WO2000076642A2

WO2000076642A2 - Mixing apparatus and method of mixing during conducting an assay

Info

Publication number: WO2000076642A2
Application number: PCT/GB2000/002259
Authority: WO
Inventors: David Andrewes; John Anthony O'dell; Anthony Stevenson; John Curtis; Adrian Richard Gray; John Philip Vessey; Felix Fernando; Mark Creswell; David Alan Percival; John Worthington Attridge
Original assignee: Provalis Diagnostics Limited
Priority date: 1999-06-10
Filing date: 2000-06-09
Publication date: 2000-12-21
Also published as: IL147008A0; MXPA01012565A; CN1384769A; JP2003501250A; KR20020016828A; BR0011444A; CA2374590A1; NZ516057A; AU5239100A; WO2000076642A3; RU2001133017A; EP1185357A2; GB9913560D0

Abstract

Apparatus, instrument, device and a method of mixing for conducting an assay. Apparatus (31) comprising a first and second inlet and an inlet port accommodating a filter and/or binder retaining means, the inlet port moveable relative to first and second inlets such that the port (9) can be brought into liquid communication with each inlet, and a sample chamber (3, 5) comprising a paddle (100) wich undergeos a reciprocating motion.

Description

DESCRIPTION

MIXING APPARATUS AND METHOD OF MIXING

The present invention relates to an improved mixing apparatus and a

method of mixing.

More particularly it relates to an improved apparatus, instrument and

device for conducting an assay and the assay methodology.

In a particularly preferred embodiment it relates to a device suitable

for use in assaying analyses, for example glaciated proteins, in samples such

as, for example, blood.

A person skilled in the art will however appreciate that the principle

behind the invention can be applied to solve a mixing problem in a number

of different apparatus, instruments or devices.

The applicant has developed an apparatus, instrument and device for

conducting an assay ,as disclosed in PCT/GB98/03586. The apparatus

comprises a first inlet, a second inlet, and an inlet port, the inlet port being

moveable relative to each of said first and second inlets such that the port

can be brought into liquid communication with each inlet in turn as required,

the inlet port accommodating a filter means and/or a binder retaining means.

In the course of conducting an assay to, for example, determine the

presence or absence of one or more analyses in a sample, the sample is

separated into a first component fraction and a second component fraction, the second component fraction being obtained by eluting a component "held"

on the binder retaining means from the binder retaining means.

The applicant has determined that the elution step in which the elutant

fills the second inlet under gravity, gives a non-homogenous sample (due to

the formation of an elution gradient in the second inlet) which results in

inaccurate readings when the sample is "read" in a measuring instrument,

such as, for example, an instrument comprising a microprocessor operable

via a keypad, one or more light emitters and one or more light detectors, a

display and driver, an analogue to digital convertor and means for connecting

the instrument to a power source.

It is an aim of the present invention to provide a simple method for

making a gravity fed fraction homogenous and more particularly to provide

a modified apparatus, instrument and/or device capable of performing such

a method.

In accordance with a first aspect of the present invention there is

provided a method of mixing a sample in a chamber comprising positioning

a paddle in the sample and causing said paddle to undergo a reciprocating

motion.

It is another and independent aim of the present invention to provide

an apparatus, instrument or device, capable of mixing a sample for an assay

in which an analyte is detected by a spectrophotometric means and/or a

component for use in achieving this aim. According to a further aspect of the present invention there is

provided, a paddle comprising a liquid moving surface and means for

supporting said paddle in or over a chamber such that the paddle can

undergo a reciprocating motion in the chamber.

Preferably the means for supporting the paddle in or over the chamber

comprises a pair of arms extending from the liquid moving surface.

Preferably the paddle is T shaped.

More preferably the fluid moving surface has an opening formed

therein through which a light beam can pass.

In one embodiment, the paddle comprises a magnetic material and is

caused to undergo a reciprocating motion by an electromagnetic means such

as a solenoid.

Of course other mechanisms could be used to effect a reciprocating

motion. For example, the paddle could comprise a piezoelectric material and

be caused to undergo a reciprocating motion using a localised current.

According to yet a further aspect of the present invention there is

provided a sample container adapted to receive a paddle said paddle being

mounted in or over said sample container such that the paddle can undergo

a reciprocating motion in the container.

Preferably the sample container comprises a base with sides

extending therefrom to define a chamber, said sides comprising means, for

example a pair of slots, which support the paddle. Preferably the sample container is an apparatus comprising an optical

chamber.

More preferably the sample container is part of a carousel or cassette.

According to yet a still further aspect of the present invention there is

provided an instrument adapted to receive a sample container comprising a

paddle, said instrument comprising means for causing said paddle to

undergo a reciprocating motion in the container.

Preferably said means for causing said paddle to undergo a

reciprocating motion is an electromagnetic means, for example a solenoid.

According to yet a still further aspect of the present invention there is

provided a device comprising a reading instrument comprising means for

driving a paddle in a reciprocating manner in an apparatus comprising an

optical chamber.

An example of an apparatus and instrument which can be adapted

in accordance with the present invention are described in International

application PCT/GB98/03586.

The apparatus and instrument described in PCT/GB98/03586 are

susceptible to a number of other problems common to devices which are

used in assays. Thus, a separate and unrelated problem with a device of the

general type described in PCT/GB98/03586, namely one in which a sample

or samples are presented to an instrument for reading, is one of accurately

positioning the sample relative to, for example, one or more of the light emitters and one or more light detectors which make up the reading means

if reading errors are to be avoided or at least minimized.

Thus it is an independent aim of the present invention to provide a

device which enables accurate readings to be taken.

According to this independent aspect of the present invention there is

provided a device, comprising an instrument for reading one or more

samples, and an apparatus for presenting the one or more samples to the

instrument, wherein the positioning of the one or more samples into a

reading position is achieved using two phased recognition.

The two phased recognition preferably utilises at least two

independent micro switches.

A first switch informs the instrument that the apparatus is within range

and a second switches confirms precise alignment.

A first micro switch on the instrument is activated by an "element" on

the apparatus and this constitutes the first phase of detection. Preferably the

element on the apparatus is a projecting member which depresses a board

mounted micro-switch via a rocker arm assembly. The rocker arm actuation

overcomes any error in the horizontal location of the switch on the circuit

board.

A second switch on the instrument serves as the "fine tune" and is

activated when the instrument reaches a precise (as opposed to general)

location on the instrument. In one embodiment the two members of the switch are a notch in the

outermost wall of the apparatus, more particularly a carousel or cassette

type apparatus, and a resilient member or arm on the instrument. When the

carousel or cassette type apparatus moves into position the resilient member

or arm moves from a position in which the member is biased to its unbiased

position thereby deactivating the switch.

This two stage recognition makes assembly easier and increases the

robustness of operation. It also improves the ease of use.

In the case of a carousel device of the type disclosed in

PCT/GB98/03586, it is preferred that there are a plurality of such switches.

More preferably there are four such switches located 90° apart.

A separate and unrelated problem with a device of the general type

described in PCT/GB98/03586 is how to achieve good readings when

quantifying two different fractions.

For example in the case of diabetes management it is desirable to

determine the percentage of blood haemoglobin (Hb) that is glaciated. This

means two assay results need to be obtained and a comparison made

between them. For example between glaciated and non glaciated

haemoglobin.

Traditionally analyses are measured at a peak frequency. In the case

of glaciated proteins containing haem pigment this peak frequency is around

405nm. The applicant has determined that there are significant advantages to be gained by making the measurements off peak, and in the case of

glaciated haemoglobin protein, at between 415-460nm, more particularly still

at about 440nm. This frequency range corresponds to be shoulder of the

absorbance verses wavelength graph for haemoglobin. The 440 nm figure

is the preferred wavelength. This extends the linear response to cover a

wider and hence more useful range of haemoglobin concentrations.

According to this independent aspect of the present invention there is

provided a method for determining the % glycation of blood comprising

separating a blood sample into a first component fraction containing one or

more non glaciated proteins, and a second component containing the one or

more glaciated proteins, and detecting/quantifying the analyte by

spectrophotometric means at between 405 nm and 460 nm, more preferably

at about 440nm.

The "off peak" measurement avoids complicated calibration

procedures, both in production and on-going in the field. It is essential for the

performance of an instrument and when comparing tests between

instruments that there is a linear response between measurement and

concentration of absorbing substance. It is equally important that the range

of linear response is wide enough so that measurement of, for example,

both glaciated and unglycated fractions can be made on a linear portion of

a response curve. The reason for this is that the slope of the linear response

will vary for a number of reasons from instrument to instrument. Also the slope of the linear response will also vary within an instrument as a function

of temperature or other environmental factors. However, the nature of the

calculation of % glycation is such that, within a given instrument, the slope

of the response cancels out. Variations in slope do not therefore effect the

result either within an instrument or between instruments, as long as

significant change does not occur over the period of an assay. Any

remaining variation between instruments can therefore be equalised using

a calibrated offset established during initial set up at manufacture.

The use of a narrow band of wavelength produces a linear response

but as this nears the absorbance maximum of haemoglobin the range of

response is reduced. This is because at this point the system is at its

maximum sensitivity. The selection of suitable band pass filters away from

the absorbance maximum de-sensitises the system and extends the working

range of the response allowing the elimination of slope factors described

above. The reduced sensitivity is then offset by achieving a high signal to

noise ratio on the detector electronics.

A separate and unrelated problem with a device of the general type

described in PCT/GB98/03586 is ensuring accuracy of readings and keeping

critical values (CV'^S) to a minimum.

The applicant has demonstrated that a major factor effecting critical

values is ensuring all of the sample is collected for measuring. Thus where

small volumes are measured as much as 8% of the total volume can be lost in a single drop.

According to this independent aspect of the present invention there is

provided an apparatus incorporating one or a plurality of means for breaking

the surface tension of a drop to ensure it leaves a first component part and

enters a second component part of an apparatus.

In one embodiment the means comprise a web or like member

situated between the first and second components parts.

More preferably the apparatus is of a type in which an inlet port is

movable relative to each of first and second inlets, said inlet port being

funnel shaped and accommodating a filter means or binder retaining means,

said web being situated across the outlet of said funnel.

A separate and unrelated problem with a device of the general type

described in PCT/GB98/03586 is the problem of ensuring the apparatus is

firmly held in position in the apparatus when readings are to be taken.

The Applicant has resolved this problem by careful design of the

apparatus and instrument.

Thus in one embodiment the carousel apparatus comprises a tapered

circumferential ring and the instrument comprises spring clips which pull the

carousel downwards preventing wobble.

According to this independent aspect of the present invention there is

provided a device comprising an instrument for reading one or more samples

and an apparatus for presenting the one or more sample to the instrument, wherein the apparatus is held firmly in position in the instrument by means

of spring clips.

The main invention and various independent aspects of the invention

will now be described, by way of example only with reference to a device of

the general type described in PCT/GB98/03586 and a method of assaying

glaciated and non glaciated haemoglobin fractions.

FIG. 1 is a perspective view of an apparatus according to one aspect

of the invention;

FIG. 2 is a partial sectional view of the Fig. 1 apparatus;

FIG. 3 is a perspective view of the base portion of the apparatus of

FIG. 2 showing the paddle of the invention; and

FIG. 4 is a perspective view of an instrument for use with an

apparatus as illustrated in FIGS 1 to 3.

Referring to Figs. 1 and 2 the carousel apparatus 31 comprises a

base section 2 of clear plastics (shown in detail in Fig. 3), a top portion 6 and

a funnel portion 32. The funnel portion 32 is made of a hydrophobic plastics

and has a relatively large aperture to simplify emptying of reagents therein.

It has an outlet 34 which directs the liquid into the optical chambers 3 and 5

when the apparatus is rotated in an instrument. The outlet 34 includes a frit

(not shown) which frit serves to retain particles such as, for example, an

amino phenyl boronate agarose affinity matrix. The funnel 32 which serves

as an inlet port has an annular rim 36 with a recessed portion 38. The rim 36 partially overlies apertures 40, 42 and 44 formed in the top portion 6 of the

apparatus such that tubes vertically disposed in the apparatus cannot pass

through the respective apertures until the apertures are aligned with the

recessed portion 38 of the annular rim. Projecting from the underside of the

funnel is a stem 48 with a female mating member via which the apparatus 31

is connected to the instrument 24 which has a male member 50 adapted to

engage it. The male member 50 holds the funnel in a fixed position relative

to the instrument 24 such that the base portion 2 and top portion 6 of the

apparatus 31 which together form a carousel rotate around the funnel, the

annular rim 36 of the funnel serving as a guide means.

The base portion 6 of the apparatus is made of a clear plastics, is

generally annular in shape and is divided into a plurality of compartments.

As can be seen from Fig. 3 there are two optical chambers 3 and 5, a third

chamber 4, for receiving waste from a wash step, which third chamber is

disposed between optical chambers 3 and 5, and three additional chambers

40", 42' and 44' each housing a reagent tube. These chambers 40', 42' and

44', which are disposed below apertures 40, 42 and 44 in the top portion 6

of the apparatus 31 , are arranged so that the reagent tubes are presented

to the user when the carousel is in the appropriate position in use. The

optical chambers have a curved outer wall 52 and a curved inner wall 54 of

optical quality, which help focus light from the LED's of the instrument 24

through the sample in the chamber to photodiodes at the other side thereof. Each optical chamber 3, 5 can be brought into liquid communication

with the outlet 34 of the funnel inlet port 9. Alternatively, the optical

chambers can be recessed. Extending outwardly from the outermost wall 56

of the base portion 2 is a guide member 58 which sits within a circumferential

channel member 60 formed on the outermost wall 62 of the annular recess

64 of the instrument 24. A communicating channel 66 which extends from

the channel member 60 in outermost wall 62 to the top face 68 of the

instrument 24 allows the guide member 58 to be inserted into the channel

member 60 when the apparatus 31 is connected to the instrument 24.

A projecting member or tab 70 on the knurled edge 72 of the top

portion 6 acts as an indicator means, denoting the position for locating the

apparatus on the instrument and serves to assist in the turning of the

apparatus.

The base portion 2 is connected to the top portion and the funnel

portion sits in a channel 76 formed by a step on the top surface 78 of the top

portion 6.

The instrument illustrated in Fig. 4 has been designed for use with a

basic apparatus as herein before described. The instrument is provided with

a power management and monitoring circuit so that the instrument can be

connected to, for example, an external dc supply or a car battery.

Additionally, the instrument is provided with a communication system such

as, for example, a RS232 thereby providing means for sending and receiving instructions and down loading data.

Significantly, the means for receiving the apparatus is an annular

recess 64 in the instrument which is defined by a floor, an outermost sidewall

62 and an innermost sidewall 80.

The floor of the annular recess comprises a ramp 82 on a part thereof.

Within the outermost sidewall 62 of the annular recess is a channel member

60 and extending therefrom to the top surface a connecting channel 66.

In use the basic apparatus is inserted into the annular recess 60 by

aligning guide member 58 of the apparatus with connecting channel 66 so

that the apparatus is connected to male mating member 50 via its female

mating member 48. The guide member 58 can thus enter channel member

60 such that it can be rotated. On rotation a first tube is directed up the ramp

82 and out of its aperture 44 since the recessed portion 38 of the annular

ring 36 is aligned with the aperture. In this position the outlet 34 is in liquid

communication with the first optical chamber 3 and the first step of the assay

can be conducted. By turning the apparatus through a further 90° a wash

solution is presented through aperture 42 for use and then on turning the

apparatus though a further 90° tube 40, the eluting solution, is presented.

In this manner the appropriate reagents are presented for each step of the

assay process.

Having briefly described the favoured basic apparatus and instrument

there follows a more detailed look at the improvements. When tested an apparatus as described in PCT/GB98/03586 showed

a critical value in the order of 6-7%. This was found to result primarily from

an elution gradient forming when the glaciated fraction was eluted off the

solid phase. In fact, it was found that the glaciated fraction was eluted off in

a decreasing concentration as the elution buffer percolated into the optical

chamber 5. Tests indicated that the first concentrated drops emerging from

the funnel 32 collected in the corners of the optical chamber 5 and did not

mix sufficiently with the more dilute drips that followed. As a result

measurements taken before mechanical mixing of the solution showed

poorer precision and an "off set" from those recorded post mixing.

To overcome this problem it proved necessary to introduce a mixing

step.

However traditional methods proved unsuitable. Thus, for example,

the apparatus could not be shaken without fear of damage to the instrument,

and the use of a rotating flea or oscillating ball bearing could damage the

optical chamber.

The applicant solved the problem using a paddle 100. A number of

approaches were used:

Retention of a stirring device was seen as the major issue to be

resolved. Attaching a stirring component to the side walls of the optical

chamber was seen as a possible approach to overcome this problem. Two

alternatives were investigated: In one embodiment the paddle was clipped over the side walls of the optical chamber and the paddle was made

to vibrate in the direction of the optical axis using an electromagnet. A hole

in the centre of the paddle provides a path for the light from the LED.

In another embodiment the paddle was clipped over one side of the

optical chamber 5 and was made to vibrate at right angles to the optical axis

away from the light path.

Both these embodiments provided adequate mixing once a resonant

frequency was found by adjusting the frequency of an oscillator driving an

electromagnet. Though attractive there were still a number of problems with

this approach.

As the paddle must retain stiffness a significant amount of energy was

required to generate the oscillation. This would have implications for any

battery operated instrument.

Furthermore resonant frequencies vary from component to component

and with the liquid level within a chamber. Some means of scanning the

frequencies would thus be required to hit resonance and thus ensure

adequate mixing. Since both components also had a 3-dimensional shape

forming was required increasing costs.

An alternative approach of using a flat paddle overcame the problems

associated with the oscillating approach described above.

Thus in a preferred embodiment and as illustrated in Fig.3 a metal

paddle 100 was retained in grooves 101 formed by building-up the side walls 102, 104 of the optical chamber 5. The paddle was able to reciprocate with

minimal friction and could be forced to swing through the solution, along the

direction of the optical axis, using an electromagnet positioned below the

photodiode on the outer circumference of the platten moulding (described

hereafter). A hole 106 is provided to enable the light from the LED to reach

the detector.

As very little force is necessary to move the paddle, significantly less

energy is required to drive the electromagnet. Experiments have shown that

fewer than 10 swings of the paddle are required to produce a visually

homogenous solution from a layered dye-water starting solution.

Effective retention of the paddle has been demonstrated by

positioning a web (not shown) on the underside of the top moulding 6, just

above the centre of the paddle.

Another improvement relates to the use of 2 micro-switches in a

phased approach. This allows the precise unambiguous detection of the

apparatus 31 in the instrument 24. One switch (not shown) at each of four

locations is activated by a feature 58 (in this case also the guide member) on

the circumference of the plastic well as it rotates. This depresses a board

mounted micro-switch (not shown) via a rocker arm assembly (110, 112, 114

& 116) at each of the four operating positions (Fig. 4). The rocker arm

actuation overcomes any error in the horizontal location of the switch on the

circuit board (not shown). This constitutes the first phase of detection. The second phase of detection is provided by a micro-switch which is

activated by the operation of a ratchet arm not shown with a respective notch

or notches 120, 122, (only two of the four are visible) in the outermost wall

56 of the carousel. A flange 131 extending from the ratchet arm contacts a

switch on the instrument. The ratchet arm is biased such that when the

carousel is in one of the four operating positions it moves into a notch in the

carousel, deactivating the switch but when it is not in one of these positions

it is acted against by the outermost wall 56 of the carousel causing the switch

to be activated. These notches are preferably shaped to allow rotation in

one direction only. These switches are only de-activated when the

instrument and apparatus are in an exact location. The two phased

approach makes assembly easier, increases robustness of operation and

improves ease of use.

Finally another improvement relates to the arrangement used to

overcome a 'wobble' problem. Any movement, however small, between the

carousel and instrument can alter the path of light during reading. By

modifying the carousel and instrument to provide a lock facility the reading

problem was overcome.

In one embodiment the carousel comprises (Fig.1) a circumferential

ring 124 comprising an inclined surface 126 and a flat surface 128.

The instrument 24 which receives the carousel comprises a casing

130, a printed circuit board 132 onto which is mounted a plateen 134 and a hold down 136 comprising four spring clips 138, 140, 142, 144. When the

carousel is inserted into the instrument, the spring clips ride up the inclined

surface 126 and their claws lock against the flat surface 128.

Claims

1. A method of mixing a sample in a chamber (3, 5) comprising

positioning a paddle (100) in the sample and causing said paddle to undergo

a reciprocating motion.

2. A method a claimed in claim 1 wherein the paddle is of a magnetic

material and comprises a liquid moving surface and means for supporting the

paddle in or over the chamber and said paddle is caused to undergo a

reciprocating motion by the action of an electromagnetic means.

3. A method as claimed in claim 2 wherein the means for supporting

the paddle in or over the chamber comprises a pair of arms extending from

the liquid moving surface which arms sit in a pair of slots in sides extending

from a base which defines the chamber.

4. A method as claimed in either claim 2 or 3 which further comprises

detecting an analyte in said sample by passing a light beam from a light

emitter through said chamber and an opening (106) formed in the liquid

moving surface of said paddle to a light detector.

5. A method as claimed in claim 4 wherein the sample is glycated

haemoglobin and is detected by a spectrophotometric means at between

405 nm and 460 nm.

6. A paddle (100) comprising a liquid moving surface and means for

supporting said paddle in or over a chamber such that the paddle can

undergo a reciprocating motion in the chamber.

7. A paddle as claimed in claim 6 wherein the means for supporting

the paddle in or over the chamber comprises a pair of arms extending from

the liquid moving surface.

8. A paddle as claimed in either claim 6 or 7 wherein the paddle is T

shaped.

9. A paddle as claimed in any of claim 6,7 or 8 wherein the liquid

moving surface has an opening (106) formed therein through which a light

beam can pass.

10. A paddle as claimed in any of claims 6 to 9 wherein the paddle

comprises a magnetic material.

11. A paddle as claimed in any of claims 6 to 9 wherein the paddle

comprises a piezoelectric material.

12. A sample container comprising a chamber (3, 5) adapted to

receive a paddle (100), said paddle being mounted in or over said chamber

such that the paddle can undergo a reciprocating motion in the chamber.

13. A sample container as claimed in claim 12 comprising a base with

sides extending therefrom to define the chamber, said sides comprising

means which support the paddle.

14. A sample container as claimed in claim 13 wherein said means

which support the paddle is a pair of slots in said sides.

15. A sample container as claimed in any of claims 12-14 in which

said chamber is an optical chamber.

16. A sample container as claimed in any of claims 12-15 which is

a carousel or cassette.

17. A carousel as claimed in claim 16, for use in an assay in which a

sample is separated into a first component fraction and a second component

fraction, which fractions are presented to an instrument, comprising a first

inlet which is or leads to a first component fraction collection chamber, a

second inlet which is or leads to a second component fraction collection

chamber, and an inlet port accommodating a filter means or a binder

retaining means, said inlet port being movable relative to each of said first

and second inlets such that the inlet port can be brought into liquid

communication with each first and second inlet in turn as required.

18. A sample container as claimed in claim 16 which is a carousel

comprising a base portion having a plurality of chambers including first and

second inlets, a top portion which together with the base portion forms the

carousel, and a funnel portion including an inlet port, said carousel being

rotatably mounted about said funnel portion.

19. An instrument adapted to receive a sample container comprising

a chamber adapted to receive a paddle, said instrument comprising means

for causing said paddle to undergo a reciprocating motion in the chamber.

20. An apparatus as claimed in claim 19 in which said means for

causing said paddle to undergo a reciprocating motion is an electromagnetic

means.

21. An apparatus as claimed in claim 20 wherein said

electromagnetic means is a solenoid.

22. A device comprising an instrument capable of detecting an

analyte in a sample which is presented thereto in a sample container

comprising a chamber adapted to receive a paddle said paddle being

mounted in or over said chamber such that said paddle can undergo a

reciprocating motion when initiated by said instrument.

23. A device, comprising an instrument for reading one or more

samples, and an apparatus for presenting the one or more samples to the

instrument, wherein the positioning of the one or more samples into a

reading position is achieved using two phased recognition.

24. A device as claimed in claim 23 in which a first switch informs the

instrument that the apparatus is within range and a second switches confirms

precise alignment.

25. A device as claimed in claim 23 or 24 wherein a first micro switch

on the instrument is activated by an "element" on the apparatus and this

constitutes the first phase of detection and a second switch on the instrument

serves as the "fine tune" and is activated when the instrument reaches a

precise location on the instrument.

26. A device as claimed in claim 25 wherein the "element" on the

apparatus is a projecting member which depresses a board mounted micro-

switch via a rocker arm assembly.

27. A device as claimed in claim 26 wherein the two members of the

switch are a notch in the outermost wall of the apparatus, and a resilient

member or arm on the instrument.

28. A device as claimed in claim 27 wherein the apparatus is a

carousel or cassette type apparatus.

29. A device as claimed in claim 28 comprising four switches located

90° apart.

30. A method for determining the percentage glycation of blood

comprising separating a blood sample into a first component fraction

containing one or more non glycated proteins, and a second component

containing the one or more glycated proteins, and detecting/quantifying

glycated haemoglobin by spectrophotometric means at between 405 nm and

460 nm.

31. A method as claimed in claim 30 wherein the

detection/quantification of glycated haemoglobin is measured at about

440nm.

32. An apparatus incorporating one or a plurality of means for

breaking the surface tension of a drop to ensure it leaves a first component

part and enters a second component part of an apparatus.

33. An apparatus as claimed in claim 32 in which the means comprise

a web or like member situated between the first and second component

parts.

34. An apparatus as claimed in claim 32 or 33 wherein the apparatus

is of a type in which an inlet port is movable relative to each of first and

second inlets which are or lead to first and second collection chambers, said

inlet port being a funnel and accommodating a filter means or binder

retaining means, said web being situated across an outlet of said funnel.

35. A device comprising an instrument for reading one or more

samples and an apparatus for presenting the one or more sample to the

instrument, wherein the apparatus is held firmly in position in the instrument

by means of spring clips.