Curiculum Vitae

MANAGEMENT OF
THROMBOSIS IN CANCER
Anticoagulant and Laboratory Tests

Usi Sukorini
 INTRODUCTION

• Cancer-associated venous thromboembolism (VTE) is

a well-recognized complication associated with
significant patient morbidity and mortality

• Malignancy is a strong risk factor for venous

thromboembolism (VTE)
• 20%-30% of incident VTE in population studies

Timp JF, Braekkan SK, Versteeg HH, Cannegieter SC. Epidemiology of cancer-associated venous thrombosis. Blood.
2013;122:1712–23
Cancer  Virchow’s triad
 Direct mechanisms involved in
cancer‐associated thrombosis.

Razak et al., Cancer-Associated Thrombosis: An Overview of Mechanisms, Risk Factors, and Treatment Cancers 2018, 10, 380;
doi:10.3390/cancers10100380
 Indirect mechanisms promoting thrombosis
in cancer

Razak et al., Cancer-Associated Thrombosis: An Overview of Mechanisms, Risk Factors, and Treatment Cancers 2018, 10,
380; doi:10.3390/cancers10100380
 Unique Challenges in Cancer-associated
Thrombosis

 These patient have both a higher risk of recurrent VTE and a

higher risk of bleeding than patients without cancer
 Patients with malignant diseases can benefit from
anticoagulant therapy and its monitoring
  prevents & reduces recurrent risk of thromboembolic
events & subsequent bleeding
 Anticoagulant
and
Laboratory Tests
 Anticoagulant therapy

Indirect: Fondaparinux
Unfractionated FXa Direct: Rivaroxaban
Heparin (UFH) inhibitors Apixaban
Edoxaban
Low-Molecular-
Weight Direct
Heparin Thrombin Hirudin
(LMWH) Inhibitors Dabigatran
(DTI)

Vitamin K
Antagonists
 Laboratory monitoring for anticoagulants
No Anticoagulants
1 Vitamin K antagonists
2 UFH, LMWH
3 FXa inhibitor
(Rivaroxaban, Apixaban, Edoxaban)
4 Direct Thrombin Inhibitor (DTI):
(Hirudin, Dabigatran etexilate)
Tests
PT (less sensitive)
INR
APTT (less sensitive)
Chromogenic anti FXa assay
APTT
Chromogenic anti FXa assay
APTT, TT
Diluted thrombin time (dTT)
Ecarin clotting time (ECT)
Ecarin chromogenic assay (ECA)
Chromogenic anti IIa assay
 • Traditional oral anticoagulant (warfarin)
• Vitamin K dependent, interfere with normal synthesis of factors
II, VII, IX and X, and protein C and S
• Warfarin cause incomplete coagulation due to lacking calcium
binding site and cannot form enzyme substrate complexes
• The onset of action: 8 – 12 hours, maximum effect: 36 hours
• Duration of action:
72 hours

Kaushansky et al., 2010; Turgeon, 2012

 Laboratory monitoring for VKA

 Prothrombin time (PT): less sensitive

 Standardized  International sensitivity index (ISI)  ≈ 1.0
 International normalized Ratio (INR)

ISI
PT patient (sec)
INR =
mean normal PT (sec)
Therapeutic INR range recommended for standard
oral anticoagulant therapy (VKA)

INR Indication
• optimal level for anticoagulant therapy
• post myocardial infarction therapy & prophylaxis
• atrial fibrillation
2–3
• treatment of VTE, PE
• prevention of systemic embolism
• tissue heart valves
• high risk of clot formation
2.5 – 3.5 • mechanical prosthetic heart valves
• recurrence embolism

<2 • may not provide adequate protection from clotting

>4 • uncontrolled bleeding risk caused by slow blood clotting

Chest, 2001; 119 (Suppl): 85-215

• Heparin is a heterogeneous mixture of highly negatively
charged, sulfated mucopolysaccharides (polysugars) also known
as glycosaminoglycans
• MW of heparin: 15,000 to 18,000 Daltons, injectable anticoag.
• Heparin:
• Unfractionated Heparin (UFH), can cause: bleeding,
thrombocytopenia, osteopenia
• Low molecular weight heparin (LMWH)
• Reduces mortality vs UH, no increase major bleeding
complications, highly cost effective
• Synthetic pentasacharide (fondaparinux)
• Inhibitor of factor Xa
Heparin: mode of action
 APTT and Anti-FXa assay: heparin

 APTT  less sensitive

 The anti–FXa assay is designed to measure plasma heparin
UFH and LMWH levels and to monitor anticoagulant therapy
 Principle:
 The activity of both UFH and LMWHs is dependent upon
binding to antithrombin (AT)
 Binding induces a conformational change in the molecule
which accelerates its inhibitory activity
 LMWHs have primarily anti-Xa activity
 UFH has both anti-Xa and anti-IIa activity
 Anti-FXa assay: heparin

• Anti-FXa assay: clotting-based or chromogenic assay

• A standard curve
• This results in the formation of an inactive AT-Xa complex and
residual Xa is measured
• The residual Xa activity is inversely proportional to the
concentration of heparin in the sample and may be
quantitated from a calibration curve
 Chromogenic anti-FXa assay: heparin assay

Ikeda & Tachibana, 2016. Clinical implication of monitoring rivaroxaban and apixaban by using anti-factor Xa assay in patients with non-valvular atrial
fibrillation, Journal of Arrhythmia 32: 42–50
 Direct Oral Anticoagulant
(DOACs)/
New Oral Anticoagulant (NOACs)

Usi Sukorini
 Direct oral anticoagulants

• Rivaroxaban • Dabigatran
• Apixaban
• Edoxaban
 in Debate

Laboratory test monitoring

during DOACs?
• frequent
International
Normalized Ratio
(INR) monitoring
• labile INR in many
studies
• numerous drug and • Anti-FXa
food interactions inhibitor
• risk of bleeding
• Direct thrombin
inhibitor (DTI)
2013
2013
 2018

Real life case

 A patient with changes in renal function
while on DOAC (real life case)

• An 78-yo woman, history of chronic renal insufficiency, peripheral artery

disease, and microcytic anemia due to blood loss from gastritis

• She developed AF: apixaban 5 mg bd was started at GFR 36 mL/min

• A few months later: microcytic anemia (again) ~ TGI blood loss
• eGFR had deteriorated to 21 mL/min, PT & aPTT normal
• Anti-Xa activity of 832 μg/L at Ctrough , elevated
(on-therapy trough apixaban levels 41-230 μg/L)

• Apixaban dose of 2.5 mg bd

• Ctrough : a persistently elevated level of 376 μg/L
• As she experienced new episodes of TGI bleeding, anticoagulation had to be
stopped completely, eventually
Mechanism of action: DOACs
 DOACs: mode of action

Rivaroxaban
Apixaban
Ieko et al. Journal of Intensive Care (2016) 4:19 Edoxaban Dabigatran
 Direct oral anticoagulants

http://www.islh.org/speaker-portal/files/handouts/HANDOUT-34-1525520837.pdf
 Expected plasma concentration of dabigatran or
rivaroxaban after treatment

Tripodi, The laboratory and the direct oral anticoagulants, Blood, 2013;121(20):4032-4035

Timing of Testing
Drugs Cpeak (hours) Ctrough (hours)
Dabigatran 2 or 3 12
RIvaroxaban 2 or 3 24

Important: communication with clinician

Tripodi A, 2013. The Laboratory and the New Oral Anticoagulants, Clinical Chemistry 59:2
Douxfils et al., 2018. Laboratory testing in patients treated with direct oral anticoagulants: a practical guide for clinicians, J Thromb Haemost; 16: 209–19.
 FXa inhibitor: Global assays

Rivaroxaban
• PT & APTT :
• Available, easy, little expertise
• Prolong PT & APTT:
• linearly and dose-dependently to the [rivaroxaban]
• responsiveness is adequate
• Standardization: concern
• PT more sensitive than APTT, but not specific
• PT is not sensitive to dabigatran

Tripodi A, 2013. The Laboratory and the New Oral Anticoagulants, Clinical Chemistry 59:2
 FXa inhibitor: Global assays

• PT are adequate to detect rivaroxaban activity within the range

of concentration observed in treated patients
• On average PT > 1.5 x (215 ng/ml rivaroxaban – 20 mg OD)
• CV interlaboratory < anti-FXa for rivaroxaban
• Strongest effect on PT:  rivaroxaban, then edoxaban and
apixaban
• PT  for screening for rivaroxaban

Tripodi A, 2013. The Laboratory and the New Oral Anticoagulants, Clinical Chemistry 59:2
Anti-FXa chromogenic assay: rivaroxaban measurement

 FXa is the target of rivaroxaban  the anti-FXa assay should

be the test of choice for this drug
 This test is based on measuring residual FXa with a synthetic
chromogenic peptide upon addition of excess FXa to the
patient’s plasma sample
 The standardization of results across reagents is a matter of
concern
 Anti-Fxa chromogenic assay: is not readily in most lab

Tripodi A, 2013. The Laboratory and the New Oral Anticoagulants, Clinical Chemistry 59:2
Chromogenic Anti-FXa assay: FXa inhibitor measurement

Ikeda & Tachibana, 2016. Clinical implication of monitoring rivaroxaban and apixaban by using anti-factor Xa assay in patients with non-valvular atrial
fibrillation, Journal of Arrhythmia 32: 42–50
 Anti-FXa clotting-based assay

• This is similar to the chromogenic assay but the anti-Xa activity is

measured by performing an APTT-based factor X assay on each
dilution rather than using a chromogenic substrate
• Standard curve
Direct thrombin inhibitor: Laboratory tests
 PT andGlobal
APTT: assays & Dabigatran
Dabigatran
• PT is an excellent test:
• readily available in clinical
laboratories, easiy, little
expertise
• Prolongation of the PT:
• is related linearly and dose-
dependently to the plasma
dabigatran concentration
• responsiveness is not very high:
• Plasma concentration 200
g/L (post 150 mg
dabigatran BD)  prolongs
the PT 1.2 x basal value
• Less sensitive
• Standardization: is a matter of concern
• APTT is readily available, easy
to run
• APTT Prolongation 
• dose-dependently
• not linearly to the dabigatran
concentration
• The responsivenessis adequate:
• a plasma dabigatran
concentration of 200g/L
prolongs the APTT by about
2.5 times the basal value
• Standardization across reagents
will be an issue
Tripodi A., 2013. The Laboratory and the New Oral Anticoagulants, Clinical Chemistry 59:2
 APTT & Dabigatran
APTT & Dabigatran

 The peak plasma concentration (Cmax) and maximal anticoagulant effect

of dabigatran is achieved: within 3 h after oral dosing
 APTT  can provide a qualitative assessment of dabigatran activity, but
the sensitivity depends on the reagent and the coagulometer

 Most patients treated with dabigatran etexilate will present with a

prolonged APTT (ratio > 1.2)
 A normal APTT excludes above on-therapy levels of dabigatran but does
not exclude the presence of dabigatran in the on-therapy range
 APTT is not recommended for low concentration of dabigatran  dTT

Douxfils et al, 2018. Laboratory testing in patients treated with direct oral anticoagulants: a practical guide for clinicians, Journal of Thrombosis and Haemostasis, 16:
 Thrombin Time (TT) & Dabigatran

 The TT is prolonged due to:

 low fibrinogen concentration
 thrombin inhibitors, such as dabigatran
• TT prolongation:
• Linearly & dose dependently to dabigatran
• High responsiveness: 175 ng/ml  TT >> 10–15 x
• The degree of prolongation poor reflects drug concentration

• a normal thrombin time (TT) excludes the presence of dabigatran

with a high negative predictive value
• Modification of TT  dilute Thrombin Time
• Choice for dabigatran: dTT or ECT
 Dilute Thrombin
Dilute ThrombinTime
Time(dTT)
(dTT)

Modifications of TT: diluting plasma samples before testing make

the TT test adequately responsive (TT is prolonged 3 x the basal
value by 200g/L dabigatran)
 Ecarin Clotting Time (ECT)

• Clot formation is obtained by a venom extract (ecarin) from the

snake Echis carinatus
• Ecarin converts FII into meizothrombin
 Ecarin Clotting Time (ECT)

• not yet widely used in clinical laboratories

• ecarin is commercially available
• could be easily run in an ordinary coagulometer
• ECT prolongations are related linearly and dose-dependently to the
dabigatran concentration
• Responsiveness: adequate (i.e., 200 g/L dabigatran prolongs the ECT by
about 3 times the basal value)
• ECT (or dTT): choice for dabigatran
• ECT: not readily available, useful in the absence of specific kit & standard
•  is not approved by FDA  not recommended

Tripodi A., 2013. The Laboratory and the New Oral Anticoagulants, Clinical Chemistry 59:2
 Ecarin Chromogenic Assay (ECA)

• Clot formation is obtained by a venom extract (ecarin) from the snake Echis
carinatus.
• Ecarin converts FII into meizothrombin, which is then measured with a specific
synthetic chromogenic substrate

• ECA can accuracy assess

dabigatran plasma
concentration in the low
concentration (<50 ng/ml)
and normal (50-150 ng/ml)
• ECA not sensitive to heparin

http://www.viapath.co.uk/news-and-press/measurement-of-direct-oral-anticoagulants

Tripodi A., 2013. The Laboratory and the New Oral Anticoagulants, Clinical Chemistry 59:2
 Ecarin Chromogenic Assay (ECA)

ECA is improved by:

 availability of liquid presentations
 Methods can be calibrated
 Without requiring skilled personnel
 Control is sufficient for validating
ECA and ECT
 Choice for Rivaroxaban & Dabigatran

Rivaroxaban Dabigatran
• Screening: • Screening:
• PT • APTT or TT
• Quantification: • Quantification:
• Anti-Fxa assay • dTT or ECT

Tripodi A., 2013. The Laboratory and the New Oral Anticoagulants, Clinical Chemistry 59:2
 Absorption and metabolism of the different
new anticoagulant drugs

http://www.islh.org/speaker-portal/files/handouts/HANDOUT-34-1525520837.pdf
Other laboratory tests

• Excluding an underlying bleeding tendency

• Renal insufficiency
• Drug effect
• Dabigatran !
• Discontinuation dabigatran: Creatinin Clearance (CrCl)
 Coagulation & fibrinolysis markers

• Prothrombin fragment 1.2 (F1 + 2,

• Thrombin-antithrombin complex (TAT)
• A2-antiplasmin
• Plasmin-antiplasmin complex (PAP)
• Fibrinopeptide A (FPA)
• D-dimer

Blood activation markers

• The presence of pro-coagulant activity induces release of blood activation
markers in the circulation
• Extracelullar vesicles (EVs)
• Platelet Factor 4 or beta-thromboglobulin
(Amiral & Seghatchian, 2017)
 Conclusion

• Assessment of the anticoagulant effect may be useful in many

circumstances
• Anticoagulation is important issue dealing with conventional
and new regimen of anticoagulant
• Varied laboratory tests are offer to assess the exposure and
effect of the anticoagulant
• The better understanding of pre- (time to testing), analytical
and post analytical problem will provide a better information
to minimize morbidity and mortality of cancer-associated
thrombosis patients, especially