U.S. patent application number 10/156462 was filed with the patent office on 2003-03-13 for method for predicting the presence of haemostatic dysfunction in a patient sample.
Invention is credited to Downey, Colin, Fischer, Timothy J., Toh, Cheng Hock.
Application Number | 20030049851 10/156462 |
Document ID | / |
Family ID | 23470305 |
Filed Date | 2003-03-13 |
United States Patent
Application |
20030049851 |
Kind Code |
A1 |
Toh, Cheng Hock ; et
al. |
March 13, 2003 |
Method for predicting the presence of haemostatic dysfunction in a
patient sample
Abstract
A method which may be used to determine haemostatic dysfunction
in a patient is carried out by (a) adding a reagent to a test
sample, wherein the test sample includes at least a component of a
blood sample from a patient; and then (b) measuring the formation
of a precipitate due to the reaction of the test sample and the
reagent, over time so as to derive a time-dependent measurement
profile, the reagent forming a precipitate in the test sample
without causing substantial fibrin polymerization.
Inventors: |
Toh, Cheng Hock; (Liverpool,
GB) ; Downey, Colin; (Liverpool, GB) ;
Fischer, Timothy J.; (Oro Valley, AZ) |
Correspondence
Address: |
MYERS BIGEL SIBLEY & SAJOVEC
PO BOX 37428
RALEIGH
NC
27627
US
|
Family ID: |
23470305 |
Appl. No.: |
10/156462 |
Filed: |
May 28, 2002 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10156462 |
May 28, 2002 |
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09372954 |
Aug 12, 1999 |
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6429017 |
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09372954 |
Aug 12, 1999 |
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09244340 |
Feb 4, 1999 |
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09244340 |
Feb 4, 1999 |
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09001647 |
Dec 31, 1997 |
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6321164 |
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09001647 |
Dec 31, 1997 |
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08859773 |
May 21, 1997 |
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6101449 |
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08859773 |
May 21, 1997 |
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08477839 |
Jun 7, 1995 |
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5708591 |
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Current U.S.
Class: |
436/69 ; 422/73;
422/82.09; 435/13; 435/7.1; 436/164; 436/501; 436/73; 436/74;
436/79; 436/84; 436/86; 600/369 |
Current CPC
Class: |
A61P 7/04 20180101; G01N
33/86 20130101; G01N 30/96 20130101; G01N 33/4905 20130101 |
Class at
Publication: |
436/69 ; 436/73;
436/74; 436/79; 436/84; 436/164; 436/501; 436/86; 422/73;
422/82.09; 600/369; 435/7.1; 435/13 |
International
Class: |
G01N 033/86 |
Claims
We claim:
1. A method comprising: a) adding a reagent to a test sample
comprising at least a component of a blood sample from a patient;
b) measuring the formation of a precipitate due to the reaction of
the test sample and the reagent, over time so as to derive a
time-dependent measurement profile, said reagent capable of forming
a precipitate in the test sample without causing substantial fibrin
polymerization.
2. The method according to claim 1, wherein said reagent comprises
a metal ion.
3. The method according to claim 2, wherein said metal ion is a
divalent metal ion.
4. The method according to claim 3, wherein said divalent metal ion
is a metal ion from the transition elements.
5. The method according to claim 2, wherein said metal ion
comprises one or more of calcium, magnesium, manganese, iron or
barium.
6. The method according to claim 1, wherein a clot inhibitor is
provided as part of said reagent or as part of an additional
reagent added to said test sample.
7. The method according to claim 6, wherein said clot inhibitor
comprises one or more of hirudin, heparin, PPACK, I2581, and
antithrombin.
8. The method according to claim 1, wherein the formation of said
precipitate is correlated to the existence of haemostatic
dysfunction in the patient.
9. The method according to claim 8, wherein the greater the
formation of said precipitate, the worse the existence of
haemostatic dysfunction in the patient which can be quantified by
constructing a reference curve to compare said patient test sample
with previous patient samples.
10. The method according to claim 1, wherein the time dependent
measurement profile is an optical transmission profile, and wherein
the greater the decrease in transmission in the test sample, the
greater the formation of said precipitate, and the greater the
haemostatic dysfunction in the patient.
11. The method according to claim 1, wherein said precipitate
comprises a protein weighing approximately 20 kD.
12. The method according to claim 11, wherein said protein is
insoluble in saline, EDTA and Imidazole, and soluble in 5 molar
urea.
13. The method according to claim 1, wherein said reagent is added
to said test sample in the absence of clot inducing reagents.
14. The method according to claim 1, wherein the formation of the
precipitate is measured at least once after time_0.
15. The method according to claim 14, wherein a single endpoint
measurement is made of precipitate formation after time_0.
16. The method according to claim 1, wherein said reagent is
capable of causing precipitate formation completely in the absence
of fibrin polymerization.
17. The method according to claim 10, wherein the amount of fibrin
polymerization in the method, if any, causes no change in optical
transmittance.
18. A method for determining whether or not a patient has
haemostatic dysfunction, comprising: a) obtaining a blood sample
from a patient; b) obtaining plasma from said blood sample; c)
adding a reagent capable of inducing the formation of a precipitate
in patients with haemostatic dysfunction without causing any
substantial fibrin polymerization; d) taking one or more
measurements of a parameter of the sample wherein changes in the
sample parameter are capable of correlation to precipitate
formation if present; e) determining that a patient has haemostatic
dysfunction if precipitate formation is detected.
19. The method according to claim 18, wherein a plurality of
measurements are made after addition of said reagent.
20. The method according to claim 18, wherein a single reagent is
used prior to taking said measurements.
21. The method according to claim 18, wherein said measurements are
measurements of optical transmission or absorbance through said
sample.
22. The method according to claim 21, wherein said reagent
comprises a metal ion.
23. The method according to claim 22, wherein said metal ion
comprises one or more of calcium, magnesium, manganese, iron or
barium.
24. The method according to claim 18, wherein a clot inhibitor is
provided as part of said reagent or as part of an additional
reagent added to said test sample.
25. The method according to claim 24, wherein said clot inhibitor
comprises one or more of hirudin, heparin, PPACK, I2581 or
antithrombin.
26. The method according to claim 18, wherein said one or more
measurements are unaffected by clot formation due to lack of fibrin
polymerization.
27. The method according to claim 18, wherein the one or more
measurements are a plurality of measurements, and wherein a rate of
change of said plurality of measurement is determined, and wherein
haemostatic dysfunction is determined based on the determined rate
of change.
28. The method according to claim 18, wherein said haemostatic
dysfunction is DIC.
29. A method for determining a patient sample the presence of a
complex of proteins comprising at least one of serum amyloid A and
C-reactive protein, comprising: a) obtaining a test sample from a
patient; b) adding an alcohol, a clot inhibitor, and a metal
cation; wherein a precipitate is formed which comprises a complex
of proteins including at least one of serum amyloid A and
C-reactive protein.
30. The method according to claim 29, wherein said patient test
sample is a sample of whole blood or a portion thereof.
31. The method according to claim 30, wherein said alcohol is
methanol or ethanol.
32. The method according to claim 30, wherein said metal ion is a
divalent metal cation selected from the group consisting of calcium
magnesium, manganese, iron and barium, and wherein said clot
inhibitor is selected from the group consisting of hirudin,
heparin, PPACK, I2581 and antithrombin.
33. A method comprising: a) adding a coagulation reagent to an
aliquot of a test sample from a patient; b) monitoring the
formation of fibrin over time in said test sample by measuring a
parameter of said test sample which changes over time due to
addition of said coagulation reagent; c) determine a rate of
change, if any, of said parameter in a period of time prior to
formation of fibrin in said test sample; d) if said determined rate
of change is beyond a predetermined threshold, then with a second
aliquot of said patient test sample, add thereto a reagent that
induces the formation of a precipitate in the absence of fibrin
polymerization; e) measuring the formation of the precipitate over
time; and f) determining the possibility or probability of
haemostatic dysfunction based on the measurement of the
precipitate.
34. The method according to claim 33, wherein said coagulation
reagent is a PT reagent or an APTT reagent.
35. The method according to claim 34, wherein said reagent that
causes precipitate formation is a divalent metal cation.
36. The method according to claim 35, wherein said divalent metal
cation is calcium, magnesium, manganese, iron or barium.
37. The method according to claim 35, wherein said reagent that
causes precipitate formation comprises a clot inhibitor.
38. The method according to claim 37, wherein said clot inhibitor
is hirudin, heparin, PPACK, I2581 or antithrombin.
39. The method according to claim 33, wherein at least one
measurement in the test of the second aliquot with the reagent
capable of forming a precipitate without causing fibrin
polymerization is at a time greater than the clotting time of the
first aliquot.
40. A method for monitoring an inflammatory condition in a patient,
comprising: a) adding a reagent to a patient test sample, said
reagent capable of causing precipitate formation in some patient
test samples without causing fibrin polymerization; b) measuring a
parameter of said test sample over time which is indicative of said
precipitate formation; c) determining the slope of said changing
parameter; d) repeating steps a) to c) at a later date or time;
wherein an increase or decrease in said slope at said later date or
time is indicative of progression or regression, respectively, of
said inflammatory condition.
41. The method according to claim 40, wherein a plurality of
measurements are taken over time so as to provide the slope of said
changing parameter.
42. The method according to claim 40, wherein said reagent
comprises a metal divalent cation.
43. The method according to claim 42, wherein said reagent
comprises calcium, magnesium, manganese, iron or barium.
44. The method according to claim 42, wherein said reagent further
comprises a clot inhibitor.
45. The method according to claim 44, wherein said clot inhibitor
is heparin, hirudin, PPACK, I2581 or antithrombin.
46. The method according to claim 40, wherein said changing
parameter is optical transmission or absorbance.
47. The method according to claim 40, wherein said inflammatory
condition is one or more of rheumatoid arthritis, sepsis, or a
condition due to surgery of trauma.
48. A method for diagnosing and treating patients with haemostaic
dysfunction, comprising: a) adding a reagent to a test sample that
causes precipitate formation without causing fibrin polymerization;
b) taking measurements over time of a parameter of the test sample
that changes due to the formation of the precipitate; c)
determining the rate of change of said parameter; d) determining
that a patient has haemostatic dysfunction if said rate of change
is beyond a predetermined limit; e) intervening with treatment for
said haemostatic dysfunction if said rate of change is beyond the
predetermined limit.
49. The method according to claim 48, wherein said treatment
includes administration of antibiotics and/or clot inhibitors.
50. The method according to claim 48, wherein said treatment
comprises identifying and correcting the underlying cause of said
haemostatic dysfunction.
51. The method according to claim 50, wherein said treatment
comprises the administration of a broad spectrum antibiotic,
evacuation of the uterus in abruptio placentae, blood replacement
therapy, administration of platelet concentrates to correct
thrombocytopenia, administration of fresh plasma, administration of
one or more blood factors, and/or treatment with interleukin-1.
52. The method according to claim 48, wherein said haemostatic
dysfunction is DIC or haemostatic dysfunction with the potential to
lead to DIC.
53. The method according to claim 48, further comprising repeating
steps a) to d) at one or more later times or dates, comparing the
later one or more rates of change of the parameter with the
earlier, and optimizing treatment based on increases or decreases
in the rates of change of the parameter.
54. The method according to claim 53, wherein said changing
parameter is changing optical transmittance through the test
sample.
55. The method according to claim 54, wherein the test sample is
whole blood or a portion thereof.
56. The method according to claim 55, wherein said test sample is a
plasma sample.
57. The method according to claim 48, wherein said reagent that
causes the precipitate formation in the absence of fibrin
polymerization comprises a divalent metal cation.
58. The method according to claim 57, wherein said reagent
comprises barium, iron, manganese, magnesium and/or calcium.
59. The method according to claim 57, wherein said reagent further
comprises a clot inhibitor.
60. The method according to claim 59, wherein said clot inhibitor
is antithrombin, I2581, PPACK, heparin and/or hirudin.
61. A method comprising: a) adding a reagent to a patient sample
capable of causing formation of a precipitate in said sample; b)
monitoring a changing parameter of said sample over time, said
parameter indicative of said precipitate formation; C) determining
the rate of change of said parameter or whether said parameter
exceeds a predetermined limit at a predetermined time; d) repeating
steps a) to c) at least once, each time at a different
plasma/reagent ratios; e) measuring the maximum, average and/or
standard deviation for the measurements in step c); and f)
determining haemostatic dysfunction based on measurements of step
e).
62. The method according to claim 61, further comprising repeating
steps a) to e) at a later time or date to monitor disease
progression or regression.
63. The method according to claim 61, wherein said haemostatic
dysfunction is DIC or haemostatic dysfunction with the potential to
lead to DIC.
64. The method according to claim 61, wherein said reagent further
comprises a clot inhibitor.
65. The method according to claim 64, wherein said reagent
comprises a metal cation capable of inducing precipitate
formation.
66. The method according to claim 65, wherein said reagent
comprises calcium, magnesium, manganese, iron or barium as divalent
cations.
67. The method according to claim 61, wherein said different
plasma/reagent ratios are a result of altering the concentration of
said reagent from test to test.
68. The method according to claim 61, wherein said different
plasma/reagent ratios are a result of altering the concentration of
said test sample from test to test.
69. The method according to claim 68, wherein said test sample is a
plasma sample that is diluted at different ratios from test to
test.
70. An immunoassay comprising: a) providing a ligand capable of
binding to C-reactive protein or the 300 kD protein in lane 5 of
FIG. 21; b) adding said ligand to a test sample from a patient and
allowing binding of said ligand to C-reactive protein or said 300
kD protein in said test sample; c) detecting the presence and or
amount of C-reactive protein or said 300 kD protein in said sample;
and d) diagnosing haemostatic dysfunction in the patient due to the
detection and/or amount of C-reactive protein or said 300 kD
protein detected.
71. The immunoassay of claim 70, wherein said C-reactive protein or
said 300 kD protein detected is part of a complex of proteins
formed upon addition of a metal divalent cation to the test
sample.
72. The immunoassay of claim 71, wherein said complex of proteins
comprises both CRP and said 300 kD protein and further comprises
serum amyloid A.
73. The immunoassay of claim 70, wherein said haemostatic
dysfunction diagnosed is DIC or haemostatic dysfunction with the
potential to lead to DIC.
74. The immunoassay according to claim 71, wherein the diagnosed
haemostatic dysfunction has the potential to lead to bleeding or
thrombosis.
75. The immunoassay of claim 71, wherein said C-reactive protein is
modified, cleaved, mutated or whole C-reactive protein.
76. A method for testing the efficacy of a new drug on a human or
animal subject with an inflammatory condition and/or haemostatic
dysfunction, comprising: a) adding a reagent to a patient test
sample, said reagent capable of causing precipitate formation in
some subject test samples without causing fibrin polymerization; b)
measuring a parameter of said test sample over time which is
indicative of said precipitate formation; c) determining the slope
of said changing parameter and/or the value of said parameter at a
predetermined time; d) administering a drug to said animal or human
subject; e) repeating steps a) to c) at a later date or time;
wherein an increase or decrease in said slope or value at said
later date or time is indicative of the efficacy of said drug.
Description
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 09/244,340, filed Feb. 4, 1999, the subject
matter of which is incorporated herein by reference. This
application also relates to U.S. Pat. No. 5,646,046 to Fischer et
al., the subject matter of which is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] Blood clots are the end product of a complex chain reaction
where proteins form an enzyme cascade acting as a biologic
amplification system. This system enables relatively few molecules
of initiator products to induce sequential activation of a series
of inactive proteins, known as factors, culminating in the
production of the fibrin clot. Mathematical models of the kinetics
of the cascade's pathways have been previously proposed.
[0003] Thrombosis and hemostasis testing is the in vitro study of
the ability of blood to form clots and to break clots in vivo.
Coagulation (hemostasis) assays began as manual methods where clot
formation was observed in a test tube either by tilting the tube or
removing fibrin strands by a wire loop. The goal was to determine
if a patient's blood sample would clot after certain materials were
added. It was later determined that the amount of time from
initiation of the reaction to the point of clot formation in vitro
is related to congenital disorders, acquired disorders, and
therapeutic monitoring. In order to remove the inherent variability
associated with the subjective endpoint determinations of manual
techniques, instrumentation has been developed to measure clot
time, based on (1) electromechanical properties, (2) clot
elasticity, (3) light scattering, (4) fibrin adhesion, and (5)
impedance. For light scattering methods, data is gathered that
represents the transmission of light through the specimen as a
function of time (an optical time-dependent measurement
profile).
[0004] Two assays, the PT and APTT, are widely used to screen for
abnormalities in the coagulation system, although several other
screening assays can be used, e.g. protein C, fibrinogen, protein S
and/or thrombin time. If screening assays show an abnormal result,
one or several additional tests are needed to isolate the exact
source of the abnormality. The PT and APTT assays rely primarily
upon measurement of time required for clot time, although some
variations of the PT also use the amplitude of the change in
optical signal in estimating fibrinogen concentration.
[0005] Blood coagulation is affected by administration of drugs, in
addition to the vast array of internal factors and proteins that
normally influence clot formation. For example, heparin is a
widely-used therapeutic drug that is used to prevent thrombosis
following surgery or under other conditions, or is used to combat
existing thrombosis. The administration of heparin is typically
monitored using the APTT assay, which gives a prolonged clot time
in the presence of heparin. Clot times for PT assays are affected
to a much smaller degree. Since a number of other plasma
abnormalities may also cause prolonged APTT results, the ability to
discriminate between these effectors from screening assay results
may be clinically significant.
[0006] The present invention was conceived of and developed for
predicting haemostatic dysfunction in a sample based on one or more
time-dependent measurement profiles, such as optical time-dependent
measurement profiles. In addition, the present invention is
directed to predicting the presence of Disseminated Intravascular
Coagulation in a patient based on a time-dependent profile, such as
an optical transmission profile, from an assay run on the patient's
blood or plasma sample.
SUMMARY OF THE INVENTION
[0007] The present invention is directed to a method for detecting
a precipitate in a test sample in the absence of clot formation.
The method includes providing a test sample and adding thereto a
reagent, the reagent alone or in combination with additional
reagents causing the formation of a precipitate. The reagent
preferably comprises a metal divalent cation and optionally
includes a clot inhibiting substance. The detection of the
precipitate can be qualitative or quantitative, and the precipitate
can be detected such as by a clotting assay, a latex agglutination
or gold sol assay, an immunoassay such as an ELISA, or other
suitable method that would allow for detection and/or quantitation
of the precipitate. The formation of the precipitate can be
detected as an endpoint value, or kinetically. This precipitate
detection allows for predicting Haemostatic Dysfunction in
patients. The present invention is useful for predicting
Haemostatic Dysfunction that can lead to bleeding or thrombosis, or
specifically to Disseminated Intravascular Coagulation (DIC).
[0008] More particularly, the present invention is directed to a
method comprising adding a reagent to a test sample having at least
a component of a blood sample from a patient, measuring the
formation of a precipitate due to the reaction of the test sample
and the reagent, over time so as to derive a time-dependent
measurement profile, the reagent capable of forming a precipitate
in the test sample without causing substantial fibrin
polymerization. The invention is also directed to a method for
determining whether or not a patient has haemostatic dysfunction,
comprising obtaining a blood sample from a patient, obtaining
plasma from said blood sample, adding a reagent capable of inducing
the formation of a precipitate in patients with haemostatic
dysfunction without causing any substantial fibrin polymerization,
taking one or more measurements of a parameter of the sample
wherein changes in the sample parameter are capable of correlation
to precipitate formation if present, and determining that a patient
has haemostatic dysfunction if precipitate formation is
detected.
[0009] The present invention is also directed to a method for
determining in a patient sample the presence of a complex of
proteins comprising at least one of a 300 kD protein, serum amyloid
A and C-reactive protein, comprising obtaining a test sample from a
patient, adding an alcohol, a clot inhibitor, and a metal cation,
wherein a precipitate is formed which comprises a complex of
proteins including at least one of a 300 kD protein, serum amyloid
A and C-reactive protein.
[0010] The invention is also directed to a method comprising adding
a coagulation reagent to an aliquot of a test sample from a
patient, monitoring the formation of fibrin over time in said test
sample by measuring a parameter of the test sample which changes
over time due to addition of the coagulation reagent, determine a
rate of change, if any, of said parameter in a period of time prior
to formation of fibrin in said test sample, if the determined rate
of change is beyond a predetermined threshold, then with a second
aliquot of the patient test sample, add thereto a reagent that
induces the formation of a precipitate in the absence of fibrin
polymerization, measuring the formation of the precipitate over
time, and determining the possibility or probability of haemostatic
dysfunction based on the measurement of the precipitate.
[0011] The invention is also directed to a method for monitoring an
inflammatory condition in a patient, comprising adding a reagent to
a patient test sample, the reagent capable of causing precipitate
formation in some patient test samples without causing fibrin
polymerization, measuring a parameter of the test sample over time
which is indicative of said precipitate formation, determining the
slope of the changing parameter, repeating he the above steps at a
later date or time, wherein an increase or decrease in the slope at
the later date or time is indicative of progression or regression,
respectively, of the inflammatory condition.
[0012] The invention is further directed to a method for diagnosing
and treating patients with haemostaic dysfunction, comprising
adding a reagent to a test sample that causes precipitate formation
without causing fibrin polymerization, taking measurements over
time of a parameter of the test sample that changes due to the
formation of the precipitate, determining the rate of change of
said parameter, determining that a patient has haemostatic
dysfunction if said rate of change is beyond a predetermined limit;
intervening with treatment for said haemostatic dysfunction if said
rate of change is beyond the predetermined limit.
[0013] The invention also is directed to a method comprising adding
a reagent to a patient sample capable of causing formation of a
precipitate in said sample, monitoring a changing parameter of said
sample over time, said parameter indicative of said precipitate
formation, determining the rate of change of said parameter or
whether said parameter exceeds a predetermined limit at a
predetermined time, repeating the above steps at least once, each
time at a different plasma/reagent ratios, measuring the maximum,
average and/or standard deviation for the measurements; and
determining haemostatic dysfunction based on the maximum, average
and/or standard deviation measurements.
[0014] The present invention is further directed to an immunoassay
comprising providing a ligand capable of binding to C-reactive
protein or the 300 kD protein in lane 5 of FIG. 21, adding said
ligand to a test sample from a patient and allowing binding of said
ligand to C-reactive protein or said 300 kD protein in said test
sample, detecting the presence and or amount of C-reactive protein
or said 300 kD protein in said sample, and diagnosing haemostatic
dysfunction in the patient due to the detection and/or amount of
C-reactive protein or said 300 kD protein detected.
[0015] The invention further relates to a method for testing the
efficacy of a new drug on a human or animal subject with an
inflammatory condition and/or haemostatic dysfunction, comprising
adding a reagent to a patient test sample, said reagent capable of
causing precipitate formation in some subject test samples without
causing fibrin polymerization, measuring a parameter of said test
sample over time which is indicative of said precipitate formation,
determining the slope of said changing parameter and/or the value
of said parameter at a predetermined time, administering a drug to
said animal or human subject, repeating the above steps at a later
date or time, wherein an increase or decrease in said slope or
value at said later date or time is indicative of the efficacy of
said drug.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIGS. 1a and 1b illustrate transmittance waveforms on the
APTT assay with FIG. 1a showing a normal appearance, and 1b showing
a biphasic appearance;
[0017] FIG. 2 illustrates transmittance levels at 25 seconds in
relation to diagnosis in 54 patients with bi-phasic waveform
abnormalities. The horizontal dotted line represents the normal
transmittance level;
[0018] FIG. 2 illustrates transmittance levels (upper panel) and
waveforms (lower panel) on a patient who developed DIC following
sepsis and recovered;
[0019] FIG. 4 illustrates serial transmittance levels (upper panel)
and waveforms (lower panel) on a patient who developed DIC
following trauma and died;
[0020] FIG. 5 illustrates ROC plots for the prediction of DIC
transmittance at 25 seconds (TR25), APTT clot time, and slope_1
(the slope up to the initiation of clot formation);
[0021] FIGS. 6 and 7 show histograms for DIC, normal and
abnormal/non-DIC populations for TR25 and slope_1;
[0022] FIGS. 8 and 10 show group distributions for slope_1 and TR25
respectively;
[0023] FIGS. 9 and 11 show partial subpopulations of the data shown
in FIGS. 8 and 10;
[0024] FIG. 12 is an optical transmission profile for an APTT
assay;
[0025] FIGS. 13 and 14 are optical transmission profiles for PT
assays;
[0026] FIG. 15 is an illustration of two waveforms where (x) is a
test run on a sample using an APTT clotting reagent and resulting
in a biphasic waveform, and (y) is a test run where a clot
inhibitor is used;
[0027] FIG. 16 is a standard curve for ELISA of CRP;
[0028] FIG. 17 shows an isolated precipitate after gel
filtration;
[0029] FIG. 18 is a graph showing the time course of turbidity in a
sample upon adding a precipitate inducing reagent;
[0030] FIG. 19 is a graph showing the relationship between maximum
turbidity change and amount of patient plasma in a sample;
[0031] FIG. 20 shows the results of anion exchange chromatography
of material recovery after fractionation of patient plasma;
[0032] FIGS. 21a and 21 show the non-reduced and reduced SDS page
of various fractions of patient plasma;
[0033] FIG. 22 shows immunoblots of CRP in normal and DIC
plasma;
[0034] FIG. 23 illustrates the turbidity change upon adding
divalent calcium to materials obtained upon Q-sepharose
chromatography in the absence of plasma (except top curve);
[0035] FIG. 24 is a table showing CRP determined by ELISA;
[0036] FIG. 25 shows the response to increasing calcium
concentrations in optical transmission profiles;
[0037] FIG. 26 shows a more pronounced slope without the use of a
clotting agent;
[0038] FIG. 27 is a calibration curve with heparin;
[0039] FIG. 28 shows CRP levels in 56 ITU patients plotted against
transmittance at 18 seconds; and
[0040] FIG. 29 shows more samples with CRP and decrease in
transmittance at 18 seconds (10000-TR18).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0041] In the present invention, not only can a particular
abnormality (Haemostatic Dysfunction) be detected, but in addition
the progression of the disease can be monitored in a single
patient. Haemostatic Dysfunction, as used herein, is a condition
evidenced by the formation of a precipitate prior to (or in the
absence of clot formation, depending upon the reagent used).
[0042] Disseminated intravascular coagulation (DIC--a type of
Haemostatic Dysfunction) prognosis has been hampered by the lack of
an early, useful and rapidly available diagnostic marker. The
invention has been found to be not only useful as an early
diagnostic and single monitoring marker of DIC, but in addition the
quantifiable and standardizable changes also allow for prognostic
applicability in clinical management.
[0043] Disseminated intravascular coagulation (DIC) is a secondary
response to a pre-existing pathology whereby the haemostatic
response becomes perturbed and disseminated as opposed to the
focused events of normal haemostasis. Despite improvements both in
the intensive care management of patients and in our basic
knowledge of haemostatic mechanisms in DIC, survival in this
patient group is still very discouraging. Fundamental to the
management of this complication is the implementation of aggressive
therapy directed at forestalling or eradicating the primary
pathology as the source of the initiating stimulus. However, in
practical terms, the problem remains one of early identification of
DIC to facilitate immediate and appropriate intervention. Although
the technological armory available to the clinical investigator has
expanded enormously, the pace of acute DIC precludes most of the
more specific tests and reliance is still placed on traditional
screening tests such as the prothrombin (PT), activated partial
thromboplastin time (APTT) and platelet count. These tests lack
specificity on an individual basis and are only useful in DIC if
they lead on to further determinations of fibrinogen and fibrin
breakdown products/D-dimers. However, changes in these parameters
may not occur all at the same time and as such, serial testing is
often needed which inevitably leads to a delay in diagnosis and
clinically useful intervention.
[0044] The normal sigmoidal appearance from an APTT transmittance
waveform (TW) changes to a "bi-phasic" appearance in DIC patients.
This represents a loss in the plateau of a normal APTT-TW, with
development of an initial low gradient slope followed by a much
steeper slope (FIGS. 1a and b). In addition, this bi-phasic pattern
can be seen even when the APTT clotting time result is normal.
[0045] Freshly collected blood samples that required a PT or an
APTT were analyzed prospectively over a two week working period.
These were in 0.105M tri-sodium citrate in the ratio of 1 part
anticoagulant to 9 parts whole blood and the platelet-poor plasma
was analyzed on the MDA (Multichannel Discrete Analyzer) 180, an
automated analyzer for performing clinical laboratory coagulation
assays using an optical detection system (Organon Teknika
Corporation, Durham, N.C., USA). In addition, to deriving the clot
times for both PT (normal 11.2-15s) using MDA Simplastin LS and
APTT (normal 23-35s) using MDA Platelin LS with 0.025M calcium
chloride (Organon Teknika Corporation, USA), an analysis of the TW
for the APTT was performed on each occasion at a wavelength of 580
nm. To quantitate the visual profile, the amount of light
transmittance at 25 seconds was recorded. A normal waveform has a
light transmittance of 100% that is represented on the analyzer and
in FIG. 1a without the decimal point as 10000. As such, a bi-phasic
change will have a reduced light transmittance of less than 10000.
As can be seen in FIG. 1b, decreasing levels of light transmittance
prior to clot formation correlate directly with increasing
steepness of the bi-phasic slope. The recording of the light
transmittance at 25 seconds also allows for standardization between
patients and within the same patient with time. If the minimum
level of light transmittance for each sample were to be used
instead, this would be affected by variations in the clot time of
the APTT and would therefore not be ideal for comparisons.
[0046] To ensure that no cases of DIC were overlooked, the
following criteria was followed. If (a) an abnormal bi-phasic TW
was encountered, or (b) a specific DIC screen was requested, or (c)
if there was a prolongation in either the PT or APTT in the absence
of obvious anticoagulant therapy, a full DIC screen was performed.
This would further include the thrombin time (TT) (normal 10.5-15.5
seconds), fibrinogen (Fgn) (normal 1.5-3.8 g/l) and estimation of
D-dimer levels (normal <0.5 mg/l) on the Nyocard D-Dimer
(Nycomed Pharma AS, Oslo, Norway). Platelet counts (Plt) (normal
150-400 10.sup.9/l) performed on an EDTA sample at the same time
were recorded. In addition, clinical details were fully elucidated
on any patient with a bi-phasic TW or coagulation abnormalities
consistent with DIC.
[0047] The diagnosis of DIC was strictly defined in the context of
both laboratory and clinical findings of at least 2 abnormalities
in the screening tests (increased PT, increased APTT, reduced Fgn,
increased TT or reduced Plt) plus the finding of an elevated
D-dimer level (>0.5 mg/l) in association with a primary
condition recognized in the pathogenesis of DIC. Serial screening
tests were also available on those patients to chart progression
and confirmation of the diagnosis of DIC as was direct clinical
assessment and management. For statistical analysis, values for the
sensitivity, specificity, positive and negative prediction of the
APTT-TW for the diagnosis of DIC were calculated employing a
two-by-two table. 95% confidence intervals (CI) were calculated by
the exact binomial method.
[0048] A total of 1,470 samples were analyzed. These were from 747
patients. 174 samples (11.9%) from 54 patients had the bi-phasic
waveform change. 22 of these 54 patients had more than 3 sequential
samples available for analysis. DIC was diagnosed in 41 patients
with 30 of these requiring transfusion support with fresh frozen
plasma, cryoprecipitate or platelets. The underlying clinical
disorders as shown in Table 1
1TABLE 1 Clinical disorders predisposing patients to DIC. Disorder
No Infections 17 Trauma or recent major surgery 16 Malignancy 2
Hepatic Disease 1 Obstetric Cause 1 Miscellaneous Additional Causes
* 4 * Includes hypoxia, acidosis, Lithium overdosage and graft
rejection
[0049] 40 of the 41 patients with DIC had the bi-phasic TW. The one
false negative result (DIC without a bi-phasic TW) occurred in a
patient with pre-eclampsia (PET) where the single sample available
for analysis showed a prolonged PT of 21.0s, APTT of 44.0s and
raised D-dimers of 1.5 mg/l. 5 other patients were identified in
this study with PET and none had either DIC or a bi-phasic TW. Of
the 14 patients with a bi-phasic TW which did not fulfil the
criteria of DIC, all had some evidence of a coagulopathy with
abnormalities in one or two of the screening tests. These abnormal
results fell short of the criterion for DIC as defined above. 4 of
these 14 patients had chronic liver disease with prolonged PT and
mild thrombocytopaenia. A further 2 patients had atrial
fibrillation with isolated elevation of D-dimer levels only. The
remaining 8 patients were on the ICU with multiple organ
dysfunction arising from trauma or suspected infection but without
the classical laboratory changes of DIC. These patient profiles
were described in the ICU as consistent with the "systemic
inflammatory response syndrome" (SIRS). Based on these figures, the
bi-phasic TW has a 97.6% sensitivity for the diagnosis of DIC with
a specificity of 98%. Use of an optical transmittance waveform was
found to be helpful in detecting the biphasic waveform.
2TABLE 2 Performance of the transmittance waveform (TW) analysis in
patients with and without DIC Biphasic Normal TW TW Total DIC
positive 40 1 41 DIC negative 14 692 706 Total 54 693 747
Sensitivity 97.6% (Cl 85.6-99.9%), Specificity 98.0% (Cl
96.6-98.9%), Positive predictive value 74.0% (Cl 60.1-84.6%),
Negative predictive value 99.9% (Cl 99.1-99.9%)
[0050] The positive predictive value of the test was 74%, which
increased with increasing steepness of the bi-phasic slope and
decreasing levels of light transmittance (Table 2 and FIG. 2). In
the first two days of the study, there were 12 patients who had an
abnormality in the clotting tests plus elevation of D-dimer levels.
These were patients who were clinically recovering from DIC that
occurred in the week preceding the study. This led to the
impression that TW changes might correlate more closely with
clinical events than the standard markers of DIC.
3TABLE 3 Serial results in a patient with sepsis Pit PT APTT TT Fgn
D-Dimer (150-400 .times. Day Time (11.2-15 s) (23-35 s) (10.5-15.5
s) (1.5-3.8 g/l) (<0.5 mg/l) 10.sup.9/l) TW 1 0923 14.7 32.9
12.0 4.7 0.00 193 B* 1 2022 20.8* 38.6* 12.4 5.7 6.00* 61* B* 2
0920 18.0* 33.0 13.0 5.2 2.00* 66* N 3 1011 16.3* 24.8 13.2 4.7
0.00 64* N PT = Prothrombin time, APTT = Activated Partial
Thromboplastin Time, TT = Thrombin Time, Fgn = Fibrinogen, Pit =
Platelet count, TW = Transmittance Waveform *Indicates abnormal
changes. B = bi-phasic; N = normal
[0051] The availability of more than 3 sequential samples in 22
patients allowed for further assessment. Table 3 illustrates one
such example with serial test results from a patient with E. coli
septicaemia.
[0052] The appearance of a bi-phasic TW preceded changes in the
standard tests for the diagnosis of DIC. It was only later in the
day that the PT, APTT, Plt and D-dimer levels became abnormal and
fulfilled the diagnostic criteria of DIC. Treatment with
intravenous antibiotics led to clinical improvement by Day 2 with
normalization of her TW in advance of the standard parameters of
DIC. D-dimers and Plt were still respectively abnormal 24 and 48
hours later.
[0053] This correlation between clinical events and TW changes was
seen in all the DIC patients where samples were available to chart
the course of clinical events. As the TW changes were quantifiable
and standardizable through recording of the transmittance level at
25 seconds, this analysis provided a handle in assessing prognostic
applicability. FIG. 3 illustrates the results of a patient who
initially presented with peritonitis following bowel perforation.
This was further complicated by gram negative septicaemia
post-operatively with initial worsening of DIC followed by a
gradual recovery after appropriate therapy. As DIC progressed
initially, there was increasing steepness in the bi-phasic slope of
the TW and a fall in the light transmittance level. A reversal of
this heralded clinical recovery. FIG. 4 illustrates the results of
a patient who sustained severe internal and external injuries
following a jet-ski accident. Although initially stabilized with
blood product support, his condition deteriorated with continuing
blood loss and development of fulminant DIC. The bi-phasic slope
became increasingly steep with falls in transmittance level as the
consequences of his injuries proved fatal.
[0054] As DIC can arise from a variety of primary disorders, the
clinical and laboratory manifestations can be extremely variable
not only from patient to patient but also in the same patient with
time. There is therefore, a need for systems that are not only
robust in their diagnosis but simple and rapid to perform. Although
it has been shown that the bi-phasic TW appeared to be sensitive
for Haemostatic Dysfunction (e.g. DIC) and was not seen in other
selected patient groups with coagulation aberrations or influenced
by either (i) pre-analytical variables, (ii) different silica-based
APTT reagents, (iii) the use of thrombin as the initiator of the
coagulation reaction or (iv) treatment in the form of heparin or
plasma expanders, the robustness of this assay for DIC could only
be addressed through a prospective study. This study has shown that
the bi-phasic TW provides diagnostic accuracy in DIC with an
overall sensitivity of 97.6% and specificity of 98%. In contrast,
none of the standard parameters on an individual basis (i.e., PT,
APTT, TT, Fgn, Plt, D-dimers) or even in combination, has ever
reached the degree of sensitivity or specificity. The ready
availability of TW data from the MDA-180 would also fulfil the
criteria of simplicity and rapidity unlike the measurements of
thrombin-antithrombin complexes or other markers that are dependent
on ELISA technology. In addition, the advantages of TW analysis are
that: (a) the bi-phasic TW change appears to be the single most
useful correlate within an isolated sample for DIC and as such,
reliance need no longer be placed on serial estimations of a
battery of tests, and (b) the appearance or resolution of the
bi-phasic TW can precede changes in the standard, traditional
parameters monitored in DIC with strong, clear correlation to
clinical events and outcome.
[0055] Although the bi-phasic TW was also seen in patients who did
not have DIC per se as defined by the above criteria, the clinical
conditions were associated with Haemostatic Dysfunction--namely
activated coagulation prior to initiation of clot formation
resulting in a biphasic waveform (for example in chronic liver
disease or in the very ill patients on the Intensive Care Unit who
had multiple organ dysfunction). It appears that bi-phasic TW is
sensitive to non-overt or compensated DIC and that a transmittance
level of less than 90% (FIG. 2) or sequential falls in that level
(FIG. 4), reflects decompensation towards a more overt
manifestation and potentially fulminant form of DIC. This line of
explanation is supported by the observation of only a mild
bi-phasic TW (transmittance level of about 95%) in 2 patients with
atrial fibrillation; a condition that is associated with mild
coagulation activation and elevated D-dimer levels. As no follow-up
samples were available on these 2 patients whose clinical details
were otherwise unremarkable, their bi-phasic TW could well have
been transient. Nonetheless, these cases illustrate that the lower
the level of light transmittance, the more likely the bi-phasic TW
becomes predictive of Haemostatic Dysfunction, particularly
DIC.
[0056] The observation of a normal TW in a patient with PET and DIC
needs further exploration as the study did not selectively aim to
examine any particular patient groups and only had a total of 6
patients with PET; the remaining 5 of which did not have DIC. One
explanation which would be supported by other findings in this
study is that the patient could have been recovering from PET and
DIC at the time of the sample. There may already have been
normalization in the bi-phasic TW in advance of the other
parameters which were still abnormal and indicative of DIC. Another
explanation is that the disturbed haemostatic process in PET is
more localized and different from the DIC that arises from other
conditions. Such patients respond dramatically to delivery of the
fetus which suggests anatomical localization of the pathological
process to the placenta despite standard laboratory clotting tests
implying systemic evidence of the condition.
EXAMPLE
[0057] Though analysis of the transmittance at a time of 25 seconds
is helpful in predicting DIC, a second embodiment of the invention
has been found that greatly improves sensitivity and specificity.
It has been found that looking at transmittance at a particular
time can result in detecting an artifact or other decrease in
transmittance at that point, even though the waveform is not a
bi-phasic waveform. For example, a temporary dip in transmittance
at 25 seconds would cause such a patient sample to be flagged as
bi-phasic, even if the waveform was normal or at least not
bi-phasic. Also, if a patient sample had a particularly short
clotting time, then if clot formation begins e.g. prior to 25
seconds (or whatever time is preselected), then the waveform could
be flagged as biphasic, even though the real reason for decreased
transmittance at 25 seconds is because clot formation has already
begun/occurred.
[0058] For this reason, it has been found that rather than analysis
of transmittance at a particular time, it is desirable to calculate
the slope of the waveform prior to initiation of clot formation.
This calculation can involve determination of clot time followed by
determination of waveform slope prior to clot time. In an
additional embodiment, the slope (not transmittance) is determined
prior to clot time or prior to a preselected time period, whichever
is less. As can be seen in FIG. 11, when transmittance is used for
determining e.g. DIC, there is poor specificity and sensitivity.
However, as can be seen in FIG. 9, when slope prior to initiation
of clot formation is used, specificity and sensitivity are greatly
improved, and are better than standard tests used in the diagnosis
of Haemostatic Dysfunction, such as DIC.
[0059] Additional testing was performed on three sets of patients.
The first set consisted of 91 APTT assays run on samples from 51
different confirmed DIC patients. The second set of data consisted
of 110 APTT assays run on samples from 81 different confirmed
normal patients. The third set of data included 37 APTT assays run
on 22 abnormal, non-DIC samples. FIG. 5 illustrates ROC plots for
the prediction of DIC for three different parameters derived from
the APTT assay using the combined data sets described: (1)
transmittance at 25 seconds (TR25), (2) APTT clot time, and (3)
slope 1 (the slope up to initiation of clot formation). Slope 1
exhibited the best predictive power, followed by TR25. It has also
been shown that transmittance at 18 seconds has predictive value,
particularly when the APTT clot time is less than 25 seconds. The
"cutoffs" associated with the highest efficiency for the three
parameters are listed in Table 4:
4 TABLE 4 Parameter Cutoff TR25 <9700 Clot Time >35 Slope 1
<-0.0003
[0060] It should be noted that these cutoffs have shifted with the
addition of the third set, and would likely shift again, depending
on the sample populations. FIGS. 6 and 7 show the histograms for
the DIC, normal and abnormal/non-DIC populations for TR25 and slope
1 respectively. Tables 5 and 6 show the data for the histograms in
FIGS. 6 and 7 respectively:
5 TABLE 5 Bins DIC Normal Abnormal/Non-DIC -0.006 3 0 0 -0.005 2 0
0 -0.004 1 0 0 -0.003 10 0 0 -0.002 24 0 0 -0.001 33 0 0 -0.0005 12
0 0 -0.0002 5 5 2 -0.0001 1 37 13 More 0 68 22
[0061]
6 TABLE 6 Bin DIC Normal Abnormal/Non-DIC 7000 34 1 0 8000 18 2 0
9000 26 6 1 9500 8 3 0 9600 3 2 1 9700 1 0 0 9800 1 3 0 9900 0 21 4
10000 0 62 30 More 0 10 1
[0062] FIGS. 8 and 10 show the group distributions for Slope 1 and
TR25 respectively; and FIGS. 9 and 11 show the group distributions
for Slope 1 and TR25 respectively. FIGS. 9 and 11 show partial
subpopulations of the data shown in FIGS. 8 and 10.
[0063] When the prediction of Haemostatic Dysfunction is performed
on an automated or semi-automated analyzer, the detected bi-phasic
waveform can be flagged. In this way, the operator of the machine,
or an individual interpreting the test results (e.g. a doctor or
other medical practitioner) can be alerted to the existence of the
biphasic waveform and the possibility/probability of Haemostatic
Dysfunction such as DIC. The flag can be displayed on a monitor or
printed out. A slope of less than about -0.0003 or less than about
-0.0005 is the preferred cutoff for indicating a bi-phasic
waveform. An increasing steepness in slope prior to clot formation
correlates to disease progression.
[0064] The above examples show that waveform analysis on the APTT
assay can identify characteristic bi-phasic patterns in patients
with haemostatic dysfunction. In the majority of cases, this
dysfunction could be labelled as DIC. This diagnostic waveform
profile was seen in all APTT reagents tested, which were either
silica or ellagaic acid-based. It has also been surprisingly found
that a bi-phasic waveform can also be seen on PT assays with
particular reagents, and that the bi-phasic waveform is likewise
indicative of haemostatic dysfunction, primarily DIC.
[0065] Using samples that give bi-phasic APTT waveforms, the PT
waveform profile was derived using PT reagents (thromboplastin),
namely Recombiplast.TM. (Ortho), Thromborel.TM. (Dade-Behring) and
Innovin.TM. (Dade-Behring). Both Recombiplast and Thromborel were
particularly good at showing bi-phasic responses. Innovin was
intermediate in its sensitivity. Using the transmittance level at
10 seconds into the PT reaction as the quantitative index,
Recombiplast and Thromborel objectively showed lower levels of
light transmittance than Innovin. Thromborel can show a slight
increase in initial light transmittance before the subsequent fall.
This may be, in part, related to the relative opaqueness of
Thromborel.
[0066] Further studies were performed comparing ABTT profiles using
Platelin.TM. and PT waveform profiles using Recombiplast.TM..
Consecutive samples over a four week period from the intensive care
unit were assessed. Visually, and on objective scores (comparing
TL18 for APTT and TL10 for PT), the APTT profile was more sensitive
to changes of haemostatic dysfunction and clinical progression than
the PT profile. This relative sensitivity can be seen in the APTT
profile of FIG. 12 (Platelin) compared to the PT profiles of FIG.
13 (Recombiplast) and FIG. 14 (Thromborel S). Invariably, at
smaller changes in light transmittance, the APTT waveform detected
abnormalites more easily than the PT waveform. Nonetheless, in
severe degrees of haemostatic dysfunction, both bi-phasic profiles
were concordant.
[0067] In a further embodiment of the invention, the time dependent
measurement, such as an optical transmittance profile, can be
performed substantially or entirely in the absence of clot
formation. In this embodiment, a reagent is added which causes the
formation of a precipitate, but in an environment where no fibrin
is polymerized. The reagent can be any suitable reagent that will
cause the formation of a precipitate in a sample from a patient
with haemostatic dysfunction, such as DIC. As an example, divalent
cations, preferably of the transition elements, and more preferably
calcium, magnesium, manganese, iron or barium ions, can be added to
a test sample. These ions cause activation of an atypical waveform
that can serve as an indicator of haemostatic dysfunction. It is
also possible to run this assay in the absence of a clotting
reagent (APTT, PT, or otherwise). As part of the reagent that
comprises the activator of the atypical waveform, or separately in
another reagent, can also be provided a clot inhibitor. The clot
inhibitor can be any suitable clot inhibitor such as hirudin,
PPACK, heparin, antithrombin, I2581, etc. The formation of the
atypical waveform can be monitored and/or recorded on an automated
analyzer capable of detecting such a waveform, such as one that
monitors changes in turbidity (e.g. by monitoring changes in
optical transmittance).
[0068] FIG. 15 is an illustration of two waveforms: waveform (x) is
a test run on a sample using an APTT clotting reagent and resulting
in an atypical (biphasic) waveform, whereas waveform (y) is a test
run on a sample where a clot inhibitor is used (along with a
reagent, such as a metal divalent cation, which causes the
formation of a precipitate in the sample). Waveform (y) is
exemplary of a waveform that can result in patients with
haemostatic dysfunction where no clotting reagent is used and/or a
clot inhibitor is added prior to deriving the time-dependent
measurement profile. Generally speaking, the greater the slope of
the waveform (the larger the drop in transmittance in the same
period of time) due to the precipitate formation, the greater
severity of the patient's haemostatic dysfunction. FIG. 16 is a
standard curve for ELISA of CRP (CRP isolated from a patient used
as the standard).
[0069] The precipitate formed in the present invention was isolated
and characterized by means of chromatography and purification. Gel
Filtration was performed as follows: A column (Hiprep Sephacryl
S-300 High resolution--e.g. resolution of 10 to 1500 kDa) was used.
The volume was 320 ml (d=26 mm, 1=600 mm), and the flow rate was
1.35 ml/min. FIG. 17 shows the isolated precipitate.
[0070] FIG. 18 is a graph showing the time course of turbidity in a
sample upon adding a precipitate inducing agent (in this case
divalent calcium) and a thrombin inhibitor (in this case PPACK) to
mixtures of patient and normal plasmas. FIG. 19 is a graph showing
the relationship between maximum turbidity change and amount of
patient plasma in one sample. 0.05 units implies 100% patient
lasma. (Data from FIG. 18).
[0071] The steps used in the purification of components involved in
the turbidity change in a patient's plasma were as follows: PPACK
(10 .mu.M) was added to patient plasma. Calcium chloride was added
to 50 mM, followed by 8 minutes of incubation, followed b the
addition of EtOH to 5%. The sample was then centrifuged
10,500.times.g for 15 inutes at 4 degrees Celsius. The pellet was
then dissolved in HBS/1 mM citrate/10 .mu.M PPACK, followed by
35-70% (NH.sub.4).sub.2SO.sub.4 fractionation. Finally, sepharose
chromatography was performed using a 5 ml bed, 0.02-0.5M NaCl
gradient and 50 ml/side, to collect 2 ml fractions. FIG. 20 shows
the results of anion exchange chromatography (Q-sepharose) of
material recovery after the 35-70% ammonium sulfate fractionation
of patient plasma.
[0072] FIGS. 21a and 21b show the non-reduced and reduced,
respectively, SDS PAGE of various fractions obtained upon
fractionation of patient plasma. The loading orientation (left to
right): 5-15% gradient/Neville Gel. (approximately 10 .mu.g protein
loaded per well). In lane 1 are molecular weight standard (94, 67,
45, 30, 20 and 14 kDa from top to bottom. In lane 2 is 35%
(NH.sub.4).sub.2SO.sub.4 pellet, whereas in lane 3 is 70%
(NH.sub.4).sub.2SO.sub.4 supernate. Lane 4 is Q-sepharose starting
material. Also shown in FIGS. 21a and b are (from FIG. 20) peaks 1,
2a, 2b and 3 in, respectively, lanes 5, 6, 7 and 8. Lane 9 is
pellet 1, whereas in lane 10 are again, molecular weight standards.
Results of NH.sub.2-terminal sequencing showed peak 3, the 22 kD
protein in lanes 8 and 9 to be C-reactive protein (CRP), and the 10
kD protein in lane 9 to be human serum amyloid A (SAA). Peak 1 in
lane 5 is a 300 kD protein which, as can be seen in FIG. 23, is
part of the complex of proteins (along with CRP) in the precipitate
formed due to the addition of a metal divalent cation to a plasma
sample.
[0073] Immunoblots of CRP were performed in normal and DIC plasma.
Blot 1 (see FIG. 22): (used 0.2 .mu.l plasmas for reducing
SDS-PAGE/CRP Immunoblotting). Loading orientation (left to right):
NHP; Pt 5; 3; 1; 2; 4; and 8. For Blot 2: Loading orientation (left
to right): NHP; Pt 9; 10; 11; 7; 6; 12. For Blot 3: (CRP purified
from DIC patient plasma)--Loading orientation (left to right; ng
CRP loaded): 3.91; 7.81; 15.625; 31.25; 62.5; 125; 250. The Blots
were blocked with 2% (w/v) BSA in PBS, pH 7.4 and then sequentially
probed with rabbit anti-human CRP-IgG (Sigma, Cat# C3527, dil
1:5000 in PBS/0.01% Tween 20) and then treated with the same
antibody conjugated to HRP (dil 1:25000 in PBS/0.01% Tween 20).
[0074] FIG. 23 illustrates the turbidity changes upon adding
divalent calcium to materials obtained upon Q-sepharose
chromatography in the absence of plasma. No single peak gave a
positive response, but a mixture of peak 1 and peak 3 materials did
give a positive response indicating the involvement of CRP, a 300
kD protein, and one or more other proteins in the precipitate (peak
3+plasma was the control). FIG. 24 is a table showing CRP, .mu.g/ml
determined by ELISA. Delta A405 nm is the maximum turbidity change
observed when patients' plasma was recalcified on the presence of
the thrombin inhibitor PPACK). FIG. 24, therefore, shows that
patients with increased absorbance have varying elevated levels of
CRP, once again indicating that more than one protein is involved
in the precipitate formation.
[0075] In one embodiment of the invention, the reagent to plasma
ratio is varied between multiple tests using a reagent that induces
precipitate formation. This variance allows for amplifying the
detection of the precipitate formation by optimization of reagent
to plasma ratio (e.g. varying plasma or reagent concentrations). In
the alternative, the slope due to the precipitate formation can be
averaged between the multiple tests. As can be seen in FIG. 25, the
response to increasing calcium concentrations is shown in optical
transmission waveform profiles. The left panels show two normal
patients where calcium concentrations were varied (no clotting
agents used), whereas the right panels show two patients with
haemostatic dysfuntion (DIC in these two cases) where the metal
cation (calcium) concentration was varied (the calcium alone being
incapable of any substantial fibrin polymerization).
[0076] Though precipitate formation is capable of being detected in
patients with haemostatic dysfunction when a clotting agent is
used, it is beneficial that the reagent used is capable of forming
the precipitate without fibrin polymerization. As can be seen in
FIG. 26, the slope is more pronounced and more easily detectable
when a reagent such as calcium chloride is used (right panel) as
compared to when a clotting reagent such as an APTT reagent (left
panel) is used. As can be seen in FIG. 27, when a clot inhibitor
was added (in this case heparin), all parameters including slope_1
gave good results, and slope_1 showed the best sensitivity. For the
above reasons, a reagent capable of precipitate formation in the
absence of fibrin polymerization and/or a clot inhibitor are
preferred.
[0077] As can be seen in FIG. 28, CRP levels from 56 ITU patients
were plotted against transmittance at 18 seconds. The dotted line
is the cut-off for an abnormal transmittance at 18 seconds. FIG. 29
shows more samples with CRP and decrease in transmittance at 18
seconds (10000-TR18). These figures indicate that patients with
abnormal transmittance levels due to precipitate formation all have
increased levels of CRP. However, not all patients with increased
levels of CRP have abnormal transmittance levels thus indicating
that more than CRP is involved in the precipitate.
[0078] In a further embodiment of the invention, the formation of
the precipitate comprising a complex of proteins including CRP is
detected and/or quantitated, by the use of a latex agglutination
assay. In this method, antibodies are raised against wither the 300
kD protein or CRP. Whether monoclonal or polyclonal antibodies are
used, they are bound to suitable latex and reacted with a patient
test sample or preferably with the precipitate itself having been
separated from the rest of the patient plasma, in accordance with
known methods. The amount of agglutination of the latex is
proportional to the amount of the CRP complex in the sample.
[0079] Alternatively, immunoassays can be performed, such as
ELISA's, according to known methods (sandwich, competition or other
ELISA) in which the existence and/or amount of the complex of
proteins is determined. For example, an antibody bound to solid
phase binds to CRP in the CRP protein complex. Then, a second
labeled antibody is added which also binds to CRP in the CRP
protein complex, thus detecting the complex of proteins. In the
alternative, the second labeled antibody can be specific for the
300 kD protein in the complex. Or, in a different assay, the
antibody bound to solid phase can bind to the 300 kD protein in the
complex, with the second (labeled) antibody binding either to the
300 kD protein or to CRP. Such immunoassays could likewise be
adapted to be specific for SAA, where antibodies, bound and
labeled, bind to SAA, or where one antibody binds to SAA and the
other either to CRP or the 300 kD protein. The above techniques are
well known to those of ordinary skill in the art and are outlined
in Antibodies, A Laboratory Manual, Harlow, Ed and Lane, David,
Cold Spring Harbor Laboratory, 1988, the subject matter of which is
incorporated herein by reference.
[0080] It is to be understood that the invention described and
illustrated herein is to be taken as a preferred example of the
same, and that various changes in the methods of the invention may
be resorted to, without departing from the spirit of the invention
or scope of the claims.
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