Method for determining the integrity of a deposit of a complex based on a biological sample and system for carrying out the method

Description

[0001] The present invention relates to the field of microbiology and in particular to the characterization of microorganisms from a sample using mass spectrometry. More particularly, the invention relates to an analysis system and a method for determining the integrity of sample-based complexes intended for analysis by mass spectrometry of the SELDI-TOF (Surface Enhanced Laser Desorption Ionization - Time of Flight) or MALDI-TOF (Matrix Assisted Laser Desorption Ionization - Time of Flight) type.

[0002] In the present invention, the term "complex" means the combination of at least one microorganism present in a taken biological sample and at least one matrix, the microorganism being able to be a bacterium, a virus, a yeast, a mold, a fungus, etc. and the matrix being able to be alpha-cyano-4-hydroxycinnamic acid (HCCA), 3,5-dimethoxy-4-hydroxycinnamic acid (or sinapinic acid, SA), 2,5-dihydroxybenzoic acid (DHB), β-carboline (9H-pyrido[3,4-b]indole) (or Norharmane), or any other substance known to ionize the complex during a MALDI-TOF analysis. In the particular case of microorganisms which are yeasts, the complex further comprises formic acid.

[0003] The deposition of complex on a support to be analyzed by a mass spectrometer, i.e. the deposition of a sample followed by a deposition of matrix, is currently carried out manually. Inconsistencies in this qualitative deposition are observed.

[0004] Indeed, the complex is sometimes incomplete due to the absence of the deposit of a sample or of the matrix or even of the two components and makes the mass spectrum unusable, the deposition method often being carried out manually and in a non-standardized manner.

[0005] This observation is quite alarming and solutions exist to overcome the total absence of complex such as the solution proposed by document US 7,145,135 B1, which describes an automated control system for the complex reception area for a mass spectrometer with a MALDI ionization source. The system described in document US 7,145,135 B1 is an automated flow system configured to identify the optimal location for the laser impact in order to ionize the complex deposited on a MALDI plate type support, the detection of the presence of a complex is therefore mandatory in order to determine where to position the laser, especially since the reception areas are very close together and therefore difficult to separate.

[0006] US2013 / 302847 A1 relates to a method for assisting manual sample deposition and a method for determining the deposition of a sample site on a sample support for matrix-assisted laser desorption ionization.

[0007] To date, no solution allows for more precise detection of the level of completeness of the complex or for alerting the technician in time to correct or at least identify the reception areas which present a failure due to the absence of one or more components of the complex, which generates distorted results and therefore can lead to an incorrect diagnosis.

[0008] The invention aims to remedy all or part of the aforementioned drawbacks and in particular to carry out a control of the quality and therefore of the integrity of each complex deposited on a support such as a MALDI-TOF plate comprising a plurality of reception zones in which a complex deposition is carried out.

[0009] To this end, the invention, as defined in the appended claim 1, relates to a method for determining the integrity of at least one complex based on at least one biological sample and at least one matrix, said complex being arranged in at least one receiving zone of a support intended to be analyzed subsequently, said method being implemented by a determination system comprising at least one imager, an analysis unit configured to cooperate with the imager, characterized in that said method comprises at least the following steps: acquisition of at least one image of at least one reception area of ​​the support, the acquisition being carried out by the imager of the determination system, sending the at least one image acquired by the imager to the analysis unit, analysis of the sent image by extracting light intensity values ​​representative of at least one spectral band, the analysis being carried out by the analysis unit, relating the light intensity values ​​to each other to obtain representative spectral data, and obtaining said representative spectral data,determination by the analysis unit of a state of integrity of the complex by comparison by similarity grouping with a determined similarity threshold of each of the representative spectral data obtained in the “relationship” step with reference spectral data contained in the analysis unit (30) and identified for each state of integrity among at least: (i) a first state of integrity corresponding to the presence of a sample deposit combined with the absence of matrix deposit, (ii) a second state of integrity corresponding to the presence of a matrix deposit combined with the absence of sample deposit, (iii) a third state of integrity corresponding to the presence of a sample deposit combined with the presence of a matrix deposit, triggering of at least a first alert, by the analysis unit, when the representative data are similar to the first state of integrity or to the second state of integrity.

[0010] Using such a method, it is easy to identify the reception areas in which there is an incomplete complex, to complete the complex if this is still possible or at least to isolate these reception areas so as not to take them into account for the rest of the analysis.

[0011] In the present invention, "integrity of the complex" means the level of completeness of the complex; a complex which is complete / integrate has at least one deposit of biological sample and at least one deposit of a fluid called matrix.

[0012] According to a characteristic of an embodiment of the invention, in the acquisition step, the imager acquires a plurality of images of at least the receiving area of ​​the support, the acquisition of the images being carried out at different determined times or over at least a determined period of time. The acquisition of a plurality of images allows for more detailed identification after the steps of depositing each component of the complex. This acquisition is all the more advantageous when the latter is carried out over a period of time, that is to say in kinetics, which makes it possible to capture the variations in states and to complete the deposits in real time when the latter are omitted.

[0013] According to a characteristic of an embodiment of the invention, the imager is an RGB imager or a multi-spectral imager or a hyperspectral imager. The imager can be of different types, the advantage of an RGB imager is that the spectral channels are already determined and this type of imager is inexpensive. The advantage of a multi-spectral or hyperspectral imager is that the "selected" channels can be different from the RGB channels and can also be more numerous, knowing that the more channels are selected, the higher the classification performance rate.

[0014] According to a characteristic of an embodiment of the invention, the list comprising the integrity states comprises at least a fourth integrity state corresponding to the absence of complex, i.e. the absence of sample deposition combined with the absence of matrix deposition. This functionality makes it possible to distinguish an empty reception zone from a reception zone with any deposit, which makes it possible to discard the empty reception zone during the analysis of the results or to fill this zone.

[0015] According to a characteristic of an embodiment of the invention, the acquisition of at least one image is carried out by manual or automatic triggering. Thus, a manual trigger makes it possible to control the image capture and to ensure that the image acquisition is carried out at the determined time. An automatic trigger is calibrated in time, which makes it possible to automate the image capture, simplifying and reducing the number of manipulations for the operator. In particular, according to a characteristic of the invention, the frequency of acquisition of at least one image is calculated as a function of the time required to deposit the sample and the time required to deposit the matrix.

[0016] According to a characteristic of an embodiment of the invention, the triggering of the image acquisition can be semi-automatic. For example, the triggering is carried out or initiated upon completion of a determined action, for example the scanning of an identifier such as a bar code.

[0017] According to a characteristic of an embodiment of the invention, the triggering of the image acquisition can be carried out by detecting an interruption of the light signal or by detecting variations by means of a piezoelectric sensor for example.

[0018] According to a characteristic of an embodiment of the invention, the method comprises a step of triggering a second alert, different from the first alert, the second alert being triggered when the representative data resembles the third integrity state, which makes it possible to validate the steps of the method of depositing the complex before inserting the support into the mass spectrometer for analysis.

[0019] According to a characteristic of an embodiment of the invention, the triggering of the second alert causes the image acquisition to be stopped when the latter is carried out over a determined period unless the analysis unit is aware of another complex deposition in progress on the support.

[0020] Indeed, it is possible to deposit several samples from different origins on the same support: several deposits will therefore be in progress simultaneously. In this case, image acquisition continues until all the deposits are finished or considered finished by the analysis unit.

[0021] Alternatively, triggering the second alert results in the acquisition of an image when the latter is carried out at specific times and in particular at the end of the support preparation process.

[0022] According to a characteristic of an embodiment of the invention, the method comprises a step of triggering a third alert, different from the first alert and the second alert, the third alert being triggered when the representative data resembles the fourth integrity state. This third alert makes it possible to warn the operator that the reception areas of the support are empty and that it is possible to remedy this defect. Advantageously, in the case of continuous image acquisition, the third alert is triggered only when the analysis unit receives information on the production of a complex deposit while said analysis unit determines the fourth integrity state.

[0023] According to a characteristic of an embodiment of the invention, the method comprises a calibration step prior to the acquisition step of the method, the calibration step consisting of the acquisition of an image, called a reference image, of the at least one reception zone, for each integrity state to be referenced.

[0024] This calibration step allows the adaptation of the method to different experimental conditions because it allows the definition of an in situ reference frame. In addition, the calibration step is used in particular when determining each integrity state and the related reference spectral data, which are implemented in the analysis unit prior to the implementation of the determination method according to the invention.

[0025] For example, for the fourth integrity state, a reference image of the empty receiving area is acquired. For the first integrity state, a reference image with a sample deposit alone is acquired. For the second integrity state, a reference image with a matrix deposit alone is acquired, and for the third integrity state, an image with a sample deposit supplemented by a matrix deposit is acquired.

[0026] When the matrix is ​​deposited in the receiving zone, a drop is formed on the surface of the receiving zone. The degree of wetting, corresponding to the degree of spreading of the liquid on this solid, varies according to the surface energy of the receiving zone. At constant volume, the projected diameter of a drop deposited on a "poorly wetting" surface, corresponding to a high contact angle (low surface energy) is smaller than the projected diameter of a drop deposited on a "more wetting" surface, corresponding to a low contact angle (high surface energy).

[0027] To quantify the quality of the wetting, we measure the diameter projected in the plane of the reception zone (the drops being deposited at constant volume.

[0028] According to a characteristic of an embodiment of the invention, the diameter of a drop of a complex according to the third state of integrity is greater than the diameter of a drop of a complex according to the second state of integrity. The contact surface is determined by Young's law, which makes it possible to determine a contact angle from the surface energies of the solid 's' (the reception zone) and the liquid 'l' (the matrix). Measuring the diameter is an indirect measurement that is simpler to implement than measuring the contact angle.

[0029] Without being bound to the interpretation, we explain this behavior by the high surface energy of the sample forming a film of bacteria (charged and hydrophilic) in comparison with the areas receiving virgin bacteria (more hydrophobic, polymer material of the MALDI receiving plate).

[0030] According to a characteristic of an embodiment of the invention, the determination method comprises a step of determining the second state of integrity by measuring the projected diameter of the drop of the complex present in the reception zone of the support.

[0031] According to a characteristic of an embodiment of the invention, the determination method may comprise a prior sub-step of measuring the diameter of the drop of the complex present in the receiving zone of the support (in the absence of sample deposition, second integrity state (0M) and a prior sub-step of comparing the measured diameter with a reference diameter corresponding to the diameter of a drop of a complex having a third integrity state (BM), said sub-steps being carried out before the step of determining the second integrity state by measuring the contact angle. Indeed, it is possible to distinguish the second integrity state (0M) of the complex from the other integrity states by measuring the projected diameter of the deposit drop in addition to or as a replacement for a measurement of the diameter of the contact angle.

[0032] In the present disclosure, the contact angle of the drop is measured by the following Young-Laplace equation: cosθ = σ Sv − σ Sl σ lv

[0033] Where Θ is the contact angle between the ambient air, the drop of complex and the support, expressed in degrees, σ sv represents the interfacial energy between the air and the support, expressed in milliNewtons / m or dynes / cm σ sl represents the interfacial energy between the drop and the support, expressed in milliNewtons / m or dynes / cm, σ lv represents the interfacial energy (interfacial tension) between the drop and the ambient air, expressed in milliNewtons / m or dynes / cm.

[0034] Advantageously, the integrity determination method is used in a method of preparing a support for analysis purposes, for example by mass spectrometer, so as to detect the nature of the microorganism(s) present in the sample(s) deposited on the support.

[0035] The invention, as defined in the appended claim 7, also relates to a method for preparing a support for analysis purposes, for example by mass spectrometer, so as to detect the nature of the microorganism(s) present in the sample(s) deposited on the support, the preparation method comprising at least the following steps: depositing a biological sample in at least one receiving area of ​​the support depositing a matrix in the at least one receiving area of ​​the support in which the sample deposit is carried out, the preparation process being characterized in that it incorporates a process for determining the integrity of the complex according to the invention.

[0036] According to a characteristic of an embodiment of the invention, each deposit is a deposit of a sample or matrix layer.

[0037] According to a characteristic of an embodiment of the invention, the step of acquiring at least one image of the determination method is carried out after the matrix deposition step.

[0038] Alternatively, according to a characteristic of an embodiment of the invention, the step of acquiring at least one image of the determination method is carried out after the step of depositing the sample and after the step of depositing the matrix.

[0039] Alternatively, according to a characteristic of an embodiment of the invention, the step of acquiring at least one image of the determination method is triggered before the sample deposition step and is stopped after the matrix deposition step, the acquisition step being carried out continuously during the steps of the preparation method.

[0040] According to a characteristic of an embodiment of the invention, at each step of depositing a sample or matrix on the receiving zone, the operator can manually notify the analysis unit of a piece of production information, the step of acquiring at least one image being carried out after this notification.

[0041] The invention, as defined in the appended claim 11, also relates to a system configured to implement the method according to the invention, comprising at least one imager, an analysis unit and at least one display device configured to display the alert(s).

[0042] Further, the display device is configured to display a graphical representation of the media illustrating the at least one receiving area of ​​the media and preferably all receiving areas of the media.

[0043] According to a characteristic of an embodiment of the invention, the system comprises a guidance device cooperating with the display device, the guidance device being integrated into the analysis unit, the guidance device being configured to indicate the receiving zone(s) intended for the deposition of a sample and / or a matrix deposition.

[0044] According to a characteristic of an embodiment of the invention, the guidance device is configured to cooperate with the imager.

[0045] According to a characteristic of an embodiment of the invention, the guidance device is configured to indicate via the display device which receiving zone is to be filled or completed depending on the progress of the preparation method and the integrity determination method. This guidance device makes it possible to guide the operator from step to step with preferably real-time monitoring which is particularly possible during the acquisition of several images according to the integrity determination method, whether at specific times or over a specific period. In addition, the guidance device makes it possible to ensure that the entire support is ready before analysis in the mass spectrometer.

[0046] According to a characteristic of an embodiment of the invention, the meaning of the information on the making of a deposit is carried out via the guidance device cooperating with the analysis unit.

[0047] Advantageously, when a complex is categorized according to the first integrity state or the second integrity state, the guidance device indicates to the operator the faulty support reception area requiring intervention via the display device, the intervention being able to be either the correction of the error or the isolation of the reception area depending on the circumstances.

[0048] In a first case, if the acquisition step of the integrity determination method is carried out over a determined period of time in real time, then the operator can correct the absence of matrix (second integrity state) or identify the absence of sample (first integrity state) and not take it into account in the subsequent analysis.

[0049] In a second case, if the acquisition step of the integrity determination process is carried out after each deposition step, it is easy to correct the missing deposition step if necessary or identify the absence of sample (first integrity state) and not take it into account in the subsequent analysis.

[0050] In a third case, if the acquisition step of the determination process is carried out once the sample deposition and the matrix deposition are supposed to have been carried out, then the operator identifies the faulty reception areas and does not take them into account in the subsequent analysis of the microorganism(s).

[0051] According to a characteristic of an embodiment of the invention, the indications of the guidance device via the display device are represented by indicators of determined geometric shape and / or indicators of determined colors.

[0052] For example, for an empty receiving area to be filled by a matrix deposit, the receiving area will have a green round indicator (for example) on the graphic representation displayed on the display device so that the operator can easily identify the receiving area to be filled. In addition, for example, if the sample deposit is missing or incorrectly made, the corresponding receiving area will have a red round indicator (for example) indicating the need to redo the deposit or to isolate said receiving area to separate it from the receiving areas to be analyzed later.

[0053] According to a characteristic of an embodiment of the invention, the imager comprises a spectral channel, preferably at least two spectral channels. For example, a first spectral channel corresponds to a first spectral band ranging from 410 nm to 427 nm and a second spectral channel corresponds to a second spectral band ranging from 761 nm to 786 nm.

[0054] According to a characteristic of an embodiment of the invention, the imager of the system is configured to read an image of the one-dimensional or two-dimensional barcode or QR code type.

[0055] According to a feature of an embodiment of the invention, the imager of the system is also configured to acquire an image of a Petri dish so as to correlate the microorganism of the sample analyzed on the support to the Petri dish from which it originates.

[0056] According to a characteristic of an embodiment of the invention, the support comprises a plurality of receiving zones, for example forty-eight receiving zones. Thus, it is possible to deposit up to forty-eight samples of different origin.

[0057] The invention will be better understood from the following description, which relates to embodiments according to the present invention, given as non-limiting examples and explained with reference to the attached schematic figures. The attached schematic figures are listed below: [ Fig. 1 ] is a schematic view of the preparation process according to a first embodiment, [ Fig. 2 ] is a schematic view of the preparation process according to a second embodiment, [ Fig. 3A ] is a schematic view of the preparation process according to a third embodiment, [ Fig. 3B ] is a schematic view of the preparation process according to a variant of the third embodiment, [ Fig. 4 ] is a series of black and white images of three sample holders according to the channels R (PC1) G (PC2) B (PC3), [ Fig. 5 ] is a graphical representation of the light intensity values ​​of the complexes of the figure 4 , after a principal component analysis. [ Fig. 6 ] is a graphical representation of the luminous intensity value of a complex as a function of time according to a wavelength, [ Fig. 7 ] is a partial photograph of the support illustrating deposited complexes presenting different states of integrity, [ Fig. 8 ] is a schematic view of the system according to one embodiment of the invention, [ Fig. 9 ] is a partial illustration of the system according to the embodiment of the invention shown in figure 8 , [ Fig. 10 ] is a representative view of the sample holder through the display device.

[0058] In the context of a method for preparing a sample support according to the invention for analysis purposes, for example by mass spectrometer, a sample support is used in the form of a card having a plurality of reception zones called "wells". In each well, a drop of a sample containing one or more microorganism(s) to be identified, for example, and at least one drop of matrix is ​​generally deposited. In the particular case of identifying a yeast, a drop of formic acid can be added between the sample deposit and the matrix deposit. The final deposit is called a "complex" and therefore comprises at least one sample layer and one matrix layer.

[0059] Before proceeding with the analysis and identification of the microorganism(s) in the sample, a method for determining the integrity of the complex is carried out according to the invention in order to verify whether the preparation of the support is carried out properly to avoid any inaccurate results. Whatever the embodiment of the preparation method according to the invention, the determination method according to one embodiment of the invention is implemented by a system 1 illustrated in figures 8 et 9 .

[0060] According to one embodiment of the invention, the system 1 comprises at least one imager 20, an analysis unit 30 and at least one display device 40 configured to display / transmit the alert(s) to the operator. The display device 40 is configured to display a graphical representation of the support 10 illustrating the at least one receiving area 11 of the support 10 and preferably all the receiving areas 11 of the support 10.

[0061] As illustrated in figure 8 in particular, the system 1 comprises a guidance device 31 cooperating with the display device 20 and with the display device 40, the guidance device 31 being integrated into the analysis unit 30.

[0062] The guidance device is configured to indicate the receiving zone(s) 11 of the support 10 intended for the deposition of a sample and / or the deposition of matrix and also to alert via the display device 40 which receiving zone 11 is to be filled or completed depending on the progress of the preparation method and the integrity determination method.

[0063] As can be seen in figure 10 , the guidance device 31 makes it possible to guide the operator from step to step with preferentially real-time monitoring which is particularly possible during the acquisition of several images according to the integrity determination method, whether at specific times or over a specific period. As can be seen in figure 8 , the guidance device can alert the operator by graphic indicators on the representation of the support displayed on the display device 40. In the illustrated example, the guidance device 31 indicates to the operator that the two reception zones in line C comprise complexes having a third integrity state (BM) by a tight dotted circle, it also indicates that in line D the first reception zone is empty (fourth integrity state 00) and is ready to receive a sample deposit (spaced dotted circle) and that in line E the first reception zone has a complex whose integrity state corresponds to the first integrity state (B0) and is therefore ready to receive a matrix or formic acid deposit (continuous circle).

[0064] The determination method and the support preparation method will now be described with reference to figures 1 à 3B .

[0065] In a first embodiment illustrated in figure 1 , the integrity determination method is carried out after steps 101, 102, 103 of depositing respectively sample, matrix, formic acid where appropriate. Said integrity determination method in the first embodiment comprises a step 201 of capturing at least one image of the sample support and more particularly of at least one receiving zone. The captured image is then sent (step 202) to the analysis unit of the system. Subsequently, the analysis unit analyzes the image by extracting light intensity values ​​representative of at least one spectral band (step 203) and relates light intensity values ​​to each other to obtain representative spectral data (step 204), and obtains said representative spectral data (step 205).By comparing (step 206) each of the representative spectral data obtained in the previous step with reference spectral data, the analysis unit determines an integrity state of the complex contained in the at least one receiving zone. It should be noted that the reference spectral data are contained in the analysis unit and are each identified for an integrity state among at least: (i) a first integrity state (B0) corresponding to the presence of a sample deposit combined with the absence of matrix deposit, (ii) a second integrity state (0M) corresponding to the presence of a matrix deposit combined with the absence of sample deposit, (iii) a third integrity state (BM) corresponding to the presence of a sample deposit combined with the presence of a matrix deposit. Following the determination of the integrity state of the complex, the analysis unit sends a corresponding alert.

[0066] A first alert is sent to the operator (step 207) when the analysis unit determines that the complex has an integrity state corresponding to the first integrity state or the second integrity state. In fact, the complex is considered incomplete and the operator must take note of it. Following this first alert, a differentiation step (step 210) of the integrity state is carried out to allow the operator to know whether it is an absence of matrix deposition or the absence of sample deposition that is detected. For this differentiation step, either a more detailed analysis of the spectral bands is carried out or the contact angle of the drop of the complex deposited on the receiving zone can also be measured.

[0067] In figure 7 are represented deposits of complexes presenting different states of integrity. We see that in line B, at the level of the first reception zone, a deposit of complex presenting a second state of integrity (0M) and in line B, at the level of the three other reception zones, deposits of complexes presenting a third state of integrity (BM). We can clearly see that the drop of the complex presenting a second state of integrity (0M) has a diameter smaller than that of the drops of the complex presenting a third state of integrity (BM). It should be noted that the diameter observed after deposition of the matrix on the virgin deposition zone is approximately 70% of the nominal value of the diameter observed after deposition of the matrix on the sample forming a bacterial film.

[0068] If it is determined that the matrix deposit is missing (first integrity state B0), the operator can remedy the oversight by depositing said matrix by returning to step 102, the determination method will be carried out again afterwards to ensure that step 102 is carried out correctly and that the complex is complete (step 104).

[0069] If it is determined that the sample deposit is missing (second integrity state 0M), the operator identifies on the display device of the analysis unit the defective reception areas and isolates them (step 211) so as not to take them into account during the subsequent analysis. It should be noted that the isolation of the defective areas (step 211) triggers the continuation of the procedure, namely the decision step 105 and depending on the decision, either the step 106 of closing the support preparation process or the initialization step 100.

[0070] A second alert is sent to the operator (step 208) when the analysis unit determines that the complex has an integrity state corresponding to the third integrity state. In fact, the complex is considered complete (step 104) and the operator must take note of this to proceed to the next stage of the process.

[0071] Optionally, and as illustrated in dotted lines, the determination method can determine a fourth integrity state (00) of the complex corresponding to the absence of sample deposition and the absence of matrix deposition. This fourth integrity state (00) is indicated by a third alert (step 209) to the operator. The operator can remedy the oversight by returning to step 101, the determination method will be carried out again afterwards to ensure that the complex is complete (step 104).

[0072] Optionally, a calibration step 200 can be performed before sample deposition (step 101).

[0073] In a second embodiment illustrated in figure 2 , the integrity determination method is triggered and more precisely the step 201 of acquiring at least one image of the support and more particularly of at least one receiving zone, after the first step 101 of depositing a sample or after the step of depositing formic acid 103 if applicable. The captured image is then sent (step 202) to the analysis unit of the system. Subsequently, the analysis unit analyzes the image by extracting light intensity values ​​representative of at least one spectral band (step 203) and relates light intensity values ​​to each other to obtain representative spectral data (step 204), and obtains said representative spectral data (step 205). By comparing (step 206), each of the representative spectral data obtained in the previous step with reference spectral data, the analysis unit determines a state of integrity of the complex contained in the at least one reception zone.It should be noted that the reference spectral data are contained in the analysis unit and are each identified for an integrity state among at least: (i) a first integrity state (B0) corresponding to the presence of a sample deposit combined with the absence of a matrix deposit, (ii) a fourth integrity state (00) corresponding to the absence of a sample deposit combined with the absence of a matrix deposit. Following the determination of the integrity state of the complex, the analysis unit sends a corresponding alert: either a first alert (step 207) indicating a first integrity state (B0) since only step 101 is carried out at this stage, or a third alert (step 209) indicating a fourth integrity state (00) so that the sample deposit step (step 101) is carried out again, steps 101, 103, 201, 202, 203, 204, 205 and 206 are carried out as long as the third alert (step 209) is triggered.Once the first integrity state (B0) is determined, the second step (102) of depositing a matrix is ​​triggered. Subsequently, steps 201, 202, 203, 204, 205 and 206 are carried out so that the analysis unit determines the integrity state of the complex: either it is the first integrity state (B0) and the first alert is triggered (step 207) to return to the matrix deposition step (step 102) and to the following steps 201, 202, 203, 204, 205 and 206, or it is the third integrity state (BM) and a second alert (step 208) is triggered and indicates that the complex is complete (step 104), which triggers the decision step 105 and depending on the decision, either the step 106 of closing the support preparation process or the initialization step 100.

[0074] In the first embodiment ( figure 1 ) and the second embodiment ( figure 2 ), the image acquisition step (step 201) is carried out punctually but in the first embodiment, it is carried out once the complex has been deposited while in the second embodiment, it is carried out after each deposition step constituting the complex. The second embodiment is more advantageous because it makes it easier to correct errors.

[0075] In a third embodiment illustrated in figure 3 , the determination method is triggered from the initialization step 100 of the support preparation method. The step of acquiring at least one image (step 201) is carried out continuously during the steps of depositing the sample (101), depositing the formic acid (103) if applicable, and depositing the matrix (102). The start of acquisition is illustrated by step 201.1, step 201n represents the plurality of image acquisitions during steps 101, 102, 103. After each image acquisition (201n), steps 202 to 206 are carried out: if the complex has an integrity state corresponding to the third integrity state (BM) then the acquisition step (201n) stops (step 201.2), the complex is considered by the analysis unit as complete (step 104) and step 105 is implemented.

[0076] If the complex has an integrity state corresponding to the first (B0) or second integrity state (0M) then the first alert is triggered (step 207) or if optionally, it is a fourth integrity state (00) then the third alert is triggered (step 209), then: in a first case, the operator decides to isolate the faulty reception area (step 211), the image acquisition (201n) stops (step 201.2) and step 105 is implemented; in a second case, the operator decides to correct the error in the case of a first determined integrity state (B0) and the image acquisition continues (step 201n) and steps 202 to 206 of the determination method continue and step 102 of the support preparation method is carried out again.

[0077] As a variant of this third embodiment, as illustrated in figure 3B , after the matrix deposition step (step 102), the image acquisition (201n) is stopped (step 201.2) and the analysis unit implements steps 202 to 206: if the complex has an integrity state corresponding to the third integrity state (BM) then the complex is considered by the analysis unit as complete (step 104) and step 105 is implemented; in all other cases (integrity states 00, B0, 0M), the operator decides to isolate the faulty reception area (step 211), and step 105 is implemented and depending on the decision, either step 106 of closing the support preparation process or step 100 of initialization.

[0078] We have illustrated an embodiment of the invention with an example shown in figures 4 et 5 . This example is of course non-limiting and allows for a better understanding of the claimed approach. In this example, three spectral channels R, G, B were used. In figure 4 a series of images of three supports with deposits of different complexes is illustrated. The first image of the three supports is an image according to the first R channel (PC1), the second image of the three supports is an image according to the second G channel (PC2) and the third image of the three supports is an image according to the third B channel (PC3). The images are in black and white. Subsequently, the different complexes are identified by points whose coordinates (PC1; PC2; PC3) correspond to their light intensity value as a function of the chosen wavelengths (RGB). After analysis by principal component, as illustrated in figure 5 , the system analysis unit groups the different points by similarity to establish "groups" of integrity states, the differentiation of integrity states being more obvious in this mode. It should be noted, however, that the analysis by principal component is not obligatory. As can be seen in figure 5 , the empty reception areas (absence of complex equivalent to the fourth integrity state 00) stand out from the lot and are represented by small circles, the complexes presenting an integrity state equivalent to the first integrity state B0 are represented by '+', the complexes presenting an integrity state equivalent to the second integrity state 0M are represented by triangles, the complexes presenting an integrity state equivalent to the second integrity state 0M are represented by crosses 'x'.

[0079] In figure 6A graphical representation of light intensity values ​​of a complex as a function of time at a given wavelength is shown. In this graph, the first peak corresponds to the tip of the deposition tool passing under the imager to deposit a sample. The difference in light intensity after this first peak is small. The second peak corresponds to the passage of a hand under the imager to deposit the matrix. The signal then reaches its maximum after drying of the matrix, which whitens as it crystallizes.

[0080] Of course, the invention is not limited to the embodiments described and shown in the appended figures. Modifications remain possible, in particular from the point of view of the constitution of the various elements or by substitution of technical equivalents, without departing from the scope of protection of the invention as defined by the appended claims.

Claims

1. Method for determining the integrity of at least one complex based on at least one biological sample and at least one matrix, said complex being arranged in at least one receiving area (11) of a support (10) to be analysed subsequently, said method being implemented by a determination system (1) comprising at least one imager (20) and an analysis unit (30) configured for interacting with the imager (20), wherein said method comprises at least the following steps: - (201, 201n) acquiring at least one image of at least one receiving area (11) of the support (10), the acquisition being executed by the imager (20) of the determination system (1), - (202) sending the at least one image acquired by the imager (20) to the analysis unit (30), - (203) analyzing the sent image by extracting the light intensity values representative of at least one spectral band, the analysis being executed by the analysis unit (30), - (204) relating the light intensity values to each other to obtain representative spectral data, and obtaining said representative spectral data, wherein - (206) determining, by the analysis unit (30), a state of integrity of the complex, by comparing (205), by similarity grouping with a specified similarity threshold, each of the representative spectral data obtained in the "relating" step with reference spectral data contained in the analysis unit (30) and identified for each state of integrity from among at least the following: (i) a first state of integrity (B0) corresponding to the presence of a sample deposit combined with the absence of a matrix deposit, (ii) a second state of integrity (0M) corresponding to the presence of a matrix deposit combined with the absence of a sample deposit, (iii) a third state of integrity (BM) corresponding to the presence of a sample deposit combined with the presence of a matrix deposit, - (207) triggering at least a first alert, by the analysis unit (30), when the representative data resemble the first state of integrity (B0) or the second state of integrity (0M).

2. Determination method according to Claim 1, wherein the representative values of light intensity are representative of at least two different spectral bands and preferably at least three different spectral bands.

3. Determination method according to either of Claims 1 and 2, wherein, in the acquisition step, the imager (20) acquires at least one plurality of images of the same receiving area (11) of the support (10), the acquisition of the images being executed at different specified instants or during at least one specified period of time.

4. Determination method according to any of Claims 1 to 3, wherein the imager (20) is an RGB imager or a multi-spectral imager or a hyperspectral imager.

5. Determination method according to any of Claims 1 to 4, wherein the list comprising the states of integrity comprises at least a fourth state of integrity (00) corresponding to the absence of a sample deposit combined with the absence of a matrix deposit.

6. Determination method according to any of Claims 1 to 5, comprising a calibration step (200) before the acquisition step of the method, the calibration step (200) consisting in the acquisition of an image, called the reference image, of at least one empty receiving area (11) and in the recording of said reference image in the analysis unit (30).

7. Method for preparing a sample support (10) for purposes of analysis, by mass spectrometer for example, in order to detect the nature of the microorganism(s) present in the sample(s) deposited on the support, the preparation method comprising at least the following steps: - (101) depositing a sample in at least one receiving area (11) of the support (10), - (102) depositing a matrix in the at least one receiving area (11) of the support (10) in which the sample has been deposited, the preparation method being characterized in that in incorporates a method for determining the integrity of the complex according to any of Claims 1 to 6.

8. Preparation method according to Claim 7, wherein the step (201) of acquiring at least one image of the determination method is executed after the step (102) of depositing the matrix.

9. Preparation method according to Claim 7, wherein the step (201) of acquiring at least one image of the determination method is executed after the step (101) of depositing the sample and after the step (102) of depositing the matrix.

10. Preparation method according to Claim 7, wherein the step (201n) of acquiring at least one image of the determination method is triggered (201.1) before the step (101) of depositing the sample and is stopped (201.2) after the step of depositing the matrix, the acquisition step being executed continuously during the steps of the preparation method.

11. System (1) configured for implementing the determination method according to any of Claims 1 to 6, comprising at least one imager (20), an analysis unit (30) and at least one display device (40) configured for displaying the alert(s).

12. System according to Claim 11, wherein the display device (40) is configured for displaying a graphic representation of the support (10), illustrating the at least one receiving area (11) of the support (10), and preferably all the receiving areas (11) of the support (10).

13. System according to Claim 12, comprising a guidance device interacting with the display device (40), the guidance device (31) being integrated into the analysis unit (30), the guidance device (31) being configured for indicating the receiving area(s) (11) designated for the deposition of a sample and / or a matrix deposit.