Assay cartridge

Abstract

An assay cartridge has a base member (26) that defines at least two wells (30, 32, 34, 36, 38), a pipette (108, 110) positionable in at least one of the wells and a cap member (86) arranged to carry the pipette. The cap member can be releasably fastened to the base member. An extension member (28) defines at least one further well (40, 42, 44) and can be fastened to the base member such that the pipette is then positionable in at least one of the wells of the base and in the further well of the extension member.

Claims

1. An assay cartridge comprising: a base member that defines at least two wells; a pipette positionable in at least one of said wells; a cap member arranged to carry the pipette; the cap member configured to be releasably fastened to the base member; and an extension member comprising walls that define at least one further well wherein one of the base member and the extension member comprises a mating projection, and the other of the base member and the extension member is arranged to define a channel for receiving the mating projection so as to fasten the extension member to the base member such that when the extension member is fastened to the base member, the extension member extends an outer dimension of the base member, and the pipette is positionable in at least one of the wells of the base member and is further positionable in the further well of the extension member and wherein one of the base member and the extension member further comprises a depression or hole in addition to and on the same wall as the mating projection or channel, and the other of the base member and the extension member comprises a sprung or flexible protrusion arranged to engage with the depression or hole so as to fasten the extension member resiliently to the base member when they are in desired alignment.

2. An assay cartridge as claimed in claim 1, wherein the extension member is formed substantially of a different material from the base member.

3. An assay cartridge as claimed in claim 1, wherein the extension member is formed substantially of a relatively opaque material, while the base member is formed substantially of a relatively transparent material.

4. An assay cartridge as claimed in claim 3, wherein a light-sensitive reagent is stored in the extension member.

5. An assay cartridge as claimed in claim 1, wherein the extension member defines three wells.

6. An assay cartridge as claimed in claim 1, wherein at least one of the wells in the extension member has a floor that slopes at an angle of between around 20 to around 40 degrees to a length axis of the well.

7. An assay cartridge as claimed in claim 1, wherein at least one of the wells in the extension member has a transparent wall so that radiation can be detected through the wall.

8. An assay cartridge as claimed in claim 1, wherein the channel is elongate and parallel to the sides of the wells in the member that defines the channel.

9. An assay cartridge as claimed in claim 1, wherein the channel is open at one end, to allow sliding entry of the mating projection into the channel.

10. An assay cartridge as claimed in claim 1, wherein the channel is defined between two guides that project outwardly from a wall of the member, wherein one or both of the guides comprises a flange to retain the mating projection of the other member when it is in the channel.

11. An assay cartridge as claimed in claim 1, wherein the mating projection comprises one or more flanges suitable for engaging with one or more flanges on projecting elements or guides of the member that defines the channel when the mating projection is in the channel defined by the other member.

12. An assay cartridge as claimed in claim 1, wherein the extension member and the base member are arranged to be resiliently fastened together at least in part due to a friction fit between the channel and the mating projection.

13. An assay cartridge as claimed in claim 1, wherein the base member and the extension member each comprise additional stabilising surfaces which are in mutual contact when the members are fastened so as to prevent relative movement between the base member and the extension member.

14. An assay cartridge as claimed in claim 1, wherein the channel comprises a stop at one end, arranged to contact the mating projection when the extension member and the base member are in a desired fastened position.

15. An assay cartridge as claimed in claim 1, wherein well openings of the base member and the extension member lie in a plane when the two members are fastened.

16. An assay cartridge as claimed in claim 1, wherein the base member defines a single line of wells, the extension member defines one well or a line of wells, and the assay cartridge is arranged such that all these wells lie in a line when the extension member is fastened to the base member.

17. The cartridge of claim 16, wherein mating surfaces of the base member and extension member are in the vertical plane whereas the openings of the wells are in the horizontal plane.

18. An assay cartridge as claimed in claim 1, wherein the pipette is a membrane-tipped pipette and is fixed permanently to the cap member.

19. An assay cartridge as claimed in claim 18, wherein the membrane-tipped pipette has a reagent impregnated in the membrane, arranged to emit radiation, detectable through a wall of one of the wells, under appropriate conditions.

20. An assay cartridge as claimed in claim 1, wherein the cap member is arranged to carry a magnetic member, positionable in at least one of the wells of the base member and also positionable in the further well of the extension member.

21. An assay cartridge as claimed in claim 20, wherein the magnetic member comprises a sleeve, closed at one end, with a permanent magnet located within the sleeve.

22. An assay cartridge as claimed in claim 21, wherein the closed end of the sleeve is planar and is inclined at an angle of between around 20 to around 40 degrees to an axis of the magnetic member and wherein the magnet has a sloping face adjacent and parallel to the angled end of the sleeve.

23. An assay cartridge as claimed in claim 1, wherein the pipette is a capillary-tipped pipette and is detachable from the cap member.

24. An assay cartridge as claimed in claim 1, wherein the pipette is a capillary-tipped pipette, the capillary tip of which comprises a hydrophobic polymer and a hydrophilizing component.

25. An assay cartridge as claimed in claim 24, wherein said hydrophilizing component has been incorporated into the polymer by co-melting.

26. An assay cartridge as claimed in claim 1, wherein the cap member is sized to cover the wells in the base member and in the extension member, when the base and extension member are fastened together.

27. An assay cartridge as claimed in claim 1, wherein the base member comprises resiliently deformable projections for engaging into depressions or holes in the cap member so as to fasten the cap member to the base member.

28. An assay cartridge as claimed in claim 1, wherein wells of both the extension member and the base member are sealed by a single foil sheet.

29. An assay cartridge as claimed in claim 1, wherein the cap member comprises a cutter comprising two blades for shearing a foil seal covering a well of the base member or extension member, wherein each blade is arranged to shear the foil adjacent a respective wall of the well.

30. An assay cartridge as claimed in claim 29, wherein the two respective walls are parallel walls on opposite sides of the well.

31. An assay cartridge as claimed in claim 29, wherein the cutter comprises a third blade extending between the two shearing blades, for cutting the foil along a line between the two walls.

32. An assay cartridge as claimed in claim 29, wherein said well has an opening that is substantially rectangular, and the cutter is arranged to cut the foil cover so as to form two rectangular flaps, each of substantially half the area of the well opening.

33. An assay cartridge as claimed in claim 1, wherein the cap member is provided with resilient material at one or more positions corresponding to the tops of some or all of the wells in the base member and extension member, such that when cap member is urged towards the base and extension members, a liquid-tight seal is formed at the well tops.

34. An assay cartridge as claimed in claim 1, wherein the cap member comprises an elongate, resiliently-deformable seal with a cross-section which comprises at least one edge.

35. An assay cartridge as claimed in claim 34, wherein the seal comprises two elongate concave faces that are asymmetrical to each other, and that are connected by a horizontal edge face arranged so as to make first contact with the base member when the cap member is urged towards the base member.

36. An assay cartridge as claimed in claim 1, wherein the extension member comprises a glass filter located within the lower part of a well in the extension member.

37. An assay cartridge as claimed in claim 1, wherein the extension member comprises a magnet located within the lower part of a well of the extension member.

38. An assay cartridge as claimed in claim 1, wherein the cap member comprises an elongate, resiliently-deformable seal with a cross-section which comprises at least two concave curved edges.

39. An assay cartridge as claimed in claim 1, wherein the extension member comprises a magnet located adjacent an inside wall of a well of the extension member.

40. A kit of parts for an assay cartridge, the kit comprising: a base member that defines at least two wells; a pipette positionable in at least one of said wells; a cap member arranged to carry the pipette wherein one or both of the base member and the cap member is configured to be releasably fastened to the other; and an extension member that defines at least one further well, wherein one of the base member and the extension member comprises a mating projection, and the other of the base member and the extension member is arranged to define a channel for receiving the mating projection so as to fasten the extension member to the base member such that when the extension member is fastened to the base member it extends an outer dimension of the base member, and the pipette is positionable in at least one of the wells of the base member and is further positionable in the further well of the extension member and wherein one of the base member and the extension member further comprises a depression or hole in addition to and on the same wall as the mating projection or channel, and the other of the base member and the extension member comprises a sprung or flexible protrusion arranged to engage with the depression or hole so as to fasten the extension member resiliently to the base member when they are in desired alignment.

41. A kit of parts as claimed in claim 40, configured to keep the extension member out of contact with the base member, or in a different atmosphere from the base member, while the kit is in storage.

42. A kit of parts as claimed in claim 41, wherein a well of one of the base member and the extension member contains a reagent that must be protected from humidity or moisture above a predetermined level, while a well of the other of the base member and the extension member contains a liquid reagent.

43. A method of manufacturing an assay cartridge, comprising: fastening an extension member to a base member, wherein the base member defines at least two wells and the extension member defines at least one further well, wherein one of the base member and the extension member comprises a mating projection, and the other of the base member and the extension member is arranged to define a channel for receiving the mating projection so as to fasten the extension member to the base member such that when the extension member is fastened to the base member, it extends an outer dimension of the base member, and a pipette is positionable in at least one of the wells of the base member and is further positionable in the further well of the extension member, and wherein one of the base member and the extension member further comprises a depression or hole in addition to and on the same wall as the mating projection or channel, and the other of the base member and the extension member comprises a sprung or flexible protrusion arranged to engage with the depression or hole so as to fasten the extension member resiliently to the base member when they are in desired alignment; and releasably fastening a cap member to the base member, wherein the cap member is sized to substantially cover the base member and the extension member, wherein the cap member carries the pipette.

44. A method comprising: performing an assay in a cartridge comprising a base member that defines at least two wells by; positioning a pipette in at least one of said wells; arranging a cap member to carry the pipette, the cap member configured to be releasably fastened to the base member; and aligning the base member with an extension member, the extension member containing a reagent, and the extension member comprising walls that define at least one further well wherein one of the base member and the extension member comprises a mating projection, and the other of the base member and the extension member is arranged to define a channel for receiving the mating projection so as to fasten the extension member to the base member such that when the extension member is fastened to the base member it extends an outer dimension of the base member, and the pipette is positionable in at least one of the wells of the base member and is further positionable in the further well of the extension member, and wherein one of the base member and the extension member further comprises a depression or hole in addition to and on the same wall as the mating projection or channel, and the other of the base member and the extension member comprises a sprung or flexible protrusion arranged to engage with the depression or hole so as to fasten the extension member resiliently to the base member when they are in desired alignment and reacting the reagent in the extension member with a sample.

Description

(1) Certain preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

(2) FIG. 1 is a perspective view of a prior-art cartridge;

(3) FIG. 2 is a perspective view of a base member fastened to an extension embodying the invention;

(4) FIG. 3 is a close-up of part of FIG. 2;

(5) FIG. 4 is a perspective view of the unconnected base member;

(6) FIG. 5 is a close-up of part of FIG. 4;

(7) FIG. 6 is a perspective view of the unconnected extension member;

(8) FIG. 7 is a further perspective view of the unconnected extension member;

(9) FIG. 8 is a perspective view of the underside of a lid member for use with the base member and extension member;

(10) FIG. 9 is a more-detailed perspective view of part of the lid member;

(11) FIG. 10 is a different perspective view of the lid member;

(12) FIG. 11 is a further perspective view of part of the lid member;

(13) FIG. 12 is a graph relating to the force required to cut a foil seal for two difference cutter designs;

(14) FIG. 13 is a cross-section through a prior-art seal;

(15) FIG. 14 is a cross-section through a seal embodying the invention;

(16) FIG. 15 is a cross-section through a different seal also embodying the invention;

(17) FIG. 16 is a graph relating to the force required to compress the seals depicted in FIG. 13 and FIG. 15;

(18) FIG. 17 is a vertical cross-section through a magnetic member for use with a cartridge embodying the invention;

(19) FIG. 18 is a graph of experimental results comparing an embodiment of the invention with available systems for determining cholesterol;

(20) FIG. 19 is a further such graph for triglycerides;

(21) FIG. 20 is a further such graph for HDL;

(22) FIG. 21 is a further such graph for computed LDL;

(23) FIG. 22 is a dose-response graph for BNP levels measured using a cartridge embodying the invention; and

(24) FIG. 23 is a BNP levels measured using a different cartridge embodying the invention.

(25) FIG. 2 shows a base member 26 and an extension member 28 which embody the invention. The base member 26 is former of a transparent plastics material. The extension member 28 is shown with different shading to aid identification, but may be made of the same transparent plastic. In alternative embodiments, one or both members may be opaque. The base member 26 defines a line of five wells 30-38, four of which 32-38 have sloping bases. The first well 30 is principally a storage area for a capillary-tipped pipette (not shown), and the second well 32 is principally a storage area for a membrane-tipped pipette (not shown).

(26) The extension member 28 defines three wells 40, 42, 44 in a line. The second 42 and third 44 of these have sloping bases. The wells 40, 42, 44 in the extension member 28 and the second to fifth wells 32-38 in the base member 26 are principally intended to contain reagents when the assay cartridge is in use and optionally when the cartridge is in storage.

(27) The base member 26 has four flexible, upward projections having flanged tips 46-52 which can snap into through-holes in a cap member (not shown) to fasten the base member 26 to the cap member.

(28) FIG. 3 shows the mating connection between the base member 26 and the extension member 28 in greater detail. Two elongate, vertical projections 54, 56 from the extension member 28 can be seen.

(29) FIG. 4 shows the base member 26 separate from the extension member 28.

(30) FIG. 5 shows, in greater detail, the elements of the mating connection which are on the base member 26. Each of the two elongate, vertical projections 54, 56 has a flange along its outer length which curves towards the other projection, thereby defining a vertical channel 62 between the projections 54, 56. A pair of upwardly extending plates 64, 66 towards the bottom of the base member 26 act both as height stops, by preventing movement of the extension member 28 below their upper faces, and as tilting stops to resist tilting movement of the extension member 28 away from the base member 26. The underside of a small projection 68 located centrally between the two vertical projections 54, 56 and approximately half way up the end face of the base member 26 acts as a locking hook for retaining the extension member 28 when it is fastened to the base member 26.

(31) FIGS. 6 and 7 show the extension member 28 separate from the base member 26. The extension member 28 also has two elongate vertical projections 70, 72, each of which has a flange 74, 76 along its outer length which curves away from the other projection. The vertical projections 70,72 are closer to each other than the projections 54, 56 on the base member 26 and are sized such that they can slide into the channel 62 defined between the base member's projections 54, 56. The two sets of projections and flanges can form an interference fit when the extension member 28 is fastened to the base member 26. The vertical projections 54, 56, 70, 72 may be only substantially vertical, and may instead taper together slightly towards the bottom of the members 26, 28 so that the strength of the interference fit increases as the extension member 28 is lowered relative to the base member 26. The interference fit may be fairly gentle in some embodiments, since other means are provided for retaining the base member 26 and extension member 28 in the desired position once fastened.

(32) FIG. 8 shows a cap member 86 lying on its side. The pipettes are not shown for ease of understanding. The cap member 86 is sized to cover the joined base member 26 and extension member 28 shown in FIG. 2 when fastened. The sides of the cap member 86 can overhang the sides of the joined base and extension members in all directions.

(33) The cap member 86 has eight through-holes in its side walls, four of which 88-94 are visible in FIG. 8. The four flanged tips 46-52 of the base member 26 can engage in either the lower 90, 94 or upper 88, 92 sets of through holes to fasten the cap member 86 to the base member 26 in either a fastened position in which the cap is relatively distanced from the base member, or in an urged-together position in which they are relatively close.

(34) A line of six downward-facing cutters 96-106 is situated along an inner horizontal surface of the cap member 86, to align with the third to fifth wells 34-38 of the base member 26 and the three wells 40-44 of the extension member 28. These six wells are initially sealed by foil sheeting (not shown), which the cutters are arranged to cut open when the cap member 86 is urged into the urged-together position.

(35) FIG. 10 shows the cap member 86 including a capillary-tipped pipette 108 and a membrane-tipped pipette 110, which are arranged to be positioned in the first and second wells 30, 32 of the base member 26 respectively when the cap member 86 is fastened to the base member 26. Also visible is an elongate, downwardly-protruding rubber seal 109 surrounding the first three cutters 96-100, and another, similar seal 111 surrounding the next three cutters 102-106. The seals may be bonded to, or formed with, a backing sheet which connects between the two.

(36) FIG. 11 shows a detailed view of the cutters, looking vertically upwards from underneath. The first, second, third, fifth and sixth cutters 96, 98, 100, 104, 106 are of identical design, while the fourth cutter 102 is different. The first cutter 96 is rectangular in outline and has three linear blades each having two cutting edges. One pair of cutting edges 112, 114 is situated along a short edge of the rectangle. The edges 112, 114 slope downwards to a point 116 away from the bulk of the cap member 26. Another pair of cutting edges 120, 122 is situated along the other short edge of the rectangle and similarly slope downwards to a point 124. The contiguous cutting edges 118, 126 of the third blade form a line joining the mid-points of the short edges; one edge 118 slopes downwards to meet the first pair of edges 112, 114 at the first point 116. The other edge 126 slopes downwards to meet the second pair of edges 120, 122 at the second point 124.

(37) The fourth cutter 102 has four cutting edges which meet at a central point 132. It acts to pierce the foil centrally. It is of different design because the corresponding well has a smaller opening, for which the three-blade cutter is less well suited.

(38) In use, the assay cartridge may be received by the end-user (e.g. a physician) with the extension member 28 fastened to the base member 26 and with the cap member 86 fastened to the base member 26 in its higher, fastened position. The third to fifth wells 34-38 of the base member 26 and the three wells 40-44 of the extension member are initially sealed by one or more foil sheets stretched across their openings and bonded to an upper surface of the walls defining the wells. Some or all of the wells may contain reagents, e.g. as described in the Examples below.

(39) A physician can remove the capillary-tipped pipette 108 from the cap member 86, collect a sample in the pipette (e.g. of blood), replace the pipette in the cap member 86, and load the cartridge into as analyser device (e.g. an Afinion AS100 Analyzer). The analyser device will urge the cap member 86 towards the base member 26. This causes the first cutter 96 to shear the foil seal along the shorter edges of the rectangular opening of the third well 34, shearing from the mid-point of the short edge outwards, as well as to cut a line along the middle of the foil, parallel to the longer edges of the rectangle. Ridges 128, 130 along the long edges of the cutter 96 push the two flaps of foil against the inside edges of the well 34. The other identical cutters act similarly. The fourth cutter 102 pierces the foil centrally and creates four flaps.

(40) The analyser device may then release the cap member 86 from the base member 26 and move it relative to the base and extension member 26, 28 so as to position the capillary-tipped and membrane-tipped pipettes 108, 110 in various of the wells as necessary for carrying out the desired assay. The operation of the analyser device in this respect is substantially as described in WO 02/090995. Once the assay is complete, the cap member 86 is again fastened to the base member 26 in the urged-together position, thereby causing the seals 109, 111 to compress and form a fluid-tight seal around the wells, preventing leakage of used reagents from the cartridge.

(41) FIG. 12 shows a graph of the urging force, against displacement, required to be exerted to move the cap member 86 into the urged-together position when the cutters are cutting the foil seals. The upper line 134 shows the force required when the cutters all have a piercing design similar to that of the fourth cutter 102. The required force exceeds 60 N. The lower line 136 shows the force required when the cutters are as described in FIGS. 10 and 11. This force never exceeds 20 N.

(42) FIG. 13 shows a prior-art seal design, as already described.

(43) FIGS. 14 and 15 show cross-sections through two different designs for the seals 109, 111, both embodying the invention. In the first design, a pair of concave, curved faces 148, 150 extend towards each other, away from the cap member 86. They are connected by a horizontal edge 152. The curves 148, 150 are mirror images. In the second design, a pair of concave, curved faces 156, 158 again extend towards each other, away from the cap member 86. They are connected by a horizontal edge 160. However, here the curves 156, 158 are not mirror images, but are defined by dissimilar equations. This asymmetry has been found to provide particularly good performance.

(44) FIG. 16 plots the urging force, against displacement, required to move the cap member 86 into the urged-together position when compressing the seals 109, 111 against the base and extension members 26, 28. The upper line 162 shows the force required when the seal has a known design as shown in FIG. 13. The lower line 164 shows the force required when the seals are as described in FIG. 15. It will be seen that the compression is significantly greater for the same force when using the design embodying the invention.

(45) FIG. 17 shows a magnetic member 110 belonging to an assay cartridge that is like the cartridge depicted in FIGS. 2-11 but with the membrane-tipped pipette 110 (shown in FIG. 10) replaced by the present magnetic member 110, which is similarly sized and shaped. The magnetic member 110 is elongate, having a wider top section 168 for connection to the cap member and a narrower sleeve section 170. The sleeve section 170 is a hollow cylinder of rectangular cross-section 168. It is closed at one end by a sloping planar face 172. The sleeve section 170 and sloping face 172 may be of plastics material. The sleeve section 170 contains within it a permanent magnet 174 that spans the widths of the sleeve and occupies the majority of the length of the sleeve section 170.

(46) Some example assays using cartridges embodying the invention are now described.

(47) The wells in the extension member 28 of FIG. 2 may carry reagents; alternatively these wells can carry calibrators (liquid or freeze-dried), harbour a blood separation unit, a wiper (to minimize cross-contaminations in assays measuring a plurality of analytes), or have other functions. The base member 26 and extension member 28 may be snapped together by the manufacturer and sealed with foil as one unit in the assembly line, or treated and sealed separately to be snapped together by the customer.

EXAMPLE 1

(48) An enzymatic assay using the base member 26 can be performed as follows. 15 L of serum or plasma is drawn into the capillary-tipped pipette 108 by capillary forces and the capillary-tipped pipette 108 placed in the cartridge. The cartridge is inserted into an Afinion analyser device. In the instrument, the capillary-tipped pipette 108 is moved to the fifth well 38 and the sample mixed with dilution buffer, typically 250 L. The third and fourth wells 34, 36 are prefilled with reagent 1 and 2 for the analysis of analytes 1 and 2, typically 100-150 L of reagent. Blank readings are performed on reagents and diluted sample. The capillary-tipped pipette 108 transfers diluted sample (typically 50 to 100 L) third and fourth wells 34, 36, and sample and reagent are mixed. Depending on analyte measured a second blank reading may be performed. Reaction mixtures are heated. Depending on measurement mode (end-point, fix-point or kinetic reading) one or several readings are performed. Readings are transformed to concentrations by interpolation from calibration curves present in the barcode of the cartridge.

EXAMPLE 2

(49) Cholesterol (CH) and triglyceride (TG) levels of nine serum samples were measured on an Afinion analyser device essentially as described in Example 1.

(50) The calibration curve used was constructed from 6 calibrators that had been previously quantified at a CRMLN laboratory (Seattle, USA). In parallel the same nine samples were measured by a commercial point-of-care analyzer, Cholestech LDX system (Lipidprofile-Glu), and on a clinical laboratory instrument ADVIA 2400, using Siemens reagents, each using a calibrator system supplied by the manufacturer. Obtained values are given in Table 1

(51) TABLE-US-00001 TABLE 1 Cholesterol (mg/dL) Triglycerides (mg/dL) Sie- Sie- Sample Afinion Cholestech mens Afinion Cholestech mens 11-1487 156 167 157 101 111 106 11-1488 162 163 162 110 104 107 11-1489 161 170 166 61 56 64 11-1491 221 220 224 191 194 182 11-1491 167 174 168 75 68 73 11-1492 202 189 204 74 59 71 11-1493 173 175 172 97 97 97 11-1494 215 241 222 285 304 277 11-1495 268 261 253 110 116 109

EXAMPLE 3

(52) CH, TG and HDL levels of 42 plasma samples were measured in an Afinion analyser device essentially as described in Example 2 with the assay cartridge extended with a three-well extension unit embodying the invention (similar to that depicted in FIG. 6). The cartridge now also contained two HDL reagents, one for blocking nonHDL and one for converting HDL to detectable product. As in Example 2, the plasma samples were also measured with the Cholestech LDX Lipidprofile-Glu and Siemens Advia systems. FIGS. 18-21 compare the three methods with respect to obtained values for CH, TG, HDL, and LDL. LDL was not directly measured but computed from the other variables using the Friedewald equation:
LDL=CHHDLTG/5 (in mg/dL).

EXAMPLE 4

(53) A whole blood sample was filtered in an extended assay cartridge embodying the invention (similar to that depicted in FIG. 2), in which one of the wells contained a glass filter attached to the well bottom. The sample was run in 6 replicates. Whole blood was drawn into a 15 L capillary-tipped pipette by capillary forces and inserted into the extended cartridge. The sample was emptied and mixed in 285 L dilution buffer. 200 L was transferred by the capillary-tipped pipette to the glass filter containing well. The membrane-tipped pipette was then positioned tightly over the glass filter. When below ambient pressure was applied to the open end of the membrane-tipped pipette the diluted whole blood flowed into the glass filter and whereas blood cells were trapped in the filter the diluted plasma flowed through the filter and the microporous membrane into the membrane-tipped pipette. When the pressure started rising (sucking air), the membrane-tipped pipette was removed from the glass filter, lowered into an empty well and forced to release the filtered plasma by applying an above ambient pressure. Blood contamination was measured after converting contaminating hemoglobin to methhemoglobin using NaNO.sub.2 and measuring the absorbance at a wavelength of 410 nm. The absorbance was converted to % Hb by interpolation from a calibration curve constructed from known concentrations of methhemoglobin. Results are shown in Table 2.

(54) TABLE-US-00002 TABLE 2 % Hb Run# contamination Mean SD 1 1.23 1.24 0.07 2 1.16 3 1.27 4 1.29 5 1.32 6 1.15

EXAMPLE 5

(55) Triglycerides and total cholesterol are determined using end-point measurements, but cholesterol associated with HDL does not produce a measurable end-point and must be determined using fix-point or kinetic measurement. Whereas end-point measurement renders the measurement largely independent of reagent activity, fix-point or kinetic measurements are highly dependent upon reagent activity. It is therefore recommended that such assays use calibrators to compensate for any reagent degradation.

(56) TABLE-US-00003 TABLE 3 Reagents and volumes of assay cartridge Well nr. Content Volume 3rd CH reagent Typically 100 L 4th TG reagent Typically 100 L 5th Dilution buffer Typically 150 L 6th HDL reagent 1 Typically 350 L 7th HDL calibrator Freeze dried 8th HDL reagent 2 Typically 100 L

(57) 15 L of serum or plasma is drawn into the capillary-tipped pipette by capillary forces and placed in the extended assay cartridge shown in FIGS. 2-11, where the extension member is transparent. The cartridge is inserted into an Afinion analyser device. In the instrument, the capillary-tipped pipette 108 is moved to the fifth well 38 and mixed with dilution buffer. 150 L HDL reagent 1 is transferred from the sixth well 40 to the seventh well 42 containing the freeze dried HDL calibrator. Diluted sample (typically 30 L each) is added to the sixth, fourth and third wells 40, 36, 34. While the enzymatic reactions which produce measurable end-points are running in the third and fourth wells 34, 36, nonHDL in sample (sixth well 40) and calibrator (seventh well 42) are being blocked by the R1 reagent. After sufficient time to block nonHDL has passed (typically 2 minutes), HDL R2 (typically 50 L) is transferred from the eighth well 44 to the seventh well 42, and nonHDL-blocked sample (typically 150 L) is transferred from the sixth well 40 to the eighth well 44. The reactions in the seventh and eighth wells 42, 44 are monitored through a transparent side of the extension member to determine the absorption increase and end-points are measured in the third and fourth wells 34, 36.

(58) Absorptions are converted to concentrations by interpolation from pre-established calibration curves supplied in the barcode of the cartridge. The absorption increase of the calibrator is used to correct the HDL calibration curve for any age related reagent degradation.

EXAMPLE 6

(59) B-type Natriuretic Peptide (BNP) levels in six samples spanning 0 to 100 pg/mL were measured in replicates in an Afinion analyser device with an extended assay cartridge embodying the invention, similar to that depicted in FIG. 2, but without the membrane-tipped pipette 110 shown in FIG. 10 and with the extension member being made from a transparent polymer material. A magnet was located in the sixth well 40. Reagents and volumes are given in Table 4. Sample (15 L) was allowed to react with the Reagent Formulation (5 L) of eighth well 44, containing anti-BNP antibody 1 conjugated to 0.5 m paramagnetic particles (1.25%) and 0.2 m latex particles coated with HRP and anti-BNP antibody 2 (0.3%). Formation Anti-BNP 1-BNP-anti BNP 2/HRP complex was allowed to proceed for 10 minutes at ambient temperature, then wash buffer (100 L) was added and the mixture transferred to the seventh well 42. The paramagnetic particles of the Reagent Formulation, including bound BNP/anti-BNP2/HRP, were attracted to the wall between seventh well 42 and the sixth well 40 by the magnet. Unbound anti-BNP 2/HRP was removed and the trapped paramagnetic particles with complexes were washed five times with 150 L of wash buffer. Finally 150 L TMB was added to the seventh well 42 and the absorption increase monitored for 750 s at 625 nm. FIG. 22 shows the dose-response graph obtained. The achieved Limit of Detection was 5 pg/mL.

(60) TABLE-US-00004 TABLE 4 Reagents and volumes of BNP assay Well nr. Content Volume Base member 3rd TMB solution 300 L 4th Wash buffer 400 L 5th Wash buffer 475 L Extension member 6th Magnet 7th Partitioning vessel 8th Reagent Formulation 5 L

EXAMPLE 7

(61) BNP levels in five samples spanning 0 to 100 pg/mL were measured in replicates in an Afinion analyser device with an extended assay cartridge embodying the invention, similar to that depicted in FIG. 2 but with the membrane-tipped pipette 110 shown in FIG. 10 replaced by a magnetic member 110 similar to that in FIG. 17. Reagents and volumes are given in Table 5. Sample (15 L) was allowed to react with the Reagent Formulation (5 L) of the seventh well 42, containing anti-BNP antibody 1 conjugated to 0.5 m paramagnetic particles (1.25%) and 0.2 m latex particles coated with HRP and anti-BNP antibody 2 (0.3%). Formation Anti-BNP 1-BNP-anti BNP 2/HRP complex was allowed to proceed for 10 minutes at ambient temperature, then wash buffer (165 L) was added and the magnetic member was introduced into the diluted reaction mixture.

(62) The paramagnetic particles of the Reagent Formulation, including bound BNP/anti-BNP2/HRP, were attracted to the magnetic member. The magnetic member, with bound BNP/anti-BNP2/HRP, was removed, while unbound anti-BNP 2/HRP remained in the well. The magnetic member was washed extensively in the third, fourth and eighth wells 34, 36, 44. Finally the magnetic member was immersed in the TMB solution of the fifth well 38 and the absorption increase monitored for 750 s at 625 nm. FIG. 23 shows the dose-response graph. The achieved Limit of Detection was 18 pg/mL.

(63) TABLE-US-00005 TABLE 5 Reagents and volumes of BNP assay Well nr. Content Volume Base member 3rd Wash buffer 300 L 4th Wash buffer 300 L 5th TMB solution 200 L Extension member 6th Wash buffer 400 L 7th Reagent formulation 5 L 8th Wash buffer 300 L

EXAMPLE 8

(64) Five different magnets were tested with respect to capture of paramagnetic microspheres and generation of colour, using the set-up and protocol described in Example 7 and a sample containing 400 pg/mL BNP. Results are depicted in Table 6. The size and shape of the magnet was found to affect the efficiency of paramagnetic microsphere capture and assay performance. The best performance was achieved using a magnet with a perfect fit to the sleeve cavity of the magnetic member (153.21.3), similar to the arrangement shown in FIG. 17.

(65) TABLE-US-00006 TABLE 6 Size Capture of Absorbance Shape (mm) No. MP (%) (mAU at 625 nm) disc 2.0 1.3 1 54 4 332 63 2 56 13 414 100 trapezoid 7.2 1.6 1.3 1 68 17 480 141 prism 10 1.6 1.3 1 89 4 512 40 15 3.2 1.3 1 98 2 569 91

(66) In summary, preferred embodiments of the invention provide a novel assay cartridge which enables a wide range of assays to be conveniently and usefully performed.