1. Coagulation Assays – Part 2
Larry Smith, PhD
Director, Coagulation/Hemostasis
Assistant Attending
2. Objectives
• Types of assays in the laboratory
• Monitoring LMWH
• Thrombophilia work-ups
• Lupus Anticoagulant Testing
• HIT
• Factor Assays
2
3. Types of Assays
• Functional Assays
Clot-based assays
Good screening assays
Based on a functioning cascade
Affected by preanalytical variables
▫ May require additional testing
Chromogenic assays
Measure the activity of a specific enzyme rather than
general biologic function
Not affected by most preanalytical variables
3
Enzyme of interest
Peptide pNA Peptide pNA
Yellow color
develops, absorbance
reading at 405 nm
(Substrate)
+
4. Types of Assays
Immunologic-based assays
LIA- or ELISA-based technologies
Most measure the amount of protein present rather than function
Some measure a functional component
• Confirmatory assays
▫ DNA-based assays
4
5. Types of Deficiencies
• Type I Deficiency
Parallel reduction in both FUNCTION and AMOUNT of
protein present
True deficiency
Functional assay (clot-based assay)
• Type II Deficiency
Represents dysproteinemia
Total amount of protein > than the activity
Functional + Antigenic assays
5
6. Anti-Xa Heparin Assay
• Specifically determines anticoagulant
activity of LMWH and UFH by
measuring ability of heparin-bound
antithrombin to inhibit a single
enzyme
• More specific than aPTT since it
measures inhibition of a single
enzyme
• Major advantage is lack of biologic
factors that affect its result
• Limitations of Heparin Assay
▫ Clinical data examining outcomes
is limited
Eikelboom JW. Thromb Haemost 2006;96:547-52.
Francis JL. Pharmacotherapy 2004;24:108S-19S.
Plasma [heparin] + Antithrombin
Excess
FXa
[AT-Heparin-Xa] + Residual FXa
Chromogenic
substrate
pNA
Inversely proportional to the anticoagulant activity
concentration in the plasma sample
7. Antithrombin Deficiency
• Single chain glycoprotein
▫ Synthesized in the liver
▫ SERPIN
• Inherited deficiencies
▫ Associated with increased risk of VTE
▫ 0.02 – 0.2% in general
▫ 1-2% of patients with VTE
• Acquired deficiencies
▫ Decreased synthesis
LD
▫ Drug-induced decreased synthesis
L-asparaginase
Heparin
▫ Increased clearance
Active thrombosis
DIC
Nephropathies
7
8. Antithrombin Assays (functional)
• The assay relies on the AT in the patient’s plasma***
▫ Reflects of the functional AT status of the patient
8
AT + Heparin [AT*Heparin]
[AT*Heparin] + FXa (excess) [AT-FXa- Heparin] + FXa (residual)
Chromogenic substrate
FXa (residual)
Peptide + pNA
Inversely proportional to the AT activity concentration in the plasma sample
9. Classification of Antithrombin Deficiency
9
Type Interpretation
Type I Quantitative
• Parallel reduction in functional and immunologic AT –
50% of normal
• Antigen = Activity
Type II Qualitative
• Greater reduction in functional assay in comparison to
immunologic assay
• Antigen > Activity
T2-HBS mutation in heparin binding domain
T2-RS mutation in reactive site of AT
T2-PL mutation in both heparin binding domain and reactive site of AT
10. Protein C Deficiency
• 1960 – Seeger described it
anticoagulant role
• 1980 – Griffin et al associated PC
deficiency with VTE
• VKD serine protease that is
activated by IIa to aPC
▫ Inhibits Va and VIIIa shuts off
thrombin generation
▫ Exhibits
Anti-inflammatory activities
Anti-apoptotic activities
▫ Inhibits PAI-1 enhanced
fibrinolysis
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http://practical-haemostasis.com/Thrombophilia%20Tests/pc_assays.html
11. Protein C Deficiency
• Congenital Deficiency
• Heterozygous PC deficiency:
• ~0.2% of the general population
• ~3% of patients with VTE
• ~85% of PC mutations are type 1
• Acquired Deficiency
▫ Decreased synthesis
LD
VKD/VKA
▫ Drug-induced decreased synthesis
L-asparaginase
▫ Increased clearance
Acute thrombosis
Acute medical illness
DIC
SCD
Trauma
11
12. Protein C Assays
• Functional assays
▫ No single test for PC is 100% sensitive and specific for abnormalities
▫ Antigenic assay
12
Clot-based Assay (functional) Chromogenic Assay (functional)
aPTT assay to measures the anticoagulant
effect of aPC due to its ability to inhibit
FV and FVIII
aPC cleaves a substrate release of
chromophore generating a color change
Subject to a number of preanalytical
variables
FVIII
FVL
Hyperlipidemia
DTI
Heparin
Lupus Anticoagulant
OAT
Subject to fewer preanalytical variables
Detects most functional defects but
not all
PF3 binding defect
May be affected by OAT
Misleadingly low
levels
False normal results
13. PC Assays
• Dilute patient’s plasma (1:10) in PC-deficient plasma
▫ Clot-based assay
▫ Chromogenic-based assay
13
Plasma + Venom incubate
aPC + substrate-pNA release of pNA
Hydrolysis of aPC on a specific chromogenic substrate
14. Protein S Deficiency
• Described in 1984, Comp
• TOTAL PS circulates in 2 forms:
▫ Bound PS—60%
C4B-BP—nonfunctional
▫ Free PS—40%-functional
• Serves as a cofactor for PC
▫ Binds aPC to the
phospholipid surface
▫ VKD glycoprotein synthesized in
the liver
Free
PS
14
C4B-BP
Total Protein S
Bound
PS
15. Protein S Deficiency
• Congenital Deficiency
• Heterozygous PS deficiency:
▫ ~2% in general population
▫ ~3-6% in recurrent thrombosis or family history
• Acquired Deficiency
▫ Decreased synthesis
LD
VKD/VKA
▫ Drug-induced decreased synthesis
L-asparaginase
OCT
HRT
▫ Increased clearance
Acute thrombosis
DIC
Acute medical illness
Trauma
15
16. Protein S Assays
Three types of assays
1. Clot-based functional PS assay—”activity” assay
Based on aPC inactivation of FVa and FVIIIa
Plasma + PNP(PS free)+ aPC + Add CaCL2 Clot
1. Antigenic-based Free PS assay
Free PS is adsorbed on the C4BP latex particle triggers an agglutination reaction with
the second latex reagent which is sensitized with a monoclonal antibody directed against
human Protein S
The degree of agglutination is directly proportional to the free PS concentration
Immunologic assay measures “free” (functional) portion of PS
3. Antigenic-based Total PS assay
Immunologic assay that measures PS bound to C4BBP + free PS
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Type PS (Activity) PS (Free) PS Total C4B-BP
I Decreased Decreased Decreased Normal
II Decreased Normal Normal Normal
III Decreased Decreased Normal Elevated
17. Preanalytical Variables Affecting PS Deficiency
• Activity assay may be misleading low
▫ FVL
▫ Elevated FVIIII
▫ VKA/VKD
▫ LD – not universal (there are extra-hepatic sites of PS synthesis)
▫ SCD
▫ Type II deficiencies are very rare
May be helpful if there is a high index of suspicion
▫ Pregnancy and some women on OCT
▫ HIV infection
▫ Acute phase response
• Marlar et al. (2012) AJCP. 137; 173-175
▫ Found increased number of falsely low PS activity when PS activity is used as first assay
• Marlar et al. (2011) Am J Hem. 86;418-421
▫ Free PS assay considered by many to be more reliable than activity
▫ Diagnoses 95-99% of PS deficiencies
1717
Type III
18. 18
aPC-Resistance—Screening assay
• aPC-resistance
▫ Dahlbäck et al in 1993
Noted a blunted response in
aPTT’s of a group of
patients with thrombophilia
when aPC was added
▫ Shuts-off thrombin
generation
Via inhibition FVa and
FVIIIa
▫ Ratio of 2 aPTT’s
__(aPTT plus aPC)__
(aPTT minus aPC)
• http://www.wardelab.com/arc_2.html
• Normal : adding aPC prolongs aPTT
• FVL: adding aPC does NOT prolong aPTT
19. aPC-Resistance—Screening assay
• “Screening assay” for FVL mutation
▫ ~95% of aPC-resistance is caused by a defect in the Factor V molecule
• Sensitivity and specificity approach 100% with modified assay
▫ Uses FV-deficient normal plasma + patient plasma
▫ Requires a normal aPTT in the patient
▫ Should not be used in patient on OAC or with LA
• Mutation later described in 1994 by Bertina et al
▫ Caused by single point mutation in the FV gene
▫ Substitution of adenine for guanine at 1691 – G1961A
▫ Changes arginine to glutamine at 506 – R506Q
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20. PG20210 Mutation
Poort et al, 1996
Single nucleotide substitution
G20210A in the 3’ UT regions of the
prothrombin gene
G ® A substitution at nucleotide
20210 in prothrombin gene
Results in elevated levels of
prothrombin (~30% increase)
No screening test available
Occurs primarily in Caucasians--~3%
in general population
2-5-fold increased risk of VTE
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21. ISTH Criteria for Lupus Anticoagulant Testing
The ISTH has defined the minimum diagnostic criteria for lupus
anticoagulants to include
1. A prolonged clotting time in a screening assay such as the aPTT
2. Mixing studies indicating the presence of an inhibitor
3. Confirmatory studies demonstrating phospholipid dependence of the
inhibitor
a. Screen – decreased amount of phospholipids prolonged
clotting time
b. Confirm—increased amount of phospholipids shortened
clotting time
4. No evidence of other inhibitor-based coagulopathies
Specific factor assays if the confirmatory step is negative or there is
evidence of a specific factor inhibitor
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22. ISTH Criteria for Lupus Anticoagulant Testing
• Updated ISTH guidelines (2009) ISTH
▫ Pengo V, Tripodi A, Reber G, Rand JH, Ortel TL, Galli M, de Groot PG. Update of
the guidelines for lupus anticoagulant detection. J Thromb Haemost 2009; 7:
1737–40
▫ Choice of tests
1. Two tests based on different principles
2. dRVVT should be the first test considered
3. Seconds test should be a sensitive aPTT (low
phospholipids and silica as activator)
4. LA should be considered as positive if one of the two tests
gives a positive result
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23. Detection of LA
• Assays
dRVVT*
SCT*
HEX
Kaolin CT
dPT
23
dPT
Why do we see so few LA’s
on the extrinsic side???
SCT
DRVVT
Clot-based assays
24. dRVVT Screen (Normal plasma)
24
X
Xa
Va
Xa
Prothrombin
dRVVT
Thrombin
Ca2+
Fibrinogen Fibrin
Phospholipid
(PF3)
25. dRVVT Screen (Lupus Anticoagulant)
25
X
Xa
Va
Xa
Prothrombin
dRVVT
Thrombin
Ca2+
Fibrinogen Fibrin
Low
Phospholipid
Content
26. dRVVT Confirm (LA)
26
X
Xa
Va
Xa
Prothrombin
dRVVT
Thrombin
Ca2+
Fibrinogen Fibrin
High
Phospholipid
Content
27. Laboratory Tests for HIT
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FFuunnccttiioonnaall ((AAccttiivvaattiioonn))
Washed
Platelets
Washed
Platelets
Serotonin release
HIPA
ATP release
Flow cytometry
Serotonin release
HIPA
ATP release
Flow cytometry
• Primarily detect IgG antibody complex
• Higher specificity for detecting clinically
relevant pathogenic antibodies
AAnnttiiggeenniicc
Serum or
Plasma
Serum or
Plasma
ELISA-based
PGI-based
ELISA-based
PGI-based
IgG-specific
IgG/M/A
28. Serotonin Release Assay (SRA)
28
14C-Serotonin
Y
Y
Y
Y Y
% 14C-Serotonin Release
0 0.1 1.0 10
Heparin Concentration (U/mL)
100
80
60
40
20
0
Heparin =
Antibody =
PF4 =
Y
30. ELISA-based Assay
Y
Patient
Plasma
Sample
PF4
coated
ELISA
plate
Wash
Tagged goat anti-human Ig+
alkaline phosphatase
Detect
absorbance
substrate
Anti-human IgG + AP
Heparin Antibody
Heparin:PF4 complex
Y
Y
YYYYY
Y Y
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31. Factor Assays
• Used to establish the % activity of a
circulating factor
• Procedure
▫ Patient’s plasma deficient in a
specific factor
▫ Dilute 1/10, 1/20/ 1/40 with a
normal plasma deficient in the
specific factor above
▫ Perform PT or aPTT and compare
the seconds to a standard curve
1/10 = 100%
1/20 = 50%
1/40 = 25%
▫ Patient is run in multiple dilutions
to check for the presence of an
inhibitor
31
Std curve
Patient
32. Summary
• Broad menu of assays that can potentially be performed as part of a
hypercoagulable workup
• These assays help to identify risk factors that may contribute to thrombosis
• Clot-based assays should be interpreted with caution
• All of the assays have their advantages and disadvantages
▫ Chromogenic PC and Free PS assays are the assays of choice to screen for these
deficiencies
▫ ISTH Subcommittee on Thrombosis and WHO recommend Free PS to screen for
PS deficiency
Ballard RB, Marques, MB. Pathology Consultation on the Laboratory Evaluation of Thrombophilia: When,
How, and Why. Am J Clin Pathol 2012;137-553-560
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This lecture is a continuation of a previous lecture on coagulation assays. Here we will discuss assays involved in thrombophilia testing.
Objectives: 1) review of thrombophilia and risk factors; 2) discuss assays that identify risk factors; 3) review preanalytical variables that interfere with these assays
What types of assays are there for identifying patients at risk and how good are they?
Functional assays for type I deficiencies
Antigenic assays for type II deficiencies
DNA-based assays for confirmation
The general principle of a chromogenic assay, in its simplest form: A protein is converted to its active formThe cleaved peptide releases the chromophore, in this example pNA, which gives off a color.
In this example, PNA is used. PNA = para-nitroaniline, which appears as a yellow color.
What types of assays are there for identifying patients at risk and how good are they?
Functional assays for type I deficiencies
Antigenic assays for type II deficiencies
DNA-based assays for confirmation
The general principle of a chromogenic assay, in its simplest form: A protein is converted to its active form. the. The cleaved peptide releases the chromophore, in this example pNA, which gives off a color.
In this example, PNA is used. PNA = para-nitroaniline, which appears as a yellow color.
For most clinical coagulation evaluations, the main goal is to test for function (preferred method) and if decreased then confirm by antigenic assay to determine the type of deficiency (quantitative or qualitative). PS may be the exception, since the PS activity assay is infulenced by a viariety of conditions.
Described by Eggberg in coworkers in 1963
Single-chain glycoprotein, 58,200 Daltons, plasma ½-life ~ 3 days
Chromosome 1 (SERPINC1) gene – wide variety of mutations have been identified
Plasma is incubated with an excess of bovine Xa in the presence of heparin (in the patient). Heparin binds to AT inactivation of Xa. Residual Xa left is measured by its ability to hydrolyze a chromogenic substrate (S-2765) added to the test tube. Absorbance is measured at 405nm and is inversely proportional to the AT activity concentration in the plasma sample.
Some methods use Factor Xa instead of IIa in reagent. This theoretically decreases the contribution from other proteins such as heparin cofactor II. IV-heparin therapy can cause lowering of the AT level in plasma by ~25%.
Not affected by HCII
VKD serine protease
Single chain glycoprotein, 62KDa, produced in the liver and converted to its active form by thrombin becomes aPC
Protein C gene (PROC) located on the long arm of chromosome 2 (2q13-q14)
VKD serine protease
Single chain glycoprotein, 62KDa, produced in the liver and converted to its active form by thrombin becomes aPC
Protein C gene (PROC) located on the long arm of chromosome 2 (2q13-q14)
Clot-based assay:
Clotting time of the aPTT (or PT) will be influenced by the amount of Va and VIIIa present in the reaction mixture and in turn this will be influenced by the activity of aPC. aPC is generated from the conversion of PC to aPC by Protac. So, if there is a reduction in circulating PC levels, then less aPC will be generated, less Va and VIIIa will be inactivated and the clotting times will be shorter.
Chromogenic assay:
Protac is added to PPP and incubated. A chromogenic substrate for aPC is added. aPC cleaves the substrate releasing pNA and the change in optical density is measured and compared to a standard reference curve. (Calcium, PF3 or coagulation activator is necessary since the test plasma serves as the only source of PC—clot formation is not necessary for this test).
A note about chromogenic protein C assays: they may overestimate the true level of protein C in patients treated with oral anticoagulants like warfarin.
This is because the non-carboxylated forms of protein C formed in warfarin-treated patients are also activated by Protac and can then cleave the chromogenic substrate.
The chromogenic assay detects only abnormalities of PC activation (that is when protein C is converted to APC) and abnormalities of the enzymatic active site.
In rare cases the patient may have defects in the part of the molecule that binds FVa and FVIIIa or the part that binds protein S or phospholipid.
These defects are not detected by the chromogenic assay. Measures anticoagulant activity of APC exerted against natural substrates – FVa & FVIIIa.
Argument for clotting based assays – measures natural substrate. Chromogenic based – measures amidolytic activity against synthetic substrate. Argument against clotting based assays – many interfering substances.
Argument for chromogenic – fewer interfering substances, but not natural substrate. Rare reported mutations that affect catalytic site which can not be detected by chromogenic assays. Venom is Protac.
Protein C assays can be clotting or chromogenic. The clotting assay is widely used, though the chromogenic assay is the method of choice.
A note about chromogenic protein C assays: they may overestimate the true level of protein C in patients treated with oral anticoagulants like warfarin. This is because the non-carboxylated forms of protein C formed in warfarin-treated patients are also activated by Protac and can then cleave the chromogenic substrate. The chromogenic assay detects only abnormalities of PC activation (that is when protein C is converted to APC) and abnormalities of the enzymatic active site. In rare cases the patient may have defects in the part of the molecule that binds FVa and FVIIIa or the part that binds protein S or phospholipid. These defects are not detected by the chromogenic assay. Measures anticoagulant activity of APC exerted against natural substrates – FVa & FVIIIa. Argument for clotting based assays – measures natural substrate. Chromogenic based – measures amidolytic activity against synthetic substrate. Argument against clotting based assays – many interfering substances.
Argument for chromogenic – fewer interfering substances, but not natural substrate. Rare reported mutations that affect catalytic site which can not be detected by chromogenic assays. Venom is Protac.
Protein S, APC’s cofactor, circulates in the plasma free or bound to the complement protein C4b binding protein. 40% of protein S is in the free, functionally active form, which is what APC binds to.
Binding affinity of C4BP high, therefore all C4BP molecules will be bound to PS. C4BP is an acute phase reactant. PEG precipitation takes out form bound to C4bBP. Free form in the supernatant. The gene (PROS1) located in chromosome 3 (3p11.1-3p11.2)
VKD serine protease
Single chain glycoprotein, 62KDa, produced in the liver and converted to its active form by thrombin becomes aPC
Protein C gene (PROC) located on the long arm of chromosome 2 (2q13-q14)
Functional assay – measures on the FREE (physiologically-active) PS.
Free PS – monoclonal ab directed against PS epitopes that are not accessible in the bound form and these are usually the residues that are involved in the binding to C4BBP. Second assay involves used PEG to separate the bound PS from C4BBP and then measures the free PS.
Total PS – measures both PS bound to C4BBP and the Free form of PS
There is a good correlation between FPS antigen (LIA) assay and functional PS activity so many labs choose only to measure FPS. HOWEVER, this may miss some rare cases in which the immunological PS level is normal but there is a functional abnormality (i.e. a true Type 2 deficiency).
In SCD PS may bind to sickle cells, however, PS is not decreased during sickle cell crisis.
Approximately 90% of APC Resistance is caused by a defect in the Factor V molecule, known as the Factor V Leiden gene mutation. This is explained by a point mutation in the coagulation factor V gene, changing arginine 506 in the factor V molecule to glutamine. So now APC can’t recognize the cleavage site at position 506, which allows for longer duration of thrombin generation and may lead to a hypercoagulable state. In other words, the FVa molecule isn’t allowing APC to do its job of inactivating FVa and ultimately inhibiting thrombin generation.
In normal patients, adding APC to the aPTT prolongs the aPTT
In patients with the FVL mutation, adding APC shortens the aPTT because they are resistant to cleavage by the APC complex
Dilute in FV deficient plasma to normalize all other plasma proteins. Theoretically could test samples from patients on OAC. Better discrimination between heterozygotes and homozygotes.
Limitations – With modified test (diluting in FV deficient plasma) – many limitations eliminated. No significant difference between fresh and frozen plasma. Lot to lot changes should be evaluated to verify cut-off range. High FVIII levels (associated with pregnancy & acute phase response) should not be an issue with modified test (unless really high!).
With classic APTT test – the baseline APTT must be within 25-40 seconds. If results from different laboratories are to be compared, it may be beneficial to normalize the APC ratio against the APTC ratio of a normal plasma pool.
Note: only 90% of APCr is due to FV Leiden mutation, so if using the modified assay alone may not pick up the 10% of people who are APCr for other reasons. High FVIII levels will lower APC ratio (found in pregnancy and inflammatory states).
Prothrombin and FX concentrations below 50% tend to produce higher APC ratios
High FVIII levels will lower APC ratio (found in pregnancy and inflammatory states).
Prothrombin and FX concentrations below 50% tend to produce higher APC ratios
High FVIII levels will lower APC ratio (found in pregnancy and inflammatory states).
Prothrombin and FX concentrations below 50% tend to produce higher APC ratios
High FVIII levels will lower APC ratio (found in pregnancy and inflammatory states).
Prothrombin and FX concentrations below 50% tend to produce higher APC ratios
Approximately 90% of APC Resistance is caused by a defect in the Factor V molecule, known as the Factor V Leiden gene mutation. This is explained by a point mutation in the coagulation factor V gene, changing arginine 506 in the factor V molecule to glutamine. So now APC can’t recognize the cleavage site at position 506, which allows for longer duration of thrombin generation and may lead to a hypercoagulable state. In other words, the FVa molecule isn’t allowing APC to do its job of inactivating FVa and ultimately inhibiting thrombin generation.
In normal patients, adding APC to the aPTT prolongs the aPTT
In patients with the FVL mutation, adding APC shortens the aPTT because they are resistant to cleavage by the APC complex
Dilute in FV deficient plasma to normalize all other plasma proteins. Theoretically could test samples from patients on OAC. Better discrimination between heterozygotes and homozygotes.
Limitations – With modified test (diluting in FV deficient plasma) – many limitations eliminated. No significant difference between fresh and frozen plasma. Lot to lot changes should be evaluated to verify cut-off range. High FVIII levels (associated with pregnancy & acute phase response) should not be an issue with modified test (unless really high!).
With classic APTT test – the baseline APTT must be within 25-40 seconds. If results from different laboratories are to be compared, it may be beneficial to normalize the APC ratio against the APTC ratio of a normal plasma pool.
Note: only 90% of APCr is due to FV Leiden mutation, so if using the modified assay alone may not pick up the 10% of people who are APCr for other reasons. High FVIII levels will lower APC ratio (found in pregnancy and inflammatory states).
Prothrombin and FX concentrations below 50% tend to produce higher APC ratios
High FVIII levels will lower APC ratio (found in pregnancy and inflammatory states).
Prothrombin and FX concentrations below 50% tend to produce higher APC ratios
High FVIII levels will lower APC ratio (found in pregnancy and inflammatory states).
Prothrombin and FX concentrations below 50% tend to produce higher APC ratios
High FVIII levels will lower APC ratio (found in pregnancy and inflammatory states).
Prothrombin and FX concentrations below 50% tend to produce higher APC ratios
The mutation (a 1691G→A substitution) removes a cleavage site of the restriction endonuclease MnII.
Molecular Cause:
Mutation of an APC cleavage site in Factor V
Mutation Site:
Amino acid #506 is Arg and mutated to Gln
DNA and protein sequence:
Amino Acid:Arg506Gln506
Nucleotide:CGACAA
Molecular Function:
APC cleaves Arg-Gly bond.
If mutated to Gln, then bond not cleaved.
FVa remains active generating more fibrin clot.
High FVIII levels will lower APC ratio (found in pregnancy and inflammatory states).
Prothrombin and FX concentrations below 50% tend to produce higher APC ratios
APC resistance due to the presence of the FV:Q506 allele is inherited as an autosomal dominant trait and has a prevalence of 2-13% in the general population. Frequencies of APC resistance among patients with venous thrombosis range from 20-60%.
APC resistance is highly prevalent. Individuals who are heterozygous for Factor V Leiden have a 2 – 5 fold increased thrombotic risk, while homozygotes have up to an 80-fold increase. Most heterozygotes are asymptomatic unless other risk factors are present.
Simple blood tests are available to detect APC Resistance and it is part of the routine thrombophilia screen
A single nucleotide polymorphism at position 20210 in the 3’-untranslated region of the prothrombin gene (FII*20210GA) is associated with elevated plasma prothrombin levels and an increased risk of venous
thrombosis. The FII*20210GA nucleotide substitution did not change the restriction map of the prothrombin gene. Poort et al (1996) designed a mutant reverse oligonucleotide that introduces a Hind III restriction site (A↓AGCTT) at nucleotide position 20214 in the mutant sequence but not in the normal sequence. The G-to-A transition at position 20210 in the 3’-untranslated region of the prothrombin gene is detected by PCR amplification and Hind III digestion.
After Hind III digestion of the amplified DNA from a person homozygous for FII*20210G (the more frequent allele), the size of the band is unchanged (lane 1). By contrast, after Hind III digestion of the amplified DNA fragment obtained from a person heterozygous for FII*202010G/A, bands of 345 bp and 322 bp are detected (lane 2). The 23 bp band is not visible in the figure. After Hind III digestion from the amplified DNA from a person homozygous for FII*20210A, the fragment is fully digested (lane 3). Lane M: size marker ФX174 DNA-Hae III digest, Lane U: undigested PCR product).
The prothrombin gene mutation is found in approximately 3% of the population and leads to an increase in prothrombin levels, which can generate excess thrombin, leading to an increased risk of VTE. It is the second most common genetic risk factor for VTE, with a relative risk of 2.8.
substitution of adenine for guanine at position 20210 of the prothrombin gene. Although this falls outside the reading frame for the protein, it leads to high levels of prothrombin and a possibly increased risk of thrombosis (Poort et al 1996
Polymorphism at bp 20210 in 3’ UT
Increases Prothrombin Levels (?)
Mechanism: longer surviving mRNA
Genetic Abnormality
Heterozygote and Homozygote
Assay: DNA based only
Relative Risk:2.0
There are two types of laboratory tests available to confirm a diagnosis of HIT: a functional assay and an antigen assay.
Functional assays detect HIT-IgG on the basis of their ability to activate platelets. Activation can be measured in a number of different ways, including SRA, platelet aggregation, ATP release, and flow cytometry. A washed platelet system is recommended. Of the available assays, the serotonin release assay has the most advantages.
The citrated plasma assay is widely used, specifically, the platelet aggregation. This assay measures the aggregation of normal donor platelets by patient serum or plasma in the presence of heparin. However, it is not recommended because of its low sensitivity and low specificity.
Antigen assays detect the binding of antibodies to immobilized PF4-heparin complexes. The most typical of the direct antigen assay performed on serum or plasma is the heparin-PF4 ELISA.
Functional assays detect HIT-IgG on the basis of their ability to activate platelets. Activation can be measured in a number of different ways, including SRA, platelet aggregation, ATP release, and flow cytometry. A washed platelet system is recommended. Of the available assays, the serotonin release assay has the most advantages.
The citrated plasma assay is widely used, specifically, the platelet aggregation. This assay measures the aggregation of normal donor platelets by patient serum or plasma in the presence of heparin. However, it is not recommended because of its low sensitivity and low specificity.
Radio-labeled, washed platelets from reactive donors are incubated with heat-treated PATIENT serum in the presence of heparin. The test is positive if 14C-serotonin release occurs at therapeutic (0.1 u/mL) but not at high (100 u/mL) heparin concentrations. Laboratory assays for HIT antibodies. Top: the SRA. Washed platelets loaded with radiolabeled 14C-serotonin are incubated with patient serum and pharmacologic concentrations of heparin. Visentin et al,5 PF4-heparin complexes formed at a heparin:PF4 molar ratio of approximately 1:2 are optimal for HIT antibody detection, and reactions of antibodies with this target are inhibited when high doses of heparin are added. A significant fraction of serum samples from patients suspected of having HIT react with the unmodified complexes, sometimes quite strongly, but fail to inhibit with excess heparin. What these antibodies recognize is uncertain, but they are not likely indicative of HIT. Findings showed that fewer than 10% of patients judged to have HIT on clinical grounds failed the confirmatory test, whereas approximately one-third of those who did not appear to have HIT did so. Blood, 9 September 2010, Vol. 116, No. 10, pp. 1761-1766.
Aggregation at the low concentration of heparin must by greater than 20% compared to normal control and 0% at the higher concentration of heparin. Measures platelet aggregation induced by IgG in serum or plasma of HIT patients treated with heparin. Donor platelets are incubated with the patient’s plasma contain IgG antibodies. The presence of HIT antibodies may cause platelet aggregation. Advantages Easily performed in most laboratories -- Specificity greater than 90%. Disadvantages --Low sensitivity: 35%–81%, Heparin can activate platelets in the absence of HIT antibodies, Reactivity varies among donor platelets.
The PF4/heparin EIA. The assay shown utilizes PF4 and heparin bound in optimal stoichiometric concentrations to detect HIT antibodies. Alternatively, PF4 can be bound to certain other polyanions, such as polyvinyl sulfonate (not shown).