OUTLINE
Abnormal Clot Retraction indicates on abnormal platelet numbers, abnormal glycoprotein amount or structure, abnormal platelet signaling, abnormal fibrinogen levels, or abnormal fibrinogen structure
PROTOCOL
- Use either the whole blood or platelet-rich plasma (PRP)
- To obtain PRP, collect 500 µl of blood on 50 µl of ACD
- Add 600 µl of PBS, centrifuge once at 10ѓC, 1200 rpm ( Joan, GR 412)
- In Glass Centrifuge Tube add 100 µl of PRP and 160µl of clot retraction buffer, and 50µl of working solution of thrombin (8 U/mL) and 5-10 µL of erythrocytes (for visualization). Vortex briefly.
- Place the Dead End Glass Rod (use Pasteur Pipette) to the bottom of the tube. Place the tube at 37ѓC. Check the CLOT FORMATION (appearance) and RETRACTION. Measure the volume of remained solution after clot retraction, notice the color of solution, take digital picture.
SOLUTIONS
- Clot Retraction Buffer: 400 mL of PBS x1 + 10 mL of PBS-MgCl2 (MgCm2=4g/L) + 10 mL PBS-CaCl2 (CaCl2= 5.3 g/L)
- Working Solution of Thrombin = 7 µl of Thrombin (1250U/mL) + 1 mL of Clot Retraction Buffer
ADDITIONAL INFO
The Clot Retraction Test may be used for screening of new anti-platelet drugs, anti-platelet-antibodies, or unexplained bleeding-problems. This test does not indicate exactly what is the problem but shows the direction to look for it.
REFERENCES
- Platelet Protocols. Research and Clinical Laboratory Procedures. McCabe White M. and Jennings L.K. Academic Press, 1999
- Azam M. et al, Molecular Cellular Biology, March 2001, p. 2213-2230, Vol. 21, No. 6
- Hodivala-Dilke KM et al, Beta3-integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival. J. Clin. Investig. 103:229-238
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