1. 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
© Copyright 2012 by the American Association for Clinical Chemistry

2. 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

3. 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:

4. 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.

5. 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

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

7. 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

8. 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

9. 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

10. 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.

11. 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:

12. 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.

13. 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.

14. 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.

15. 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?

16. 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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