1. 1 >> How to handle samples for coagulation analyses Anne-Mette Hvas Department of Clinical Biochemistry Center for Haemophilia and Thrombosis Aarhus University Hospital, Skejby Denmark

2. 2 >> For the next 15 minutes  Preparation of the patient  Specimen collection Venipuncture Vascular access device Tubes and anticoagulant  Specimen transport and processing  Sample Storage

3. 3 >> Preparation of the patient – the ideal Standardized time of the dayOvernight fast or a light meal No alchohol during the No coffee during the past 18-24 hpast 1 h No smoking during the 20 minutes rest past 1 hbefore blood sampling Specified posture (sitting/lying)

4. 4 >> Preparation of the patient – possible and acceptable Routine laboratory PT/INR, APTTNo preparation of the patient

5. 5 >> Preparation of the patient – possible and acceptable Routine laboratory PT/INR, APTTNo preparation of the patient Specialised coagulation laboratory Coagulation factors, natural anticoagulants, No preparation of the patient von Willebrand factor Markers of fibrinolysis Standardized time of the day Tissue-type plasminogen activator (t-PA) Overnight fast or a light meal Plasminogen activator inhibitor (PAI-1) No alchohol during the past 18-24 h Plasminogen activity No coffee during the past 1 h Plasmin-inhibitor No smoking during the past 1 h Specified posture (sitting/lying) 20 minutes rest before blood sampling

6. 6 >> Specimen collection - what is the problem? As soon as the blood vessel is entered:  Tissue factor is released activation of extrinsic pathway  Platelets are activated  Coagulation factors are activated  Natural anticoagulants are activated

7. 7 >> Specimen collection  Venipuncture Needle gauge from 21 to 19 Max 25 mL (20-gauge needle), max 50 mL (19-gauge needle) Torniquet application for max 1 min with minimal stasis Collected directly into the tube containing the anticoagulant Discard the first 5 mL collected (except for PT/INR or APTT) The tube must be correctly filled

8. 8 >> Specimen collection  Vascular access device Avoid air leaks Avoid heparin contamination and specimen dilution  Flush the line with 5 mL saline and discard the first 5 mL or six dead space volumes

9. 9 >> Specimen collection  Tubes and anticoagulant Tubes of non-reactive materials (polypropylene or siliconized glass) 3.2% trisodium citrate The proportion of blood:trisodium citrate are usually 9:1

10. 10 >> Specimen transport and processing  Transport Vibration might cause haemolysis and activation of coagulation Ideally, tubes should be held upright during transport  Processing – centrifugation To obtain platelet poor plasma (platelet count < 10 x 10 9 /L) PT/INR, APTT, Fibrinogen, Fibrin D-dimer, Antithrombin Factors: II, V, VII, VIII, IX, XI, XII Protein C and S, Von Willebr.factor Gravity2000 g2800 g Time10 min.25 min.

11. 11 >> Sample storage PT/INRAPTT, Thrombin time, Fibrinogen, Fibrin D-dimer, Antithrombin Factors: II, V, VII, VIII, IX, XI, XII Protein S and C Plasminogen, Von Willebr. factor Whole blood  21  C 24 h1 h Plasma  21  C 24 h4 h2 h  -20  C NCCLS: 2 weeks Woodhams: 3-24 months  -70  C NCCLS: 6 months Woodhams: 6-24 months Thaw Rapidly thawed at 37  C in water bath and tested immediately

12. 12 >> Summary Patient preparationAttention when analysing markers for fibrinolysis Tubes and anticoagulantTubes of non-reactive material with 3.2 % citrate VenipunctureMinimize stasis, max 1 min Discard first tube except for PT/INR or APTT Collect directly into the tube containing anticoagulant Vascular access deviceFlush the line with 5 mL saline and discard the first 5 mL or 6 x dead space PT/INRAPTT, Thrombin time, Fibrinogen, Fibrin D- dimer, Antithrombin Factors: II, V, VII, VIII, IX, XI, and XII, Protein C and S, Von Willebr.factor Storage, whole blood,  21  C 24 h1 h Centrifugation2000 g, 10 min 2800 g, 25 min Storage, plasma  21  C 24 h4 h2 h  -20  C 3-24 months  -70  C 6-24 months Thaw Rapidly thawed at 37  C in water bath and tested immediately

13. 13 >> Main documentation  National Committee for Clinical Laboratory Standards. Fourth edition 2003. Document H21-A3  Woodhams et al. Blood Coagulation and Fibrinolysis 2001;12:229-236  Laboratory Techniques in Thrombosis. A Manual. 2nd revised edition of ECAT Assay Procedures Edited by J Jespersen, RM Bertina and F Haverkate