Microparticle-associated tissue factor activity measured with the Zymuphen MP-TF kit and the calibrated automated thrombogram assay.
There is increasing clinical interest for measuring microparticle (MP)-associated tissue factor (TF) activity owing to its possible role as a prothrombotic biomarker in a variety of diseases. However, the methods used are to various extents hampered by lack of (pre)analytical standardization as well as limited published documentation. The objective of this study was to evaluate the performance of the Zymuphen MP-TF kit and the calibrated automated thrombogram (CAT) assay in measuring MP-associated TF activity in plasma using a Neisseria meningitidis (Nm)-stimulated whole blood model. In addition, (pre)analytical variables like centrifugation procedures, freezing/thawing and the effect of addition of exogenous phosphatidylserine in plasma were evaluated in the CAT assay. Citrate-anticoagulated blood was stimulated with Nm bacteria for 4 h before platelet-poor plasma (PPP) or platelet-free plasma (PFP) were prepared and assayed with either of the two methods. Nm dose-dependently (10-10 bacteria/ml) induced TF-specific activity, measured as decreased lagtimes, in the CAT assay. The Zymuphen MP-TF kit also detected TF activity, although much higher Nm doses (10 bacteria/ml) were required to achieve measurable levels. Neither freezing/thawing nor the use of PPP vs. PFP influenced the TF activity, measured over a broad range of lagtimes, in the CAT assay. In conclusion, changes in lagtime in the CAT assay reflected levels of MP-associated TF activity in a more sensitive manner than the Zymuphen MP-TF kit did, in our Nm-stimulated whole blood system.
Blood Cell Research Group, Section for Research, Department of Medical Biochemistry, Oslo University Hospital, Ullevål, Norway. m.s.hellum@medisin.uio.no
Marit
M
Hellum
Reidun
R
Øvstebø
Anne-Marie S
AM
Trøseid
Jens P
JP
Berg
Petter
P
Brandtzaeg
Carola E
CE
Henriksson
D057205:Automation, Laboratory; D001681:Biological Assay; D001777:Blood Coagulation; D001792:Blood Platelets / Q000737:chemistry; D002138:Calibration; D055252:Cell-Derived Microparticles / Q000737:chemistry*; D006801:Humans; D009345:Neisseria meningitidis / Q000737:chemistry; D011933:Reagent Kits, Diagnostic; D013048:Specimen Handling; D013917:Thrombin / Q000378:metabolism; D013925:Thromboplastin / Q000032:analysis*; D014914:Whole Blood Coagulation Time
D011933:Reagent Kits, Diagnostic; D013925:Thromboplastin; D013917:Thrombin