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Description  |
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BACKGROUND OF THE INVENTION
This invention relates to a multianalyte test vehicle which may be used in
diagnostics and monitoring particularly optical immunodiagnostics.
In the fields of diagnosis and monitoring e.g. patient health care, there
have been two main approaches to the analysis of samples from patients The
first approach is concerned with a generally qualitative evaluation of
whether an analyte is present or whether the level of analyte in a test
sample deviates from acceptable limits while the second approach is
concerned with the quantitative evaluation of the amount of analyte in a
sample.
Usually the diagnostic devices used in the first approach are relatively
inexpensive and disposable. An example of such a device is the so-called
dipstick device used to test for glucose in the urine of diabetics. The
dipstick device comprises a test area which is usually loaded with several
enzymes and a chromogen. In the example of testing for the presence of
glucose, a liquid sample, usually urine, is applied to the test area and
results in a colour change of the test area in only a few seconds. The
colour change after a given time is broadly divided into three categories
which are discernable by the naked eye in comparison with a colour chart,
viz. normal, glucose present but below a certain concentration, and
glucose present in unacceptable concentrations. It is relatively easy to
see if a sample falls squarely within any one of the categories but it is
difficult to decide on borderline samples especially as the sensitivity of
such devices are seriously affected by their storage conditions
(temperature, humidity etc). Nevertheless such devices are useful as they
can give a qualitative answer with respect to a sample, their simplicity
allows for their use by a person suffering from a chronic disorder or
someone monitoring the presence of a particular substance and their
inexpensiveness allows for their regular use. However, in many fields
there is a need to make a quantitative assessment of the levels of analyte
or different analytes in a sample.
In the past quantitative tests were performed individually by a skilled
technician working in a laboratory under carefully controlled conditions.
The high level of labour involved in effecting such tests made them very
expensive; consequently attempts have been made to automate or partially
automate these tests.
Many attempts at providing a multianalyte test apparatus have relied on
metered sub-division of a sample into a number of aliquots; each aliquot
being tested for a different analyte. Expensive pumping equipment and
complicated purging systems were needed in these apparatus to control the
consistent division of the sample and to avoid problems of contamination
caused by earlier samples. The cost and complexity of this sort of
apparatus has meant that it is usually located at hospitals, if concerned
with medical samples, or central laboratories removed from the site where
monitoring is needed e.g. when monitoring a food production line or river
for contamination. The remoteness of the apparatus from the place where
the sample is taken causes a delay in effecting the test and obtaining a
result. Sometimes the delay is unacceptable. Thus there is a general need
to provide a multianalyte test apparatus which avoids the disadvantages
associated with prior art apparatus and which has some of the elements of
simplicity and ease of use associated with disposable diagnostic devices.
Much work has been done in the field of optical biosensors in an effort to
simplify multianalyte test apparatus. An optical biosensor is a small
device which, together with its measuring instrument, uses optical
principles quantitatively to convert chemical or biochemical
concentrations or activities of interest into electrical signals. The
sensor may incorporate biological molecules, such as antibodies or enzymes
to provide a transducing element giving the desired specificity. The range
of application of such sensors is vast although many requirements, such as
working temperature range, sterilizability or biocompatibility, have
limited range.
Recently, an optical biosensor for immunoassays, the fluorescence
capillary-fill device (FCFD) has been proposed. The device is based on an
adaptation of the technology used to mass manufacture liquid-crystal
display (LCD) cells. The device uses 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 in the assay. 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 an
immunometric 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 in less
than 5 minutes in the case of a competition assay.
FCFD cells avoid the need for separation steps and/or washing steps by
using an optical phenomenon known as evanescent wave coupling. Basically,
the fluorescence from unbound reagent molecules in solution enters the
waveguide which comprises the baseplate of the FCFD at relatively large
angles (e.g. more than 44.degree. for a serum sample) 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 into
all angles within the waveguide. By measuring the intensity of
fluorescence at smaller angles to the axis of the guide (e.g. less than
44.degree. for a serum sample), it is possible to assess the quantity of
reagent bound to the surface thereby allowing the amount of analyte in the
sample to be measured. The principles involved in FCFDs are described in
more detail in U.S. Pat. No. 4,978,503.
As mentioned earlier the ligand bound to the waveguide is selected to suit
the FCFD to a particular assay. Also, FCFDs allow for rapid tests without
the need for accurate measurement of sample or reagent(s) and without the
need for separation and washing steps. These factors suggest that FCFDs
will be useful in simplifying multianalyte test apparatus. However, there
is a need to provide an arrangement whereby the timing of the contact of
sample with the FCFDs is controlled, since timing is important in rapid
assays, and where the various FCFDs can be brought into alignment with
both the light source acting as the fluorescence pump and the fluorescence
detector which needs to be aligned with the end of the waveguide.
Moreover, there is a need to avoid contamination of the optical surfaces
of the FCFDs by stray sample or other matter which would affect optical
quality.
SUMMARY OF THE INVENTION
Viewed from one aspect the invention provides a multianalyte test vehicle
comprising a sample receiving reservoir, a plurality of test stations each
comprising an FCFD or other capillary fill sensor cell, and means for
providing fluid communication between the reservoir and a conduit (or spin
collection chamber) with which the inlets ends of said cells communicate
such that in use sample from the reservoir may be fed to the plurality of
cells substantially simultaneously.
Thus, in accordance with the invention a plurality of different assay types
may be run from one sample.
A test vehicle according to the invention in a multianalyte test apparatus
also has the advantages that addition of the sample to each cell is
governed by the apparatus and not the user and that time zero for each
assay is known. This aspect of the invention is particularly applicable to
FCFD cells, but the apparatus may comprise other sensors which take up
fluid by capillary action.
Advantageously, the test cells are arranged about the outer periphery of
the reservoir. The vehicle is preferably configured such that it has at
least one plane of symmetry passing through an axis of rotation. For
example, eight test cells may be equi-angularly spaced about the outer
periphery of the reservoir (i.e. arranged concentric with and parallel to
the axis of rotation). They may form a cylinder around the reservoir. They
may also be arranged such that they form a cone. Preferably however they
are horizontally disposed in a vane-like manner, extending outwardly from
an axis of rotation of the device. The vehicle may include two or more
reservoirs each arranged to feed sample to a plurality of FCFD cells
whereby different samples could be accommodated. Thus, in the preferred
arrangements discussed above, a cylindrical reservoir, for example, may
include an internal dividing wall. In the presently preferred embodiments,
however, the vehicle includes only a single reservoir.
Preferably, the means providing fluid connection between the reservoir and
the test stations comprises at least one pore in or adjacent a side wall
of the reservoir; the conduit may be in the form of a trough or well
extending around, or around and under, the reservoir and communicating
with the pore(s). The pore(s) may be at or near the base of the reservoir
although, in one preferred embodiment, a pore is formed in an eccentric
step in the reservoir. In the latter embodiment, the step assists in
preventing sample reaching the pore until the device is rotated (as will
be described later).
In one embodiment the conduit comprises an annular trough having an outer
retaining wall with an inwardly facing "C" shape in vertical cross-section
to provide an overhang for improved fluid retention. In another
embodiment, the conduit comprises a well formed by a spin collection
chamber which is preferably annular and concentric with the reservoir, and
a shallow sump, which may extend under the reservoir. The shallow sump
preferably contains an absorbent material to absorb excess sample. The
spin collection chamber preferably includes vanes or baffles to aid
partitioning of sample.
The pore or pores are preferably of a size so that surface tension of the
liquid in the reservoir normally prevents the liquid from escaping whereby
release of fluid from the reservoir may be achieved when desired by
rotating the apparatus so that liquid moves by centrifugal force from the
reservoir to the conduit. For example, with regard to the trough
embodiment, the additional force exerted when the apparatus 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 trough retaining wall. Slowing rotation causes the sample to fall into
the trough(s) in which the end portions of FCFD cells extend. A gentle
reversing action at this stage will ensure that the sample is evenly
distributed to all the cells substantially simultaneously. The pore(s)
is/are positioned in a gap between the FCFD cells so as to allow
uninhibited passage of the sample from the pore(s) to the retaining wall.
In an alternative preferred embodiment comprising a step and spin
collection chamber as aforesaid, sample is firstly forced onto the step
upon rotation of the device. Sample then passes through the pore and is
forced against an outer wall of the spin collection chamber. An inwardly
facing lower lip preferably extends from this wall to prevent sample
reaching the FCFD devices or the like until the device has stopped
rotating. High speed rotation of the device causes sample to be evenly
distributed around the outer wall of the chamber. When the speed of
rotation of the device is decreased, sample tends to settle and is
partitioned by the vanes or baffles. Stopping the device suddenly causes
the sample to drop towards the FCFDs.
In order to improve the flow of sample in this embodiment, the riser of the
step and lower portions of the wall of the spin collection chamber may
slope up and away from the axis of rotation. Such an arrangement of the
wall of the spin collection chamber leads to a more even distribution of
liquid around the circumference of the chamber at a given speed of
rotation and the wider upper portions of the chamber mean that the liquid
can be more easily accommodated. Additionally, smaller volumes of sample
are required.
A wall may be provided in the reservoir in order to funnel sample towards
the pore. The funnelling of sample towards the pore leads to a more
efficient transfer of liquid through the pore during rotational
acceleration of the vehicle.
Advantageously, some form of air vent to the reservoir is provided so that
a partial vacuum is not formed in the reservoir; a potential vacuum would
inhibit outflow of sample. Preferably the air vent communicates with the
conduit and thereby provides a pressure balancing port.
Instead of providing a small pore or pores it would be possible to provide
suitable valve means opened by rotation of the device or opened
mechanically, for example. Both of these arrangements though are more
complicated than providing the simple, narrow bore pore or pores.
The test vehicle preferably comprises a plurality of parts made by
injection moulding. For example, a two part embodiment may have an inner
or base part which comprises the reservoir and part of the retaining wall
while an outer or upper part may comprise (in embodiments having a
cylindrical configuration) an FCFD cell support structure having windows
for illumination and detection optics, a filling aperture and an upper
part of the retaining wall. It will be clear to a skilled person that the
more complex the construction of the vehicle the larger the number of
subparts. For example, the embodiment comprising the step and spin
collection chamber comprises three injection moulded parts. Once tests
cells have been inserted into subassemblies, parts may be joined by, for
example, ultrasonic welding.
Ribs may be provided adjacent to the windows to discourage finger contact
with the optical surfaces and surfaces may be provided for the attachment
of labels and bar codes.
Preferably surface irregularities at the optical edge of each FCFD i.e. the
end of the waveguide from which emerging light is detected, are avoided
since they will give rise to some degree of light scattering or dispersion
and consequent mixing of the narrow angle light emission (attributable
only to surface-bound fluorescent material) and the broader angle
emissions. Such mixing inevitably degrades the signal quality and overall
performance of optical assay techniques using FCFD's. Advantageously each
optical edge is maintained in intimate contact with an index matching
substance which itself also forms or intimately contacts a further optical
component, such as a optical flat or lens.
Suitable liquid index matching substances, for example those having a
refractive index in the range 1.35-1.65, include microscopy immersion
fluids such as cedar oil and Canada balsam, and other liquids such as
silicones, ethyl alcohol, amyl alcohol, aniline, benzene, glycerol,
paraffin oil and turpentine. Appropriate gels include, for example,
silicone gels. Suitable precursors for solids include adhesives such as
epoxy and acrylate systems, and optical cements as well as plastics
materials (including thermoplastics) with appropriate refractive index,
for example silane elastomers. Alternatively, readily meltable solids e.g.
naphthalene, may be applied in molten form and then allowed to cool and
solidify.
The sub-parts are designed so that simple two part tooling may be used in
their construction, thus lowering the tooling cost and improving quality.
A preferred method of producing the pore includes the provision of a pin
on a mould tool which results in the pore being formed during moulding.
Alternatively, the pore or pores may be formed by a small core. Such a
core may be removed before assembling the vehicle or it can be an inert
plug which will dissolve when the liquid sample makes contact therewith.
Another option is to provide the pore or pores after moulding e.g. by
drilling or using a 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. There are a variety of ways of controlling the amount of
liquid which can leave the reservoir. Firstly, one can control the amount
of liquid 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-moulded bellows
pipette which can only be inserted into the reservoir to a predetermined
depth. Squeezing and releasing the bulb in this position causes all of the
contents of the pipette to be ejected into the device, but any excess will
be drawn back into the pipette.
Another way of controlling the amount of liquid which will pass from the
reservoir involves locating a disc with a central hole in the reservoir
such that the volume below or above the disc, as appropriate,
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; one portion will flow out of the
reservoir via the pore while the other portion remains separated from the
pore by the disc.
In view of the fact that most samples will be biological and, in some
instances may contain pathogens, it is desirable that excess sample is
absorbed. To this end, an absorbent, such as a sponge may be provided.
The preferred method of communicating a sample with one or more test
station(s) as discussed above combines structural simplicity with ease of
operation, and may have applications where only a single FCFD cell is used
or indeed in other assay types whether involving capillary fill cells or
not.
Accordingly, viewed from a second aspect the invention provides a method of
communicating a fluid sample with one or more sample test stations,
comprising introducing the sample into a reservoir having at least one
passageway in a wall or base thereof, the passageway being adapted such
that release of sample from the reservoir is prevented in a stationary
condition, and then rotating the reservoir and sample in such a way and at
such speed whereby sample flows to the test station(s).
It is preferred that each passageway is a pore of such a size that surface
tension of the sample is effective to prevent release of sample from the
reservoir in a stationary, non-pressurised condition.
Viewed from a third aspect the invention provides a multianalyte test
vehicle comprising a sample receiving reservoir, at least one test station
and means for providing fluid communication between the reservoir and the
test station(s), which means includes at least one pore in a wall of the
reservoir, the pore being of a size such that surface tension of a liquid
in the reservoir normally prevents egress of the liquid through the pore.
BRIEF DESCRIPTION OF THE DRAWINGS
Some embodiments of the invention will now be described, by way of example,
with reference to the accompanying drawings, in which:
FIG. 1 is an exploded perspective view of one embodiment of a multianalyte
test vehicle according to the invention;
FIG. 2 is a transverse section towards the base of the embodiment shown in
FIG. 1;
FIGS. 3(a) to 3(c) are schematic sectional elevations of the embodiment of
FIG. 1 in use;
FIGS. 4(a) and 4(b) are top plan and side elevational views of a second
embodiment of the invention;
FIG. 5 is an exploded sectional view of a third embodiment of a test
vehicle according to the invention;
FIG. 6 is a stylised sectional view of the vehicle shown in FIG. 5 taken
through two planes;
FIG. 7 is a schematic plan showing the arrangement of parts of the
embodiment of a test vehicle shown in FIGS. 5 and 6;
FIGS. 8A to 8C are a plan and sectional views of portions of a further
embodiment according to the invention; and
FIGS. 9 and 10 are respectively a plan and a sectional view of further
embodiments of reservoirs for a test vehicle according to the invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Similar reference numerals are used throughout for like parts of the
different embodiments.
The embodiment of the vehicle according to the invention shown in FIG. 1
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 7 are
equi-angularly spaced around the top 6. A hole 8 is provided in the top 6
to allow insertion of a liquid sample. 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.
An optional filter 2 may be 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 (a spin
collection chamber) 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 4.
A cylindrical wall 20, formed by the outer surface of the upstanding lip
16 provides an area upon which labels, such as a bar code 21, may be
applied.
A pore 22 is provided in the wall of the reservoir 15. As can be seen in
FIG. 2, the pore 22 is positioned in a gap 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 optional filter 2 is also
located in the upper part 1. The upper and lower parts 1 and 4 are then
brought into engagement; the lips 11 and 16 abutting each other and
defining the retaining wall 12. The parts 1 and 4 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 has been added to the vehicle via the hole 8, the vehicle is
then located on a rotatable head of a multianalyte test instrument (not
shown) by means of the lugs 18 and guides 19 on the lower part 14. The
head of the instrument is rotatable at about 300 to 500 rpm and can also
be rotated in a stepping mode at low speed to bring each FCFD cell into
alignment with the light source and with the fluorescence detector which
aligns with the respective optical edge window 7 on the top of the vehicle
Turning to FIG. 3, where some parts of the vehicle are not shown for the
sake of clarity, it can be seen in FIG. 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 FIG. 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.
Slowing the rotation of the vehicle allows the sample 23 to sink into the
trough, formed by the web 17, and then be drawn up the FCFD cells 3 by
capillary action in the direction indicated by the arrows in FIG. 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
source and fluorescence detector
FIGS. 4(a) and 4(b) show, schematically, a second embodiment of the test
vehicle. This again includes a central sample receiving reservoir 15
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
of the cells communicate with the trough via slit like apertures in the
retaining wall such that sample is 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.
The embodiment depicted in FIGS. 5 to 7 comprises upper and lower casings
1' to 4' between which FCFDs are radially disposed in a vane-like manner
(i.e. perpendicular to the axis of rotation), as shown schematically in
FIG. 7. The upper casing 1' has a central filling hole 8, defined by a
depending wall 24, and a pair of walls 25, 26 which co-operate with a
moulding 27. The moulding 27 provides the sample reservoir 15' and a spin
collection chamber 28. The reservoir includes an eccentric step 29 which
has the pore 22 passing therethrough. The spin collection chamber 28 is,
in part, defined by an outer retaining wall 12' connected to the reservoir
15' by four vanes 30. An inwardly facing lower lip 31 extends from the
bottom of the retaining wall 12'. A sponge 32 is located below the
moulding 27 in a shallow sump 37. The sponge 32 is formed with a central
hole 33, in which a boss 34 of the lower casing 4' locates, and an
indented periphery. Each FCFD 3 has a portion of sponge 32 in close
proximity thereto.
It can be seen in FIGS. 5 and 6 that the upper casing 1' is provided with
vents 35 to allow air to escape from the sample chamber during filling
while the lower casing 4' has splines 36 inside the boss 34. The splines
co-operate with a spindle of a multianalyte test instrument (not shown).
To fill the test vehicle with sample, a filling device (not shown) may be
used which, for example, may cooperate with the depending wall 24 to
provide a partial seal and avoid the possibility of spillage. As mentioned
earlier, vents 35 are provided to allow for the escape of air as sample is
introduced into the reservoir 15'.
The multianalyte test vehicle is mounted on the spindle of a multianalyte
test instrument and rotated. Upon rotation of the device, sample is forced
outwardly and upwardly. Due to the eccentric placement of the step 29, the
sample gathers on the step 29 and is forced through the pore 22. Sample
which has passed through the pore 22 impacts on the retaining wall 12' of
the spin collection chamber 28. The inwardly facing lip 31 prevents sample
descending into the shallow sump 37. As more sample leaves the reservoir
15' and impacts on the retaining wall 12' it spreads out, passing over the
vanes 30 and becomes evenly distributed on the retaining wall 12'.
Decreasing the speed of rotation of the device causes the sample on the
retaining wall 12' to sag; the vanes 30 helping to partition it into equal
aliquots. The device is then stopped suddenly. The inertia of the sample
causes it to impact on the vanes 30, which are now stationary, and then
descend. The sample flows over the inwardly facing lip 31 and passes over
the inner ends of the FCFDs. Some of the sample is drawn into the FCFDs by
capillary action. Excess sample descends into the shallow sump 37 and is
absorbed by the sponge 32. The FCFDs can then be indexed to a test station
of the instrument.
A multianalyte test vehicle according to the invention may be modified so
as to improve the flow of liquid therein. For example the second
embodiment described above may have certain components replaced by those
shown in FIGS. 8 to 10.
FIGS. 8A to 8C illustrate an arrangement of reservoir 15' and spin
collection chamber 28 in which the walls taper towards the axis of
rotation. The tapering improves the flow of sample onto the step 29 and,
once through the pore 22, the distribution of sample in the spin
collection chamber 28. The sample tracks upwardly and outwardly against
the wall of the chamber 28 and becomes evenly distributed. Better
distribution of sample in the chamber may lead to less sample being
required.
An internal wall 38 may be provided in the reservoir 15', as shown in FIG.
9, in order to assist in the movement of sample onto the step 29 and
through the pore 22. When the reservoir is rotated in a clockwise
direction sample is funnelled by the wall 38 and the outer wall of the
reservoir towards the step 29. This funnelling of sample increase initial
flow through the pore 22 during acceleration of the vehicle. This
embodiment also includes a sloping riser for the step 29.
FIG. 10 shows a further embodiment of the reservoir 15' which includes a
sloping step 29 having a pore 22 therein and an air vent 39. The vent 39
includes a pore 40 which is too small to allow liquid to escape but will
allow air into the reservoir to, for example, equilibrate the pressures in
the reservoir and the spin collection chamber (not shown) on transfer of
sample to the latter.
Vehicles according to the embodiments described above thus provide a simple
and inexpensive arrangement for supplying sample to FCFD or other test
cells. Modifications which fall within the scope of the present invention
will be apparent to the skilled person.
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