal plasma is prolonged (>100s) due to the inactivation of FVa and FVIIIa by protein C.
- The clotting time of PC deficient plasma is normal.
How is APCr/ Factor V Leiden tested for
1) Clot based assay: APTT measured before and after administration of APC and expressed as a ratio (APTT+ APC)/ (APTT- APC)
- This can be normalised by dividing by an APTT derived from pooled normals + and - APC addition.
- Can be further modified by pre-diluting in FV deficient plasma
- This reduces the number of exogenous confounding factors that might affect the APTT but makes it very sensitive to the effects of LAC.
- This modification makes the assay specific only for mutations within Factor V whereas the original APTT measures APC resistance from any cause.
2) Genotyping/ PCR (>90% of cases result from a polymorphism encoding a single amino acid change)
What are the causes of acquired APC resistance?
Elevated FVIII levels
Pregnancy or oestrogen exposure.
What are the causes of acquired protein S deficiency?
Vitamin K deficiency Vitamin K antagonists Liver disease Pregnancy Oral contraception SLE HIV Infection (varicella)
