LU80809A1 - METHOD FOR MEASURING THE REGENERATION TIME OF BLOOD PLATES AND DOSING DISPSITIVE WHICH MAY BE USED FOR THIS MEASUREMENT - Google Patents

Description

*. "

DESCRIPTIVE MEMORY

3 | filed in support of an application for

j: PATENT OF INVENTION

! on behalf of the public limited company known as: "Consulting and Technical Achievements", abbreviated as "C.R.T." and

Roméo RONCUCCI for: "Method for measuring the regeneration time of I-blood platelets and dosing device which can possibly be used for this measurement".

Inventors: Aloîs, Christian MENCIK, Roméo RONCUCCI, I Jacqueline LANSEN, Patrice d1OULTREMONT.

The present invention relates to a method for measuring the regeneration time of blood platelets (TRP) by the determination of malondialdehyde in various samples of blood inhibited by a single dose of an agent inhibiting the platelet cyclooxygenase. , such as acetylsalicylic acid. / '»Ί - 2 -

The principle of this method, based on the irreversible action of acetylsalicylic acid on blood platelets, is as follows:

It is known that blood platelets are capable of producing a large amount of endoperoxides from arachidonic acid. This transformation takes place thanks to an * enzymatic system of platelet cyclooxygenase irreversibly inhibited after contact with acetylsalicylic acid. The assay of one of the metabolic derivatives of endoperoxides, j malondialdehyde (or MDA) produced after ex vivo induction of platelet aggregation before and after oral administration of a single dose of acetylsalicylic acid, constitutes a possibility of '' assess the platelet regeneration time in humans or experimental animals. Indeed, the pla-j watch having been "affected" in vivo by acetylsalicylic acid | . permanently lose their ability to normally produce endoperoxides and therefore MDA. This inhibition phenomenon does not occur for the platelets which enter the blood circulation after the elimination of acetylsalicylic acid and, in! 1 by plotting appropriate graphs, one can extrapolate the platelet regeneration time.

One of the essential aims of the present invention is to present a method which can be made fully automatic for measuring the regeneration time of blood platelets by the determination of platelet malondialdehyde. / d * - 3 -

The process according to the invention is characterized in that, for each blood sample, after having anti-coagulated it and having essentially separated the red blood cells 1 therefrom, the following is carried out: | i - a count or a count of blood platelets by | turbidimetric measurement; î: - a separation of blood platelets from their plastic medium; i1 - a colorimetric assay of MDA for blood platelets i | ij; j after having induced the phenomenon of platelet aggregation by ί I * ji an adequate inducing agent.

i, i, il The invention also relates to a particular metering device | l, which in particular makes it possible to automatically apply the above process.

j In this respect, according to the invention, this device comprises, for each sample, at least two test tubes - eccentrically mounted on a rotor, means for driving this rotor, means for transferring liquid to pass the sample from one test piece to another, means for introducing liquid into at least one of the test pieces and means for discharging the content of at least one of the test pieces, possibly through a measuring device.

In a preferred embodiment of the device according to the invention, the rotor is arranged so as to rotate around a substantially vertical axis and has / a

Ir, - 4 -} I housings for at least two test tubes, the means for transferring, introducing and discharging liquid comprising at least one pipette mounted on a movable arm making it possible to move the pipette from one test tube to the other and can be connected! at a suction and / or blowing source, positioning means being provided for placing the test pieces and the pipette; ‘In correspondence with each other and to order them! > ♦! means for transferring, introducing and discharging liquid j according to a programmed cycle.

Other details and particularities of the invention will emerge from the description given below, by way of nonlimiting example, with reference to the appended drawings, of a j: particular embodiment of a method and a device for automatic measurement of the regeneration time of the plates! 1 blood watch, according to the invention.

Figure 1 schematically shows the different successive stages I of this particular embodiment of the method according to the invention.

Figure 2 is a schematic cross-sectional and axial view of the particular embodiment of the arrangement; sitive according to the invention.

i I1 Figure 3 is a view along line III-III of I __ Figure 2.

In the various figures, the same reference numerals designate analogous or identical elements. / i. - 5 -, j

The automatic measurement of the platelet regeneration time takes place in two main phases, namely 1) the determination of malondialdehyde on blood samples taken from a subject at time t ^ before and at times t ^, ... t ^ after the taking, by a subject, of a single dose of an agent irreversibly altering the platelet cyclooxygenase system and [2) the computer processing of the result of each assay, so as to deduce therefrom the platelet regeneration time.

Phase 1, that is to say, the automatic dosage of! malondialdehyde according to this particular embodiment! of the method according to the invention, comprises the following steps, which are shown schematically in FIG. 1:! 1 ° - The fresh blood sample 1, for example of the order of i j 10 ml, is anticoagulated by the addition of approximately 1 ml of a | solution of trisodigue citrate Ojl29 M and is submitted, in one! test tube 2, at centrifugation 7 of 200 x g, for approximately | 10 minutes, if you want to obtain, on the one hand, a light fraction

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! 3 3 which is platelet rich blood plasma (PRP), and, | on the other hand, a heavy fraction 4 containing mainly 1. red blood cells.

jtj 2 ° - From this light fraction 3, the volume of which is approximately d 5 ml, a constant volume V is extracted by pumping into the test tube 2. This light fraction passes through a photo-sensitive camera 5 for the platelet count by Ü „measurement.

'turbidimetric. The number N of plaguettes counted in this /

! i volume V is stored in a memory. / ~ \ J

u - 6 - • t 3 ° - This volume V having passed through the apparatus 5 is then injected into a test tube 6 which is subjected to centrifugation 7 'of 2000 xg for 15 minutes until a new fraction is obtained light 8, called platelet-poor plasma, and a heavy fraction 9 essentially comprising platelets. 4 ° - This second light fraction is sent again to a photo-sensitive camera. 10 with counting cell.

! The purpose of this second measurement is to correct the first turbidimetric measurement carried out on the light fraction 3 ([or PRP ^ for possible turbidity due to a high content of plasma lipids. Thus, the difference between the result obtained by the measurement in the cell counting device 5 and that obtained in the counting cell of device 10 makes it possible to determine, for the sample considered, the net number of platelets P.

5 ° - The pH of this heavy fraction is maintained at a value of the order of 7.4, for example by means of 2 ml of a phosphate buffer, and 0.1 ml of an agent is additionally added. inducer of the phenomenon of plaguettary aggregation, such as N-ethyl- | maleimide or arachidonic acid.

; This aggregating agent promotes the production by '' platelets, of malondiadéhyde.

'The addition of the phosphate buffer and the aggregating agent is represented diagrammatically by the arrow 11.

6 ° - The mixture is incubated at 37 ° C and is subjected to the action of ultrasound, as shown schematically by the arrows 12, / f $ - 7 - i! î | to promote aggregation of platelets.

7 ° - The proteins in suspension are precipitated by adding to the mixture containing the malondialdehyde, thus released, an acid, for example perchloric acid, as shown by arrow 13. To promote complete precipitation of the proteins, this mixture is preferably heated to about 82 ° C for 10 minutes.

8 ° - The precipitated proteins are eliminated by a third centrifugation 15, at 2000 × g for approximately 10 minutes, until a light fraction 16 and a heavy fraction 17 are again obtained.

9 ° - Then add a colorigen reagent, preferably a solution of 2-thiobarbituric acid at 0.8% (W / V), as shown by arrow 14, to the light fraction 16. This colorimetric reagent could possibly be added before the third centrifugation.

10 ° - After the addition of thiobarbituric acid to the light fraction, the mixture is incubated for 10 minutes at 82 ° C, in order to form a colored complex between MDA and thiobarbituric acid.

; 11 ° - After this incubation period, the solution is aspirated i through the cell of a colorimeter 19 and the value C measured in the latter is also stored in memory.

As already indicated above, the automatic dosing of malondialdehyde is an operation which must be repeated at times t, t, t, ... t in order to obtain the results / i.

I. - 8 -

The MDA level determined in each PRP sample is corrected for the number of blood platelets present in this same sample. By comparing MDA rates! platelet, after a. single intake of acetylsalicylic acid ^ in a subject to those obtained before taking this acid, one can quantify the percentage of inhibition of production j in platelet MDA.

<j A linear or exponential relationship between this for-

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j inhibition centering and the time makes it possible to calculate the time it takes for platelet regeneration.

As already indicated above, the invention also relates to a metering device which can in particular be used for the automatic implementation of the pro-! assigned described above.

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A preferred embodiment of such! j device was illustrated by Figures 2 and 3 attached.

I ’This embodiment of the device according to the in! vention was built to process ten different samples simultaneously.

! For this reason, it includes ten groups, a, b, c, d,; e, f, g, h, i and j of specimens mounted eccentrically on a | j rotor 21 rotating around a vertical axis 22.

; In the present case, each group consists of three test pieces 23, 24 and 25 which are freely placed in corresponding housings provided in supports 26 distributed uniformly on the periphery of the rotor 21 and each pivoting around / - 9 -% an axis 27 perpendicularly crossing the axis 22.

The test pieces of the same group are radially offset relative to each other.

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The rotor 1 can be driven by a motor 28 at different speeds and accelerations.

The device according to the invention further comprises means for transferring liquid for passing a sample from one determined test piece to another from the same group, means for introducing liquid into at least one of the test pieces and means for evacuation of the content of at least one of the test pieces, possibly through a measuring device, such as a photo-sensitive cell device dnnt question above, but not shown in Figures 2 and 3.

In the embodiment shown in these two figures, these transfer, introduction and evacuation means comprise a pipette 29 mounted on a movable arm 30 articulated around an axis 31 on a fixed frame 32 by means of 'a telescopic upright 33. Thus, this arm 30 can move in a horizontal plane, as shown by the arrow 34, and undergo an upward and downward movement, as shown by the arrows 35.

In addition, this arm is connected to a suction source, not shown, for example by a flexible pipe 36 or to a blowing source, not shown any more, by a flexible pipe 37.

The rotor 21 is mounted in a tank 38 which can contain j S - 10 -

This fluid is preferably constituted by a liquid, such as water, and can be introduced into the tank by a pipe 40 terminating in the side wall of the tank; a pipe 21 starting from the inclined bottom of the tank allows the evacuation of this liquid.

Ultrasonic emitting heads 42 are mounted in the bottom of the tank 38 and make it possible to act, by means of the liquid 39, on the content of the test tubes 23 to 25.

Thanks to the arm 30, it is possible to transfer calibrated volumes of liquid from a test tube to another test tube of the same group or another group, to introduce calibrated volumes of reagent into well-determined test tubes and to extract therefrom calibrated volumes of solution.

This pipetting system is made fully automatic by controlling the motor 28 and the drive motor of the arm 30 incorporated for example in the frame 32 but not shown in the figures, by means of an appropriate computer, in particular of a "mini processor".

Thanks to this computer, the acceleration and deceleration of the rotation of the rotor can be controlled in a fully programmed manner, as well as the sequences of positioning of the groups of test pieces relative to the arm 30.

The motor 28 is therefore programmed so as to allow the angular position to be adjusted when the rotor stops relative to / with the pipette 29. / - 11 - 11 j The use of the following metering device 11in- i || Vention is therefore not necessarily limited to the measurement of the regeneration time of blood platelets according to the pro-! assigned described above but can advantageously be used for any metering or other operation. In this regard, it suffices to adapt the computer program for controlling the cycle of operations of the motor 28, of the arm 30, of the heads i emitting ultrasound 42, of the heating or cooling i | liquid 39, etc.

i This is how the device according to the invention could advantageously be used for the automatic dosing of aldehydes in general. In such a case, the process to be applied could be limited to turbidimetric measurement in the photo-sensitive camera. 10 shown in Figure 1.1a precipitation of extra proteins! pension by the addition of an acid as shown by arrow 13 in FIG. 1 and to the colorimetric determination of the aldehyde with the addition of a suitable colorigen reagent as indicated by arrow 14.

The stages prior to the turbimetric measurement in the apparatus 10 being intended in particular for a sample; therefore could be omitted for this application.

| | Another case that could still arise is a | platelet count in human or animal plasma samples. In such a case, the operations illustrated in FIG. 1 can be stopped after the first turbimetric measurement in the apparatus 5 or possibly after the second turbimetric measurement in the apparatus 10 which could. . - 12 -

Yet another possibility offered by the device i according to the invention is the dosage of malondialdehyde in the platelet-poor plasma without prior induction of platelet aggregation. This method in fact also contributes to the evaluation of a prothrombotic state of the sample donor subject. For such an assay, the control program of the device according to the invention could be adapted to subject the light fraction 8, after the turbimetric measurement in the apparatus 10, to an assay by colorimetry of the malon-I dialdehyde of the platelets of this fraction. slight after? induces the phenomenon of platelet aggregation by an appropriate agent.

Yet another possibility offered by the device according to the invention is the differential count of blood platelets in plasma samples of the same subject "prepared with two different preservation mixtures.

Such a count makes it possible to estimate the presence of a circulating micro-aggregate. In this case, by the device according to the embodiment shown in FIGS. 2 and 3, it would be possible to process the samples of five different subjects simultaneously, {(since the test pieces of a group should be used for the prepared sample with a first preservation mixture and another group of test tubes should be used to process the sample prepared with the second preservation mixture for two blood samples from the same subject, taken at the same time. Such an assay will therefore not require a color measurement / é - * / 7 / I - 13 -

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of live platelets, so that the operation could be stopped after counting the total number of platelets by the turbidimetric measurement. r

Finally, the device according to the invention could still be used for the preparation of pad suspensions. your washed to be used for experimental or other purposes.

I This would therefore not require turbidimetric or! <| colored trique.

D1

It is understood that the invention is not limited to the particular embodiment of the method and the device described above and illustrated by the appended figures and that many variants could still be envisaged without, 1 | outside the scope of the present invention.

The microprocessor of which question above and which controls the cycle of the operations of the device according to the invention advantageously serves for the computer processing of the results of measurement by the photo-sensitive cameras and by the colorimeter and can be connected in addition at the entrance to printers, tape or magnetic recording devices, etc.

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! In addition, the number of housings or specimens per support can be very variable. Thus, for the implementation of | a method for measuring the regeneration time of blood platelets, the number of test pieces per support 26 could be limited to two. In this case, the test piece 23 of Figures 2 and 3 could * i correspond to the test piece 2 of Figure 1 and the test piece 24 to î * the test piece 6 of the latter figure. One could even provide a single test piece per support 26, so that in such a case

Claims (19)

1. Method for measuring the regeneration time of blood platelets for the determination of platelet malondialdehyde in various blood samples from a subject previously treated with a single dose of an agent inhibiting platelet cyclooxygenase, such as acid acetylsalicylic acid, characterized in that, for each sample, after having anticoagulated them and having essentially separated the red blood cells there, a count of the total number of platelets is carried out by turbidimetric measurement, in that the blood platelets are separated from their plasma medium and in that the malondialdehyde of these platelets is dosed, by colorimetry, after having induced the phenomenon of aggregation by a suitable agent. 2. Method according to claim 1, characterized in that one carries out, on the same sample, the counting of the total number of platelets by a double turbidimetric measurement, a first measurement taking place on the fraction of the sample i! - essentially free of red blood cells, the second turbidimetric measurement taking place on the part of this fraction essentially free of plasma lipids, the result of this second measurement being subtracted from the result of the first measurement so as to obtain a value corresponding to the net number of platelets contained in the considered sample /! I - 15 - 3, - Method according to either of claims 1 and 2, characterized in that, to separate the red blood cells from a sample, the latter is subjected to centrifugation I by forming a light fraction containing the blood plasma ri- i ehe in platelets and a heavy fraction mainly containing the red blood cells and in that the above-mentioned counting is carried out on this light fraction. 4. Method according to claim 3, characterized H | i in that, for the above-mentioned counting, j: j is passed continuously; the light fraction across the field of a sensitive camera, and in that this fraction is then subjected to a further further centrifugation which is such as to form a second light fraction or plasma poor in platelets and a second fraction heavy containing essentially the platelets, in that we pass this second light fraction to! across the field of a photo-sensitive camera and in determining the difference between the two values recorded by the photo-sensitive camera to obtain the total net number of platelets. 5.- Method according to claim 4, characterized in that a phosphate buffer is added to the second heavy fraction, to maintain the pH at 7.4, and the above-mentioned aggregating agent to promote the production of the malondialdehyde, in that we release the malondialdehyde regenerated by the platelets, in that the proteins are precipitated in solution by the addition of an acid, in that we separate, by centrifugation, the I! .. - 16 - ί precipitated proteins, in that a colorigen reagent is added to the light fraction resulting from this centrifugation, in that this mixture is heated to promote the reaction between the malondialdehyde and the colorigen reagent and which is then passed through the solution through a colorimeter. 6. Method according to claim 5, characterized in that the malondialdehyde is released by subjecting the solution \ j to the action of ultrasound, in that the proteins are precipitated by the addition of perchloric acid, in that thiobarbituric acid is used as the colorigen reagent and that the light fraction resulting from centrifugation and the thiobarbituric acid are subjected to incubation at approximately 82 ° C. for approximately 10 minutes before passing to the colorimeter. 7. Method according to any one of claims 4 to 6, characterized in that the sample is centrifuged, to separate the red blood cells, at a centrifugal force between 150 xg and 250 xg, preferably of the order of 200 xg,. for a period of between 15 and 5 minutes, preferably of the order of 10 minutes, and in that the light fraction resulting from this first centrifugation is centrifuged at a force of between 1500 xg and 2500xg, preferably from order of - 2000x g for a period of between 20 and 10 minutes, preferably of the order of 15 minutes, in each of these two centrifugations, the shortest durations corresponding / to the greatest centrifugal forces. / "/ {• · - 17 - d 8, - A method according to any one of claims 5 to 7 / characterized in that the solution containing the precipitated proteins is centrifuged at a force compri-se between 1500X g and 2500x g, preferably of the order of 2000X. g,] for a period of between 15 and 5 minutes, preferably of the order of 10 minutes, the shortest durations corresponding to the greatest centrifugal forces. 9. Method according to any one of claims 5 to 8, characterized in that, after the addition of the aggregating agent | j, the mixture is subjected simultaneously to the action Λ I of ultra -sons and incubation at a temperature between! "ij 36 and 38 ° C, and preferably of the order of 37 ° C, for about j! 15 minutes. !; 10. Method for measuring the regeneration time jj j | of blood platelets as described above or resulting from 13 | attached drawings. π * lïj J] 11, - Dosing device, in particular for measuring the! regeneration time of blood platelets by the assay j of malondiadéhyde of blood platelets before and after j oral administration of an agent capable of inhibiting irreversible! | the platelet cyclooxygenase system, such as acetylsalicylic acid, characterized in that it comprises, for each sample, at least two test tubes mounted * 1 eccentrically on a rotor, means for driving the ro tor, means for transfer of liquid to pass the liquid - 18 - in at least one of the test pieces and means for evacuating the contents of at least one of the test pieces, if necessary, through a measuring device. 12. Device according to claim 11, charac-! terized in that the rotor is arranged so as to rotate j I about a substantially vertical axis and has housings for at least two test pieces, the means for transferring, introducing and discharging liquid comprising at least one pi - t; pipette mounted on a movable arm allowing the pipette to be moved from one test tube to the other and which can be connected to an i- source of suction and / or blowing, position-ing means being provided for placing the test tubes and the pipette | in correspondence with each other and to order them! means of transfer, introduction and evacuation of liquids! following a programmed cycle. 13. Device according to claim 12, character- I. laughed in that it comprises a motor making it possible to drive the rotor around the above-mentioned vertical axis and programmed in such a way as to allow, on the one hand, to adjust the angular position; when the rotor stops relative to the pipette and, on the other hand, j to subject the rotor, and consequently the placed test pieces | in the abovementioned dwellings, at predetermined speeds and accelerations during predetermined times. 14.- Device according to any one of claims 1 to 3, characterized in that it comprises heating and / or cooling means for bringing and maintaining / \ * * - 19 - 3 ϊ ji the temperature of the samples at a certain temperature. 15. Device according to claim 14, characterized in that the heating and / or cooling means! comprise a bath of a heating or cooling fluid at least partially surrounding the aforementioned test pieces. 16. Device according to claim 13, characterized in that the rotor is mounted in a tank which can contain the above-mentioned bath formed of a liquid, at least one head emitting ultrasound being provided to act, via liquid, on the contents of the test pieces. 17. Device according to any one of claims 11 to 16, characterized in that the aforementioned evacuation means comprise, for the solution to be passed through the colorimeter, a pipe of which one of the ends can plunge into a test tube, at a certain distance from the bottom of the latter, and the other end of which is connected to the cell of the colorimeter, this cell being connected to a suction source. 18. Device according to any one of claims 11 to 17, characterized in that the liquid transfer means are connected to at least one photo-sensitive measuring device for passing the sample from a test tube to î ; the other through this device. 19. Device according to any one of claims 12 to 18, characterized in that the housings are provided in a support pivoting about an axis crossing substantially ^ / perpendicularly the axis of rotation of the rotor. 20.- Metering device as described above or shown in the accompanying drawings. ! “-; n ': —1 Γ -' :: Πγ ^ 3 f i 3Ö r. ' -VU / --- pane da G3rt- | 2 '...... JC pact description j! 3 P ^ s of claims I 17 descriptive abstract LuxernboitpJe 0Λ. i. The j (^ - <iiaries München i: i [j A