ITERATIVE LIQUID ASPIRATION

Abstract

A method for aspirating a first liquid medium of two liquid media of different density from a sample container comprises: lowering a pipette of the laboratory automation device into the sample container until a pipette tip of the pipette has passed a lowering distance from the surface of the first liquid medium in the sample container, wherein the lowering distance is chosen, such that the pipette tip passes at least an aspiration volume in the sample container; aspirating liquid from the sample container during the lowering of the pipette by generating an underpressure in the pipette, wherein the first liquid medium is aspirated, and after the interface and the pipette tip pass each other, the second liquid medium of the two liquid media is aspirated; measuring a pressure in the pipette during the lowering of the pipette and detecting a position of the interface, when a slope of the pressure changes; when the lowering distance has been passed and no interface is detected, aspirating the aspiration volume from the first liquid medium and dispensing the aspiration volume of the first liquid medium into a further sample container.

Claims

1. A method for aspirating a first liquid medium of two liquid media of different density from a sample container, the method comprising: lowering a pipette of the laboratory automation device into the sample container until a pipette tip of the pipette has passed a lowering distance from the surface of the first liquid medium in the sample container, wherein the lowering distance is chosen, such that the pipette tip passes at least an aspiration volume in the sample container; aspirating liquid from the sample container during the lowering of the pipette by generating an underpres sure in the pipette, wherein the first liquid medium (l) is aspirated, and after the interface and the pipette tip pass each other, the second liquid medium of the two liquid media is aspirated; measuring a pressure in the pipette during the lowering of the pipette and detecting a position of the interface, when a slope of the pressure changes; when the lowering distance (l) has been passed and no interface is detected, aspirating the aspiration volume from the first liquid medium and dispensing the aspiration volume of the first liquid medium into a further sample container.

2. The method of claim 1, when the aspiration volume of the first liquid medium has been disposed into the further sample container, repeating of: lowering of the pipette for the lowering distance (l) corresponding to the aspiration volume, detection of the interface and, when no interface is detected, aspirating the aspiration volume.

3. The method of claim 1, wherein, when the lowering distance (l) has been passed and no interface is detected, withdrawing the pipette for a safety distance (d) before aspirating the aspiration volume.

4. The method of claim 1, wherein, when the lowering distance has been passed and no interface is detected, stopping the pipette before the aspiration volume is aspirated.

5. The method of claim 1, wherein a first aspiration rate during the lowering of the pipette for the lowering distance is lower than a second aspiration rate, after the lowering distance has been passed.

6. The method of claim 1, wherein an aspiration rate of the aspirated liquid medium is adjusted, such that a movement speed of a liquid level in the sample container is slower than a movement speed of the pipette tip; wherein a movement speed of the pipette tip is at least 10% faster, in particular 2 to 10 times faster than the movement speed of the liquid level.

7. The method of claim 1, wherein the pipette tip is solely moved, when a device generating the underpressure or the overpressure operates at a constant volume rate.

8. The method of claim 7, wherein the device comprises a plunger and the pipette is solely moved, when the plunger is moved with constant speed.

9. The method of claim 1, after detection of the interface, withdrawing the pipette from the sample container and discarding an amount of liquid from the pipette such that an aspirated amount of second liquid medium is discarded.

10. The method of claim 1, after detection of the interface, stopping the pipette tip at the detected position and generating overpressure to dispense an amount of the second aspirated liquid medium and/or an amount of the first aspirated liquid medium from the pipette.

11. The method of claim 1, further comprising: after the aspiration volume of the first liquid medium has been dispensed into the further sample container, lowering the pipette tip in the sample container to a level at which the pipette tip was at an end of a previous movement, in which interface detection has been performed, and from this level continuing lowering the pipette into the sample container until the pipette tip has passed the lowering distance (l) from the surface of the first liquid medium corresponding to the aspiration volume.

12. The method of claim 11, wherein the pipette tip is lowered to a level), which is a safety distance away from the level at which the previous detection movement was stopped.

13. A computer program for aspirating a first liquid medium of two liquid media of different density from a sample container, which computer program, when being executed by a processor, is adapted to carry out the steps of the method of one of claim 1.

14. A computer-readable medium, in which a computer program according to claim 13 is stored.

15. A laboratory automation device, comprising: a pipetting arm for carrying a pipette; a pressure device for changing a pressure in a volume connected to the pipette for aspirating and dispensing a liquid medium in the pipette; a pressure sensor for pressure measurements in the volume connected to the pipette; a control device for controlling the pressure device and the pipetting arm and for receiving a pressure signal from the pressure sensor; wherein the control device is adapted for performing the method of one of claims.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Below, embodiments of the present invention are described in more detail with reference to the attached drawings.

[0058] FIG. 1 schematically shows a laboratory automation device according to an embodiment of the invention.

[0059] FIG. 2 shows a flow diagram for a method for aspirating a liquid medium according to an embodiment of the invention.

[0060] FIG. 3 shows a sample container illustrating a repeated aspiration process used during an embodiment of the invention.

[0061] FIG. 4 shows a diagram with a pressure over time generated during a method according to an embodiment of the invention.

[0062] FIG. 5A to 5D show diagrams with pipette tip positions and liquid levels over time generated during a method according to an embodiment of the invention.

[0063] FIG. 6 shows a diagram with a pipette tip and pump speeds generated during a method according to an embodiment of the invention.

[0064] FIG. 7 shows pipettes, which may be used in a method according to an embodiment of the invention.

[0065] The reference symbols used in the drawings, and their meanings, are listed in summary form in the list of reference symbols. In principle, identical parts are provided with the same reference symbols in the figures.

DETAILED DESCRIPTION

[0066] FIG. 1 schematically shows a laboratory automation device 10, which comprises an automatically movable pipetting arm 12 to which a pipette 14 is attached. As shown in FIG. 1, the pipette tip 16 of the pipette 14 may be lowered into a sample container 18 via the movable pipetting arm 12. For example, the container 18 may be a test tube with a centrifuged blood sample.

[0067] The pipetting arm 12 may move the pipette 14 and the pipette tip 16 in three dimensions, may lower the pipette tip 16 into the sample container 18 and may retract the pipette tip 16 therefrom.

[0068] In the sample container 18, liquid media 20a, 20b are contained, which have a different density and which separate themselves in a vertical direction, for example under gravity or centrifugal forces. The two liquid media 20a, 20b are separated by an interface 22. The interface 22 may be seen as a layer or plane between the liquid media 20a, 20b. It has to be noted that there is a second interface 24, which is present between the liquid media 20a and the air of the environment.

[0069] For example, the first liquid media 20a may be blood plasma and the second liquid media 20b may contain blood cells, such as red and white blood cells. As shown in FIG. 1, the red and white blood cells also may be separated from each other and the white blood cells may form a small layer 26 between the plasma and the red blood cells. This layer 26 may be seen as a part of the interface 22.

[0070] As a further example, the two liquid media 20a, 20b may be two unmixable liquids, which are used for liquid-liquid extraction.

[0071] The laboratory automation system 10 furthermore comprises a pump 28, which is connected via a hose 30 with the pipette 14. With the pump 28, a pressure may be applied to the hose 30 and to the pipette 14, which causes the pipette 14 to aspirate or dispense a liquid medium 20a, 20b or any other fluid. For example, the pump 28 comprises a plunger 29, which is moved for generating underpressure and overpressure in the hose 30 and the pipette 14.

[0072] A pressure sensor 32, which may be attached to the hose 30 and/or the pipette 14, is adapted for measuring a pressure in the hose 30 and/or the pipette 14.

[0073] A control device 34 of the laboratory automation device 10, which may be a part of the laboratory automation device 10 or connected thereto, may control the pipetting arm 12, the pump 28 and may receive a pressure signal from the pressure sensor 32.

[0074] In general, liquid may be aspirated from the sample container 18 with the pipette 14 by generating an underpressure in the pipette 14, wherein the first liquid medium 20a of the two liquid media is aspirated and after the interface 22 and the pipette tip 16 pass each other, the second liquid medium 20b of the two liquid media is aspirated. A pressure in the pipette 14 is measured with the pressure sensor 32, while the pipette tip 16 and the interface 22 move with respect to each other and the position of the interface 22 is detected, when a slope of the pressure changes.

[0075] FIG. 1 also shows a further sample container 36 into which the first liquid medium 20a will be dispensed as described below.

[0076] FIG. 2 shows a flow diagram for aspirating the first liquid medium 20a and for transporting the first liquid medium 20a into the further sample container 36. The method may be performed by the laboratory automation device 10 controlled by the control device 34.

[0077] In step S10, the pipette tip 16 is moved into the container 18 and lowered into the container 18, until the liquid level 24 being the boundary between the first liquid medium 20a and air is reached. The liquid level 24 may be detected with a capacitive method. After that, the pipette tip 16 is moved in the sample container 18 along at least a part of a path 38 intersecting the interface 22 between the first liquid medium 20a and the second liquid medium 20b. As shown, the pipette tip 16 may be moved along a vertical direction, however, more complicated paths 38 are possible, which may have components in a horizontal direction and/or inclined sections.

[0078] In step S12, when the pipette tip is immersed in the first liquid 20a, liquid is aspirated from the sample container 18 with the pipette 14 by generating an underpressure in the pipette 14 with the pump 28. The first liquid medium 20a is then aspirated, when the pipette tip 16 is above the interface 22, and after passing the interface 22, when the pipette tip 16 is below the interface 22, the second liquid medium 20b is aspirated by the pipette tip 16.

[0079] However, with the method it may be tried to aspirate as less second liquid medium 20b as possible, as for example described below.

[0080] In step S12, the amount of first liquid medium 20a in the pipette 14 is estimated, which may be determined from the volume rate of the pump 28. When a aspiration volume of the pipette 14 has been filled, the method continues in step S14, where the content of the pipette is dispensed into the second sample container 36.

[0081] Also during step S12, the pressure sensor 32 measures a pressure signal 40 (see FIG. 4) in the pipette 14, while the pipette tip 16 is moved. When the position of the interface 22 is detected by evaluating a slope of the pressure signal 40, the method continues in step S16.

[0082] Otherwise, the method continuous in step S14, where the pipette 14 is withdrawn from the sample container 18 and the first liquid medium 20a in the pipette 14 is dispensed into the further sample container 36.

[0083] After that, the method continues in step S12, where again the pipette tip 16 is lowered into the sample container 18. For example, blood samples usually have a volume of 9 mL or 6 mL, where the plasma content is 53-59%, a usual pipette 14 will be full before reaching the interface 22.

[0084] Again, the air-liquid interface, i.e. the level 24, may be detected, for example with a capacitive method. After the first filling of the pipette 14, the plasma is dispensed into the container 36 and the pipette 14 can aspirate more plasma. This may be repeated until the interface 22 to the red blood cells is reached.

[0085] In step S16, a post-detection procedure may be performed, when the interface 22 has been detected. In particular, after the detection of the interface 22, the pipette 14 may be withdrawn from the sample container 18 and/or an amount of liquid from the pipette 14 may be dispensed such that an aspirated amount of second liquid medium 20b and if desired a fraction of the first liquid medium 20a is discarded.

[0086] After step S16, the method may continue with dispensing the first liquid medium 20a in the pipette 14 into the further sample container 36 and may stop afterwards. It also may be that the pipette 14 moves back to the first liquid container 18 and aspirates the remaining amount of the first liquid medium 20a with interface detection. This is possible since the position 22 of the interface position is now known.

[0087] In FIG. 3, the movement path 38 of the pipette tip 16 during step S12 is shown, when liquid is aspirated. In a first pass, the pipette tip 16 is moved from a level z.sub.0, where the liquid level is at the starting time, for a lowering distance Ito the level z.sub.2, where the pipette 14 has been filled up or is filled to its aspiration volume. The movement from z.sub.0 to z.sub.2, may be done rather fast and/or with a speed, such that the aspirated liquid medium 20a, 20b is enough for interface detection. At z.sub.2, when no interface 22 has been detected, the movement may be stopped and first liquid medium 20a may be aspirated until the pipette 14 is filled up to the aspiration volume.

[0088] It also may be that before the pipette 14 is filled up to the aspiration volume, the pipette tip is retracted for a safety distance d from the level z.sub.2 to the level z.sub.1 and that there, the pipette 14 is filled up to the aspiration volume.

[0089] During the second pass and optionally all subsequent passes, the pipette tip 16 in the sample container 18 may be lowered to a level at which the pipette tip 16 was at an end of a previous detection movement. The pipette tip 16 also may be lowered to a level z.sub.1, which is a safety distance d above the level z.sub.2 at which the previous detection movement was stopped. The benefit of the safety distance d will be described with respect to the following figures. The retraction safety distance after the distance I is reached may be different from the lowering safety distance, which is used during again lowering the pipette into the container.

[0090] FIG. 4 shows a diagram with a pressure signal or pressure curve 40, which has been recorded during a movement of the pipette tip 16 through the interface 22. For example, the pressure 40 shown in FIG. 4 may be the one as the last detection movement performed in step S12, when in the end, the interface 22 is detected.

[0091] FIG. 4 is an example of a pressure signal 40, which was recorded during aspiration of blood plasma, when entering from the plasma into the erythrocytes. When the pipette tip 16 goes through the interface 22, the slope of the pressure 40 changes.

[0092] In particular, the diagram shows the time t on the horizontal axis and pressure p on the vertical axis. At time t=0, the underpressure is generated and in a first time interval 42, the first liquid medium 20a starts to enter the pipette tip 16. In this time interval 42, the pressure 40 varies, since the flow in the pipette tip 16 has not yet stabilized. After the stabilization phase in the time interval 43, the slope of the pressure 40 is constant. The volume rate of the pump 28 has been adjusted to the amount of first liquid medium 20a flowing into the pipette 14. At point 44, the pipette tip 16 enters the second liquid medium 20b, which is more viscous as the first liquid medium 20a and in the following time interval 46, the slope of the pressure 40 changes (it deceases). This slope change is detected by the controller 34, which then stops the pump 28 and the movement of the pipette tip 16. After the stop of the pump 28, the pressure starts to increase again.

[0093] Due to the first time interval 42, it may be beneficial to introduce a waiting time t.sub.w after starting the pump 28, before the interface detection 22 becomes active, to ignore the pressure change when the first liquid medium 20 starts to enter into the empty pipette tip 16. Furthermore, it may be beneficial to stop the z-movement of the pipette tip 16 and the pump 28 upon interface detection to avoid aspiration of a large amount of the second liquid medium 20b and to minimize a contamination risk.

[0094] To ensure that the blind phase defined by the waiting time t.sub.w takes place in a safety distance d it follows that v.sub.z.Math.t.sub.w<d, where v.sub.z is the z-axis speed of the pipette tip 16. Furthermore, the speed vi of the liquid level should be smaller than the speed v.sub.z of the pipette tip 16. It has to be noted that v.sub.z may only be the z-component of the speed of the pipette tip 16 and that speed of the pipette tip 16 also may have components in another direction.

[0095] This results in an upper and lower boundary for the speed v.sub.z of the pipette tip 16 during detection movement

v.sub.I<v.sub.z<d/t.sub.w

[0096] FIG. 5A to 5D show diagrams indicating the z-coordinate respectively the level 50 of the pipette tip 16 and the liquid level 52 in the sample container 18 during lowering and retreating the pipette 14 in the sample container 18 in different scenarios.

[0097] The diagrams all start at time to, where it has been detected that the pipette tip 16 has touched the liquid level 24 in the sample container 18 and the pump 28 is started.

[0098] In FIG. 5A and 5B, the pipette 16 is lowered into the sample container 18 with a constant speed for the distance I until a time t.sub.1 is reached. During this movement interface detection is performed. In FIG. 5A and 5B no interface is detected.

[0099] The pipette 16 is withdrawn by a safety distanced and stopped there. Then the rest of the aspiration volume is aspirated until a time t.sub.3. Afterwards, the pipette is completely removed from the sample container 18 and dispensed in the further sample container 36.

[0100] In FIG. 5A, the liquid medium 20a is aspirated with a constant aspiration rate between the times t.sub.0 and t.sub.3. The liquid level 52 drops with a constant rate between these two time points.

[0101] In FIG. 5B, the liquid medium 20a is aspirated with a first aspiration rate during the lowering of the pipette 14 between t.sub.0 and t.sub.2. This first aspiration rate is lower than a second aspiration rate between t.sub.2 and t.sub.3, where the rest of the aspiration volume of the liquid medium 20a is aspirated.

[0102] FIG. 5C and 5D show scenarios, where the interface 22 is detected at a time {tilde over (t)}.sub.t, before the pipette 14 has been moved for the distance I.

[0103] In FIG. 5C, the pipette 14 is then stopped and withdrawn from the sample container 18. After that the aspirated amount of the second liquid medium 20b may be discarded by dispensing a small content of the pipette 14. The remaining content may be dispensed in the further sample container 36. It also may be that then the complete content of the pipette 14 is discarded.

[0104] In FIG. 5D, after the interface detection at time {tilde over (t)}.sub.1 an amount of the content of the pipette 14 is dispensed until time {tilde over (t)}.sub.2. This dispensing is done at the level, where the interface has been detected. After that the pipette 14 is withdrawn from the sample container 18 and, for example, may be dispensed into the further sample container 36.

[0105] In the detection movements of FIG. 5A to 5D, an aspiration rate of the aspirated liquid medium 20a has been adjusted, such that a movement speed of the liquid level 52 in the sample container 18 is slower than a movement speed of the pipette tip 16.

[0106] aspiration FIG. 6 shows a diagram with a speed/volume rate v.sub.p of the pump 28, which may be proportional to the speed of the plunger 29 and a speed v.sub.z of the pipette tip. Both quantities are depicted over time t.

[0107] To improve the interface detection, the pipette tip 16 solely moves when the pump 28 has a constant speed and/or volume rate v.sub.p. If the pump 28 is in an acceleration or deceleration phase, then the interface detection may be more difficult and/or not so accurate. It may be that the pipette tip 16 is solely moved, when a pump 28 generating the underpressure operates at a constant volume rate v.sub.p. When the pump 28 comprises a plunger 29, the pipette 14 may be solely moved, when the plunger 29 is moved with constant speed v.sub.p.

[0108] FIG. 7 schematically shows two tip designs for pipettes 14, which may be used in the laboratory automation device 10, when the method is performed. The upper pipette 14 has an elongated nozzle 54 with a diameter along the extension of the nozzle, which varies at least 10% from a mean diameter of the nozzle 54. The mean diameter of the nozzle 54 may be smaller than the mean diameter of the rest of the pipette, i.e. the pipette body 56, for example more than 3 times. An elongated nozzle 54 may be beneficial in washing dispensing (see description of step S16) the undesired second liquid medium 20b from the pipette tip 16.

[0109] The lower pipette 14 has a conical pipette tip with a small orifice 58, which, for example, may have a diameter of less than 10% of a diameter of the pipette body 56. A smaller orifice 58 may allow to have a higher sensitivity and trigger a faster stop when the pipette tip 16 enters into the second liquid medium 20b.

[0110] Also the upper pipette 14 with the nozzle 54 may have such a small orifice 58.

[0111] After the interface detection, there usually is an amount of second liquid medium 20b in the pipette tip 16. The amount of second liquid medium 20b may be calculated from the pressure-time curve with the knowledge of the aspiration speed. For better washing of the second liquid medium 20b, for example in a nozzle 54, an additional amount of first liquid medium 20a may be dispensed.

[0112] After interface detection, an under-pressure may exist within the pipette 14. Therefore, a further amount of second liquid medium 20b may flow inside the pipette tip, although the pump 28 has been stopped. This may be minimized by reversing the operation of the pump 28 directly after interface detection and/or switching to dispense instead of only stopping the movement. After detection of the interface 22, the pipette tip 16 may be stopped at the detected position and an overpressure may be generated in the pipette 14 to dispense an amount of the second aspirated liquid medium 20b from the pipette 14.

[0113] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art and practising the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. A single processor or controller or other unit, such as an FPGA, may fulfil the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.