thrombophilia testing in anticoagulated patients · thrombophilia testing in anticoagulated...

1

Thrombophilia testing in

anticoagulated patients

Katrien Devreese, MD, PhD

Ghent University Hospital, Belgium

Coagulation Laboratory

NTHC 7the annual meeting 30/3/2017

2 2

Investigation of thrombophilia

R. Virchow,

1856

3 3

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

4 4

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

6 6

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

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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

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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

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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

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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

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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

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aPTT and thrombophilia testing

Resistance to activated protein C (aPC-r)

Missing aPC-r

Mannucci and Franchini, Thromb Haemost 2015; 114: 885-889

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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

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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

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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

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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

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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

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DOAC and thrombophilia testing

Antithrombin

Mannucci and Franchini, Thromb Haemost 2015; 114: 885-889

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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

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Douxfils et al, Thromb Haemost 2016; 115: 368-381

Increase of AT in Xa based chromogenic assay by edoxaban

DOAC and thrombophilia testing

Antithrombin

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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

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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

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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

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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%

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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