Fibrinogen

Plasminogen Plasmin Fibrin

FDPD-Dimer

tPA

V

II

PSAPC

ATPAI-1

Activation pathways Inhibition pathways

Coagulation

Fibrinolysis

PSAPC

PAI-1

AT

AT

AT

TF

TFPITFPI

PSAPC

PSAPC

PC

ActivatedPlateletXaVa

IXa

AT

IIa

XaXa

IIa

TM

vWF

XI VIIIa VIII

IXXIa

X

VIIa AT

H A E M O S T A S I S

At the Heart of Haemostasis

Diagnostica Stago S.A.S.RCS Nanterre B305 151 4099, rue des Frères Chausson92600 Asnières sur Seine (France)Ph.: +33 (0)1 46 88 20 20Fax: +33 (0)1 47 91 08 91webmaster@stago.comwww.stago.com

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AT THE HEART OF HAEMOSTASISQuick Guide To Haemostasis

Screening assays in Haemostasis

Anticoagulant therapy monitoring (1): Vitamin K antagonists

Anticoagulant therapy monitoring (2): Heparin

Assays for other anticoagulant therapy (3)

Disseminated Intravascular Coagulation (DIC)

Thrombophilia

D-Dimer assay for the exclusion of venous thromboembolism (VTE)

Lupus anticoagulants / Antiphospholipid antibodiesFor more information, visit our website at www.stago.com

Screening assays in Haemostasis1) Questionnairel Personal and familial historyl Treatmentsl Diseasesl Clinical symptoms

2) Physical examination

3) Pre-operative Haemostasis screening assaysl Prothrombin time (PT) n Screening test for the extrinsic and common pathways of coagulation

(factors II, V, VII, X). Limited sensitivity to fibrinogen. n Usual normal ranges: 12 - 13 sec (may vary between reagents, please refer to

manufacturer’s insert)

l Activated Partial Thromboplastin Time (aPTT) n Screening test for the intrinsic and common coagulation pathways of coagulation (factors VIII, IX, XI, XII, V and II). Limited sensitivity to fibrinogen. n May be normal in some forms of von Willebrand disease n Usual normal range: ratio patient/reference ≤ 1.2

l Thrombin time n Exploration of fibrin formation

n Usual normal range: < 21 seconds (may vary between reagents, please refer to manufacturer’s insert)

Effects of coagulation factors and protein levels on clotting assays

1. Approximate level in the case of a single factor deficiency. These levels may vary according to different clinical settings, e.g.

haemophiliacs undergoing surgery will require higher levels of FVIII2. Results vary as a function of sensitivity of reagents and single or multiple factor levels3. PT: Prothrombin Time4. aPTT: activated Partial Thromboplastin Time5. TT: Thrombin time6. Sex and age-dependent

XII 60 - 150% - N N

XI 60 - 150% 20 - 30% N N

VIII 60 - 150% 30 - 40% N N

IX 60 - 150% 30 - 40% N N

VII 55 - 170% 10 - 20% N N

X 70 - 120% 30 - 40% N

V 70 - 120% 30 - 40% N

II 70 - 120% 30 - 40% N

Fibrinogen 2 - 4 g/L 0.5 - 1 g/L N or N or VWF 50 - 160% 40% N N or N Antithrombin 80 - 120% - N N N Protein C 70 - 130% - N N N Protein S 60 - 140%6 - N N N

FACTORS NORMAL VALUESMINIMUM LEVEL

FOR LOW BLEEDING RISK

INFLUENCE OF COAGULATION TESTS2

PT3 aPTT4 TT5

Screening assays in Haemostasisl Fibrinogen level n Quantitative assay of fibrinogen level n Usual normal range: 2-4 g/L (200-400 mg/dL) n Elevated fibrinogen levels are observed in inflammatory syndrome (acute or

chronic)

l Platelet count nNumber of circulating platelets

n Usual normal range: 150-400 G/L

4) Screening assays for Disseminated Intravascular Coagulation (DIC)PT, aPTT and fibrinogen levels, as well as D-Dimers, are generally abnormal in acute DIC but may be normal during chronic and subacute DIC. These screening tests are thus of limited specificity and sensitivity for the diagnosis of DIC (see section on DIC). Recent studies suggest that fibrin monomer may constitute early and more specific markers of DIC.

5) Screening assays for thrombophilial There are as yet no global tests available to explore all thrombophilia factors.l First-line activity assays are required for monitoring of inhibitors (see Thrombophilia

section).

6) Screening tests for Lupus Anticoagulants (LA)l The screening assays are phospholipid-dependent function tests. At least two

different tests are required for detection of LA (see Lupus Anticoagulants section).

Bibliography: l Simple coagulation tests survival prediction in patients with septic shock. Lissalde Lavigne G et al. J Thromb Haemost 2008, 6:645-653l Guidelines for the diagnosis and management of DIC. Levi M. et al. Br J Haematol, 2009, 145, 1:24-33l DIC diagnosed based on the Japanese association for acute medicine criteria is a dependant continuum to overt DIC in patient with sepsis. Gando S. et al. Thromb Res, 2009, 123, 5:715-718l Conduites pratiques en hémostase, Charles Marc Samama, LFB monograph, 1996l Hémostase & thrombose, La Simare éditions impression, 1994l Hemostasis, a case oriented approach, D. A. Triplett, Igaku-Shoin Medical publishers, 1985l Recommandations pour une juste prescription des examens d’hémostase en pratique médicale courante, M. Gouault-Heilmann, STV, 2006; vol.18, No1, pages 29-42l Laboratory Techniques in Thrombosis - A Manual, Second revised edition of the ECAT Assay Procedures, Jespersen J, Bertina RM, Haverkate F, 1999, pages 1-308

Anticoagulant therapy monitoring (1)Oral anticoagulant therapy with vitamin K antagonists (VKA*)l Vitamin K antagonists reduce synthesis of both vitamin K-dependent coagulation factors (factors II, VII, IX, X) and some proteins (Protein C and Protein S). The deficiency in factors II, VII, IX and X induced by the absorption of vitamin K antagonists results in prolongation of PT and of aPTT.

l The Prothrombin Time is the reference test of choice for monitoring of vitamin K antagonist therapy. It is expressed as a coagulation time, using the ratio between the patient and a control, or as % prothrombin time, leading to wide inter-laboratory result variability due to the use of different analysers/reagents combination.In order to standardise the results obtained for patients on vitamin K antagonists, results are expressed as INR.

l PT results expressed as INR (International Normalized Ratio)n The INR is the ratio of PT to the power ISI (International Sensitivity Index).It represents the ratio of PT that would have been obtained if an international thromboplastin reference material had been used for the test.n The ISI is a value calculated for each batch of thromboplastin reagent and varies according to the type of analyser used. ISI values are specific for pairs of reagents and instruments. The international recommendations suggest the use of a reagent with an ISI value of less than 1.7.n The control time is the geometric mean of PT values measured on at least 20 fresh plasma samples from healthy subjects (with no pathology or treatment that might interfere with coagulation).

l Anticoagulant therapy monitoring n Heparin followed by vitamin K antagonists l Monitoring of treatment with vitamin K antagonists with the INR (using a

reagent insensitive to heparin at therapeutic doses) l Monitoring of heparin treatment - aPTT (indicates the combined effect of vitamin K antagonists and unfractionated heparin) - Specific anti-Xa assay (specific monitoring of anti-Xa activity) l Treatment with heparin and vitamin K antagonists may be given concomi-

tantly for 4 to 5 days until the desired therapeutic range is achieved in terms of INR (i.e. two consecutive INR of two consecutive days unchanged and in the therapeutic range).

nStable patients on vitamin K antagonists treatment The INR value should be checked weekly then monthly, and more frequently in the event of a dose change, unbalanced assay results or suspected interference by other factors.

*Antivitamins K or vitamin K antagonists

INR = Patient Time (sec) ISI

Control Time (sec)( )

Anticoagulant therapy monitoring (1)

Bibliography:l Guidelines on oral anticoagulation (warfarin): third edition - 2005 update. Baglin T.P., Keeling D.M., Watson H.G. for the British

Commitee for Standards in Haemotalogy. Br J Haematol, 2005; 132: 277-285

l Evidence-Based Management of Anticoagulant Therapy. Antithrombotic Therapy and Prevention of Thrombosis, 9th Ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Holbrook A., Schulman S., Witt D.M., Vandvik P.O., Fish J., Kovacs M.J., Svensson P.J, Veenstra D.L., Crowther M., Guyatt G.H. Chest, 2012; 141: 152S-184S

1 ACCP: American College of Chest Physicians2 BCSH: British Committee for Standards in Haematology3 GEHT: Groupe d’Etude sur l’Hémostase et la Thrombose

Prophylaxis of venous thrombosis.

Treatment for deep venous thrombosis (DVT) and pulmonary embolism (EP). Cardiac valve disease. Myocardial infarction. Atrial fibrillation.

Antiphospholipid syndrome associated with recurrent DVT or additionnal risk factors.

INDICATION

2.0 - 3.0

2.0 - 3.0

2.5 - 3.5

INR

ACCP1, BCSH2, GEHT3

2.5

2.5

3.5

l N.B.: n Wide inter-patient variability n Interferences due to: l Other treatments (many drugs can interfere with vitamin K antagonists) l Other diseases l Food and drinks rich in vitamin K

l Recommended therapeutic ranges (expressed in INR)

Range Target

Anticoagulant therapy monitoring (2)Unfractionated Heparin monitoring

Unfractionated heparin (UFH) is a heterogeneous mix of polysaccharide chains ofvariable molecular weight. One-third of the chains have a pentasaccharide patternthat has a strong binding affinity to Antithrombin.Molecular weight: 2,000 to 40,000 Dalton (average: 15,000).

They are used for both therapeutic and prophylactic therapy

lLimitation: n Short half-life, dose-dependent n Non-specific binding n Random dose-response effect n Major bleeding risk

lSample collection and treatment: n A standardised protocol should be followed for the collection and processing of the

samples. n Collection: tube containing an anticoagulant - sodium citrate 0.105/0.109 M or

CTAD (Citrate, Theophylline, Adenosine, Dipyridamole). n Centrifugation within an hour of collection. n Stability at +20°C: 2 hours in a citrate tube or 4 hours in a CTAD tube

lAssays: naPTT l Widely used for monitoring UFH treatment but sensitive to certain coagulation

abnormalities (deficiency or specific coagulation disorders, lupus anticoagulant) and to certain drugs such as vitamin K antagonists (during heparin-VKA therapy switch) or thrombolytic drugs

Note: Therapeutic ranges may vary between reagents n Anti-Xa activity: standardised measurement using an international standard

(see Tables 1 and 2)

n Platelet count: for the detection of heparin-induced thrombocytopenia (HIT) l HIT, Type II : - Immunoallergic reaction to the drug - Up to 3% of treated patients(1)

- Generally occurs after the 5th day of treatment - Severe thrombocytopenia, followed by life-threatening thrombotic complications

n Antithrombin (AT): for the exclusion of antithrombin deficiency in the case of heparin resistance.

Bibliography: l (1) Treatment and Prevention of Heparin-Induced Thrombocytopenia. Antithrombotic Therapy and Prevention of Thrombosis,

9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Linkins L.A., Dans A.L., Moores L.K., Bona R., Davidson B.L., Schulman S., Crowther M. Chest, 2012; 141: 495S-530S

Anticoagulant therapy monitoring (2)

continuous intravenous infusion

discontinuous subcutaneous or intravenous infusion

any time after fourth hour of treatment

at the midpoint between 2 injections

Table 1 : Unfractionated heparin: curative therapeutic range

1.5-3.5 times

1.5-3.5 times 0.3 to 0.7 IU/mL

0.3 to 0.7 IU/mL

ADMINISTRATION MODEaPTT Ratio*

patient/referenceAnti-Xa activity

SAMPLING

Curative treatment

ADMINISTRATION MODEaPTT Ratio*

patient/referenceAnti-Xa activity

SAMPLING

Preventive treatment

subcutaneous or at the midpoint intravenous infusion between 2 injections

Tableau 2 : Unfractionated Heparin: preventive therapeutic range

1.2-1.3 times 0.1 to 0.2 IU/mL

*The extent of aPTT prolongation differs between reagent. It is recommended that every laboratory establish their own aPTT therapeutic range according to their own operating procedure

Bibliography:l Parenteral anticoagulants. Antithrombotic Therapy and Prevention of Thrombosis.

9th ed: American College of Chest Physicians Evidence-based Clinical Practice Guidelines. Garcia D.A., Bagin T.P. Witz J., Samama M.M. Chest, 2012; 141: e24S-e43S

Low Molecular Weight Heparin (LMWH) monitoring

l Low Molecular Weight Heparin is obtained by enzymatic or chemical depolymerisation of unfractionated heparin.Molecular weight: 2.000 to 12.000 Daltons (mean: 5.000 Da)

lAnticoagulant profile: n Ratio of anti-factor Xa / anti-factor IIa activity: - UFH: 1:1 - LMWH: 2 to 5:1 (and more for same LMWH preparation)

lAdvantages: n More predictable anticoagulant response n Better bioavailability at low doses n Non-dose-dependent clearance n Longer half-life

lTests:n The result of global coagulation tests (aPTT) are not correlated with the anti-

coagulant activity of LMWHn The only tests currently available are those that specifically measure anti-Xa

activity in plasman An international standard for LMWH is availablen Anti-Xa activity for LMWH dose adjustment should be assayed at least 48 hours

after the initial injection (curative treatment)n Monitoring is not necessary during prophylactic treatment (except in the event

of renal impairement, weight gain, bleeding or thrombosis risk)n Monitoring is particularly recommended in children and elderly subjectsn Platelet count should be measured: - within the first 24h of treatment - thereafter, twice weekly throughout treatment.

IU = International UnitsDVT: Deep Vein Thrombosis; PE: Pulmonary EmbolismINN: International Nonproprietary Name1NB: mean values measured in subjects receiving treatment with each LMWH;2Threshold values above which dose reduction can be considered;3For patients weighing between 50 and 100 kg; 5 mg/24h for patients weighing < 50 kg; 10 mg/24h for patients weighing > 100 kg

Table 3: Heparin derivatives (LMWH and fondaparinux) available in France at curative doses in 2012 - (1, 16, 18)

Product Indications Dosage

Peak anti-Xa activity APTT prolongation(if measured)Mean

values1 m±sd

Overdose threshold2

LMWH: twice-daily injection regimen: sample taken at peak activity, 3 to 4 h after injection

LOVENOX® (INN enoxaparin)

DVT with or without PE Acute coronary syndrome

100 IU/kg/12h (1 mg/kg/12h)

1.20 ± 0.17 IU/mL ND Moderate

prolongation

FRAGMINE® (INN dalteparin)

Established DVT Unstable angina

Non-Q-wave myocardial infarction

100 to 120 IU/kg/12h

0.6 ± 0.25 IU/mL

1.0 IU/mL

Moderate prolongation

FRAXIPARINE® (INN nadroparin) 85 IU/kg/12h 1.0 ± 0.2

IU/mL ND Moderate prolongation

LMWH: once-daily injection regimen: sample taken at peak activity, 4 to 6 h after injection

INNOHEP® (INN tinzaparin)

Established DVT Non-serious PE 175 IU/kg/24h 0.87 ± 0.15

IU/mL < 1.5 IU/mL Prolongation

FRAXODI® (INN nadroparin) Established DVT 171 IU/kg/24h 1.34 ± 0.15

IU/mL < 1.8 IU/mL Moderate prolongation

Fondaparinux: once-daily injection regimen: sample taken at peak activity, 2 to 3 h after injection

ARIXTRA® (INN fondaparinux)

Established DVT Non-serious PE 7.5 mg/24h3 1.41 µg/mL ND No

prolongation

ARIXTRA® (INN fondaparinux) Acute coronary syndrome 2.5 mg/24h 0.45 µg/mL ND No

prolongation

Assays for other anticoagulant therapy (3)

Replacement therapy monitoring

Hirudin - desirudin (Revasc®) A protein produced by the salivary glands of leeches.Direct thrombin inhibitor.Recombinant hirudins are currently commercially available.

lIndications (depend on drug and country) n Treatment of patients with heparin-induced thrombocytopenia (HIT) n Prevention of thromboembolic complications after orthopaedic surgery (total

hip or knee replacement) in patients with a history of HIT

lLaboratory testsn Treatment l aPTT: there is a direct relationship between the aPTT prolongation and the plasma hirudin concentration, except at high concentrations. l Ecarin clotting time l Ecarin chromogenic assay (ECA): for the quantitative determination of hirudin and its analogues n ProphylaxisNo recommendation for monitoring, except in the case of renal or hepatic failure or combined treatment with oral anticoagulants.

Danaparoid Sodium (Orgaran®)Mixture of heparan sulphate (~ 84%), dermatane sulphate (~ 12%) and chondroitin sulphate (~ 4%). Molecular weight: approximately 5 500 Daltons.Specific inhibitor of factor Xa.

lIndications (according to country) n Treatment of patients presenting HIT n Prophylaxis of post-operative deep venous thrombosis following total hip

or knee replacement surgery (in patients with a previous history of HIT)

lLaboratory testsn Monitoring not necessary for prophylactic treatments, but recommended for

curative treatment. - mild effect on time-based assays (PT and aPTT) - anti-Xa assays (specific protocol)

Assays for other anticoagulant therapy (3)New anticoagulants monitoring

Fondaparinux (Arixtra®) Fondaparinux, a pentasaccharide, is a synthetic and selective inhibitor of factor Xa.The administration of this drug is simple: 1 subcutaneous injection per day at a fixed dose regardless of patient characteristics.The half-life varies according to age and is between 17 and 21h, with a peak concentration 2 to 3h after injection

lIndications n Prevention of thrombosis in orthopaedic surgery n Prevention of thrombosis in high-risk abdominal surgery n Prevention of thromboembolic events in patients at risk and bedridden for an

acute medical problem (cardiac failure, respiratory problems, etc) n Curative treatment of acute deep venous thrombosis n Curative treatment of acute pulmonary embolism

lTherapeutic dosage n Preventive treatment: 2.5 mg (or 1.5 mg for renal failure patients) / day n Curative treatment: 7.5 mg/day for patients weighing between 50 and 100 kg

lLaboratory testsAlthough no monitoring is officially recommended, certain clinical situations may require a dosage: n Haemorrhagic or thrombotic accidents n Additional surgery n Renal failure n etc. Anti-Xa activity assay with dedicated controls and calibrator for the monitoring of Fondaparinux (STA® - Fondaparinux Control and Calibrator) n Measuring range: 0.1 to 2 µg/mL l The assay measuring range covers all doses

Direct factor Xa inhibitors Rivaroxaban (Xarelto®) - Apixaban (Eliquis®) Rivaroxaban and apixaban are synthetic direct factor Xa inhibitors. They have a short half-life and are almost immediately bioavailable upon oral administration. There is currently no antidote available. It may be necessary to measure anticoagulant activity in certain patients or in certain clinical situations.

lIndications n VTE prophylaxis after total knee or hip replacement surgery n Atrial fibrillation n DVT and PE treatment (rivaroxaban) n Secondary prevention of VTE (rivaroxaban) n Acute coronary events, in combination with antiplatelet drugs lSpecific test: rivaroxaban anti-Xa activity is assayed using specific controls and calibrators (STA®-Rivaroxaban Control & Calibrator) l For specific determination of rivaroxaban anticoagulant activity: l Test to be performed 2 to 4 h after latest intake l Working range: 20 to 500 ng/mL

Direct Thrombin Inhibitors (DTIs) DTIs (Dabigatran -Pradaxa®-, Argatroban®, Bivalirudine®, etc) are direct inhibitors of thrombin with varied therapeutic range.They have a short half-life and practically immediate bioavailability.DTIs have no antidotes. It may be necessary to measure anticoagulant activity for certain drugs and in certain patients.

lIndications (according to drug and country) n Treatment of patients with heparin-induced thrombocytopenia (Argatroban®,

Bivalirudine®) n Prevention of thrombosis n Prevention of stroke in atrial fibrillation n Prevention of thromboembolic complications following orthopaedic or cardiac

surgery. lLaboratory tests n aPTT: there is a direct relationship between the aPTT prolongation and the DTIs concentration in plasma, however: l This test is greatly affected by different variables (presence of lupus

anticoagulants, factor deficiencies, etc) and concomitant treatments (vitamin K antagonists, heparin, etc)

l The measuring range is unsuitable for DTIs l There is poor linearity with high doses of DTIs n Ecarin clotting time (ECT): this is considered as the reference test, but: l It is dependent on the patient’s thrombin and fibrinogen levels. l There is no standardisation n Ecarin chromogenic assay (ECA): is a specific and quantitative assay of DTIs levels

Fibrinogen

D-Dimers

FXIII

+

Solubles complexes = Fibrin monomer + FDP Fibrin monomer + FDPn Fibrin monomer + fibrinogen

+

Fibrinogen degradationproducts (FDP)

Fibrin degradation products(FDPn)

Stabilised fibrin clot

+

Thrombin

Plasmin

Plasmin

PlasminFibrin Monomer

EE

E

E E

EE

EE

E

E

E

E

E

E

DD

D

D DDD

DD

D

DD

D

DD

DDD

D

D

D

D

DD

D D

Disseminated intravascular coagulation (DIC)l DIC is an acquired syndrome characterised by excessive systemic intravascular activation of coagulation, with no specific site and due to a variety of different causes and origins (ISTH 2001).

l Diagnosis of DIC is based on clinical criteria (presence of underlying disease or specific medical conditions such as septicaemia, trauma, malignant tumour, etc) and laboratory criteria.

l There are 2 types of DIC: nCompensated or «non-overt» DIC nDecompensated or «overt» DIC

Non-overt DIC is defined as DIC with no signs of haemorrhage or thrombosis. Diagnosis of compensated DIC is based primarily on laboratory criteria.

Overt DIC consists of established, manifest and serious DIC. Diagnosis is based on a combination of clinical signs and different laboratory tests.

Diagnosis of DICClinical diagnosis Clinical signs of DIC are either thrombotic signs or haemorrhagic signs, or both at the same time.Patients are classified according to 3 different physiological clinical scoring systems.

Laboratory diagnosis There are no laboratory tests truly specific for DIC.

PT, aPTT and fibrinogen are generally abnormal in acute DIC, but they may be nor-mal in chronic or subacute DIC. These screening tests are thus of limited specificity and sensitivity for diagnosis of DIC. Recent studies show assay of fibrin monomers to be an early marker for DIC.

Determination of DIC defined by the ISTH is based on the presence of underlying disorders and on clotting tests, which allow the identification of patients presenting non-overt and overt DIC.

Bibliography: l Disseminated Intravascular Coagulation. Levi M, Cate H. ten. NEJM, 1999, 586-592 l Diagnostic Criteria and Laboratory Tests for Disseminated Intravascular Coagulation. Kaneko T., Wada H. J. Clin. Exp.

Hematopathol. 2011; 51: 57-76 l Diagnosis of Disseminated Intravascular Coagulation and related consumptive coagulopathies. Mammen

EF. Caduceus Medical Publishers Inc., Patterson, NY, USA, 1992l Laboratory diagnosis of DIC and activated coagulation. Schmaier AH., DIC assessment ABC monography,1989, 2-18l Towards Definition, Clinical and Laboratory Criteria, and a Scoring System for Disseminated Intravascular Coagulation (ISTH),

Taylor F.B., Toh C.H., Hoots W.K., Wada H., Levi M., Thromb Haemost, 2001; 86: 1327-30l Diagnostic criteria and laboratory tests for disseminated intravascular coagulation. Kaneko T, Wada H. J. Clin. Exp. Hematopathol

2011; 51: 67-76

PRESENCE OF AN UNDERLYING DISORDER ASSOCIATED WITH DIC

YES: proceed with ISTH overt DICscore PT, APTT, Fibrinogen, platelet count, fibrin-related markers

NO: stop

Score :

l Platelet count: > 100 G/L = 0 — < 100 G/L = 1 — < 50 G/L = 2l Fibrinolysis: (e.g: D-Di, FDP, FM): No increase = 0 — moderate increase = 2 high increase = 3l PT prolongation: < 3 sec. = 0 3 sec. < PT < 6 sec. = 1 — > 6 sec. = 2l Fibrinogen: > 1 g/L = 0 — > 1 g/L = 1

Overall score :

≥ 5 compatible with overt-DIC (repeat daily)

< 5 non-suggestive of overt DIC (repeat the next 1-2 days)

Thrombophilial Thrombophilia may be defined as the tendency to develop thrombosis: n Hereditary thrombophilia (congenital or juvenile), is a genetically determined

tendency to develop venous thrombosis. n Acquired thrombophilia.

Classification

l Hereditary thrombophilia (congenital) n Associated with a family history of venous thrombosis (in most cases). n Recurrent thrombosis in certain patients. n Not always associated with a clear triggering factor. n Affects young subjects: < 60 years.

l Acquired thrombophilia n No associated family history (generally). n Associated with transient triggering event such as: l Traumatic injury. l Pregnancy - in particular during the post-partum period. l Immobilisation (i.e., stasis, long flights, etc). l Post-operative period. l Advanced age (first event > 60 years). n Oestrogen therapy. n Antiphospholipid antibodies. n Can also be associated with other clinical disorders such as: l Cancer. l Heparin-induced thrombocytopenia.

Diagnostic tests for thrombophilia

l Prelimary Screen: n Antithrombin, Protein C, Protein S, resistance to activated Protein C / Factor V Leiden, prothrombin gene mutation, lupus anticoagulants (LA) and antiphospholipid antibodies (APA).

l Secondary Screen:n Fibrinogen, homocysteine.n Investigation of fibrinolysis (tPA, etc.)n Assay of factor VIII, etc.

Thrombophilia

CAUSES

l Other testsn Other Haemostasis elements.

l Sample collection procedure Samples used for confirmation of thrombophilia must be collected at least 3 months after the latest thrombotic event and in accordance with the time limits associated with the test to be performed after the end of treatment.

n Samples should not be taken in the following cases: lDuring acute episodes of DVT/PE l During oral anticoagulant treatment with vitamin K antagonists, PC and

PS levels are reduced by 50% and levels of activity are reduced. l It is necessary to wait one month after discontinuation of treatment with

vitamin K antagonists before performing Protein C and Protein S assays. l In the case of heparin treatment, which could reduce antithrombin levels l In pregnant women and for up to 3 months after childbirth l In patients on oestrogen therapy until 2 cycles after treatment discontinuation

(e.g. contraceptive pills) l In patients treated with L-asparaginase l In patients with nephritic syndrome, hepatic impairment, DIC or an

inflammatory condition. l In the case of positive screening tests for LA, further screening should

be repeated after 12 weeks.

Low levels of PC and PS are seen in normal paediatric samples.In tests performed on paediatric samples, normal values for patients of the same age group should be established before the diagnosis is established.

Bibliography : l Inherited thrombophilia: part 1. Lane D et al. Thromb Haemost. 1996; 76: 651.l High prevalence of antithrombin III, protein C and S deficiency, but not factor V Leiden mutation in venous thrombophilic Chinese patients in Taiwan. Shen MC, Lin JS, Tsay W, Thromb Res. 1997; 87: 377-385.l Risk factors for venous thrombotic disease. Rosendaal FR. Thromb Haemost. 199; 82: 610-19.l Protein C and protein S deficiencies are the most important risk factors associated with thrombosis in Chinese venous thrombophilic patients in Taiwan. Thromb Res. 2000; 99: 447-52.l Management of thrombophilia, Bauer K. A., J Thromb Haemost. 2003; 1: 1429-1434l Screening for thrombophilia: a laboratory perspective, Jennings I., Cooper P., Br J Biomed Sc. 2003; 60: 39-51

RELATIVE RISK

Antithrombin

80 - 120% 10-20 in heterozygous 1-2% in patients

patients with VTE

Protein C

70 - 130%

6 3% in patients

with VTE

Protein S*

60 - 140%

5 in patients

0.05-1% in the general population 2-3% in thrombotic subjects 5% in the general FV leiden 3-5 heterozygous patients population >20% in thrombotic subjects

FII genous polymorphism 2-3 heterogenous patients 2% in the European population

αβ2Gp1 + ACL 2.2

USUAL VALUES PREVALENCE

*depends on age and sex

Prevalence in thrombotic subjects

l It has been shown that only D-Dimer is of diagnosis value in ruling out deep venous thrombosis or pulmonary embolism. D-Dimer fragments are formed through the degradation of polymerised fibrin by plasmin.

N.B.: High levels of D-Dimers do not necessarily imply DVT/PE since they may be associated with many other causes.Elevated D-Dimers may also be found in samples: n from pregnant patients n from older subjects n from patients with infection n following traumatic injury n during the post-operative period n from patients with cancer n from hospitalised patients (who generally have a higher baseline D-Dimer level

than ambulatory patients) n from patients with inflammatory syndrome

l Value of D-Dimers in the diagnosis of deep venous thrombosis:n Negative Predictive Value (NPV) close to 100% n Use of D-Dimers combined with clinical pretest prohability score clinical pretest prohability score (Wells score) lWith a score < 2 for patients with suspected DVT

lWith a score ≤ 4 for patients with suspected PE lMay be used for all patients with suspected DVT n Non-invasive method n Limits the number of tests required as well as the need for imaging n Improves diagnosis

D-Dimer assay for exclusion of Venous ThromboEmbolism (VTE)

l Several studies performed with STA®-Liatest® D-Di have shown a negative predictive value (NPV) pretty to close to 100% in ambulatory patients using a threshold value for D-Dimers of 500 ng/mL (i.e. 0.5 µg/mL).

Bibliography: l Evaluation of a new, rapid, and quantitative D Dimer test in patients with suspected pulmonary embolism. Oger E. and al.,

Am. J. Respir. Crit. Care Med. 1998; 158: 65-70. l Performances of a new, rapid and automated microlatex D Dimer assay for the exclusion of pulmonary embolism in symptomatic outpatients. Reber G. et al., Thromb Haemost. 1998; 80: 719-720. l D Dimer testing in patients with suspected pulmonary embolism. Comparison of 2 rapid quantitative assays STA Liatest DDi

and vidas ddimer with an ELISA. Lecourvoisier C. et al., ASH 2000. l Comparative study of 4 new and rapid DDimer assays to exclude deep venous thrombosis or pulmonary embolism. Breton E.

et al., Thromb Haemost. 1997; suppl 1 844: 720.

Number of patients Pathologies NPV STA®-Liatest® D-Di

386 EP 100%

501 EP 99.4%

222 EP 100%

114 TVP/EP 100%

Clinical validation of STA®-Liatest® D-Di in ruling out diagnosis of DVT and PE.

D-Dimer assay for exclusion of Venous ThromboEmbolism (VTE)

NR: Normalized Ratio; CT: Clotting Time* If OAT patient, mix 1:1 plasma patient - Pool Norm®

** according to Rosner Index or ICA calculation (1)

Screening test

prolonged time*

> 1.2patient time

referent time

Mixing studies

STA©-Staclot©

DRVV Screen (dRVVt)

PTT-LA(APTT)

Staclot© LA(APTT)

LA NegativeFactor assays

LA Positive

Confirmatory test

ORAND

CT correction ∆ CT > 8 sec NR > 1.2

STA©-Staclot©

DRVV Confirm (dRVVt)

Diagnostic decision tree for screening for LA according to ISTH recommendations (1)

Ratio

prolonged time no Patient plasma

+Pool Norm©

Lupus Anticoagulants (LA) / Antiphospholipid antibodies (APA)l Antiphospholipid antibodies are a heterogeneous family of antibodies directed against proteins bound to negatively charged phospholipids. Lupus-type inhibitors belong to this family of antibodies as do anticardiolipin antibodies. The presence of antiphospholipid antibodies is a biological marker for antiphospholipid syndrome (APS).

l APS is an autoimmune disease associated with high risk for thrombosis, recurrent spontaneous miscarriage, thrombocytopenia and numerous other clinical manifestations.

l Lupus inhibitor is also known as «antiprothrombinase anticoagulant antibody» or «lupus anticoagulant» (LA).

n LA is an acquired abnormality affecting between 1 and 5% of the general population. However, only 15 to 20% of these individuals are positive for control testing performed several months afterwards.

n In vitro, LA results in prolonged coagulation times in phospholipid-dependent tests (e.g. aPTT or PT as well as certain APCR tests). Diluted Russell’s viper venom time (dRVVT) is a specific test used for LA screening.

n In vivo, LA is associated with a risk of thrombosis that may result in obstetric, neurological stroke, renal or pulmonary complications.

l A single test is not sufficient to allow the diagnosis of this syndrome.

l In some patients, transient antiphospholipid antibodies may be detected due to infection or to another treatment, despite the absence of any clinical signs.

Lupus Anticoagulant (LA) /Antiphospholipid antibodies (APA)Laboratory diagnosisDiagnosis may only be made on the basis of two types of test: immunological tests with screening for APA and/or coagulation tests specific for LA.The recommendations of the «Scientific Subcommittees of the International Society on Thrombosis and Haemostasis» (ISTH) are as follows:

1) Lupus Anticoagulants (LA)Must be detected in plasma on at least two occasions separated by a minimum interval of 12 weeks:

lScreening step: detection of increased coagulation time during a phospholipid-dependent test;

n At least 2 screening tests must be performed before LA can be ruled out: l The first-line test is the DRVV test. l The second-line test consists of sensitised aPTT containing silica activator

and a low concentration of phospholipids.

lIdentification step for the presence of a coagulation inhibitor: 50/50 mix of patient + control (a commercially available pool may be used).

lConfirmation step indicating that the inhibition is phospholipid-dependent; the selected test contains a high phospholipid concentration. It must be based on the same principle as the screening test.

Exclusion of associated clotting disorder.

2) Anticardiolipin antibody (aCL) (IgG and/or IgM) May be present in serum or plasma, in moderate to high concentrations (e.g. > 40 GPL or MPL, or > 99th percentile), on at least two occasions, separated by a minimum interval of 12 weeks, determined using a standardised ELISA method.

3) Anti-ß2 glycoprotein I antibodies (IgG and/or IgM)May be present in serum or plasma (titre > 99th percentile), on one or two occasions, separated by a minimum interval of 12 weeks, determined using a standardised ELISA method, in accordance with the recommended operating procedure.

Bibliography: l Pengo A, Update of the guidelines for lupus anticoagulant detection. J. Thromb Haemost. 2009; 7: 137-40.l Miyakis S et al. International consensus statement on an update of the classification criteria for definite anti-

phospholipid syndrome (APS). J Thromb Haemost. 2006; 4: 295-306.

Fibrinogen

Plasminogen Plasmin Fibrin

FDPD-Dimer

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PSAPC

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Activation pathways Inhibition pathways

Coagulation

Fibrinolysis

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ActivatedPlateletXaVa

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H A E M O S T A S I S

At the Heart of Haemostasis

Diagnostica Stago S.A.S.RCS Nanterre B305 151 4099, rue des Frères Chausson92600 Asnières sur Seine (France)Ph.: +33 (0)1 46 88 20 20Fax: +33 (0)1 47 91 08 91webmaster@stago.comwww.stago.com

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AT THE HEART OF HAEMOSTASIS

At the Heart of HaemostasisAt the Heart of Haemostasis

Diagnostica Stago S.A.S.RCS Nanterre B305 151 4093, allée Thérésa 92600 Asnières sur Seine FrancePh.: +33 (0)1 46 88 20 20Fax: +33 (0)1 47 91 08 91webmaster@stago.comwww.stago.com

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