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

Abstract

The present invention mainly 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, which comprises at least the following steps: – acquiring at least one image, – analyzing the transmitted image by extracting light intensity values ​​representing at least one spectral band, – correlating the light intensity values ​​with each other to obtain representative spectral data, – comparing each representative spectral data by similarity grouping according to a determined similarity threshold to determine the integrity state of the complex, – triggering at least a first alarm by an analysis unit when the representative data approximates a first integrity state or a second integrity state.

Description

Technical Field

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

[0002] In the present invention, "complex" refers to a combination of at least one microorganism present in a collected biological sample and at least one matrix. The microorganism may be bacteria, viruses, yeast, mold, fungus, etc., and the matrix may be α-cyano-4-hydroxycinnamic acid (HCCA), 3,5-dimethoxy-4-hydroxycinnamic acid (or sinapinic acid, SA), 2,5-dihydroxybenzoic acid (DHB), β-carboline (9H-pyridinium [3,4-b] indole) (or Norharmane), or any other known substance used for ionizing the complex in MALDI-TOF analysis. In the specific case where the microorganism is yeast, the complex also includes formic acid.

[0003] The deposition of the complexes on the support to be analyzed by mass spectrometry is currently performed manually, ie the sample deposition is followed by the matrix deposition. Qualitative inconsistencies were found in this deposition.

[0004] This is because complexes are sometimes incomplete due to missing samples, matrix or even both components, rendering mass spectra unusable, and deposition methods are often performed manually in a non-standardized manner.

[0005] This finding is quite worrying, and there are solutions for remedying the complete absence of complexes, such as the solution proposed in document US7291835, which describes a system for automatically monitoring the complex receiving area for a mass spectrometer using a MALDI type ionization source. The system described in document US7291835 is an automated flow system configured to identify the optimal positioning of the laser strike for the purpose of ionizing the complexes deposited on the MALDI plate carrier; therefore, detecting the presence of the complex is necessary to determine where to position the laser, especially when the receiving areas are very close and difficult to separate.

[0006] Until now, there has been no solution to more finely detect the degree of integrity of the composite or to alert the technician in time to enable correction or at least to identify receiving areas that exhibit malfunctions due to the composite being missing one or more components, which leads to erroneous results and therefore to misdiagnosis. Summary of the invention

[0007] The object of the present invention is to overcome some or all of the above disadvantages and in particular to provide monitoring of the quality and thus the integrity of each complex deposited on a carrier (eg a MALDI-TOF plate) comprising a plurality of receiving areas in which the complexes are deposited.

[0008] To this end, the invention proposes a method for determining the integrity of at least one complex based on at least one biological sample and at least one matrix, the complex being arranged in at least one receiving area of ​​a carrier to be subsequently analyzed, the method being implemented by a determination system comprising at least one imager and an analysis unit configured to interact with the imager, the method comprising at least the following steps:

[0009] - a step of acquiring at least one image of at least one receiving area of ​​the carrier is performed by an imager of the determination system;

[0010] - a step of sending at least one image acquired by the imager to an analysis unit;

[0011] - a step of analyzing the transmitted image by extracting light intensity values ​​representative of at least one spectral band, performed by an analysis unit;

[0012] - a step of correlating said light intensity values ​​to obtain representative spectral data and obtaining representative spectral data;

[0013] - a step of determining the integrity state of the composite by comparing, by means of an analysis unit, each representative spectral data obtained in the "correlating" step with reference spectral data, by similarity grouping at a specified similarity threshold, the reference spectral data being contained in said analysis unit (30) and being identified for each of at least the following integrity states:

[0014] (i) a first integrity state corresponding to the presence of sample deposits and the absence of matrix deposits,

[0015] (ii) a second integrity state corresponding to the presence of matrix deposits and the absence of sample deposits,

[0016] (iii) a third integrity state corresponding to the presence of sample deposition and the presence of matrix deposition;

[0017] - When the representative data approximates the first integrity state or the second integrity state, at least a first alarm is triggered by the analysis unit.

[0018] Thanks to this method, it is easy to identify recipient areas where there are unfinished complexes and, if still possible, to complete the complexes, or at least to isolate these recipient areas so that they are not considered in the rest of the analysis.

[0019] In the present invention, "completeness of the complex" means the degree to which the complex is complete; a completed / complete complex has at least one deposit of a biological sample and at least one deposit of a fluid referred to as a matrix.

[0020] According to a feature of the invention, in the acquisition step, the imager acquires a plurality of images of at least the receiving area of ​​the carrier, the acquisition of the images being performed at different specified moments or within at least one specified period of time. By acquiring a plurality of images, a more refined identification can be provided after the step of depositing each component of the composite. It is further advantageous when the acquisition is performed over a period of time, i.e. in a dynamic manner, which enables the capture of changes in state and the completion of the deposition in real time when it is omitted.

[0021] According to a feature of the present invention, the imager is an RGB imager or a multispectral imager or a hyperspectral imager.

[0022] Imagers can be of different types, the advantage of RGB imagers is that the spectral channels are already determined and this type of imager is cheap. The advantage of multispectral imagers or hyperspectral imagers is that the "selected" channels may be different from the RGB channels and may also be more numerous (since the classification performance rate improves as more channels are selected).

[0023] According to a feature of the invention, the list of integrity states includes at least a fourth integrity state corresponding to a composite absence, i.e., a sample deposit absence mixed with a matrix deposit absence. This feature can distinguish between empty receiving areas and receiving areas with deposits, thereby enabling the empty receiving areas to be ignored when analyzing the results, or enabling the empty receiving areas to be filled.

[0024] According to a feature of the present invention, the acquisition of at least one image is performed by manual or automatic triggering. Thus, the manual trigger can monitor the image capture and ensure that the image acquisition is performed at a specified time. The automatic trigger is calibrated in time to automate the image capture, simplify and reduce the number of operator operations. Specifically, according to a feature of the present invention, the acquisition frequency of at least one image is calculated based on the time required to deposit the sample and the time required to deposit the substrate.

[0025] According to the features of the present invention, the triggering of image acquisition can be semi-automatic, for example, the triggering is executed or started when a specified action (such as scanning a barcode or other identification) is completed.

[0026] According to a feature of the invention, triggering of the image acquisition may be performed, for example, using a piezoelectric sensor, by detecting an interruption of the light signal or by detecting a change.

[0027] According to a feature of the invention, the method includes the step of triggering a second alarm different from the first alarm, the second alarm being triggered when the representative data approximates a third integrity state, so that the step of the method of depositing the composite is verified before the carrier is inserted into the mass spectrometer for analysis.

[0028] According to a feature of the invention, the triggering of the second alarm causes the acquisition of images to be stopped when it has been carried out for a certain period of time, unless the analysis unit is aware of the ongoing deposition of another compound on the carrier.

[0029] This is because multiple samples obtained from different sources can be deposited onto the same carrier, so multiple depositions will be performed simultaneously. In this case, image acquisition will continue until all depositions are terminated or are considered to have been terminated by the analysis unit.

[0030] Furthermore, if the action of acquiring an image is performed at a specific moment, in particular at the end of a method for preparing a carrier, the triggering of the second alarm may result in the acquisition of an image.

[0031] According to a feature of the invention, the method comprises the step of triggering a third alarm, different from the first alarm and the second alarm, the third alarm being triggered when the representative data approximates a fourth integrity state. The third alarm can be used to warn the operator that the receiving area of ​​the carrier is empty and that this defect can be remedied. Advantageously, in the case of continuous image acquisition, the third alarm is triggered only when the analysis unit receives execution information of the composite deposition when the fourth integrity state is determined by the analysis unit.

[0032] According to a characteristic of the invention, the method comprises, prior to the acquisition step of the method, a calibration step consisting in acquiring, for each integrity state to be referenced, an image, called reference image, of at least one reception area.

[0033] This calibration step enables the method to be adapted to different experimental conditions, since it allows the reference frame to be defined on site.In addition, in particular, a calibration step is used in the determination of each integrity state and the reference spectral data associated therewith implemented in the analysis unit before implementing the determination method according to the invention.

[0034] For example, for the fourth integrity state, a reference image of an empty receiving area is acquired. For the first integrity state, a reference image with only sample deposition is acquired. For the second integrity state, a reference image with only sample deposition is acquired, and for the third integrity state, an image with sample deposition and completed matrix deposition is acquired.

[0035] When the substrate is deposited on the receiving area, droplets are formed on the surface of the receiving area. The degree of wetting, which corresponds to the degree of spreading of the liquid on the solid, varies according to the surface energy of the receiving area. At constant volume, the projected diameter of the droplets deposited on the "less wettable" surface (corresponding to a large contact angle (low surface energy)) is smaller than the projected diameter of the droplets deposited on the "more wettable" surface (corresponding to a small contact angle (high surface energy)).

[0036] To quantify the quality of wetting, the projected diameter in the plane of the receiving area (droplet deposited at constant volume) was measured.

[0037] According to a feature of the invention, the diameter of the droplet of the composite according to the third integrity state is greater than the diameter of the droplet of the composite according to the second integrity state. The contact surface is determined by Young's law, which determines the contact angle from the surface energies of the solid "s" (receiving area) and the liquid "I" (substrate). The measurement of the diameter is an indirect measurement and is simpler than the measurement of the contact angle.

[0038] Without being limited by the interpretation, we can explain this behavior by the high surface energy of the sample forming the bacterial film (charged and hydrophilic) in comparison with the bacteria-free receiving area (more hydrophobic, polymer material of the MALDI receiving plate).

[0039] According to a characteristic of the invention, the determination method comprises a step of determining the second integrity state by measuring the projected diameter of a droplet of the composite present in the receiving area of ​​the support.

[0040] According to a characteristic of the invention, the determination method may comprise a preliminary sub-step of measuring the diameter of the droplet of the composite present in the receiving area of ​​the support in the second integrity state (OM) in the absence of sample deposition, and a preliminary sub-step of comparing the measured diameter with a reference diameter corresponding to the diameter of the droplet of the composite having a third integrity state (BM), said preliminary sub-step being performed before the step of determining the second integrity state by measuring the contact angle. This is because, by measuring the projected diameter of the deposited droplet, in addition to or instead of the measurement of the diameter of the contact angle, the second integrity state (OM) of the composite can be distinguished from the other integrity states.

[0041] In the present invention, the contact angle of the droplet is measured by the Young–Laplace equation as follows:

[0042] [Mathematics 1] Young–Laplace equation:

[0043]

[0044] where θ is the contact angle between the ambient air, the droplet of the composite and the carrier, expressed in degrees,

[0045] σ sv It represents the interfacial energy between air and carrier, in millinewtons / meter or dynes / cm.

[0046] σ sl represents the interfacial energy between the droplet and the carrier in millinewtons per meter or dynes per centimeter, and

[0047] σ lv It represents the interfacial energy (interfacial tension) between the droplet and the ambient air, with units of millinewtons per meter or dynes per centimeter.

[0048] Advantageously, the method for determining integrity is used in a method for preparing a carrier for analysis, for example by mass spectrometry, in order to detect the properties of the microorganisms present in a sample deposited on the carrier.

[0049] The present invention also provides a method for preparing a carrier for use in, for example, analysis by a mass spectrometer to detect the properties of microorganisms present in a sample deposited on the carrier, the preparation method comprising at least the following steps:

[0050] – depositing the biological sample in at least one receiving area of ​​the carrier;

[0051] - depositing the matrix in at least one receiving area of ​​the carrier on which the sample has been deposited,

[0052] The production method is characterized in that it comprises the method according to the invention for determining the integrity of a complex.

[0053] According to a feature of the invention, each deposition is a deposition of a layer of a sample or matrix.

[0054] According to a feature of the invention, the step of acquiring at least one image of the determination method is carried out after the step of depositing the substrate.

[0055] Furthermore, according to a feature of the invention, the step of acquiring at least one image of the determination method is performed after the step of depositing the sample and after the step of depositing the substrate.

[0056] Furthermore, according to a feature of the invention, the step of acquiring at least one image of the determination method is triggered before the step of depositing the sample and is stopped after the step of depositing the substrate, the acquisition step being performed continuously during the steps of the preparation method.

[0057] According to a feature of the invention, in each step of sample or matrix deposition on the receiving area, the operator can manually notify the analysis unit of execution information, the step of acquiring at least one image being performed after the notification.

[0058] The invention further proposes a system for carrying out the method according to the invention, comprising at least one imager, an analysis unit and at least one display device configured to display an alarm.

[0059] The display device is further configured for displaying a graphical representation of the carrier showing at least one receiving area of ​​the carrier, and preferably showing all receiving areas of the carrier.

[0060] According to a feature of the invention, the system comprises guide means interacting with the display means, the guide means being integrated into the analysis unit, the guide means being configured to indicate a receiving area designated for the deposition of the sample and / or the deposition of the matrix.

[0061] According to a feature of the invention, the guiding device is configured to interact with the imager.

[0062] According to a feature of the invention, the guiding device is configured to indicate by the display device the receiving area to be filled or filled, depending on the progress status of the preparation method and the integrity determination method. The guiding device can be used to guide the operator step by step by monitoring, preferably in real time, which is particularly possible when a plurality of images are acquired at a specified time or within a specified period according to the integrity determination method. The guiding device can also ensure that the carrier is fully prepared before analysis in the mass spectrometer.

[0063] According to a feature of the invention, the notification of the deposited execution information is provided by means of a guiding device interacting with the analysis unit.

[0064] Advantageously, when classifying the composite according to the first integrity state or the second integrity state, the guidance means indicate to the operator, through the display means, the reception areas of the carrier requiring intervention, which may be the correction of errors or the isolation of reception areas, as the case may be.

[0065] In the first case, if the acquisition step of the integrity determination method is performed in real time within a specified time period, the operator can correct the absence of matrix (second integrity state) or identify the absence of sample (first integrity state) and ignore it in subsequent analysis.

[0066] In the second case, if the acquisition step of the integrity determination method is performed after each deposition step, it is easy to correct the missing deposition step as appropriate, or to identify the absence of a sample (first integrity state) and ignore it in subsequent analysis.

[0067] In the third case, if the acquisition step of the determination method is performed after sample deposition and matrix deposition are assumed to have been performed, the operator recognizes defective receiving areas and ignores them in the subsequent microbiological analysis.

[0068] According to a feature of the present invention, the indication of the guiding means through the display means is represented by an indication signal of a specified geometric shape and / or an indication signal of a specified color.

[0069] For example, for an empty receiving area to be filled with a matrix deposit, the receiving area will have an indication signal, for example a green circle, on the graphical representation displayed on the display device, so that the operator can easily identify the receiving area to be filled. In addition, if, for example, the sample deposition is missing or performed poorly, the corresponding receiving area will have an indication signal, for example a red circle, indicating that the deposition must be repeated or that the receiving area must be isolated to be removed from the receiving area to be analyzed later.

[0070] According to a feature of the invention, the imager comprises a spectral channel, or preferably at least two spectral channels, for example, a first spectral channel corresponding to a first spectral band of 410 nm to 427 nm, and a second spectral channel corresponding to a second spectral band of 761 nm to 786 nm.

[0071] According to a feature of the invention, the imager of the system is configured to read a one-dimensional barcode or a two-dimensional barcode or QR code type image.

[0072] According to a feature of the invention, the imager of the system is further configured to acquire an image of a Petri dish in order to associate the microorganisms of the analysis sample on the carrier with the Petri dish from which they came.

[0073] According to a feature of the present invention, the carrier includes a plurality of receiving areas, for example 48 receiving areas, so that up to 48 samples from different sources can be deposited. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] The present invention will be more easily understood with the aid of the following description, which relates to some preferred embodiments of the present invention, which are provided by way of non-limiting examples and explained with reference to the accompanying schematic diagrams. The attached schematic diagrams are listed below:

[0075] Figure 1 is a schematic diagram of a preparation method according to a first embodiment;

[0076] Figure 2 is a schematic diagram of a preparation method according to a second embodiment;

[0077] Figure 3A is a schematic diagram of a preparation method according to a third embodiment;

[0078] Figure 3B is a schematic diagram of a preparation method according to a variation of the third embodiment;

[0079] Figure 4It is a series of black and white images of three sample vectors according to the R (PC1), G (PC2), and B (PC3) channels;

[0080] Figure 5 After principal component analysis, Figure 4 A graphical representation of the light intensity values ​​of the complex;

[0081] Figure 6 is a graphical representation of the light intensity values ​​of the complex according to wavelength over time;

[0082] Figure 7 are partial photographs of a support showing deposited composites with different states of completeness;

[0083] Figure 8 is a schematic diagram of a system according to the present invention;

[0084] Fig. 9 yes Figure 8 A partial diagram of a system according to the present invention is shown;

[0085] Fig.10 is a representative view of a sample carrier through a display device. DETAILED DESCRIPTION

[0086] In the context of the method according to the invention for preparing a sample carrier for analytical purposes (e.g. by mass spectrometry), a sample carrier in the form of a card with a plurality of receiving areas, called "wells" is used. For example, a droplet of sample contains one or more microorganisms to be identified, and in each well at least one droplet of matrix is ​​usually deposited. In the specific case of identifying yeasts, a drop of formic acid can be added between the sample deposition and the matrix deposition. The final deposition is called a "complex" and therefore comprises at least one layer of sample and one layer of matrix.

[0087] Before carrying out the analysis and identification of the microorganisms in the sample, the method for determining the integrity of the complex is carried out according to the invention to check whether the preparation of the support has been properly carried out, thus avoiding any false results. Regardless of the mode of execution of the preparation method according to the invention, the method for determining the integrity of the complex is carried out according to the invention by Figure 8 and Fig. 9 The system 1 shown is implemented.

[0088] According to 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 an alarm and send it to an operator. The display device 40 is configured to display a graphical representation of the carrier 10, showing at least one receiving area 11 of the carrier 10, and preferably showing all receiving areas 11 of the carrier 10.

[0089] like Figure 8As shown, in particular, the system 1 comprises a guiding device 31 interacting with the display device 20 and the display device 40 , and the guiding device 31 is integrated in the analysis unit 30 .

[0090] The guiding device is configured to indicate the receiving areas 11 of the carrier 10 where the sample and / or matrix is ​​to be deposited and is also configured to provide an alarm via the display device 40 showing which receiving areas 11 are to be filled or are full based on the progress status of the preparation method and the integrity determination method.

[0091] like Fig.10 As shown, the guiding device 31 enables the operator to be guided step by step by monitoring (preferably in real time), which is particularly possible when a plurality of images are acquired at a specified time or within a specified period according to the integrity determination method. Figure 8 As shown, the guiding device can remind the operator by means of a graphical indication signal on the carrier representation displayed on the display device 40. In the example shown, the guiding device 31 indicates to the operator by means of a circle of closely spaced dots that the two receiving areas in row C contain composites having the third integrity state (BM); it also indicates that on row D the first receiving area is empty (fourth integrity state 00) and is ready to receive sample deposition (a circle of widely spaced dots), and that on row E the first receiving area has composites whose integrity state corresponds to the first integrity state (B0) and is therefore ready to receive deposition of matrix or formic acid (continuous circle).

[0092] Now refer to Figures 1 to 3B Describe the determination method and vector preparation method.

[0093] exist Figure 1In the first embodiment shown, the integrity determination method is performed after the steps 101, 102 and 103 of depositing the sample, the matrix and, if necessary, the formic acid, respectively. The integrity determination method in the first embodiment includes capturing at least one image of the sample carrier is step 201, and more specifically, capturing an image of at least one receiving area. The captured image is then sent (step 202) to an analysis unit of the system. The analysis unit then analyzes the image by extracting light intensity values ​​representing at least one spectral band (step 203), associating the light intensity values ​​with each other for obtaining representative spectral data (step 204), and obtaining the representative spectral data (step 205). By comparing each representative spectral data obtained in the previous step with the reference spectral data (step 206), the analysis unit determines the integrity state of the complex included in the at least one receiving area. It should be noted that reference spectral data are included in the analysis unit and the reference spectral data are respectively identified for at least the following integrity states: (i) a first integrity state (B0) corresponding to the presence of sample deposition and the absence of matrix deposition; (ii) a second integrity state (OM) corresponding to the presence of matrix deposition and the absence of sample deposition; (iii) a third integrity state (BM) corresponding to the presence of sample deposition and the presence of matrix deposition. After determining the integrity state of the composite, the analysis unit sends a corresponding alarm.

[0094] When the analysis unit determines that the composite has an integrity state corresponding to the first integrity state or the second integrity state, a first alarm is sent to the operator (step 207). This is because the composite is considered to be incomplete and the operator must be aware of this. After this first alarm, a differentiation step of the integrity state is performed (step 210) so that the operator knows whether a matrix deposition deficiency or a sample deposition deficiency is detected. For this differentiation step, either a more refined analysis of the spectral bands is performed or, alternatively, the contact angle of the droplet of the composite deposited on the receiving area can be measured.

[0095] Figure 7 Deposition of complexes with different integrity states is shown. It can be seen that there is deposition of complexes with the second integrity state (0M) in the first receiving area on row B, while there is deposition of complexes with the third integrity state (BM) in the other three receiving areas on row B. Clearly, the diameter of the droplets of complexes with the second integrity state (0M) is smaller than the diameter of the droplets of complexes with the third integrity state (BM). It should be noted that the diameter observed after deposition of the matrix on the unused deposition area is approximately 70% of the nominal value of the diameter observed after deposition of the matrix on the sample forming the bacterial film.

[0096] If it is determined that substrate deposition was missing (first completeness state B0), the operator may remedy the omission by returning to step 102 to deposit the substrate, thereafter repeating the determination method to ensure that step 102 was performed correctly and the composite is complete (step 104).

[0097] If it is determined that the sample deposition is missing (second integrity state 0M), the operator identifies defective receiving areas on the display device of the analysis unit and isolates them (step 211) so that they are ignored in the subsequent analysis. It should be noted that the isolation of the defective areas (step 211) triggers the rest of the procedure, namely the decision step 105 and the end step 106 or the start step 100 of the carrier preparation method according to the decision.

[0098] When the analysis unit determines that the composite has an integrity state corresponding to the third integrity state, a second alert is sent to the operator (step 208). This is because the composite is considered complete (step 104), and the operator must be aware of this in order to proceed with the rest of the method.

[0099] Alternatively, and as shown in dashed lines, the determination method can determine a fourth integrity state (00) for the composite corresponding to a lack of sample deposition and a lack of matrix deposition. The fourth integrity state (00) is communicated to the operator via a third alarm (step 209). The operator can remedy the omission by returning to step 101 and then repeating the determination method to ensure that the composite is complete (step 104).

[0100] Optionally, the calibration step 200 may be performed prior to sample deposition (step 101 ).

[0101] exist Figure 2In the second embodiment shown, the integrity determination method is triggered; more precisely, after the first step 101 of depositing the sample, or if applicable after the step 103 of depositing formic acid, a step 201 of acquiring at least one image of the carrier and, more specifically, of acquiring at least one image of at least one receiving area is triggered. The captured image is then sent (step 202) to an analysis unit of the system. The analysis unit then analyzes the image by extracting light intensity values ​​representative of at least one spectral band (step 203), associating the light intensity values ​​with each other to obtain representative spectral data (step 204), and obtaining the representative spectral data (step 205). By comparing each representative spectral data obtained in the previous step with reference spectral data (step 206), the analysis unit determines the integrity state of the composite included in the at least one receiving area. It should be noted that the reference spectral data are included in the analysis unit and the reference spectral data are respectively identified for at least the following integrity states: (i) a first integrity state (B0) corresponding to the presence of sample deposition and the absence of matrix deposition, (ii) a fourth integrity state (00) corresponding to the absence of sample deposition and the absence of matrix deposition. After determining the integrity state of the composite, the analysis unit sends a corresponding alarm: either a first alarm (step 207) indicating a first integrity state (B0), since only step 101 has been performed at this stage, or a third alarm (step 209) indicating a fourth integrity state (00), so that the sample deposition step (step 101) is performed again, and since the third alarm (step 209) is triggered, steps 101, 103, 201, 202, 203, 204, 205 and 206 are performed. When the first integrity state (B0) has been determined, the second step (102) of depositing the matrix is ​​triggered. Steps 201, 202, 203, 204, 205 and 206 are then performed so that the analysis unit determines the integrity state of the composite: this state is either a first integrity state (B0), which returns to the matrix deposition step (step 102) and the following steps 201, 202, 203, 204, 205 and 206 when the first alarm is triggered (step 207); or a third integrity state (BM), which indicates that the composite is completed (step 104) when the second alarm is triggered (step 208), which triggers the decision step 105, and according to the decision, triggers the end step 106 or the start step 100 of the carrier preparation method.

[0102] In the first embodiment ( Figure 1 ) and the second embodiment ( Figure 2 ), the image acquisition step (step 201) is performed once, but in the first embodiment it is performed when the composite has been deposited, whereas in the second embodiment it is performed after each deposition step to form the composite. The second embodiment is more advantageous because it allows errors to be corrected more easily.

[0103] In a third embodiment shown in FIG3 , the determination method is triggered at the start step 100 of the method for preparing a carrier. The step of acquiring at least one image (step 201) is performed continuously during the steps of sample deposition (101), formic acid deposition (103) (if applicable) and matrix deposition (102). Step 201.1 indicates the start of acquisition, and steps 201n represent multiple image acquisitions during steps 101, 102, 103. After each image acquisition (201n), steps 202 to 206 are performed: if the composite has an integrity state corresponding to the third integrity state (BM), the acquisition step (201n) stops (step 201.2), the analysis unit considers the composite complete (step 104), and step 105 is implemented.

[0104] If the composite has an integrity state corresponding to the first integrity state (B0) or the second integrity state (0M), a first alarm is triggered (step 207), or if, optionally, the state is a fourth integrity state (00), a third alarm is triggered (step 209), then, in the first case, the operator decides to isolate the defective receiving area (step 211), the image acquisition (201n) is stopped (step 201.2) and step 105 is implemented; in the second case, the operator decides whether to correct the error, and if the first integrity state (B0) is determined, the image acquisition is continued (step 201n), followed by steps 202 to 206 of the determination method, and step 102 of the carrier preparation method is performed again.

[0105] In a variation of the third embodiment, Figure 3B As shown, after the matrix deposition step (step 102), the image acquisition is stopped (201n) (step 201.2), and the analysis unit implements steps 202 to 206: if the composite has an integrity state corresponding to the third integrity state (BM), the analysis unit considers the composite to be complete (step 104) and implements step 105; in all other cases (integrity states 00, B0, 0M), the operator decides to isolate the defective receiving area (step 211), implements step 105 and, depending on the decision, implements the end step 106 of the carrier preparation method or the start step 100.

[0106] The present invention Figure 4 and Figure 5 The example shown is explained. This example is obviously non-limiting and makes it easier to understand the claimed method. In this example, three spectral channels R, G, B are used. Figure 4A series of images of three supports with deposition of different complexes is shown. The first image of the three supports is an image according to the first channel R (PC1), the second image of the three supports is an image according to the second channel G (PC2), and the third image of the three supports is an image according to the third channel B (PC3). The images are in black and white. The different complexes are then identified by the points whose coordinates (PC1; PC2; PC3) correspond to the light intensity values ​​according to the selected wavelength (RGB). After principal component analysis, as Figure 5 As shown in , the analysis unit of the system groups different points by similarity in order to establish a "grouping" of integrity states for them. In this mode, the distinction of integrity states is more obvious. However, it should be noted that principal component analysis is not necessary. Figure 5 As shown, empty receiving areas (compounds missing, equivalent to the fourth integrity state 00) are separated from the batch and represented by small circles, compounds with an integrity state equivalent to the first integrity state B0 are represented by a "+" sign, compounds with an integrity state equivalent to the second integrity state 0M are represented by triangles, and compounds with an integrity state equivalent to the second integrity state 0M are represented by a cross "x".

[0107] Figure 6 A graphical representation of the light intensity values ​​of the composite according to the specified wavelength over time is shown. In this figure, the first peak corresponds to the movement of the point of the deposition tool under the imager for depositing the sample. The light intensity difference after this first peak is small. The second peak corresponds to the movement of the hand under the imager for depositing the matrix. Then, the signal reaches its maximum value after the matrix dries and crystallizes and turns white.

[0108] Obviously, the invention is not limited to the embodiments described and represented in the drawings. Modifications may be made without departing from the scope of protection of the invention, in particular in the composition of the various elements or by substitution by equivalents.

Claims

1. A method for determining the integrity of at least one complex based on at least one biological sample and at least one matrix, the complex being arranged in at least one receiving area (11) of a carrier (10) to be subsequently analyzed, the method being implemented by a determination system (1) comprising at least one imager (20) and an analysis unit (30) configured to interact with the imager (20), characterized in that The method comprises at least the following steps: - a step (201, 201n) of acquiring at least one image of said at least one receiving area (11) of said carrier (10), said acquisition being performed by said imager (20) of said determination system (1); - a step (202) of sending the at least one image acquired by the imager (20) to the analysis unit (30); - a step (203) of analyzing said image sent by extracting light intensity values ​​representative of at least one spectral band, said analysis being performed by said analysis unit (30); - a step of correlating said light intensity values ​​to obtain representative spectral data and obtaining said representative spectral data (204); - a step (206) of determining the integrity state of the composite by comparing (205) each of the representative spectral data obtained in the associating step with reference spectral data, the reference spectral data being included in the analyzing unit (30) and being identified for each of at least the following integrity states, by performing similarity grouping at a specified similarity threshold: (i) a first integrity state (B0), corresponding to the presence of sample deposits and the absence of matrix deposits, (ii) a second integrity state (OM), corresponding to the presence of matrix deposition and the absence of sample deposition, (iii) the third integrity state (BM), which corresponds to the presence of sample deposition and the presence of matrix deposition; - a step (207) of triggering at least a first alarm by the analysis unit (30) when the representative spectral data approximate the first integrity state (B0) or the second integrity state (0M).

2. The determination method according to claim 1, wherein: The light intensity values ​​represent at least two different spectral bands.

3. The determination method according to claim 1, wherein: The light intensity values ​​represent at least three different spectral bands.

4. The determination method according to any one of claims 1 to 3, wherein: In the acquisition step, the imager (20) acquires at least a plurality of images of the same receiving area (11) of the carrier (10), and the acquisition of the images is performed at different designated moments or within at least one designated time period.

5. The determination method according to any one of claims 1 to 3, wherein the imager (20) is an RGB imager or a multispectral imager or a hyperspectral imager.

6. Determination method according to any one of claims 1 to 3, wherein the list of integrity states comprises at least a fourth integrity state (00) corresponding to absence of sample deposition mixed with absence of matrix deposition.

7. The determination method according to any one of claims 1 to 3 comprises a calibration step (200) prior to the acquisition step of the method, the calibration step (200) comprising acquiring an image, referred to as a reference image, of at least one empty reception area (11) and recording the reference image in the analysis unit (30).

8. A method for preparing a sample carrier (10) for analysis by mass spectrometry to detect the properties of microorganisms present in a sample deposited on the carrier, the preparation method comprising at least the following steps: - a step (101) of depositing a biological sample in at least one receiving area (11) of the carrier (10); - a step (102) of depositing a matrix in the at least one receiving area (11) of the carrier (10) on which the sample has been deposited, The preparation method is characterized in that the preparation method comprises the method for determining the integrity of the at least one complex according to any one of claims 1 to 7.

9. The preparation method according to claim 8, wherein the step (201) of acquiring at least one image of the determination method is performed after the step (102) of depositing the matrix.

10. The preparation method according to claim 8, wherein the step (201) of acquiring at least one image of the determination method is performed after the step (101) of depositing the sample and after the step (102) of depositing the matrix.

11. The preparation method according to claim 8, 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 substrate, the acquisition step being performed continuously during each step of the preparation method.

12. A system (1) configured to implement the determination method according to any one of claims 1 to 7, the system (1) comprising at least one imager (20), an analysis unit (30) and at least one display device (40) configured to display an alarm.

13. The system of claim 12, wherein the display device (40) is configured to display a graphical representation of a carrier (10) showing at least one receiving area (11) of the carrier (10).

14. The system of claim 12, wherein the display device (40) is configured to display a graphical representation of the carrier (10) showing all receiving areas (11) of the carrier (10).

15. A system as claimed in claim 13 or 14, comprising a guiding device interacting with the display device (40), the guiding device (31) being integrated into the analysis unit (30), the guiding device (31) being configured to indicate a receiving area (11) designated for deposition of a sample and / or deposition of a matrix.

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