GB2232766A - Blood separation - Google Patents

Abstract

A method of analyzing a sample containing liquid and cellular components comprises feeding sample to be analyzed to a conduit having an annular outer retaining wall 5 and having at least one test station 3 communicating therewith, rotating the conduit and sample at a speed selected such that the sample is separated by centrifugation and cellular components of the sample collect against the outer retaining wall 5, reducing the speed of rotation so that the liquid component flows to a region of the conduit which is spaced radially inwardly from the retaining wall 17, such region communicating with the test station(s) 3 whereby the liquid component flows from the conduit to the test station(s) 3 for subsequent analysis. The sample is preferably whole blood which may comprise an anti-coagulant. <IMAGE>

Description

Blood Separation This invention relates to a method of separating blood and the use of a device in performance of the method.

Blood samples can provide much useful data for a physician. However many diagnostic tests on blood, such as immunoassays, require either serum or plasma for quantitative assays because the use of whole blood often gives unreliable results. The unreliability inherent in performing immunoassays and the like using whole blood is caused by inter alia uncontrollable partitioning of the analyte of interest between the cellular and liquid components of the blood and changes in sample volume due to varying haematocrit levels from sample to sample.

As described in EP-A-171148, a recently proposed device for performing immunoassays is the so-called FCFD or Fluorescence Capillary Fill Device which is based on an adaptation of the technology used to mass manufacture liquid-crystal display (LCD) cells. The device relies on the principles of optical fibres and waveguides to reduce the need for operator attention and it avoids the need for physical separation methods or washing steps.

An FCFD cell typically comprises two pieces of glass which are separated by a narrow gap. One piece of glass is coated with a ligand and acts as a waveguide. The other piece is coated with a dissoluble fluorescent reagent which has affinity for the ligand (in competition assays) or the analyte (in non-competitive labelling assays). When a sample is presented to one end of the FCFD cell it is drawn into the gap by capillary action and dissolves the reagent. In a competitive assay the reagent and analyte compete to bind to the ligand on the waveguide and the amount of bound reagent is inversely proportional to the concentration of analyte. In a labelling assay, the amount of reagent which becomes bound to the waveguide is directly proportional to the amount of analyte in the sample.As the gap between the pieces of glass is narrow (typically 0.1 mm) the reaction will usually go to completion in a short time, probably less than 5 minutes in the case of a competition assay.

FCFD cells avoid the need for separation steps or washing steps by using an optical phenomenon known as evanescent wave coupling. Basically, reagent molecules in solution fluoresce at relatively large angles (e.g.

more than 47 ) relative to the plane of the waveguide and emerge from the waveguide at the same large angles in accordance with Snell's Law of Refraction. On the other hand, reagent molecules bound to the surface of the waveguide emit light at smaller angles within the waveguide. By measuring the intensity of fluorescence at smaller angles to the axis of the guide, it is possible to assess the quantity of reagent bound to the surface.

In common with other immunoassay types, when analyzing blood samples, with FCFD's it is necessary to provide either serum or plasma to the cell if reliable results are to be obtained.

Previously, separation of blood samples has been carried out by initial centrifugation, so that either serum or plasma is provided for subsequent analysis.

However, this adds to the overall time involved and it would be particularly beneficial for example, in a doctor's office or other non-laboratory environments, to avoid the need to produce an initial serum or plasma sample.

Viewed from one aspect the invention provides a method of analyzing a sample containing liquid and cellular components, the method comprising feeding sample to be analyzed to a conduit having an annular outer retaining wall and having at least one test station communicating therewith, rotating the conduit and sample at a speed selected such that the sample is separated by centrifugation and cellular components of the sample collect against the outer retaining wall, reducing the speed of rotation so that the liquid component flows to a region of the conduit which is spaced radially inwardly from the retaining wall, such region communicating with the test station(s) whereby the liquid component flows from the conduit to the test station(s) for subsequent analysis.

Thus, liquid sample is supplied to the test station without the need for the operator to perform sample manipulations involved in initial sample separation. In a particularly preferred embodiment, the conduit is in the form of a generally annular trough which extends around a reservoir for initially receiving sample to be analyzed, flow communication being provided between the reservoir and trough such that, upon rotation, sample flows from the reservoir to the trough by centrifugal force. In one such embodiment, which avoids the need for any complex valve means, at least one pore is provided in a peripheral wall or base of the reservoir and communicates the reservoir with the trough, the pore(s) being of such size that surface tension normally prevents escape of sample, and the centrifugation effect of initial rotation causes liquid to be forced from the reservoir into the trough.It is preferred that only means for performing the method is sealed prior to rotation.

The method is particularly applicable to the separation and analysis of blood. Preferably, a blood sample is first treated with an anti-coagulant.

The retaining wall preferably has an inwardly facing generally "C" shape in vertical cross-section to provide an overhang for improved retention of the cellular component which packs against the wall in use.

The method of the invention is particularly applicable to FCFD analysis as discussed above, or to other assays involving capillary fill sensors, and therefore the or each test station communicating with the said inwardly spaced region of the conduit preferably comprises an FCFD or other capillary fill cell.

A preferred test vehicle for carrying out the method of the invention forms part of the subject of our copending PCT application of even date entitled "Multianalyte Test Vehicle". The preferred arrangement comprises a plurality of test stations arranged around a central reservoir.

The apparatus is preferably configured such that it has at least one plane of symmetry passing through an axis of rotation. For example, eight test stations may be equi-angularly spaced about the outer periphery of the reservoir. They may form a cylinder around the reservoir. They may also be arranged such that they form a cone. They preferably, however, extend horizontally in a vane-like arrangement.

As discussed above, a pore connecting the reservoir with the trough is of a size so that surface tension of the liquid in the reservoir normally prevents the liquid from escaping. The additional force exerted when the device rotates quickly, say 300 to 500 rpm, is sufficient to break the surface tension and allow the liquid to flow out. The increase in centrifugal force with radius causes sample which has exited through a pore to be forced against the retaining wall. An increase in rotational speed then causes the sample to separate and the cellular component to pack against the retaining wall. Slowing rotation causes the liquid component fall into the trough in which the bases of FCFD cells are standing. A gentle reversing action at this stage will ensure that the liquid component is evenly distributed to all the cells. The or each pore may be positioned in a gap between the FCFD cells so as to allow uninhibited passage of the sample from the pore to the retaining wall.

The test vehicle is preferably in two parts and made by injection moulding. An inner or base part comprises the reservoir and part of the retaining wall while the outer or upper part comprises (in the generally cylindrical arrangement discussed above) an FCFD cell support structure having windows for illumination and detection optics, a filling aperture and an upper part of the retaining wall. Once tests cells have been inserted into the upper part the two halves may be joined by, for example, ultrasound.

Ribs may be provided adjacent the windows to discourage finger contact with the optical surfaces and cylindrical surfaces may be provided for the attachment of labels and bar codes.

The upper and lower parts are designed so that simple two part tooling may be used in their construction, thus lowering the tooling cost and improving quality. The pore or pores may be formed by a small side core. Such a core may be removed before assembling the vehicle or it can be an inert plug which will dissolve when the sample is added. Alternatively, the pore or pores may be provided after moulding e.g. by drilling or using a drill or laser.

It is preferred to form the vehicle such that there is a space above the sample reservoir to receive an anti-splash filling aperture.

Although each FCFD cell will only take up a precise amount of liquid by capillary action there is a need to limit the amount of sample passing from the reservoir to the rest of the device otherwise unwanted flooding will occur. The are a variety of ways of controlling the amount of sample which can leave the reservoir.

Firstly, one can control the amount of sample initially placed in the reservoir by using a pipette. The pipette may be graduated but the overall desire to provide a disposable device means that it is preferable to provide a blow-inoulded bellows pipette which can only be inserted into the reservoir to a pre-determined depth.

Squeezing and releasing the bulb in this position causes all of the contents of the pipette to be ejected into the disposable, but any excess will be drawn back into the pipette.

Another way of controlling the amount of sample which will pass from the reservoir involves locating a disc with a central hole in the reservoir such that the volume below the disc substantially equals the volume to be dispensed. When the test vehicle is spun, the sample will be flung out against the wall of the reservoir and the disc will divide the sample; the lower portion will flow out the pore while the upper portion remains above the disc until the vehicle slows and then stops spinning.

Some embodiments of a test vehicle for use in the method according to the invention will now be described, by way of example, with reference to the accompanying drawings, in which: Figure 1 is an exploded perspective view of a preferred embodiment of a multianalyte test vehicle; Figure 2 is a transverse section towards the base of the preferred embodiment shown in Figure 1; Figures 3(a) to 3(c) are schematic sectional elevations of the preferred embodiment in use according to the method of the invention; and Figures 4(a) and 4(b) are top plan and side elevation views of a second embodiment of test vehicle.

A preferred embodiment of the vehicle is shown in Figure 1 and comprises an outer or upper part 1, a filter 2, a plurality of FCFD cells 3, and an inner or lower part 4. The upper part 1 is a generally cylindrical cap-shape having a wall 5 and a top 6.

Windows are equi-angularly spaced around the top 6. A hole 8 is provided in the top 6 to allow insertion of a liquid sample and a plug (not shown) may be used to seal the hole 8. The wall 5 has a plurality of windows 9 which are aligned with respective windows 7 in the top 6. Elongate projections 10 are provided next to the windows 9 so as to limit finger contact with the FCFD cells located in the vehicle. The wall 5 has a depending and outwardly projecting lip 11 which forms part of a retaining wall 12, as will be described later.

A filter 2 is provided to stop particulate or gelatinous matter passing into the vehicle.

The lower or inner part 4 comprises a wall 14 defining a central cylindrical sample reservoir 15 a circumferential trough defined by part of the outer wall of the reservoir 15, a circumferential upstanding lip 16 and a web 17 which forms the base of the trough.

Locating lugs 18 and guides 19 project from the lower part 14. A cylindrical wall 20, formed by the outer surface of the upstanding lip 16 provides an area upon which labels, such as bar codes 21, may be applied.

A pore 22 is provided in the wall of the reservoir 15. As can be seen in Figure 2, the pore 22 is positioned in a group between the FCFD cells 3 so as to allow uninhibited passage of sample from the pore 22 to the retaining wall 12. The pore will be described in more detail below after the assembly of the vehicle has been described.

A plurality of FCFD cells ready for use are located in the upper part 1 in alignment with the windows 7 and windows 9. The filter 13 is also located in the upper part 1. The upper and lower parts 1 and 14 are then brought into engagement; the lips 11 and 16 abutting each other and defining the retaining wall 12. The parts 1 and 14 are then secured together, preferably by the use of ultrasound but glue or tape may be used. The device is now ready for use.

After a sample 2, e.g. blood and an anti-coagulant, has been added to the vehicle via the hole 8, the plug (not shown) is inserted and the vehicle is then located on a rotatable head of a multianalyte test instrument by means of the lugs 18 and guides 19 on the lower part 14.

The head of the instrument is rotatable at a first speed of about 300 to 500 rpm, a second higher speed at which separation takes place and can also be rotated in a stepping mode to bring each FCFD cell into alignment with the light pump and with the fluorescence detector which aligns with the respective optical edge window 7 on the top of the vehicle.

Turning to Figure 3, where some parts of the vehicle are not shown for. the sake of clarity, it can be seen in Figure 3(a) that a sample 23 is in the reservoir 15. The pore 22 is so sized that surface tension of the sample 23 normally prevents the sample from escaping through the pore 22.

As the vehicle is rotated, as shown by the arrow in Figure 3(b), the sample 23 is forced through the pore 22 by centrifugal force. The increase in centrifugal force with increasing radius causes each droplet of sample 23 which has exited through the pore 22 to be forced against the retaining wall 12.

Once the sample 23 has left the reservoir and become forced against the retaining wall 12 the vehicle is accelerated to a suitable, higher speed so that the cellular components of the sample pack against the wall 12.

Carefully slowing the rotation of the vehicle allows the liquids cell-free part of the sample 23 to flow into the trough, formed by the web 17, and then be drawn up the FCFD cells by capillary action in the direction indicated by the arrows in Figure 3(c). The time when the vehicle is slowed and stopped are known so it follows that time zero for each FCFD cell is also known. The instrument can then step the vehicle to bring each FCFD cell into alignment with the light pump and fluorescence detector.

Figures 4(a) and 4(b) show, schematically, a second embodiment of test vehicle suitable for carrying out the method of the invention. This again includes a central sample receiving reservoir communicating with a trough bounded by a retaining wall 12 of "C" shape cross-section via a small pore (not shown) in a manner similar to the first embodiment. In the second embodiment, the FCFD cells 3 extend radially outwardly in a vane like arrangement on a disc 30. The inner ends (not shown) of the cells communicate with a region of the trough which is spaced inwardly from the retaining wall via slit like apertures in the retaining wall such that after initial rotation and separation, the liquid component of the sample may be drawn from the trough by capillary action in a horizontal plane. In this way any adverse effect gravity may have on the performance of the cells may be avoided. The disc 30 may include windows aligned with the cells for illumination thereof.

Claims (10)

Claims:

1. A method of analyzing a sample containing liquid and cellular components, the method comprising feeding sample to be analyzed to a conduit having an annular outer retaining wall and having at least one test station communicating therewith, rotating the conduit and sample at a speed selected such that the sample is separated by centrifugation and cellular components of the sample collect against the outer retaining wall, reducing the speed of rotation so that the liquid component flows to a region of the conduit which is spaced radially inwardly from the retaining wall, such region communicating with the test station(s) whereby the liquid component flows from the conduit to the test station(s) for subsequent analysis.

2. A method as claimed in claim 1, wherein the conduit is in the form of a generally annular trough which extends around a reservoir for initially receiving sample to be analyzed, flow communication being provided between the reservoir and trough such that, upon rotation, sample flows from the reservoir to the trough by centrifugal force.

3. A method as claimed in claim 2, wherein at least one pore is provided in a peripheral wall or base of the reservoir and communicates the reservoir with the trough, the pore(s) being of such size that surface tension normally prevents escape of sample, and the centrifugation effect of initial rotation causes liquid to be forced from the reservoir into the trough.

4. A method as claimed in any preceding claim wherein means containing the sample for performing the method is sealed prior to rotation.

5. A method as claimed in any preceding claim wherein the sample comprises blood.

6. A method as claimed in claim 5, wherein the blood is first treated with an anti-coagulant.

7. A method as claimed in any preceding claim wherein the or each test station comprises a capillary fill device.

8. A method as claimed in any preceding claim wherein the or each device is a fluorescent capillary fill device.

9. A method of analyzing a sample substantially as hereinbefore described with reference to Figures 1 to 3 of the drawings.

10. A method of analyzing a sample substantially as hereinbefore described with reference to Figures 4 of the drawings.