U.S. patent application number 17/182964 was filed with the patent office on 2021-07-29 for detection of reversal of an anticoagulant using a clotting assay.
The applicant listed for this patent is Haemonetics Corporation. Invention is credited to Marc Doubleday, Fowzia S. Zaman.
Application Number | 20210230663 17/182964 |
Document ID | / |
Family ID | 1000005523138 |
Filed Date | 2021-07-29 |
United States Patent
Application |
20210230663 |
Kind Code |
A1 |
Zaman; Fowzia S. ; et
al. |
July 29, 2021 |
Detection of Reversal of an Anticoagulant Using a Clotting
Assay
Abstract
In some embodiments, the invention provides methods for
detecting the reversal an anticoagulant at a therapeutically
relevant amount or higher in a patient, including subjecting a
sample of a control blood component (known not to contain the
anticoagulant) to a clotting assay in the presence of an ecarin
reagent to obtain a control ecarin clotting measurement; subjecting
a sample of a control blood component (known not to contain the
anticoagulant) to a clotting assay in the presence of a Factor Xa
reagent to obtain a control Factor Xa clotting measurement;
subjecting a sample of a blood component from a patient suspected
to contain a reversal agent to a clotting assay in the presence of
the ecarin reagent to obtain a patient ecarin clotting measurement;
subjecting a sample of a blood component from the patient suspected
to contain the reversal agent to a clotting assay in the presence
of the Factor Xa reagent to obtain a patient Factor Xa clotting
measurement; and comparing the control ecarin clotting measurement
to the patient ecarin clotting measurement and comparing the
control Factor Xa clotting measurement to the patient Factor Xa
measurement.
Inventors: |
Zaman; Fowzia S.; (Aurora,
IL) ; Doubleday; Marc; (Cary, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Haemonetics Corporation |
Boston |
MA |
US |
|
|
Family ID: |
1000005523138 |
Appl. No.: |
17/182964 |
Filed: |
February 23, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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16674334 |
Nov 5, 2019 |
10954549 |
|
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17182964 |
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14813939 |
Jul 30, 2015 |
10501773 |
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16674334 |
|
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62031371 |
Jul 31, 2014 |
|
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62111376 |
Feb 3, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2333/96444
20130101; G01N 33/86 20130101; C12Q 1/56 20130101 |
International
Class: |
C12Q 1/56 20060101
C12Q001/56; G01N 33/86 20060101 G01N033/86 |
Claims
1. A method for detecting reversal of an anticoagulant at a
therapeutically relevant amount or higher in a patient having an
anticoagulant, the method comprising: (a) subjecting a sample of a
control blood component known to lack the anticoagulant to a
clotting assay in the presence of an ecarin reagent to obtain a
control ecarin clotting measurement; (b) subjecting a sample of the
control blood component known to lack the anticoagulant to a
clotting assay in the presence of a Factor Xa reagent to obtain a
control Factor Xa clotting measurement; (c) subjecting a sample of
a blood component from a patient suspected to contain a reversal
agent to a clotting assay in the presence of the ecarin reagent to
obtain a patient ecarin clotting measurement; (d) subjecting a
sample of the blood component from the patient suspected to contain
the reversal agent to a clotting assay in the presence of the
Factor Xa reagent to obtain a patient Factor Xa clotting
measurement; and (e) comparing the control ecarin clotting
measurement to the patient ecarin clotting measurement and
comparing the control Factor Xa clotting measurement to the patient
Factor Xa measurement, wherein the patient ecarin clotting
measurement less than or equal to the control ecarin clotting
measurement and/or the patient Factor Xa clotting measurement less
than or equal to the control Factor Xa clotting measurement
identifies the blood component from the patient as having a
reversal agent that has reversed the anticoagulation activity of
the anticoagulant in the patient.
2. The method of claim 1, wherein the clotting assay is selected
from the group consisting of a prothrombin time assay, an activated
partial thromboplastin time assay, an activated clotting time
assay, and a viscoelastic analysis clotting assay.
3. The method of claim 2, wherein the clotting assay is a
viscoelastic analysis clotting assay and wherein the viscoelastic
analysis is performed using a container containing the sample on an
interior of the container.
4. The method of claim 3, wherein the viscoelastic analysis is
performed using the container and a pin, wherein the pin moves
relative to the container or wherein the container moves relative
to the pin.
5. The method of claim 3, wherein the container lacks a bottom
surface.
6. The method of claim 1, wherein the patient is a human.
7. The method of claim 1, wherein the patient is undergoing a
condition selected from the group consisting of surgery, trauma,
bleeding, stroke, a thromboembolic event, and combinations
thereof.
8. The method of claim 1, wherein the anticoagulant is an oral
anticoagulant.
9. The method of claim 1, wherein the control Factor Xa clotting
measurement is a range of at least two Factor Xa clotting
measurements of at least two control blood components known to lack
the anticoagulant, and wherein the control ecarin clotting
measurement is a range of at least two ecarin clotting measurements
of at least two control blood components known to lack the
anticoagulant.
10. The method of claim 1, wherein the reversal agent reverses the
anticoagulation activity of a direct thrombin inhibitor.
11. The method of claim 1, wherein the reversal agent reverses the
anticoagulation activity of a Factor Xa inhibitor.
12. The method of claim 1, wherein the reversal agent is
andexanet.
13. The method of claim 1, wherein the reversal agent is a
prothrombin complex concentrate.
14. The method of claim 1, wherein the reversal is a complete
reversal of the anticoagulation activity of the anticoagulant in
the patient.
15. The method of claim 1, wherein the reversal is a partial
reversal of the anticoagulation activity of the anticoagulant in
the patient.
16. The method of claim 8, wherein the oral anticoagulant is
selected from the group consisting of a direct thrombin inhibitor,
a Factor Xa inhibitor, and combinations thereof.
17. The method of claim 1, wherein the reversal agent is
idarucizumab.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/674,334, filed Nov. 5, 2019, which is a
continuation of U.S. patent application Ser. No. 14/813,939, filed
Jul. 30, 2015, now U.S. Pat. No. 10,501,773, which claims priority
to U.S. Provisional Application No. 62/031,371, filed Jul. 31,
2014, and to U.S. Provisional Application No. 62/111,376, filed
Feb. 3, 2015, each of which is hereby incorporated by reference in
its entirety.
BACKGROUND
[0002] The present invention relates to the field of coagulation
and hemostasis.
[0003] With the aging of the population the risk of diseases
involving the heart and circulation system has become a growing
concern. Anticoagulants have been used to combat and/or manage
(e.g., prevent) syndromes including atrial fibrillation, pulmonary
embolism, deep vein thrombosis, venous thromboembolism, congestive
heart failure, stroke, myocardial infarction, and
hypercoagulability in patients. In the past, the anticoagulant drug
warfarin, which reduced the functional levels of all of the vitamin
K-dependent clotting factors, was often used. However, recently
improved anticoagulants have been developed that specifically
target certain factors in the coagulation cascade.
[0004] For example, the introduction of oral anticoagulants has
changed the management of patients with venous and arterial
thromboembolic diseases. Unlike traditional oral vitamin K
antagonists (VKA), the recently developed oral anticoagulant that
do not universally reduce vitamin K-dependent factors are given at
fixed doses and have a lower potential for drug and food
interactions, thus eliminating the requirement for routine
laboratory monitoring (Ansell J. et al., Chest 133: 160S-198S,
2008; Ageno W. et al., Chest 141: e44S-88S, 2012). These novel
agents show similar or improved efficacy and safety profiles
compared with VKA drugs such as warfarin and established parenteral
agents including unfractionated heparin and low molecular weight
heparin.
[0005] However, these new oral anticoagulants present management
challenges to both clinicians and laboratory personnel when
patients develop bleeding diatheses (e.g., due to a traumatic
injury or during surgery). Lack of a readily available method to
determine the degree of anticoagulation creates a major challenge
to clinicians treating bleeding patients who are potentially
receiving an anticoagulant. In some cases, there are no useful
methods to detect and monitor these agents (see Miyares and Davis,
Am. J. Health. Syst. Pharm. 69: 1473-1484, 2012).
[0006] It would be useful to have a rapid method to detect the
presence of an anticoagulant in a sample taken from the patient. It
would also be useful to identify which type of anticoagulant is in
the patient's sample.
SUMMARY OF THE EMBODIMENTS
[0007] In some embodiments, the invention provides a rapid and
accurate method to detect the presence and reversal of an
anticoagulant in a sample taken from the patient. In some
embodiments, the invention provides a rapid and accurate method to
identify which type of anticoagulant is in the sample.
[0008] Accordingly, in a first aspect, the invention provides a
method for detecting an anticoagulant at least at a therapeutically
relevant amount (i.e., a therapeutically relevant amount or higher)
in a patient suspected of having an anticoagulant. The method
comprises (a) subjecting a control sample of a control blood
component, the control sample known not to contain the
anticoagulant, to a clotting assay in the presence of a Factor Xa
reagent to obtain a clotting measurement of the control sample; and
(b) subjecting a patient sample of a blood component from the
patient suspected of having the anticoagulant to the clotting assay
in the presence of the Factor Xa reagent to obtain the clotting
measurement of the patient sample, wherein the clotting measurement
of the patient sample greater than the clotting measurement of the
control sample indicates the presence of the anticoagulant at a
therapeutically relevant amount in the patient and wherein the
clotting measurement of the patient sample less than or equal to
the clotting measurement of the control sample indicates the
absence of the anticoagulant at a therapeutically relevant amount
in the patient. In some embodiments, the anticoagulant is an oral
anticoagulant. The oral anticoagulant may be a direct thrombin
inhibitor or may be a Factor Xa inhibitor.
[0009] In some embodiments, the method further comprising
classifying the anticoagulant identified as being present in the
patient by (a) subjecting a control sample of the control blood
component to a clotting assay in the presence of an ecarin reagent
to obtain a control ecarin clotting measurement; and (b) subjecting
a patient sample of the blood component from the patient to the
clotting assay in the presence of the ecarin reagent to obtain a
patient ecarin clotting measurement, wherein the patient ecarin
clotting measurement greater than the control ecarin clotting
measurement identifies the anticoagulant as a direct thrombin
inhibitor (DTI) in the patient and wherein the patient ecarin
clotting measurement less than or equal to the control ecarin
clotting measurement identifies the anticoagulant as an anti-Factor
Xa anticoagulant in the patient.
[0010] In another aspect, the invention provides a method for
detecting and/or classifying an anticoagulant at a therapeutically
relevant amount or higher in a patient suspected of having an
anticoagulant comprising subjecting a first sample of a blood
component from the patient to a clotting assay in the presence of a
Factor Xa reagent to obtain a patient Factor Xa clotting
measurement, subjecting a second sample of the blood component from
the patient to a clotting assay in the presence of an ecarin
reagent to obtain a patient ecarin clotting measurement; and
comparing the patient Factor Xa clotting measurement to a control
Factor Xa clotting measurement from a control blood component known
to lack the anticoagulant and comparing the patient ecarin clotting
measurement to a control ecarin clotting measurement from the
control blood component; wherein the patient Ecarin clotting
measurement greater than the control ecarin clotting measurement
identifies the presence of the anticoagulant at or above a
therapeutically relevant amount in the patient and identifies the
anticoagulant as a Direct Thrombin Inhibitor (DTI), and wherein the
patient Factor Xa clotting measurement of greater than the control
Factor Xa clotting measurement of identifies the presence of the
anticoagulant at or above a therapeutically relevant amount in the
patient and identifies the anticoagulant as an anti-Factor Xa
anticoagulant, and wherein the patient Factor Xa clotting
measurement less than or equal to the control Factor Xa clotting
measurement identifies absence of an anticoagulant at or above a
therapeutically relevant amount in the patient.
[0011] In another aspect, the invention provides a method for
detecting and/or classifying an anticoagulant at a therapeutically
relevant amount or higher than a therapeutically relevant amount in
a blood component from a patient, comprising: detecting the
presence of an anticoagulant in the blood component by (i)
subjecting a first portion of a control blood component known not
to contain the anticoagulant, to a clotting assay in the presence
of a Factor Xa reagent to obtain a control Factor Xa clotting
measurement; and (ii) subjecting a first portion of the blood
component from the patient to the clotting assay in presence of the
Factor Xa reagent to obtain a patient Factor Xa clotting
measurement, wherein the patient Factor Xa clotting measurement
that is greater than the control Factor Xa clotting measurement
indicates the presence of the anticoagulant at a therapeutic level
in the patient blood component and wherein the patient Factor Xa
clotting measurement that is less than or equal to the control
Factor Xa clotting measurement indicates the absence of the
anticoagulant at a therapeutically relevant amount in the patient
blood component and classifying the anticoagulant, if present, in
the patient blood component, by (i) subjecting a second portion of
the control blood component to a clotting assay in the presence of
an ecarin reagent to obtain a control ecarin clotting measurement;
and (ii) subjecting a second portion of the patient blood component
to the clotting assay in the presence of the ecarin reagent to
obtain a patient ecarin clotting measurement, wherein the patient
ecarin clotting measurement that is greater than the control ecarin
clotting measurement identifies the anticoagulant as a direct
thrombin inhibitor (DTI) and wherein the patient ecarin clotting
measurement that is less than or equal to the control ecarin
clotting measurement identifies the anticoagulant as an anti-Factor
Xa anticoagulant.
[0012] In some embodiments, the clotting assay is selected from the
group consisting of a prothrombin time (PT) assay, an activated
partial thromboplastin time (APTT) assay, and the activated
clotting time (ACT) assay.
[0013] In some embodiments, the clotting measurement of the control
sample is a range of at least two clotting measurements of at least
two control samples from at least two control blood components
known to lack the anticoagulant.
[0014] In some embodiments, the clotting assay is a viscoelastic
analysis clotting assay. In some embodiments, the viscoelastic
analysis is performed using a container containing the sample on an
interior of the container. In some embodiments, the viscoelastic
analysis is performed using the container and a pin, wherein the
pin moves relative to the container. In some embodiments, the
viscoelastic analysis is performed using the container and a pin,
wherein the container moves relative to the pin. In some
embodiments, the container lacks a bottom surface.
[0015] In various embodiments, the patient is a human. In some
embodiments, patient is undergoing a condition including surgery,
trauma, bleeding, stroke, or a thromboembolic event.
[0016] In some embodiments, the anticoagulant is an oral
anticoagulant (e.g., an anti-Factor Xa anticoagulant or a DTI
anticoagulant).
[0017] In various embodiments, the patient Factor Xa clotting
measurement that is at least 1.25 times greater than the control
Factor Xa clotting measurement identifies the presence of the
anticoagulant at a therapeutically relevant amount or higher in the
patient. In some embodiments, the patient Factor Xa clotting
measurement that is at least 1.5 times greater than the control
Factor Xa clotting measurement identifies the presence of the
anticoagulant at a therapeutically relevant amount or higher in the
patient.
[0018] In various embodiments, the patient ecarin clotting
measurement that is at least 1.25 times greater than the control
ecarin clotting measurement identifies the presence of the
anticoagulant at a therapeutically relevant amount or higher in the
patient. In some embodiments, the patient ecarin clotting
measurement that is at least 1.5 times greater than the control
ecarin clotting measurement identifies the presence of the
anticoagulant at a therapeutically relevant amount or higher in the
patient.
[0019] In various embodiments, the patient identified as comprising
the presence of the anticoagulant at a therapeutically relevant
amount or higher is administered a therapeutically relevant amount
of a reversal agent. In some embodiments, the reversal agent is
prothrombin complex concentrates. In some embodiments, the patient
identified as comprising the presence of a DTI anticoagulant at a
therapeutically relevant amount or higher is administered a
therapeutically relevant amount of a reversal agent that reverses
the DTI anticoagulant (e.g., idarucizumab). In some embodiments,
the patient identified as comprising the presence of an anti-Factor
Xa anticoagulant at a therapeutically relevant amount or higher is
administered a therapeutically relevant amount of a reversal agent
that reverses the anti-Factor Xa anticoagulant (e.g.,
andexanet).
[0020] In another aspect, the invention provides a method for
detecting and/or classifying an anticoagulant at a therapeutically
relevant amount (or higher) in a patient suspected of having an
anticoagulant. The method includes (a) detecting the presence of an
anticoagulant in a blood component from the patient, comprising the
steps of: (i) subjecting a first portion of a sample of a control
blood component, the control blood component known not to contain
the anticoagulant, to a clotting assay in the presence of a Factor
Xa reagent to obtain a Factor Xa clotting measurement of the
control sample (i.e., a control Factor Xa clotting measurement);
and (ii) subjecting a first portion of a sample of a blood
component from the patient, the patient suspected of having an
anticoagulant, to the clotting assay in the presence of the Factor
Xa reagent to obtain the Factor Xa clotting measurement of the
patient sample (i.e., a patient Factor Xa clotting measurement),
wherein the Factor Xa clotting measurement of the patient sample
greater than the Factor Xa clotting measurement of the control
sample indicates the presence of the anticoagulant at a
therapeutically relevant amount in the patient and wherein the
Factor Xa clotting measurement of the second sample less than or
equal to the Factor Xa clotting measurement of the control sample
indicates the absence of the anticoagulant at a therapeutically
relevant amount in the patient; and (b) classifying the
anticoagulant, if detected as being present, in the patient,
comprising the steps of: (i) subjecting a second portion of the
sample of the control blood component to the clotting assay in the
presence of an ecarin reagent to obtain the ecarin clotting
measurement of the control sample (i.e., the control ecarin
clotting measurement); and (ii) subjecting a second portion of the
sample of the blood component from the patient to the clotting
assay in the presence of the ecarin reagent to obtain the ecarin
clotting measurement of the patient sample (i.e., the patient
ecarin clotting measurement), wherein the ecarin clotting
measurement of the patient sample greater than the ecarin clotting
measurement of the control sample identifies the anticoagulant as a
direct thrombin inhibitor (DTI) and wherein the ecarin clotting
measurement of the patient sample that is less than or equal to the
ecarin clotting measurement of the control sample identifies the
anticoagulant as an anti-Factor Xa reagent.
[0021] In some embodiments, the clotting assay is selected from the
group consisting of a prothrombin time (PT) assay, an activated
partial thromboplastin time (APTT) assay, and the activated
clotting time (ACT) assay.
[0022] In some embodiments, the clotting measurement of the control
sample is a range of at least two clotting measurements of at least
two control samples from at least two control blood components
known to lack the anticoagulant. For example, in some embodiments,
the control Factor Xa clotting measurement is a range of at least
two Factor Xa clotting measurements of at least two control blood
components known to lack the anticoagulant. In some embodiments,
the control ecarin clotting measurement is a range of at least two
ecarin clotting measurements of at least two control blood
components known to lack the anticoagulant.
[0023] In some embodiments, the clotting assay is a viscoelastic
analysis clotting assay. In some embodiments, the viscoelastic
analysis is performed using a container containing the sample on an
interior of the container. In some embodiments, the viscoelastic
analysis is performed using the container and a pin, wherein the
pin moves relative to the container. In some embodiments, the
viscoelastic analysis is performed using the container and a pin,
wherein the container moves relative to the pin. In some
embodiments, the container lacks a bottom surface.
[0024] In various embodiments, the patient is a human. In some
embodiments, patient is undergoing a condition including surgery,
trauma, bleeding, stroke, or a thromboembolic event.
[0025] In some embodiments, the anticoagulant is an oral
anticoagulant (e.g., an anti-Factor Xa anticoagulant or a DTI
anticoagulant).
[0026] In various embodiments, the patient Factor Xa clotting
measurement that is at least 1.25 times greater than the control
Factor Xa clotting measurement identifies the presence of the
anticoagulant at a therapeutically relevant amount or higher in the
patient. In some embodiments, the patient Factor Xa clotting
measurement that is at least 1.5 times greater than the control
Factor Xa clotting measurement identifies the presence of the
anticoagulant at a therapeutically relevant amount or higher in the
patient.
[0027] In various embodiments, the patient ecarin clotting
measurement that is at least 1.25 times greater than the control
ecarin clotting measurement identifies the presence of the
anticoagulant at a therapeutically relevant amount or higher in the
patient. In some embodiments, the patient ecarin clotting
measurement that is at least 1.5 times greater than the control
ecarin clotting measurement identifies the presence of the
anticoagulant at a therapeutically relevant amount or higher in the
patient.
[0028] In various embodiments, the patient identified as comprising
the presence of the anticoagulant at a therapeutically relevant
amount or higher is administered a therapeutically relevant amount
of a reversal agent. In some embodiments, the reversal agent is
prothrombin complex concentrates. In some embodiments, the patient
identified as comprising the presence of a DTI anticoagulant at a
therapeutically relevant amount or higher is administered a
therapeutically relevant amount of a reversal agent that reverses
the DTI anticoagulant (e.g., idarucizumab). In some embodiments,
the patient identified as comprising the presence of an anti-Factor
Xa anticoagulant at a therapeutically relevant amount or higher is
administered a therapeutically relevant amount of a reversal agent
that reverses the anti-Factor Xa anticoagulant (e.g.,
andexanet).
[0029] In another aspect, the method for detecting reversal of an
anticoagulant at a therapeutically relevant amount or higher in a
patient suspected of having an anticoagulant. The method includes
by (a) subjecting a sample of the control blood component known to
lack (i.e., have the absence of) an anticoagulant to a clotting
assay in the presence of an ecarin reagent to obtain a control
ecarin clotting measurement; (b) subjecting a sample of the control
blood component known to lack (i.e., have the absence of) an
anticoagulant to a clotting assay in the presence of a Factor Xa
reagent to obtain a control Factor Xa clotting measurement; (c)
subjecting a blood component from a patient known or suspected to
contain a reversal agent or antidote (e.g., idarucizumab or
andexanet) in the presence or absence of an anticoagulant to a
clotting assay in the presence of an Ecarin reagent to obtain a
patient Ecarin clotting measurement and (d) subjecting a blood
component from a patient known or suspected to contain a reversal
agent or antidote (e.g., idarucizumab or andexanet) in the presence
or absence of an anticoagulant to a clotting assay in the presence
of a Factor Xa reagent to obtain a patient Factor Xa clotting
measurement, and comparing the control ecarin clotting measurement
to the patient ecarin clotting measurement and comparing the
control Factor Xa clotting measurement to the patient Factor Xa
measurement. When the patient Ecarin clotting measurement and/or
the patient Factor Xa clotting measurement is less than or equal to
the control Ecarin clotting measurement and/or the control Factor
Xa clotting measurement, the patient is identified as comprising a
reversal agent that has reversed the anticoagulation activity of
the anticoagulant in the patient. In some embodiments, the reversal
is complete reversal of the anticoagulation activity of the
anticoagulant in the patient. In some embodiments, the reversal is
partial reversal of the anticoagulation activity of the
anticoagulant in the patient. In some embodiments, the reversal
agent is a prothrombin complex concentrate (PCC) or an active
substance (such as a monoclonal antibody or peptide) specific for
reversing a direct thrombin inhibitor (e.g., idarucizumab) or for
reversing a Factor Xa inhibitor (e.g., andexanet) or an agent that
reverses both Direct Thrombin Inhibitors and Factor Xa
inhibitors.
[0030] In some embodiments, the anticoagulant is an oral
anticoagulant. The oral anticoagulant may be a direct thrombin
inhibitor or may be a Factor Xa inhibitor.
[0031] In some embodiments, the clotting assay is selected from the
group consisting of a prothrombin time (PT) assay, an activated
partial thromboplastin time (APTT) assay, and the activated
clotting time (ACT) assay.
[0032] In some embodiments, the clotting measurement of the control
sample is a range of at least two clotting measurements of at least
two control samples from at least two control blood components
known to lack the anticoagulant.
[0033] In some embodiments, the clotting assay is a viscoelastic
analysis clotting assay. In some embodiments, the viscoelastic
analysis is performed using a container containing the sample on an
interior of the container. In some embodiments, the viscoelastic
analysis is performed using the container and a pin, wherein the
pin moves relative to the container. In some embodiments, the
viscoelastic analysis is performed using the container and a pin,
wherein the container moves relative to the pin. In some
embodiments, the container lacks a bottom surface.
[0034] In various embodiments, the patient is a human. In some
embodiments, patient is undergoing a condition including surgery,
trauma, bleeding, stroke, or a thromboembolic event.
[0035] In some embodiments, the Factor Xa clotting measurement of
the control sample is a range of at least two Factor Xa clotting
measurements of at least two control samples from at least two
control blood components known to lack the anticoagulant. In some
embodiments, the ecarin clotting measurement of the control blood
component is a range of at least two ecarin clotting measurements
of at least two control blood components known to lack the
anticoagulant.
[0036] In another aspect, the invention provides a method for
detecting an anticoagulant at a therapeutically relevant amount or
higher in a patient suspected of having an anticoagulant, the
method comprising subjecting a patient sample of a blood component
from the patient to a clotting assay in the presence of a Factor Xa
reagent to obtain a clotting measurement of the patient sample,
wherein the clotting measurement of the patient sample greater than
a clotting measurement of a control sample of a control blood
component known to lack the anticoagulant identifies the presence
of the anticoagulant at a therapeutically relevant amount in the
patient.
[0037] In some embodiments, the clotting measurement of the patient
sample that is at least 1.25 times greater than a clotting
measurement of the control sample identifies the presence of the
anticoagulant at a therapeutically relevant amount in the patient.
In some embodiments, the clotting measurement of the patient sample
that is at least 1.5 times greater than a clotting measurement of
the control sample identifies the presence of the anticoagulant at
a therapeutically relevant amount in the patient.
[0038] In some embodiments, the clotting assay is selected from the
group consisting of a prothrombin time (PT) assay, an activated
partial thromboplastin time (APTT) assay, and the activated
clotting time (ACT) assay.
[0039] In some embodiments, the clotting measurement of the control
sample is a range of at least two clotting measurements of at least
two control samples from at least two control blood components
known to lack the anticoagulant.
[0040] In some embodiments, the clotting assay is a viscoelastic
analysis clotting assay. In some embodiments, the viscoelastic
analysis is performed using a container containing the sample on an
interior of the container. In some embodiments, the viscoelastic
analysis is performed using the container and a pin, wherein the
pin moves relative to the container. In some embodiments, the
viscoelastic analysis is performed using the container and a pin,
wherein the container moves relative to the pin. In some
embodiments, the container lacks a bottom surface.
[0041] In various embodiments, the patient is a human. In some
embodiments, patient is undergoing a condition including surgery,
trauma, bleeding, or a thromboembolic event.
[0042] In some embodiments, the clotting measurement of the control
sample is a range of at least two clotting measurements of at least
two control samples from at least two control blood components
known to lack the anticoagulant.
[0043] In some embodiments, the anticoagulant is an oral
anticoagulant. The oral anticoagulant may be a direct thrombin
inhibitor or may be a Factor Xa inhibitor.
[0044] In yet a further aspect, the invention provides a method for
classifying an anticoagulant at a therapeutically relevant amount
or higher in a patient suspected of having an anticoagulant. The
method includes (a) subjecting a first patient sample of a blood
component from the patient to a clotting assay in the presence of a
Factor Xa reagent to obtain a Factor Xa clotting measurement of the
patient blood component, wherein the Factor Xa clotting measurement
of the patient blood component greater than a Factor Xa clotting
measurement of a control blood sample of a control blood component
known to lack the anticoagulant identifies the presence of the
anticoagulant at a therapeutically relevant amount in the patient;
and (b) subjecting a second patient sample of the blood component
from the patient to the clotting assay in the presence of an ecarin
reagent to obtain the ecarin clotting measurement of the patient
blood component; wherein the ecarin clotting measurement of the
second patient sample greater than an ecarin clotting measurement
of a control sample of the control blood component identifies the
anticoagulant as a direct thrombin inhibitor (DTI) and wherein the
ecarin clotting measurement of the second patient sample less than
or equal to the ecarin clotting measurement of the control sample
identifies the anticoagulant as an anti-Factor Xa reagent.
[0045] In another aspect, the invention provides a method for
detecting and classifying an anticoagulant at a therapeutically
relevant amount or higher in a patient. The method includes
subjecting a control sample of a control blood component (known not
to contain the anticoagulant), to a clotting assay in the presence
of an ecarin reagent to obtain a control ecarin measurement; and
subjecting a sample of a blood component from the patient suspected
of having the anticoagulant to the clotting assay in the presence
of the ecarin reagent to obtain a patient ecarin clotting
measurement, wherein the clotting measurement of the patient sample
greater than the clotting measurement of the control sample
indicates the presence of the anticoagulant at a therapeutically
relevant amount or higher in the patient and classifies it as a
DTI.
[0046] The invention also provides a method for detecting and
classifying an anticoagulant at a therapeutically relevant amount
or higher in a patient suspected of having an anticoagulant. The
method includes (a) subjecting a first patient sample of a blood
component from the patient to a clotting assay in the presence of
an ecarin reagent to obtain an ecarin clotting measurement of the
patient blood component, wherein the ecarin clotting measurement of
the patient blood component greater than an ecarin clotting
measurement of a control blood sample of a control blood component
known to lack the anticoagulant identifies the presence of the
anticoagulant at a therapeutically relevant amount in the patient
and identifies the anticoagulant as a direct thrombin inhibitor
(DTI); and (b) subjecting a second patient sample of the blood
component from the patient to the clotting assay in the presence of
a Factor Xa reagent to obtain the Factor Xa clotting measurement of
the second patient sample; wherein the Factor Xa clotting
measurement of the second patient sample greater than a Factor Xa
clotting measurement of a control sample of the control blood
component identifies the presence of the anticoagulant at a
therapeutically relevant amount in the patient and identifies the
anticoagulant as an anti-Factor Xa and wherein the Factor Xa
clotting measurement of the second patient sample less than or
equal to the Factor Xa clotting measurement of the control sample
identifies the sample as having no anticoagulant.
[0047] In some embodiments, the Factor Xa clotting measurement of
the patient sample that is at least 1.25 times greater than a
Factor Xa clotting measurement of the control sample identifies the
presence of the anticoagulant at a therapeutically relevant amount
in the patient. In some embodiments, the Factor Xa clotting
measurement of the patient sample that is at least 1.5 times
greater than a Factor Xa clotting measurement of the control sample
identifies the presence of the anticoagulant at a therapeutically
relevant amount in the patient.
[0048] In some embodiments, the ecarin clotting measurement of the
patient sample that is at least 1.25 times greater than the ecarin
clotting measurement of the control sample identifies the
anticoagulant as a direct thrombin inhibitor (DTI). In some
embodiments, the ecarin clotting measurement of the patient sample
that is at least 1.5 times greater than the ecarin clotting
measurement of the control sample identifies the anticoagulant as a
direct thrombin inhibitor (DTI).
[0049] In some embodiments, the anticoagulant is an oral
anticoagulant. The oral anticoagulant may be a direct thrombin
inhibitor or may be a Factor Xa inhibitor.
[0050] In some embodiments, the clotting assay is selected from the
group consisting of a prothrombin time (PT) assay, an activated
partial thromboplastin time (APTT) assay, and the activated
clotting time (ACT) assay.
[0051] In some embodiments, the clotting measurement of the control
sample is a range of at least two clotting measurements of at least
two control samples from at least two control blood components
known to lack the anticoagulant.
[0052] In some embodiments, the clotting assay is a viscoelastic
analysis clotting assay. In some embodiments, the viscoelastic
analysis is performed using a container containing the sample on an
interior of the container. In some embodiments, the viscoelastic
analysis is performed using the container and a pin, wherein the
pin moves relative to the container. In some embodiments, the
viscoelastic analysis is performed using the container and a pin,
wherein the container moves relative to the pin. In some
embodiments, the container lacks a bottom surface.
[0053] In various embodiments, the patient is a human. In some
embodiments, patient is undergoing a condition including surgery,
trauma, bleeding, stroke, or a thromboembolic event.
[0054] In some embodiments, the Factor Xa clotting measurement of
the control sample is a range of at least two Factor Xa clotting
measurements of at least two control samples from at least two
control blood components known to lack the anticoagulant. In some
embodiments, the ecarin clotting measurement of the control blood
component is a range of at least two ecarin clotting measurements
of at least two control blood components known to lack the
anticoagulant.
[0055] In various embodiments, the patient identified as comprising
the presence of the anticoagulant at a therapeutically relevant
amount or higher is administered a therapeutically relevant amount
of a reversal agent. In some embodiments, the reversal agent is
prothrombin complex concentrates. In some embodiments, the patient
identified as comprising the presence of a DTI anticoagulant at a
therapeutically relevant amount or higher is administered a
therapeutically relevant amount of a reversal agent that reverses
the DTI anticoagulant. In some embodiments, the reversal agent that
reverses the DTI anticoagulant is idarucizumab. In some
embodiments, the patient identified as comprising the presence of
an anti-Factor Xa anticoagulant at a therapeutically relevant
amount or higher is administered a therapeutically relevant amount
of a reversal agent that reverses the anti-Factor Xa anticoagulant.
In some embodiments, the reversal agent that reverses the
anti-Factor Xa anticoagulant is andexanet.
BRIEF DESCRIPTION OF THE DRAWINGS
[0056] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0057] The foregoing features of embodiments will be more readily
understood by reference to the following detailed description,
taken with reference to the accompanying drawings, in which:
[0058] FIG. 1 is a schematic diagram showing the clotting cascade
that leads eventually to the formation of a fibrin clot made of
cross-linked fibrin. Both an anti-Factor Xa reagent (or drug) and a
direct thrombin inhibitor will affect both the extrinsic and
intrinsic pathways.
[0059] FIGS. 2A-2C are a series of flow charts showing different
types of clotting assays that are useful in embodiments of the
invention. FIG. 2A depicts the prothrombin time (PT) clotting
assay; FIG. 2B shows the activated Partial Thromboplastin Time
(aPTT) clotting assay; FIG. 2C shows the activated clotting time
(ACT) assay.
[0060] FIG. 3A is schematic diagram showing a TEG tracing measured
throughout the clot's lifespan of a blood component sample taken
from a normal human. The R (reaction time) and ACT (activated
clotting time) is the time of formation of the fibrin strand
polymers, K (coagulation time) is a measurement of time until a
certain clot strength is attained, .alpha. (alpha angle) is the
slope of a line drawn from R tangent to the curve, and MA (maximum
amplitude, measured in mm) is the strength of the clot. The LY30 is
a percent lysis measurement which is measured until 30 minutes
after MA is defined.
[0061] FIG. 3B is a schematic diagram representing a Thrombus
Generation Curve (V-Curve in green) overlaying a TEG tracing. The
V-curve is plotted from the first derivative of changes in clot
resistance expressed as a change in clot strength per unit of time
(dynes/cm2/s), representing the maximum velocity of clot formation.
MRTG stands for Maximum Rate of Thrombus Generation; and TMRTG
stands for Time to Maximum Rate of Thrombus Generation.
[0062] FIG. 4 is schematic diagram showing a TEMogram tracing. CT
indicates clotting time, CFT indicates clot formation time, alpha
is the alpha-angle, lambda-angle is the lysis rate, MCF is the
maximum clot firmness, and ML is maximum lysis.
[0063] FIG. 5 is a schematic diagram of the clotting cascade
showing the stages at which various ecarin reagents (which includes
ecarin and similar enzymes) interfere with the cascade.
[0064] FIG. 6 is a schematic diagram of a decision tree showing the
steps involved in a non-limiting aspect of the invention. A blood
component taken from a patient suspected of being on an
anticoagulant is first subjected to a detection step (to see if
there is an anticoagulant in the test blood component) and then
subjected to a classification step to determine if the
anticoagulant is a DTI or an anti-Factor Xa anticoagulant.
[0065] FIGS. 7A-7F are line graphs. FIGS. 7A-7C show the results of
the TEG kaolin test R time sensitivity as a function of different
concentrations of drug. FIG. 7A shows rivaroxaban; FIG. 7B shows
apixaban; and FIG. 7C shows dabigatran. FIG. 7D-7F show the results
of Rapid TEG test ACT time sensitivity as a function of different
concentrations of drug. FIG. 7D shows rivaroxaban; FIG. 7E shows
apixaban; and FIG. 7F shows dabigatran. R times for all doses of
dabigatran as well as the highest concentration of apixaban and
rivaroxaban tested were significantly higher than the non-spiked
blood sample. ACT times for all concentrations of dabigatran as
well as the medium and higher concentrations of apixaban and
rivaroxaban tested significantly higher than the non-spiked blood
sample for ACT. Dotted parallel bars show the normal ranges of R
and ACT. Statistically significant between: --higher dose and
medium dose; --higher dose and lower dose; .diamond.--medium dose
and lower dose; *--control. In FIGS. 7A-7F, 1 symbol p<0.05; 2
symbols p<0.01; 3 symbols p<0.001. Error bars represent the
standard error of three independent experiments measured in
triplicate.
[0066] FIGS. 8A-8C are line graphs showing the results of TEG
kaolin test R time as a function of drug concentrations in the
presence or absence of ecarin for rivaroxaban (FIG. 8A), apixaban
(FIG. 8B) and dabigatran (FIG. 8C). Rivaroxaban and apixiban both
show an equivalent and significant shortening of R time due to a
the presence of ecarin despite concentration of drug. Dabigatran
has a concentration dependent decrease in R time. Dotted parallel
bars show the normal ranges of R for normal donors. Statistically
significant between: --higher dose and medium dose; --higher dose
and lower dose; .diamond.--medium dose and lower dose; *--control.
.sctn.--Statistically significant between paired sample with or
without ecarin, p<0.001. Error bars represent the standard error
of three independent experiments measured in triplicate.
[0067] FIGS. 9A-9C are TEG tracings and line graphs showing
clotting measurements of blood spiked with 50 ng/ml, 200 ng/ml, or
500 ng/ml of dabigatran in a standard Kaolin test. FIG. 9A shows
the increase in R time as the dosage of dabigatran increases. FIG.
9B (which is an enlargened view of FIG. 7C) shows the increase in R
time of the dabigatran-spiked blood as compared the Kaolin R-time
of control (unspiked) blood. FIG. 9C (which is an enlargened view
of FIG. 8C) shows that when the dabigratran-spiked blood is
subjected to the clotting assay in the presence of ecarin and
Kaolin, a decrease in the R time results.
[0068] FIG. 10 is a schematic diagram showing standard TEG tracings
which show the rate of thrombus generation in blood spiked with 50
ng/ml of dabigatran (pink, far leftline), 200 ng/ml of dabigatran
(green, center line), or 500 ng/ml of dabigatran (white, far right
line) using Kaolin.
[0069] FIGS. 11A-11C are line graphs showing clotting measurements
of blood spiked with 22 ng/ml, 89 ng/ml, or 500 ng/ml of
rivaroxaban (a Factor Xa inhibitor anticoagulant) in a standard
Kaolin test. FIG. 11A shows an increase in R time at the highest
dosage of rivaroxaban. FIG. 11B shows the increase in R time of
rivaroxaban-spiked blood as compared to the Kaolin R time of
control (unspiked) blood. FIG. 11C shows that when the
rivaroxaban-spiked blood is subjected to the clotting assay in the
presence of ecarin, a dramatic decrease in the R time results, such
that at even the highest dosage, the R time of the
rivaroxaban-spiked blood was shorter than the lower boundary of the
Kaolin normal range for R time (from control, unspiked blood).
[0070] FIG. 12 is a line graph showing the R times in the presence
of the FXa reagent from blood spiked with apixaban ("AP";
diamonds), blood spiked with rivaroxaban ("RV"; squares), and blood
spiked with dabigatran ("DB"; triangles). The dotted horizontal
line represents the R range for control blood (i.e., taken from a
donor known not to be taking an anticoagulant and not spiked with
any anticoagulant).
[0071] FIG. 13 is a bar graph showing the R times in presence of
ecarin from blood from the indicated donors spiked with 0 ng/ml
dabigatran, 50 ng/ml dabigatran, 150 ng/ml dabigatran, or 300 ng/ml
dabigatran. The horizontal dotted line represents the R range for
control blood (blood without anticoagulant).
[0072] FIG. 14 is a line graph showing the R times of rivaroxaban
or apixaban-containing blood from four donors in the presence of
ecarin. The horizontal dotted line represents the R range for
control blood (without any anticoagulant).
[0073] FIGS. 15A-15C are a series of bar graphs depicting the
detection and classification steps of blood obtained from a patient
before and after the patient had been administered an
anticoagulant. FIG. 15A shows that for the "before" samples, the
detection and the classification R times were within the normal R
range from control blood. FIG. 15B shows that in the "after sample
in the detection step (i.e., in the presence of the FXa reagent),
the R time lengthened such that it was no longer in the normal R
range from the control blood. FIG. 15C shows that in the
classification step (i.e., in the presence of the ecarin reagent),
the R time was within the normal range. These results identified
that there was an anticoagulant in the "after" sample, and that the
anticoagulant was an anti-Factor Xa anticoagulant.
[0074] FIG. 16 is a bar graph demonstrating elongation of R-time in
presence of a Direct thrombin Inhibitor (DTI, red bars) followed by
shortening of the R-time to baseline when a reversal agent (green
bars) was administered in swine plasma samples. Baseline is shown
as a blue bar.
[0075] FIG. 17 shows the TEG Kaolin test coagulation parameters'
sensitivity in healthy donor spiked samples with different doses of
apixaban, rivaroxaban and dabigatran in the presence or absence of
ecarin. In FIG. 17, R--Reaction Time; MRTG--Maximum Rate to
Thrombus Generation; TMRTG--Time to Maximum Rate of Thrombus
Generation. Statistically significant between: --higher dose and
medium dose; --higher dose and lower dose; .diamond.--medium dose
and lower dose; *--the control. .sctn.--paired sample with or
without Ecarin. SDR--standard error of the mean of three
independent experiments measured in triplicate. 1 symbol p<0.05;
2 symbols p<0.01; 3 symbols p<0.001.
[0076] FIG. 18 shows the Rapid TEG test coagulation parameters'
sensitivity in healthy donor spiked samples with different doses of
apixaban, rivaroxaban and dabigatran in the presence or absence of
ecarin. Statistically significant between: .sctn.--paired sample
with or without Ecarin. SDR--standard error of the mean of three
independent experiments measured in triplicate. 1 symbol p<0.05;
2 symbols p<0.01; 3 symbols p<0.001.
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS
[0077] In some embodiments, the invention provides methods and
reagents (e.g., cups) for detecting the presence of an
anticoagulant in a blood component (e.g., from a patient). In some
embodiments, the invention provides methods and reagents for
identifying which type of anticoagulant is present in the blood
component. In some embodiments, the invention also provides methods
and reagents for demonstrating reversal of the anticoagulant effect
with a reversal agent or antidote.
[0078] The publications (including patent publications), web sites,
company names, and scientific literature referred to herein
establish the knowledge that is available to those with skill in
the art and are hereby incorporated by reference in their entirety
to the same extent as if each was specifically and individually
indicated to be incorporated by reference. Any conflict between any
reference cited herein and the specific teachings of this
specification shall be resolved in favor of the latter.
[0079] In a first aspect, the invention provides a method for
detecting an anticoagulant at a therapeutically relevant amount in
a patient suspected of having an anticoagulant, the method
comprising (a) subjecting a control sample of a control blood
component, the control sample known not to contain the
anticoagulant, to a clotting assay in the presence of a Factor Xa
reagent to obtain a clotting measurement of the control sample; (b)
subjecting a patient sample of a blood component from the patient
suspected of having the anticoagulant to the clotting assay in the
presence of the Factor Xa reagent to obtain the clotting
measurement of the patient sample, and (c) comparing the clotting
measurement of the patient sample to the clotting measurement of
the control sample wherein the clotting measurement of the patient
sample greater than the clotting measurement of the control sample
indicates the presence of the anticoagulant at a therapeutically
relevant amount in the patient and wherein the clotting measurement
of the patient sample less than or equal to the clotting
measurement of the control sample indicates the absence of the
anticoagulant at a therapeutically relevant amount in the
patient.
[0080] In another aspect, the invention provides a method for
detecting an anticoagulant at a therapeutically relevant amount (or
higher than a therapeutically relevant amount) in a patient
suspected of having an anticoagulant, the method comprising
subjecting a patient sample of a blood component from the patient
to a clotting assay in the presence of a Factor Xa reagent to
obtain a clotting measurement of the patient sample, wherein the
clotting measurement of the patient sample greater than a clotting
measurement of a control sample of a control blood component known
to lack the anticoagulant identifies the presence of the
anticoagulant at a therapeutically relevant amount in the
patient.
[0081] The degree of elongation of the clotting measurement depends
on the concentration of the oral anticoagulant in the blood
component. The higher the concentration, the greater will be the
clotting measurement compared to blood components known to lack any
oral anticoagulant. In some embodiments, the clotting measurement
of the test blood component that is at least 1.25 times greater
than a clotting measurement of a blood component known to lack the
anticoagulant identifies the presence of the anticoagulant at a
therapeutically relevant amount in the blood component. In some
embodiments, the clotting measurement of the test blood component
that is at least 1.5 times greater than a clotting measurement of a
blood component known to lack the anticoagulant identifies the
presence of the anticoagulant at a therapeutically relevant amount
in the blood component. In some embodiments, the clotting
measurement of the test blood component that is at least 1.75 times
greater than a clotting measurement of a blood component known to
lack the anticoagulant identifies the presence of the anticoagulant
at a therapeutically relevant amount in the blood component. In
some embodiments, the clotting measurement of the test blood
component that is at least two times greater than a clotting
measurement of a blood component known to lack the anticoagulant
identifies the presence of the anticoagulant at a therapeutically
relevant amount in the blood component.
[0082] In some embodiments, the clotting measurement of the control
sample may be an average, a median, or a range of at least two
clotting measurements of at least two control samples from at least
two control blood components known to lack the anticoagulant. For
example, a range (which may be referred to as a reference range)
may be created from control samples from multiple control blood
components (e.g., from multiple donors known to lack the
anticoagulant). If an average or mean is used, the clotting
measurements from multiple control samples are averaged, and that
average number is used as the clotting measurement of the control
sample.
[0083] In some embodiments, the invention utilizes a clotting assay
to assess the functioning of the clotting cascade in the blood
component from the patient.
[0084] The clotting cascade (or coagulation cascade) is a tightly
regulated process by which blood changes from liquid to a solid
clot. This process is called coagulation or clotting. FIG. 1
provides a schematic diagram of the clotting cascade. Clotting can
be triggered by the extrinsic tissue factor pathway (e.g., by
injury or damage to a blood vessel) or by the intrinsic contact
activation pathway. The two pathways join in the activation of
Factor Xa which then activates prothrombin to thrombin.
[0085] By "blood component" is meant one or more components of
blood taken, for example, from a patient, where the blood component
contains a sufficient quantity of plasma to form a fibrin mediated
clot. The blood component may contain at least about 8% plasma on a
volume basis (i.e., 8% v/v plasma). The blood component may contain
at least about 10% v/v plasma, or at least about 12% v/v plasma, or
at least about 15% v/v plasma, or at least about 20% v/v plasma.
The patient may be a human, but may also be any other animal (e.g.,
veterinary animal or exotic animal). Blood is the circulating
tissue of an organism that carries oxygen and nutritive materials
to the tissues and removes carbon dioxide and various metabolic
products for excretion. Blood includes a pale yellow or gray yellow
fluid, plasma, in which are suspended red blood cells, white blood
cells, and platelets. Blood (sometimes referred to as whole blood)
can be fractionated into various components or fractions following
density gradient centrifugation. Thus, a blood component includes,
without limitation, whole blood (which may be simply referred to as
blood), white blood cells including at least about 10% volume
plasma, red blood cells including at least about 10% volume plasma,
platelets including at least about 10% volume plasma, plasma, and
various fractions of blood including at least about 10% volume
plasma including the platelet fraction, the red blood cell fraction
(e.g., comprised of a majority of red blood cells, and a minority
of some white blood cells and plasma), and the buffy coat fraction
(e.g., comprised of a majority of white blood cells and platelets,
and a minority of some red blood cells and plasma). A blood
component also includes any of the above-listed components that
also includes a substance (e.g., citric acid or citrate, or
heparin) added after the blood component is obtained from the
patient that prevents or reduces the coagulation of the blood
component.
[0086] By "anticoagulant" is meant a substance (i.e., a reagent or
a drug) that prevents or reduces coagulation (i.e., clotting) that
is present in a blood component of the patient if that substance is
taken by or administered to the patient prior to obtaining the
blood component from the patient. Such administration may be by any
route including oral, parenteral, intravenous, intraperitoneal,
intramuscular, subcutaneous, etc. Note that a substance (e.g.,
heparin or citrate) that is added to a blood component after the
blood component is obtained from the patient is not an
anticoagulant within this definition.
[0087] In some embodiments, the anticoagulant is administered to
the patient orally. The orally administered anticoagulant may be
referred to as an oral anticoagulant.
[0088] By "reversal agent" is meant a substance (e.g., a reagent,
antibody, protein or a drug) that reverses the effect of
anticoagulation (e.g., the reversal agent reverses the bleeding
effected by the anticoagulant) that is present in a blood component
of the patient if that substance is taken by or administered to the
patient prior to obtaining the blood component from the patient.
Such administration may be by any route including oral, parenteral,
intravenous, intraperitoneal, intramuscular, subcutaneous, etc.
[0089] In some embodiments, the reversal agent is administered to
the patient orally.
[0090] By a "patient suspected of having an anticoagulant" is meant
that the patient (e.g., a human patient) is suspected of having
taken an anticoagulant (e.g., through oral administration) before
the blood component is obtained from the patient. For example, an
unconscious patient may be brought into the emergency room. During
surgery, one of the methods described herein may be performed on a
sample of the patient's blood component to determine if he/she has
taken an anticoagulant by detecting the presence or absence of the
anticoagulant in the blood component sample. This information is
helpful in attending to the needs of the patient and for reversing
the anticoagulant effect as required. A blood component from a
patient suspected of having an anticoagulant may be referred to as
a "blood component suspected of having an anticoagulant". A sample
of a blood component from a patient suspected of having an
anticoagulant may be referred to as a "sample suspected of having
an anticoagulant".
[0091] In some embodiments, the patient from whom the blood
component is obtained is a patient suspected of having bleeding
diatheses. In some embodiments, the bleeding diatheses may be due
to any cause including a congenital hemophilia condition, a vitamin
K deficiency. In some cases, the bleeding diatheses may be due to
the intake of an anticoagulant by the patient such that it is not
known if the patient has bleeding diatheses and, if the patient
does have bleeding diatheses, it is not known why the patient has
bleeding diatheses. Using the methods described herein, the
identification and classification of an anticoagulant (if the
patient has taken the anticoagulant) and reversal (if a reversal
agent has been administered to the patient) can be determined. In
some embodiments, the patient is undergoing (or will shortly be
undergoing) a condition that may involve bleeding. For example, the
patient may be undergoing surgery, may be being prepared for
surgery, may be injured or wounded, may be bleeding, or may have
had or is currently having or is suspected to imminently have a
thromboembolic event including, without limitation, a stroke, a
venous thromboembolic event (VTE), a heart attack, heart failure,
an arterial thromboembolic event, and a pulmonary embolism. The
patient may be a trauma patient and/or may have internal
bleeding.
[0092] The recently developed oral anticoagulants that do not
universally inhibit all vitamin K-dependent clotting factors can be
classified generally into two categories, depending upon the
particular clotting factor that the anticoagulant targets.
[0093] In some embodiments, the oral anticoagulant is a direct
thrombin inhibitor, and may be referred to as a DTI. Thrombin
(Clotting Factor IIa) is a central player in the blood clotting
process (see FIG. 1). Thrombin plays multiple roles including (a)
converting soluble fibrinogen to fibrin; (b) activating factors VI,
VIII, XI, and XIII and (c) stimulating platelets. By activating
Factors XI and XIII, thrombin generates more thrombin and favors
formation of crosslinked fibrin molecules, thereby strengthening
the blood clot.
[0094] A DTI is an anticoagulant that binds thrombin and blocks
thrombin's interaction with its substrates. DTIs may be bivalent
(blocking thrombin at the active site and one of the exosites) or
univalent (blocking thrombin at the active site). Bivalent DTIs
include, without limitation, hirudin and bivalirudin. Univalent
DTIs include, without limitation, argatroban, melagatran,
ximelagatran, and dabigatran. Dabigatran is sold commercially by
Boehringer Ingelheim International GmbH, Ingelheim, Germany under
the name PRADAXA.RTM.. Dabigatran is an oral direct inhibitor of
thrombin (Factor IIa) that is "not permanent," selective and
competitive. Dabigatran is licensed in Europe and the USA to reduce
the risk of venous thromboembolism (VTE) in orthopedic surgical
patients as well as stroke and systemic embolism in patients with
non-valvular atrial fibrillation.
[0095] In some embodiments, the oral anticoagulant is an inhibitor
of Factor Xa and may be referred to as an anti-Factor Xa reagent, a
Factor Xa inhibitor, or an xaban. An xaban acts directly upon
Factor Xa in the blood clotting cascade (see FIG. 1A). Two
non-limiting commercially available inhibitors of Factor Xa are
Rivaroxaban (sold under the name of XARELTO.RTM. by Bayer Pharma
AG, Leverkusen, Germany and Janssen Pharmaceuticals, Inc.,
Titusville, N.J.) and Apixaban (sold under the name ELIQUIS.RTM. by
Bristol-Myers Squibb, New York, N.Y. and Pfizer EEIG Sandwich,
United Kingdom). Rivaroxaban and Apixaban are licensed in Europe
and the USA to reduce the risk of venous thromboembolism (VTE) in
orthopedic surgical patients as well as stroke and systemic
embolism in patients with non-valvular atrial fibrillation.
Rivaroxaban is also approved in the EU for the secondary prevention
of acute coronary syndrome. Rivaroxaban can be administered in
combination with acetylsalicylic acid (ASA) or with ASA plus
clopidogrel or ticlopidine for the prevention of thrombotic events
in adult patients with elevated cardiac biomarkers after a coronary
event according to the product information provided by Bayer
Pharma.
[0096] Additional non-limiting inhibitors of Factor Xa include
betrixaban (LY517717; Portola Pharmaceuticals), darexaban (YM150;
Astellas), edoxaban (Lixiana; DU-176b; Daiichi), TAK-442 (Takeda),
and eribaxaban (PD0348292; Pfizer).
[0097] In various embodiments, the methods described herein involve
the use of a Factor Xa reagent. By "Factor Xa reagent" is meant
Factor Xa (FXa) and/or any combination of clotting Factors that
include Factor Xa. This Factor Xa reagent may contain other
substances for performance and/or stability improvement (including
salts, buffers, sugars etc.). Factor Xa reagent is added to a blood
component after that blood component has been obtained from the
patient. Alternatively, the Factor Xa reagent may be prepared from
the Factor X endogenous in the blood component sample by the
addition of another reagent such as Russel's Viper venom that
activates the Factor X zymogen (precursor of active Factor Xa).
[0098] By "clotting measurement" is a measurement of clot
formation. This measurement can be taken any time during the
formation of a clot including, without limitation, the time of the
initial formation of fibrin or the time the clot achieves a certain
level of strength. If the clotting assay used to determine the
clotting measurement is a thromboelastography (TEG) assay (e.g.,
performed on the thromboelastograph coagulation analyzer model 5000
platform), the time of the initial formation of fibrin is the "R"
time and the time the clot achieves a certain level of strength is
the "K" time. If the clotting assay used to determine the clotting
measurement is a thromboelastometry (TEM) assay (e.g., performed on
the ROTEM platform), then the time of the initial formation of
fibrin is the "Reaction Time (RT)" and the time the clot achieves a
certain level of strength is the "Clot Formation Time (CFT)". It
should be noted that the clotting measurement can be taken on a
blood component taken directly from a patient, or a blood component
that has been treated with a clotting activator such as kaolin or a
clotting inhibitor such as citrate that has been suitably reversed
by the addition of calcium.
[0099] By "clotting assay" is meant any type of assay that can be
used to measure the ability of blood or a blood component to form a
clot. Clotting assays including, without limitation, a viscoelastic
assay (including a thromboelastography (TEG) assay or a
thromboelastometry (TEM) assay), a prothrombin time (PI') assay, an
activated partial thromboplastin time (aPTT) assay, and an
activated clotting time (ACT) assay.
[0100] In some embodiments, the clotting assay is a prothrombin
time (PT) clotting assay. FIG. 2A schematically depicts the
prothrombin time (PT) clotting assay. The PT assay is performed by
adding a thromboplastin reagent that contains tissue factor (which
can be recombinant in origin or derived from an extract of brain,
lung, or placenta) and calcium to plasma (or other blood component,
which may be citrated, for example) and measuring the clotting
time. The prothrombin time (PT) varies with reagent and
coagulometer but typically ranges between 10 and 14 second (see
Bates and Weitz, Circulation 112: e53-e60, 2005; White et al.,
"Approach to the bleeding patient", In: Colman R W et al. (eds)
Hemostasis and Thrombosis: Basic Principles and Clinical Practice.
3rd ed. Philadelphia, Pa.: JB Lippincott Co. 1134-1147, 1994). The
PT value may be used as a non-limiting clotting measurement in
accordance with the methods described herein. The PT may be
abnormal (e.g., may be prolonged) when the blood being tested
contains an anticoagulant (e.g., an anticoagulant that was orally
administered to the person whose blood is being tested) or when the
blood being tested is from a patients with a deficiency in a
clotting factor (e.g., a deficiency of factors VII, X, and V,
prothrombin, or fibrinogen). This test also is abnormal in patients
with inhibitors of the fibrinogen-to-fibrin conversion reaction,
including high doses of heparin and the presence of fibrin
degradation products.
[0101] In some embodiments, the clotting assay is the activated
partial thromboplastin time (aPTT) clotting assay. FIG. 2B
schematically depicts the steps involved in the activated partial
thromboplastin time (aPTT) clotting assay. The aPTT assay is also
known as the Kaolin Cephalin Clotting Time assay (cephalin is a
platelet phospholipid substitute) or a partial thromboplastin time
with Kaolin (PTTK) assay. The aPTT is typically performed by first
adding a surface activator (e.g., kaolin, celite, ellagic acid, or
silica) and diluted phospholipid (e.g., cephalin) to citrated
plasma (FIG. 2B). The phospholipid in this assay is called partial
thromboplastin because tissue factor is absent. After incubation to
allow optimal activation of contact factors (e.g., factor XII,
factor XI, prekallikrein, and high-molecular-weight kininogen) and
the generation of Factor IXa, calcium is then added, and the
clotting time is measured. Thus, the APTT time is the time taken
from the addition of calcium to the formation of a fibrin clot.
Although the clotting time varies according to the reagents (e.g.,
type of surface activator) and coagulometer used, the aPTT
typically ranges between 22 and 40 seconds (see Bates and Weitz,
supra). The aPTT time may be used as a non-limiting clotting
measurement in accordance with the methods described herein. The
aPTT may be abnormal (e.g., may be prolonged) when the blood being
tested contains an anticoagulant (e.g., an anticoagulant that was
orally administered to the person whose blood is being tested). The
aPTT may be prolonged with deficiencies of contact factors; factors
IX, VIII, X, or V; prothrombin; or fibrinogen. Specific factor
inhibitors, as well as nonspecific inhibitors, may also prolong the
aPTT. Fibrin degradation products and anticoagulants (e.g.,
heparin, direct thrombin inhibitors, or warfarin) also prolong the
aPTT.
[0102] In some embodiments, the clotting assay is an activated
clotting time (ACT) assay. FIG. 2C schematically depicts the steps
involved in the activated clotting time (ACT) assay. Typically,
whole blood is collected into a tube or cartridge containing a
coagulation activator (e.g., celite, kaolin, or glass particles)
and a magnetic stir bar. Once thrombin is generated, it induces
both platelet aggregation and fibrin formation, and the time taken
for the blood to dot is then measured (see also Van Cott and
Laposata, "Coagulation". In: Jacobs D S et al. (eds), The
Laboratory Test Handbook, 5th ed. Cleveland, Ohio: Lexi-Comp;
2001:327-358, 2001). The reference value for the ACT ranges between
70 and 180 seconds. The ACT value may be used as a non-limiting
clotting measurement in accordance with the methods described
herein. The ACT time is prolonged when the blood being tested
contains an anticoagulant (e.g., an anticoagulant that was orally
administered to the person whose blood is being tested).
[0103] In some embodiments, the clotting assay is a viscoelastic
assay. By "viscoelastic analysis" is meant any analysis method that
measures the characteristics of elastic solid (e.g., fibrin solids)
and fluids. In other words, viscoelastic analysis allows the study
of properties of a viscous fluid, such as blood, plasma, or a blood
sample. In some embodiments, the viscoelastic analysis is performed
under conditions that mimic the conditions in vivo that result in
haemostasis. For example, the condition may include a temperature
that mimics a body temperature (e.g., a temperature of 37.degree.
C.). The condition may also include clot formation and dissolution
at flow rates that mimic those found in blood vessels.
[0104] In some embodiments, viscoelastic analysis of a blood sample
may include subjecting the blood sample to analysis on a hemostasis
analyzer instrument. One non-limiting viscoelastic analysis method
is the thromboelastography ("TEG") assay. Thus in some embodiments,
the viscoelastic analysis includes subjecting a blood sample to
analysis using thromboelastography (TEG), which was first described
by Helmut Hartert in Germany in the 1940's.
[0105] Various devices that perform thromboelastography, and
methods for using it are described in U.S. Pat. Publication Nos.
5,223,227; 6,225,126; 6,537,819; 7,182,913; 6,613,573; 6,787,363;
7,179,652; 7,732,213, 8,008,086; 7,754,489; 7,939,329; 8,076,144;
6,797,419; 6,890,299; 7,524,670; 7,811,792; 20070092405;
20070059840; 8,421,458; US 20120301967; and 7,261,861, the entire
disclosures of each of which are hereby expressly incorporated
herein by reference.
[0106] Thromboelastography (TE) monitors the elastic properties of
blood as it is induced to clot under a low shear environment
resembling sluggish venous blood flow. The patterns of changes in
shear elasticity of the developing clot enable the determination of
the kinetics of clot formation, as well as the strength and
stability of the formed clot; in short, the mechanical properties
of the developing clot. As described above, the kinetics, strength
and stability of the clot provides information about the ability of
the clot to perform "mechanical work," i.e., resisting the
deforming shear stress of the circulating blood. In essence, the
clot is the elementary machine of hemostasis. Haemostasis
instruments that measure haemostasis are able to measure the
ability of the clot to perform mechanical work throughout its
structural development. These haemostasis analyzers measure
continuously all phases of patient hemostasis as a net product of
whole blood components in a non-isolated, or static fashion from
the time of test initiation until initial fibrin formation, through
clot rate strengthening and ultimately clot strength through clot
lysis.
[0107] In some embodiments, the viscoelastic analysis and/or the
haemostais analyzer comprises a container which is in contact with
the blood.
[0108] As used herein, by "container" is meant a rigid surface
(e.g., a solid surface), a portion of which contacts a portion of a
blood sample placed into the container at any point during the
viscoelastic analysis. The portion of the container that contact
the portion of blood sample may also be referred to as the
"interior" of the container. Note that the phase "into the
container" does not mean that the container has a bottom surface
which is in contact with the portion of the blood sample. Rather,
the container can be a ring-shaped structure, where the inside of
the ring is the interior of the container, meaning that the inside
of the ring is the portion of the ring-shaped container that
contacts a portion of the blood sample. A blood sample can flow
into the container and be held there, for example, by vacuum
pressure or surface tension.
[0109] Still additional types of containers that are included in
this definition are those present on plates and cassettes (e.g., a
microfluidic cassette), where the plate or cassette has multiple
channels, reservoirs, tunnels, and rings therein. Each of the
contiguous channels (comprising, for example, a channel, a
reservoir, and a ring) is a container, as the term is used herein.
Hence, there may be multiple containers on one cassette. U.S. Pat.
No. 7,261,861 (incorporated herein by reference) describes such a
cassette with multiple channels or containers. Any of the surfaces
in any of the channels or tunnels of the cassette may be an
interior of the container if that surface comes into contact with
any portion of the blood sample, at any time during the
viscoelastic analysis.
[0110] One non-limiting haemostasis analyzer instrument is
described in U.S. Pat. No. 7,261,861; US Patent Publication No. US
US20070092405; and US Patent Publication No. US20070059840.
[0111] Another non-limiting haemostasis analyzer instrument that
performs viscoelastic analysis using thromboelastography is the
TEG.RTM. thromboelastograph hemostasis analyzer system sold
commercially by Haemonetics, Corp. (Braintree, Mass.).
[0112] Thus, the TEG assay may be performed using the TEG
thromboelastograph hemostasis analyzer system that measures the
mechanical strength of an evolving blood clot. To run the assay,
the blood sample is placed into a container (e.g., a cup or a
cuvette), and a plastic pin goes into the center of the container.
Contact with the interior walls of the container (or addition of a
clot activator to the container) initiates clot formation. The TEG
thromboelastograph hemostasis analyzer then rotates the container
in an oscillating fashion, approximately 4.45 degrees to 4.75
degrees, every 10 seconds, to imitate sluggish venous flow and
activate coagulation. As fibrin and platelet aggregates form, they
connect the inside of the container with the plastic pin,
transferring the energy used to move the container in the pin. A
torsion wire connected to the pin measures the strength of the clot
over time, with the magnitude of the output directly proportional
to the strength of the clot. As the strength of the clot increases
over time, a classic TEG tracing curve develops (See FIG. 3A). The
curve depicted is from a normal human patient who is known to be
not taking any anticoagulants.
[0113] The rotational movement of the pin is converted by a
transducer to an electrical signal, which can be monitored by a
computer including a processor and a control program. The computer
is operable on the electrical signal to create a hemostasis profile
corresponding to the measured clotting process. Additionally, the
computer may include a visual display or be coupled to a printer to
provide a visual representation of the hemostasis profile. Such a
configuration of the computer is well within the skills of one
having ordinary skill in the art. As shown in FIG. 3A, the
resulting hemostasis profile (i.e., a TEG tracing curve) is a
measure of the time it takes for the first fibrin strand to be
formed, the kinetics of clot formation, the strength of the clot
(measured in millimeters (mm) and converted to shear elasticity
units of dyn/cm2) and dissolution of clot. See also Donahue et al.,
J. Veterinary Emergency and Critical Care:15(1): 9-16. (March
2005), herein incorporated by reference.
[0114] The descriptions for several of these measured parameters,
any of which can be used as a clotting measurement in accordance
with the methods described here, are as follows:
[0115] R is the period of time of latency from the time that the
blood was placed in the thromboelastography analyzer until the
initial fibrin formation. This typically takes about 30 second to
about 10 minutes; however the R range will vary based on the
particular TEG assay performed (e.g., type of blood component being
tested, whether the blood component is citrated or not, etc.). For
example, in Example 1 below, the normal R range (i.e., from a
citrated blood component in the presence of Kaolin) is between
about 5 minutes to about 10 minutes. For patients in a
hypocoagulable state (i.e., a state of decreased coagulability of
blood), the R number is longer which indicates slower clot
formation, while in a hypercoagulable state (i.e., a state of
increased coagulability of blood), the R number is shorter. In the
methods described herein, the R value (in minutes or seconds) that
can be used as a non-limiting clotting measurement. Note that in
FIG. 3A, the R value is labeled as "R/ACT" because when the TEG
assay is performed with a kaolin-Tissue Factor treated blood
component, the R value is sometimes referred to as the ACT value
(the activated clotting time value) in reference to the older
activated clotting time (ACT) assay described above and in FIG.
2C.
[0116] K value (measured in minutes) is the time from the end of R
until the clot reaches 20 mm and this K value represents the speed
of clot formation. This K value is typically about 0 to about 4
minutes (i.e., after the end of R). In a hypocoagulable state, the
K number is longer, while in a hypercoagulable state, the K number
is shorter. This K value may be used as a non-limiting clotting
measurement in accordance with the methods described herein.
[0117] .alpha. angle (or simply .alpha.) measures the rapidity of
fibrin build-up and cross-linking (clot strengthening). It is angle
between the line formed from the split point tangent to the curve
and the horizontal axis. This angle is typically about 47.degree.
to 74.degree.. In a hypocoagulable state, the .alpha. degree is
lower, while in a hypercoagulable state, the .alpha. degree is
higher. This a angle value may be used as a non-limiting clotting
measurement in accordance with the methods described herein.
[0118] MA or Maximum Amplitude in mm, is a direct function of the
maximum dynamic properties of fibrin and platelet bonding and
represents the ultimate strength of the fibrin-platelet clot. This
number is typically from about 54 mm to about 72 mm, and the MA
occurs typically between about 15 to about 35 minutes after the
start of the viscoelastic assay. Note that if the blood sample
tested has a reduced platelet function, this MA represents the
strength of the clot based on fibrin only. Decreases in MA may
reflect a hypocoagulable state (e.g., with platelet dysfunction or
thrombocytopenia), whereas an increased MA (e.g., coupled with
decreased R) may be suggestive of a hypercoagulable state. This MA
value may be used as a non-limiting clotting measurement in
accordance with the methods described herein.
[0119] LY30 is a measurement of tracing area--reduction 30 minutes
after MA. The LY30 is a percentage decrease in amplitude 30 minutes
after the MA. This number is typically 0% to about 8%--a
non-limiting clotting measurement in accordance with the methods
described herein. When no fibrinolysis occurs, the amplitude value
at the MA tracing stays constant or may decrease slightly due to
clot retraction. However, as fibrinolysis occurs (e.g., in a
hypocoagulable state), the curve of the TEG tracing starts to
decay. The resultant loss in potential area-under-the-curve in the
30 minutes following Maximum Amplitude in the TEG assay is called
the LY30 (see FIG. 3A). LY30, the percentage of lysis 30 minutes
after the maximum amplitude point (expressed as a percentage of the
clot lysed) indicates the rate of clot lysis.
[0120] It should be noted that modifications of the TEG assay can
be performed, such as the modified TEG assays described below in
the Examples section. For example, in Example 1 below, the RapidTEG
(rTEG) test incorporates both tissue factor and kaolin to generate
the conventional kaolin parameters as well as the TEG-ACT
parameter, which is measured in seconds. The TEG-ACT is equivalent
to the Activated Clotting Time (see Chavez J J., Anesth. Analg. 99:
1290-1294, 2004). A prolonged TEG-ACT time (as compared to the ACT
time from a normal blood component) indicates slower clot
formation.
[0121] Finally, it should be noted that velocity curves can be
derived from the kaolin TEG tests and RapidTEG tests (incorporating
both kaolin and tissue factor). These velocity curves can be
plotted using TEG software. These curves represent the speed of
clot propagation (MRTG, Maximum Rate of Thrombus Generation; and
TMRTG, Time to Maximum Rate of Thrombus Generation) (see FIG. 3B).
Either or both of the MRTG or the TMRTG may be used as a
non-limiting clotting measurement in accordance with the methods
described herein.
[0122] Viscoelastic measurements of coagulation provided by devices
such as TEG are increasingly being employed to assess trauma
patients who arrive in shock secondary to massive bleeding as well
as for acute care of surgical patients with bleeding diatheses. TEG
is widely used as a management tool for cardiac surgery and
transplant patients and provides information to guide
administration of blood products (see Holcolmb J. B. et al., Ann.
Surg. 256: 476-486, 2012). TEG is able to detect both low molecular
weight and unfractionated heparin and, with the use of a heparinase
cup, can illustrate whether the effects of these agents have been
completely reversed. Furthermore, the TEG PlateletMapping Assay is
used to quantify the response to antiplatelet therapies including
clopidogrel and aspirin that can be used in combination with an
oral anticoagulant. TEG assays using ecarin have been employed to
monitor recombinant hirudin as well as bivalirudin during cardiac
surgery (Koster, A. et al., J. Card. Surg. 23: 321-323, 2008; Choi,
T. S. et al., Am. J. Clin. Path. 125: 290-295, 2006).
[0123] Another viscoelastic hemostasis assay that can be used is
the thromboelastometry ("TEM") assay. This TEM assay may be
performed using the ROT EM Thromboelastomewy Coagulation Analyzer
(TEM International GmbH, Munich, Germany), the use of which is well
known (See, e.g., Sorensen, B., et al., J. Thromb. Haemost., 2003.
1(3): p. 551-8. Ingerslev, J., et al., Haemophilia, 2003. 9(4): p.
348-52. Fenger-Eriksen, C., et al. Br J Anaesth. 2005. 94(3): p.
324-9]. In the ROTEM analyzer, the blood sample is placed into a
container (also called a cuvette or cup) and a cylindrical pin is
immersed. Between pin and the interior wall of the container there
is a gap of 1 mm which is bridged by the blood. The pin is rotated
by a spring to the right and the left. As long as the blood is
liquid (i.e., unclotted), the movement is unrestricted. However,
when the blood starts clotting, the clot increasingly restricts the
rotation of the pin with rising clot firmness. The pin is connected
to an optical detector. This kinetic is detected mechanically and
calculated by an integrated computer to the typical tracing curves
(TEMogram) and numerical parameters (see FIG. 4).
[0124] In the ROTEM Thromboelastometry Coagulation Analyzer, the
movement of the pin can be monitored by a computer including a
processor and a control program. The computer is operable on the
electrical signal to create a hemostasis profile corresponding to
the measured clotting process. Additionally, the computer may
include a visual display or be coupled to a printer to provide a
visual representation of the hemostasis profile (called a
TEMogram). Such a configuration of the computer is well within the
skills of one having ordinary skill in the art. As shown in FIG. 5,
the resulting hemostasis profile (i.e., a TEM tracing curve) is a
measure of the time it takes for the first fibrin strand to be
formed, the kinetics of clot formation, the strength of the clot
(measured in millimeters (mm) and converted to shear elasticity
units of dyn/cm2) and dissolution of clot. The descriptions for
several of these measured parameters, any of which can be used as a
clotting measurement in accordance with the methods described here,
are as follows:
[0125] CT (clotting time) is the period of time of latency from the
time that the blood was placed in the ROTEM analyzer until the clot
begins to form. This CT time may be used as a non-limiting clotting
measurement in accordance with the methods described herein.
[0126] CFT (Clot formation time): the time from CT until a clot
firmness of 20 mm point has been reached. This CFT time may be used
as a non-limiting clotting measurement in accordance with the
methods described herein.
[0127] alpha-angle: The alpha angle is the angle of tangent at 2 mm
amplitude. This alpha angle may be used as a non-limiting clotting
measurement in accordance with the methods described herein.
[0128] MCF (Maximum clot firmness): MCF is the greatest vertical
amplitude of the trace. MCF reflects the absolute strength of the
fibrin and platelet clot. The MCF value may be used as a
non-limiting clotting measurement in accordance with the methods
described herein.
[0129] A10 (or A5, A15 or A20 value). This A10 value describes the
clot firmness (or amplitude) obtained after 10 (or 5 or 15 or 20)
minutes and provide a forecast on the expected MCF value at an
early stage. Any of these A values (e.g., A10) may be used as a
non-limiting clotting measurement in accordance with the methods
described herein.
[0130] LI30 (Lysis Index after 30 minutes). The LI30 value is the
percentage of remaining clot stability in relation to the MCF value
at 30 min after CT. This LI30 value may be used as a non-limiting
clotting measurement in accordance with the methods described
herein. When no fibrinolysis occurs, the amplitude value at the MCF
on a TEM tracing stays constant or may decrease slightly due to
clot retraction. However, as fibrinolysis occurs (e.g., in a
hypocoagulable state), the curve of the TEM tracing starts to
decay. LI30 corresponds to the LY30 value from a TEG tracing.
[0131] ML (Maximum Lysis). The ML parameter describes the
percentage of lost clot stability (relative to MCF, in %) viewed at
any selected time point or when the test has been stopped. This ML
value may be used as a non-limiting clotting measurement in
accordance with the methods described herein.
[0132] Thus, parameters of interest in TEG or TEM assays, each of
which can be used as a clotting measurement in accordance with the
methods described herein, include the maximum strength of the clot
which is a reflection of clot strength. This is the MA value in the
TEG assay, and the MCF value in the TEM assay. The reaction time
(R) in TEG (measured in seconds or minutes) and clotting time (CT)
in TEM is the time until there is first evidence of clot; clot
kinetics (K, measured in minutes) is a parameter in the TEG test
indicating the achievement of clot firmness; and .alpha. in TEG or
alpha-angle in TEM is an angular measurement from a tangent line
drawn to the curve of the TEG tracing or TEM tracing starting from
the point of clot reaction time that is reflective of the kinetics
of clot development. (See Trapani, L. M. Thromboelastography:
Current Applications, Future Directions", Open Journal of
Anesthesiology 3(1): Article ID: 27628, 5 pages (2013); and Kroll,
M. H., "Thromboelastography: Theory and Practice in Measuring
Hemostasis," Clinical Laboratory News: Thromboelastography 36(12),
December 2010; instruction manuals for the TEG instrument
(available from Haemonetics, Corp.), and the instruction manual for
the ROTEM instrument (available from TEM international GmbH), all
of which documents are herein incorporated by reference in their
entireties.
[0133] In some embodiments, the parameters (and hence the clotting
measurements) are recorded by observation of different excitation
levels of the sample as coagulation occurs. For example, where the
container is a microfluidic cassette or a particular channel in the
cassette, the blood component sample may be excited at a resonant
frequency and its behavior observed by an electromagnetic or light
source as coagulation occurs. In other embodiments the blood
component sample's clotting measurement may be observed for changes
with a light source without exciting the sample.
[0134] Because a single cassette may have multiple containers
(e.g., different channels in the cassette), the different samples
(e.g., portions of the blood component from the patient) are easily
directly comparable one another. For example, one channel may be
untreated, one channel may be treated with the Factor Xa reagent,
and one channel may be treated with the ecarin reagent. In another
example, blood components from different individuals can be
measured in the different channels, and the results from the
different individuals obtained simultaneously from a single
cassette.
[0135] By "therapeutically relevant amount" is meant an amount of
an anticoagulant in the blood component being tested that is within
the therapeutically effective concentration range for the
anticoagulant. The therapeutically relevant amount will differ for
each anticoagulant, and is affected by the bioavailability of the
anticoagulant and also the half-life of the anticoagulant following
ingestion by the patient. For example, dabigatran has a half-life
of 12-17 hours which is lengthened in patients with renal
dysfunction (Boehringer Ingelheim International G. Pradaxa
(dabigatran etexilate) product information). Apixaban and
rivaroxaban have shorter half-lives than dabigatran. However,
apixaban has also an increased half-life of up to 44% in patients
with severe renal impairment compared to healthy volunteers (see
Dager et al., Crit. Care Med. 41: e42-46, 2013). The anticoagulant
effect of apixaban or rivaroxaban can be expected to persist for at
least 10-30 hours after the last dose, i.e. for about two
half-lives. Generally, however, the therapeutically relevant amount
of an anticoagulant is between about 75 ng/ml to about 500 ng/ml in
the blood (or blood component). For example, for apixaban, a
therapeutically relevant amount is between about 275 to about 775
ng/ml, or between about 300 to about 650 ng/ml, or between about
400 to about 600 ng/ml, or at about 500 ng/ml in the blood or blood
component. For rivaroxaban, a therapeutically relevant amount is
between about 40 to about 350 ng/ml, or between about 55 to about
250 ng/ml, or between about 70 to about 150 ng/ml, or at about 89
ng/ml in the blood or blood component. For dabigatran, a
therapeutically relevant amount is between about 100 to about 350
ng/ml, or between about 150 to about 300 ng/ml, or between about
175 to about 250 ng/ml, or at about 200 ng/ml in the blood or blood
component.
[0136] In another aspect, the invention provides a method for
classifying an anticoagulant at a therapeutically relevant amount
or higher than a therapeutically relevant amount in a blood
component from a patient, the method comprising: (a) identifying
the presence of an anticoagulant in the blood component, comprising
the steps of (i) subjecting a control blood component sample (known
not to contain anticoagulant) to a clotting assay in the presence
of a Factor Xa reagent to obtain a control clotting measurement;
and (ii) subjecting a blood component sample from the same donor
(unknown regarding presence of anticoagulant) to the clotting assay
in presence of the Factor Xa reagent to obtain the clotting
measurement of the second blood component, wherein the clotting
measurement of the second sample that is greater than the clotting
measurement of the first sample indicates the presence of the
anticoagulant at a therapeutic level in the blood component and
wherein the clotting measurement from the second sample that is
less than or equal to the clotting measurement of the first sample
indicates the absence of the anticoagulant at a therapeutically
relevant amount in the blood component and (b) classifying the
anticoagulant in the blood component, comprising the steps of: (i)
subjecting a second portion of the blood component (known to
contain anticoagulant according to the steps described in (a) to a
clotting assay in the presence of an ecarin reagent to obtain the
clotting measurement of the second portion; and (ii) subjecting a
second portion of the blood (known not to contain anticoagulant) to
the clotting assay in the presence of the ecarin reagent to obtain
the clotting measurement of this portion, wherein the clotting
measurement of the portion (known to contain anticoagulant) that is
greater than the clotting measurement of the portion (known not to
contain anticoagulant) identifies the anticoagulant as a direct
thrombin inhibitor (DTI) and wherein the clotting measurement of
the portion (known to contain anticoagulant) that is less than or
equal to the clotting measurement of the portion (known not to
contain anticoagulant) identifies the anticoagulant as an
anti-Factor Xa anticoagulant.
[0137] In yet another aspect, the invention provides a method for
classifying an anticoagulant at a therapeutically relevant amount
or higher than a therapeutically relevant amount in a patient
suspected of having an anticoagulant, the method comprising: (a)
identifying the presence of an anticoagulant in a blood component
from the patient, comprising the steps of: (i) subjecting a control
sample of a control blood component, the control blood component
known not to contain the anticoagulant, to a clotting assay in the
presence of a Factor Xa reagent to obtain a Factor Xa clotting
measurement of the control sample; and (ii) subjecting a patient
sample of a blood component from the patient to the clotting assay
in the presence of the Factor Xa reagent to obtain the Factor Xa
clotting measurement of the patient sample, wherein the Factor Xa
clotting measurement of the patient sample greater than the Factor
Xa clotting measurement of the control sample indicates the
presence of the anticoagulant at a therapeutically relevant amount
in the patient and wherein the Factor Xa clotting measurement of
the second sample less than or equal to the Factor Xa clotting
measurement of the control sample indicates the absence of the
anticoagulant at a therapeutically relevant amount in the patient;
and (b) classifying the anticoagulant in the patient, comprising
the steps of: (i) subjecting a second control sample of the control
blood component to the clotting assay in the presence of an ecarin
reagent to obtain the ecarin clotting measurement of the second
control sample; and (ii) subjecting a second patient sample of the
blood component from the patient to the clotting assay in the
presence of the ecarin reagent to obtain the ecarin clotting
measurement of the second patient sample, wherein the ecarin
clotting measurement of the second patient sample greater than the
ecarin clotting measurement of the second control sample identifies
the anticoagulant as a direct thrombin inhibitor (DTI) and wherein
the ecarin clotting measurement of the second patient sample that
is less than or equal to the ecarin clotting measurement of the
second control sample identifies the anticoagulant as an
anti-Factor Xa reagent.
[0138] In another aspect, the invention provides a method for
classifying an anticoagulant at a therapeutically relevant amount
or higher than a therapeutically relevant amount in a patient
suspected of having an anticoagulant, the method comprising: (a)
subjecting a first patient sample of a blood component from the
patient to a clotting assay in the presence of a Factor Xa reagent
to obtain a Factor Xa clotting measurement of the patient blood
component, wherein the Factor Xa clotting measurement of the
patient blood component greater than a Factor Xa clotting
measurement of a control blood sample of a control blood component
known to lack the anticoagulant identifies the presence of the
anticoagulant at a therapeutically relevant amount in the patient;
and (b) subjecting a second patient sample of the blood component
from the patient to the clotting assay in the presence of an ecarin
reagent to obtain the ecarin clotting measurement of the second
patient sample; wherein the ecarin clotting measurement of the
second patient sample greater than an ecarin clotting measurement
of a control sample of the control blood component identifies the
anticoagulant as a direct thrombin inhibitor (DTI) and wherein the
ecarin clotting measurement of the second patient sample less than
or equal to the ecarin clotting measurement of the control sample
identifies the anticoagulant as an anti-Factor Xa reagent.
[0139] In another aspect, the invention provides a method for
identifying and classifying an anticoagulant at a therapeutically
relevant amount or higher in a patient suspected of having an
anticoagulant (or known to have an anticoagulant), the method
comprising subjecting a first patient sample of a blood component
from the patient to a clotting assay in the presence of an ecarin
reagent to obtain ecarin clotting measurement of the patient blood
component, wherein the ecarin clotting measurement of the patient
blood component greater than an ecarin clotting measurement of a
control blood sample of a control blood component known to lack the
anticoagulant identifies the presence of the anticoagulant at a
therapeutically relevant amount in the patient and classifies it as
a DTI. In some embodiments, the method further includes subjecting
a second patient sample of the blood component from a patient known
or suspected to contain anticoagulant to the clotting assay in the
presence of a FXa reagent to obtain the FXa clotting measurement of
the second patient sample; wherein the FXa clotting measurement of
the second patient sample greater than the FXa clotting measurement
of a control sample of the control blood component known to lack
the anticoagulant identifies the presence of an anticoagulant at a
therapeutically relevant amount and classifies it as a FXa
inhibitor and wherein the FXa clotting measurement of the second
patient sample less than or equal to the FXa clotting measurement
of the control sample indicates lack of any anticoagulant.
[0140] In some embodiments, the Factor Xa clotting measurement of
the patient sample that is at least 1.25 times greater than the
Factor Xa clotting measurement of the control sample identifies the
presence of the anticoagulant at a therapeutically relevant amount
in the patient. In some embodiments, the Factor Xa clotting
measurement of the patient sample that is at least 1.5 times, or at
least 1.75 times, or at least 2.0 times, or at least 2.25 times
greater than the Factor Xa clotting measurement of the control
sample identifies the presence of the anticoagulant at a
therapeutically relevant amount in the patient.
[0141] In some embodiments, the ecarin clotting measurement of the
patient sample that is at least 1.25 times greater than the ecarin
clotting measurement of the control sample identifies the
anticoagulant as a direct thrombin inhibitor (DTI). In some
embodiments, the ecarin clotting measurement of the patient sample
that is at least 1.5 times, or at least 1.75 times, or at least 2.0
times, or at least 2.25 times greater than the ecarin clotting
measurement of the control sample identifies the anticoagulant as a
direct thrombin inhibitor.
[0142] By "ecarin reagent" is meant a molecule that activates a
prothrombin zymogen (precursor of active thrombin) and produces an
activated form with thrombin-like enzymatic activity. In some
embodiments, an ecarin reagent also includes enzymes similar to
ecarin. Some non-limiting ecarin reagents are shown in FIG. 5. In
some embodiments, the ecarin reagent activates the prothrombin
zymogen (precursor of active thrombin) and is derived from the
venom of the saw-scaled viper, Echis carinatus. In some
embodiments, the ecarin reagent is textarin.
[0143] FIG. 6 schematically diagrams the decision tree involved in
this non-limiting aspect of the invention. As shown in FIG. 6, step
(a) of the above-described method may be referred to as the
"detection" step. Using a clotting assay in the presence of the
Factor Xa reagent, if the clotting measurement (the "R-time" in
FIG. 6) of the tested blood component is within the normal range
(where the normal range is based on a blood component from a donor
known not to be taking an anticoagulant), the patient from whom the
tested blood component was obtained is identified as not being on
an anticoagulant (i.e., the patient was not being administered an
anticoagulant prior to the test blood component being obtained).
If, however, the clotting measurement (the "R-time" in FIG. 6) of
the tested blood component is not within the normal range (e.g.,
has a R-time longer than the normal range), then patient from whom
the tested blood component was obtained is identified as being on
an anticoagulant. Step (b) of the above-described method may be
referred to as the "classification" step (see FIG. 6). Once the
test blood component is identified as being obtained from a patient
on an anticoagulant (i.e., a patient being administered an
anticoagulant), using an ecarin reagent in a clotting assay, the
anticoagulant can be identified as being either a DTI or an
anti-Factor Xa anticoagulant depending upon whether or not the
clotting measurement is within the ecarin normal range. As shown in
FIG. 6, if the R time in a clotting assay using ecarin is within
the normal range (i.e., the range of R times in the presence of
ecarin of blood component from a healthy donors known not to be on
an anticoagulant), then the anticoagulant in the patient is
identified as being an Factor Xa inhibitor. However, if the R time
in a clotting assay using ecarin is longer than (i.e., outside of)
the normal range (i.e., the range of R times in the presence of
ecarin of blood component from donors known not to be on an
anticoagulant), then the anticoagulant in the patient is identified
as being a DTI inhibitor.
[0144] In some embodiments, once the patient has been identified as
patient being administered an anticoagulant, if desired, the
patient can be treated with a reversal agent (e.g., in a
therapeutically relevant amount). For example, if the patient is
identified as having a dabigatran anticoagulant (a DTI
anticoagulant), a non-limiting reversal agent that can be
administered to the patient to reverse the anticoagulant effect of
the dabigatran is Idarucizumab (Boehringer Ingelheim). Similarly,
if the patient is identified as having a Factor Xa inhibitor
anticoagulant, a non-limiting reversal agent that can be
administered to the patient to reverse the anticoagulant effect of
the Factor Xa inhibitor is andexanet alfa (Portola
Pharmaceuticals). Another non-limiting reversal agent that can be
administered to the patient to reverse the anticoagulant effect of
a DTI or a Factor Xa inhibitor anticoagulant is prothrombin complex
concentrates (PCC) (for example, the PCC-4 factor sold under the
name KCENTRA.RTM. (registration owned by CSL Behring GmbH),
OCTAPLEX.RTM. (registration owned by Octapharma AG
AKTIENGESELLSCHAFT), and BERIPLEX.RTM..
[0145] It should be noted that FIG. 6 is merely one example of the
disclosed methods. In some embodiments, the classification steps
and detection steps occur simultaneously. For example, if the
clotting assay is an assay performed using the method and apparatus
where multiple clotting assays are performed simultaneously (using,
for example, the TEG method and apparatus disclosed in U.S. Pat.
No. 7,261,861), one can easily envision the scenario where, if a
single cassette contains four channels, the four channels may
contain (a) control blood component from a donor known not to be
taking an anticoagulant in the presence of a Factor Xa reagent, (b)
test blood component in the presence of a Factor Xa reagent, (c)
control blood component in the presence of an ecarin reagent, and
(d) test blood component in the presence of an ecarin reagent.
Where the normal ranges of the control blood component is
pre-determined, the four channels may be, for example, (a) test
blood without Factor Xa reagent, (b) test blood with Factor Xa
reagent, (c) test blood without ecarin reagent, and (d) test blood
with ecarin reagent. Of course, the routinely skilled practitioner
can utilize any clotting assay to determine the information needed
to determine if the patient is taking an anticoagulant and, if so,
whether that anticoagulant is a DTI or an antiFactor Xa
anticoagulant.
[0146] The following examples are provided which are meant to
illustrate but not limit the various embodiments of the invention
described herein.
Example 1
[0147] The TEG clotting assay was used to determine if low, normal,
and high doses of dabigatran, rivaroxaban, and apixaban could be
detected in blood spiked with these compounds. Three oral
anticoagulants (OAC), namely dabigatran, rivaroxaban, and apixaban,
were spiked into blood obtained from 14 healthy volunteer human
donors. For each OAC tested, citrated blood from three donors was
spiked with three different concentrations of the active drug
(i.e., the compound). The spiked blood samples and control samples
spiked with diluent were tested with the TEG.RTM. 5000
Thrombelastograph.RTM. Hemostasis Analyzer (Haemonetics
Corporation, Braintree, Mass., USA) using the Kaolin and
RapidTEG.RTM. reagents (Haemonetics). Each sample was run in
triplicate. All samples were tested with and without ecarin
(purchased from Enzyme Research Laboratories, South Bend, Ind.).
This study was IRB approved and all donors were over 18 and signed
informed consent forms.
[0148] Sample Preparation
[0149] Blood was drawn using standard venipuncture technique and a
Becton Dickinson Vacutainer Push Button Collection set with a
21-gauge needle. Blood was spiked and tested within two hours of
being drawn.
[0150] Dabigatran stock was prepared from the active dabigatran
moiety (Alsachim, France) by dissolution in 0.1M HCl and further
dilution in 1:1 DMSO:H20. The final stock used to spike the blood
had a concentration of 20 ng/pL in 0.1M HCl/DMSO/H20. Tubes of
citrated blood were spiked with this dabigatran stock to create
final concentrations of 500, 200, and 50 ng/mL of citrated whole
blood.
[0151] Dabigatran is approved for prevention of venous
thromboembolism (VTE) following elective knee or hip replacement
(220 mg/day for patients without renal impairment and 150 mg/day
for patients with moderate renal impairment and for prevention of
stroke in patients with renal impairment and atrial fibrillation
(AF) in the US (at a reduced 75 mg/day dose). (Boehringer Ingelheim
International, dabigatran etexilate product and prescribing
information) A 150 mg oral dose of dabigatran has a maximum plasma
concentration (C.) of 110 ng/mL (see Stangier et al., Clin.
Pharmacokinet. 47: 285-295, 2008; Mueck, W., et al., Thrombosis
Journal 11:10 (2013).
[0152] Rivaroxaban stock was prepared by agitation of a 20 mg
Xarelto tablet (Jannsen, Titusville, N.J.) in a 1:1 DMSO: H.sub.2O
solution, which was diluted to a final concentration of 20 ng/uL
rivaroxaban in 1:1 DMSO:H20. Tubes of citrated blood were spiked
with this rivaroxaban stock to create final concentrations of 500,
89, and 22 ng/mL in citrated whole blood. Rivaroxaban is approved
for the prevention of stroke and systemic embolism in adults with
non-valvular AF (20 mg/day; EU and US), for the treatment of deep
venous thrombosis (DVT) and pulmonary embolism (PE) and for the
prevention of recurrent DVT and PE in adult patients (15 mg twice
daily for 3 weeks followed by 20 mg/day; EU and US) (see Wong et
al., J. Thromb. Haemost. 6: 820-829, 2008; Janssen Pharmaceuticals,
rivaroxaban prescribing information. An oral dose of 10 mg of
rivaroxaban has a C.sub.max of 141 ng/mL (see Mueck, W., et al.,
Thrombosis Journal 11:10, 2013; Kubitza D. et al., Clin Pharmacol
Ther. 78:412-421, 2005). Apixaban stock was prepared in a similar
manner from a 2.5 mg Eliquis tablet (Bristol-Myers Squibb, New
York, N.Y.), with final concentrations of 1000, 500, and 250 ng/mL
in whole blood. Apixaban is approved for prevention of VTE in
elective hip or knee replacement surgery (2.5 mg BID) and for
prevention of stroke and systemic embolism in patients with
non-valvular AF (5 mg BID) (see Bristol-Myers S P, EEIG. apixaban
summary of product characteristics). An oral dose of 20 mg of
apixaban has a Cmax of 460 ng/mL mL (see Mueck, W., et al.,
Thrombosis Journal 11:10, 2013; Raghavan N et al., Drug Metab
Dispos. 37:74-81, 2009). Control samples, prepared for each tested
drug, included a solvent control containing only citrated blood and
the diluent used to dilute the drug stock, and an unadulterated
citrate blood tube.
[0153] Thromboelastography
[0154] Testing was performed on TEG-5000 analyzers (Haemonetics
Corp., Braintree, Mass., USA) using Kaolin vials, 0.2M CaCl2,
RapidTEG (rTEG) vials, diluent water, and disposable clear cups and
pins provided by the manufacturer (Haemonetics, Braintree, Mass.),
and ecarin (Enzyme Research Laboratories, South Bend, Ind.). All
testing was performed in triplicate at each dose, and allowed to
continue until the MA (maximum amplitude) parameter defined. The
various components of the TEG tracing are depicted in FIG. 3A. The
Kaolin test generates an R parameter, which is measured in minutes,
and is the time elapsed from the initiation of the test until the
point where the onset of clotting provides enough resistance to
produce a 2 mm amplitude reading on the TEG tracing. This parameter
represents the initiation phase of coagulation related to the
function of enzymatic clotting factors. The R parameter has a
normal range of 5 to 10 min for kaolin. A prolonged R time
indicates slower clot formation. K is a measurement of the time
interval from the split point to the point where fibrin
cross-linking provides enough clot resistance to produce a 20 mm
amplitude reading. The .alpha. angle is the angle formed by the
slope of a tangent line traced from the R to the K time and a
central line measured in degrees. K time and the a angle denote the
rate at which the clot strengthens, and is representative of
thrombin's cleaving of the available fibrinogen into fibrin. The MA
indicates the point at which clot strength reaches its maximum
amplitude, measured in millimeters on the TEG tracing, and reflects
the end result of maximal platelet-fibrin interaction via the
GPIIb-IIIa receptors (see Khurana S et al., J Lab Clin Med.
130:401-411, 1997).
[0155] The RapidTEG (rTEG) test incorporated both tissue factor and
kaolin to generate the conventional kaolin parameters as well as
the TEG-ACT parameter, which is measured in seconds. The TEG-ACT is
equivalent to the Activated Clotting Time (see Chavez J. J. et al.,
Anesth. Analg. 99:1290-1294, 2004) and has a normal range of 86 to
118 seconds. A prolonged TEG-ACT time indicates slower clot
formation. In addition, velocity curves derived from the above
mentioned kaolin and rTEG tests were plotted using TEG software.
These curves represent the speed of clot propagation (MRTG, Maximum
Rate of Thrombus Generation; and TMRTG, Time to Maximum Rate of
Thrombus Generation) (see FIG. 3B).
[0156] For the kaolin test, 1 mL of citrated blood sample was mixed
with kaolin and 340 microliters (.mu.L) of this blood was added to
a TEG cup containing 20 .mu.L of 0.2M CaCl2 for recalcification.
The kaolin with ecarin test was performed in a similar fashion,
using 20 .mu.L of an ecarin/CaCl2 solution (0.16 M CaCl2; 19 EU/mL
ecarin).
[0157] For the RapidTEG (rTEG) test, the reagent was reconstituted
with 20 .mu.L diluent water, and allowed to stand for 5 minutes per
manufacturer instructions. Ten .mu.L of this reconstituted reagent
was added to the TEG cup with 20 .mu.l 0.2M CaCl2 for
recalcification.
[0158] 340 .mu.L of the citrated blood sample was added to the cup
with these two reagents, and the contents of the cup were mixed 3
times by drawing the contents of the cup up into the pipette and
redispensing it into the cup. The test was started immediately
after mixing and allowed to run until the MA parameter had defined.
The rTEG with ecarin test was performed as the rTEG test above,
using 20 .mu.L of an ecarin/CaCl2 solution (0.16M CaCl2; 19 EU/mL
ecarin).
[0159] Statistical Analysis
[0160] Statistical analyses were done using a two-tailed Student's
t test. For all analyses, a P value of <0.05 was deemed
statistically significant.
[0161] Results:
[0162] The results of the Kaolin test are shown in Table 1 and in
FIGS. 7A, 7B, and 7C.
[0163] FIG. 17 shows the TEG Kaolin test coagulation parameters'
sensitivity in healthy donor spiked samples with different doses of
apixaban, rivaroxaban and dabigatran in the presence or absence of
ecarin. In FIG. 17, R--Reaction Time; MRTG--Maximum Rate to
Thrombus Generation; TMRTG--Time to Maximum Rate of Thrombus
Generation. Statistically significant between: --higher dose and
medium dose; --higher dose and lower dose; .diamond.--medium dose
and lower dose; *--the control. .sctn.--paired sample with or
without Ecarin. SDR--standard error of the mean of three
independent experiments measured in triplicate. 1 symbol p<0.05;
2 symbols p<0.01; 3 symbols p<0.001.
[0164] The R, K, .alpha., and MRTG parameters in the kaolin test
only achieved statistical significance for the higher
concentrations of rivaroxaban (FIG. 17, FIG. 7A) but were able to
detect the presence of all tested concentrations of Apixaban
(P<0.045) (FIG. 17, FIG. 7B) and Dabigatran (P<0.038) (FIG.
17, FIG. 7C). In addition, for all drugs the TMRTG parameter was
statistically different between the control group and all tested
concentrations. Furthermore, the R, .alpha., and TMRTG parameters
for the dabigatran samples were significantly different between all
concentrations indicating an appropriate dose response (FIG. 17,
FIG. 7C). Finally, the MA values from the kaolin test for
rivaroxaban and dabigatran did not change with the addition of the
studied NOAC when compared to the control, illustrating the lack of
effect of these agents on platelet/fibrin contribution to clot
strength. However, apixaban tracing at a concentration of 250 ng/mL
demonstrated that the MA was significantly different from the
control group (P<0.001), however was still within normal range
(data not shown).
[0165] FIG. 18 shows the Rapid TEG test coagulation parameters'
sensitivity in healthy donor spiked samples with different doses of
apixaban, rivaroxaban and dabigatran in the presence or absence of
ecarin. Statistically significant between: .sctn.--paired sample
with or without Ecarin. SDR--standard error of the mean of three
independent experiments measured in triplicate. 1 symbol p<0.05;
2 symbols p<0.01; 3 symbols p<0.001
[0166] The TEG ACT parameter for all tested drugs in the RapidTEG
test was significantly different between the control group and all
tested concentrations of rivaroxaban, apixaban, and dabigatran. The
results of this test are shown in FIG. 18 and in FIGS. 7D, 7E, and
7F) with the exception of the rivaroxaban concentration of 22 ng/mL
(P=0.576) (see FIG. 18, FIG. 7D).
[0167] Furthermore, the TEG ACT parameter was able to distinguish
between concentrations of rivaroxaban (See the results in FIG. 18
and in FIG. 7D) and dabigatran (FIG. 18, FIG. 7F) indicating a good
dose response curve. The K, .alpha. and MRTG parameters for both
apixaban and rivaroxaban from the RapidTEG test did not show any
statistical difference between the control or between studied
concentrations. However, the K parameter for the dabigatran group
was statistically different from control for the lower tested
concentrations (200 ng/mL, P=0.003; 50 ng/mL, P=0.003) but not for
the concentration of 500 ng/mL (P=0.438) and the .alpha. parameter
from the dabigatran group was statistically different from control
for the concentration of 500 ng/mL (P<0.01) and 50 ng/mL
(P<0.001) but not for the concentration of 200 ng/mL (P=0.383).
In addition, both K and .alpha. parameters were able to
differentiate between the highest dabigatran concentration from the
other concentrations (500 ng/mL vs 200 ng/mL, P=0.002; 500 ng/mL vs
50 ng/mL, P<0.001). Furthermore, the MRTG parameter was
sensitive to the two lowest concentrations of dabigatran (500
ng/mL, P=0.061; 200 ng/mL, P=0.0015; 50 ng/mL, P<0.001). The
TMRTG parameter from the RapidTEG test is sensitive to the presence
of both rivaroxaban and dabigatran but not apixaban. Furthermore,
the TMRTG parameter is able to differentiate between concentrations
of dabigatran (500 ng/mL vs 200 ng/mL, P<0.001; 200 ng/mL vs 50
ng/mL, P<0.001). Finally, the MA values of the RapidTEG test for
rivaroxaban and apixaban did not change with the addition of the
studied drug concentrations when compared to control and only the
MA values of the dabigatran 500 ng/mL concentration were
significantly different from the control group (P<0.01), however
was still within normal range (data not shown).
[0168] Ecarin is derived from the venom of the saw-scaled viper,
Echis carinatus. Ecarin activates prothrombin (the precursor of
thrombin--see FIG. 5). This activation of prothrombin by ecarin
produces meizothrombin, a prothrombin-thrombin intermediate which
has a low level of procoagulant enzymatic activity.
[0169] As can be seen in FIG. 17 and also in FIGS. 8A-8C, the
addition of ecarin to the kaolin test caused a significant decrease
of the R, K and TMRTG values for both treated and control groups
(apixaban P<0.001; rivaroxaban P<0.003; dabigatran
P<0.004) and a significant increase of the a and MRTG values for
both treated and control groups (apixaban P<0.001; rivaroxaban
P<0.008; dabigatran P<0.008) (see FIG. 17). Furthermore, the
addition of ecarin to the kaolin test in the presence of
anti-Factor Xa drugs (also simply called anti-Xa drugs) severely
decreases the R values to the hypercoagulable range (<5 min)
with no statistical difference from the control with the exception
of the higher studied dosages (Apixaban: 1000 ng/mL, P=0.026; 500
ng/mL, P=0.756; 250 ng/mL, P=0.054), Rivaroxaban (500 ng/mL,
P=0.0017; 89 ng/mL, P=0.079; 22 ng/mL, P=0.898) while in the
presence of dabigatran there is only a dose related decrease of the
R (FIG. 17, see also FIG. 8C). The addition of ecarin to the kaolin
test did not change the MA values of the samples for dabigatran or
rivaroxaban relative to samples run without ecarin, but in the
presence of apixaban the MA values were statistically different
(1000 ng/mL, P=0.020; 500 ng/mL, P=0.009; 250 ng/mL, P<0.001)
from samples run without ecarin, however was still within normal
range (data not shown).
[0170] As can be seen in FIG. 18, the addition of ecarin to the
RapidTEG test significantly decreases the TEG-ACT times for both
anti-Xa and DTI drugs (apixaban, P<0.001; rivaroxaban,
P.ltoreq.0.001; dabigatran, P<0.001) as well as the TMRTG times
in the presence of both rivaroxaban and dabigatran (rivaroxaban,
P<0.001; dabigatran, P<0.001) increasing the hypercoagulable
status (see FIG. 18). On the other hand, the RapidTEG a angle did
not change for any of the studied concentrations when ecarin was
added in the presence of rivaroxaban or apixaban. However, for the
lowest concentrations of dabigatran there was a decrease of the
angle value (200 ng/mL, P=0.047; 50 ng/mL, P=0.016). The RapidTEG K
values significantly decreased for the highest and lowest
concentrations of apixaban (1000 ng/mL, P=0.017; 250 ng/mL, P=0.17)
and the middle concentration of rivaroxaban (89 ng/mL, P=0.044) but
increased in the middle concentration of dabigatran (200 ng/mL,
P=0.004) when ecarin was added. Finally, the addition of ecarin to
the RapidTEG test only significantly decreased the MA value of the
200 ng/mL concentration of dabigatran (p<0.05), however this was
still within normal range (data not shown).
Example 2
[0171] Healthy human volunteers (who are not being administered any
anticoagulant), donated blood. Following donation, the blood
collected from the donors (which was added to citrate to prevent
clotting) was divided into portions, and portions of the blood were
spiked with either nothing, dabigatran, or rivaroxaban. The
portions were then assayed using thromboelastography, including
assays performed in the presence of ecarin.
[0172] For these studies, blood from three healthy human volunteers
was collected. For each dose, three thromboelastography assays were
run, and the reported number indicates the averaged result. The
direct thrombin inhibitor used was dabigatran. The stock of
dabigatran was 1 ml DMSO+750 ul saline (0.9% NaCl)+1 mg active
dabigatran moiety. The anti-Factor Xa drug used was rivaroxaban.
The stock of rivaroxaban was a 20 mg tablet dissolved in 1 ml
saline plus 9 ml DMSO.
[0173] Three dosages of each of dabigatran and rivaroxaban were
used, namely a low dose, a normal dose, and a high dose. For
dabigatran, the low dose was 50 ng/ml, the normal dose was 200
ng/ml, and the high dose was 500 ng/ml. For rivaroxaban, the low
dose was 22 ng/ml, the normal dose was 89 ng/ml, and the high dose
was 500 ng/ml.
[0174] FIG. 9A shows the kaolin TEG tracing from blood components
spiked with 50 ng/ml dabigatran (red line), 200 ng/ml dabiggatran
(green line) and 500 ng/ml dabigatran (black line). As can be seen
in FIG. 9A, the presence of dabigatran increases the R value of the
tested blood components in a dose dependent manner. FIG. 9B is a
plot of the R values of the spiked blood components compared to the
R value of control blood component (i.e., taken from a volunteer
known to not be taking an anticoagulant and whose blood was not
spiked with an anticoagulant). Note that FIG. 9B is an enlargened
view of FIG. 7C. As FIG. 9B shows, the reference range of the R
value for the control blood was between about 5 minutes to about 10
minutes, where the average R value was about 7.9 minutes. The
lowest dosage of dabigatran (50 ng/ml) resulted in an R value that
was outside of the normal (i.e., control) reference range.
Interestingly, when the blood components are treated with ecarin,
the R value of the dabigatran-spiked blood is shortened and
approaches the normal reference range (see FIG. 9C). Note that FIG.
9C is an enlargened view of FIG. 8C.
[0175] FIG. 10 shows that the presence of dabigatran decreases the
rate and time to achieve thrombus generation. These results are
reflected in Table 1 below, measuring the MRTG (mm/min) and TMRTG
(min) of the spiked blood. (See FIG. 3B for a description of these
parameters).
TABLE-US-00001 TABLE 1 Dabigatran (ng/ml) MRTG (mm/min) TMRTG (min)
500 7 22 200 12 18 50 13 13
[0176] FIG. 11A shows the kaolin TEG tracing from blood components
spiked with 22 ng/ml rivaroxaban (red line), 89 ng/ml rivaroxaban
(green line) and 500 ng/ml rivaroxaban (black line). As can be seen
in FIG. 11A, the presence of the high dosage of rivaroxaban
increases the R value of the tested blood components in a dose
dependent manner. FIG. 11B is a plot of the R values of the
rivaroxaban-spiked blood components compared to the R value of
control blood component (i.e., taken from a volunteer known to not
be taking an anticoagulant and whose blood was not spiked with an
anticoagulant). As FIG. 11B shows, the reference range of the R
value for the control blood was between about 5 minutes to about 10
minutes, where the control R value was about 8.2 minutes. The
highest dosage of rivaroxaban (500 ng/ml) resulted in an R value
that was outside of the normal (i.e., control) reference range,
while the lower two doses were within the control reference range.
Interestingly, when the blood components are treated with ecarin,
the R value of the rivaroxaban-spiked blood is dramatically
shortened and all three dosage levels fall outside of the control
reference range (see FIG. 11C).
Example 3
[0177] This method was performed to determine if TEG could be used
to identify the presence of an anticoagulant in a blood
component.
[0178] Blood from healthy volunteers known not to be taking any
oral anticoagulant and without any other significant health issues
are analyzed using the FXa or the Ecarin reagent in a TEG.RTM.
hemostasis analyzer.
[0179] The FXa reagent (i.e., the Factor Xa reagent) is used to
detect the presence (or absence) of an anticoagulant in the blood
component being tested. In absence of any oral anticoagulant, the
FXa reagent will accelerate the clotting process and result in a
very short R-time. In presence of direct thrombin inhibitors and/or
anti-Factor Xa anticoagulant(s), the process is slowed down and
will cause elongation of the R-time. This difference in R-time is
utilized to detect the presence of the oral anticoagulant.
[0180] If before and after blood sample is available then the
significant difference in R-time will be used to detect presence.
However in an actual patient population it is assumed that before
(drug) samples will not be readily available. In such a case a
reference range generated from a sample healthy population will be
used for detection. If the R-time from a blood sample is within
normal range then it is concluded that no oral anticoagulation is
present. If the R-time is out of range then the result will
indicate the presence of an oral-anticoagulant in that
person/donor.
[0181] The ecarin reagent is used to classify the type of
anticoagulant. The R-time from the Ecarin assay is used to
differentiate between the two available classes (Direct thrombin
inhibitors and anti-Xa inhibitors). This ecarin reagent may be used
in combination with the results from the FXa reagent to make a
decision. A reference range will be generated from a sample healthy
volunteer population (not on any drug) for this reagent.
[0182] According to this non-limiting embodiment of the invention,
if the FXa reagent indicates presence of an oral anticoagulant then
the Ecarin reagent R-time will be analyzed against the Ecarin
reference range for classification purposes. If the R-time is
within Ecarin normal range then the anticoagulant present will be
an anti-Xa. An R-time that is out of (Ecarin normal) range will
indicate the presence of a direct thrombin inhibitor.
[0183] The R-time generated from multiple donors was used to
construct a reference range. Blood was spiked with either direct
thrombin inhibitors or anti-Xa(s) at different concentrations and
analyzed using the FXa reagent or the Ecarin reagent. The drug
concentrations chosen represent the published therapeutic ranges
for these two classes of drugs. This assay was repeated in multiple
donors to account for possible donor variations.
[0184] FIG. 12 shows the reference ranges for R time (in minutes)
in the presence of the FXa reagent. The dotted line in FIG. 12
represents the normal reference range for the FXa reagent (i.e., R
time of a blood component known to lack an anticoagulant in the
presence of the FXa reagent. In absence of any oral anticoagulant
the R-time will fall within this range (i.e., will be below
approximately 1.8 minutes). In FIG. 12, AP and RV refer to Apixaban
and Rivaroxaban respectively and is representative of two
non-limiting anti-Xa drugs. A non-limiting direct thrombin
inhibitor (DTI) is represented by DB or Dabigatran in FIG. 12. In
vitro spiking of the blood from the same donor (who is known not to
be administered with an anticoagulant) with either one of the
anti-Xa drugs (i.e., AP or RV) or with the direct thrombin
inhibitor (i.e., DB) resulted in a dose dependent elongation of the
R-time compared to the reference range.
[0185] In some embodiments, the reference range of control blood
(i.e., taken from a donor to whom an anticoagulant has not been
administered) in the presence of FXa is between about 1.0 min to
about 2.0 minutes. In some embodiments, the reference range of
control blood (i.e., taken from a donor to whom an anticoagulant
has not been administered) in the presence of FXa is between about
1.5 min to about 1.9 minutes. In some embodiments, the reference
range of control blood (i.e., taken from a donor to whom an
anticoagulant has not been administered) in the presence of FXa is
about 1.8 minutes.
[0186] As shown in FIG. 12, a concentration of 100 ng/ml of drug in
the blood component (where this amount is a therapeutically
relevant amount of AP, RV, and DB) raises the R time by at least
1.25 times, or at least 1.5 times, or at least 1.75 times, or at
least 2 times above the reference range of approximately 1.8
minutes.
[0187] Using the ecarin reagent, the presence of a direct thrombin
inhibitor anticoagulant can be detected in a blood component using
thromboelastography. FIG. 13 shows the R time measurements on blood
components from four donors, whose blood (after being collected
from the donor) was spiked with dabagatran at the indicated amounts
and measured using thromboelastography in the presence of the
ecarin reagent. The R time (in minutes) was compared to the R time
obtained from donor blood with no oral anticoagulant (who is known
not to be administered with an anticoagulant) treated with the
ecarin reagent and measured using thromboelastography.
[0188] In some embodiments, the reference range of control blood
(i.e., taken from a donor to whom an anticoagulant has not been
administered) in the presence of the ecarin reagent is between
about 1.0 minutes to about 3.5 minutes. In some embodiments, the
reference range of control blood (i.e., taken from a donor to whom
an anti-coagulant has not been administered) in the presence of the
ecarin reagent is about 1.5 minutes to about 3.25 minutes. In some
embodiments, the reference range of control blood (i.e., taken from
a donor to whom an anticoagulant has not been administered) in the
presence of the ecarin reagent is between about 1.9 minutes to
about 3 minutes. In some embodiments, the reference range of
control blood (i.e., taken from a donor to whom an anti-coagulant
has not been administered) in the presence of the ecarin reagent is
about 3.1 minutes.
[0189] As FIG. 13 shows, the reference range from a particular
donor can vary. For example, donor 126 has a low untreated R-time
(i.e., when the blood from donor 126 is not spiked with DB).
Spiking the blood of donor 126 with 50 ng/ml DB, and measuring the
R time using thromboelastography in the presence of the ecarin
reagent resulted in an increase in R time that was two times longer
than the untreated R time of unspiked donor blood 126 (i.e.,
untreated or control blood). As shown in FIG. 13, this was true for
the other donors as well as compared to unspiked blood from the
same donors. Furthermore, using an R time of about 3.1 minutes as a
collective reference range, the blood from donors 117, 127, and 146
spiked with 50 ng/ml DB (which is lower than the therapeutically
relevant amount of 110 ng/ml of DB--see Muek et al., supra) in the
presence of ecarin was at least 1.25 times, or at least 1.5 times
greater than the reference range of 3.1 minutes. When looking at
the R times of 150 ng/ml DB (which is a therapeutically relevant
amount of DB), spiked blood from all four donors had an R time (in
the presence of ecarin) that exceeded the collective reference
range of 3.1 minutes by at least 1.25 times, or at least 1.4
times.
[0190] Using the ecarin reagent, the presence of an anti-Factor Xa
anticoagulant drug can be detected in a blood component using
thromboelastography.
[0191] FIG. 14 shows the R time measurements on blood components
from four donors, whose blood (after being collected) was spiked
with rivaroxaban (an anti-Factor Xa drug) at the indicated amounts
and measured using thromboelasography in the presence of the ecarin
reagent. The R time (in minutes) was compared to the R time
obtained from donor blood with no oral anticoagulant (i.e., taken
from a patient who is known not to be administered with an
anticoagulant) treated with the ecarin reagent and measured using
thromboelastography.
[0192] In the assay performed in FIG. 14, the R time (in minutes)
of control blood (i.e., taken from a donor to whom an anticoagulant
has not been administered) in the presence of ecarin is between
about 1 min to about 4.5 minutes (data not shown). In some
embodiments, the reference range of control blood (i.e., taken from
a donor to whom an anticoagulant has not been administered) in the
presence of ecarin is between about 3.9 min to about 4.2 minutes.
In some embodiments, the reference range of control blood (i.e.,
taken from a donor to whom an anticoagulant has not been
administered) in the presence of ecarin is about 4.1 minutes.
[0193] As shown in FIG. 14, a concentration varying from 50-350
ng/ml of the RV drug in the blood component (where this represents
a therapeutically relevant amount of RV) results in an R time that
is within the reference range of approximately 1 to 4.1 minutes.
Note that the reference range of the R time of the control blood
will depend upon the patient from whom the blood was taken. In some
embodiments, the R time of the RV-spiked blood is at least 1.25
times, or at least 1.5 times, or at least 1.75 times, or at least 2
times smaller slower than the upper boundary R time of the control
blood.
[0194] A similar result is found for another anti-Factor Xa drug,
namely apixaban (data not shown).
[0195] These results in FIG. 14 show that ecarin can be used to
classify an anticoagulant as a Factor Xa molecule.
Example 4
[0196] Detection and Classification of an anticoagulant using the
Factor Xa reagent and ecarin.
[0197] Blood from a single human volunteer was analyzed using
thromboelastography with Factor Xa and ecarin before and after the
volunteer was orally administered an anticoagulant.
[0198] For these studies, the normal ranges for the particular
reagent (i.e., Factor Xa or ecarin) were determined using multiple
donors known not to be taking any anticoagulant.
[0199] The human volunteer was known not to be taking (i.e., was
not being administered) an anticoagulant. In this example, blood
was taken from the volunteer before the volunteer was administered
any anticoagulant. This is the "before" blood component sample. The
volunteer was orally administered 20 mg rivaroxaban, an anti-Factor
Xa anticoagulant. Two hours and thirty minutes after oral
administration of the rivaroxaban, blood was drawn again from the
volunteer. This is the "after" blood component sample.
[0200] The "before" and "after" samples were analyzed using
thromboelastography in the presence of Factor Xa or in the presence
of ecarin. When the R time is determined in the presence of Factor
Xa, that time is called the detection time. When the R time is
determined in the presence of ecarin, that time is called the
classification time.
[0201] FIG. 15A shows the results of the "before" analyses (i.e., R
times taken before the donor is administered an anticoagulant
drug). In FIG. 15A, the solid horizontal line at approximately 1.5
minutes (in the detection time) and the solid horizontal line at
approximately 4.1 minutes (in the classification time) indicates
the upper boundary of the reference range for Factor Xa and ecarin,
respectively, as determined from multiple human donors known to not
be taking an anticoagulant (i.e., control donors). As shown in FIG.
15A, in the absence of any anticoagulant drug in the volunteer's
blood (i.e., in the "before" blood samples), the R-time for the
detection reagent (i.e., FXa) falls within normal range (i.e., is
below the horizontal line) of the detection time As the R times was
within the normal range (i.e., within the range of blood from
control donors known not to be taking an anticoagulant), these
findings confirmed the absence of any anticoagulant in the
volunteer's blood. (See also the decision tree in FIG. 6).
[0202] FIG. 15B shows the results of the "before" and "after"
analyses (i.e., "after" analysis is the R times taken after the
donor has been administered an anticoagulant drug) using
thromboelastography in the presence of the FXa reagent. Blood was
drawn 2 hours and 30 minutes after the donor patient was orally
administered 20 mg Rivaroxaban, an anti-Xa drug. As in FIG. 15A,
the horizontal line at approximately 1.5 minutes is the upper
boundary of the reference range from multiple donors known to not
be taking an anticoagulant in the presence of the FXa reagent
(i.e., this is the reference range for the detection time). As
shown in FIG. 15B, the "before" detection time is within (i.e.,
shorter than) the reference range of approximately 1.5 minutes.
However, the "after" detection time (i.e., 2.5 hours after the
donor was orally administered Rivaroxaban) is outside of the
reference range of 1.5 minutes. In fact, the "after" detection time
is approximately 4.4 minutes; accordingly in this study, the
"after" detection time is 2.93 times longer than the reference
range R time of 1.5 minutes. The "after" detection time (the R
time) being outside of the reference range in the detection time
indicates the presence of an anticoagulant (refer to FIG. 5).
[0203] FIG. 15C shows the results of the "before" and "after"
analyses (i.e., "after" analysis is the R times taken after the
donor has been administered an anticoagulant drug) using
thromboelastography in the presence of the ecarin reagent. Blood
was drawn 2 hours and 30 minutes after the donor patient was orally
administered 20 mg Rivaroxaban, an anti-Xa drug. As in FIG. 15A,
the horizontal line at approximately 4.1 minutes is the upper
boundary of the reference range in the presence of the ecarin
reagent (i.e., this is the reference range for the classification
time) as determined from multiple donors known to not be taking an
anticoagulant. As shown in FIG. 15C, because the R results of the
volunteer's "after" blood was within the Ecarin reference range of
control blood (i.e., blood from donors known not to be taking an
anticoagulant), the anticoagulant in the volunteer's blood was
identified as being an anti-Factor Xa reagent and identified as not
being a direct thrombin inhibitor (See also the decision tree in
FIG. 6).
[0204] Thus, in FIGS. 15A-15C, using the FXa reagent and the ecarin
reagent in combination with the thromboeastography clotting assay,
analysis of blood from a volunteer taking (i.e., being
administered) an anticoagulant was able to confirm that (a) the
volunteer was taking an anticoagulant (as evidenced by the
increased R time in the detection time in the presence of Factor Xa
as compared to control blood) and (b) that the anticoagulant the
volunteer was taking was an anti-Factor Xa reagent and was not a
direct thrombin inhibitor reagent (as evidenced by an R-time within
the reference range of control blood in the presence of
Ecarin).
Example 5
[0205] New oral anticoagulants (OAC) do not require routine
monitoring however measuring drug levels may be needed in clinical
situations such as trauma and emergent surgery. Clinical or
laboratory whole blood assays are not established for dabigatran.
Thrombelastography (TEG.RTM.) has shown promising results in
detecting and following dabigatran through changes in hemostatic
parameters.
[0206] This study evaluated the effect of dabigatran and its
specific reversal agent with the next generation fully automated
TEG.RTM.6S system.
[0207] The TEG.RTM.6S system (Haemonetics Corp., Braintree, Mass.)
is based on viscoelasticity measurements using resonance frequency
and disposable multi-channel microfluidic cartridges. Blood and
plasma from healthy volunteers were spiked with dabigatran in the
therapeutic range (in increasing concentrations) and tested with an
OAC cartridge with an ecarin based, direct thrombin inhibitor (DTI)
channel. This channel was also evaluated with porcine plasma from
an experimental trauma model (n=6) where dabigatran levels were
measured with dilute TT (Hemoclot.RTM.). TEG 6s R-time (reaction
time) was correlated to the drug levels.
[0208] Results: In vitro: R-time was highly correlated with
dabigatran levels in whole blood (r.sup.2>0.95). Significant
correlation was also observed in plasma (r.sup.2>0.6)
[0209] Trauma model: R-time was significantly correlated with
dabigatran plasma levels (r.sup.2=0.7). R-time at baseline was
(3.4.+-.0.6 min, n=4). Following dabigatran administration,
significant elongation of R-time was observed pre and post trauma
with R-times .gtoreq.21.+-.5.9 min. Administration of the reversal
agent idarucizumab returned R-time to baseline (3.6.+-.0.6
min).
[0210] Table 2 summarizes how R time from different channels is
analyzed to detect and classify the anticoagulants.
TABLE-US-00002 TABLE 2 Factor Xa Reagent Ecarin Reagent Result
Result Condition Long R Short R Anti-Factor Xa Long R Long R DTI
Short R Short R Healthy Normal
[0211] The bar graph shown in FIG. 16 shows how in five separate
swine plasma samples, R time is elongated in the presence of a DTI
anticoagulant (red bars); however, upon administration of the
idarucizumab reversal agent, the R time (green bar) returned to
baseline (blue bars). Thus, when a sample is analyzed using the
methods described herein, the identification and classification of
an anticoagulant in the sample allows selected reversal of that
anticoagulant.
[0212] Conclusion: TEG.RTM. 6s has the potential to measure the
effect of dabigatran on the hemostasis system effectively in whole
blood as well as plasma in a clinical setting. This novel
technology is fully automated and can provide clinically relevant
results with whole blood in as little as 5 minutes.
[0213] The embodiments of the invention described above are
intended to be merely exemplary; numerous variations and
modifications will be apparent to those skilled in the art. All
such variations and modifications are intended to be within the
scope of the present invention as defined in any appended
claims.
* * * * *