U.S. patent application number 11/324257 was filed with the patent office on 2006-05-25 for devices for motile sperm separation.
Invention is credited to David Brickwood.
Application Number | 20060110821 11/324257 |
Document ID | / |
Family ID | 9885741 |
Filed Date | 2006-05-25 |
United States Patent
Application |
20060110821 |
Kind Code |
A1 |
Brickwood; David |
May 25, 2006 |
Devices for motile sperm separation
Abstract
Device (10, 110) for separating motile sperm from a sample (100,
200), comprises a vessel (15, 115) having an inlet port (20, 120),
an outlet port (35, 135) which is initially closed, a medium (40,
140) into which motile sperm in the sample (100, 200) can migrate
into the vessel (15, 115) via the inlet (20, 120), and an actuator
(50, 150), the operation of which opens the outlet (35, 135),
thereby allowing the medium (40, 140) to flow out of the vessel
(15, 115) through the outlet (35, 135). The medium (40, 140) in the
vessel (15, 115) is initially prevented from flowing through the
outlet (35, 135), thus allowing an incubation period which allows
motile sperm sufficient time to migrate from the sample (100, 200)
into the medium (40, 140) before the medium (40, 140) leaves the
vessel (35, 135). The device (10, 110) preferably comprises a
spermatozoa detector in communication with the outlet. In devices
for separating sperm, capillary flow takes place through
non-fibrous material, such as the space between sheets (81a and
81b; 181a and 181b) of closely-juxtaposed material. A device for
separating motile and non-motile sperm includes a temperature
sensor, a heat source (70, 170) and, optionally, a temperature
regulator.
Inventors: |
Brickwood; David;
(Kingston-Upon-Thames, GB) |
Correspondence
Address: |
WENDEROTH, LIND & PONACK, L.L.P.
2033 K STREET N. W.
SUITE 800
WASHINGTON
DC
20006-1021
US
|
Family ID: |
9885741 |
Appl. No.: |
11/324257 |
Filed: |
January 4, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11080928 |
Mar 16, 2005 |
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11324257 |
Jan 4, 2006 |
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10203318 |
Oct 16, 2002 |
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PCT/GB01/00654 |
Sep 16, 2001 |
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11080928 |
Mar 16, 2005 |
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Current U.S.
Class: |
435/287.2 ;
435/287.7; 435/288.2 |
Current CPC
Class: |
C12N 5/0612 20130101;
G01N 2333/70596 20130101; G01N 2800/367 20130101; G01N 33/689
20130101 |
Class at
Publication: |
435/287.2 ;
435/287.7; 435/288.2 |
International
Class: |
C12M 1/34 20060101
C12M001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 16, 2000 |
GB |
0003596.4 |
Claims
1. A device for separating and detecting motile spermatozoa in a
liquid sample, the device having: a sample separation vessel
comprising:(a) a sample inlet port, which is initially closed or
sealed; (b) a separation medium into which motile spermatozoa in
the sample can flow via the sample inlet port (c) an actuator
operable to open the sample inlet port a spermatozoa detection
zone, integral with the device, in which the presence of
spermatozoa can be detected.
2. The device of claim 1, wherein the inlet port is the open end of
a cylinder.
3. The device of claim 1, wherein the spermatozoa flow by capillary
action through a non-fibrous material.
4. The device of claim 1, wherein the spermatozoa are labelled with
a fluorescent tag.
5. The device of claim 1, wherein the device provides a visible
signal to indicate the presence of motile spermatozoa in the
sample.
6. The device of claim 1, wherein the visible signal is visible
from the exterior of the device.
7. The device of claim 1, wherein the device includes a receptacle
for the sample.
8. The device of claim 1, wherein the device includes a foil
cutter.
9. The device of claim 1, wherein the device includes a
battery.
10. The device of claim 1, wherein the device includes a
temperature sensor.
11. The device of claim 1, wherein the device includes a heat
source.
12. The device of claim 1, wherein the device includes a
temperature sensor and a heat source.
13. The device of claim 12, wherein the device also includes
temperature regulation means.
14. A device for separating and detecting motile spermatozoa in a
liquid sample, wherein the device includes a temperature sensor and
a heat source.
15. The device of claim 14, further comprising temperature
regulation means.
16. The device of claim 14, wherein the device has a sample
separation vessel and a spermatozoa detection zone.
17. The device of claim 16, wherein the sample separation vessel
comprises: (a) a sample inlet port, which is initially closed or
sealed; (b) a separation medium into which motile spermatozoa in
the sample can flow via the sample inlet port; and (c) an actuator
operable to open the sample inlet port.
18. The device of claim 17, wherein the inlet port is the open end
of a cylinder.
19. The device of claim 14, wherein the spermatozoa flow by
capillary action through a non-fibrous material.
20. The device of claim 14, wherein the spermatozoa are labelled
with a fluorescent tag.
21. The device of claim 14, wherein the device provides a visible
signal to indicate the presence of motile spermatozoa in the
sample.
22. The device of claim 21, wherein the visible signal is visible
from the exterior of the device.
23. The device of claim 14, wherein the device includes a foil
cutter.
24. The device of claim 14, wherein the device includes a
battery.
25. The device of claim 14, wherein the device includes a
receptacle for the sample.
26. In a device for separating and detecting motile spermatozoa in
a liquid sample, the improvement consisting of including a
temperature sensor and a heat source in the device.
27. A device for separating and detecting motile spermatozoa in a
liquid sample, wherein sperm flow by capillary action takes place
through non-fibrous material.
28. The device of claim 27, wherein the device has a sample
separation vessel and a spermatozoa detection zone.
29. The device of claim 28, wherein the sample separation vessel
comprises: (a) a sample inlet port, which is initially closed or
sealed; (b) a separation medium into which motile spermatozoa in
the sample can flow via the sample inlet port; and (c) an actuator
operable to open the sample inlet port.
30. The device of claim 29, wherein the inlet port is the open end
of a cylinder.
31. The device of claim 27, wherein the spermatozoa are labelled
with a fluorescent tag.
32. The device of claim 27, wherein the device provides a visible
signal to indicate the presence of motile spermatozoa in the
sample.
33. The device of claim 32, wherein the visible signal is visible
from the exterior of the device.
34. The device of claim 27, wherein the device further comprises a
receptacle for the sample.
35. The device of claim 27, wherein the device includes a foil
cutter.
36. The device of claim 27, wherein the device includes a
battery.
37. The device of claim 27, wherein the device includes a
temperature sensor and a heat source.
38. The device of claim 37, wherein the device also includes
temperature regulation means.
39. In a device for separating and detecting motile spermatozoa in
a liquid sample, wherein spermatozoa flow by capillary action, the
improvement consisting of having the spermatozoa flow through
non-fibrous material.
Description
[0001] All documents cited herein are incorporated by reference in
their entirety.
TECHNICAL FIELD
[0002] This invention is in the field of male fertility tests, more
specifically tests for separating and detecting motile spermatozoa
in a semen sample.
BACKGROUND ART
[0003] Approximately 15% of couples attempting to conceive fail to
do so within one year of unprotected intercourse. Fertility
specialists define these couples is being infertile. 40% of these
cases result from male factors. In a substantial proportion of
these treatment, is available to ameliorate or relieve the
condition which leads to infertility.
[0004] Other conditions also exist in which it is desirable to test
for the presence or otherwise of viable spermatozoa in a sample.
For example, vasectomies are now frequently carried out as a method
of contraception, but it is necessary to confirm that ejaculate is
free of viable spermatozoa after the operation.
[0005] A number of methods exist for assessing the motility and
number of spermatozoa in a sample. One such method is microscopic
analysis, which is typically carried out in a hospital or
commercial laboratory. More recently, however, a number of
proposals have been made for test kits which are intended to
simplify the detection of spermatozoa.
[0006] WO97/40386 discloses a kit which is based on immunodetection
of the 35 kDa human epididymal spermatozoa protein. Spermatozoa in
a sample are washed three times by centrifugation in
Dulbecco-phosphate buffered saline. The samples are then heat
denatured at 95.degree. C., centrifuged at 14000 g. and the
supernatants are then used for analysis.
[0007] WO95/29188 describes a test based on antibodies to the SP-10
antigen of human spermatozoa.
[0008] EP-A-0387873 discloses a kit which uses solid beads to which
are bound antibody specific to the human spermatozoon acrosome. The
beads are mixed with a sample, incubated, separated, washed, and
the number of spermatozoa bound to the beads is measured,
preferably by examination with the aid of a microscope.
[0009] A disadvantage of these test kits is that they do not
distinguish between motile and non-motile spermatozoa. This
distinction is the most predictive indicator of male infertility.
Moreover, they involve procedures which do not lend themselves to
home use (e.g. centrifugation, microscopy), thus requiring
implementation by a skilled practitioner.
[0010] These disadvantages are addressed by the device of
WO00/09648 (Genosis Limited), which discloses an apparatus for
separating motile spermatozoa from non-motile spermatozoa in a
liquid sample, the apparatus comprising (i) a vessel having a
sample receiving inlet, a filtered sample outlet and a sample
separation filter mounted therebetween, the sample separation
filler having a sample-receiving surface and an opposed surface,
and the sample separation filler being effective substantially to
prevent flow of the sample therethrough, but permitting passage of
motile spermatozoa therethrough when said opposed surface of said
sample separation filter is placed in contact with a liquid medium
and (ii) means for supplying a liquid to said opposed surface of
said filter.
[0011] Further improvements in sperm separation detection and
analysis are disclosed herein.
DISCLOSURE OF THE INVENTION
[0012] The present invention provides a device for separating
motile spermatozoa from a liquid sample, the device having a sample
separation vessel comprising: [0013] (a) a sample inlet port;
[0014] (b) a sample outlet port, which is initially closed; [0015]
(c) a sample separation medium into which motile spermatozoa in the
sample can migrate via the sample inlet port; and [0016] (d) an
actuator operable to open the sample outlet port, thereby allowing
the sample separation medium to flow out of the vessel through the
sample outlet port.
[0017] In this device, the separation medium in the vessel is
initially prevented from flowing through the outlet port. This
allows an incubation period which allows motile sperm sufficient
time to migrate from the sample into the sample separation medium
before it exits the vessel. On operation of the actuator, the
outlet port is opened and the separation medium (including motile
spermatozoa) can migrate through the outlet port and leave the
vessel e.g. For analysis.
[0018] In order for motile spermatozoa in the sample to enter the
separation medium, the inlet port and the separation medium must be
in liquid communication. Preferably, however, such communication is
initially prevented, thus preventing contamination of the medium
via the inlet port before the device is used. This may also prevent
the medium from escaping from the vessel via the inlet port,
although this may be achieved anyway due to, for instance,
viscosity or surface tension of the medium.
[0019] In this embodiment, therefore, the vessel comprises (e) a
second actuator, operable to bring the separation medium into
communication with a sample via the inlet port. This might be
achieved by storing the medium remotely from the inlet port until
the second actuator is operated or, alternatively, by having an
inlet port which is initially closed or sealed, which is opened
using the actuator.
[0020] During use of this device, therefore, the second actuator is
operated, thereby bringing the separation medium and the inlet port
into communication. This allows motile spermatozoa in the sample to
migrate from the sample into the separation medium. After a period
of incubation, the (first) actuator is operated, thereby allowing
the separation medium (now containing motile spermatozoa) to leave
the vessel. It is preferred that the first actuator cannot be
operated until the second actuator has been operated.
[0021] It will be appreciated that feature (e) can operate
independently from feature (d).
[0022] The vessel may be of circular cross-section e.g. conical or
cylindrical.
[0023] The inlet and outlet ports can take a variety of forms. For
instance, the inlet port might be an open end of a cylinder or
cone. This open end can be placed in a sample and, when the
separation medium and the sample are in communication, spermatozoa
can enter the vessel via the open end. It may he desirable to cover
the open end by a mesh or grid, to assist in retaining sample
separation medium within the vessel (via surface tension) without
preventing the entry of spermatozoa from the sample.
[0024] The outlet port is preferably an opening, hole or aperture
in the vessel's wall, which may continue into a pipe, tube or
conduit leading away from the vessel.
[0025] The inlet and outlet ports are preferably arranged such
that, during use, the outlet port is above the inlet port.
Spermatozoa thus have to swim against gravity in order to exit the
vessel, enhancing the preferential migration of motile vs.
non-motile sperm.
[0026] Any suitable actuators can be used in the device e.g.
buttons, switches etc. Where the vessel is of circular
cross-section, the (first) actuator is preferably a rotatable
collar containing an opening. Rotation of the collar from a closed
position aligns openings in the collar and vessel wall, thereby
allowing sample separation medium to leave the vessel via the
outlet port.
[0027] Another preferred form of (first) actuator acts to withdraw
fluid into a barrel or tube e.g. it withdraws the plunger of a
syringe. The plunger will initially block the outlet port but, on
withdrawal of the plunger, sample separation medium can flow
through the outlet port and into the internal space of the syringe.
A suitable actuator for effecting withdrawal of the syringe plunger
is a rotatable knob which is attached to the plunger e.g. by a rack
and pinion mechanism.
[0028] Where the device includes a second actuator, this is
preferably a button. When pressed, sample separation medium stored
within the device is released so that it reaches (e.g. by gravity)
the inlet port. This can be achieved by, for instance, including a
reservoir of medium within the vessel, sealed by a foil wall,
together with a foil cutter. Operation of the second actuator
causes the cutter to pierce the foil, releasing the medium.
[0029] A preferred arrangement of reservoir is that disclosed in
United Kingdom patent application 0021665.5, in which the
separation medium is held within a hermetically sealed reservoir.
The device will include a venting needle for venting the medium
from the reservoir, the venting needle comprising a fluid delivery
portion and a reservoir-venting portion, the reservoir-venting
portion being distal along the needle of the delivery portion, the
delivery portion and the venting portion each defining a channel
extending from the respective portion through the sidewall of the
needle. The channels allow the delivery portion, in use, to deliver
fluid from the reservoir, for example by venting air at one end and
dispensing fluid at the other. In this arrangement, the venting
needle will preferably be at least partially a cannula having a
cannulated point at the delivery portion for controlled,
pipette-like delivery. In a needle with a proximal portion and a
distal portion, the distal portion may comprise both the venting
portion and the delivery portion. The needle preferably has a
C-shaped section at the venting portion and the delivery portion.
The two channels are preferably unitary the channel extending from
the venting portion to the delivery portion.
[0030] In a venting position, the venting needle preferably extends
through two wall portions of the reservoir such that the venting
portion forms a passage through the first wall portion to allow the
container to be vented with air and the delivery portion forms a
passage through the second wall portion to allow fluid to be
dispensed from the reservoir.
[0031] Using this sort of arrangement, the needle is operated by
the second actuator (e.g. it is attached to a button or a
screw-advance mechanism).
[0032] The separation medium allows motile spermatozoa, in
preference to non-motile sperm, to migrate therethrough. This can
be achieved using any suitable buffer (e.g. HEPES, EBSS etc.), as
motile spermatozoa will be able to migrate through the medium
actively whereas, over the relevant time-scale, non-motile
spermatozoa will at best enter passively by diffusion. It is
preferred, however, to use a medium that enhances the migration of
motile spermatozoa from the sample. Suitable media include cervical
mucus [e.g. Keel & Webster (1988) Fertil. Steril. 49:138-143],
polyacrylamide gel [e.g. Lorton et al. (1981) Fertil. Steril.
35:222-225], hyaluronic acid [e.g. Aitken et al. (1992) J. Androl.
13:44-54], or a cellulose derivative [e.g. international patent
application PCT/GB00/03130 (Genosis Limited)] such as
methylcellulose.
[0033] The medium may be in the form of a solution or a gel.
[0034] Advantageously, the separation medium also serves to wash
the sample, in order to remove components such as seminal
plasma.
[0035] In addition to separating motile and non-motile spermatozoa,
it is preferred that the device can detect motile spermatozoa after
separation. Accordingly, the device of the invention preferably
comprises spermatozoa detection means in communication with the
vessel outlet port. The detection means may be integral with the
device, or may be provided as a separate component for inserting
into the device before, during or after sample separation.
[0036] Accordingly, the invention provides a device for separating
and detecting motile spermatozoa in a liquid sample, the device
having: [0037] a sample separation vessel comprising: [0038] (a) a
sample inlet port; [0039] (b) a sample outlet port, which is
initially closed; [0040] (c) a sample separation medium into which
motile spermatozoa in the sample can migrate via the sample inlet
port; and [0041] (d) an actuator operable to open the sample outlet
port, thereby allowing the sample separation medium to flow out of
the vessel through the sample outlet port, [0042] spermatozoa
detection means comprising: [0043] (i) an application zone in
communication with the outlet port; [0044] (ii) a detection zone,
in which the presence of spermatozoa can be detected; and [0045]
(iii) a reagent zone containing a reagent which is capable of
reacting with spermatozoa to facilitate their detection in the
detection zone, [0046] these zones being arranged to permit
capillary flow of spermatozoa from the application zone to the
detection zone.
[0047] Preferably, the detection zone is a zone past which
spermatozoa cannot flow (a `trapping zone`). During operation, flow
of spermatozoa is thus prevented beyond the trapping zone, and
sperm are immobilised for detection. Preferably, the trapping zone
utilises the principles disclosed in WO00/20866--the zone is porous
with a pore size such that spermatozoa cannot enter. During flow
into the trapping zone, therefore, sperm are captured a( the
entrance, as sperm concentration increases, the amount retained
there also increases.
[0048] The trapping zone can be made from any suitable porous
material (e.g. HDPR, nitrocellulose) through which spermatozoa
cannot migrate. This requirement is reflected in the pore size of
the trapping zone. The head of a human spermatozoon is typically
3-5 .mu.m in diameter, and tail length is approximately 50-60
.mu.m. The pore size should be selected accordingly (e.g.
nitrocellulose, with a nominal pore size between 5-8 .mu.m). and an
appropriate pore size may be determined empirically by simple
experimentation.
[0049] It will be apparent that any porous material encountered
before the trapping zone must, in contrast to the trapping zone,
have a pore size large enough to allow spermatozoa to move
relatively freely.
[0050] As is well known to those in the art, the nominal pore size
of a porous material can be determined by hard particle challenge
testing i.e. by determining the maximum diameter of spherical
particles which can pass through the material. Alternatively, the
pore size of a material may be determined by measuring its `bubble
point`. The bubble point is the pressure required to force air
through a (water) wet membrane, and correlates with the pore size
as measured by particle retention (although at extremes of pressure
and pore size, the correlation may be weaker). The bubble point is
generally easier to measure than particle retention and is thus the
preferred test when assessing pore size.
[0051] The entrance to the trapping zone is preferably narrow, such
that sperm are focused to give a sharper signal.
[0052] As a less-preferred detection zone, immobilised anti-sperm
antibody may be utilised.
[0053] In one embodiment of the detection zone, detection is based
on acrosin. The detection zone will contain a reagent for detecting
acrosin (e.g. see Mortimer, Practical Laboratory Andrology (1994),
page 90) and the reagent zone will include a reagent such as
proteinase K or the calcium ionophore A23187 [Perry et al. (1997)
J. Exp. Zool. 279:284-290; Perry et al. (1997) J. Exp. Zool.
279:291-300 (1997); Perry et al. (1996) Human Reprod. 11:1055-1062;
Perry et al. (1995) Fertil. Steril. 64:150-159] which causes the
acrosomes to open. A preferred reagent for lysing acrosomes is a
lysis buffer comprising 2% SDS, 100 .mu.g/ml proteinase K in 10 mM
Tris-HCl and 0.1 M EDTA. During migration, therefore, acrosin is
released from the sperm and is detected downstream at the detection
zone.
[0054] More usually, the reagent zone will contain a reagent which
binds to intact spermatozoa or to one or more of the components
thereof, and this binding reaction is used to generate a visual
signal. Preferably, therefore, the reagent zone is a `labelling
zone`, containing a label capable of binding to spermatozoa. This
is preferably used in conjunction with a trapping zone.
[0055] The label within the labelling zone can be any suitable
reagent that can bind to spermatozoa, preferably giving a visible
signal. Because only motile spermatozoa will reach the outlet port,
the label need not be specific for sperm.
[0056] The label is typically an antibody which can bind to
spermatozoa and which has been suitably tagged. It is preferred to
use a visible tag, such as colloidal gold (which is visible as a
pink colour), although fluorescent, luminescent or radioactive tags
can also be used. It will be appreciated that the tern `antibody`
may include polyclonal and monoclonal antibodies, as well as
antibody fragments (e.g. F(ab).sub.2, Fc etc.), provided that
anti-sperm reactivity is retained.
[0057] Preferred labels recognise a surface antigen which is
present on the majority of a population of spermatozoa, rather than
a subset. Whilst any sperm antigen may be used (e.g. P34H
(WO97/40836), SP-10 (WO95/29188), see also EP-A-0387873),
`universal` antigens such as CD59 may also used. As an alternative,
a stain, such as eosin, may be used.
[0058] It is preferred that the label is not activated (e.g.
re-hydration of dehydrated label) until sample separation medium
leaves the vessel. This can be achieved by arranging the labelling
zone such that label therein is not activated until the (first)
actuator has been operated. Label therefore remains static until
motile spermatozoa have left the sample and entered the sample
separation medium.
[0059] The labelling zone may be arranged in any suitable position
such that its label can contact spermatozoa in the detection zone
(e.g. sperm retained at the trapping zone). It may be upstream or
downstream of the application zone, or may be integral therewith.
If it is upstream, sample separation medium leaving the vessel
should be able to contact the labelling zone separately from its
contact with the application zone, in order to activate the label;
if it is downstream or integral, flow will automatically activate
the label.
[0060] Where the detection means includes both a `trapping zone`
and a `labelling zone`, the pore size of the trapping zone will be
such that free label (not bound to spermatozoa) can flow
therethrough, whereas label which is bound to spermatozoa cannot.
During flow into the trapping zone, therefore, bound label is
captured at the entrance. As sperm concentration increases, the
amount of capture label also increases. It will be apparent that
the label must be smaller than spermatozoa, such that free label is
not retarded by the trapping zone.
[0061] Where the detection means utilises a label, it preferably
includes a zone downstream of the detection zone which retains
unbound label (the `label control` zone). This will typically
comprise immobilised antibody which can bind to unbound label (e.g.
if the label is a murine monoclonal antibody, the label control
zone may utilise anti-mouse antibody). Label which passes through
the detection zone (e.g. which is not captured on entry to the
trapping zone) is thus retained within the label control zone,
where it can be measured. A comparison of the amount of label in
the detection zone and the label control zone allows
semi-quantitative measurement of the amount of spermatozoa in the
sample.
[0062] The application zone is where the sample separation medium
leaving the vessel comes into contact with the spermatozoa
detection means. It can be formed from any material suitable for
allowing capillary flow of spermatozoa therethrough e.g. fibrous
material, such as a pad of HDPE material, bonded polyester fibre,
glass fibre, or the like.
[0063] It has been found, however, that spermatozoa tend to be
retained within fibrous materials, thus reducing sensitivity. It is
therefore preferred to use a non-fibrous application zone. Suitable
examples include capillary tubes, the space between two or more
sheets of material juxtaposed in close proximity such that
capillary flow can occur therebetween, or a series of parallel
capillary channels or grooves. In such arrangements, if the
labelling zone and the application zone are integral, this is
preferably formed from more than one piece of material such that
label can be applied during assembly. Where juxtaposed sheets are
used, for instance, label can be applied to one or more sheets, and
these can then be brought into proximity to permit capillary flow.
Similarly, where parallel channels or grooves are used the label
can be applied to the channelled material and, optionally, covered
by a further sheet.
[0064] In a similar arrangement, capillary flow takes place past a
fibrous material rather than through it. Whilst flowing past,
liquid can enter the fibrous material (e.g. to re-hydrate a label
impregnated therein), but the main capillary flow through the
application zone of sperm is not within the fibrous material.
[0065] The application zone and the sample outlet port may be
essentially unitary.
[0066] In preferred embodiments, flow of a sample to the detection
zone is assisted by a downstream wick, to aid capillary
movement.
[0067] During use of preferred devices, therefore, the migration
path of motile sperm in the sample is: entry to the separation
medium via the vessel's inlet port, exit from the separation medium
via the vessel's outlet port (upon operation of the actuator after
an incubation period), entry to the application zone, and capillary
flow to the trapping zone, in which further migration is prevented.
A visible signal at the trapping zone indicates the presence of
motile spermatozoa in the sample
[0068] The visible signal provided by the detection means is
preferably external to the vessel, and more preferably one which is
also visible from the exterior of the device.
[0069] The detection means is preferably in the form of a lateral
flow test strip mounted on the outside of the vessel.
[0070] It will be appreciated that the vessel may include more than
one sample outlet port, each of which may lead to separate
detection means. This allows independent tests on the same sample
e.g. an assay for motile sperm on one strip and an assay for
acrosome reaction on another strip. This can also be achieved by
using a single outlet port that leads downstream to several
separate detection means.
[0071] To enhance the separation of motile and non-motile
spermatozoa (e.g. EP-A-0437508), it is preferred that the device
should be operated at an essentially fixed temperature. This will
typically be between 30.degree. C. and 44.degree. C., preferably
between 35.degree. C. and 39.degree. C., and more preferably fixed
at or around 37.degree. C. Preferably, therefore, the device
includes a temperature sensor.
[0072] The temperature sensor may give a simple signal to indicate
when operating temperature has been reached. The device could be
held in a user's hand, or inserted in a heated water bath, until
the signal is given.
[0073] More preferably, however, the device also includes its own
heat source, which can preferably be regulated e.g. using
thermostatic control. The heat source should supply heat primarily
to the region of the vessel which contains the sample separation
medium. Power and control circuitry for the beat source may
conveniently be concealed within an actuator button, or within the
body of the device.
[0074] It will be appreciated that the inclusion of temperature
regulation in the device can operate independently from the
device's other features.
[0075] The device of the invention may be used with any suitable
liquid sample, which is preferably a semen sample. The device may
include a receptacle for the sample. Preferably, this is in the
form of a cup or sloping surface onto which a sample can be
deposited, after which it collects at the base (e.g. in a well).
The sample inlet can then be placed into contact with the collected
sample. The receptacle may also include an overflow near its base.
The receptacle may be integral with the device, or may be provided
as a separate component to which the device may be attached before,
during or after sample deposition and/or collection.
[0076] Advantageously, the interface between the receptacle and the
vessel's inlet port (e.g. where the vessel attaches to the well) is
of a defined area, thereby aiding assay reproducibility.
[0077] The device of the invention can be produced simply and
cheaply. Furthermore, it can be used very easily, for instance by
the home user. The invention thus provides an assay device which
can be used at home as a basic screen of, for instance, male
fertility.
[0078] The invention also provides corresponding processes. Thus
the invention provides a process for separating motile spermatozoa
from a liquid sample, the process comprising the steps of: [0079]
(a) providing a vessel having a sample inlet port, a closed sample
outlet port, and a sample separation medium; [0080] (b) allowing
spermatozoa in the sample to migrate into the sample separation
medium over a period of between 5 and 60 minutes (preferably around
30 minutes); and [0081] (c) allowing the sample separation medium
to leave the vessel by opening the sample outlet port.
[0082] The process may initially comprise the step of opening the
inlet port.
[0083] Preferably, the process also comprises steps for detecting
spermatozoa in the medium once it has exited the vessel. Thus the
invention provides a process for separating and detecting motile
spermatozoa in a liquid sample, comprising the further steps of:
[0084] (i) allowing sample separation medium that leaves the outlet
port to flow by capillary action into material capable of
immobilizing spermatozoa; [0085] (ii) contacting spermatozoa with a
label: and [0086] (iii) detecting label that is retained at the
entrance to said material.
[0087] The invention further provides a process for separating
and/or detecting spermatozoa, wherein sperm flow by capillary
action takes place through non-fibrous material.
[0088] The invention further provides a process for separating
motile and non-motile- spermatozoa, wherein said process takes
place at an essentially fixed temperature, preferably around
37.degree. C.
BRIEF DESCRIPTION OF DRAWINGS
[0089] FIG. 1 shows a device according to the invention.
[0090] FIG. 2 shows this device after insertion into a
receptacle.
[0091] FIGS. 3 to 6 show the operation of this device.
[0092] FIG. 7 shows an overview of using this device.
[0093] FIG. 8 shows a second device according to the invention,
prior to operation.
[0094] FIG. 9 shows the same device separated into its two
constituent pieces, with
[0095] FIG. 10 showing an exploded view of the top piece.
[0096] FIG. 11 shows the construction of the test strip assembly
used in the device.
[0097] FIGS. 12 to 16 show an overview of using this device.
MODES FOR CARRYING OUT THE INVENTION
[0098] The device (10) shown in FIG. 1 comprises: a cylindrical
plastic vessel (15); a sample inlet port (20), covered by a nylon
mesh (21) formed from 0.15 mm strands spaced by 0.25 mm: a hole
(30) in its side, shown in the open position: a solution (40) of
EBSS supplemented with 0.88 mg/ml hyaluronic acid and 0.45% BSA; a
plastic rotatable collar (50), including a hole (51) which is shown
aligned with hole (30) to form an outlet port (35); a button (60,
shown depressed), which houses a battery and circuitry for powering
a circumferential heat source (70) and attached to which is a
solution reservoir (65, shown empty), a foil seal (66, shown
broken), and a hollow foil cutter (68); and an externally mounted
test strip (80).
[0099] FIG. 2 shows the FIG. 1 device (10) attached to a semen
receptacle (90) with sloped walls. A semen sample (100) has
collected in the well (99) at the base of the receptacle (90), but
some of it has overflowed into the receptacle overflow (95).
[0100] FIG. 3 shows the device (10) immediately prior to use. A
semen sample (100) has collected in the well (99) and is in contact
with the inlet port (20). The outlet port (35) is closed because
the hole (51) in the collar (50) is not aligned with the hole (30)
in the vessel wall. Solution (40) is held in reservoir (65) by foil
seal (66), away from the inlet port (20). To initiate the test,
button (60) is depressed, as shown in FIG. 4A. Foil cutter (68)
pierces seal (66), releasing solution (40). Motile sperm in the
sample (100) are now in liquid communication with solution (40) and
are able to migrate into it through the inlet (20), as shown in
FIG. 4B, whereas non-motile sperm and seminal plasma remain in
sample (100). Button (60) also activates heat source (70), bringing
the temperature of solution (40) to 37.degree. C.
[0101] After a period of about 30 minutes, during which motile
sperm have migrated into the solution (40), collar (50) is rotated,
as shown in FIG. 5, so that its hole (51) aligns with hole (30),
thereby opening the outlet port (35). Solution (40), now containing
motile sperm, is free to leave the vessel and contact
externally-mounted test strip (80).
[0102] As shown in FIG. 6, the solution flows by capillary action
through closely-juxtaposed plastic strips (81a & b) at the base
of the test strip (80). Between the strips (81a & b) is an area
(82) of dehydrated gold-labelled murine anti-CD59. As the solution
passes area (82) between the strips (81a & b), the antibody is
re-hydrated and is able to bind to spermatozoa in the solution.
Further downstream, the solution reaches nitrocellulose strip (83).
The pore size of the strip (83) is too small to allow the
spermatozoa to enter, so they are captured at its entrance (84).
Free label continues to flow until it is captured downstream at a
line (85) of immobilised anti-mouse antibody.
[0103] As shown in FIG. 7, entrance (84) and line (85) are visible
through windows. In FIG. 7E, two lines are visible, indicating the
presence of motile spermatozoa in the original sample. In contrast,
only the control line (85) is visible in FIG. 7F, indicating an
absence of motile sperm in the original sample.
[0104] The device (110) shown in FIGS. 8 and 9 comprises an upper
piece (191) and a lower piece (190) which fit together. The base of
lower piece (190) contains a well (199) into which a semen sample
(200) is deposited. Upper piece (191) includes a recessed window
(113), a button (160) and a rotatable knob (150). Knob (15(0) and
button (160) are shaped such that knob (150) cannot be rotated
until button (160) has beer depressed.
[0105] In FIG. 10, the internal components of upper piece 191) are
shown in an exploded view. Upper piece (191) is formed from a top
piece (192) which engages a seating (193). On the bottom of button
(160) is a needle (168) and, when button (160) is operated, needle
(168) pierces reservoir (165) which, prior to operation, contains a
solution (140) of EBSS supplemented with 0.88 mg/ml hyaluronic acid
and 0.45% BSA. Reservoir (165) sits in plastic housing (166), which
has a neck portion (115) and a head portion (120). The side of neck
(115) contains a hole (130) which engages the tube portion (186) of
test strip assembly (180). The base of head portion (120) is
covered by a circular nylon mesh (121) formed from 0.15 mm strands
spaced by 0.25 mm. In the assembled device (110), mesh (121) is in
contact with sample (200) within well (199). Knob (150) is attached
via a rack and pinion mechanism to plunger (155) which, prior to
use, passes through tube (186) and towards hole (130). Seating
(193) contains a battery (194) which powers heat source (170). When
assembled, heat source (170) surrounds neck (115)
circumferentially, except in the region of hole (130).
[0106] Exploded and assembled views of test strip assembly (180)
are shown in FIG. 11. In the assembled device (110), tubular
portion (186) communicates directly with hole (130) and, prior to
use, plunger (155) engages and fills tube (186), thereby preventing
liquid flow therethrough. As plunger (155) is withdrawn by
operation of knob (150) in the direction of the arrow in FIG. 11,
tube (186) opens to form, together with hole (130), an outlet port
(135) through which liquid can flow. Tube (186) and plunger (155)
therefore operate in the manner of syringe. Liquid flows through
tube (186) into the capillary space between clear plastic housings
(181a; 181b) and passes under a pad (182) containing dehydrated
gold-tagged murine anti-CD59. As liquid passes pad (182), the
antibody is re-hydrated and can pass into the liquid, where it is
able to bind to spermatozoa. The liquid continues to flow towards
and into nitrocellulose strip (183), aided by a wick (188). The
pore size of strip (183) is too small to allow the spermatozoa to
enter, so they are captured at its entrance (184). Antibody can
bind captured spermatozoa at entrance (184) and form a pink line.
Any free antibody continues to flow until it is captured downstream
at a line (185) of immobilised anti-mouse antibody.
[0107] The device is used as illustrated in FIGS. 12 to 16: [0108]
In FIG. 12, a semen sample (200; e.g. obtained by masturbation) is
placed into lower piece (190) and collects in well (.199) whilst
lower piece (190) rests on a flat surface. [0109] After 30 minutes,
upper piece (191) is assembled with lower piece (190) as shown in
FIG. 13, to form device (110). [0110] Button (160) is then
depressed, as shown in FIG. 14. This releases solution (140) and
also activates heat source (170), bringing the temperature of
solution (140) to 37.degree. C. [0111] After around 30 minutes, to
allow entry of sperm into solution (140) and also temperature
equilibration, LED (116) indicates that knob (150) should be
rotated, as in FIG. 15. [0112] This withdraws plunger (155) and
allows motile sperm from sample (200) which have swum into medium
(140) to pass into the tube portion (186) of test strip (180). The
solution (140) flows through the test strip (180) by capillary
action towards wick (188). Sperm in the solution are retained at
the entrance (184) of nitrocellulose strip (183). Free gold-tagged
antibody continues to flow until it is captured downstream at a
line (185) of immobilised anti-mouse antibody. The line at (184) in
FIG. 16 indicates a positive result. The line at (185) indicates
that the test has operated correctly.
[0113] It will be understood that the invention has been described
by way of example only and modifications may be made whilst
remaining within the scope and spirit of the invention.
* * * * *