PERFUSION DEVICE, CORRESPONDING APPARATUS USING SAID PERFUSION DEVICE AND METHOD TO ANALYZE THE THROMBOTIC-ISCHEMIC AND HEMORRHAGIC PATHOLOGY

Perfusion device for the dynamic analysis of the thrombotic-ischemic and hemorrhagic pathology, comprising at least one micro-channel (54) able to be connected to a circuit (13) and in which a fluid is able to flow, such as a biological fluid, like blood or other hematic fluids, whether they are animal or human fluids and mixtures of said fluids with additive substances, or a non-biological fluid, and in which at least one reactive substrate is present, intended for the analysis to be carried out, such as a cytoadhesive substrate, in order to simulate a damaged vasal surface and to reproduce hemostasis phenomena and processes. The perfusion chamber is made of a material which allows the optical acquisition in fluorescence light and/or in visible light of images or videos of the flow of fluid inside the at least one micro-channel (54).

FIELD OF THE INVENTION

The present invention concerns a perfusion device, a corresponding apparatus which uses said perfusion device and a method to analyze the thrombotic-ischemic and hemorrhagic pathology, and in particular allows the “in vitro” study of primary hemostasis and the formation of a thrombus. In particular the apparatus according to the present invention provides an analysis in video images, for example in fluorescence, of a fluid, such as a biological fluid, for example, blood, or other hematic fluids, whether they are animal or human fluids and mixtures of said fluid with additive substances, or a non-biological fluid.

BACKGROUND OF THE INVENTION

The coagulation process is the physiological mechanism, which stops the loss of blood from damaged blood vessels and is essential to guarantee the integrity of a person in the event of hemorrhages.

Thrombosis is an unwanted formation of a hemostatic plug or a thrombus inside a blood vessel or the cardiovascular system.

As is known, the adhesive and aggregative capacity of the platelets and the formation of fibrin normally control the repair of damaged tissues. The activation of the platelets causes the formation of a mass of aggregated platelets trapped in a reticulum of fibrin, which form a plug to stop hemorrhaging.

Sometimes, the platelets can exasperate the repair process, being activated in an unsuitable manner if there is a pathological change in the hemostatic process, for example atherosclerosis which can cause a dramatic event leading to obstruction of the blood vessel: thrombosis.

A pathological hemostatic event, whether it is thrombotic-ischemic, due to a sudden lack of hematic fluid in a blood vessel, as in the case of coronaries in myocardium infarct, or hemorrhagic, is a severe problem for the cardiovascular system. These problems derive from cardiovascular alterations acquired over time, such as atherosclerosis, aneurisms, arterio venous shunts, vasculitis, anomalies of the cardiac valves, atrial fibrillation and conduction diseases, venous and arterial thromboses or other.

Moreover, these pathological events are difficult to diagnose as they are also present in subjects who in normal life have never shown any pathological occurrences and who, for example, in the event of a surgical operation, can incur serious hemorrhagic or thrombotic-ischemic problems.

To this purpose it is of fundamental importance to have, in a clinical environment, an “ex-vivo” functional test, which allows to identify potential thrombotic and hemorrhagic risks in “silent” subjects for cardiovascular pathological events.

To this end the European patent application 06819957.9 in the name of the present Applicant is known, which concerns an apparatus for the diagnosis, prognosis and pharmacological monitoring of the thrombotic-ischemic and hemorrhagic pathology in the cardio-vascular apparatus.

In particular, this known apparatus comprises a chamber, also called perfusion chamber, for the passage or flow of hematic liquids. The perfusion chamber is provided with micro-channels into which, by means of pumping means, the blood previously taken from the patient in a test tube is made to flow, suitably treated with anti-coagulants such as heparin, citrate or similar, and with fluorescent probes which act as optical markers, such as for example quinacrine, phycoerythrin, or similar.

Optical acquisition means coordinated with said markers for analyses of fluorescence acquire images relating to the development of the hemostatic processes which lead to the formation of thrombi inside the micro-channels.

A processing unit processes the images acquired and supplies indications relating to the behavior of the hemostasis of the patient.

The conformation of the perfusion chamber is of fundamental importance for the acquisition and the analysis, and to be able to carry out measurements with high degrees of reliability. The perfusion chamber is typically made of transparent material, substantially not autofluorescent, in order to allow the acquisition of images by the optical acquisition means. Its geometry must be suitable to simulate the fluid-dynamic conditions desired, such as for example the degree of sliding or shear rate, and the laminar flow.

In particular, the perfusion chamber must guarantee a high hydraulic seal, in order to preserve the fluid-dynamic conditions desired and not invalidate the reliability of the analysis.

The scientific article by Barstad et al. is known: “A perfusion chamber developed to investigate thrombus formation and shear profiles in flowing native human blood at the apex of well-defined stenoses” Arteriosclerosis and Thrombosis, American Heart Association, US, vol. 14, no. 12, 1 Dec. 1994. The article provides a perfusion chamber formed by two parallel plates connected by screws and which has a housing to insert a support for a “cover-slip” provided with an eccentric stenosis, that is a reduction in the passage cross section of the channel of the blood flow, with reduction values up to 89%. The perfusion chamber is configured to operate in an electro-medical machine directly connected to the patient. The perfusion chamber is put in line with the blood sample taken from the patient, which is aspirated by a peristaltic roll pump located downstream. The test is carried out on the patient's native blood and the flow of blood through the stenosis causes the formation of thrombi. However, the analysis of the thrombi is not carried out in real time on the blood flow in the perfusion chamber, but is carried out after the test, by means of normal-light photo-microscopy on representative sections of the stenosis zone, in order to analyze the conformation of the thrombi that have formed. Moreover, the fluidic seal between the parallel plates of the perfusion chamber is not optimal, inasmuch as the connection by screws cannot be reliable.

The prior art document WO-A-2006/065739 describes a method and an apparatus to aggregate, display and analyze the formation of thrombi. The prior art document WO-A-2006/066008 provides the identification of the response to aspirin in a patient, using a similar apparatus to WO-A-2006/065739.

The prior art document US-A-2005/0255601 describes a cartridge and a method to determine a coagulation time based on a difference in pressure inside a capillary, which includes a transparent base plate, thick and made of rigid thermoplastic material, and a thin lid made of a sheet plastic such as Mylar®. The base plate is shaped to define a spiral capillary and is attached to the lid by means of an adhesive, which can be activated by pressure or by heat. However, the spiral configuration of the capillary might not allow a valid model of laminar flow, nor guarantee the fluidic seal in closing the base plate and the thin lid.

One purpose of the present invention is to obtain a perfusion device, an apparatus and a method which allow to carry out the complete screening of the coagulation process in a reliable way and to dynamically monitor the process of formation and stabilization of the thrombus, in order to predict, diagnose and prevent thrombotic-ischemic events which are at the base of cardiovascular pathologies.

Another purpose of the present invention is to obtain an economic device of the disposable type, while still provided with high analytical reliability, and in particular which, after use, does not need to have washed either its parts or parts of the apparatus it is coupled with, in order to carry out subsequent analyses.

Moreover another purpose is to obtain a perfusion device, which guarantees the hydraulic seal of its components.

Another purpose is to obtain an apparatus and perfect a corresponding method which allows the “in vitro” diagnosis of the thrombotic-ischemic and hemorrhagic pathology, optimizing the pharmacological monitoring of the anti-aggregating and anti-coagulant therapies performed.

Another purpose of the present invention is to obtain an apparatus which allows to acquire the coagulation process in its entirety and complexity, substantially in real time, and to analyze it, at the same time or subsequently, with a high degree of reliability and precision.

Another purpose is to perfect a method to analyze the thrombotic-ischemic and hemorrhagic pathology which is reliable and precise, and allows to monitor, acquire in real time and analyze in dynamic mode the formation of thrombi, where by thrombus we mean both complex thrombus, platelet thrombus alone, and also the fibrin that has formed.

SUMMARY OF THE INVENTION

In accordance with the above purposes, a perfusion device according to the present invention comprises a perfusion chamber provided with at least one micro-channel, advantageously a plurality of micro-channels, in which there is at least one reactive substrate, intended for the analysis to be carried out, such as a cytoadhesive substrate, like collagen, which simulates a damaged vasal surface. A fluid is able to flow in one or more of the micro-channels provided, such as a biological fluid, like blood or other hematic fluids, whether they are animal or human fluids or mixtures of said fluids with additive substances, including possible optical markers for fluorescence analyses, or a non biological fluid.

The perfusion chamber is made of a material which allows the optical acquisition, in fluorescence and/or visible light, of suitable quality images or videos of the flow of fluid inside the at least one micro-channel by means of optical acquisition means of images or videos, typically coordinated with the possible optical markers present in the fluid flowing.

In some forms of embodiment, a transparent and substantially non autofluorescent material is used, such as for example glass or plastic, such as COC (Cyclic Olefin Copolymer), COP (Cyclo Olefin Polymer), conventional polystyrene, polycarbonate PC with high optical level. Such materials, even said super-optical materials, are selected to preferably satisfy the requisites of homogenous thickness, substantially no optical aberration, high transparency, suitability for analysis with fluorescence microscopy, in particular with negligible or no autofluorescence with respect to the quality of image desired and, real time acquisition.

Another fundamental property is the high capacity of said materials to bind the reactive substances to be used as substrate for platelet adhesion.

The perfusion chamber is therefore studied to reproduce hemostasis phenomena and processes, effectively simulating the fluid-dynamic conditions of the blood in blood vessels of different caliber.

According to the present invention, the perfusion chamber can be defined by a cartridge coupled with a covering plate, between which there is at least one micro-groove, in the micrometrical size range, made open on at least one of either said cartridge or said covering plate in order to define said at least one micro-channel.

In accordance with some aspects of the present invention, the cartridge comprises, integrated therewith in a single body:

a plurality of containing elements connected to said at least one micro-channel and configured to contain a biological fluid to be analyzed and/or one or more auxiliary fluids, for example for priming and/or washing;

a selection valve configured to selectively put in fluidic communication or connection one or the other of the containing elements with said at least one micro-channel;

a suction pump device located downstream of said at least one micro-channel and configured to aspirate the flow of fluid through said at least one micro-channel in controlled and selectively variable fluid-dynamic conditions.

In accordance with some forms of embodiment, said cartridge comprises, integrated in a single body, a light wave guide configured to be positioned facing toward said at least one micro-channel.

In accordance with some forms of embodiment, the at least one micro-groove has a mainly rectilinear development.

In accordance with some forms of embodiment, the cartridge is provided with at least one surface or coupling surface on which, during use, a coordinated adhesion surface of the covering plate is suitable to be disposed. The at least one micro-groove is made on one or the other of said surfaces and is provided with two ends, respectively an entry end and an exit end, for the fluidic connection to a circuit to feed/discharge the fluid.

In a coordinated manner, one or other of the surfaces of the covering plate and/or the cartridge are configured to close the at least one micro-groove and to delimit the micro-channel through which the fluid to be analyzed is made to pass.

In particular, it is advantageous to provide that the surfaces on which the reactive substances are sprinkled are suitably treated to confer upon them hydrophile properties, and properties promoting the bond with the cytoadhesive substrate, such as collagen, and the repeatability of said bond. It is indeed preferable that the bond of the cytoadhesive substrate, such as collagen, is stable over time, reliable and quantifiable. It is possible to provide, for example, that said surfaces are treated with ethanol which has good degreasing capacities and renders the surface hydrophile.

According to one form of embodiment, the micro-channel is made on the surface of the cartridge and the coupling surface of the covering plate closes the micro-channel at the top.

In a variant embodiment, the two entry and exit ends are fluidically connected to said circuit by means of respective channels made at least transversely through the thickness of the cartridge.

In one form of embodiment, one of the channels, that is, the one which is directly connected to the entry end, is associated with an auxiliary pipe made in the cartridge for the introduction of additive substances into the fluid to be analyzed, which can be bio-markers, calcium or other.

In another form of embodiment, the channel which is directly connected to the entry end of the micro-groove is provided with at least one tank for mixing the fluid to be analyzed with the additive substances. In particular, the mixing tank determines a wider section which generates a turbulence which promotes the mixing of the fluid with the additive substances.

It is advantageous to provide, in some forms of embodiment, that the micro-groove, advantageously obtained by removing material, is defined by walls which extend substantially orthogonal and parallel with respect to the coupling surface of the cartridge. This solution allows to limit the effects of optical distortion and reflection which can occur during the image acquisition.

It is however quite clear that in other forms of embodiment, the micro-groove or micro-grooves can be defined by walls which extend transversely with respect to the coupling surface of the cartridge. It is indeed not excluded that the micro-grooves can have a trapezoid shaped section for example.

Advantageously, the coupling surface of the cartridge and the adhesion surface of the covering plate are substantially coplanar with respect to each other and geometrically mating to obtain a coplanar coupling thereof, both to increase the hydraulic seal of the perfusion chamber and also to reduce the optical disturbance effects during the acquisition of images.

In another form of embodiment, the cartridge is provided, at least partly integrated therein, with a suction pump device disposed downstream of the channel which is directly connected to the exit end of the micro-channel.

The suction pump device comprises at least one containing element of the fluid analyzed and a suction element provided to aspirate the fluid, making it pass through the micro-channel, and to keep the coupling surface of the cartridge and the adhesion surface of the covering plate adherent with respect to each other by means of a sucker effect which guarantees the hydraulic seal of the perfusion chamber.

According to some aspects of the present invention, the perfusion device described herein is of the disposable type and does not need washing after use, thus avoiding any condition in which the operator can come into contact with potentially infected or dangerous fluid.

In other forms of embodiment, the perfusion device comprises integrated pressure means suitable to maintain the surface of the cartridge in contact against the adhesion surface of the covering plate.

The advantage of the pressure means is that they promote the adherence of the coupling and adhesion surfaces of the cartridge and the covering plate, to obtain a hydraulic seal between the cartridge and the covering plate of the perfusion device.

For example it is possible to provide that said pressure means comprise mechanical clamping devices or magnetic elements which, through reciprocal attraction, maintain a coordinated contact between the covering plate and the cartridge.

The present invention also concerns an apparatus for dynamic analysis in real time of the thrombotic-ischemic and hemorrhagic pathology which comprises a perfusion device of the type described above.

The apparatus also comprises a circuit in which the fluid is able to flow, and possibly, in some forms of embodiment, a suction pump device, if it is not at least partly integrated in the perfusion device, in order to move the fluid through the circuit and the micro-channel in controlled and selectively variable fluid-dynamic conditions.

The circuit, as referred to here, is intended as closed with respect to the external environment, that is, such as to prevent any leakage or loss of fluids, which could cause contamination, or come into contact with the operator.

Optical acquisition means of images or videos are also provided, and electronic processing means suitable respectively to acquire, advantageously at high frequency, in the range of about 25-30 frames/second, and to process images of the flow moving from the at least one micro-channel of the perfusion chamber.

In some forms of embodiment, the optical acquisition means of images or videos advantageously comprise at least one optical module for fluorescence microscopy, connected in series to filming means, for example a video camera or photo camera. The filming means are, for example, but not only, of the CCD type, and have a recording/acquisition system integrated, connected to electronic processing means, such as a computerized digital system which has a computer program memorized inside it. The program, when executed in the memory of the computerized system, is able to analyze and acquire in real time the morphological, kinetic and biochemical variations inside and outside the platelets and the formation of the thrombus and its stabilization, in response to different sliding forces applied and different cytoadhesive surfaces.

According to another form of embodiment, other pressure means can be provided, suitable to press and keep pressed to each other the covering plate and the cartridge, to guarantee the hydraulic seal of the perfusion chamber.

According to another form of embodiment, the pressure means comprise a support element associated with an elastic element and a covering element. The perfusion chamber is disposed in use on the elastic element and the covering element is suitable to keep the covering plate pressed respectively against the support element and the support element pressed against the elastic element, thus absorbing possible non parallelisms between the contact surfaces and guaranteeing a substantially continuous adherence between the two surfaces of cartridge and plate.

In another form of embodiment in which the suction pump device may not be provided integrated in the perfusion device, it can be disposed downstream of the perfusion chamber and is configured to aspirate the fluid, making it pass through said at least one micro-channel.

In any case, one advantage of working under suction is that, when the suction pump device is working, it determines the suction of the covering plate against the cartridge, generating a sucker effect, which guarantees the hydraulic seal of the perfusion chamber.

With the present invention it is therefore possible to carry out a test on platelet functionality and the coagulation process in dynamic conditions, which cannot be assessed by devices existing in the state of the art. Unlike the apparatuses commonly used in the sector, the present invention allows to simulate the vascular ambient of the human or animal body and to acquire in real time the information relating to the formation of thrombi.

Indeed the present invention allows an optical or video acquisition in real time which, when processed, returns dynamic information on the test for the formation of the thrombus, unlike the end point analysis returned by the majority of diagnostic devices, which study the functionality of platelets and/or coagulation.

In order to emulate physiological conditions during the test with maximum accuracy, the shear rate of the hematic flow is advantageously known and controlled in all the micro-channels of the perfusion chamber, as is the working temperature which must be advantageously equal to the physiological temperature (37° C. in the case of humans). This is particularly important and effective for monitoring anti-coagulant or anti-aggregating therapies.

Moreover with the present invention it is possible to use whole blood without the addition of substances which artificially induce aggregation. The presence of all the hematic components and the absence of any chemically induced aggregation renders the test more accurate (or nearer to the real value) and, at the same time, reduces the biological risk to which operators are subjected, since the blood sample undergoes less handling.

The present invention also concerns a method to analyze thrombotic-ischemic and hemorrhagic pathology which comprises a first step in which a fluid, such as blood, hematic or biological fluids, whether they are animal or human, and mixtures of said fluids with additive substances, is introduced and moved in controlled and variable fluid-dynamic conditions through a circuit by means of a suction pump device. In particular the biological fluid is made to pass through at least one micro-channel of a perfusion chamber in which a reactive substrate is present, intended for the analysis to be carried out, such as a cytoadhesive, able to simulate a damaged vasal surface and to reproduce hemostasis phenomena and processes. The method also comprises at least one second step of optical acquisition and processing of the images or videos acquired from the at least one micro-channel, by means of acquisition means of images or videos, and processed by electronic processing means. The perfusion chamber is advantageously made of a material which allows the optical acquisition of images or videos of the flow of hematic fluid inside the at least one micro-channel by said optical acquisition means of images or videos.

According to aspects of the method, during the first step the fluid is introduced into the perfusion chamber which is defined by at least one cartridge coupled to a covering plate between which there is at least one micro-groove to define said micro-channel. The micro-groove is made open on at least one of either said cartridge or said covering plate with a desired depth. The cartridge comprises, integrated therewith in a single body:

a plurality of containing elements connected to said at least one micro-channel and configured to contain a biological fluid to be analyzed and/or one or more auxiliary fluids, for example for priming and/or washing;

a selection valve configured to selectively put one or the other of the containing elements in fluidic communication with said at least one micro-channel;

a suction pump device, located downstream of said at least one micro-channel.

In some forms of embodiment, the flow of biological liquid is introduced into the circuit through suction, so as to create a sucker effect between covering plate and cartridge, increasing both the adherence between a coupling surface of the cartridge and a coordinated adhesion surface of the covering plate, and also the hydraulic seal between them.

According to further aspects of the method, during the first step the covering plate is compressed by pressure means against the cartridge, making both the covering and adhesive surfaces of the cartridge and of the covering plate adhere so as to guarantee the desired hydraulic seal.

According to further aspects, before said first step where the biological fluid flows, a priming step is provided in which a solution of biologically inert liquid, typically physiological solution, is introduced through the circuit and made to pass through the micro-channel of the perfusion chamber to flood the micro-channel and the areas surrounding the micro-channel. The priming step determines the formation of a liquid sealing packing between covering plate and cartridge, in order to generate a hydraulic seal between them and to guarantee in this way that afterward, during the passage of the biological fluid through the micro-channels, the fluid remains confined inside the micro-groove with no danger of leaking from the perfusion chamber, contamination of the other micro-channels or influence on the reliability of the shear-rate measurement.

Some forms of embodiment can provide that the solution of biologically inert liquid is contained in at least one of the containing elements possibly provided in the cartridge.

The combination of the sucker effect described above with the making of a liquid packing, with the coplanarity between covering plate and cartridge and with the pressure exerted by the pressure means, allows to guarantee a high hydraulic seal, suitable for the analyses to be carried out, therefore allowing a high reliability and reproducibility of the same.

The present invention also concerns a method to diagnose the thrombotic-ischemic and hemorrhagic pathology which uses an apparatus and a method of analysis as described in the attached claims.

To facilitate comprehension, the same reference numbers have been used, where possible, to identify identical common elements in the drawings.

DESCRIPTION OF SOME FORMS OF EMBODIMENT

With reference toFIG. 1, an apparatus for analyzing the thrombotic-ischemic and hemorrhagic pathology is indicated in its entirety by the reference number10and comprises a device11to feed the fluids to a perfusion device comprising a perfusion chamber defined by a cartridge36and a covering plate, or slide37, a press14, an optical acquisition device15of images or videos and a processing unit16to process the images acquired by the optical acquisition device15.

The apparatus10may include a hydraulic circuit13and the fluid feed device11can be connected to the hydraulic circuit13, which provides to feed the fluid into the perfusion chamber12. The fluid feed device11comprises a plurality of pipes19,20,21, for example a first pipe19to introduce the fluid to be analyzed, in the case of a test of blood coagulation, a second pipe20associated to a first container21containing a solution of biologically inert liquid, typically a physiological solution, for example, and a third pipe22associated to a second container23containing a washing solution.

The fluid to be analyzed can be biological fluid, blood, hematic fluids whether they are animal or human fluids, and mixtures of said fluid with additive substances, or a non-biological fluid.

The fluid to be analyzed, such as for example blood, can for example already be anti-coagulated and mixed with reagents useful for the analysis to be carried out, for example markers such as fluorescent probes and/or anti-fibrin antibody or possible compounds which interact with the anti-coagulation process such as calcium or similar components.

Each of the three pipes19,20,22is provided with interception valves, such as electro-valves25, advantageously servo controlled, for the selective introduction of the fluid to be analyzed, of the physiological solution or of the washing solution to the perfusion chamber12.

The first, second and third pipe19,20,22converge in a distribution node26from which three shunts27branch off (FIG. 2).

In other forms of embodiment it is possible to provide a single pipe for the introduction both of the physiological solution and of the washing solution.

The interception valves25select whether the flow passes or not, and also prevent a reflux of the fluid which is being introduced from one pipe, toward the other two pipes.

Each shunt27is provided with an interception valve31, like an electro-valve, controlled by the processing unit16to allow the selective passage through it of the blood, the physiological solution or the washing solution.

In particular each interception valve31is of the clamping type, made of biocompatible materials, and such as not to induce in the fluid passing through it an increase in temperature which could invalidate the analysis.

The shunts27connect to the perfusion chamber12in proximity to respective entry seatings32, as will be described hereafter.

The perfusion chamber12comprises the cartridge36, also called base element, and the slide37, also called covering plate of the cartridge36and which during use is disposed in contact against a surface, hereafter called coupling surface39of the cartridge36.

The cartridge36(FIGS. 2-4) has a substantially parallelepiped shape and is provided, in a first side41, with said entry seatings32and in a second side42, opposite the first side41, with corresponding three exit seatings43.

Both the entry seatings32and the exit seatings43are threaded internally: the shunts27and pipes45to discharge the fluids are connected directly and respectively to them.

The entry32and exit43seatings are made substantially blind, and have abutment surfaces33which end substantially orthogonal with respect to their lateral surfaces.

Moreover, the threadings inside the entry32and exit43seatings extend substantially for the entire length of the latter so that when the shunts27and the discharge pipes45are connected to the entry seatings32and respectively to the exit seatings43, their ends abut against the abutment surface33, preventing stagnation zones of the fluid to be analyzed from being generated on the bottom of the seatings, in which zones unwanted phenomena can be generated, such as the formation of clots.

This particular formation of the seatings32and43is such as to guarantee the hydraulic seal between the perfusion chamber12and the hydraulic circuit13, to allow an optimal cleaning thereof after the execution of the tests, and to prevent the fluid to be analyzed from going into contact for example with metal parts or other non biocompatible materials.

In other preferential forms of embodiment, the connection between the shunts27and the entry32and exit43seatings are made by means of suitable connectors of the screw or snap-in type.

Channels46are made in proximity to the abutment surfaces33of the seatings32and43(FIG. 4), each comprising a first segment47which extends substantially parallel to the direction of extension of the seatings32and43, and a second segment48which extends substantially orthogonal to the first segment47and ends at the upper part toward the coupling surface39with a flaring49which opens toward the outside.

The channels46can have any section whatsoever and sizes of about 1 mm in diameter, even if the sizing of the channels46is made to prevent the formation of unwanted stagnations and accumulations of fluid, and at the same time compatible with the reductions in section of the entry channels. In particular it is necessary to prevent the flow of fluid from being obstructed because of the sudden reduction in section.

The coupling surface39has very restricted geometric tolerances of planarity, to cooperate effectively with the covering plate37so as to obtain the desired hydraulic seal.

In some forms of embodiment, one or more micro-grooves51are provided, with micrometric transverse sizes, for example made on the coupling surface39, which are open at the top and are configured for the passage of the fluid.

In this case three micro-grooves51, peripherally closed, are made inside the coupling surface39in correspondence to the flarings49, and each of them connects respectively one of the entry seatings32with one of the corresponding exit seatings43, in proximity to an entry end51aand an exit end51b. To this purpose the entry seatings32are disposed on the first side41in a position opposite the exit seatings43disposed on the second side42.

The covering plate37, disposed in contact against the coupling surface39, closes the micro-grooves51defining respective closed micro-channels54through which during operations the fluids to be analyzed are made to pass, guaranteeing the hydraulic seal of the perfusion chamber12.

In possible example embodiments, the micro-grooves51are disposed parallel with respect to each other, and can have equal lengths and widths, and possibly different depths.

In particular, the length of the micro-grooves51is less than that of the cartridge36, extending for a desired partial segment of the cartridge36itself, so that, when the covering plate37is positioned, it cooperates directly and with continuity against the coupling surface39of the cartridge36, obtaining the hydraulic seal on all the peripheral profile that surrounds the area where the micro-grooves51are made.

In some forms of embodiment, the micro-grooves51can have at least one main part with a rectilinear development. For example, the micro-grooves51can be completely rectilinear, or they can have one or more rectilinear segments and possibly curved segments. The micro-grooves51can be parallel with each other, or, in other example embodiments, one or more of the micro-grooves51can cross and intersect with respect to each other, depending on the type of phenomenon that is to be simulated.

The geometry of the individual micro-grooves51and the pattern which they form in the perfusion chamber12depends on the type of phenomenon to be simulated and on the type of test to be carried out.

The micro-grooves51have characteristic sizes of the micrometric type, for example depth and width different from each other and they can vary from about 100 μm to about 2000 μm.

The shape of the cross section of the three micro-grooves51is advantageously rectangular or square, in order to optimize the acquisition of images by the optical acquisition device15, and together with the pressure with which the fluid to be analyzed is introduced, define the flow rate of the fluid inside the micro-grooves51.

It is advantageous to provide that the lateral walls of the micro-grooves51extend substantially parallel to each other, and orthogonal to the coupling surface39, in order to prevent reflection phenomena during the acquisition of images.

Moreover, the cross section of the micro-grooves51has substantially constant sizes along the whole extension, to keep the flow of fluid in a laminar flow condition and prevent phenomena of turbulence which can cause the formation of thrombi and therefore invalidate the tests. The covering plate37substantially consists of a thin plate with a thickness which, merely by way of example, is comprised between 0.1 mm and 4 mm, advantageously between 0.1 and 2 mm, and in any case compatible with the field depth capacity of the optical acquisition device15. For example, the covering plate37can be formed by an element of the slide type, or similar or comparable.

The covering plate37is provided with a first face52, or adhesion surface which, during use, is in contact against the coupling surface39, and a second face53opposite the first face52.

The first face52, like the coupling surface39, has a very restricted geometric tolerance of planarity, so as to render, during use, the covering plate37adherent to it.

Moreover, to prevent problems of reflection during the acquisition of the images, the first52and the second53face are made substantially coplanar with respect to each other.

In order to not create optical distortion effects and/or noise during the acquisition of the images, both the cartridge36and the covering plate37are made of material with a high degree of transparency and a reduced level of autofluorescence during the analytic conditions of use.

The material used to make the cartridge36and the covering plate or slide37is chosen from a group comprising glass, COC (Cyclic Olefin Copolymers), COP (Cyclo Olefin Polymers), polycarbonate with a high optical degree or similar.

In one form of embodiment, the cartridge36is obtained starting from a block of polycarbonate, and by means of subsequent workings by removing material, it is provided to make the entry32and exit43seatings, the channels46and the micro-grooves51. The removal of material, compared to other types of working, allows to easily obtain the desired properties of planarity and coplanarity of the surfaces, of parallelism between the faces of the micro-grooves51, as well as the desired optical properties for an accurate analysis of the tests. More specifically, the removal of material must be such as not to alter the optical properties of the material.

The hydraulic circuit13disposed downstream of the perfusion chamber12comprises at least one suction pump device55suitable to aspirate the fluids through the perfusion chamber12, and a discharge tank56suitable to receive the fluids processed after the execution of the tests.

In particular the suction pump device55exerts a depression action which aspirates the fluid from the shunts27and makes it pass through the micro-channels54with a defined and controlled pressure. The suction pump device55not only guarantees the flow of the fluid to be analyzed but also exerts on the covering plate37a sucker effect which renders it adherent to the cartridge36.

A further pressure action is exerted by the press14which provides to maintain the covering plate37under pressure against the coupling surface39of the cartridge36, and therefore guarantees the hydraulic seal of the perfusion chamber12securely.

In some forms of embodiment, the depression action exerted by the suction pump device55is sufficient to keep the covering plate37properly adherent to the cartridge36, and so the use of the press14is not necessary.

The press14(FIGS. 5 to 9) comprises a support or cartridge bearer element59, an elastic element60disposed on the bottom of the cartridge bearer element59and on which, during use, the cartridge36is disposed, and a covering element62which, during use, is disposed in contact against the covering plate37.

The covering element62comprises actuation means, not shown in the drawings, which provide to exert a pressure on the covering plate37and keep the latter adherent against the coupling surface39of the cartridge36.

The elastic element60allows to compensate possible different inclinations of the cartridge36due to the action of the actuation means to obtain a desired parallelism between the various adhesion faces.

The covering element62(FIG. 9) can be made of metal or plastic material, non-transparent to radiations, and is provided with an aperture63to allow the passage of luminous radiations, visible light and/or fluorescent light, to detect the images from the perfusion chamber12.

In particular, the images are acquired substantially in the rectilinear segment, that is, in areas where the flow of fluid is not turbulent, such as for example in proximity to the flarings49where the fluid enters/exits from the micro-channels54.

The optical acquisition device15can be a camera or a video camera associated to optical microscopy modules.

The optical acquisition device15can for example be an apparatus for the analysis of epifluorescence, to highlight the platelets and the fibrin marked with a suitable fluorescent probe which adheres to the cytoadhesive substrate, or an apparatus for another type of test based on a different biomarker.

The optical acquisition device15can comprise an optical module65to detect the image of the thrombi adhering on the cytoadhesive substrate, which in this case is able to slide along a guide66(FIGS. 5 and 6) to move into proximity to one of the micro-channels54and acquire the images.

The optical module65comprises sources of light radiation of the mercury, xenon or led lamps type.

In some preferential forms of embodiment, the optical module65comprises led lamps of the strobe type, which is selectively fed to emit light radiations only in determinate instants of time when the images are acquired. In this way the heat energy irradiated is considerably reduced and at the same time the risks of activating and coagulating the blood are reduced.

More specifically the optical module65is suitable to acquire a sequence of images, advantageously at high frequency in the range of about 25-30 frames/second, or a video film, which allow to analyze the kinetics of thrombi or hemorrhagic formation.

The processing unit16is suitable to process the images or the film acquired by the optical module65to determine the evolution of the test.

The apparatus10according to the present invention can advantageously comprise thermostating means to keep the perfusion chamber12at a desired temperature, advantageously at the physiological temperature which in the case of the human body is about 37° C.

The method for analyzing the thrombotic-ischemic and hemorrhagic pathology using an apparatus10as described above is described hereafter.

The method comprises at least one step in which a sample of hematic fluid is taken from a patient and suitably prepared in ways which vary depending on the tests to be obtained.

More specifically at least one sample of venous blood is required, correctly carried out, that is without hematic decantation, breakages of the veins or similar, the temporary conservation of the blood in a test tube containing anti-coagulation substances such as for example heparin, citrate or suchlike, and the addition of fluorescent substances such as quinacrine, anti-fibrin phytoerythinate antibody and, according to the type of tests, of other biomarkers or also the above biomarkers.

The apparatus10is in the configuration shown inFIG. 1, that is, in the condition in which the perfusion chamber12is disposed on the cartridge bearing element59, the press14keeps the covering plate37under pressure against the cartridge36and the shunts27and the discharge pipes45are connected to the cartridge36respectively in the entry32and exit43seatings.

The covering plate37and possibly also the coupling surface39or the micro-grooves51are lined with collagen substances, or cytoadhesive substances totally similar to those normally present in a damaged blood vessel. The contact between the blood and the cytoadhesive substance causes the formation and stabilization of a thrombus. The cytoadhesive substances used can be for example those cited in the European patent application 06819957.9, included here in its entirety as a reference.

In this condition a preparatory step of the perfusion chamber12is provided, also called priming step, in which the physiological solution contained in the first container21is aspirated, using the suction pump device55, through the channels46to fill the micro-channels54by capillarity and to partly flood the zones surrounding the micro-channels54.

The physiological solution, both due to the effect of the suction action of the pumping devices55, and also the effect of the action of the actuation devices of the press14, both of which guarantee the adhesion of the covering plate37to the coupling surface39, generates between the first face52of the covering plate37and the coupling surface39of the cartridge36a liquid packing effect which guarantees the hydraulic seal and prevents hematic contamination between one micro-channel54and another.

A step to introduce the fluid contained in the test tube into the perfusion chamber12is subsequently provided, through the first pipe19.

The fluid is aspirated by means of the pumping devices55and passes through the micro-channels54, entering into contact with the cytoadhesive substance present therein.

As soon as the platelet aggregation begins, when the blood comes into contact with the cytoadhesive substance and the platelets are activated and begin to aggregate, a step is started to acquire the images, in which the optical module65acquires a film and/or a sequence of images in real time, which will allow to analyze the formation kinetics of the thrombi on the evolution of the process by the processing unit16.

More specifically the fluorescent substances introduced into the fluid are highlighted using the lamps described above, in order to supply an image which is suitably collected by the optical module65.

The formation test of the thrombus can be carried out in visible light and in fluorescent light. However, by suitably modifying the optical module65, the perfusion chamber12itself can also be used for different tests which use illumination with different wavelengths from the current ones. It is also possible to carry out a simultaneous analysis of the signal on several wavelengths.

A subsequent processing step provides that the processing unit16, by means of dedicated software, dynamically evaluates the evolution of the platelet aggregation process and the coagulation process over time, determining two primary curves which represent the area percentage of the micro-channels54covered by thrombi as well as the dimension of the thrombi depending on the acquisition time.

In particular, the comparison of the curve representing the area covered by thrombi is necessary in order to discriminate, given the same area covered, the presence of many small-sized thrombi or the presence of a few large-sized thrombi, which give different clinical information.

The processing unit16supplies a series of data, information and/or graphs of the development of the coagulation process also as a function of the time.

Merely by way of example,FIG. 10shows a graph showing the development of the percentage of covering of platelet aggregates of the adhesion surface of the micro-channel as a function of time, in three different clinical behaviors.

In particular the curve indicated by A has a substantially rectilinear development and identifies a patient with values within the norm; curve B has a substantially exponential development and identifies a patient with a predisposition to “thrombotic” pathologies, and curve C has a substantially logarithmic development and identifies a patient with a predisposition to “hemorrhagic” pathologies.

It is clear that modifications and/or additions of parts may be made to the apparatus for the analysis of thrombotic-ischemic and hemorrhagic pathologies as described heretofore, without departing from the field and scope of the present invention.

For example,FIG. 11shows another form of embodiment of a perfusion chamber12, and in particular a cartridge136substantially similar to that described before with reference to cartridge36, and also comprising a mixing tank169which is disposed in an intermediate position along the channel46, directly connected to the entry end51aof the micro-groove51, and a suction pump device155integrated in the cartridge136and directly connected to the other channel46.

The mixing tank169generates a wider section of the channel46and therefore a turbulence in the fluid to be analyzed, which allows to mix in additive substances which can be added through a pipe170. The pipe170can be connected to the outside by means of connections similar to those provided for the entry seatings32, or with stoppers which can be perforated by syringes with which the additive substance is introduced. The additive substances added can be biomarkers, anti-coagulant substances such as ACD or calcium solutions, which activate enzymatic reactions in the fluid to be analyzed.

On the other channel46, instead of providing the exit seatings43, the fluid analyzed and passed through the micro-channel or micro-channels54, remains inside the suction pump device155. More specifically the suction pump device155comprises a containing element, such as a cylindrical jacket171, inside which a plunger172is made to slide, selectively sliding advantageously by means of suitable actuation means, not shown in the drawings.

By driving the plunger172a depression is generated inside the micro-channel or micro-channels54, which as well as aspirating the fluid from the entry seating32generates a sucker effect between the coupling surface39of the cartridge36,136and the first face52of the slide37in order to guarantee the hydraulic seal and prevent any contact between the operator and the fluid analyzed.

In other forms of embodiment (FIG. 12), instead of providing the tank169for mixing the fluid with the additive substances, the cartridge136is provided with another auxiliary pipe175which connects with its convergence176in proximity to the first segment47of the introduction channel46of the fluid to be analyzed. It is therefore possible to introduce said additive substances through the auxiliary pipe175.

In another simplified form of embodiment of the perfusion chamber12(FIG. 13), it can be provided that the micro-groove51is made longitudinally through its whole coupling surface39and that the entry51aand exit51bends terminate, open toward the outside, in proximity to the sides41,42of the cartridge36. Suitable flarings or housing seatings, made in proximity to the entry51aand exit51bends, allow to connect pipes to introduce/discharge the fluid.

In yet other forms of embodiment, instead of providing to use a press14to keep the slide37and the cartridge36,136adherent to each other, it is possible to provide that pressure means are interposed between them, which exert a compression action between said two parts, at the same time guaranteeing the hydraulic seal. In a particular form of embodiment (FIG. 14) it can be provided that the pressure means comprise magnets68which are associated, advantageously in proximity to the corners of the cartridge36,136and the slide37, and disposed with respect to each other in mating positions in order to exert an attraction action between the latter two parts.

It is advantageous to combine what has been described with reference to FIG.11with what has been described with reference toFIG. 14, to make pre-packed kits, ready for use, and of the disposable type. In particular, the coupling surface39of the cartridge136and the first face52of the slide37are suitably pre-treated to make them hydrophile, on the coupling surface39a reactive substrate is laid, intended for the analysis to be carried out, such as cytoadhesive, in order to simulate a damaged vasal surface, and by means of the magnets68it is possible to couple the cartridge136and the slide37so as to constitute the perfusion chamber12ready for use. The perfusion chamber12thus obtained can be suitably packed in a controlled environment to render it available as needed, without requiring further preparation operations.

After use the perfusion chamber12can be suitably disposed of without requiring washing or cleaning and thus preventing the operator coming into contact with the processed fluid. Indeed the analyzed fluid remains confined inside the containing element171of the suction pump device155.

FIGS. 15-23are used to describe a plurality of forms of embodiment, all combinable with forms of embodiment already described, of an apparatus10for the analysis of thrombotic-ischemic and hemorrhagic pathology which includes a perfusion chamber12as described here, defined by a cartridge236coupled to a covering plate37. Some forms of embodiment of the apparatus10include a cartridge236, similar to that described above, which comprises, in a single body, a plurality of containing elements or tanks182,184,186,188, a possible housing189for a possible light wave guide190to cooperate with the optical acquisition device15, a suction pump device191comprising a plunger192, for example a plunger of a syringe pump, insertable sliding in one of the containing elements182,184,186,188and a selection valve194.

For example, the containing elements182,184,186,188, like the possible housing189for a possible light wave guide190, can be obtained by means of milling or similar working by removing material, or directly by molding the cartridge236.

Advantageously, the cartridge236and the slide37coupled together constitute a perfusion device180which constitutes a diagnostic kit which is completely disposable. Indeed, all the elements which come into contact with the biological fluid, in particular blood, are integrated in the perfusion device180and, consequently, it is a finished disposable element which does not need sanitation interventions on the parts of the hydraulic-mechanical circuit of the device.

The cartridge236is coupled to the slide37, as described above, in an essentially overturned configuration with respect for example to that described inFIGS. 1,5-8,13and14, that is, having the optical acquisition device15aligned with the light wave guide190and disposed below the perfusion device180.

a plurality of internal containing elements or tanks182,184,186,188, to store the liquids to be analyzed and the necessary auxiliary fluids, for example for priming and/or washing,

a selection valve194to select the liquid to be passed through the perfusion chamber12defined by the cartridge236and by the slide37, advantageously connected to a motor195which allows a linear or circular movement or a combination of the two;

a possible light wave guide190, to illuminate the perfusion chamber12;

a plunger192, to generate a flow of liquid along the perfusion chamber12, advantageously connected to a motor193which allows a linear or circular movement or a combination of the two.

Advantageously, the perfusion device180can be easily connected and disconnected to/from the drive shaft of each of the motors193and195which drive the selection valve194and the plunger192, making the replacements rapid and safe in the light of a disposable device as above.

The perfusion device180formed by the cartridge236and slide37can be provided with a number of containing elements182,184,186,188, for example comprised between 1 and 5, which can be at entry to or exit from the perfusion chamber12, according to the various functionalities required. The containing elements182,184,186,188can contain fluids, such as blood or other biological liquids, to be analyzed, other fluids, such as blood or other biological liquids, already analyzed, reagents to be mixed inside the sample, buffer solutions needed for priming or for the test to be carried out or for washing. The perfusion device180provided with a plurality, for example two, three, four, five or even more than five, of containing elements182,184,186,188allows different test configurations with a high number of reagents or auxiliary fluids, for example for priming and/or washing

The containing elements182,184,186,188have passageways which can be selectively connected to the one or more micro-channels54. For example, it is possible to provide that some containing elements182,184,186are connected at exit to one or more hydraulic inlets or connections of the one or more micro-channels54, and for example that one containing element188is connected at entrance to the exit of the one or more micro-channels54.

In particular, the selection valve194allows to choose which of the containing elements182,184,186,188must be fluidically connected on each occasion to the perfusion chamber12, in particular to the one or more micro-channels54. The selection valve194can be driven by means of a linear movement or a rotation.

As we said, one of the containing elements182,184,186,188of the cartridge236comprises the plunger192, sliding inside it, which allows to generate a known and constant flow of fluid, by moving the plunger192. The containing element, for example identified by the reference number188, which houses the plunger192, can act at the same time as a deposit for the liquids already analyzed.

As we said, the plunger192can be connected to a motor193, in particular to the corresponding drive shaft, for movement, which can be either linear or rotational. The linear movement to all purposes simulates a syringe, while a rotational movement provides that the inside of the tank, for example the tank188, and the plunger192, are threaded.

In some forms of embodiment, where provided, the light wave guide190allows the light to reach, substantially without optical aberrations, in proximity to the zone of the perfusion chamber12, in particular of the micro-channel or micro-channels54, where the test occurs, and allows to carry out the operations in which an illumination in the range of visible light is needed, for example a first focusing on the test micro-channel.

FIG. 15is used to describe some forms of embodiment, which can be combined with all the forms of embodiment described here, in which the perfusion device180includes a cartridge236which comprises, for example, three containing elements182,184,188, of which a first containing element182to contain the fluid to be analyzed, a second containing element184for an auxiliary fluid, and a third containing element188to house the plunger192, downstream of the one or more micro-channels54, and a housing189is also provided for the light wave guide190. The cartridge236also comprises the selection valve194which is formed, for example by a mobile stopper rod197sliding in a channel199made in the cartridge236. The mobile stopper rod197, which can be shaped with a transverse passage channel197a, is drivable, linearly or in rotation, by a suitable motor195, linear or rotational, and couples with the corresponding drive shaft. The channel199is typically positioned below the containing elements182,184,186,188, transverse to the longitudinal axis of development thereof, so as to affect the exit of each of them. For example, the channel199can be obtained by means of milling or similar process of removing material, or directly by molding the cartridge236.

The plunger192is formed for example by a plate192a, of a size and shape mating with those of the cross section of the third containing element188, and by a mobile rod192b, drivable linearly or in rotation by a suitable motor193, linear or rotational.

FIGS. 16 and 17are used to describe forms of embodiment which can be combined with all the forms of embodiment described here, of the perfusion device180with three containing elements, in two operating conditions. In a first operating condition (for example seeFIG. 16), the selection valve194is driven linearly and puts the first containing element182in communication with the micro-channel or micro-channels54of the perfusion chamber12, and in a second operating condition (for example seeFIG. 17), the selection valve194is driven linearly and puts the second containing element184in communication with the micro-channel or micro-channels54of the perfusion chamber12, for the passage of auxiliary fluid, for example for priming and/or washing

In some forms of embodiment, described usingFIGS. 16 and 17, an entry54aand an exit54bcan be provided for the micro-channel54, or for each of the micro-channels54. The selection valve194is configured to selectively put one of the exits of the containing elements182,184into fluidic communication with the entry54aof the micro-channel54.

FIGS. 18,19and20are used to describe forms of embodiment which can be combined with all the forms of embodiment described here, of the perfusion device180with four containing elements, in three operating conditions. In a first operating condition (for example seeFIG. 18), the selection valve194is driven linearly and puts the first containing element182in communication with the micro-channel or micro-channels54of the perfusion chamber12; in a second operating condition (for example seeFIG. 19), the selection valve194is driven linearly and puts the second containing element184in communication with the micro-channel or micro-channels54of the perfusion chamber12, for the passage of a first auxiliary fluid, reagent or additive; and in a third operating condition (for example seeFIG. 20), the selection valve194is driven linearly and puts a fourth containing element186in communication with the micro-channel or micro-channels54of the perfusion chamber12, for the passage of a second auxiliary liquid, reagent or additive.

In forms of embodiment described usingFIGS. 18,19and20two hydraulic entries or connections can be provided, indicated respectively by54aand54c, in the micro-channel54, or for each of the micro-channels54, including an entry for the fluid, for example blood, and an entry for one of the auxiliary fluids, for example for priming. The selection valve194is configured to selectively put one of the exits of the containing elements182,184, into fluidic communication with one or another of the entries54aand54c.

FIGS. 21,22and23are used to describe forms of embodiment which can be combined with all the forms of embodiment described here, of the perfusion device180and similar to the forms of embodiment described usingFIGS. 18,19and20, in which, however, the selection valve194is driven in rotation to selectively put one of the containing elements182,184,186,188into fluidic communication with the micro-channel or micro-channels54of the perfusion chamber12. Moreover, with reference toFIGS. 21,22and23for example only one entry54aand one exit54bof the micro-channel54are provided, as inFIGS. 16 and 17.