Patent EP-4016083-A1 (EP)
Inventor: RUGGERI ZAVERIO M
IPC: G01N33/86
Filed: 2021-11-03
Abstract: The present invention relates to a method for evaluating the risk of thrombotic and hemorrhagic diseases linked to blood coagulation processes. 
     In particular, the present invention relates to an ex  vivo  method for analyzing the coagulation function of a subject's blood sample, comprising the following steps: 
a) providing a whole-blood sample taken from a subject and added with an anti-coagulant substance; 
b) treating the blood sample of step a) with a solution consisting of fluorescent probes for marking platelets and for marking fibrin capable of binding more or less specifically to platelets and fibrin and emitting fluorescent light on two different emission frequencies and of a recalcification solution of the blood sample, wherein the two aforesaid solutions are separated or combined in a single reagent; 
c) introducing said recalcified blood sample in an analytical device having at least one perfusion chamber, in which at least one wall consists of a transparent material comprising a cytoadhesive substance and on which there are reference marks configured so as to identify the height at which the surface on which the adhesion phenomenon of the platelets and fibrin will occur, the blood sample being made to flow into the perfusion chamber under known and measurable flow conditions; 
d) during the blood flow in the perfusion chamber, alternately acquiring at least two series of fluorescence images, so that at least one series of fluorescence images is acquired at an emission wavelength characteristic of the fluorescent probe for platelets and at least one other series of fluorescence images is acquired at another emission wavelength characteristic of the fluorescent probe for fibrin; 
e) binarizing the pixels of the acquired images for the platelets and for the fibrin, so as to generate a first curve of area covered by illuminated pixels vs. time (t) for the formation of a platelet aggregate and a second curve of area covered by illuminated pixels vs. time for the fibrin formation; 
f) generating a first curve of average fluorescence intensity vs. time for the formation of the platelet aggregate and a second curve of average fluorescence intensity vs. time for the fibrin formation; 
the method comprising at least one of the following steps: 
g) generating a first integrated density curve defined as the product of area and fluorescence intensity vs. time for the formation of the platelet aggregate and a second integrated density curve defined as the product of area and fluorescence intensity vs. time for the fibrin formation; 
h) generating a curve of particle number vs. time, in which the number of particles is given by the number of platelet aggregates consisting of more than 2 illuminated pixels; 
i) generating a curve of particle size vs. time, in which the particle size is given by the average size of the platelet aggregates consisting of more than 2 illuminated pixels; 
j) generating values of maximum slope, defined as the relative maximum points of the first derivative function calculated for the curves of the parameter obtained in one or more of steps e), f), g), h) and i); 
k) generating values of maximum acceleration, defined as the relative maximum points of the second derivative function calculated for the curves of the parameter obtained in one or more of steps e), f), g), h) and i); 
l) generating values of F/P ratio, defined as the ratio of the AUCs of fibrin to the AUCs of the platelets of the parameter obtained in one or more of steps e), f) and g); 
wherein the method further comprises the steps of: 
m) generating a lag time value representing the fibrin formation time defined as the time corresponding to the first occurrence of five consecutive images in which the value of the measured area related to fibrin is > 1% of the maximum peak value of the curve; 
n) generating the AUC (Area Under the Curve) values given by the area under the curve of the parameter obtained in one or more of steps e), f), g), h) and i).