Clotting tests. June 2009 V Diagnostica Stago - All right reserved

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1 7 Clotting tests June 2009 V2

2 Reagents Technologies Clotting tests Chromogenic assays Enzyme Immuno Assays (ELISA) Latex Immuno Assays (LIA) Latex slide agglutination Flow cytometry 2

3 Clotting tests Samples Plasma or diluted plasma Assay From 1 to 4 reagents Last reagent: start reagent, after an incubation time End point Detection of a in-vitro clot Raw data: time (seconds) 3

4 Clotting detection system June 2009 V2

5 Manual detection (1) Water bath at 37 C Endpoint is the formation of a clot Early method for clot detection (1) The manual tilt method of Quick 5 M. Heins et al, Automation in coagulation testing, JIFCC 1996

6 Manual detection (2) Water bath at 37 C Endpoint is the ability of a hook to lift the clot out of the sample Early method for clot detection (2) The hook method of Koller 6 M. Heins et al, Automation in coagulation testing, JIFCC 1996

7 Optical detection systems M. Heins et al, Automation in coagulation testing, JIFCC Detection can be performed with a filter photometer, or with a laser diode, by photometry, turbidimetry or nephelometry Drawbacks Interference with lipemic, turbid samples and turbid reagents

8 Mechanical detection systems M. Heins et al, Automation in coagulation testing, JIFCC Example of KC (Amelung) The ball is kept stationary by gravity while the tube is rotating. Clot formation moves the ball and an electromagnet stops the clotting time Drawbacks: weak clots, precision Advantage: no interference by lipemiae or turbidity

9 Viscosity-based detection system ST4, STart, STA, STA Compact, STA Compact CT, STA-R, STA-R Evolution June 2009 V2

10 Viscosity-based detection system (1) Disposable: cuvette + ball Side view The detection is based on the increase of the viscosity of the plasma being tested 10

11 Viscosity-based detection system (2) Amplitude of the ball movement Activating coil Activating coil When the start reagent is added, the detection starts. The ball motion is activated by 2 coils, working alternatively to maintain a natural oscillation 11

12 Amplitude Viscosity-based detection system (3) 12 The amplitude of the pendular movement ball is monitored during the entire clotting process It remains constant while no clot is present

13 Amplitude Viscosity-based detection system (4) As the clot forms, viscosity increases and amplitude decreases The timer is stopped, based on calculation with different algorithms, even if the clot is weak and the ball, not stopped 13

14 Viscosity-based detection system (5) Signal measured Attraction* / release + Release / attraction *25 ms / ms 14 Upper view Signal transmitted

15 Viscosity-based detection system (6) Amplitude Weak clot 1 Normal clot A measure every 5 ms Mean 1: all measures (moving mean) Mean 2: 4 last measures (weak/normal clot) 2 15

16 Viscosity-based detection system 16

17 Viscosity-based detection system Maximum sensitivity for Weak clot Working range for fibrinogen ( g/l, STart ) No interference by Hemolysed, lipemic or turbid plasma Turbid reagents Standardised All clotting tests The entire range of Stago instruments 17

18 STart : presentation 18

19 Clotting tests 7 PT* APTT* Fibrinogen assay* Thrombin Time Reptilase time Factor assays Staclot / STA -Staclot PC, PS drvvt and LA Heparin and LMWH Activated factor VII (Research kit) 7 19 * 2 lines: STA -Reagents and MultiPurpose Reagents (MPR) Others reagents: 1 line

20 Clotting tests PT APTT Fibrinogen assay Néoplastine CI/CI plus STA - Néoplastine CI/CI plus STA - Néoplastine R PTT Automate / C.K. Prest STA -PTTA / STA - C.K. Prest STA - Cephascreen Fibri-Prest Automate STA -Fibrinogen 5 / STA -Fib 2 20

21 Clotting tests 21

22 Prothrombin Time June 2009 V2

23 Prothrombin Time Prothrombin time (PT) is a screening test Prothrombin time (PT) explores the extrinsic and common coagulation pathways, i.e. coagulation factors VII, X, V, II and fibrinogen PT consists of the use of calcium thromboplastin to measure the clotting time of the patient s plasma 23

24 XII XI IX XIIa Prothrombin XIa Thromboplastin Ca++ IXa FP3 VIIIa Ca ++ VIIa - TF Time X Xa FP3 Va Ca ++ VII - TF II IIa PT sensitive to factors II, V, VII, X and low fibrinogen 24 Fibrinogen Fibrin

25 Néoplastine CI Plus 25 Freeze dried thromboplastin from rabbit brain Contains an heparin inhibitor Reconstitution with a solvent containing calcium Stability after reconstitution 8 hours at 37 C 2 days at 20 ± 5 C 8 days at 2-8 C Refer to the package insert included in the kit for more information

26 Néoplastine CI 26 Freeze dried thromboplastin from rabbit brain Contains an heparin inhibitor Reconstitution with a solvent containing calcium Stability after reconstitution 8 hours at 37 C 24 hours at 20 ± 5 C 8 days at 2-8 C Refer to the package insert included in the kit for more information

27 STA -Néoplastine R 27 Freeze-dried thromboplastin prepared from human recombinant tissue factor and from phospholipids. Contains an heparin inhibitor Reconstitution with a solvent containing calcium Stability after reconstitution Stable for 5 days on STA Compact and STA-R only Refer to the package insert included in the kit for more information

28 Prothrombin Time Sample: Citrated plasma centrifugation 15 min. at g Stability: 8 hours at 20 ± 5 C do not keep at 2-8 C (cold activation of factor VII) 28

29 Prothrombin Time protocols Protocol (STart ) Plasma 50 µl Incubation at 37 C for 60 seconds Néoplastine (37 C) 100 µl With a white mixing rod Note clotting time Half volume Refer to the Standardized Operating Protocols (SOP) 29

30 Prothrombin Time protocols Protocol (Automated instruments) Plasma 50 µl Incubation at 37 C for 240 seconds Néoplastine (37 C) 100 µl With a white mixing rod Note clotting time Half volume Refer to the Standardized Operating Protocols (SOP) 30

31 Prothrombin Time protocols Protocol Plasma Incubation at 37 C 120 seconds Néoplastine (37 C) 200 µl mixed (manual technique*) 100 µl Note clotting time *Full volume 31

32 Prothrombin Time results The PT test results can be given in different units according to the country As a time (seconds) As a ratio of the patient's PT to that of a normal PT As a percentage of the normal activity (requires a standard curve) As INR (International Normalised Ratio) 32

33 PT results in seconds Normal values vary depending on instrumentation and reagent Each laboratory must determine its own normal values Expected normal values for Néoplastine CI Plus ~13 seconds 33

34 PT results in ratio Results are compared to the Mean Normal PT* Patient s PT Ratio = MNPT * see INR 34

35 PT results in % Calibration (PT %) Unicalibrator Undiluted: value on the insert 1:2 dilution: half 1:3 dilution: one-third 1:4 dilution: one-fourth Etaloquick 3 levels Same calibration as long as the same batch (+ daily QC) 35

36 Calibration (PT %) Seconds* 35 * given as an indication only Slope /dilution % 36

37 Clinical use of a PT Screening test before an operation Screening for a factor deficiency Congenital deficiency Acquired deficiency Haemorrhagic disease of the new-born Intestinal reabsorption disorders Liver diseases (cirrhosis, hepatitis, jaundice) Disseminated Intravascular Coagulation (DIC) Monitoring of Oral Anticoagulant Therapy 37

38 Laboratory monitoring of Oral Anticoagulant Therapy OAT = Vitamin K Antagonist = VKA = "Warfarin" June 2009 V2

39 Why Vitamin K Antagonist? OAT = Antivitamin K Vitamin K structure Derivatives from hydroxycoumarine (Warfarin, Coumadin ), or from indane 1-2 dione (Phenindione, Pindione ) 39

40 Oral Anticoagulant blocks Vitamin K Coagulation factor H -C -COOH COO - Vit.K Ca ++ platelet - phospholipids* * Platelet factor 3 Coagulation factors II, VII, IX, X require a carboxyl group to be fully functional 40

41 Oral Anticoagulant blocks Vitamin K Coagulation factor H -C -COOH Coagulation factor H -C -COOH COO - Vit.K H Ca ++ Ca ++ platelet - phospholipids* * Platelet factor 3 platelet - phospholipids* Coagulation factors II, VII, IX, X require a carboxyl group to be fully functional 41

42 Vitamin K mechanism of action Carboxylase Precoagulation factor CH2 CH2 COOH O 2, CO 2 Reduced vitamin K vitamin K epoxide coagulation factor CH2 CH2 COOH PIVKA Protein Induced by Vitamin K Antagonist Vitamin K epoxyde reductase COOH VKA blocks 42

43 Effect of OAT on factors Coagulation factor VII IX X II Factor levels 100 Half-life* (hours) Time required for effect (~3 half-lives) *the time required for something to fall to half its initial value VII IX X II days

44 Oral Anticoagulant therapy OAT reduces the levels of factors II, VII, IX, X, as well as protein C and protein S Oral preparation: Bioavailability close to 100 % Delayed activity : At least 5 days for full effect when starting therapy At 10 days to completely clear up after stop 44

45 Indication for OAT 45

46 Need for a monitoring of OAT A common therapy but OAT is associated with an increased risk of bleeding In France, ~ hospitalised patients per year on patients treated with OAT*» STV, october 2000 Major haemorrhage at 24 months: 10.6 %» Gitter MJ, Mayo clin proc, 70, 725, 1995 A lower level of anticoagulation is associated with an increased risk of thrombosis 46 * about 1% of the population

47 Need for a monitoring of OAT The goal of the monitoring is to maintain the patient within a narrow therapeutic range The individual response to treatment is quite variable and unpredictable Individual susceptibility to OAT Genetic Acquired 47

48 Individual susceptibility to OAT Genetic factors: Hereditary resistance to warfarin (rare) Race Acquired factors Variations in the metabolisms of vitamin K, OAT and coagulation factors Pathologies (e.g. renal insufficiency) Age and weight Drugs and diet 48

49 Drugs interfering with OAT 49 Inducing drugs Amiodarone Anabolisants stéroïdiens Anti-inflammatoires non stéroïdiens Antiplaquettaires (aspirine, clopidogrel, etc.) Céphalosporines Cimétidine Erythromycine Fibrates Hormones thyroïdiennes Isoniazide Métronidazole Miconazole (danger++) Phénylbutazone Quinidine Simvastatine Sulfamides Sulfinpyrazone Inhibitory drugs Barbituriques Carbamazépine Cholestyramine Griséofulvine Nafcilline Oestrogènes Phénytoïne Rifampicine Sucralphate Vitamine K

50 Vitamin K content of food Food Vitamin K content µg/100g Food Booth SL et al, J agric food chem, 43, 1574, 1995 Vitamin K content µg/100g Beer/wine <0.001 Potatoes 1.1 Orange <0.01 Banana 0.2 White bread 1.9 Milk 0.2 Tomatoes 3 Spinach 360 Asparagus 80 Beef steak 1.8 Broccoli 113 Brussels sprout, cabbage

51 Need for a monitoring of OAT The goal of the monitoring is to maintain the patient within a narrow therapeutic range Effect of OAT on factors % Risk of thrombosis Safety / efficacy zone Risk of bleeding Approximate therapeutic range days 51 VII IX X II

52 Need for a monitoring of OAT The goal of the monitoring is to maintain the patient within a narrow therapeutic range PT % Risk of thrombosis PT Safety / efficacy zone Risk of bleeding Approximate therapeutic range days 52

53 Prothrombin Time results Major differences are observed when PT are performed on the same patient by different PT methods Different results are obtained when using seconds, ratio or % PT reagent dependant therapeutic range A solution: the INR system? 53

54 INR International Normalised Ratio power ISI patient's PT INR = MNPT With MNPT : Mean Normal Prothrombin Time ISI : International Sensitivity Index 54

55 Value of the International Sensitivity Index (ISI) Low ISI high sensitivity ISI < 1.5 Medium ISI 1.5 < ISI < 2.0 High ISI low sensitivity ISI > 2.0 (STA -) Néoplastine CI Plus ISI ~ 1.3 STA -Néoplastine R ISI ~ (STA -) Néoplastine CI Standardisation against International Thromboplastin Preparations

56 Therapeutic ranges Therapeutic ranges have been recommended in INR by different representative groups The most widely used come from the ACCP 1, the BSCH 2 or the GEHT 3 Therapeutic ranges given in range or in target INR 1. American College of Chest Physicians 2. British Committee for Standards in Haematology 3. Groupement d Etude Hémostase et Thrombose 56

57 Therapeutic ranges 57 Given as an example, refer to the current guidelines

58 Determination of the ISI For each lot of reagent by the manufacturer Fresh specimen from 20 healthy individuals and 60 patients with OAT PT to be calibrated against a Reference Standard, species dependant (Rabbit Brain Thromboplastin) PT (seconds) plotted on log / log scales Manual reference method Slope of the orthogonal regression line = ISI 58

59 Determination of the ISI ISI: slope of a regression line between a given thromboplastin and the Reference Thromboplastin 59

60 Goals for INR / ISI Improve patients management International recommendations for therapeutic ranges according to the different indications Increase safety for the patients Improve inter-laboratory comparison Improve quality assessment of all labs 60

61 Use of the INR system Initially restricted to patients on stable oral anticoagulant treatment (at least 2 weeks of medication) Further studies lead to a more widely use When starting the treatment After a change of dose And in very few countries, for any clinical situations including normal samples 61

62 INR What can influence the calculation of the INR? 62

63 INR What can influence the calculation of the INR? Mean Normal PT ISI 63

64 Determination of the MNPT 64 Each laboratory should determine their own Mean Normal PT MNPT = geometric mean* of at least 20 freshly prepared normal plasmas samples from healthy persons, collected over a period no longer than 5 days A correct MNPT is of fundamental importance for accuracy of INR A control plasma or a pool of normal plasma cannot be used *

65 ISI: effect of instruments Several studies showed that instruments have a significant effect on the PT and the ISI Differences between INR measured with different instruments may be sufficiently great to influence patient management» Poller et at, Am J Clin Pathol, 103, 358,

66 ISI: effect of instruments ISI variation due to instruments, from the ISI given by the manufacturer* From -7.5 % to % INR variation due to instruments, from - 15 % to +7% (INR 2.7) * Compared to the ISI (1.08) given by the manufacturer» Hemolab, STA, CA 5000, MLA 1000, ACL 300, BFA, KC 10, Schnitzer & Gross C. Caron,

67 ISI: effect of instruments Since instruments have a marked and variable effect on the ISI of the thromboplastins, a correction may be therefore necessary The use of systems (reagents + instruments) is a recommendation Local system calibration gives a reliable correction» Poller et al, Br J Haematol, 86, 112,

68 ISI: effect of instruments 1. ISI specifically determined for a couple reagent / instrument (System ) e.g STA -Néoplastine CI Plus / STA or STA Compact or STA-R No need of correction Safe 2. Direct INR calibration (seconds/inr)? Under investigation Main issue: availability of INR Standards 68

69 ISI: effect of instruments 3. Use of a standard Diagnostica Stago Use of Etaloquick (plasmas with assigned INR value) for a local ISI calculation! INR values valid only with Néoplastine on semi automated instruments such as STart Use of the local MNPT 69

70 Instrument's specific ISI INR = patient's PT ISI MNPT log INR = log patient's PT ISI MNPT log INR = ISI log patient's PT MNPT 70

71 Instrument's specific ISI ISI = log INR log (patient's PT / MNPT) ISI = log INR log ratio 71

72 Instrument's specific ISI 1st step: run Etaloquick as sample If the INR of Etaloquick 2 and 3 is within ± 10 % of the target INR, use the given ISI If > 10%, calculate ISI, as shown next page 72

73 Instrument's specific ISI log INR (Etaloquick 2) ISI 2 = log (Etaloquick 2 PT / MNPT) log INR (Etaloquick 3) ISI 3 = log (Etaloquick 3 PT / MNPT) ISI = mean of the ISI values (ISI 2 # ISI 3) 73

74 Instrument's specific ISI Example Néoplastine CI +, lot , ISI = 1.30 Clotting time Etaloquick 2 (EQ2): INR = sec. Etaloquick 3 (EQ3): INR = sec. MNPT: 13 sec. ISI 2 = log 2.1 / log ( 24 / 13) = 1.21 ISI 3 = log 3.3 / log ( 34 / 13) = 1.24 ISI =

75 Monitoring of Oral anticoagulant Therapy 75 Due to delayed action, OAT starts in conjunction with heparin therapy Patient receives heparin for at least 4 days PT reagent (insensitive to heparin) results indicate the action of OAT only PT are performed 36 to 48 hours after OAT initiation, then regularly until the PT reaches the therapeutic range in 2 successive samples (heparin should not be discontinued before) APTT checks the overall hypocoagulability Also performed if bleeding despite PT within the therapeutic range

76 XII XI IX XIIa Prothrombin XIa Thromboplastin Ca++ IXa FP3 VIIIa Ca ++ VIIa - TF Time X Xa FP3 Va Ca ++ VII - TF Heparin inhibitor II IIa - 76 Fibrinogen Fibrin

77 To be or not to be PT-sensitive Example to heparin Day PT (sec.) APTT (sec.) Néoplastine Heparin + OAT OAT PT heparin sensitive reagent Day PT (sec.) ? APTT (sec.)

78 Oral anticoagulant therapy Long term therapy Ultimately, weekly then monthly checks ensure that PT remain within the therapeutic range Results in INR allow the interpretation of the results independently of the reagent Precautions: More frequent checks are necessary if: Unstable biological results, change of dosage, change in associated therapy, pathology 78

79 Kits presentation MPR / STA June 2009 V2

80 Kits presentation / MPR Néoplastine CI Plus 2, 6 x 2 ml,cat n 00374, for 60 tests (full volume) or 120 tests (halfvolume) Néoplastine CI Plus 5,cat n 00375, 6 x 5 ml, for 150 or 300 tests Néoplastine CI Plus 10, cat n 00376, 12 x 10 ml, for 600 or 1200 tests Néoplastine CI 5, 6 x 5 ml, cat n Néoplastine CI 10, 12 x 10 ml, cat n Refer to the package insert included in the kit for more information 80

81 Kits presentation / STA reagents STA -Néoplastine R*, 12 x 15 ml, cat n * availability depending upon the country STA -Néoplastine CI plus 5, 6 x 5 ml, cat n STA -Néoplastine CI plus 10, 12 x 10 ml,cat n STA -Néoplastine CI 5, 6 x 5 ml, cat n STA -Néoplastine CI 10, 12 x 10 ml, cat n Refer to the package insert included in the kit for more information 81

82 Standards Unicalibrator, 6 x 1 ml, cat n Buffer: STA -Owren-Koller, for dilution Etaloquick, 4 x 3 x 0.5ml, cat n Etaloquick 1 ~90 % Etaloquick 2 ~30 % and INR ~3 Etaloquick 3 ~20 % and INR~4 Refer to the package insert included in the kit for more information 82

83 Quality controls System control N+P, 2 x 12 x 1 ml, cat n Coag control N+P, 2 x 12 x 1 ml, cat n Coag-Norm, 100 x 1ml, cat n Coag-Path, 100 x 1ml, cat n Non assayed controls Need for at least a daily Quality Control Refer to the package insert included in the kit for more information 83

84 STA standard and STA controls STA -Néoplastine CI and CI Plus are precalibrated Same calibration for all STA instruments STA -Unicalibrator 6x1 ml, cat n STA -System control N+P 2 x 12 x 1 ml, cat n STA -Coag control N+P 2 x 12 x 1 ml, cat n Refer to the package insert included in the kit for more information 84

85 85 STA standard and STA controls STA -Néoplastine R is precalibrated Same calibration for STA-R and STA Compact instruments Stable 5 days on STA-R and STA Compact Dedicated to STA line of analysers STA -System control N+P 2 x 12 x 1 ml, cat n STA -Coag control N+P 2 x 12 x 1 ml, cat n Refer to the package insert included in the kit for more information

86 Néoplastine CI Plus Key points ISI in the recommended range ISI < 1.5 or ISI < 1.7 (CAP) Reconstitution with a solvent STA system Bar-coded Reagent / ISI / Standard / QC / instrumentation MPR system Reagent / Standard / QC / calculation of instrument specific ISI 86

87 Néoplastine CI Plus Key points 87 Stability Heparin and LMWH non-sensitivity up to 1 IU/ml and 1.5 anti-xa IU/ml Packaging Performances characteristics Intra-assay reproducibility CV ~ 1% with normal and abnormal plasma Inter-assay reproducibility CV ~ 1% with normal plasma, CV ~ 2 % with abnormal plasma

88 PT? June 2009 V2

89 APTT Activated Partial Thromboplastin Time June 2009 V2

90 APTT The APTT is a general coagulation screening test of the intrinsic and common pathways (factors XII, XI, IX, VIII, X, V, II and I) The APTT involves the recalcification of plasma in the presence of a standardised amount of platelet substitute and a specific activator 90

91 PTT Automate The reagent is a preparation of brain phospholipids in a buffered suspension of particulate activator of the siliceous type Reconstitution with distilled water of good quality Stability after reconstitution 24 hours at 20 ± 5 C 7 days at 2-8 C Refer to the package insert included in the kit for more information 91

92 C.K. Prest Reagent 1: platelet substitute, freeze dried Reagent 2: Buffered suspension of kaolin (5 mg/ml) Reconstitution: Shake a vial of R2 and transfer the content into a vial of R1. Stabilise and swirl gently. C.K. Prest is a turbid reagent red mixing rod Stability after reconstitution 2 days at 20 ± 5 C; 7 days at 2-8 C Refer to the package insert included in the kit for more information 92

93 STA - Cephascreen Made of original Polyphenolic activator (patented) Liquid Ready to use Once opened, the STA - Cephascreen remains stable for 14 days at 2-8 C 24 hours at 20 ± 5 C 93 Refer to the package insert included in the kit for more information

94 APTT Additional reagent STA -CaCl M Sample: Citrated plasma Stability: 4 hours at 20 ± 5 C If on heparin therapy, plasma remains stable for 2 hours (4 hours in CTAD tubes) 94

95 APTT protocol Protocol Plasma APTT reagent Incubation at 37 C CaCl2 (37 C) Note clotting time 1/2 vol. 50 µl 50 µl Full vol. 100 µl 100 µl for 180 seconds exactly 50 µl 100 µl Automated instruments 240 seconds Refer to the SOP 95

96 XII XI IX XIIa XIa IXa VIIIa Activator + Phospholipids FP3 Ca ++ VIIa - TF VII - TF APTT CaCl2 X Xa Va FP3 Ca ++ II IIa APTT sensitive to factors XII, XI, IX, VIII, X, II, V and low fibrinogen 96 Fibrinogen Fibrin

97 APTT results 97 APTT results can be given as a clotting time or a ratio to the normal time Expected values: Mean times: 29.6 seconds*, SD 2.4 sec. (C.K. Prest ), 33.5 sec.*, SD 2.3 sec (PTT Automate); 29.2 sec.*, SD 2.8 sec. (STA - Cephascreen ) May vary according to local conditions APTT lengthened in young subject and shorter times are found in older population

98 Clinical use of an APTT June 2009 V2

99 Clinical use of an APTT (1) Screening test before an operation Screening for a factor deficiency Congenital deficiency If the PT is normal, the factor VIII, factor IX, factor XI or factor XII may be deficient Abnormal APTT if factor VIII ~ < 35% Von Willebrand disease (low factor VIII) Acquired deficiency Liver diseases, DIC,... 99

100 Clinical use of an APTT (2) Monitoring of heparin therapy Heparin has provided an effective therapy and prophylaxis of thromboembolism for more than 50 years, but the need to monitor therapy is still a fundamental problem Monitoring is possible Using non-specific test Using specific assays 100

101 Monitoring of heparin therapy 1. Unfractionated Heparin 2. Low Molecular Weight Heparin 3. Fondaparinux June 2009 V2

102 Unfractionated Heparin UFH June 2009 V2

103 Structure of unfractionated heparin Extracted from porcine or bovine intestinal mucosa or bovine lungs Heterogeneous mixing of polysaccharide chains, with variable molecular weights 5,000 to 40,000 Dalton average: 15,000 Da monosaccharide units 103

104 Structure of unfractionated heparin One third of the chains carry a pentasaccharide with a strong affinity to Antithrombin Binding of the pentasaccharide to AT causes a conformational change that accelerates its interaction with thrombin and factor Xa by ~1,000 times AT AT AT AT Xa IIa 104

105 Mechanism of action Action on factors IIa and Xa Activation of the clotting system Prothrombin 1. Antithrombin pathway 2. Anti-Xa pathway 3. Antiprothrombinase activity Factor Xa factor Xa Ca++ factor Va factor V 2. AT-heparin 1. thrombin

106 Clinical use of heparin Venous thromboembolism Prophylaxis of DVT and PE Treatment of DVT Coronary heart disease Unstable angina or acute MI without thrombolytic therapy Acute MI after thrombolytic therapy DVT: Deep Vein Thrombosis PE: Pulmonary Embolism MI: Myocardial Infarction 106

107 Clinical use of heparin 107

108 Limitations of heparin Main limitations: to bind to positively charged proteins and surfaces plasma proteins, proteins released from platelets, and endothelial cells resulting in variable anticoagulant response 108

109 Pharmacokinetic of heparin 109 Administration Bioavailability (SC) Elimination pathways Half-life Complications Injection (IV or SC) 30 % 1. Cellular uptake saturable uptake 2. Renal elimination non saturable uptake Dose-dependent, 1-3 hours Bleeding, thrombosis, thrombocytopenia (HIT) and osteopenia

110 Low Molecular Weight Heparin (LMWH) June 2009 V2

111 Structure of LMWH LMWH are derived from heparin by enzymatic or chemical depolymerisation MW: 2000 to Da (average 5000 Da) One third of the chains carry the pentasaccharide They are given by SC injection 111

112 112

113 Mechanism of action Action on factors IIa and Xa Activation of the clotting system Prothrombin 1. Antithrombin pathway 2. Anti-Xa pathway 3. Antiprothrombinase activity Factor Xa factor Xa Ca++ factor Va factor V 2. AT-heparin 1. thrombin

114 Molecular Weight dependent interaction with IIa and Xa AT Xa IIa P MW < 5400 Da (<18 monosaccharides units) P: pentasaccharide AT Xa AT IIa 114 P P MW > 5400 Da ( 18 monosaccharides units)

115 Molecular Weight dependent AT P interaction with IIa and Xa Xa MW < 5400 Da (<18 monosaccharides units) MW = 5400 Da IIa P: pentasaccharide AT Xa AT IIa 115 P P MW > 5400 Da ( 18 monosaccharides units)

116 MW = 5400 Da (18 monosaccharides units) LMWH 116

117 MW = 5400 Da (18 monosaccharides units) LMWH 117

118 MW = 5400 Da (18 monosaccharides units) LMWH 118

119 LMWH Anti-Xa to anti-iia activity ratio >>1 119

120 Unfractionated heparin Anti Xa activity = anti IIa activity MW = 5400 Da AT Xa AT IIa P P 120 MW > 5400 Da MW > 5400 Da

121 LMWH 121 Features Easier management More predictable anticoagulant response Better availability at low doses Doses-independent clearance mechanism Longer half-life Less complication Mechanism Less binding to proteins Less binding to endothelium Less binding to macrophages Lower Molecular Weight Action anti Xa > IIa

122 Fondaparinux (Arixtra) June 2009 V2

123 UFH, LMWH, Fondaparinux UFH LMWH Fondaparinux Pentasaccharide with a strong affinity to Antithrombin 123

124 Structure of Fondaparinux Developed by Sanofi, sold to GSK (Jan. 05) 1 st selective synthetic inhibitor of factor Xa Indirect (antithrombin-dependent) synthetic specific factor Xa inhibitor Pentasaccharide MW : 1728 Da Administration Given by SC injection 124

125 Structure of Pentasaccharide OH OSO 3 Na COONa OSO 3 Na O O - - O OH O OH O -O- OSO 3 Na NHSO OH 3 Na NHSO 3 Na O COONa O OH OSO 3 Na OSO 3 Na O OH OMe NHSO 3 Na Structure of the specific pentasaccharide 125

126 Mechanism of action Reversible binds to AT Cause a conformation change Anticoagulant activity of AT towards factor Xa is accelerated ~ 300 times Pure anti-xa activity AT AT AT Xa P P 126 P FONDAPARINUX P

127 Clinical use of Fondaparinux Unique dose Prophylaxis of DVT in major orthopedic surgery Dose : 2,5 mg (1,5 mg for renal insufficiency) Concentration : 0,1 to 0,5 µg/ml Treatment of DVT and coronary heart disease Dose : 7.5 mg for kg Concentration : 0,6 to 1,5 µg/ml DVT: Deep Vein Thrombosis 127

128 Pharmacokinetic of Fondaparinux Administration Bioavailability (SC) Elimination pathways Half-life Complications SC Injection, once daily 100 % Renal elimination non saturable uptake Dose-independent, hours Bleeding, thrombosis 128

129 Unfractionated heparin therapy monitoring June 2009 V2

130 Unfractionated heparin therapy monitoring A need for monitoring +++ Short half life, dose dependent, non-specific binding: unpredictable dose response Complications Heparin has provided an effective therapy for more than 50 years, but the need to monitor therapy is a fundamental problem 130

131 Side effects of heparin therapy Major side effect is bleeding: Frequency of severe bleeding accidents during therapeutic use of heparin ~ 5% Other side effects includes thrombosis Failure to achieve the lower limit of the therapeutic range by 24 hours in many patients with a standardised protocol associated with a 23.3% frequency of VTE* Vs about 5% for those within the range * Venous Thromboembolism 131» Hull RD, Arch Intern Med, 157, 2562, 1997

132 Heparin therapy monitoring Sample collection and treatment Standardise a protocol for specimen collection and processing Sampling: citrate anticoagulant or CTAD Centrifugation within 1 hour Stability: 2 hours at room temperature or 4 hours (CTAD) 132

133 Heparin level IU/ml Sample collection (UFH) injection hours sample collection at peak or at the minimum protective level 133 Sub cutaneous (SC) injection 3 injections per day

134 Sample collection (UFH) Heparin level IU/ml sample collection any time hours 134 Intra Venous Infusion (IV)

135 UFH therapy monitoring Which tests? APTT Heparin anti-xa assays Platelet count Antithrombin 135

136 UFH therapy monitoring Which tests? APTT Heparin anti-xa assays Platelet count Antithrombin 136

137 UFH therapy monitoring APTT tests for the overall hypocoagulability as also sensitive to other coagulation abnormalities There is a poor correlation between APTT results and specific anti-xa assay results 137

138 APTT 75 seconds Anti Xa????? 138

139 UFH therapy monitoring APTT tests for the overall hypocoagulability as also sensitive to other coagulation abnormalities Responsiveness to heparin of different APTT reagents is highly variable Heparin sensitivity cannot be determined with plasma samples spiked with heparin 139

140 UFH therapy monitoring APTT tests for the overall hypocoagulability as also sensitive to other coagulation abnormalities Responsiveness to heparin of different APTT reagents is highly variable They are clinical situations in which APTT monitoring may cause inappropriate dosage adjustment 140

141 UFH therapy monitoring Clinical situations in which APTT is not suitable Coagulation factor deficiency Lupus Anticoagulants Inflammatory syndrome Pregnancy 141

142 APTT and heparin (1)* Administration mode continuous Intravenous route Intermittent intravenous Sub cutaneous Time of sampling Any time 1 hour before next injection Between 2 injections Before the next injection PTTA (ratio**) 3 to 4 2 to 3 3 to 4 2 to 3 * Given as an indication, results usually observed ** Patient's APTT / Mean normal APTT 142

143 APTT and heparin (2)* Administration mode continuous Intravenous route Intermittent intravenous Sub cutaneous Time of sampling Any time 1 hour before next injection Between 2 injections Before the next injection CK Prest (ratio**) 2 to to 2 2 to to 2 * Given as an indication, results usually observed ** Patient's APTT / Mean normal APTT 143

144 UFH therapy monitoring Which tests? APTT Heparin anti-xa assays Platelet count Antithrombin 144

145 UFH therapy monitoring Heparin anti-xa assays: Specific assay for heparin Clotting or chromogenic assays, based on factor Xa inhibition Stachrom heparin STA -Rotachrom heparin* STA -Staclot heparin Standardised against an International Standard, results in IU/ml Therapeutic ranges, NOT reagent-dependant * no added Antithrombin 145

146 Anti-Xa assays Recommended when: Discrepancies expected APTT / Heparin APTT not enough prolonged with usual or large large doses of heparin APTT too prolonged with usual or low dose Factor deficiency or Lupus Anticoagulants Low dose prophylactic heparin Paediatric use Combined heparin and warfarin therapy or thrombolytic therapy 146

147 Anti-Xa assays Cost/test higher, but Fewer monitoring tests Fewer change of heparin dose Less nurse & pharmacy involvement Standardised Safer Specific for heparin Reflects the true heparin circulating in blood * T.K. Rosborough, Pharmacotherapy

148 Anti-Xa therapeutic ranges* Administration mode continuous Intravenous route Sub cutaneous Time of sampling Any time Between 2 injections Before the next injection Heparin (IU/ml) * Depending upon local recommendations 148

149 Heparin therapy monitoring APTT Specific anti-xa assays Platelet count To detect any Heparin-Induced Thrombocytopenia HIT, Type I Directly caused by heparin, high incidence (5-30 %), early onset, moderate and reversible, with no clinical complications HIT, type II 149

150 Heparin Induced Thrombocytopenia, type II Immunoallergic drug reaction Antibodies against PF4-heparin complexes Severe thrombocytopenia, with life threatening thrombotic complications Low incidence (< 1%) 150

151 Diagnosis of HIT, type II Thrombocytopenia Platelet <100x10 9 /litre or decrease >30% Developing after 5-15 days of heparin therapy, earlier if re-exposure Normalisation of platelet count 2-5 days, sometimes later, after heparin withdrawal Difficult diagnosis, includes clinics and biological tests: Functional (platelet-activation tests with / withoutheparin Antigenic (Asserachrom HPIA) 151

152 Heparin therapy monitoring APTT Specific anti-xa assays Platelet count AT In case of heparin resistance To detect any antithrombin deficiency 152

153 LMWH therapy monitoring June 2009 V2

154 LMWH therapy monitoring Global coagulation assays (APTT) May be normal or prolonged, with no correlation with LMWH level The only tests currently available are the specific anti-xa activity in plasma Based on factor Xa inhibition Same assays as for heparin, with different protocol, standardised against an international standard for LMWH, results in Anti-Xa IU/ml Therapeutic ranges, NOT reagent-dependant 154

155 LMWH therapy monitoring The anti-xa assay is necessary to adjust the dose of LMWH at least 48 hours after the first injection (therapeutic treatment) Monitoring is not necessary for prophylactic treatment (except in case of kidney problems, overweight, risk of haemorrhage or thrombosis) Strongly recommended for children 155

156 156

157 LMWH therapy monitoring? 157 "In this cohort of patients with UA / NSTEMI patients, low anti-xa activity on Enoxaparin treatment is associated with 30-days mortality, which highlight the need of achieving at least the minimum prescribed anti-xa level of 0.5 IU/ml " UA: Unstable Angina; NSTEMI: non ST-segment elevation MI

158 To answer market needs... June 2009 V2

159 UFH and LMWH therapy monitoring International recommendations use of dedicated calibrations curves for UFH and LMWH monitoring But, development of a unique "hybrid calibration" more practical not validated by the international authorities 159

160 Hybrid Calibration STA Hybrid Hep Calibrator (cat. n 00687) STA -Rotachrom Heparin STA -R Evolution / STA -Compact Packaging : 5 levels x 4 vials x 1 ml H0-H3-H6-C9-C18 1 point points UFH + 2 points LMWH 160

161 Hybrid calibration integration STA Rotachrom Heparin heparin 4/ Heparin 8 ref : / ref : Method LMWH UFH Hybrid calibration Calibrators STA Calibrator LMWH STA Hepanorm H STA Hybrid Hep Calibrator Ref : Ref : Ref : Controls STA Quality LMWH Ref : STA Heparin Control Ref : STA Quality LMWH Ref : STA Heparin Control Ref : 00683

162 Fondaparinux therapy monitoring June 2009 V2

163 Fondaparinux therapy monitoring No coagulation monitoring Longer half-life, dose-independent Predictable dose-response 100 % synthetic = more "safe" = biological risks are limited No pb of availability & lot-to-lot reproducibility Major side effect is bleeding: Severe bleeding accidents during therapeutic use of Fondaparinux out of the recommendations 163

164 Fondaparinux therapy monitoring Which tests? APTT Heparin anti-xa assays Platelet count 164

165 Fondaparinux therapy monitoring Which tests? APTT : Global coagulation assays APTT is of no use Heparin anti-xa assays Platelet count 165

166 Fondaparinux therapy monitoring Which tests? APTT Heparin anti-xa assays Platelet count 166

167 Fondaparinux therapy monitoring In practice, specific anti-xa activity in plasma recommended in particular groups of patients Renal insufficiency, pregnancy, body weight <50kg, elderly Same assays as for heparin, with different protocol STA -Rotachrom heparin Results in µg/ml Therapeutic ranges, NOT reagentdependant 167

168 Anti-Xa assays 168 STA Fondaparinux Calibrator & Control Human plasma containing Fondaparinux at different levels STA -Rotachrom Heparin STA -R Evolution / STA -Compact STA Fondaparinux Calibrator (ref : 00354) packaging : 3 levels (0; 0.9; 1.8 µg/ml) x 2 vials x 1 ml STA Fondaparinux Control (ref : 00355) packaging : 2 levels (0.5; 1.4 µg/ml) x 3 vials x 1 ml

169 Fondaparinux therapy Which tests? monitoring APTT Heparin anti-xa assays Platelet count "No episodes of Heparin-Induced Thrombocytopenia have been reported in Phase 2 and 3 clinical trials of fondaparinux." "The ACCP did not give a graded recommendation to fondaparinux stating that "minimal data supporting the efficacy of fondaparinux in HIT and other thrombocytopenic situations precludes us from making any recommendation"". 169

170 Heparin? 170

171 Clinical use of an APTT (3) Screening test before an operation Screening for a factor deficiency Congenital deficiency Acquired deficiency Monitoring heparin therapy Lupus Anticoagulants (LA) See "thrombophilia" 171

172 APTT Kits presentation 172

173 C.K. Prest C.K. Prest 2, cat n x 2 ml, for 120 tests or 240 tests C.K. Prest 5, cat n x 5 ml, for 300 tests or 600 tests Additional reagent STA -CaCl M, cat n Refer to the package insert included in the kit for more information 173

174 PTT Automate PTT Automate 5, cat n x 5 ml,for 600 tests or 1200 tests PTT Automate 10, cat n x 10 ml, for 1200 tests or 2400 tests Additional reagent STA -CaCl M, cat n Refer to the package insert included in the kit for more information 174

175 APTT STA -PTT Automate 5, cat n x 5 ml (12 x 100 tests) Stability: 24 hours (STA analysers) STA -C.K. Prest 5, cat n x 5 ml (6 x 100 tests) Stability: 24 hours (STA and STA-R ) and 48 hours (STA Compact ) Additional reagent STA -CaCl M, cat n Refer to the package insert included in the kit for more information 175

176 STA - Cephascreen STA Cephascreen, cat n x 4 ml vials ml, for 480 tests or 960 tests STA Cephascreen, cat n x 10 ml vials, for 1200 tests or 2400 tests Adapted to fully automated instruments (STA line) and STart Additional reagent STA -CaCl M, cat n Refer to the package insert included in the kit for more information 176

177 STA - Cephascreen Once opened, the STA Cephascreen remains stable for 14 days at 2-8 C Extended on board stability (on STA line of instruments) 7 days for the 4 ml vials 10 days for the 10 ml vials Refer to the package insert included in the kit for more information 177

178 Quality controls System control N+P, 2 x 12 x 1 ml, cat n Coag control N+P, 2 x 12 x 1 ml, cat n Coag Norm, 100 x 1ml, cat n Coag Path, 100 x 1ml, cat n Non assayed controls Need for at least a daily Quality Control Refer to the package insert included in the kit for more information 178

179 STA -controls STA -System control N+P 2 x 12 x 1 ml, cat n STA -Coag control N+P 2 x 12 x 1 ml, cat n Refer to the package insert included in the kit for more information 179

180 APTT reagents - key points Validated reagents, with well-defined characteristics Therapeutics ranges Performance characteristics (CV < 2%) Systems Batch to batch consistency Positioning 180

181 APTT reagents - key points- Positioning Sensitive to Heparin Factors Lupus Anticoagulants PTT Automate C.K. Prest STA -Cephascreen PTT LA Silica Kaolin polyphenolic Silica Polyvalent activity Pre surgery screening 181

182 APTT reagents - key points - positioning If main interest of laboratory is screening before surgery in order to detect bleeding risk through surgery No need to detect the LA not associated with hemorrhagic risk and with no incidence on surgery STA -CK Prest STA - Cephascreeen 182

183 APTT reagents - key points - positioning If the main interest of laboratory is to detect any coagulation pathologies that could lead to bleeding or thrombosis (including Lupus Anticoagulants) STA-PTT Automate 183

184 APTT? 184

185 Practical session 185

186 Clotting tests PT * APTT * Fibrinogen assay * Thrombin Time Reptilase time Factor assays Staclot / STA -Staclot PC, PS APC-Resistance drvvt and LA Heparin and LMWH Activated factor VII (Research kit) * 2 lines: STA -Reagents and MultiPurpose Reagents (MPR) Others reagents: 1 line 62

187 Thrombin time 187 The thrombin time (TCT) is a test designed for the assessment of fibrin formation Thrombin time may also be performed as a screening test, in complement to PT and APTT Principle In the presence of a predetermined quantity of thrombin, a normal plasma will clot in a finite and constant time

188 STA -Thrombin STA and MPR reagent Contains titrated calcium human thrombin (~1.5 NIH* unit/ml), freeze-dried Reconstitution with distilled water Stability after reconstitution 2 days at 20 ± 5 C 7 days at 2-8 C * NIH: National Institute of Health Refer to the package insert included in the kit for more information 188

189 Thrombin time Sample: Citrated plasma Stability: 8 hours at 20 ±5 C If on heparin therapy, plasma remains stable for 2 hours at 20 ±5 C or 4 hours on CTAD 189

190 Thrombin time protocol Protocol Plasma 1/2 vol. 100 µl Full vol. 200µl Incubation at 37 C for 60 sec. 120 sec. STA -Thrombin Note clotting time N < 21 seconds 100 µl 200 µl Refer to the Protocols - tests setting, cat n

191 Thrombin time Reagent: Thrombin Fibrinogen fibrin monomer fibrin polymer 191

192 Prolonged thrombin time Reagent: Thrombin Fibrinogen fibrin monomer 1 Low fibrinogen Dysfibrinogenemiae fibrin polymer 192

193 Prolonged thrombin time Reagent: Thrombin 2 Heparin in sample Fibrinogen fibrin monomer 1 Low fibrinogen Dysfibrinogenemiae fibrin polymer 193

194 Prolonged thrombin time Reagent: Thrombin 2 Heparin in sample Fibrinogen fibrin monomer 1 Low fibrinogen Dysfibrinogenemiae 3 fibrin polymer Fibrinogen Degradation Products 194

195 Clinical use of a thrombin time Prolongation of the thrombin time indicates (and/or) An abnormality of fibrinogen Qualitative (dysfibrinogenaemia) Quantitative: Congenital hypofibrinogenaemia or afibrinogenaemia acquired hypofibrinogenaemia (liver disease, DIC) The presence of heparin, hirudin or argatroban The presence of FDP 195

196 Thrombin time STA -Thrombin 2, cat n x 2 ml for 120 tests or 240 tests (half volume) Stable 8 hours on STA STA -Thrombin 10, cat n x 10 ml for 600 tests or 1200 tests Stable 7 days on STA Coag Control N/ STA -Coag Control N System Control N/ STA -System Control N Refer to the package insert included in the kit for more information 196

197 Thrombin time? 197

198 Reptilase Time Reptilase is a thrombin-like enzyme extracted from the venom of Bothrops atrox Reptilase converts fibrinogen into fibrin, but unlike thrombin, is not affected by heparin Principle of the assay In the presence of a predetermined quantity of Reptilase, a normal plasma will clot in a finite and constant time. 198

199 STA -Reptilase STA and MPR reagent Contains Reptilase (~20 BU), freezedried Reconstitution with distilled water Stability after reconstitution 8 hours at 37 C 5 days at 20 ± 5 C 48 hours on the STA instruments Refer to the package insert included in the kit for more information 199

200 Reptilase Time Other required reagents STA -Owren-Koller (buffer) Sample: Citrated plasma Stability: 8 hours at 20 ± 5 C 200

201 STA -Reptilase Protocol Plasma STA -Owren-Koller Full vol. 100 µl 100 µl Incubation at 37 C for 120 seconds STA -Reptilase (37 C) 200 µl 1/2 vol. 50 µl 50 µl 100 µl Note clotting time N < 20 seconds Refer to the Protocols - tests setting, cat n

202 Reptilase Time Reagent: Reptilase Fibrinogen ~ fibrin monomer fibrin polymer 202

203 Prolonged Reptilase Time Reagent: Reptilase Fibrinogen ~ fibrin monomer 1 Low fibrinogen Dysfibrinogenemiae 2 fibrin polymer FDP 203

204 Thrombin time & Reptilase Time Reagent: Reptilase Prolonged Thrombin Time + Normal Reptilase Time: Heparin in sample Fibrinogen ~ fibrin monomer 1 Low fibrinogen Dysfibrinogenemiae 2 fibrin polymer FDP 204

205 Reptilase time STA -Reptilase, cat n x 2 ml for 60 tests or 120 tests (half volume) STA -Owren-Koller System control N STA -System control N Refer to the package insert included in the kit for more information 205

206 Reptilase time? 206

207 Fibrinogen assay Quantitative determination of fibrinogen by the clotting method of Clauss In the presence of an excess of thrombin, the clotting time of a diluted plasma has a direct bearing on the level of plasma fibrinogen 207

208 Fibri-Prest Automate Titrated calcium human thrombin (~80 NIH unit/ml), freeze-dried Contains a specific heparin inhibitor Reconstitution with distilled water Stability after reconstitution 7 days at 20 ± 5 C 14 days at 2-8 C Refer to the package insert included in the kit for more information 208

209 Fibrinogen assay Other required reagents STA -Owren-Koller Unicalibrator for calibration Quality control Sample: Citrated plasma Stability: 8 hours at 20 ± 5 C Refer to the package insert included in the kit for more information 209

210 Fibri-Prest Automate protocol Protocol (STart ) Sample* (1:20 dilution) 1/2 vol. 100 µl Incubation at 37 C for 60 seconds Fibri-Prest Automate 50 µl Note clotting time Calibration curve: results in g/l or mg/dl * plasma and control Refer to the Protocols - tests setting, cat n

211 Calibration Unicalibrator At least 3 dilutions 1:20 dilution: value on the insert 1:40 dilution: half of the value 1:10 dilution: value x 2! 1:7 dilution: value x (20/7) Same calibration as long as the same batch (+ daily QC) 211

212 Seconds* 35 If < 0.9 g/l sample with a 1:10 dilution Calibration Fibrinogen assay Working range g/l ( mg/dl) If > 10 g/l sample with a 1:40 dilution g/l Dilutions 1:40 1:20 1:10 1:7 log/log graph * given as indication only

213 Protocol on other instruments Protocol 1/2 vol. Full vol. Sample* (1:10 dilution) 100 µl 200 µl Incubation at 37 C for 60 sec. 120 sec. Fibri-Prest Automate 50 µl 100 µl! * With other instruments, plasma sample dilution is 1:10 or 1:16 Reduced linearity of assay, more samples dilutions * plasma and control 213

214 Clinical use of a fibrinogen assay Normal range of fibrinogen (adults): 2-4 g/l ( mg/dl) Screening before surgery Diagnosis Qualitative defect (dysfibrinogenaemia) Quantitative deficiency (congenital or acquired) Increase of fibrinogen level Inflammatory syndrome, diabetes, obesity, involved in the pathogenicity of thrombotic events 214

215 Reagents for fibrinogen assay Fibri-Prest Automate 2, cat n x 2 ml for 240 tests or 480 tests Fibri-Prest Automate 5, cat n x 5 ml for 600 tests or 1200 tests Fibri-Prest 2, cat n x 2 ml, for 120 manual tests Refer to the package insert included in the kit for more information 215

216 Reagents for fibrinogen assay STA -Fibrinogen 5, cat n x 5 ml for 1200 tests New set-up: Linearity range: g/l 5 days stability on board STA -Fib 2, cat n x 2 ml for 240 tests Linearity range: g/l 4 days stability on board Refer to the package insert included in the kit for more information 216

217 Standard and controls Unicalibrator, 6x1 ml, cat n System control N+P, 2 x 12 x 1 ml, cat n Coag control N+P, 2 x 12 x 1 ml, cat n Coag Norm, 100 x 1ml, cat n Coag Path, 100 x 1ml, cat n Refer to the package insert included in the kit for more information 217

218 STA standard and STA controls Precalibration (STA instruments) STA -Unicalibrator 6x1 ml, cat n STA -System control N+P 2 x 12 x 1 ml, cat n STA -Coag control N+P 2 x 12 x 1 ml, cat n Refer to the package insert included in the kit for more information 218

219 Fibri-Prest Automate - Key points Large working range Long stability Heparin inhibitor STA -Fibrinogen : Precalibration on automated instruments 219

220 Fibrinogen assay? 220

221 Factor assays June 2009 V2

222 1.Extrinsic & common factor assays Quantitative determination of an extrinsic/common factor by the clotting method The assay consists of the measurement of the clotting time, in the presence of a PT reagent, of a system in which all the factors are present, constant and in excess, except the factor to be measured which is derived from the sample being tested 222

223 STA-Deficient plasma Extrinsic & common factors STA and MPR STA-Deficient plasma are depleted in a coagulation factor Reconstitution with distilled water Stability after reconstitution 8 hours at 20 ± 5 C and on the STA Refer to the package insert included in the kit for more information 223

224 STA -Deficient plasma Extrinsic & common factors STA -Deficient X, cat n STA -Deficient VII, cat n STA -ImmunoDef II, cat n Immunodepleted (mandatory with STA -Néoplastine R) STA -Deficient II, cat n STA -Deficient V, cat n x1 ml for 60 tests or 120 tests (half volume) Refer to the package insert included in the kit for more information 224

225 Extrinsic & common factor assays Other required reagents Néoplastine Unicalibrator System Control N+P STA -Owren-Koller Sample: Citrated plasma Stability 4 hours at 20 ± 5 C (refer to package inserts) Refer to the package insert included in the kit for more information 225

226 Factor VII assay Protocol STA -Deficient VII Sample* (1:10 dilution) Incubation at 37 C for 60 or 120 sec. (240 sec. STA) Néoplastine (37 C) 1/2 vol. Full vol. 50 µl 100µl 50 µl 100µl 100 µl 200µl * Plasma and control Note clotting time Calibration curve: results in % or U/ml Refer to the Protocols - tests setting, cat n

227 Calibration Unicalibrator At least 3 dilutions 1:10 dilution: value on the insert ~100 % 1:20 dilution: half of the value 1:40 dilution: one-fourth 1:80 dilution: one-eightieth 227

228 Seconds* If < 5 % sample with a 1:5 dilution 35 Calibration Factor VII assay Working range % ( U/ml) If > 150 % sample with a 1:40 dilution % Dilutions 1:80 1:40 1:20 1:10 * given as indication only log/log graph 228

229 Extrinsic & common factor assay 229 Normal range for factors (adults): VII: % X: % V: % II: % Deficiencies Very rare congenital deficiencies Acquired deficiencies ( deficiencies of vitamin-k*, liver damage, DIC) * except factor V

230 2.Intrinsic factor assays Quantitative determination of an intrinsic factor by the clotting method The assay consists of the measurement of the clotting time, in the presence of an APTT reagent, of a system in which all the factors are present, constant and in excess, except the factor to be measured which is derived from the sample being tested 230

231 STA -Deficient plasma Intrinsic factors STA and MPR STA -Deficient plasma are depleted in a coagulation factor Reconstitution with distilled water Stability after reconstitution 4 or 8 hours at 20 ± 5 C and 4 hours on the STA Refer to the package insert included in the kit for more information 231

232 STA -Deficient plasma Intrinsic factors STA -Deficient VIII, cat n STA -Deficient IX, cat n Immunodepleted STA -Deficient XI, cat n STA -Deficient XII, cat n x1 ml for 60 tests or 120 tests (half volume) Refer to the package insert included in the kit for more information 232

233 Intrinsic factor assays Other required reagents PTT A, C.K. Prest or STA -Cephascreen STA -CaCl2 Unicalibrator & System Control N+P STA -Owren-Koller Sample: Citrated plasma Stability 4 hours at 20 ± 5 C (refer to the package insert) Refer to the package insert included in the kit for more information 233

234 Factor VIII assay Protocol STA -Deficient VIII Sample* (1:10 dilution) APTT reagent Incubation at 37 C for 180 seconds (240 seconds STA) STA -CaCl 2 (37 C) Note clotting time Calibration curve: results in % or U/ml 1/2 vol. Full vol. 50 µl 100 µl 50 µl 100 µl 50 µl 100 µl 50 µl 100 µl * plasma and control 234

235 Calibration Unicalibrator At least 3 dilutions 1:10 dilution: value on the insert ~100 % 1:20 dilution: half of the value 1:40 dilution: one-fourth 1:80 dilution: one-eightieth 235

236 115 Seconds* If < 5 % sample with a 1:5 dilution Calibration Factor VIII assay Working range % ( U/ml) If > 150 % sample with a 1:40 dilution , % Dilutions 1:80 1:40 1:20 1:10 log/log graph * given as indication only

237 Intrinsic factor assay 237 Normal range for factors (adults): VIII: % IX: % XI: % XII: % Deficiencies Congenital deficiencies Acquired deficiencies (vitamin K deficiencies*, liver damage, DIC) * Factor IX, II and X

238 Factor assay? 238

239 Practical session 239

240 STA -QCE Program Diagnostica Stago External Quality Assessment June 2009 V2

241 STA -QCE Presentation QCE website Benefits for participants and distributors Promotion campaign 241

242 STA -QCE Presentation June 2009 V2

243 What is an EQA* program? ISO Definition : System designed to objectively assess the quality of results obtained by laboratories, by means of an external agency Synonyms : Proficiency Testing, interlaboratory QC,» * EQA: external quality assessment 243

244 Objectives of an EQA (1) EQA complements the internal Quality Control used to monitor within- and between analytical run variability Ensures harmonization of results & comparability between laboratories Identifies possible deficiencies in laboratory practice 244

245 Objectives of an EQA (2) Provides continuing education Increases results confidence and patient safety Collecting information for the purpose of lab. accreditation (certification of quality) Analytical method assessment through peer group (=reagent / instrument combination) 245

246 EQA features International, national, regional and increasingly commercial schemes Each type can have different important functions (comparability, method assessment, ) Mandatory in some countries or remains a voluntary activity in others 246

247 STA -QCE program 247 Dedicated to Stago international customers Only requirement: STA analyzers 2 surveys/year 2 levels of freeze-dried human plasma (STA -QCE ) Up to 3 days testing Up to 6 parameters / level PT, APTT, Fibrinogen, Factors, Inhibitors, D-Di, VWF, Heparins

248 Next STA -QCE program The next STA-QCE & 2 program, April 2009: STA QCE 2009/1: PT, APTT, Fibrinogen, Antithrombin, Protein C and Protein S STA QCE 2009/2: PT, APTT, Fibrinogen, Antithrombin, Protein C and Protein S 248

249 STA -QCE organisation STA -QCE kits have to be ordered within 4 weeks STA -QCE kits dispatched to customers via Stago subsidiary or distributors around 6 weeks to run the tests and submit the results via website 3 weeks for analysis of the results Individual report for each participating laboratory 249

250 STA -QCE participation STA -QCE programs began in 1998 Latest campaign : 22nd survey, STA -QCE 2008/1 & laboratories 46 countries Subscription fee : 50 Euros /kit /campaign 250

251 STA -QCE Customer's reports June 2009 V2

252 Customer's report General report Customised report Individual result sheet "Youden Plot" graph Laboratory results recap 252

253 General Report General comments Participation of laboratories Reagents repartition Details Distribution of results for each test and level 253

254 General Report: Reagents repartition 254

255 General Report: Distribution of the results 255

256 Customised report Sent to customers via Stago subsidiary or distributors Include : Individual result sheet "Youden Plot" graph Laboratory results recap 256

257 Customised report : Analysis of the results Calculation of the mean ± 2 SD (Standard Deviation) only on Stago reagents (same base of calculation over programs) Laboratory results versus overall results : Grade A : within Stago mean ± 1 SD Grade B : within Stago mean ± 2 SD Grade C : > ± 2SD / out of consensus 257

258 Analysis of the results 258

259 Individual result sheet

260 Individual result sheet 1 Synthetic table For each test, classification in letter and fast visualisation of the performances 260

261 Individual result sheet 2 Table: Laboratory results Results brought back by the laboratory during the 3 days 261

262 Individual result sheet 3 Table: global results Means and standard deviation calculated with the Stago reagents are indicated In the event of several Stago reagents, we mention which one is used as comparison 262

263 Youden Plot graph ±2 SD ±1 SD Level 2 Laboratory ±1 SD A B C Level 1 Laboratory results Others 263

264 Laboratory Results Recap Results' recap of the 5 last QCE programs 264

265 @QCE website June 2009 V2

266 @QCE website available for the QCE participants Dedicated internet website for distributors and customers Access also possible from the Diagnostica Stago site heading "STA -QCE " 266

267 @QCE Homepage 267

268 Connection Customers: Direct registration on website ID connection will be automatically and immediately sent by directly to the customers Distributors: ID connection will be sent to you by by administrator 268

269 @QCE Organisation CD Rom sent with the STA-QCE kits Dispatch to every new customers CD Rom Content: User manual Interactive tutorial (demonstration) 269

270 @QCE Benefits Access website via one click for easiest data submission and loading reports Save time for distributors and customers Gathering data Availability of the reports Available in several languages Now available in English, French, Chinese and Spanish 270

271 @QCE Benefits 271 Two types of access: Distributors: Management and constant follow up of your customers View of the results / "C" Listing / Listing of software version not updated Customers: Submit results / 5 last programs on line (including reports) / Certificates of participation on line available / Traceability of results

272 STA -QCE Benefits for participants and distributors June 2009 V2

273 Benefits for participants (1) 273 International External Quality Control program dedicated to Stago instruments Great number of participants Comparison of homogenous systems Similar performances in term of CV inter laboratories for the groups "systems" Specialized tests Customised follow-up of the laboratory Suggested troubleshooting if out of consensus ("C") results Suggested corrective action

274 Benefits for participants (2) Monitoring of reagents and methodology performances over time website for easy data submission and loading of the reports Ensure harmonisation of results and comparability between laboratories Help to answer to regulatory requirements Collects information for laboratory accreditation purposes 274

275 Benefits for distributors Management and follow up of customers Stago system performances Help for customers assistance 275

276 STA -QCE Promotion campaign June 2009 V2

277 STA -QCE Press release HAEMOSTASIS QUALITY ASSESSMENT AT YOUR FINGERTIPS 277

278 Brochure STA -QCE June 2009 V2

279 Objectives Provide support to summarize STA -QCE features Help distributors to sell and promote STA -QCE Inform about STA -QCE improvements 279

280 Brochure presentation 5 items: EQA goals Programme website Reports content Customer's benefits Available in English (ref 29319) and French (ref 29318) 280

281 Practical session 281

282 7 94 Calibration and Quality Control plasmas Refer to the package insert included in the kit for more information June 2009 V2

283 Calibration Plasmas Refer to the package insert included in the kit for more information June 2009 V2

284 Unicalibrator Cat n x 1 ml + 1 ml distilled water Stability after reconstitution: 4 hours at 20 ± 5 C PT (%) Fibrinogen Factors AT antigen PC and PS clotting tests 284

285 STA -Unicalibrator Cat n x 1 ml + 1 ml distilled water Stability after reconstitution: 4 hours on the STA analysers PT (%) Fibrinogen Factors AT activity PC activity PS activity Plasminogen Antiplasmin 285

286 Other standards 286 Etaloquick, cat n x 3 x 0.5 ml Stability: 4 hours at 20 ± 5 C STA -vwf Calibrator, cat n x 1 ml Stability: 4 hours on STA analysers For STA -Liatest vwf STA -Liatest FM Calibrator, cat n For STA -Liatest FM (for research use only)

287 Other standards (Heparin) Hepanorm H, cat n x 3 x 1 ml Stability: 2 hours at 20 ± 5 C / 4 hours at 2-8 C STA -Hepanorm H, cat n x 3 x 1 ml Stability: 4 hours on STA 287

288 Other standards (LMWH) Hepanorm HBPM/LMWH, cat n 00517, 4 x 3 x 1 ml Stability: 2 hours (20 ± 5 C) / 4 hours (2-8 C) STA -Calibrator HBPM/LMWH, cat n 00685, 4 x 3 x 1 ml Stability: 4 hours on STA STA -Hepanorm HBPM/LMWH, cat n 00681, 4 x 3 x 1 ml Stability: 4 hours on STA 288

289 Other standards (Fondaparinux & Hybrid) Fondaparinux STA -Fondaparinux Calibrator, cat n 00354, 3 levels x 2 x 1 ml Stability: 4 hours on STA Hybrid calibration STA -Hybrid Hep Calibrator, cat n 00687, 5 levels x 4 x 1 ml Stability: 4 hours on STA 289

290 Quality Control plasmas Refer to the package insert included in the kit for more information June 2009 V2

291 Coag Control N+P Cat n Normal and abnormal levels 2 x 12 x 1 ml + 1 ml distilled water Stability after reconstitution: 4 hours at 20 ± 5 C PT (seconds) PT (%) APTT Fibrinogen Thrombin Time (N) 291

292 System Control N+P Cat n Normal and abnormal levels 2 x 12 x 1 ml + 1 ml distilled water Stability after reconstitution: 4 hours at 20 ± 5 C Id. Coag Control N+P + Factors Reptilase Time (N) Liatest AT III PC and PS clotting tests 292

293 STA -Coag Control N+P Cat n Normal and abnormal levels 2 x 12 x 1 ml + 1 ml distilled water Stability after reconstitution: 8 hours on the STA analysers PT (seconds) PT (%) APTT Fibrinogen Thrombin Time (N) AT activity 293

294 STA -System Control N+P Cat n Normal and abnormal levels 2 x 12 x 1 ml + 1 ml distilled water Stability after reconstitution: 8 hours* on the STA analysers * 4 hours for FVIII + Id. STA -Coag Control N+P Factors Reptilase Time (N) PC activity PS activity Plasminogen Antiplasmin 294

295 Other controls (1) 295 STA -Control LA 1+2, cat n 00201, 2 x 3 x 1 ml Stability: 8 hours on STA and at 20 ±5 C For STA -Staclot drvv Screen, STA - Staclot drvv Confirm and Staclot LA STA -Liatest control N+P, cat n 00526, 12 x 2 x 1 ml Stability: 8 hours on STA For STA -Liatest vwf, STA -Liatest D-Di and STA -Liatest Free PS

296 Other controls (2) Coag-norm, cat n 00522, 100 x 1 ml, Coag-path, cat n 00523, 100 x 1 ml Stability: 4 hours at 20 ± 5 C Unassayed controls for PT, APTT and fib. STA -Liatest FM Control, cat n For STA -Liatest FM (for research use only) 296

297 Other controls (Heparin) Heparin Control, cat n Heparin protocols Stability: 8 hours at 20 ± 5 C STA - Heparin Control, cat n Heparin protocols Stability: 4 hours on STA 297

298 Other controls (LMWH) STA -HBPM/LMWH Control, cat n 0068, 6 x 2 x 1 ml Stability: 4 hours on STA STA -Quality HBPM/LMWH, cat n 00682, 6 x 2 x 1 ml HBPM / LMWH Control, cat n 00537, 6 x 1 ml Stability: 8 hours at 20 ± 5 C 298

299 Other controls (Fondaparinux) STA -Fondaparinux Control, cat n 00355, 2 levels x 3 x 1 ml Stability: 4 hours on STA 299

300 Diagnostica Stago assay range for Heparin Application Kit STA -Staclot Heparin STA - Rotachrom Heparin Stachrom Heparin (MPR only) Unfractionated Heparin STA -Hepanorm H STA -Heparin Control Hepanorm H (MPR) Heparin Control(MPR) STA -Hepanorm H STA -Heparin Control Hepanorm H (MPR) Heparin Control(MPR) Low Molecular Weight Heparin STA -Hepanorm LMWH STA -LMWH Control Hepanorm LMWH (MPR) LMWH Control (MPR) STA -Calibrator LMWH STA -Quality LMWH Hepanorm LMWH (MPR) LMWH Control (MPR) 300 Standards Controls

301 Diagnostica Stago assay range for Heparin STA Rotachrom Heparin Heparin 4/ Heparin 8 ref : / ref : Method LMWH UFH Hybrid calibration Fondaparinux Calibrators STA Calibrator LMWH Ref : STA Hepanorm H Ref : STA Hybrid Hep Calibrator Ref : STA Fondaparinux Calibrator Ref : Controls 301 STA Quality LMWH Ref : STA Heparin Control Ref : STA Quality LMWH Ref : STA Heparin Control Ref : STA Fondaparinux Control Ref : 00355

302 page 101 Algorithms Proposal for a diagnosis approach Given for information only / single defect June 2009 V2

303 Algorithms (1) Abnormal PT Normal APTT 303

304 Algorithms (1) Normal Abnormal PT Check blood Sampling APTT Abnormal Factor VII deficiency Initiation of OAT See next page 304

305 Conclusion (2) 305

306 Algorithms (2) Abnormal PT and abnormal APTT 306

307 Algorithms (2) Normal Abnormal PT and abnormal APTT Check blood Sampling Thrombin Time Abnormal Mixing study (APTT) Low fibrinogen Correction No correction See LA 307 Check factors II, X and V Acquired or congenital deficiency, OAT

308 Conclusion (2) 308

309 Algorithms (3) Abnormal APTT Normal PT 309

310 Algorithms (3) Normal Abnormal APTT Check blood Sampling PT Abnormal Thrombin Time See abnormal PT and APTT Normal Abnormal Heparin (fibrinogen) Mixing study Correction No correction See L.A. 310 Deficiency to F VIII or IX or XI or XII

311 Conclusion (3) 311