WO2019020932A1 - Method for processing a sputum sample and method for the rheometric measurement of a preprocessed sample of this kind - Google Patents

Abstract

The present invention relates to a method for processing a sputum sample (1) comprising a saliva phase (2) and a sputum phase (3) subjacent to the saliva phase (2) and containing mucus plugs (6), said method comprising the following steps: - separating the sputum phase (3) and the saliva phase (2) by drawing off the sputum phase (3) and transferring it to a recipient (10) having a flat bottom; and homogenizing the sputum (3) by fractionating the mucus plugs (6) by vortex shaking of the sputum (3), the shaking speed being adjusted such that in the recipient (10) the shape of the sputum (3) is generally toroidal in a plane parallel to the bottom of said recipient (10).

Description

 METHOD FOR TREATING A SPUTUM SAMPLE AND METHOD FOR RHEOMETRIC MEASUREMENT OF SUCH A PRE-TREATED SAMPLE

FIELD OF THE INVENTION

 The present invention applies to the analysis of bronchopulmonary secretions of a subject. The present invention more specifically relates to a method of treating a sample of bronchopulmonary secretions of a subject and a rheometric measurement method made from the sample previously treated by the treatment method.

STATE OF THE ART

 Bronchopulmonary secretions, also called "sputum", are obtained by expectoration of a subject. Their rheometric analysis can potentially detect a large number of diseases in subjects, track their evolution over time as well as evaluate the effectiveness of treatments administered to the subject or the treatment effect applied in vitro on sputum.

 In particular, it may be useful to perform rheomeric measurements on sputum in several cases, particularly in the context of screening for pathologies with pulmonary involvement. This may include, for example, early detection of chronic obstructive pulmonary disease (COPD), monitoring of COPD or cystic fibrosis progression over time to predict the occurrence of exacerbation monitoring the effectiveness of antibiotic treatment in the event of bronchial infection, or the qualification of drug candidates in clinical trials in the biotechnology and pharmaceutical industry. In basic or therapeutic research, the in vitro treatment of sputum followed by its analysis is a source of information, from the physiopathology to the screening of molecules (pharmacodynamics and efficacy studies).

A first obstacle to making rheometric measurements on a sample of sputum is the repeatability of these measurements. At present, it is not possible to perform repeatable rheometric measurements on a sample of sputum directly obtained by expectoration of a subject without prior treatment of this sample. Indeed, the sample taken from the subject contains a variable amount and sometimes not negligible and uncontrollable saliva in addition to the sputum. With reference to FIG. 1, the sample 1 then comprises a saliva phase 2 and an underlying sputum phase 3 and in contact with the saliva phase. The boundary between these two phases is represented by curve 4 on Figure 1. Because saliva has different rheological properties than sputum, a rheometric measure of the sample would be distorted by the presence of saliva. In particular, saliva induces undesirable dilution of the sputum, in addition to including bubbles.

 A second obstacle to making rheometric measurements on a sputum sample is the presence of "mucous plugs" in the sputum. These mucosal plugs are dense and thick secretions of the bronchial mucosa that are in the form of large clumps relative to the biological content of the sample. These plugs are composed in particular of partially dehydrated mucus (by the repeated action of the passage of air on static mucus during respiration), bacteria, biofilms, pus, and blood cells, and their composition varies in subject function. They are particularly numerous in sputum samples taken from subjects suffering from chronic respiratory diseases such as the BCPO and cystic fibrosis, for example.

 The mucous plugs are heterogeneously distributed in the sputum, the latter being a medium with viscoelastic properties about ten to one hundred times lower than those of the mucous plugs. In addition, the size of the mucous plugs is very variable. This results in a strong heterogeneity of the sputum.

 In a rheometric measurement of a sputum sample by a suitable rheometric measuring device, the high heterogeneity of the sputum results in uneven distribution of the mucous plugs on the measurement geometry of the apparatus.

 However, in a well-known manner, a conventional rheometry apparatus commonly used for carrying out such a measurement comprises a measuring cell comprising a rotor and a stator. The rotor comprises a "low" or "high" rotor geometry and the stator comprises a "low" or "high" geometry of the stator (depending on the different technologies), along a vertical axis corresponding to a current use of the apparatus these two geometries being coaxial and in contact with the sample when the latter is introduced into the measuring cell. The rotor and stator geometries may have different geometric shapes from one device to another, such as a cone or a cylinder for example.

Sample 1 of sputum is placed on the low geometry 7 shown in Figure 2, the surface of which is generally disk-shaped. The rotor applies shear to the sample via the controlled rotation of one of the geometries, and the apparatus measures the resultant stress applied by the sample via the torque exerted on one of the geometries. The ratio of the measured torque (from which the stress is deduced) and the rotational speed of the rotor (of which one deduce the shear) allows to deduce the rheological characteristics or viscoelastic properties of the sample.

 Thus, the most sensitive area of the measurement is the peripheral zone 18 of the measurement cell (the torque being directly proportional to the lever arm), which extends between the schematic circle 8 and the border 9 of the cell. However, the distribution of the mucous plugs 6 in the cell is almost random and the proportion of mucous plugs in the peripheral zone 18 is not controllable, the latter may be more or less high than the average proportion of mucous plugs in the sample . This results either in overestimating the viscoelastic properties of the sample or in underestimating them. This results in a large disparity of rheometry measurements on the same sample and therefore very poor repeatability of the measurements.

 A third obstacle to making rheometric measurements on a sputum sample is the large size of the mucous plugs compared to that of the measuring cell. In fact, by applying the continuous medium hypothesis, which is a basis for rheological calculations, the characteristic size of the particles or inclusions present in a material must be negligible compared to the dimensions of the measuring cell of the rheometry apparatus. . In theory, a factor of 100 is commonly accepted, but in practice it is necessary that a factor of at least ten exists between the size of the inclusions and the size of the air gap of the measuring cell.

 In view of the foregoing, it is therefore necessary to have a method of treating a sputum sample allowing the subsequent realization of precise and repeatable rheometric measurements, and this especially in the case where the sputum of the sample contains mucous plugs.

 WO 94/10567 proposes a method for measuring the centrifugation pellet of a sputum sample obtained on a subject suffering from a respiratory disease, in which the sample is centrifuged in order to split into a so-called phase pellet phase. And a supernatant. "Pellets" are mucous plugs which are then measured according to the described method.

In this document, the centrifugation simply makes it possible to concentrate the solid constituents in the "phase pellet", and thus to separate several phases of the sample according to their density. This method is therefore specifically adapted to the realization of rheometric measurements on the mucous plugs, but not on the entire sputum including such mucous plugs. SUMMARY OF THE INVENTION

 The object of the invention is therefore to overcome the drawbacks of the prior art by proposing a sputum sample processing method making it possible to obtain precise and repeatable rheometric measurements later, and this especially in the case where the sputum sample contains mucous plugs.

 Another object of the invention is to propose such a treatment method, the production of which does not degrade the viscoelastic properties of the sputum.

 The aim of the invention is also to propose a process for the rheometric measurement of a sample of sputum treated according to the preceding treatment method, the realization of which enables precise and repeatable rheometric measurements to be obtained, and this especially in the case where the Sample sputum contains mucous plugs.

 To this end, according to a first aspect, the invention provides a method of treating a sputum sample from a subject comprising a saliva phase and a sputum phase underlying the saliva phase and comprising plugs. mucosa, the process comprising the following steps:

 Separating the sputum phase and the saliva phase by taking the sputum phase and transferring it to a container equipped with a flat bottom,

Homogenization of the sputum by fractionation of the mucous plugs by means of agitation of said sputum by vortex, the stirring speed being adjusted so that the sputum takes in the container a general toric shape which extends in a parallel plane at the bottom of said container.

 A torus designates in geometry the surface generated by the revolution of a circle around a non-diametral line coplanar with the circle, and by extension, the volume limited by this surface. The term "toric general shape" reflects the fact that in practice, according to the method of the invention, the sputum adheres to the side wall and the bottom of the container in which it is located. It then marries at least in part the contours of the side wall and the bottom of the container, which causes a slight deformation of the torus. The torus may be open if the amount of sputum is low or if the viscoelastic properties of the sputum are very pronounced (high viscosity) so that the sputum forms a strongly cohesive whole. Otherwise, the torus is closed.

 For the sake of brevity, the general toric shape is designated by the term "torus" in the remainder of the description.

According to other aspects, the proposed treatment method has the following different characteristics taken alone or according to their technically possible combinations: the container advantageously has a circular section with a diameter of between 30 millimeters and 50 millimeters, at the level of the sputum stirring zone; the stirring speed is preferably less than or equal to 6000 rpm; the sputum is homogenized by imposing a predetermined sputum shear rate, the value of which is less than a critical value below which the viscoelastic properties of the sputum are not degraded;

the critical value of the shear rate is about 50 s ^-1 ;

 during the implementation of the step of separating the sputum phase and the saliva phase, the sputum is subjected to a shear rate whose value is lower than the critical value described above. In other words, the separation step is performed in such a way as not to impose on the sputum a shear rate whose value is greater than the critical value;

 the sputum phase is completely removed and transferred to the container with a positive displacement pipette;

 The invention also proposes a method for rheometric measurement of a sputum sample, comprising the following steps:

 The treatment of the sputum sample according to the treatment method described above, in order to obtain a homogeneous sputum,

 The transfer of the homogenized sputum into a measuring cell of a rheometric measuring apparatus,

 Performing one or more rheometric measurements of at least one rheological parameter of the sputum.

 According to other aspects, the proposed rheometric measurement method has the following different characteristics taken alone or according to their technically possible combinations:

 the step of transferring the homogenized sputum into the measuring cell is carried out by imposing on the sputum a shear rate the value of which is less than a critical value below which the viscoelastic properties of the homogenized sputum are not degraded;

the critical value of the shear rate is equal to approximately 50 s ^-1 ;

the homogenized sputum is transferred to the measuring cell with a positive displacement pipette. DESCRIPTION OF THE FIGURES

 Other advantages and characteristics of the invention will appear on reading the following description given by way of illustrative and nonlimiting example, with reference to the appended figures which represent:

 - Figure 1, an image of a sample of sputum taken from a subject with cystic fibrosis;

 Figure 2, a diagram of a low geometry of a rheometric measuring cell containing a non-homogenized sputum, according to the state of the art;

 - Figure 3, a diagram illustrating the agitation of a sputum, previously separated from the saliva, by vortex, the sputum taking the form of a torus;

 Figure 4 is a diagram of a rheometric measuring cell containing a sputum treated according to the treatment method of the invention;

 Figure 5 is a diagram of a low geometry of a rheometric measuring cell containing a sputum homogenized by the treatment method of the invention;

 FIG. 6, a graph illustrating the evolution of the viscoelastic modulus of sputum as a function of the shear rate for five samples, according to a first measurement protocol;

 FIG. 7, a graph illustrating the evolution of the viscoelastic module of the sputum as a function of the shear rate for a sample, according to a second measurement protocol.

DETAILED DESCRIPTION OF THE INVENTION

 A first aspect of the invention relates to a method of processing a sputum sample. Referring to Figure 1, a sample 1 of sputum comprises two superimposed phases, a first overlying phase composed of saliva 2 and an underlying phase composed of sputum 3. The boundary between these two phases is represented by curve 4 in Figure 1.

 The saliva phase 2, or more simply "saliva", mainly comprises saliva and is found systematically in the sputum sample obtained by expectoration of a subject.

However, it is possible to reduce the amount of saliva in the sample taken, for example by placing cotton protective elements in the subject's mouth during the sampling, cotton to absorb a large amount of saliva, or in asking the patient to spit as much saliva as possible, or to drink a glass of water and then spit again, before sputum (the sensation must be to have a dry mouth during sputum).

 As an indication, spontaneous expectoration and induced sputum are commonly used sampling methods, induced sputum consisting of inspiring a mixture of air and fine particles of hypertonic saline between 3 and 5% ten to twenty minutes using a nebulizer, with the aim of slightly irritating the bronchi and producing a greater secretion of sputum.

 The phase of sputum 3, or more simply "sputum", mainly comprises sputum, that is to say, bronchopulmonary secretions. When the subject on which the sample is made is suffering from a disease causing bronchopulmonary complications, such as COPD or cystic fibrosis, for example, the sputum phase comprises a liquid phase in which mucous plugs 6 are exposed under the form of granules or clusters whose size is large enough to be visible to the naked eye.

 The presence of mucosal plugs 6 confers on sputum 3 a strong heterogeneity, which distorts any subsequent measurement of rheometry of the sample. The proposed method solves this problem.

 From a sample 1 of sputum taken from a healthy subject or suffering from a respiratory disease, sputum 3 is first separated from saliva 2. To do this, sputum 3 is aspirated using of a sampling device, without sucking saliva supernatant. The suction port of the sampling device is positioned in the sputum, below the border between sputum and saliva. It is best to position the suction port of the device at the bottom of the sample at the base of the sputum, to ensure that the saliva is not sucked with the sputum. The sputum 3 is then transferred in its entirety into a suitable container 10, such as a sterile container for example (Figure 3).

 An important aspect of this sampling step is to aspirate the mucous plugs 6. In fact, it is advisable not to simply take the liquid phase of sputum by "sorting" the mucous plugs (effect related to pipetting, which aspires preferentially the less consistent liquid phase), but to collect the sputum 3 in its entirety.

It will therefore be ideal to use a sampling device adapted to this purpose, and in particular a device of this kind provided with a suction orifice whose characteristic size (generally the diameter, in the case of an orifice circular) is at least of the order of magnitude of the characteristic size of the mucous plugs in order to allow the passage of the mucous plugs through the suction port. Indeed, by their highly viscoelastic nature, mucous plugs tend to remain intact and relatively deformed during aspiration. If their characteristic size is much greater than that of the suction port, they are not preferentially aspirated and are sorted. Thus, the proportion of mucous plugs in the sputum subsequently transferred into the container provided for this purpose is low and not representative of that initially present in the sample taken from the subject.

 Advantageously, use will be made of a pipette provided with a nozzle with a large suction port to collect the sputum, and more particularly a positive displacement pipette provided with a nozzle with a large suction orifice.

 A positive displacement pipette (not shown) has the advantage of transferring the collected sputum with minimal loss. Such a pipette is specially designed for pipetting dense and viscous liquids. It comprises a piston in direct contact with the liquid that moves in a nozzle, and the positive wiping action of the piston against the inner wall of the nozzle prevents a portion of the liquid remains adhered thereto. It is indeed necessary to minimize the losses of sputum during sample processing in order to ensure the good feasibility of a subsequent rheometric measurement, especially in the relatively frequent case where the amount of sample taken from the subject is weak and just sufficient to achieve the rheometric measurement. Moreover, in a traditional pipette, suction being done by depression created by the displacement of a piston which is not in direct contact with the sample, the air column which overhangs said sample may experience variations of volume more or less important because of its high compressibility. As a result, the volume of a very consistent aspirated fluid depends on its mechanical properties, which can make successive pipetting non-repeatable. A positive displacement pipette eliminates this problem since the piston is in direct contact with the sample, thus eliminating the air buffer volume.

 Sputum freed from saliva is then homogenized by stirring by means of a stirring device. Homogenization consists in imposing a shear to the sputum, which corresponds to a predetermined shear rate, in order to split the mucous plugs. This results in a sputum containing the sputum fluid in which mucosal plug fractions well distributed in said liquid, and whose size is considerably smaller than that of the mucous plugs from which they are derived, are found.

The homogenization step is carried out by means of vortexing. This type of stirring makes it possible to increase the occurrence of effective secondary flows which allow a better homogenization of the sputum compared to stirring at room temperature. by means of other mechanical agitators (rotary or shaking) or by means of magnetic stirrers, and without requiring the introduction of a mobile. In addition, the vortex agitation offers the advantage of being able to visually ascertain that the sputum is optimally homogenized. In more detail, an optimal homogenization of the sputum by vortex results in the sputum 3 forming a torus in the container 10, as shown in FIG. 3.

 The torus 3 rotates about its axis of revolution A, which corresponds to the axis of revolution of the container (which is perpendicular to the bottom of the container 10), according to a flow called "primary flow" represented by the arrow 1 1 in a plane P1, and also rotates on itself in a transverse movement (or meridian) called "secondary flow" in a plane P2 substantially orthogonal to the plane P1 and oriented from the center of the torus outwardly of the torus and inversely according to the arrows 12 and 13.

 The presence of the secondary flow 12, 13 improves the mass transfers within the sputum 3. This results in a better homogenization of the latter.

Shaking the sputum by vortex means shearing it, which corresponds to a value of shear rate. And in general, the shear rate is also called "velocity gradient", and allows to measure the shear applied within a fluid. For a viscous fluid such as sputum, the shear rate γ ^'is expressed in s ^-1 (inverse second) and is related to the shear stress τ and the viscosity η of the fluid by the following relation: τ = η χ ^γ. Furthermore, the shear stress τ corresponds to applying a tangential force on a fluid surface.

 It is therefore understood that the shear rate imposed on the sputum depends on the stirring speed, the structural properties of the container 10 which contains the sputum (in particular the shape and the volume of the container), and the physico-chemical properties of the sputum (especially the volume of sputum in the container and its viscoelastic properties).

 Therefore, the stirring speed is adjusted, taking into account the structural properties of the container and the physico-chemical properties of the sputum, until a torus 3 is obtained.

 Optionally, the stirring speed can then be further varied to provide a speed range for which the sputum forms a torus. As long as the stirring speed is chosen to be included in this range, the sputum is optimally homogenized.

 When the torus is formed, the stirring is carried out for a period generally of between 20 seconds and a minute, in particular a time of 20 seconds, to properly homogenize the sputum.

It is specified that in certain cases, especially when the sample is bulky (more than 5 ml), it may be useful to remove a certain volume of sputum from the container 10 in order to be able to form a torus when its formation does not take place at a given stirring speed, and in particular when its formation does not take place for all the stirring speed values of the range.

 The container 10 in which is transferred and homogenized the sputum has meanwhile a circular section and is advantageously in the form of a cylinder. It is provided with a flat bottom and preferably has a diameter of between 30 and 55 millimeters at the sputum stirring zone, and therefore the formation of the torus. The presence of a flat bottom (as opposed to a conical bottom) makes it possible to obtain a homogeneous fluid flow on the surface of the bottom, which makes it a characteristic necessary for the formation of a torus. A diameter of between 30 and 55 millimeters makes it possible to form a torus at reasonable stirring speeds, that is to say, stirring speeds which are not too high and within the speed range allowed by the apparatus. conventional homogenization so that homogenization can be performed under standard laboratory analysis conditions.

 As previously described, adjusting the stirring speed of the sputum makes it possible to adjust the shear rate. And shaking the sputum by imposing a predetermined shear rate allows to split mucosal plugs, on the one hand so that they have a sufficiently small characteristic size for the assumption of continuous media detailed above is satisfied during a measurement rheometric subsequent sputum and homogenized, and secondly so that they are evenly distributed on the measurement geometry, which ensures the feasibility, reliability and reproducibility of the measurement.

 The shear rate is preferably adjusted so that its value is less than a critical value below which the sputum, and in particular its viscoelastic properties, are not degraded. Indeed, it is desirable that the mucous plugs are fractionated to allow a subsequent rheological analysis, while ensuring that the sputum retains its initial viscoelastic properties. In doing so, in addition to ensuring the feasibility of a rheometric measurement on a sputum, the proposed treatment method achieves rheometric results consistent with the state of the subject.

To do this, the stirring speed of the sputum is adjusted so that the sputum forms a torus and the shear rate imposed on it is less than the critical value. In detail, the sputum is agitated with a given stirring speed until a torus is obtained and the corresponding shear rate is determined. If this shear rate is below the critical value, the process is continued. Otherwise, continue adjusting the stirring speed in a speed range allowing the formation of a torus, until a shear rate lower than the critical value.

A critical value of shear rate equal to about 50 s ^-1 will preferably be chosen. Shaking the sputum by imposing a shear rate equal to or less than this value of 50 s ^-1 makes it possible to obtain a good homogenization of the sputum. avoiding to damage it. The choice of this value was made following the realization of experimental tests detailed in the example at the end of the text.

 It is also specified that the critical value is decisive in the step of separating the sputum phase and the saliva phase described above. During the implementation of the separation step, the sputum is subject to a shear rate which is imposed upon it during its sampling. Care should be taken that the sputum is removed and that the two phases are well separated while ensuring that the sputum shear rate is below the critical value defined above.

 Obtaining a torus, without degrading the viscoelastic properties of the sputum, therefore results from a judicious adjustment of the stirring speed, based on: the structural properties of the container which contains the sputum, the physico-chemical properties of the sputum, and the rate shear value whose value must be less than the critical value.

As an indication, for a container whose volume is of the order of a few tens to a few hundred milliliters, a stirring speed of between 200 rpm and 6000 rpm will be chosen. For example, a volume of 3 milliliters of sputum in a sterile pot of 40 milliliters and 12 millimeters radius, agitated at a speed of 4000 revolutions per minute on a commercial vortex, is likely to undergo a shear rate of about 10 s ^"1. Similarly, a volume of 3 milliliters of sputum in a sterile container of 40 milliliters and 12 mm radius, stirred at a speed of 6000 revolutions per minute, is capable of undergoing a shear rate of about 15 s ^"1 . It should be noted that in both cases the value of the shear rate is less than the preferred critical value of 50 s ^-1 .

 After shaking, it is visually examined whether mucous plugs are still present in the sputum. If this is the case, we renew the agitation as many times as necessary until we no longer see mucous plug. Otherwise, the sputum is sufficiently homogeneous to make a rheometric measurement.

This visual examination is relevant because the human eye is on average able to detect a minimum granulosity corresponding to a tenth of a millimeter. Therefore, if the mucosal plugs are not detectable to the naked eye, this means that they have been fractionated into particles whose characteristic size is less than one-tenth of a millimeter. The homogenized sputum is then transferred to a measuring cell of a rheometric meter. Any suitable sampling device may be used, such as those used for the initial sputum removal step in the sample taken from a subject that has been previously described. A positive-displacement pipette with a wide mouthpiece will be preferred in order firstly to limit the loss of sputum, and secondly to ensure that, in the case where mucous plugs whose reduced size makes them no detectable to the naked eye, they are well transferred into the measuring cell with the rest of the sputum.

 With reference to FIG. 4, the suction orifice is placed in the bottom of the container 10 in order not to generate air bubbles during aspiration, and the sputum 3 is sucked in order to fill a cell 15 with measuring the rheometric measuring device, that is to say in order to fill the air gap 17 of the measuring cell. The volume of the gap 17 corresponds to the volume between the high geometry 16 and the low geometry 7 of the measuring cell 15.

 Advantageously, an air gap 17 with a characteristic size of about 1 millimeter is provided for the rheometric measurement. As a result, there is a factor equal to ten between the air gap 17 and the particle size limit from which the particles are detectable to the naked eye, and we are at the upper empirical limit of the hypothesis of continuous environments.

 Sputum 3 is injected into the measurement cell in a given region of the cell depending on the type of cell. For example, the sputum is injected at the center of the cell for a "plane-plane" or "cone-plane" type cell, and at the bottom of the cell for "duvet" type cells. It should be ensured that the sputum is evenly distributed in the cell, and not creating air bubbles during the injection, so as not to distort the measurement.

 When the volume of sputum 3 injected corresponds to the volume of the cell 15, the sputum 3 occupies the entire volume of the air gap 17 and its periphery is at the right of the high geometry 16 and the low geometry 7 of the cell.

 When too much sputum has been injected into the measuring cell, the periphery of the sputum protrudes outside the high geometry and low geometry of the cell. In this case it is possible to deburr, which consists of scraping the periphery of the sputum with a spatula in order to better distribute the sputum within the cell.

With reference to FIG. 5, the homogeneous sputum is in the form of a liquid in which mucosal plug fractions of very small size swim in front of that of the mucous plugs from which they originate. The sputum 3 then extends homogeneously over the whole of the low geometry 7 of the measuring cell 15, and particularly in the peripheral zone 18, thus allowing a precise and repeatable rheometric measurement of the sputum.

EXAMPLE

Evolution of the viscoelastic modulus of the sputum as a function of the shear rate and determination of the critical value of the shear rate.

 The tests presented below were conducted to determine a critical shear value above which the viscoelastic properties of the sputum are likely to be deteriorated. To do this, the viscoelastic module of five sputum samples, which were previously separated from the saliva and homogenized according to the method described above, was measured before and after the application of shear stresses.

 The five samples are each placed in a measuring cell of a "DHR3" type rheometer marketed by the company "TA INSTRUMENTS", the cell being of rough plan-plane type 25 millimeters in diameter. The cell is maintained at approximately 37 ° C. ± 0.5 ° C. thanks to a Peltier element.

 Subsequently, for each of the five samples, the following successive steps are carried out:

 the oscillation module of the sample is measured in oscillation,

a shear rate of 0.43 s ^-1 is applied to the sample,

 the oscillation module of the sample is measured in oscillation,

a shear rate of 4.3 s ^-1 is applied to the sample,

 the oscillation module of the sample is measured in oscillation,

a shear rate of 43 s ^-1 is applied to the sample,

 the viscoelastic module of the sample is measured in oscillation.

 The viscoelastic module measured previously for different imposed shear values is written according to the following expression: G = G '+ iG "in which:

 - G 'represents the real part of G, and characterizes the elastic behavior of the sample. G 'is generally designated as a preservation module;

G "represents the imaginary part of G, and characterizes the viscous behavior of the sample G" is generally referred to as a loss module or dissipation module.

The results of the measurements of the viscoelastic module of the sample as a function of the imposed shear rate are represented by the graph of FIG. 6. Each value of shear rate corresponds to a value of the conservation modulus G 'and a value of Loss modulus G "The values of G 'and G" are the average values obtained for the five samples.

We hypothesize that there is shear deterioration of the viscoelastic properties of the sample when the conservation modulus G 'decreases below its reference value obtained before any shear (0 s ^"1 ), with a deviation greater than or equal to 10% of said reference value after shear The value of 10% corresponds to the estimated accuracy of the measurement in rheometry.

From the results obtained, it is found that the conservation modulus G 'goes from about 23 Pa for a shear rate of 0 s ^-1 to about 21.5 Pa for a shear rate of 0.43 s ^-1 a decrease of about 6.5% compared to the reference value of 23 Pa at 0 s ^-1 , which is less than 10%.

In addition, the conservation modulus G 'increases to about 30 Pa for a shear rate of 4.3 s ^-1 , and about 25 Pa for a shear rate of 4.3 s ^-1 , compared to the value of reference of 23 Pa at 0 s ^"1 .

Thus, there is no decrease of G 'or G "by more than 10% of the reference value of 23 Pa to 0 s ^" .

Therefore, a shear rate of up to 43 s ^-1 does not degrade the viscoelastic properties of the sample.

 To observe the behavior of the sample at greater shear, a single sample is placed in a measuring cell of a rheometer, the cell being of the rough planar plane type of 25 millimeters in diameter. The cell is maintained at approximately 37 ° C. ± 0.5 ° C. thanks to a Peltier element. These measurement conditions correspond to those of the five samples of the previous protocol.

 For this single sample, the following successive steps are carried out:

 the oscillation module of the sample is measured in oscillation,

a shear rate of 1 s ^-1 is applied to the sample,

 the oscillation module of the sample is measured in oscillation,

a shear rate of 10 s ^-1 is applied to the sample,

 the oscillation module of the sample is measured in oscillation,

a shear rate of 100 s ^-1 is applied to the sample,

 the viscoelastic module of the sample is measured in oscillation.

 The results of the measurements of the viscoelastic module of the sample as a function of the imposed shear rate are represented by the graph of FIG. 7.

From the results obtained, it can be seen that the conservation modulus G 'increases between 0 s ^-1 and 10 s ^-1 , from approximately 6.3 Pa for a shear rate of from 0 s ^-1 to approximately 7.5 Pa for a shear rate of 1 s ^-1 , then about 9.5 Pa for a shear rate of 10 s ^-1 .

On the other hand, the conservation modulus G 'strongly decreases between 10 s ^-1 and 100 s ^-1 , from approximately 9.5 Pa for a shear rate of 10 s ^-1 to approximately 5.2 Pa for a shear rate of 100 s ^-1 , a decrease of about 17.5% from the reference value of 6.3 to 0 s ^-1 which is greater than 10%.

Thus, a shear rate of 100 s ^-1 degrades the viscoelastic properties of the sample.

Therefore, the critical value of shear rate beyond which the viscoelastic properties of the sample are degraded is between 43 s ^-1 and 100 s ^-1 . Given the high value of the conservation modulus G 'at 10 s ^"1 and the large difference between the shear rate values of 10 s ^{" 1} and 100 s ^"1 , it is considered that a rate value Shear rate of about 50 s ^-1 corresponds to a critical value not to be exceeded in order not to degrade the viscoelastic properties of the sputum sample during homogenization.

REFERENCES

 · WO 94/10567

Claims

A method of treating a sputum sample (1) comprising a saliva phase (2) and a sputum phase (3) underlying the saliva phase (2) and comprising mucosal plugs (6), the method comprising the following steps:  separating the sputum phase (3) and the saliva phase (2) by removing the sputum phase (3) and transferring it to a container (10) provided with a flat bottom,  homogenizing the sputum (3) by fractionation of the mucous plugs (6) by agitating said sputum (3) by vortexing, the stirring speed being adjusted so that the sputum (3) takes up in the container ( 10) a generally toroidal shape in a plane (P1) parallel to the bottom of said container (10). Process according to Claim 1, characterized in that the container (10) has a circular cross-section of diameter between 30 millimeters and 50 millimeters at the level of the stirring zone of the sputum. Process according to one of the preceding claims, characterized in that the homogenization of the sputum (3) is carried out by imposing a predetermined shear rate on the sputum (3), the value of which is less than a critical value below which the properties viscoelastic sputum (3) are not degraded. Process according to claim 3, characterized in that the critical value of the shear rate is about 50 s ^-1 . Process according to claim 3, characterized in that during the implementation of the separation step of the sputum phase (3) and the saliva phase (2), the sputum (3) is subjected to a rate of shear value below the critical value. 6. Method according to one of the preceding claims, characterized in that the sputum phase (3) is completely removed and transferred into the container (10) with a positive displacement pipette. 7. A method for rheometric measurement of a sample (1) of sputum, characterized in that it comprises the following steps:  treating the sample (1) of sputum according to the treatment method according to one of the preceding claims, in order to obtain a homogeneous sputum (3),  transferring homogenized sputum (3) into a measuring cell (15) of a rheometric measuring apparatus,  performing one or more rheometric measurements of at least one rheological parameter of the sputum (3). 8. Method according to claim 7, characterized in that the step of transferring the sputum (3) homogenized in the measuring cell (15) is performed by imposing on the sputum (3) a shear rate whose value is less than a critical value below which the viscoelastic properties of homogenized sputum (3) are not degraded. 9. Process according to claim 8, characterized in that the critical value of the shear rate is equal to approximately 50 s ^-1 . 10. Method according to one of claims 7 to 9, characterized in that the homogenized sputum (3) is transferred into the measuring cell (15) with a positive displacement pipette.