U.S. patent application number 14/234136 was filed with the patent office on 2014-06-05 for reader device for luminescent immunoassays.
This patent application is currently assigned to BIOSENSIA PATENTS LIMITED. The applicant listed for this patent is BIOSENSIA PATENTS LIMITED. Invention is credited to Diarmuid Flavin, Shane Moynihan.
Application Number | 20140154792 14/234136 |
Document ID | / |
Family ID | 47146452 |
Filed Date | 2014-06-05 |
United States Patent
Application |
20140154792 |
Kind Code |
A1 |
Moynihan; Shane ; et
al. |
June 5, 2014 |
READER DEVICE FOR LUMINESCENT IMMUNOASSAYS
Abstract
The present disclosure, among other things, describes a reader
system for analyzing one or more analytes in a fluid sample,
comprising a casing (204) with at least one port (201) leading to a
holster (301) which is configured to receive a cartridge (107)
comprising a vertically oriented immunoassay device for analyzing
one or more analytes in a fluid sample, an optical system (121)
with excitation optics comprising a light source (302) and an
excitation lens (305) configured to transmit light from the light
source (302) and thereby excite a region of the vertically oriented
immunoassay device when a cartridge (107) is placed in the holster
(301), and collection optics comprising a photosensor (306) and a
collection lens (307) configured to collect emitted light from the
vertically oriented immunoassay device when a cartridge (107) is
placed in the holster (301), an electromechanical motor system
(106) configured to move the holster (301) in a vertical direction
with respect to the optical system (121) so that the optical system
(121) can interrogate different regions of the vertically oriented
immunoassay device when a cartridge (107) is placed in the holster
(301), and one or more digital processors (104a, 104b) and
associated electronics configured to receive data from and control
the optical system (121) and to control the electromechanical motor
system (106).
Inventors: |
Moynihan; Shane;
(Ballsbridge, IE) ; Flavin; Diarmuid;
(Ballsbridge, IE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIOSENSIA PATENTS LIMITED |
Dublin |
|
IE |
|
|
Assignee: |
BIOSENSIA PATENTS LIMITED
Dublin
IE
|
Family ID: |
47146452 |
Appl. No.: |
14/234136 |
Filed: |
July 20, 2012 |
PCT Filed: |
July 20, 2012 |
PCT NO: |
PCT/IB2012/001907 |
371 Date: |
January 21, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61510779 |
Jul 22, 2011 |
|
|
|
Current U.S.
Class: |
435/287.2 ;
422/69 |
Current CPC
Class: |
G01N 21/6486 20130101;
G01N 2021/6441 20130101; G01N 21/278 20130101; G01N 21/6452
20130101; G01N 2021/6421 20130101; G01N 2201/0627 20130101; G01N
2201/0624 20130101; G01N 21/645 20130101; G01N 2021/6471 20130101;
G01N 21/8483 20130101; G01N 2201/0648 20130101; G01N 2021/7786
20130101; G01N 2201/0693 20130101; G01N 2021/6419 20130101; G01N
33/5302 20130101 |
Class at
Publication: |
435/287.2 ;
422/69 |
International
Class: |
G01N 21/64 20060101
G01N021/64; G01N 33/53 20060101 G01N033/53 |
Claims
1. A reader system for analyzing one or more analytes in a fluid
sample, comprising: a. a casing with at least one port leading to a
holster which is configured to receive a cartridge comprising a
vertically oriented immunoassay device for analyzing one or more
analytes in a fluid sample; b. an optical system with excitation
optics comprising a light source and an excitation lens configured
to transmit light from the light source and thereby excite a region
of the vertically oriented immunoassay device when a cartridge is
placed in the holster, and collection optics comprising a
photosensor and a collection lens configured to collect emitted
light from the vertically oriented immunoassay device when a
cartridge is placed in the holster; c. an electromechanical motor
system configured to move the holster in a vertical direction with
respect to the optical system so that the optical system can
interrogate different regions of the vertically oriented
immunoassay device when a cartridge is placed in the holster; and
d. one or more digital processors and associated electronics
configured to receive data from and control the optical system and
to control the electromechanical motor system.
2. The reader system of claim 1, further comprising non-volatile or
volatile digital memory for storing data generated by the optical
system.
3. The reader system of any one of the preceding claims, wherein
the casing further comprises a display screen, a data entry device,
or a combination thereof.
4. The reader system of claim 3, wherein the data entry device
comprises an integrated keypad or a touch-screen.
5. The reader system of any one of the preceding claims, wherein
the light source is a surface mounted light emitting diode.
6. The reader system of claim 5, wherein the surface mounted light
emitting diode comprises a collimation lens.
7. The reader system of any one of the preceding claims, wherein
the collection lens is configured to collect emitted light from an
entire excited region.
8. The reader system of claim 7, wherein emitted light is directed
onto the central portion of a photodiode.
9. The reader system of any one of the preceding claims, wherein
the excitation optics further comprises an optical excitation
filter.
10. The reader system of claim 9, wherein the optical excitation
filter is a band-pass or short-pass optical filter.
11. The reader system of claim 9, wherein the optical excitation
filter operates by interference or by absorption.
12. The reader system of any one of the preceding claims, wherein
the collection optics further comprises an optical collection
filter.
13. The reader system of claim 12, wherein the optical collection
filter is mechanically actuated.
14. The reader system of claim 12, wherein the optical collection
filter is a band-pass or long-pass optical filter.
15. The reader system of claim 12, wherein the optical collection
filter operates by interference or by absorption.
16. The reader system of any one of the preceding claims, wherein
the excitation optics further comprises a plate with one or more
optical apertures.
17. The reader system of claim 16, wherein the plate is absorptive
or reflective.
18. The reader system of claim 16, wherein at least one dimension
of the one or more apertures is in a range of 0.1-2 mm, 0.7-0.8 mm,
or 0.3-0.4 mm.
19. The reader system of any one of the preceding claims, wherein a
cartridge comprising a vertically oriented immunoassay device is
located within the holster.
20. The reader system of claim 19, wherein the vertically oriented
immunoassay device comprises one or more immunoassay channels.
21. The reader system of claim 20, wherein the one or more
immunoassay channels each independently comprise one or more test
lines for analyzing one or more analytes.
22. The reader system of any one of the preceding claims, wherein
the vertically oriented immunoassay device comprises multiple
immunoassay channels and the excitation optics comprises multiple
light sources, each with different central wavelengths.
23. The reader system of claim 22, wherein the collection optics
comprises multiple photosensors, and the light sources and
photosensors are configured in pairs so that they transmit light to
and collect emitted light from different immunoassay channels.
24. The reader system of claim 22, wherein the pairs of light
sources and photosensors are configured so that different
immunoassay channels are interrogated at different points in
time.
25. The reader system of claim 22, wherein electronic frequency
filtering is used to filter signals from the multiple
photosensors.
26. The reader system of claim 22, wherein the multiple immunoassay
channels are spatially separated such that cross talk between
different immunoassay channels and different photosensors is
substantially absent when the multiple immunoassay channels are
interrogated simultaneously.
27. The reader system of claim 1, wherein the excitation optics
comprises a photosensor that monitors the light source.
28. The reader system of claim 27, wherein measurements from the
photosensor that monitors the light source are used to control the
emission intensity of the light source.
29. The reader system of any one of the preceding claims, wherein
the optical collection plane of the collection optics and the
optical excitation plane of the excitation optics form an angle
that is offset from normal.
30. The reader system of any one of claims 19-29, wherein the one
or more digital processors collect data generated when the optical
system scans the vertically oriented immunoassay device.
31. The reader system of claim 30, wherein the one or more digital
processors process the data to quantify an amount of one or more
analytes in a fluid sample that was applied to the immunoassay
device before the cartridge was placed in the holster.
32. The reader system of any one of the preceding claims, further
comprising one or more quality controls checks that are actualised
in software on the one or more digital processors.
33. The reader system of claim 32, wherein the one or more quality
control checks are selected from the group consisting of checks of
control line development, checks of channel clearance, checks as to
the size and position of peaks, checks as to the time of an assay
run as compared to expiry data of the immunoassay device, and any
combination thereof.
34. The reader system of any one of the preceding claims, further
comprising at least one sensor for recognizing cartridge insertion
or removal.
35. The reader system of claim 34, wherein the sensor is an optical
or mechanical sensor.
36. The reader system of any one of the preceding claims, further
comprising a barcode reading system.
37. The reader system of claim 36, wherein the barcode reading
system is one-dimensional or two-dimensional.
38. The reader system of claim 37, wherein the two-dimensional
barcode reading system encodes information selected from the group
consisting of identification of cartridge type or lot data, lot
manufacture and expiry dates, analyte names, cartridge expected
response, lot parameters, peak finding parameters, calibration
parameters, and any combination thereof.
39. The reader system of claim 1, further comprising a physically
separate quality control component with substantially the same
external dimensions as a cartridge.
40. The reader system of claim 39, wherein the quality control
component comprises photoluminescent materials that exhibit
characterised levels of photoluminescence upon excitation by the
light source.
41. The reader system of claim 40, wherein the photoluminescent
materials are or comprise plastics impregnated with
photoluminescent dyes, nanocrystals, quantum dots or any
combination thereof.
42. The reader system of claim 40, wherein photoluminescent areas
are defined on the quality control component using masked
materials, coated materials, multilayer etched materials or any
combination thereof.
43. The reader system of claim 42, wherein the photoluminescent
areas are localised at the optical excitation plane of the
excitation optics within the reader system when the quality control
component is placed in the holster.
44. The reader system of claim 42, wherein the photoluminescent
areas are patterned such that optical misalignments within the
reader system lead to predictable changes in the pattern or
intensity of emitted light.
45. The reader system of claim 44, wherein the one or more digital
processors collect data generated when the optical system scans the
quality control component and use the data to validate the reader
system for further use.
46. The reader system of claim 44, wherein the one or more digital
processors collect data generated when the optical system scans the
quality control component and use the data to modify internal
calibration factors of the reader system.
47. The reader system of claim 44, wherein the one or more digital
processors collect data generated when the optical system scans the
quality control component and use the data to calculate a degree of
optical misalignment between the optical system and the
holster.
48. The reader system of claim 47, wherein the one or more digital
processors control the electromechanical motor system to actuate
and thereby bring the optical system and the holster into optical
alignment.
49. The reader system of any one of the preceding claims, wherein
the electromechanical motor system comprises an encoder which is
used to detect and report a relative position of the holster.
50. The reader system of any one of the preceding claims, wherein
the reader system comprises travel sensors that are used to detect
and report a relative position of the holster.
51. The reader system of claim 50, wherein the travel sensors are
optical or mechanical.
52. The reader system of any one of the preceding claims, further
comprising a printer.
53. The reader system of any one of the preceding claims, further
comprising components and protocols for external wireless access,
such as by Wi-Fi, ANT or Bluetooth.
54. The reader system of any one of the preceding claims, further
comprising components and protocols for wired connectivity, such as
by serial, USB or Ethernet cable.
55. The reader system of any one of the preceding claims, further
comprising alignment features within the holster for ensuring
alignment of the cartridge.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional
application Ser. No. 61/510,779, filed Jul. 22, 2011, the entirety
of which is hereby incorporated herein by reference.
BACKGROUND
[0002] Immunoassays may be used for the determination of clinical
decisions. As such, accuracy, reliability, and repeatability of
immunoassay interpretation are largely important. With regard to
the field of use, this analysis is important in a point-of-care or
mobile setting. In these settings, tests may be carried out by
unskilled technicians or patients themselves, while still requiring
the maintenance of traceability and accuracy. In addition,
communication of test and auditing data is important; insofar as it
may be, for example, examined by remote healthcare professionals,
integrated with hospital LIMS systems, or used to verify device
operation.
SUMMARY OF CERTAIN EMBODIMENTS
[0003] Various embodiments of the present invention utilize
photoluminescence based methodologies to provide accurate,
non-subjective interpretation of labelled mobilizable reagent
binding in immunoassays, and quantization of analyte presence or
concentration in fluid test samples. Bespoke algorithms and devices
compensate for assay and optical variability. Embodiments of the
invention combine portability, automation and communication
technologies to cater for use by an unskilled technician in a
point-of-care setting.
[0004] Embodiments of the present invention concern an immunoassay
analysis system for the recording and interpretation of
photoluminescent immunoassays, herein termed the reader system.
Embodiments of the invention quantify photoluminescence from one or
more capture zones of an immunoassay, and thereby determines a
quantitative or qualitative measurement of analyte presence within
a fluid sample.
[0005] In brief, a reader system according to embodiments of the
present invention may comprise a casing, an optical system, en
electromechanical motor system, and one or more digital processors.
The casing may include at least one port leading to a holster which
is configured to receive a cartridge comprising a vertically
oriented immunoassay device for analyzing one or more analytes in a
fluid sample. The optical system may include excitation optics
comprising a light source and an excitation lens configured to
transmit light from the light source and thereby excite a region of
the vertically oriented immunoassay device when a cartridge is
placed in the holster, and collection optics comprising a
photosensor and a collection lens configured to collect emitted
light from the vertically oriented immunoassay device when a
cartridge is placed in the holster. The electromechanical motor
system may be configured to move the holster in a vertical
direction with respect to the optical system so that the optical
system can interrogate different regions of the vertically oriented
immunoassay device when a cartridge is placed in the holster. The
one or more digital processors may be associated electronics
configured to receive data from and control the optical system and
to control the electromechanical motor system
[0006] A reader system, in some embodiments, includes non-volatile
or volatile digital memory for storing data generated by the
optical system.
[0007] In some embodiments, the casing of a reader system further
includes a display screen, a data entry device, such as a keypad or
display integrated touch-screen, or a combined device that acts as
a display screen and a data entry device.
[0008] In some embodiments, a light source is a light emitting
diode (LED) surface mounted device. The light source may also
include integrated lens for collimation of LED emitted light. In
some embodiments, a reader system includes multiple excitation
sources, for example, LEDs with various central emission
wavelengths, with matched optical filters.
[0009] In some embodiments, excitation optics includes an plate
(e.g., absorptive or reflective plate) with an optical aperture.
The aperture may be aligned within the optical excitation path; and
defined to form a specific, regular excitation area upon the
immunoassay device. In an implementation, a excitation area is 0.3
mm-3 mm in width and 0.2 mm to 2 mm in height.
[0010] In some embodiments, a collection lens collects emitted
light from an entire excited region. The collection lens may
integrate the emitted light and direct it onto the central portion
of a corresponding photosensor for detection.
[0011] In some embodiments, excitation or collection optics each
includes an optical filter. For example, the optical filer can be a
band-pass or short-pass optical filer. The optical filer may
operate by interference or by absorption. An optical filter in the
excitation optics tunes optical excitation wavelengths experienced
by an immunoassay. An optical filter in the collection optics
passes wavelengths associated with the photoluminescent label
emission of an immunoassay, while blocking wavelengths associated
with optical excitation. Additionally or alternatively, an optical
collection filter may be mechanically actuated.
[0012] In some embodiments, a cartridge comprising a vertically
oriented immunoassay device is located within a holster. The
vertically oriented immunoassay device may include one or multiple
parallel, vertically oriented immunoassay channels. The one or more
immunoassay channels may each independently comprise one or more
test lines for analyzing one or more analytes. A reader system may
include multiple light sources and multiple photosensors, with one
of each being dedicated to each individual immunoassay channel. The
multiple light sources may each have different central wavelength.
Electronic frequency filtering may be used to filer signals from
the multiple photosensors. For example, electronic filtering may be
applied to the photosensor signal to register signals associated
with the duty cycle of immunoassay excitation and thus the
immunoassay photoluminescence, while blocking low or high frequency
system noise.
[0013] In certain embodiments, the pairs of light sources and
photosensors are configured so that different immunoassay channels
are interrogated at different points in time. In certain
embodiments, the multiple immunoassay channels are spatially
separated such that cross talk between different immunoassay
channels and different photosensors is substantially absent when
the multiple immunoassay channels are interrogated
simultaneously.
[0014] In some embodiments, an aperture plate includes an aperture
for each light source. There may be a dedicated collection lens
associated with each immunoassay channel and photosensor. The plate
may be absorptive or reflective. At least one dimension (e.g.,
width, or height) of each of one or more apertures is in a range of
0.1-2 mm, 0.7-0.8 mm, or 0.3-0.4 mm.
[0015] In some embodiments, optical emission intensity of the light
source is controlled and stabilised through a cartridge scan. In
this case, the excitation optics may include a dedicated excitation
source monitoring photosensor. The light source emission intensity
can be monitored by analysis of the monitoring photosensor
electronic signal. Feedback of this monitoring signal to the
excitation source may act to stabilize the emission of the
excitation source across all scans. Feedback stabilization may be
carried out throughout a scan, for each duty cycle of each light
source's emission. Alternatively, feedback stabilization may be
independently carried out for each light source prior to the
commencement of each cartridge scan. In certain embodiments, a
reader system includes a proportional-integral-derivative control
algorithm to optimally stabilise light source emission at a desired
intensity by analysis of the monitoring photosensor signal.
[0016] In some embodiments, there is an angular offset between the
optical collection plane of the photodiode/lens assembly, and the
optical excitation plane. The specific angular offset and
configuration can be selected in order to inhibit direct reflection
of excitation light into the detector assembly, while maintaining
efficient excitation and collection. For example, the optical
excitation is normal to the cartridge surface, while detection is
offset by 35 degrees.
[0017] In some embodiments, one or more digital processors collect
data generated when the optical system scans the vertically
oriented immunoassay device. One or more digital processors can
process the data to quantify an amount of one or more analytes in a
fluid sample that was applied to the immunoassay device before the
cartridge was placed in the holster.
[0018] For example, digital processors may use an algorithm to
characterise the presence or amount of an analyte within a fluid
sample, according to assay specific calibration parameters. An
algorithm provides either a quantitative, semi-quantitative or
qualitative estimate of analyte concentration within the fluid
sample. In certain embodiment of this invention, multiple
photoluminescent immunoassays assays are present in the cartridge
device, and the algorithm provides independent quantitative,
semi-quantitative or qualitative estimations of analyte
concentration for all tested analytes within the sample.
[0019] In some embodiments, a reader system includes one or more
quality controls checks that are actualised in software on the one
or more digital processors. Quality controls checks may include: a
quality control check of scan data, including a check of control
line development; a check of channel clearance; and checks as to
the size and position of peaks. Additionally, the reader software
verifies the time of test as being within the expiry date of a
particular assay.
[0020] In some embodiments, a reader system includes a barcode
reading system. A barcode may be encoded on a cartridge. A barcode
may be encoded with assay specific calibration data relating to an
assay cartridge batch. Upon introduction of the cartridge to the
reader system, the assay specific calibration data may be read,
interpreted and copied to internal reader memory. For example, a
barcode reading system may be one dimensional, or two dimensional.
Exemplary information that can be encoded in a barcode reading
system includes, but is not limited to, identification of cartridge
type or lot data, lot manufacture and expiry dates, analyte names,
cartridge expected response, lot parameters, peak finding
parameters, calibration parameters, and any combination
thereof.
[0021] In some embodiments, a read system includes at least one
sensor for recognizing cartridge insertion or removal. The sensor
may be an optical or mechanical sensor. For example, one or more
optically emissive sources and corresponding optical sensors can be
included. These sensors may be held within the cartridge holster,
and their positioning corresponds to locations which define the
cartridge insertion or removal of the cartridge. In this case, the
optical sensor may be a light source and photosensor couple. This
may be located in close proximity to the mouth of the holster. Upon
insertion, the cartridge blocks propagation of light from the
sensor light source to its corresponding photosensor. The sensor
registers full removal of the cartridge by the resumption of light
propagation from the sensor light source to its corresponding
photosensor. A mechanical switch sensor may be located at the base
of the holster. Upon full insertion of the cartridge into the
holster, this switch is actuated by the cartridge, enabling the
detection of cartridge insertion. In some embodiments, there exists
a physically separate quality control component with substantially
the same external dimensions as a cartridge. This component may
include photoluminescent materials that exhibit characterised
levels of photoluminescence upon excitation by the light source.
For example, photoluminescent materials can be or comprise plastics
impregnated with photoluminescent dyes, nanocrystals, quantum dots
or any combination thereof. Generally, the photoluminescent areas
of a quality control component are localised at the optical plane
of the reader, at a similar position to that of immunoassay surface
in a given cartridge. Further, reader scans of this quality control
component may be carried out in a similar method to that of the
immunoassay cartridge. Photoluminescent areas may be defined on the
quality control component using masked materials, coated materials,
multilayer etched materials or any combination thereof.
Photoluminescent areas may be patterned such that optical
misalignments within the reader system lead to predictable changes
in the pattern or intensity of emitted light.
[0022] In some embodiments, the one or more digital processors of a
read system collect data generated when the optical system scans
the quality control component and use the data to validate the
reader system for further use. The one or more digital processors
may collect data generated when the optical system scans the
quality control component and use the data to modify internal
calibration factors of the reader system. The one or more digital
processors may collect data generated when the optical system scans
the quality control component and use the data to calculate a
degree of optical misalignment, such as the direction and degree of
lateral misalignment, degree of focus or defocus, or optical system
tilt, between the optical system and the holster. In this case, the
one or more digital processors may control the electromechanical
motor system to actuate and thereby bring the optical system and
the holster into optical alignment.
[0023] In some embodiments, a reader system includes travel sensors
that are used to detect and report a relative position of the
holster. The travel sensors may be optical or mechanical.
[0024] In some embodiments, a reader system includes a printer for
the printout of hardcopies of scan results and data following the
reading of an immunoassay cartridge, or data from stored memory.
This printer may be incorporated within the casing of the reader
device, or provided as a separate component. In the case of the
printer being a separate component, the printer may be interfaced
with the reader using a USB, Ethernet or serial port connection.
Power may be provided to the printer device directly from the
reader system or via a separate power supply component.
[0025] In some embodiments, a reader system includes a processing
algorithm for the verification of immunoassay batch responses. This
algorithm analyses the response of one or more immunoassay
cartridges of the specified batch, run with control liquids of
specified concentrations. The algorithm compares expected responses
with those found from these cartridges, and verifies the
immunoassay batch as operating to a given specification. Further,
this algorithm also may conduct the optimisation and correction of
immunoassay batch specific calibration parameters, as stored within
reader memory, to compensate for time-related changes in
immunoassay photoluminescence response. In this case, following the
analysis of one or more immunoassay cartridges of the specified
batch, run with control liquids of specified concentrations,
internal calibration parameters are then updated to provide a
best-fit result to control responses.
[0026] In some embodiments, a reader system includes components and
protocols for external wireless access, such as by Wi-Fi, ANT or
Bluetooth. In an implementation, this connectivity enables remote
reader operation diagnostics, firmware or software updates and data
transfer.
[0027] In some embodiments, a reader system includes components and
protocols for wired connectivity, such as by RS-232 serial,
universal serial bus (USB) or Ethernet cable. In an implementation,
this connectivity enables remote reader operation diagnostics,
firmware or software updates and data transfer.
[0028] In some embodiments, a reader system includes alignment
features within a cartridge holster. These features hold the
cartridge in position and ensure that the immunoassay surface is
localised at the optical plane. In certain embodiments, spring
loaded dowels are located in positions corresponding to recesses in
the assay cartridge when the cartridge is correctly localised
within the holster. In certain embodiment, physical alignment
features prevent the mis-insertion of the cartridge, by blocking
full insertion of the cartridge at an incorrect rotation.
[0029] In some embodiments, a reader system includes an internal
battery which supplies the reader with electrical power when not
connected to a mains power supply.
[0030] In some embodiments, a reader system includes electronic
memory and a digital file management system for the storage of
data, operation parameters, and software and user interface
details. Files stored within this electronic memory may include:
Scan files, calibration files, quality control run files, user
lists, settings and change logs, scan logs, calibration run logs,
or user logs. In order to review a potentially large number of scan
files, search functionality may be implemented. This search
functionality generally consists of user interface options enabling
the user to filter scans results by date, operator, patient ID or
test.
[0031] The present disclosure include, among other thing, methods
of using reader systems described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 shows a reader system top level diagram.
[0033] FIGS. 2(a) and 2(b) show reader system casing diagrams: (a)
side A and (b) side B.
[0034] FIGS. 3(a) and 3(b) show reader optics diagrams.
[0035] FIG. 4 shows a reader optics diagram for a multiple
wavelength system.
[0036] FIG. 5 shows a reader use procedure block diagram for a test
scan.
[0037] FIG. 6 shows a reader use procedure block diagram for a
quality control scan.
[0038] FIG. 7 shows a reader use procedure block diagram for liquid
controls scans.
[0039] FIG. 8 shows a scan processing algorithm block diagram.
[0040] FIG. 9 shows a test scan calibration algorithm block
diagram.
[0041] FIG. 10 shows a quality control algorithm block diagram.
[0042] FIG. 11 shows a liquid controls calibration adjustment
algorithm block diagram for a qualitative tests.
[0043] FIG. 12 shows a liquid controls calibration adjustment
algorithm block diagram for a quantitative test.
[0044] FIG. 13 shows an example printout of test data following a
test scan.
[0045] FIG. 14 shows a schematic of sample light source emission
power feedback algorithm.
[0046] FIGS. 15(a) and 15(b) show exemplary optical-path schematics
of a reader system: (a) side view and (b) top view.
[0047] FIG. 16 shows an example optical scan taken by an reader
system.
DEFINITIONS
[0048] Assay--As used herein, the term "assay," refers to an in
vitro analysis carried out to determine the presence or absence of
one or more target analytes in a fluid sample. In certain
embodiments the assay may be quantitative and determine the amount
of the one or more target analytes in the fluid sample. In general,
an assay includes at least one pair of reagent components where at
least one of the reagent components has a high binding affinity for
the other. In certain embodiments, the assay is an immunoassay
(e.g., a sandwich, competitive or inhibition immunoassay).
Generally, an immunoassay includes an antibody component which
binds with high affinity to another antibody component or to an
antigen component. In certain embodiments, the assay is a molecular
assay and includes a pair of nucleic acid components which
hybridize to form a complex.
[0049] Target analyte--As used herein, the term "target analyte" or
"analyte" refers to the substance or substances that an assay is
designed to detect. Examples of analytes include, but are not
restricted to proteins (e.g., antibodies, hormones, enzymes,
glycoproteins, peptides, etc.), nucleic acids (e.g., DNA, RNA,
etc.), lipids, small molecules (e.g., drugs of abuse, steroids,
environmental contaminants, etc.) and infectious disease agents of
bacterial or viral origin (e.g., E. coli, Streptococcus, Chlamydia,
Influenza, Hepatitis, HIV, Rubella, etc.).
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0050] A reader system for the recording and interpretation of
photoluminescent immunoassays is described herein in connection
with embodiments of the present invention. Various embodiments of
the invention quantify photoluminescence from one or more capture
zones of said immunoassay, and determines a quantitative or
qualitative measurement of analyte presence within a fluid
sample.
[0051] In brief, a reader system may include a casing,
incorporating a display screen, or a data entry device, such as a
keypad or display integrated touch-screen, or a combined device
that acts as a display screen and a data entry device. This casing
may also incorporate a fluidic immunoassay device access port, and
contains a holster receptacle for receiving an external immunoassay
fluidic device. Said external immunoassay fluidic device comprises
one or more immunoassays oriented in a substantially vertical
configuration, configured for the analysis of a fluid sample; and
is herein referred to as the cartridge. Further, the reader
comprises an optical system within the casing, consisting of
excitation and collection optics within a single optical block; and
an electromechanical motor system, such as a stepper motor, whereby
the holster is moved in a vertical direction with respect to the
optical block. Also, the reader system incorporates a digital
processor and electronics for the actuation and control of
readings, and non-volatile digital memory for the storing of data.
It is to be understood that the reader system may incorporate
multiple processor components, with a division of processing
occurring between separate components. For example a single
processor may handle sensing and time critical tasks, while an
additional processor may control screen display, user interface
operations, communications, and additional processing.
Additionally, the reader may incorporate communication ports or
wireless connectivity, internal batteries and an internal or
external printer unit (such as a thermal printer) for the printout
of hardcopies of scan results following the reading of an assay, or
from stored memory.
[0052] With regard to the particulars of the various reader
assemblies and components, these are further detailed, below.
[0053] In some embodiments, the cartridge holster of a reader
system includes alignment features to ensure the correct insertion
and placement of the cartridge within the reader. In certain
embodiments, spring loaded dowels are located in positions
corresponding to recesses in the assay cartridge when the cartridge
is correctly localised within the holster. Upon correct insertion
of the cartridge, the dowels register with recesses in the
cartridge. This locks the cartridge in position, and ensures that
the immunoassay surface is localised at the optical plane until a
force is applied to remove said cartridge. In certain embodiments,
physical alignment features prevent the mis-insertion of the
cartridge, by blocking insertion of the cartridge at an incorrect
rotational alignment. In certain embodiments, the cartridge holster
incorporates fluidic flow channels to ensure any liquid spillages
from the cartridge or into the reader port flow along a defined
path to a spill receiver area. This area may be upon, or otherwise
connected to an additional removable cover on the underside of the
reader casing, enabling access to and cleaning of this spill area
without disassembly of the reader device.
[0054] In some embodiments, a reader system includes optically
emissive sources and corresponding optical sensors for the
recognition of cartridge insertion. These sources and sensors can
generally be placed within the cartridge holster opposite the
optics block. The positioning of these optical components
corresponds to absorptive or reflective features upon the
cartridge, generally defined on the distal surface of the cartridge
to that of the assay channels. Upon insertion of the cartridge into
the cartridge holster, the response registered by the optical
sensors varies as light emitted by the optically emissive sources
interact with the absorptive or reflective features upon the
cartridge. Analysis of the sensor response during the insertion of
the cartridge enables recognition of the cartridge movement
direction, and verification of full cartridge insertion. Additional
absorptive or reflective features may be incorporated upon the
cartridge may, encoding information relating to the identification
of cartridge type or immunoassay cartridge batch data. In this
case, the reader may also incorporate additional optically emissive
source and corresponding optical sensor components for the
registration of these features. In certain embodiments, the reader
system may incorporate optical or mechanical sensors, which
register full cartridge insertion or removal. In this case, the
reader may automatically initiate travel of the holster without
further user intervention upon full cartridge insertion or removal.
For example, the reader system may initiate a scan or move of the
holster to a rest position, upon insertion or removal of the
cartridge, respectively.
[0055] In some embodiments, a reader system includes a one
dimensional or two dimensional barcode reader, as known in the art,
within the reader casing. Generally, these register and read
barcode structures disposed upon the assay cartridge. In an
embodiment of this invention, the barcode encodes information for
the identification of cartridge type or lot data. In certain
embodiments, the barcode is a two dimensional barcode and encodes
information corresponding to any of the following: identification
of cartridge type or lot data, lot manufacture and expiry dates,
analyte names, cartridge expected response, lot parameters, peak
finding parameters, and calibration parameters for the immunoassay
cartridge batch.
[0056] In some embodiments, a reader system includes a radio
frequency identification (RFID) reader. This registers and reads
RFID chips present in or on the assay cartridge. In an embodiment
of this invention, the RFID chip encodes information relating to
any of: identification of cartridge type or lot data, lot
manufacture and expiry dates, analyte names, cartridge expected
response, lot parameters, peak finding parameters, and calibration
parameters for the cartridge lot.
[0057] In some embodiments, the motor component integrates an
encoder system which detects and reports the relative motor
actuation position. Alternatively, holster relative position may be
determined by calculation from motor speed and time of travel. In
each case, holster position may be determined with reference to
this relative measurement and signals received from particular
optical or mechanical travel sensors located relative to specific
positions in the holster travel. In the case of optical travel
sensors, the holster component incorporates beam blocking features,
which break an optical beam sensed by the optical travel sensors,
indicating holster position at these locations. Alternatively, the
holster component may incorporate reflective features, which direct
an optical beam to the optical travel sensors, indicating holster
position at these locations.
[0058] An example reader system optical block and optical paths is
schematically represented in FIGS. 15 (a) and 15 (b). In an
embodiment of the present invention, the excitation optics within
the optical block include: one or more light sources [302] and an
excitation lens [305]. In particular embodiments of the present
invention, an excitation light source may comprise any of, for
example: an inorganic light emitting diode (LED), or an organic
LED, or a laser. Generally, the light sources have emission
wavelengths compatible with the excitation spectra of
photoluminescent labels associated with the mobilizable or control
reagents of the assay. In an embodiment of the present invention,
optical excitation is derived from six surface mounted device LEDs,
each with an integrated lens which serves to partially collimate
emitted light.
[0059] Generally, one or more excitation lenses [305] are located
within the light paths of excitation, and direct light source
emitted optical energy to the surface of the immunoassay device. An
excitation lens may also act to collimate or spread excitation
light. An excitation lens may be formed of one of a variety of
optically transparent materials; including glass, fused silica or
organic polymers (for example: polymethylmethacrylate,
polycarbonate, polytetrafluoroethylene, polystyrene, or cyclic
olefin co-polymer). Generally, an excitation lens is of a light
converging form, with the design of the lens being one of, for
example: convex, bi convex, spherical, plano-convex, positive
meniscus, or aspheric. Lens parameters include focal length,
numerical aperture, material and optical coatings. These are
selected to optimise the optical design and have transparency
corresponding to the wavelengths of excitation.
[0060] In particular embodiments of the reader system, excitation
optics incorporate an absorptive or reflective plate with one or
more optical apertures [303]. The aperture plate may be coated with
a stable absorptive material to ensure scattering and reflections
are limited. Defined areas of stable diffusely reflective material
may be further coated onto the aperture plate, causing a portion of
light source emitted light to be back-reflected to light-source
monitoring photosensors. Optical apertures restrict excitation
light rays to those passing through the aperture. Each aperture is
aligned to an optical excitation path of a single corresponding
light source, and is shaped and sized to block specific light rays.
An aperture forms a specific, regular excitation area upon the
immunoassay device by blocking light corresponding to optical rays
which would illuminate sections outside this area. The placement
and size of this optical excitation area may be tuned by modifying
the corresponding aperture's position and dimensions. Apertures may
be designed to ensure that all excitation areas are regular and of
similar size. In particular, the aperture plate may be curved,
ensuring optical light paths are of similar lengths following
passing through the aperture plate. In particular embodiments,
apertures act to collimate the excitation light by selecting light
rays originating from the central, more homogenous angles of light
source emission. This may be important for subsequent optical
filtering using an interference type filter, as the pass and stop
bands of these filters are dependent on the angle of incidence of a
light beam. In certain embodiments, apertures are 0.2 mm-2 mm in
width and 0.1 mm to 1 mm in height, and each excitation area is 0.3
mm-3 mm in width and 0.2 mm to 2 mm in height.
[0061] In particular embodiments of the present invention,
excitation optics also comprise one or more optical filters [304].
An optical excitation filter shapes the spectral profile of
excitation light experienced by the immunoassay device. This filter
may act to ensure spectral separation between excitation light and
luminophore emitted light. An optical excitation filter may, for
example, be of band-pass or short-pass variety, and may operate by
interference or absorptive mechanisms. Generally, an excitation
filter is selected such that the filter pass-band corresponds to
some portion of the excitation spectrum of the photoluminescent
labels associated with the mobilizable or control reagents of the
assay, and that the filter stop-band corresponds to some portion of
the emission spectrum of these photoluminescent labels. The Stokes'
shift between a photoluminescent label's excitation and emission
spectra defines the maximum filter transition band. In an
embodiment, a short pass interference optical filter is
selected.
[0062] Generally, collection optics include: one or more collection
lenses [307] for the collection of light emitted from the
immunoassay surface; and one or more photosensors [306] for the
detection and transduction of this luminescence to an electrical
signal.
[0063] Generally, each photosensor is a device which transduces
optical energy directed at the surface of this sensor into an
electrically registered signal. Photosensors are selected to be
responsive to optical emission wavelengths of the photoluminescent
assay labels. These photosensors may be selected from, for example:
photodiodes, phototransistors, photo-resistors, charge coupled
devices, or photon multiplier tubes.
[0064] In an embodiment of the present invention, a collection lens
is located within the collection light path and collects light
emitted from an excitation area, that area of the immunoassay
surface illuminated by the excitation optical assembly, and directs
this light towards a corresponding photosensor. In a specific
embodiment of the present invention, the collection lens directs
light emitted from the full excitation area to the central portion
of a corresponding photosensor. In this case, selection of the size
of the excitation area and the integration of the optical signal
over the full excitation area is carried out to afford resilience
to local inhomogeneity in the assay materials. Also, as light is
directed towards the centre of the sensor, the system may withstand
some misalignment of the optics before light is transferred to an
area outside the active area of the photosensor. A collection lens
may be formed of one of a variety of transparent materials;
including glass, fused silica or organic polymers (e.g.,
polymethylmethacrylate, polycarbonate, polytetrafluoroethylene,
polystyrene or cyclic olefin co-polymer). Generally a collection
lens is of a light converging form, with the design of the lens
being one of, for example: convex, bi-convex, spherical,
plano-convex, positive meniscus or aspheric. Lens parameters
include focal length, numerical aperture, material and coatings.
These are selected to optimise the optical design and conform to
the wavelengths of collection.
[0065] In particular embodiments of the present invention,
collection optics also comprise one or more optical filters [308].
Such a filter is located within the optical collection path. The
optical collection filter shapes the spectral profile of collected
light prior to detection by the photosensor. This filter acts to
ensure that residual excitation light is not transmitted to the
photosensor. An optical collection filter may, for example, be of
band-pass or long-pass variety, and may operate by interference or
absorptive mechanisms. The filter specification is selected to have
a stop-band including the spectral bandwidth of the excitation
light source, following optical filtration by any excitation
filter, and a pass-band including some portion of the emission
wavelengths of the photoluminescent labels. In an embodiment, a
long pass absorptive optical collection filter is selected. In the
particular case of an interference-type collection filter, an
additional collection lens may be present in the reader device. In
this case, the first collection lens may be located within the
collection light path and collects light emitted from an excitation
area, that area of the immunoassay surface illuminated by the
excitation optical assembly, and acts to collimate or partially
collimate this light. The interference-type collection filter can
be placed between this first collection lens, and the second
collection lens. The second collection lens can direct the filtered
light towards a corresponding photosensor. In a particular
embodiment of the present invention, there may be an angular offset
between the plane of optical collection paths of the collection
optics, and the plane of optical excitation paths of the excitation
optics. The angular position of these planes and their specific
offset can be selected in order to inhibit direct reflection of
excitation light into the detector assembly. In an embodiment, as
shown in FIG. 15(a), the optical excitation plane is normal to the
cartridge surface, while detection is offset by 35 degrees.
[0066] In particular embodiments of the present invention, the
reader system is capable of selecting one of multiple optical
wavelength bands for optical excitation of the sample,
corresponding to the excitation wavelengths of one of multiple
photoluminescent labels associated with the mobilizable or control
reagents of immunoassays. Likewise, the reader system is capable of
selecting one of a variety of various optical wavelength bands for
detection of the optical signals corresponding to the emission
wavelengths of one of multiple photoluminescent labels associated
with the mobilizable or control reagents of immunoassays. In this
regard, the reader may incorporate multiple excitation sources;
consisting of multiple banks of LEDs or other excitation light
sources, each bank emitting optical radiation at a particular
central wavelength. The specific bank of excitation sources used
for optical excitation of the sample during a scan is selected with
regard to the excitation maxima of the photoluminescent labels.
Each bank of excitation light sources may have an associated
optical excitation filter and aperture plate. The banks of
excitation light sources may be placed at 90 degrees to one
another, at similar distances from the sample plane. Light emitted
from one bank of excitation light sources may be normal to the
sample plane.
[0067] In an embodiment of the reader system, as shown in FIG. 4,
two banks of LEDs [401], [402] are used with directions of emission
normal to and parallel to the sample surface. A half-reflective
mirror [403] is placed in the excitation light paths at 45 degrees
from each of the LED bank's nominal light paths, such that some
portion of light emitted from each bank is directed to produce
excitation areas upon the immunoassay surface at similar positions
and dimensions. The reader's nominal excitation wavelength is then
selected by activating only the LED bank of this wavelength.
[0068] In an alternative embodiment of the reader system, two banks
of LEDs are used, with directions of emission normal to and
parallel to the sample surface. A mechanically actuated mirror is
placed such that it may be moved into an "engaged" position within
the excitation light paths, at 45 degrees from each of the LED
bank's nominal light paths. In this case, light originating from
the LED bank with emission normal to the sample surface has its
emission blocked, while that with emission parallel to the assay
surface has its light reflected to form excitation areas on the
assay surface. Thus, actuation to the "engaged" position ensures
excitation of the sample using a first wavelength. In a
"non-engaged" position, the mirror is in neither of the optical
light paths. In this case, light originating from the LED bank with
emission normal to the sample surface forms excitation areas on the
assay surface, while light originating from the LED bank with
emission parallel to the assay surface does not reach the assay
surface. Thus, actuation to the "non-engaged" position ensures
excitation of the sample using the second wavelength.
[0069] In particular embodiments of the present invention, the
reader may select between specific wavelength ranges of sensitivity
in detection. In this regard, a mechanically actuated selection of
optical filters is present within the collection assembly, such as
a motor turned filter wheel. Selection of a particular filter
ensures that this filter lies within the collection optical path
for a particular scan. In this regard, collected light with
energies corresponding to the pass-range of this filter is
transmitted to the optical detector, determining the optical
wavelength response of the reader. The particular filter selected
in an assay scan is selected to ensure transmission of some portion
of the light emitted by the photoluminescent labels associated with
the mobilizable or control reagents of immunoassays, and exclusion
of stray excitation light. Alternatively, multiple light collection
assemblies may be present within the reader system with various
spectral sensitivities. For example, the reader system may
incorporate a second set of lens, filer and detector elements in a
mirrored layout to the previously specified assemblies. This may be
located at an angle above the plane of cartridge excitation. In
this case the collection filters may be each long-pass or bandpass
in character, and may be tuned to pass substantially different
wavelengths bands of light. By simultaneous, or temporally
separated monitoring of photodetector signals or each detector
assembly emission from multiple spectrally separated
photoluminescent labels may be distinguished within a single scan.
In this regard, the reader instrument is able to address and
separately register multiple different sets of overlapping emissive
features within a single channel.
[0070] In an embodiment of the present invention, the reader system
incorporates one or more digital processors and electronics for the
actuation and control of readings. Generally, these control the
operation of motors, optical electronic components, display
components, and scan processing. Digital processors also interpret
data entry and communications protocols. Additionally, the digital
processors control any internal digital memory; enabling the
writing, reading, search and transfer of data. The digital
processors carry out scan processing and interpretation algorithms,
and controls the various electronic components of the reader
devices.
[0071] In some embodiments, a reader system includes non-volatile
or volatile digital memory for the storing of data. Generally, such
data may include collected scan data, and corresponding patient
details and assay results; user details and passwords; events and
error logs; calibration parameters; reader settings; user interface
screens; interface and communications parameters; and reader
operation programs. This memory may consist of one of, or multiple
instances of, for example, internal flash memory, magnetic
hard-drives, and SD-card components.
[0072] In some embodiments, a reader system includes one or more
communications ports. Components and protocols are incorporated for
wired connectivity, such as universal serial bus (USB), Ethernet
(IEEE 802.3), and serial recommended standard 232 (RS-232). These
facilitate communication to devices external to the reader, such as
personal computers or mobile devices. These may also enable control
and powering of external devices, such as barcode readers or
printers. These may also facilitate connections to hospital or
laboratory information management systems. In an embodiment, this
connectivity enables remote diagnostics, firmware or software
updates and data transfer, and control of an external barcode
reader device.
[0073] In some embodiments, a reader system includes components and
protocols for external wireless access, such as by Wi-Fi (IEEE
802.11), ANT or Bluetooth. These facilitate communication to, or
control of, devices external to the reader. These may also
facilitate connections to hospital or laboratory information
management systems. In an embodiment, this connectivity enables
remote diagnostics, firmware or software updates and data
transfer.
[0074] In some embodiments, a reader system includes a printer for
the printout of hardcopies of scan results and associated audit
data following the reading of an assay, or from stored memory.
Additional printable data may include: user lists, reader settings,
events or error logs, installed calibrations, quality control
results, etc. This printer may be of a type including: thermal,
ink-jet, laser, or dot-matrix. In an embodiment, this reader is
within the reader casing and is of thermal type.
[0075] In some embodiments, a reader system is portable, being
intended for bench- or table-top point-of-care use. In an
embodiment of the present invention, the reader includes an
internal rechargeable battery, which may power the reader in
situations where the system is not connected to mains power
supplies. This battery is rechargeable, and charges while the
reader is connected to a mains power supply. Electronics and the
digital processor may monitor battery charge, reporting this to the
user, and regulating such details as: charge speed, battery
temperature, and minimum charge levels before the unit is
automatically shut down. In certain embodiments of the reader
system, batteries may be held in a removable battery pack, or be
insertable into a dedicated battery compartment by the user. In
certain embodiments, a reader system incorporates a speaker for
transmission of auditory alarms, or auditory feedback or user
actions. In certain embodiments, the reader comprises an internal
clock. This clock is generally powered by a separate, long life
battery component.
[0076] In an embodiment of the present invention, a Secure Digital
(SD) card component holds assay specific calibration data relating
to an assay cartridge batch. The SD card may be introduced into the
reader system, and the assay specific calibration data copied to
internal reader memory. In an embodiment of the present invention,
the SD card is a secure write-once, read many times form. This card
may be encoded with identification data corresponding to unique
characteristics of the particular card, enabling security of
written data and recognition of the correct card type prior to
transfer of information.
[0077] In an embodiment of the current invention, an SD card may
hold firmware or software updates for the reader device.
Alternatively, a standard SD card may be inserted into the SD card
slot, and the user may transfer saved data (such as scans, results,
settings, calibrations or quality control data) from the internal
device to the SD card for back up or subsequent transport.
[0078] In an embodiment of the present invention, the excitation
source activation and emission timings and photosensor read timings
are tuned in accordance with the positions and numbers of assay
channels within the immunoassay cartridge. These parameters may be
stored in the batch calibration file, and the excitation and read
logic of the reader system is modified with regard to the cartridge
structure. For example, in a reader system with six channels, the
system is presented with a three channel immunoassay cartridge. The
reader system is informed of the positions of the present assay
channels, and acts to only excite and read from these channels,
modifying the relevant timings accordingly.
[0079] In an embodiment of the present invention, the excitation
source emission timings and photosensor read timings are tuned such
that only one test is being excited at a specific time, ensuring
that optical crosstalk between channels is minimised.
Alternatively, multiple channels may be illuminated and read
simultaneously. However, these channels may be spatially separated
in order to ensuring that optical crosstalk between channels is
minimised.
[0080] In an embodiment of the present invention, electronic
frequency filtering is applied to the photosensor signal. The
pass-band of this electronic filter is tuned to the frequency of
the excitation source duty cycle, and serves to amplify the
detected photoluminescence while attenuating noise signals. Such
noise may be associated with constant (low frequency) light leakage
into the system, or high frequency electronic noise. Such a filter
may be comprised of multiple high-pass and low-pass electronic
filters placed in series.
[0081] In an embodiment of the present invention, a reader system
records a dark count, corresponding to detected signal from each
channel without activation of the corresponding light source for an
equivalent time to the defined excitation source emission time and
at a time close to each excitation duty cycle. In this case, the
photosensor signals may be corrected by subtraction of this dark
count from that acquired during activation of the corresponding
light source. This enables compensation for light leakage into the
device, interference from light generation within the device, or
thermal or electronic noise.
[0082] In an embodiment of this invention, time resolved detection
of photoluminescence may be employed in the reader system. In this
regard, an excitation source may be activated briefly, and
corresponding photoluminescence recording initiated some time
(generally at least tens to hundreds of nanoseconds) after the
light source has been deactivated. In this manner,
photoluminescence from long emissive lifetime (for example,
lanthanide labels with emissive lifetimes of multiple microseconds)
photoluminescent labels may be discriminated from shorter lifetime
background fluorescence (generally termed in nanoseconds).
[0083] In an embodiment of this invention, the light source optical
emission intensity is controlled and stabilised through the
cartridge scan. In this case, the excitation optics incorporates a
dedicated excitation source monitoring photosensor. The light
source emission intensity is thereby monitored by analysis of the
monitoring photosensor electronic signal Feedback of this
monitoring signal to the excitation source may ensure that the that
the emission of the excitation source remains constant across all
scans. Feedback stabilization may be carried out throughout a scan,
across each duty cycle of each light source's emission.
Alternatively, feedback stabilization may be independently carried
out for each light source prior to the commencement of each
cartridge scan. In an embodiment of this invention, the reader
incorporates a proportional-integral-derivative control algorithm
to optimally stabilise light source emission at a desired intensity
by analysis of the monitoring photosensor signal.
[0084] In some embodiments, a reader system uses an algorithm for
the detection of optical emission peaks from each optical scan.
Algorithm parameters may include such details as expected numbers
of peaks, expected peak scan positions, expected widths of peaks,
expected ranges of peak heights. Peak detection algorithms may
include background subtraction; compensating for background
fluorescence derived from the assay materials, stray background
light, unbound labelled assay materials or other sources. This may
be realised by subtracting the minima of a scan, or estimation and
subsequent subtraction of background fluorescence at the point of
the peak maximum. In an embodiment, such an estimation is carried
out by registering fluorescence levels at particular scan positions
at a defined distance to either side of the peak position, then
determining a linear fit to the background versus scan position,
and finally estimating the level of background fluorescence at a
scan position at a position corresponding to the peak maximum.
[0085] In particular embodiments of the present invention, sets of
quality controls are actualised in software to ensure that the
assay progressed in a defined manner. These may include: quality
control checks of scan data, including a check of control line
development, a check of channel clearance, and checks as to the
size and position of peaks. Additionally, controls may verify the
time of test as being within the expiry data of a particular assay.
In particular, the level of detected luminescence is characterised
at a particular scan position, defined in calibration parameters
for the assay in question, at which no capture or control zones are
present, and which generally corresponds to background
fluorescence. If the magnitude of this photoluminescence is found
to be above a certain level defined in calibration parameters for
the assay in question, the unbound luminescent materials is not
taken to have achieved full clearance, and the particular assay is
termed a "Missrun". Control zone peaks are further analysed: if
these are not found, or are of insufficient magnitude, the assay is
likewise is considered to have not fully developed, and is likewise
termed a "Missrun".
[0086] In an embodiment of the present invention, the reader
includes a calibration algorithm for the qualification or
quantification of analyte presence within an immunoassay fluid
sample. These algorithms take as input the following: calibration
parameters specific to the assay batch and peak heights as
determined by a peak detection algorithm for each of the capture
and control zones. For each analyte, the algorithm processes the
corresponding capture zone peak height, according to the
calibration parameters. Alternative algorithms may alternatively
normalize the capture zone peak height by the control zone peak
height, compensating for flow related or assay component related
variability. Generally for qualitative tests, the algorithm
compares the peak height versus a threshold value, and reports a
positive or negative result. Alternatively for quantitative tests,
the algorithm characterises the concentration of an analyte within
the test sample, according to assay specific calibration
parameters. In this case, the algorithm may report that the
concentration is greater or less than particular limits of
quantization, respectively. Finally, for semi-quantitative tests,
the algorithm characterises the concentration of an analyte within
the test sample to be within specific ranges, according to assay
specific calibration parameters. It should be understood that a
multiplex assay panel may consist of a selection of qualitative,
quantitative and semi-quantitative assays, all within a single
cartridge, being read and interpreted simultaneously.
[0087] In the case where the assay batch includes parallel tests
for a particular analyte with similar sensitivities, an alternative
calibration algorithm may take as input the following: calibration
parameters specific to the assay batch and peak heights as
determined by a peak detection algorithm for each of the capture
and control zones for each of the parallel tests. For each analyte,
the algorithm processes the corresponding capture zone peak
heights, according to the calibration parameters. Estimation error
in the quantitative or quantitative estimate of analyte presence
may be minimised by averaging multiple results, or by discarding
results with outlying values or corresponding peak heights.
[0088] In the case where the assay batch includes parallel tests
for a particular analyte with varying sensitivities and
corresponding linear ranges, an alternative calibration algorithm
may take as input the following: calibration parameters specific to
the assay batch and peak heights as determined by a peak detection
algorithm for each of the capture and control zones for each of the
parallel tests. For each analyte, the algorithm processes the
corresponding capture zone peak heights, according to the
calibration parameters. The algorithm then selects a result
predicted by one test in which the quantitative measurement is
within the linear range of the test. Additionally, where the
measurement is within the linear range of multiple tests, the
algorithm may report the result as the weighted average of the
analyte concentrations estimated from each such test.
[0089] Finally, in the case where the assay batch includes various
tests related to single or multiple clinical decisions, a secondary
algorithm may take as input the quantitative or qualitative
estimates from each individual test as provided by the calibration
algorithm. This secondary algorithm processes the various
calibration results and reports a single diagnostic result or
multiple diagnostic result.
[0090] In an embodiment of the present invention, a physically
separate quality control component, of external dimensions similar
to that of the assay cartridge is incorporated. This component
incorporates materials exhibiting specific, characterised
efficiencies of photoluminescence upon optical excitation at a
wavelength corresponding to the reader excitation source. In an
alternative embodiment of the current invention, the quality
control component may be disposed on each assay cartridge, at a
position separate from the assays. In another embodiment of the
current invention, the quality control component may be integrated
within the reader system itself. In particular embodiments of the
current invention, the quality control component may be integrated
within the reader's cartridge holster, being automatically actuated
to and from the optical plane upon removal and insertion of the
test cartridge, respectively.
[0091] In embodiments of the present invention, the quality control
component's photoluminescent areas are defined using masked
photoluminescent materials, coated non-fluorescent materials or
multilayer etched materials. Photoluminescent materials may consist
of plastics impregnated with fluorescent dyes, nanocrystals or
quantum dots.
[0092] In embodiments of the present invention, the quality control
component's photoluminescent areas may be localised at the optical
plane within the reader. Photoluminescent areas are patterned in a
defined manner, such that optical misalignments will lead to
predictable changes in scan responses.
[0093] In a first embodiment of the present invention, a processing
algorithm for the analysis of quality control component scans is
incorporated. This algorithm will compare the expected response
from fluorescent areas with those of received responses, and
validate the reader for the analysis of assays. In a second
embodiment of the present invention, the algorithm will compare the
expected response from fluorescent areas with those of received
responses, and compensate for changes in system response by the
modification of internal calibration factors. In a third embodiment
of the present invention, the algorithm compares the reader
response with the expected response from the patterned fluorescent
component. This algorithm then calculates the type and degree of
optical misalignment, such as: the direction and degree of lateral
misalignment, degree of focus or defocus, or optical system tilt.
In this case, the reader may incorporate motor driven alignment of
the optical stage. Reader algorithms analyse the quality control
component scan, and automatically adjust the position of the
optical stage for optimal system alignment.
[0094] In an embodiment of this invention, the reader incorporates
a processing algorithm for the verification of immunoassay batch
response. This algorithm analyses the response of one or more
immunoassay cartridges of the specified batch, run with control
liquids of specified concentrations. The algorithm compares
expected responses with those found from these cartridges, and
verifies the immunoassay batch as operating to a given
specification. Further, this algorithm also may conduct the
optimisation and correction of immunoassay batch specific
calibration parameters, as stored within reader memory, to
compensate for time-related changes in immunoassay
photoluminescence response. In this case, following the analysis of
one or more immunoassay cartridges of the specified batch, run with
control liquids of specified concentrations, internal calibration
parameters are then updated to provide a best-fit result to control
responses.
[0095] Embodiments of the present invention include procedures and
methods for updating reader software and firmware. In certain
embodiments, an update may be initiated by the user selecting
particular menu options of the reader's user interface. The reader
system may receive data corresponding to the compiled firmware or
software code from a variety of sources, including but not limited
to: an SD card inserted into the SD card port, a USB flash drive
inserted into a USB port, an external connected personal computer,
or a wireless connection. In certain embodiments of the reader
system, firmware or software updates have "roll-back"
functionality, affording a reset to factory settings or a previous
firmware or software version if the update does not proceed
correctly.
Test Scan Procedure
[0096] In an exemplary embodiment of the present invention, an
operation procedure for the conducting of test scans may be
summarised as follows (shown in FIG. 5): The user adds a fluid
sample to the assay cartridge, and the assay cartridge is left for
sufficient time for the assay to develop [501]. Next, the user
selects the "run test" option of the reader's user-interface [502].
The reader system lifts the cartridge holster to a cartridge access
position, opening the reader lid [201], and prompts the user to
insert the assay cartridge [503]. Upon insertion of the cartridge,
the holster is brought to a "home" position, and the user is
prompted to enter a patient identification (via the integrated text
entry device, or external barcode reader) [504]. The system carried
out a scan of the assay panel, and calculates assay results [505].
The cartridge holster is brought back to the access position,
opening the reader lid, and the user is prompted to remove the
cartridge [506]. Finally, results are displayed on the reader
screen, and are automatically saved [507].
Liquid Calibrator Scan Procedure
[0097] In an exemplary embodiment of the present invention, the
operation procedure for the conducting of liquid calibrator scans
to account for assay changes over time may be summarised as follows
(shown in FIG. 7): The user adds a "level one" liquid control
sample (with a characterised concentration of each analyte) to a
standard assay cartridge of the batch to be corrected, and the
assay cartridge is left for sufficient time for the assay to
develop. Next, the user selects the "run liquid control" option of
the reader's user-interface [701]. The reader system brings the
cartridge holster to an access position, opening the reader lid,
and prompts the user to insert the assay cartridge [702]. The
system carried out a scan of the cartridge assay panel, and
processes the raw data [703]. The cartridge holster is brought back
to the access position, opening the reader lid, and the user is
prompted to remove the cartridge [704].
[0098] If the assay batch corresponds to a quantitative assay
panel, the user is then prompted to add a "level two" liquid
control sample (with a second characterised concentration of each
analyte) to a second standard assay cartridge of this batch. The
user leaves the assay cartridge for sufficient time for the assay
panel to develop, before inserting the assay cartridge into the
reader system [705]. The system carried out a scan of the assay
panel, and processes the raw data [706]. The cartridge holster is
brought back to the access position, opening the reader lid, and
the user is prompted to remove the cartridge [707].
[0099] In the case of either a qualitative or quantitative assay
batch, results are then calculated from the processed raw data,
displayed on the reader screen, and automatically saved [708].
Scan Processing
[0100] In embodiments of the present invention, raw scan response
data acquired during either test scans or liquid control scans are
processed prior to estimation of analyte presence or concentration.
This processing calibrates the data to account for reader response,
which may be somewhat different between reader channels, or between
reader models. Following this, peaks are detected in the reader
calibrated data, according to peak detection parameters stored
within corresponding batch calibration files. Finally, the data is
checked for read or assay run errors.
[0101] In an exemplary embodiment of the present invention, the
algorithm utilised for the processing of raw scan data, following a
test scan or liquid calibrator scan, but prior to assay calibration
or calculation of liquid calibrator results may be summarised as
follows (as shown in FIG. 8): Initially the raw data, corresponding
to optical energy collected by a reader photosensor at points
across the scan length [801], is scaled by an internal reader
calibration function [802]. This function is generally of the form
of a linear equation, and normalises each point of the data set
accounts for inter- or intra-reader variability, and is stored in
non-volatile memory within the reader system itself.
[0102] Next, analysis of peaks within the reader calibrated scan
data is carried out. A scan of a single cartridge channel may
incorporate a control capture zone peak and any number of
mobilizable reagent capture peaks. Generally, each mobilizable
reagent capture peak is associated with a separate assayed analyte
within the fluid sample. An example calibrated scan is shown in
FIG. 16 for a single channel. This figure shows a peak
corresponding to the control reagent capture zone [1601], and a
peak corresponding to photoluminescence from the mobilizable
reagent capture zone [1602]. Generally, the reader system
incorporates an algorithm for the detection of optical emission
peaks, corresponding to labelled mobilizable or control reagent
capture zones from each optical scan. Algorithm parameters may
include such details as expected numbers of peaks, expected scan
positions of peaks, expected widths of peaks, and expected ranges
of peak heights. Peak detection algorithms may include background
subtraction; compensating for background fluorescence derived from
the assay materials, stray background light, unbound labelled assay
materials or other sources [1603]. This may be realised by
subtracting the minima of a scan, or estimation of background
luminescence at the scan position of the photoluminescence peak
maximum. In an embodiment, such estimation is carried out by
registering luminescence levels at particular scan positions at a
defined distance to either side of the peak position, determining a
linear fit to the background versus scan position, and then
estimating the level of background fluorescence at a scan position
corresponding to the peak maximum.
[0103] In a particular embodiment of the current invention, an
algorithm is incorporated which searches for specific peaks within
the optical scan data. The block-diagram operation of this
algorithm is shown in FIG. 8. Parameters for the peak recognition
are provided in the lot calibration file, which is generally stored
in reader memory. Initially, the algorithm processes the reader
calibrated data [802] by determining the differential of this data
[803]. Next, the differential is smoothed using a Savitsky Golay
smoothing filter [804]. Following this, each peak is detected by
searching for corresponding rising edges, falling edges and
zero-crossing points within the smoothed differential data; with
search parameters in accordance with those stored within the batch
calibration file [805]. The algorithm analysis derives the
positions of the scan maxima for each peak. These positions may be
further refined by interpolation. Subsequently, a maximum response
value is searched for in the original scan response data [806].
This search is carried out between scan positions corresponding to
the smoothed differential's rising and falling edges,
respectively.
[0104] A background luminescence baseline is estimated by fitting a
linear function between two points [807]. These points correspond
to average values of luminescence about two positions at set
distances either side of the corresponding peak maximum. Finally,
the background corrected peak height is estimated by subtracting
the value of this linear function at the position of the scan
maximum from the peak maximum itself [808]. This algorithm is
carried out for each peak in the scan data.
[0105] In particular embodiments of the present invention, prior to
running a scan, the reader software initially verifies the date of
testing as being prior to the expiry date of a particular assay,
and that the test date and time being within a set period of time
since the reader optical quality control checks, or liquid control
verification or calibration corrections of the cartridge batch in
question. In addition, sets of quality controls may be actualised
in software to verify that the assay ran in a defined manner. These
may include: quality control check of scan data, including a check
of control line development, a check of channel clearance, and
checks as to the size and position of peaks. In particular, the
level of detected luminescence is characterised at a particular
scan position, defined in calibration parameters for the assay in
question, at which no capture or control zones are present, and
which generally corresponds to background fluorescence. If this
luminescence is found to be above a certain level defined in
calibration parameters for the assay in question, the unbound
luminescent materials is not taken to have achieved full clearance,
and the particular assay is termed a "Missrun" [809]. Control zone
peaks are further analysed: if these are not found, or are of
insufficient magnitude, the assay is likewise is considered to have
not fully developed, and is likewise termed a "Missrun" [810]. In
either case, no estimate is made of analyte concentration or
presence. Following this, the algorithm verifies that a capture
zone peak was detected. If this is not the case, the algorithm
interprets the magnitude of the capture zone peak to be negligible
and a capture zone peak height value of "0" is utilised for assay
calculations [811]. Finally, the results are output for analysis by
the relevant test scan calibration algorithm, or liquid control
algorithm [812].
Algorithm for Calibration of Test Scans
[0106] In an embodiment of the present invention, the reader
includes a calibration algorithm for the qualification or
quantification of luminescence from active areas of an assay scan.
This algorithm takes as input the following: calibration parameters
specific to the assay batch and peak heights as determined by a
peak detection algorithm for each of the capture and control zones.
For each analyte, the algorithm processes the corresponding capture
zone peak height, according to the calibration parameters.
Generally for qualitative tests, the algorithm compares a scaled
peak height versus a threshold value, and reports a "positive" or
"negative" test result. Alternatively for quantitative tests, the
algorithm characterises the concentration of an analyte within the
test sample, according to assay specific calibration parameters. In
this case, the algorithm may report that the concentration is less
than a particular limits of detection, or greater or less than
particular limits of quantization, respectively. Finally for
semi-quantitative tests, the algorithm characterises the
concentration of an analyte within the test sample to be within
specific ranges, according to assay specific calibration
parameters. It should be understood that a multiplex assay panel
may consist of a selection of qualitative, quantitative and
semi-quantitative assays, all within a single cartridge, being read
and interpreted simultaneously.
[0107] In an exemplary embodiment of the present invention, the
test scan calibration algorithm may be summarised by FIG. 9. Taking
as inputs the peak height and error check data [901], and assay
calibration parameters as given in the corresponding cartridge
batch calibration file, the algorithm determines the nature of the
assay--either as a quantitative or qualitative assay [902]. In the
case of a qualitative assay, the algorithm may use a linear
equation for calibrating the capture zone peak height to account
for variations in the assay batch response over time [903]. The
coefficients of this calibration function are stored in the
corresponding cartridge batch calibration file. The scaled response
is subsequently compared with the response threshold value [905],
which is also stored in the corresponding cartridge batch
calibration file. In the case of a competitive assay, if the
response is below this threshold, the assay is reported as positive
for the analyte in question. Otherwise, the assay is reported as
negative. In the case of a sandwich assay, if the response is below
this threshold, the assay is reported as negative for the analyte
in question. Otherwise, the assay is reported as positive.
[0108] In the case of a quantitative assay, the algorithm may use a
5-parameter log-logistic equation to estimate the analyte
concentration from the capture zone peak height.
[0109] Initially, the capture zone peak height is analysed to
ensure it is within the range of the calibration 5-parameter
log-logistic curve equation [904]. If the capture zone peak height
is within the range of the calibration equation, calibration is
carried out with regard to the 5-parameter log-logistic equation,
and the estimated analyte concentration determined [906].
Otherwise, the concentration may be reported as beyond the
respective limit of quantization.
[0110] The estimated analyte concentration is next compared against
the lower concentration limit of quantization, and the upper
concentration limit of quantization, as given in the cartridge
batch calibration file for the assay in question [907]. If the
estimated concentration is outside one of these bounds, reporting
is carried out as: If the calculated concentration is below a
stipulated lower limit of quantization, the result is given as
below this limit, rather than the estimated concentration.
Conversely, if the calculated concentration is above a stipulated
upper limit of quantization, the result is given as above this
limit, rather than the estimated concentration.
[0111] Finally, the respective results, specifically run errors and
presence or concentration estimation results are reported to the
user, and these results saved [908].
Liquid Control Algorithms
[0112] In an embodiment of the present invention, the reader
includes a processing algorithm for the updating of assay specific
calibration parameters to compensate for assay-related changes in
mobilizable reagent capture zone luminescence response. This
algorithm analyses the response of one or more assay cartridges of
the specified batch run with control liquids of specified analyte
concentrations. Internal calibration parameters are then updated to
provide a best-fit result to control responses.
[0113] In an embodiment of the present invention, liquid
calibrators are used to account for minor assay and reader changes
over time. These are generally run and analysed by the
corresponding liquid control algorithm on a monthly basis for each
batch of assays. However, the required frequency of this correction
may be set by the administrator level user.
[0114] In an exemplary embodiment of the present invention, one
stable control liquid is run to recalibrate a single assay batch
for qualitative assays. This control liquid contains defined
concentrations of the analytes of interest. Generally, these
concentrations are selected to correspond to the concentration
thresholds for each of the analytes. A specific quantity of each
control liquid is run in an individual, standard cartridge of that
batch. Conversely for quantitative assays, two stable control
liquids are run to recalibrate a single assay batch. Each of the
two liquid controls contains defined concentrations of the analytes
of interest. Generally, the two concentrations of each analyte are
selected to correspond with defined low and high concentrations,
respectively, with mobilizable reagent capture zone luminescence
responses within the linear range of the 5-parameter log-logistic
curve equation. A specific quantity of each control liquid is run
in an individual, standard cartridge of that batch.
[0115] An example of a liquid control calibration adjustment
algorithm for a qualitative test is shown in FIG. 11. Upon
acquisition and processing of scan data from the "level one" liquid
control [1101], this data is verified to ensure that no miss-run
has occurred, and that the capture zone peak of each assay analyte
is within an expected range [1102]. If either of these checks is
failed, the user is prompted to repeat the liquid control
calibration adjustment.
[0116] If these checks are passed for all assays, a new linear
equation for calibrating the capture zone peak height to account
for variations in the assay batch response over time is
calculated.
[0117] Finally, the calibration file is updated with the new value
of the linear equation, and the result of the liquid calibration
displayed for the user.
[0118] An example of a liquid control calibration adjustment
algorithm for a quantitative test is shown in FIG. 12. Upon
acquisition and processing of scan data from the "level one" (e.g.,
low concentration levels) liquid control [1201], this data is
verified to ensure that no missrun has occurred. Next, the
estimated concentration of analyte is calculated; in an identical
manner to that of a standard test scan. For this purpose, the
original values for 5-parameter log logistic equation are used, as
given in the batch calibration file. This equation is prior to
corrections carried out in previous liquid controls, and this step
verifies the assay is still operating in a similar manner to that
of the freshly manufactured batch. This estimated concentration is
then validated, verifying this is within a set range, of the actual
concentration of the "level one" liquid control (also given in the
batch calibration file). If no error has occurred, and the
estimated concentration is within the expected range, the raw and
processed peak data is saved, and the "level two" liquid control
calibrator is called for. Alternatively, the user is prompted to
repeat the "level one" liquid control calibrator [1202].
[0119] Upon acquisition and processing of scan data from the "level
two" (e.g., high concentration levels) liquid control [1203], this
data is verified to ensure that no missrun has occurred. Next, the
estimated concentration of analyte is calculated; in an identical
manner to that of a standard test scan. For this purpose, the
original values for 5-parameter log logistic equation are used, as
given in the batch calibration file. This estimated concentration
is then validated, verifying this is within a set range of the
actual concentration of the "level two" liquid control (also given
in the batch calibration file). If an error has occurred, or if the
estimated concentration is outside the expected range, the user is
prompted to repeat the "level two" liquid control calibrator
[1204]. Alternatively, the algorithm calculates updated calibration
parameters as below [1205].
[0120] Generally, the calculation of liquid control updated
5-parameter log logistic parameters is carried out by the
minimisation of error residuals.
[0121] Upon calculation of optimised calibration parameters, the
batch calibration file is updated to include these parameters
[1206], and the success of the liquid controls calibration
adjustment algorithm is reported to the user [1207].
Quality Control Check
[0122] In particular embodiments of the present invention, the
reader system incorporates an optical quality control algorithm.
This algorithm compares the response from a quality control
component scan with the expected response, and thereby validates
the reader for the analysis of assays. This optical quality control
may be required to be run at specific intervals, such as to ensure
daily optical checking of the reader operation. Parameters for this
optical self check are stored in the system's internal memory, and
contain quality control verification parameters for the specific
reader system, as determined during reader verification. This
optical quality control data file comprises specific information,
such as: quality control cartridge barcode identifier, scan
positions of quality control features, and expected response range
at each quality control feature. Data corresponding to this file
may be encoded on a 2-D barcode on the quality control component,
or encoded within an RFID chip associated with the quality control
component. This data may be thereby read by the reader system, and
stored in internal memory.
[0123] In an exemplary embodiment of the present invention, a
separate bar-coded optical quality control component is used and
the optical self check procedure may be described as follows (as
shown in FIG. 6): Upon initiation of the optical self check by the
system operator [601] and insertion of the corresponding quality
control device [602], the system checks the device barcode to
ensure this corresponds with the quality control barcode stored in
the quality control parameters file in memory. If this file do not
exist, or if the cartridge is incorrect the check is halted.
[0124] The system then initiates a scan, recording the optical
photoluminescence from the quality control cartridge according to
the standard scan procedure [603]. The optical quality control
algorithm examines the detected photoluminescence response at each
of the defined quality control features scan positions and compares
these responses to the expected response range at each quality
control feature, respectively. If every response is within the
expected response range, the quality control test is reported as a
"pass". If any level falls outside these thresholds, the test is a
failure, and the scan and tests are repeated. Scans and analyses
are repeated up to three times. If one of these scans is a "pass",
this is reported. If all these fail, the quality control test is
reported as a "fail". The user is prompted to remove the QC
cartridge [604]. Results are then displayed and scan details for
the final scan are stored within reader memory [605], including:
header details (such as: time/date, cartridge identifier, user
identifier, scan parameters and final number of scans taken), a
copy of the parameter file, the detailed "pass"/"fail" status for
each quality control feature and original scan data.
[0125] The optical quality control algorithm is shown in FIG. 10,
and may be summarised as follows: The algorithm takes as inputs the
raw scan data [1001], and quality control parameters (as given in a
quality control file, specific to the reader and quality control
device). The algorithm records the detected photoluminescence
response at a scan position nominated within the quality control
data file. This position corresponds to defined quality control
feature on the quality control device. Next, the algorithm compares
the response to the expected response range for quality control
feature [1002]. This range is specific to the feature, quality
control device, and reader device; and is specified in the quality
control data file. If the response is within this range, the
optical setup is considered to be well aligned to this feature, and
the result is a "pass". Otherwise, the quality control test is a
"fail". This process is repeated for all features on all read
channels of the quality control chip [1003]. The algorithm then
outputs results corresponding to the "pass"/"fail" properties of
each test [1004].
LED Feedback Control
[0126] In an embodiment of the present invention, the excitation
source intensity is controlled through the measurement scan. This
is carried out by monitoring the excitation source intensity using
one or more dedicated photosensors. Active feedback of this
intensity signal ensures that the emission power of the excitation
source remains constant through the scan. This is important as
changes in excitation source emission power lead to direct changes
in assay photoluminescence which may create inaccuracies in
measurements of analyte presence.
[0127] In an embodiment of the present invention, the reader has
excitation sources being a bank of six similar LEDs. In this case,
excitation power variation may be caused by, for example: power
regulation variation, LED degradation over time, and thermal
responses. Of these, a thermal response is particularly notable. As
temperature increases or decreases linearly, the emission intensity
of an LED decreases or increases exponentially, respectively. In
this case, two sets of three LEDs each are optically isolated from
each other using a baffle. A single photodiode is placed within
each LED chamber, and monitors three corresponding LEDs. During the
scan of a cartridge, only one LED in each isolated set of three
LEDs is active at a specific time. Some portion of the emission
from each LED is back-scattered or reflected from the aperture
plate. This is monitored by the relevant photodiode. The LED
emission power is thus detected by observing the photodiode signal.
Monitoring and optimisation of the LED emission is carried out
prior to the acquisition of assay luminescence, for each activation
pulse of the corresponding LED. For example, optimisation of LED
power may proceed for 25 ms, and then acquisition of assay
luminescence may proceed for 15 ms for each scan data point.
Alternatively, LED stabilisation may be carried out for each LED
prior to the commencement of a scan, an LED applied voltage is
maintained at the relevant stabilized setpoint for each LED
throughout the scan.
[0128] Such optimisation of LED emission power is carried out as
follows.
[0129] The applied voltage to each of the LEDs is individually
controlled by the reader software. The voltage applied to each LED
is optimised by the LED feedback algorithm to stabilise the optical
emission power at an expected level. Initial levels for LED control
voltages [1401] and expected phototransistor response for the
desired LED emission power are stored in the reader calibration
file for each LED.
[0130] Initially, the LED is set at the initial default voltage
[1401], and the LED monitoring optical signal read [1402].
Optimisations of LED control voltages are calculated using a
control algorithm, such as a proportional-integral controller,
separately for each LED [1404].
Print-Out of Data
[0131] In an embodiment of the present invention, the reader system
incorporates a printer [110] for the printout of hardcopies of scan
results and associated audit data following the reading of an
assay, or from stored memory. A typical sample print-out following
a scan of a qualitative, six analyte, drugs of abuse panel is shown
in FIG. 13. Additional printable data may include: user lists,
reader settings, events or error logs, installed calibrations, or
quality control results.
[0132] In an embodiment of the present invention, touch-sensitive
screen elements may be provided on the touchscreen interface [102]
which initiate the printing of data, or the feeding of paper
through the printer. Further, prompts may be provided on screens in
the user interface containing printable material. These prompts
inform the user that these touch-sensitive screen elements may be
used to initiate printing of data.
System Self-Check
[0133] In embodiments of this invention, the reader incorporates
software and electronics for the initiation and interpretation of
self-check tests. Generally, these tests may be initiated
automatically at start up of the reader device, or initiated by
selection of user interface options by the user. Upon initiation of
the self-check, the reader verifies the operation of various
internal and peripheral components. For example, verification may
be carried out on memory devices, wireless communications, port
connections, motor operation, various control sub-systems,
excitation sources and detectors, printer operation, internal and
external barcode sensors, battery operation, and power supply
operation.
[0134] Further connectivity may be provided for password protected
access to device test menus incorporating these and further test
operations. These may aid an engineer in the identification and
resolution of errors occurring in device operation.
[0135] Upon encountering an error, this may be reported to the
user, and a detailed report included in an internal events or error
log file. The reader may also be prevented from initiating tests
while components have been found to be in an error state. Further
reader functionality may be likewise restricted should associated
components be detected to be in an error state.
Security
[0136] In embodiments of the present invention, various strategies
may be provided to achieve security of data. For example, various
user access levels may be provided; each with specific levels of
data access and control rights. One implementation of the current
invention has two access levels, being "administrator" and "user".
Generally, the "user" level has a subset of the "administrator"
level rights. Specifically; scan, quality control, calibration,
record review and printing functionality is available to all
operators. In addition to these rights, the "administrator" level
operator has access to additional functionality, including the
transfer of records to external devices, deletion of records,
initiating of firmware or software updates, and setting of reader
options. In an embodiment of the present invention, the
"administrator" level user can create and manage user accounts, set
requirements for entering passwords at log-on, and further set
these passwords for each user.
[0137] In an embodiment of the present invention, event audit logs
are maintained of all settings changes, scans and system warnings
and errors. Each system event is uniquely identifiable, and is
linked to the time of the event, and the user logged into the
device.
[0138] In an embodiment of the present invention, batch calibration
files may only be acquired from specific secure, non-rewritable
chips. These calibration files may be encoded, to prevent
interpretation outside the reader device.
[0139] In an embodiment of the present invention, connectivity to
the reader, and access to reader connection menus may be further
password controlled. Wireless connections from the reader device
may require setup using the reader user interface by an
"administrator" level user.
Reader Settings
[0140] In embodiments of the present invention, the reader user
interface provides settings sub-menus which enable an
"administrator" level user to set or modify various reader settings
and parameters. These may include: configuration of user IDs and
passwords; requirements for, or required frequency of, optical
quality control or liquid control scans for the running of test
scans; language settings; display brightness; configuration of
functions enabling the reader timing of assay development, and
subsequent automatic initiation of scans; setting of wireless
connections and settings; handling of error reports; volume levels
of integrated speakers; management and/or deletion of saved results
and calibrations; or activation of ports and data communication
settings.
Memory and Files
[0141] In embodiments of the present invention, the reader system
incorporates memory and a file management system for the storage of
essential data, operation parameters, and software and user
interface details. Files stored within this memory may include:
Scan files, calibration files, quality control run files, user
lists, settings and change logs, scan logs, calibration run logs,
or user logs. In order to review a potentially large volume of scan
files, search functionality may be implemented. This allows the
user to filter scans results by date, operator, patient ID or test.
Generally, original scan data and calibration parameters are
included in each scan results file, in addition to salient reader
information for quality control of scans.
[0142] In a particular embodiment of the present invention, the
reader system may store five thousand patient scan records in
internal memory, with the oldest records being deleted once new
ones are taken.
EXAMPLES
[0143] The following examples serve to further illustrate the
methods and devices of the present disclosure. These examples are
in no way intended to limit the scope of the invention.
Example 1
Six Channel Fluorescence Reader System
[0144] This example describes an example reader system for the
recording and interpretation of fluorescence from immunoassays,
according to the present invention. This reader system receives a
cartridge, comprising six vertical channels. Each channel comprises
an immunoassay configured for the detection of a specific analyte
within a single fluid sample. The reader system captures
fluorescence from the surface of each immunoassay, quantifies the
fluorescent response the single capture zone of each immunoassay,
and determines a quantitative or qualitative measurement each
analyte's presence within said sample.
[0145] A system diagram of the reader is shown in FIG. 1. In
addition, a diagram of the reader and the reader's optical
components are shown in FIGS. 2 and 3, respectively. In brief, the
reader system comprises a casing [204], incorporating a
touch-screen display and data entry device [102] an on/off switch
[101], a multicolor status indicator light [202] and a device
access port with rotating lid [201]. The casing contains a holster
receptacle [301] for receiving an external immunoassay device
[107]. Further, the reader comprises an optical system within the
casing, consisting of excitation and collection optics within a
single optical block; and an electromechanical motor system, such
as a stepper motor, whereby the holster is moved with respect to
the optical block [106].
[0146] Also, the reader system incorporates digital processors
[104] and electronics for the actuation and control of readings,
and non-volatile digital memory for the storing of data [105].
Finally, the reader incorporates communication ports and wireless
connectivity [114], a power management system [113], an internal
battery [111] and an external thermal printer [124] for the
printout of hardcopies of scan results following the reading of an
assay, or from stored memory. This printer interfaces with and is
powered by the reader via a communication port.
[0147] With regard to the particulars of the reader assembly and
components, these are further detailed, below.
[0148] Within the cartridge holster [301], spring loaded dowels are
located in positions corresponding to recesses in the assay
cartridge when the cartridge is correctly localised within the
holster. Upon correct insertion of the cartridge, the dowels
register with the recessed features of the cartridge. This locks
the cartridge in position, ensuring assays are localised at the
optical plane until a force is applied to remove said cartridge.
Also, physical alignment features prevent the mis-insertion of the
cartridge, by blocking insertion of the cartridge at an incorrect
rotational alignment.
[0149] The reader incorporates optical and mechanical sensors,
which register full cartridge insertion and removal. These sensors
are held within the cartridge holster, and their positioning
corresponds to locations which define the cartridge insertion or
removal of the cartridge. In this case, the optical sensor is a
light source and photosensor couple. This is be located in close
proximity to the mouth of the holster. Upon insertion, the
cartridge blocks propagation of light from the sensor light source
to its corresponding photosensor. The sensor registers full removal
of the cartridge by the resumption of light propagation from the
sensor light source to its corresponding photosensor. A mechanical
switch sensor is located at the base of the holster. Upon full
insertion of the cartridge into the holster, this switch is
actuated by the cartridge, enabling the detection of cartridge
insertion.
[0150] The motor component integrates an encoder system which
detects and reports the relative motor actuation position. Holster
position may be determined with reference to this signal and
signals received from particular optical travel sensors [120a]
located relative to specific positions in the holster travel. The
holster component incorporates beam blocking features, which break
an optical beam sensed by the optical travel sensors, indicating
holster position at these locations.
[0151] The reader system incorporates an internal 2D barcode camera
system [120] for the reading of barcode information encoded on the
cartridge. This camera system further incorporates a light source
for illumination of the barcode within the reader.
[0152] The excitation optics within the optical block include: six
light sources [302], an aperture plate [303], an interference
filter [304], and a single excitation lens [305].
[0153] Light sources consist of surface mounted device LEDs, each
with an integrated lens which serves to partially collimate emitted
light [302]. The LEDs have an optical emission wavelength of circa
606 nm (LOE63B; Osram GmBH). The aperture plate is a thin flat
metal shim, etched with six rectangular optical apertures which
restrict excitation light rays to those passing through the
aperture [303]. Each aperture is aligned to emission from a single
LED. In the reader system, apertures are 0.7 mm-0.8 mm in width and
0.3-0.4 mm in height, and each excitation area is 1.2 mm in width
and 0.6 mm in height.
[0154] The excitation filter [304] is located within the light
paths of excitation and centred within these light paths using a
tube construction system. The optical excitation filter shapes the
spectral profile of excitation light experienced by the immunoassay
device. This filter acts to ensure spectral separation between
excitation light and photoluminescent label emitted light. An
optical excitation filter may, for example, be of band-pass or
short-pass variety, and may operate by interference or absorptive
mechanisms. Generally, the excitation filter is selected such that
the filter pass-band corresponds to some portion of the excitation
spectrum of the photoluminescent label, and that the filter
stop-band corresponds to some portion of the emission spectrum of
the photoluminescent label. The Stokes' shift between the
photoluminescent label's excitation and emission spectra defines
the maximum filter transition band. In this case, a band-pass
interference optical filter is selected, with a central pass
wavelength of 590 nm, and a transparent bandwidth of 60 nm
(BK-590-60; Interferenzoptik GmbH).
[0155] The excitation lens [305] is located within the light paths
of excitation and centred within these light paths using a tube
construction system. This lens directs light source emitted optical
energy to the surface of the immunoassay device. The lens is
biconvex aspheric in design, and is formed of transparent cyclic
olefin co-polymer material (Zeonex 480 R).
[0156] Generally, the light sources have emission wavelengths
compatible with the excitation spectra of photoluminescent labels
associated with the mobilizable or control reagents of the assay.
Such labels may include dark red emitting fluorophores, such as
DyLight.RTM. 650 (Thermo-Fischer Scientific), Alexa Fluor.RTM. 647
(Invitrogen Corporation) or Cy5.
[0157] Collection optics within the optical block include: six
collection lenses [307], a glass absorptive filter [308] and six
photodiodes for the detection and quantification of this
luminescence [306]. Each collection lens [307] collects light from
an individual excitation area, an area of the immunoassay surface
illuminated by the excitation optical assembly; and directs this
light towards the central portion of a corresponding
photodiode[306]. All six lenses are identical in design, being
biconvex aspheric, and are formed of transparent cyclic olefin
co-polymer material (Zeonex 480 R). A single long-pass glass
absorptive optical filter [308] is used to filter excitation light
from all channels. This filter has optical pass-band beyond a
wavelength of ca. 665 nm (ZVL050; Knight Optical (UK) Ltd.). This
filters out residual reflected or scattered excitation light, and
passes light associated with fluorescence of the labelled
conjugates.
[0158] There is an angular offset between the plane of optical
collection paths of the collection optics, and the plane of optical
excitation paths of the excitation optics; with the optical
excitation plane is normal to the cartridge surface, while
detection is offset by 35 degrees. The angular position of these
planes and their specific offset are selected in order to inhibit
direct reflection of excitation light into the detector
assembly.
[0159] The reader system incorporates digital processors and
electronics for the actuation and control of readings. Generally an
operations processor [104a] controls time critical sensing and
control operations, such as the operation of motors, optical
electronic components, sensors, and scan processing. An additional
interface processor controls display and interface components,
interpreting data entry and communications protocols. Additionally,
this processor control internal digital memory [105]; enabling the
writing, reading, search and transfer of data.
[0160] The reader system incorporates non-volatile digital memory
for the storing of data [105]. Generally, such data includes
collected scan data, and corresponding patient details and assay
results; user details and passwords; events and error logs;
calibration parameters; reader settings; user interface screens;
interface and communications parameters; and reader operation
programs. This memory consists of: internal flash memory and an
internal SD-card.
[0161] The reader system incorporates communications ports [114].
In particular, components and protocols are incorporated for wired
connectivity, including USB, and Ethernet. These facilitate
communication to, and control of, devices external to the reader.
Specifically, this connectivity enables remote diagnostics,
firmware or software updates and data transfer, and control of an
external barcode reader device [115]. The reader also includes
components and protocols for external wireless access by WI-FI.
Specifically, this connectivity enables remote diagnostics,
firmware or software updates and data transfer.
[0162] The reader system incorporates an external thermal printer
[124] for the printout of hardcopies of scan results and associated
audit data following the reading of an assay, or from stored
memory. This printer is communicates and is powered by the reader
via a connection to one of the readers communication ports [114].
The reader system is portable, being intended for bench- or
table-top point-of-care use, and includes an internal battery
[111], which can power the reader in situations where the system is
not connected to a power supply [112]. This battery is
rechargeable, and recharges while the reader is connected to a
mains power supply. A power system [113] monitors battery charge,
reporting this to the user, and regulating such details as: charge
speed, battery temperature, and minimum charge levels before the
unit is automatically shut down.
[0163] In some embodiments, an SD card component holds assay
specific calibration data relating to an assay cartridge batch. The
SD card may be introduced into a reader system socket, and the
assay specific calibration data copied to internal reader memory.
SD card devices are of a secure write-once, read many times form.
Additionally, a standard SD may be inserted into the SD slot, and
the user may transfer saved data (such as scans, results, settings,
calibrations or quality control data) from the internal device to
the SD card for back up or subsequent transport.
[0164] With regard to system control, the LED emission timings and
photodiode read timings are tuned such that only one test is being
excited at a specific time, ensuring that optical crosstalk between
channels is minimised. Further, the LED emission intensity for each
LED is stabilized to a standard setpoint prior to the commencement
of each scan through the measurement scan. This is carried out by
monitoring the excitation source intensity using two dedicated
photodiodes. Active feedback of this intensity signal ensures that
the emission of the excitation source remains constant across all
scans.
[0165] Also, prior to each LED emission pulse, the reader system
also records a dark count, corresponding to detected signal without
activation of the corresponding LED. This is integrated over an
equivalent time to the LED pulse time. Following the recording of
all scan points, the received signals are corrected by subtraction
of each dark count from that acquired during activation of the
corresponding LEDs. This enables compensation for light leakage
into the device, interference from light generation within the
device, or thermal or electronic noise. The reader includes an
algorithm for the detection of optical emission peaks from each
optical scan. Algorithm parameters may include such details as
expected numbers of peaks, expected peak scan positions, expected
widths of peaks, expected ranges of peak heights. The peak
detection algorithm includes background subtraction; compensating
for background fluorescence derived from the assay materials, stray
background light, unbound labelled assay materials or other
sources. This is realised by estimation and subtraction of
background fluorescence at the point of the peak maximum. Such
estimation is carried out by registering fluorescence levels at
particular scan positions at a defined distance to either side of
the peak position, then determining a linear fit to the background
versus scan position, and then estimation of the level of
background fluorescence at a scan position at a position
corresponding to the peak maximum.
[0166] Sets of quality controls are actualised in software to
verify that an assay panel ran in a defined manner. These include:
quality control check of scan data, including a check of control
line development, a check of channel clearance, and checks as to
the size and position of peaks. Additionally, the software verifies
the time of test as being within the expiry data of a particular
assay. In particular, the level of detected luminescence is
characterised at a particular scan position, defined in calibration
parameters for the assay in question, at which no capture or
control zones are present, and which generally corresponds to
background fluorescence. If this luminescence is found to be above
a certain level defined in calibration parameters for the assay in
question, the unbound luminescent materials is not taken to have
achieved full clearance, and the particular assay is termed a
"Missrun". Control zone peaks are further analysed: if these are
not found, or are of insufficient magnitude, the assay is likewise
is considered to have not fully developed, and is likewise termed a
"Missrun".
[0167] The reader includes a calibration algorithm for the
qualification or quantification of luminescence from active areas
of an assay scan. This algorithm takes as input the following:
calibration parameters specific to the assay batch and peak heights
as determined by a peak detection algorithm for each of the capture
and control zones. For each analyte, the algorithm processes the
corresponding capture zone peak height, according to the
calibration parameters. Generally for qualitative tests, the
algorithm compares the peak height versus a threshold value, and
reports a positive or negative result. Alternatively for
quantitative tests, the algorithm characterises the concentration
of an analyte within the test sample, according to assay specific
calibration parameters. Should the estimated concentration be
outside the assay's bounds of quantization, the algorithm reports
that the concentration is greater than or less than particular
limits of quantization, respectively.
[0168] A physically separate quality control component [108], of
external dimensions similar to that of the assay cartridge is
incorporated. This component incorporates materials exhibiting
specific, characterised levels of fluorescence. The quality control
component's fluorescent areas are defined using masked fluorescent
PVC materials. The quality control component's fluorescent areas
may be localised at the optical plane within the reader.
Fluorescent areas are patterned in a defined manner, such that
optical misalignments lead to predictable changes in scan
responses. A processing algorithm for the analysis of quality
control component scans is incorporated in the reader. This
algorithm compares expected responses from fluorescent areas with
those of received responses, and validates the reader for the
analysis of assays.
[0169] The reader also includes a processing algorithm for the
verification of assay batch responses or optionally, the updating
of assay specific calibration parameters to compensate for assay
based changes in response. This algorithm analyses the response of
one or more assay cartridges of the specified batch run with
control liquids of specified concentrations. Assay responses are
verified to be within expected limits. Additionally, internal
calibration parameters may then be updated to provide a best-fit
result to control responses.
* * * * *