Hemostasis Testing Involves:
- PT (prothrombin time), PT INR
- PTT (activated partial thromboplastin time)
- PT/PTT mixing study
- Thrombin time
- Coagulation factor assay
Lupus anticoagulant assay
Platelet function test
- Platelet aggregation study
A functional assay is performed using what part of the blood?
What is citrate? How does it work?
Citrate is an anticoagulant that works by chelating calcium
- It is used in the performance of a functional assay to separate out the plasma
Process of performing a functional assay on plasma?
- Use citrate as anticoagulate (chelates Ca)
- Centrifuge -> supernatant = platelet poor plasma (PPP)
- PPP contains very small amt of platelets
Antigenic assay is usually performed using what part of the blood?
- Serum contains very small amt of coagulation factors and platelets
What is the difference between plasma and serum?
- Plasma contains coagulation factors and complements
- Serum contains very few coagulation factors and complements since they go to a clot
Describe the coagulation cascade
PT measures what coagulation factors? PTT?
PTT: (8, 9, 11, 12)
Which pathway is more sensitive to common pathway factors?
- When fibrinogen is 80 mg/dL, PT is prolonged but PTT is not
- When fibrinogen is 25 mg/dL, PTT is also prolonged
What is INR? Why is it needed?
International Normalized Ratio
- Designed to monitor pts who take warfarin as an anticoagulate
- PT reagents differ in their sensitivity towards FII, FVII, and FX (2, 7, 10); PT from one lab may not be comparable to PT from another lab
What is thrombin time? When would it be prolonged?
Measures the conversion of fibrinogen to fibrin after addition of excess thrombin to undiluted plasma
Reasons for prolongation:
- Positive FSP (or D-dimer)
Process of fibrinogen assay?
- Clauss assay (thrombin time based assay) is widely used = function fibrinogen
- Thrombin and phospholipid is added to 1:10 diluted pt plasma
- Initiate clotting by addition of Ca
- Functional fibrinogen is inversely proportional to thrombin time
How does functional fibrinogen correspond to thrombin time?
Functional fibrinogen is inversely proportional to thrombin time
What is the normal range of functional fibrinogen?
- For surgery or delivery, minimum fibrinogen required is 200 mg/dL
What is FDP? FSP?
- FDP- fibrin degradation prodcuts
- FSP- fibrin split products
Causes of Prolonged PT/PTT (broad list)?
1. Factor deficiency
- Decreased synthesis
- Increased consumption
- Dilutional coagulopathy (massive transfusion)
2. Circulating inhibitor
3. Adsorption of FX by amyloid
What are factor deficiencies that involve decreased synthesis?
- Congenital factor deficiency (usually 1 factor)
- Liver failure (all factors except FVIII and FXIII (8 and 13))
- Vitamin K deficiency (FII, FVII, FIX, FX) (2, 7, 9, 10)
- Warfarin (FII, FVII, FIX, FX) (2, 7, 9, 10)
- Transient deficiency
What are factor deficiencies that involved increased consumption?
- DIC (all factors)
- Massive thrombosis (all factors)
What are some examples of circulating inhibitor contributing to prolonged PT/PTT?
- Factor specific inhibitor
- Lupus anticoagulant
- Anticoagulant such as heaprin, LMW heparin, and thrombin inhibitor
What are the steps you should take in a prolonged PT/PTT work up?
- Repeat in an optimal condition
- Rule out possible anticoagulant use (heparin removal)
- PT/PTT mixing study
- Individual factor assay
- Assay for lupus anticoagulant
Principles of a mixing study?
1. If an inhibitor is present, it will inhibit normal and pt plasma, AND
2. That 50% of any factor is enough to yield a normal test result. In fact, 20-40% of the normal level of coagulation factor is generally sufficient to give a normal PT and PTT
Since they are screening tests, the result should be confirmed by factor assay and/or lupus anticoagulant assay
What is Lupus Anticoagulant (LAC)?
Misnomer; not an anticoagulant and not always associated with SLE
- Interferes with phospholipid-dependent coagulation tests such as PT and PTT
- Does not specifically neutralize any coagulation factors -> LAC does NOT cause any bleeding in spite of prolonged PTT
Presence of LAC is a risk factor for what?
LAC combined with what may cause bleeding?
LAC with concurrent factor II deficiency or immune thrombocytopenia
Describe the assay for Lupus Anticoagulant
There is no "lupus anticoagulation test"
It is recommended to perform 2 tests for LAC:
1. DRVVT- dilute Russell's viper venom test
2. Hexagonal phase phospholipid neutralization test
No gold standard for LAC
Describe the Dilute Russell's Viper Venom Test (DRVVT).
What are you looking for?
Looking for LAC
- RVV activates F. X directly
- The test is made more sensitive to LAC by dilute RVV and dilute phospholipids.
- Contains more phospholipids which may neutralize LAC.
- Take ratio of screen and confirmatory.
Describe the Hexagonal Phase Phospholipid Neutralization Test. What are we looking for?
Looking for LAC
- Tube 1 is a PTT
- Tube 2 contains hexagonal phase phosphatidyl ethanolamine (PE) which may neutralize LAC
- Equal volume of patient’s plasma, normal plasma, and PE are incubated for 5 min and then measure PTT.
- Addition of normal plasma can correct factor deficiency
- 37 yo male with pre-op screening
- PT: 13.2 s (normal 11.4-11.9)
- PTT: 66.0 s (normal 23-37)
In the PTT mixing study:
- 0 min 47.3 s - 90 min 51.3 s
- No correction at 0 min and after 90-min incubation.
- The results are suggestive of the presence of a circulating inhibitor, most likely lupus anticoagulant
Lupus Anticoagulant Assay performed
- Screen: 76.7 s
- Confirm: 37.6 s
- What is S/C?
Positive or negative test result?
Hexagonal phase phospholipid neutralization test?
- Tube 1: 96.5 s
- Tube 2: 68.7 s
- Tube 1- Tube 2?
Positive or negative?
- S/C: 2.04 s
- Positive result (normal under 1.35)
Hexagonal phase phospholipid neutralization test
- Tube 1 - Tube 2: 227.8 s
- Positive result (normal under 8s)
What is the use of specific coagulation factor assays be done?
Determine the extent to which the patient's plasma corrects the clotting time of plasma deficient only in a particular clotting factor
- Example: add patient plasma to factor VIII-deficient plasma and perform an PTT on the mix; compare result to standard
Factor VIII assay example:
- Calibration curve
- Pt plasma
Overview of platelet function
Steps in platelet activation?
- Shape change
- Secretion (fibrinogen, vWF, ADP, Ca)
What factors may activate platelets?
What substances are secreted by activated platelets?
What is platelet Lumi-aggregometry?
Platelet Aggregation Tracing?
What does a typical platelet aggregation study look like?
What may result in abnormal results of a platelet aggregation study?
Thrombocytopenia Platelet function tests:
- Drugs, uremia, myeloproliferative disorders
- Bernard-Soulier syndrome (ristocetin)
- Glanzmann thrombasthenia (all agonists except ristocetin)
- Storage pool disease (epinephrine and ADP)
- von Willebrand disease (ristocetin)
- Basic coagulation tests are PT, PTT, and fibrinogen.
- PT is more sensitive to fibrinogen level and other common pathway factor levels.
- Lupus anticoagulant is a risk factor for thrombosis.
- Platelet aggregation study is one of the tests for platelet function.
T/F: When PT is prolonged, but PTT is normal, only possibility is low factor VII
- Even if FX is low, PT may be low but PTT may be normal (due to sensitivity differences)
T/F: Presence of lupus anticoagulant is a risk for bleeding
T/F: When platelet aggregometry shows decreased maximal aggregation, you can conclude that the patient has platelet function defects
- Need to rule out thrombocytopenia