AUTOMATIC SAMPLING METHOD FOR HANDLING WHOLE BLOOD

Abstract

A method that enables handling whole blood for assays where the whole blood is directly taken from a patient without any prior treatment and being put in a collection tube or other container ready to use for further analysis. The method can enable simple processing of the whole blood while avoiding any clots or aggregates and without damaging white cells in a reproducible and automatable way. The method can furthermore enable an automatic and reproducible pipetting of whole blood cells inside several wells to be tested.

Claims

1. An automatic sampling method for non-homogenous biological sample contained in a container, the automatic sampling method comprising the following steps: providing a device comprising at least one pipetting unit equipped with at least one pipette with a tip, a control unit cooperating and controlling the pipetting unit, loading a container containing the non-homogenous biological sample inside the device, starting automatic homogenization inside the device, of the non-homogenous biological sample, the homogenization comprising the following substeps: the tip of the pipette dives into the container at a first predetermined depth under the surface of the non-homogenous biological sample, the pipette operates a plurality of cycles of aspirating and dispensing the non-homogenous biological sample into the container, wherein in each cycle the pipette aspirates the non-homogenous biological sample at the first predetermined depth, the tip end of the pipette is being kept under the surface of the non-homogenous biological sample at the first predetermined depth during the plurality of cycles, sampling a predetermined volume of non-homogenous biological sample once the plurality of cycles is over, by: a first phase of aspirating the predetermined volume of non-homogenous biological sample from the container, wherein the tip is immerged at the first predetermined depth and a second phase of dispensing the predetermined volume previously aspirated, in a well distinct from the container containing the non-homogenous biological sample.

2. The automatic sampling method according to claim 1, wherein during homogenization step, the aspirating is carried out at a first predetermined speed and the dispensing is carried out at a second predetermined speed, the first predetermined speed being different from the second predetermined speed.

3. The automatic sampling method according to claim 1, comprising at least one step of liquid level tracking consisting in: detecting the liquid height of the sample inside the container by a sensor, and determining theoretically how deep the tip end must dive and aspirate and dispense non-homogenous biological sample into the container based on the liquid height and/or based on the volume of non-homogenous biological sample to be aspirated/dispensed, and/or based on the volume and size of the container, the determination being carried out by the control unit, monitoring movements of the tip end inside the container during homogenization step and/or sampling step and following the liquid height variation inside the tube.

4. The automatic sampling method according to claim 3, wherein the at least one step of liquid level tracking can be carried out during aspiration and/or dispensing of homogenization step and/or during aspiration phase of sampling step.

5. The automatic sampling method according to claim 1, comprising at least one step of monitoring comprising the following substeps: a detecting substep, wherein at least one sensor is configured to detect pressure or vacuum generated inside the tip end of the pipette during the homogenization step and/or the sampling step, and to send to control unit at least one pressure value corresponding to detected pressure or vacuum, a comparison substep, wherein the control unit compares the at least one pressure value sent with pressure thresholds corresponding to at least one distinctive pressure indicator representative of a characteristic sampling defect, the pressure thresholds being embedded in the control unit and being predetermined by the control unit, a determination substep, wherein the control unit determines, based on the comparison substep, if there is a failure of the homogenization step or the sampling step.

6. The automatic sampling method according to claim 5, wherein for each distinctive pressure indicator correspond at least one pressure threshold.

7. The automatic sampling method according to claim 5, wherein during the detecting substep, the control unit runs an algorithm that monitors the selected distinctive pressure indicators.

8. The automatic sampling method according to claim 5, wherein some selected distinctive pressure indicators are considered characteristic of sampling defects occurring during aspiration and some others are considered characteristic of sampling defects occurring during dispensing and some others are for both, according to the step aspiration or dispensing during which the monitoring step is carried out, the control unit selects what distinctive pressure indicators are the best to be monitored.

9. The automatic sampling method according to claim 5, wherein if the control unit determines that at least one pressure value measured by the sensor, during sampling step, is over at least one determined pressure threshold corresponding to at least one pressure indicator, the control unit triggers an alarm and sampling step is automatically stopped and if the control unit determines that at least one pressure value measured by the sensor, during sampling step, is equal or under at least one determined pressure threshold corresponding to at least one pressure indicator, the sampling step goes on until the control unit considers that the automatic sampling method is completed and considered achieved.

10. The automatic sampling method according to claim 5, wherein the step of monitoring is carried out during aspiration of homogenization step and/or during dispensing of homogenization step and/or during aspiration phase of sampling step and/or during dispensing phase of sampling step.

11. The automatic sampling method according to claim 1, wherein the control unit is configured to command a retry mode, consisting in retrying an identified step of the automatic sampling method, the step being identified by the control unit if the step is determined failed by the control unit, because of the correspondence of measured pressure value with at least one determined pressure threshold.

12. The automatic sampling method according to claim 11, wherein in the retry mode, if an aspiration step is to be retried, the tip of pipette dives into the non-homogenous biological sample at a second determined depth, the second predetermined depth being deeper than the first predetermined depth at which the tip dives in for the aspiration of the homogenization step or for the aspiration phase of the sampling step.

13. A device configured to carry out the automatic sampling method according to claim 1, the device comprising at least: a first workstation wherein a container containing a non-homogenous biological sample is loaded, a second workstation wherein at least one well, different from the container, is loaded, the well being configured to receive a predetermined volume of the non-homogenous biological sample, at least one pipetting unit comprising a pipette equipped with a tip, the pipette being configured to move inside the device between the at least two workstations, at least one control unit of the device configured to cooperate and control the pipette unit.

14. The automatic sampling method according to claim 1, wherein the method is implemented, for detecting and/or quantifying an analyte in the non-homogenous biological sample.

15. The automatic sampling method according to claim 1, wherein the method is implemented to quantify interferon (IFN-y) in a Tuberculosis Interferon Gamma Release Assay.

Description

BRIEF DESCRIPTION OF FIGURES

[0095] FIG. 1 is a diagram illustrating a first embodiment of the automatic sampling method according to the invention,

[0096] FIG. 2 is a diagram illustrating a second embodiment of the automatic sampling method according to the invention,

[0097] FIG. 3 is a diagram illustrating a method for detection and/quantification of analytes in a whole blood sample according to the invention,

[0098] FIG. 4 illustrates schematically a device according to the invention,

DETAILED DESCRIPTION

[0099] The invention relates mainly to an automatic sampling method of non-homogenous biological sample intended to be implemented in a method for detection and/quantification of analytes in a non-homogenous biological sample. For these methods, devices such as the ones commercialized by the applicant under the brand VIDAS® can be used.

[0100] The automatic sampling method can be carried out immediately after blood collection or can be postponed later, slightly differences in the steps of the method are to be performed and will be identified in the detailed description.

[0101] The automatic sampling method will now be detailed in reference with FIG. 1 and FIG. 2.

[0102] The automatic sampling method according to the invention was conceived for handling non-homogenous biological sample directly without any prior treatment such as centrifugation, or filtration.

[0103] In this description, the non-homogenous biological sample is whole blood sample.

[0104] The automatic sampling method 100 comprises at least a step 101 of providing a device 1 illustrated schematically in FIG. 4. Said device 1 comprises at least a first workstation 11 wherein a container 2, for example a collection tube, containing a biological sample 3, preferentially whole blood sample, is loaded, a second workstation 12 wherein at least one well 5, different from the container 2, is loaded, the well 5 being configured to receive a predetermined volume of the sample 3. The device 1 according to the invention further comprises at least one pipetting unit 6 comprising a pipette 61 equipped with a tip 62, the pipette 6 being configured to move inside the device 1 between the at least two workstations 11, 12. According to the invention and as illustrated in FIG. 4, the device 1 also comprises a control unit 7 configured to control the pipette unit 6. The device 1 further comprises at least one sensor 8, preferably a pressure sensor, arranged on the body of the pipette 61 and configured to sense and monitor pressure or vacuum inside the tip 62 of the pipette 61.

[0105] The automatic sampling method 100 comprises at least a step 103 of loading the container 2 inside the device 1. Previously to the step 103 of loading, a step of manual inversion 102 is recommended to carry out in order to ensure at least that the coated lithium heparin, which can be applied inside the container 2, came into contact with the whole blood sample 3 in an efficient manner. The manual inversion step 102 is illustrated with dotted line in FIG. 1 and FIG. 2, because manual inversion could be not carried out in another embodiment.

[0106] After loading the container 2 (step 103), a step of homogenization 104 is automatically started. The homogenization step 104 comprises at least a first substep 104.1 consisting in the dive of tip 62 inside the container 2 into the biological sample 3 at a first predetermined depth (determined during liquid level monitoring step 105, described below). For example, the first predetermined depth is comprised between 1.6 mmm to 15 mm for a maximum volume of the tip of 300 μL under the first determined depth. In parallel of the homogenization step 104, a liquid level tracking step 105 is carried out to ensure that the tip 62 end is always immerged in the biological sample 3 during homogenization step 104.

[0107] During first substep 104.1 of homogenization step 104, a substep 105.1 of liquid level tracking step 105 is carried out and consists in detecting the liquid height of the biological sample 3 inside the container 2 by the sensor 8.

[0108] When the tip 62 reach the first predetermined depth, the control unit 7 orders pipette unit 6 to start several cycles of aspiration and dispensing (substep 104.2 of homogenization step 104) of the biological sample 3 inside the container 2. During the cycles of aspiration and dispensing 104.2, a substep 105.2 and a substep 105.3 of liquid level tracking step 105 are carried out, the substep 105.2 consisting in determining theoretically the first predetermined depth, by the control unit 7, based on at least liquid height and the substep 105.3 consisting in monitoring movements of the tip 62 end inside the container 2, by the control unit 7, so as ensure that the tip 62 moves following the liquid level of the biological sample 3 and stay immerged.

[0109] As shown in FIGS. 1 and 2, the at least one step of liquid level tracking 105 is carried out during aspiration and/or dispensing 104.2 of homogenization step 104. Optionally, the at least one step of liquid level tracking 105 can be carried out during aspiration phase 106.1 of sampling step 106 (shown on dotted lines the figures).

[0110] When the cycles are ended, the control unit 7 orders automatically and directly a sampling step 106 to the pipette unit 6. The sampling step 106 consists in a first phase 106.1 during which the pipette unit 6 aspirates a predetermined volume of biological sample 3 from the container 2 and a second phase 106.2 of dispensing the predetermined volume previously aspirated, in a well 5 distinct from the container 2, the tip 62 of the pipette 61 being the same for the homogenization step 104 and for sampling step 106. In the framework of a TB-IGRA assay, the biological sample is a whole blood sample and the volume of said whole blood sample to be aspirated is around 300 μL for each well 5, for instance in this assay there are three wells in three different supports (strips for example) intended to contain 300 μL of whole blood sample.

[0111] As illustrated in FIGS. 1 and 2, the automatic sampling method 100 further comprises at least one step of monitoring 107 comprising the following substeps: [0112] a detecting substep 107.1, wherein at least one sensor 8 is configured to detect pressure or vacuum generated inside the tip 62 end of the pipette 61 during the homogenization step 104 (see in dotted line) or the sampling step 106, and to send to control unit 7 at least one pressure value and preferably several pressure values corresponding to detected pressure or vacuum, [0113] a comparison substep 107.2, wherein the control unit 7 compares the at least pressure value sent with pressure thresholds corresponding to at least one distinctive pressure indicator representative of a characteristic sampling defect, said pressure thresholds being embedded in the control unit 7 and being predetermined by the control unit 7 prior to the automatic sampling method, [0114] a determination substep 107.3, wherein the control unit determines, based on the comparison substep 107.2, if there is a failure of the homogenization step 104 or the sampling step 106.

[0115] In FIGS. 1 and 2 the steps of monitoring will be illustrated only for sampling step 106 but could be applied to homogenization step 104 as well and in particular for monitoring if total clogging of the tip 62 is happening.

[0116] During the detecting substep 107.1, the control unit 7 runs an algorithm that monitors the selected distinctive pressure indicators. If the control unit determines that at least one pressure value measured by the sensor 8, during sampling step 106, is over (Y) at least one determined pressure threshold corresponding to at least one pressure indicator, the control unit 7 triggers an alarm and sampling step 106 is automatically stopped 108. Accordingly, if the control unit 7 determines that at least one pressure value measured by the sensor 8, during sampling step 106, is equal or under (N) at least one determined pressure threshold corresponding to at least one pressure indicator, the sampling step 106 goes on until the control unit 7 considers that the automatic sampling method 100 is completed and considered achieved 109. The monitoring step is carrying out during aspiration phase 106.1 of sampling step as well as during dispensing phase 106.2 of sampling step 106 as it can be seen in FIGS. 1 and 2.

[0117] In FIG. 2 is illustrated automatic sampling method 100 with a retry mode 200. Accordingly, the control unit 7 is configured to command a retry mode 200, consisting in retrying a step (104, 106) of the automatic sampling method 100 if the control unit 7 has determined that the said step (104, 106) is a failure because of the correspondence of measured pressure values with at least one distinctive pressure indicator.

[0118] As shown in FIG. 2, in the retry mode 200, the tip 62 of the pipette 61 is changed or cleaned (step 201) before retrying (202) the failed step. During retry mode 200, the steps of liquid level tracking 105 and monitoring 107 are carried out as well, as illustrated in FIG. 2. Furthermore, during retry mode 200, in case of retrying aspiration phase 106.1 of the sampling step 106, the tip 62 of pipette 61 dives into the biological sample at a second determined depth, the second predetermined depth being deeper than the first predetermined depth at which the tip 62 dived in for the aspiration 104.2 of the homogenization step 104 or for the aspiration phase 106.1 of the sampling step 106 that failed.

[0119] Moreover, if during monitoring step 107 of the retrying step 202, the control unit 7 considers (step 107.3) that there is still a failure (Y), the automatic sampling method 100 is definitely stopped and considered failed (step 110), the automatic sampling method 100 is then restarted if needed at the beginning with the manual inversion step 102 (optional) or with the homogenization step 104 as illustrated in FIG. 2. If during monitoring step 107 of the retrying step 202, the control unit 7 considers (step 107.3) that there is no more failure (N), the automatic sampling method 100 continues to the next step (dotted lines in FIG. 2).

[0120] The detection/quantifying method according to the invention will now be described in reference with FIG. 3 and according to the detailed automatic sampling method 100 of the invention. The method 300 of the invention is a method for detecting and/or quantifying analytes in a whole blood sample, after cell stimulation, wherein the automatic sampling method 100 according to the invention is implemented.

[0121] According to the invention, the detection and/or quantification method 300 comprises at least one step of loading 301 at least one support 4 comprising at least one well 5 configured to receive a predetermined volume of whole blood sample 3, aspirated from the container 2 inside the device 1.

[0122] After the step of loading 301, the step of equipping 302 the pipette 61 with a first tip 62 is carried out, the first tip 62 being configured to pierce (step 303) at least one sealing protection foil 41 covering the at least one well 5 of the support 4. It should be noted that the step 303 of piercing the protective foil is optional since, in some assay, wells are not equipped with such protective foil and are completely opened. The step 303 is then considered optional and is represented in dotted lines in FIG. 3. Furthermore, the detection and/or quantification method 300 further comprises a step of removing 304 said first tip 62 and positioning 305 a second tip 62 on the pipette 61, the second tip 62 being configured to be used in the automatic sampling method 100 during the step of homogenization 104 and for the sampling step 106. After the automatic sampling method 100, the following steps are carried out: removing 306 second tip 62 and equipping 307 the pipette 61 with a third tip and aspirating 308 a stimulant reagent contained in specific vials and dispensing 309 it into the well 5 containing the whole blood sample 3 sampled, ejecting 310 the third tip 62.

[0123] Alternatively, in another embodiment (not illustrated) where the tip 62 is not removable, for example the tip being a needle : after the step of loading 301, the step of equipping 302 the pipette 61 with a first tip 62 is carried out, the first tip 62 being configured to pierce (step 303) at least one protection foil 41 covering the at least one well 5 of the support 4. Furthermore, the detection and/or quantification method 300 further comprises a step of cleaning (not illustrated) said first tip 62 in order to ensure that the first tip 62 can be used in the automatic sampling method 100 during the step of homogenization 104 and for the sampling step 106.

[0124] After the automatic sampling method 100, the following steps are carried out: cleaning first tip 62 and aspirating 308 a stimulant reagent contained in specific vials and dispensing 309 it into the well 5 containing the whole blood sample 3 sampled.

[0125] Alternatively, in another embodiment (not illustrated) where the well(s) is/are not protected by a protective foil: after the step of loading 301, the step of equipping 302 the pipette 61 with a first tip 62 is carried out, said first tip 62 being configured to be used in the automatic sampling method 100 during the step of homogenization 104 and for the sampling step 106. After the automatic sampling method 100, the following steps are carried out: removing 306 first tip 62 and equipping 307 the pipette 61 with a second tip or cleaning first tip 62, and then aspirating 308 a stimulant reagent contained in specific vials and dispensing 309 it into the well 5 containing the whole blood sample 3 sampled, ejecting 310 the second tip 62 or keeping first tip 62.

[0126] Of course, the invention is not limited to the embodiments described and shown in the appended figures. Modifications remain possible, notably from the point of view of the constitution of the various elements or by substitution of technical equivalents, without thereby departing from the scope of protection of the invention defined by the claims.