Fibrinolysis Testing

Question 1

What situations in the patient’s clinical

history would warrant a fibrinolysis workup ?

Answer 1

• bleeding associated with cardiopulmonary bypass, liver transplant or trauma
• clinical history of unexplained bleeding
  
  • PAI-activity
  • PAI antigen
  • tPA antigen
  • antiplasmin
• clinical history of unexplained arterial thrombosis
  
  • PAI activity
  • PAI antigen
  • tPA antigen
  • CRP

Question 2

What is the usual presentation of someone

presenting with fibrinolysis associated bleeding ?

Answer 2

• delayed bleeding often associated with
  
  • recurrent, excessive wound hematomas after surgery or trauma
• history of unexplained arterial thrombosis
  
  • absence of arterial risk factors:
    
    • HTN, DM
    • Hyperlipidemia, smoking etc

Question 3

What is important when drawing samples for evaluation

of the fibrinolytic pathway ?

Answer 3

• needed citrate anticoagulated whole blood
• affected by
  
  • circadian rhythms
  • acute phase responses
• samples should be drawn in the morning around 8 am usually
  
  • with references based on the time of day it was drawn

Question 4

What other lab test should be drawn/evaluated

to determine that tPA and PAI-1 levels are not

due to an acute phase response?

Answer 4

• CRP
  
  • if normal then it suggests that the tPA response is not occurring

Question 5

Which fibrinolytic test cannot use

citrated anticoagulated tubes?

Answer 5

• tPA activity
  
  • needs acidified citrated plasma

Question 6

When testing for PAI-1, what

needs to be taken into consideration ?

Answer 6

• cannot activate platelets
  
  • this would result in a release of PAI-1 from alpha granules
  • blood should be drawn into a citrate anticoagulated tube and kept at room temperature until the platelets can be removed.

IMP: EDTA can stimulate platelet release and should not be used as an anticoagulant in fibrinolytic studies

Question 7

How can fibrinogen, fibrin, and degradation products

be measured?

Answer 7

• immunologically using specific antibodies
• currently most common D-dimer assay measures two fibrin D-domains which are cross-linked by factor XIIIa
  
  • these are released by plasmin

Question 8

What are things to consider when

evaluating D-dimer assays?

Answer 8

• results depend on the target of the antibody used
  
  • in vivo, there are many different fibrin degradation products
• also different companies use different units to report findings
• d-dimer fragment is approximately half the molecular weight of fibrin
• most suffer from interference by human anti-mouse monoclonal antibodies
  
  • rheumatoid factors (false negative or positive)

read p. 124

Question 9

What is one situation where a

false increase in D-dimer can occur ?

Answer 9

• excessive levels of plasmin or plasminogen activators
• can lead to in vitro formation of fibrin fragments

Question 10

How are plasminogen levels in plasma

usually measured ?

Answer 10

• measured by activity assay
  
  • usually by addition of steptokinase to plasma
  • binds to plasminogen and forms a plasmin like complex
  • a plasmin sensitive substrate (chromogenic) is added to the sample and absorption is measured
• reported as % of normal plasma
• can also measure antigen by using polyclonal or monoclonal antibodies with immunoassays

Note:

• streptokinase-plasminogen complex is inhibited by alpha-antiplasmin

Question 11

How is alpha 2 antiplasmin measured ?

Answer 11

• using a back titration
• excess plasmin is added to the mixture and incubated to allow the plasmin to react to completion with alpha 2 antiplasmin
  
  • then a plasmin-sensitive chromogenic substrate is added to measure the amount of remaining residual plasmin activity
• reported as % of normal plasma

IMP: these assays are not truly specific for alpha 2 antiplasmin

• they will detect anything in plasma that inhibits plasmin

Question 12

What synthetic inhibitors of plasmin can

produce falsely elevated results of alpha 2 antiplasmin activity ?

Answer 12

• epsilon aminocaproic acid
• tranexamic acid
• aprotinin

This is important because anti-fibrinolyitic drugs are often used during cardiopulmonary bypass surgery

Question 13

When could you see falsely low results for alpha 2

antiplasmin?

Answer 13

• if samples contain other enzymes that can cleave the plasmin-sensitive chromogenenic substrate
  
  • makes plasmin activity look increased

Question 14

How does the level of PAI-1 affect the levels of tPA in the blood ?

Answer 14

• increased PAI-1 increases the fraction of tPA-PAI-1 but does actually lowers the levels of active tPA in the blood
• in vivo
  
  • PAI-1 can never completely block the activity of tPA because new tPA is constantly being released by the endothelium
• ex vivo
  
  • if the PAI-1 activity is not blocked after the blood is drawn then the levels of tPA will be low
    
    • this is done by drawing the sample into acidified citrate solution

Question 15

How is tPA measured in the lab ?

Answer 15

• measure the activity of tPA converting plasminogen to plasmin
  
  • then plasmin activity is evaluated using a plasmin sensitive chromogen
  • need a catalyst for tPA to function
    
    • usually fibrin or fibrinogen is added to the assay

Question 16

What is the major problem to overcome

in tPA assays?

Answer 16

• the presence of active PAI-1 or alpha-2 antiplasmin

Question 17

What is the normal activity of tPA in vivo ?

Answer 17

• peak activity in the evening around 1800
• nadir of activity in the morning around 0600

Note: this is the opposite for total tPA antigen because it measures also what is bound to PAI-1

see page 128 for more details

Question 18

What is a common TPA immunoassay

inhibitor or interference ?

Answer 18

• rheumatoid factors
• human anti-mouse antibodies

Question 19

In vivo, PAI-1 is produced where ?

Answer 19

• liver
• abdominal adipose tissue
• platelet alpha granules

Question 20

What is the preferred way to measure

PAI-1 activity ?

Answer 20

• bioimmunoassay
• see p. 128 for details