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55th Annual Meeting Society of Thrombosis and Haemostasis Research – [PS]

Aim: Protein S (PS) is an anticoagulant factor, and its deficiency leads to increased thrombotic risk . Although PS clotting assays are well described and many commercial kits available, pitfalls are met in current laboratory practice, such as limited reagent stability (usually 4h), high variability (sometimes lot to lot for a same reagent) and possible interference from other plasma factors. A new PS clotting assay was developed to overcome those limits and facilitate current use.

Method: The diluted tested plasma (1:20) is mixed with a PS deficient « substrate » plasma. A clotting mixture containing optimized concentrations of Activated Protein C (APC), FIXa with trace amounts of F Xa, and phospholipids (R2) is then added. Following a 2 min incubation step, clotting is initiated with 0.025M Ca++, and clotting time (CT) recorded.

Results: A linear dose response is obtained in the range 10-200% PS (r²>0.995). PS deficiencies and high PS concentrations are directly and accurately measured. Designed with only 2 reagents, this assay is simple and fully automatable on the main coagulation analyzers. A high stability of at least 48h at 2-8°C, 24h at RT (and possibility to freeze) makes its use easy and consistent with current laboratory practice throughout the working day. Intra and inter assay variability evaluated on STAR were of 4-8% and 7-10% on the range 40 to 130 %. Inter lots consistency was verified (r²>0.98) on N=50 normal and pathological plasmas (deficiencies of PS or other factors, thrombosis risk check up, dicumarol treatment, cirrhosis, Factor V-L…) ranging from 0 to 150%, and results well correlated with another commercial assay (r>0.94). The assay is insensitive to heparin (UFH or LMWH up to 2 IU/ml). Heparinized plasmas at usual therapeutic ranges can be tested.

Conclusions: This improved PS clotting assay is proposed as a new efficient and reliable tool for current PS testing, and could introduce an easier and safer clinical laboratory practice.

 

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