U.S. patent application number 09/155406 was filed with the patent office on 2002-03-14 for automatic diagnostic apparatus.
Invention is credited to CASALIN, PAOLA, CONNOLLY, PATRICIA, COUPE, NEVILLE, FRIEDLANDER, URI, LISSANDRELLO, FABIO.
Application Number | 20020031446 09/155406 |
Document ID | / |
Family ID | 27268215 |
Filed Date | 2002-03-14 |
United States Patent
Application |
20020031446 |
Kind Code |
A1 |
FRIEDLANDER, URI ; et
al. |
March 14, 2002 |
AUTOMATIC DIAGNOSTIC APPARATUS
Abstract
An automatic diagnostic apparatus (1) comprising: a controller
(3) for controlling operation of the apparatus (1) and for
processing data; a sensing system (15) operably connected to the
controller (3) for performing an assay, preferably an
electrochemical assay (more preferably an electrochemical
immunoassay), of a sample and communicating data from said assay to
said controller (3); voltage supply means for applying a potential
difference to said sensing system (15); and output means (11.23)
for communicating processed data to a user. operation of the
apparatus (1) and not processing data; a sensing system (15)
operably connected to the controller (3) for performing an assay,
preferably an electrochemical assay (more preferably an
electrochemical immunoassay), of a sample and communicating data
from said assay to said controller (3); voltage supply means for
applying a potential difference to said sensing system (15); and
output means (11, 23) for communicating processed data to a
user.
Inventors: |
FRIEDLANDER, URI; (LONDON,
GB) ; COUPE, NEVILLE; (WEST SUSSEX, GB) ;
LISSANDRELLO, FABIO; (MILANO, IT) ; CASALIN,
PAOLA; (MILAN, IT) ; CONNOLLY, PATRICIA;
(GLASGOW, GB) |
Correspondence
Address: |
JACOBSON PRICE HOLMAN & STERN
400 SEVENTH STREET N W
WASHINGTON
DC
20004
|
Family ID: |
27268215 |
Appl. No.: |
09/155406 |
Filed: |
December 2, 1998 |
PCT Filed: |
March 27, 1997 |
PCT NO: |
PCT/GB97/00888 |
Current U.S.
Class: |
422/68.1 ;
422/72; 435/287.2; 436/177; 436/178; 436/45; 436/63 |
Current CPC
Class: |
Y10T 436/111666
20150115; Y10T 436/255 20150115; G01N 33/49 20130101; G01N 35/00603
20130101; Y10T 436/25375 20150115; G01N 35/00594 20130101 |
Class at
Publication: |
422/68.1 ;
422/72; 204/403; 435/287.2; 436/45; 436/63; 436/177; 436/178 |
International
Class: |
G01N 035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 29, 1996 |
GB |
9606728.5 |
Nov 1, 1996 |
GB |
9622853.1 |
Mar 13, 1997 |
GB |
9705243.5 |
Claims
1. An automatic diagnostic apparatus comprising: a controller for
controlling operation of the apparatus and for processing data; a
sensing system operably connected Lo the controller for performing
an assay, preferably an electrochemical assay (more preferably an
electrochemical immunoassay), of a sample and communicate data from
said assay to said controller, voltage supply means for applying a
potential difference to said sensing System; and output means for
communicating processed data to a user.
2. An apparatus according to claim 1, further comprising sample
holding means for holding said sample.
3. An apparatus according to claim 2 wherein said sample holding
means comprises a container having a first base and a second base,
said second base being raised from said first base and having a
depression provided therein, such that when material comprising a
heavier component and a lighter component is placed within said
container and spun, said heavier component is forced towards said
first base and said lighter component is forced towards and onto
said second base and subsequently retained within said
depression.
4. An apparatus according to claim 2 or claim 3, wherein said
apparatus Ambler comprises a centrifuge for spinning said sample
holding means.
5. An apparatus according to any one of claims 2 to 4, wherein said
sample holding means further comprises reagent holding means.
6. An apparatus according to claim 5, wherein said reagent holding
means is a reagent cartridge comprising a body with at least one
depression therein: and a removable cover sealed over said
depression: wherein at least a reagent is provided within each of
said at least one depression and said removable cover is provided
with a bar-code on an outer side thereof, said bar-code being
usable to identify said reagent(s) and/or a diagnostic test
requiring said reagent(s).
7. An apparatus according to claim 5 or claim 6, comprising heating
means for heating said reagent holding means, said heating means
being controlled by said controller.
8. An apparatus according to any one of claims I to 7, comprising
input means for inputting data into said controller.
9. An apparatus according to claim 8 wherein said input means
comprises a keypad, and a scanner for scanning bar-code data.
10. An apparatus according to any preceding claim wherein said
apparatus has a lid and wherein said controller is operably
connected to a lid sensor for sensing whether the apparatus's lid
is open or closed.
11. An apparatus according to any preceding claim wherein said
controller is operably connected to a sample sensor for sensing
whether a sample is present.
12. An apparatus according to any preceding claim wherein said
sensing system comprises: a electrochemical immunoassay biosensor
for performing an electrochemical immunoassay of a sample; and
means for generating flow of said sample through said
biosensor.
13. An apparatus according to claim 12 wherein said biosensor
comprises., a sensor body having a sensor outlet in a part thereof,
a counter electrode having a first aperture operably connected to
said sensor outlet: a working electrode having a second aperture
operably connected to said sensor outlet; a solid phase system
operably located within said working electrode, and an inlet means
to provide a sample onto said solid phase system.
14. An apparatus according to claim 13, wherein said sensor body is
manufactured from a plastics material and said working and counter
electrodes are manufactured from an electrically conductive
plastics material.
15. An apparatus according to claim 12 or claim 13, wherein said
biosensor is that of GB-A-2289339.
16. An apparatus according to any one of claims 12 to 15 wherein
said means for generating said flow is a syringe.
17. An apparatus substantially as hereinbefore described and as
shown in the accompanying drawings.
18. Use of an apparatus according to any one of clam 1 to 17 to
diagnose and monitor a clinical condition, in particular acute
myocardial infarction.
19. A method of automatic diagnosis, the method comprising the
steps of: (a) placing a sample within an automatic diagnostic
apparatus: (b) generating instructions with a controller for
instructing a voltage supply means to apply a voltage to a sensing
system; (c) controlling said sensing system with said controller to
perform an assay, preferably an electrochemical assay (more
preferably an electrochemical immunoassay), of said sample and to
generate data for output to said controller (d) processing said
data in said controller to generate processed data, and (e)
controlling with said controller an output means to output said
processed data to a user.
20. A method of automatic diagnosis according to claim 19,
conducted with an automatic diagnostic apparats according to any
one of claims 1 to 17.
21. A disposable electrochemical immunoassay biosensor comprising:
a sensor body with a depression therein and a sensor outlet in said
depression; an apertured counter electrode provided in abutment
with one side of said depression such that said counter electrode
aperture communicates with said outlet, an apertured working
electrode provided in abutment with another side of said depression
such that said working electrode aperture communicates with said
sensor outlet, an immunoassay system provided in close proximity to
said working electrode, and an apertured sensor inlet means also
provided within said working electrode and in communication with
said immunoassay system; wherein said sensor body is manufactured
from a plastics material and said working and counter electrodes
are manufactured from an electrically conductive plastics
material.
22. A disposable electrochemical immunoassay biosensor according to
claim 21 wherein said immunoassay system is within said working
electrode.
23. A prepacked disposable diagnostic testing kit sealed with a
removable cover, the kit comprising at least one disposable sample
holding means, at least of disposable electrochemical biosensor, at
least one disposable through-flow producing means and at least one
disposable reagent cartridge, wherein said each of said at least
one disposable reagent cartridge is prepared with at least one
reagent for the performance of at least one diagnostic test and
then sealed with a removable seal.
24. A kit according to claim 23, wherein said sample holding means
comprises a container as defined in claim 3.
25. A kit according to claim 23 or claim 24, wherein said
electrochemical biosensor comprises a biosensor according to claim
21 or claim 22.
26. A kit according to any one of claims 23 to 25, wherein said
through flow producing means is a syringe.
27. A kit according to any one of claim 23 to 26, wherein said at
least one reagent cartridge is a cartridge as defined in claim
6.
28. A container having a first base and a second base, said second
base being raised from said first base and having a depression
provided therein, such that when material comprising a heavier
component and a lighter component is placed within said container
and spun, said heavier component is forced towards said first base
and said lighter component is forced towards and onto said second
base and subsequently retained within said depression.
29. A disposable reagent cartridge comprising a body with at least
one depression therein, and a removable cover sealed over said
depression; wherein at least one reagent is provided within said
depression and said removable cover is printed with a bar-code on
an outer side thereof, said bar-code being usable to identify said
reagent and/or a diagnostic test requiring that reagent.
30. A reagent cartridge according to claim 29 comprising at least
one depression filled with buffer solution.
31. A reagent cartridge according to claim 30 comprising at least
one depression filled with a dried substrate that is dissolvable by
mixing with said buffer solution.
32. A reagent cartridge according to claim 31 wherein said
substrate is naphthyl phosphate.
33. A reagent cartridge according to any of claims 29 to 32
comprising at least one depression filled with a wash solution.
34. A reagent cartridge according to any of claims 29 to 33
comprising at least one depression filled with a conjugate
solution.
35. A reagent cartridge according to claim 34 wherein said
conjugate is alkaline phosphatase, preferably having associated
therewith an antibody.
36. A disposable reagent cartridge for diagnostic testing of
myocardial infarction, the cartridge comprising a plastic body with
four depressions therein and a removable cover sealed over said
depressions; wherein a first depression is filled with a buffer
solution, a second depression is filled with a wash solution, a
third depression is filled with dried naphthyl phosphate, a fourth
depression is filled with alkaline phosphatase, preferably
associated with an antibody, and said removable cover is printed
with a barcode on an outer side thereof, said bar-code being usable
to identify said contents within one or more of the depressions
and/or the diagnostic rest.
37. A method of automatically diagnosing myocardial infarction, the
method comprising monitoring ex vivo levels of one or more
detectable cardiac marker proteins, such as any one or more of CK,
CK-MM, CK-MB, myoglobin, cardiac myosin light chain(s), Troponin T
or Troponin I, or a cardiac marker suitable for the diagnosis of
acute myocardial infarction.
38. A method according to claim 36 accomplished with the apparatus
according to any one of claims 1 to 17.
39. A conducting plastic electrode suitable for use in a diagnostic
apparatus.
40. Use of a conducting plastic electrode for an electrochemical
immunoassay.
41. An automatic diagnostic apparatus comprising: a controller for
controlling operation of the apparatus and for processing dam; a
sensing system for performing an assay of a sample, and for
communicating sensed information to the controller: and output
means for communicating processed data to the user.
42. Apparatus according E claim 42, fisher comprising means for
supplying a power or voltage signal to the sensing system.
43. Apparatus according to claim 41 or 42, wherein the controller
is operable to control at least partly the operation of the sensing
system.
44. A self contained diagnostic apparatus comprising: a centrifuge;
a system for collecting and temporarily storing material from the
centrifuge after spinning; means for transferring the collected
material to or through a sensor for performing an assay on the
collected material; means for transferring one or more other
materials to or through the sensor; an electronic controller for
controlling operation of the apparatus and for processing output
information from the sensor.
45. Apparatus according to claim 44, further comprising means for
receiving a cartridge containing said one or more ocher materials
for the sensor, and wherein said means for transferring said one or
more other materials, compress means for obtaining said materials
from the cartridge and, preferably, for temporarily storing said
material.
46. Apparatus according to claim 44 or 45, comprising multi-channel
collecting means for handling and/or creating a plurality of
samples.
47. Apparatus according to claim 44, 45 or 46 wherein the sensor is
interchangeable.
48. A method of automatic diagnosis, the method comprising the
steps of: operating a sensing system under the control of a
controller to perform an assay of a sample and to generate output
information to the controller; processing said information in said
controller, and outputting information from the controller to the
user.
49. A method according to claim 48, comprising the steps of
applying a power or voltage signal to the sensing system under the
control of the controller.
50. A carrier for carrying material in a centrifuge and having
first and second regions such that, in use, during spinning in a
centrifuge a heavier component of the material collects in one of
die regions, and a lighter component of the I collects in the other
regions, the carrier being configured to obstruct re-mixing of the
component after spinning.
51. A carrier according to claim 50, wherein the carrier has a
barrier wall between the first and second regions for obstructing
mixing of the components.
52. A carrier according to claim 51, wherein the first region
comprises a depression, a wall thereof forming the barrier
wall.
53. A disposable reagent cartridge substantially as hereinbefore
described with reference to FIGS. 6 and 7 of the accompanying
drawings.
54. A container substantially as hereinbefore described with
reference to FIG. 5 of the accompanying drawings.
55. A biosensor substantially as hereinbefore described with
reference to FIG. 8 of the accompanying drawings.
56. A kit substantially as hereinbefore described with reference to
FIG. 9 of the accompanying drawings.
57. A method of automatic diagnosis substantially as hereinbefore
described.
58. A method of automatically diagnosing myocardial infarction
substantially as hereinbefore described.
Description
[0001] This invention relates generally to an automatic diagnostic
apparats.
[0002] When a patient is treated by a physician, it is not uncommon
for the physician to take samples of body fluids to be sent on to a
laboratory for analysis. The testing often has to be done manually
and thus, inevitably, some delay is incurred in the processing of
these samples which also delays the point at which the results can
be communicated to the patient.
[0003] Even in Hospitals, where the condition of the patients can
be extremely serious, the samples still have to be sent away to an
"in-house" laboratory for testing. It can often take a matter of
hours for the results of these tests to be communicated to the
physician in charge of that patient. Accordingly, it is not
uncommon for the physician to begin treating a patient without
knowing the results of any requested testing.
[0004] In situations where the patient is seriously ill, the delay
incurred in testing samples could conceivably put the well-being of
that patient at risk.
[0005] One might consider that a suitable way to overcome his
problem would be for the physician in charge of a particular
patient to conduct the testing himself/herself, without sending the
samples away to a laboratory. However, the testing of samples is
often a complex process which must be carried Out by highly skilled
personnel if the results are to be reliable and hence of any real
use to the physician.
[0006] Therefore, there is a need in the art for an apparatus which
can be quickly and reliably operated by a user (who will sometimes
referred to as an operator) to test samples, particularly samples
obtained from patients.
[0007] In accordance with the present invention, there is provided
an automatic diagnostic apparatus comprising: a controller for
controlling operation of the apparatus and for processing data; a
sensing system operably connected to the controller for performing
an assay, preferably an electrochemical assay (more preferably an
electrochemical immunoassay). of a sample and communicating data
from said assay to said controller; optionally voltage supply means
for applying a potential difference to said sensing system; and
output means for communicating processed data to a user.
[0008] The present invention therefore provides an automated
apparatus for the testing of samples, especially patient samples.
If patient samples are tested then the results of this testing can
be made available to a physician within a mater of minutes and thus
provide an early and rapid diagnosis of a patient's condition.
[0009] In accordance with the present invention, there is also
provided a method of automatic diagnosis, the method comprising the
steps of:
[0010] (a) placing a sample within an automatic diagnostic
apparatus;
[0011] (b) optionally general instructions with a controller for
instructing a voltage supply means to apply a voltage to a sensing
system;
[0012] (c) controlling said sensing system with said controller to
perform an assay, preferably an electrochemical assay (more
preferably an electrochemical immunoassay), of said sample and to
generate data for output to said controller,
[0013] (d) processing said data in said controller to generate
processed data; and
[0014] (e) outputting said processed data to a user.
[0015] The automatic diagnostic apparatus, and the method of
operating the same, is particularly useful for the testing of acute
myocardial infarction and for the monitoring of reperfusion.
[0016] Accordingly, in accordance with a preferred embodiment of
the present invention there is provided a method of automatically
diagnosing myocardial infarction, the method comprising the steps
of: monitoring ex vivo levels of one or more detectable cardiac
marker proteins, such as any one or more of CK, CK-MM, CK-MB,
myoglobin, cardiac myosin light chain(s), Troporin T or Troponin I
or a cardiac marker suitable for the diagnosis of acute myocardial
infarction. Advantageously, this method enables a quantitative
assay to be conducted for these protein combinations.
[0017] Preferably the above method is accomplished with the above
mentioned apparatus.
[0018] However, it will of course be understood, that whilst the
present invention is preferably used for diagnostic testing for
myocardial infarction, other testing (such as for any other
clinical condition) may alternatively be conducted. Thus, the
present disclosure is not to be read as being limited to the
diagnostic testing of myocardial infarction only.
[0019] Prior to the testing of a patient's condition, it is often
necessary to separate the sample from the patient into its
constituent components. This separation is usually accomplished by
placing the sample in a test tube, for example, and spinning the
test tube at high speed in a centrifuge.
[0020] Throughout the spinning process, the sample separates into
its constituent components with the heavier components moving
towards the bottom of the test tube and the lighter components
moving towards the top of the test tube. For example, if a sample
of blood is taken and spun as described above, the heavier red
blood cells move towards the bottom of the tube and the lighter
plasma moves towards the top of the test tube.
[0021] The required portion of the sample may then be removed from
the test rube. However, the operator must be careful to ensure that
the tube is not subject to any further agitation, as such agitation
may cause the components to recombine. The operator must also be
careful to ensure that when he/she withdraws the required component
of the sample, that component is not contaminated with any of the
other component in the tube. Thus the withdrawing of separated
components from a spun sample can be problematic.
[0022] In accordance with the present invention, there is also
provided a container having a first base and a second base, said
second base being raised from said first base and having a
depression provided therein, such that when material comprising a
heavier component and a lighter component is placed within said
container and spun said heavier component is forced towards said
first base and said lighter component is forced towards and onto
said second base and subsequently retained within said
depression,
[0023] In this way, the lighter component of a separated material
may be easily withdrawn from the depression by an operator (which
may be a mechanical or an electromechanical operator). Furthermore,
the risk of that operator accidentally contaminating the lighter
component with the heavier component, either by agitating the
container or accidentally withdrawing any of the heavier component,
is significantly reduced.
[0024] United Kingdom Patent Application No. 9409449.7 (published
as GB-A-2 289 339) discloses an electrochemical through-flow
immunoassay biosensor. The biosensor comprises a solid phase
immunoassay system, a porous working electrode, a counter electrode
and a means for producing a fluid flow through the biosensor.
Whilst this arrangement produces excellent results in the
laboratory, it could suffer from a number of drawbacks when used in
a clinical environment requiring rapid analysis of a number of
samples. The most significant of these may be associated with the
fact that the biosensor must be thoroughly cleaned before it can be
used again, or used to test another sample. Conceivably, the
biosensor could be thrown away immediately after use. However, the
relatively expensive material from which the preferred biosensor
body and preferred biosensor electrodes are manufactured could
quickly make such a strategy uneconomic. In addition, the
associated equipment used with the biosensor would still have to be
thoroughly cleaned and so, any time saving attained by the disposal
of the biosensor would be counteracted by the time needed to clean
the associated equipment.
[0025] In accordance with the present invention, there is also
provided a disposable electrochemical immunoassay biosensor
comprising: a sensor body with a depression therein and a sensor
outlet in said depression; an apertured counter electrode provided
in abutment with one side of said depression such that said counter
electrode aperture communicates with said outlet, an apertured
working electrode provided in abutment with another side of said
depression such that said working electrode aperture communicates
with said sensor outlet; an immunoassay system provided in close
proximity to said working electrode, and an apertured sensor inlet
means also provided within said working electrode and in
communication with said immunoassay system; wherein said sensor
body is manufactured from a plastics material and said working and
counter electrodes are manufactured from an electrically conductive
plastics material.
[0026] Preferably, the immunoassay system is provided within the
working electrode.
[0027] Alternatively or additionally, at least one of the
electrodes may include other conventional electrode materials, such
as silver (Ag) / silver chloride (AgCl).
[0028] In this way, the biosensor of the present invention may be
manufactured from relatively inexpensive materials and, thus, a new
biosensor may be used for each test and the old biosensor may be
disposed of. The use of such a biosensor removes the need for
extensive time-consuming cleaning of the biosensor.
[0029] In accordance with another embodiment of the invention,
there is also provided a conducting plastic electrode suitable for
use in a diagnostic apparatus. The present invention also provides
for use of a conducting plastic electrode for an electrochemical
immunoassay.
[0030] In order to perform an electrochemical immunoassay with
conventional techniques, the operator would first have to prepare a
suitable reagent. The preparation of this reagent may be a
relatively complex process that would probably have to be repeated
on each occasion that a diagnostic test was to be undertaken. By
way of example, for a physician operating in his/her surgery, the
preparation of suitable reagents would require the physician to
keep stocks of necessary chemicals and to waste valuable time
making up suitable reagents,
[0031] Furthermore, each preparation of a suitable reagent by the
physician may be subject to minor variations that could cause doubt
to be cast on tests made on the same patient, but with different
sets of reagents.
[0032] Also, if a physician were to prepare a number of different
reagents for use with different diagnostic tests, it is conceivable
that these reagents could become contaminated with each other or,
more seriously, one reagent could be mistaken for another.
[0033] Thus, there is a need in the art for a suitable means for an
operator, such as a physician, to prepare consistent reagents
without having to waste time and without having to maintain a large
stock of chemicals. The means must also enable the physician to
tell quickly and easily one reagent from another.
[0034] In accordance with the present invention, there is provided
a disposable reagent cartridge comprising a body with at least one
depression therein; and a removable cover sealed over said
depression; wherein at least a reagent (which may be the same or
different) is provided within each of said at least one depression
and said removable cover is provided with a bar-code on an outer
side thereof, said bar-code being usable to identify said
reagent(s) and/or a diagnostic test requiring said reagent(s).
[0035] As mentioned above the present invention may be used for the
monitoring and the diagnosis of acute myocardial infarction.
Accordingly, the present invention provides a disposable reagent
cartridge for diagnostic testing of myocardial infarction, the
cartridge comprising a plastic body with four depressions therein
and a removable cover sealed over said depressions; wherein a first
depression is filled with a buffer solution, a second depression is
filled with a wash solution, a third depression is filled with
dried naphthyl phosphate a fourth depression is filled with dried
enzyme substrate (which may be alkaline phosphatase) (preferably
associated with an antibody, more preferably an antibody for an
antigen associated with a clinical condition - such as acute
myocardial infarction) and said removable cover is printed with a
bar-code on an outer side thereof, said bar-code being usable to
identify said contents within one or more of the depressions
(reagents) and/or the diagnostic test.
[0036] In accordance with the present inventions there is also
provided a prepacked disposable diagnostic testing kit sealed with
a removable cover, the kit comprising at least one disposable
sample holding means, at least one disposable electrochemical
biosensor, at least one disposable trough-flow producing means and
at least one disposable reagent cartridge, wherein said each of
said at least one disposable reagent cartridge is prepacked with at
least one reagent for the performance of at least one diagnostic
test and then sealed with a removable seal.
[0037] In order to perform a diagnostic test, the operator (e.g.
physician) need only tear off a removable cover from the kit and
operate the content thereof to perform the test. As the rest may be
performed with only the contents of the kit, the operator does not
have to waste time cleaning any other pieces of equipment.
[0038] Embodiments of the present invention will now be described,
by way of example only, with reference to the accompanying
drawings, in which like numerals represent like parts, and in
which:
[0039] FIG. 1 is a schematic representation of an automatic
diagnostic apparatus,
[0040] FIG. 2 is a schematic representation of a syringe and
biosensor system as shown in FIG. 1;
[0041] FIG. 3 is a schematic representation of a rack and platform
system also as shown in FIG. 1:
[0042] FIG. 4 is a flow diagram generally illustrating the
operation of the apparatus depicted in FIGS. 1, 2 and 3 under
control of a controller;
[0043] FIG. 5 is a schematic representation in cross-section of a
container:
[0044] FIG. 6 is an elevation of a reagent cartridge;
[0045] FIG. 7 is a plan view of the reagent cartridge of FIG.
6;
[0046] FIG. 8 is a schematic representation in cross-section of an
electrochemical biosensor;
[0047] FIG. 9 is a plan view of a disposable diagnostic kit;
[0048] FIG. 10 is a graph;
[0049] FIG. 11 is a series of electrophorerograms and is taken from
FIG. 5 of "A Study on the Dimeric Structure of Creatine Kinase" by
R. A. Wevers, H. P. Olthuis, J. C. C. van Niel, M. G. M van
Wilgenburg and J. B. J. Soons, published in Clinica Chimica Acta,
75 (1977) pp 377-385;
[0050] FIG. 12 is a graph and is taken from FIG. 6 of "Two-Site
Monoclonal Antibody Assays for Human Heart- and Brain- Type
Creatine Kinase" by A. P. Jackson, K. Siddle and R. J. Thompson,
published in Clinical Chemistry, Vol.30 No.7 (1984), pp 1157-1162,
and
[0051] FIG. 13 presents two graphs and is tan from FIG. 1 of "Acute
Myocardial Infarction and Coronary Reperfusion" by F. S. Apple,
published in Clinical Chemistry (Review Article), A. J. C. P.
February 1992 Volume 92, No.2.
[0052] FIGS. 14 and 15 are graphs.
[0053] FIG. 1 shows a schematic representation of an automatic
diagnostic apparatus 1. The apparatus 1 comprises a controller 3
for controlling operation of the apparatus and all of the
components thereof. The apparatus 3 is powered from a power supply
unit 5 which includes a transformer 7. A user input 9 in this case
a 16-key keypad, enables a user to input instructions and data to
the controller 3. Data and instructions for the user are displayed
on a display 11. Also provided for the input of data into the
controller 3 is a bar code scanner 13.
[0054] The controller is connected by ribbon cables 16 to a syringe
and biosensor system 15 and a rack and platform system 17. It is
these systems that manipulate samples taken from a patient and
generate readings therefrom.
[0055] Also provided for the output of dam to a user are an RS232
port 19 and a printer interface 21 which is in turn connected to a
printer 23. The RS232 port 19 may be connected to a Personal
Computer (PC) if desired.
[0056] The controller is also connected to a lid sensor 25 which
senses whether the apparatus's lid is open or closed. The
controller will not allow the apparatus to operate until the lid of
the apparatus has been closed.
[0057] FIG. 2 is a schematic representation of a syringe and
biosensor system 15 as shown in FIG. 1. As shown, the system 15
comprises three sets of syringes 27 and associated biosensors 29.
It will be appreciated of course, that the number of sets may be
varied at will. In one example, the system may be used as a means
for diagnosing myocardial infarction by varations in three
parameters. Tests for alternative ailments may require a fewer or
greater number of sets.
[0058] The biosensors 29 are electrochemical immunoassay
biosensors, and may be constructed from plastic material at a
reduced unit cost. The reduced cost of these biosensors 29 enables
them to be disposed of after each test without prohibitively
increasing the cost of operating the apparatus. The construction of
an example of the biosensor will be described later in conjunction
with FIG. 8. Conventional electrochemical immunoassay biosensors
could, of course, alternatively be provided.
[0059] The syringes 27 are, in this embodiment, simple commonplace
syringes which comprise a plunger 31 and a syringe body 33, and are
used to generate a fluid flow through the biosensors 29. It will be
understood, that whilst syringes have been described, other
flow-flow producing means may alternatively be provided. For
example, a fluid flow could conceivably be generated by drawing
fluid through the biosensors with a pump. The pump could be
connected to each of the biosensors by a disposable pipe, for
example, which could be discarded after a test has been
conducted.
[0060] As shown in FIG. 2, one end of the plunger 31 is connected
to an arm 35 of a biosensor motor 37. During use of the apparatus,
the motor 37 may be operated by a biosensor motor control board 39
to move the arm 35 and attached plunger 31 in and out of the
syringe body 33 thereby to generate a flow through a biosensor 29
attached to an opposite apertured end of the syringe body 33. The
biosensor motor control board 39 is in turn controlled by the
controller 3. Three syringe sensors 41 are provided that enable the
controller 3 to sense whether a syringe 27 and attached biosensor
29 has been correctly placed in the apparatus before the testing is
commenced.
[0061] A biosensor control board 43 under control of the controller
3 is provided. The board 43 is provided with contacts 45 for each
biosensor 29 of the apparatus and is operable under instruction of
the controller 3 to apply a voltage to each biosensor 29 as
required. The biosensor control board 43 measures a current flowing
through each biosensor 29, digitises the data and outputs it to the
controller 3. In common with other through-flow immunoassay
biosensors, the current through the biosensor is indicative of the
quantity of material-to-be-sensed in a sample under test. In this
embodiment, the controller 3 is an EPROM microcontroller with a 32
KB (kilobyte) ROM (Read Only Memory) and a 32 KB (kilobyte) RAM
(Random Access Memory), although other arrangements are
conceivable.
[0062] FIG. 3 is a schematic representation to a rack and platform
system 17 also as shown in FIG. 1 The rack and platform system
comprises a block 47 with three shaped apertures 49 each for
securely holding a reagent cartridge (not shown). A suitable
reagent cartridge will be later described in relation Lo FIGS. 6
and 7. The block also includes an electrical heater 51 which may be
used as required to heat the cartridges in the rack and platform
system 17. The block 47 is provided with a heat sensor 53 which
relays temperature data to the controller 3, which responds by
switching on or switching off the heater 51 as required.
[0063] Whilst the apparatus of FIG. 3 illustrates three apertures
for holding three cartridges, it will be appreciated that a greater
or lesser number of apertures and cartridges may alternatively be
provided, In each of the apertures 49, a cartridge sensor 55, under
control of the controller 3, is provided that senses whether a
cartridge has been correctly placed in the aperture 49. If a
cartridge is missing from one of the apertures 49, the controller 3
senses the absence of that cartridge and will not generate any data
for the sensing system associated with that cartridge position.
[0064] Also provided is a rotor motor 57 which is operable to spin
a sample container (not shown) placed in operable communication
therewith, A suitable sample container is later described in
relation to FIG. 5. The rotor motor 57 is under the control of a
motor control board 59 which is in turn controlled by the
controller 3. The rack and platform system 17 is provided with a
rotor sensor 61 which senses whether a sample container has been
correctly placed in communication with the rotor motor 57 and
communicates this information to the controller 3. The motor
control board 59 also controls a rotor index motor 63 which is
operable to align the rotor motor 57 and attached sample container
with each sensing system of the apparatus.
[0065] The rack and platform system 17 is also provided with an
up/down motor 65 and a forward/back motor 67 for moving the rack
and platform system 17 in any of the aforementioned directions. The
up/down and forward/back motors are controlled by the motor control
board 59 in the rack and platform system 17. A pair of home sensors
69 are provided which sense when the block 47 is at it's "home"
position in the forward/back and/or up/down directions. The "home"
position is when the block 47 is at its furthest point from the
sensing system in a forward/back and up/down direction The home
sensors 69 communicate position data to the controller 3.
[0066] At this juncture, it is appropriate to provide a brief
general description of the manner in which the apparatus operates
and is operated. Typically, a user decides, as a first step, which
test they wish to perform for a particular patient. An appropriate
diagnostic kit is selected and the various components removed
therefrom Next, a bar-code on the reagent cartridge (or any other
part of the kit) is read with the bar-code scanner 13 and the
cartridge is placed in the block aperture 49. In accordance with
the barcode, the controller 3 displays on the display 11 the type
of test to be conducted and sets up the apparatus vis-a-vis the
number of reagent compartments required and the testing routine to
be undertaken. The user may then visually inspect the display 11 to
check that they are indeed about to conduct the desired test.
[0067] Next, the user takes a fluid sample from a patient and
places the sample in a container provided in the kit. The cover is
then placed in operable communication with the rotor motor 57 in
the rack and platform system 17. The rack and platform system 17
is, at this stage, at its "home" position - i.e. at its furthest
position from the sensing system 15--so as to improve user
accessibility to the apparatus.
[0068] It will be apparent that bar-codes may also be provided on
any of the biosensor, container and syringe.
[0069] Next, the user takes a biosensor 29 and a syringe 27 from
the kit, and fits them together (alternatively, the biosensor and
syringe may be supplied pre-fitted together). The connected
biosensor 29 and syringe 27 are then placed in the sensing system
15 with one end of the syringe's plunger 31 in communication with
the biosensor system motor arm 35. The other end of the plunger 31
internally abuts the syringe's base. The biosensor 29 is fitted
within the sensing system 15 in such a manner that the sensing
system contacts 45 electrically connect with electrodes in the
biosensor 29. The apparatus is now primed and ready for testing the
sample.
[0070] The controller 3, via the various sensors, senses that the
container cartridge, biosensor and syringe have been correctly
placed in the apparatus and waits until the closing of the
apparatus lid has been sensed by the lid sensor 25. When the lid
has been closed the controller 3 begins the testing process,
[0071] Firstly, the controller 3 instructs the rack and platform
system motor control board 59 to operate the forward/back motor 67
so that the block 47 is withdrawn into the apparatus in such a
fashion that each cartridge container is positioned below each
biosensor 29.
[0072] Next, the controller 3 instructs the rack and platform
system motor control board 59 to operate the rotor motor 57 and so
to spin the container placed in communication therewith. The
centrifuging of the sample in the container continues at
approximately 400 revolutions per minute for some four minutes
until the sample is properly separated (ocher rotational speeds may
be adopted if desired). Whilst the sample is being spun, the
controller 3 instructs the rack and platform system motor control
board 59 to move the block 47 towards the biosensor 29 until the
tip of the biosensor protrudes into a compartment of the reagent
cartridge.
[0073] If the reagents need to be made up from constituents in the
reagent cartridge, the controller 3 may then instruct the sensing
system motor control board 39 to operate the biosensor motor 37 to
move the attached syringe plunger 31 in and out of the syringe body
33 thereby to draw fluid into and to expel fluid from the biosensor
29. In addition, the controller 3 may simultaneously instruct the
rack and platform system motor control board 59 to move the block
47 and hence the reagent cartridge up, down, forward or back so
that reagents may be mixed between compartments of the reagent
cartridge until a final desired reagent is achieved.
[0074] Optionally, the controller 3 may then instruct the biosensor
motor 37 to withdraw the plunger 31 from the syringe body 33 and
draw an amount of reagent provided in the reagent cartridge through
the biosensor 29. Simultaneously, the controller 3 may instruct the
biosensor control board 43 to apply a voltage to the biosensor 29
and measure the current flowing in the biosensor 29 If the current
is below a predetermined threshold, the controller 3 determines
that the integrity of the reagent has been maintained If, however,
the current is above the threshold, then the controller determines
that the integrity of the reagent has been compromised and the
apparatus is halted and a suitable message displayed to the user
requesting the user to replace the reagent cartridge with another
reagent cartridge. The example later described below will exhibit
such a step.
[0075] Next, the controller 3 instruct the rack and platform motor
control board 59 to move the sample container so that the container
3 is directly below the biosensor 29. The controller 3 then
instructs the rack and platform motor control board 59 to move the
container so that the biosensor 29 dips into a lighter portion of
die separated sample. The controller 3 then instructs the biosensor
motor 37 via the biosensor motor control board 39 to move the
plunger 31 and draw a quantity of separated sample into the
biosensor 29. The controller 3 then instructs the rack and platform
motor control board 59 to cause the movement of the cartridge until
Me cartridge is directly below the biosensor 29 and the biosensor
29 dips into the reagent in the reagent cartridge. The controller 3
then instructs the biosensor motor control board 39 to move the
plunger 31 and draw a quantity of reagent (which may be rehydrated
substrate) through the biosensor 29. AS an additional step, the
controller 3 may then instruct the rack and platform motor control
board 39 to cause the cartridge to be moved again so that the
biosensor 29 once more dips into the cartridge and a wash solution
is drawn through the biosensor 29 to wash any excess reagent from
the biosensor 29.
[0076] Then the controller 3 instructs the biosensor control board
43 to apply a voltage to the biosensor 29 and to measure the
produced current. The current value is then communicated to the
controller 3 as testing data via the ribbon cable 16.
[0077] The controller 3 then processes the testing data and outputs
the processed data to the user. The controller may also store the
data so that a plurality of results may be stored over time for a
particular patient. The results may then be outputted to the user
in the form of a graph via the printer 23,
[0078] One example of data collection and processing will now be
described. The current flowing through the sensor Is recorded at
precise intervals. The typical current response after applying the
potential to the sensor is a decay curve. When the substrate
reaches the sensor the decay quickly becomes an exponential growth
curve to a peak plateau. Typically, a quantity of electrical charge
is estimated initially, which is taken as the area between the two
curves the lower curve being interpolated beneath the growth curve
by examination of the decay race. The turning point where decay
becomes growth is called the start of peak and is determined by
software in the controller by looking for a trend when the average
rate of change over a number of samples reaches a threshold value.
The assay result required is the concentration of analyte which is
obtained by the formula: 1 conc = charge - b a
[0079] where a and b are parameters read from the bar code or
database.
[0080] FIG. 4 is a flow diagram generally illustrating the
operation of the apparatus depicted in FIGS. 1, 2 and 3 under
control of a controller. With reference to FIG. 4, the stages
undertaken by the apparatus are as follows.
[0081] In a first step 71, the controller 3 waits for the input of
barcode information or the input of keypad information regarding
the test to be undertaken. In a second step 73, the controller 3
uses the connected sensors to sense whether the container,
cartridge, syringe and biosensor have been correctly placed in the
apparatus. If so, then in a third step 75, the controller 3 uses
the lid sensor 25 to sense whether the lid is open or closed. If
the lid is closed, then the controller, in a fourth step 77, causes
the spinning of the sample in the container. The controller 3, in a
fifth step 79, then prepares the reagent(s) in accordance with the
inputted bar-code or keypad information. In a sixth step 81, the
controller 3 instructs the apparatus to draw separated sample
through the biosensor 29 and then, in a seventh step 83, instructs
he apparatus to draw the reagent(s) through the biosensor 29. In an
eighth step 85, the controller 3 instructs the apparatus to apply a
voltage to the biosensor 29 and, in ninth step 87, to measure the
current flowing in the biosensor 29. In a tenth step 89, the sensed
current data is digitised and outputted to the controller 3 for
processing in an eleventh step 91. In a final twelfth step 93, the
processed data is outputted to the user.
[0082] As mentioned above, the apparatus may be used to diagnose
myocardial infarction by testing three blood parameters. In such an
example, the reagent cartridge would contain the following reagents
in four separate compartments. The first, largest compartment would
contain a buffer solution. The second compartment, smaller than the
first compartment, would contain a wash solution. The third
compartment, smaller than the second compartment, would contain a
dried substrate (which in one example may be naphthyl phosphate).
The fourth compartment, smaller than the second compartment, would
contain a conjugate (which in one example may be the enzyme
Alkaline Phosphatase (ALP), preferably associated with an antibody,
more preferably an antibody for an antigen associated with a
clinical condition - such as acute myocardial infarction).
[0083] When using such a cartridge, the buffer solution would be
used to rehydrate the dried substrate and the integrity of the
substrate would then be checked by way of the biosensor 29 in Me
above described manner. The wash solution would be used to remove
any excess conjugate from the biosensor 29. In this example, the
controller 3 would instruct the apparatus to perform the above
mentioned additional step of testing, the integrity of the
rehydrated substrate by passing rehydrated substrate through the
biosensor 29 whilst applying a voltage thereto. If the detected
current is less than substantially 80 nA (nanoamperes), the
controller 3 determines that the substrate integrity is maintained,
A current level above this threshold causes the controller 3 to
determine that the substrate integrity has been compromised.
[0084] The biological processes being undertaken in the biosensor
have already been described in United Kingdom Patent Publication
No. 2 289 339 mentioned above, and so will not be described in any
great detail herein. However, to further illuminate the operation
of the present invention, a brief summary will now be given,
[0085] FIG. 5 is a schematic representation in cross-section of a
container 100. The container 100 comprises a substantially
frusto-conical outer wall 101, with a lip 103 at its narrow end.
The outer wall 101 connects at its broader end with a substantially
planar annular first base 105. A second substantially conical inner
wall 107 connects at its broader end with an inner edge of the
annular first base 105. The inner wall 107 connects at its narrow
end with a depression 109. The annular first base 105 is provided
with a lip 111 on its outer edge to enable better communication of
the rotor motor 57 with the container 100.
[0086] Prior to use of the apparatus, a sample of patient fluid is
placed within the container 100 and the container is placed in
communication with the rotor motor 57. Operation of the rotor motor
57 causes the container 100 to be spun about a central axis of the
outer wall 101. Spinning of the container 100 causes heavier
components of patient fluid to move towards the first base 105 and
lighter components to move up the inner wall 107 to the depression
109, The lighter components are then contained within the
depression 109 for facilitated removal thereof.
[0087] It will be apparent that the external configuration of the
above mentioned container 100 is not essential for the function
which the container 100 is to perform, namely the separation of
fluid components. It is the provision of a raised depression 109
that eases the separation of fluid components when centrifuged.
Thus, the container herein described is not to be read as being
limited by its external configuration or shape.
[0088] FIG. 6 is an elevation of a reagent cartridge 200. As shown,
the reagent cartridge comprises a substantially planar body 201
with four reagent compartments (203,205,207,209) depending
therefrom. The reagent compartments are open at the surface of the
planar body 201. At one end of the cartridge, there is provided a
tube 211 sized so as to accept an inlet of a biosensor therein. In
this way, the reagent cartridge and the biosensor may be fitted
together so that they occupy a smaller volume when packaged prior
to use.
[0089] FIG. 7 illustrates a top plan view of the cartridge depicted
in FIG. 6. As shown, the four reagent compartments are open at the
planar body 201 and increase in volume from a smallest compartment
203 to a largest compartment 209. Of course, the size of the
compartments may be varied at will. One end of the tube 211 is also
shown in FIG. 7. The first compartment 203 has an approximately
circular cross section and the second 205, third 207 and fourth 209
compartments have substantially elliptical cross-sections of
increasing focal spacing.
[0090] The cartridge 200 of FIGS. 6 and 7 is initially filled with
reagents for a particular diagnostic test that is to be undertaken.
An example of a set of reagents for the testing of myocardial
infarction (see earlier and later discussions). Once the
compartments have been filled with reagent, then the cartridge 200
is sealed. Sealing of the cartridge 200 may be accomplished by
adhering a removable metal foil cover to the planar body 201.
[0091] The cartridge 200 may thus be sealed and transported with a
reduced risk of reagents becoming contaminated with each other, and
with a reduced risk of reagents becoming spoiled. Immediately prior
to use, the user can remove the cover to reveal the compartments
and reagents. Alternatively, the reagent cartridge cover may be
left in place and the biosensor tip may be arranged to pierce the
cover where appropriate prior to removal of the cartridge contents,
In either case, the user is provided with a set of reagents for a
particular test without having to waste time preparing those
reagents.
[0092] The cover (not shown) of the cartridge 200 may be provided
with a bar-code. The bar-code gives information regarding the
reagents contained within the cartridge 200 and may give
information regarding the type of testing to be conducted with that
cartridge 200.
[0093] As mentioned above, the apparatus of the present invention
may be used for the diagnosis of myocardial infarction by
electrochemical immunoassay. In this case, the cartridge 200 of
FIG. 6 and FIG. 7 could be provided with the following reagents,
for example. The first compartment 203 would be filled with a
conjugate (which may be the enzyme ALP). the second compartment 205
would be filled with a dried substrate (which may be naphthyl
phosphate), the third compartment 207 would be filled with a wash
solution and the fourth compartment 209 would be filled with a
buffer solution. In use, buffer solution would be taken from the
fourth compartment 209 and added to the dried substrate to
reconstitute the substrate solution. Other enzyme-substrate pairs
are mentioned below.
[0094] FIG. 8 is a schematic representation in cross-section of an
electrochemical biosensor. With reference to FIG. 8, the biosensor
comprises a counter electrode 301, a working electrode contact 303,
a biosensor body 305, a biosensor inlet 307 and a solid phase
immunoassay site comprising a porous spacer disk 309, a porous PVDF
disk 311 and a porous graphite disk 313 as a working electrode. The
spacer disk may be a Loprosorb.TM. disk, for example, and the
graphite disk may be a Toray.TM. disk (Toray Industries,
Japan).
[0095] As mentioned above, the biosensor may be used for conducting
an immunoassay by testing parameters of a patient's blood sample.
In an example of such a test, plasma is first separated from the
patient sample--preferably by use of the container of the present
invention--and then drawn into the biosensor by way of a syringe
attached to the counter electrode 301 As the plasma passes from the
biosensor inlet 307 through the biosensor, it traverses the porous
PVDF disk 311. The porous PVDF disk 311 is impregnated with a
particular antibody and the drawing of plasma through the disk
causes the capture of an antigen under test on the disk 311.
[0096] Next, the syringe is used to draw a quantity of tracer
antibody (preferably an antibody for an antigen associated with a
clinical condition--such as acute myocardial infarction) conjugated
to alkaline phosphatase (ALP) through the biosensor. As the
conjugate passes through the PVDF disk 311, the antibody marks the
antigen captured on the disk 311.
[0097] Next, the syringe draws up a quantity of wash solution which
is used to wash any excess conjugate from the biosensor. Next, the
syringe draws up a quantity of rehydrated substrate and a potential
difference is then applied to the counter and working electrodes
301, 313 and a current is produced that is indicative of the
quantity of antigen captured on the disk 311.
[0098] This process functions due to the electrochemical nature of
the ALP and substrate. As the ALP marks the antigen captured on the
disk 311, the substrate (naphthyl phosphate) is converted to
naphthol which is drawn through the biosensor 29 and oxidised on
the porous graphite disk 313 by the potential difference applied
thereto by the working electrode contact 303. Oxidation of the
naphthol on the graphite disk 313 causes a now of electrons (ie a
current flowing in an electrical circuit comprising the counter
electrode 301, aqueous solution, the working electrode 313, the
working electrode contact 303 and connected devices) between the
working electrode contact 303 and the counter electrode 301, the
magnitude of the produced current being indicative of the quantity
of naphthol oxidised at the graphite disk 313 and hence indicative
of the quantity of antigen under test in the patient sample.
[0099] Whilst the above has been described in relation to an ALP
enzyme and naphthyl phosphate pair, it will be understood that any
enzyme-substrate combination may be used that produces a readily
oxidisable or reducible species, For example, amimophenyl phosphate
could be used as a substrate with ALP. Other examples of
enzyme-substrate pairs are beta-galactosidase with
p-Aminophenyl-beta-D-galactosidase to produce the electroactive
species amimophenyl, glucose oxidase with glucose to produce the
electroactive species hydrogen peroxide and lactate dehydrogenase
with lactate in the presence of NAD+ to produce the electroactive
species NADH.
[0100] FIG. 9 is a plan view of a disposable diagnostic kit 400.
The kit 400 is particularly suitable for use with the apparatus of
FIG. 1. As shown in FIG. 9, the kit 400 comprises a container 401
within which there is provided a disposable sample container 100, a
disposable syringe 27, a disposable biosensor 29 and a disposable
reagent cartridge 200. The kit container 401 is provided with a
removable sealed cover (not shown) which allows the sterility of
the components to be maintained up to their point of use. As
mentioned above, the kit 400 and its components may be manufactured
at a relatively low cost from plastic material.
[0101] One highly preferred embodiment of the apparatus according
to the invention will now be described. The temperature controlled
block which holds the reagent strips and acts as a support for the
centrifuge mechanism may be made from aluminum.
[0102] Each cartridge sensor may be a reflective optical device
connected to the controller for indicating the presence of a
cartridge to the microcontroller. The entire block is lifted by an
up/down motor to enable sample or reagent to be drawn from the
container or cartridge as required. This motor is mounted onto the
a base of the apparatus.
[0103] The centrifuge is mounted on a sliding mechanism and
positioned under each sensor by an index motor, The centrifuge
consists of a holder into which the container is placed by the user
and a guard ring to contain the container. A light sensing device
is placed under the holder and interfaced to the controller to
detect the presence of the sample (e.g. blood) filled container
through light level changes.
[0104] The cartridges and container are positioned by a motor in a
front to back direction. Since the sensor rip is fixed all samples
are presented to the tip by the combination of motions of the
forward/back motor, index motor and up/down motor.
[0105] The sensor system has a motor for each biosensor which
drives the syringe piston through a direct linkage, in either
direction as required by the controller. The lower part of the
drive assembly holds the biosensor in a fixed position and provides
a guard for the electrical contacts to the biosensor. An LED
indicator is positioned adjacent each biosensor to inform the user
of that biosensor's status. The electrical contacts are mounted
directly onto a signal processing board which interfaces with the
controller and provides a voltage to the biosensors during an
assay
[0106] The apparatus is operated by selecting pre-programmed
options presented by menus which appear on the display. Bar-codes
on the syringe and cartridge also provide a means of selecting test
type, batch or kit calibration data etc.. The user is required to
confirm the selection by keypad. A printer provides a hard copy of
the result in either a text or graphical format. Should an error
occur a single red LED lights and an audible alarm beeps while an
error message is displayed.
[0107] A CCD (charge coupled device) type scanner reads information
from bar-codes on the kit components such as the biosensor/syringe
and cartridge.
[0108] The kit also has a bar-code label for entering other data.
Should the label be unreadable then data is entered manually
through the keypad.
[0109] Data, sensor and control signal inputs are read by the
controller and processed to determine the control and data output
response. The apparatus is based upon a microcontroled integrated
circuit which requires external data and program memory with extra
I/O (input/output) capability. The data memory is non-volatile RAM
(NVR) so patient identity and results are preserved as a database
when the apparatus is shut down. A real time clock is resident
within the NVR to provide date and time reference during testing
The various positioning and syringe drive motors are enabled and
stepped by the controller via motor drive interfaces on the motor
drive boards. Biosensor power and data conversion is carried out by
the biosensor signal board under microcontroller supervision. Data
for printout are sent to a printer interface board and which
manages the printer operation. Block temperature is controlled by
the microcontroller via the block heater which contains a
temperature sensor and heater power control.
[0110] The apparatus and its components and signal elaboration
software operate from mains power supplies via an IEC type inlet:
The entire works and kit components are enclosed during the assay
to prevent tampering.
[0111] The biosensor contains a porous disk which is impregnated
with an assay specific material. Another disk in the biosensor
(preferably a graphite disk) is in contact with conductive plastic
parts which provide a path for current applied by the instrument
during the test. Test kit reagents and sample are successively
drawn through the cell by the action of the syringe piston. The
speed of piston movement determines the flow rate which is
controlled precisely by the controller. An air pocket inside the
syringe damps drive movement to produce a smooth liquid flow
through the cell. A potential is applied across the cell and the
current flow measured. Analysis of this current gives the result of
the assay. A bar code label is placed on the syringe to identify
the assay, calibration data, batch/lot data and expiry.
[0112] The container is filled with sufficient sample (e.g. blood)
to guarantee sufficient sample for three assays. High speeds are
employed to produce a packed cell consistency to the haematocrit
leaving plasma to be sampled. The shape of the centre of the
container allows plasma to flow to the centre of the container for
retention while keeping haematocrit in the outer region.
[0113] The reagent cartridge contains four compartments which hold
the reagent for the assay. The reagent is sealed into the strip by
a foil membrane which is pierced by the sensor tip during the
assay. A bar-code is put on the strip to identify the type of assay
and lot number.
[0114] Use of the Apparatus to assess Acute Myocardial Infarction
(AMI)
[0115] The cardiac marker proteins are proteins highly specific to
myocardial tissue which are released into serum during AMI tissue
damage. Some of these, such as CK-MB and Myoglobin, have now been
clinically validated by many studies as specific and sensitive
markers for AMI. Others e.g. Troponin are growing in popularity and
there are many groups involved in trying to discover earlier and
more sensitive markers
[0116] Table 1 (below) summarises the most popular of the markers
currently available and their main characteristics. Each of these
markers has something slightly different to offer in diagnosis and
therapy. Myoglobin with a molecular weight of 17,000 daltons is one
of the first to appear in serum or plasma after the AMI event.
However it returns to normal levels within 24 hours so is not
useful in diagnosing a patient who has presented some time after
the symptoms commenced but would help in the decision to start
thrombolytic therapy for a patient who presents early.
1TABLE 1 AMI ANALYTE PANEL Return to Normal Enzyme Rise (h) Peak
(h) (h) Notes CK 4-6 24 48 Indicator of reperfusion CK-MB 4-6 24 48
Specific for myocardium Indicator of reperfusion Myoglobin 1-3 4-8
24 Very rapid Cardiac Myosin 2 high levels 240 Related to infarct
Light Chains stable for size (cMLC) several days Elevated in
Unstable Angina Troponin 4-6 48-72 240 Highly specific (T and I)
for myocardium Indicator of reperfusion
[0117] To select the ideal parameters for a particular patient it
is necessary to consider the general time course of these proteins
in blood and their other characteristics FIG. 10 shows typical
behaviour with time of these markers in a patient's serum. In this
regard FIG. 10 shows the concentration variation in serum with time
after AMI for currently popular cardiac markers (see also FIG. 14
and FIG. 15).
[0118] The apparatus of the present invention will offer the
possibility to log and to present the parameters in this graphical
format which allows the clinician to closely follow the patient
therapy, to monitor for second infarction and to detect successful
reperfusion.
[0119] In a preferred embodiment, the first panel of instrument
will have the parameters Myoglobin and CK-MB in the now acceptable
(and increasingly preferred) mass format (.mu.g/L). Many clinicians
would traditionally request a total CK test as well as the CK-MB.
Comparing CK-MB ratio to CK (when both are U/L) is a recommended
criterion of the World Health Organisation (AMI if CK-MB
CK>4%).
[0120] The apparatus of the present invention provides a means of
determining total CK as an activity measurement or an estimate of
total CK. In this regard, the total CK content of serum is largely
composed of the isoforms CK-MM and CK-MB and the brain enzyme CK-BB
is not present in significant quantities unless there is severe
head injury. For example see FIG. 11 which is an electrophoresis
separation to illustrate CK isoforms in serum and brain extract. In
these CK electrophoretograms, a=total brain extract: b=serum sample
from a patient with an infarction: c=extract from the cortex of the
brain: d=extract from the medulla of the brain: e=extract from the
cerebellum (agarose electrophoresis 50 mM sodium barbital buffer
(pH 8.0). 85 V). Thus in effect the measurement of CK-BB is not
effective during AMI. A graph of CK-MB levels and CK-BB levels
against time in the serum of a patient during AMI in FIG. 12 also
illustrates his. In this regard, FIG. 12 is an illustration of
CK-MB and CK-BB levels measured by two site immunoassay over time
in serum from a patient suffering from AMI.
[0121] In particular FIG. 12 shows a typical curve showing increase
in serum CK-MB with time after myocardial infarction As can be
seen, both CK-MM and CK-MB elevate during AMI although the
proportion of CK-MB to MM rises due to the high amounts of CK-MB in
heart tissue. CK-MM however can also be elevated after muscle
trauma as can CK-MB to a lesser extent. In practice measurement of
CK-MM+CK-MB will effectively give the total CK in serum. Normally
total CK is measured
[0122] There are also been studies of the various isoforms in serum
and how they change with time. Three types of MM exist - namely
MM1, MM2 and MM3--and two types of MB exist--namely MB1 and M2.
These are normally quantified by high voltage electrophoresis and
fluorescent staining but some immunoassays are becoming available.
The ratios of the MB1/MB2 and MM1/MM3 also help in the early
diagnosis of AMI but some studies claim that total CK-MB
measurement is just as effective. Thee seem to be no studies of the
total CK-MB/CK-MM ratio. However, the apparatus of the present
invention would be capable of performing such a study and which in
theory would be very specific for AMI (setting a threshold ratio
for positive diagnosis).
[0123] For the preferred apparatus and cartridge of the present
invention the most convenient method will be to supply tests for
myoglobin, CK-MB and CK-MM all as mass assays (via two-site
immunoassay). It is quite possible that the users will use only
CK-MB and Myoglobin for the majority of the patients but if they
require total CK they have the option of loading to load both CK-MB
and CK-MM, cartridges in one ran. The instrument will give back
values for CK-MM, CK-MB and estimate total CK and the CK-MB total
CK ratio, Alternatively CK-MM can be measured on its own by the
instrument.
[0124] Myoglobin remains the parameter of choice for early
diagnosis of AMI - increasing in the first 1-3 hours after AMI,
peaking around 6 hours after and returning to normal within 24
hours. The current threshold for AMI with Myoglobin is >90
.mu.g/L although this could be clinically verified using the
apparatus of the present invention.
[0125] CK-MB threshold levels for AMI have been set at around 5
.mu.g/L in other manufacturer's kits.
[0126] Both CK-MB and Myoglobin can be used to monitor reperfusion.
FIG. 13 shows the difference between reperfused and non-reperfused
CK-MB levels in two patients
[0127] In this regard. FIG. 13 illustrates CK-MB measurement with
time in reperfused patients and non-reperfused patients, wherein
serial total CK (left) and CK-MB (right) values for two patients
following myocardial infarction: one successfully reperfused after
recombinant tissue-type plasminogen activator (.pi.-PA) therapy
(reperfusion): one not reperfused.
[0128] In summary, therefore, the biosensor system of the present
invention allows sensitive immunoassays to be performed in less
than 15 minutes in the ward or satellite laboratory. The present
invention is particularly of use in the areas of emergency
cardiology, critical care units and other departments concerned
with the diagnosis and treatment of acute myocardial infarction
(AMI). In a preferred embodiment, the system is capable of
performing up to three immunoassay parameters simultaneously on one
patient sample in less than fifteen minutes. In the cardiology
sector the instrument will act as a diagnostic aid for AMI and as a
means of monitoring reperfusion. In a preferred embodiment, the
three parameters offered on the first panel will be myoglobin,
CK-MB and CK-MM (for total CK).
[0129] The instrument of the present invention can be small and
light, and can be easily carried around a ward to different
locations or suitable for transportation on a small trolley.
Typically, an operator will load 3 mls of heparinised blood from
the patient into a disposable plastic rotor which is then placed in
the machine. For each parameter there is a small syringe and
reagent cartridge which will be packaged together and bar coded for
a specific test (myoglobin, CK-MB etc.). The operator uses a wand
type bar code reader to swipe the details from the side of the
syringe and the machine lights up an LED where the syringe is to be
loaded and checks on the display that the operator wants to test
this parameter for the current patient sample. This is repeated for
the cartridge, One, two or three parameters can be run for any
patient sample in one cycle of the machine.
[0130] When the lid of the instrument is closed the apparatus goes
into its routine Typically, the blood is centrifuged for 4 minutes
and during that time the instrument is priming and checking the
electrochemical biosensors.
[0131] At the end of the period typically 250 .mu.l of plasma is
aspirated directly from the disposable rotor into each of the
syringe heads. In a preferred embodiment the plasma passes through
the syringe head it traverses a porous antibody-coated membrane and
the antigen being tested is captured. The syringe then goes to the
cartridge and typically draws up 500 .mu.l of tracer antibody
conjugated to alkaline phosphatase (ALP). This passes through the
membrane marking the captured antigen.
[0132] In this preferred embodiment, the syringe next draws up wash
solution (1 ml) and then goes to the enzyme substrate well on the
cartridge. Inside the syringe head (behind the antibody-coated
membrane) is a porous electrode with a second return electrode
located further along the head. The ALP substrate used is
electrochemical in that contact with ALP converts the substrate
(naphthyl phosphate into an electroactive product (naphthol) which
is easily oxidised on the porous electrode. The assay is calibrated
for each antigen so that current at the electrode corresponds to
antigen concentration.
[0133] Typically, all three parameters are completed within 15 mins
and the instrument will display concentrations, print out
concentrations on request and also print graphs for each parameter
against time if previous values have been stored for that
patient.
[0134] In a preferred embodiment, the instrument is capable of
storing 24 values for each of the three parameters for up to a
maximum of 15 patients.
[0135] Thus, the apparatus of the present invention uses an in
vitro electrochemical assay technique to determine heart attacks by
measuring the levels of specific markers in a patient's or victim's
blood sample. The levels of markers indicate the time and severity
of the attack and also the progress of recovery.
[0136] Thus also, the apparatus of the present invention is an
instrument into which one use disposable kit components and blood
sample are loaded in order to obtain a result. The kit components
consist of an electrochemical cell and syringe, a reagent snip and
a sample holder (otherwise known as a centrifuge rotor).
[0137] The syringe and strip are bar-coded for correct
identification and assay/calibration data. Each marker requires a
specific type of cell.
[0138] The apparatus of the present invention performs the assay
automatically once the assay kit components have been loaded and
verified by the bar-code matching and the operators confirmation.
The patients blood is measured into the rotor and loaded onto the
instrument at the begs of the test. The assay is performed
automatically and results are stored internally for display or
printout as required.
[0139] It will be understood that the present invention has been
described herein by way of example only and that modifications and
additions may be made within the scope of the invention.
* * * * *