thrombophilia testing in anticoagulated patients · thrombophilia testing in anticoagulated...
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1
Thrombophilia testing in
anticoagulated patients
Katrien Devreese, MD, PhD
Ghent University Hospital, Belgium
Coagulation Laboratory
NTHC 7the annual meeting 30/3/2017
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Investigation of thrombophilia
R. Virchow,
1856
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Consolidated evidence
Loss-of-function mechanisms Antithrombin deficiency
Protein S deficiency
Protein C deficiency
Gain-of-function mechanisms Factor V Leiden (aPC-r)
Prothrombin G202010A
High levels of FVIII
Acquired mechanisms Lupus anticoagulants
Anticardiolipin antibodies
Anti-β2 glycoprotein I antibodies
Investigation of thrombophilia Haemostatic abnormalities associated with thrombophilia
Franchini et al, Thromb Haemost 2016; 115: 25-30
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Thrombophilia= anticoagulation
Traditional antivitamin K therapy (AVK)
Direct oral anticoagulants (DOAC)
dabigatran
rivaroxaban, apixaban, edoxaban
Influence of anticoagulation (AVK and DOAC) on
test results
Investigation of thrombophilia
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Case
Male patient, °1966
Recurrent DVT (2 episodes)
Treated with AVK, stopped in 02/2012
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Case
Sample for thrombophilia screening send to our lab
Thrombophilia screening 9/5/2012
Antithrombin 100.0% 80.0-120.0%
Protein C activity 120.0% 70.0-130.0%
Protein S antigen (free) 118.0% 50.0-134.0%
APC-r Not interpretable >2.00 (ratio)
FVIII 53.0% 60-120%
Lupus anticoagulants positive /
Protrombin mutation negative
FV Leiden negative
Reference value
Non
blood
group O
7 7
Case
FVIII one stage clotting assay
Standard predilution sample 1:10
Predilution in 1:40 or 1:100
FVIII 53% 92%
8 8
aPC-r
1. aPTT based method with FV deficient plasma (1:5
predilution in FV def plasma)
APTT (+APC)
APTT (-APC) Normal: ratio > 2
FV Leiden ratio <2
2. aPTT based method with FV deficient plasma (1:40,
1:100 predilution in FV def. plasma)
e.g. in presence of LAC interfering with aPTT
Not
interpretable
aPTT(-APC)
prolonged
aPC-r 2.2
Case
9 9
Case
LAC
aPTT
dRVVT
screening 76.7 s <45.6 s
2.13 <1.28
Mixing Rosner index 29.6 <10.1
confirmation 0.5 s delta <8 s
screening 136.6 s <46.1 s
3.46 <1.23
Mixing Rosner index 42.4 <7.8
Confirmation
(scr/cfr ratio)
1.32 1.26
LAC present
10 10
Case
aPTT 58.6 s 28.9-38.1 s
INR 1.3 0.9-1.1
PT 73% 60-120%
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Case
Asked for more information
Patient was treated by cardiologist
Atrial fibrillation
Treated with Pradaxa (dabigatran) 150 mg twice daily
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trombine
XII XII a
XI XI a
IX
II
X X a
V a
PL
Ca++
TF
VII a
PL
Ca++ IX a
VIII a
PL
Ca++
neg geladen oppervlak
fibrine
aPTT
PT
Direct Xa
inhibitor
Direct IIa
inhibitor
Rivaroxaban
Apixaban
Edoxaban
Dabigatran
13 13
Influence of dabigatran on aPTT and PT
- aPTT and the PT are influenced by dabigatran depending on
the reagents used
-aPTT and PT showed a concentration dependent prolongation
-PT reagents were less sensitive than aPTT
Douxfils et al, Thromb Haemost 2012; 107:985-997
14 14
Van Blerk et al, Thromb Haemost 2015; 113: 154-164; Van Blerk, WIV global report 2012
12
14
16
18
20
22
PT(sec)
Dabigatran (ng/mL)
Me
dia
n P
T(s
ec)
0 100 250
Innovin
Neoplastin CI PLUS
Neoplastin R
Recombiplastin 2G
Thromborel S
-wide variation in responsiveness between reagent/instrument
combinations
-concentration dependent prolongation of aPTT and PT
-PT was less sensitive to dabigatran
Influence of dabigatran on aPTT and PT
15 15
15
20
25
30
35
PT(sec)
Rivaroxaban (ng/mL)
Me
dia
n P
T(s
ec)
0 120 290
Innovin
Neoplastin CI PLUS
Neoplastin R
Recombiplastin 2G
Thromborel S
Influence of rivaroxaban on aPTT and PT
30
35
40
45
50
55
60
aPTT(sec)
Rivaroxaban (ng/mL)
Me
dia
n a
PT
T(s
ec)
0 120 290
Actin FS
Actin FSL
APTT-SP
CK PREST
STA-Cephascreen
STA-PTTA
SynthasIL
-concentration dependent prolongation of aPTT and PT
-more effect of rivaroxaban on PT
Van Blerk et al, Thromb Haemost 2015; 113: 154-164; Van Blerk, WIV global report 2012
16 16
-aPTT and PT are influenced by rivaroxaban depending on the
reagents used.
- PT shows a more sensitive concentration-dependent
prolongation than aPTT
Ctrough Cmax
Ctrough Cmax
Douxfils et al, Thromb Res 2012; 130:956-966
Influence of rivaroxaban on aPTT and PT
17 17
30
35
40
45
aPTT(sec)
Apixaban (ng/mL)
Media
n a
PT
T(s
ec)
0 41 94 225
SynthasIL
CK PREST
STA-Cephascreen
STA-PTT A
Actin FS
Actin FSL
APTT-SP
Influence of apixaban aPTT and PT
Van Blerk et al, Int J Lab Hematol 2017; doi:10.1111/ijlh.12640; Van Blerk, WIV global report
2014
12
13
14
15
16
17
PT(sec)
Apixaban (ng/mL)
Media
n P
T(s
ec)
0 41 94 225
Recombiplastin 2G
Neoplastin CI PLUS
Neoplastin R
Innovin
Thromborel S
PT and aPTT were barely influenced at the concentrations tested
18 18
Influence of edoxaban on aPTT and PT
Douxfils et al, Thromb Haemost 2016; 115: 368-381
-aPTT and PT are influenced by edoxaban depending on the
reagents used
- PT shows a more sensitive concentration-dependent
prolongation than aPTT
19 19
aPTT and PT in thrombophilia testing
One stage clotting assay FVIII
Underestimate FVIII
Douxfils et al,
Thromb Haemost 2016; 115: 368-381
Jacquemin et al,
J Thromb Haemost 2015; 13: 2087-292
20 20
aPTT and thrombophilia testing
One stage clotting assay FVIII
Underestimate FVIII
Missing elevated FVIII as thrombophilia marker
FVIII > 150% (>90th percentile)
5x higher risk thrombosis
6-11x higher risk for recurrent VTE
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aPTT and thrombophilia testing
Resistance to activated protein C (aPC-r)
Prolonged aPTT in modified Dahlbäck test
Elevated ratio, falsely normal result
Johnson et al, Am J Hematol 2012; 87: S108-112
aPTT based method with FV deficient
plasma (1:5 predilution in FV def
plasma)
APTT (+APC)
APTT (-APC)
Normal: ratio > 2
FV Leiden: ratio <2
22 22
aPTT and thrombophilia testing
Resistance to activated protein C (aPC-r)
Prolonged aPTT in modified Dahlback test
Elevated ratio, falsely normal result
Johnson et al, Am J Hematol 2012; 87: S108-112
NPP dabigatran rivaroxaban
100 250 120 290 ng/ml
23 23
aPTT and thrombophilia testing
Resistance to activated protein C (aPC-r)
Prolonged aPTT in modified Dahlbäck test
Clot based (prothrombinase) assays
Elevated ratio, falsely normal result
Douxfils et al, Thromb Haemost 2016; 115: 368-381
Jacquemin et al,
J Thromb Haemost 2015; 13: 2087-292
24 24
aPTT and thrombophilia testing
Resistance to activated protein C (aPC-r)
Missing aPC-r
Mannucci and Franchini, Thromb Haemost 2015; 114: 885-889
25 25
Screening Mixing Confirmation
Presence of aPL Presence of an inhibitor;
exclusion of factor
deficiency
Demonstrate the PL-
dependent character
Low concentration of PL
Mix PP and NPP 1:1 Excess of PL
Prolongation No correction Shortening compared to
screening test
aPTT and thrombophilia testing
Lupus anticoagulant testing
Functional assays to detect antiphospholipid antibodies
Sensitive aPTT and dRVVT
aPTT prolonged by DOAC
Falsely positive LAC
26 26
dRVVT (LAC) and thrombophilia testing
Lupus anticoagulant testing
dRVVT prolonged
Falsely positive LAC
Depreter and Devreese, Clin Chem Lab Med 2017; 55:91-101.
Apixaban, dabigatran and rivaroxaban:
Concentration dependent prolongation of dRVVT screening and
confirmation test; ratio false positive for rivaroxaban
27 27
dRVVT (LAC) and thrombophilia testing
Lupus anticoagulant testing
dRVVT prolonged
Falsely positive LAC
Edoxaban:
Concentration dependent prolongation of dRVVT screening and
confirmation test Douxfils et al, Thromb Haemost 2016; 115: 368-381
28 28
Screening assay Mixing test Confirmation assay
Ratio screen/confirm
PL-dependent
coagulation test
Mix 1 PP: 1 NPP PL-dependent
coagulation test
Prolonged by DOAC DOAC= inhibitor
No correction
Prolonged by DOAC
Positive Positive Positive/negative
Final conclusion LAC: false positive
DOAC and LAC in thrombophilia testing
Lupus anticoagulant testing
dRVVT and aPTT prolonged by DOAC
Falsely positive LAC
29 29
DOAC and LAC in thrombophilia testing
False positives
Dabigatran 12.5 ng/ml aPTT and dRVVT 43%
Rivaroxaban 7.5 ng/ml dRVVT 30%
Apixaban 18.75 ng/ml dRVVT 21%
Ratzinger et al, Thromb Haemost 2016; 116: 235-240
Lupus anticoagulant testing
Even at trough levels: false LAC positive
Falsely positive results in aPTT and dRVVT system
Taipan snake venom time/Ecarin clotting time as
alternative in AVK and direct Xa inhibitors
Not standardized, not widely available
van Os et al, J Thromb Haemost 2011;9:1657-9
Hoxha et al, Autoimmun Rev. 2017;16:173-178
30 30
DOAC and LAC in thrombophilia testing
Lupus anticoagulant testing
Falsely positive results in aPTT and dRVVT system
Neutralization of dabigatran by antidote Idarucizumab
(Praxbind)
Jacquemin et al,
J Thromb Haemost 2015; 13: 2087-292
31 31
DOAC and thrombophilia testing
Lupus anticoagulant testing
-DOACs lead to false-positive LAC detection in both aPTT
and dRVVT test systems
-It is not advisable for the moment to carry out LAC testing
during DOACs treatment. Hoxha et al, Autoimmun Rev. 2017;16:173-178
-for valid tests patients have to be off DOACs for 48 hours
Moll, J Thromb Thrombolysis 2015; 39: 367-378
32 32
DOAC and thrombophilia testing
Antithrombin
Mannucci and Franchini, Thromb Haemost 2015; 114: 885-889
33 33
DOAC and thrombophilia testing Antithrombin
Van Blerk et al, Thromb Haemost 2015; 113: 154-164; Van Blerk, WIV global report 2012, 2014
Increase of AT in IIa based chromogenic assay by dabigatran
Increase of AT in Xa based chromogenic assay by rivaroxaban and apixaban
80
85
90
95
10
01
05
Antithrombin(%)
Rivaroxaban (ng/mL)
Me
dia
n A
ntith
rom
bin
(%)
0 120 290
Berichrom Antithrombin III
Chromogenix Coamatic Antithrombin
IL HemosIL Liquid Antithrombin
Stachrom AT III 3
80
85
90
95
10
0
Antithrombin(%)
Dabigatran (ng/mL)
Me
dia
n A
ntith
rom
bin
(%)
0 100 250
Berichrom Antithrombin III
Chromogenix Coamatic Antithrombin
IL HemosIL Liquid Antithrombin
Stachrom AT III 3
90
100
110
120
Antithrombin(%)
Apixaban (ng/mL)
Media
n A
ntith
rom
bin
(%)
0 41 94 225
Liquid Antithrombin
Innovance Antithrombin
Antithrombin chromogenic assay
IIa based Stachrom Berichrom
Xa based HemosIL (--) Chromogenix Innovance
34 34
Douxfils et al, Thromb Haemost 2016; 115: 368-381
Increase of AT in Xa based chromogenic assay by edoxaban
DOAC and thrombophilia testing
Antithrombin
35 35
Antithrombin
chromogenic assay
Dabigatran Rivaroxaban Apixaban Edoxaban
IIa based Increase No effect No effect No effect
Xa based No effect Increase Increase Increase
DOAC and thrombophilia testing
Antithrombin
36 36
DOAC and thrombophilia testing
Protein C and Protein S
Mannucci and Franchini, Thromb Haemost 2015; 114: 885-889
37 37
DOAC and thrombophilia testing
Protein C and Protein S
Assay Influence of DTI Influence of antiXa
Protein C
Clot based False decrease False decrease
Chromogenic No effect No effect
Protein S
Clot based False decrease False decrease
Free antigen No effect No effect
Total antigen No effect No effect
Johnson et al, Am J Hematol 2012; 87: S108-112
Undas et al, Adv Clin Exp Med 2016; 26: 1321-1330
38 38
AVK and thrombophilia testing
Protein C
Protein S
Assay Influence of AVK
Protein C
Clot based Decrease
Chromogenic Decrease
Protein S
Clot based Decrease
Free antigen Decrease
Total antigen Decrease
-Avoid testing in patients during
AVK therapy
Discontinuing anticoagulation 3-4
weeks for reliable result
-bridging with LMWH
39 39
AVK and thrombophilia testing
Lupus anticoagulants
AVK
check INR
Analysis of LAC After AVK, INR<1.5
Mix 1:1 PPP/NPP
No analysis INR >3
Interference aPTT and dRVVT FP and FN
Pengo et al, J Thromb Haemost 2009; 7: 1737-40
40 40
AVK and thrombophilia testing
Lupus anticoagulants
Pengo et al, J Thromb Haemost 2009; 7: 1737-40
Screening assay Mixing test Confirmation assay
Ratio screen/confirm
PL-dependent
coagulation test
Mix 1 PP: 1 NPP PL-dependent
coagulation test
Prolonged by AVK Correction of the
factor deficiency
Prolonged by AVK
Positive Negative/positive Positive/negative
Final conclusion LAC: false positive or false negative
Depreter and Devreese, Clin Chem Lab Med 2017; 55:91-101.
Devreese and de Laat, J Thromb Haemost 2015 ; 13:1475-8.
41 41
Effects of antico in thrombophilia tests
Test Dabi Riva Apixa Edoxa AVK
Antithrombin Xa-based / + + + /
IIa-based + / / / /
Protein S Free antigen / / / / -
Protein C chromogenic / / / / -
F V Leiden
(aPC-r)
Clot based ++ + + + /
Prothrombin
G202010A
DNA analysis / / / / /
FVIII one stage clot - - - - - /
LAC aPTT, dRVVT ++ ++ ++ ++ + or -
aCL Solid phase / / / / /
aβ2GPI Solid phase / / / / /
+: increase -: decrease /: none
42 42
Case
Thrombophilia screening not interpretabale during treatment
with DOAC
Antithrombin (Xa based) 100.0% 80.0-120.0%
Protein C activity
(chromogenic)
120.0% 70.0-130.0%
Protein S antigen (free) 118.0% 50.0-134.0%
APC-r Not interpretable >2.00 (ratio)
FVIII 53.0% 60-120%
Lupus anticoagulans positive /
Protrombine mutation negative
FV Leiden negative
Reference value
aPTT 58.6 s 28.9-38.1 s
INR 1.3 0.9-1.1
PT 73% 60-120%
43 43
Conclusion
Treatment of acute thrombosis is not influenced by test
result: postpone testing
Test for PT to exclude AVK, which will reduce Protein C and
protein S
Chromogenic assays for protein C are are no subject for
interference of DOAC
Immunoreactive assays for protein S are no subject for
interference of DOAC
Deficiencies should not be diagnosed on one result
Clotting assays (aPC-r, factor dosage, LAC) are influenced
by all anticoagulants
Collaboration in thrombophilia testing: under
supervision of experienced lab staff and interpreted by
experienced clinician
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Thank you for your attention