Whole Blood Thrombin Generation Monitored with a Calibrated Automated Thrombogram Based Assay M. Ninivaggi, R. Apitz-Castro, Y. Dargaud, B. de Laat, H.C. Hemker, and T. Lindhout August 2012 www.clinchem.org/cgi/content/article/58/8/1252.full © Copyright 2012 by the American Association for Clinical Chemistry

Introduction A balanced interaction between vasculature, blood cells and plasma proteins prevents bleeding on the one hand and thrombosis on the other Hemostasis: no bleeding no thrombosis

Introduction (cont.) Blood cells and plasma proteins interact in a complicated web of positive and negative feedback loops to generate thrombin (f IIa) FXI FXIa FIX FIXa FVIII FVIIIa FX FXa FVa FV Prothrombin Fibrinogen Fibrin TF FVII TF:FVII TF:FVIIa Thrombin Ref: Tanaka KA et al. Blood coagulation: hemostasis and thrombin regulation. Anest Analg. 2009;108:1433-46

Introduction (cont.) Thrombin is a multifunctional protease Plays a crucial role in hemostasis: the more thrombin the less bleeding but the more thrombosis; the less thrombin the more bleeding but the less thrombosis Also has other important “non-hemostatic” functions Calibrated Automated Thrombogram (CAT) assay Allows quantitative assessment of thrombin formation in platelet-rich and platelet poor plasma Can distinguish between normal, hypo-, and hypercoagulable states Thrombosis time thrombin Normal Bleeding Ref: Tripodi A. The long-awaited whole-blood thrombin generation test. Clin Chem 2012; 58: xxx-xxx.

Introduction (cont.) The CAT assay 1. Thrombin cleaves the substrate that in turn emits fluorescence II IIa ZGGR-AMC AMC* 2. Thrombin concentration is calculated from the fluorescence that develops

Question What makes the CAT assay superior to conventional tests such as the PT and aPTT?

Introduction (cont.) Why perform thrombin generation (TG) assays in whole blood? Closer to physiology Less laboratory manipulation (no centrifugation, thereby reducing experimental errors and time needed to perform the assay) Possibility of direct measurement from non-anticoagulated blood (fingerprick) Impediments to performing the CAT assay with whole blood The fluorescent signal of the cleaved substrate is quenched by hemoglobin The red blood cells sediment, cluster, and retract with the formed clot, leading to highly erratic signals

Introduction (cont.) Whole blood (WB) CAT-based assay To overcome the problems of performing TG assays in WB, the following adaptations were made: - The use of a rhodamine-based thrombin substrate for which the excitation and emission wavelength are less affected by hemoglobin than an AMC-based substrate - The use of a porous filter paper to create a thin layer of blood, resulting in the entrapment of the red blood cells

Materials & Methods Whole blood (WB) drawn on citrate 5µl of activated blood (50% WB + 50% reagents) TG: 0.3mM P2Rho, 0.5pM tissue factor (TF), 16.7mM CaCl2 Calibration: 0.3mM P2Rho, 100nM calibrator 40µl of mineral oil 40µl oil filter paper with 5µl sample Filter paper Filter paper + sample Filter paper + sample + oil

Results No substrate consumption and inner filter effect Figure 1. Reaction profiles. A) Fluorescence tracings show the response of the WB-CAT system to TG in activated PPP (dashed line) and whole blood (solid line). B) Fluorescence tracing of the reaction response (dashed line, n = 3) with the standard deviation (solid lines) between P2Rho substrate and α2M-thrombin calibrator. The dotted line is the first derivative of the calibrator slope.

Results (cont.) Figure 2. Typical thrombogram. The dotted line represents the raw TG data, the dashed line is the raw data with subtraction of the α2M-thrombin activity and the solid line is the TG curve (first derivative of the dashed line). Ref: Wagenvoord R et al. The limits of simulation of the clotting system. J Thromb Haemost 2006;4:1331-8.

Results (cont.) We recommend performing the assay with low TF concentration to include the contribution of the intrinsic pathway Intra- and inter-assay precision of the peak height is sufficient for useful application of the WB-CAT assay (respectively, 6.5% and 10.7% for thrombin peak) Table 1. Tissue factor dependency. The thrombogram parameters as function of the tissue factor (TF) concentration. ETP, endogenous thrombin potential; LT, lag time; TP, thrombin peak; TTP, time-to-peak.

Results (cont.) No need to add phospholipid vesicles (PV) for TG in WB Procoagulant phospholipids are provided by red blood cells B Figure 3. Phospholipid dependency. (A) TG in whole blood (WB) with and without the addition of phospholipid vesicles (PV). (B) Hematocrit dependency.

Results (cont.) The WB-CAT assay is sensitive to the FVIII concentration, which is one of the most important determinants of thrombin generation Figure 4. Factor VIII dependency. Correlation between the whole blood thrombogram parameters ETP (A) and thrombin peak (B) with plasma FVIII concentrations of hemophilia A patients.

Conclusion We developed a method that accurately measures TG in WB by the use of a thin layer of blood in a multiwell microtiter plate Question: The use of filter paper requires some precautions. What are they? Question: What further future work will be need to make the WB-CAT applicable in clinical practice?

Editorial Slide The long-awaited whole blood thrombin generation test (TGT) TGT is more suitable than the traditional coagulation tests (PT and APTT) to assess the pro- and anti-coagulant balance operating in vivo. Until recently TGT was performed in platelet-poor or platelet-rich plasma. Ninivaggi et al (Clin Chem, 2012), now describe a new TGT that can be performed in whole-blood, thus mimicking much better than the previous TGT what occurs in vivo.

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