IMAGING SYSTEM AND ASSOCIATED METHOD FOR DETECTION OF
PROTEIN CONTAMINATION
Field of the Invention
The invention relates to an apparatus for measuring protein contamination,
specifically on surgical instruments, and to associated methods. In particular,
the invention relates to an instrument that is designed to be used in conjunction
with a stain comprising a reagent composition that fluoresces in the presence of
protein matter (such as intact proteins and/or their subunit amino acids and
peptides), such as the compositions described in the co-pending application
entitled "IN-SITU REAGENT", filed on the same date under attorney reference
KS.P49074GB.
Background to the Invention
In view of increased concerns relating to the role of proteins in the transmission
of diseases, healthcare authorities might impose mandatory protein
contamination detection requirements on the use of all surgical instruments.
Currently, detection of protein residues on surgical instruments is carried out
using a standard Ninhydrin assay. However, this standard assay has been
shown to be unreliable, because it is ineffective in detecting all but two amino
acids which are water soluble and which would in any event rarely present a
problem. Moreover, the Ninhydrin test is often carried out by 'swabbing'
instruments and testing the swab. It is difficult to swab all portions of an
instrument, particularly in areas that are most prone to collect protein residues,
such as corners and recesses.
A further consideration is cost. Currently, a pack of four Ninhydrin tests with
positive controls costs £25. The test is also relatively time-consuming to carry
out and requires trained personnel.
The present invention aims to address these issues and to provide a quick,
accurate method for detecting protein contamination over an entire instrument.
The inventive detection method is many times more sensitive than the Ninhydrin
technique.
A stain comprising a reagent composition has been developed that fluoresces in
the presence of protein. Fluorophors in the stain are capable of emitting light of
an emitted type when and only when both excited by light of an excitation type
and in contact with a protein. Details of the stain are disclosed in co-pending
application entitled "REAGENT", filed on the same date under attorney reference
KS.P49074GB. The components of the stain are readily available and
inexpensive.
Summary of the Invention
According to a first aspect of the invention, there is provided an imaging system
for detection of protein contamination on a specimen that has been treated with
a fluorescing stain, wherein fluorophors in the stain are capable of emitting light
of an emitted type when both excited by light of an excitation type and in contact
with a protein, the system comprising:
a chamber for receiving the specimen;
a first light source adapted to illuminate the specimen with light of the
excitation type when, in use, the specimen is received in the chamber;
a first image capture device adapted to capture a second image, of
patterns of fluorescence emitted by the fluorophors in the stain on the specimen,
corresponding to protein contamination, when illuminated by the second light
source; and
means for indicating, dependent on said first image capture, whether the
specimen is contaminated with protein.
The imaging system is able to provide a quick, accurate determination of
whether a specimen is contaminated with protein.
The system may further comprise means for determining the level of protein
contamination. By determining the level of protein contamination, it is possible
to make a more informed decision as to whether the specimen is contaminated
to such an extent that it must be unfit for use. Moreover, healthcare authorities
might set standard acceptable threshold levels, whereby if contamination levels
are determined to be above a particular threshold the specimen must be
sterilised or scrapped. The means for determining the level of protein
contamination may comprise a processor and associated analysis software.
The system may further comprise means to detect extraneous signals and
means to compensate for any such signals.
The system may further comprise a filter between the specimen and the first
image capture device, the filter adapted to transmit light of said emitted type and
to prevent transmission of light of said excitation type. The filter is preferably
adapted to transmit only light having a wavelength in the range of 430nm to
450nm.
The light of the excitation type may typically be in the range of 270nm to 370nm.
Preferably, the light of the excitation type has a 3 2nm peak wavelength.
The specimen may typically comprise a surgical instrument. The invention has
particular implementation in the context of surgical instruments, due to the
critical need for these to be verified as sterile prior to use. Should the system
indicate that the instrument is contaminated, the instrument could be sent to be
sterilised or discarded.
The system may further comprise:
a second light source adapted to illuminate the specimen with visible light
when, in use, the specimen is received in the chamber; and
a second image capture device adapted to capture a second image, of
the specimen, when illuminated by the second light source. The system may yet
further comprise an image combiner adapted to combine the first and second
images. The addition of a visible image of the specimen, particularly when
combined with the fluorescing image, offers several advantages. Firstly, the
combined captured images may be displayed to provide a clear visual indication
to a user as to whether protein contamination is present. Secondly, as explained
below, the visible image of the specimen may be used as a mask so as to ignore
any (extraneous) signals than might occur in the fluorescing image outside of the
area of the specimen.
Preferably, the first image capture device comprises a digital camera.
Preferably, the second image capture device comprises a digital camera. A
single digital camera may function as both the first image capture device and the
second image capture device. A single piece of equipment adapted to carry out
dual functions might be the most cost-effective solution. However, it can be
seen that by having separate, more specialised equipment for each distinct
image capture task more accurate results might be achieved.
Where the system further comprises means for determining the level of protein
contamination, said means for indicating may comprise an indicator adapted to
indicate whether the level of protein contamination is below or above a
predetermined threshold, thus providing a simple pass/fail indication. Such an
indicator would provide a simple, clear way to identify whether the specimen has
passed or failed the contamination test. Optionally, the indicator might further be
adapted to indicate whether the level of protein contamination is close to the
predetermined threshold, which might require the test to be re-run. Such an
indicator might, for example comprise a 'traffic light' system having a red light for
a 'fail', a green light for a 'pass' and an amber light for 'further attention'.
Preferably, the stain comprises a protein and/or amino acid detecting
composition comprising:
(a) o-phthaldialdehyde,
(b) a C3-C6 thiol,
(c) a buffer in the range of pH from 7.5 to 0, and
(d) a surfactant,
wherein the composition further comprises (e) a thiol reducing compound.
Further preferably, the composition is prepared by combining:
(a) about 0.1 mmol/L to about 10mmol/L of o-phthaldialdehyde,
(b) 1mM to 20mM of a C3-C6 thiol,
(c) 10mM to 100mM of a buffer in the pH range from 7.5 to 10,
(d) 0.01% v/v to 2% v/v of a surfactant, and
(e) about 0.05mmol/L to about 5mmol/L of the thiol reducing
compound.
According to a second aspect of the invention, there is provided a method of
detecting protein contamination on a specimen, the method comprising the steps
of:
treating the specimen with a fluorescing stain, wherein fluorophors in the
stain are capable of emitting light of an emitted type when both excited by light of
an excitation type and in contact with a protein;
placing the treated specimen within a chamber;
illuminating the specimen with light of the excitation type, and, when so
illuminated, capturing a first image, of patterns of fluorescence emitted by the
fluorophors in the stain on the specimen, corresponding to protein
contamination; and
dependent on said first image capture, indicating whether the specimen is
contaminated with protein.
The method may further comprise a step of determining the level of protein
contamination.
The method may further comprise the step of illuminating the specimen with
visible light, and, when so illuminated, capturing a second image of the
specimen. The method may even further comprise the step of combining the
first and second images. As noted above, the addition of a visible image of the
specimen, particularly when combined with the fluorescing image, offers several
advantages.
The method may further comprise the steps of:
detecting extraneous signals; and
compensating for any such extraneous signals;
wherein the detecting step comprises measuring a background signal
level, and wherein the compensating step comprises subtracting the background
signal level from the first image. In one embodiment, said measuring comprises
summing the grey level values in the first image. In another embodiment, said
measuring comprises low pass filtering the first image. In yet another
embodiment, said measuring comprises measuring the minimum signal level in
the first image, and said compensating comprises subtracting said minimum
signal level from every point in the second image. In a different embodiment,
said measuring comprises the steps of:
illuminating the chamber with light of the excitation type, with no
specimen present in the chamber; and
when so illuminated, capturing a background image of the chamber;
and wherein said compensating comprises subtracting said background
image from the first image on a pixel by pixel basis.
Where the method further comprises the step of illuminating the specimen with
visible light, and, when so illuminated, capturing a second image of the
specimen, that second image may be used in a masking step in which all signals
emanating from an area of the first image outside of the specimen, as captured
in the second image, are rejected. 'Foreign matter', such as bits of tissue or dust
or other debris can fluoresce. One way to eliminate extraneous signals due to
such foreign matter within the chamber is to identify the shape of the
contaminated specimen from the visible image and then to limit the
measurements to within that shape. Thus, any signals from outside the detected
specimen shape can be rejected as "not from contamination on the specimen".
The method may further comprise a calibration step comprising carrying out the
steps on a specimen having a known standard amount of protein.
Brief Description of the Drawings
The invention will be described, by way of example, with reference to the
accompanying drawings, in which:
Fig. 1 is a schematic representation of the system of the invention, showing the
interior of the chamber in cross section;
Fig. 2 is an illustration of an exemplary surgical instrument for protein
contamination measurement, corresponding to the second image of the
invention;
Fig. 3 is an illustration of the same instrument, corresponding to the first image
of the invention, in which protein contamination patches are visible; and
Fig. 4 is a composite image, comprising a combination of the images of Figs 2
and 3.
Detailed Description
Figure 1 shows, schematically, an imaging system 10 for detection of protein
contamination on a specimen 100. Preferably, the specimen 100 is a surgical
instrument. The system and associated method rely on a stain that fluoresces in
the presence of protein, as discussed in the 'Background to the Invention'.
The specimen 100 is coated with the stain, such as by dipping or spraying.
The system comprises a housing 12, such as a cabinet, the interior of which
defines a chamber 14. The chamber 14 is impervious to light, so providing a
totally dark imaging space. The internal walls 16 of the chamber 14 are treated
with non-reflective material to enhance the darkness and improve system
performance. One example of a suitable treatment is to paint the walls 16 with
matt black paint. Another suitable treatment is to line the interior walls 16 with
matt black anodised aluminium foil.
The interior of the cabinet 12 is accessible via an access opening such as a door
or a drawer (not shown). The edges of the access opening are adapted to
prevent ambient light from entering the chamber 14, for example including
flexible seals and/or light tight labyrinths. The specimen 100 can be placed
through the door or into the drawer for location inside the chamber 14 for
imaging. The specimen 100 may rest on a tray 110, the tray and specimen
together being placed on a bottom wall 16a of the chamber 14. Alternatively, the
specimen 100 may be placed directly on the bottom wall 16c.
Where the system includes a drawer, the specimen might be sprayed with the
stain after having been placed in the drawer.
Light sources are located on opposite side walls 16b, 16c on the inside of the
chamber 14. Visible light sources 20 are located and oriented to evenly
illuminate the specimen 100. The visible light sources 20 emit broad spectrum
light in the range of 380-750 nm. In this embodiment, excitation light sources 22
are located directly above the respective visible light sources 20 and are capable
of exciting the stain on the coated specimen 100. However, alternative lighting
positions are possible.
The excitation light sources 22 emit light in the range of 270nm to 370nm, which
is optimal for excitation of the stain. Optionally, an excitation filter 24 is
associated with each respective excitation light source 22 so that the excitation
light sources 22 do not emit light within the emission spectra of the stain. In one
embodiment, the excitation light sources 22 emit mid wave or long wave ultra
violet light that is filtered.
The top wall 16d of the housing 12 includes an aperture 18. A digital camera 30
and lens 32 are located on the exterior of the top wall 16d, outside the chamber
14 and aligned with the aperture 18. The specimen 100 is included in the
camera's field of view. An emission filter 34 is located between the specimen
100 and the camera 30. The emission filter 34 is adapted to transmit light of
said emitted type and to prevent transmission of light of said excitation type,
thereby to improve the sensitivity of the system by rejecting any signal other than
that emitted by the stain, such as leakage from the excitation lights 22.
In use, a user coats the surgical instrument 100 to be inspected with the stain
and places it inside the chamber 14, closing the drawer or door to make the
chamber 14 light tight. The excitation light sources 22 are turned on to illuminate
the instrument. Whilst so illuminated, the camera 30 captures a first image of
patterns of fluorescence 50 emitted by the stain where in contact with protein on
the instrument 100 (see Fig 3). As an example, with a mid wave UV excitation
light source, 4 s is an appropriate exposure time.
Next, the excitation light sources 22 are turned off and the visible light sources
20 are turned on. Whilst so illuminated, the camera 30 captures a second,
visible image of the instrument 100 (see Fig. 2). As an example, with a white
light source, 80 ms is an appropriate exposure time. It will be appreciated that
this exposure time and that for the visible light source are merely exemplary and
that the required exposure times will depend on a number of factors, including
the stain, the lens 32, the camera 30 and the emission filter 34.
The non-reflective material covering the internal walls 16 of the chamber 14
doesn't fluoresce when illuminated by the excitation source 22.
It should be noted that the order of capturing the first and second images may be
reversed, such that the 'second', visible image is in fact captured before the
'first', fluorescent image.
The camera 30 is connected to a processor (not shown) that is programmed with
analysis and measurement software. The first and second images are overlaid
and displayed by the software. In the combined image (see Fig. 4), the user can
see areas of contamination 50 highlighted on the instrument 100. One method
of overlaying the images would be to display the visible image in "red" and the
fluorescent image in "green". This display gives the user a quick visual
indication as to the presence or otherwise of protein residues on the instrument
100.
The analysis and measurement software of the system 10 can also analyse the
fluorescent image to measure the total volume of stained protein 50 visible in the
image. If the measured amount of stained protein 50 is greater than a precalibrated
threshold, the system will flag the specimen 100 as being
contaminated and unfit for use. One implementation would be to use a "traffic
light" indicator, with "red" for contaminated and "green" for OK. An amber
indicator might be used to indicate that the volume of protein is close to the
threshold.
Measurements of the amount of stained protein 50 on the surgical instruments
100 under test are thus derived from the "fluorescent image". This first image
should only contain signals corresponding to the emission from the stained
protein. However it may contain some background signal level due to the
camera 30 or imaging conditions.
To improve the accuracy of the measurements, the results may be corrected for
any such extraneous signals. The measurement process thus consists of:
summing the grey level values in the image; and background correcting the
results to account for any background signal level or offset in the camera
digitisation. Note that the background correction can be applied to the image
data before measurement or to the results after measurement.
The background correction can be implemented in several ways. In a first
embodiment, the first, fluorescent image is low pass filtered to produce a
background image which is then subtracted from the original first image. In a
second embodiment, the minimum signal level in the first, fluorescent image is
measured and this minimum signal is then subtracted from the signal value at
every point in the original first image. In a third embodiment, a background first
image is captured without a specimen 100 inside the chamber 14 but with the
excitation lights 22 turned on. This image would be subtracted on a pixel-by-pixel
basis from each subsequent specimen image.
The measurement process may be fully automated, being initiated by a user
once the specimen 100 has been loaded in to the chamber 14 and continuing
until the measurement steps have all been completed. Alternatively, the system
may be semi-automated, requiring user input at certain stages. It is also
possible for the system to be fully manual, the user initiating each of the series of
required steps in turn.
Improved accuracy might be achieved by repeating any or all of the image
capture steps and the measurement and analysis steps. Also, the instrument
100 could be turned over after a first run so as to repeat the process on the
reverse side.
In order to verify the accuracy of the system, it may be calibrated by testing a
specimen having a known standard amount of protein contamination.
It will be appreciated that the specific locations and orientations of the light
sources are exemplary and that alternative arrangements having no visible light
source 20, just a single visible light source 20 or more than two visible light
sources 20 are also possible. Likewise, there may be just a single excitation
light source 22 or more than two excitation light sources 22. The excitation light
sources 22 do not have to be located above respective visible light sources 20.
The consideration is to provide as even illumination of the specimen 100 as
possible.
Rather than a single camera 30 to capture both the first (fluorescing) image and
the second (visible light) image, two separate cameras. Moreover, it will be
understood that a digital camera 30 and associated lens 32 is just one example
of an image capture device. Other devices capable of capturing the respective
first and second images will be known to the skilled person. Furthermore, it will
be understood that the image capture device(s) might be partially or fully located
within the chamber 14.
The display of the first and second images and their combination is optional. It
will be understood that for the purposes of determining whether a surgical
instrument or other specimen is contaminated with protein it would be sufficient
to provide a contamination indication, which might be visible or audible. At its
most basic, the indication might be a light or an audible alert message that is
turned on if patterns of fluorescence are detected in the first image.
CLAIMS
1. An imaging system for detection of protein contamination on a specimen
that has been treated with a fluorescing stain, wherein fluorophors in the stain
are capable of emitting light of an emitted type when both excited by light of an
excitation type and in contact with a protein, the system comprising:
a chamber for receiving the specimen;
a first light source adapted to illuminate the specimen with light of the
excitation type when, in use, the specimen is received in the chamber;
a first image capture device adapted to capture a second image, of
patterns of fluorescence emitted by the fluorophors in the stain on the specimen,
corresponding to protein contamination, when illuminated by the second light
source; and
means for indicating, dependent on said first image capture, whether the
specimen is contaminated with protein.
2. The system of claim 1, further comprising means for determining the level
of protein contamination.
3. The system of claim 2, wherein the means for determining the level of
protein contamination comprises a processor and associated analysis software.
4. The system of any preceding claim, further comprising means to detect
extraneous signals and means to compensate for any such signals.
5. The system of any preceding claim, further comprising a filter between
the specimen and the first image capture device, the filter adapted to transmit
light of said emitted type and to prevent transmission of light of said excitation
type.
6. The system of claim 5, wherein the filter is adapted to transmit only light
having a wavelength in the range of 430nm to 450nm.
7. The system of any preceding claim, wherein the light of the excitation
type has a wavelength in the range of 270nm to 370nm,
8. The system of claim 7, wherein the light of the excitation type has a
3 12nm peak wavelength.
9. The system of any preceding claim, wherein the specimen comprises a
surgical instrument.
10. The system of any preceding claim, further comprising:
a second light source adapted to illuminate the specimen with visible light
when, in use, the specimen is received in the chamber; and
a second image capture device adapted to capture a second image, of
the specimen, when illuminated by the second light source.
11. The system of claim 10, further comprising an image combiner adapted to
combine the first and second images.
12. The system of any preceding claim, wherein the first image capture
device comprises a digital camera.
13. The system of any preceding claim, when dependent on claim 9, wherein
the second image capture device comprises a digital camera.
14. The system of claim 13, wherein a single digital camera functions as both
the first image capture device and the second image capture device.
15. The system of claim 2, or any claim dependent thereon, wherein said
means for indicating comprises an indicator adapted to indicate whether the
level of protein contamination is below or above a predetermined threshold.
16. The system of any preceding claim, wherein the stain comprises a protein
and/or amino acid detecting composition comprising:
(a) o-phthaldialdehyde,
(b) a C3-C6 thiol,
(c) a buffer in the range of pH from 7.5 to 10, and
(d) a surfactant,
wherein the composition further comprises (e) a thiol reducing compound.
17. A method of detecting protein contamination on a specimen, the method
comprising the steps of:
treating the specimen with a fluorescing stain, wherein fluorophors in the
stain are capable of emitting light of an emitted type when both excited by light of
an excitation type and in contact with a protein;
placing the treated specimen within a chamber;
illuminating the specimen with light of the excitation type, and, when so
illuminated, capturing a first image, of patterns of fluorescence emitted by the
fluorophors in the stain on the specimen, corresponding to protein
contamination; and
dependent on said first image capture, indicating whether the specimen is
contaminated with protein.
18. The method of claim 17, further comprising a step of determining the level
of protein contamination.
19. The method of claim 17 or claim 18, further comprising the step of
illuminating the specimen with visible light, and, when so illuminated, capturing a
second image of the specimen.
20. The method of claim 19, further comprising the step of combining the first
and second images.
2 1. The method of any of claims 17 to 20, further comprising the steps of:
detecting extraneous signals; and
compensating for any such extraneous signals;
wherein the detecting step comprises measuring a background signal
level, and wherein the compensating step comprises subtracting the background
signal level from the first image.
22. The method of claim 2 , wherein said measuring comprises summing the
grey level values in the first image.
23. The method of claim 2 1, wherein said measuring comprises low pass
filtering the first image.
24. The method of claim 2 1, wherein said measuring comprises measuring
the minimum signal level in the first image, and wherein said compensating
comprises subtracting said minimum signal level from every point in the first
image.
25. The method of claim 2 1, wherein said measuring comprises the steps of:
illuminating the chamber with light of the excitation type, with no
specimen present in the chamber; and
when so illuminated, capturing a background image of the chamber; and
wherein said compensating comprises subtracting said background
image from the first image on a pixel by pixel basis.
26. The method of any of claims 17 to 25, when dependent on claim 18,
further comprising a masking step in which all signals emanating from an area of
the first image outside of the specimen, as captured in the second image, are
rejected.
27. The method of any of claims 17 to 26, further comprising a calibration
step comprising carrying out the steps on a specimen having a known standard
amount of protein.
28. The method of any of claims 17 to 27, wherein the stain comprises a
protein and/or amino acid detecting composition comprising:
(a) o-phthaldialdehyde,
(b) a C3-C6 thiol,
(c) a buffer in the range of pH from 7.5 to 10, and
(d) a surfactant,
wherein the composition further comprises (e) a thiol reducing compound.