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

© Copyright 2009 by the American Association for Clinical Chemistry

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

IntroductionIntroduction

© Copyright 2009 by the American Association for Clinical Chemistry

Introduction (cont.)Introduction (cont.)

Blood cells and plasma proteins interact in a complicated web of positive and negative feedback loops to generate thrombin (f IIa)

Ref: Tanaka KA et al. Blood coagulation: hemostasis and thrombin regulation. Anest Analg. 2009;108:1433-46

FXI FXIaFIX

FIXaFVIII FVIIIa

FX

FXaFXaFVa FV

Prothrombin

FibrinogenFibrin

TFTFFVII

TF:FVII

TF:FVIIa

ThrombinThrombin

© Copyright 2009 by the American Association for Clinical Chemistry

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

Introduction (cont.)Introduction (cont.)

Thrombosis

time

thrombin

Normal

Bleeding

Ref: Tripodi A. The long-awaited whole-blood thrombin generation test. Clin Chem 2012; 58: xxx-xxx.

© Copyright 2009 by the American Association for Clinical Chemistry

Introduction (cont.)Introduction (cont.)

II

IIa

ZGGR-AMC

AMC*

2. Thrombin concentration is calculated from the fluorescence that develops

1. Thrombin cleaves the substrate that in turn emits fluorescence

The CAT assay

© Copyright 2009 by the American Association for Clinical Chemistry

QuestionQuestion

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

© Copyright 2009 by the American Association for Clinical Chemistry

Introduction (cont.)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

© Copyright 2009 by the American Association for Clinical Chemistry

Introduction (cont.)Introduction (cont.)

Whole blood (WB) CAT-based assayTo 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

© Copyright 2009 by the American Association for Clinical Chemistry

Materials & MethodsMaterials & Methods

40µl oil

filter paper with 5µl sample

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

Filter paper

Filter paper + sample

Filter paper + sample + oil

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

ResultsResults

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.

No substrate consumption and inner filter effect

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Results (cont.)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.

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Results (cont.)Results (cont.)

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.

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)

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Results (cont.)Results (cont.)

Figure 3. Phospholipid dependency. (A) TG in whole blood (WB) with and without the addition of phospholipid vesicles (PV). (B) Hematocrit dependency.

No need to add phospholipid vesicles (PV) for TG in WBProcoagulant phospholipids are provided by red blood cells

B

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Results (cont.)Results (cont.)

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.

The WB-CAT assay is sensitive to the FVIII concentration, which is one of the most important determinants of thrombin generation

© Copyright 2009 by the American Association for Clinical Chemistry

ConclusionConclusion

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?

© Copyright 2009 by the American Association for Clinical Chemistry

Editorial SlideEditorial 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.

© Copyright 2009 by the American Association for Clinical Chemistry

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