Automated sampling method for handling whole blood

Abstract

The object of the present invention is to provide a method that allows for the handling of whole blood for testing, said whole blood being drawn directly from a patient without any pretreatment and placed in a blood collection tube or other ready-to-use container for further analysis. The object of this method is to allow for a simple, reproducible and automatable treatment of whole blood, without damaging white blood cells and preventing any clots or aggregates. Furthermore, the object of the present invention is to allow for the automated and reproducible pipetting of whole blood cells into several wells to be examined.

Description

Technical Field

[0001] The present invention relates to an automatic sampling method for handling heterogeneous biological samples such as whole blood. More specifically, the automatic sampling method of the present invention enables sampling of a heterogeneous biological sample and distributing it in a reproducible manner after homogenizing the biological sample for further analysis, and thus standardizes the situation and state of the sample before processing with an automatic device.

[0002] The automatic sampling method according to the present invention can preferably be implemented in a device for in vitro detection and / or quantification of at least one analyte in a biological sample. The present invention can be used in an automated device for in vitro diagnosis in a clinical setting.

[0003] For example, the present invention can be applied to a known test (interferon γ release test) that involves a manual stimulation process of contacting a patient's whole blood cells with a stimulatory peptide that mimics Mycobacterium tuberculosis. In the case of a preliminary contact with a pathogen, the patient's blood leukocytes will induce the secretion of interferon γ during a latency period of 16 to 24 hours at 37°C. After precipitation of the stimulated cells, the last step of the test consists of examining the released interferon γ (IFN-γ).

[0004] Nevertheless, the present invention can be applied to any field related to any test that has to handle whole blood or heterogeneous biological samples that tend to precipitate and require homogenization before further analysis.

Background Art

[0005] A major challenge when handling whole blood or heterogeneous biological samples for analysis is the tendency of samples to precipitate. In whole blood precipitates containing plasma and solid components such as red blood cells, white blood cells, and platelets, red blood cell decantation occurs at the bottom of the collection tube, and white blood cells rise to the boundary between plasma and red blood cells. Therefore, if you intend to sample whole blood, homogenization is essential, especially if the first tube has been waiting for a long time and precipitation has begun. Without homogenization, there is a risk that the volume of white blood cells will differ depending on the depth of sample collection between several blood aspirates. Blood precipitation can be very fast or slow depending on the characteristics of the patient's blood, such as hematocrit levels, so homogenization to accommodate the resuspension of elements is particularly important. Furthermore, while whole blood management usually refers to obtaining plasma from the first tube, for some tests, such as testing for interferon-gamma, it is necessary to avoid damage to cells, mainly white blood cells, so homogenization must be done carefully and reproducibly to maintain the volume of white blood cells. Finally, it is essential to consider that whole blood and other heterogeneous biological samples belong to the category of liquids that are difficult to handle (due to their heterogeneity), and moreover, they are in small volumes of about a few hundred microliters and have never been used in this manner before, especially without automated preparation / pre-processing.

[0006] According to the present invention, the term "tip" is used and should be understood as any means that is placed on a pipette, in contact with a sample, and used to aspirate / dispense the sample, and the tip may be a needle or tube or any similar element having the same function, and may be removable or not. [Overview of the Initiative]

[0007] The object of the present invention is to provide a method for handling whole blood for testing, said whole blood being collected directly from a patient without pretreatment and placed in a blood collection tube or other prepared container for further analysis. The object of this method is to enable simple, reproducible, and automated processing of whole blood without damaging leukocytes and preventing any clots or aggregates. Furthermore, the object of the present invention is to enable automated and reproducible pipetting of whole blood cells within several wells to be tested.

[0008] Therefore, the present invention relates to an automated sampling method for heterogeneous biological samples contained in containers such as blood collection tubes, wherein the automated sampling method is: - A step of preparing an apparatus comprising at least one pipette operating unit having at least one pipette having a tip, and a control unit that cooperates with and controls the pipette operating unit, - A step of loading a container containing heterogeneous biological samples into the device, - A step in which the automatic homogenization of the heterogeneous biological sample is initiated inside the apparatus, wherein the homogenization is performed • A substep of lowering the pipette tip into the container to a first predetermined depth below the surface of the heterogeneous biological sample, A step to initiate automated homogenization, comprising: a substep of causing a pipette to perform multiple cycles of aspirating and distributing the heterogeneous biological sample into a container, wherein in each cycle, the pipette preferentially aspirates the biological sample to the entire volume of the pipette tip at a first predetermined depth before returning it to the container, and the tip of the pipette is maintained at the first predetermined depth below the surface of the heterogeneous biological sample throughout the multiple cycles; - Once the above cycles are completed, A first step of aspirating a predetermined volume of heterogeneous biological sample from a container, wherein the tip is submerged to a first predetermined depth, • The second step involves distributing a predetermined volume previously aspirated into a well different from the container containing the heterogeneous biological sample. This involves the step of sampling a heterogeneous biological sample of a predetermined volume, Includes.

[0009] According to one of the features of this invention, the heterogeneous biological sample is whole blood.

[0010] The method according to the present invention is - In a container, the aspirating / dispensing cycle at a predetermined depth below the surface of the heterogeneous biological sample reduces or even prevents the effects of clumping, agglomeration, bubbles, and decantation. - By keeping the tip of the pipette below the surface, it becomes possible to aspirate only homogeneous samples. - Multiple cycles of the homogenization step create turbulent zones within the container, allowing for the resuspension of cells present in heterogeneous biological samples and the removal of aggregates. - During sampling, a first step of aspirating to a predetermined depth ensures that only air-free, homogeneous samples are aspirated, and these homogeneous samples are then distributed into wells for further analysis. - The homogenization step wets the tip edge, minimizing sliding friction and improving the accuracy of the distributed capacitance. - A homogenization step performed in the container, with an optimal rate and an optimal delay between aspiration and distribution, enables the aspiration of a reproducible amount of blood cells without damaging the cells. In many ways, this is extremely advantageous.

[0011] Advantageously, the reproducibility of the distribution in terms of the number of white blood cells is demonstrated by comparative measurements of hematocrit values ​​for red blood cells across several wells. Comparative measurements of hematocrit values ​​are also used to optimize the number and rate of aspiration / distribution cycles, as well as the delay between aspiration and distribution in the homogenization step.

[0012] According to one of the features of the present invention, the pipette tip is the same for both the homogenization step and the sampling step.

[0013] According to one feature of the present invention, with respect to a 4 mL container and a 300 μL tip volume, the pipette operating unit preferably performs 3 to 12 aspiration / dispensing cycles during the homogenization step, more preferably 6 to 20 times.

[0014] According to one feature of the present invention, the homogenization step includes a substep of equipping a pipette operating unit with a tip located in front of a container of heterogeneous biological samples.

[0015] According to one feature of the present invention, during the homogenization step, aspiration is performed at a first predetermined speed that can prevent damage to blood cells. According to one feature of the present invention, during homogenization, distribution is performed at a second predetermined speed that can prevent damage to blood cells. Preferably, the first predetermined speed and the second predetermined speed are different. Advantageously, the first predetermined speed and the second predetermined speed are independent of each other.

[0016] According to one of the features of this invention, the pipette tip is replaced or washed after each sampling step, thereby enabling precise control of sampling conditions to ensure that external conditions are identical for several samples in separate wells, and ensuring a reproducible and accurate method. Furthermore, replacing or washing the tip prevents contamination of the tip.

[0017] Alternatively, the pipette tip could be the same for several sampling steps, and then only replaced or washed to prevent contamination of the tip when another well to be filled is empty.

[0018] According to one of the features of the present invention, this method includes a step of manually inverting the container prior to the step of loading the container into the apparatus. The manual inversion is optional and can improve the homogenization step.

[0019] According to one of the features of the present invention, the sampling step is performed immediately after the homogenization step in order to utilize the turbulent flow zone generated by the homogenization step and also so as not to aspirate the aggregates that concentrate towards the tip of the chip.

[0020] According to one of the features of the present invention, the volume of the inhomogeneous biological sample aspirated to be dispensed in the wells for further analysis can vary as a function of the type of analysis to be performed and also as a function of the volume of the chip equipped on the pipette. For example, the volume of the chip may be from 200 μL to 400 μL, and more particularly may be about 300 μL. It should be noted that if the volume of the chip and / or the volume to be sampled is less than 300 μL, then accordingly, the number of cycles, speed and delay have to be optimized (possibly increased) to fit.

[0021] According to one of the features of the present invention, the aspiration during the sampling step can be performed one or several times depending on the volume determined to be required and the volume of the pipette tip. For example, the decantation of whole blood can be quite fast. If the pipetting operation is set to 300 μL at one time, the period of aspiration in separate pipetting operations before starting the analysis can be shortened compared to aspirating 200 μL twice to obtain 400 μL.

[0022] According to one of the features of the present invention, at least one step of liquid level tracking further included in the automatic sampling method is - detecting the liquid height of the inhomogeneous biological sample in the container by means of a sensor; - theoretically determining a first predetermined depth by the control unit based on the liquid height, the volume of the inhomogeneous biological sample to be aspirated / dispensed, and / or the volume and size of the container. - During the homogenization step and / or the sampling step, monitor the movement of the tip of the chip in the container to follow changes in the liquid level in the tube, and exists in.

[0023] According to one of the features of the present invention, the term "size" of the container should be understood as the diameter and length, mainly as the diameter.

[0024] According to one of the features of the present invention, at least one step of liquid level tracking can be performed during the suction and / or distribution of the homogenization step. According to one of the features of the present invention, the inhomogeneous biological sample is a liquid.

[0025] According to this configuration, during the homogenization step, the tip of the chip is always submerged below the surface of the inhomogeneous liquid biological sample in the container, making good use of the generated turbulent flow zone to suck and distribute the homogenized liquid and prevent the suction of air. Also, during the homogenization step, when the pipette sucks a part of the inhomogeneous biological sample, the tip of the chip follows the level drop / level rise of the sample so as to be kept below the surface. In other words, when the pipette sucks the sample, the level of the sample in the container drops, so the tip of the chip sinks deeper into the inhomogeneous biological sample in the container, and when the pipette distributes the inhomogeneous biological sample into the container, the level of the inhomogeneous biological sample in the container rises and the tip of the chip rises and returns.

[0026] According to one of the features of the present invention, at least one step of liquid level tracking can be performed during the suction stage of the sampling step.

[0027] According to one of the features of the present invention, at least one monitoring step further included in the automatic sampling method is - A sensing substep is configured to send to a control unit at least one pressure value, preferably several pressure values, corresponding to the detected pressure or vacuum, which is configured to detect pressure or vacuum occurring inside the tip of a pipette during a homogenization step or a sampling step, and to send at least one pressure value, preferably several pressure values, corresponding to the detected pressure or vacuum. - A substep in which the control unit compares at least one received pressure value with a pressure threshold predetermined and built into the control unit, which corresponds to at least one characteristic pressure index representing a characteristic sampling loss. - A control unit determines, based on the substep being compared, whether there is a problem in the homogenization step or the sampling step, and a determination substep. Includes.

[0028] According to one feature of the present invention, the monitoring step is performed during the aspiration of the homogenization step, during the distribution of the homogenization step, during the aspiration stage of the sampling step, and / or during the distribution stage of the sampling step.

[0029] According to one feature of the present invention, a pressure threshold is predetermined and incorporated into the control unit, preferably to perform an automated sampling method during feasibility testing.

[0030] According to one feature of the present invention, the pressure threshold corresponds to at least one characteristic pressure index selected from a plurality of possible pressure indices, the selection being based on the sensitivity of the pressure index to represent sampling defects. In other words, the selected characteristic pressure index best represents sampling defects, which are potential defects that occur during the sampling or homogenization steps and affect the liquid processing. For example, sampling defects may be lumps, air leaks, aggregates, liquid films, bubbles, foam, etc.

[0031] For advantage, at least one pressure threshold corresponds to each characteristic pressure index.

[0032] According to one feature of the present invention, the sensor used for pressure measurement during the homogenization step is the same as the one used during the sampling step. More specifically, the sensor used for pressure measurement during suction is the same as the one used during distribution.

[0033] According to one feature of the present invention, during a detection substep, the control unit executes an algorithm that monitors selected characteristic pressure indicators. Preferably, the algorithm monitors only some of the selected characteristic pressure indicators. Preferably, some of the selected characteristic pressure indicators take into account the characteristics of sampling loss occurring during suction, some take into account the characteristics of sampling loss occurring during distribution, and some relate to both. Advantageously, according to the suction step or the distribution step, during which the monitoring step is performed, the control unit selects the best characteristic pressure indicator to monitor.

[0034] According to one feature of the present invention, the characteristic pressure index may be a pressure value or a pressure value calculated by an algorithm incorporated into the control unit. For example, the characteristic pressure index may be the maximum pressure reached during suction, which may be specific to clogging during suction, or a scoring check calculated from the pressure value may be a characteristic of leaks, air intakes, bubbles and / or foam.

[0035] According to the present invention, the control unit triggers an alarm and automatically stops the homogenization step if it determines that at least one pressure value measured by the sensor during the homogenization step exceeds at least one determined pressure threshold corresponding to at least one pressure index. Therefore, if the control unit determines that at least one pressure value measured by the sensor during the homogenization step is less than or equal to at least one determined pressure threshold corresponding to at least one pressure index, it continues the homogenization step cycle to the end and then triggers the sampling step.

[0036] According to the present invention, if the control unit determines that at least one pressure value measured by the sensor during the sampling step exceeds at least one determined pressure threshold corresponding to at least one of the selected characteristic pressure indices, it triggers an alarm and the sampling step is automatically stopped. Therefore, if the control unit determines that at least one pressure value measured by the sensor during the sampling step is less than or equal to at least one determined pressure threshold corresponding to at least one pressure indices, it continues the sampling step until the automatic sampling method is deemed to be completed and achieved.

[0037] According to one feature of the present invention, the control unit is configured to command a retry mode that retries an identified step of an automated sampling method, the step being identified by the control unit if it determines that a failure has occurred because the measured pressure value corresponds to at least one determined pressure threshold. Advantageously, the retry mode prevents time loss in running the entire method from the beginning, and the retry mode is advantageous in terms of the productivity of the automated system and in saving sample volume because the sample is distributed and returned before execution.

[0038] In the present invention, the specified “identified step” may be a homogenization step (suction and / or distribution) or the suction step of a sampling step.

[0039] According to one feature of the present invention, when the aspiration step is retried in retry mode, the pipette tip is submerged into the biological sample to a second predetermined depth, which is deeper than a first predetermined depth to which the tip is submerged for homogenization aspiration or for the aspiration stage of the sampling step. This configuration makes it possible to prevent the tip from moving to an area of ​​the sample container different from where defects that would adversely affect the handling of the previous sample were found, in order to ensure homogeneous volume aspiration, and also makes it possible to prevent the aspiration of clumps / aggregates that may have been previously aspirated.

[0040] Advantageously, the control unit determines a second predetermined depth based on a first predetermined depth.

[0041] According to one feature of the present invention, in retry mode, the pipette tip is replaced or cleaned before retrying the failed step.

[0042] According to one feature of the present invention, the retry mode includes at least one monitoring step, wherein the monitoring step includes the same substeps used during the homogenization step and / or sampling step.

[0043] According to one feature of the present invention, in retry mode, if a failure occurs in the sampling step, the homogenization step is repeated, allowing the tip end to remain wet and improving the accuracy of aspiration. Alternatively, in retry mode, if a failure occurs in the sampling step, the homogenization step is not repeated to prevent further stress on the cells.

[0044] Another object of the present invention is an apparatus configured to perform an automated sampling method according to the present invention, wherein at least, - For example, a first workstation loaded with a container such as a blood collection tube containing a heterogeneous biological sample, such as a whole blood sample, - A second workstation configured to accept a predetermined volume of heterogeneous biological sample and loaded with at least one well distinct from the container, - A pipette operating unit comprising a pipette equipped with a tip, configured to move between at least two workstations within the device, - At least one control unit configured to cooperate with and control the pipette operating unit, It is a device equipped with [a certain feature].

[0045] According to one of the features of the present invention, since the chip is removable, it is easy to replace the chip and contamination can be prevented.

[0046] According to one of the features of the present invention, the chips can be disposable, and contamination between steps of the automated sampling method can be prevented.

[0047] According to one feature of the present invention, the pipette operating unit comprises at least one sensor configured to sense and monitor the pressure or vacuum inside the pipette tip, and is preferentially located on the body of the pipette.

[0048] According to one of the features of the present invention, at least one sensor may be a pressure sensor.

[0049] According to one of the features of the present invention, at least one sensor is placed on the body of the pipette.

[0050] Advantageously, the sensor is configured to detect the retention of the tip on the pipette in order to detect the presence and level of liquid in the container. More specifically, at least one sensor is configured to control suction in terms of agglomeration, bubbles, foam, membranes, air aspiration, etc.

[0051] Specifically, at least one sensor can detect that the device is precisely penetrating the foil during the penetration step.

[0052] According to one of the features of the present invention, the apparatus is configured to perform a detection and / or quantification method, including an automated sampling method according to the present invention.

[0053] According to one feature of the present invention, the inner wall of the sample container is coated with lithium heparin. Advantageously, heparin is a commonly used anticoagulant, particularly in clinical biochemistry and chemical measurement tests. Heparin is a preferred anticoagulant for blood chemical measurements or plasma tests due to its minimal chelating properties, minimal interference with water (and relatively low cation concentration). In fact, EDTA (ethylenediaminetetraacetique), which is also used as an anticoagulant, cannot be used in certain tests because it inhibits irritation.

[0054] Another object of the present invention is a method for carrying out the automated sampling method according to the present invention for the detection and / or quantification of a specimen in a heterogeneous biological sample, such as a whole blood sample, wherein the detection and / or quantification method is carried out by an apparatus configured to carry out the automated sampling method according to the present invention, the apparatus according to the present invention comprises at least, - For example, a first workstation loaded with a container such as a blood collection tube containing a heterogeneous biological sample, such as a whole blood sample, - A second workstation configured to accept a predetermined volume of heterogeneous biological sample and loaded with at least one well distinct from the container, - A pipette operating unit comprising a pipette equipped with a tip, configured to move between at least two workstations within the apparatus, It is equipped with.

[0055] According to one feature of the present invention, a detection and / or quantification method includes at least one step of loading at least one support having at least one well configured to receive a predetermined volume of heterogeneous biological sample aspirated from a container inside the device.

[0056] According to one feature of the present invention, a detection and / or quantification method includes at least one step of equipping a pipette with a first tip. Advantageously, if at least one well is protected by a protective foil, the first tip is configured to penetrate the protective foil positioned on the well of at least one support. In this case, if the first tip is removable, it is replaced with a second tip before the homogenization step. If the first tip is not removable, it can be washed instead.

[0057] According to one feature of the present invention, the support is a strip and comprises at least one well, preferably a plurality of wells adjacent to each other.

[0058] According to one feature of the present invention, the method includes at least one step of inserting a first tip of a pipette into at least one protective foil placed on at least one well of the apparatus.

[0059] According to one feature of the present invention, a detection and / or quantification method further includes the step of removing the first tip and placing a second tip on the pipette, the second tip being configured to be used in an automated sampling method for use during a homogenization step and for use in a sampling step. It is advantageous to replace the first tip between insertion into the protective foil and the automated sampling method. In fact, the tip may be damaged or deformed during insertion, which could impair the accuracy of the volume of blood aspirated or sample distributed through the automated sampling method during aspiration. Alternatively, a detection and / or quantification method further includes the step of washing the first tip, which is configured to be used in an automated sampling method for use during a homogenization step and for use in a sampling step.

[0060] According to one feature of the present invention, the substeps distributed by the sampling step are performed in an empty, dedicated well of the support without replacing the first or second chip.

[0061] According to one of the features of the present invention, the detection and / or quantification method is - The steps of removing the second tip from the pipette and installing the third tip, or cleaning the first tip, - A step of aspirating an irritating reagent contained in a special glass bottle and distributing it into a well containing the sampled heterogeneous biological sample, It also includes.

[0062] Sampling of heterogeneous biological samples is performed before pipetting of irritating reagents. Since each well dedicated to whole blood samples is already opened and empty by the first tip and no carryover occurs, this has the advantage of using identical conditions for the tip between dispensing separate biological samples into wells of separate supports.

[0063] The detection and / or quantification method may be used to quantify interferon (IFN-y) in the TB-IGRA test (tuberculosis interferon-gamma release assay). In this application, at least three separate supports, each equipped with wells, are loaded inside the apparatus. Each support may be in the form of a strip, preferably made of plastic, and contains multiple adjacent wells, one of which is dedicated to the whole blood sample.

[0064] Another object of the present invention is the use of an automated sampling method according to the present invention to enable the quantification of interferon (IFN-y) in a tuberculosis interferon-gamma release assay in a method according to the present invention. [Brief explanation of the drawing]

[0065] [Figure 1] This figure shows a first embodiment of the automatic sampling method according to the present invention. [Figure 2] This figure shows a second embodiment of the automatic sampling method according to the present invention. [Figure 3] This figure shows a method for detecting and / or quantifying a specimen in a whole blood sample according to the present invention. [Figure 4] This is a schematic diagram of the apparatus according to the present invention. [Modes for carrying out the invention]

[0066] The present invention relates primarily to an automated sampling method for heterogeneous biological samples, intended to be used in methods for detecting and / or quantifying specimens within heterogeneous biological samples. For these methods, devices such as those commercialized by the applicant under the trademark VIDAS® may be used.

[0067] The automated sampling method may be performed immediately after blood collection or postponed, resulting in slight differences in the steps of the method, which are identified in the embodiments for carrying out the invention.

[0068] Next, the automatic sampling method will be described in detail with reference to Figures 1 and 2.

[0069] The automated sampling method according to the present invention is designed for directly handling heterogeneous biological samples without pretreatment such as centrifugation or filtration.

[0070] In this explanation, heterogeneous biological samples refer to whole blood samples.

[0071] The automated sampling method 100 includes at least a step 101 of preparing an apparatus 1 schematically shown in Figure 4. The apparatus 1 comprises at least a first workstation 11 loaded with a container 2, such as a blood collection tube containing a biological sample 3, for example, a whole blood sample is preferred, and a second workstation 12 configured to receive a predetermined volume of sample 3 and loaded with at least one well 5, different from the container 2. The apparatus 1 according to the present invention further comprises at least one pipette operating unit 6 equipped with a pipette 61 fitted with a tip 62, the pipette 6 being configured to move between at least two workstations 11 and 12 within the apparatus 1. According to the present invention, the apparatus 1 also comprises a control unit 7 configured to control the pipette operating unit 6, as shown in Figure 4. The apparatus 1 further comprises at least one sensor 8, preferably a pressure sensor, configured to sense and monitor the pressure or vacuum in the tip 62 of the pipette 61, located on the body of the pipette 61.

[0072] The automated sampling method 100 includes at least a step 103 of loading a container 2 into the device 1. Prior to the loading step 103, it is recommended to perform a manual inversion step 102 to at least ensure that the coated lithium heparin that may be applied to the inside of the container 2 comes into effective contact with the whole blood sample 3. In another embodiment, manual inversion could not be performed, so the manual inversion step 102 is shown by a dotted line in Figures 1 and 2.

[0073] After container 2 is loaded (step 103), the homogenization step 104 is automatically initiated. The homogenization step 104 includes at least a first substep 104.1 which consists of submerging the tip 62 into the biological sample 3 in container 2 to a first predetermined depth (determined during step 105, which is described below, and which monitors the liquid level). For example, the first predetermined depth is 1.6 under the first determined depth with respect to the maximum volume of the 300 μL tip. mm It is contained within ~15 mm. During the homogenization step 104, step 105 is performed in parallel to track the liquid level in order to ensure that the end of the tip 62 is always submerged in the biological sample 3.

[0074] During the first substep 104.1 of the homogenization step 104, a substep 105.1 of the liquid level tracking step 105 is performed, which involves detecting the liquid level of the biological sample 3 in the container 2 using a sensor 8.

[0075] When the tip 62 reaches a first predetermined depth, the control unit 7 instructs the pipette operating unit 6 to begin several cycles (substep 104.2 of the homogenization step 104) of aspirating and dispensing the biological sample 3 in the container 2. During the aspiration and dispensing cycle 104.2, substeps 105.2 and 105.3 of the liquid level tracking step 105 are performed, with substep 105.2 consisting of the control unit 7 theoretically determining the first predetermined depth based at least on the liquid height, and substep 105.3 consisting of the control unit 7 monitoring the movement of the tip 62's edge in the container 2 to ensure that the tip 62 moves and remains submerged in line with the liquid level of the biological sample 3.

[0076] As shown in Figures 1 and 2, at least one of the steps 105 for tracking the liquid level is performed during the aspiration and / or distribution 104.2 of the homogenization step 104. Optionally, at least one of the steps 105 for tracking the liquid level is performed during the aspiration stage 106.1 of the sampling step 106 (shown by a dotted line in the figure).

[0077] When the cycle is complete, the control unit 7 automatically commands the pipette operating unit 6 to directly perform the sampling step 106. The sampling step 106 consists of a first stage 106.1 in which the pipette operating unit 6 aspirates a predetermined volume of the biological sample 3 from the container 2, and a second stage 106.2 in which the previously aspirated predetermined volume is distributed into a well 5 different from the container 2, and the tip 62 of the pipette 61 is the same as that of the homogenization step 104 and the sampling step 106. In the framework of the TB-IGRA test, the biological sample is a whole blood sample, and the volume of the whole blood sample to be aspirated is approximately 300 μL per well 5. For example, in this test, there are three wells intended to contain 300 μL of whole blood sample in three separate supports (e.g., strips).

[0078] As shown in Figures 1 and 2, the automatic sampling method 100 further includes at least one monitoring step 107, - A sensing substep 107.1 is configured to send to the control unit 7 at least one pressure value, preferably several pressure values, corresponding to the detected pressure or vacuum, which occurs inside the tip 62 of the pipette 61 during the homogenization step 104 (see dotted line) or the sampling step 106, and to send to the control unit 7 at least one pressure value, preferably several pressure values. - Prior to the automatic sampling method, the control unit 7 compares the received pressure values ​​with pressure thresholds predetermined and incorporated into the control unit 7, corresponding to at least one characteristic pressure index representing a characteristic sampling loss, in a comparison substep 107.2. - The control unit determines, based on the comparison substep 107.2, whether there is a problem in the homogenization step 104 or the sampling step 106, in a determination substep 107.3. Includes.

[0079] In Figures 1 and 2, the monitoring step is shown only for the sampling step 106, but it can also be applied to the homogenization step 104, and in particular to monitor whether overall clogging is occurring in the chip 62.

[0080] During the detection substep 107.1, the control unit 7 executes an algorithm to monitor the selected characteristic pressure index. If the control unit determines that at least one pressure value measured by the sensor 8 during the sampling step 106 exceeds (Y) at least one determined pressure threshold corresponding to at least one pressure index, the control unit 7 triggers an alarm and the sampling step 106 is automatically stopped at 108. Thus, if the control unit 7 determines that at least one pressure value measured by the sensor 8 during the sampling step 106 is less than or equal to (N) at least one determined pressure threshold corresponding to at least one pressure index, the sampling step 106 continues until the automatic sampling method 100 is deemed to be completed and achieved (109). The monitoring steps are performed during the suction stage 106.1 of the sampling step and during the distribution stage 106.2 of the sampling step 106, as shown in Figures 1 and 2.

[0081] Figure 2 shows the automatic sampling method 100 with a retry mode 200. Thus, the control unit 7 is configured to command the retry mode 200, which consists of retrying steps (104, 106) of the automatic sampling method 100, if it determines that a failure occurred in steps (104, 106) because the measured pressure value matches at least one characteristic pressure index.

[0082] As shown in Figure 2, in retry mode 200, the tip 62 of pipette 61 is replaced or washed (step 201) before the failed step is retried (step 202). As shown in Figure 2, during retry mode 200, steps 105 to track the liquid level and step 107 to monitor the liquid level are also performed. Furthermore, if the aspiration stage 106.1 of sampling step 106 is retried during retry mode 200, the tip 62 of pipette 61 is submerged in the biological sample to a second predetermined depth, the second predetermined depth being deeper than the first predetermined depth to which the tip 62 was submerged for aspiration 104.2 of homogenization step 104 or for the failed aspiration stage 106.1 of sampling step 106.

[0083] Furthermore, if, during the monitoring step 107 of the retry step 202, the control unit 7 still considers there to be a fault (Y) (step 107.3), the automatic sampling method 100 is of course stopped and considered to have failed (step 110), and then restarted, as shown in Figure 2, first using the (optional) manual inversion step 102 or homogenization step 104, if necessary. If, during the monitoring step 107 of the retry step 202, the control unit 7 considers there to be no further faults (N) (step 107.3), the automatic sampling method 100 proceeds to the next step (shown as a dotted line in Figure 2).

[0084] Next, the detection / quantification method according to the present invention will be described in detail according to the automated sampling method 100 of the present invention with reference to Figure 3. Method 300 of the present invention is a method for detecting and / or quantifying a specimen in a whole blood sample after cell stimulation, in which the automated sampling method 100 of the present invention is carried out.

[0085] According to the present invention, the detection and / or quantification method 300 includes at least one step 301 of loading at least one support 4 having at least one well 5 configured to receive a predetermined volume of whole blood sample 3 aspirated from a container 2 inside the device 1.

[0086] Following the loading step 301, a step 302 is performed in which a first tip 62 is fitted onto the pipette 61, the first tip 62 being configured to penetrate into at least one sealed protective foil 41 covering at least one well 5 of the support 4 (step 303). Note that in some tests, there is no such protective foil in the well and it is completely open, so step 303 of penetrating into the protective foil is optional. Thus, step 303 is considered optional and is represented by a dotted line in Figure 3. Furthermore, the detection and / or quantification method 300 further includes a step 304 of removing the first tip 62 and a step 305 of placing a second tip 62 on the pipette 61, the second tip 62 being configured to be used in the automated sampling method 100, both during the homogenization step 104 and for the sampling step 106.

[0087] Following the automated sampling method 100, the following steps are performed: step 306 to remove the second tip 62; step 307 to equip the pipette 61 with the third tip; step 308 to aspirate the irritant reagent contained in a special glass vial; step 309 to distribute the irritant reagent into the wells 5 containing the three sampled whole blood samples 3; and step 310 to remove the third tip 62.

[0088] Alternatively, in another embodiment (not shown) in which the tip 62 is a needle and cannot be removed, a step 302 is performed after the loading step 301 to equip the pipette 61 with the first tip 62, and the first tip 62 is configured to penetrate into at least one protective foil 41 covering at least one well 5 of the support 4 (step 303). Furthermore, the detection and / or quantification method 300 further includes a step (not shown) of washing the first tip 62 to ensure that the first tip 62 can be used in the homogenization step 104 and for the sampling step 106 in the automated sampling method 100.

[0089] Following the automated sampling method 100, the following steps are performed: step 308, which involves washing the first tip 62 and aspirating the irritant reagent contained in a special glass vial; and step 309, which involves distributing the irritant reagent into the well 5 containing the sampled whole blood sample 3.

[0090] Alternatively, in another embodiment (not shown) in which the wells are not protected by a protective foil, a step 302 is performed after the loading step 301, in which a first tip 62 is fitted onto the pipette 61, the first tip 62 being configured to be used in the homogenization step 104 and for the sampling step 106 in the automated sampling method 100.

[0091] Following the automated sampling method 100, the steps of removing the first tip 62 (step 306), equipping the second tip to the pipette 61 (step 307) or washing the first tip 62 (step 308), aspirating the irritant reagent contained in a special glass vial (step 309), distributing the irritant reagent into the well 5 containing the sampled whole blood sample 3 (step 309), and removing the second tip 62 (step 310) or retaining the first tip 62 (step 310) are performed.

[0092] Of course, the present invention is not limited to the embodiments shown and described in the accompanying drawings. Modifications are still possible, particularly in terms of the structure of various elements, or by substituting technical equivalents, without departing from the scope of protection of the present invention as defined by the claims.

Claims

1. An automated sampling method (100) for heterogeneous biological samples (3) contained in a container (2), - Step (101) of preparing an apparatus (1) comprising at least one pipette operating unit (6) having at least one pipette (61) having a tip (62), and a control unit (7) that cooperates with and controls the pipette operating unit (6), - Step (103) of loading the container (2) containing the heterogeneous biological sample into the device (1), - Step (104) of automatically homogenizing the heterogeneous biological sample (3) inside the apparatus (1), - A first substep (104.1) of submerging the tip (62) of the pipette (61) into the container (2) to a first predetermined depth below the surface of the heterogeneous biological sample (3), - A second substep (104.2) in which the pipette is made to perform multiple cycles of aspirating the heterogeneous biological sample (3) and distributing it into the container (2), wherein in each cycle, the pipette (61) aspirates the heterogeneous biological sample (3) to the entire volume of the tip (62) at a first predetermined depth, then distributes it back into the container (2), and the end of the tip (62) of the pipette (61) is maintained at the first predetermined depth below the surface of the heterogeneous biological sample (3) throughout the multiple cycles, Step (104) includes an automated homogenization step, - Once the multiple cycles described above have been completed, the heterogeneous biological sample (3) of a predetermined volume is used. - A first step (106.1) in which a predetermined volume of the heterogeneous biological sample (3) is aspirated from the container (2), wherein the tip (62) is submerged to the first predetermined depth, - A second step (106.2) of distributing the previously aspirated predetermined volume into a well (5) different from the container (2) containing the heterogeneous biological sample (3), The step of sampling by (106), An automated sampling method (100) including the above.

2. The automatic sampling method according to claim 1, wherein during the step (104) of performing the automatic homogenization, the suction is performed at a first predetermined speed and the distribution is performed at a second predetermined speed different from the first predetermined speed.

3. At least one step (105) of tracking the liquid level, - A step (105.1) in which the sensor (8) detects the liquid level of the heterogeneous biological sample (3) in the container, - The control unit (7) theoretically determines the depth to which the tip (62) should be submerged based on the liquid height and / or the volume of the heterogeneous biological sample (3) to be aspirated / distributed, and / or the volume and size of the container (2), and aspirates the heterogeneous biological sample (3) and distributes it into the container (2) (105.2), - Step (105.3) of monitoring the movement of the tip (62) in the container (2) and tracking the change in the liquid height in the container (2) during the automatic homogenization step (104) and / or the sampling step (106), An automated sampling method according to claim 1 or 2, comprising at least one step (105) of tracking the liquid level present in the sample.

4. The automated sampling method according to claim 3, wherein the at least one step (105) of tracking the liquid level may be performed during the aspiration (104.2) and / or dispensing (104.2) of the automated homogenization step (104), and / or during the first step (106.1) of the sampling step (106).

5. At least one monitoring step (107), - A sensing substep (107.1) is configured to have at least one sensor (8) detect pressure or vacuum occurring in the tip (62) of the pipette (61) during the automatic homogenization step (104) and / or the sampling step (106), and to send at least one pressure value corresponding to the detected pressure or vacuum to the control unit (7), - A comparison substep (107.2) in which the control unit (7) compares the at least one pressure value that has been sent with a pressure threshold value that has been predetermined by the control unit (7) and incorporated into the control unit (7), which corresponds to at least one characteristic pressure index that represents a characteristic sampling loss, - The control unit (7) performs a substep (107.3) based on the comparison substep (107.2) to determine whether there is a problem in the automatic homogenization step (104) or the sampling step (106), An automated sampling method according to any one of claims 1 to 4, comprising at least one monitoring step (107), including the above.

6. The automatic sampling method according to claim 5, wherein at least one pressure threshold corresponds to each characteristic pressure index.

7. The automatic sampling method according to claim 5 or 6, wherein during the detection substep (107.1), the control unit (7) executes an algorithm for monitoring the selected characteristic pressure index.

8. The automatic sampling method according to any one of claims 5 to 7, wherein, according to suction (104.2, 106.1) or distribution (104.2, 106.2) performed in parallel with the monitoring step (107), some characteristic pressure indicators selected are considered to be characteristics of sampling loss occurring during suction (104.2, 106.1), some others are considered to be characteristics of sampling loss occurring during distribution (104.2, 106.2), and some others relate to both, and the control unit (7) selects the best characteristic pressure indicator to monitor.

9. The automatic sampling method according to any one of claims 5 to 8, wherein the control unit (7) determines that at least one pressure value measured by the sensor (8) during the sampling step (106) exceeds at least one pressure threshold corresponding to at least one pressure index, triggers an alarm and automatically stops the sampling step (106) (step 108), and if it determines that at least one pressure value measured by the sensor (8) during the sampling step (106) is less than or equal to at least one pressure threshold corresponding to at least one pressure index, it continues the sampling step (106) until it is deemed that the automatic sampling method (100) has been completed and achieved (step 109).

10. The automated sampling method according to any one of claims 5 to 9, wherein the monitoring step (107) is performed during the aspiration (104.2) of the automated homogenization step (104) and / or during the distribution (104.2) of the automated homogenization step (104) and / or during the first stage (106.1) of the sampling step (106) and / or during the second stage (106.2) of the sampling step (106).

11. The automatic sampling method according to any one of claims 1 to 10, wherein the control unit (7) is configured to command a retry mode (200) which consists of retrying identified steps (104, 106) of the automatic sampling method (100), and the control unit (7) identifies when it determines that the automatic homogenization step (104) and the sampling step (106) have failed on the grounds that the measured pressure value matches at least one pressure threshold.

12. The automated sampling method according to claim 11, wherein if the aspiration step (104.2, 106.1) is retried in the retry mode, the tip (62) of the pipette (61) is submerged in the heterogeneous biological sample (3) to a second predetermined depth, the second predetermined depth being greater than the first predetermined depth to which the tip (62) is submerged for the aspiration (104.2) of the automated homogenization step (104) or for the first step (106.1) of the sampling step (106).

13. The automated sampling method according to any one of claims 1 to 12, wherein the heterogeneous biological sample (3) is a whole blood sample.

14. An apparatus (1) configured to perform an automatic sampling method (100) according to any one of claims 1 to 13, wherein at least, - A first workstation (11) loaded with a container (2) containing a heterogeneous biological sample (3), - A second workstation (12) configured to accept a predetermined volume of the heterogeneous biological sample (3), and loaded with at least one well (5) different from the container (2), - A pipette operating unit (6) comprising a pipette (61) equipped with a tip (62), wherein the pipette (61) is configured to move between the first workstation (11) and the second workstation (12) within the apparatus (1), - At least one control unit (7) configured to cooperate with and control the pipette operating unit (6), A device (1) equipped with the following:

15. A method for detecting and / or quantifying a sample in a heterogeneous biological sample (3), wherein an automated sampling method (100) according to any one of claims 1 to 13 is performed, and the detection and / or quantification method is performed by an apparatus (1) according to claim 14 configured to perform the automated sampling method (100).

16. Use of an automated sampling method (100) according to any one of claims 1 to 13 to enable quantification of interferon (IFN-y) in a tuberculosis interferon-gamma release assay according to the method of claim 15.

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