U.S. patent application number 15/166699 was filed with the patent office on 2017-01-26 for low-volume coagulation assay.
The applicant listed for this patent is Theranos, Inc.. Invention is credited to Samartha Anekal, Mark Dayel, Ian Gibbons, Elizabeth A. Holmes, Paul Patel.
Application Number | 20170023594 15/166699 |
Document ID | / |
Family ID | 49949383 |
Filed Date | 2017-01-26 |
United States Patent
Application |
20170023594 |
Kind Code |
A1 |
Dayel; Mark ; et
al. |
January 26, 2017 |
LOW-VOLUME COAGULATION ASSAY
Abstract
Compositions and methods for measuring coagulation parameters
using very small volumes of blood are provided. Advantageously, the
methods described herein can be performed from a single drop of
blood (about 20 .mu.L) while generally leaving enough sample to
perform other measurements, optionally in a multiplexed format. The
methods and devices do not require a skilled operator and can be
performed at the point of service, which can be an important
feature for managing blood coagulation disorders and treatments
thereof.
Inventors: |
Dayel; Mark; (Palo Alto,
CA) ; Anekal; Samartha; (Palo Alto, CA) ;
Patel; Paul; (Palo Alto, CA) ; Gibbons; Ian;
(US) ; Holmes; Elizabeth A.; (Palo Alto,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Theranos, Inc. |
Palo Alto |
CA |
US |
|
|
Family ID: |
49949383 |
Appl. No.: |
15/166699 |
Filed: |
May 27, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13944863 |
Jul 17, 2013 |
9500639 |
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15166699 |
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61673227 |
Jul 18, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 21/84 20130101;
G01N 33/86 20130101; G01N 33/582 20130101; G01N 2333/75 20130101;
C12Q 1/56 20130101; G01N 33/4905 20130101 |
International
Class: |
G01N 33/86 20060101
G01N033/86; G01N 33/58 20060101 G01N033/58; C12Q 1/56 20060101
C12Q001/56 |
Claims
1. A method for measuring coagulation time of a blood sample of a
subject, comprising: (a) initiating a coagulation reaction of the
blood sample of the subject; (b) obtaining a set of more than one
image of said coagulation reaction; and (c) analyzing said set of
images to measure the coagulation time of said blood sample.
2. The method of claim 1, wherein the amount of time for carrying
out steps (a) to (c) is less than or about 1 hour.
3.-4. (canceled)
5. The method of claim 1, wherein an individual image of said set
of images is pixilated and comprises at least 10,000 pixels.
6. The method of claim 1, further comprising diluting said blood
sample such that the coagulation time of the blood sample after
dilution is between about 1 minute and about 10 minutes.
7. The method of claim 6, further comprising adding fibrinogen to
the coagulation reaction.
8.-9. (canceled)
10. The method of claim 1, wherein said blood sample is obtained
via a non-venous route.
11. The method of claim 1, wherein said images are light scattering
images of the coagulation reaction.
12. The method of claim 1, wherein the coagulation time is measured
based on a transition of the intensity of said scattered light.
13. The method of claim 1, further comprising adding a plurality of
beads to said blood sample prior to obtaining the set of
images.
14. The method of claim 13, wherein said plurality of beads
comprise beads of at least two different sizes.
15.-17. (canceled)
18. The method of claim 13, wherein said step of analyzing said set
of images comprises locating a time point when said beads become
substantially motionless.
19. The method of claim 13, wherein said step of analyzing said set
of images comprises locating the time point when a transition of
the mobility of said beads occurs.
20.-22. (canceled)
23. The method of claim 13, wherein said beads are labeled.
24. The method of claim 23, wherein said beads are labeled with a
fluorescent label.
25-53. (canceled)
Description
CROSS REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/673,227 filed Jul. 18, 2012, which application
is incorporated herein by reference in its entirety.
BACKGROUND
[0002] Coagulation is a complex process by which blood or blood
plasma forms clots. It is an important part of homeostasis, the
cessation of blood loss from a damaged vessel, wherein a damaged
blood vessel wall is covered by a platelet and fibrin-containing
clot to stop bleeding and begin repair of the damaged vessel. Since
inadequate coagulation can lead to an increased risk of bleeding
(hemorrhage) and excessive coagulation can lead to obstructive
clotting (thrombosis), the coagulation process is tightly
controlled and highly conserved throughout biology.
[0003] Blood coagulation disorders are very dangerous, and the
therapeutic means to treat them and to control coagulation are
difficult to manage and also dangerous. In addition, many patients
are chronically treated with anticoagulant drugs such as warfarin
after receiving replacement heart valves and need to be monitored.
It is increasingly advantageous to be able to monitor coagulation
parameters, in particular prothrombin time ("PT"; also expressed as
the mathematical transform, International Normalized Ratio "INR")
and Activated Partial Thromboplastin Time ("aPTT") as part of a
more comprehensive health and therapy monitoring program in which
biomarkers and other therapeutic agents are measured. PT, INR and
aPTT can be measured in clinical laboratories using conventional
methods requiring relatively large volumes of blood or plasma,
typically 5 mL collected into fixed volume vacuum tubes. In order
to improve monitoring of patient blood coagulation parameters,
improvements in performing coagulation assays and measuring
coagulation parameters are needed.
SUMMARY
[0004] The inventors have recognized a need for and provided a
solution to the challenge of measuring coagulation parameters using
very small volumes of blood samples. Advantageously, the methods
described herein can be performed with a small quantity of blood or
plasma derived from a single drop of blood (about 20 .mu.L) while
generally leaving enough sample to perform other measurements,
optionally in a multiplexed format. The methods and devices do not
require a skilled operator and can be performed at the point of
service, which can be an important feature for managing blood
coagulation disorders and treatments thereof by providing
information useful in adjusting dosage and frequency of
medication.
[0005] In one embodiment, provided herein is a method for measuring
coagulation time of a blood sample of a subject, wherein the method
includes: (a) initiating a coagulation reaction of the blood sample
of the subject; (b) obtaining a set of more than one of image of
said coagulation reaction; and (c) analyzing said set of images to
measure the coagulation time of said blood sample.
[0006] In another embodiment, provided here is a method for
measuring coagulation time of a blood sample of a subject, wherein
the method includes (a) obtaining a blood sample from the subject
of 1 ml or less; (b) initiating a coagulation reaction of said
blood sample; (c) obtaining a set of more than one image of said
coagulation reaction; and (d) analyzing said set of images to
measure the coagulation time of said blood sample, wherein the
amount of time for carrying out steps (a) to (d) is less than or
about 1 hour.
[0007] In another embodiment, provided herein is a method for
measuring a plurality of coagulation parameters of a blood sample
of a subject, wherein the method includes: (a) obtaining the blood
sample of the subject, wherein said blood sample is less than or
about 1 ml; and (b) performing a plurality of assays using said
sample to measure said plurality of coagulation parameters.
[0008] In another embodiment, provided herein is a device for
measuring coagulation time of a blood sample of a subject,
comprising (a) a component configured to add a coagulation
initiation reagent to the blood sample from the subject under a
condition suitable for clot formation, thereby initiating the
coagulation reaction; (b) a component configured to obtain set of
more than one image of said coagulation reaction; and (c) a
component that analyzes said set of images to measure the
coagulation time of said blood sample.
[0009] In another embodiment, provided herein is a system for
measuring coagulation time of a blood sample of a subject,
comprising: (a) a device configured to add a coagulation initiation
reagent to the blood sample from the subject under a condition
suitable for clot formation, thereby initiating the coagulation
reaction; (b) a camera configured to obtain a set of more than one
image of said coagulation reaction; and (c) a computer configured
to analyze said set of images to measure the coagulation time of
said blood sample.
[0010] In another embodiment, provided herein is a method for
measuring coagulation time of a blood sample of a subject,
comprising: (a) initiating a coagulation reaction of the blood
sample of the subject; (b) obtaining a video of said coagulation
reaction; and (c) analyzing said video to measure the coagulation
time of said blood sample.
[0011] In some embodiments, methods for assaying coagulation
include: A) monitoring coagulation in a very small volume of blood
sample (e.g. 20 .mu.l or less), and/or B) diluting all or part of a
blood sample and using the diluted blood sample for an assay, thus
reducing the total amount of sample used for the assay.
[0012] In one aspect, various techniques are provided for
performing coagulation assays with diluted or undiluted samples. In
one embodiment, a coagulation assay is performed in a small
container which has a high surface to volume ratio which aids in
the adhesion of an incipient clot to the surface of the container.
In another embodiment, an exogenous material (for example,
fibrinogen) which increases clot strength and/or the turbidity (due
to light scattering) generated during the clotting process is added
to a sample. In another embodiment, small beads are added to a
blood sample, and video imaging is used to track the movement of
the beads as they settle by gravity and then reduce or cease
movement upon clot formation. In another embodiment, small
fluorescent beads are added to a blood sample, and fluorescent
microscopy is used to track the movement of beads as they are moved
by Brownian motion, convention and/or airflow and reduce or cease
movement upon clot formation. In another embodiment, a blood sample
is propelled through a container by a force, and video imaging is
used to track the movement of the sample and the reduction or
cessation in movement of the sample in the container upon clot
formation.
[0013] In one aspect, provided herein is a method for measuring
coagulation time of a blood sample of a subject. The method
comprises (a) initiating a coagulation reaction of the blood sample
of the subject, (b) obtaining a set of images of the coagulation
reaction, and (c) analyzing the set of images to measure the
coagulation time of the blood sample.
[0014] In one aspect, provided herein is a method for measuring
coagulation time of a blood sample of a subject. The method
comprises (a) obtaining a blood sample from the subject from blood
obtained from the subject via a non-venous route, (b) initiating a
coagulation reaction of the blood sample, (c) obtaining a set of
images of the coagulation reaction, and (d) analyzing the set of
images to measure the coagulation time of the blood sample wherein
the amount of time for carrying out steps (a) to (d) is less than
or about 1 hour. In some embodiments, the amount of time for
carrying out steps (a) to (d) is less than or about 30 minutes. In
some embodiments, the amount of time for carrying out steps (a) to
(d) is less than or about 10 minutes.
[0015] In some embodiments, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,
10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 .mu.l or less of blood sample is
used for the coagulation assay.
[0016] In some embodiments, the volume of the coagulation reaction
is less than or about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,
9, 8, 7, 6, 5, 4, 3, 2, or 1 .mu.l.
[0017] In some embodiments, an individual image of the set of
images is pixilated and comprises at least 1, 10, 100, 500, 1000,
2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000
pixels.
[0018] In some embodiments, the method further comprises diluting
the blood sample such that the coagulation time of the sample after
dilution is between about 30 seconds and about 10 minutes.
[0019] In some embodiments, the method further comprises adding
fibrinogen to the coagulation reaction.
[0020] In some embodiments, the initiating step comprises adding a
coagulation initiation reagent to the blood sample.
[0021] In some embodiments, the blood sample is a plasma
sample.
[0022] In some embodiments, the blood sample is obtained using a
finger prick.
[0023] In some embodiments, the images are light scattering images
of the coagulation reaction.
[0024] In some embodiments, the coagulation time is measured based
on a transition of the intensity of the scattered light.
[0025] In some embodiments, the method further comprises adding a
plurality of beads to the blood sample prior to obtaining the set
of images. For example, at least 2, 3, 4, 5, 10, 100, or 1000 beads
may be added to the blood sample.
[0026] In some embodiments, the plurality of beads include beads of
at least two different sizes.
[0027] In some embodiments, the plurality of beads have sizes from
about 1 .mu.m to 5 .mu.m.
[0028] In some embodiments, the plurality of beads have sizes from
about 5 .mu.m to 50 .mu.m.
[0029] In some embodiments, the plurality of beads include one or
more of polystyrene, latex, acrylic, or glass. In certain aspects,
the beads may have a different refractive index from the reaction
medium (e.g. higher or lower by about 1, 2, 3, 4, 5, 8, 10, 15, 16,
20, 25, 30, 35, 40, or 50%), or may be opaque. In addition, the
beads may have a density different from the reaction medium (e.g.
higher or lower by about 1, 2, 3, 4, 5, 8, 10, 15, 16, 20, 25, 30,
35, 40, or 50%). In some assays, the reaction medium has a density
of about 1.01 g/cc.
[0030] In some embodiments, the step of analyzing the set of images
comprises locating a time point when the beads become substantially
motionless.
[0031] In some embodiments, the step of analyzing the set of images
comprises locating the time point when a transition of the mobility
of the beads occurs.
[0032] In some embodiments, the transition of the mobility of the
beads is evidenced by deceleration of the settling of the beads in
the coagulation reaction.
[0033] In some embodiments, the step of analyzing the set of images
comprises comparing two images of the set of images to measure the
motion of the beads.
[0034] In some embodiments, the beads are substantially motionless
if the two images are substantially the same.
[0035] In some embodiments, the beads are labeled.
[0036] In some embodiments, the beads are labeled with a
fluorescent label.
[0037] In another aspect, a method is provided for measuring a
plurality of coagulation parameters of a blood sample of a subject.
The method comprises (a) obtaining the blood sample of the subject,
wherein the blood sample is less than or about 500, (b) preparing a
plasma sample from the blood sample, and (c) performing a plurality
of assays using the plasma sample to measure the plurality of
coagulation parameters, wherein at least one of the plurality of
parameters is coagulation time.
[0038] In some embodiments, one of the coagulation parameters is
selected from the group consisting of: Activated Partial
Thromboplastin Time (aPTT), prothrombin time (PT), International
Normalized Ratio (INR), bleeding time, coagulation factor,
anti-phospholipid antibody, dilute Russell's viper venom time
(dRVVT), and platelet function, thromboelastography (TEG or
Sonoclot), and euglobulin lysis time (ELT).
[0039] In some embodiments, less than about 10 .mu.l of the plasma
sample is utilized for an individual assay of the plurality of
assays. In some embodiments, less than about 2 .mu.l of the plasma
sample is utilized for an individual assay of the plurality of
assays.
[0040] In some embodiments, the reaction volume of some or each of
the plurality of assays is about or less than 6 .mu.l. In some
embodiments, the reaction volume of some or each of the plurality
of assays is about or less than 100.
[0041] In some embodiments, the amount of time for carrying out an
assay to measure coagulation time is less than or about 1 hour, 30
minutes, 10 minutes, 5 minutes, or 1 minute.
[0042] In some embodiments, obtaining a set of images of a
coagulation reaction includes (i) obtaining a set of images of at
least one of the plurality of assays and (ii) analyzing the set of
images to measure the coagulation time.
[0043] In one aspect, a device for measuring coagulation time of a
blood sample of a subject is provided. The device comprises (a) a
component configured to add a coagulation initiation reagent to the
blood sample from the subject under a condition suitable for clot
formation, thereby initiating the coagulation reaction, (b) a
component configured to obtain a set of images of the coagulation
reaction, and (c) a component that analyzes the set of images to
measure the coagulation time of the blood sample.
[0044] In one aspect, a system for measuring coagulation time of a
blood sample of a subject is provided. The system comprises (a) a
device configured to add a coagulation initiation reagent to the
blood sample from the subject under a condition suitable for clot
formation, thereby initiating the coagulation reaction (b) a camera
that obtains a set of images of the coagulation reaction, and (c) a
computer that analyzes the set of images to measure the coagulation
time of the blood sample.
[0045] In some aspects, a blood sample that is anticoagulated may
be used with any of the assays provided herein. To prepare an
anticoagulated blood sample for use with a coagulation assay, a
reagent which reverses the effect of an anticoagulant is added in
excess over the anticoagulant. For the anticoagulants
ethylenediaminetetraacetic acid (EDTA), citrate, and oxalate, a
suitable reagent is calcium (Ca.sup.2+); for the anticoagulant
heparin, a suitable reagent is polybrene.
[0046] This Summary is provided to introduce a selection of
concepts in a simplified form that are further described below in
the Detailed Description. This Summary is not intended to identify
key features or essential features of the claimed subject matter,
nor is it intended to be used to limit the scope of the claimed
subject matter.
INCORPORATION BY REFERENCE
[0047] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference. Incorporated publications include U.S. Patent
Publication Number 2010/0081144 A1; U.S. Pat. No. 7,888,125; U.S.
Patent Publication Number 2011/0093249 A1; U.S. Patent Publication
Number 2009/0318775 A1; U.S. Pat. No. 7,291,497; U.S. Pat. No.
8,012,744; U.S. Patent Publication Number 2006/0264783 A1; U.S.
Patent Publication Number 2007/0224084 A1; and U.S. application
Ser. No. 13/244,947.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] FIG. 1 shows an increase in light scattering from before
coagulation (left panel) to after coagulation (right panel) of a
plasma sample.
[0049] FIG. 2 shows a plot of mean light scattering signal versus
time during coagulation of a plasma sample.
[0050] FIG. 3 shows a plot of mean light scattering signal versus
time fit to a four-parameter log-logistic function progress
curve.
[0051] FIG. 4 shows a representative reaction time course analyzed
by the PSNR method.
[0052] FIG. 5 shows a schematic of a bead settling embodiment.
[0053] FIG. 6 shows a schematic of a fluorescent microscopy
embodiment.
[0054] FIG. 7 shows a schematic of a cuvette suitable for
microscopy embodiments.
[0055] FIG. 8 shows a plot of correlation factor as a function of
time for measurement of PT activation factor of a plasma
sample.
[0056] FIG. 9 shows a method for excluding background from video
images.
[0057] FIG. 10 shows exemplary results of measuring PT by light
scattering.
[0058] FIG. 11 shows exemplary results of measuring aPTT by light
scattering.
[0059] FIG. 12 shows exemplary results of measuring PT by bead
settling.
[0060] FIG. 13 shows exemplary results of measuring aPTT by bead
settling.
[0061] FIG. 14 shows images acquired in an exemplary fluorescent
microscopy embodiment.
[0062] FIG. 15 shows exemplary results of measuring PT by
fluorescent microscopy.
[0063] FIG. 16 shows exemplary results of the effect of added
heparin to human plasma samples on coagulation time measured by
light scattering.
[0064] FIG. 17 shows the calibrated aPTT dose-response to heparin
based on the results of FIG. 16.
[0065] FIG. 18 shows exemplary results of measuring PT in human
plasma samples (normal subjects and subjects on Warfarin therapy)
by light scattering.
[0066] FIG. 19 shows a plot of mean light scattering signal versus
time fit to a bilinear curve.
DETAILED DESCRIPTION
[0067] Before the embodiments of the invention are described, it is
to be understood that such embodiments are provided by way of
example only, and that various alternatives to the embodiments of
the invention described herein may be employed in practicing the
invention. Numerous variations, changes, and substitutions will now
occur to those skilled in the art without departing from the
invention.
[0068] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. In
case of conflict, the patent specification, including definitions,
will control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting. Numerous
variations, changes, and substitutions will now occur to those
skilled in the art without departing from the invention.
DEFINITIONS
[0069] The articles "a", "an" and "the" are non-limiting. For
example, "the method" includes the broadest definition of the
meaning of the phrase, which can be more than one method.
[0070] A "subject" may be a human or animal. The subject may be
living or dead. The subject may be a patient, clinical subject, or
pre-clinical subject. A subject may be undergoing diagnosis,
treatment, and/or disease prevention. The subject may or may not be
under the care of a health care professional.
[0071] A "blood sample" is a sample of blood or any blood fraction,
blood derivative, and the like. Plasma is an example of a blood
fraction. The blood sample can have any suitable volume, be
obtained by any suitable method, be collected from any part of the
subject at any time, be collected in any suitable vessel, and the
like. Blood is a specialized bodily fluid in animals (including
humans) that delivers necessary substances such as nutrients and
oxygen to the cells and transports metabolic waste products away
from those same cells. Blood samples may have any suitable
materials added, optionally one or more anti-coagulants. "Blood
sample" also includes blood samples that are diluted.
[0072] "Plasma" is the liquid component of blood in which the blood
cells in whole blood are normally suspended. It is the
intravascular fluid part of extracellular fluid (all body fluid
outside of cells). It is mostly water (about 93% by volume) and may
contain dissolved proteins, glucose, clotting factors, mineral
ions, hormones and carbon dioxide (plasma being the main medium for
excretory product transportation). Blood plasma may be prepared by
spinning (centrifuging) a tube of blood containing an
anti-coagulant in a centrifuge until the blood cells sediment to
the bottom of the tube. The blood plasma is then aspirated or drawn
off.
[0073] "Blood serum" is blood plasma without fibrin, fibrinogen or
the other clotting factors (i.e., whole blood minus both cells and
clotting factors).
[0074] Blood samples may be obtained by a "non-venous route",
meaning that the blood is not drawn from the veins and arteries of
the body with a needle. Non-venous route does not limit the blood
sample to being either venous blood (deoxygenated blood) or
arterial blood (oxygenated blood). Both venous blood and arterial
blood are suitable. Obtaining blood from capillaries of the body is
one example of a non-venous route.
[0075] A "finger prick", "fingerstick", or similar is one example
of a method suitable for obtaining a blood sample by a non-venous
route. Here, a sharp point or edge may be used to penetrate the
skin of the finger (or any other part of the body), causing blood
to emanate from the body. A fingerstick may also be performed on
the heel, optionally on the heel of a baby for example. The blood
may be collected using a capillary tube, pipette, swab, drop, or
any other mechanism known in the art.
[0076] The terms "clotting" and "coagulation", as well as their
grammatical variants are used interchangeably. They refer to any
process in which a fluid or any portion of a fluid solidifies
and/or becomes highly viscous. In general, as used herein
"clotting" and "coagulation" refer to the biological process by
which blood forms clots as described above.
[0077] A "coagulation reaction" is any process by which a fluid
coagulates, including any process by which blood coagulates. The
coagulation reaction may be natural, unmodified, and the like, or
may be modified, manipulated, controlled, and the like as described
herein.
[0078] A "coagulation initiation reagent" is any material added to
a blood sample to initiate a coagulation reaction. As described
herein, exemplary coagulation initiation reagents include
thromboplastin and calcium plus thromboplastin.
[0079] The "coagulation time" is the amount of time that elapses
between an initiation event such as the addition of a coagulation
initiation reagent and the formation of a clot. As described
herein, in one version of the assay formation of a clot can be
detected by adding beads and observing when they become
substantially motionless and/or undergo a transition of
mobility.
[0080] The terms "beads" and "particles" are used interchangeably
to mean small (as described herein), solid elements of matter,
suitable for imaging as described herein.
[0081] "Substantially motionless" means that the objects such as
beads do not move in any direction relative to the coagulation
reaction medium (i.e. are suspended in the reaction medium). A
small amount of motion is allowable including about 0.01%, about
0.1%, or about 1% of the rate of motion of the beads prior to the
coagulation time.
[0082] Coagulation time may also extend from an initiation event to
a "transition of the mobility" of the beads, generally meaning a
deceleration of the movement of the beads.
[0083] "Coagulation parameters" are any quantitative or qualitative
measure of any property of a coagulation reaction. Coagulation
parameters may be relatable to coagulation time as described herein
or known in the art.
[0084] "Images" are any artifact, for example a two-dimensional
picture, set of pictures, or video that has a similar appearance to
some physical object. Images may involve the capture of light by a
camera.
[0085] Images may be "pixilated", meaning that they comprise
pixels.
[0086] A "point of service" is any location that may receive or
analyze a sample from a subject, any location where the health of a
subject may be monitored, any location where a subject may receive
a medical treatment, or any location where a subject may receive an
answer or resolution to a health-related question or issue. In some
embodiments, a point of service is a subject's location (e.g.,
home, business, sports event, security screening, combat location),
the location of a healthcare provider (e.g., doctor), a pharmacy or
retailer, a clinic, a hospital, an emergency room, a nursing home,
a hospice care location, or a laboratory. A retailer may be a
pharmacy (e.g., retail pharmacy, clinical pharmacy, hospital
pharmacy), drugstore, chain store, supermarket, or grocer. In some
situations, a point of service is any location that is designated
for use by a certifying or licensing entity (e.g., a government
certifying entity). In other situations, a point of service a
transportation vehicle, such as a car, boat, truck, bus, airplane,
motorcycle, van, traveling medical vehicle, mobile unit, ambulance,
fire engine/truck, critical care vehicle, or other vehicle
configured to transport a subject from one point to another. In
some embodiments, a point of service is a "point of care." A "point
of care" is a point of service where a subject may receive medical
treatment. A "point of care" can include any location at or near
the site where a subject may receive medical treatment, including,
without limitation, hospitals, doctor's offices, transportation
vehicles, or a subject's home. A "point of care" can include
locations such as grocery stores, pharmacies, or businesses, if
such locations are configured to provide any form of medical
treatment, and/or are configured to host medical personnel that
provide medical treatment to a subject.
[0087] "Video" images are a series of images collected sequentially
over time. Video images may be collected, for example, at at least
1 frame/minute, at least 1 frame/10 seconds, at least 1
frame/second, at least 10 frames/second, at least 20 frames/second,
at least 30 frames/second, at least 40 frames/second, at least 50
frames/second, at least 100 frames/second, or at least 200
frames/second.
Coagulation Parameters
[0088] Coagulation is highly conserved throughout biology. In
mammals, coagulation typically involves both a cellular (platelet)
and a protein (coagulation factor) component (although in some
circumstances, plasma can clot without platelets being present).
The system in humans has been the most extensively researched and
is therefore the best understood.
[0089] Coagulation begins almost instantly after an injury to the
blood vessel has damaged the endothelium lining a vessel. Exposure
of the blood to molecules such as tissue factor initiates changes
to blood platelets and the plasma protein fibrinogen, a clotting
factor. Platelets immediately form a plug at the site of injury.
This is typically referred to as primary hemostasis. Secondary
hemostasis typically occurs simultaneously. Proteins in the blood
plasma, such as coagulation factors or clotting factors, respond in
a complex cascade to form fibrin strands, which strengthen the
platelet plug. This cascade involves at least about 13 clotting
factors, a defect in any of which can result in a coagulation
disorder. Furthermore, the coagulation cascade comprises a tissue
factor pathway (also known as the extrinsic pathway) and a contact
activation pathway (also known as intrinsic pathway). Clinical
tests are often designed to eliminate the complexity of the
underlying coagulation process and report a single, easily utilized
parameter.
[0090] In general, coagulation parameters are determined by
measuring a "coagulation time", which is the time between
initiation of a coagulation event and the formation of a clot.
Coagulation parameters encompass the measured coagulation time. In
some instances, the coagulation parameter is the coagulation time
in the presence of certain reagents, at a certain temperature, and
the like.
[0091] Numerous tests and/or assays have been developed to assess
the function of the coagulation system, any of which may be
suitable for measurement using the methods described herein. Common
coagulation parameter assays include aPTT, PT, and INR as
introduced above and described in more detail below. Other
coagulation parameter assays commonly known in the art include
fibrinogen testing, which is often performed by the Clauss method,
platelet count assays, and platelet function testing which is often
performed with a PFA-100.TM. analyzer from Siemens Corporation.
Further coagulation assays and/or clinical procedures known in the
art include thrombin clotting time (TCT) testing, bleeding time
assays, mixing test (whether an abnormality corrects if the
patient's plasma is mixed with normal plasma), coagulation factor
assays, antiphosholipid antibody assays, D-dimer test, genetic
tests (e.g. factor V Leiden, prothrombin mutation G20210A), dilute
Russell's viper venom time (dRVVT) assay, miscellaneous platelet
function tests, thromboelastography assays (TEG or Sonoclot), and
euglobulin lysis time assays (ELT).
[0092] The contact activation (intrinsic) pathway is initiated by
activation of the "contact factors" of plasma, and can be measured
by the activated partial thromboplastin time (aPTT) test (formerly
called the Kaolin cephalin clotting time, "KccT"). The method
historically involves collection of blood into a vessel with
oxalate or citrate to arrest coagulation by binding calcium. The
intrinsic pathway is activated by adding phospholipid, an activator
(such as silica, celite, kaolin, ellagic acid), and calcium to
reverse the effect of the oxalate or citrate. Time is measured
until a clot forms. The test is termed "partial" due to the absence
of tissue factor from the reaction mixture. Values below 25 seconds
or over 39 seconds are generally abnormal. In certain embodiments,
the present method involves dilution of the sample, which prolongs
the clotting time. The equivalent range of clotting times in an
undiluted sample can be easily determined by preparation of a
calibration curve.
[0093] An abnormal aPTT time can be indicative of either the
presence of a clotting inhibitor or a deficiency in quantity or
function of certain clotting factors. Tests can be performed to
distinguish the case, wherein the sample is diluted (initially
about 50:50) with normal plasma. If the abnormality does not
disappear, the sample likely contains an inhibitor such as heparin,
antiphospholipid antibodies, or coagulation factor specific
antibodies. If the abnormal aPTT time is corrected, there may be a
deficiency in factors VIII, IX, XI, XII and/or von Willebrand
factor. The present disclosure encompasses both aPTT measurements
and mixing tests involving aPTT measurement.
[0094] The tissue factor (extrinsic) pathway is initiated by
release of tissue factor (a specific cellular lipoprotein), and can
be measured by the prothrombin time (PT) test. PT results are often
reported as a ratio (INR value) to monitor dosing of oral
anticoagulants such as warfarin, indicate liver damage, or indicate
vitamin K status. PT measures coagulation factors I, II, V, VII and
X. The method historically involves collection of blood into a
vessel with citrate and centrifugation to separate blood cells from
plasma. Typically, an excess of calcium is added to the
EDTA/citrate/oxalate anti-coagulated plasma, tissue factor (also
known as factor III) is added, and the time the sample takes to
clot is observed. The clotting time can vary substantially
according to the analytical system employed and variations between
different batches of manufacturer's tissue factor used to perform
the test. The INR was devised to standardize the results. As seen
in Equation 1, the INR is the prothrombin ratio (prothrombin time
for a patient divided by the result for average normal control
plasmas) raised to the power of the ISI. The ISI (International
Sensitivity Index) indicates how a particular batch of tissue
factor compares to an international reference tissue factor. The
ISI is usually between 1.0 and 2.0 and is reported by the
manufacturer of the tissue factor.
INR = ( PT test PT normal ) ISI ( Equation 1 ) ##EQU00001##
[0095] A high INR level such as about 5.0 indicates that there is a
high chance of bleeding. A low INR level such as 0.5 indicates that
there is a high chance of forming a clot. The normal range for a
healthy person is generally between about 0.9 and 1.3. The normal
range for persons on warfarin therapy is generally between about
2.0 and 3.0, although the target INR may be higher for those with a
mechanical heart valve for example.
[0096] Quantitative and qualitative screening of patients for
fibrinogen disorders may be achieved by measuring by the thrombin
clotting time (TCT). Measurement of the exact amount of fibrinogen
present in the blood is generally done using the Clauss method for
fibrinogen testing. Many analyzers are capable of measuring a
"derived fibrinogen" level from the graph of the Prothrombin time
clot. The methods and devices described herein can similarly be
used to measure the quantity and/or quality of fibrinogen.
[0097] If a coagulation factor is part of the contact activation or
tissue factor pathway, a deficiency of that factor will not
necessarily affect all coagulation parameter tests. For example,
hemophilia A is a deficiency of factor VIII, which is part of the
contact activation pathway. Hemophilia A therefore results in an
abnormally prolonged aPTT test but a normal PT test. It can be
advantageous that the methods and devices described herein allow
multiple tests, including various coagulation tests from a single
drop of blood in a multiplexed, easy to use format.
Coagulation Measurement Methods, Devices and Systems
[0098] The methods, devices and systems described herein may be
used to measure any of the above-referenced coagulation parameters
and/or monitor the effects of drug dosage in persons medicated with
anti-coagulants. Anti-coagulants inhibit clotting and increase the
time in which blood clots, and can be used as medication for
thrombic disorders or in medical devices. Exemplary anti-coagulants
include coumadins such as warfarin, acenocoumarol, phenprocoumon,
and phenindione; heparin and its derivative substances such as low
molecular weight heparin; synthetic pentasaccharide inhibitors of
factor Xa such as fondaparinux and idraparinux; direct thrombin
inhibitors including argatroban, lepirudin, bivalirudin,
ximelagatran and dabigatran; direct factor Xa inhibitors such as
rivaroxaban and apixaban; and other types such as batroxobin and
hementin.
[0099] In some embodiments, provided herein are methods, devices
and systems for measuring coagulation time of a blood sample of a
subject. In some embodiments, the methods include adding a
coagulation initiation reagent to the blood sample from the subject
under a condition suitable for clot formation, thereby initiating
the coagulation reaction; obtaining a set of images of the
coagulation reaction; and analyzing the set of images to measure
the coagulation time of the blood sample. In some embodiments, the
methods further include obtaining a blood sample from the subject
via a non-venous route.
[0100] In one embodiment, a device includes a component capable of
adding a coagulation initiation reagent to the blood sample from
the subject under a condition suitable for clot formation, thereby
initiating the coagulation reaction; a component capable of
obtaining a set of images of the coagulation reaction; and a
component capable of analyzing the set of images to measure the
coagulation time of the blood sample. The component capable of
analyzing a set of images to measure the coagulation time of a
blood sample may be part of the same apparatus within the device as
the component that is configured to obtain more than one image of
the coagulation reaction. The component capable of analyzing a set
of images to measure the coagulation time of a blood sample may be
embedded within the device. The component capable of analyzing a
set of images to measure the coagulation time of a blood sample may
be configured to perform multiple types of analysis and/or it may
be used for multiple applications within the device. A component
capable of analyzing a set of images to measure the coagulation
time of a blood sample may be located remotely from the device. A
component capable of analyzing a set of images to measure the
coagulation time of a blood sample may be located in a cloud
computing infrastructure (e.g. cloud computing). A component
capable of analyzing a set of images to measure the coagulation
time of a blood sample may be located in the cloud, and the device
may be configured to be dynamically controlled from the cloud. In
some embodiments, the device is configured to affect a secondary
procedure based on the results of a coagulation assay analysis. In
some embodiments, a device capable of performing a coagulation
assay as described herein may be configured as a device described
in, for example, U.S. Ser. No. 13/244,947, which is herein
incorporated by reference in its entirety.
[0101] The subject systems may include a device capable of adding a
coagulation initiation reagent to the blood sample from the subject
under a condition suitable for clot formation, thereby initiating
the coagulation reaction; a camera capable of obtaining a set of
images of the coagulation reaction; and a computer capable of
analyzing the set of images to measure the coagulation time of the
blood sample. The computer configured to analyze a set of images to
measure the coagulation time of a blood sample may be part of the
same apparatus within the system as the camera that is configured
to obtain a set of more than one image of the coagulation reaction.
The computer configured to analyze a set of images to measure the
coagulation time of a blood sample may be embedded within the
system. The computer configured to analyze a set of images to
measure the coagulation time of a blood sample may be configured to
perform multiple types of analysis and/or it can be used for
multiple applications within the system. The computer configured to
analyze a set of images to measure the coagulation time of a blood
sample may be located remotely from a camera configured to obtain a
set of more than one image of a coagulation reaction. The computer
configured to analyze a set of images to measure the coagulation
time of a blood sample may be located in the cloud. The computer
configured to analyze a set of images to measure the coagulation
time of a blood sample may located in the cloud, and the system may
be configured to be dynamically controlled from the cloud. The
system may be configured to affect a secondary procedure based on
the results of a coagulation assay analysis. In some embodiments, a
system capable of performing a coagulation assay as described
herein may be configured as a system described in, for example,
U.S. Ser. No. 13/244,947, which is herein incorporated by reference
in its entirety.
[0102] In one aspect, the methods, devices and systems described
herein measure coagulation time using small volumes of blood or
plasma. The blood can be obtained by a finger-stick, where a drop
with a volume of about 20 .mu.L is generally obtained. The
coagulation measurement methods described herein can use this
entire amount (or even more than 20 .mu.L, including about 2, about
3, or about 4 drops). The methods can also use less than one drop
of blood. In some instances, a single drop of blood is used in
several measurements, coagulation or otherwise, optionally in a
multiplexed format.
[0103] The volume of blood or plasma used in the methods, devices
and systems described herein can be any suitable amount. In some
embodiments, the volume is about 1 ml, about 500 .mu.L, about 400
.mu.L, about 300 .mu.L, about 200 .mu.L, about 100 .mu.L, about 75
.mu.L, about 50 .mu.L, about 40 .mu.L, about 20 .mu.L, about 10
.mu.L, about 9 .mu.L, about 8 .mu.L, about 7 .mu.L, about 6 .mu.L,
about 5 .mu.L, about 4 .mu.L, about 3 .mu.L, about 2 .mu.L, about 1
.mu.L, about 0.8 .mu.L, about 0.6 .mu.L, about 0.4 .mu.L, about 0.2
.mu.L, about 0.1 .mu.L, about 0.05 .mu.L, about 0.01 .mu.L, and the
like. In some embodiments, the volume is at most about 1 ml, at
most about 500 .mu.L, at most about 400 .mu.L, at most about 300
.mu.L, at most about 200 .mu.L, at most about 100 .mu.L, at most
about 75 .mu.L, at most about 50 .mu.L, at most about 40 .mu.L, at
most about 20 .mu.L, at most about 10 .mu.L, at most about 9 .mu.L,
at most about 8 .mu.L, at most about 7 .mu.L, at most about 6
.mu.L, at most about 5 .mu.L, at most about 4 .mu.L, at most about
3 .mu.L, at most about 2 .mu.L, at most about 1 .mu.L, at most
about 0.8 .mu.L, at most about 0.6 .mu.L, at most about 0.4 .mu.L,
at most about 0.2 .mu.L, at most about 0.1 .mu.L, at most about
0.05 .mu.L, at most about 0.01 .mu.L, and the like.
[0104] In some embodiments, the device or system includes a
microscope and/or camera. The camera may be a video camera. The
microscope may be configured for brightfield, darkfield, or
fluorescence microscopy. The device or system may be further
configured to receive a cartridge. The cartridge may contain a
blood sample and/or reagents for performing coagulation assays. The
device or system may contain integrated sample processing
mechanisms and/or the device or system may have automated sample
processing mechanisms. In some aspect, the device or system may be
configured to receive a cartridge containing a blood sample and to
perform an automated coagulation assay.
[0105] In some embodiments, a user may introduce a volume of blood
sample from a subject into a cartridge. The volume of blood may be
a small amount, such as 1 ml or less, 500 .mu.L or less, 400 .mu.L
or less, 300 .mu.L or less, 200 .mu.L or less, 100 .mu.L or less,
75 .mu.L or less, 50 .mu.L or less, 40 .mu.L or less, 30 .mu.L or
less, 20 .mu.L or less, 15 .mu.L or less, 10 .mu.L or less, 9 .mu.L
or less, 8 .mu.L or less, 7 .mu.L or less, 6 .mu.L or less, 5 .mu.L
or less, 4 .mu.L or less, 3 .mu.L or less, 2 .mu.L or less, 1 .mu.L
or less, 0.8 .mu.L or less, 0.6 .mu.L or less, 0.5 .mu.L or less,
0.4 .mu.L or less, 0.3 .mu.L or less, 0.2 .mu.L or less, or 0.1
.mu.L or less. The cartridge may contain an anticoagulant which
mixes with the blood sample. The cartridge may be introduced into a
device. Either within the cartridge or within the device, the blood
sample may be separated into a plasma portion and packed portion
containing red blood cells. Alternatively, the blood sample may
remain whole blood. The blood sample may be distributed within the
device to one or more different assay units, and used for one or
more different assays. The blood sample may be distributed within
the device by a fluid transfer device. The blood sample may be
diluted. The blood sample may be mixed with one or more reagents.
The reagents may perform one or more of the following functions: A)
reverse the effect of an anticoagulant (for example, addition of
calcium ions to a sample containing EDTA may reverse the
anti-coagulant effects of EDTA); B) promote coagulation of the
sample (for example, phospholipid, silica, celite, kaolin, ellagic
acid, etc.); C) facilitate visualization of the coagulation
reaction (for example, small beads or other particles which may be
observed); D) increase the strength and/or mass of a coagulation
clot (for example, fibrinogen); or E) reduce non-specific binding
of analytes of a blood sample within reaction vessels (for example,
detergents or proteins). The reagents may perform additional
functions, as well. The reagents may be in liquid or dry form.
Reagents such as sucrose, trehalose, polyethylene glycol, or
albumin may be formulated in any of a variety of dry forms, such as
in an erodible film formulated for rapid dissolution. The mixture
of blood sample with reagents may be observed by any device capable
of obtaining one or more image correlated with the coagulation
reaction. Video images may be obtained. Images of the coagulation
reactions may be analyzed, in order to determine the coagulation
time of the reaction. Analysis a coagulation reaction may be
performed with the aid of a computer or other component of the
device. The coagulation time of the reaction may then be used in
the analysis of the medical condition of a subject. One or more the
steps of the method may be performed at point of service or point
of care location. Performance of methods disclosed herein a point
of care location may enable medical personnel to rapidly make a
treatment decision for a subject based on assay data related to the
specific subject.
Sample Handling and Reaction Chambers
[0106] Samples, reagents, and coagulation assays described herein
can be handled and contained by a variety of reaction vessel types.
A sample handing device and a reaction vessel can be, for example,
a well, a tube, an open ended tip, which may also be a cuvette, or
rectangular or square section capillaries. As used herein, a tip
can also be referred to as a sample tip, a cuvette tip, a reaction
chamber, a cuvette, a capillary, a sample handing device, or a
sample transfer device. Samples may be collected from a source into
a tip or a tube. The tips may be sealed. Such seals may be
permanent or reversible. Once the assay is ready for reading, the
coagulation reaction can be presented to an optical system for
image analysis or other types of reading. Many assays can be
processed in parallel. Assay readout can be serial or simultaneous
depending on the assay protocol and/or incubation time. For assays
involving measurement of a rate of change, the assay element can be
presented to the optical system more than once at different
times.
Fluid and Material Handling Devices
[0107] A fluid transfer apparatus can be part of a device. A fluid
transfer device can be part of a system. The fluid transfer device
or apparatus can comprise a plurality of heads. Any number of heads
may be part of the fluid transfer device. In an example, a fluid
transfer device has about eight heads mounted in a line and
separated by a distance. In an embodiment, the heads have a tapered
nozzle that engages by press fitting with a variety of tips. The
tips can have a feature that enables them to be removed
automatically by the instrument and disposed into in a housing of a
device after use. In an embodiment, the assay tips are clear and
transparent and can be similar to a cuvette within which an assay
is run that can be detected by an optical detector such as a
photomultiplier tube or camera sensor.
[0108] In an example, a programmable processor of a system can
comprise instructions or commands and can operate a fluid transfer
device according to the instructions to transfer liquid samples by
either withdrawing (for drawing liquid in) or extending (for
expelling liquid) a piston into a closed air space. Both the volume
of air moved and the speed of movement can be precisely controlled,
for example, by the programmable processor.
[0109] Mixing of samples (or reagents) with diluents (or other
reagents) can be achieved by aspirating components to be mixed into
a common tube and then repeatedly aspirating a significant fraction
of the combined liquid volume up and down into a tip. Dissolution
of reagents dried into a tube can be done is similar fashion.
Removal of samples and reagents can be achieved by expelling the
liquid into a reservoir or an absorbent pad in a device. Another
reagent can then be drawn into the tip according to instructions or
protocol from the programmable processor.
[0110] A system can comprise a holder or engager for moving the
assay units or tips. An engager may comprise a vacuum assembly or
an assembly designed to fit snugly into a boss of an assay unit
tip. For example, a means for moving the tips can be moved in a
manner similar to the fluid transfer device heads. The device can
also be moved on a stage according to the position of an engager or
holder.
[0111] In an embodiment, an instrument for moving the tips is the
same as an instrument for moving a volume of sample, such as a
fluid transfer device as described herein. For example, a sample
collection tip can be fitted onto a pipette head according to the
boss on the collection tip. The collection tip can then be used to
distribute the liquid throughout the device and system. After the
liquid has been distributed, the collection tip can be disposed,
and the pipette head can be fitted onto an assay unit according to
the boss on the assay unit. The assay unit tip can then be moved
from reagent unit to reagent unit, and reagents can be distributed
to the assay unit according to the aspiration- or pipette-type
action provided by the pipette head. The pipette head can also
perform mixing within a collection tip, assay unit, or reagent unit
by aspiration- or syringe-type action.
[0112] In another embodiment, tips containing liquids including
coagulation assays can be disconnected from the pipetting device
and "parked" at specific locations within the instrument or within
a disposable unit. If needed, tips can be capped using a seal to
prevent liquids from draining out. In some embodiments, the seal
can be a vinyl seal. Any variations in the fluid and material
handling devices disclosed herein or described in U.S. Ser. No.
13/244,947 or U.S. Pat. No. 8,088,593 which are incorporated herein
in their entirety, can be adopted
Exemplary Sample Tips
[0113] A variety of container shapes can be utilized as sample
tips, reaction chambers, capillaries and cuvettes. For example, a
cuvette can be circular, cylindrical, square, rectangular, cubical,
conical, pyramidal, or any other shape capable of holding a sample
of fluid. Rectangular cuvettes where a light beam impinges at right
angles [or other angle (other than 0 degrees) to the light beam] to
the cuvette surfaces can be employed. In such rectangular cuvettes,
the liquid sample that is illuminated is also rectangular and is
defined by the cuvette. Cuvettes with circular cross-sections can
also be used.
[0114] Variable pathlength cuvettes can be used to optimize and
extend the assay response and minimize the volume of sample
required to measure the assay. Cuvettes can be longer in relation
to their cross-section in at least one region.
[0115] In some embodiments, one version of the assay cuvette has a
circular cross-section in the direction of the light beam. The use
of a cuvette with a circular cross-section may have several
advantages, including, but not limited to the following:
[0116] 1. The optical pathlength can be precisely defined.
Dimensional precision of injection-molded parts have been found to
be better than 1-2% Coefficient of Variation (CV) In conventional
microtiter plates the unconstrained liquid meniscus can introduce
imprecision in pathlength.
[0117] 2. The open-ended character and circular section of the tips
confers excellent fluid handling characteristics, making aspiration
of liquids very precise.
[0118] 3. The optical image of the tips provides for the ability to
identify the tip location and boundaries of the liquid column and
to locate very precisely the center of the tip where the signal is
maximal.
[0119] 4. More than one liquid sample can be incubated and analyzed
in the same tip. This is because in the narrow part of the tip,
very little material transfer occurs (in the axial direction)
between adjacent "slugs" of liquid.
[0120] Any variations in cuvettes or imaging systems or methods
disclosed herein or described in U.S. Ser. No. 13/355,458 which is
incorporated herein in its entirety, can be adopted.
[0121] An exemplary tip may have the following general
features:
[0122] Tip length: 0.5-4 cm.
[0123] Tip outer diameter: 0.2-1.0 cm.
[0124] Tip inner diameter: 0.1-0.5 cm.
[0125] Tip capacity for liquids: 0.5-50 .mu.L.
[0126] Tip dimensional precision: generally better than 2% or
+1-0.001 cm.
[0127] Tip configuration: The tip will generally have a feature
that engages with a pipette (cylindrical) so as to form a fluid
tight seal. There is a region generally cylindrical or conical
which is used for imaging. The lower end of the tip will typically
be narrow so as to aid in retention of vertical liquid columns
under gravity.
[0128] Tip material: Preferably clear plastic (polystyrene,
polypropylene etc.) (for example, which transmits light in the
visible >80, 60, 40, 20%). Other suitable materials are not
excluded.
[0129] In one example, at the upper end of the cylinder is a
truncated cylindrical "boss" fluidically connected to the cylinder
and adapted so as to be able to engage with the tapered feature of
a pipetter. The lower end of the tip may be narrowed to provide a
feature that enables the tip to hold its liquid contents when
oriented vertically and not attached to the pipetter. The external
shape of the lower end of the tip is typically also somewhat
pointed with the diameter being reduced from the main part of the
cylindrical shaft toward the end so as to be capable of being
fluidically sealed with a flexible (vinyl) cap into which the tip
end is press fit. Tips are usually made of molded plastic
(polystyrene, polypropylene and the like). The tips can be clear or
translucent such that information about the sample can be acquired
by imaging.
[0130] In some embodiments, the tip is configured with (1) an upper
feature that can engage to form an air tight seal with a pipette
head, (2) a basically cylindrical (or conical with a very slight
draft angle) shaft and a narrow, pointed lower tip. This tip can
form a liquid-tight seal with a cap. The pointed shape aids in
getting good conformance with the cap under moderate force. The tip
material may be injection-molded polystyrene.
[0131] Sealing can be achieved using a cap made of vinyl or other
materials which is easily press-fitted to the narrow end of the
sample containment means using force generated by motion of the
instrument stage in the z-direction.
Reagents and Reactions
[0132] Any user may perform the methods described herein. The user
can be the subject himself or herself. The user can be a medically
trained person such as a doctor or nurse, but this is not
necessarily required. The user may have undergone general or
special technical training in order to perform the methods
described herein, but this is not necessarily required. The methods
may also be performed by more than one user, for example various
users may perform various steps.
[0133] The methods described herein may begin with a previously
drawn blood and/or plasma sample. In such embodiments, the sample
will often have an anticoagulant added, typically EDTA. The methods
described herein may also begin with obtaining blood from a
subject. The blood may be obtained from a non-venous route (e.g.
from capillaries, e.g. not involving a needle). The blood may be
obtained from a finger-stick, for example. The blood may be
collected into any suitable vessel. In some embodiments, the blood
is collected into a cartridge. The vessel and/or collection
cartridge may contain an anticoagulant, typically EDTA. The
anticoagulant may spontaneously mix with and/or dissolve in the
blood sample.
[0134] In some embodiments, the sample (generally containing an
anticoagulant) is centrifuged. Centrifugation may be performed for
any suitable combination of time and centrifugal force such that
the blood separates into packed formed elements and plasma. The
formed elements generally consist predominantly of red blood cells
and the plasma is generally substantially free of cells.
Centrifugation is not always necessary. Some embodiments can be
performed with whole blood. The whole blood may be diluted. Some
embodiments may also prepare plasma from blood without the use of
centrifugation, such as by addition of a reagent that aggregates
blood cells for example.
[0135] All or a portion of the plasma may be used in the assay. The
plasma may also be distributed among several assays in a
multiplexed format. Suitable methods for pipetting low volumes of
plasma are disclosed in, for example, U.S. Ser. No. 13/244,947 and
U.S. Pat. No. 8,088,593. In some embodiments, the plasma is diluted
as described below. The plasma can be diluted in any suitable
fluid. Exemplary fluids include HEPES buffered saline (HBS),
phosphate buffered saline (PBS), tris buffered saline (TBS),
imidazole, ethylenediamine, N-ethyl morpholine, triethanlolamine,
and other buffering agents in the neutral range (i.e. about pH
5-9). The plasma and/or plasma sample may be diluted any one or
more of before, during, or after measuring the coagulation
parameter and/or distributing the plasma among the assays of a
multiplexed format.
[0136] A small volume of the optionally diluted sample is then
mixed with certain reagents. This mixing is generally performed
rapidly, such as in less than about 10 seconds, less than about 5
seconds, or less than about 1 second. The reagents generally
include (a) if needed, a reagent which reverses the effect of the
anti-coagulant, (b) a reagent which promotes coagulation, and (c)
for diluted samples one or more ancillary reagents such as
fibrinogen, or proteins as described below. In some instances, a
dispersion of fluorescent or other particles may be added to
determine coagulation time by imaging as described below.
[0137] The reagent that reverses the effect of the anti-coagulant
can be any suitable reagent. For example, calcium ions may be added
to reverse the effect of EDTA. The calcium ions, optionally in the
form of CaCl.sub.2, may be added in excess.
[0138] The reagents that promote coagulation ("coagulation
initiation reagent") can vary depending on the coagulation
parameter being measured. As described above, measurement of PT
and/or INR involves the addition of prothrombin reagent comprising
tissue factor and lipids. The aPTT assay uses phospholipid plus an
activator such as silica, celite, kaolin or ellagic acid.
[0139] In some embodiments, one or more of the reagents are present
in concentrated or dried form. Concentrated reagents may reduce the
amount of sample dilution. Regarding dried reagents, mixing the
sample with the dried reagents rapidly dissolves and/or disperses
the reagents in the sample. The dried reagents may be dissolved
and/or dispersed in the sample by repeated aspiration or other
means of mixing. Any reagent may be concentrated or dried, provided
that the reagent is stable in concentrated or dried form. In fact,
some reagents may have increased stability in dried form,
potentially avoiding the need for refrigeration, preservatives and
the like. In particular, the PT reagent, aPTT reagent, and/or
CaCl.sub.2 may be concentrated and/or dried. All of the reagents
can be combined into one dried reagent. For example, the reagents
can be in the form of an erodible film formulated for rapid
dissolution including formulations with sucrose, trehalose,
polyethylene glycol, albumin, and the like. One formulation of
dried reagents suitable for use in PT and/or INR measurement can be
found in U.S. Pat. No. 5,164,598.
[0140] In some embodiments, the plasma mixed with reagents is then
incubated, generally at a controlled temperature. In some
embodiments, the temperature is about 15.degree. C., about
20.degree. C., about 25.degree. C., about 30.degree. C., about
35.degree. C., about 40.degree. C., about 45.degree. C., about
50.degree. C., and the like. In some embodiments, the temperature
is between about 15.degree. C. and about 50.degree. C., between
about 30.degree. C. and about 40.degree. C., and the like. In some
instances the incubation step may be omitted. For example, the aPTT
assay may not require incubation.
Methods of Dilution
[0141] In some embodiments, the blood or plasma sample is diluted.
Diluting the sample may confer at least three potential advantages.
First, dilution may reduce the amount of sample required. In some
embodiments, once an aliquot of diluted plasma has been reserved
for measurement of coagulation parameters, the remainder can be
used for other assays. Also, for example, dilution of the sample
10-fold allows the method to use 10-fold less sample. Secondly,
dilution may reduce light scattering from lipemic samples (samples
with a high fat content that may appear milky white). Thirdly,
dilution increases the coagulation time. This may be advantageous
in making the assay less time-sensitive. That is, steps such as
mixing the sample with reagents or moving the camera do not have to
be performed so rapidly. Performing such steps can be particularly
challenging when using small sample volumes where flow is laminar,
the camera has to be aligned to a small volume, and the like.
[0142] The coagulation time may be any suitable time, optionally
long enough to make the procedure less time sensitive and more
precise and/or accurate, and optionally short enough to be
performed at the point of care and give near real-time results. In
some embodiments, the coagulation assay is performed in parallel
(i.e. multiplexed) with measurement of other biomarkers or
therapeutic agents. In these embodiments, it may be advantageous
and/or practical to dilute the sample such that the coagulation
time is similar to the other measurement times being performed in
the multiplexed assay. Optionally the coagulation time is similar
to or shorter than the longest of the other biomarker or
therapeutic agent assays.
[0143] In some embodiments, the coagulation time, using either
diluted or non-diluted sample, is about 1 minute, about 2 minutes,
about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes,
about 7 minutes, about 8 minutes, about 9 minutes, about 10
minutes, about 15 minutes, about 20 minutes, about 25 minutes,
about 30 minutes, and the like. In some embodiments, the
coagulation time, using either diluted or non-diluted sample, is
less than about 1 minute, less than about 2 minutes, less than
about 3 minutes, less than about 4 minutes, less than about 5
minutes, less than about 6 minutes, less than about 7 minutes, less
than about 8 minutes, less than about 9 minutes, less than about 10
minutes, less than about 15 minutes, less than about 20 minutes,
less than about 25 minutes, less than about 30 minutes, and the
like. In some embodiments, the coagulation time, using either
diluted or non-diluted sample, is between about 1 minute and about
2 minutes, between about 1 minute and about 5 minutes, between
about 2 minutes and about 10 minutes, between about 5 minutes and
about 8 minutes, and the like.
[0144] The sample may be diluted to any suitable extent, optionally
diluted to achieve any suitable coagulation time. In some
embodiments, the sample is diluted accurately and precisely by 1.2
fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 7.5 fold, 10 fold,
20 fold, 30 fold, 40 fold, 50 fold, 100 fold, and the like. In some
embodiments, the sample is diluted by at least about 1.2 fold, at
least about 1.5 fold, at least about 2 fold, at least about 3 fold,
at least about 4 fold, at least about 5 fold, at least about 7.5
fold, at least about 10 fold, at least about 20 fold, at least
about 30 fold, at least about 40 fold, at least about 50 fold, at
least about 100 fold, and the like. In some embodiments, the sample
is diluted by at most about 1.2 fold, at most about 1.5 fold, at
most about 2 fold, at most about 3 fold, at most about 4 fold, at
most about 5 fold, at most about 7.5 fold, at most about 10 fold,
at most about 20 fold, at most about 30 fold, at most about 40
fold, at most about 50 fold, at most about 100 fold, and the like.
In some embodiments, the sample is diluted by between about 1.2
fold and about 2 fold, between about 2 fold and about 5 fold,
between about 5 fold and about 20 fold, between about 5 fold and
about 50 fold, and the like.
[0145] In some embodiments, it may be desirable to complete the
entire coagulation assay in a short time as a short time may be
desirable for using the assay at the point of care in order to
achieve a near real-time result. The total assay time may extend
from drawing blood, optionally by a finger-stick to adding certain
reagents to the blood to capturing images to analyzing images to
reporting a coagulation parameter. The total assay time may also
extend to using the reported coagulation parameter to calculate
and/or administer a dose of anti-coagulant to a patient in need
thereof. In some embodiments, the total assay time is about 1
minute, about 3 minutes, about 5 minutes, about 10 minutes, about
15 minutes, about 20 minutes, about 30 minutes, and the like. In
some embodiments, the total assay time is less than about 1 minute,
less than about 3 minutes, less than about 5 minutes, less than
about 10 minutes, less than about 15 minutes, less than about 20
minutes, less than about 30 minutes, and the like.
[0146] In some embodiments, dilution of the sample under certain
conditions may reduce the mechanical strength of the clot and/or
turbidity of the sample upon coagulation. In certain instances this
may be disadvantageous if the clot strength is decreased such that
it becomes difficult to determine the time at which clotting
occurs. The present disclosure encompasses a number of optional
methods for compensating for dilution.
[0147] In some embodiments, the method may be performed in small
containers and/or containers that have a high surface area to
volume ratio. This may aid in adhesion of an incipient clot to the
surface of the container. Container size and/or surface area may be
a consideration in embodiments where coagulation time is determined
by imaging bulk reciprocating movement of the sample as described
below. The volume of the container and/or surface area to volume
ratio of the container may be any suitable value such that the
methods described herein can be reliably and accurately
performed.
[0148] In some embodiments, ancillary reagents may be added such as
small particles, fibrinogen, surfactants, and/or proteins. Such
ancillary reagents may support the formation of a stronger clot
and/or may increase sample turbidity upon coagulation.
[0149] In some embodiments, small particles may be added to the
sample to visualize changes in sample viscosity. A reduction in
movement of the small particles may be correlated with an increase
in sample viscosity, and may provide an indication of sample
coagulation.
[0150] Without being held to any particular theory, it is believed
that in many versions of the present methods gravitational force is
too weak to overcome the viscous resistance to movement of the
particles once coagulation has occurred, even for coagulation in
dilute samples. Imaging of the particles may be used to determine
coagulation time as described herein.
[0151] In some embodiments, fibrinogen may be added to the sample.
Additional fibrinogen can provide for a more substantial clot
and/or provide for increased turbidity when a clot forms. The
fibrinogen is optionally derived from animals, optionally being
bovine fibrinogen. The amount of fibrinogen to add can be any
amount such that a clot of suitable mechanical strength and/or
turbidity is formed. In various embodiments, the diluted sample is
supplemented with about 1 mg/mL, about 2 mg/mL, about 3 mg/mL,
about 4 mg/mL, about 5 mg/mL, or about 10 mg/mL fibrinogen. In
various embodiments, the diluted sample is supplemented with at
least about 1 mg/mL, at least about 2 mg/mL, at least about 3
mg/mL, at least about 4 mg/mL, at least about 5 mg/mL, or at least
about 10 mg/mL fibrinogen.
[0152] It is also possible to use other methods to compensate for
dilution and/or to use the methods described herein in combination
with each other or in combination with other methods. For example,
one can add fibrinogen and particles in order to aid in the
determination of coagulation time in diluted samples.
[0153] In some instances, dilution of blood and/or plasma samples
may lead to loss of clotting factors from the sample, for example
by adsorption to surfaces such as tubes or pipette tips. In some
embodiments, surfactants and/or proteins may be added to the sample
to prevent and/or reduce the loss of clotting factors. An exemplary
surfactant suitable for this purpose is Triton X-100. An exemplary
protein suitable for this purpose is bovine serum albumin (BSA).
The concentration of surfactant and/or protein may be any suitable
concentration such that clotting factor loss is reduced to any
acceptable level.
Images
[0154] The devices and systems described herein may include any
imaging devices, such as a camera, radio recorder, and any other
handheld, benchtop, or larger devices with imaging capabilities.
The methods described herein may use optical methods to measure a
coagulation parameter. In some embodiments, coagulation of the
sample increases the turbidity and light scattering of the sample.
The addition of fibrinogen is one method for increasing the
turbidity and light scattering of the clot such that it can be
detected as described herein.
[0155] In other embodiments, the sample is initially turbid and/or
is made to be initially turbid by addition of suspended particles
often individually invisible to the naked eye but producing a
turbid haziness in bulk. An initially turbid sample may become less
turbid over time as the particles settle from the bulk fluid, but
coagulation of the sample may halt or substantially slow the
settling of particles and/or decrease in turbidity. In some
embodiments, coagulation time can be determined by the cessation or
slowing of the rate of decrease in bulk turbidity of the sample.
The determination of coagulation time by turbidity is an optical
technique (involves light) and may involve capture of images, but
does not necessarily require the use of images. In some
embodiments, the images are light scattering images, and are
optionally not pixilated. Having a long path of light through the
sample, or increasing the sample path length is one means for
increasing the sensitivity of measurements involving changes in
turbidity.
[0156] The methods described herein may use images to measure a
coagulation parameter. The images can be video images or a time set
of photographic images. The images may be captured by optical
devices such as cameras, mirrors, lenses, microscopes, and the
like. The images can be two-dimensional or three-dimensional. The
images can be black and white, gray-scale or color. The images can
be digital or analog, including digitization of analog images. The
images can be pixilated, meaning that it comprises a plurality of
pixels which may distinguish the images from other optical
phenomena including various forms of spectroscopy. A two
dimensional image may be pixilated by dividing the image into a
plurality of rows and columns, wherein each element (row and column
position) defines a pixel.
[0157] The images can be pixilated into any suitable number of
pixels. In one embodiment, the image is divided into 512 rows and
512 columns, defining 262,144 pixels. In some embodiments, the
image comprises about 10,000 pixels, about 50,000 pixels, about
60,000 pixels, about 100,000 pixels, about 200,000 pixels, about
500,000 pixels, about 1,000,000 pixels, about 5,000,000 pixels,
about 10,000,000 pixels, about 50,000,000 pixels, and the like. In
some embodiments, the image comprises at least about 10,000 pixels,
at least about 50,000 pixels, at least about 60,000 pixels, at
least about 100,000 pixels, at least about 200,000 pixels, at least
about 500,000 pixels, at least about 1,000,000 pixels, at least
about 5,000,000 pixels, at least about 10,000,000 pixels, at least
about 50,000,000 pixels, and the like. In some embodiments, a pixel
density and/or resolution may be reported in which a given area
comprises a certain number of pixels.
[0158] In one aspect, the method describes determining a
coagulation time using a time series of images. Images can be
captured at any suitable rate. The rate can be constant, or can
vary over the time course of coagulation, optionally with more
images being captured around the time when the clot forms in the
sample. In some embodiments, images are captured at a rate of about
1 frame per second, about 5 frames per second, about 10 frames per
second, about 15 frames per second, about 20 frames per second,
about 30 frames per second, about 50 frames per second, about 100
frames per second, about 500 frames per second, and the like. In
some embodiments, images are captured at a rate of at least about 1
frame per second, at least about 5 frames per second, at least
about 10 frames per second, at least about 15 frames per second, at
least about 20 frames per second, at least about 30 frames per
second, at least about 50 frames per second, at least about 100
frames per second, at least about 500 frames per second, and the
like. In some embodiments, interpolation methods can be used to
estimate a coagulation time that falls between frame captures.
[0159] The images can be stored on a computer readable medium. The
images can be processed in real-time or processed at a later time.
The images can be processed manually or using methods implemented
by a computer.
Optical Setup for Imaging
[0160] Coagulation analysis can be performed using an optical
setup. The optical setup can include a light source, an aperture,
and a sensor or a detector. The setup may include a light source, a
camera, and a camera sensor. The camera may further include a lens.
In some embodiments, the camera can be a webcamera, the camera
sensor can be CMOS or CCD chip, the lens can be glass with a
standard object distance webcam lens (e.g. anywhere from 5-100 mm,
including 35 mm), and the light source may be a white light source.
Camera images can be taken in a sequence where 1, 2, 3 4, or more
tips are moved by an x-y-z stage into the optical path.
[0161] In an embodiment, the detector is a reader assembly housing
a detection assembly for detecting coagulation assays, and
optionally, other assay types. The detection assembly may be above
a reaction vessel or at a different orientation in relation to the
reaction vessel based on, for example, the type of assay being
performed and the detection mechanism being employed. The detection
assembly can be moved into communication with the reaction vessel
or the reaction vessel can be moved into communication with the
detection assembly.
[0162] The sensors can be PMTs, wide range photo diodes, avalanche
photodiodes, single frequency photo diodes, image sensors, CMOS
chips, and CCDs. The illumination sources can be lasers, single
color LEDs, broad frequency light from fluorescent lamps or LEDs,
LED arrays, mixtures of red, green, and blue light sources,
phosphors activated by an LED, fluorescent tubes, incandescent
lights, and arc sources, such as a flash tube.
[0163] In many instances, an optical detector is provided and used
as the detection device. Non-limiting examples include a
photodiode, photomultiplier tube (PMT), photon counting detector,
avalanche photo diode, or charge-coupled device (CCD). In some
embodiments a pin diode may be used. In some embodiments a pin
diode can be coupled to an amplifier to create a detection device
with sensitivity comparable to a PMT. In some embodiments a
detection assembly could include a plurality of fiber optic cables
connected as a bundle to a CCD detector or to a PMT array. The
fiber optic bundle could be constructed of discrete fibers or of
many small fibers fused together to form a solid bundle. Such solid
bundles are commercially available and easily interfaced to CCD
detectors.
[0164] A detector can also comprise a light source, such as a bulb
or light emitting diode (LED). The light source can illuminate an
assay in order to detect the results. The detector can also
comprise optics to deliver the light source to the assay, such as a
lens or fiber optics.
[0165] The sample vessel can be back lit, front lit, or oblique
(side) lit. Back lighting can be used for the purpose of detecting
light scattering (nephelometry). The optical arrangement may take
the form of a broad, evenly illuminated rear field, and a
specifically shaped beam that is interrupted by the subject.
Front-lit illumination may also be used.
[0166] In some embodiments, the optical set up for imaging
coagulation is configured to measure scattered light. In one
aspect, a set up for measuring scattered light includes a light
source and a detector. The light source may provide diffuse light.
The sample is held inside of a vessel such as an optically clear
tip or other holder. The vessel may be made of a clear material
such as polystyrene or acrylic. The light path of the vessel (inner
diameter) may be, for example, about 0.05, 0.1, 0.25, 0.5, 0.75, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more millimeters. The
vessel is placed in front of a detector, typically at a spacing of
about 20-50 millimeters from the detector (although other spacing
may be used). The detector can be an imaging detector such as a CCD
or CMOS sensor, or can be a photon counter/detector such as a PMT
or photodiode. The detector may or may not include optical
components such as lenses and filters. Filters may be used to
reduce background by eliminating any stray light from the
surroundings. The sample is illuminated at right angles with
respect to the detector (or any other non-zero angle to the
illuminating beam). The illumination source can either be oriented
from the side (at approximately 90 degrees from the detector), or
from the top. The light source can be any light source, diffuse or
non-diffuse, and be of any wavelength, or a combination of
wavelengths. The wavelength of the light source can be chosen to
match the maximum spectral sensitivity of the detector.
[0167] In fluorescence excitation, subjects can be illuminated from
the front for the purpose of fluorescence illumination. The light
sources may be single color lights, most commonly lasers. Oblique
lighting can also be used in fluorescence excitation. The subjects
are often excited at an angle, usually 90 degrees, from which the
emitted photons will appear. This form of lighting enables scatter
detection directly behind the subject (back lit) as well as the
fluorescence emissions exiting from the side.
[0168] In some embodiments, fluorescent emission is imaged at 90
degrees to the excitation beam. In some embodiments, a photon
source, typically a high-intensity LED, passes through a beam
diffuser and a shaping lens, producing a collimated or gradually
diverging excitation beam. The excitation beam passes through a
band-pass filter and illuminates the sample, consisting of a vessel
(tube, cuvette, or pipette tip) containing a solution with a
fluorescently-labeled sample. Isotropically-emitted fluorescence is
spectrally separated from excitation light with a long- or
band-pass filter appropriate to pass Stokes-shifted fluorescence.
Light is then imaged through a lens onto a digital camera or other
detector. Fluorescence intensity is extracted from the resulting
images via image analysis.
[0169] In other embodiments, transmitted light is imaged after
optical filtering to remove the light at the exciting wavelength.
In some embodiments, a photon source, typically a high-intensity
LED, passes through a beam diffuser and a shaping lens, producing
slowly divergent, elliptical excitation beam. The excitation beam
passes through a band-pass filter and illuminates the samples,
presented as one or more sample vessels (tube, cuvette, or pipette
tip), each containing a solution with a fluorescently-labeled
material. Isotropically-emitted fluorescence is spectrally
separated from excitation light with a long- or band-pass filter
appropriate to pass Stokes-shifted fluorescence. Light is then
imaged through a camera lens onto a digital camera. Fluorescence
intensity is extracted from the resulting images via image
analysis. The optical setup can be used to produces array images of
multiple tubes simultaneously.
[0170] In some embodiments, imaging may occur using fluorescence,
darkfield illumination, or brightfield illumination.
[0171] Darkfield illumination may be achieved by the use of a
ringlight (located either above or below the sample), a darkfield
Abbe condenser, a darkfield condenser with a toroidal mirror, an
epi-darkfield condenser built within a sleeve around the objective
lens, or a combination of ringlight with a stage condenser equipped
with a dark stop. Fundamentally, these optical components create a
light cone of numerical aperture (NA) greater than the NA of the
objective being used. The choice of the illumination scheme depends
upon a number of considerations such as magnification required,
mechanical design considerations, size of the imaging sensor etc. A
ringlight based illumination scheme generally provides uniform
darkfield illumination over a wider area while at the same time
providing sufficient flexibility in mechanical design of the
overall system.
[0172] Brightfield illumination may be achieved by the use of a
white light source along with a stage-condenser to create Koehler
illumination.
[0173] In some embodiments, an automatic filter wheel may be
employed. The automatic filter wheel allows control of the imaging
optical path to enable imaging of multiple fluorophores on the same
field of view.
[0174] In some embodiments, image based auto-focusing may take
place. An image-based algorithm may be used to control the
z-position (e.g., vertical position) of an objective (i.e., its
distance from the sample) to achieve auto-focusing. Briefly, a
small image (for example, 128.times.128 pixels) is captured at a
fast rate using darkfield illumination. This image may be analyzed
to derive the auto-focus function which is measure of image
sharpness. Based on a fast search algorithm the next z-location of
the objective is calculated. The objective may be moved to the new
z-location and another small image may be captured. This
closed-loop system does not require the use of any other hardware
for focusing. The microscope stage may be connected to
computer-controlled stepper motors to allow translation in the X
and Y directions (e.g., horizontal directions). At every location,
the desired number of images is captured and the stage is moved to
the next XY position.
[0175] A camera with a CCD, EMCCD, CMOS or in some cases a
photo-multiplier tube can be used to detect the signal.
Light Scattering Method
[0176] In some embodiments, the coagulation time can be determined
by light scattering. The reaction mixture can be drawn into a
pipette tip or capillary tube by either capillary force or by
aspiration. The tip or capillary can be made of any optically clear
material including but not limited to glass or a clear plastic such
as polystyrene or polymethylmethacrylate (acrylic). In some
embodiments, the pipette tip or capillary is long and thin. For
example, if 1 .mu.L of reaction mixture is drawn into a capillary
of 0.5 mm diameter, a liquid column of about 5.1 mm results.
[0177] The pipette tip or capillary can then be sealed or capped,
optionally to prevent sample evaporation during the assay. One
suitable method for sealing the tip or capillary is to further
aspirate a small volume of mineral oil. The pipette tip or
capillary can also be coated in oil to improve imaging by reducing
light scatter from the surface of the tip or capillary.
[0178] The pipette tip or capillary can then be suspended in front
of a dark background and illuminated. In general, the direction of
illumination is such that the light illuminates the tip or
capillary but does not directly enter the camera or other
photometric detector. For example, if the camera is in front of the
tip or capillary, the light may enter from the side.
[0179] The tip or capillary is optically monitored. A simple
photometric detector such as a photodiode can be used. A camera can
also be used, optionally a video camera. Use of a camera may be
advantageous in some embodiments in that it allows one to determine
the position of the sample by image processing, thereby making the
method less sensitive to pipetting errors and sample placement. In
some embodiments, the tip or capillary is monitored by reflectance
or absorption spectroscopy.
[0180] When coagulation occurs, the fibrinogen polymerizes and
increases the turbidity of the sample. Additional light is
scattered from the reaction mixture, which is registered by the
camera or photometric detector as an increase in the amount of
illumination light scattered into the detector. FIG. 1 shows the
reaction mixture before 100 and after 105 coagulation. Note the
increase in turbidity from indicated positions 100 to 105. Also, in
FIG. 1, the lower parts of the tips 101 are coated with oil,
whereas the upper parts of the tips are not coated with oil 102.
Note the lower amount of light scattering from the oil-coated part
101 of the tip as compared to the non-oil-coated part 102.
[0181] Image analysis allows one to analyze only the light which
passes through the assay mixture, which is especially relevant to
very small assay volumes.
[0182] The time of onset of the increase in light scattering is the
coagulation time for the diluted sample and may be appropriately
transformed to give the appropriate coagulation parameter.
Transformation of the diluted coagulation time to the coagulation
parameter may be done through calibration of the system with
independently characterized samples.
Analysis of Light Scattering Data
[0183] In embodiments where scattered light is measured as
described above, data of the type shown in FIG. 2 may be obtained.
As depicted, the mean signal is plotted against time. In this case
time is on the horizontal axis and extends from just less than 50
seconds to just greater than 300 seconds on the right. Mean signal
intensity is plotted on the vertical axis and extends from just
less than 0.2 mean intensity units at the beginning of the reaction
to just more than 0.45 units at the end. FIG. 3 shows the data from
FIG. 2 fit to a four-parameter log-logistic function progress
curve.
[0184] In some embodiments, the coagulation time can be estimated
from any defined part of the plotted data in FIG. 2 and/or the
progress curve as depicted in FIG. 3. For example, the coagulation
time can be defined to be where the light scattering reaches about
10%, about 50%, or about 80%, and the like of the maximum light
scattering. The optimum point of the curve to use could be
determined by correlation of the results with those of other
accepted predicate methods for clinical samples with coagulation
parameter values spanning the range of clinical concern.
[0185] In some embodiments, the scatter light intensity data may be
fit to a curve as shown in FIG. 19. As depicted, the mean signal is
plotted against time. In this case time is on the horizontal axis
and mean signal intensity is plotted on the vertical axis. In FIG.
19, the sigmoid curve is the mean intensity data, and it is fit to
the bilinear curve. In some embodiments, the coagulation time can
be estimated from any defined part of the bilinear fit as depicted
in FIG. 19. For example, the coagulation time can be defined where
the light scattering signal reaches about 10%, about 20%, about
30%, about 40%, about 50%, about 60%, about 70%, about 80% or about
90% above the baseline level of light scattering.
Particle Bead Settling Method
[0186] In some embodiments, the coagulation time can be determined
by the cessation or slowing of the rate of settling of microscopic
particles or beads in the sample upon clot formation. Beads may be
added to the sample at any suitable concentration at any time prior
to coagulation. In some embodiments, the beads are a part of a
concentrated or dried mixture of reagents that are mixed,
suspended, and/or dissolved and re-suspended in the sample.
[0187] Turning to FIG. 5, the method involves drawing the sample
comprising beads 500 into a transparent vessel such as a capillary
or pipette tip 505. The capillary or pipette type may have a narrow
diameter, optionally about 0.5 mm. The capillary is typically
oriented in the vertical such that the beads settle by force of
gravity along the longest dimension of the tip or capillary. The
settling of the beads is imaged over time by a camera 510. The
camera may be a video camera and/or be coupled with a microscope
capable of imaging the particles. In some embodiments, the
visualization method is video microscopy. In some embodiments, the
camera is a webcam. In some embodiments, the webcam is mounted
about 10 mm from the capillary or tip.
[0188] The terms "beads" and "particles" are used interchangeably.
The beads may have any size such that they settle at a suitable
rate, which is slowed or ceases upon coagulation. In some
embodiments, the beads have a diameter of about 5 .mu.m, about 10
.mu.m, about 20 .mu.m, about 25 .mu.m, about 30 .mu.m, about 35
.mu.m, about 40 .mu.m, about 45 .mu.m, about 50 .mu.m, about 60
.mu.m, about 100 .mu.m, and the like. In some embodiments, the
beads have a diameter of at most about 5 .mu.m, at most about 10
.mu.m, at most about 20 .mu.m, at most about 25 .mu.m, at most
about 30 .mu.m, at most about 35 .mu.m, at most about 40 .mu.m, at
most about 45 .mu.m, at most about 50 .mu.m, at most about 60
.mu.m, at most about 100 .mu.m, and the like. In some embodiments,
the beads have a diameter of between about 5 .mu.m and about 100
.mu.m, between about 20 .mu.m and about 60 .mu.m, between about 25
.mu.m and about 40 .mu.m, and the like. The beads may be made of
any suitable material including polystyrene or latex, they may be
of any shape including spherical, and they may have any suitable
density.
[0189] Beads of various sizes, of various shapes, having various
densities, made of various materials, and the like may settle at
different rates and/or be retained in a clot to various extents. In
some embodiments, a mixture of beads having a distribution of sizes
may be used. In some embodiments, a mixture of beads having a
plurality of different sizes may be used. As shown in FIG. 5, a
mixture of beads having a diameter of 25 .mu.m 515 and beads having
a diameter of 45 .mu.m 520 may be used. A mixture of beads having
various shapes, having various densities, made of various
materials, and the like may also be used. In one aspect, mixtures
of beads may be used. A mixture of beads may provide for a
plurality of settling times and/or clot retention properties in
order to improve the sensitivity of the method, reproducibility of
the method, the range of clotting time measurable by the method,
and the like.
[0190] As time progresses, the beads will settle under force of
gravity as shown in FIG. 5, indication 525. In some embodiments,
the bead motion may be driven by any other suitable force such as
convection, air flow, magnetic fields, Brownian motion, and the
like, optionally in combination with gravity. The beads could also
float in the medium under gravity, if they have a density less than
the medium. Without being held to any particular theory, even weak
clots in diluted plasma are generally sufficient to overcome these
weak forces such as gravity and prevent bead motion. In some
embodiments, the strength of the clots can be increased by addition
of exogenous fibrinogen as described above. In some embodiments,
the bead settling method may use less exogenous fibrinogen than the
light scattering method. The clotting time may be the time at which
the beads cease to move under the weak force and/or the time at
which the rate of movement under the weak force decreases
substantially.
[0191] In some embodiments, the clotting time may be determined by
analyzing the images to determine when bead motion ceases. In some
embodiments, the image analysis may be automated, optionally by any
suitable algorithm. For example, a difference parameter such as the
mean squared difference between each pixel of each frame and the
final frame of the video may be calculated. When clotting occurs
and bead motion ceases, the final frame of the video will
approximately resemble all frames between clotting and the end of
the video. In this example, the difference parameter will drop to
near zero as soon as clotting has occurred. The time that this drop
begins represents the clotting time, which can be transformed into
a coagulation parameter as appropriate.
[0192] In another embodiment, the peak signal-to-noise ratio
("PSNR") can be used to determine the coagulation time. PSNR
assesses the difference between images "I" and "K" by the mean
squared error ("MSE") as defined in Equation 2
MSE = 1 mn i = 0 m - 1 j = 0 n - 1 [ I ( i , j ) - K ( i , j ) ] 2
( Equation 2 ) ##EQU00002##
wherein PSNR is defined in Equation 3
PSNR = 10 log 10 ( MAX I 2 MSE ) = 20 log 10 ( MAX I MSE ) (
Equation 3 ) ##EQU00003##
with MAXI being the maximum intensity of the image, and "m" and "n"
being the image dimensions in pixels (width by length).
[0193] FIG. 4 shows a representative reaction time course analyzed
by the PSNR method. The PSNR progress curve is shown with the PSNR
value along the vertical scale extending from 15 to just above 35
at the top. The time is shown on the horizontal axis extending from
just below 50 to just above 300 seconds on the right. In some
embodiments, the PSNR data are fit with a simple function such as a
linear or quadratic function and the mid-point of the increase can
be determined and related to a coagulation parameter though
calibration as described.
[0194] In some embodiments, the coagulation time can be determined
by microscopy. In one embodiment, the coagulation of whole blood
can be measured by observing the movement of red blood cells under
microscopy. The whole blood sample may be diluted. In assays
containing red blood cells, the red blood cells perform a similar
function as the beads described in the bead settling method
above.
Fluorescent Microscopy Method
[0195] In some embodiments, the coagulation time can be determined
by observing the movement of fluorescent beads by fluorescent
microscopy. Suitable fluorescent beads include carboxylate-modified
microspheres. Suitable carboxylate-modified microspheres may be
obtained from Life Technologies Inc., Carlsbad Calif., under the
trade name FluoSpheres, catalog #F-8816. The fluorescent beads can
fluoresce at any suitable wavelength, including in the crimson part
of the spectrum. The fluorescent beads may also be any suitable
size. In some embodiments, the beads have a size such that they do
not settle by gravity or settle slowly by gravity in the reaction
medium, and that they cease to move when the sample coagulates. In
one embodiment, the fluorescent spheres have a diameter of about 1
.mu.m.
[0196] As in the bead settling method described above, the
fluorescent beads may be at any suitable concentration, added along
with other dried reagents, and the like. As shown in FIG. 6, the
sample 600 is not necessarily drawn into a capillary or pipette tip
and may be placed as a drop on a slide 605. The sample comprising
fluorescent beads 610 may be of any suitable volume, including
about 2 .mu.L. The microscope objective piece 615 images the sample
at a focal plane 620. The position of the slide 605 relative to the
microscope objective piece 615 can be varied to change the depth of
the focal plane 620 and/or image various areas of the sample. In
some embodiments, the slide can be systematically moved relative to
the microscope objective piece so that several fields of view
within the assay reaction volume are captured. The relative
position of the slide and the microscope objective piece can also
insure that only the sample is visualized and not background
areas.
[0197] In some embodiments, the sample is placed on a cuvette. The
cuvette may be imaged by positioning it in relation to the
microscope objective 615, or the cuvette may be placed on a slide
605. An exemplary cuvette is depicted in FIG. 7 in a top view 700
and a side view 705. The exemplary cuvette has two layers. A top
layer 710 may be an acrylic spacer material and the bottom layer
715 can be a standard glass cover-slip. The top layer may have any
suitable thickness, including approximately 80 .mu.m. The top layer
may also have ports 720, optionally about 2 mm in diameter,
optionally created with a laser cutter. The acrylic material
comprising the top layer 710 may have an adhesive side which sticks
to the bottom layer 715. The top layer and bottom layer are
assembled such that miniature sample wells 720 of about 2 mm
diameter and 80 .mu.m depth are created, which are capable of
holding about 0.25 .mu.L of sample.
[0198] In some embodiments, the cuvette is made from thin slabs of
light-transmissive acrylic with machined ports for sample holding.
In some embodiments, the cuvette is made of injection molded
plastic with a plasma-etched surface to render the cuvette
hydrophilic.
[0199] The movement of the fluorescent beads is driven by a mixture
of air flow (FIG. 6, indication 625), convection and Brownian
motion. In some embodiments, the sample is illuminated at the
excitation wavelength of the fluorescent beads. In some
embodiments, the sample is illuminated with a xenon arc lamp. The
fluorescent beads emit radiation at an emission wavelength, which
may be observed by a microscope. In some embodiments, the
microscope may be an inverted fluorescence microscope where the
microscope objective 615 is below the sample 600. The microscope
may have any suitable power of magnification such that the
fluorescent beads are imaged and their motion can be analyzed. In
one embodiment, a 20.times. objective lens is used. The motion of
the beads is recorded by a camera. The camera may be a cooled CCD
camera. The images may be taken at any suitable rate, including at
a rate of about 5 frames per second. In some embodiments, the
images are acquired at the emission wavelength of the fluorescent
beads.
[0200] The clotting time may be determined by analyzing the
recorded images to determine when motion of the fluorescent beads
ceases. In some embodiments, 100 to 200 images are acquired. The
images can be analyzed as described herein to determine coagulation
time, which is related by calibration to a coagulation parameter as
described herein.
[0201] In one embodiment, the coagulation of whole blood can be
measured by observing the movement of red blood cells under
microscopy. The whole blood sample may be diluted. In some
embodiments, the red blood cells may be fluorescently labeled. The
red blood cells may be observed by regular microscopy, or, in the
event they are fluorescently labeled, by fluorescent microscopy. In
assays containing red blood cells, the red blood cells perform a
similar function as the beads described in the fluorescent
microscopy method above.
Propelled Liquid Column Method
[0202] In some embodiments, the coagulation time can be determined
by imaging of the bulk movement of a sample. In this method, the
coagulated sample adheres to the interior of a vessel such as the
inside of a capillary and ceases to move following coagulation. The
sample can be moved by any method including pneumatic means.
[0203] The movement can be in any manner including reciprocating,
circular, and the like. The movement can be regular or irregular
and traverse any suitable distance. In some embodiments, the bulk
movement is on the order of several millimeters, such as about 1
mm, about 2 mm, about 4 mm, about 6 mm, about 8 mm, about 10 mm,
about 20 mm, about 30 mm, and the like. In some embodiments, the
bulk movement is at least about 1 mm, at least about 2 mm, at least
about 4 mm, at least about 6 mm, at least about 8 mm, at least
about 10 mm, at least about 20 mm, at least about 30 mm, and the
like. In some embodiments, the bulk movement is at most about 1 mm,
at most about 2 mm, at most about 4 mm, at most about 6 mm, at most
about 8 mm, at most about 10 mm, at most about 20 mm, at most about
30 mm, and the like.
[0204] The movement can have any suitable frequency or occur on any
suitable time scale. In some embodiments, the bulk movement is on
the order of seconds such as about 0.5 s, about 1 s, about 2 s,
about 4 s, about 6 s, about 8 s, about 10 s, and the like. In some
embodiments, the bulk movement is on a time scale of at most about
0.5 s, at most about 1 s, at most about 2 s, at most about 4 s, at
most about 6 s, at most about 8 s, at most about 10 s, and the
like.
[0205] The sample may be blood or plasma. In the propelled liquid
column methods, microscopic imaging may not be necessary. The
relevant image is generally the position of the bulk fluid sample
in relation to the capillary vessel. The vessel is generally
transparent so, in the case of blood, the sample will be easily
distinguished by its red color. Plasma is generally essentially
transparent, so detection of a bulk plasma sample may be more
difficult in some embodiments. In some embodiments, plasma samples
can be imaged due to refraction of light from the liquid-air
meniscus at the end of the sample in the capillary tube. If the
menisci are difficult to locate by imagery in a particular
embodiment, a dye or other suitable material may be added to the
sample to improve visualization of the menisci. In another example,
even if not initially visible, the menisci may become visible due
to light scattering once coagulation has occurred.
[0206] The sample can be diluted or not diluted. Some examples,
potentially including embodiments utilizing diluted plasma may not
form a suitably strong clot in some cases. However, the clot can be
made suitably strong by reinforcing it with addition of exogenous
fibrinogen or a high volume fraction of neutrally buoyant beads.
Without being bound by theory, the beads provide a greater surface
area for the incipient clot to bind to, which stiffens or increases
the effect of increasing viscosity of the reagent and sample
mixture and allows the clot formed to adhere to the capillary and
stop moving. The volume fraction can be any suitable fraction such
that the clot adheres to the capillary. In some embodiments, the
volume fraction is about 20%, about 30%, about 40%, about 50%,
about 60%, about 70%, about 80%, about 90%, and the like. In some
embodiments, the volume fraction is at least about 20%, at least
about 30%, at least about 40%, at least about 50%, at least about
60%, at least about 70%, at least about 80%, at least about 90%,
and the like. The beads can be larger in the propelled liquid
column methods, for example about 10 .mu.m in diameter. The
addition of beads may have another advantageous effect in that they
increase the effective sample volume and thus may aid in
visualization of small sample volumes without contributing to
dilution of the sample.
Image Processing and Analysis
[0207] Images may be acquired by the small volume coagulation
measurement methods described herein. In general, the images are
acquired over a period of time, generally longer than the
coagulation time. The last image captured is generally an image of
the coagulated sample.
[0208] The pixilation of images, including exemplary numbers of
pixels is described above. In some embodiments, each image
comprises an array of 512 by 512 pixels (262,144 total pixels).
Each image may be divided into 16 strips of dimension 512 by 32
(row by column, or column by row). Each strip may then be converted
into its respective Fourier-transformed image and re-assembled to
form a modified 512 by 512 image with Fourier-transformed strips.
In some embodiments, a reference image is created by taking the
last image acquired and transforming it in the same manner.
[0209] In some embodiments, such as analysis of the particle bead
settling method and the fluorescence microscopy method (described
above), the analysis involves calculating the correlation
coefficients of each column of a transformed image with the
corresponding column of the transformed reference image. The
correlation coefficient may be calculated according to Equation
4,
.rho. = i = 1 n ( x i - x _ i ) ( x i ref - x i ref _ ) i = 1 n ( x
i - x _ i ) 2 i = 1 n ( x i ref - x i ref _ ) 2 ( Equation 4 )
##EQU00004##
where x.sub.i.sup.ref and x.sub.i.sup.ref are the i.sup.th element
of a column of an image and the reference image respectively.
Values of the correlation factor vary from 0 to 1. The correlation
factor for the final image (correlated to itself) would be 1.0.
[0210] A correlation factor may be obtained for each column, and
the overall correlation of the two images is the median value of
the correlation factor calculated over all columns. Thus, for every
image, a single value quantifying its correlation with the
reference image may be calculated.
[0211] In some embodiments, the correlation factors are plotted as
a function of time as shown in FIG. 8. In this example for
measurement of PT in a plasma sample, the correlation factor is on
the vertical axis and ranges from 0.0 to 1.0 at the top. Time is on
the horizontal axis and ranges from 0 to 100 seconds on the right.
At times less than the coagulation time, the images are poorly
correlated with the final image, potentially due to particle
movement by both convective and Brownian diffusive forces. At the
onset of coagulation, the fluid transitions into a gel and the
particles become locked in place. As seen in FIG. 8, this phase
transition is indicated by a dramatic rise in the calculated
correlation factor, since beyond coagulation, the particle
positions do not change significantly and are hence strongly
correlated with the final image. In this example the coagulation
time appears to be around 40 seconds. In an automated set-up a
sigmoidal curve could be fitted to the data to estimate the
inflection point indicative of coagulation time.
[0212] In some embodiments, coagulation times measured for diluted
samples are longer than those obtained for other methods that do
not use diluted samples. The results for diluted samples can be
made to conform with results of non-diluted samples by correlating
the results of the different methods and applying a mathematical
correction designed to give results from diluted samples which are
equivalent to results from a method for non-diluted samples.
Video Imaging Considerations
[0213] In some embodiments, video imagery is used to determine the
coagulation time. For instances where the settling of particles or
changes in light scattering are measured, one may need to image a
significant fraction of the reaction volume while excluding
substantially all background. This objective may be achieved by
using feature recognition software.
[0214] Turning to FIG. 9, an image of the assay cuvette 900 is
made. The walls of the cuvette are identified due to refractive
index differences 905 and excluded from the image 910. The menisci
of the reaction volume are also identified 915 and excluded from
the image 920. The final cropped image 920 may then be used for
image analysis.
Measurement of Clotting Factors
[0215] Methods, devices, and systems for measuring the
concentration and/or activity of a clotting factor are also
provided herein. Methods for measuring concentration and/or
activity of a clotting factor may be performed in a multiplexed
manner, or in a device or system capable of multiplexed analysis,
optionally wherein multiple assays are performed with a single drop
of blood. In some aspects, one or more clotting time assays are
performed with blood sample from a subject, and one or more assays
directed to the concentration and/or function of a clotting factor
is also performed. In some aspects, if the coagulation time for a
blood sample from a subject is outside of a certain range, a blood
sample from the subject will also be analyzed for the concentration
and/or function of a clotting factor. This procedure may be carried
out in a system or device that is programmed to perform an assay
for a clotting factor if a blood sample has a certain clotting
time. The system or device may also be programmed remotely, such as
by a cloud-computing infrastructure.
[0216] Certain coagulation parameters may be measurable by methods
other than by a coagulation time. For example, described herein are
methods, devices and systems for measuring the concentration and/or
function of any of the clotting factors and/or regulators thereof.
Exemplary clotting factors and/or regulators thereof include von
Willebrand factor, Factor I (fibrinogen), Factor Ia (fibrin),
Factor II (prothrombin), Factor IIa (thrombin), Factor V, Factor
Va, Factor VII, Factor VIIa, Factor VIII, Factor VIIIa, Factor IX,
Factor IXa, Factor X, Factor Xa, Factor XI, Factor XIa, Factor XII,
Factor XIIa, Factor XIII, Factor XIIIa, collagen, platelets,
platelet-activating factor, platelet factor 4, thromboxane A.sub.2,
protein kinase C, phospholipase A.sub.2, tissue factor,
high-molecular-weight kinninogen, prekallikrein, kallikrein,
protein C, thrombomodulin, calcium, vitamin K, protein S,
antithrombin, tissue factor pathway inhibitor (TFPI), plasmin,
tissue plasminogen activator (t-PA), prostacyclin, and the like. In
the clotting factor nomenclature, the lowercase "a" indicates the
active form. For example Factor XIIa is the active form of Factor
XII. In some embodiments, the methods described herein distinguish
between active and inactive forms of clotting factors.
[0217] In some embodiments, the concentration and/or activity of a
clotting factor may be measured by enzyme-linked immunosorbent
assay (ELISA). Performing an ELISA involves at least one antibody
with specificity for a particular antigen (e.g. a clotting factor).
The sample with an unknown amount of antigen is immobilized on a
solid support either non-specifically (via adsorption to the
surface) or specifically (via capture by another antibody specific
to the same antigen, in a "sandwich" ELISA). After the antigen is
immobilized, the detection antibody is added, forming a complex
with the antigen. The detection antibody can be covalently linked
to an enzyme, or can itself be detected by a secondary antibody
that is linked to an enzyme through bioconjugation. Between each
step, the immobilized materials are typically washed with a mild
detergent solution to remove any proteins or antibodies that are
not specifically bound. After the final wash step, the plate is
developed by adding an enzymatic substrate to produce a visible
signal, which indicates the quantity of antigen in the sample.
Methods for performing ELISA reactions with small volumes are
described in, for example, U.S. Pat. No. 8,088,593, which is herein
incorporated by reference. As described therein, colorimetric
methods such as ELISA may benefit from multi-color imaging and
multiple light pathways when performed in small volumes.
Multi-Color Images and Multiple Light Pathways
[0218] One aspect described herein provides for coagulation
analysis using image-based analysis. The system can include a
camera that can measure an optical signal using one or more
detection spectrum regions. For example, a camera can measure an
optical signal using red, green, and blue detection spectrum
regions. The measured signal can include three measured values that
can be interpreted using one or more algorithms described herein.
The use of more than one detection spectrum region can increase the
dynamic range of an assay and can increase the accuracy of a
measurement as compared to measurements using a single detection
spectrum region.
[0219] Also provided herein are systems, devices, and methods for
performing optical measurements on samples and assay reaction
products that are contained within reaction chambers, each with a
plurality of distinct path lengths. The reaction chambers can have
a plurality of distinct path lengths such that a greater or lower
amount of light absorbance is observed. The plurality of distinct
path lengths allows for an increase in the dynamic range of a
selected assay protocol. The image of the reaction chamber can be
analyzed as described herein to obtain information on the sample or
the assay reaction products. The combination of utilizing the
plurality of available path lengths within a single reaction
chamber and the use of three channel detection spectrum regions
greatly enhances the dynamic range of a given assay.
Computer Implementation
[0220] The methods, devices and systems described herein may be
implemented with aid of a programmable computer. For example, the
image pixilation, the analysis of light scattering data, the image
processing and analysis, video image processing methods, and the
like may be programmed into a computer and/or performed by a
programmed computer. Computer assistance may be preferred for
achieving a rapid, automated method, device or system.
[0221] For example, a computer-assisted method for characterizing
an analyte suspected to be present in a sample may be used. The
computer-assisted method may comprise obtaining a digital image of
the sample, wherein the digital image comprises at least a
two-dimensional array of pixels, and wherein each pixel comprises a
plurality of intensity values, each of which corresponds to a
distinct detection spectral region; correlating, with the aid of a
programmable device, the obtained intensity values with a
predetermined set of values that define a dynamic range of each
detection spectral region; and predicting the presence and/or
quantity of the analyte in the sample based on the correlating of
the obtained intensity values with a predetermined set of
values.
Additional Assays
[0222] The methods, devices, and systems described herein may be
used for analyzing any assay that results in the change in
viscosity or solid/liquid state of a sample. For example, as
described herein, beads may be added to any assay that results in
the change in viscosity or solid/liquid state of a sample, and
image analysis of the movement of the beads may be used to
determine the time of change in viscosity or solid/liquid state of
the sample. In other examples, as described herein, the change in
turbidity of a sample may be monitored by light scattering. Any
assay that results in the change in viscosity or solid/liquid state
of a sample and/or a change in light scattering may be monitored by
methods provided herein. In other examples, as described herein,
the change in viscosity or solid/liquid state of a sample may be
monitored by imaging the movement of the sample through a column.
Assays that may be analyzed by the methods provided herein include
assays that do not involve blood coagulation factors, as long as
the assay results in the change in viscosity or solid/liquid state
or light scattering of a sample. For example, in some embodiments,
agglutination assays may be analyzed by methods provided herein.
Other examples of assays that may be analyzed by methods provided
herein include: (1) platelet aggregation assays, (2) nephelometric
immunoassays, (3) particle-enhanced nephelometric immunoassays, (4)
turbidometric immunoassays, (4) latex agglutination immunoassays,
and (5) Limulus Amebocyte Lysate (LAL) test (for example, for
detecting bacterial endotoxins and bacterial diseases).
EXAMPLES
Example 1
PT Measurement by Light Scattering
[0223] Measurement of the PT coagulation parameter by light
scattering was performed with the following materials: [0224]
Plasma samples: QuikCoag.TM. Control Level 1 (Normal Coagulation
Plasma Control); QuikCoag.TM. Control Level 2 (Low Abnormal
Coagulation Plasma Control); and QuikCoag.TM. Control Level 3 (High
Abnormal Coagulation Plasma Control) [0225] Bovine fibrinogen
(Sigma-Aldrich) 10 mg/ml stock in Hepes Buffered Saline (HBS) pH
7.4 [0226] Reconstituted PT reagent (QuikCoag.TM. PT plus Calcium,
BioMedica Diagnostics Inc., Nova Scotia, Canada) [0227] 1.times.
Hepes Buffered Saline (HBS) [0228] 0.02 M CaCl.sub.2
[0229] These materials were used in the following procedure to
measure the PT coagulation parameter by light scattering. All steps
were performed at room temperature, using an automated liquid
handler. [0230] 1) Fibrinogen was dissolved 2.5 mg/ml in PBS
(solution A) [0231] 2) Mixed 0.2 volumes of each plasma sample with
0.8 volumes solution A (i.e. each plasma sample was diluted 5-fold)
[0232] 3) At t=0, mixed 1 volume of diluted plasma with 1 volume PT
reagent and aspirated 2 .mu.l of the mixture into tip. [0233] 4)
Aspirated 1 .mu.l mineral oil, dipping tip deep enough in oil to
cover viewing area 5) Moved to camera/photodetector and began
recording [0234] 6) Stopped recording after clotting occurs
(typically <10 minutes) [0235] 7) Repeated steps 3-6 four
additional times Exemplary results are shown in FIG. 10. Here, the
clotting time is shown on the vertical axis and extends from 0 to
120 seconds at the top. Levels 1, 2, and 3 on the horizontal axis
refer to QuikCoag.TM. Control Level 1, 2, and 3 samples,
respectively. The five different points for each level show the
separate result of replicate assays with each of the Level 1, 2,
and 3 plasma samples. The mean value provided on the graph
indicates the mean clotting time in seconds for the five assays
performed with each of the Level 1, 2, and 3 plasma samples. The PT
coagulation parameter was determined by light scattering.
Example 2
aPTT Measurement by Light Scattering
[0236] Measurement of the aPTT coagulation parameter by light
scattering was performed with the following materials: [0237]
QuikCoag.TM. Control Level 1 (Normal Coagulation Plasma Control)
and QuikCoag.TM. Control Level 3 (High Abnormal Coagulation Plasma
Control) [0238] Bovine fibrinogen (Sigma-Aldrich) 10 mg/ml stock in
Hepes Buffered Saline (HBS) pH 7.4 [0239] Reconstituted aPTT
reagent (QuikCoag.TM. APTT, BioMedica Diagnostics Inc., Nova
Scotia, Canada) [0240] 1.times. Hepes Buffered Saline (HBS) [0241]
0.02 M CaCl.sub.2
[0242] These materials were used in the following procedure to
measure the aPTT coagulation parameter by light scattering. All
steps were performed at room temperature, using an automated liquid
handler. [0243] 1) Fibrinogen was dissolved 5 mg/ml in PBS
(solution A) [0244] 2) Mixed 0.2 volumes of each plasma sample with
0.8 volumes solution A (i.e. each plasma sample was diluted 5-fold)
[0245] 3) Mixed 1 volume of diluted plasma with 1 volume of aPTT
reagent and incubated for 3 minutes [0246] 4) At t=0, mixed 1 .mu.l
of the mixture with 1 .mu.l of 0.2M CaCl.sub.2 and aspirated into
tip. [0247] 5) Aspirated 1 .mu.l mineral oil, dipping tip deep
enough in oil to cover viewing area [0248] 6) Moved to
camera/photodetector and began recording [0249] 7) Stopped
recording after clotting occurred (typically <10 minutes) [0250]
8) Repeated steps 3-7 two (Level 3 sample) or four (Level 1 sample)
more times Exemplary results are shown in FIG. 11. Here, the
clotting time is shown on the vertical axis and extends from 0 to
6:29 at the top. Levels 1 and 3 on the horizontal axis refer to
QuikCoag.TM. Control Level 1 and 3 samples, respectively. The
different points for each level show the separate result of
replicate assays with each of the Level 1 and 3 plasma samples. The
aPTT coagulation parameter was determined by turbidity.
Example 3
PT Measurement by Bead Settling
[0251] Measurement of the PT coagulation parameter by bead settling
was performed with the following materials: [0252] Plasma samples:
QuikCoag.TM. Control Level 1 (Normal Coagulation Plasma Control);
QuikCoag.TM. Control Level 2 (Low Abnormal Coagulation Plasma
Control); and QuikCoag.TM. Control Level 3 (High Abnormal
Coagulation Plasma Control) [0253] Reconstituted PT reagent
(QuikCoag.TM. PT plus Calcium, BioMedica Diagnostics Inc., Nova
Scotia, Canada) [0254] Bead slurry (e.g. 1:1 mixture of 25 um and
45 um diameter beads (e.g. Polybead Microspheres, Polysciences,
PA), washed in HBS, centrifuged, and excess liquid removed) [0255]
1.times.HBS (Hepes Buffered Saline pH7.4) [0256] 0.02 M
CaCl.sub.2
[0257] These materials were used in the following procedure to
measure the PT coagulation parameter by bead settling. All steps
were performed at room temperature, using an automated liquid
handler. [0258] 1) Each plasma sample was diluted 5-fold with HBS
[0259] 2) Mixed beads 1:4 with PT reagent (e.g. 10 .mu.l beads+40
.mu.l PT reagent) [0260] 3) At t=0, mixed 1 .mu.l of diluted plasma
with 1.25 .mu.l of bead/PT reagent mixture and aspirated into tip
[0261] 4) Aspirated 1 .mu.l mineral oil, dipping tip deep enough in
oil to cover viewing area [0262] 5) Moved to camera and begin
recording [0263] 6) Stopped recording after clotting occurred
(typically <10 minutes) [0264] 7) Repeated steps 3-6 one (Level
1 sample), three (Level 2 sample), or two (Level 3 sample) more
times
[0265] Exemplary results are shown in FIG. 12. Here, the clotting
time is shown on the vertical axis and extends from 0 to 120
seconds at the top. Levels 1, 2, and 3 on the horizontal axis refer
to QuikCoag.TM. Control Level 1, 2, and 3 samples, respectively.
The different points for each level show the separate result of
replicate assays with each of the Level 1, 2, and 3 plasma samples.
The mean value provided on the graph indicates the mean clotting
time in seconds for the assays performed with each of the Level 1,
2, and 3 plasma samples. The PT coagulation parameter was
determined by the bead sedimentation assay.
Example 4
aPTT Measurement by Bead Settling
[0266] Measurement of the aPTT coagulation parameter by bead
settling was performed with the following materials: [0267] Plasma
samples: QuikCoag.TM. Control Level 1 (Normal Coagulation Plasma
Control) and QuikCoag.TM. Control Level 2 (Low Abnormal Coagulation
Plasma Control) [0268] Reconstituted aPTT reagent (QuikCoag.TM.
APTT, BioMedica Diagnostics Inc., Nova Scotia, Canada [0269] Bead
slurry (e.g. 1:1 mixture of 25 um and 45 um diameter beads (e.g.
Polybead Microspheres, Polysciences, PA), washed in HBS,
centrifuged, and excess liquid removed) [0270] 1.times.HBS (Hepes
Buffered Saline pH7.4) [0271] 0.02 M CaCl.sub.2
[0272] These materials were used in the following procedure to
measure the aPTT coagulation parameter by bead settling. All steps
were performed at room temperature, using an automated liquid
handler. [0273] 1) Each plasma sample was diluted 5-fold with HBS
[0274] 2) Mixed beads 1:1 with 0.2M CaCl.sub.2 (e.g. 10 .mu.l
beads+10 .mu.l 0.2 M CaCl.sub.2) [0275] 3) Mix 1 .mu.l of diluted
plasma with 1 .mu.l of aPTT reagent and incubate for 3 minutes
[0276] 4) At t=0, mixed 1 .mu.l of the mixture with 1 .mu.l of
bead/CaCl.sub.2 mix, and aspirated into tip. [0277] 5) Aspirated 1
.mu.l mineral oil, dipping tip deep enough in oil to cover viewing
area [0278] 6) Moved to camera and begin recording [0279] 7)
Stopped recording after clotting occurred (typically <10
minutes) [0280] 8) Repeated steps 3-7 three more times
[0281] Exemplary results are shown in FIG. 13. Here, the clotting
time is shown on the vertical axis and extends from 0 to 300
seconds at the top. Levels 1 and 2 on the horizontal axis refer to
QuikCoag.TM. Control Level 1 and 2 samples, respectively. The
different points for each level show the separate result of
replicate assays with each of the Level 1 and 2 plasma samples. The
aPTT coagulation parameter was determined by the bead sedimentation
assay.
Example 5
aPTT Measurement by Fluorescent Microscopy
[0282] Measurement of the aPTT coagulation parameter by fluorescent
microscopy is performed with the following materials: [0283] Plasma
samples: QuikCoag.TM. Control Level 1 (Normal Coagulation Plasma
Control); QuikCoag.TM. Control Level 2 (Low Abnormal Coagulation
Plasma Control); and QuikCoag.TM. Control Level 3 (High Abnormal
Coagulation Plasma Control) [0284] Reconstituted aPTT reagent (e.g.
QuikCoag.TM. APTT, BioMedica Diagnostics Inc., Nova Scotia, Canada)
[0285] 1.times.HBS (Hepes Buffered Saline pH7.4) [0286] 0.02 M
CaCl.sub.2+0.3% fluorescent beads by volume (e.g. FluoSpheres
carboxylate-modified microspheres, 1.0 .mu.m, crimson fluorescent
(625/645) (Life Technologies #F-8816))
[0287] These materials are used in the following procedure to
measure the aPTT coagulation parameter by fluorescent microscopy.
All steps are performed at room temperature, using an automated
liquid handler. [0288] Dilute plasma 5-fold (with HBS) [0289] Add 1
.mu.l CaCl.sub.2 with fluorescent beads to slide [0290] Focus
objective to view .about.20% into drop above slide surface [0291]
Mix 1 ul of diluted plasma with 1 ul of aPTT reagent and incubate
for 3 minutes [0292] At t=0, add 1 .mu.l of this mixture to
CaCl.sub.2/bead mixture on slide, mix well and begin recording
images [0293] Stop recording after clotting occurs (typically
<10 minutes)
Example 6
PT Measurement by Fluorescent Microscopy
[0294] Measurement of the PT coagulation parameter by fluorescent
microscopy was performed with the following materials: [0295]
Plasma samples: QuikCoag.TM. Control Level 1 (Normal Coagulation
Plasma Control); QuikCoag.TM. Control Level 2 (Low Abnormal
Coagulation Plasma Control); and QuikCoag.TM. Control Level 3 (High
Abnormal Coagulation Plasma Control) [0296] Reconstituted PT
reagent (QuikCoag.TM. PT plus Calcium, BioMedica Diagnostics Inc.,
Nova Scotia, Canada)+0.2% fluorescent beads by volume (e.g.
FluoSpheres carboxylate-modified microspheres, 1.0 .mu.m, crimson
fluorescent (625/645) (Life Technologies #F-8816)) [0297]
1.times.HBS (Hepes Buffered Saline pH7.4) [0298] 0.02 M
CaCl.sub.2+0.3% fluorescent beads by volume (e.g. FluoSpheres
carboxylate-modified microspheres, 1.0 .mu.m, crimson fluorescent
(625/645) (Life Technologies #F-8816))
[0299] These materials were used in the following procedure to
measure the PT coagulation parameter by fluorescent microscopy. All
steps at room temperature, using an automated liquid handler.
[0300] 1) Each plasma sample was diluted 5 or 10-fold with HBS
[0301] 2) Added 1.5 .mu.l PT reagent with fluorescent beads to
slide [0302] 3) Focused objective to view drop .about.20% into
liquid column above slide surface [0303] 4) At t=0, mixed 1.5 .mu.l
of diluted plasma with bead/PT reagent mixture on slide and began
recording images [0304] 5) Stopped recording after clotting
occurred (typically <10 minutes) [0305] 6) Repeated steps 4-5
two (Level 1 sample), three (Level 2 sample), or four (Level 3
sample) more times
[0306] Exemplary microscopy results are shown in FIG. 14. Here,
sample images were acquired after deposition of a sample with
plasma (diluted 10.times.) and PT activation factor into a sample
well. The images were taken at 5 seconds 1400, 25 seconds 1405, 50
seconds 1410, and 100 seconds 1415. The images for 50 seconds and
100 seconds were nearly identical, signifying that there was
limited particle motion between 50 and 100 seconds. This implies
that the motion of the particles was arrested at or before 50
seconds. Image analysis was used to determine the coagulation time
using methods described above.
[0307] FIG. 15 shows exemplary results of PT measurement by
fluorescent microscopy of 1:5 diluted plasma samples. Here, the
clotting time is shown on the vertical axis on a logarithmic scale
ranging from 10 to 1000 seconds. Levels 1, 2, and 3 on the
horizontal axis refer to QuikCoag.TM. Control Level 1, 2, and 3
samples, respectively. The different points for each level show the
separate result of each individual assay with each of the Level 1,
2, and 3 plasma samples. The mean value provided on the graph
indicates the mean clotting time in seconds for the assays
performed with each of the Level 1, 2, and 3 plasma samples.
Example 7
aPTT Measurement by Light Scattering
[0308] Measurement of the aPTT coagulation parameter by light
scattering was performed with the following materials: [0309] Human
plasma containing EDTA [0310] Bovine fibrinogen (Sigma-Aldrich) 10
mg/ml stock in Hepes Buffered Saline (HBS) pH 7.4 [0311]
Reconstituted aPTT reagent (QuikCoag.TM. APTT, BioMedica
Diagnostics Inc., Nova Scotia, Canada) [0312] 1.times. Hepes
Buffered Saline (HBS) [0313] 0.02 M CaCl.sub.2 [0314] Porcine
heparin (Heparin lithium salt from porcine intestinal mucosa, Sigma
h0878)
[0315] These materials were used in the following procedure to
measure the (1) onset of turbidity due to coagulation, and (2) aPTT
by light scattering. All steps were performed at room temperature,
using an automated liquid handler. [0316] 1) Fibrinogen was
dissolved 5 mg/ml in PBS (solution A) [0317] 2) Prepared various
samples of human plasma containing different concentrations of
heparin. To prepare these samples, heparin was added to different
human plasma aliquots to yield samples containing heparin in
concentrations ranging from 0-1 U/ml. [0318] 3) Mixed 0.2 volumes
of each plasma sample with 0.8 volumes solution A (i.e. each plasma
sample was diluted 5-fold) [0319] 4) Mixed 5 .mu.l of each diluted
plasma with 5 .mu.l of aPTT reagent and incubated for 3 minutes
[0320] 5) At t=0, mixed 1 .mu.l of each mixture of diluted
plasma/aPTT reagent with 1 .mu.l of 0.2M CaCl.sub.2, and aspirated
into a tip. [0321] 6) Aspirated 1 .mu.l mineral oil into each tip,
dipping tip deep enough in oil to cover viewing area [0322] 7)
Moved to camera/photodetector and began recording [0323] 8) Stopped
recording after clotting occurred (typically <10 minutes)
[0324] Exemplary results are shown in FIG. 16. Here, the clotting
time is shown on the vertical axis in seconds. The concentration of
heparin in different plasma samples in U/ml is shown on the
horizontal axis. The coagulation parameter (onset of turbidity) was
determined by turbidity measurements over time.
[0325] The dose-response of the assay of FIG. 16 was calibrated to
provide an estimate of heparin concentration. The results shown in
FIG. 17 confirm that the assay gave an excellent result for heparin
concentration over the range of clinical interest.
[0326] In addition to performing assays as described above, aPTT
assays with the plasma samples were also performed with the Helena
Cascade aPTT system (Helena Laboratories, Beaumont, Tex.),
according to the manufacturer's protocols. When the data from FIG.
16 were calibrated to provide an estimate of aPTT and the results
compared with results using the same plasma samples on the Helena
Cascade aPTT system, the following correlation was obtained: aPTT
(present method)=1.00*Helena aPTT; R.sup.2=0.82 indicating good
agreement between the two methods.
[0327] Coagulation parameters determined by the Helena method were
also correlated with the known heparin concentrations. The
following was observed: y (Helena aPTT)=x (heparin
concentration)*341+13.9; R.sup.2=0.73 which is a poorer correlation
than that of the results with the method described above (i.e. in
Example 7).
Example 8
PT Measurement by Light Scattering
[0328] Measurement of the PT coagulation parameter by light
scattering was performed with the following materials: [0329]
Various samples of human plasma containing EDTA, including samples
from subjects on warfarin therapy and subjects not on warfarin
therapy [0330] Bovine fibrinogen (Sigma-Aldrich) 10 mg/ml stock in
Hepes Buffered Saline (HBS) pH 7.4 [0331] 1.times. Hepes Buffered
Saline (HBS) [0332] Reconstituted PT reagent (QuikCoag.TM. PT plus
Calcium, BioMedica Diagnostics Inc., Nova Scotia, Canada) (for
assay disclosed herein) [0333] 0.02 M CaCl.sub.2
[0334] These materials were used in the following procedure to
measure the PT coagulation parameter by light scattering. All steps
were performed at room temperature, using an automated liquid
handler. [0335] 1) Fibrinogen was dissolved 2.5 mg/ml in PBS
(solution A) [0336] 2) Mixed 0.2 volumes of different plasma sample
with 0.8 volumes solution A (i.e. each plasma sample was diluted
5-fold) [0337] 3) Prepared 5 .mu.l aliquots of the diluted plasmas
[0338] 4) At t=0, mixed this 5 .mu.l of each diluted plasma with 5
.mu.l of either Helena Laboratories Thromboplastin Reagent (for
reference method assays) or QuikCoag.TM. PT plus Calcium PT reagent
(for assay method disclosed herein), and aspirated 2 .mu.l of the
mixture into tip. [0339] 4) Aspirated 1 .mu.l mineral oil, dipping
tip deep enough in oil to cover viewing area [0340] 5) Moved to
camera/photodetector and began recording [0341] 6) Stopped
recording after clotting occurs (typically <10 minutes)
[0342] In addition to performing assays as described above, PT
assays with the plasma samples were also performed with the Helena
Cascade PT system, according to the manufacturer's protocols.
Exemplary results comparing the methods are shown in FIG. 18. Each
point on the graph represents a different sample. Here, PT
(QuikCoag.TM. PT plus Calcium PT reagent)=1.00(PT Helena Cascade);
R.sup.2=0.92.
[0343] As would be understood by a person of skill in the art, it
is possible to use various alternatives, modifications and
equivalents to the embodiments disclosed herein. Therefore, the
scope of the present invention should be determined not with
reference to the above description but should, instead, be
determined with reference to the appended claims, along with their
full scope of equivalents. Any feature, whether preferred or not,
may be combined with any other feature, whether preferred or not.
The appended claims are not to be interpreted as including
means-plus-function limitations, unless such a limitation is
explicitly recited in a given claim using the phrase "means for."
It should be understood that as used in the description herein and
throughout the claims that follow, the meaning of "a," "an," and
"the" includes plural reference unless the context clearly dictates
otherwise. Also, as used in the description herein and throughout
the claims that follow, the meaning of "in" includes "in" and "on"
unless the context clearly dictates otherwise. Finally, as used in
the description herein and throughout the claims that follow, the
meanings of "and" and "or" include both the conjunctive and
disjunctive and may be used interchangeably unless the context
expressly dictates otherwise. Thus, in contexts where the terms
"and" or "or" are used, usage of such conjunctions do not exclude
an "and/or" meaning unless the context expressly dictates
otherwise.
* * * * *