How to handle samples for coagulation analyses

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

4. Preparation of the patient – possible and acceptable Routine laboratory PT/INR, APTT No preparation of the patient

5. Preparation of the patient – possible and acceptable • Routine laboratory • PT/INR, APTT No 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 activityNo coffee during the past 1 h • Plasmin-inhibitorNo smoking during the past 1 h • Specified posture (sitting/lying) • 20 minutes rest before blood sampling

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. 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. 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. 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. 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 109/L)

11. Sample storage

12. Summary

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