Thrombin generation assays

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Thrombin generation assays!

• Anything new?
• Dr Roger Luddington
• Clinical Scientist, Addenbrookes
• CQAS 2008

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The idea is not new
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1953
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Hows it done?

• Plasma
• Tissue factor
• Phospholipid /-
• Calcium
• Thrombin substrate
• Something to measure it in.

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The thrombin generation curve
TF
no activator
ETP
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Quick catch-up

• 1967 2-stage prothrombin assay
• 1986 Subsampling method into chromogenic
  substrate
• 1993 SQ68 no subsampling
• 2000 Fluorochrom
• 2003 CAT assay
• 2005 Dade Behring
• 2008 TGA assay

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Hows it different to standard screening tests?
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Standard Screening tests

• Clot after 5 thrombin generated
• End-point at 1st sign of fibrin polymerisation
• Do not reflect physiological conditions

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Whats Physiological ?
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Physiological

• Low tissue factor trigger
• Platelets
• White cells
• Red cells
• Vessel wall
• Shear stress

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Low tissue factor

• PT reagent 6000x physiological

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The Story

• 1988 Reduced TG with heparin
• 1989 Attempt at PRP-PPP comparison
• 1994 Applied to whole blood
• 1997 DVT and CAD
• 1999 General haemostatic balance
• Pregnancy , exercise
• 2002 Clinical haemophilias
• 2002 Antiplatelet therapy
• 2003 Ximelagatran
• 2003 Everything else !?!
• 2004 Corn Trypsin Inhibitor

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Contact Inhibition
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Haemophilia A and B

• Studies have suggested bleeding tendency better
  shown by TG

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Haemophilia A and B

• Studies have suggested bleeding tendency better
  shown by TG
• PRP or PPP?

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Haemophilia A and B

• Studies have suggested bleeding tendency better
  shown by TG
• PRP or PPP?
• Inhibitor bypass therapy

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Novo 7
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FEIBA
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We dont need a fancy test for this!
FEIBA
VIIa
Nought
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Non-fancy test ½ hr later.
FEIBA
VIIa
Nought
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Haemophilia A and B

• Studies have suggested bleeding tendency better
  shown by TG
• PRP or PPP?
• Inhibitor bypass therapy
• Replacement therapy?

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Replacement therapy
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Thrombophilia

• APC / thrombomodulin resistance
• Hypercoagulability?
• Recurrent risk?

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Recurrence
188 patients Ann.risk 4.9
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Recurrence
188 patients Ann.risk 4.9
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Anticoagulant therapy

• Heparin Unfractionated and LMWH
• Novel therapeutics pre trial
• Drug monitoring?
• PCCs warfarin reversal

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Heparin
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So what has thrombin generation ever done for us?
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Apart from-

• Being able to assess the impact of FEIBA
• Being able to assess the impact of VIIa
• Potential to monitor thrombin inhibitors
• Showing influence of OCs on APCsr
• Ability to predict recurrent thrombotic risk

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ICU Case 1

• PT 13.5 secs
• APTT 36.7 secs
• VIII 2.99 iu/ml
• IX 1.69 iu/ml
• XI 0.92 iu/ml
• XII 0.20 iu/ml
• Anti Xa 0
• Lagtime 3.3 min
• ETP 1365 nmol.min
• Peak 232 nmol

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ICU Case 2

• PT 12.4 secs
• APTT 36.4 secs
• VIII 3.25 iu/ml
• IX 1.95 iu/ml
• XI 1.13 iu/ml
• XII 0.26 iu/ml
• Anti Xa 0.16
• Lagtime 11.5 min
• ETP 686 nmol.min
• Peak 49 nmol

• PT 12.5 secs
• APTT 36.7 secs
• VIII 2.97 iu/ml
• IX 1.90 iu/ml
• XI 1.18 iu/ml
• XII 0.28 iu/ml
• Anti Xa 0
• Lagtime 7.5 min
• ETP 942 nmol.min
• Peak 75 nmol

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ICU Case 3

• PT 12.8 secs
• APTT 33.2 secs
• Fgn 5.17g/L
• DD 3171 ugFEU/L
• VIII 2.77 iu/ml
• IX 1.41 iu/ml
• XI 0.70 iu/ml
• XII 0.89 iu/ml
• Anti Xa 0.57
• Lagtime 10.2 min
• ETP 279 nmol.min
• Peak 18 nmol

• PT12.7 secs
• APTT 30.4 secs
• Fgn 5.30 g/L
• DD 3312 ugFEU/L
• VIII 2.77 iu/ml
• IX 1.43 iu/ml
• XI 1.87 iu/ml
• XII 0.91 iu/ml
• Anti Xa 0
• Lagtime 6.7 min
• ETP 683 nmol.min
• Peak 124 nmol

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One assay!

• PPP / PRP / whole blood
• Tissue factor
• Lipids
• Contact inhibition
• Chromogenic / fluorogenic
• Time of assay
• alpha2macroglobulin correction

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Where are we at ?