EP3384275A2 - Verfahren zur bestimmung von hämostase unter querlast - Google Patents

Verfahren zur bestimmung von hämostase unter querlast

Info

Publication number
EP3384275A2
EP3384275A2 EP16804763.7A EP16804763A EP3384275A2 EP 3384275 A2 EP3384275 A2 EP 3384275A2 EP 16804763 A EP16804763 A EP 16804763A EP 3384275 A2 EP3384275 A2 EP 3384275A2
Authority
EP
European Patent Office
Prior art keywords
binding partner
flow
platelet
flow cell
reaction zone
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP16804763.7A
Other languages
English (en)
French (fr)
Inventor
Remco VERDOOLD
DE Susanne WITT
Judith COSEMANS
Johan HEEMSKERK
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universiteit Maastricht
Academisch Ziekenhuis Maastricht
Original Assignee
Universiteit Maastricht
Academisch Ziekenhuis Maastricht
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universiteit Maastricht, Academisch Ziekenhuis Maastricht filed Critical Universiteit Maastricht
Publication of EP3384275A2 publication Critical patent/EP3384275A2/de
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/05—Flow-through cuvettes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/05—Flow-through cuvettes
    • G01N2021/056—Laminated construction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7756—Sensor type
    • G01N2021/7763—Sample through flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7786—Fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7793—Sensor comprising plural indicators

Definitions

  • the present invention relates to a method for monitoring of coagulation status and haemostasis in the perioperative setting.
  • the invention also relates to an apparatus for applying said method, and to a cartridge applicable in said method, said cartridge being a flow cell.
  • the invention relates to the use of said method, said apparatus and said cartridge for assessing coagulation status or thrombus formation in a whole blood sample.
  • the invention also relates to comparing test results obtained with the method of the invention with test results in a database.
  • the invention relates to a kit of parts comprising disposables required for applying said method of the invention with the apparatus of the invention.
  • Blood loss in the operative context requires acute and adequate intervention, most preferably within a time-span of minutes.
  • the underlying cause of the blood loss is divers and available therapies that could be applied are equally divers.
  • application of several therapies is diversified with regard to the underlying cause of the blood loss and with regard to the coagulation status of the bleeding patient.
  • the available tests do not provide results in a time- span shortly enough to wait for, before treatment of the patient can be started [van de Kerkhof & Herold, 2012; Bosch et al., 2014].
  • Added to these shortcomings of currently available tests are further shortcomings related to the requirement of sample preparation (of a too large volume of test sample, i.e. blood) before certain tests can be started, which is time-costly, and the required level of skills for executing the measurement and subsequently for analyzing and interpreting the data [Bosch et al., 2014].
  • light-transmission platelet aggregation requires a relative large volume of platelet-rich plasma, and this assay takes a relatively long time, whereas it is doubted whether the assay results provide information relevant to the blood coagulation status in the bleeding patient. For example, routine coagulation tests require 20-30 minutes before test results become available; a time-span that is too long to wait for while the bleeding patient requires acute treatment.
  • Examples of nowadays clinical tests used in the perioperative setting are platelet aggregation tests such as the PFA-100 platelet function analysis with citrated whole blood, and viscoelastic whole blood clot formation tests, such as the ROTEM clot formation test.
  • PFA-100 test implies whole blood under shear conditions, it has been concluded that the test is not applicable for excessively bleeding patients.
  • the PFA- 100 test has been judged as being not of importance in directing transfusion therapy strategy when excessive bleeding occurs after cardiac surgery [Forestier et al., 2002].
  • the ROTEM test is not able to detect impairment in platelet function induced by anti-platelet agents.
  • the ROTEM test run under static conditions, therefore under non- physiological conditions by definition, requires about 23 minutes for delivering test results.
  • Flow perfusion chamber technology allows for assessment of platelet function and coagulation status at the same time, under conditions of (patho)physiological shear rates present in the circulation.
  • Flow perfusion measurements provide information on a panel of parameters, such as platelet adhesion, platelet activation, thrombus growth, fibrin formation, coagulation, at selected venous or arterial shear rates. Platelet adhesive activity and thrombotic tendency of the blood of a patient can be analyzed while using various platelet-adhesive substrates as an activating surface.
  • the international application WO2006065739 provides an example of flow perfusion chamber technology meeting some though not all above mentioned limitations of haemostasis tests, and describes a device for aggregating, imaging and analyzing thrombi.
  • An instrument is described comprising a plurality of channels which are partially or fully coated with a material that induces blood cell aggregation, and an imaging assembly and an analyzer for quantifying at least one characteristic of the aggregation.
  • the device is aimed at providing image data in less than 30 minutes, related to for example platelet adhesion and aggregation parameters for an individual.
  • a video of thrombus formation is provided. For assessing brightfield
  • WO2006065739 provides a first analytical system, whereas for assessing fluorescence signal, WO2006065739 provides a second analytical system, thereby limiting the number of output parameters that can be obtained with a single measurements.
  • WO2015102726 A different example of a flow perfusion chamber technology is provided in international patent application WO2015102726.
  • a blood coagulation monitoring technology is described which takes into account shear stress and which requires minimal sample preparation and operator training.
  • changes in pressure in micro-channels is recorded upon formation of a thrombus on a substrate, from blood.
  • WO20101023335 yet another microfluidics device is presented, which provides real-time monitoring of platelet aggregation of a biological sample.
  • the device of WO2010102335 comprises a flow cell comprising a flow channel with a protrusion.
  • a biological sample comprising platelets flows through the flow channel and platelets are activated and aggregate once having contacted the protrusion while flowing.
  • the platelet aggregation is assessed e.g. by optical detection means.
  • an alternative microfluidic device for determining clotting time in a fluid medium such as blood.
  • the blood flows through a first flow channel of a flow cell, comprising a region containing a reagent that reacts with the blood.
  • the blood flows through a second flow channel of a flow cell, not comprising a region containing a reagent that reacts with the blood.
  • the measured difference in blood flow over time between the channels is a measure for the clotting of blood in the flow channel comprising the reagent.
  • Total thrombus-formation analysis system is a flow perfusion system for quantitative assessment of thrombus formation under flow conditions.
  • the system either provides information on thrombus growth, or, in a separate measurement, on platelet adhesion and aggregation.
  • Test results are obtained either upon applying a surface of collagen in the T-TAS, or a surface of collagen with thromboplastin.
  • Measurements with blood relate to pressure differences in occluding capillaries comprising the coated surface, on which e.g. activated platelets accumulate.
  • T-TAS may perhaps complement viscoelastic whole blood clot formation tests in the clinic, such as the ROTEM clot formation test, although T-TAS still needs to be validated [Schott & Johansson, 2013].
  • flow perfusion chamber technology is a proven tool for application in research laboratories, suitable to derive information on e.g. platelet function and the influence of anti-thrombotic therapy on haemostasis status with prospective value, up till now the technology has been unable to repay expectations regarding beneficial implementation of the technology in the clinic [Westein et al., 2012; de Witt et al, 2014], e.g. during perioperative patient care. Entrance of the application of the technology in the clinic is severely hampered by a lack of standardization in chamber construction and in the use of the test method. The requirements of complex microscopic imaging analysis when applying the technology demands for highly trained personnel. In addition, the required volume of blood for running tests with the technology can be relatively large.
  • the quick test should demand a minimal level of skills relating to operating the test and a minimal level of skills relating to analyzing and interpreting the data.
  • the test should be applicable for test samples without laborious sample preparation required, e.g. applicable for small volumes of freshly drawn ((non-)anti-coagulated) whole blood.
  • the inventors have developed a method for assessing coagulation status and
  • a first aspect of the present invention therefore relates to an automated method for optically measuring complex formation under flow at physiological shear rate between a mobile binding partner and an immobilized binding partner, comprising the steps of:
  • a flow cell 4, 4', 4" comprising at least one flow channel 43, which flow channel comprises at least one reaction zone 45 located at a portion of the flow channel that is transparent for light, and wherein said reaction zone comprises an immobilized binding partner;
  • the light imaging assembly comprises a light source 30 and a light sensitive sensor 37, wherein the light sensitive sensor records in a time- lapse manner transmitted light signal from the light source 30 through the reaction zone;
  • the invention provides said method further comprising step aa) following step a):
  • step cc) preceding step d):
  • the light sensitive sensor records in a time-lapse manner light signal emitted by
  • the light emitting label is selected from a fluorescent dye, a fluorescently labeled binding moiety, or combinations thereof.
  • the invention offers the advantage that it has now reduced to practice a method to retrieve information within a short time-frame most relevant to haemostasis status of a patient, whereas a minimal level of skills and intervention is required for retrieving said information.
  • a second aspect of the invention is an automated analytical system, comprising: (i) an imaging assembly (50) comprising seen along the direction of an optical path:
  • a fluidic system 60, 60', 60" for feeding an aqueous sample comprising a
  • mobile binding partner to the flow channel 43 of a flow cell 4, 4', 4" comprising: a) at least one high-precision pump 1 , 1 ', connected with
  • aqueous solution A-C, D-F comprising an aqueous solution A-C, D-F, wherein the aqueous solution is selected from Ca 2+ /Mg 2+ solution, physiological salt solution, buffer solution, wash buffer, rinse buffer, solution comprising a fluorescent label,
  • a flow cell 4, 4', 4" comprising at least one flow channel 43, 43' in connection with an inlet 42, 42' and an outlet 44, 44', wherein the flow channel comprises at least one reaction zone 45.
  • the automated analytical system of the invention is particularly suitable for carrying out the method according to the invention.
  • a third aspect of the invention is a flow cell 4, 4' for use in the automated analytical system according to the invention, comprising at least one flow channel 43, preferably two flow channels 43, 43', said flow channel comprising at least one reaction zone 45 located at a portion of the flow cell that is transparent for light, and said reaction zone comprising an immobilized binding partner, and said flow channel having an inlet 42, 42' positioned at an angle of 5-90° relative to the longitudinal dimension (length) and/or the transversal dimension (width) of the at least one reaction zone in the flow channel, preferably at an angle of 10° to 20°, more preferably 1 1 ° to 12°, most preferably about 1 1 °, and an outlet 44 positioned at about the same angle as the angle of the inlet relative to the longitudinal dimension (length) and/or the transversal dimension (width) of the reaction zone, and having a cross-sectional area of 0.075 mm 2 to 0.30 mm 2 , preferably with a width of about 2 mm or about 3 mm and
  • a fourth aspect of the invention is the use of the automated analytical system according to the invention and a flow cell according to the invention for measuring haemostasis with the method of the invention by measuring at least one parameter selected from platelet deposition (thrombus surface area), thrombus build up, number of thrombi per surface area of immobilized binding partner, multilayer, P-selectin expression, phosphatidylserine exposure, fibrinogen binding, static platelet adhesion under non- coagulating conditions, and/or by measuring platelet deposition (thrombus surface area), thrombus build-up, time-to-fibrin formation, fibrin formation under coagulating conditions, and/or by measuring fibrinolysis under coagulating conditions, platelet-fibrin interaction, platelet aggregation, platelet adhesion, annexin A5 binding, granule secretion, formation of thrombin, platelet accumulation, thrombus volume,
  • a fifth aspect of the invention is a kit of parts comprising:
  • physiological salt solution buffer solution, wash buffer, rinse buffer, solution comprising a fluorescent label
  • sample holder 2 wherein the sample holder is a syringe for blood draw, said syringe pre-filled with citrate and optionally pre-filled with at least one light emitting label according to the invention and with PPACK,
  • time-lapse recording has its normal scientific meaning and refers to time-lapse imaging with a digital camera, wherein images are captured at predetermined regular time intervals, i.e. the capture rate.
  • coagulating condition has its normal scientific meaning and refers to a fluid e.g. whole blood, that is kept in a state that allows for coagulation.
  • a coagulating condition for a whole blood sample is a volume of whole blood mixed initially with buffer solution comprising for example citrate, and then subsequently mixed with a solution comprising calcium cations.
  • non-coagulating condition has its normal scientific meaning and refers to a fluid e.g. whole blood, that is kept in a state that prevents coagulation.
  • an anti-coagulating condition for a whole blood sample is a volume of whole blood mixed with citrate, or citrate and PPACK.
  • automated as used herein has its normal scientific meaning and refers to a method or process that does not require any intervention by a (skilled) person up to the moment that a test resulted is provided by the method or process, once a test sample has been subjected to the method or process by the person.
  • real-time e.g. in 'real-time processing'
  • real-time results in the almost instantaneous availability of test results of the method of the invention, coinciding with the run-time of the complex formation under flow and the time-lapse recording of signal data.
  • FIG. 1 A flow cell 4 with two flow channels 43 and 43' according to the invention
  • Flow-cell 4 Top part 40; Bottom part 41 ; Inlet 42, 42'; flow-channels 43, 43'; Outlet 44, 44'; multiple reaction zones 45 comprising immobilized binding partner.
  • the top part 40 and the bottom part 41 are in intimate contact (e.g. by means of sealing, gluing, pressing, etc.) according to the invention and when part of the automated analytical system of the invention.
  • B. A flow cell 4' with a single flow channel 43 according to the invention.
  • C A flow cell 4" according to the invention, further comprising a sample holder 2'.
  • FIG. 1 Drawings schematically showing an imaging assembly 50, as part of an automated analytical system according to the invention.
  • the imaging assembly 50 of an automated analytical system according to the invention comprises, seen in the direction of an optical path defined by light traveling through the imaging assembly 50: Top light source 30 for trans-illumination; Flow cell 4' (or 4, 4"); Microscope objective lens 31 ; Mirror 32; Bottom light sources 33; Semi-transparent mirror 34; Wavelength filter 35; Imaging lens 36; Imaging sensor 37.
  • A. transmission is measured and signal data is time-lapse recorded upon illuminating the flow-cell 4' from above, with light source 30.
  • B. the fluorescence mode for measuring emitted light from a label which fluoresces is outlined.
  • the flow cell 4' is irradiated by the bottom light-source 33.
  • the socket 19 is depicted with thermostat 9 and xyz stage controller 10, and the flow-cell holder 20 for receiving a flow cell.
  • the socket 19 intimately receives the flow-cell holder, for efficient heat transfer from the socket to the flow cell (heating the flow cell) or vice versa (cooling of the flow cell).
  • the socket with the received flow cell holder comprising the flow cell is also depicted in A. and B.
  • FIG. 3 A fluidic system 60 according to the invention, as part of the automated analytical system of the invention. For clarity reasons, the imaging assembly 50 of the automated analytical system is not depicted (see Figure 2 for the relative orientation of the imaging assembly with regard to a flow cell 4').
  • a schematic drawing of fluidic system 60 of the invention is outlined, wherein the fluidic system comprises a flow cell 4' having a single flow channel 43.
  • Sample holder 2 here a container, e.g. a syringe, for an aqueous sample, e.g.
  • T-connector inlet connector 3 with an inlet 3' in connection with the outlet of the sample holder 2 and with an inlet 3" in connection with the outlet 5' of a flow-through micro pump 5 (see below); flow cell 4' with a single channel 43; flow through micro pump 5 having an outlet 5' connected to the T-connector inlet connector inlet 3", and having an inlet 5" in connection with the outlet 6' of a three-way selector valve 6 (see below); three-way selector valve 6, having an outlet 6' in connection with the inlet 5" of the pump 5, and having three inlets 6" in connection with three reservoirs 11 containing further aqueous solutions A, B, and C; outlet connector 7 in connection with the flow channel 43 of the flow cell 4; optionally, a waste container 8 in connection with the outlet connector 7; optionally a thermostat 9 for keeping at least the flow cell 4 at a predetermined temperature during complex formation and data collection; an xyz stage controller 10.
  • T-connector inlet connector 3 with an inlet 3' in connection with
  • the fluidic system comprises a flow cell 4 having two flow channels 43, 43'.
  • the two-channel flow cell in Figure 3B comprises a further set of pumps, valves, etc.
  • Sample holder 13 here a container, e.g. a syringe, for an aqueous sample, e.g.
  • T-connector inlet connector 14 with an inlet 14' (not shown) in connection with the outlet of the sample holder 13 and with an inlet 14" (not shown) in connection with the outlet 15' (not shown) of a flow-through micro pump 15 (see below); flow chamber 4 with two channels 43, 43'; flow through micro pump 15 having an outlet 15' (not shown) connected to the T-connector inlet connector inlet 14" (not shown), and having an inlet 15" (not shown) in connection with the outlet 16' (not shown) of a three- way selector valve 16 (see below); three-way selector valve 16, having an outlet 16' (not shown) in connection with the inlet 15" (not shown) of the pump 15, and having three inlets 16" (not shown) in connection with three reservoirs 17 containing further aqueous solutions D, E, and F; outlet connector 7 in connection with a first flow channel 43 of the flow cell 4; outlet connector 18 in connection with a second flow channel
  • FIG. 3C provides the schematic drawing of another embodiment of the invention, with yet an alternative fluidic system 60" according to the invention, wherein the fluidic system comprises a flow cell 4" having a single flow channel 43 and a sample holder 2'.
  • the fluidic system comprises a precision (syringe) pump 1 '.
  • the micropump 5 is now connected to the inlet 42 of the flow cell, via connector 3a.
  • Figure 4 Effect of storage of immobilized binding partner on reaction zones 45 under freezing conditions, regarding the extent of complex formation with platelets as the mobile binding partner in whole blood as the aqueous sample.
  • FIG. 5 The effect of a tyrosine kinase inhibitor (TKI; anti-cancer drug) on thrombus formation.
  • TKI tyrosine kinase inhibitor
  • the effect of TKI in a whole blood sample is compared with a whole blood sample comprising vehicle only.
  • Figure 6 Test results of time-lapse recording of fluorescent signal from fluorescent labels bound to platelets, fibrinogen, phosphatidylserine as mobile binding partners in the complex, showing thrombus formation on collagen I +/- tissue factor as the immobilized binding partners, as measured with the method of the invention using an automated analytical system according to the invention.
  • FIG. 7 Fibrin formation on an immobilized binding partner, with whole blood of a patient with hemophilia B as the source of fibrinogen/fibrin, in comparison to fibrin formation with whole blood of a healthy control.
  • Figure 8 Figures A-C show three different embodiments of the invention regarding time- lapse recording of transmitted light and fluorescence.
  • time-lapse recordings are performed at a single reaction zone with a flow cell comprising a single flow channel with a single reaction zone.
  • Time-lapse recordings of brightfield transmitted light and fluorescence at three different wavelengths is sequentially alternated.
  • First, for a time window transmittance is recorded and analyzed. While signal is analyzed, the filter wheel repositions to a filter for a second wavelength.
  • processed transmitted light data is stored and displayed. Subsequently, the first fluorescence data is processed while the filter wheel repositions to a third wavelength, for a second fluorescence recording etc.
  • a two-channel flow cell is used.
  • time-lapse recordings are performed at a single reaction zone with a flow cell comprising a single flow channel with a single reaction zone.
  • Time-lapse recordings of brightfield transmitted light is executed, only.
  • transmittance is recorded and analyzed.
  • Signal is analyzed and processed transmitted light data is stored and displayed, before a second time-lapse recording of brightfield transmitted light is executed, etc., up till 4 minutes or six minutes of complex formation are completed.
  • test results should be sufficiently and reliably associated with the therapy to be selected for the (acute, severe) bleeding patient in need of the therapy, wherein applying said test and analyzing said test results should not require a significant level of skills from the test operator.
  • limitation related to the required amount of test sample i.e. blood of the patient, and related to test sample processing conditions in the test methods, which test method should closely mimic physiological circumstances.
  • a first aspect of the present invention relates to an automated method for optically measuring complex formation under flow at physiological shear rate between a mobile binding partner and an immobilized binding partner, comprising the steps of:
  • a flow cell 4, 4', 4" comprising at least one flow channel 43, which flow channel comprises at least one reaction zone 45 located at a portion of the flow channel that is transparent for light, and wherein said reaction zone comprises an immobilized binding partner;
  • the light imaging assembly comprises a light source 30 and a light sensitive sensor 37, wherein the light sensitive sensor records in a time- lapse manner transmitted light signal from the light source 30 through the reaction zone;
  • the inventors developed a method that provides for a sufficiently short flow time, preferably about 4 minutes or about 6 minutes, required to allow for sufficient formation of a complex between the mobile binding partner and the immobilized binding partner.
  • the inventors also found that despite the short flow time in the method of the invention, it is possible to record a sufficient amount of relevant imaging data of sufficient intensity and contrast and sufficient detail, in order to provide the required test results in a shortly enough time-frame, upon processing the recorded signal data and subsequently displaying processed data as numerical data and/or in graphical form. Of course, within the same time span, also recorded images can be displayed, if required.
  • the method of the invention by combining the short flow time with time-lapse imaging mode (time-lapse signal data recording) at a carefully selected capture rate, the inventors found that this combination now allows for real-time processing of a sufficiently large set of recorded data signal, providing the required test results in a shortly enough time-frame at sufficient level of quality and detail.
  • the invention addresses the current limitations as listed above by providing the relevant test results within a meaningful short time-frame.
  • the inventors were also able to automate the whole process from test sample introduction to delivery of the test results as numerical data and/or in graphics.
  • the method of the invention not only provides for a sufficiently fast process for assessing complex formation, including data acquisition and data processing, but the method of the invention also only requires a minimal level of skills.
  • the inventors managed to align and shorten the time lines required for
  • complex formation is for example visualized as accumulating aggregates of complexed mobile binding partner and immobilized binding partner in the reaction zone 45 surface area.
  • This imaging technique is only one example of the many imaging techniques available today, and fit for the purpose of the method of the invention.
  • images derived from fluorescence microscopy techniques are equally suitable for application in the method of the invention.
  • Fluorescence imaging is a routine technique, and virtual any fluorescent label that can be coupled to a mobile binding partner is suitable for application in the method of the invention.
  • the method comprises a fluorescently labeled mobile binding partner.
  • a mobile binding partner is provided with a fluorescent label by means of introducing a fluorescently labeled binding moiety, preferably a fluorescently labeled affinity molecule which binds to the mobile binding partner once exposed to it.
  • a fluorescent label or the fluorescently labeled affinity molecule with affinity for the mobile binding partner is provided in the aqueous sample comprising the mobile binding partner, before complex formation.
  • said fluorescent label or said fluorescently labeled affinity molecule is bound to the mobile binding partner after the mobile binding partner and the immobilized binding partner have formed a complex.
  • the initial aqueous sample comprising the mobile binding partner has then first be replaced by a further aqueous solution, e.g. wash buffer, and the complex in the flow channel 43 has first been washed with said wash buffer, before the fluorescent label or the fluorescently labeled affinity molecule is bound to the mobile binding partner in the complex.
  • the method of the invention further comprises a step aa) following step a):
  • step cc) preceding step d):
  • the light imaging assembly comprises a further light source 33, wherein the light sensitive sensor records in a time-lapse manner light signal emitted by the light emitting label bound to the mobile binding partner in the complex in the reaction zone upon irradiation of the complex by the light source 33, wherein the light emitting label is selected from a fluorescent dye, a
  • fluorescently labeled binding moiety or combinations thereof.
  • the fluorescent label is selected from a fluorescent dye selected from 3,3'-dihexyloxacarbocyanine iodide, DAPI, FITC, TRITC and CY5
  • the fluorescently labeled binding moiety is selected from a labeled antibody, antibody V domain, Fab fragment, ScFv, antibody chain, or combinations thereof.
  • mobile binding partners that are particularly suitable for labeling with a light emitting label are phospatidylserine (PS), P-selectin, fibrinogen, fibrin, thrombin, allb33, von Willebrand factor (vWF), thrombospondin-1 , Factor V, Factor XII, Factor VIII, Factor IX, Factor X, and more preferably phospatidylserine, P-selectin, fibrinogen, fibrin, thrombin, allb33, or combinations thereof.
  • PS phospatidylserine
  • P-selectin fibrinogen, fibrin, thrombin, allb33
  • vWF von Willebrand factor
  • thrombospondin-1 thrombospondin-1
  • Factor V Factor XII, Factor VIII, Factor IX, Factor X
  • phospatidylserine P-selectin, fibrinogen, fibrin,
  • activated platelet surface e.g. phosphatidylserine
  • endothelial cell surface e.g. P-selectin
  • other mobile binding partners e.g. such as those present in whole blood, involved in complexing with the immobilized binding partner are optionally labeled with a light emitting label, according to the invention.
  • the labeled mobile binding partner is thus preferably selected from phospatidylserine, P-selectin, fibrinogen, fibrin, thrombin, allb33, von Willebrand factor, thrombospondin-1 , Factor V, Factor XII, Factor VIII, Factor IX, Factor X, or combinations thereof, more preferably phospatidylserine, P-selectin, fibrinogen, fibrin, thrombin, allb33, or combinations thereof.
  • the light emitting label is selected from a fluorescent dye selected from 3,3'-dihexyloxacarbocyanine iodide, DAPI, FITC, TRITC and CY5, a fluorescently labeled binding moiety selected from a labeled antibody, antibody V domain, Fab fragment, ScFv, antibody chain, or
  • phospatidylserine P-selectin, fibrinogen, fibrin, thrombin, allb33, von Willebrand factor, thrombospondin-1 , Factor V, Factor XII, Factor VIII, Factor IX, Factor X, or combinations thereof, preferably phospatidylserine, P-selectin, fibrinogen, fibrin, thrombin, allb33, or combinations thereof.
  • time-lapse recording of transmitted light signal and/or of emitted light signal is performed according to the invention by time-lapse imaging during complex formation under flow, and/or at at least one time point at which complex has been formed and flow is (temporarily) reduced to 0 ml/hour, e.g. for an end-point recording and/or in a stop-flow set-up, known in the art.
  • the formed complex is first washed with an aqueous solution such as a wash buffer, before transmitted light signal and/or of emitted light signal is recorded.
  • Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium is exposed to the circulation.
  • the platelet adhesion cascade takes place in the presence of shear flow.
  • Flow-chamber, or perfusion-chamber, based assays to measure thrombus formation in vitro under conditions of shear flow are applied in the clinical research setting. These currently applied assays are laborious in nature. Applying the assays requires in-depth knowledge of the technology and biological mechanism underlying the assay and require a high level of skills in order to be able to properly operate such assays and interpret test results correctly.
  • test results of the method of the invention are not only provided on short notice, but are optionally also related to a database comprising reference values.
  • reference values preferably relate to previously acquired patient data, previously acquired test results with test samples reflecting a wide array of possible haemostatic conditions in the patient, etc.
  • reference values optionally also relate to historical values obtained with test samples retrieved from the very same patient and measured with the method of the invention. This way, haemostatic condition of a patient is monitored over time with the method of the invention.
  • the pro- thrombotic status of a subject is assessed with the method according to the invention, by comparison of retrieved test results with a whole blood sample comprising at least platelets as a mobile binding partner, by comparison of the test results with reference control test results, i.e. reference values, stored in a database.
  • the bleeding status of a subject is assessed with the method according to the invention.
  • the aqueous sample is preferably anti-coagulated citrated whole blood
  • the immobilized binding partner is typically collagen or collagen with tissue factor.
  • the processed data is related to a reference value, such that the amount and/or type and/or complex-forming capacity of mobile binding partner in the aqueous sample and/or in the complex can be determined, and/or such that the amount or type of formed complex or the rate of complex formation can be determined.
  • shear rates are between about 75 s "1 and 2.000 to 10.000 s "1 , or at about 150 s "1 for veins and at about 1.000 to 1 .600 s "1 for arteries. Therefore, preferably in the method according to the invention, shear rates are applied that fall within these mentioned physiological ranges.
  • shear rates between about 150 s “1 and about 1 .600 s “1 , preferably selected from about 150 s “1 , about 1.000 s “1 , about 1 .600 s “ 1 , therewith closely mimicking the physiological conditions in veins and in arteries of the body.
  • the shear rate is 150 s “1 to 1 .600 s “1 , preferably selected from about 150 s “1 , about 1 .000 s "1 , 1 .600 s '
  • the flow rate is optimized for meeting the requirements of desired shear rate and simultaneously for facilitating complex formation within a short time frame and with sufficiently high quality suitable for providing high quality test results with the method of the invention.
  • flow of the aqueous sample comprising the mobile binding partner or of a subsequently applied aqueous solution, e.g. the subsequent buffer; see above) is provided at between 0 ml/hour (when e.g.
  • the flow is at about 0.6 ml/hour to about 225 ml/hour, more preferably, the flow is about 0.675 to 10 ml/hour, most preferably about 7.2 ml/hour or about 0.675 ml/hour or about 4.5 ml/hour.
  • the shear rate is about 1 .600 s "1 , according to the invention.
  • the shear rate is about 150 s "1 , according to the invention.
  • the shear rate is about 1 .000 s "1 , according to the invention.
  • the flow is 0.6 to 225 ml/hour, more preferably 0.675 to 10 ml/hour, more preferably about 7.2 ml/hour, or about 0.675 ml/hour, or about 4.5 ml/hour.
  • an ingenious balance is found between at one hand time-lapse recording of images suitable for capturing enough signal and enough detail of the complex at a selected capture rate, providing high-quality signal data for subsequent processing (i.e. with a shutter time of the imaging sensor 37 of about 10 ms to about 200 ms), and at the other hand creating a time-gap between consecutive steps of time-lapse data signal recording, which time gap is just long enough for allowing storage of acquired data, processing said data, storing and displaying processed data (numerical, graphical, image) and optionally relating the processed data (i.e. the test result) to a reference value.
  • the imaging and subsequent storing, processing and displaying is in real time.
  • the complex formation is within 4 to 6 minutes, preferably in about 4 minutes or in about 6 minutes. Recording of images during complex formation and all subsequent image processing steps, eic, all together are also finished within these 4 to 6 minutes.
  • the method of the invention can also provide test results within 30 seconds to 4 minutes, if complex formation is also established within these same 30 seconds to 4 minutes.
  • the time gap is a time interval of between 500 ms to 60 seconds between consecutive time-lapse recordings, preferably 600 ms to 30 seconds, preferably 600 ms to 6 seconds, most preferably about 800 ms to 990 ms, or about 1 second, or about 2 seconds, or about 6 seconds.
  • recorded signal data i.e. an image
  • time for each recording i.e. the shutter time
  • time for each image is about 10 ms to about 200 ms for each image recorded.
  • signal is time-lapse recorded with a shutter time of the imaging sensor 37 of about 10 ms to about 200 ms, and with a time gap of 500 ms to 60 seconds, preferably 600 ms to 30 seconds, more preferably 800 ms to 990 ms.
  • Example 17 and Figures 8A-C provide embodiments of the invention, showing various routines for measuring complex formation by time lapse imaging and
  • time lapse recordings and subsequent storing, processing, etc. are performed in consecutive order.
  • first transmittance is recorded for 10- 200 ms, and subsequently signal is stored, processed, etc.
  • the filter wheel is turned to a new position allowing recording of fluorescence of a first label which signal is stored, processed, etc.
  • the filter wheel is again turned to a new position allowing recording of fluorescence of a second label which signal is stored, processed, etc.
  • the filter wheel is turned to the starting position allowing again recording of transmitted light which signal is stored, processed, etc.
  • time lapse imaging at least time lapse signal (brightfield) of transmitted light is recorded, and preferably also time lapse signal of at least one fluorescent label is time lapse recorded, preferably of one, two, three or four, or more different fluorescent labels. Most preferably, transmittance is recorded and fluorescent signal at 1 to 4 wavelengths, relating to 1 to 4 different fluorescent labels is recorded. See Figure 8 for preferred embodiments of the invention, related to time lapse recordings of transmitted light and fluorescence, with a flow cell having a single flow channel or having two flow channels, for example.
  • the method of the invention provides this guidance by providing the relevant test results (numerical data, graphical data, if required supplemented with one or a few recorded images) within a time-frame short enough for application in the perioperative setting of the operation theater, e.g. within 4 to 6 minutes.
  • the method according to the invention provides the test results within 30 seconds to 30 minutes, preferably 2 minutes to 15 minutes, more preferably 3 minutes to 10 minutes, even more preferably 4 minutes to 6 minutes, most preferably at about 4 minutes or at about 6 minutes.
  • steps a) to d) or steps a) to e) together take 30 seconds to 30 minutes, more preferably 2 minutes to 15 minutes, more preferably 3 minutes to 10 minutes, most preferably 4 minutes to 6 minutes, or about 4 minutes or about 6 minutes.
  • the flow is 0.6 to 225 ml/hour, more preferably 0.675 to 10 ml/hour, more preferably about 7.2 ml/hour, or about 0.675 ml/hour or about 4.5 ml/hour, wherein the shear rate is 150 s "1 to 1.600 s "1 , preferably selected from about 150 s "1 , about 1 .000 s "1 , about 1 .600 s "1 , wherein in steps c) and cc) signal is time-lapse recorded and stored at time intervals of 600 ms to 60 seconds, preferably 600 ms to 30 seconds, most preferably about 600 ms, or about 1 second, or about 2 seconds, or about 6 seconds, and wherein steps a) to d) or steps a) to e) together take 30 seconds to 30 minutes, preferably 2 minutes to 15 minutes, more preferably 3 minutes to 10 minutes, even more preferably 4 minutes to 6 minutes, most preferably about
  • the method of the invention allows for assessing complex formation in at least one reaction zone and up to about 20 reaction zones 45, preferably 1 to 9 reaction zones 45, most preferably 3 to 5 reaction zones 45, in a single application run of the method.
  • a flow cell with two or multiple flow channels, with each flow channel comprising multiple reaction zones even more parameters are assessable within a single measurement according to the method of the invention. This opens the way for applying the method of the invention in a mode wherein multiple parameters and/or multiple conditions that influence complex formation, are assessed simultaneously, within a single measurement.
  • a concentration series of immobilized binding partner divided over multiple reaction zones 45 is assessed with the method of the invention, and/or various individual immobilized binding partners are analyzed in separate reaction zones 45, and/or various combinations of different immobilized binding partners are analyzed in separate reaction zones 45, or combinations thereof.
  • These modes of the method according to the invention optionally widens the window of test results that is provided.
  • the flow channel 43 comprises one reaction zone 45 with immobilized binding partner or comprises multiple reaction zones 45 with immobilized binding partner, preferably 1 to 20 reaction zones 45, more preferably 1 to 9 reaction zones 45, most preferably 3 to 5 reaction zones 45.
  • the flow cell 4 comprises two or more reaction zones 45 comprising a concentration series of an immobilized binding partner. It is part of the invention that if multiple reaction zones 45 in the method of the invention are applied in the flow channel 43 in consecutive manner (See Figure 1 B for an exemplifying embodiment of the invention), as opposed to an equally suitable parallel arrangement of reaction zones 45, or combinations thereof, the immobilized binding partners are immobilized at the multiple reaction zones 45 in an order from relative low complex-forming capability to high complex forming ability, with respect to the direction of the flow. This way, aqueous sample entering the flow channel 43 first contacts
  • a first reaction zone 45 comprises the immobilized binding partner collagen and a second reaction zone 45 comprises immobilized binding partner tissue factor combined with collagen, wherein the aqueous sample is whole blood and the mobile binding partner is a platelet and/or fibrinogen and/or a blood coagulation factor, or combinations thereof.
  • proteins and peptides are suitable immobilized binding partners.
  • cells of any type as immobilized binding partners in the method of the invention are also combinations of proteins and peptides, and cells are optionally applied as immobilized binding partners in the method of the invention.
  • Cells particularly suitable for application as immobilized binding partner in the method of the invention are cells that contact whole blood in the circulation of the body under physiological conditions or under pathophysiological conditions.
  • a preferred cell as immobilized binding partner is for example a platelet, a red blood cell, a vascular cell, a tissue cell, a macrophage, a T-cell, a B-cell, a NK-cell, a monocyte, a neutrophil, an endothelial cell, a muscle cell, a vascular smooth muscle cell, a fibroblast, or combinations thereof, more preferably an endothelial cell, a vascular smooth muscle cell, or a cell related to inflammation selected from a monocyte, a neutrophil, or combinations thereof.
  • the immobilized binding partner is selected from a single protein, a mixture of proteins, a single cell type, a mixture of cell types, a complex-inducing material, or a combination thereof.
  • the immobilized binding partner is selected from a single protein or a mixture of proteins and wherein the mobile binding partner is selected from a single protein, a mixture of proteins, a single cell type, a mixture of cell types, or a combination thereof.
  • the immobilized binding partner comprises a material such as an artificial surface material, wherein said material activates blood platelets and induces complex formation and platelet aggregation.
  • a material such as an artificial surface material
  • the immobilized binding partner comprises a material such as an artificial surface material, wherein said material activates blood platelets and induces complex formation and platelet aggregation.
  • materials applicable in the method of the invention are materials applied in stents, surface materials of any apparatus or part that contacts the circulation in the body, a negatively charged surface material, a mechanical valve, and the like.
  • Preferred materials for use as an immobilized binding partner according to the invention are for example a polyurethane, a polyvinylchloride, a polymethylmethacrylate, or combinations thereof.
  • the method of the invention provides a fast and reliable method for assessing complex formation between a mobile binding partner and an immobilized binding partner under conditions of flow at physiological shear rate
  • the method of the invention is of course particularly suitable for assessing formation of complexes as is occurring in the circulation of the body, under (patho)physiological conditions.
  • the immobilized binding partner is thus preferably selected from at least a molecule or at least a cell, or combinations thereof, that are also present in immobilized form in the circulation or that are exposed to the circulation under pathophysiological conditions.
  • the immobilized binding partner comprises at least one protein selected from laminin, proteoglycan, tissue factor (TF), fibrin, fibrinogen, fibronectin, vitronectin, osteopontin, collagen, collagen type I, collagen-derived peptide, collagen peptide mimetic, rhodocytin, von Willebrand factor, tissue thromboplastin, activated protein C, factor XII, P-selectin, annexin A5, integrin a2, integrin ⁇ 3, integrin ⁇ 5 ⁇ 1 , CD36, integrin allbp3, glycoprotein VI, integrin ⁇ 2 ⁇ 1 , glycoprotein Iba, CLEC-2, coagulation factors or combinations thereof, or a(n artificial) material that triggers a platelet activation pathway or that triggers a platelet inhibitory pathway, or combinations thereof, more preferably selected from collagen, von Willebrand factor, tissue thromboplastin, activated protein C, factor X
  • the aqueous sample is preferably selected from culture medium, blood serum, blood plasma, buffer solution or a cell suspension selected from whole blood, plasma, synovial fluid, cerebrospinal fluid, lymph, interstitial fluid, cells in culture medium, cells in buffer solution, or any dilutions thereof and/or any combinations thereof, more preferably, whole blood
  • the mobile binding partner comprises a cell selected from a platelet, a red blood cell, a cell of the vascular lining, a human umbilical vein endothelial cell, a patient-derived blood outgrowth endothelial cell, a tissue cell, a macrophage, a T-cell, a B-cell, a NK-cell, a monocyte, a neutrophil, a leukocyte, a progenitor cell, an endothelial cell, a muscle cell, a vascular smooth muscle cell, a fibroblast, or a combination thereof, more preferably, a platelet, and even more preferably the a platelet, and
  • the whole blood sample is typically an anti-coagulated whole blood sample according to the invention.
  • at least one anti-coagulant is added to a whole blood sample, selected from D-Phenylalanyl-L-propyl- L-arginine chloromethylketone (PPACK), fragmin, hirudin, heparin, citrate, corn trypsin inhibitor (CTI) or other anticoagulants, or combinations thereof.
  • PPACK D-Phenylalanyl-L-propyl- L-arginine chloromethylketone
  • fragmin fragmin
  • hirudin hirudin
  • heparin citrate
  • CTI corn trypsin inhibitor
  • other anticoagulants or combinations thereof.
  • citrated whole blood samples are used as aqueous samples according to the invention ('coagulating' conditions).
  • aqueous samples according to the invention are whole blood samples provided with citrate and PPACK at concentrations known in the art ('anti- coagulating' conditions).
  • any method to be applied that requires a substantial volume of blood from the patient is heavily undesired.
  • any required sample handling and preparation which takes (too much) time is also limiting the applicability of any test aimed at facilitating patient care.
  • the method of the current invention addresses both limitations adequately.
  • the method of the invention is directly applicable with whole blood samples of the patient, obtained following routine practice in the operation theater, without the need of any sample preparation before the start of the method of the invention.
  • the method according to the invention only requires less than a milliliter of blood, preferably 200 microliter to 1 milliliter, most preferably about 500 microliter.
  • the aqueous sample is a sample of 200 microliter to 1 ml, preferably about 500 microliter.
  • the aqueous sample is 200 microliter to 1 ml whole blood, preferably about 500 microliter.
  • the aqueous sample is 200 microliter to 1 ml citrated whole blood provided with calcium and magnesium ions, preferably about 500 microliter ('coagulating' conditions).
  • the aqueous sample is 200 microliter to 1 ml citrated whole blood provided with calcium and magnesium ions and with PPACK, preferably about 500 microliter ('anti-coagulating' conditions).
  • the temperature applied in the method of the invention contributes to the high degree of mimicking physiological conditions as seen with the method of the invention.
  • the temperature during complex formation and during data acquisition is 4°C to 42°C, preferably 10°C to 39°C, most preferably ambient temperature to about 37°C.
  • the temperature during complex formation is at ambient temperature to about 37°C.
  • the temperature during steps a)-d) or a)-e) of the method of the invention is 4°C - 42°C, preferably 10°C - 39°C, most preferably at ambient temperature to about 37°C.
  • the aqueous sample is 200 microliter to 1 ml whole blood, preferably about 500 microliter
  • the mobile binding partner is selected from a platelet, a blood coagulation factor or combinations thereof
  • the immobilized binding partner comprises at least one protein selected from laminin, proteoglycan, tissue factor, fibrin, fibrinogen, fibronectin, vitronectin, osteopontin, collagen, collagen type I, collagen-derived peptide, collagen peptide mimetic, rhodocytin, von Willebrand factor, tissue thromboplastin, activated protein C, factor XII, P-selectin, annexin A5, integrin a2, integrin ⁇ 3, integrin ⁇ 5 ⁇ 1 , CD36, integrin allbp3, glycoprotein VI, integrin ⁇ 2 ⁇ 1 , glycoprotein Iba, CLEC-2, coagulation factors or combinations thereof, or a(n artificial) material
  • the immobilized binding partner in the reaction zone 45 comprises collagen type I or collagen type I and tissue factor, wherein the mobile binding partner comprises a platelet, wherein the aqueous sample comprises anti-coagulated whole blood, wherein a further solution is a reaction mixture comprising an anti-coagulant, wherein the light emitting label moiety is 3,3'-dihexyloxacarbocyanine iodide, wherein the flow is 0.6 to 30 ml/hour, preferably 0,675 to 10 ml/hour, more preferably about 7.2 ml/hour, and wherein steps a) and onwards take 4 to 6 minutes.
  • the temperature during complex formation and during time-lapse recording of signal data is at about ambient temperature to about 37°C.
  • the aqueous sample is citrated whole blood comprising PPACK, calcium ions and magnesium ions (non-coagulating conditions). Then, preferably, steps a) and onwards, up to d) or e) last about 4 minutes, according to the method of the invention. Equally preferred is when the aqueous sample is citrated whole blood comprising calcium ions and magnesium ions (coagulating conditions). Then, preferably, steps a) and onwards, up to step d) or step e) last about 6 minutes, according to the method of the invention.
  • the method of the invention has been proven to be adequately sensitive for detecting variations in platelet count in a whole blood sample. Also coagulation defects that influence coagulation status of a subject suffering from such defect, is adequately measured with the method according to the invention. Moreover, when whole blood samples from subjects who receive anti-platelet therapy or subjects who receive antithrombotic therapy, are subjected to the method of the invention, sensitive measurements reveal meaningful and informative test results.
  • the method according to the invention provides the relevant information on the activation of platelets, formation of a thrombus, platelet-based coagulation with platelets of a patient. More preferably, the method according to the invention provides test results relating to the platelet aggregation status, platelet adhesion status and/or platelet- fibrin interaction status in a whole blood sample of a patient.
  • the method according to the invention is typically suitable for providing parameter values regarding platelet aggregation, thrombus size, granule secretion, fibrinogen binding, clotting time and maximum lysis.
  • These parameters provide guidance to for example the ones responsible for selecting at least one adequate therapy for a patient suffering from (severe, acute) bleeding or suffering from (acute) thrombosis, for example in the perioperative or postoperative context, for example during or after surgery in the operation theater.
  • a second aspect of the invention is an automated analytical system, comprising:
  • an imaging assembly 50 comprising:
  • a socket 19 comprising an xyz stage controller 10 and a thermostat 9, for receiving a flow-cell holder 20;
  • aqueous solution A-C, D-F comprising an aqueous solution A-C, D-F, wherein the aqueous solution is selected from Ca 2+ /Mg 2+ solution, physiological salt solution, buffer solution, wash buffer, rinse buffer, solution comprising a fluorescent label,
  • a flow cell 4, 4', 4" comprising at least one flow channel 43, 43' in connection with an inlet 42, 42' and an outlet 44, 44', wherein the flow channel comprises at least one reaction zone 45 with the reaction zone having a surface area of 1 .25 mm 2 to 15 mm 2 , preferably with a longitudinal dimension (length) of 0.8 mm to 3 mm and a transversal dimension (width) of 2 mm to 3 mm.
  • the flow cell of the automated analytical system of the invention preferably has outer dimensions of about 24 mm x 60 mm x 5 mm (longitudinal width x transversal length x height), but other dimensions are equally preferred.
  • Conventional glass cover slips are suitable for use as a bottom part 41 of a flow cell of the invention, comprising the at least one reaction zone. These conventional cover slips are 24 mm x 60 mm (longitudinal width x transversal length). Further embodiments of flow cells according to the invention are provided in Example 1 and Figure 1.
  • a preferred embodiment of a fluidic system of the automated analytical system of the invention is provided in Example 3 and Figure 3.
  • the fluidic system of the invention preferably comprises said at least one reservoir 11 , 17 for containing a further aqueous solution, for example for the purpose of providing a wash buffer or a solution comprising a fluorescent label to formed complex.
  • Said at least one reservoir also comprises a container comprising a solution comprising calcium ions and magnesium ions
  • flushing the flow channel 43 with wash buffer once complex has been formed is beneficial before a (time-lapse) recording of signal is applied, for example before an end-point recording is applied.
  • the fluidic system of the invention comprises tubings, connectors, interior of pumps, etc., which are made of non-adhesive material.
  • connectors are preferably made of Teflon.
  • Teflon surfaces When the aqueous sample is citrated whole blood and the mobile binding partner are platelets, use of Teflon surfaces in the fluidic system of the invention has the advantage that platelets contacting these surfaces are minimally pre-activated before being contacted with immobilized binding partner in the flow channel.
  • the inner lining of the flow channel is made of non- adhesive material.
  • the inner lining of the flow channel is also made of non- activating material, when platelets and coagulation factors are concerned.
  • Light source 30 of the imaging assembly of the automated analytical system of the invention is preferably a diffuse white LED, suitable for brightfield transmission recordings.
  • the LED is preferably a non-modulated LED or a pulsed LED.
  • Light source 33 of the imaging assembly of the automated analytical system of the invention is preferably a diffuse RGB LED. Equally preferable is a plurality of fluorescence lamps, for example four fluorescence lamps. Preferable, the light source 33 is applicable for fluorescent excitation of dyes in the range of 390 nm, 465 nm, 488 nm, 549 nm and 647 nm.
  • a filter wheel 35 is part of the imaging assembly, said filter wheel preferably comprising for example filters: 1 ) dapi, long pass, > 435 nm; 2) DIOC (488 nm), 525 nm/50 nm; 3) Cy3 605 nm/70 nm; 4) Cy5 700 nm/75 nm.
  • the automated analytical system of the invention comprises an imaging assembly according to the invention with a light source 30. Then, light source 33, mirror 34, filters 35 and lens 36 are not comprised by the imaging assembly.
  • Such an automated analytical system is suitable for time lapse recording of transmitted light and provides test results suitable for application in the perioperative setting.
  • a preferred automated analytical system of the invention comprises a fluidic system which comprises filters 35 for filtering light of 2-6 wavelengths, preferably 5 wavelengths, more preferably 4 wavelengths emitted by the at least one light emitting label.
  • these filters 35 which are commonly known in the art, are typically suitable for selecting wavelengths applicable for measurements involving recording at least one emitted light according to the invention.
  • the xyz stage controller of the imaging assembly according to the invention is a conventional xyz stage controller known in the art.
  • the xyz stage controller is preferably fixed to the socket which receives the flow cell holder comprising the flow cell.
  • the xyz controller controls the focusing of the reaction zone (z-direction), and controls the positioning of a selected reaction zone in the area where light of light source 30 or 33 reaches the reaction zone and transmitted light or fluorescent signal from the complex in the reaction zone can reach lens 31 (xy positioning).
  • the xyz stage controller may also be fixed to for example lens 31 in an alternative embodiment of the invention.
  • the flow cell holder is preferably a holder separate from the socket. Once a flow cell is received by the flow cell holder, it is easily mounted in the socket.
  • the holder and socket have a close fit, allowing efficient thermal conduction between the thermostat mounted on the socket and the flow cell in the flow cell holder.
  • a preferred embodiment of the invention is a socket with a fixed flow cell holder.
  • a preferred embodiment of a socket and a separate flow cell holder is provided in Figure 2 and Example 2.
  • the imaging assembly of the invention comprises a microscope objective lens.
  • the lens preferably magnifies 40x or 60x although other magnifications are also applicable in the automated analytical system of the invention.
  • the imaging sensor is a CCD sensor known in the art or a CMOS sensor known in the art.
  • Preferred embodiments of microscope objective lenses and imaging sensors are provided in Figure 2 and Example 2. Now that no conventional microscope is required for time lapse recording of images of light transmitted through complex and/or of fluorescence emitted by label in the formed complex, according to the invention, a small and compact automated analytical system of the invention is provided. Furthermore, obeying a microscope has the further advantage that the risk for recording blurry images reduced with the use of the imaging assembly of the invention.
  • the automated analytical system comprises means 9, i.e. a thermostat to allow complex formation according to the invention at a temperature selected from 4°C - 42°C, preferably between 10°C - 39°C, most preferably at ambient temperature to about 37°C.
  • a temperature selected from 4°C - 42°C, preferably between 10°C - 39°C, most preferably at ambient temperature to about 37°C.
  • Preferred is the ability to maintain the temperature at ambient temperature or at 37°C during complex formation.
  • the automated analytical system of the invention is preferably small-size, e.g. with dimensions not exceeding about 40 cm x about 40 cm x about 40 cm (depth x width x height), preferably not exceeding about 30 cm x about 30 cm x about 30 cm (depth x width x height), more preferably the automated analytical system of the invention is an integrated and miniaturized system with dimensions of about 25 cm x about 25 cm x about 25 cm (depth x width x height). That is to say, the automated analytical system of the invention is preferably 'miniaturized'. Such a miniaturized system according to the invention bears the benefit of occupying a limited surface area of valuable space in e.g. the operation theater.
  • the automated analytical system comprises a housing comprising the imaging assembly, the fluidic system and the flow cell according to the invention. In an even more preferred
  • said housing has a dimension of about 30 cm x about 30 cm x about 30 cm (depth x width x height).
  • the housing further comprises computer assisted means configured for manually starting the imaging assembly and the fluidic system, wherein the imaging assembly further comprises computer assisted automated means configured for time-lapse recording transmitted light and/or emitted light, and computer assisted automated means configured for real-time storing and processing time-lapse recorded data, and displaying said processed data, and wherein the fluidic system further comprises computer assisted automated means configured for driving the high-precision pump and the flow-through pump.
  • said computer assisted means of the invention are not comprised in the housing comprising the automated analytical system, though are positioned proximate to said automated analytical system.
  • the automated analytical system further comprises computer assisted means configured for manually starting the imaging assembly and the fluidic system
  • the imaging assembly further comprises computer assisted automated means configured for time-lapse recording transmitted light and/or emitted light
  • computer assisted automated means configured for real-time storing and processing time-lapse recorded data, and displaying said processed data
  • the fluidic system further comprises computer assisted automated means configured for driving the high-precision pump and the flow-through pump.
  • the computer assisted means according to the invention comprise at least two cores with two threads per core, or comprise at least four cores, for fast and
  • FIGs 1 -3 provide an overview of typical examples of embodiments of the invention, i.e. the parts of an automated analytical system of the invention applicable for use in the method of the invention, i.e. flow cells according to the invention, and an imaging assembly according to the invention, and fluidic systems according to the invention.
  • These figures also provide exemplified flow cells 4 according to the invention, such as the flow cells 4 comprised by the automated analytical system according to the invention.
  • Figure 2 provides a detailed scheme of the imaging assembly part of the automated analytical system of the invention.
  • a further aspect of the invention is flow cell 4, 4' for use in the automated analytical system according to the invention, comprising at least one flow channel 43, preferably two flow channels 43, 43', said flow channel comprising at least one reaction zone 45 located at a portion of the flow cell that is transparent for light, and said reaction zone comprising an immobilized binding partner, and said flow channel having an inlet 42, 42' positioned at an angle of 5-90° relative to the longitudinal dimension (length) and/or the transversal dimension (width) of the at least one reaction zone in the flow channel, preferably at an angle of 10° to 20°, more preferably 1 1 ° to 12°, most preferably about 1 1 °, and an outlet 44 positioned at about the same angle as the angle of the inlet relative to the longitudinal dimension (length) and/or the transversal dimension (width) of the reaction zone, and having a cross-sectional area of 0.075 mm 2 to 0.30 mm 2 , preferably with a transversal width of about 2 mm or about 3 mm and
  • the angle between the inlet (and outlet) and the surface of reaction zone provides for an optimal flow of the aqueous sample.
  • a flow cell with an inlet and an outlet with an angle of about 10° to 20°, preferably about 1 1 ° according to the invention provides the advantage of a constant viscosity of the aqueous sample flowing through the flow channel, over the whole length of the flow channel.
  • a further advantage of these selected angles according to the invention is the minimized collision and interaction of mobile binding partner with the walls of the inlet and outlet of the flow channel and the wall of the flow channel, when entering and exiting the flow channel.
  • the mobile binding partner is a platelet
  • minimalized collision and interaction with the inlet and the flow channel surface prevents pre-activation of the platelet, before it is activated and binding to immobilized binding partner of the reaction zone in the flow channel.
  • the cross-sectional area of the flow channel of the invention is selected for the optimized flow conditions achieved with such area, according to the invention.
  • the selected cross-sectional area allows for a desired physiological shear rate in the flow channel with a minimized volume of aqueous sample required to achieve the desired shear rate.
  • minimizing aqueous sample volume is beneficial for, for example, a patient awaiting test results for personalizing the therapy to be selected, when the aqueous sample is whole blood.
  • the combination of flow and cross-sectional area is also selected such that the width of the channel is optimal for laminal flow with a homogeneous shear rate along the full width of the flow channel.
  • the reaction zone comprising the immobilized binding partner is rectangular with a longitudinal dimension (length) of about 0.8 mm to about 3 mm, according to the invention.
  • a longitudinal dimension (length) of about 0.8 mm to about 3 mm.
  • Applying rectangular reaction zones has several advantages according to the invention. First, rectangular reaction zones are easily provided in a highly reproducible manner, for example using a pipetting robot. Second, the longitudinal dimension or length of the reaction zones is easily fine-tunable with regard to the minimal length required in order to allow for complex formation with a mobile binding partner. For example, platelets in whole blood require a minimal reaction zone length comprising collagen of about 0.8 mm, when the flow of the aqueous sample, i.e. the blood, is such that physiological shear rate is achieved in the flow channel.
  • This minimal longitudinal length is for example for platelets determined by a minimal path length for contacting an activating immobilized binding partner while still (partly) in the aqueous solution, and a subsequent minimal path length for adhering to the immobilized binding partner (complex formation).
  • a reaction zone comprises about 1 ⁇ g to 20 ⁇ g immobilized binding partner, more preferably about 1 .5 ⁇ g to 15 ⁇ g, most preferably about 1.5, 3, 6, 9, 12, 15 ng.
  • the inventors surprisingly found a combination of features related to the process of complex formation under flow and physiological shear rates within a sufficiently short time-frame, i.e. within 30 minutes, preferably within 4 to 6 minutes, and related to acquiring high-quality time-lapse recorded signal data combined with time- efficient data processing, addressing the aforementioned limitations in the art.
  • the size of the surface area of the reaction zone 45 makes a contribution.
  • the inventors found that typically reaction zones 45 with a surface area of 1.25 mm 2 to 10 mm 2 , preferably about 1 .5 to 6 mm 2 are suitable in the method of the invention.
  • reaction zones 45 are rectangular in shape, with a longitudinal dimension (length) of about 2 mm and a transversal dimension (width) of about the width of the flow channel 43, preferably about 2 mm or about 3 mm.
  • Such reaction zones 45 of the invention expose immobilized binding partner in a manner typically suitable for efficient and timely complex formation with mobile binding partner in the method according to the invention.
  • These sizes and shapes of the reaction zone 45 in the flow cell 4 of the invention are particular suitable for the method according to the invention in connection with a flow channel 43 having a cross-sectional area of 0.075 mm 2 to 0.30 mm 2 , preferably with a width of about 2 mm or about 3 mm and a depth of about 50 micrometer, according to the invention.
  • Particularly these combinations of cross-sectional area and surface area and shape of the reaction zone 45 enable the beneficial features of the method of the invention with regard to the short time line to relevant data acquisition due to sufficiently fast complex formation under flow and physiological shear rate.
  • the invention provides for a balanced combination of an imaging assembly, a flow cell and a fluidic system, enabling the simultaneous complex formation under flow at physiological shear rate and recording, storing and processing of images.
  • the microscope objective lens for example magnifying 60x and for example a CMOS sensor, for example 2 megapixel images are recorded, with an image showing a surface area of the reaction zone of 160 x 200 ⁇ 2 .
  • the resolution is 144 pixels per squared micrometer. This resolution suffices for retrieving the relevant parameters upon processing the recorded images for determining the haemostatic status of the patient.
  • This exemplified image size for example allows for sufficiently fast image data storage and processing, given a processor speed of a selected computer assisted means for real-time storing and processing time-lapse recorded image data.
  • a processor speed of a selected computer assisted means for real-time storing and processing time-lapse recorded image data.
  • applying a processor which has a higher speed allows for higher resolution images, for example 8 megapixel images.
  • the resolution of 144 pixels per squared micrometer provides sufficiently high quality input images for processing and
  • One of the important aims of the present invention was the provision of a method for optically measuring complex formation under flow at physiological shear rate between a mobile binding partner and an immobilized binding partner, which would require a minimal level of steps before test results are obtainable, and which would require as much low-tech sample handling as possible.
  • One way to facilitate this aim is providing a flow cell 4" according to the invention for use in the method according to the invention, in which the flow cell 4" is provided with a sample holder 2'.
  • a preferred examples of such a flow cell 4" comprising a sample holder is provided in Figure 1 C.
  • the flow cell 4" comprises a sample holder 2' in connection with the flow channel 43, 43', with the inlet 42, 42' for the aqueous sample in connection with a connector 3a for connecting the flow cell to the flow-through micro-pump 5, 15 of the fluidic system according to the invention, and with the outlet 44, 44' connected to the high-precision pump 1 '.
  • the flow cell 4 of the invention comprising at least one reaction zone 45 with immobilized binding partner, is readily conserved for at least six months at frozen conditions, e.g. at -20°c to -30°C or at about -70° to -85°C, preferably at about -20°C or at about -80°C according to the invention.
  • the at least one reaction zone 45 with immobilized binding partner is also readily conserved for at least 6 months when the immobilized binding partner is lyophilized once immobilized at the reaction zone 45.
  • the flow cell comprising a reaction zone with immobilized binding partner is stored frozen.
  • storage conditions are under liquid nitrogen or liquid helium.
  • Storing flow cells frozen according to the invention provides the opportunity to fabric large quantities of flow cells with reaction zones comprising immobilized binding partner in a single batch, providing flow cells with constant specifications. Furthermore, the risk for expiry of the reaction zone of the flow cells before application of the flow cells in stock is reduced. See for a typical embodiment of the invention Figure 4 and Example 7.
  • Typical materials and surfaces suitable for application as a reaction zone 45 according to the invention are conventional glass and polydimethylsiloxane (PDMS).
  • PDMS polydimethylsiloxane
  • other materials routinely applied in the field of complex formation between a mobile binding partner in an aqueous sample and an immobilized binding partner are equally applicable for use as a reaction zone 45 in the flow cell 4 of the invention and in the method according to the invention.
  • Further examples of materials applicable for use as a reaction zone 45 are polymers routinely used in ELISA based assays, mica, saffire, and the like.
  • the top part 40 of a flow cell 4 according to the invention is made of glass, preferably PDMS, or other (transparent) materials such as polycarbonate, polyethylene (PE), polystyrene (PS).
  • the method of the invention has been proven to be exceptionally suitable for use in the assessment of haemostatic conditions and coagulation status of a subject, preferably a human subject.
  • the use of the method according to the invention provides the relevant information on the activation of platelets, formation of a thrombus, platelet-based coagulation with platelets of a patient. More preferably, the use of the method according to the invention provides test results relating to the platelet aggregation status, platelet adhesion status and/or platelet-fibrin interaction status in a whole blood sample of a patient.
  • Use of the method according to the invention is typically suitable for providing parameter values regarding platelet aggregation, thrombus size, granule secretion, fibrinogen binding, clotting time and maximum lysis. These parameters provide guidance to for example the ones responsible for selecting at least one adequate therapy for a patient suffering from (severe, acute) bleeding or from (acute) thrombosis, for example in the perioperative or postoperative context, for example during or after surgery in the operation theater.
  • thrombus surface area thrombus build up
  • number of thrombi per surface area of immobilized binding partner multilayer, P-selectin expression, phosphatidylserine exposure, fibrinogen binding, platelet adhesion under non-coagulating conditions
  • platelet deposition thrombus surface area
  • thrombus build-up time-to- fibrin formation, fibrin formation under coagulating conditions
  • fibrinolysis fibrinolysis under coagulating conditions
  • coagulation or combinations thereof, preferably selected from platelet activation, platelet adhesion, thrombus formation, platelet-dependent fibrin formation under coagulating conditions, platelet-based coagulation, wherein the aqueous sample is whole blood.
  • the method of the invention is for bed-side monitoring of haemostasis conditions and coagulation status, aiding in improved care. Also the influence of antithrombotic agents on haemostasis can be assessed under flow conditions at physiological shear rate, with the method, the analytical system and the flow cell 4 of the invention.
  • the invention provides for use of the analytical system and a flow cell 4 according to the invention in drug-screening programs aimed at determining the influence of tested drugs or druggable compounds on the one or more parameters attributing to haemostasis and coagulation status in a subject, preferably a human subject.
  • the analytical system and a flow cell 4 according to the invention are particularly suitable for use in screening aqueous samples, such as blood samples, for defects related to impairment of haemostasis in the subjects.
  • these further applicable uses of the method according to the invention and of the analytical system and a flow cell 4 according to the invention are with whole blood, although other aqueous samples comprising a suitable binding partner are also applicable.
  • Other aqueous samples comprising a mobile binding partner applicable for use in the method according to the invention are platelet-rich plasma, platelet free plasma, serum, isolated blood components, such as leukocytes, neutrophils, progenitor cells.
  • use of the analytical system according to the invention and a flow cell 4 according to the invention is preferred for determining the influence of a protein mutation or a protein modification and/or for determining the influence of a cell defect in the mobile binding partner or in the immobilized binding partner and/or for determining the influence of the concentration of the mobile binding partner or the immobilized binding partner, on complex formation under flow at physiological shear rate between the mobile binding partner in an aqueous sample and an immobilized binding partner.
  • the aqueous sample is whole blood, and the whole blood is selected from a subject who is healthy subject, or who is a patient:
  • cardiovascular disease a vascular disease, an inflammatory disease, a known platelet defect, an unknown platelet defect, a haemophilia, a cancer, diabetes, obesity, a congenital disease such as Noonan;
  • a coagulation factor such as Factor VIII and von Willebrand factor
  • a whole blood sample is measured, wherein the whole blood sample is derived from a patient suffering from cancer, thrombocytopenia before and after transfusion, a patient who receives tyrosine kinase inhibitor therapy, a SCID patient, a haemophilia B patient, or from a patient that underwent a bone marrow transplantation.
  • the whole blood sample is derived from a patient suffering from cancer, thrombocytopenia before and after transfusion, a patient who receives tyrosine kinase inhibitor therapy, a SCID patient, a haemophilia B patient, or from a patient that underwent a bone marrow transplantation.
  • Preferred embodiments of the invention in this regard are provided in Figures 5-7 and Examples 8, 12, 15 and 16.
  • the method of the invention is particularly suitable to screen for patients and select patients suffering from any of the diseases or health issues related to von Willebrand factor disease, Bernard Soulier syndrome, clopidogrel therapy, platelet function disorders, fibrinogen deficiency, allb33 inhibitors, coagulation factor deficiency, hyperfibrinolysis, heparin / hirudin effects, or combinations thereof.
  • the analytical system according to the invention and a flow cell 4 according to the invention is used for determining the influence of a compound on protein- protein-, protein-cell-, cell-cell interactions.
  • the analytical system according to the invention and a flow cell 4 according to the invention is used for determining the influence of a gene mutation or a gene defect, protein mutation or protein modification, or cell defect on protein-protein-, protein-cell-, cell-cell interactions.
  • the method is also equally suitable for screening purposes, e.g. high- throughput screening purposes.
  • series of druggable compounds can be screened routinely, or populations can be screened for hereditary or acquired defects at the DNA level and/or at the protein level of subjects, influencing haemostasis in the subjects, and test results are for example compared to reference values in a database.
  • the invention also provides for a kit comprising the essential elements for routine use of the method according to the invention.
  • kit of parts comprising:
  • sample holder 2 wherein the sample holder is a syringe for blood draw, said syringe pre-filled with citrate and optionally pre-filled with at least one light emitting label according to the invention and with PPACK,
  • the kit of parts according to the invention comprises a disposable flow cell 4 and disposable holders.
  • the kit of parts according to the invention comprises the disposables required for applying the method of the invention with the apparatus of the invention.
  • the method according to the invention is equally applicable for the assessment of complex formation, which complex formation relates to animal health and disease.
  • the method of the invention is also applicable as an alternative or as an additive to pre-clinical and clinical animal studies.
  • animal studies are for example model studies that mimic a certain complex formation in the human body.
  • the method of the invention replaces animal studies that are model studies for assessing human coagulation status and haemostatic balance.
  • the immobilized binding partner comprises a cell, preferably a cell selected from the aforementioned list of cells selectable as immobilized binding partner.
  • the method of the invention typically replaces current animal models that requiring testing in for example dogs, rabbits, mice, pigs, monkeys, Guinea pigs, etc.
  • the use of the method of the invention for replacing animal studies and animal testing is part of the invention.
  • the method of the invention is also preferably used as a standardized research tool with respect to gathering human and animal coagulation data and haemostasis data in the academic setting.
  • test results applicable in the clinic were obtained related to: Thrombus formation with whole blood from healthy donors or patients;
  • Thrombus formation with said whole blood under non-coagulated conditions Thrombus formation with said whole blood under non-coagulated conditions; and Thrombus formation with said whole blood under coagulating conditions.
  • FIG. 1A The illustrative example of a flow cell 4 according to the invention comprising two flow channels 43, 43' is provided in Figure 1A and is constructed from two main parts, the top 40 and bottom part 41.
  • the top part 40 consists of a single component, though may also be a multi-component part.
  • the inlet 42, 42' is positioned at an angle between 5° to 90°, preferably about 1 1 ° related to the longitudinal dimension (length) and/or the transversal dimension (width) or the surface of the reaction zone 45, which allows aqueous sample, e.g. blood to flow through the flow channels 43, 43' under stress-free conditions to prevent e.g.
  • the outlet 44, 44' is positioned at an angle between 5° to 90°, preferably about 1 1 ° related to the longitudinal dimension (length) and/or the transversal dimension (width) or the surface of the reaction zone 45. Most preferable, the two angles are the same or similar.
  • the bottom part 41 of the flow cell 4 is made of glass or of any optically transparent plastic or other material known in the art and comprises the at least one reaction zone 45 comprising the immobilized binding partner according to the invention. Most preferably, the bottom part 41 and the top part 40 of the flow cell 4 is made of a material that is at least transparent at the location of the reaction zone(s).
  • this material does not or only to a very tiny extent have an influence on complex formation.
  • top part 40 and the bottom part 41 are depicted with a spacing in between, indicated with the dashed lines.
  • the top part 40 and the bottom part 41 are in intimate contact.
  • the parts are glued together, or fixed to each other by any other means known in the art.
  • This flow cell 4 is for example suitable as a part in the analytical system according to the invention, as depicted in Figure 3B.
  • a flow cell 4' is depicted, which flow cell comprises a single flow channel 43.
  • This flow cell 4' is suitable as a part in the analytical system according to the invention, as depicted in Figure 3A.
  • a flow cell 4" is depicted, having a sample holder 2' in connection with the inlet 42 of the at least one flow channel 43 of the flow cell. According to the invention, flow cell 4" is applied in the automated analytical system according to the invention for which a preferred embodiment is provided as Figure 3C.
  • the inlet and the outlet of the flow channel of the flow cell according to the invention are also applicable for use as the outlet and the inlet of the flow channel, respectively, according to the invention.
  • the order in which mobile binding partner contacts subsequent immobilized binding partners may have an influence on test result outcomes of measurement.
  • the top light source 30 is a wide spectrum visible light source that emits light in the visible range (300 nm to 750 nm).
  • the top light source 30 is a diffuse white LED, suitable for brightfield transmission recordings, such as a non-modulated LED or a pulsed modulated LED.
  • Light passes through the flow-cell 4' of the analytical system according to the invention, in which the complex formation occurs, e.g. in which thrombus formation is assessed, and the light is passed on to the (commercially available) microscope objective lens 31.
  • This lens is preferably an oil lens, such as the Olympos 60x magnifying oil lens. Magnification of 40x also suffices, and also lenses with other magnifications are suitable.
  • Other suitable lenses are for example water lenses or air lenses known in the art.
  • the magnified light is reflected on a mirror 32, which is preferably a high-reflectance mirror.
  • this mirror minimally reflects 99%, and preferably this mirror is a silver mirror, or the like.
  • the bottom light source 33 is mainly used for emission of narrow spectrum light for fluorescent excitation of dyes in the range of 390, 465, 488, 549 and 647 nm (all +/- 20 nm).
  • a broad spectrum lamp 33 is applied, preferably a diffuse RGB LED known in the art, or up to four fluorescence lamps 33 each emitting a selected wavelength are applied.
  • Lamp 33 is preferably either a non-modulated LED, or a pulsed modulated LED.
  • This emitted light is reflected onto the flow-cell by a semi-transparent mirror 34 and the high reflectance mirror 32.
  • a different mirror is mounted with each filter mirror optimally matching the excitation and emission wavelengths of the fluorescent labels used according to the invention.
  • a multi dichroic mirror is applied, capable of filtering for example up to four wavelengths.
  • set-ups with mirrors suitable for more than four excitation and emission wavelengths are equally suitable, for the method according to the invention.
  • a wide spectrum visible light is used at position 33, to achieve reflection microscopy.
  • a narrow band filter 35 allows for selectively focusing on the fluorescent label used.
  • the filter 35 is exchangeable and is be matched with the wavelength selected at 33, using a filter-wheel (automated).
  • the filter 35 is an emission filter wheel and preferably comprises 1 to 4 wavelength filters, although it may also comprise more than 4 wavelength filters.
  • the emitted and transmitted light is projected on the imaging sensor 37 using an imaging lens 36.
  • Signal data is time lapse recorded and stored on a data carrier coupled to a computer device, e.g. a(n external) hard disk (not shown).
  • the imaging sensor 37 is preferably a CCD sensor known in the art, or a CMOS sensor known in the art, such as a Sony IMX-174 CMOS sensor or a Sony IMX-249 CMOS sensor.
  • the senor is equipped with a global shutter, although a sensor with a non-global shutter is also suitable for use in the method of the invention, if lamp 30 and lamp 33 are non-modulated LEDs.
  • the shutter of the sensor according to the invention preferably has a shutter time of about 10 milliseconds (ms) to about 200 ms.
  • FIG 2A trans-illumination of the flow cell 4' is shown.
  • the top light source 30 is emitting light and illuminates the reaction zone 45 in the flow cell 4' with diffuse light. Transmitted light is captured by the objective lens 31 positioned below the flow cell and is forwarded onto the mirror 32.
  • the suitable semi-transparent mirror 34 By selecting the suitable semi-transparent mirror 34 a substantial amount of light is passed through and imaged onto the imaging sensor 37. In this configuration there is no filter 35 in use.
  • fluorescence via bottom illumination of the flow cell 4' is outlined, as used for recording fluorescence signal data (images).
  • the top light source 30 is not emitting light.
  • the bottom light 33 is on, the emitted light is reflected by the semi- transparent mirror 34 and passed onto the mirror 32 and then projected in the objective lens 31 and onto the reaction zone in the flow channel.
  • the emitted light excites the fluorescent label in the reaction zone 45 in the flow-cell.
  • the fluorescent label emits light signal into any direction, and some of said emitted light is captured by the objective lens 31 , reflected by the mirror 32, passed through the semi-transparent mirror 34.
  • the filter 35 filters out remaining excitation light emitted by the bottom light source 33 and allows the light emitted by the fluorescent label to pass through, which is projected by the lens 36 onto the imaging sensor 37. Subsequently, recorded image data is stored and processed, and optionally compared with reference values for assessed parameters relating to coagulation status and haemostasis, and processed data is displayed in numbers or in graphs.
  • the imaging assembly of the analytical system according to the invention also comprises conventional automated means for data storage, data processing and automated display means for at least numerical values and graphical output data, known in the art.
  • these means for data storage, data processing and display means are not shown in the Figures 2A and 2B, but these means are an integral part of the analytical system according to the invention.
  • the socket 19 is provided with a thermostat 9, capable of efficiently keeping a flow cell received by a flow cell holder 20 which holder is in intimate contact with the socket, at a predetermined temperature. See Figure 2C.
  • Socket 19 is an integrated part of the imaging assembly according to the invention.
  • the socket is also provided with an xyz stage controller 10, as shown in Figure 2A-C. The controller allows for bringing a reaction zone to be imaged in focus, and for positioning a selected reaction zone in a selected flow channel to be imaged.
  • Figure 3A and 3B and 3C provide examples of preferred embodiments showing three fluidic systems of the automated analytical systems according to the invention, suitable for application in the method according to the invention.
  • the imaging assembly of the analytical system according to the invention as outlined in Figure 2A-C is omitted from the Figures 3A-C.
  • the automated means for data storage, data processing and automated display means are not shown in the Figures 2A and 2B and the Figures 3A-C, but it has to be understood that these means are an integral part of the analytical system according to the invention.
  • a fluidic system of the invention is exemplified with a system having a flow cell 4' with one flow channel 43 (Figure 3A), and with an analytical system having a flow cell 4 with two flow channels 43, 43' ( Figure 3B).
  • Figure 3C yet a different fluidic system according to the invention is shown ( Figure 3C).
  • analytical systems comprising a fluidic system having a flow cell 4 with a plurality of flow channels 43 of over 2, for example 3 to 6 flow channels are also part of the invention.
  • the containers A, B, and C of reservoir 11 and containers D, E, F of reservoir 17 contain the further aqueous solutions recalcification buffer comprising Ca 2+ and Mg 2+ (A, D), post-perfusing buffer (B, E), and rinse / cleaning buffer (C, F).
  • at least one container comprises a fluorescent label having affinity for a mobile binding partner.
  • the aqueous sample analyzed in the experiments was whole blood from human subjects.
  • blood was drawn with a conventional vacuum system known in the art, in 3.2% sodium citrate (0.129 M) +/- PPACK (40 ⁇ ), and kept at 37°C for 5 minutes before start of a measurement according to the method of the invention.
  • Ca 2 7 Mg + mix was mixed with the drawn whole blood sample, to obtain as an aqueous sample whole blood which is able to coagulate under fibrin formation.
  • Immobilized binding partner was immobilized in a reaction zone 45 by applying either of two techniques, as depicted below.
  • Immobilized binding partner having a circular surface area
  • a circular surface area of an immobilized binding partner was achieved by manual pipetting or by using a robot, on coverslips which are degreased with 2 M HCI in 50% ethanol. Volumes of 0.5 ⁇ were for example pipetted on a coverslip. When a robot is used a MicroGrid II robot is used using a printing pin of >700 ⁇ . Immobilization methods ('coating') result in a circular surface area of 1 .5 mm 2 . For both methods coated coverslips are kept in a humid chamber with >50% humidity for 60 minutes. Blocking of coverslips is performed with 1 % BSA (in HEPES, pH 7.45). After blocking, coverslips are rinsed with saline and mounted on the flow cell. When applying a volume of 2 ⁇ of binding partner onto the coverslip, a circular surface area with a diameter of 2 mm is obtained.
  • Typical immobilized binding partners according to the invention are listed in Table 1.
  • a reaction zone comprises about 1 ⁇ g to 20 ⁇ g immobilized binding partner, more preferably about 1 .5 ⁇ g to 15 ⁇ g, most preferably about 1.5, 3, 6, 9, 12, 15 ⁇ g.
  • Immobilized binding partner having a rectangular surface area
  • Rectangular surface areas of immobilized binding partner were obtained by so-called 'stream coating'.
  • Coverslips were selected as the bottom part 41 of the flow cell 4 according to the invention, and coverslips are degreased with 2 M HCI in 50% ethanol.
  • Ten ⁇ of solution comprising the binding partner to be immobilized in a reaction zone 43 was perfused (10 ⁇ volume) through a coating channel perpendicular to the longitudinal direction of the flow channel 43. This assembly was kept in a humid chamber with >50% humidity for 60 minutes. After 60 minutes the flow channel 43 comprising the immobilized binding partner was perfused with saline. Blocking of coverslips is performed with 1 % BSA (in HEPES, pH 7.45).
  • Table 1 an overview of experiments is provided, which provides examples of combinations of immobilized binding partners and aqueous samples comprising the mobile binding partner and applied shear rates, that allowed for the provision of at least one test result suitable for use in the clinic, when applied in the method according to the invention.
  • the combinations of immobilized binding partner, surface areas of the immobilized binding partner and applied shear rates imaging data is recorded and processed in a time short enough to be of clinical relevance, i.e. within about 6 minutes from the start of the measurements using the method of the invention with the analytical system according to the invention.
  • n.c non-coagulating conditions
  • c coagulating conditions
  • mQ ultrapure water
  • flow cells 4 constructed from either hard plastic material, or from soft PDMS material.
  • flow channel 43 dimensions were either 2 mm or 3 mm (width) and 50 ⁇ (height).
  • the flow cell 4 inlet 42 and outlet 44 have an angle of 1 1 ° relative to the longitudinal dimension (length) and/or the transversal dimension (width) or the surface area of the reaction zone 45, unless indicated otherwise.
  • test results are automatically provided within 6 minutes, the following examples are provided.
  • test results are provided with collagen as the immobilized binding partner and either whole blood under non-coagulating conditions, or whole blood under coagulating conditions, as the aqueous sample.
  • the test results show that when the method of the invention is run using an automated analytical system according to Figure 3B, measuring blood under non-coagulating conditions and whole blood under coagulating conditions in parallel in the two flow channels 43, all test results are available within 6 minutes.
  • Table 3 provides an overview of test results that were obtained with these aqueous samples.
  • test results for the whole blood sample applied under coagulating conditions were obtained within 6 minutes.
  • Test results for the whole blood sample applied under non-coagulating conditions were obtained after 4 minutes of blood perfusion through the flow channel, followed 2 minutes of rinsing buffer / label / cleaning buffer perfusion, thus also within 6 minutes.
  • Test results were automatically made available as numerical values, as a mean value +/- 2 SD, and as color images. Furthermore, test results were automatically compared with reference test results acquired from whole blood samples of healthy control subjects. All blood samples were from human subjects.
  • Non-coagulating conditions assessing measures for the rate of platelet adhesion, aggregation and activation
  • Platelet adhesion in time area under the curve (AUC) + slope) to a combination of platelet receptors / via different pathways.
  • Integrated feature size See below
  • fibrinogen binding measure for capability of platelets to aggregate
  • PS Phosphatidylserine
  • the integrated feature size was determined as a parameter, taking into account a proportional contribution of large and small thrombi on microspots. It was defined as described in De Witt et al., 2014, page 12, left column, "Quantitative analysis of recorded images", which is incorporated by reference.
  • Ivophilization Coverslips comprising immobilized binding partner in reaction zones 45 are kept at -20°C for 9 days.
  • Whole blood was the aqueous sample, platelets were the mobile binding partner.
  • the immobilized binding partner was as indicated below. Thrombus formation was measured prior to freezing and after freezing, with the method according to the invention.
  • Blood collection Blood was drawn in Vacutainer tubes containing 3.2% of sodium citrate. Prior to each experiment blood was recalcified with 40 ⁇ PPACK and Ca 2 7 Mg 2+ mix (3.75 mM/7.5 mM).
  • Immobilized binding partners were applied in flow cells 4 made of hard plastic material with flow channel 43 dimensions of 3 mm (width) and 50 ⁇ (height). Inlet 42 and outlet 44 of the flow channel 43 make an angle of 1 1 ° relative to the longitudinal dimension (length) and/or the transversal dimension (width) or the surface area of the reaction zones 45.
  • coagulation factors e.g. factor VIII, von Willebrand factor
  • the following experiment demonstrates the use of the method of the invention for assessing complex formation between platelets from whole blood and immobilized binding partners, immobilized in a series of reaction zones 45 in the flow channel 43.
  • Blood collection Blood was drawn from a healthy donor in Vacutainer tubes containing 3.2% of sodium citrate. Prior to each experiment blood was recalcified with 40 ⁇ PPACK and Ca 2 7 Mg 2+ mix (3.75 mM/7.5 mM).
  • Coatings were applied in flow cells 4 constructed out of hard plastic material with flow channel 43 dimensions of 3 mm width and 50 ⁇ height.
  • Inlet 42 and outlet 44 have an angle of 1 1 °, relative to the longitudinal dimension (length) and/or the transversal dimension (width) or the surface area of the reaction zones 45.
  • Perfusion experiment Performed at room temperature. Recalcified whole blood samples were put in a syringe (container 2) rinsed with rinse buffer and connected to the pump 1 and the flow cell 4. Blood was perfused for 4 minutes at 1 .600/s. Imaging was performed after 1 minutes rinsing with rinse buffer in order to discard red blood cells. End point measurements (brightfield) were performed for assessing platelet area coverage (Table 4). Table 4 indicates that different adhesive surfaces (immobilized binding partners) give different amounts of surface area coverage (SAC) of platelet (mobile binding partner) and thrombi.
  • SAC surface area coverage
  • Blood collection (non-coagulating conditions). Blood was drawn from a healthy donor in Vacutainer tubes containing 3.2% of sodium citrate.
  • a stream coating was made with collagen type I, collagen type III or vWF-fibrinogen (immobilized binding partners) on a degreased coverslip (Table 5) using a volume of 2 ⁇ _. Spots were coated in the same order as described in Example 9 with size of 1 mm (length) x 2 mm (width).
  • flow cell Type of flow cell. Coatings were applied in flow cells 4 constructed out of PDMS material with flow channel 43 dimensions of 2 mm width and 50 ⁇ height. Inlet 42 and outlet 44 have an angle of 90°, relative to the longitudinal dimension (length) and/or the transversal dimension (width) or the surface area of the reaction zones.
  • Perfusion experiment Performed at room temperature, with the method of the invention, applying an automated analytical system of the invention.
  • Anticoagulated whole blood samples (aqueous samples) were put in a syringe (container 2) rinsed with rinse buffer and connected to the pump 1 and the flow cell 4. Blood was perfused for 4 minutes at a shear rate of 1 .600 s ' Imaging recording was performed after 1 minutes rinsing with rinse buffer in order to discard the red blood cells.
  • End point measurements (brightfield) were performed for assessing platelet area coverage (Table 5) and Annexin-A5 binding was determined as a measure of the procoagulant activity.
  • Table 5 indicates that different adhesive surfaces (immobilized binding partners) result in different amounts of surface area coverage (SAC) of platelet and thrombi. These data show that stream coating is adequate to determine more than one thrombus formation marker on different platelet adhesive surfaces as the immobilized binding partners.
  • Type of flow cell Coatings (immobilized binding partners) were applied in flow cells 4 constructed of hard plastic material with flow channel 43 dimensions of 3 mm width and 50 ⁇ height. Inlet 42 and outlet 44 have an angle of 1 1 °, relative to the longitudinal dimension (length) and/or the transversal dimension (width) or the surface area of the reaction zone 45.
  • Perfusion experiment Performed at room temperature. Recalcified whole blood samples were put in a syringe (container 2) rinsed with rinse buffer and connected to the pump 1 and the flow cell 4. Blood was perfused for 4 minutes at a shear rate of 1.600 s ' Image recording was performed after 1 minutes rinsing with rinse buffer in order discard the red blood cells.
  • Thrombus formation at venous shear rate (effect of bone marrow transplantation)
  • Blood collection aqueous sample was drawn from a healthy donor and from a patient with severe immunodeficiency syndrome (SCID) after bone marrow
  • flow cell Type of flow cell. Coatings were applied in flow cells 4 constructed of hard plastic material with flow channel 43 dimensions of 3 mm width and 50 ⁇ height. Inlet 42 and outlet 44 have an angle of 1 1 °, relative to the longitudinal dimension (length) and/or the transversal dimension (width) or the surface area of the reaction zones 45.
  • Perfusion experiment Performed at room temperature. Recalcified whole blood samples (aqueous sample) were put in a syringe (container 2) rinsed with rinse buffer and connected to the pump 1 and the flow cell 4. Blood was perfused for 6 minutes at a shear rate of 150 s ' Image recording was performed after 1 minutes rinsing with rinse buffer containing fluorescent labels for Annexin-A5 binding (procoagulant activity), fibrinogen binding and P-selectin expression (platelet activation markers) in order to discard the red blood cells. End point measurements (brightfield + fluorescence images) were performed concerning platelet area coverage with respect to brightfield and the various fluorescent markers (Table 8).
  • Table 8 shows that at venous shear rate thrombus formation is decreased with respect to platelet adhesion, platelet activation and procoagulant activity, for the blood sample of the patient, indicating that the bone marrow transplantation did not work properly. This was confirmed by other clinical tests, showing the predictive power of the method of the invention. Table 8. Multiparameter thrombus formation in a patient with SCID (single representative experiment)
  • Thrombus formation under influence of a drug used in the clinic Thrombus formation under influence of a drug used in the clinic: tyrosine kinase inhibitor Blood collection. Blood was drawn from a healthy donor in Vacutainer tubes containing 3.2% of sodium citrate. Prior to each experiment blood was recalcified with 40 ⁇ PPACK and Ca 2 7 Mg 2+ mix (3.2 mM/6.3 mM). A tyrosine kinase inhibitor (TKI), used in the clinical setting as a drug (cancer patients) was added prior to whole blood perfusion.
  • TKI tyrosine kinase inhibitor
  • reaction zone 45 had a diameter of 2 mm. Spots (reaction zones 45) were blocked after 1 hour incubation in a humid chamber (> 50% humidity) to prevent further adhesion of the coated protein. After blocking the coverslip (bottom part 41 of flow cell 4) was mounted on the top part 40 of a flow cell 4.
  • Coatings (immobilized binding partner) were applied in flow cells 4 constructed of hard plastic material with flow channel 43 dimensions of 3 mm width and 50 ⁇ height.
  • Inlet 42 and outlet 44 have an angle of 1 1 °, relative to the longitudinal dimension (length) and/or the transversal dimension (width) or the surface area of the reaction zones 45.
  • Blood collection Blood (aqueous sample) was drawn in Vacutainer tubes containing 3.2% of sodium citrate from a healthy donor. During each experiment blood was recalcified Ca 2 7 Mg 2+ mix (6.3 mM/3.2 mM) at 1 :10 volume ratio.
  • Perfusion experiment Performed at room temperature. Whole blood samples were put in a syringe (container 2) rinsed with rinse buffer (without heparin) and connected to the pump 1 and the flow channel 43 of a flow cell 4. During each experiment blood was recalcified with Ca 2 7 Mg 2+ mix (6.3 mM/3.2 mM) at 1 :10 volume ratio. Blood was perfused at a shear rate of 1.000 s ' Image recording was performed in real-time (time frames of 15 seconds). Time-lapse measurements (DiOC6 fluorescence signal recording), annexin-A5 binding (coagulation activity) and fibrinogen binding (platelet activation and fibrin formation) were performed during 1 1 minutes of whole blood perfusion (Table 9).
  • TTF time-to-fibrin formation
  • TF tissue factor
  • PS phosphatidylserine
  • Figure 6 shows the time curve of the three parameters (SAC, PS exposure and fibrinogen binding).
  • SAC serum-binding protein
  • PS fibrinogen binding
  • Blood collection Blood was drawn in Vacutainer tubes containing 3.2% of sodium citrate from a healthy donor or from a patient with hemophilia B (Factor IX defect). During each experiment blood was recalcified with Ca 2 7 Mg 2+ mix (6.3 mM/3.2 mM) at 1 :10 volume ratio.
  • a spot of collagen type I (immobilized binding partner) was applied on a degreased glass coverslip (bottom part 41 of a flow cell 4) (2 ⁇ _, giving a diameter of spots of 2 mm) (Table 3). After one hour of incubation, spots were dried using N 2 and 2 ⁇ _ of tissue factor as the second immobilized binding partner was applied on top of the initial coated spots. Afterwards the spot was blocked after 1 hour incubation in a humid chamber (> 50% humidity) to prevent further adhesion of the coated protein. After blocking the coverslip was mounted on a flow chamber.
  • Perfusion experiment Performed at room temperature. Whole blood samples were put in a syringe (container 2) rinsed with rinse buffer (without heparin) and connected to the pump 1 and the flow cell 4. During each experiment blood was recalcified by adding Ca 2 7 Mg 2+ mix (6.3 mM/3.2 mM) at 1 :10 volume ratio. Blood was perfused for 6 minutes at a shear rate of 1 .000 s ' Image recording was performed in real-time (time frames of 15 seconds). Time-lapse measurements (DiOC6 fluorescent intensity) and fibrinogen binding (amount of fibrin formation) were performed during 7 minutes of whole blood perfusion.
  • Blood collection Blood (aqueous sample) was drawn in Vacutainer tubes containing 3.2% of sodium citrate from a patient with thrombocytopenia before and after platelet transfusion. During each experiment blood was recalcified by adding Ca 2 7 Mg 2+ mix (6.3 mM/3.2 mM) at 1 :10 volume ratio.
  • Perfusion experiment Performed at room temperature. Whole blood samples were put in a syringe (container 2) rinsed with rinse buffer (without heparin) and connected to the pump 1 and the flow cell 4. During each experiment blood was recalcified by adding Ca 2 7 Mg 2+ mix (6.3 mM/3.2 mM) at 1 :10 volume ratio. Blood was perfused for 10 minutes at a shear rate of 1 .000 s ' Platelet deposition (complex formation) was determined at endpoint and the amount of fibrinogen was assessed as a measure of the amount of fibrin present. Platelet surface area coverage and fibrin formation was increased after transfusion (Table 10) which was confirmed by the ratio that is routinely determined in the clinic: the corrected count increment (CCI). This ratio provides an indication of the effect of transfusion. Table 10. Parameters before and after platelet transfusion
  • the flow cell according to Figure 1 C was applied in the automated analytical system according to the invention, for measuring surface area coverage of a collagen I (immobilized binding partner) coated reaction zone by platelets (mobile binding partner) with the method of the invention.
  • the aqueous sample was citrated whole blood (human), without calcium ions added, without magnesium ions added (thus, without recalcification), and without PPACK added.

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