H A E M O S T A S tPA FDP I S 1 D-Dimer - PAI Plasminogen Plasmin Fibrin T M IIa

PC IIa

T A APC PS II

PS AP T V C A Activated Va Xa vWF X

A T

XI IXa VIIIa APC PS VIII

XIa IX T A

Activation pathways Inhibition pathways

Coagulation PAI-1 AT VIIa AT All rights reserved - 04/2013 Ref. 27646 Xa TFPI AGO - APC PS Xa TFPI S T TF © 2009 DIAGNOSTIC A 4614

Diagnostica Stago S.A.S. RCS Nanterre B305 151 409 9, rue des Frères Chausson 92600 Asnières sur Seine (France) AT THE HEART OF HAEMOSTASIS Ph.: +33 (0)1 46 88 20 20 Quick Guide Fax: +33 (0)1 47 91 08 91 [email protected] At the Heart of Haemostasis www.stago.com To Haemostasis Screening assays in Haemostasis

Anticoagulant therapy monitoring (1): Vitamin K antagonists

Anticoagulant therapy monitoring (2):

Assays for other anticoagulant therapy (3)

Disseminated Intravascular (DIC)

Thrombophilia

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

Lupus / Antiphospholipid antibodies

For more information, visit our website at www.stago.com Screening assays in Haemostasis 1) Questionnaire l Personal and familial history l Treatments l Diseases l Clinical symptoms

2) Physical examination

3) Pre-operative Haemostasis screening assays l (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 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

MINIMUM LEVEL INFLUENCE OF COAGULATION TESTS2 FACTORS NORMAL VALUES FOR LOW BLEEDING RISK PT3 aPTT4 TT5

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 6 ­ Protein S 60 - 140% - N N N 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 FVIII 2. Results vary as a function of sensitivity of reagents and single or multiple factor levels 3. PT: Prothrombin Time 4. aPTT: activated Partial Thromboplastin Time 5. TT: 6. Sex and age-dependent Screening assays in Haemostasis l 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 n Number of circulating 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 thrombophilia l 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, 6:645-653 l Guidelines for the diagnosis and management of DIC. Levi M. et al. Br J Haematol, 2009, 145, 1:24-33 l 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, 5:715-718 l Conduites pratiques en hémostase, Charles Marc Samama, LFB monograph, 1996 l Hémostase & thrombose, La Simare éditions impression, 1994 l Hemostasis, a case oriented approach, D. A. Triplett, Igaku-Shoin Medical publishers, 1985 l Recommandations pour une juste prescription des examens d’hémostase en pratique médicale courante, M. Gouault-Heilmann, STV, 2006; vol.18, No1, pages 29-42 l 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).

ISI Patient Time (sec) INR = ( Control Time (sec) ) 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). n Stable 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 Anticoagulant therapy monitoring (1) 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) INR

INDICATION ACCP1, BCSH2, GEHT3

Range Target

Prophylaxis of venous thrombosis. 2.0 - 3.0 2.5

Treatment for deep venous thrombosis (DVT) and pulmonary embolism (EP). Cardiac valve disease. Myocardial infarction. 2.0 - 3.0 2.5 Atrial fibrillation.

Antiphospholipid syndrome associated with recurrent DVT or 3.5 additionnal risk factors. 2.5 - 3.5

1 ACCP: American College of Chest Physicians 2 BCSH: British Committee for Standards in Haematology 3 GEHT: Groupe d’Etude sur l’Hémostase et la Thrombose

Bibliography: l Guidelines on oral anticoagulation (): 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 Anticoagulant therapy monitoring (2) Unfractionated Heparin monitoring

Unfractionated heparin (UFH) is a heterogeneous mix of polysaccharide chains of variable molecular weight. One-third of the chains have a pentasaccharide pattern that 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 l Limitation: n Short half-life, dose-dependent n Non-specific binding n Random dose-response effect n Major bleeding risk l Sample 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 l Assays: n aPTT l Widely used for monitoring UFH treatment but sensitive to certain coagulation abnormalities (deficiency or specific coagulation disorders, ) 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)

Table 1 : Unfractionated heparin: curative therapeutic range

Curative treatment

aPTT Ratio* Anti-Xa ADMINISTRATION MODE SAMPLING patient/reference activity

any time after fourth continuous intravenous infusion 1.5-3.5 times 0.3 to 0.7 IU/mL hour of treatment

discontinuous subcutaneous at the midpoint between 1.5-3.5 times 0.3 to 0.7 IU/mL or intravenous infusion 2 injections

Tableau 2 : Unfractionated Heparin: preventive therapeutic range Preventive treatment

aPTT Ratio* Anti-Xa ADMINISTRATION MODE SAMPLING patient/reference activity

subcutaneous or at the midpoint 1.2-1.3 times 0.1 to 0.2 IU/mL intravenous infusion between 2 injections

*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 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) l Anticoagulant 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) l Advantages: n More predictable anticoagulant response n Better bioavailability at low doses n Non-dose-dependent clearance n Longer half-life l Tests: n The result of global coagulation tests (aPTT) are not correlated with the anti- coagulant activity of LMWH n The only tests currently available are those that specifically measure anti-Xa activity in plasma n An international standard for LMWH is available n 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 subjects n Platelet count should be measured: - within the first 24h of treatment - thereafter, twice weekly throughout treatment.

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

Peak anti-Xa activity APTT prolongation Product Indications Dosage Mean (if measured) Overdose values1 threshold2 m±sd

LMWH: twice-daily injection regimen: sample taken at peak activity, 3 to 4 h after injection LOVENOX® DVT with or without PE 100 IU/kg/12h 1.20 ± 0.17 Moderate ND (INN enoxaparin) Acute coronary syndrome (1 mg/kg/12h) IU/mL prolongation

® FRAGMINE Established DVT 100 to 120 IU/ 0.6 ± 0.25 1.0 Moderate (INN dalteparin) Unstable angina kg/12h IU/mL IU/mL prolongation FRAXIPARINE® Non-Q-wave myocardial 1.0 ± 0.2 Moderate 85 IU/kg/12h ND (INN nadroparin) infarction IU/mL prolongation LMWH: once-daily injection regimen: sample taken at peak activity, 4 to 6 h after injection INNOHEP® Established DVT 0.87 ± 0.15 175 IU/kg/24h < 1.5 IU/mL Prolongation (INN tinzaparin) Non-serious PE IU/mL FRAXODI® 1.34 ± 0.15 Moderate Established DVT 171 IU/kg/24h < 1.8 IU/mL (INN nadroparin) IU/mL prolongation Fondaparinux: once-daily injection regimen: sample taken at peak activity, 2 to 3 h after injection ARIXTRA® Established DVT No 7.5 mg/24h3 1.41 µg/mL ND (INN fondaparinux) Non-serious PE prolongation ARIXTRA® No Acute coronary syndrome 2.5 mg/24h 0.45 µg/mL ND (INN fondaparinux) prolongation

IU = International Units DVT: Deep Vein Thrombosis; PE: Pulmonary Embolism INN: International Nonproprietary Name 1NB: 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 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. l Indications (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 l Laboratory tests n Treatment l aPTT: there is a direct relationship between the aPTT prolongation and the plasma concentration, except at high concentrations. l Ecarin clotting time l Ecarin chromogenic assay (ECA): for the quantitative determination of hirudin and its analogues

n Prophylaxis No 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. l Indications (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) l Laboratory tests n 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 l Indications 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 l Therapeutic 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 l Laboratory tests Although 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.

l Indications 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 l Specific 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. l Indications (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. l Laboratory 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

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: n Compensated or «non-overt» DIC n Decompensated 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.

Fibrinogen E D D

Fibrinogen degradation Thrombin products (FDP) Plasmin E E + D + E Fibrin Monomer D D D Plasmin

FXIII

Stabilised fibrin clot E E D D D D E D D D D D D E E E E D D D D

Plasmin Fibrin degradation products (FDPn) Solubles complexes = Fibrin monomer + FDP D-Dimers D D D Fibrin monomer + FDPn E E + Fibrin monomer + fibrinogen D D D E E Diagnosis of DIC

Clinical 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.

PRESENCE OF AN UNDERLYING DISORDER ASSOCIATED WITH DIC

YES: proceed with NO: stop ISTH overt DIC score PT, APTT, Fibrinogen, platelet count, fibrin-related markers

Score : Overall score : l Platelet count: 5 compatible ≥ > 100 G/L = 0 — < 100 G/L = 1 — < 50 G/L = 2 with overt-DIC l Fibrinolysis: (e.g: D-Di, FDP, FM): (repeat daily) No increase = 0 — moderate increase = 2 high increase = 3 l PT prolongation: < 3 sec. = 0 < 5 non-suggestive 3 sec. < PT < 6 sec. = 1 — > 6 sec. = 2 of overt DIC l Fibrinogen: > 1 g/L = 0 — > 1 g/L = 1 (repeat the next 1-2 days)

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, 1992 l Laboratory diagnosis of DIC and activated coagulation. Schmaier AH., DIC assessment ABC monography,1989, 2-18 l 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: 1327-30 l Diagnostic criteria and laboratory tests for disseminated intravascular coagulation. Kaneko T, Wada H. J. Clin. Exp. Hematopathol 2011; 51: 67-76 Thrombophilia l 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 Prevalence in thrombotic subjects

CAUSES USUAL VALUES RELATIVE RISK PREVALENCE

10-20 in heterozygous 1-2% in patients Antithrombin 80 - 120% patients with VTE 3% in patients Protein C 70 - 130% 6 with VTE 0.05-1% in the Protein S* 60 - 140% 5 in patients 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 2Gp1 + ACL 2.2 αβ *depends on age and sex

l Other tests n 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: l During 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: 1429-1434 l Screening for thrombophilia: a laboratory perspective, Jennings I., Cooper P., Br J Biomed Sc. 2003; 60: 39-51 D-Dimer assay for exclusion of Venous ThromboEmbolism (VTE)

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) l With a score < 2 for patients with suspected DVT l With a score ≤ 4 for patients with suspected PE l May 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).

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

Number of patients Pathologies NPV STA®-Liatest® D-Di 386 EP 100% 501 EP 99.4% 222 EP 100% 114 TVP/EP 100%

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. 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. Diagnostic decision tree for screening for LA

according to ISTH recommendations (1)

Screening test Mixing studies Confirmatory test

prolonged time* STA©-Staclot© STA©-Staclot© DRVV DRVV Screen (dRVVt) Confirm (dRVVt)

AND OR

prolonged time no Patient plasma PTT-LA Staclot© LA + (APTT) (APTT) Ratio patient time > 1.2 Pool Norm© referent time CT correction ∆ CT > 8 sec NR > 1.2

LA Negative LA Positive Factor assays

NR: Normalized Ratio; CT: Clotting Time * If OAT patient, mix 1:1 plasma patient - Pool Norm® ** according to Rosner Index or ICA calculation (1) Lupus Anticoagulant (LA) / Antiphospholipid antibodies (APA) Laboratory diagnosis

Diagnosis 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: l Screening 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. l Identification step for the presence of a coagulation inhibitor: 50/50 mix of patient + control (a commercially available pool may be used). l Confirmation 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. H A E M O S T A S tPA FDP I S D-Dimer PAI-1 Plasminogen Plasmin Fibrin TM

Fibrinogen IIa

PC IIa

AT APC PS II

PS APC V AT Activated Va Xa Platelet vWF X

AT

XI IXa VIIIa APC PS VIII

XIa IX

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Activation pathways Inhibition pathways Coagulation VIIa AT PAI-1 AT Fibrinolysis Xa TFPI APC PS Xa TFPI TF © 2009 DIAGNOSTICA STAGO - All rights reserved - 04/2013 Ref. 27646 - STAGO © 2009 DIAGNOSTICA 4614

Diagnostica Stago S.A.S. RCS Nanterre B305 151 409 9, rue des Frères Chausson 92600 Asnières sur Seine (France) AT THE HEART OF HAEMOSTASIS Ph.: +33 (0)1 46 88 20 20 Fax: +33 (0)1 47 91 08 91 [email protected] At the Heart of Haemostasis www.stago.com agenceL2R.com - ©2001 Diagnostica Stago All rights reserved Non-contractual illustration 07/2015 Ref. 28171

For further information, please contact:

Diagnostica Stago S.A.S. RCS Nanterre B305 151 409 3, allée Thérésa 92600 Asnières sur Seine France Ph.: +33 (0)1 46 88 20 20 Fax: +33 (0)1 47 91 08 91 [email protected] At the Heart of Haemostasis www.stago.com