U.S. patent application number 13/716246 was filed with the patent office on 2014-06-19 for kits, compositions and methods for detecting a biological condition.
This patent application is currently assigned to LEUKODX LTD.. The applicant listed for this patent is LEUKODX LTD.. Invention is credited to YEHOSHUA BRODER, BRUCE DAVIS, YAEL HIMMEL, HARVEY LEE KASDAN, JULIEN MEISSONNIER, MICHA ROSEN, YOAV ZUTA.
Application Number | 20140170678 13/716246 |
Document ID | / |
Family ID | 50931350 |
Filed Date | 2014-06-19 |
United States Patent
Application |
20140170678 |
Kind Code |
A1 |
KASDAN; HARVEY LEE ; et
al. |
June 19, 2014 |
KITS, COMPOSITIONS AND METHODS FOR DETECTING A BIOLOGICAL
CONDITION
Abstract
The present invention provides kits, apparatus and methods for
determining a biological condition in a mammalian subject, the
method includes incubating a specimen from a patient with at least
one composition in a kit for a predetermined period of time to form
at least one reaction product, when the subject has said biological
condition, and receiving an indication of the at least one reaction
product responsive to at least one reporter element in the kit
thereby providing the indication of the biological condition in the
subject.
Inventors: |
KASDAN; HARVEY LEE;
(JERUSALEM, IL) ; MEISSONNIER; JULIEN; (JERUSALEM,
IL) ; ZUTA; YOAV; (JERUSALEM, IL) ; DAVIS;
BRUCE; (JERUSALEM, IL) ; ROSEN; MICHA;
(JERUSALEM, IL) ; HIMMEL; YAEL; (JERUSALEM,
IL) ; BRODER; YEHOSHUA; (JERUSALEM, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LEUKODX LTD. |
Jerusalem |
|
IL |
|
|
Assignee: |
LEUKODX LTD.
JERUSALEM
IL
|
Family ID: |
50931350 |
Appl. No.: |
13/716246 |
Filed: |
December 17, 2012 |
Current U.S.
Class: |
435/7.24 ;
422/554 |
Current CPC
Class: |
B01L 3/502 20130101;
B01L 2300/041 20130101; B01L 2300/0867 20130101; B01L 2200/10
20130101; B01L 3/5027 20130101; B01L 3/50273 20130101; G01N 33/6872
20130101; B01L 2200/025 20130101; G01N 33/582 20130101; B01L
2300/0654 20130101; B01L 2300/0883 20130101; B01L 2300/123
20130101; G01N 33/68 20130101; G01N 2333/70535 20130101; G01N
2800/26 20130101; B01L 3/502715 20130101; B01L 2400/0406 20130101;
G01N 33/5091 20130101; G01N 33/5302 20130101; B01L 2300/0816
20130101; G01N 33/56972 20130101; B01L 2300/0877 20130101; G01N
2333/70596 20130101; G01N 33/54386 20130101; B01L 2400/0481
20130101; G01N 33/569 20130101; B01L 2300/18 20130101 |
Class at
Publication: |
435/7.24 ;
422/554 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Claims
1-13. (canceled)
14. A method for determining a biological condition in a subject,
the method comprising: a. incubating a specimen from said subject
in the kit of claim 1 for a predetermined period of time; and b.
receiving an indication responsive to said at least one reporter
element thereby providing the indication of the biological
condition in said subject.
15. A method according to claim 14, wherein the biological
condition is selected from blood diseases such as leukemia,
thrombocytopenia, immune system disorders, local infections,
urinary tract disorders, autoimmune diseases and sepsis.
16. A method according to claim 14, wherein said indication is
quantitative.
17. A method according to claim 14, wherein the sample is of a
volume of less than 200 microliters (.mu.L).
18. A method according to claim 14, wherein the method is completed
within twenty minutes.
19. A method according to claim 18, wherein the method is completed
within fifteen minutes.
20. A method for determining a biological condition in a mammalian
subject, the method comprising: a. incubating a specimen from the
subject with at least one composition in a kit for a predetermined
period of time to form at least one reaction product, when said
subject has said biological condition; and b. receiving an
indication of said at least one reaction product responsive to at
least one reporter element in said kit thereby providing the
indication of the biological condition in said subject.
21. An automated method of determining the presence or absence of
sepsis in a subject, comprising: a. contacting a blood sample from
the subject with a fluorescently-labeled binding moiety specific to
a sepsis marker, wherein the volume of the blood sample is 50 .mu.L
or smaller; b. detecting the presence, absence or level of the
binding moiety in the sample, thereby determining the presence or
absence of sepsis in the subject within twenty minutes.
22. The method of claim 21, wherein the sepsis marker is CD64.
23. The method of claim 21, wherein the sepsis marker is CD163.
24. The method of claim 21, further comprising contacting the blood
sample with a second fluorescently-labeled binding moiety specific
for a second sepsis marker.
25. The method of claim 24, wherein the sepsis marker is CD64 and
the second sepsis marker is CD163.
26. The method of claim 21, wherein the binding moiety is an
antibody.
27. The method of claim 21, wherein the detecting step is performed
in a device capable of receiving the sample and capable of
detecting the binding moiety.
28. The method of claim 24, further comprising the step of
calibrating the device by detecting a population of the
fluorescently-labeled particles.
29. The method of claim 28, wherein said particles comprise the
same fluorescent label as the fluorescently-labeled binding
moiety.
30. The method of claim 29, further comprising a second population
of particles that comprise the same fluorescent label as the second
fluorescently-labeled binding moiety.
31. The method of claim 28, further comprising performing an
internal calibration after said detecting said
fluorescently-labeled binding moiety.
32. The method of claim 31, wherein the calibration is completed in
less than 5 minutes.
33. The method of claim 32, wherein the particles are
microbeads.
34. The method of claim 21, wherein said method is performed in
less than 15 minutes.
35. The method of claim 31, wherein the particles are
microbeads.
36. The method of claim 21, further comprising the step of
determining the presence of at least one cell population in the
sample that is not bound by the binding moiety, thus providing an
internal negative control for the sample.
37. A disposable kit for rapid evaluation of a biological condition
in a subject, the kit comprising: a static disposable cartridge
adapted to receive a biological specimen from said subject, said
cartridge including at least one treatment composition chamber
adapted to house at least one composition, a treatment chamber
comprising at least one of a vortexing element and tortuous mixing
means adapted to provide a rapid reaction of said specimen, said at
least one composition to form a reaction product, when said subject
has said biological condition, a detection chamber adapted to
receive said reaction product to provide an indication of said
reaction product indicative of said biological condition, and a
plurality of valveless microfluidic channels, each adapted to
convey at least one of said at least one treatment composition,
said reaction product, at least one detector moiety and said
reporter element between any two of said chambers, wherein said
chambers, elements and channels are activated according to a
predefined sequence.
38. A kit according to claim 37, further comprising instructions
for obtaining an indication of the biological condition by using
the kit.
39. A kit according to claim 37, wherein said disposable cartridge
comprises at least one of the following elements: a reservoir; a
pump; a conduit; a miniaturized flow cell; a transport channel; a
reading channel; a microfluidic element; a compressed gas holding
element; a compressed gas releasing element; a nozzle element; and
a mixing element.
40. A kit according to claim 39, wherein said disposable
microfluidics cartridge comprises at least two of the elements.
41. A kit according to claim 40, wherein said disposable
microfluidics cartridge comprises at least three of the
elements.
42. A kit according to claim 37, wherein said at least one
composition disposed in said cartridge comprises at least one of:
at least one target antibody; at least one positive control
identifying antibody; and at least one negative control identifying
detection moiety.
43. A kit according to claim 37, wherein said at least one
composition disposed in said cartridge comprises at least one
reference composition comprising at least one of: a target signal
reference composition; and a reference identifier composition.
44. A kit according to claim 37, wherein said at least one
composition disposed in said cartridge comprises: a positive
control moiety; and a negative control moiety.
45. A kit according to claim 37, wherein said at least one
composition comprises a sepsis biomarker.
46. A kit according to claim 45, wherein said biomarker comprises
at least one of CD64 and CD163.
47. A kit according to claim 37, wherein said predefined sequence
is adapted to occur within fifteen minutes.
48. A kit according to claim 47, wherein said rapid evaluation is
adapted to occur within fifteen minutes.
49. A kit according to claim 37, wherein the biological condition
is selected from blood diseases such as leukemia, thrombocytopenia,
immune system disorders, local infections, urinary tract disorders,
autoimmune diseases and sepsis.
50. A kit according to claim 49, wherein the biological condition
is sepsis.
51. A kit according to claim 37, wherein said indication is a
visual indication.
52. A kit according to claim 50, wherein said visual indication is
indicative of said biological condition.
53. A kit according to claim 51, wherein said visual indication is
provided within fifteen minutes.
54. A kit according to claim 37, wherein the sample of said
biological specimen is of a volume of less than 200 microliters
(.mu.L).
55. A kit according to claim 37, wherein said at least one
composition is of a volume of less than 200 microliters (.mu.L).
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The disclosures of the co-pending US Provisional Patent
Application to Kasdan, et al, filed on Nov. 17, 2012, and titled
"Kits, Compositions and Methods for Detecting a Biological
Condition" and the co-pending US Provisional Patent Application to
Kasdan, et al, filed on Nov. 17, 2012, and titled "Kits,
Compositions and Methods for Rapid Chemical Detection" are
incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to apparatus and
methods for detecting a biological condition, and more specifically
to methods and apparatus for detecting a biological condition in
small fluid samples.
BACKGROUND OF THE INVENTION
[0003] There are numerous medical conditions which are hard to
diagnose. Often diagnosis by a physician is based on the
physician's observation of combinations of symptoms in a patient.
This sometimes leads to misdiagnosis. Furthermore, the patient's
response to a treatment, whether drug or other modality is often
followed up by physician's observation.
[0004] Many laboratory tests are performed in the diagnostic arena
on a bodily specimen or fluid to determine a biological condition
in a patient. However, these tests are performed off-line in
diagnostic laboratories. Often, the laboratory services are only
provided during a single 8-hour shift during the day and tend to be
labor intensive. Some prior art publications in the field include,
inter alia, U.S. Pat. No. 8,116,984, US2006215155 and
US2012187117.
[0005] Despite the inventions mentioned hereinabove, there still
remains an unmet need to provide improved apparatus and methods for
detecting and diagnosing biological conditions in a patient.
SUMMARY OF THE INVENTION
[0006] It is an object of some aspects of the present invention to
provide improved apparatus and methods for detecting and diagnosing
biological conditions in a patient.
[0007] In some embodiments of the present invention, improved
methods, apparatus and kits are provided for detecting and
diagnosing a biological condition in a patient.
[0008] In other embodiments of the present invention, a method and
kit is described for providing rapid detection of biological
moieties in a sample from a patient.
[0009] In further embodiments of the present invention, a method
and kit is disclosed for providing detection of biological moieties
in a small fluid sample from a patient.
[0010] There is thus provided according to an embodiment of the
present invention, a kit for evaluating a biological condition in a
patient, the kit comprising; [0011] a) a disposable element for
receiving a biological specimen and for combining said specimen
with at least one composition; [0012] b) at least one composition
comprising at least one detector moiety adapted to react with said
specimen to form a reaction product, when said patient has said
biological condition; and [0013] c) at least one reporter element
adapted to provide an indication of reaction product thereby
providing the indication of the biological condition.
[0014] Additionally, according to an embodiment of the present
invention, the kit further comprises; [0015] d) instructions for
using the kit.
[0016] Furthermore, according to an embodiment of the present
invention, the disposable element is a disposable cartridge.
[0017] Moreover, according to an embodiment of the present
invention, the disposable cartridge is a disposable microfluidics
cartridge.
[0018] Additionally, according to an embodiment of the present
invention, the disposable microfluidics cartridge comprises at
least one of the following elements: [0019] a) a reservoir; [0020]
b) a pump; [0021] c) a valve; [0022] d) a conduit; [0023] e) a
motor; [0024] f) a miniaturized flow cell; [0025] g) a transport
channel; [0026] h) a microfluidic element; [0027] i) a compressed
gas holding element; [0028] j) a compressed gas releasing element;
[0029] k) a nozzle element; [0030] l) a mixing element; [0031] m) a
bellows element; [0032] n) software adapted to activate said
elements according to a specific sequence; and [0033] o) hardware
to activate said elements according to a specific sequence.
[0034] Additionally, according to an embodiment of the present
invention, the disposable microfluidics cartridge comprises at
least two of the elements.
[0035] Additionally, according to an embodiment of the present
invention, the disposable microfluidics cartridge comprises at
least three of the elements.
[0036] Additionally, according to an embodiment of the present
invention, the disposable microfluidics cartridge comprises at
least four of the elements.
[0037] Additionally, according to an embodiment of the present
invention, the disposable microfluidics cartridge comprises at
least five of the elements.
[0038] Additionally, according to an embodiment of the present
invention, the disposable microfluidics cartridge comprises at
least ten of the elements.
[0039] Additionally, according to an embodiment of the present
invention, the disposable microfluidics cartridge comprises at
least twenty of the elements.
[0040] Additionally, according to an embodiment of the present
invention, the disposable microfluidics cartridge comprises at
least thirty of the elements.
[0041] According to an embodiment of the present invention, the
microfluidics kit is configured to provide the rapid indication
with one hour.
[0042] According to another embodiment of the present invention,
the micro fluidics kit is configured to provide the rapid
indication with thirty minutes.
[0043] According to another embodiment of the present invention,
the micro fluidics kit is configured to provide the rapid
indication with fifteen minutes.
[0044] According to another embodiment of the present invention,
the micro fluidics kit is configured to provide the rapid
indication with ten minutes.
[0045] According to another embodiment of the present invention,
the micro fluidics kit is configured to provide the rapid
indication with five minutes.
[0046] According to another embodiment of the present invention,
the micro fluidics kit is configured to provide the rapid
indication with one minute.
[0047] According to another embodiment of the present invention,
the micro fluidics kit is configured to provide the rapid
indication with thirty seconds.
[0048] According to another embodiment of the present invention,
the micro fluidics kit is configured to provide the rapid
indication with ten seconds.
[0049] According to another embodiment of the present invention,
the micro fluidics kit is configured to provide the rapid
indication with one second.
[0050] There is thus provided according to an embodiment of the
present invention, a microfluidics assay kit for performing a rapid
biological assay, the kit comprising; [0051] a) a disposable
element comprising a reactant, the disposable element being adapted
to receive a sample comprising a biological entity and for
combining said reactant with said biological entity to form a
reaction product; and [0052] b) at least one reporter element
adapted to provide a rapid indication of disappearance of said
reactant thereby providing rapid assay of the biological
entity.
[0053] There is thus provided according to an embodiment of the
present invention, a microfluidics assay kit for performing a rapid
assay of a biological entity, the kit comprising; [0054] a) a
disposable element comprising a reactant, the disposable element
being adapted to receive a sample comprising the biological entity
and for combining said reactant with said biological entity to form
a reaction product; and [0055] b) at least one reporter element
adapted to provide a rapid indication of appearance of said
reaction product thereby providing rapid assay of the biological
entity.
[0056] There is thus provided according to an embodiment of the
present invention, a composition for evaluating a biological
condition, the composition comprising; [0057] a. a sample
composition comprising at least one of; [0058] i. a bodily specimen
comprising a target moiety; [0059] ii. a positive control moiety;
and [0060] iii. a negative control moiety; [0061] b. a detection
composition comprising at least one of; [0062] i. at least one
target antibody; [0063] ii. at least one positive control
identifying antibody; and [0064] iii. at least one negative control
identifying detection moiety or characteristic; and [0065] c. at
least one reference composition comprising at least one of; [0066]
i. a target signal reference composition; and [0067] ii. a
reference identifier composition.
[0068] There is thus provided according to another embodiment of
the present invention a composition for evaluating a biological
condition, the composition comprising; [0069] a. a sample
composition comprising at least one of; [0070] iii. a bodily
specimen comprising a target moiety; [0071] iv. a positive control
moiety; and [0072] v. a negative control moiety; [0073] b. an
antibody composition comprising at least one of; [0074] vi. at
least one target antibody (CD64 antibody); [0075] vii. at least one
positive control identifying antibody (CD163); and [0076] viii. at
least one negative control identifying antibody or characteristic;
and [0077] c. at least one reference composition comprising at
least one of; [0078] ix. a target signal reference composition; and
[0079] x. a reference identifier composition.
[0080] Additionally, according to an embodiment of the present
invention, the composition further comprises at least one
conditioning moiety comprising; [0081] d. at least one lysis
reagent; and [0082] e. at least one diluent.
[0083] Furthermore, according to an embodiment of the present
invention, the biological condition is selected from a group
consisting of blood diseases such as leukemia, thrombocytopenia
immune system disorders, local infections, urinary tract disorders,
autoimmune diseases and sepsis.
[0084] Moreover, according to an embodiment of the present
invention the bodily specimen is selected from a group consisting
of blood, serum, plasma, urine, saliva, cerebrospinal fluid (CSF),
serous fluid, peritoneal fluid and synovial fluid.
[0085] According to another embodiment of the present invention,
the target moiety includes a CD64 surface antigen on
neutrophils.
[0086] Additionally, according to a further embodiment of the
present invention, the positive control moiety includes monocytes
and the negative control includes lymphocytes.
[0087] Additionally, according to an embodiment of the present
invention, the target moiety is CD64 on neutrophils, the positive
control moiety includes CD64 expression on monocytes, and the
negative control moiety includes lymphocytes without CD64
expression.
[0088] Further, according to an embodiment of the present
invention, the target indicator is bound to a signaling moiety on
the at least one target antibody.
[0089] Yet further, according to an embodiment of the present
invention, the at least one reference composition includes
beads.
[0090] Additionally, according to an embodiment of the present
invention, the beads include polystyrene microbeads.
[0091] Moreover, according to an embodiment of the present
invention, the target antibody reference composition includes a
first fluorescent signal and the reference identifier composition
includes a second fluorescent signal.
[0092] Furthermore, according to an embodiment of the present
invention, the first fluorescent signal includes FITC and the
second fluorescent signal includes Starfire Red fluor.
[0093] There is thus provided according to an embodiment of the
present invention, a method of quantifying a biomarker in a sample,
comprising; [0094] a. contacting the sample with a
fluorescently-labeled binding moiety that specifically binds to the
biomarker; [0095] b. detecting a first fluorescent signal from at
least a portion of the labeled sample; [0096] c. detecting a second
fluorescent signal from a population of fluorescently-labeled
particles, wherein the population includes a known fluorescent
intensity over a fixed time; and [0097] d. normalizing the first
fluorescent signal to the second fluorescent signal, thereby
quantifying the biomarker, wherein the normalizing includes using a
device comprising software capable of comparing the first and
second fluorescent signal.
[0098] Furthermore, according to an embodiment of the present
invention, the biomarker is a sepsis biomarker.
[0099] Moreover, according to an embodiment of the present
invention, the biomarker is CD64 or CD163.
[0100] Additionally, according to an embodiment of the present
invention, the sample is a blood sample.
[0101] According to another embodiment of the present invention,
the fluorescent label of the binding moiety and the fluorescent
label of the particles is the same fluorescent label.
[0102] Further, according to an embodiment of the present
invention, the binding moiety is an antibody.
[0103] According to an embodiment of the present invention, the
software is capable of recognizing a specific lot of
fluorescently-labeled particles.
[0104] Moreover, according to an embodiment of the present
invention, the individual fluorescent signals include at least one
first fluorescent signal and at least one second fluorescent
signal.
[0105] Additionally, according to an embodiment of the present
invention the fluorescently-labeled binding moiety targets a first
cell population and a second cell population in the sample.
[0106] According to another embodiment of the present invention the
detection of binding of the binding moiety to the second cell
population provides an internal positive control for the
sample.
[0107] Furthermore, according to an embodiment of the present
invention, the binding moiety is anti-CD64 antibody and the first
cell population includes polymorphonuclear leukocytes.
[0108] Yet further, according to an embodiment of the present
invention, the second cell population includes monocytes.
[0109] According to an embodiment of the present invention, the
method further comprises the step of determining the presence of at
least one cell population in the sample that is not bound by the
binding moiety, thus providing an internal negative control for the
sample.
[0110] There is thus provided according to another embodiment of
the present invention, a composition for evaluating a biological
condition, the composition comprising; [0111] a. a sample
comprising at least one of; [0112] i. a bodily specimen comprising
a target moiety; [0113] ii. a positive control moiety; and [0114]
iii. a negative control moiety; [0115] b. an antibody composition
comprising at least one of; [0116] iv. at least one target
antibody; [0117] v. at least one positive control identifying
antibody; and [0118] vi. at least one negative control identifying
antibody or characteristic; and [0119] c. at least one reference
composition comprising at least one of; [0120] vii. a target
antibody reference composition; and [0121] viii. a reference
identifier composition.
[0122] According to an embodiment of the present invention, the
composition further comprises at least one conditioning moiety
comprising; [0123] a) at least one lysis reagent; and [0124] b) at
least one diluent.
[0125] There is thus provided according to another embodiment of
the present invention, a method of determining the presence or
absence of sepsis in a subject, the method including; [0126] a)
contacting a blood sample from the subject with a
fluorescently-labeled binding moiety specific to a sepsis marker,
wherein the volume of the blood sample is 50 .mu.L or smaller; and
[0127] b) detecting the presence, absence or level of the binding
moiety in the sample, thereby determining the presence or absence
of sepsis in the subject.
[0128] There is thus provided according to another embodiment of
the present invention, a method of quantifying a biomarker in a
sample, comprising; [0129] a) contacting the sample with a
fluorescently-labeled binding moiety that specifically binds to the
biomarker; [0130] b) detecting a first fluorescent signal from at
least a portion of the labeled sample; [0131] c) detecting a second
fluorescent signal from a population of fluorescently-labeled
particles, wherein the population includes a known fluorescent
intensity over a fixed time; and [0132] d) normalizing the first
fluorescent signal to the second fluorescent signal, thereby
quantifying the biomarker, wherein the normalizing includes using a
device comprising software capable of comparing the first and
second fluorescent signal.
[0133] According to some embodiments, the sample may be liquid,
according to other embodiments, the sample may be a colloid or
suspension. According to further embodiments, the sample may be a
solid, such as in a powder or crystal form.
[0134] Typical turnaround times for diagnostic prior art assays are
30-120 minutes. Often, the time lost in waiting for laboratory
results can lead to a further deterioration in a patient, and
sometimes death. In some cases, the physician has to act without
having the laboratory results. This can lead to providing the
patient with the wrong treatment. The present invention provides
rapid assays to save lives and provide fast correct treatments to a
patient.
[0135] There is thus provided according to an embodiment of the
present invention automated method of determining the presence or
absence of sepsis in a subject, including; [0136] a) contacting a
blood sample from the subject with a fluorescently-labeled binding
moiety specific to a sepsis marker, wherein the volume of the blood
sample is 50 .mu.L or smaller; and [0137] b) detecting the
presence, absence or level of the binding moiety in the sample,
thereby determining the presence or absence of sepsis in the
subject within twenty minutes.
[0138] Additionally, according to an embodiment of the present
invention, the sepsis marker is CD64.
[0139] Furthermore, according to an embodiment of the present
invention, a second sepsis marker is CD163.
[0140] Moreover, according to an embodiment of the present
invention, the method further includes contacting the blood sample
with a second fluorescently-labeled binding moiety specific for a
second sepsis marker.
[0141] Further, according to an embodiment of the present
invention, the sepsis marker is CD64 and the second sepsis marker
is CD163.
[0142] Additionally, according to an embodiment of the present
invention, the binding moiety is an antibody.
[0143] Moreover, according to an embodiment of the present
invention, the detecting step is performed in a device capable of
receiving the sample and capable of detecting the binding
moiety.
[0144] Additionally, according to an embodiment of the present
invention, the method further includes the step of calibrating the
device by detecting a population of the fluorescently-labeled
particles.
[0145] According to another embodiment of the present invention,
the particles include the same fluorescent label as the
fluorescently-labeled binding moiety.
[0146] Additionally, according to an embodiment of the present
invention, the method further includes a second population of
particles that include the same fluorescent label as the second
fluorescently-labeled binding moiety.
[0147] Moreover, according to an embodiment of the present
invention, the method further includes performing an internal
calibration after the detecting the fluorescently-labeled binding
moiety.
[0148] Notably, according to an embodiment of the present
invention, the calibration is completed in less than 5 minutes.
[0149] According to some embodiments, the particles are
microbeads.
[0150] Additionally, according to an embodiment of the present
invention, the method is performed in less than 15 minutes.
[0151] Furthermore, according to an embodiment of the present
invention, the method, further includes the step of determining the
presence of at least one cell population in the sample that is not
bound by the binding moiety, thus providing an internal negative
control for the sample.
[0152] The present invention will be more fully understood from the
following detailed description of the preferred embodiments
thereof, taken together with the drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0153] The invention will now be described in connection with
certain preferred embodiments with reference to the following
illustrative figures so that it may be more fully understood.
[0154] With specific reference now to the figures in detail, it is
stressed that the particulars shown are by way of example and for
purposes of illustrative discussion of the preferred embodiments of
the present invention only and are presented in the cause of
providing what is believed to be the most useful and readily
understood description of the principles and conceptual aspects of
the invention. In this regard, no attempt is made to show
structural details of the invention in more detail than is
necessary for a fundamental understanding of the invention, the
description taken with the drawings making apparent to those
skilled in the art how the several forms of the invention may be
embodied in practice.
[0155] In the drawings:
[0156] FIG. 1 is a simplified schematic illustration showing an
apparatus for detecting a biological condition, in accordance with
an embodiment of the present invention;
[0157] FIG. 2 is a simplified flow chart of a method for detecting
a biological condition, in accordance with an embodiment of the
present invention;
[0158] FIG. 3 is a simplified schematic illustration showing a
methodology for detecting a biological condition associated with a
CD64 cell surface antigen, in accordance with an embodiment of the
present invention;
[0159] FIG. 4 is a simplified flow chart of a method for detecting
a biological condition associated with a CD64 cell surface antigen,
in accordance with an embodiment of the present invention;
[0160] FIG. 5A is a graphical output of a fluorescent detection
assay of a non-activated neutrophil signature associated with the
method of FIGS. 3-4, in accordance with an embodiment of the
present invention;
[0161] FIG. 5B is a graphical output of a fluorescent detection
assay of an activated neutrophil signature, associated with the
method of FIGS. 3-4, in accordance with an embodiment of the
present invention;
[0162] FIG. 5C is a graphical output of a fluorescent detection
assay of a monocyte signature, associated with the method of FIGS.
3-4, in accordance with an embodiment of the present invention;
FIG. 5D is a graphical output of a fluorescent detection assay of a
reference bead signature, associated with the method of FIGS. 3-4,
in accordance with an embodiment of the present invention;
[0163] FIG. 6 is a simplified flow chart of a method for
differentiating between different particles, in accordance with an
embodiment of the present invention;
[0164] FIG. 7 is a graphical output of fluorescence from reference
beads in eight wavebands, in accordance with an embodiment of the
present invention;
[0165] FIG. 8 is a graphical output of data from FIG. 7 after a
first mathematical manipulation, in accordance with an embodiment
of the present invention;
[0166] FIG. 9 is a graphical output of data from FIG. 7 after a
second mathematical manipulation, in accordance with an embodiment
of the present invention;
[0167] FIG. 10 is a graphical output of data from FIG. 7 after a
third mathematical manipulation, in accordance with an embodiment
of the present invention; and
[0168] FIG. 11 is a graphical output of an event locator, based on
data from FIG. 8-10, in accordance with an embodiment of the
present invention.
[0169] In all the figures similar reference numerals identify
similar parts.
DETAILED DESCRIPTION OF THE INVENTION
[0170] In the detailed description, numerous specific details are
set forth in order to provide a thorough understanding of the
invention. However, it will be understood by those skilled in the
art that these are specific embodiments and that the present
invention may be practiced also in different ways that embody the
characterizing features of the invention as described and claimed
herein.
[0171] International patent application publication no.
WO2011/128893 to Kasdan et al., describes a device, system and
method for rapid determination of a medical condition and is
incorporated herein by reference.
[0172] The microfluidic cartridges of the present invention may be
any suitable cartridge as shown in the figures or any of the prior
art cartridges described or cited herein, such as, but not limited
to, those described in U.S. Pat. No. D669,191 S1, US20120266986 A1,
EP1846159 A2, US2012275972, WO11094577A, US2007292941A and
EP1263533 B1.
[0173] Reference is now made to FIG. 1, which is a simplified
schematic illustration showing an apparatus 100 for detecting a
biological condition, in accordance with an embodiment of the
present invention.
[0174] Apparatus 100 is a kit comprising a cartridge 102 and a
number of chemical/biochemical reactants termed herein, treatment
compositions. The treatment compositions are adapted to react, at
least in part, with biological specimen, such as a body specimen,
to be introduced to the apparatus. The body specimen may be a
bodily fluid such as, but not limited to, blood, serum, plasma,
urine, saliva, cerebrospinal fluid (CSF), serous fluid, peritoneal
fluid and synovial fluid. Additionally or alternatively, the body
specimen may be a solid such as a hair, a tooth part, a bone part
or a piece of cartilage.
[0175] Apparatus 100 comprises a specimen receiving element 118,
adapted to transfer the specimen to a sample composition chamber
104. The sample composition chamber comprises on or more transfer
elements 105, adapted to transfer the specimen from the sample
composition chamber to one or more other locations in the
cartridge. In the non-limiting example shown in FIG. 1, transfer
element 105 is a conduit in fluid connection with a treatment
chamber 112.
[0176] Additionally, the cartridge comprises a number of treatment
composition chambers 106, 108, 110, adapted to respectively house a
corresponding number of treatment compositions 120, 122, 124. These
treatment compositions may be liquid, solid or combinations
thereof. Apparatus 100 is typically sold commercially as a kit with
the treatment compositions disposed therein. In some cases, the
apparatus 100 may be adapted for a one-off test and may be
disposable. In other cases, the apparatus may be re-used. A
re-usable apparatus may be adapted to receive additional external
compositions (not shown) or may have a plurality of treatment
compositions, wherein only a portion is used for each test.
[0177] The apparatus may be constructed and configured such that
the treatment composition comprises proteins attached to a surface,
such as to beads. A plurality of beads or other structural elements
with proteins attached to their surfaces can be made by any one or
more of the following methodologies: -- [0178] simple attachment
such as by adsorption via electrostatic or hydrophobic interactions
with the surface, entrapment in immobilized polymers, etc. [0179]
non-covalent or physical attachment; [0180] covalent bonding of the
protein to the bead surface [0181] biological recognition (e.g.,
biotin/streptavidin). [0182] requires two steps: a first layer is
formed by silane chemistry such that the surface presents a
reactive group (e.g., epoxy, amino, thiol, etc.), and a second
layer (e.g., the protein to be immobilized or a linker molecule) is
covalently attached via the immobilized reactive groups. [0183]
covalent attachment to functionalized polymer coatings on the
interior of the device or linkage to the free end of a
self-assembled monolayer (SAM) on a gold surface.
[0184] The reaction type may include any one or more of
antigen-antibody binding, sandwich (such as
antibody--antigen-antibody), physical entrapment, receptor-ligand,
enzyme-substrate, protein-protein, aptamers, covalent bonding or
biorecognition.
[0185] Cartridge 102 further comprises at least one transfer
element 107, 109, 111 in fluid communication with each respective
of treatment composition chamber, each transfer element also being
in fluid communication with treatment chamber 112. These elements
are typically micro fluidic channels and may be designed for
mixing, such as being tortuous in shape.
[0186] Various methodologies for transferring the contents of the
treatment composition chambers and the sample composition chamber
via the transfer elements to the treatment chamber may be employed,
some of which are known in microfluidics technologies. These
include air blowing, suction, vacuuming, mechanical transfer,
pumping and the like.
[0187] Cartridge 102 further comprises at least one transfer
element 113 in fluid communication with treatment chamber 112 and
with an evaluation chamber 114.
[0188] Optionally, evaluation chamber 114 is further in fluid
communication with a transfer element 115, adapted to remove the
contents of the evaluation chamber for disposal outside the
cartridge. Alternatively, the evaluation chamber may have no
external disposal means.
[0189] Table 1 shows some representative applications of apparatus
100 and methods of the present invention.
TABLE-US-00001 TABLE 1 Applications of the apparatus and methods of
this invention. Typical Prior This Relevant Art Laboratory
invention Figures in Turnaround Turnaround Type of this time (TAT)-
time Application Test invention see references (TAT) References
Application #1 - Surface FIGS. 1-2 4 hours 10 minutes U.S. Pat. No.
8,116,984, CD64 Infection Marker and 3-5D Davis, B H et al, &
Sepsis (2006) 1 - Fetal Plasma FIGS. 1-2 4 hours 10 minutes
Dziegiel et al. Hemoglobin Protein and 6-8D (2006) Test 2 - Low
Platelet Surface FIGS. 1-2 4 hours 10 minutes Segal, H. C., et al.
Count Marker and 3-5D (2005): 3 - Resolving Surface FIGS. 1-2 4
hours 10 minutes Guerti, K., et al. BLAST Flag for Marker and 3-5D
hematology Lab 4 - CD34 Stem Surface FIGS. 1-2 4 hours 10 minutes
Sutherland et al. Cell Marker and 3-5D (1996) Enumeration Assay 5 -
Platelets Surface FIGS. 1-2 4 hours 10 minutes Graff et al. (2002)
Activation Marker and 3-5D Divers, S. G., et al. Assay CD62 (2003)
6 - D-dimer Plasma FIGS. 1-2 4 hours 10 minutes Stein et al. (2004)
(Bead based Protein and 6-8D Rylatt, D. B., et al. protein) (1983):
7- Surface FIGS. 1-2 4 hours 10 minutes Hillier et al. (1988)
Chorioamnioitis Marker and 3-5D CD64 8 - CD20 Cell Surface FIGS.
1-2 4 hours 10 minutes Rawstron et al. Quantitation Marker and 3-5D
(2001) (Therapy Cheson et al. Monitoring (1996) 9 - CD52 Cell
Surface FIGS. 1-2 4 hours 10 minutes Rawstron et al. quantitation
Marker and 3-5D (2001) (Therapy Monitoring) 10 - Circulating
Surface FIGS. 1-2 4 hours 10 minutes Cristofanilliet al. Tumor
Cells Marker and 3-5D (2004 11 - Reticulated Surface FIGS. 1-2 4
hours 10 minutes Matic et al. (1998) Platelet Assay Marker and 3-5D
Ault et al (1993) Wang et al. (2002) 12 - Bacteria 4 hours 10
minutes Blajchman et al Detection in (2005) platelet packs McDonald
et al. (2005) 13 - Platelet Surface FIGS. 1-2 4 hours 10 minutes
Michelson (1996) Associated Marker and 3-5D Antibodies 14 -
Residual Surface FIGS. 1-2 4 hours 10 minutes Bodensteiner,
Leukocyte Marker and 3-5D (2003) Count in blood products 15 - CD4
HIV Surface FIGS. 1-2 4 hours 10 minutes Rodriguez (2005). AIDS
Marker and 3-5D Dieye et al. (2005) 16 - Leukemia Surface FIGS. 1-2
4 hours 10 minutes Drexler et al (1986) Panels - Very Marker and
3-5D complex 17 - Bladder Surface FIGS. 1-2 4 hours 10 minutes
Ramakumar et al Cancer Marker and 3-5D (1999) Screening in Lotan et
al. (2009) Urine - Urine sample 18 - HLA DR Surface Figs. 1-2 4
hours 10 minutes Hershman et al. Sepsis and Marker and 3-5D (2005)
Immuno- Perry et al (2003) suppression 19 - RECAF Plasma FIGS. 1-2
4 hours 10 minutes Moro et al. (2005). Protein for Protein and 6-8D
Canine and other Cancers 20 - CytoImmun - 4 hours 10 minutes
Hilfrich et al. Cervical (2008) Screening 21 - Plasma FIGS. 1-2 4
hours 10 minutes Assicot et al. Procalcitonin Protein and 6-8D
(1993) (Bead Based Christ-Grain et al. Protein) + (2004)
Feasibility
[0190] Reference is now made to FIG. 2, which is a simplified flow
chart 200 of a method for detecting a biological condition, in
accordance with an embodiment of the present invention.
[0191] It should be understood that each of the steps of the method
may take a predetermined period of time to perform, and in between
these steps there may be incubation and/or waiting steps, which are
not shown for the sake of simplicity.
[0192] In a sample transferring step 202, a sample, such as a
bodily specimen is transferred from outside apparatus 100 via
receiving element 118 into sample composition chamber 104 and then
to the treatment chamber 112. According to some embodiments, the
volume of the specimen or sample is less than 200 .mu.L, less than
100 .mu.L, less than 50 .mu.L, less than 25 .mu.L or less than 11
.mu.L.
[0193] Thereafter, treatment composition 120 is transferred via
transfer element 107 to the treatment chamber in a composition
transfer step 204. In some cases, there may be a treatment
composition disposed in the treatment chamber.
[0194] Depending on the nature of the treatment composition and
sample/specimen type, there may be a requirement to mix or agitate
the treatment chamber contents in an optional mixing step 206. This
may be performed by using a small stirbar (not shown) disposed in
the chamber. Additionally or alternatively, this may be effected by
the fluid dynamics of kit. Additionally or alternatively, stirbars
may be disposed in any of the other chambers in the apparatus.
[0195] Typically, the total sample volumes are in the range of 10
to 1000 .mu.L, 100 to 900 .mu.L, 200 to 800 .mu.L, 300 to 700
.mu.L, 400 to 600 .mu.L, or 420 to 500 .mu.L.
[0196] According to some embodiments, the volume of the treatment
composition chambers 106, 108, 110 (also called blisters) is from
about 1 .mu.L to 1000 .mu.L. According to other embodiments, the
volume of the specimen is from about 10 .mu.L to 200 .mu.L.
According to other embodiments, the volume of the specimen is about
1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,
200, 250, 300, 350, 400, 450, or 500 .mu.L.
[0197] According to some embodiments, the volume of the treatment
compositions 120, 122, 124 is at most about 500 .mu.L. According to
other embodiments, the volume of the specimen is at most about 200
.mu.L. According to other embodiments, the volume of the specimen
at most about 500, 450, 400, 350, 300, 250, 200, 180, 160, 140,
120, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 1 .mu.L.
[0198] According to some embodiments, the volume of a reactant is
at least about 1 .mu.L. According to other embodiments, the volume
of the specimen is from about 10 .mu.L. According to other
embodiments, the volume of the specimen is at least about 1, 10,
20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250,
300, 350, 400, 450, or 500 .mu.L.
[0199] The sequence of transfer of the various treatment
compositions may be important to the reaction sequence and is
typically predefined. Steps 204-206 may be performed, for example
on treatment composition chamber 106, thereafter on treatment
composition chamber 108 and thereafter on treatment composition
chamber 110. In some cases, some of these steps may be performed
concurrently.
[0200] In a checking step 208, it is ascertained whether all the
compositions required for the sample treatment have been
transferred to the treatment chamber. If any compositions remain,
then steps 204-206 are performed on the subsequent treatment
composition chamber(s). If no further treatment compositions
require transfer, then the sample/specimen is transferred from
chamber 104 into the treatment chamber.
[0201] Thereafter, in a second sample transfer step 210, the sample
is transferred from the sample composition chamber into the
treatment chamber.
[0202] According to some embodiments, step 210 may be performed
before steps 204-208.
[0203] If required, an optional mixing step 212 to the contents of
the treatment chamber may be performed.
[0204] In a transferring step 214, the contents of the treatment
chamber are transferred to the evaluation chamber.
[0205] The evaluation chamber 114 is configured and constructed for
one or more evaluation steps 216. These may include any of the
following, or combinations thereof: [0206] a) transfer of radiation
there-through, [0207] b) impinging radiation thereupon; [0208] c)
detecting reflected, refracted, and/or transmitted radiation,
[0209] d) detecting emitted radiation; [0210] e) capturing one or
more images thereof; [0211] f) performing image analysis on the
captured images; [0212] g) measuring electrical characteristics of
the treated specimen; [0213] h) impinging sonic energy thereon;
[0214] i) detecting sonic energy therefrom; and [0215] j) analyzing
the outputs of any one or more of the above steps.
[0216] According to some embodiments, the cartridge is introduced
into a system as described in International patent application
publication no. WO2011/128893 to Kasdan et al., incorporated herein
by reference.
[0217] The results of the evaluation step are then outputted in a
results outputting step 218. According to some embodiments; the
apparatus may have on-board means for showing a result, such as a
colorimetric strip (not shown). Additionally or alternatively, the
results are displayed in a display unit, separate and remote from
apparatus 100.
[0218] Reference is now made to FIG. 3, which is a simplified
schematic illustration showing a methodology 300 for detecting a
biological condition associated with a CD64 cell surface antigen,
in accordance with an embodiment of the present invention.
[0219] According to some embodiments, the method is carried out in
the apparatus shown in FIG. 1 and as described herein. A biological
specimen, such as a blood sample, is aspirated via specimen
receiving element 118 to sample composition chamber 104, and then
to treatment chamber 112. The sample is typically of a volume in
the range of 10-200 .mu.L.
[0220] The blood sample is typically whole blood recently removed
from a patient. The whole blood comprises mainly red blood cells
(also called RBCs or erythrocytes), platelets and white blood cells
(also called leukocytes), including lymphocytes and neutrophils
Increased number of neutrophils, especially activated neutrophils
are normally found in the blood stream during the beginning (acute)
phase of inflammation, particularly as a result of bacterial
infection, environmental exposure and some cancers.
[0221] A cocktail 304 comprising antibodies to CD64 and antibodies
to CD163 is introduced to the treatment chamber (see Davis et al.
(2006)). Each antibody type is typically tagged by a specific
fluorescent tag.
[0222] The contents of the chamber are incubated and/or mixed as is
required to bind the activated blood neutrophils with the CD64
tagged antibody (also called a marker) to form activated
neutrophils with CD64 marker 310, and/or monocyte with a CD64
tagged antibody and a CD163 tagged antibody 312. Lymphocytes with
no markers 314 are present in the contents, as well as unaffected
RBCs 316.
[0223] Thereafter, a lysis reagent or diluent 306 is introduced
into treatment chamber 112. In the case of a lysis reagent, it is
adapted to lyse red blood cells to form lysed red blood cells 324.
Additionally, reference/calibration beads 308 are added to the
treatment chamber. These are used to calibrate the outputs, as is
explained with reference to FIGS. 5A-5D hereinbelow.
[0224] CD64 (Cluster of Differentiation 64) is a type of integral
membrane glycoprotein known as an Fc receptor that binds monomeric
IgG-type antibodies with high affinity. Neutrophil CD64 expression
quantification provides improved diagnostic detection of
infection/sepsis compared with the standard diagnostic tests used
in current medical practice.
[0225] CD163 (Cluster of Differentiation 163) is a human protein
encoded by the CD163 gene. It has also been shown to mark cells of
monocyte/macrophage lineage.
[0226] Reference is now made to FIG. 4, which is a simplified flow
chart 400 of a method for detecting a biological condition
associated with a CD64 cell surface antigen, in accordance with an
embodiment of the present invention.
[0227] According to some embodiments, the method is carried out in
the apparatus shown in FIG. 1 and as described herein. In a first
transferring step 402, a biological specimen, such as a blood
sample is aspirated via specimen receiving element 118 to sample
composition chamber 104. The sample is typically of a volume in the
range of 10-200 .mu.L.
[0228] Typically, the total sample volumes are in the range of 10
to 1000 .mu.L, 100 to 900 .mu.L, 200 to 800 .mu.L, 300 to 700
.mu.L, 400 to 600 .mu.L, or 420 to 500 .mu.L.
[0229] According to some embodiments, the volume of the treatment
composition chambers 106, 108, 110 (also called blisters) is from
about 1 .mu.L to 1000 .mu.L. According to other embodiments, the
volume of the specimen is from about 10 .mu.L to 200 .mu.L.
According to other embodiments, the volume of the specimen is about
1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,
200, 250, 300, 350, 400, 450, or 500 .mu.L.
[0230] According to some embodiments, the volume of the treatment
compositions 120, 122, 124 is at most about 500 .mu.L. According to
other embodiments, the volume of the specimen is at most about 200
.mu.L. According to other embodiments, the volume of the specimen
at most about 500, 450, 400, 350, 300, 250, 200, 180, 160, 140,
120, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 1 .mu.L.
[0231] According to some embodiments, the volume of a reactant is
at least about 1 .mu.L. According to other embodiments, the volume
of the specimen is from about 10 .mu.L. According to other
embodiments, the volume of the specimen is at least about 1, 10,
20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250,
300, 350, 400, 450, or 500 .mu.L.
[0232] In an addition step 404, a cocktail of tagged antibodies to
CD64 and to CD163 is added to the treatment chamber 112 and is
incubated with the blood sample. In the incubation phase of this
step, the antibodies bind activated neutrophils with CD64 marker
310, and/or monocytes activated with a CD64 tagged antibody and a
CD 163 tagged antibody 312.
[0233] In a lysis reagent addition step 406, the lysis reagent is
added to the treatment chamber and thereby lyses at least some of
the RBCs in the chamber.
[0234] At any suitable time, typically following lysis step 406,
reference beads are added to the contents of the treatment chamber
in a reference bead adding step 408.
[0235] After a predefined period of time, an analysis step 410 is
performed to analyze the fluorescent emission signatures from the
contents. This is described in further detail with reference to
FIGS. 5A-5D. According to some examples, the evaluation chamber 114
is constructed and configured to allow cells to pass through a
reading zone 130 such that each cell passing therethrough is
analyzed individually. The assay sensitivity is around 86% and its
specificity is around 87% (Hoffmann, 2011).
[0236] The time required to complete an assay using apparatus 100
of the present invention varies depending on a number of factors,
with non-limiting examples that include described herein. In some
embodiments, the time required to complete an assay is from about
0.5 to 100 minutes. In other embodiments, the time required to
complete an assay is from about 1 to 20 minutes. In still other
embodiments, the time required to complete an assay is from about 1
to 10 minutes. In some examples, the time required to complete an
assay is from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,
35, 40, 50, 60, 80, or 100 minutes.
[0237] Reference is now made to FIG. 5A, which is a graphical
output of a fluorescent detection assay of a non-activated
neutrophil signature 500 associated with the method of FIGS. 3-4,
in accordance with an embodiment of the present invention. The
non-activated tagged neutrophils each emit a signal 502 at
wavelength W1 of an intensity I.sub.1. The wavelengths shown in
FIGS. 5A-5D represent a peak wavelength of waveband outputs
detected, as are shown in FIGS. 7-11.
[0238] FIG. 5B shows a graphical output of a fluorescent detection
assay of an activated neutrophil signature 510, associated with the
method of FIGS. 3-4, in accordance with an embodiment of the
present invention. Each activated tagged neutrophil emits an
activated neutrophil signature 512 at wavelength W1 of an intensity
I.sub.2. Typically I.sub.2 is greater than I.sub.1. In some cases
the difference in signatures 512 and 510 may be detected by an
image analysis, a fluorescent emission radiation count or by other
qualitative or quantitative methods known in the art. The current
example is not meant to be limiting.
[0239] Turning to FIG. 5C, there can be seen a graphical output of
a fluorescent detection assay of a monocyte signature 520,
associated with the method of FIGS. 3-4, in accordance with an
embodiment of the present invention. The monocyte signature
comprises a first signal 522 at a first wavelength W1 of an
intensity I.sub.3 and a second signal 524 at a second wavelength W2
of an intensity I.sub.4.
[0240] FIG. 5D shows a graphical output of a fluorescent detection
assay of a reference bead signature 530, associated with the method
of FIGS. 3-4, in accordance with an embodiment of the present
invention. The reference bead signature comprises a first signal
532 at a first wavelength W1 of an intensity I.sub.1 (similar or
equal to non-activated tagged neutrophils' signal 502) and a second
signal 534 at a second wavelength W3 of an intensity I.sub.5.
[0241] This methodology enables the identification and
quantification of activated neutrophils by intensity of signature
512 of the CD64 tag. Monocytes are identified by the double signal
signature 522, 524, acting as a positive control. Reference beads
are identified by the unique signal 534 at wavelength W3. The
intensity of signal 532 at wavelength W1 provides a reference level
of CD64 for the comparison of intensity of 512 of the
neutrophils.
[0242] Lymphocytes with no markers 330 (FIG. 3) act as a negative
control and should provide no fluor signature, but may be detected
by their scattering or other characteristics. Further details of
some embodiment of this assay procedure are described in U.S. Pat.
No. 8,116,984 and in Davis, B H et al., (2006).
[0243] Reference is now made to FIG. 6, which is a simplified flow
chart of a method 600 for differentiating between different
particles, in accordance with an embodiment of the present
invention.
[0244] The input to the processing is a time series from each of
the channels in the eight channel photomultiplier array 601. In
addition, data from multiple scatter channels 609 is introduced.
Each fluorescent time series and scatter time series may be
processed individually employing respective spectral
crosscorrelation algorithm 606 and scatter algorithm 607 to smooth
it and minimize noise. Two possible processing methods are boxcar
averaging algorithm 602 and matched filtering algorithm 604. In
addition, groups of individual channels may be correlated to yield
a multiple spectral crosscorrelations 606. One or more of these
derived time series may be used to determine event locations.
[0245] Once an event is located in the eight channel time series
the composition of that event in terms of known fluorophore
signatures is determined using a minimum mean square error fit 610.
The event is now described in terms of its composition of known
fluors. Each event thus described is stored in an event store, i.e.
memory, together with the data from the eight time series for that
event and its description 612. Based on the fluor composition for
each event in the data store, it is possible to determine the type
of particle. For example, a neutrophil 616 is characterized by the
single fluor attached to the CD64 antibody shown in FIG. 5 as W1.
Thus events that are preponderantly characterized by the single
fluor attached to the CD64 antibody are identified as
neutrophils.
[0246] Similarly, monocytes 618 are characterized by fluors W1 and
W2 so that an event with both of these fluor signatures is
identified as a monocyte. Similarly, a bead 620 is characterized by
an event that has fluors W1 and W3. Lymphocytes 622 do not express
significant fluorescence but are identified by their scatter as
events. Events that do not match any of the known combinations of
the fluorophores are identified as rejects 626.
[0247] Given the population of identified events, the median
intensity of the neutrophil population and the median intensity of
the bead population are determined. The ratio of the neutrophil
median to the bead median is the desired Leuko64 index. The
positive control value is determined as the median intensity of the
CD64 fluorophore bound to monocytes divided by the median intensity
of the same fluorophore on the bead population. The negative
control value is determined by the median intensity of the CD64
fluorophore bound to lymphocytes. These are the key steps in
performing the Leuko64 assay.
[0248] FIG. 7 is a graphical output 700 of fluorescence from
reference beads in eight wavebands, in accordance with an
embodiment of the present invention. This figure shows the smoothed
signals from the eight channel PMT array for two reference beads.
The amplitude for each waveband is shown on the same graph. The
corresponding wavelength range is shown for each plot 702, 706,
708, 710, 712, 714, 716, 718 in the legend box. The two
fluorophores signatures present in this plot are 702,706 and 708
for FITC, which is the fluorophore attached to the CD64 antibody
and 710, 712 for Starfire Red, which is the fluorophore identifying
the reference beads.
[0249] Reference is now made to FIG. 8, which is a graphical output
800 of data from FIG. 7 after a first mathematical manipulation, in
accordance with an embodiment of the present invention. FIG. 8
shows the cross correlation of wave bands one two and three
corresponding to wavelength 500 to 525, 525 to 550, and 552 to 575
nm. This cross correlation is computed by multiplying the boxcar
smoothed time series corresponding to these wavelengths. This
signal will have a high-value when an event containing the FITC
fluorophore is present.
[0250] FIG. 9 is a graphical output 900 of data from FIG. 7 after a
second mathematical manipulation, in accordance with an embodiment
of the present invention. FIG. 9 shows the cross correlation of
wave bands 3, 4 and 5 corresponding to wavelengths 550 to 575, 575
to 600, and 600 to 625 nm. This signal will have a high-value when
an event containing the PE fluorophore is present.
[0251] FIG. 10 is a graphical output 1000 of data from FIG. 7 after
a third mathematical manipulation, in accordance with an embodiment
of the present invention. FIG. 10 shows the cross correlation of
wave bands 7 and 8 corresponding to wavelengths 650 to 675, and 675
to 700 nm. This signal will have a high-value when an event
containing the Starfire Red fluorophore is present.
[0252] FIG. 11 is a graphical output 1100 of an event locator,
based on data from FIG. 8-10, in accordance with an embodiment of
the present invention. FIG. 11 shows the event locations determined
from the cross correlations computed in FIGS. 8, 9 and 10. The
solid fill area 1102 corresponds to the region where any of the
cross correlations 802, 902 and 1002 exceeded a predefined
threshold. Similarly, the solid fill area 1104 corresponds to the
region where any of the cross correlations 804, 904 and 1004
exceeded a predefined threshold. This then completes the event
location process.
Example
Application No. 1--CD64 Infection & Sepsis
[0253] A cartridge 102 (FIG. 1) is prepared for receiving a blood
sample. The cartridge comprises a number of treatment composition
chambers 106, 108, 110, adapted to respectively house a
corresponding number of treatment compositions 120, 122, 124. These
compositions are described in further detail in U.S. Pat. No.
8,116,984 and in Davis, B H et al., (2006)), incorporated herein by
reference. In brief, Reagent A comprises a mixture of murine
monoclonal antibodies (contains buffered saline), Reagent
B--10.times. Concentrated Trillium Lyse solution (contains ammonium
chloride), Reagent C--suspension of 5.2 .mu.m polystyrene beads
labeled with Starfire Red and fluorescein isothiocyanate (FITC),
(contains <0.1% sodium azide and 0.01% Tween 20).
[0254] In a sample transferring step 202 (FIG. 2), a 10 uL blood
sample, is transferred from outside apparatus 100 via receiving
element 118 into sample composition chamber 104 and then on to
treatment chamber 112 in a transferring step 214.
[0255] An antibody composition (Reagent A) 120 comprising CD64
antibodies is transferred via transfer element 107 to the treatment
chamber 112 in a composition transfer step 204.
[0256] These two steps combined with mixing step 206 take around
four minutes using cartridge 102 of the present invention.
[0257] A lysis buffer (Reagent B) 122 is also added and mixed with
the resultant mixed composition. This step and mixing all the
compositions takes around three minutes using cartridge 102 of the
present invention. Reference beads (Reagent C) 308 are added to the
treatment chamber.
[0258] The evaluation chamber 114 is configured and constructed for
one or more evaluation steps 216.
[0259] According to some embodiments, the cartridge is introduced
into a system as described in International patent application
publication no. WO2011/128893 to Kasdan et al., incorporated herein
by reference. This system has software associated therewith for
computing the CD64 and CD163 indices on leukocytes.
[0260] The results of the evaluation step are then outputted in a
results outputting step 218. According to this example, the time
taken from the introduction of the small blood sample to obtaining
an indication of sepsis is less than 15 minutes, typically around
10 minutes.
[0261] From a user point of view, the following steps are
performed: [0262] 1) The user adds drop of blood to the cartridge
102 and seals it. (10 .mu.L are metered out by microfluidics).
[0263] 2) Blister A (106) is pressed, releasing 100 .mu.L of
Reagent A. Mixing in the cartridge is controlled by the cartridge
handling unit (CHU), followed by a 4-minutes incubation. [0264] 3)
Blister B (108) is pressed, releasing .about.250 .mu.L of Reagent
B. Mixing in the cartridge is controlled by the CHU, followed by a
3-5-minutes incubation. [0265] 4) Magnetic stirbar is activated,
stirring the bead suspension (Reagent C). [0266] 5) Blister C (110)
is pressed, releasing 100 .mu.L of Reagent C. Mixing in the
cartridge is controlled by the CHU. According to one example,
Reagent A is a mixture of murine monoclonal antibodies--diluted 1:5
in buffered saline (PBS+0.5% BSA); Reagent B is a Trillium Lyse
solution (at working concentration); Reagent C is a suspension of
5.2 .mu.m polystyrene beads labeled with Starfire Red and FITC,
diluted 1:100 in PBS+0.01% Tween 20. [0267] 6) The sample is read
by the optoelectronics core, and collected to the reading below.
[0268] 7) Data is analyzed automatically and result is presented.
[0269] 8) The cartridge is disposed as biohazard.
TABLE-US-00002 [0269] TABLE 2 Comparison of Prior art methodology
with the methodology of the present invention for detecting sepsis
using CD64 and CD163 antibodies. LeukoDx device- present invention
Volume Step Description (uL) Duration (min) comments 1 Mixing blood
and Blood- 10 4 antibodies Abs- 50 2 Adding RBC lysis 250 3 Might
require buffer heating the buffer to 37 C. 3 Incubating, 3
Vortexing 4 Adding 2 Less than 1 normalization beads 5 Reading Less
than 1 Total 312 10
[0270] In the case of sepsis, by "normalization" is meant taking
the ratio of the median of the target population fluorescence
emission to the median of the reference bead population
fluorescence emission.
[0271] According to some embodiments, the readout may comprise an
optoelectronics core, which enables identification and detection of
fluorescent signals.
[0272] The CCD in the core, used for focusing, can also be used to
read chemiluminescent signals. The readout to user may also
indicate where the result falls relative to reference ranges.
[0273] The contents of these publications are incorporated by
reference herein where appropriate for teachings of additional or
alternative details, features and/or technical background.
[0274] It is to be understood that the invention is not limited in
its application to the details set forth in the description
contained herein or illustrated in the drawings. The invention is
capable of other embodiments and of being practiced and carried out
in various ways. Those skilled in the art will readily appreciate
that various modifications and changes can be applied to the
embodiments of the invention as hereinbefore described without
departing from its scope, defined in and by the appended
claims.
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