Introduction to Special Coagulation a survey of the coagulation system and a review of clotting vs c

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Introduction to Special Coagulation a survey of
the coagulation system and areview of clotting
vs chromogenic assays
S. Rosen (Rossix) and J Kiblinger (Diapharma)
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Coagulation Cascade Then Now

• Waterfall theory developed in the 1960s
• Clotting occurs through a series of reactions in
  which serine protease zymogens are converted into
  active enzymes in a step-wise process.
• For many years, the intrinsic pathway was
  believed to be the more important clotting
  mechanism nowadays the significance of the
  extrinsic pathway (TF/FVIIa) is realised.

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.it all relates to generation of thrombin for
conversion of fibrinogen to fibrin and thereby to
clot formation
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The Common Pathway
Involves Factors V, X, II (prothrombin), XIII,
Fibrinogen
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Extrinsic Pathway
Extrinsic Pathway Tissue Factor
PathwayTF-FVIIa Initiated when blood is
exposed to TF released from damaged
endothelium.
Measured with PT clotting assay (Quick or Owren)
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The Intrinsic Pathway

• Intrinsic Pathway
• Contact Activation
• Initiated by the activation of FXII and other
  contact factors on negatively-charged
  phospholipid surfaces (glass or kaolin in vitro)
• Factors XII, XI, IX, VIII, prekallikrein, HMW
  kininogen
• Measured with aPTT clotting assay

Contact Activation
IX
X
XI
Prekallikrein HMW Kininogen
Ca2
XIIa
XIa
PL, Ca2
(Tenase)
IXa
VIIIa
Xa
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Intrinsic Pathway - APTT

• The Activated Partial Thromboplastin Time (APTT)
  Provides clotting time in seconds of a mixture of
  citrated plasma, Ca2, contact activator, and
  phospholipid
• Tests for deficiencies of pro-coagulant factors
  in the Intrinsic and Common pathways
• Heparin, Warfarin, Factor Inhibitors, Lupus
  Anticoagulant can prolong the APTT

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The Coagulation Cascade
Extrinsic Pathway
Intrinsic Pathway
Common Pathway
XI
TF-VIIa
Prekallikrein HMW Kininogen
PL
XIIa
XIa
(Tenase)
(Prothrombinase)
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The Protein C Anticoagulation Pathway
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Activated Protein C (APC) cofactors

• APC cofactors
• 1. Protein S
• Protein S enhances binding of APC to platelet
  phospholipids and to endothelial cells.
• Only free protein S has APC cofactor function.
  60 of protein S is bound to C4bBP.
• 2. FV
• FV together with Protein S increases APC
  inactivation rate of FVIIIa.

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FVIIIa
FVa
FIIa
FIXa
FX/Xa
FII
APC
Protein S
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The Fibrinolytic Pathway
PAI-1
Plasminogen
Tissue Plasminogen Activator (t-PA) Urokinase
(uPA)
Exogenous streptokinase
Plasmin Inhibitor
XL-Fibrin, fibrinogen
Plasmin
XL- fibrin degradation products (FDP)
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Review of clotting vs chromogenic methods
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General method requirements
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Chromogenic vs. Clotting Assays

• In general, chromogenic assays are more specific,
  accurate, and precise and less susceptible to
  pre-analytical variables
• Clot-based assays are typically fast and
  sometimes less expensive
• Clot-based assays are subject to interference by
  other coagulation factor levels, heparin,
  warfarin, other anticoagulants, as well as the
  presence of lupus anticoagulant
• The relatively high dilution used in chromogenic
  assays are sometimes raised as a concern
• Both clotting and chromogenic assays can
  generally be applied on automated analyzers

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General principle of chromogenic assays
fordetermination of the activity of a proenzyme
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Antithrombin assays
ACTIVITY
IMMUNOLOGICAL measures only type I deficiency
Clotting Plasma samples require
defibrination Slow assay, high variability
Chromogenic High precision, simple to use
FXa The most used assay, recommended by
ISTH Higher diagnostic accuracy
FIIa Influenced by heparin cofactor II and by
thrombin inhibitors.
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Protein C clotting assays

• Clotting assays are based on activation of
  Protein C by the snake venom enzyme Protac and
  the APC activity is usually determined as
  prolongation of the aPTT clotting time
• Clotting assays seem to be influenced by FV
  Leiden and may result in false positives.
• Furthermore, high levels of FVIII may
  underestimate, and presence of Lupus
  anticoagulant overestimate, the true level of PC.

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Protein C chromogenic assays

• Use of the same activation principle (Protac )
  as in clotting assays for activation of Protein
  C.
• Preferred method since subject to fewer
  pre-analytical variables and very high precision
  and accuracy. Not influenced by heparin.
• Detects abnormalities of PC activation and
  abnormalities of the enzymatic active site. Does
  not detect defects in binding of FVa and FVIIIa
  or inability to bind Protein S or phospholipids
  (minority of patients), i.e. Type II deficiency.
• When plasma samples from patients on
  streptokinase therapy, suffering from DIC or
  receiving oral contraceptives are tested, a
  sample blank should be included since such
  plasmas may to some extent cleave the chromogenic
  substrate due to contact activation.

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Protein S

• Vitamin K dependent protein
• Acts as a cofactor to Protein C
• Expresses some anticoagulant activity independent
  of APC
• Circulates in plasma in the free form or bound
• to C4bBP
• Free Protein S is the functionally active form of
  the protein and represents the stoichiometric
  excess over C4bBP

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Protein S Assays

• Functional protein S is measured with clot-based
  assays or by measuring free protein S antigen
  levels with an immunological assay. No
  chromogenic assay is available.
• Total protein S antigen can be measured using
  immunological assays.
• APTT based Protein S activity assays, like
  Protein C assays, may show interference by
  presence of FV Leiden, elevated FVIII levels as
  well as the presence of lupus anticoagulant.
• Measurement of free Protein S antigen seems to be
  most suitable for thrombophilia screening
  purposes.

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Warfarin Monitoring Chromogenic Factor X Assay

• Lupus anticoagulants may produce prolonged
  prothrombin times, which result in an INR that
  does not accurately reflect the level of
  anticoagulation
• The chromogenic Factor X assay may be used to
  more accurately monitor warfarin therapy in
  patients with LA
• The therapeutic range must be determined by each
  lab, but is in the general range of 20 40,
  corresponding to an INR of about 2.0 3.5
• Another application is during transition period
  on switching patients from Argatroban to warfarin

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Measurement of FVIII-
Hemophilia Patients- Potency assignment of
FVIII concentrates- Elevated FVIII
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Elevated Factor VIII

• FVIII activity gt 1.5 IU/mL results in 5-6-fold
  higher risk for DVT, especially recurrent DVT,
  than FVIII activity lt 1.0 IU/mL
• Confirmation of risk not associated with acute
  phase response
• Elevated FVIII persistent over time
• Familial trait observed no explanation so far

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?!
FVIII
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One-stage Clotting Assay

• Principle
• APTT based assay
• Diluted sample
• FVIII Def. Plasma
• PL, Ca2, Surface
• activator
• time for clot formation

• Most widely used method
• Cheap, rapid and simple
• but..........
• Accuracy and precision influenced by a large
  number of variables
• Sensitive to pre-activation of the coagulation
  cascade
• Requires considerable amount of FVIII deficient
  plasma

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Two-stage Clotting Assay

• Stage 1
• FIXa, Ca2, PL
• FX FXa FVIII
• FXa Ca2, FV, PL
  Complex
• Stage 2
• Complex
• Prothrombin Thrombin
• Fibrinogen

Less variation than the one-stage assay No need
of FVIII deficiency plasma It was earlier the
method of choice by the British and European
Pharmacopoeia but it has been replaced by
the chromogenic method
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Basic considerations in developing a chromogenic
FVIII kit in the early 1980s
Use of non-human coagulation factors to decrease
the risk of viral infections due to hepatitis B
and C and later to HIV. No use of FVIII
deficiency plasma for the same reason.
Use of a relatively high sample dilution to
minimize interferences.
Use of a method design which mimics the part of
coagulation where FVIII plays its role.
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Chromogenic FVIII method principle
FIXa, Ca2, PL 1. FX
FXa
FVIIIa FXa 2.
S-2765 Peptide pNA
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Criterion Insensitivity to preactivation by
thrombin
One-stage
Coatest FVIII
Thromb Haemost 54, 818-823 (1985)
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Thromb Haemost 54, 818-823 (1985)
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Chromogenic method recommended by SSC/ISTH
1993 and adopted as the Ph Eur reference method
1995 for assignment of FVIII potency in FVIII
concentrates. Important features to ensure
optimal assay conditions Predilution to 1 IU/mL
with FVIII deficiency plasma with normal vWF
content Final dilution in buffer with 1 BSA
New challenges rFVIII Full length
rFVIII Recombinate (Baxter), Advate (Baxter),
Kogenate (Bayer) Truncated rFVIII ReFacto
(Wyeth)
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Are the species of FIXa and FX of importance in
determination of FVIII activity?
Examples of other Ph Eur reference
methods Antithrombin, anti-IIa bovine
thrombin Prothrombin Ecarin FVII bovine or
human FX FX RVV
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Study purpose Compare FVIII potency
assignments of FVIII concentrates when
using combinations of bovine and human FIXa and
FX and thrombin or prothrombin FV.
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Materials
Bovine and human FIXa, bovine and human FX
(Haematologic Inc.) Bovine FII and human FII
(Enzyme Research) Bovine FV (gift from B
Dahlbäck), human FV (Alexis) Human thrombin
(Biovitrum), bovine thrombin (Instrumentation
Laboratory) Phospholipid emulsion
(Instrumentation Laboratory) S-2765
(Chromogenix) I-2581 (Chromogenix) Octonativ-M
(Octapharma) Recombinate (Baxter) ReFacto
(Wyeth) 6th WHO FVIII Concentrate Standard, Lot
97/616 (NIBSC)
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Conc. during FX activationCa2 6.25
mmol/LPhospholipid (PC, PS, SM) 15
mmol/LThrombin 0.2 U/mL (ca 2
nmol/L)orexcess of FII and FV relative to
sample contributionFIXa 0.04 U/mL (4
nmol/L)FX 0 0.35 mmol/LFVIII 0.6 pmol/L
(presence of FII, FV) and 2 pmol/L (presence of
thrombin)
Fixed parameters in Km determinations
Medium for Km determinations 0.05 mol/L Tris-HCl
pH 7.3, I 0.15, 1 BSA
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Manual microplate assay FIXa PL FIIa
/ FII FV 50 mL FX 50 mL Octonativ-M 50
mL Ca2 50 mL Activation 2 or 5 min,
37oC S-2765/I-2581 50 mL Hydrolysis x min,
37oC HAc, 20 50 mL
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Apparent Km values and relative FXa activities
( r H-Wmean 0.9986 )
B. Thrombin (Coamatic FVIII, Immunochrome
FVIIIC, Factor VIII Chromogenic)
( r H-Wmean 0.9992 )
Porcine PL Km 3 nmol/L (all bovine)
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FVIII potency assignments vs 6th IS with all
bovine and all human species
of FIXa and FX and of thrombin or prothrombin FV
Sample Bovine FII FV Human FII FV Bovine
FIIa Human FIIa IU/mL CV, IU/mL CV,
IU/mL CV, IU/mL CV,
Octonativ-M 41 4.6 40 8.1 41 4.6 42
8.8 Recombinate 86 8.1 88 4.3 87
4.3 92 6.7 ReFacto 83 8.0 81 7.7
87 1.1 87 5.2
rmean 0.998 (range 0.991-1.00) n 58
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FVIII Assays

• One stage clotting assays may give different
  results from two-stage clotting and chromogenic
  assays
• For FVIII concentrates, the difference between
  methods is often more pronounced for products of
  higher purity such as rFVIII concentrates.
• One-stage assays often give underestimation of
  FVIII levels in rFVIII concentrates and
  overestimation in plasma derived concentrates in
  cases of slight preactivation or increased
  activatability.
• The discrepancies, sometimes up to 50, create
  problems when determining therapeutic dosages.

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Measuring Elevated Factor VIII

• The chromogenic assay has been recommended as the
  optimal assay for measuring elevated FVIII levels
• The chromogenic assay precision is typically
  better than that of one-stage clotting assay at
  high FVIII levels
• No interference from heparin, direct thrombin
  inhibitors, or lupus anticoagulant
• The assay is automatable