WO1988004051A1 - Biological treatment method by using ultrasound, particularly for immuno-hematological tests - Google Patents

Abstract

A microtitration plate (21) is immersed into a liquid (29) contained in a container (20). A high frequency generator (23) energizes a transducer (24) of which the ultrasound radiation is reflected by a movable reflector (25) to the rows of capsules (22-i) provided on the microplate (21). With a ultrasound frequency of the order of one Megahertz, there is a considerable acceleration of the aggregation effects on suspended particles by fixation of the antigen to the antibody searched for in immunological tests.

Description

 Method of biological treatment by ultrasound, in particular for immuno-hematological tests.

The invention relates to biological treatments, and in particular immuno-hematological tests.

Agglutination, a basic technique in immunology, is a complex phenomenon leading to the clustering of cells such as red blood cells or bacteria or even viruses. One can also use particles other than living, the best known example being that of latex microbeads, or particles of carbon.

These particles are suspended in a biological liquid medium, which can be defined as comprising water associated with a suitable buffer electrolyte (physiological saline), and containing proteins in solution.

An immunoassay also involves antigens (or antigenic sites) and antibodies. The antigens can be the elements in suspension themselves, in which case the antibodies are present in solution in the liquid medium. Conversely, antibodies. can be carried by suspended particles, the antigens then being in solution. Materially, the biological liquid to be tested is contained in containers which are sometimes still test tubes, or, better, a microtiter plate, defining a two-dimensional network of capsules capable of each receiving a sample.

The response to the test essentially depends on the distribution of the optical density existing in each capsule, after a more or less long time, depending on the reaction. We know how to make an automatic measurement of this image. We also know how to automate some of the preparation phases of immunological tests.

The agglutination phase proper, can be carried out by using medium devices, such as enzymes, macromolecules, polycations, antiglobulins, or others. Currently, we can either allow the red cells (or others) to sediment under the effect of gravitation, or centrifuge the micro-titration plate.

The free sedimentation technique requires a fairly long time, which we have been able to reduce a little by using stair-shaped capsule plates (OLYMPUS).

Centrifuging the microtiter plates further accelerates the reaction; however, the centrifugation effect causes systematic red cell sedimentation - agglutinated or not - at the bottom of the capsules. We can then use different complementary techniques, which take time: tilting the plates at a slightly higher temperature, or resuspending the free red cells, for example by successive agitations at slow speed. These techniques, which are difficult to implement, cannot be automated.

In addition, they are likely to introduce a loss of sensitivity, especially when a medium is centrifuged. is the site of a weak reaction, where the agglutinates are small.

The time of sedimentation and therefore of image formation, therefore limits the performance of the test devices currently used.

The present invention improves the situation.

A first object of the invention is to make it possible to accelerate and / or amplify agglutination.

Another object of the invention is to further accelerate the sedimentation, to avoid recourse to centrifugation, as much as possible.

Another object of the invention is to allow red cells (or others) to be resuspended, by simple and automatable means, in cases where such an operation is necessary.

The invention makes use of ultrasound for this purpose, in a particular way.

The use of ultrasound in a living environment has already been the subject of several observations.

The formation of clusters of red blood cells, all half-length wavelengths, in a living chicken embryo, was noted in the article: "A microscope viewing ultrasonic iradiation chamber", JB POND, B. WOODWARD and MARY DYSON, Phys. Med. Biol., 1971, Vol. 16, No. 3, pp 521-524. A numerical simulation of the formation of the clusters was proposed later: "Blood cell banding in ultrasonic standing wave fields: a physical analysis" GAIL TER HAAR and SJ WYARD, Ultrasound in Med. & Biol., Vol. 4, pp 111, 123, Pergamon Press, 1978. These publications take into account:

- the so-called "radiation pressure" force, known for a long time, at least when it is a question of the effect of a standing acoustic wave on a sphere in a non-viscous medium;

- the viscosity effect ("viscous drag");

- the interparticle forces, which exist when several neighboring bodies are subjected to the stationary ultrasonic field; and

- side effects due to the presence of gas bubbles, distortions to the acoustic wave (Oosen forces, or "Oosen-type forces"), as well as variations in viscosity as a function of temperature.

More recently, the article "Interparticle forces on red blood cells in a standing wave field" MAH WEISER and RE APFEL, Acustica 56 (2), pp 114-128, 1984, proposed an analysis of the intercellular force in a field of stationary acoustic waves, and deduced therefrom the equilibrium position of two red cells for a determined acoustic frequency.

However, these calculations are only valid in the case of long interparticle distances.

The observation of POND, WOODWARD and DYSON, and the analyzes which it gave rise to, relate to in vivo experiments.

Besides this, an in vitro observation has been made public: "Segregation and sedimentation of red blood cells in ultrasonic standing waves", N. VASHON BAKER, Nature 239, Oct (13), 1972, pp 398-399. The author notes that, in polystyrene containers containing whole blood or cultures thereof, the natural sedimentation of red blood cells is greatly accelerated, under an acoustic field of 3W / cm ² at 1 MHz. On the one hand are also noted the fluence of the red blood cell density of the suspension, on the other hand the fact that accelerated sedimentation is observed only when the sound field propagates horizontally (segregation according to vertical sedimentation bands). NV BAKER draws from this that segregation is not due to Bernouilli forces, as suggested by POND, WOODWARD and DYSON, but rather to convection currents caused by the periodic variation of the density of cell suspensions.

The present invention is based on observations of another nature.

The particles suspended in a liquid medium are electrically charged.

The elementary forces that come into play are:

- electrostatic repulsion due to the electric double layer; and

- the LONDON - VAN DER WAALS type attraction.

In the example of human blood, these forces cause the red blood cells to aggregate, in the presence of macromolecular bypasses, in the manner of "stacks of plates".

For its part, the agglutination of erythrocytes (or erythrocytes) occurs when the suspension allows antibodies to be attached to antigenic structures.

It has now been found that, taking into account the electrical and physicochemical properties of red blood cells, erythrocyte agglutination reactions are substantially improved under the effect of ultrasound.

The invention applies first of all to a biological treatment process, in which a container is placed a biological liquid medium containing particles in suspension, this medium and these particles. being associated one with antibodies, the other with antigens, so that an immunological reaction can be established between the antibodies and the antigens and possibly lead to agglutination of the particles. According to the invention, a coherent beam of ultrasound is applied to the liquid medium, at a frequency between 0.1 and 100 MHz, preferably between 0.5 and 20 MHz, approximately, capable of producing radiation forces favoring the grouping of these particles.

These radiation forces can thus be used to promote an agglutination reaction of the particles or even to promote the exchange of the biological liquid medium, for washing purposes, the particles being grouped together.

The power density of the ultrasound beam can range from 0.1 to 100 W / cm ² , preferably from 0.5 to 20 W / cm ² , approximately.

In practice, the container (test tube, or better, microtiter plate) is placed externally in contact with an ultrasound transfer medium, in principle a liquid such as water. As a variant, it is possible to use, as transfer medium, a flexible medium such as a silicone-based material.

Advantageously, the direction of the ultrasonic field in the container is substantially aligned on the vertical thereof.

More restrictively, the liquid medium is water associated with a pH buffer and containing proteins in solution, and the particles are cells or microparticles capable of forming reaction substrate.

In a particular application, the liquid medium is a solution of red blood cells at low concentration. In another application, the particles are latex microparticles.

According to one aspect of the invention, the ultrasound beam is applied so as to establish a substantially stationary regime in a direction of space, but in a non-uniform or non-continuous manner (in space and / or in the time).

In other words, it must be a "non-progressive" sound field, that is to say one that does not propagate indefinitely. Although the proper modes are very difficult to define, the container will constitute a sort of resonant cavity for ultrasound.

As for the coherence of the acoustic field, it can be defined as follows: the coherence time of the acoustic field must be greater than the time of the waves going back and forth in the container.

It is possible to operate by repeated interruptions of the acoustic field.

During the application of the field, the red cells aggregate in bundles, trapped in the vibration bellies, and which are the seat of the agglutination reaction. When these packets reach a certain critical size, they fall or sediment, creating a pellet at the bottom of the container. At this time, the application of the sound field can be stopped.

However, the pellet has agglutinated red cells (practically irreversible reaction) at the same time as simply aggregated red cells (substantially reversible reaction).

Another particularly interesting way of operating is to wobble slightly. the frequency of the sound field. Preferably, this wobulation is carried out so as to slowly lower the vibration bellies towards the bottom of the container. It can also be moved in the opposite direction, if one wishes to carry out a resuspension of the red cells, agglutinated or not.

Those skilled in the art will understand that it is therefore necessary to choose the amplitude and the speed of this wobulation as a function of the speed of the agglutination reaction (s) in the presence of which one is.

Another very important observation has been made: the invention makes it possible to accelerate the agglutination reactions, without altering the results. The reagents and procedures used to date can therefore be retained.

On the other hand, it seems that the sensitivity of the tests is increased, in particular for weak agglutination reactions.

Another aspect of the invention relates precisely to such weak reactions.

Current techniques use centrifugation to accelerate sedimentation. The implementation of this centrifugation is in itself delicate; in particular, a large radius of gyration is required, so that centrifugation produces homogeneous effects on all the containers (microplate tubes or capsules).

But centrifugation sediments all the red cells, aggregated or agglutinated. A re-solution of the aggregated red cells - also a very delicate operation - is therefore necessary.

The invention also makes it possible to carry out this solution solution by ultrasound. Although the same type of acoustic field can be used for this purpose as previously, it currently seems preferable for the ultrasound beam to be focused, on the liquid medium contained in the container.

Thus, the invention makes it possible in most cases to avoid centrifugation. But, when this remains necessary, the re-solution can be obtained in a simple manner by ultrasound, avoiding breaking the small aggregates.

Certain immuno-hematological procedures, such as the COOMBS test (reaction amplified by antiglobulins) require one or more washes of the red cell suspension. A conventional washing comprises the following stages: centrifugation, aspiration of the supernatant, redistribution of the washing liquid, resuspension.

Low frequency ultrasound has already been used for the homogenization and dispersion of suspensions. But the effect obtained is accompanied by cavitation, which causes the destruction (lysis) of part of the cells.

It has been discovered that with an ultrasonic beam according to the invention (typically, frequency from 1 to 10 MHz), the red blood cells are resuspended almost instantaneously without any effect of lysis by cavitation.

This opens the way to complete automation of tests requiring washes, such as the COOMBS test (indirect COOMBS reaction), which is known to be useful for determining fetal-maternal Rhesus incompatibilities or other immuno-hematological reactions.

The invention also relates to a biological treatment installation, which comprises an ultrasound cell, suitable for applying a coherent beam of ultrasound, of frequency between 0.1 and 100 MHz, to at least one container containing a biological liquid medium with some by suspended particles, the beam being capable of producing radiation forces favoring the regrouping of these particles.

Other characteristics and advantages of the invention will appear on examining the detailed description below, and the appended drawings, in which:

- Figure 1 is a block diagram recalling the different phases of an immunohematology test;

- Figure 1A is a block diagram similar to Figure 1, for the case where the implementation of the test requires centrifugation;

- Figure 2A is a simplified diagram, in section, of a first experimental device for implementing the invention;

- Figure 2B is a simplified diagram, also in section, of a second device for implementing the invention;

- Figure 3 is a time diagram showing a wobulated signal as a function of time;

- Figures 3A and 3B are spatial diagrams showing the effect of the wobulation of Figure 3;

- Figure 4 is a simplified diagram, in section, of another device for implementing the invention;

- Figure 5 is a simplified diagram, also in section, of yet another experimental device for the implementation of the invention; and

FIG. 6 is a simplified diagram, in section, of a variant of the experimental device of FIG. 5. As already noted, the invention can be applied to different types of biological suspensions, in which the elements in suspension can be living elements, such as red blood cells, bacteria or viruses, or else inert particles such as latex or carbon microbeads. Unless otherwise stated, this detailed description concerns the case where the elements in suspension are red blood cells or red blood cells.

In addition, immunological tests can be carried out with test tubes for containers. We will now assume that these are microtiter plates, which we will more briefly call "microplates". A common model of microplates has 8 x 12, or 96 elementary containers, also called capsules. One could also use any other container comprising a multiplicity of capsules or cells arranged in a two-dimensional network.

In an ordinary immuno-hematological test, the first step 11 (FIGS. 1 and 1A) consists in the automatic filling of the microplates, which comprises:

- the distribution of the sample in each capsule;

- diluting the sample in each capsule, by adding a suitable diluting agent, such as physiological saline;

- the distribution of the reagent (s) in controlled quantity in the different capsules.

Now, a microsyringe dispenser can, in less than a minute, dilute and distribute the reagents in the 96 capsules of the microplate.

The next step 12 is the incubation, during which the agglutination reaction takes place or not. When the reaction involved is a strong reaction, either by itself or by the fact that we are using medium devices (enzymes, macromolecules, polycations, antiglobulin, for example), we can be satisfied with simply sedimenting red cells, under the effect of gravitation, as illustrated by step 17 of Figure 1.

This so-called free sedimentation technique requires a fairly long time, which usually exceeds half an hour. Ordinarily, the quantitative measurement of agglutination is done with microplates whose bottom is in U or V. With a new type of microplates, whose capsules have a bottom in the shape of a staircase, the company OLYMPUS has made it possible to reduce a little sedimentation time, which drops to around 20 minutes.

The results are then read in step 18. If there is agglutination, a sedimentation pellet is present at the bottom of the capsule. Otherwise, it is covered in a uniform manner. The acquisition of the measurements can therefore be automated, since it is a priori a measurement of optical density through the capsule at different points.

Then, generally using a microcomputer, the measurements are processed and the results are presented. It is necessary to take into account the cases where the result is not clear, as well as the particular conditions specific to each test.

FIG. 1A relates to the case of a slow or weak agglutination reaction. After incubation, or during it, centrifugation 13 is usually carried out, which accelerates the reaction, concentrating all the red cells, agglutinated or not, at the bottom of the capsules. Of course, additional steps are necessary to read the age. A first way of proceeding consists in tilting the microplates at an angle of approximately 75 ° relative to the horizontal, for a time of approximately 5 minutes, in order to observe different images which make it possible to quantify the agglutination. This process is tricky to implement, especially when full automation is desired.

Another way of proceeding consists in performing successive agitations of the microplates, at slow speed, to resuspend the free red cells, that is to say those which have been sedimented, but are not agglutinated. Again, the handling is delicate, and there is often a loss of sensitivity due to agitation, in the case where the reaction is weak, and where consequently the agglutinates are small. In addition, this process is also difficult to automate, because it requires manipulation of the microplates.

Then the optical density readings and the processing of the results can be carried out according to steps 18 and 19 similar to those of FIG. 1.

Reference is now made to Figure 2A. We see a microplate 21, mounted in the upper part of a container 20 filled with a liquid 29 such as water. A high-frequency generator 23 excites an ultrasonic transducer 24, which applies an acoustic field to a reflector 25 positioned to return this field to the row of capsules 22i of the microplate 21. As illustrated by arrow 26, the reflector 25 can be moved, so as to allow the insonification of any one of the rows of capsules 22-1 to 22-n that the microplate 21 comprises.

A variant of this device is illustrated in FIG. 2B. We see a high frequency generator 23 which feeds a series of transducers 24-1 to 24-n, each insonating a row 22-1 to 22-n of the capsules of the microplate 21. The microplate can be incorporated into the ultrasonic cell thus formed without any modification. Its movement between the filling machine and the image reader can be ensured using a conveyor belt, controlled by a predetermined program. In the ultrasonic cell, the transducer (s) 24 and possibly the reflector 25 are immersed in the liquid 29, while the microplate 21 is held on the surface of the liquid. The liquid 29 ensures the propagation of the ultrasonic waves in the capsules of the microplate 21. The liquid air interface, both in the microcapsule and for the liquid 29, serves as an almost perfect reflector for establishing a stationary ultrasonic field.

FIGS. 2A and 2B have shown that the ultrasonic generator can consist either of pairs (piezoelectric elements / reflector) associated with a motor to insonify each cell, or of a battery of piezoelectric elements which ensures this insonification in a direct manner, and simultaneously, for each cell.

Electronic focusing and / or orientation of the beam can be carried out from a matrix of transducers.

Although a wider frequency range can be used, it is preferred that the frequency of the ultrasound is between 0.5 and 20 MHz, approximately. In most cases, this frequency will be between 1 and

10 MHz. The choice of frequency depends on the type of reaction, as well as the acoustic impedance and the thickness of the microplate.

The surface power of the ultrasound beam can vary within wide limits depending on the applications. It will preferably be, at the level of the transducers, from 0.5 to 20 watts / square centimeter, most often about 1 to 5 watts / square centimeter.

The generator 23 will produce either a monochromatic, continuous or intermittent signal, or a wobulated frequency signal.

Monochromatic excitation allows rapid agglomeration of the particle clusters, into two bands each separated by half a wavelength. Stopping the acoustic emission causes rapid sedimentation by simple gravitation, because the sedimentation rate is greater the larger the particle size.

It will be understood later that the intermittent monochromatic excitation is advantageous for certain applications. The reason is that it makes it possible to obtain effects of resuspension of sedimented but not agglutinated red cells, which can then react again, and agglutinate, again in an accelerated manner, taking into account the application of ultrasound.

Furthermore, in the above, the ultrasonic field applied to the capsules is vertical. With a vertical field, it seems preferable that the insonification is intermittent.

If a horizontal ultrasonic field could be used for microplate tests, then the insonification could more easily be made continuous.

Figure 3 shows a very interesting variant of the invention. This variant makes use of a downward frequency wobulation. More precisely, the excitation frequency varies from f ₂ to f ₁ , symmetrically around a central value fg, and this with a repetition period, T _r . This downward wobulation of frequency makes it possible to move the nodes and the bellies of the ultrasonic field and consequently the heaps of red blood cells towards the bottom of the capsules. This is illustrated schematically in the figures 3A and 3B, where we see that the last node, which is in position A in FIG. 3A, descends in position B at the bottom of the capsule in FIG. 3B, when we go from time to to time t _O + T _r .

This significantly accelerates spontaneous sedimentation in the microplate.

It also seems that by choosing the repetition frequency Fr = 1 / T _r properly, it is possible to carry out differential sedimentation, that is to say that particles of different sizes will sediment at different speeds.

The amplitude of wobulation, that is to say the difference f ₂ -f ₁ , essentially depends on the volume of suspension to be insonified, and on the distance between the ultrasonic probe or transducer 24 and the liquid / air interface.

In practice, it will be necessary to determine the acoustic intensity, the working frequency, the repetition frequency and the insonification time for each type of reaction.

We know that, taking into account the difference between agglutinated and free red cells as regards adhesion to a surface, a positive reaction is characterized by a veil image, while a negative reaction is characterized by a ring or a point. round at the bottom of the capsules.

The image reader will therefore analyze the central optical intensities, and a peripheral network of optical intensity, in each capsule. The ratio of these intensities makes it possible to classify the reaction as positive, negative or doubtful.

Reading can also be done only by a network image analysis system, covering the entire surface of the capsule. Of course, the threshold values of this report depend on the type of reaction concerned, and these threshold values are previously entered in the corresponding analysis program.

The means of the invention allow a substantial acceleration of the carrying out of immunological tests, and above all a complete automation thereof.

When we know that the current dispenser used for step 11 allows the capsules to be filled in less than a minute, and that the automatic image reading also takes very little time, it is very advantageous to use the means of the invention, which considerably lower the intermediate time necessary for the reading to take place.

Examples of reactions carried out according to the invention will be given below.

EXAMPLE 1 (TABLE 1)

This example concerns an ABO blood grouping test.

The antigens are carried by red blood cells from a group Al donor. The dilution is carried out at 1% in physiological saline (NaCl) at 9%.

The antibody is an anti-A test serum, as supplied by the National Center for Blood Transfusion.

The results are shown in the attached Table 1, in which:

V denotes the excitation voltage of the transducers in volts,

f denotes the central excitation frequency in MHz, T ₁ indicates the duration of insonification, in seconds,

T ₂ indicates the duration of sedimentation, in seconds and / or minutes, as the case may be.

A control sample was prepared according to the methods of the prior art. The sedimentation time was 30 minutes. The results were strongly positive or positive up to the 1/128 dilution, doubtful for the 1/256 dilution, and negative for the other weaker dilutions.

Two experiments were carried out with insonification, the duration of sedimentation being 12 minutes in the first case, and 1 minute 30 seconds in the second. The results are the same for these two experiments, since these results are strongly positive up to the 1/256 dilution, doubtful for 1/512, and negative for the weaker dilutions.

It is observed first of all that the duration of sedimentation can be made very short, since there is no difference after insonification between the sedimentation of 12 minutes and that of the 30 ".

We also observe that the results obtained with insonification are completely consistent with those obtained by means of the prior art.

Finally, we observe that the application of ultrasound gives the method a better sensitivity, since the questionable sample at dilution 1/256 now becomes positive, the doubt no longer remaining until dilution 1/512.

EXAMPLE 2 (TABLE 2)

This example concerns a test relating to hepatitis B, known as "HBS AG research". The HBS antigen is sought in the serum of three subjects, denoted respectively Tra, Nal, Dix.

The antibodies are carried by sensitized red cells, as well as control antibodies, available in the so-called Hematest VKOl kits sold by the American company Wellcome.

The parameters of the ultrasound application are defined as above.

According to the usual method in this type of test, each sample is subjected to the test antibody, then to the control reagent, and this first without ultrasound, then with ultrasound, as indicated in Table 2.

For the Tra antigen, a negative response is observed in all cases, with or without ultrasound.

for the Nal antigen, the response is positive for the test and negative for the control, with a sedimentation time of 40 minutes.

The attached Table 3 shows that, after applying ultrasound at 3.5 MHz for 15 seconds, the same result can be obtained after only 2 '30 ".

Regarding the third sample, the conventional method gave a positive result up to the 1/128 dilution, doubtful for 1/256, and negative for 1/512.

Still after application of ultrasound at 3.5 MHz for 15 seconds and after a sedimentation time of only 2.5 minutes (instead of 40 minutes), the application of the invention makes it possible to obtain exactly the same result.

You can immediately see the considerable practical advantages that can be gained from such speed, combined with the possibility of fully automating the test.

EXAMPLE 3 (TABLE 3)

This example relates to phenotyping in the Rhesus RH groups, and will be described with reference to Table 3.

The antigen consists of the red cells of a donor referenced D EE. These red cells are diluted in suspension at 1% in physiological saline NaCl at 9%.

Antibodies are Anti serum tests

Anti E and Anti C, sold by the company Ortho Diagnostic Systems.

Table 3 shows that the application of ultrasound at the frequency of 3.5 MHz for 20 seconds allows the reading after 2 minutes, with results in accordance with the donor's phenotype.

Comparatively, this reading would have required 20 minutes, using the means of the prior art.

Due to its great speed, the method according to the invention in many cases makes it possible to avoid the centrifugation phase, and the complications which result therefrom.

Furthermore, there are reactions, including the so-called indirect COOMBS reaction tests, which require one or more washes of the red cell suspension, the conventional washing imperatively comprising: centrifugation, aspiration of the supernatant, redistribution of the liquid washer and resuspension.

When the test container is a microplate, such washing requires delicate handling of this microplate, during the centrifugation and agitation steps. This prevents automation of the test. As already mentioned, it has been proposed to homogenize and disperse suspensions by ultrasound. However, this solution cannot be used in most biological tests, because the homogenization and dispersion effect is linked to the cavitation created by low frequency ultrasound, which causes the destruction of part of the cells.

The present invention proposes to operate differently.

According to the invention, the capsules of the microplate are sonicated with an ultrasonic beam of relatively high frequency, typically 1 to 10 MHz. It has been observed that, under these conditions, the ultrasonic wave does not cause cavitation, therefore no hemolysis of the cells. Provided that the ultrasonic beam is correctly positioned, so as to create a vortex in the capsules, there is an almost instantaneous resuspension of the red blood cells. Unlike the stirring methods of the prior art, this process is very fast, and easily automated.

This can be implemented using the experimental devices of Figures 2A and 2B.

However, it will often be preferable to use a focused ultrasound beam, as will be seen now.

The reflector 25 is now in FIG. 4 a concave reflector 25C, which allows the ultrasonic beam to be focused on a row of capsules, in such a way that the beam is convergent inside the capsule.

As before, the different caps can be insonified by moving the reflector 25-C according to arrow 26.

A variant also consists in providing a battery of trans ductors (similar to that of FIG. 2B), but arranged either individually or in combination with each other, to produce in a convergent or wobulated beam at the level of each of the rows of capsules.

Of course, instead of moving the reflector 25 (Figure 2A) or 25-C (Figure 4), we may prefer to move the plate 21 itself, note that the device will most often include a treadmill to bring this plate at the level of the ultrasonic cell.

It was observed above that the invention can be implemented either with a single ultrasonic transducer associated or not with a movable reflector, or with a network of ultrasonic transducers, each of which is capable of exciting, for example one of the capsules of the microplate.

The advantage of frequency wobulation of the ultrasonic signal has also been indicated, which makes it possible to produce a coherent, but slowly mobile stationary ultrasonic field. It is also possible to obtain such a coherent and slowly moving stationary ultrasonic field by using two (or more) transducers, to which signals are made which have between them a variable phase shift with time. This can lead to providing several transducers for each capsule.

The repetition frequency (of the wobulation, or the phase shifts varying with time) is advantageously chosen so that a new cycle of variations begins when the particles which were located at the first node corresponding to the starting frequency of the wobulation reached the first belly corresponding to the frequency of the end of the wobulation, as can be seen from Figures 3A and 3B.

Thus, the invention allows automated and accelerated hemagglutination in microtiter plates, for testing following:

- ABO and rhesus grouping,

- determination of phenotypes,

- search for irregular antibodies,

- HB antigen (hepatitis B) test for syphilis and tetanus antibodies, or other common antibodies,

- passive hemagglutination reactions.

The invention also makes it possible to carry out tests of the Coombs type, without any centrifugation or mechanical agitation.

Furthermore, resuspension can easily be achieved by producing spherical, that is to say convergent, ultrasound beams directed towards the wall of the capsules. A vortex is thus obtained in the capsules, and an almost instantaneous resuspension of the free particles, that is to say those which are not agglutinated. The advantage is that by operating at high frequency, the homogenization and the resuspension are done without cavitation or destruction of the cells.

This is applicable to an ultrasonic washing process, in microtiter plates or in test tubes. This washing is combined with sedimentation and resuspension by ultrasound.

The automatic device useful for this purpose consists of a distributor, an ultrasonic washing unit, and a supernatant aspirator.

It will also have been observed that the invention allows accelerated sedimentation of latex particles or other particles. carrier cules usable in artificial agglutination reaction, or radioimmunological, or for immuno-enzymatic reactions.

Reference is now made to FIGS. 5 and 6 for the description of variants of the invention. The container 30 here consists of a tank, having an inlet port 31 in the lower part, on the left, for receiving, for example, blood. This tank has two outlet ports on the right, at the top an outlet 32H for the plasma, and at the bottom an outlet 32L for the sedimentation pellet.

Along the left wall of the tank 30 is provided an ultrasonic transducer 34 excited by a generator 33. The opposite wall 35 of the tank 30 is arranged as a reflector.

A coherent stationary field is thus developed inside the tank 30. It allows the sedimentation at the level of the 37V bellies of the ultrasonic field. This results in a convection movement, which results in a rise in the locations of the nodes 37N of the ultrasonic field.

Given the attenuation of ultrasound in the suspensions, here the blood, the optimal width of the sedimentation cell 30 depends on the ultrasonic frequency used.

At the frequency of 3MHz, at which the radiation force of the ultrasound makes it possible to agglomerate the red cells in a few seconds, this width is 0.5 cm for whole blood.

The assembly of Figure 5 allows, for a concentrated suspension to perform a preliminary separation or pre-separation.

The final separation can then take place, after this pre-separation, or directly if the initial suspension was not very concentrated. The assembly of Figure 6 allows for this final separation.

In this arrangement, the container 40 has, on the left, an inlet 41 for the blood, on the right, exits 42H at the top for the plasma and 42L at the bottom for the base.

A generator 43 excites a transducer 44 brought into contact with the upper surface of the liquid present in the container 40. In contrast, the bottom of the container 40 receives at 45 either a reflector or another ultrasonic transducer, as will be seen below. -after.

The member 45 being a pure reflector, the signal produced by the generator 43 is wobbled under the conditions indicated above. The agglomerated clusters are then moved downwards due to this frequency wobulation, or else thanks to an intermittent excitation of the generator, in which case there is alternation of the agglomeration and sedimentation phases.

If the member 45 is another transducer, the latter is then excited with a signal of the same frequency as the transducer 44, but with a phase shift variable in a linear or quasi-linear fashion over time.

This phase shift creates an almost continuous displacement of the nodes and the interference bellies, and causes a displacement of the red blood cells downwards, an effect reinforced by the acceleration of gravity, that is to say gravitation.

An assembly similar to that of FIG. 6, but in which the two transducers are mounted horizontally, on either side of a membrane, makes it possible to carry out the final separation with this filtration membrane.

The advantage of using ultrasound is that in this case the polarization layer usually encountered on the membrane can be greatly reduced or even destroyed by the force of ultrasonic radiation. It depends on the balance between hydraulic forces, viscous forces and forces related to the pressure of ultrasonic radiation.

In such a case, the choice of the ultrasonic frequency depends on the size of the particles to be filtered. The required acoustic intensity depends on the characteristics of the suspension and the transmembrane pressure. Preferably, the two transducers used work at the same frequency, but with a quasi-linear phase shift over time, and a repetition rate chosen as indicated above.

Claims

Claims.

1. Biological treatment method in which a biological liquid medium (22) containing particles in suspension is placed in a container (21), this medium and these particles being associated one with antibodies, the other with antigens, so that an immunological reaction can be established between the antibodies and the antigens and possibly lead to agglutination of the particles, characterized in that a coherent beam of ultrasound (23, 24, 25), at a frequency between 0.1 and 100 MHz, capable of producing radiation forces favoring the regrouping of these particles.

2. Method according to claim 1, characterized in that the radiation forces produced by the ultrasonic beam are used to promote an agglutination reaction of the particles.

3. Method according to claim 1, characterized in that the radiation forces produced by the ultrasound beam are used to promote the exchange of the biological liquid medium, for washing purposes, the particles being grouped together.

4. Method according to one of claims 1 to 3, characterized in that the frequency of the ultrasound is between 0.5 and 20 MHz, approximately.

5. Method according to one of claims 1 to 4, characterized in that the frequency of the ultrasonic field is wobbled.

6. Method according to one of claims 1 to 5, characterized in that the power density of the ultrasound beam is 0.1 to 100 W / cm ² , preferably 0.5 to 20 W / cm2, approximately .

7. Method according to one of claims 1 to 6, characterized in that the container (21) is placed externally in contact with an ultrasound transfer medium, in particular a liquid (29) such as water, or else a flexible medium such as a silicone-based material.

8. Method according to claim 7, characterized in that the container (21) comprises a multiplicity of capsules or cells and is constituted, for example, by a microtitration plate.

9. Method according to one of claims 1 to 8, characterized in that the direction of the ultrasonic field in the container is substantially aligned on the vertical thereof.

10. Method according to one of claims 1 to 9, characterized in that the liquid medium (22) is water associated with a pH buffer and containing proteins in solution, and in that the particles are cells or microparticles suitable for forming reaction substrate.

11. Method according to claim 10, characterized in that the liquid medium (22) is a solution of red blood cells at low concentration.

12. Method according to claim 10, characterized in that the particles are latex microparticles.

13. Method according to one of claims 10 to 12, characterized in that the ultrasound beam is applied so as to establish a substantially stationary regime in a direction of space.

14. Method according to one of claims 1 to 9, characterized in that the ultrasound beam is focused on the liquid medium, which allows the particles to be put back into solution without the lysis effect associated with cavitation.

15. Biological treatment installation, characterized in that it comprises an ultrasonic cell, suitable for applying a coherent beam of ultrasound, of frequency between 0.1 and 100 MHz, to at least one container containing a biological liquid medium with particles in suspension, the beam being capable of producing radiation forces favoring the regrouping of these particles.