Blood Coagulation - Screening assays and single factor assessment -

1
Blood Coagulation- Screening assays and single
factor assessment -

• Jørn Dalsgaard Nielsen
• Thrombosis Centre
• Gentofte Hospital
• Copenhagen, Denmark

JDN
2
SUBENDOTHELIAL TISSUE
ENDOTHELIAL CELLS
ADP
Ca
Serotonin
Prostacyclin Nitric oxide
Inhibition
Activated platelet
Tromboxane A2
PF4
Activation
PF3
GP Ib-IX
Platelet adhesion og activation
GP IIb-IIIa
IL-1 TNF
GP Ib-IX von Willebrand factor Collagen
Tissue factor
Released from TNF or IL-1-activated endothelial
cells
F VII
F VIIa
F IX
TFPI
Inactivates F VIIa F X tissue factor
-kompleks
F X
Endothelial damage
F XIa
PF3, Ca, F VIIIa
F XIa
F Xa
Contact activation
F XIIa
F VIIIi
F VIII
Thrombomodulin
PS
PCa
PC
Kallikrein
F V
F Vi
t-PA
PF3, Ca, F Va
Prothrombin
Thrombin
PAI-1
Plasminogen
Plasmin
Inhibits serine proteases
a2-antiplasmin
Fibrinogen
F XIII
Antithrombin
Fibrinogen/-Fibrin degradation products
Heparin Cofactor II
Polymerizing fibrin
Proteoglycans
F XIIIa
Inhibits thrombin
Crosslinked fibrin
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Is testing of single factors necessary in
patients with suspected haemostatic dysfunction ?

• It depends on who you are addressing
• A surgeon
• Will the patient bleed ?
• Can I stop bleeding with fresh-frossen plasma ?
• A haematologist
• Single factor assessment is often necessary to
  establish a correct diagnosis

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Indications for evaluation of haemostatic function

• Clinical problem ? Biochemical defect?
• Biochemical defect ? Clinical problem?

Prophylaxis/ treatment indicated? or not?
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The challenge of evaluation of clotting
abnormalities

• In vitro assessment of haemostasis is difficult
  because the important interaction between the
  endothelium and blood components cannot be
  evaluated in a single assay.
• So-called global tests can be used to test the
  haemostatic capacity of blood components (plasma
  and blood cells) but not the influence of
  antithrombotic properties of the endothelium.
• Thrombelastography may give a clue of a clotting
  defect, platelet dysfunction, or
  hyperfibrinolysis but will not give the final
  diagnosis.

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Thrombelastography
Increased in patients with clotting defects
Decreased in patients with platelet dysfunction
or defect fibrin formation
TEG
Start
Minutes
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Thrombelastography

• LA and HIT-2 are associated with a high risk of
  thrombosis
• A shortened reaction time might, therefore, be
  expected but is not seen because the thrombotic
  predisposition is provoked by endothelial
  dysfunction

TEG
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The challenge of evaluation of clotting
abnormalities

• As global tests of haemostasis neither give a
  consice diagnosis nor results that reliably
  reflect the clinical problem, more specific
  assays are often needed for the evaluation of
  thrombotic and haemorrhagic disturbances of the
  haemostatic system.

However, separation of the complex network of
reactions may result in a number of other
pitfalls and impede a comprehensive view.
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The challenge of evaluation of clotting
abnormalities

• Among laboratory testing, coagulation assays are
  the most influenced by the inaccurate
  standardization of the pre-analytical phase.
• Clotting times are influenced by
• time of tourniquet placement (lt60 sec
  recommended)
• needle size (19-22 gauge recommended)
• citrate concentration (105-109 mM recommended)
• incomplete filling of tubes (PTlt80, APTTlt90)
• platelet count (lt10109/l recommended)
• haemolysis and lipaemia
• temperature and G-force during centrifugation
• temperature and duration of storage until testing

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The challenge of evaluation of clotting
abnormalities

• Screening methods of coagulation should optimally
  be sensitive to all coagulation defects.
• This is not the case but by combination of simple
  procedures we can get close to the final
  diagnosis.

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Exploring coagulation

• The present theory of the function of the
  coagulation system is based on numerous studies
  performed in the 20th century.
• The history of the discovery of clotting factors
  and development of assays may help understanding
  the use of screening assays of coagulation.

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The theory of blood coagulationYear 1900 the
four factor theory
Known factors
Cellular damage
Prothrombin Ca
Fibrinogen
Thromboplastin
Thrombin
Fibrin

• Hammerstein. Hoppe-Seylers Zeitschrift für
  physiologische Chemie 1899 28 98.
• Morawitz, P. Ergebnisse der Physiologie
  biologischen Chemie und Experimental
• Pharmakologie 1905 4 307.

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The theory of blood coagulationYear 1935 the
Quick test
Determination of the clotting time of citrated
plasma after addition of thromboplastin and
calcium chloride
Prothrombin Ca
Fibrinogen
Thromboplastin
Thrombin
Fibrin

• Quick AJ. J Biol Chem 1935 109 LXXIII

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The theory of blood coagulationYear 1947 Factor
V
Cellular damage
Prothrombin Ca
Fibrinogen
Factor V
Thromboplastin
Thrombin
Fibrin

• Owren PA. Acta Med Scand 1947 Suppl 194

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The theory of blood coagulationYear 1947 Factor
V
Cellular damage
Prothrombin Ca
Fibrinogen
Factor V
Thromboplastin
Thrombin
Factor V deficiency showed to be a rare
disease, and the discovery of FV did not explain
the puzzle that the standard coagulation test
the Quick test, was normal in most patients with
congenital bleeding tendency.
Fibrin

• Owren PA. Acta Med Scand 1947 Suppl 194

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Mixing assays

• Whole blood clotting time and plasma clotting
  time are prolonged in haemophiliac patients and
  can be normalized by mixing patient blood/plasma
  with equal parts of normal blood/plasma.
• Both tests, however, have high CV.

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First description of APTT
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The theory of blood coagulationYear 1953 APTT

• Langdell et al. J Lab Clin Med 195341637-47.

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The theory of blood coagulationYear 1959 the
Roman numerical nomenclature
Factor Synonyms I Fibrinogen II Prothrombin III Th
romboplastin IV Calcium V Accelerator globulin
proaccelerin labile factor VI Factor V
derivative (not used now) VII Proconvertin
stable factor autoprothrombin I VIII Antihaemophi
lic factor A platelet cofactor 1 IX Plasma
thromboplastin component (PTC) Christmas factor
antihaemophilic factor B autoprothrombin II
platelet cofactor 2 X Stuart-Prower
factor XI Plasmathromboplastin antecedent
(PTA) XII Hageman factor XIII Fibrin stabilizing
factor

• suggested by an international committee under
  the chairmanship of Dr. IS Wright

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The theory of blood coagulationYear 1964 the
cascade scheme
Problems
?
VII ?
?

• Macfarlane, RG. Nature 1964 202 498

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The theory of blood coagulationYear 1975 the
classic coagulation system
Internal pathway
External pathway
VIIa VII
Ca
Phospholipid, Ca, VIII
X Xa
X
Phospholipid, Ca, V

• Austen DEG Rhymes. A laboratory manual of
  blood coagulation. 1975.

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The theory of blood coagulationdiscoveries of
the last decades

• The major in vivo importance of the
• external pathway
• Acceleration of coagulation by positive
• feed-back mechanisms
• Inibitory mechanisms of blood
• coagulation

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The theory of blood coagulation today
Tissue factor
VIIa
VII
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EXPRESSION OF TISSUE FACTOR
CONSTITUTIVEe.g.epithelial cellsglial cells
INDUCEDe.g.monocytic cellsendothelial cells
PROHIBITEDe.g.lymphocyteserythrocytes
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The theory of blood coagulation today
Tissue factor
Activation by a serine protease, e.g. hepsin
VIIa
VII
XI
XIa
IX
IXa
VIII
VIIIa
X
Xa
V
Va
XIII
II
IIa
XIIIa
Fibrinogen
Fibrin
XL-Fibrin
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The theory of blood coagulation today
Tissue factor
Activation by a serine protease, e.g. hepsin
XII ?
VIIa
VII
XI
XIa
IX
IXa
VIII
VIIIa
X
Xa
V
Va
XIII
II
IIa
XIIIa
Fibrinogen
Fibrin
XL-Fibrin
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Activated platelet
Endothelial damage
Zn2
F XIa
F XIIa
activation
Kallikrein
prourokinase
urokinase
t-PA
PAI-1
Plasminogen
Plasmin
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Natural inhibitors of blood coagulation
Tissue factor
VIIa
VII
XI
XIa
IX
IXa
HC-II
VIII
VIIIa
Endothelial cell
PCa
X
Xa
PS
V
Va
XIII
PC
IIa
II
XIIIa
Fibrinogen
Fibrin
XL-Fibrin
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The classic coagulation system
APTT
Prothrombin time
VIIa VII
Ca
Phospholipid, Ca, VIII
X Xa
X
Phospholipid, Ca, V

• Austen DEG Rhymes. A laboratory manual of
  blood coagulation. 1975.

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Clotting defects and bleeding

• Coagulation factor deficiencies seldom cause
  bleeding if the level of the deficient factor is
  gt40.
• APTT is normal when the level of coagulation
  factors is gt40.
• Therefore, APTT determined in a mixture of equal
  parts of normal plasma and plasma from a
  haemophiliac patient will be normal.
• Unless an inhibitor is present.

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Antibodies against coagulation factors

• Two types
• Alloantibodies
• Patients with hereditary coagulopathy may
  develope antibodies against the deficient factor
  when treated with plasma-derived or recombinant
  factor concentrates
• Autoantibodies
• Aquired antibodies, most often against factor
  VIII and typically in patients with autoimmune
  diseases, malignancy and in women during
  pregnancy and post partum. In half of the cases
  no underlying disease can be found. Incidence
  1-5 per 1.000.000.

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APTT-based inhibitor test
Add APTT reagents
Determine APTT
mix
Patient plasma
Normal plasma
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APTT-based inhibitor test
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Treatment of bleeding in patients with antibodies
against coagulation factors

• In some patients (low responders) the
  neutralizing effect of the antibody can be
  overcome by increasing the dose of factor
  concentrate
• In patients with high titers of antibody
  recombinant factor VIIa can be used to obtain
  haemostasis

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The by-passing effect of factor VIIa
FVII FVIIa
FIX
TF
FXIa FXI
INITIATION
FIXa
AMPLIFICATION
FVIIIa FVIII
FX FXa
FVa FV
Thrombin
Prothrombin
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30-year old female with refractory bleeding
Aquired factor VIII deficiency with a progressive
inhibitor to factor VIII. Bethesda titer 5.5 BU.
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Algoritm for evaluation of prolonged APTT

• Exclude preanalytical factors causing spuriously
  prolonged APTT
• Underfilled tubes, delayed testing, venipuncture
  above heparin lock etc.
• Is the patient receiving antithrombotic
  treatment?
• E.g. heparin, thrombin inhibitors, vitamin K
  antagonists, fibrinolytics
• Defect fibrin formation?
• Determine fibrinogen concentation thrombin time
• If not then test for inhibitors
• Lupusinhibitor (Thrombophilia)
• Antibodies against a coagulation factor
  (Haemophilia, aquired/cong.))
• If neg. inhibitor test Coagulation factor
  deficiency
• Contact factor deficiency (No bleeding)
• Deficiency of other clotting factors
  (Haemophilia)

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Algorithm for evaluation of prolonged APTT
Fibrinogen lt 3 ?M
Explore hypofibrinogenaemia
Explore possible AC treatment Heparin Thrombin
time? Vitamin K antagonist INR?
Yes
No
PtNP 11 mix immediate APTT
Corrects APTT
PtNP 11 mix Incub 2h ? APTT
Fails to correct APTT
Fails to correct APTT
Corrects APTT
Phospholipid dependent
Inhibitor present
Factor deficiency
Procoagulant factor deficiency
Lupus inhibitor
Fails to correct APTT
PtAPTT-reagent Incub 10 minutes
Phospholipid independent
Corrects APTT
Contact factor deficiency
Specific inhibitor
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Prolonged preincubation with APTT reagent
PK-deficient plasma
Asmis et al. Thromb Res 2002105463-70
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Algorithm for evaluation of prolonged APTT
Fibrinogen gt 3 ?M
Explore hypofibrinogenaemia
Explore Thrombin time? Heparin?
INR? Vitamin K antagonist?
No
Yes
PtNP 11 mix immediate APTT
Corrects APTT
PtNP 11 mix Incub 2h ? APTT
Fails to correct APTT
Fails to correct APTT
Corrects APTT
Phospholipid dependent
Inhibitor present
Factor deficiency
Procoagulant factor deficiency
Lupus inhibitor
Fails to correct APTT
PtAPTT-reagent Incub 10 minutes
Phospholipid independent
Corrects APTT
Contact factor deficiency
Specific inhibitor
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Evaluation of 177 consecutive cases of prolonged
APTT
Results
Chng et al. 2005
42
Evaluation of 177 consecutive cases of prolonged
APTT
No obvious cause
LA
Chng et al. 2005
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Factor XIII deficiency

• In FXIII deficiency the APTT, PT and thrombin
  time are normal.
• Moderate to severe FXIII deficiency can be
  diagnosed by the clot solution test.
• A fibrin clot prepared from patient plasma is
  placed in 8 M urea.
• Dissolution of the clot within 24 hours is
  suggestive of FXIII deficiency.

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• Blood coagulation
• Screening assays and single factor assessment
• Jørn Dalsgaard Nielsen
• Thrombosis Centre
• Gentofte Hospital
• Copenhagen, Denmark
• E-mail jdn_at_dadlnet.dk