WO2010013033A1

WO2010013033A1 - Improvements in and relating to extraction of a selected fraction from a fractionated sample

Info

Publication number: WO2010013033A1
Application number: PCT/GB2009/050917
Authority: WO
Inventors: Melvyn Whiteside
Original assignee: Rts Life Science Limited
Priority date: 2008-07-28
Filing date: 2009-07-24
Publication date: 2010-02-04
Also published as: GB0813733D0

Abstract

Disclosed is an automated method of extracting a selected fraction in a fractionated sample, the method comprising the steps of: providing the fractionated sample in a receptacle; inserting an extraction means into the selected fraction at a first position and extracting part of the selected fraction; withdrawing the extraction means from the selected fraction; inserting the extraction means into the selected fraction at a second position and extracting a further part of the selected fraction. Also disclosed is a corresponding apparatus. The method and apparatus find particular, but not exclusive, use in extracting the "buffy coat" from a fractionated blood sample.

Description

IMPROVEMENTS IN AND RELATING TO EXTRACTION OF A SELECTED FRACTION FROM A FRACTIONATED SAMPLE

The present invention relates to a method and apparatus for automatically determining the boundaries between fractions in a fractionated sample and to a method and apparatus for automatically extracting fractions from a fractionated sample and, in particular, to such a method and apparatus wherein the fractionated sample is a fractionated blood sample.

Blood samples are often analysed or processed by fractionation to separate particular components. This allows particular components of interest to be extracted from a sample. Fractionating is achieved by centrifugation of the sample, which results in the sample having a number of layers or fractions, each fraction consisting of particular components of the blood. Typically, the centrifuged sample comprises three fractions: the uppermost fraction, which contains, amongst other components, plasma; the lowermost fraction, which contains, amongst other components, red blood cells; and the middle fraction, known as the "buffy coat" containing, amongst other components, white blood cells.

The fractions are typically extracted in turn by a pipette means under manual control. The fraction containing the component of interest is retained and the other fractions may be retained or disposed of as desired. Manually controlled extraction in this manner is time consuming and expensive. It also requires considerable skill as, to the naked eye, the boundaries between fractions can be difficult to distinguish. These problems are exacerbated if the "buffy coat" is the faction of interest, for instance, if DNA analysis of the sample is required, as the buffy coat is typically relatively thin in relation to the other fractions.

It is therefore an object of embodiments of the present invention to provide a method and apparatus for automating some or all of this process, and to improve the extraction of the buffy coat in particular.

According to the present invention there is provided an apparatus and method as set forth in the appended claims. Other features of the invention will be apparent from the dependent claims, and the description which follows.

For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying diagrammatic drawings in which: Figure 1 is a schematic diagram showing a fractionated blood sample in a test tube together with features of an apparatus for identifying and extracting certain selected fractions of the sample;

Figure 2 is a schematic diagram of certain features of an apparatus for identifying and extracting certain selected fractions from a plurality of samples;

Figure 3 is a schematic block diagram of the control circuitry for such apparatus; and

Figure 4 is an overhead view of selected pipette positions for extracting a selected fraction.

Referring to figure 1 , a fractionated blood sample is provided in a test tube 10. The sample has three fractions, an uppermost or "plasma" fraction 12, a lowermost or "red blood cell" fraction 16 and an intermediate or "buffy coat" fraction 14, which contains white blood cells.

The test tube 10 of this embodiment is entirely transparent such that the entire test tube acts as a viewing window. As an alternative, the test tube 10 could have a portion of the tube acting as a viewing window. The window need not be completely transparent but may be at least partially transparent.

In order to determine the position of the fraction boundaries, the test tube 10 is held vertically and positioned with its lower end in contact with a datum plate 18. An image of the test tube 10 is then captured by digital camera 20. The image is then processed by suitable processing means 100 to determine the positions of the fraction boundaries relative to the datum plate 18. The processing means 100 typically achieves this by the use of edge detection algorithms.

In order that a better quality image can be captured, the test tube 10 may be illuminated in white, coloured or filtered light as required. A calibration means 24 may be provided adjacent to the test tube 10. The calibration means 24 may be provided with suitable markings to allow the focus, colour, brightness or contrast levels or alignment of camera 20 to be adjusted. A filter 36 may be provided to improve the contrast between fractions in the sample.

The test tube 10 is of known dimensions. Thus, from the determined position of each fraction boundary, the volume of each fraction can be calculated by the processing means 100. The processing means may then control an automatic pipette means 22 as will be described shortly. The pipette means can be inserted into the sample to a desired position and used to aspirate a volume equal to the calculated volume of a particular fraction. Typically, the fractions are aspirated in turn starting with the uppermost. Those fractions which are not of particular interest may be discarded after aspiration and those fractions which are of particular interest may be dispensed into other receptacles for storage or further analysis.

It is often desired to extract the buffy coat 14 for further analysis, as this is rich in genetic material. However, a particular problem in extracting the buffy coat is that it is not only much thinner than the surrounding layers (typically less than 1 mm thick compared to the surrounding layers which may be many tens of millimetres thick), but it also has a different viscosity to the surrounding layers and therefore behaves in a different manner to those layers when aspirated with a pipette. Pipetting of this very thin buffy coat layer is therefore very difficult. Prior art techniques whereby a pipette enters the buffy coat layer at the centre of the tube and begins aspiration, nearly always results in "piercing" of the buffy coat layer, which leads to the aspiration of part of the lowermost blood cell layer also. This can adversely affect the quality of the buffy coat sample which is collected.

Embodiments of the present invention use different techniques to extract the buffy coat layer from the fractionated sample. In particular, a multi-point pick up is utilised. In order to successfully aspirate the buffy coat, the uppermost plasma layer is removed by pipetting. The plasma layer is aspirated to just above the buffy coat layer, leaving a very small amount of plasma in situ.

It has been found empirically that a multi-stage technique is more effective in removing the buffy coat.

Figure 4 shows a cross-sectional view through the test tube 10. In a first embodiment, three aspirations are performed to remove the buffy coat. The three locations for pipetting are shown, each one being separated from the others by approximately 120° and being located towards the outer edge of the test tube. The first aspiration occurs at point 151 and this process results in approximately one third of the buffy coat being retrieved before the buffy coat "tears" leaving the remaining two thirds intact. After the first aspiration at point 151 , the pipette is withdrawn and moved to align with point 152, and the aspiration process is repeated. After the aspiration at point 152, the pipette is withdrawn again and moved to align with point 153 and the aspiration process is repeated again. Once aspiration at the three points has completed, it is found that essentially all of the buffy coat has been extracted, and the quality of the extracted sample is superior to that available using the prior art techniques.

It is found that due to the physical properties of the buffy coat layer, aspiration from towards the edge of the test tube 10 results in better retrieval than attempting to recover the buffy coat directly from the centre, as known from the prior art. Aspiration from the centre tends to result in "piercing" of the buffy coat which, it is believed, is due to the manner in which the buffy coat adheres to the side of the tube. When aspiration occurs from closer to the edge of the test tube 10, the buffy coat is pulled away from the interior wall of the test tube more easily, which acts to prevent piercing of the buffy coat and consequent contamination of the sample. Aspiration from towards the edge tends to result in a constant drawing of the buffy coat towards the tip of the pipette.

To further improve the aspiration process, the tip of the pipette is caused to track downwards as the buffy coat is withdrawn. This downward tracking ensures that the tip of the pipette remains in contact with the thin buffy coat layer, rather than pipetting from the thin plasma layer positioned above the buffy coat.

It is found that the speed of pipetting is of some importance to ensure maximum recovery of the buffy coat. Empirically, it has been found that pipetting the plasma layer 12 at a rate of 300μl_/sec and the buffy coat 14 at a rate of 100μl_/sec produces acceptable results. In general it has been found that aspirating more slowly results in less "tearing" of the buffy coat. If aspiration of the buffy coat is too rapid, then the buffy coat tends to puncture, resulting in the aspiration of red blood cells from the lowermost layer 16 in preference.

In a second embodiment of the invention, the multi-point pickup is further adapted to improve the extraction of the buffy coat from the fractionated sample. In this embodiment, the top layer of plasma 12 is removed or mostly removed to just above the buffy coat 14. The buffy coat is then removed in conjunction with the relatively small amount of plasma 12 which remains.

In the same way as the first embodiment uses three pickup points, the second embodiment uses substantially the same three pickup points, located in substantially the same positions with respect to the tube. However, rather than aspirating and removing the buffy coat in three operations, this embodiment moves the pipette to 22 to the first position 151 , and aspiration at this point commences. As the aspiration proceeds, the tip of the pipette tracks the buffy coat layer down the tube so that the tip of the pipette is always located, as far as possible, in the buffy coat layer 14. Approximately 600μl_ is aspirated in this operation. Once the aspiration is complete, the mixture of buffy coat, plasma and some red blood cells is dispensed back into the tube at the same location, with the pipette tracking upwards to ensure that the tip stays, as far as possible, in the buffy coat layer.

The act of dispensing the liquid back into the tube, immediately following aspiration, allows the buffy coat, plasma and red blood cells to be mixed. Due to the speed of aspiration and dispensing, the mixing only occurs in the upper portion of the red blood cell layer and therefore the whole tube does not become an homogeneous mixture. The act of aspirating and dispensing is performed at second and third points 152 and 153. Optionally, the three aspiration and dispensing operations may be repeated a further time at the first, second and third points.

After aspiration and dispensing has been performed at least once at each of the three points 151 , 152 and 153, then the pipette 22 moves to position at or near the geometric centre of the tube i.e. equidistant from points 151 , 152 and 153. Once the tip of the pipette is in this position and located in the buffy coat layer, which is now mixed thoroughly with the remaining plasma and some of the red blood cells, aspiration commences, with the tip of the pipette tracking downwards to ensure contact with the wanted material. Aspiration continues until all of the material of interest has been removed. This is of the order of 1 ml_ of material.

The approach adopted by the second embodiment appears at first sight to be counter-intuitive, as it involved mixing the wanted buffy coat, with the unwanted vestigial plasma, but it has been found empirically that this allows a maximal amount of buffy coat to be extracted and mitigates some of the problems associated with the 'tearing' of the buffy coat experienced in the prior art.

The speed of pipetting is found to be important. If the dispensing step is performed too quickly, then the mixing effect will occur too far into the red blood cell layer, meaning that when the final extraction step takes place, the amount of buffy coat which is extracted will not be maximised. Figure 2 illustrates schematically how this method and apparatus may be applied to process a plurality of fractioned samples. In figure 2, a plurality of samples are each retained in test tubes 10. Test tubes 10 are held in a tube rack 30. The tube rack 30 may optionally be provided with releasable rack clamps 32 to further secure the test tubes 10.

Back light 44 illuminates an optical booth 42 with white, coloured or filtered light as required. Back light 44 is used generally to illuminate the top level of the samples and the orientation of the test tubes 10. Light 34 is provided with both the samples and is used to identify the buffy coat fraction 14.

A tube pop-up actuator 28 may be used to raise one or more test tubes 10 to a position whereby the tube can be gripped by tube gripping means 26. This is facilitated by utilisation of an X-Y table 40 enabling the plurality of tubes to be positioned suitably corresponding to the actuator 28. The tube gripping means has gripper jaws 38 ensuring vertical compliance of the tube 10. The tube gripping means 26 is then operable to position the tubes 10 on a datum plate 18. The gripper jaws 38 enable the tubes 10 to be held securely until reaching the datum plate 18, at which point they are able to rest on the datum plate 18. The datum plate 18 may itself be moved from a stowed position to an operational position to facilitate the movement of the tubes 10 and the tube gripping means 26 into position on the datum means 18. After the fraction boundaries are determined, and any desired fractions are extracted, the tube gripping means 26 is operable to either return the tubes 10 to their original racks 30 or to place the tubes 10 in a different tube rack 30. The tube gripping means 26 is moved between the required positions by an actuator (not shown).

In the case wherein more than one tube 10 is selected from the rack, the tubes 10 may be imaged, and have their fractions extracted simultaneously or in turn. If the imaging is to be carried out simultaneously, either individual cameras 20 may be provided for each tube, or alternatively one camera may be provided for imaging a plurality of tubes. Additionally, if fractions in a plurality of tubes 10 are to be extracted simultaneously, the pipette means 22 is provided with a plurality of pipette heads, each pipette head being individually controllable. This allows the pipette heads to extract the correct amount of material from each test tube 10.

Figure 3 shows how those components of the apparatus may be connected. The processing means 100 is connected directly to the camera 20, the pipette means 22, the tube gripping means 26, the pop-up actuators 28 and the datum plate moving means 1 18. The processing means may also be provided with a user interface 102. The processing means may also be connected to means 1 10 for controlling the illumination of the tubes 10 during image capture. In one preferred embodiment, the processing means and the user interface may be provided by a computer or computer system.

Attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

Each feature disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

Claims

1 . A method of extracting a selected fraction in a fractionated sample, the method comprising the steps of:

providing the fractionated sample in a receptacle; inserting an extraction means into the selected fraction at a first position and extracting part of the selected fraction; withdrawing the extraction means from the selected fraction; inserting the extraction means into the selected fraction at a second position and extracting a further part of the selected fraction.

2. A method as claimed in claim 1 wherein after extracting part of the selected fraction, the extracted sample is dispensed back into the receptacle.

3. A method as claimed in claim 2 wherein after extracting and dispensing from the first and second position, a final extraction is performed.

4. A method as claimed in claim 3 wherein the final extraction is taken from a position substantially equidistant from the first and second positions.

5. A method as claimed in any preceding claim further comprising the step of inserting the extraction means into the selected fraction at a third position and extracting a still further part of the selected fraction.

6. A method as claimed in claim 5 wherein the first, second and third positions are located, respectively, 120^s apart, and positioned generally towards the interior surface of the receptacle.

7. A method as claimed in any preceding claim wherein the fractionated sample is a fractionated blood sample.

8. A method as claimed in claim 7 wherein the selected fraction is a 'buffy coat', positioned between an upper plasma layer and a lower red blood cell layer.

9. A method as claimed in any preceding claim wherein the receptacle comprises at least a transparent window and further comprising: positioning the receptacle in a known position relative to datum means; capturing an image of the receptacle and datum means; and processing the image to determine the position of boundaries between the fractions in the sample relative to the datum means.

10. The method of claim 9 , wherein the step of capturing an image includes using a digital imaging means.

1 1 . The method of either claim 9 or 10, wherein the step of processing the image includes using an edge detection algorithm to determine the position of the boundaries between the fractions.

12. The method of any of any of claims 9-1 1 , wherein the step of processing the image further comprises a step of calculating the volume of each fraction.

13. The method of any of claims 9-12, further comprising a step of illuminating the receptacle by white, coloured or filtered light, thereby to improve the quality of the captured image or the contrast between fractions.

14. The method of any of claims 9-13, wherein the step of capturing an image includes capturing the image through a filter thereby to improve the contrast between fractions.

15. The method of any of claims 9-14, wherein the datum means is a datum plate upon which the receptacle is positioned and the datum plate is moveable between an operational position and a stowed position.

16. The method of claim 15, wherein the step of positioning the receptacle includes holding the receptacle at a known orientation and position on the datum plate while the datum plate is in the operational position thereby enabling the position of the lower end of the receptacle to be determined and the relative positions of the fraction boundaries in the sample to be determined.

17. The method of claim 15 or 16, further comprising a step of providing a calibration means on the datum plate.

18. An apparatus for extracting a selected fraction in a fractionated sample, the apparatus comprising:

means for lowering an extraction means into a receptacle containing the fractionated sample to contact the selected fraction at a first position; means for withdrawing the extraction means and re-lowering it to contact the selected fraction at a second position; and

means for extracting part of the selected fraction at said first and second positions.

19. The apparatus of claim 18 wherein the extraction means is further arrange to dispense the extracted sample into the receptacle.

20. The apparatus of claim 19 wherein the extraction means is further arranged to perform a final extraction from a position substantially between the first and second positions.

21 . The apparatus of any of claims 18-20 being further adapted to perform the method of any of claims 1 -17.