Patent ID: 12211681

In the context of a method for preparing a sample support according to the invention for the purpose of analysis, by mass spectrometer for example, a sample support in the form of a card having a plurality of receiving areas, called “wells”, is used. A drop of a sample containing one or more microorganisms to be identified, for example, and at least one drop of matrix are usually deposited in each well. In the particular case of yeast identification, a drop of formic acid may be added between the sample deposition and the matrix deposition. The final deposit is called a “complex”, and therefore comprises at least one layer of sample and one layer of matrix.

Before proceeding to the analysis and identification of the microorganism(s) in the sample, a method of determining the integrity of the complex is executed according to the invention, in order to check whether the preparation of the support has been executed appropriately, to avoid any incorrect results. Regardless of the mode of execution of the preparation method according to the invention, the determination method according to the invention is implemented by a system1shown inFIGS.8and9.

According to the invention, the system1comprises at least one imager20, an analysis unit30and at least one display device40configured for displaying the alert(s) and transmitting them to the operator. The display device40is configured for displaying a graphic representation of the support10, illustrating the at least one receiving area11of the support10, and preferably all the receiving areas11of the support10.

As shown inFIG.8, notably, the system1comprises a guidance device31interacting with the display device20and with the display device40, the guidance device31being integrated into the analysis unit30.

The guidance device is configured for indicating the receiving area(s)11of the support10in which a sample and/or a matrix is to be deposited, and also for providing an alert via the display device40showing which receiving area11is to be filled or topped up, on the basis of the state of progress of the preparation method and the method of integrity determination.

As may be seen inFIG.10, the guidance device31enables the operator to be guided from step to step with monitoring, preferably in real time, which is, notably, possible when a plurality of images are acquired according to the integrity determination method, either at specified times or over a specified period. As may be seen inFIG.8, the guidance device may alert the operator via graphic indicators on the representation of the support displayed on the display device40. In the illustrated example, the guidance device31indicates to the operator, by a ring of closely spaced dots, that the two receiving areas in line C comprise complexes having a third state of integrity (BM); it also indicates that, on line D, the first reception area is empty (fourth state of integrity00) and is ready to receive a sample deposit (ring of widely spaced dots) and that, on line E, the first reception area has a complex whose state of integrity corresponds to the first state of integrity (B0) and is therefore ready to receive a deposit of matrix or formic acid (continuous ring).

The method of determination and the method of support preparation will now be described with reference toFIGS.1to3B.

In a first embodiment shown inFIG.1, the method of integrity determination is executed after steps101,102, and103of depositing a sample, a matrix, and if necessary formic acid, respectively. Said method of integrity determination in the first embodiment comprises a step201of capturing at least one image of the sample support, and more particularly an image of at least one reception area. The captured image is then sent (step202) to the analysis unit of the system. The analysis unit then analyzes the image by extracting values of light intensity representative of at least one spectral band (step203), relates the light intensity values to each other for the purpose of obtaining representative spectral data (step204), and obtains said representative spectral data (step205). By comparing (step206) each of the representative spectral data obtained in the preceding step with reference spectral data, the analysis unit determines a state of integrity of the complex contained in the at least one receiving area. It should be noted that the reference spectral data are contained in the analysis unit, and are each identified for a state of integrity among at least: (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.

Following the determination of the state of integrity of the complex, the analysis unit sends a corresponding alert.

A first alert is sent to the operator (step207) when the analysis unit determines that the complex has a state of integrity corresponding to the first state of integrity or to the second state of integrity. This is because the complex is considered to be incomplete, and the operator must be aware of this. Following this first alert, a step of differentiation (step210) of the state of integrity is executed to enable the operator to know whether it is an absence of matrix deposit or an absence of sample deposit that has been detected. For this differentiation step, either a finer analysis of the spectral bands is performed, or, alternatively, the contact angle of the drop of the complex deposited on the receiving area may be measured.

FIG.7shows deposits of complexes having different states of integrity. It can be seen that in the first receiving area on line B there is a deposit of complex having a second state of integrity (0M), and in the other three receiving areas on line B there are deposits of complex having a third state of integrity (BM). It will be apparent that the drop of the complex having a second state of integrity (0M) has a smaller diameter than that of the drops of the complex having a third state of integrity (BM). It should be noted that the diameter observed after the deposition of the matrix on the unused deposition area is about 70% of the nominal value of the diameter observed after the deposition of the matrix on the sample that forms a bacterial film.

If it is determined that the matrix deposit is missing (first state of integrity B0), the operator may remedy the omission by depositing said matrix by returning to step102, after which the determination method is repeated to ensure that step102has been correctly executed and that the complex is complete (step104). If it is determined that the sample deposit is missing (second state of integrity0M), the operator identifies the defective receiving areas on the display device of the analysis unit, and isolates them (step211) so that they are disregarded in the subsequent analysis. It should be noted that the isolation of the defective areas (step211) triggers the rest of the procedure, that is to say the decision step105and, depending on the decision, either step106of concluding the support preparation method or step100of initialization.

A second alert is sent to the operator (step208) when the analysis unit determines that the complex has a state of integrity corresponding to the third state of integrity. This is because the complex is considered to be complete (step104), and the operator must be aware of this in order to move on to the rest of the method. Optionally, and as shown in dotted lines, the determination method may determine a fourth state of integrity (00) of the complex corresponding to the absence of a sample deposit and the absence of a matrix deposit. This fourth state of integrity (00) is notified to the operator by a third alert (step209). The operator may remedy the omission by returning to step101, after which the determination method is repeated to ensure that the complex is complete (step104).

Optionally, a calibration step200may be executed before the sample deposition (step101).

In a second embodiment shown inFIG.2, the integrity determination method is triggered; more precisely, the step201of acquiring at least one image of the support, and more particularly of at least one reception area, is triggered after the first step101of depositing a sample, or after the step of depositing formic acid103if appropriate. The captured image is then sent (step202) to the analysis unit of the system. The analysis unit then analyzes the image by extracting values of light intensity representative of at least one spectral band (step203), relates the light intensity values to each other for the purpose of obtaining representative spectral data (step204), and obtains said representative spectral data (step205). By comparing (step206) each of the representative spectral data obtained in the preceding step with reference spectral data, the analysis unit determines a state of integrity of the complex contained in the at least one receiving area. It should be noted that the reference spectral data are contained in the analysis unit, and are each identified for a state of integrity among at least: (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 fourth state of integrity (00) corresponding to the absence of a sample deposit combined with the absence of a matrix deposit. Following the determination of the state of integrity of the complex, the analysis unit sends a corresponding alert: either a first alert (step207) indicating a first state of integrity (B0) because only step101has been executed at this stage, or a third alert (step209) indicating a fourth state of integrity (00) so that the step of sample deposition (step101) is executed again, steps101,103,201,202,203,204,205and206being executed because the third alert (step209) is triggered. When the first state of integrity (B0) has been determined, the second step (102) of depositing a matrix is triggered. Steps201,202,203,204,205and206are then executed, so that the analysis unit determines the state of integrity of the complex: this state is either the first state of integrity (B0), when the first alert is triggered (step207) to return to the step of matrix deposition (step102) and the following steps201,202,203,204,205and206, or the third state of integrity (BM), when a second alert (step208) is triggered, indicating that the complex is complete (step104), which triggers the decision step105and, depending on the decision, either step106of concluding the support preparation method or step100of initialization.

In the first embodiment (FIG.1) and the second embodiment (FIG.2), the step of acquisition of image(s) (step201) is executed on a one-off basis, but in the first embodiment it is executed when the complex has been deposited, while in the second embodiment it is executed after each deposition step forming the complex. The second embodiment is more advantageous, because it allows errors to be rectified more easily.

In a third embodiment shown inFIG.3, the determination method is triggered at step100of initialization of the method for preparing the support. The step of acquiring at least one image (step201) is executed continuously during the steps of sample deposition (101), formic acid deposition (103) if appropriate, and matrix deposition (102). The start of acquisition is illustrated by step201.1, step201nrepresenting the plurality of image acquisitions in the course of steps101,102,103. After each image acquisition (201n), steps202to206are executed: if the complex has a state of integrity corresponding to the third state of integrity (BM), then the acquisition step (201n) stops (step201.2), the analysis unit considers the complex to be complete (step104), and step105is implemented.

If the complex has a state of integrity corresponding to the first (B0) or second state of integrity (0M), then the first alert is triggered (step207), or if, optionally, the state is a fourth state of integrity (00), then the third alert is triggered (step209), and then, in a first case, the operator decides to isolate the defective receiving area (step211), the image acquisition (201n) stops (step201.2), and step105is implemented; in a second case, the operator decides whether to correct the error, if a first state of integrity (B0) is determined, the image acquisition continues (step201n), and steps202to206of the determination are followed and step102of the support preparation method is executed again.

In a variant of this third embodiment, as illustrated inFIG.3B, after the step of matrix deposition (step102), the image acquisition (201n) is stopped (step201.2) and the analysis unit implements steps202to206: if the complex has a state of integrity corresponding to the third state of integrity (BM), then the analysis unit considers the complex to be complete (step104), and step105is implemented; in all other cases (states of integrity00, B0,0M), the operator decides to isolate the defective receiving area (step211) and step105is implemented, as well as, depending on the decision, either step106of concluding the support preparation method or step100of initialization.

The invention is illustrated with an example shown inFIGS.4and5. This example is, evidently, non-limiting and enables the claimed approach to be understood more readily. In this example, three spectral channels R, G, B are used.FIG.4shows a series of images of three supports having deposits of different complexes. 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 points whose coordinates (PC1; PC2; PC3) correspond to their light intensity values as a function of the chosen wavelengths (RGB). After principal component analysis, as shown inFIG.5, the analysis unit of the system groups the different points by similarity in order to establish “groups” of states of integrity for them, the differentiation of the states of integrity being more obvious in this mode. However, it should be noted that the principal component analysis is not obligatory. As may be seen inFIG.5, the empty receiving areas (absence of complex, equivalent to the fourth state of integrity00) are separated from the batch and are represented by small circles, the complexes having a state of integrity equivalent to the first state of integrity B0are represented by “+” signs, the complexes having a state of integrity equivalent to the second state of integrity0M are represented by triangles, and the complexes having a state of integrity equivalent to the second state of integrity0M are represented by crosses, “×”.

FIG.6shows a graphic representation of the light intensity values of a complex as a function of time according to a specified wavelength. In this graph, the first peak corresponds to the movement of the point of the deposition tool under the imager for depositing a sample. The difference in light intensity after this first peak is small. The second peak corresponds to the movement of a hand under the imager for depositing the matrix. The signal then reaches its maximum after the drying of the matrix, which becomes white as it crystallizes.

Clearly, the invention is not limited to the embodiments described and represented in the appended figures. Modifications may be made, notably in terms of the composition of the various elements, or by substitution of equivalent methods, without thereby departing from the scope of protection of the invention.