JPS5837536A - Inspection cell for blood platelet - Google Patents

Abstract

PURPOSE:To measure a blood platelet function especially adhesion, discharge and agglutination functions separately and exactly, by depositing a blood platelet adhering and discharge causing substance at the inner surface of a vial having light transmission characteristics, in which a blood platelet suspension is poured. CONSTITUTION:After a blood platelet suspension is poured into an inspection cell 10 for the blood platelet which deposits 3 spiral fiber collagen as a blood platelet adhering and discharge causing substance, this is placed and fixed on a cell holder 12. When a motor 20 is rotated reciprocatively, the cell holder 12 which is borne freely rotatably by a bearing 16 at a fixed substrate 14 through a belt 18 is rotated reciprocatively and stirring is performed, and the blood platelet is adhered with the blood platelet adhering and discharge causing substance and ATP is discharged. The increase of light transmittance of the blood platelet suspension caused by adhesion of the blood platelet is measured by a photomultiplier 28 provided at the opposite side to a light source 26 against the cell 10, and the light emission caused by ATP, etc. is detected by a photomultiplier 30 provide in the rectangular direction thereto.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a platelet test cell, and more particularly to an improved platelet test cell that can independently quantify platelet adhesion, release ability, and aggregation ability with high accuracy and reproducibility.

In order for blood to perform normal hemostasis in the body, platelets must fully perform their functions, and it is known that platelets have three functions: adhesive ability, release ability, and aggregation ability. It is being Therefore, in order to test the hemostatic function of blood collected from a subject, it is necessary to test and measure at least these three types of functions separately and accurately. It becomes possible to accurately diagnose.

Of the three functions mentioned above, the platelet aggregation needle (ablibometer) is known as a means for quantitatively measuring aggregation ability, and a platelet aggregation needle (ablibometer), which uses the absorbance method, is recognized as a practical routine testing method. There is.

1.(2) However, no quantitative measurement method has been established for the hemostatic effect, especially the initial reaction of forming a hemostatic frame, and the important adhesive ability in 17, and in the past, only this adhesive ability has been accurately measured. It was almost impossible to do that.

The most common conventional method for measuring adhesion is Jel.
The LEM method, the Salzman method, and variations thereof are used, and the principle is that platelets in blood are made to adhere to a glass surface, and the amount of adhesion is measured using a microscope, an automatic blood cell measuring device, or the like. That is, whole blood or blood to which an anticoagulant has been added is passed through a column in which a large number of glass beads are packed in a plastic tube, and the number of platelets is measured before and after the blood flows through the column. However, with this method,
Since glass is used as the surface to which platelets adhere, it is non-physiological and involves platelet aggregation, so it has the disadvantage that only the amount of adhesion cannot be accurately measured.

As another conventional method, a method (L(a
umgartner method) has also been proposed, but it has the disadvantage that such a surface to be adhered lacks stability that allows a certain standardization, and lacks reproducibility and precision.

Furthermore, as a conventional measurement method, so-called lumia bribometry, in which a suspension of collagen fibers is added to platelets and the aggregation ability and release ability are measured simultaneously, is known as a practical routine testing method.

However, with this test method, platelet adhesion to collagen is followed by release and aggregation reactions, and these series of reactions are detected simultaneously, so it is difficult to determine which reaction stage the test value is due to. The only drawback is that it cannot be done. In other words, the above-mentioned reaction is stimulated by adhesion to Kolaken, and platelets become dense b.
This includes the release reaction of ADP, ATP, etc. from Ody, and the agglutination reaction due to the released ADI. Therefore, since these reactions are detected redundantly,
Even when abnormalities were found in test values, it was not possible to determine which process was abnormal. Furthermore, the mechanism of collagen aggregation in such a suspended state is different from that of platelet aggregation caused by water-soluble aggregation-inducing agents such as ADP and adrenaline, and even when diagnosing platelet pathologies, it is difficult to understand whether the symptoms are abnormal or not. In some cases, the results of platelet function tests show normal results, and there are several problems in adopting such conventional collagen aggregation as a clinical test method for platelets. As described above, conventional collagen fiber suspensions have unstable testing capabilities, including the reproducibility of test values, cannot withstand long-term storage, and degenerate in a relatively short period of time. The drawback was that it was difficult to maintain stable activity.

As described above, it has not been possible to obtain sufficiently satisfactory results using conventional platelet adhesion measuring means.

The present invention has been made in view of the above-mentioned conventional problems, and its purpose is to accurately measure platelet function, particularly adhesive ability, release ability, and aggregation ability separately, and to be able to use it as a practical clinical test method. The objective is to provide a highly reliable platelet testing cell.

In order to achieve the above object, the present invention provides a vial having light transmitting properties and having a platelet adhesion and release-inducing substance such as three-helical fibrous collagen supported on at least a part of the inner surface of a vial into which a platelet suspension is injected. It is characterized by the presence of

As described above, the present invention provides three-helical fibrous collagen with a periodic structure of 700X, which has extremely high activity against platelets, on at least a part of the inner surface of a vial made of glass or plastic, which has light-transmitting properties, to induce platelet adhesion and release. It forms cells that support the substance, and in the cells obtained in this way, platelet suspension,
For example, injecting platelet-rich plasma or washed platelet suspension;
The platelets and three-helical fibrous collagen are made to adhere.

By performing luminescence analysis using the luferrin/luciferase method to measure the amount of ATP released when platelets adhere to the surface of three-helical fibrous collagen, it is possible to quantify platelet functions, particularly adhesive ability. Become.

In the present invention, the three-helical fibrous collagen is supported uniformly and standardized on the inner bottom surface of the vial and the surrounding inner wall surface in the vicinity of the inner bottom surface of the vial. It is preferable to carry out 3
It becomes possible to stabilize and fix the active structure of the present helical fibrous collagen for a long period of time.

In the present invention, the platelet adhesion and release-inducing substance can be not only the three-helical fibrous collagen described above, but also a charged synthetic polymer, and can be supported not only by the freeze-drying method but also by any other suitable method. You can use the mutual support method. That is, as a method for supporting these, there is a method of immobilization by crosslinking with the activity of collagen, and crosslinking with ultraviolet light or radiation is also possible.

The platelet test cell according to the present invention obtained as described above was injected with a platelet suspension, and the amount of platelets was measured, as shown in FIGS. 1 and 2.

A device is shown that measures the amount of platelets by photoelectric conversion.

The platelet test cell 10 is placed and fixed on a cell holder 12 with a platelet suspension injected therein, and the platelet suspension is stirred within the cell 10 by reciprocating rotation of the cell holder 12. That is, the cell holder 12 is rotatably supported by a fixed base plate 14 with a bearing 16, and the cell holder 12 itself is connected to the main shaft 20a of the motor 20 via a belt 18, so that the cell holder 12 can reciprocate by rotating the motor 2o. Therefore, the cell holder 12, that is, the platelet test cell 10, can be rotated back and forth to perform a desired stirring action. A plate-shaped heater 22 is fixed to the fixed substrate 14, and uniform contact between the platelet suspension and the platelet adhesion and release-inducing substance supported on the inner surface of the vial is achieved by liquid stirring at a desired heating temperature. It is done under certain conditions.

As described above, when the platelet suspension uniformly contacts the platelet adhesion and release-inducing substance within the cell 10, the platelets adhere to the platelet adhesion and release-inducing substance, and then ATP
can be released and their adhesion ability and release ability can be measured, and in the illustrated embodiment, it is possible to measure such adhesion ability and release ability simultaneously and with good reproducibility.

That is, the measuring device is housed in a light-shielding box (not shown), and the opening 2 provided in a part of the plate-shaped heater 22 described above.
2a, a slit plate 24 is disposed facing each other, and a desired light source 26 is provided via the slit plate 24. An adhesion detection angle photomultiplier tube 28 is provided on the side opposite to the light source 26 with respect to the platelet test cell 10, and the photomultiplier tube 28 ensures that the light transmittance of the platelet suspension increases due to the adhesion of the platelets. Furthermore, a photomultiplier tube 30 for detecting emission is provided in a direction perpendicular to the light source 26 and the photomultiplier tube, so that the philtrum released from platelets can be detected and measured electrically. This photomultiplier tube 3 emits light caused by P, etc.
0 electrically detected.

In normal cases, the transmitted light from the light source 26 is significantly larger than the emitted light at the time of release, so when detecting release, the light source 26 is turned off or the light is blocked by moving the slit 24, and when detecting loose adhesion, the light source 26 is connected to the platelet testing cell 10. In this case, it is preferable to shield the light incident part of the photomultiplier tube 30 for emission detection.

According to the present invention, it is possible to accurately measure the adhesion ability and release ability of platelets. However, in order to prevent the aggregation reaction following release, the platelet test cell 10 contains a suitable reagent such as EGTA.
(Glycol ether diamine tetraacetic acid) or C1)
- It is preferable to add CPK (clebutin phosphate creatate/phosphokinase) etc. to the platelet suspension,
It becomes possible to measure and evaluate each release or aggregation function of platelets separately, and it becomes possible to reliably improve the shortcomings of conventional force lumia ribometry.

As a first preferred example of the platelet adhesion and release-inducing substance supported in the vial described above, three-helical fibrous collagen having a 700X periodic structure with extremely high activity against platelets is optimal, and the supporting method is A freeze-drying method is preferable.

That is, the inner surface of a cylindrical glass cell with an inner diameter of IcIrL and a volume of 3 ml was coated with a 0.2% aqueous solution of three-helical fibrous collagen prepared from a cow's breast (a 0.2% aqueous solution of collagen with a weak -NHC).
This was then centrifuged to remove unpolymerized collagen fibers, and only the collagen fibers were collected and redispersed in distilled water). Obtained.

Before using this reaction cell, add 0.1 molar phosphate buffer (p
After moistening for several minutes with H7,4, 290mos) 1. Add 0.2 ml of luiferin/luciferase and 1 ml of platelet-rich plasma (PRP), apply the test device as shown in Figs. The liquid was stirred, and the amount of adhesion was determined as the increase in transmittance, and the amount of ATP released was determined as the amount of luminescence.

Table 1 shows the quantitative results, and the transmittance and luminescence amount were recorded continuously with a recorder over time, and the maximum values are shown.

Table 1 Variation between cell lots in platelet adhesion activity of collagen-supported cells (30'C, stirring at 1000 rpm) *However, PRP permeability is 0% and plasma permeability is 10%.
It was set to 0%. Note that S and D are standard deviations, and cpm is cou
nt/mlnute.

As is clear from Table 1, the variation in lot fe''J of collagen coated cells is about 5 inches, and it is clear that good measurement results comparable to those of the conventional alibometer can be obtained.

Table 2 shows the results of adhesion and release using the platelet test cell prepared according to the first example described above, and separating the release ability from the aggregation ability. Suwachi,
In the platelet test cell, 3mM EGTA was added to PRI' to suppress the aggregation reaction associated with platelet ADP release.
Adhesion to the collagen surface and the resulting ATP release have been measured.

Table 2 Results of platelet adhesion test on collagen-supported surface (30°C, 1000 rpm in the presence of 3mM EGTA)
As is well known, when EGTA is added to a platelet suspension,
Ca' ions are removed and the aggregation reaction caused by A and DP' is inhibited. Therefore, as is clear from Table 2, 3m
In the presence of BQTA of M, the amount of change in permeability was 33%, and the amount of luminescence decreased to 68%, and this decrease was due to the aggregation secondary to ADP released from the cells by adhesion and release. This is considered to correspond to a release. In this way, it is clear that by adding an aggregation inhibitor, adhesive ability and aggregation ability can be clearly distinguished and quantified separately.

Table 3 shows the relationship between the amount of ATP released and the platelet concentration when the platelet concentration was variously changed by centrifugation.

Table 3 Relationship between ATP release amount and floating platelet concentration (3 m
As is clear from Table 3, the platelet concentration range of normal people (
3-6 x 10 platelets/μl),
It is clear that the amount of luminescence is almost constant, and that there is no need to measure the number of platelets or adjust the platelet concentration before measurement.

In Table 4, the sensitivity of the platelet test cell of the present invention is measured, and after incubating PRP at 30°C, ATP
Measurement of released amount over time] 2. The measurement conditions at this time are the same as those for the separation and determination of adhesive ability and aggregation ability.

Table 4 Relationship between platelet function changes and ATP release amount (3mM
(in the presence of EGTA, 30°C, 1000 rpm stirring) As is clear from Table 4, it is clear that even a slight functional decline that cannot be detected with a conventional alibometer can be detected with high sensitivity and sensitivity.

Table 5 shows the change in platelet adhesion activity over time when the platelet test cell obtained in the first example was stored at room temperature for one year.

Table 5 Activity change over time of collagen-supporting cells As is clear from Cloth 5, it is understood that the activity of the collagen-supporting cells in the present invention hardly changes over at least one year.

The present invention provides the platelet adhesion and release-inducing substance [7]
In addition to triple-helical fibrous collagen, it is also possible to use charged synthetic polymers, and as a second embodiment of the present invention, platelets carrying such charged synthetic polymers in a vial by air drying. A test cell is shown.

That is, the inner surface of the glass cell is coated with poly L-lysine, polyethyleneimine, polyarginine/polyionene, quaternized polyvinylpyridine, quaternized polyvinylpyridine, tetronic, polystyrene sulfonic acid, and styrene maleic anhydride copolymer hydrolyzate. A platelet test cell is obtained by moistening with a 1% aqueous solution of , styrene acrylic acid copolymer, styrene methacrylic acid copolymer, polymethacrylic acid, polyacrylic acid, polyitaconic acid, and polyglutamic acid and air-drying this. Table 6 shows the results of measuring the amount of ATP released using the platelet test cell in this second example. This measurement was carried out under the same conditions as in the case of separating and quantifying the adhesion ability and aggregation ability.

Table 6 Platelet adhesion test using various polymer-supported cells *Tetronic is a block copolymer of ethylene oxide and propylene oxide crosslinked with ethylene diamine.

As is clear from Table 6, the substance that induces platelet adhesion and release in the present invention is not limited to three-helical fibrous collagen, but can be replaced by other charged synthetic polymers.

In the present invention, each adhesion and release reaction is measured by photoelectric detection, but these measurements can also be performed by labeling with a radioactive substance. That is, platelets are 5I
The amount of platelets double-labeled with C and +4C-serotonin and adhered to the collagen surface can be counted using 51C, and the release reaction at this time can be counted using 14C.

As explained above, according to the blood and platelet testing cell according to the present invention, platelet samples used for testing can be prepared by just one centrifugation, and the operation is extremely simple and practical. It is useful as a platelet function test.

In addition, the required volume of the sample is small, and in normal cases, 2 ml is sufficient for the entire surface.

The platelet test cell of the present invention has particularly stable and excellent sensitivity, extremely high reproducibility, and has the advantage of being extremely stable (7) and being able to maintain unchanged activity over a long period of time. has.

[Brief explanation of the drawing]

FIG. 1 is a schematic front view showing an apparatus for testing the adhesive ability and release ability of platelets using a platelet testing cell according to the present invention, and FIG. 2 is a plan view of the apparatus shown in FIG. 1. 10... Platelet test cell 28... Photomultiplier tube for adhesion detection 30... Photomultiplier tube for emission detection. Figure 1: 2t! 1

Claims (1)

[Scope of Claims] (1) A platelet testing cell characterized in that a platelet adhesion and release-inducing substance is supported on at least a portion of the inner surface of a vial having light transmission properties and into which a platelet suspension is injected. (2. The platelet testing cell according to claim (1), wherein the platelet adhesion and release-inducing substance is composed of three-helical fibrous collagen. (3) Claim (2) (4) In the platelet testing cell described in claim (1), the platelet testing cell is characterized in that the three-helical fibrous collagen is supported by freeze-drying. A platelet testing cell characterized in that the adhesion and release-inducing substance is composed of a charged synthetic polymer. (5) In the platelet testing cell according to claim (4), the charged synthetic polymer is poIJ-L- Lysine, polyethyleneimine, polyarginine, polyione/, quaternized polyvinylpyridine, quaternized polyvinylimidazole, Tetronic, polystyrene sulfonic acid, styrene maleic anhydride copolymer hydrolyzate, styrene acrylic acid copolymer, styrene A platelet test cell comprising a methacrylic acid copolymer, polymethacrylic acid, polyacrylic acid, polyitaconic acid, and polyglutamic acid.