Method for measurement of thrombocyte function

Abstract

With a method for measurement of thrombocyte function, a solution is created, by which the sensitivity of individual thrombocytes can be measured with the least possible apparatus effort, with high throughput, by passing a liquid thrombocyte solution, in which the thrombocytes are present in isolated form, into a microfluidic chamber and brought into contact with at least one stimulant, wherein an electrical field directed transverse to the entry direction of the thrombocyte solution is applied to the chamber, and the movement path of the thrombocytes in the electrical field is observed and evaluated, in such a manner that thrombocytes having a movement path directed in the direction toward the minus pole of the electrical field are classified as non-activated thrombocytes, and thrombocytes having a movement path directed in the direction toward the plus pole of the electrical field are classified as activated thrombocytes.

Claims

1. A method for measurement of thrombocyte function comprising (a) passing a liquid thrombocyte solution comprising thrombocytes in isolated form into a microfluidic chamber; (b) applying to the microfluidic chamber an electrical field directed transverse to an entry direction of the thrombocyte solution; (c) first bringing the thrombocyte solution into contact with a first stimulant directly before or during action of the electrical field; (d) subsequently, downstream, bringing the thrombocyte solution into contact with a second stimulant in the microfluidic chamber; and (e) observing and evaluating a respective movement path of each individual thrombocyte in the electrical field so that a thrombocyte having a movement path directed toward a minus pole of the electrical field is classified as an activated thrombocyte and a thrombocyte having a movement path directed toward a plus pole of the electrical field is classified as a non-activated thrombocyte.

2. The method according to claim 1, wherein the microfluidic chamber is a microfluidic free-flow electrophoresis chamber.

3. The method according to claim 1, wherein the liquid thrombocyte solution is first brought together with a stimulant solution containing the first stimulant to form a mixed thrombocyte/stimulant solution, and subsequently the mixed thrombocyte/stimulant solution is passed into the microfluidic chamber.

4. A method for measurement of thrombocyte function comprising (a) passing a liquid thrombocyte solution comprising thrombocytes in isolated form into a microfluidic chamber; (b) applying to the microfluidic chamber an electrical field directed transverse to an entry direction of the thrombocyte solution; (c) bringing the thrombocyte solution into contact with at least one stimulant directly before or during action of the electrical field; and (d) observing and evaluating a respective movement path of each individual thrombocyte in the electrical field so that a thrombocyte having a movement path directed toward a minus pole of the electrical field is classified as an activated thrombocyte and a thrombocyte having a movement path directed toward a plus pole of the electrical field is classified as a non-activated thrombocyte; and (e) wherein the liquid thrombocyte solution and a stimulant solution containing the at least one stimulant are passed into the microfluidic chamber in parallel.

5. A method for measurement of thrombocyte function comprising (a) passing a liquid thrombocyte solution comprising thrombocytes in isolated form into a microfluidic chamber; (b) applying to the microfluidic chamber an electrical field directed transverse to an entry direction of the thrombocyte solution; (c) bringing the thrombocyte solution into contact with at least one stimulant directly before or during action of the electrical field; and (d) observing and evaluating a respective movement path of each individual thrombocyte in the electrical field so that a thrombocyte having a movement path directed toward a minus pole of the electrical field is classified as an activated thrombocyte and a thrombocyte having a movement path directed toward a plus pole of the electrical field is classified as a non-activated thrombocyte; and (e) wherein before introduction of the thrombocyte solution into the microfluidic chamber, stimulant particles of the at least one stimulant are immobilized on the bottom of the chamber.

6. The method according to claim 1, wherein the first stimulant comprises an activator selected from the group consisting of adenosine diphosphate, collagen, thrombin and prostaglandin.

7. The method according to claim 1, wherein the second stimulant comprises an inhibitor selected from the group consisting of acetylsalicylic acid, convulxin, clopidogrel, prasugrel, ticagrelor, and prostacyclin.

8. The method according to claim 1, wherein the movement path of the thrombocytes in the chamber is imaged using an imaging method or an optical detection method.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Other objects and features of the invention will become apparent from the following detailed description considered in connection with the accompanying drawings. It is to be understood, however, that the drawings are designed as an illustration only and not as a definition of the limits of the invention.

(2) In the drawings,

(3) FIG. 1 is a schematic representation of the fundamental surface structure of non-activated and activated thrombocytes,

(4) FIG. 2 is a top view of a free-flow electrophoresis chamber with a first embodiment of the thrombocyte feed,

(5) FIG. 3 shows a free-flow electrophoresis chamber in a top view, with a second embodiment of the feed to the solution, and

(6) FIG. 4 shows a free-flow electrophoresis chamber in a side view, with a third embodiment of the thrombocyte feed.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(7) In FIG. 1, non-activated thrombocytes, configured in round manner, are indicated with T.sub.NA, on a greatly enlarged scale. These non-activated thrombocytes T.sub.NA have a negative surface charge at their surface S1.

(8) If these non-activated thrombocytes T.sub.NA are activated by a stimulant or an agonist, as indicated by the arrow Pf, the activated thrombocytes T.sub.A assume a different, namely an irregular surface, structure and membrane components arrange themselves differently in the thrombocyte membrane, so that the electrical charge at the surface S2 of the activated thrombocytes T.sub.A is less negative to positive. The method according to the invention makes use of this different surface charge distribution of non-activated thrombocytes T.sub.NA and of activated thrombocytes T.sub.A.

(9) In FIG. 2, a microfluidic chamber in the form of a free-flow electrophoresis chamber 1 is shown, to which an electrical field is applied transverse to the main flow direction, which extends in the longitudinal direction of the chamber 1, which field is indicated with a plus pole P and a minus pole M. Suitable electrodes are disposed in the edge regions of the chamber 1, on both sides, accordingly, or are integrated into the chamber 1, if the electrodes are imprinted electrodes.

(10) The free-flow electrophoresis chamber 1 has a plurality of parallel entry channels 2 and exit channels 2a. In the exemplary embodiment according to FIG. 2, a Y-shaped feed line 3 is connected to a central entry channel, through the one inflow 4 of which a thrombocyte solution 8 with individual non-activated thrombocytes T.sub.NA is fed in, and through the other inflow 5 of which a stimulant solution 9 is fed in; these solutions mix in a mixing region 6 of the feed line 3, and get into the chamber 1 in mixed form. Parallel to this entry, a running buffer 7 is passed into the chamber 1 through the further channels 2, so that flow through the chamber 1 takes place over its entire width and length.

(11) The thrombocyte solution 8 preferably consists of a Hepes buffer, aside from the non-activated thrombocytes T.sub.NA contained in the solution in isolated manner; the running buffer 7 can also be a Hepes buffer, but can also be a stimulant solution, if applicable.

(12) In the mixing region 6 of the Y-shaped feed line 3, the stimulant solution 9 acts on the thrombocytes, thereby causing some thrombocytes to be activated and their surface charge to change from negative to less negative to positive.

(13) As a result, the movement path B.sub.NA of the non-activated thrombocytes T.sub.NA extends in the direction of the plus pole P in the influence region of the electrical field, within the chamber 1, while the movement path B.sub.A of the activated thrombocytes T.sub.A extends in the direction of the minus pole M.

(14) According to the invention, the respective movement path of each individual thrombocyte is detected, for example by means of a microscope; thrombocytes with a movement path directed toward the plus pole are classified and evaluated (in other words qualified) as non-activated thrombocytes T.sub.NA, and thrombocytes with a movement path B.sub.A directed toward the minus pole as activated thrombocytes T.sub.A.

(15) In the exemplary embodiment according to FIG. 3, the feed of the thrombocyte solution 8 and of the stimulant solution 9 into the chamber 1 is different, as compared with the exemplary embodiment according to FIG. 2; for the remainder, the exemplary embodiment does not differ from the exemplary embodiment according to FIG. 2. In the exemplary embodiment according to FIG. 3, the stimulant solution 9 is centrally introduced into the inlet channels 2, and flows through the chamber 1 essentially along the cross-hatched region. The thrombocyte solution 8 is introduced through an entry channel 2′ disposed adjacent thereto. By means of the action of the electrical field in the chamber 1, the thrombocytes T.sub.NA that are at first not activated are deflected in the direction of the plus pole, and thereby come into contact with the stimulant solution 9. Those thrombocytes that remain non-activated, in other words the thrombocytes T.sub.NA, move along a movement path shown as B.sub.NA, in the direction of the plus pole P. In contrast, thrombocytes T.sub.A that have been activated by coming into contact with the stimulant solution change their movement path; ultimately, their movement path B.sub.A is directed in the direction of the minus pole M. Therefore, these thrombocytes can be classified in this exemplary embodiment, as well, by observation of the movement path B.sub.NA, B.sub.A of the individual thrombocytes; thrombocytes with a movement path B.sub.NA that is ultimately directed toward the plus pole P are non-activated thrombocytes T.sub.NA, and thrombocytes with a movement path B.sub.A that is ultimately directed toward the minus pole are activated thrombocytes T.sub.A.

(16) In FIG. 4, a further embodiment is shown; the free-flow electrophoresis chamber 1 is rotated by 90° as compared with the representation according to FIGS. 2 and 3, and shown from the side. In this embodiment, no stimulant solution is introduced into the chamber; instead, stimulant particles are immobilized on the bottom surface of the chamber 1.

(17) The thrombocyte solution 8 is fed in through the inlet channels 2, just like a separation buffer, which is not shown in FIG. 4. In addition, an additional buffer for dynamic focusing is introduced into the chamber 1 through an inlet 10 on the top side.

(18) Thrombocytes that are activated are once again shown with T.sub.A; their movement path is then deflected transverse to the plane of the drawing of FIG. 4, in the direction of the minus pole. The evaluation of the movement paths of the individual thrombocytes in the chamber 1 takes place as described above.

(19) Of course, the invention is not restricted to the exemplary embodiments shown. Further embodiments are possible without departing from the fundamental idea. For example, the thrombocyte solution can first be brought into contact with a first stimulant, and subsequently, downstream, into contact with a second stimulant, in the chamber. The first stimulant is then an activator, for example, and can be brought into contact with the individual thrombocytes in the manner described in FIGS. 2 to 4. The second stimulant is then an inhibitor, for example, which is immobilized on the bottom of the chamber 1, preferably downstream from the entry into the chamber 1, or is introduced into the chamber 1 downstream from the entry into the chamber 1. The movement path of the thrombocytes in the entry region of the chamber 1 then reflects the efficacy of the activator on the thrombocytes and the movement path of the thrombocytes downstream reflects the efficacy of the inhibitor on the thrombocytes.

(20) Although only a few embodiments of the present invention have been shown and described, it is to be understood that many changes and modifications may be made thereunto without departing from the spirit and scope of the invention.