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Educational Material: Thrombophilia Tests
Contents
• Introduction in thrombophilia
• Specific parameters
•
•
•
•
AT III
Protein C and protein S
Factor V Leiden and APC resistance
Other thrombophilia markers, incl. D-Dimer and
antiphospholipid syndrome
• Heparin assays and their applications
DiaMed Education: Thrombophilia
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Thrombophilia
•
Thrombophilia is a disorder in which the blood clots easily or
excessively.
•
Most disorders that cause thrombophilia increase the risk of blood clot
formation in veins; some increase the risk of clot formation in both
arteries and veins.
•
Underlying problem: Enhanced generation of thrombin which induces
excessive fibrin formation
•
Thrombin activates other coagulation factors and platelets, and finally
produces fibrin clots which are stabilized by F XIIIa (activated also by thrombin)
Consequences: Enhanced clot formation  thromboembolic disease
DiaMed Education: Thrombophilia
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Causes for thrombophilia
•
Some of the disorders that cause thrombophilia are inherited, otheres are
acquired.
•
Examples for congenital causes:
•
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Activated protein C resistance (Factor V Leiden mutation)
Prothrombin 20210 mutation a specific mutation in the prothrombin gene
Deficiency of protein C, protein S, or antithrombin (AT III)
Rare disorders: Deficiency of heparin cofactor II, plasminogen, PAI-1 polymorphisms,
disorders In methionine metabolism, PNH etc.
Acquired disorders that cause thrombophilia
•
•
•
Paralysis, prolonged sitting (e.g. in a plane or car), prolonged bed rest, plaster casts etc.,
which lead to immobilization
The presence of the lupus "anticoagulant," and/ or antiphospholipid antibody syndrome
Recent surgery, and heart attack. heart failure (a condition in which the blood is not pumped sufficiently
through the bloodstream)
•
•
•
•
Increased pressure on veins, including obesity and pregnancy
Inflammatory conditions (leading to elevated concentrations of FVIII and fibrinogen, elevation of PAI-1 etc.)
Hyperhomocysteinemia
Disseminated intravascular coagulation (often associated with cancer or with sepsis)
DiaMed Education: Thrombophilia
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Symptoms and Complications
•
Most of the inherited disorders do not cause an increased risk of clotting until young
adulthood, although thrombi can form at any age (including in newborns).
•
This is primarily the case in heterozygous cases in which only one version of the protein is
abnormal, but the other version is functional. Homozygous forms, however, can lead to severe
spontaneous events.
•
Combination with other risk factors (e.g. pregnancy, high blood pressure, immobilization)
during adulthood may precipitate thromboembolic disease, for example a deep vein clot (deep
vein thrombosis) in the legs, which can result in leg swelling.
•
Formation of a deep leg thrombus may be followed by the very dangerous pulmonary
embolism.
•
Less commonly, a thrombus may form in veins of an arm or in the abdomen, and in veins
inside the skull.
•
Hyperhomocysteinemia and the antiphospholipid syndrome may result in venous or arterial
clots. When clots obstruct blood flow in arteries, tissues lose their blood supply and may be
damaged or destroyed.
•
Potential consequences: Myocardial infarction, stroke
DiaMed Education: Thrombophilia
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Incidence and progression
• Incidence: 1- 5 per 1000 individuals, increases strongly with age
• Higher incidence when combined with specific diseases
(especially with cancer), with immobilization, or certain treatments
• Often thrombosis starts in the distal leg veins, but may progress
further.
• About 20% remain only in the distal vein, and about 30%
progress to proximal leg veins
• Up to 15% may embolize to lung  Pulmonary embolism (PE)
• High morbidity
• High mortality
• Thrombosis may be “silent” until embolization
DiaMed Education: Thrombophilia
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Diagnosis and Treatment
•
A person who has had at least two separate instances of a thrombotic event without an
apparent predisposing factor may have an inherited thrombophilia disorder.
•
An inherited disorder may also be suspected there is a family history of thromboembolic
disease. A young – otherwise healthy- person who develops an initial thrombus for no
apparent reason may have an inherited disorder.
•
Laboratory tests are used to identify specific inherited disorders which cause thrombophilia.
This includes tests for APC-resistance, protein C or S, and AT III deficiency, and sometimes
others. These tests are usually more accurate when performed after a thrombus has been
treated.
•
Inherited disorders that cause thrombophilia are incurable*. Most patients with thrombosis are
initially treated with normal or low molecular heparin (LMWH). They are often treated with oral
anticoagulants for some time, in recurrent cases for the rest of their lives.
•
However, the treatment should be started only after a carefully made diagnosis, There can be
high risk situations in which therapy may either induce new thromboembolic disease (e.g.
necrosis induced by oral anticoagulants, heparin induced thrombocytopenia- HIT) or does not
work at all (inefficient heparin therapy in patients with low AT III activity)
*except by liver transplantation
DiaMed Education: Thrombophilia
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Antithrombin (AT III)
Data on AT III
Function
Inhibitor of F IIa, F Xa and F IXa
Inhibits also other coagulation factors
Is an essential cofactor for therapy with
heparin, LMW heparin or
pentascaccharide (fondaparinux)
May have also anti-inflammatory activity
Concentration
in Plasma
About 0.12 mg/l = 2.3 µM
Normal range
80 – 120 %
(somehow method dependent)
Plasma half live
~ 3 days
AT III molecule
model
DiaMed Education: Thrombophilia
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Antithrombin III: Overview
•
The serpin antithrombin (AT III) is a major
inhibitor of the coagulation factors FXa,
FIXa, and thrombin.
•
AT III forms a stable complex with its
target molecules, in which both AT III and
the active coagulation factor lose their
activity ( consumption!).
•
This process is accelerated heparin like
molecules on the vasculature surface, and
of course by heparin therapy.
•
AT III deficiency can be associated with
thrombosis
•
A low activity of AT III may lead to
inefficient therapy with anticoagulants such
as heparin, low molecular weight heparin
(LMWH), or pentasaccharide, and may
have also other adverse effects.
Serpin= serine protease inhibitor
A class of plasma proteins
Examples: AT III, Antiplasmin, C-1-Inhibitor etc.
FIXa
↓ |— AT III /heparin
FXa
↓ |— AT III/ heparin
FIIa
↓ |— AT III/ heparin
Fibrin formation,
Platelet activation
Activation of F V, FVIII, FXI
Activation of TAFI
AT III
FXa
Heparane sulphate
Endothelial cell surface
DiaMed Education: Thrombophilia
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Functional assays for Antithrombin: Principle
1. AT III(sample) + F Xaexcess + Heparin  AT-FXa inactive + F Xaresidual
2. F Xaresidual + chromogenic Substrate  pNA (yellow) + peptide
Note:
Similar tests via FIIa instead of FXa
are used as well.
However, FXa based tests do not
show interference from other
plasmatic inhibitors such as HC II, or
during heparin or DTI therapy
DTI = direct thrombin inhibitors, e.g. bivalirudin,
agatroban, hirudin
DiaMed Education: Thrombophilia
Example of a calibration curve
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Determination of Antithrombin: Indications
• Venous thrombosis or pulmonary embolism
• Congenital AT deficiency (incidence ~ 1/ 5000)
• Acquired AT deficiency (may require substitution with concentrates)
•
•
•
•
DIC / sepsis / SIRS (systemic inflammatory response syndrome)
Liver disease (AT is synthesized in the liver)
Renal disease (loss through increased pore size)
Hormonal therapy (incl. anticontraceptive drugs)
• No or inadequate effect of heparin therapy (when aPTT does not show
the expected response, AT III may be too low)
• Decision if AT III- concentrate therapy (alternatively FFP) is needed
• Monitoring of substitution therapy with AT III concentrates or FNP
DiaMed Education: Thrombophilia
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AT III assays: Other methods
• Immunoassays (ELISA or immunoturbidimetry) and clotting assays are
not very common.
• Limitation of immunoassays for AT III: No detection of abnormal forms
of AT III, e.g. type II deficiency (normal antigen, decreased function).
However useful for characterization of the nature of a specific defect
• Clotting tests for AT III are difficult to standardize
DiaMed Education: Thrombophilia
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Protein C and Protein S
Data on Protein C
Function
Vitamin K dependent plasma protein (MW 60.000).
In its activated form an inhibitor of clotting via inactivation of
FVa and FVIIIa (catalyzed by the free form of protein S,
phospholipids and calcium).
Concentration
in plasma
1.8- 3.8 mg/l
Normal range
70 – 130 %, (method dependent)
Plasma half live
~ 6 -8 h
DiaMed Education: Thrombophilia
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Protein C: Overview
• After activation, activated protein C (APC)
regulates thrombin generation, primarily by
proteolytic inactivation of FVa and FVIIIa, a
reaction which accelerated by free protein S.
• PC has also important anti-inflammatory and
cytoprotective functions.
APC
F IIa
PC

APC
F VIIIi
TM
EPCR

F Vi
F VIIIa
F Va
Activated Platelet
Endothel
Subendothel
• A deficiency of PC is often associated with
thromboembolic disease, especially in the
homozygous form, or when other risk factors
are present.
DiaMed Education: Thrombophilia
Biological funbction of Protein C (simplified)
PC = protein C zymogen, APC = Activated protein C, EPCR =
Endothelial Protein C receptor, TM = Thrombomodulin, F Vi =
inactivated F V, F VIIIi = inactivated F VIII
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DiaChrom Protein C: Principle
 APC
1.
Protein C(Sample) + Protac®
2.
APC + chromogenic substrate  pNA (yellow) + peptide
Protac® = a proteolytic
enzyme from snake venom
(Agkistrodon contortrix)
Example of a calibration curve
DiaMed Education: Thrombophilia
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Overview Protein C assays
•
Methods:
• Functional methods
• Chromogenic tests (like DiaChrom)
• Clot based assays
• Immunochemical assays
• ELISA
• Turbidimetric
•
Functional methods, especially chromogenic substrate based ones, should be
preferably used as a first line assay
•
Specific disadvantage of clotting assays for protein C function
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•
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Somehow less specific
Interference from Lupus anticoagulants or by heparin
Influenced by F V-L mutation or other causes for APC resistance
Interference from alterations of other coagulation factors
May underestimate the PC activity in patients with oral anticoagulants
Immunoassays will not pick up defective protein C (e.g. deficiency type II).
However they are useful for characterization of defective forms of protein C
DiaMed Education: Thrombophilia
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Protein C: Indications
•
Venous thrombosis or pulmonary embolism
•
Purpura fulminans (especially in newborns with severe deficiency
of PC)
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Congenital PC deficiency
•
Acquired PC deficiency (may require substitution with concentrates)
•
•
•
•
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DIC / sepsis / SIRS (systemic inflammatory response syndrome)
Vitamin K antagonists
Liver disease (PC is synthesized in the liver)
Dilution coagulopathy (blood losses, trauma, burns)
Asparaginase therapy
•
Decision if concentrate therapy (alternatively FFP) is needed
•
Monitoring of substitution therapy with concentrates or FNP
DiaMed Education: Thrombophilia
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A determination of protein C is useful before oral anticoaguation
• Due to its short half live of only 6-8 h, protein C falls rapidly In the
induction phase with vitamin K antagonists
• At the end of day one or even faster, PC may be already quite low –
while other factors, especially prothrombin are relatively high
• In this situation, and especially in cases of protein C deficiency, the
patient has a risk for the development of cumarin induced skin necrosis
% activity
FII
FX
PC
•
Coumadin induced skin necrosis
During oral anticoagulation a determination of protein C is difficult because as a
vitamin K dependent protein it is decreased by therapy as well
DiaMed Education: Thrombophilia
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Data on protein S
Function
Vitamin K dependent plasma protein (MW 60.000).
An inhibitor of clotting by catalyzing the inactivation of FVa
and FVIIIa by activated protein C in the presence of
phospholipids and calcium).
Protein S has no enzymatic activity.
Concentration in
plasma
~ 17 – 35 mg/l
Normal range
About 60 – 140 %
(strongly method dependent)
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Overview Protein S
•
A vitamin K dependent plasma protein
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A cofactor for APC, involved in the inactivation of FVa and FVIIIa- the motors of
thrombin generation
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Only the free form of protein S is active.
•
About 65 % of protein S in plasma is bound the the C4b binding protein
(C4bBP), an acute phase protein.
•
Inflammatory disease, pregnancy, or diabetes mellitus increase C4bBP. This
can lead also to a kind of acquired protein S deficiency, in which not the
absolute amount of protein S is deficient, but the active free part,
•
Protein S seems to have also other inhibitory functions, e.g. independent from
APC, or as an inhibitor of the activation of TAFI* by APC
*TAFI = thrombin activatable fibrinolysis inhibitor, a protein which stabilizes fibrin against premature fibrinolysis
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Protein S deficiency
• Patients with protein S deficiency have an elevated risk for thrombosis,
especially deep venous thrombosis
• Situations with protein S deficiency are DIC or oral anticoagulation,
liver disease, infectious disease (e.g. AIDS), and other diseases
• Type I deficiency: Low concentration, low activity
• Type 2 deficiency: Normal total antigen, low activity
• Type 3 deficiency: Low activity, low free antigen level, normal total
antigen
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Principle of protein S assays
1.
Functional tests:
•
•
Various assay principles are used in commercial tests
In general the sample is diluted into protein S deficient plasma (as a source
of coagulation factors) and activated protein C is added
After a short incubation time coagulation is triggered by adding a coagulation
activator or an activated coagulation factor, together with phospholipids and
Ca-ions
The prolongation of clotting time is proportional to the activity of protein S
The protein S induced effect on APC is quite weak, and thus the clotting tests
often show only minor clotting time differences in their calibrated ranges
•
•
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2. Immunological tests (either for total protein S or only the free form)
•
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ELISA
Immunoturbidity
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FV- Leiden and APC resistance
APC Resistance
• A clinical state characterized by resistance of plasma against added or
induced activated protein C (APC)
• Under normal conditions, APC adds as a coagulation inhibitor by
inactivation of FVa and FVIIIa, thus prolonging the clotting time
• In patients with APCR, this inhibitory effect is either absent or
weakened
• The majority of cases show APC resistance due to a polymorphism in
factor V (“Factor V- Leiden” mutation, 1691G/A)
• “APC Resistance” in some clotting assays can have also other causes
than the factor V Leiden polymorphism, for example elevated F VIII or
FII (for example in carriers of the prothrombin 20210 polymorphism), lupus
anticoagulants, or pregnancy.
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APC Resistance / Factor V Leiden (FV-L)
•
Factor V-Leiden = A polymorphism in the FV molecule. It makes FVa much
more resistant against Activated Protein C (APC), a natural anticoagulant.
•
On top of that also the cofactor function of FV in the inactivation (!) of FVIIIa
and FVa is diminished.
•
Consequences:
•
•
•
•
More thrombin is generated  more platelet activation and fibrin formation
Carriers of this polymorphism have an increased risk of thrombosis, especially
when other risk factors are present
Homozygotes may have spontaneous thrombosis, e.g. during pregnancy, with
anti-baby pills, during hormone therapy or without additional stimulus
On the contrary, the mutation protects against blood losses (e.g. during child
birth, or during surgery)
•
For Europe, the Mediterranean countries, and in many areas of the Middle
East, F V-L seems to be the most frequent hereditary cause for
thrombosis. In Northern EU up to more than 10 % of the population have FV-L
•
The polymorphism seems to be absent or extremely rare in Japan,
DiaMed Education: Thrombophilia
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Determination of APCR in the laboratory (1)
Clotting Tests („APC resistance“)
•
First Generation (After Dahlbaeck et al)
– 2 x aPTT : With or without added APC
 calculation of a ratio of these two tests. Example 75 sec/ 35 sec= 2.14
– Low ratio in patients with APCR, high ratio in F V “wildtype”
– Absolute values of ratio are strongly instrument and reagent dependent
– Not very specific, multiple interference possible
– Strong influence of blood sampling and handling technique
•
Screening tests (Activation of protein C in plasma with Protac®
• Activation of coagulation and PC in the presence of phospholipids and calcium,
e.g. via aPTT (ProC Global/ Dade Behring) or via the diluted Russel Viper venom
time (dRVVT) test system
• Test is sensitive for FVL and several other causes, e.g. protein C or protein S
deficiency, high levels of FVIII, prothrombin 20210 mutation, pregnancy, lupus
anticoagulants
• Is more a kind of global assay for the protein C pathway
• Can be made quite specific for FV-L by diluting the sample into an excess of FV
deficient plasma
DiaMed Education: Thrombophilia
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Determination of APCR in the laboratory (2)
Clotting Tests („APC resistance“)
2nd Generation:
• With added FV deficient plasma
• Dilute sample into FV deficient plasma (excess of all other coag
factors enhanced specificity for FV Leiden)
– aPTT or other clotting assay (e.g. with snake venoms/ Pentapharm)
with or without added APC  calculation of a ratio (Low ratio in patients
APCR, high ratio in FV “wildtype”
– Quite specific for FV L, sometimes overlap between homo- and
heterozygous form
•
PCR
– Specific test for the mutation
– Different techniques possible
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DiaClot FV L: a simplified method in F V-L testing
Principle:
•
•
Clotting assay. Sample is highly diluted into a
mixture of purified clotting factors, APC and
fibrinogen
Under these conditions, only the FV-L
(sample) activity determines the clotting time,
no ratio required
F VL
FV
APC /PS
F Xa + Va L /
Ca2+
F Va
F Va (inactive)
PL
•
•
•
The clotting time is converted into „%-FV-L“
from a calibration curve obtained with the
DiaClot FV-L Calibrator Set
No interference from lupus anticoagulants,
high FVIII, oral anticoagulation, heparin etc.
Normal range (in F VL activity-%)
< 10 % :
25 – 75 %
> 75 %
Wildtype (Normal F V)
Heterozygous for F V-L
Homozygous for F V-L.
DiaMed Education: Thrombophilia
2 Reagent System:
F II
F IIa
Fgen
Fibrin
Reagent 1:
Coagulation factor
reagent (Human
Fibrinogen,
Prothrombin, Protein
S, APC, heparin
neutralizer)
Reagent 2 : Factor
Xa, phospholipids
(both lyophilised)
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Other rare causes for thrombosis
•
Dysfibrinogenenmia
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•
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Paroxysmal nocturnal haemoglobinuria (PNH)
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•
•
•
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A disorder induced by dysfunctional regulation of the complement system
Reduced expression of CD55 and CD59 on erythrocytes
Thrombosis in cerebral or intra-abdominal veins
Can be detected by the ID-PNH assays or by flow cytometry
Plasminogen deficiency
•
•
•
a disorder leading for example to defective fibrinolysis
Often not detected via PT, aPTT but via thrombin time or reptilase time assays
A disorder resulting in lowered fibrinolysis
Can be detected with specific chromogenic substrate assays
Deficiency of (or autoantibody) against ADAMTS 13
•
•
•
•
ADAMTS 13 (a protease) regulates von Willebrand factor (vWF) by proteolytic cleavage. VWF is
essential for primary haemostasis
In the absence of ADAMTS 13, ultra-large and very adhesive forms of VWF lead to massively
enhanced platelet adherence which may indirectly induce also thrombin formation
In thrombotic thrombocytopenic purpura a low ADAMTS 13 activity leads to thrombosis, in spite
of thrombocytopenia
A diagnosis requires platelet function tests (e.g. DiaMed Impact R) and specific tests for ADAMTS
13
DiaMed Education: Thrombophilia
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Heparin Induced thrombocytopenia
• Ordinarily, heparin prevents clotting and does not affect the platelets,
However, heparin is immunogenic.
• Triggered by the immune system, heparin may induce a low platelet
count, heparin induced thrombocytopenia (HIT)
• Two distinct types of HIT can occur:
• Non-immune form: Occurs most frequently; is characterized by a mild
decrease in the platelet count and is not harmful.
• The second type, immune-mediated HIT, occurs much less frequently but is
very dangerous. Immune-mediated HIT causes much lower platelet counts,
most frequently 5 to 14 days after first beginning heparin therapy
• Paradoxically, despite a very low platelet count, patients who suffer
from HIT are at risk for major thromboembolic problems.
• HIT may affect up to a few % of patents exposed to heparin
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Pathomechanism
• Antibodies can form against a
complex of heparin and a
platelet factor 4, or "PF4" that is
released by platelets.
• Antibodies may activate platelets
via the fc-γ- receptor, inducing
thrombin generation on
monocytes and endothelial cells
• In rare cases also antibodies
against IL-8 or against NAP-2
(neutrophiles) may induce HIT
DiaMed Education: Thrombophilia
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Therapy of HIT
•
Patients with HIT have a significantly higher
thrombotic risk than patients with other well
characterized risk factor for thrombosis
•
Therefore an immediate anticoagulation is
required, of course with a different drug than
heparin
•
Drugs used in this situation are
45
5
HIT
AT III deficiency
10
Protein C deficiency
15
Dysfibrinogenemia
20
Factor V Leiden
25
Prothrombin G-20210A
30
Lupus Anticoagulant
35
Protein S deficiency
40
•
•
•
•
•
0
1
Odds ratio for risk of thrombosis (HIT : range 20 -40)
(Modified after Erentin TE Can J Cardiol 1995 and
Warkentin TE Thromb Res. 2003)
•
Agathroban
Bivalirudin
Hirudin (lepirudin)
Orgaran®
(Fondparinux)
Problems with these drugs
•
•
•
High expenses (may exceed 1000 USD/day)
Difficult to monitor
Bleeding not uncommon
DiaMed Education: Thrombophilia
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Diagnosis of HIT
• The diagnosis of HIT
requires a combination
of clinical data and
laboratory tests
• The clinical data can be
successfully
standardized with the
„4-T-Score“
• Lab methods include
• Immunochemical tests
• Functional tests
DiaMed Education: Thrombophilia
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Lab tests for heparin induced thrombocytopenia
Functional tests (the most accurate tests for confirmation of HIT)
• Serotonin release assay (SRA)
•
•
Limitations: Requires access to fresh platelets and radioactivity, is very
time consuming
Heparin induced platelet aggregation
•
Limitations: Requires access to fresh platelets, is very time consuming
Immunochemical methods
• ELISA
•
•
•
ID-PaGIA assay (gel centrifugation)
•
•
•
Limitations: Time consuming, high blank values, many falsely positive
values, not a good method for individual test requests, limited value for
diagnosis
Advantages: Good negative predictive value
Limitations: May give falsely positive values, limited value for diagnosis
Advantages: Very simple and fast, excellent negative predictive value
(>99%, Pouplard et al, JTH 2007), suitable for individual requests,
PIFA tests (immune filtration, Akers Biosciences): Gave unreliable
results in several clinical trials
DiaMed Education: Thrombophilia
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Other thrombophilia markers
Prothrombin mutation 20210
• Risk factor for thrombophilia
• Induced by a mutation in the promoter of the prothrombin gene
• Induces an enhanced synthesis of prothrombin with higher plasma
levels, typically > 125 % of normal activity
• Consequences
• Venous or other thrombosis
• Increased risk for obstetric omplications such as preeclampsia, abruptio
placentae, fetal retardation or still birth
• Probably also increased risk for coronary heart disease
• Risk may be increased by the use of oral contraceptives
DiaMed Education: Thrombophilia
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Lupus Anticoagulants / Antiphospholipid Syndrome
• Autoantibodies (IgG, IgA, IgM) directed against phospholipids (PL) or
the PL- binding domain of plasma proteins
• Some of them prolong PL dependent clotting tests such as the aPTT or
the diluted Russel Viper venom time, influence on PT is rather mild
• Target antigens are
• Phospholipids
• Cardioplin
• Phosphatidyl serine
• Proteins:
•
•
•
•
2-glycoprotein 1
Prothrombin
Protein C
others
DiaMed Education: Thrombophilia
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Clinical consequences of the antiphospholipid syndrome
• Venous or arterial thromboembolic disease
•
•
•
•
DVT
Stroke
Coronary artery disease
Arterial graft occlusion
• Obstetric omplications (maternal and fetal)
•
•
•
•
Intrauterine fetal death
Recurrent abortions
Fetal Growth retardation
Neonatal thrombosis
• Others
DiaMed Education: Thrombophilia
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D- Dimer
•
A marker for enhanced activity of both coagulation and fibrinolysis.
•
D-Dimer is not a defined analyte. It represents a group of “cross linked fibrin
split products” derived from fibrin.
•
Covalent cross-linking of polymerized fibrin monomers by activated Factor XIII
stabilizes the fibrin clot. D-dimers are generated by plasmin lysis of this crosslinked fibrin.
•
Tests detect split products of various size and may partly react also with
fibrinogen degradation products (hyperfibrinolysis) or split products derived
from elastase during inflammation (depends on the antibody specificity).
•
Increased D-Dimer values* indicate a situation with increased fibrin formation
and lysis, e.g. like in acute thromboembolism, pulmonary embolism, acute
myocardial infarction, during surgery or in trauma patients, in cancer, DIC etc.
* Normal values are age dependent
DiaMed Education: Thrombophilia
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Clinical application of D-Dimer tests
• Elevated levels are found in many clinical situations.
• Therefore the finding of an elevated D-Dimer does not provide very
specific information. It rather demonstrates or excludes a clinical state
with elevated thrombin formation.
• The value of D-Dimer is more the negative result which can be used for
exclusion of a thromboembolic state (negative predictive value).
• Major applications:
• Exclusion of thrombosis, e.g. deep venous thrombosis (DVT)
• Exclusion of pulmonary embolism (PE)
• Exclusion of disseminated intravascular coagulatiion (DIC)
DiaMed Education: Thrombophilia
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Potential savings when using D-Dimer tests
•
More than 6 Mio of cases with suspected DVT or PE present in the US to
physicians per year, and ~ 200,000 cases of PE are diagnosed. Each case
carries a 20% risk of sudden death.
•
Rapid diagnosis and early treatment of DVT are critical to prevent subsequent
morbidity or disabling conditions to the patients
•
A negative D- Dimer test may prevent more expensive and sometimes invasive
methods such as
•
•
•
•
•
CT Pulmonary Angiography/ Spiral-Computed Tomography
Ventilation-Perfusion Scan (VQ Scan)
Compression ultrasound of lower extremities
Venography (a radio-imaging method where contrast dye is injected and blood flow
can be visualized)
It is imperative to exclude the diagnosis of DVT for various reasons. There is an
inherent risk of hemorrhage associated with unnecessary anticoagulant
treatment, or the severe risk of heparin induced thrombocytopenia
DiaMed Education: Thrombophilia
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Some precautions when using D- Dimer assays
• D-Dimer values show age dependency (higher values
in elderly patients)
• A high does hook effect (with falsely low values) may
affect certain kits. Very high concentrations of D-Dimer
are found in some tumor patients and during
thrombolytic therapy.
Signal
• The assays are only partly standardized. Depending on
the calibration and the antibodies selected, values may
be quite different between various kits.
D-Dimer
High dose hook or prozone effect
• In hospitalized patients: Numerous diseases and
invasive procedures can elevate D-dimer levels without
the presence of VTE.
VTE = venous thromboembolism
DiaMed Education: Thrombophilia
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Determination of heparin
Background: Heparin
•
A negatively charged, sulphated glycosaminoglycan, composed of uronic and
glucuronic acid residues.
•
Commercial heparins are isolated from porcine intestinal mucosa or bovine
lung
•
Heterogeneous mixtures of polysaccharide chains (molecular weight from 3000
to 30 000, with a mean MW of 15 000)
•
Low molecular weight heparin (LMWH) is prepared from unfractionated
heparin. It has a molecular weight which is different between brands
•
Heparin and UFH exert their anticoagulant effect by activating antithrombin
(AT); the heparin-antithrombin (H-AT) complex then inactivates thrombin,
activated factor X (FXa) and other activated clotting factors.
•
LMWH is more efficient against FXa.
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Important adverse effects of heparin
• Heparin induced thrombocytopenia (HIT)
• Development of antibodies against the complex of heparin and platelet
factor 4 (PF4, a protein released from platelets)
• Patients may develop severe thromboembolic disease with high mortality
• May affect up to a few percent of patients on heparin or LMWH
• Requires immediately a change in the type anticoagulant (e.g.
anticoagulation with a direct thrombin inhibitor)
• Bleeding (when overdosed).
• Occurrence is approximately 10% overall but may increase up to 20% in
patients treated with high dose therapy.
• Risk of bleeding increases with APTT ratios above the therapeutic range
range
• Cave: aPTT is a poorly standardized test. Clotting times at the same
concentration of heparin may be quite different.
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Overdosage of heparin increases the bleeding risk
Relative risk of bleeding
60
50
40
Bleeding (%) 30
20
10
0
R1
<1
>1
Heparin concentration/ Anti- FXa level
1
These frequencies are
statistically significantly different
( 2 test, P <0.037).
Source:
Use of Anti-Xa Activity as a Marker for Heparin-Induced Bleeding in Patients
with APTT >180 s
Alexander Haliassosa, Helen Melita-Manolis, Despina Aggelaki, Despina
Tassi and George Terzoglou, Clinical Chemistry 43: 1781-1782, 1997;
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Problems in the application of heparin
•
The clinical effectiveness of heparin is dependent on achieving an in vitro defined
anticoagulant effect (e.g. a certain prolongation of the aPTT, or a certain plasma level of
heparin as measured with specific tests).
•
The dose-response relationship of heparin is not linear
•
Considerable in vivo variation in response to a fixed dose of heparin between individuals.
This is partly reflective of the pharmacokinetic limitations of heparin, caused by the
binding of heparin to plasma proteins including platelet factor 4 , fibrinogen, factor VIII
and histidine-rich glycoprotein, and probably other factors
•
The phenomenon known as ‘heparin resistance’ should be taken into consideration to
prevent the over-administration of heparin, with potential haemorrhagic consequences,
particularly postoperatively and in the setting of cardiac bypass surgery.
•
In the context of venous thromboembolism, heparin resistance is defined as the need for
more than 35 000 U /24 h to prolong the aPTT into the therapeutic range.
•
A poor response to heparin can of course be related to deficiency of AT III.
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Heparin assay versus aPTT (1)
•
Most often heparin therapy is monitored via aPTT, with a target range of a 1.52-fold prolongation of the initial aPTT during heparin therapy. However, the
aPTT has limitations:
•
Results are strongly reagent (and coagulometer) dependent, e.g. the aPTT at
the same heparin concentration can be
• 70 sec with reagent A
• 90 sec with reagent B
• Unclottable with reagent C
•
The measuring range is limited (often unclottable when still in the therapeutic
range of heparin therapy [0.3- 0.7 IU/ml]):
• Overdose not reliably detectable, bleeding risk
• Reducing the dose as a consequence of an unclottable test may lead to subtherapeutic
levels of heparin with elevated thrombotic risk
•
Sometimes an initial value is not available:
•
1.5 fold prolongation of initially 25 sec gives only 37.5 sec (almost within the normal
range) while 1.5-fold prolongation of initially 35 sec results in an aPTT of 52.5 sec.
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Heparin assay versus aPTT (2)
• APTT values are often prolonged because of underlying disease (for
example severe liver disorders, myocardial infarction, or infection, lupus
anticoagulants, haemodilution)
• Results dependent on may other factors, e.g. a high FVIII (after
surgery, inflammatory disease) may artificially shorten the clotting time
(„heparin resistance“)
• aPTT does not reliably respond on LMWH (with major differences
between brands). There are no therapeutic ranges defined for aPTT.
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Clinical indications for specific heparin assays
Patients on LMWH
- With renal disease (incl. late
stage diabetes II)
- With obesity
- With high age
- Multimorbidity
Risk of accumulation by reduced clearance 
bleeding risk
Children on LMWH
Dosing is difficult due to altered pharmacokinetics in
small children
Patients on UFH, especially
when there are other
coagulation abnormalities (e.g.
lupus anticoagulant, liver
disease etc.)
Risk of under- or overdosage  thromboembolic or
bleeding risk
Heparin Resistance: No
adequate prolongation of aPTT
during heparin (UFH) infusion
Specific assay allows to detect if there is indeed a
resistance (aPTT influenced by many other factors)
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Specific determination of Heparin: Principle of the anti-FXa test
1. F Xaexcess + Heparin/ AT III
 AT-FXa inactive + F Xaresidual
2. F Xaresidual + chromogenic substrate  pNA (yellow) + peptide
In most assays individual calibration curves are required for UFH or LMWH
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Other methods for the determination of heparin
Heptest and similar clotting tests :
• A clotting test based on the inhibition of excess FXa, folowed by adding of a
coagulation factor / fibrinogen reagent and Ca-ions
• Difficult to automate or to run on optical instruments due to weak clot
signals
• Partly dependent on the level of coagulation factors in the plasma
PICT = Prothrombinase Induced Clotting time
•
•
•
Similar concept as Heptest, but with an additional snake venom based activator for
FV
Probably less specific than anti-FXa chromogenic assays due to more influence of
plasmatic coagulation factors and thrombin inhibitors
Not widely used
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