U.S. patent application number 11/244197 was filed with the patent office on 2006-08-03 for methods and kits for detecting heparin/platelet factor 4 antibodies.
Invention is credited to Susan Andrelczyk, David Milunic.
Application Number | 20060172438 11/244197 |
Document ID | / |
Family ID | 36010988 |
Filed Date | 2006-08-03 |
United States Patent
Application |
20060172438 |
Kind Code |
A1 |
Milunic; David ; et
al. |
August 3, 2006 |
Methods and kits for detecting heparin/platelet factor 4
antibodies
Abstract
Methods for determining the presence of heparin/platelet factor
4 antibodies in a sample suspected to contain heparin/platelet
factor 4 antibodies are provided, along with apparatus suitable for
performing the methods. The method depends upon a color
visualization indicating the presence or absence of
heparin/platelet factor 4 antibodies in the sample. Preferred
methods comprise contacting the sample with particles being
complexed to platelet factor 4 (PF4) and which particle-complexed
PF4 reacts specifically with heparin/platelet factor 4 antibodies,
passing the sample/particle mixture through a filter, and then
analyzing the color of the filtrate. The presence of
heparin/platelet factor 4 antibodies in the sample is established
where the color of the filtrate is substantially different from the
color of the receptor-bearing particles.
Inventors: |
Milunic; David; (Deptford,
NJ) ; Andrelczyk; Susan; (West Deptford, NJ) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST ST
NEW YORK
NY
10017
US
|
Family ID: |
36010988 |
Appl. No.: |
11/244197 |
Filed: |
October 4, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60615622 |
Oct 4, 2004 |
|
|
|
Current U.S.
Class: |
436/524 ;
436/526 |
Current CPC
Class: |
B01L 2300/0681 20130101;
G01N 33/6893 20130101; G01N 2400/40 20130101; G01N 33/54313
20130101; G01N 33/6863 20130101; G01N 2333/522 20130101; B01L
2200/16 20130101; G01N 33/86 20130101; B01L 3/5023 20130101; B01L
2400/0683 20130101; G01N 2800/222 20130101; G01N 33/564
20130101 |
Class at
Publication: |
436/524 ;
436/526 |
International
Class: |
G01N 33/551 20060101
G01N033/551; G01N 33/553 20060101 G01N033/553 |
Claims
1. A method of detecting heparin/platelet factor 4 antibodies to
diagnose heparin-induced thrombocytopenia (HIT) in a subject
suspected of having HIT, said method comprising: (a) contacting a
sample obtained from the subject with particles to form a test
mixture, said particles being complexed to platelet factor 4 (PF4),
which particle-complexed PF4 reacts specifically with
heparin/platelet factor 4 antibodies, and said particles forming
specific aggregates upon contacting heparin/platelet factor 4
antibodies; and (b) analyzing the test mixture to detect said
specific aggregates using a particulate immunofiltration assay,
wherein the presence of said specific aggregates indicates HIT in
the subject.
2. The method of claim 1, wherein the particulate immunofiltration
assay comprises: (a) passing the test mixture through a filter
means comprising apertures which are larger than the particles but
smaller than the specific aggregates, thereby producing a filtrate;
and (b) passing said filtrate through a wicking means adjacent and
in fluid communication with the filter means, said wicking means
separating any non-specifically aggregated particles and/or any
unaggregated particles from said specific aggregates, wherein the
unaggregated particles migrate horizontally through said wicking
means at a rate faster than said specific aggregates, wherein the
presence of non-specifically aggregated or unaggregated particles
in the filtrate indicates the absence of heparin/platelet factor 4
antibodies in the sample and the absence of non-specifically
aggregated or unaggregated latex particles in the filtrate
indicates the presence of the heparin/platelet factor 4 antibodies
in the sample.
3. A method for detecting the presence of heparin/platelet factor 4
antibodies in a sample, said method comprising: (a) forming a test
mixture by incubating the sample with particles complexed to
platelet factor 4 (PF4), which particle-complexed PF4 reacts
specifically with heparin/platelet factor 4 antibodies, and said
particles forming specific aggregates upon contacting
heparin/platelet factor 4 antibodies; (b) passing the test mixture
through a filter means comprising apertures which are larger than
the particles but smaller than the specific aggregates, thereby
producing a filtrate; and (c) passing said filtrate through a
wicking means adjacent and in fluid communication with the filter
means, said wicking means separating any non-specifically
aggregated particles and/or any unaggregated particles from the
specific aggregates, wherein the unaggregated particles and
non-specifically aggregated particles migrate horizontally through
said wicking means at a rate faster than said specific aggregates,
wherein the presence of non-specifically aggregated or unaggregated
particles in the filtrate indicates the absence of heparin/platelet
factor 4 antibodies in the sample and the absence of
non-specifically aggregated or unaggregated particles in the
filtrate indicates the presence of heparin/platelet factor 4
antibodies in the sample.
4. The method of claim 2 or 3, wherein the sample comprises a
mammalian bodily fluid.
5. The method of claim 2 or 3, wherein the sample comprises a human
bodily fluid.
6. The method of claim 2 or 3, wherein the sample is selected from
the group consisting of blood, serum, plasma, urine, and
saliva.
7. The method of claim 2 or 3, wherein the particles have a
visually recognizable color which can be ascertained by the unaided
eye.
8. The method of claim 2 or 3, wherein the particles have a mean
diameter from about 0.1 micrometers to about 10 micrometers.
9. The method of claim 8, wherein the particles have a mean
diameter from about 0.2 micrometers to about 0.6 micrometers.
10. The method of claim 2 or 3, wherein the particles are
microspheres.
11. The method of claim 2 or 3, wherein the particles are
non-spherical.
12. The method of claim 2 or 3, wherein the particles are
anionic.
13. The method of claim 2 or 3, wherein the particles comprise
polystyrene.
14. The method of claim 2 or 3, wherein the particles comprise
latex.
15. The method of claim 14, wherein the particles comprise latex in
the concentration of from about 0.01% w/v to about 2% w/v.
16. The method of claim 15, wherein the particles comprise latex in
the concentration of from about 0.3% w/v to about 0.4% w/v.
17. The method of claim 2 or 3, wherein the particles comprise
metal colloid.
18. The method of claim 17, wherein the metal colloid is gold
colloid.
19. The method of claim 2 or 3, wherein the particles have been
dried.
20. The method of claim 2 or 3, wherein the particles are sealed in
a glass ampoule.
21. The method of claim 2 or 3, wherein particles are stabilized
with a colloidal stabilizer.
22. The method of claim 21, wherein the colloidal stabilizer
comprises sodium tripolyphosphate.
23. The method of claim 22, wherein the sodium tripolyphosphate is
in the concentration range of from about 0.001% w/v to about 0.1%
w/v.
24. The method of claim 23, wherein the sodium tripolyphosphate is
in the concentration range of from about 0.01% w/v to about 0.1%
w/v.
25. The method of claim 21, wherein the colloidal stabilizer
comprises an anionic detergent.
26. The method of claim 25, wherein the anionic detergent comprises
sodium dodecyl sulphate.
27. The method of claim 25, wherein the anionic detergent comprises
sodium laurel sarcosine.
28. The method of claim 25, wherein the anionic detergent comprises
polyoxyethylene sorbitan monolaureate.
29. The method of claim 28, wherein the polyoxyethylene sorbitan
monolaureate is in the concentration range of from about 0.0001%
w/v to about 0.1% w/v.
30. The method of claim 29, wherein the polyoxyethylene sorbitan
monolaureate is in the concentration range of from about 0.001% w/v
to about 0.01% w/v.
31. The method of claim 21, wherein the colloidal stabilizer
comprises sodium polymetaphosphate, sodium phosphate glass, sodium
pyrophosphate or other polyphosphate molecules.
32. The method of clam 21, wherein the colloidal stabilizer
comprises nonionic detergents.
33. The method of claim 21, wherein the colloidal stabilizer
comprises a polyphosphate and one or more detergents.
34. The method of claim 2 or 3, wherein a reaction enhancer
solution is further added to the test mixture.
35. The method of claim 34, wherein the reaction enhancer solution
has a pH of 7.2.
36. The method of claim 34, wherein the reaction enhancer solution
comprises polyethylene glycol, sodium chloride, and glycine.
37. The method of claim 36, wherein the polyethylene glycol is
polyethylene glycol 8000.
38. The method of claim 37, wherein the polyethylene glycol 8000 is
in the range of from about 5% w/v to about 15% w/v.
39. The method of claim 38, wherein the polyethylene glycol 8000 is
in the range of from about 8% w/v to about 12% w/v.
40. The method of claim 36, wherein the sodium chloride is in the
range of from about 0.0% w/v to about 1% w/v.
41. The method of claim 40, wherein the sodium chloride is in the
range of from about 0.0% w/v to about 0.1% w/v.
42. The method of claim 36, wherein they glycine is in the range of
from about 0.01 molar to about 0.2 molar.
43. The method of claim 42, wherein they glycine is in the range of
from about 0.02 molar to about 0.1 molar.
44. The method of claim 2 or 3, wherein the filter means comprises
a controlled pore polycarbonate membrane.
45. The method of claim 44, wherein the controlled pore
polycarbonate membrane has a pore size from about 2 micrometers to
about 12 micrometers.
46. The method of claim 45, wherein the controlled pore
polycarbonate membrane has a pore size of about 3 micrometers.
47. The method of claim 2 or 3, wherein the wicking means comprises
non-woven fibers of glass or synthetic polymeric material
48. The method of claim 2 or 3, wherein the presence of the
heparin/platelet factor 4 antibodies is visually determined by the
color of the filtrate.
49. The method of claim 2 or 3, wherein the particulate
immunofiltration assay is performed in a reaction cell.
50. A kit for detecting heparin/platelet factor 4 antibodies in a
liquid sample from a subject to diagnose heparin-induced
thrombocytopenia (HIT) in said subject suspected of having HIT,
said kit comprising: (a) a reaction cell comprising particles
complexed to platelet factor 4 (PF4), which particle-complexed PF4
reacts specifically with heparin/platelet factor 4 antibodies, and
said particles forming specific aggregates upon contacting
heparin/platelet factor 4 antibodies; and (b) an assay plate
comprising: (i) a top member having a filter well and an
observation well that is at a fixed distance from said filter well,
said filter well being adapted to receive the liquid sample; (ii) a
filter means adjacent the top member and extending across the
filter well comprising a controlled pore membrane for effecting a
first separation of said specific aggregates from any
non-specifically aggregated particles and/or any unaggregated
particles, wherein said non-specifically aggregated and/or said
unaggregated particles migrate vertically through the filter means;
(iii) a wicking means adjacent and in fluid communication with the
filter means and extending the length of the filter well and the
observation well, said wicking means consisting essentially of a
plurality of fibers for effecting a second separation of said
specific aggregates which passed through said filter means from
said non-specifically aggregated particles and/or said unaggregated
particles, wherein any unaggregated particles migrate horizontally
through said wicking means at a rate faster than said specific
aggregates do, and said unaggregated particles are directly
visually detectable through said observation well; and (iv) a
bottom member adjacent the wicking means; wherein said assay plate
is free of non-mobilizable ligand-specific binder, wherein the
detection of the absence of unaggregated particles in said
observation well indicates the presence of heparin/platelet factor
4 antibodies in said subject thereby diagnosing HIT in the
subject.
51. The kit of claim 50, wherein the sample comprises a mammalian
bodily fluid.
52. The kit of claim 50, wherein the sample comprises a human
bodily fluid.
53. The kit of claim 50, wherein the sample is selected from the
group consisting of blood, serum, plasma, urine, and saliva.
54. The kit of claim 50, wherein the particles have a visually
recognizable color which can be ascertained by the unaided eye.
55. The kit of claim 50, wherein the particles have a mean diameter
from about 0.1 micrometers to about 1.0 micrometers.
56. The kit of claim 55, wherein the particles have a mean diameter
from about 0.2 micrometers to about 0.6 micrometers.
57. The kit of claim 50, wherein the particles are
microspheres.
58. The kit of claim 50, wherein the particles are
non-spherical.
59. The kit of claim 50, wherein the particles are anionic.
60. The kit of claim 50, wherein the particles comprise
polystyrene.
61. The kit of claim 50, wherein the particles comprise latex.
62. The kit of claim 61, wherein the particles comprise latex in
the concentration of from about 0.01% w/v to about 2% w/v.
63. The kit of claim 62, wherein the particles comprise latex in
the concentration of from about 0.3% w/v to about 0.4% w/v.
64. The kit of claim 50, wherein the particles comprise metal
colloid.
65. The kit of claim 64, wherein the metal colloid is gold
colloid.
66. The kit of claim 50, wherein the particles have been dried.
67. The kit of claim 50, wherein the particles are sealed in a
glass ampoule.
68. The kit of claim 50, wherein particles are stabilized with a
colloidal stabilizer.
69. The kit of claim 68, wherein the colloidal stabilizer comprises
sodium tripolyphosphate.
70. The kit of claim 69, wherein the sodium tripolyphosphate is in
the concentration range of from about 0.001% w/v to about 0.1%
w/v.
71. The kit of claim 70, wherein the sodium tripolyphosphate is in
the concentration range of from about 0.01% w/v to about 0.1%
w/v.
72. The kit of claim 68, wherein the colloidal stabilizer comprises
an anionic detergent.
73. The kit of claim 72, wherein the anionic detergent comprises
sodium dodecyl sulphate.
74. The kit of claim 72, wherein the anionic detergent comprises
sodium laurel sarcosine.
75. The kit of claim 72, wherein the anionic detergent comprises
polyoxyethylene sorbitan monolaureate.
76. The kit of claim 75, wherein the polyoxyethylene sorbitan
monolaureate is in the concentration range of from about 0.0001%
w/v to about 0.1% w/v.
77. The kit of claim 76, wherein the polyoxyethylene sorbitan
monolaureate is in the concentration range of from about 0.0.001%
w/v to about 0.01% w/v.
78. The kit of claim 68, wherein the colloidal stabilizer comprises
sodium polymetaphosphate, sodium phosphate glass, sodium
pyrophosphate or other polyphosphate molecules.
79. The kit of clam 68, wherein the colloidal stabilizer comprises
nonionic detergents.
80. The kit of claim 68, wherein the colloidal stabilizer comprises
a polyphosphate and one or more detergents.
81. The kit of claim 50, wherein the reaction cell further
comprises a reaction enhancer solution is further added to the test
mixture.
82. The kit of claim 81, wherein the reaction enhancer solution has
a pH of 7.2.
83. The kit of claim 81, wherein the reaction enhancer solution
comprises polyethylene glycol, sodium chloride, and glycine.
84. The kit of claim 83, wherein the polyethylene glycol is
polyethylene glycol 8000.
85. The kit of claim 84, wherein the polyethylene glycol 8000 is in
the range of from about 5% w/v to about 15% w/v.
86. The kit of claim 85, wherein the polyethylene glycol 8000 is in
the range of from about 8% w/v to about 12% w/v.
87. The kit of claim 83, wherein the sodium chloride is in the
range of from about 0.0% w/v to about 1% w/v.
88. The kit of claim 87, wherein the sodium chloride is in the
range of from about 0.0% w/v to about 0.1% w/v.
89. The kit of claim 83, wherein they glycine is in the range of
from about 0.01 molar to about 0.2 molar.
90. The kit of claim 89, wherein they glycine is in the range of
from about 0.02 molar to about 0.1 molar.
91. The kit of claim 50, wherein the filter membrane comprises a
controlled pore polycarbonate membrane.
92. The kit of claim 91, wherein the controlled pore polycarbonate
membrane has a pore size from about 2 micrometers to about 12
micrometers.
93. The kit of claim 92, wherein the controlled pore polycarbonate
membrane has a pore size of about 3 micrometers.
94. The kit of claim 50, wherein the wicking means comprises
non-woven fibers of glass or synthetic polymeric material
95. The method of claim 2 or 3, wherein the particulate
immunofiltration assay is performed in a reaction cell.
96. A kit for detecting heparin/platelet factor 4 antibodies in a
sample to diagnose heparin-induced thrombocytopenia (HIT) in a
subject suspected of having HIT, said kit comprising: (a) a
reaction cell comprising particles complexed to platelet factor 4
(PF4), which particle-complexed PF4 reacts specifically with
heparin/platelet factor 4 antibodies, and said particles forming
specific aggregates upon contacting heparin/platelet factor 4
antibodies; and wherein the presence of heparin/platelet factor 4
antibodies indicates HIT in the subject.
97. An apparatus for detecting heparin/platelet factor 4 antibodies
in a fluid sample comprising: (a) a tower comprising at least one
foot and a block channel foot; (b) an ampoule support having at
least one slot for holding an ampoule and a reagent well; (c) a
cover with an opening for receiving the tower such that the at
least one foot crushes a first ampoule in the ampoule support while
the block channel foot blocks the flow of fluid flow to the reagent
well; and (d) a bottom plate with an indentation for holding a test
strip, the bottom plate suitable for coupling to the ampoule
support, which in-turn holds an ampoule and is covered by the
cover; whereby the tower is depressed into the cover to crush the
first ampoule to allow a first reagent in the first ampoule to flow
out while the block channel foot blocks the flow of the first
reagent to the reagent well, and wherein withdrawing the tower
allows the first reagent to flow to the reagent well.
98. The apparatus of claim 97, wherein the tower further comprises
a spur to crush a second ampoule.
99. The apparatus of claim 97, wherein a second reagent in the
second ampoule is mixed with the first reagent prior to flowing
into the reagent well.
100. The apparatus of claim 97, wherein the spur is adjacent to the
block channel foot.
101. The apparatus of claim 97, wherein the ampoule support
comprises a channel connected to the reagent well.
102. The apparatus of claim 97, wherein the bottom plate, the test
trip, the ampoule support, and the cover are engaged to form a test
device operable by depressing and withdrawing the tower.
Description
1. FIELD OF THE INVENTION
[0001] The present invention relates to methods and kits useful for
detecting heparin-induced thrombocytopenia (HIT) in a subject
suspected of having HIT. In particular, the invention relates to
methods and kits for the detection of heparin/platelet factor 4
antibodies in a sample by particulate immunofiltration assays,
which are faster, simpler, and less expensive to operate than those
previously known in the art.
2. BACKGROUND OF THE INVENTION
[0002] Thrombocytopenia is a disorder in which the number of
platelets in the blood is abnormally low. Drug-induced immune
thrombocytopenia is a condition where the use of certain drugs
leads to the formation of antibodies against platelets. These
antibodies can cause a decrease in platelet count, resulting in the
potential for increased bleeding and decreased ability for
clotting. If these antibodies are formed during pregnancy, they may
pass from the mother to the fetus.
[0003] Heparin is the most widely used intravenous anticoagulant
and one of the most widely prescribed drugs in the United States.
More than 1 trillion units are administered annually to
approximately 12 million patients. Intravenous heparin is commonly
used for the prophylaxis and treatment of thromboembolic disease,
as well as numerous other applications including certain types of
lung and heart disorders, and during or after a variety of surgery
including open heart, bypass, dialysis and orthopedic procedures.
Heparin is also used for diagnostic and therapeutic interventional
radiologic procedures. Due to the widespread use of unfractionated
and low molecular weight heparins, heparin-induced thrombocytopenia
(HIT) is considered to be the most frequent (and potentially the
most devastating) drug-induced thrombocytopenia (Picker S. M. et
al. Pathophysiology, epidemiology, diagnosis and treatment of
heparin-induced thrombocytopenia (HIT). Eur J Med Res. 2004 Apr.
30;9(4):180-5. Review).
[0004] HIT is classified into Type I and Type II, Type I being
benign and Type II severe. Type I HIT occurs early after heparin
initiation, the platelet levels are reduced only slightly and
usually return to normal even when heparin treatment is continued.
Thromboembolic complications are rare and Type I HIT is not
antibody-mediated.
[0005] In contrast, Type II HIT is caused by antibody formation to
heparin-platelet factor 4 complexes (Harenberg J. et al.
Heparin-induced thrombocytopenia: pathophysiology and new treatment
options. Pathophysiol Haemost Thromb. 2002
September-December;32(5-6):289-94. Review). Type II HIT typically
develops between 5 and 14 days after heparin therapy is started.
The hallmark symptoms of Type II HIT are a drastic fall in platelet
count and thrombosis, which can lead to limb gangrene (requiring
leg amputation) or even death. Other symptoms of Type II HIT may
include cutaneous reactions, from a simple allergic reaction to
lesions to necrosis.
[0006] Type II HIT occurs in approximately 1-5% of patients treated
with heparin (Goor Y. et al. Heparin-induced thrombocytopenia with
thrombotic sequelae: a review. Autoimmun Rev. 2002
August;1(4):183-9. Review). More alarmingly, 25-50% of post-cardiac
surgery patients develop these heparin-dependent antibodies during
the next 5 to 10 days (Warkentin T. E. et al. Heparin-induced
thrombocytopenia and cardiac surgery. Ann Thorac Surg. 2003
December;76(6):2121-31. Review). The rate of mortality and
amputation in Type II HIT is estimated to be 30% and 20%,
respectively (Picker S. M. et al. supra.).
[0007] Early diagnosis of HIT is essential to reduce morbidity and
mortality. Currently, there are three methods of detecting
heparin-induced antibodies: (1) functional tests such as the
14C-serotonin release assay (Sheridan D. et al. A diagnostic test
for heparin-induced thrombocytopenia. Blood. 1986
January;67(1):27-30); (2) platelet aggregation tests (Chong B. H.
et al. The clinical usefulness of the platelet aggregation test for
the diagnosis of heparin-induced thrombocytopenia. Thromb Haemost.
1993 Apr. 1;69(4):344-50); and (3) immunoassays such as
enzyme-linked immunosorbent assay (ELISA). Although serologic
assays to detect and identify platelet-reactive antibodies have
progressed from less sensitive and specific Phase I tests (e.g.,
those that measure platelet functional endpoints) through more
sensitive Phase II assays (e.g., those that detect
platelet-associated immunoglobulins), to highly specific Phase III
assays (e.g., those that detect antibodies bound to alloantigens
located on isolated platelet surface glycoproteins), these tests
are used primarily as confirmation of HIT after the symptoms are
seen in a patient and take many hours to perform. A more efficient,
sensitive and specific assay to diagnose HIT remains elusive.
3. SUMMARY OF THE INVENTION
[0008] To achieve the aforementioned objectives, the inventors have
invented methods and kits for detecting heparin/platelet factor 4
antibodies in a variety of substances. The methods and kits depend
upon a color visualization methodology indicating the presence or
absence of heparin/platelet factor 4 antibodies. Preferably, the
color visualization does not require the use of complicated
instrumentation or equipment such that all color changes are
readily detected by the naked human eye.
[0009] The invention is based, in part, on the inventors'
surprising discovery that complexing isolated platelet factor 4
(PF4) to a particle, preferably a spherical particle, induces a
conformational change in the PF4 molecule such that it reacts
specifically with heparin/platelet factor 4 antibodies (i.e.,
antibodies that detect PF4 complexed to heparin) in patient
samples. The surprising discovery is the basis for methods and kits
comprising isolated PF4 complexed to colored particles, preferably
spherical particles or beads, in rapid, efficient, sensitive,
specific particulate immunofiltration assays to detect HIT.
[0010] In certain embodiments, the invention provides methods that
use a particulate immunofiltration assay (PIFA.RTM.) (Akers
Biosciences, Inc., Thorofare, N.J.) to detect heparin/platelet
factor 4 antibodies in a liquid sample. The preferred method of the
invention comprises incubating the sample with colored,
particularly color detectable by the naked eye, particles being
complexed to platelet factor 4 (PF4) ("PF4-complexed particles")
and which particle-complexed PF4 reacts specifically with
heparin/platelet factor 4 antibodies such that the particles have
the capacity to form specific aggregates upon contacting
heparin/platelet factor 4 antibodies. As used herein, the term
"specific aggregates" refer to aggregates that form because of this
antibody-antigen interaction. The sample/particles mixture is then
passed through a filter having apertures which are larger than the
particles but smaller than the specific aggregates, in order to
remove any specific and/or non-specific aggregates from the
filtrate. The filtrate is then passed through a wicking membrane
that is adjacent to the filter, in order to separate unaggregated
particles from any non-specifically aggregated particles as well as
any remaining specific aggregates, the unaggregated particles being
able to migrate horizontally through the wicking membrane at a rate
faster than both the non-specifically aggregated particles and
specific aggregates. The color of the filtrate is then analyzed,
wherein the absence of color suggests the presence of
heparin/platelet factor 4 antibodies and the presence of color
suggests the absence of heparin/platelet factor 4 antibodies. In
one embodiment, the color of the filtrate is analyzed by comparing
with a visual standard corresponding to a known concentration of
the particles, wherein the presence of heparin/platelet factor 4
antibodies is established where the color of the filtrate is
substantially different from the color of the visual standard, and
the absence of heparin/platelet factor 4 antibodies is established
where the color of the filtrate is substantially similar to the
color of the visual standard.
[0011] In a preferred embodiment, the particles are spherical,
preferably microspheres. In another specific embodiment, the
particles are non-spherical.
[0012] In one embodiment, after complexing with PF4, the particles
are dried and/or sealed in a glass ampoule after being complexed to
PF4.
[0013] In another embodiment, the particles have a mean diameter
from about 0.01 micrometers to about 100 micrometers, preferably
about 0.1 micrometers to about 10 micrometers, more preferably
about 0.2 micrometers to about 0.6 micrometers, most preferably
about 0.3 micrometers.
[0014] In one embodiment, the particles are anionic compounds and
have negative charges on their surfaces. In preferred embodiments,
the particles are made of polyanionic compounds or have polyanionic
charges on their surfaces. In a preferred embodiment, the particles
comprise latex. In a specific embodiment, the particles comprise
latex in the concentration of from about 0.01% w/v to about 2% w/v,
preferably from about 0.3% w/v to about 0.4% w/v.
[0015] In another preferred embodiment, the particles comprise
polystyrene. In a specific embodiment, the particles comprise
polystyrene or styrene primary amino latex.
[0016] In another preferred embodiment, the particles comprise a
metal colloid. In a specific embodiment, the particles comprise
gold colloid.
[0017] In certain embodiments, the particles are stabilized with a
colloidal stabilizer. In a specific embodiment, the colloidal
stabilizer comprises sodium tripolyphosphate in the concentration
range of from about 0.001% w/v to about 0.1% w/v, preferably from
about 0.01% w/v to about 0.1% w/v.
[0018] In another specific embodiment, the colloidal stabilizer
comprises one or more anionic detergents selected from the group
consisting of sodium dodecyl sulphate, sodium laurel sarcosine,
polyoxyethylene sorbitan monolaureate, sodium polymetaphosphate,
sodium phosphate glass (i.e., sodium hexametaphosphate), sodium
pyrophosphate, and other polyphosphate molecules. In preferred
embodiments, the one or more anionic detergents are in the
concentration range of from about 0.0001% w/v to about 0.1% w/v,
preferably from about 0.001% w/v to about 0.01% w/v.
[0019] In yet another specific embodiment, the colloidal stabilizer
comprises a non-ionic detergent.
[0020] In specific embodiments, the PF4 complexed to the particles
reacts specifically with heparin/platelet factor 4 antibodies such
that the particles have the capacity to form aggregates upon
contacting heparin/platelet factor 4 antibodies. The particles are
incubated with the sample for a length of time sufficient for
aggregates to form, preferably for about 5 minutes. More
preferably, a reaction enhancer solution is added to the
sample/particles mixture to optimize speed and sensitivity of the
aggregation reaction.
[0021] In one embodiment, the reaction enhancer solution has a pH
of 7.2. In a specific embodiment, the reaction enhancer solution
comprises polyethylene glycol, sodium chloride, and glycine. In a
more specific embodiment, the reaction enhancer solution comprises
polyethylene glycol 8000 in the range of from about 5% w/v to about
15% w/v, preferably from about 8% w/v to about 12% w/v. In another
more specific embodiment, the reaction enhancer solution comprises
either no sodium chloride (i.e., about 0.0%) or up to about 1% w/v,
preferably about 0.1% w/v. In yet another more specific embodiment,
the reaction enhancer solution comprises glycine in the range of
from about 0.01 molar to about 0.2 molar, preferably from about
0.02 molar to about 0.1 molar.
[0022] In one embodiment, the filter comprises a controlled pore
membrane. In a specific embodiment, the filter comprises a
controlled pore polycarbonate membrane. In preferred embodiments,
the filter has apertures which are larger than the particles but
smaller than the aggregates. In a specific embodiment, the
apertures are from about 5 to about 15 larger than the particles.
In another specific embodiment, the apertures are from about 10 to
about 12 larger than the particles.
[0023] In one embodiment, the filter separates a majority of any
specific aggregates from any non-specifically aggregated particles
and/or any unaggregated particles. In a specific embodiment, the
filter separates about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%,
99%, or all specific aggregates from any non-specifically
aggregated particles and/or any unaggregated particles. In a
preferred embodiment, the filter separates more than 90% of
specific aggregates from any non-specifically aggregated particles
and/or any unaggregated particles. The specific aggregates may be
separated from the non-specifically aggregated particles and
unaggregated particles based on, for example, size and/or weight,
and the level of separation can be determined and/or confirmed by
separation methods known to one skilled in the art (e.g., mass
spectroscopy, chromatography, etc.).
[0024] In one embodiment, the wicking membrane comprises a
polymeric material. In a specific embodiment, the wicking membrane
comprises non-woven fibers of glass or synthetic polymeric
material. In a preferred embodiment, the wicking membrane comprises
polyester.
[0025] In one embodiment, the wicking membrane separates a majority
of any unaggregated particles from any non-specifically aggregated
particles and/or any aggregates. In a specific embodiment, the
wicking membrane separates about 50%, 60%, 70%, 80%, 90%, 95%, 96%,
97%, 98%, 99%, or all unaggregated particles from any
non-specifically aggregated particles and/or any specific
aggregates. In a preferred embodiment, the wicking membrane
separates more than 90% of unaggregated particles from any
non-specifically aggregated particles and/or any specific
aggregates. The unaggregated aggregates may be separated from the
non-specifically aggregated particles and specific aggregates based
on, for example, size and/or weight, and the level of separation
can be determined and/or confirmed by separation methods known to
one skilled in the art (e.g., mass spectroscopy, chromatography,
etc.).
[0026] In one embodiment, the unaggregated particles migrate
horizontally through the wicking membrane at a rate faster than the
non-specifically aggregated particles. In a specific embodiment,
the unaggregated particles migrate horizontally through the wicking
membrane at a rate from about 2 to about 10 times (i.e., 2, 3, 4,
5, 6, 7, 8, 9, and 10) faster than the non-specifically aggregated
particles. In a preferred embodiment, the unaggregated particles
migrate horizontally through the wicking membrane at a rate greater
than 10 times (e.g., greater than 10, 11, 12, 13, 14, 15, 20, 30,
50, 100, etc.) faster than the non-specifically aggregated
particles.
[0027] In another embodiment, the unaggregated particles migrate
horizontally through the wicking membrane at a rate faster than the
specific aggregates. In a specific embodiment, the unaggregated
particles migrate horizontally through the wicking membrane at a
rate from about 2 to about 10 times (i.e., 2, 3, 4, 5, 6, 7, 8, 9,
and 10) faster than the specific aggregates. In a preferred
embodiment, the unaggregated particles migrate horizontally through
the wicking membrane at a rate greater than 10 times (e.g., greater
than 10, 11, 12, 13, 14, 15, 20, 30, 50, 100, etc.) faster than the
specific aggregates.
[0028] In certain embodiments, the color of the filtrate is
compared visually, without aid by a machine. In certain other
embodiments, the color of the filtrate is compared optically, with
aid by a machine. In a specific embodiment, the color of the
filtrate is compared, visually or optically, against a standard
corresponding to a known concentration of the particles.
[0029] In one embodiment, the presence of heparin/platelet factor 4
antibodies is established where the color of the filtrate is
substantially different from the color of the particles (e.g., the
filtrate lacks color because particles aggregate and are retained).
In another embodiment, the absence of heparin/platelet factor 4
antibodies is established where the color of the filtrate is
substantially similar to the color of the particles. In certain
embodiments, the color of the filtrate and the color of the
particles are quantified and compared by optical means, a
reflectometer, or other methods well known to one skilled in the
art.
[0030] In one embodiment, the sample is a liquid sample obtained
from a subject. In a specific embodiment, the sample comprises a
mammalian bodily fluid. In a preferred embodiment, the sample
comprises a human bodily fluid such as blood, serum, plasma or
urine. As used herein, a subject is preferably a mammal such as a
non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a
primate (e.g., monkey and human), most preferably a human.
[0031] The present invention also provides kits comprising a
reaction cell. The kits can also comprise an assay plate suitable
for performance of the methods disclosed. The reaction cells
comprise PF4-complexed particles. The assay plates comprise a top
member having a filter well and an observation well that is at a
fixed distance from said filter well, a filter means adjacent the
top member and extending across the filter well, a wicking means
adjacent and in fluid communication with the filter means and
extending the length of the filter well and the observation well,
and a bottom adjacent the wicking means.
[0032] In certain embodiments, the kits optionally include a blood
separator apparatus as described in U.S. Patent Application No.
60/599,803, filed Aug. 5, 2004, attorney docket no. 8189-027-888,
which is incorporated herein by reference in its entirety.
[0033] In certain embodiments, the reaction cell comprises
PF4-complexed particles. In one embodiment, the sample is mixed
with the PF4-complexed particles in the reaction cell. In one
embodiment, the reaction cell comprises a breakable vessel that
contains PF4-complexed particles. In one embodiment, the reaction
cell further comprises a kill solution having the capacity to
biologically inactivate any infectious agents in the sample.
[0034] In certain embodiments, the assay plates comprise a top
member, a filter means, a wicking means, and a bottom member. In a
specific embodiment, the top member comprises a filter well and an
observation well that is at a fixed distance from said filter well.
In a specific embodiment, the filter means is adjacent the top
member and extending across the filter well. In a specific
embodiment, the wicking means is adjacent and in fluid
communication with the filter means and extending the length of the
filter well and the observation well. In a specific embodiment, the
bottom member is adjacent the wicking means. In preferred
embodiments, the top member, filter means, wicking means, and
bottom member are held in position with an appropriately applied
adhesive.
[0035] In one embodiment, the top member comprises a material that
is substantially impermeable to aqueous solutions such as those
associated with the human body. In a preferred embodiment, the top
member comprises polystyrene. In certain embodiments, the top
member receives the sample/particles mixture.
[0036] In one embodiment, the filter means comprises a controlled
pore membrane. In a specific embodiment, the filter means comprises
a controlled pore polycarbonate membrane. In preferred embodiments,
the filter means has apertures which are larger than the particles
but smaller than the aggregates. In a specific embodiment, the
apertures are from about 5 times to about 15 times larger than the
particles. In another specific embodiment, the apertures are from
about 10 times to about 12 times larger than the particles. In yet
another specific embodiment, the apertures are from about 2
micrometers to about 12 micrometers, preferably about 3
micrometers.
[0037] In one embodiment, the filter means separates a majority of
any specific aggregates from any non-specifically aggregated
particles and/or any unaggregated particles. In specific
embodiments, the filter means separates about 50%, 60%, 70%, 80%,
90%, 95%, 96%, 97%, 98%, 99%, or all specific aggregates from any
non-specifically aggregated particles and/or any unaggregated
particles. In a preferred embodiment, the filter means separates
more than 90% of specific aggregates from any non-specifically
aggregated particles and/or any unaggregated particles. The
specific aggregates may be separated from the non-specifically
aggregated particles and unaggregated particles based on, for
example, size and/or weight, and the level of separation can be
determined and/or confirmed by separation methods known to one
skilled in the art (e.g., mass spectroscopy, chromatography,
etc.).
[0038] In one embodiment, the wicking means comprises a polymeric
material. In a specific embodiment, the wicking membrane comprises
non-woven fibers of glass or synthetic polymeric material. In a
preferred embodiment, the wicking membrane comprises polyester. In
preferred embodiments, the wicking means separates a majority of
any unaggregated particles from any non-specifically aggregated
particles and/or any aggregates. In specific embodiments, the
wicking means separates about 50%, 60%, 70%, 80%, 90%, 95%, 96%,
97%, 98%, 99%, or all unaggregated particles from any
non-specifically aggregated particles and/or any specific
aggregates. In a preferred embodiment, the wicking means separates
more than 90% of unaggregated particles from any non-specifically
aggregated particles and/or any specific aggregates. Preferably,
the unaggregated particles migrate horizontally through the wicking
membrane at a rate faster than the non-specifically aggregated
particles and specific aggregates. In a specific embodiment, the
unaggregated particles migrate horizontally through the wicking
membrane at a rate from about 2 to about 10 times (i.e., 2, 3, 4,
5, 6, 7, 8, 9, and 10) faster than the non-specifically aggregated
particles or specific aggregates. In a preferred embodiment, the
unaggregated particles migrate horizontally through the wicking
membrane at a rate greater than 10 times (e.g., greater than 10,
11, 12, 13, 14, 15, 20, 30, 50, 100, etc.) faster than the
non-specifically aggregated particles or specific aggregates.
[0039] In one embodiment, the bottom member comprises a relatively
non-rigid material. In a specific embodiment, the bottom member
comprises a vinyl polymer.
[0040] In one embodiment, the assay plate further comprises a
substrate positioned between the top member and the filter means
and extending across the filter well. In a preferred embodiment,
the substrate is a glass substrate that contains PF4-complexed
particles and one or more reagents that promote the agglutination
(i.e., aggregation) reaction.
[0041] In one embodiment, the assay plate further comprises a
barrier positioned between the wicking means and the bottom member
and is as long as the wicking means. In a preferred embodiment, the
barrier prevents the wicking means from coming into direct contact
with the bottom member.
[0042] In one embodiment, an apparatus for detecting
heparin/platelet factor 4 antibodies in a fluid sample comprises a
tower having at least one foot, a block channel foot, an ampoule
support with a slot for holding an ampoule and a reagent well into
which a reagent from the ampoule flows following crushing of the
ampoule by the tower. The apparatus also comprises a cover with an
opening for receiving the tower such that the foot crushes the
ampoule in the ampoule support while the block channel foot blocks
the flow of fluid flow to the reagent well. Thus, when the tower is
withdrawn, the block channel foot is also withdrawn and allows flow
of reagent via the channel to the reagent well. A bottom plate
holding a test strip is below the reagent well for receiving the
reagent onto the test strip. Preferably, the bottom plate couples
to the ampoule support, which in-turn holds an ampoule and is
covered by the cover. When the tower is depressed into the cover to
crush the ampoule the reagent in the ampoule is released while the
block channel foot blocks the flow of the first reagent flow to the
reagent well via the channel.
[0043] Further, another ampoule may be simultaneously be crushed by
a second foot or a spur on the block channel foot to allow mixing
of two or more reagents prior to flowing on to the reagent well via
the channel. Preferably, the spur is adjacent to the block channel
foot and the ampoule support comprises the channel connected to the
reagent well.
[0044] Preferably, the bottom plate, the test trip, the ampoule
support, and the cover are engaged to form a test device operable
by depressing and withdrawing the tower.
[0045] The many advantages and details of the invention are
described further by the following illustrative figures and the
detailed description.
4. FIGURES
[0046] FIGS. 1-7 are taken from U.S. Pat. No. 5,565,366, which is
incorporated herein by reference in its entirety.
[0047] FIG. 1 is a top plan view of an assay plate useful according
to the preferred methods and kits of the of the present
invention.
[0048] FIG. 2 is an expanded sectional view of an assay plate
useful according to the preferred methods and kits of the of the
present invention.
[0049] FIG. 3 is an expanded sectional view of a preferred assay
plate useful according to the preferred methods and kits of the of
the present invention having a barrier between the wicking means
and the bottom member.
[0050] FIG. 4 is an expanded sectional view of an assay plate
useful according to the preferred methods and kits of the present
invention having a substrate beneath the filter well.
[0051] FIG. 5 is a perspective view of a reaction cell useful
according to the preferred kits of the present invention.
[0052] FIG. 6 is a perspective view of a reaction cell useful
according to the preferred kits of the present invention comprising
"kill" solution in a compartment.
[0053] FIG. 7 is a perspective view of a reaction cell useful
according to the preferred kits of the present invention comprising
"kill" solution in a breakable vessel.
[0054] FIG. 8 is an exploded view of an assembly suitable for
performing an assay in accordance with the present invention.
[0055] FIG. 9 is a second exploded view of an assembly suitable for
performing an assay in accordance with the present invention.
5. DETAILED DESCRIPTION OF THE INVENTION
[0056] Type II HIT is mediated by an antibody that recognizes an
epitope on the platelet protein designated "platelet factor 4"
(PF4) that is created when PF4 is complexed to heparin (Horsewood
P. et al. The epitope specificity of heparin-induced
thrombocytopenia. Br J Haematol. 1996 October;95(1): 161-7). When
heparin binds to PF4, a conformational change in the PF4 molecule
occurs and as a result, exposes neo-epitopes that act as immunogens
(Reilly R. F. The pathophysiology of immune-mediated
heparin-induced thrombocytopenia. Semin Dial. 2003
January-February;16(1):54-60. Review). Many polyanionic compounds
other than heparin can form complexes with PF4 and cause similar
conformational change in the molecule (Visentin G. P. et al.
Heparin is not required for detection of antibodies associated with
heparin-induced thrombocytopenia/thrombosis. J Lab Clin Med. 2001
July;138(1):22-31).
[0057] The invention is based, in part, on inventors' surprising
discovery that complexing isolated platelet factor 4 (PF4) to a
particle, preferably a spherical particle, more preferably a
polyanionic particle (e.g., polystyrene), induces a conformational
change in the PF4 molecule such that it reacts specifically with
heparin/platelet factor 4 antibodies in patient samples. The
surprising discovery is the basis for methods and kits comprising
isolated PF4 complexed to colored particles, preferably spherical
particles or beads, in rapid, efficient, sensitive, specific
particulate immunofiltration assays to detect HIT.
[0058] The methods of the invention comprise incubating a sample
with particles complexed to platelet factor 4 (PF4) ("PF4-complexed
particles"), passing the sample/particles mixture through a filter,
and analyzing the color of the filtrate. The kits of the invention
comprise a reaction cell for mixing and/or incubating the
PF4-complexed particles with the sample. The kits also include an
assay plate for filtering and analyzing the sample/particles
mixture.
[0059] The invention further encompasses using the methods and kits
described above for detecting heparin-induced thrombocytopenia
(HIT) in a subject suspected of having HIT. The methods and kits
can be used in a variety of settings, for example, hospitals,
clinics, physician's offices, clinical laboratories, etc.
[0060] For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the
subsections which follow.
[0061] 5.1 Particulate Immunofiltration Assay
[0062] The particulate immunofiltration assay (PIFA.RTM.) (Akers
Biosciences, Inc., Thorofare, N.J.) is a system that offers
significant advantages in terms of accuracy, ease of use, and
rapidity of results. Its principles are based on the selective
filtration of particles in response to antibody/antigen binding.
Dyed particles coated with antigen or antibody provide the visual
signal for the results of the assay: the presence of a
corresponding antibody or antigen in the test sample will result in
the formation of a matrix of aggregated particles. The ability of
matrixed (aggregated) or non-matrixed (unaggregated) particles to
move through a filter medium is the measure of the positive or
negative reactivity of the test sample.
[0063] In practice, the technology involves combining a test sample
(e.g., blood, serum, plasma, urine, or saliva) with a reagent,
which consists of particles coated with antigen or antibody. During
a period of incubation, the reagent is allowed to react with the
test sample. Test samples containing corresponding antigens or
antibodies (positive samples) will cause the particles to form a
matrix (i.e., aggregates); test samples without these substances
(negative samples) will leave the particles unaggregated. Once the
reagent has reacted with the sample, the mixture is introduced into
a device designed to separate aggregated particles from
non-specifically aggregated particles (i.e., those particles that
form a matrix with each other, or another substance, but not with
the corresponding antigens or antibodies) and/or unaggregated
particles. This device is a composite of several membranes
laminated together, combining controlled pore and liquid flow
dynamic properties. By careful control of pore size and density,
and liquid flow through a fibrous meshwork, aggregated particles
can be efficiently filtered and separated from the rest of the
reaction mixture and prevented from entering the inner layers of
the device. Conversely, unaggregated particles can penetrate the
controlled pore membranes and migrate through the inner membrane
layers.
[0064] Thus, a positive sample produces aggregated particles that
are filtered by the controlled pore membranes; no particles, and
hence no color, are able to migrate into successive membrane
layers. In this case color is only visible in a first viewing
window of the device (see, e.g., (14) of FIG. 1). Conversely, a
negative sample does not produce aggregated particles; the dyed
unaggregated particles pass through the controlled pore membrane
and into successive membrane layers, where they become visible
through a second viewing window in the device.
[0065] Specific embodiments of PIFA.RTM. technology and related
kits are described in fuller detail in U.S. Pat. Nos. 5,231,035;
5,565,366; and 5,827,749, all of which are incorporated by
reference herein in their entireties.
[0066] Specifically, the present invention encompasses the use of
PIFA.RTM. technology to detect heparin/platelet factor 4 antibodies
in a variety of substances. The substance may be of a biological
source or a non-biological source exposed to biological material.
The preferred methods of the invention comprise incubating the
sample with particles being complexed with platelet factor 4
("PF4-complexed particles"). Once complexed with the particles, the
PF4 reacts specifically with heparin/platelet factor 4 antibodies
such that the PF4-complexed particles have the capacity to form
specific aggregates upon contacting heparin/platelet factor 4
antibodies. The sample/particles mixture is then passed through a
filter having apertures which are larger than the particles but
smaller than the specific aggregates, in order to remove any
specific aggregates from the filtrate. The filtrate is then passed
through a wicking membrane that is adjacent to the filter, in order
to separate any unaggregated particles from any non-specifically
aggregated particles as well as any remaining specific aggregates.
The unaggregated particles migrate horizontally through the wicking
membrane at a rate faster than both the non-specifically aggregated
particles and specific aggregates.
[0067] After filtration through the filter and wicking membrane,
the filter is analyzed for color changes, preferably, by comparing
with a visual standard corresponding to a known concentration of
the particles. The presence of heparin/platelet factor 4 antibodies
is established where the color of the filtrate is substantially
different from the color of the particles; the absence of
heparin/platelet factor 4 antibodies is established where the color
of the filtrate is substantially similar to the color of the
particles.
[0068] The particles may be of any lattices which are known or
believed to be employable for latex agglutination, such as
exemplified by the homopolymers and copolymers produced from
styrene or its derivatives such as methylstyrene, ethylstyrene, and
chlorostyrene, olefins such as ethylene and propylene, acrylic acid
or its esters such as methyl acrylate and ethyl acrylate,
methacrylic acid or its derivatives such as ethyl methacrylate,
acrylonitrile, and acrylamide, dienes such as butadiene,
chloroprene, and isoprene, vinyl chloride, vinylidine chloride, and
vinyl acetate. The lattices of homopolymers or copolymers made of
styrene, chlorostyrene, acrylic acid, vinyl toluene, methyl
methacrylate are advantageously used.
[0069] Preferably, the particles the particles are made up of
polyanionic compounds. In specific embodiments, the particles
comprise polyanionic compounds in the concentration of about 0.001%
w/v, about 0.005% w/v, about 0.01% w/v, about 0.05% w/v, about 0.1%
w/v, about 0.2% w/v, about 0.3% w/v, about 0.4% w/v, about 0.5%
w/v, about 0.6% w/v, about 0.7% w/v, about 0.8% w/v, about 0.9%
w/v, about 1.0% w/v, about 1.1% w/v, about 1.2% w/v, about 1.3%
w/v, about 1.4% w/v, about 1.5% w/v, about 1.6% w/v about 1.7% w/v,
about 1.8% w/v, about 1.9% w/v, about 2.0% w/v, about 2.1% w/v,
about 2.2% w/v, about 2.3% w/v, about 2.4% w/v, about 2.5% w/v,
about 2.6% w/v, about 2.7% w/v, about 2.8% w/v, about 2.9% w/v,
about 3.0% w/v, about 5.0% w/v, about 10% w/v, or more. Preferably,
the particles comprise polyanionic compounds in the concentration
of from about 0.01% w/v to about 2% w/v, more preferably from about
0.3% w/v to about 0.4% w/v.
[0070] In a preferred embodiment, the particles comprise latex.
Other useful particles comprise polystyrene, preferably
carboxylated polystyrene, with or without reactive groups to
facilitate reaction with the receptor, such as amino groups, thiol
groups, carboxyl groups or other reactive groups. Butadiene/styrene
copolymers such as carboxylated styrene butadiene or acrylonitrile
butadiene styrene are also useful. In another preferred embodiment,
the particles comprise polystyrene or styrene primary amino
latex.
[0071] Inorganic particles, such as silicas, clay, carbons such as
activated charcoal, and other materials on which the
heparin/platelet factor 4 antibodies can be complexed can be used
in the present invention.
[0072] In another preferred embodiment, the particles comprise
metal colloid such as gold colloid. In a specific embodiment, the
metal colloid is charged.
[0073] In certain embodiments, the particles are stabilized with a
colloidal stabilizer. In specific embodiments, the colloidal
stabilizer comprises sodium tripolyphosphate in the concentration
of about 0.001% w/v, about 0.005% w/v, about 0.01% w/v, about 0.05%
w/v, about 0.1% w/v, about 0.2% w/v, about 0.3% w/v, about 0.4%
w/v, about 0.5% w/v, about 0.6% w/v, about 0.7% w/v, about 0.8%
w/v, about 0.9% w/v, about 1.0% w/v, about 1.1% w/v, about 1.2%
w/v, about 1.3% w/v, about 1.4% w/v, about 1.5% w/v, about 1.6% w/v
about 1.7% w/v, about 1.8% w/v, about 1.9% w/v, about 2.0% w/v,
about 2.1% w/v, about 2.2% w/v, about 2.3% w/v, about 2.4% w/v,
about 2.5% w/v, about 2.6% w/v, about 2.7% w/v, about 2.8% w/v,
about 2.9% w/v, about 3.0% w/v, about 5.0% w/v, about 10% w/v, or
more. Preferably, the colloidal stabilizer comprises sodium
tripolyphosphate in the concentration of from about 0.001% w/v to
about 0.1% w/v, more preferably from about 0.01% w/v to about 0.1%
w/v.
[0074] In another specific embodiment, the colloidal stabilizer
comprises one or more anionic detergents selected from the group
consisting of sodium dodecyl sulphate, sodium laurel sarcosine,
polyoxyethylene sorbitan monolaureate, sodium polymetaphosphate,
sodium phosphate glass (i.e., sodium hexametaphosphate), sodium
pyrophosphate, and other polyphosphate molecules. In specific
embodiments, the colloidal stabilizer comprises the anionic
detergents in the concentration of about 0.0001% w/v, about 00.001%
w/v, about 0.005% w/v, about 0.01% w/v, about 0.05% w/v, about 0.1%
w/v, about 0.2% w/v, about 0.3% w/v, about 0.4% w/v, about 0.5%
w/v, about 0.6% w/v, about 0.7% w/v, about 0.8% w/v, about 0.9%
w/v, about 1.0% w/v, about 1.1% w/v, about 1.2% w/v, about 1.3%
w/v, about 1.4% w/v, about 1.5% w/v, about 1.6% w/v about 1.7% w/v,
about 1.8% w/v, about 1.9% w/v, about 2.0% w/v, about 2.1% w/v,
about 2.2% w/v, about 2.3% w/v, about 2.4% w/v, about 2.5% w/v,
about 2.6% w/v, about 2.7% w/v, about 2.8% w/v, about 2.9% w/v,
about 3.0% w/v, about 5.0% w/v, about 10% w/v, or more. Preferably,
the colloidal stabilizer comprises the anionic detergents in the
concentration of from about 0.0001% w/v to about 0.1% w/v, more
preferably from about 0.001% w/v to about 0.01% w/v.
[0075] In certain other embodiments, the colloidal stabilizer
comprises a non-ionic detergent. Examples of non-ionic detergents
include, but are not limited to, Triton.RTM. X-100 (alkylaryl
polyether alcohol or octyl phenol ethoxylate), Triton.RTM. X-114
(octylphenol-polyethylene glycol ether), octylthioglucoside,
Nonidet.RTM. P-40 ([octylphenoxy]polyethoxyethanol), and
N-octyl-BD-glucopyranoside.
[0076] In one specific preferred embodiment, the particles are
spherical, preferably microspheres. In another specific embodiment,
the particles are non-spherical.
[0077] It is important that the particles are approximately the
same diameter, so that they will easily pass through the same size
filter aperture. In specific embodiments, the particles have mean
diameters of about 0.0001 micrometers, about 0.001 micrometers,
about 0.01 micrometers, about 0.05 micrometers, about 0.1
micrometers, about 0.2 micrometers, about 0.3 micrometers, about
0.4 micrometers, about 0.5 micrometers, about 0.6 micrometers,
about 0.7 micrometers, about 0.8 micrometers, about 0.9
micrometers, about 1.0 micrometers, about 2.0 micrometers, about
3.0 micrometers, about 4.0 micrometers, about 5.0 micrometers,
about 10 micrometers, about 20 micrometers, about 30 micrometers,
about 40 micrometers, about 50 micrometers, about 100 micrometers,
about 200 micrometers, about 300 micrometers, about 400
micrometers, about 500 micrometers, about 1,000 micrometers, or
larger.
[0078] In preferred embodiments, the particles have mean diameters
of about 0.01 micrometers to about 100 micrometers, preferably
about 0.01 micrometers to about 10 micrometers, and more preferably
about 0.2 micrometers to about 0.6 micrometers. Most preferably,
the mean diameter of the particles is about 0.3 micrometers and the
diameters of the particles do not vary from the mean by more than
30%, preferably not by more than 15%, 10%, or 5%.
[0079] The particles preferably have a visually recognizable color
produced by the addition of dyes, pigments, or coatings. For
example, the preparation of dyed polyacrylamide particles is
disclosed in U.S. Pat. No. 4,108,974 in the names of Wegfahrt et
al., which is incorporated herein by reference. It is preferred
that the color be relatively dark, preferably black or dark
blue.
[0080] Preferred particles are the small, uniform diameter colored
polystyrene latex spheres available in a variety of diameters from
Bangs Laboratories (Carmel, Ind.) and Seradyn, Inc. (Indianapolis,
Ind.).
[0081] The complexation of particles with platelet factor 4 to
expose neo-epitopes that react specifically with heparin/platelet
factor 4 antibodies can be effected by any method known in the art
for complexing proteins to particles. The treatment conditions will
understandably vary to some degree depending upon the
physicochemical properties of the selected particles. In a specific
embodiment, PF4 can be bound to particles by adsorption, a process
which is mediated by hydrophobic and ionic interactions between PF4
and the surface of the particles. In accordance with preferred
embodiments, PF4 is covalently bound to the particles. In a
specific embodiment, PF4 can be covalently bound to
carboxylate-modified particles in the presence of, for example,
water-soluble carbodiimide
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC).
[0082] While PF4 can also be chemically bonded to the particles,
the particles may alternatively be coated with a substance to which
PF4 will adhere, so long as the coating does not interfere with the
binding between PF4 and the heparin/platelet factor 4 antibodies.
The amounts of immobilized PF4 and particles are preferably
adjusted so that each of the PF4-complexed particles will aggregate
with one or more of the heparin/platelet factor 4 antibodies when
mixed together for a reasonable time.
[0083] In specific embodiments, the PF4 complexed to the particles
reacts specifically with heparin/platelet factor 4 antibodies such
that the particles have the capacity to form aggregates upon
contacting heparin/platelet factor 4 antibodies. The PF4-complexed
particles are incubated with the sample for a length of time
sufficient for aggregates to form. In certain embodiments, the
PF4-complexed particles are incubated with the sample for at least
30 seconds, at least 1 minute, at least 2 minutes, at least 3
minutes, at least 4 minutes, at least 5 minutes, at least 10
minutes, at least 15 minutes, at least 20 minutes, at least 30
minutes or longer for aggregates to form. In certain embodiments,
the PF4-complexed particles are incubated with the sample for no
longer than 30 minutes, no longer than 20 minutes, no longer than
15 minutes, no longer than 10 minutes, no longer than 5 minutes, no
longer than 4 minutes, no longer than 3 minutes, no longer than 2
minutes, no longer than 1 minute, no longer than 30 seconds or less
for aggregates to form. In a preferred embodiment, the
PF4-complexed particles are incubated with the sample for about 30
seconds to about 5 minutes. More preferably, a reaction enhancer
solution is added to the sample/particles mixture to optimize speed
and sensitivity of the aggregation reaction. In one embodiment, the
particles are dried and/or sealed in a glass ampoule after being
complexed to PF4.
[0084] In one embodiment, the sample is a liquid sample obtained
from a subject. As used herein, a subject is preferably a mammal
such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats,
etc.) or a primate (e.g., monkey and human), most preferably a
human. In a specific embodiment, the sample comprises a mammalian
bodily fluid. Preferably, the sample comprises a human bodily fluid
such as blood, serum, plasma, or urine.
[0085] The sample of interest and the PF4-complexed particles may
be contacted in a number of ways. In a preferred method, the sample
is mixed with a solution that contains the PF4-complexed particles
and other reagents helpful to promote the aggregation reaction,
forming a test mixture. An interval of time is permitted to pass
which is sufficient for aggregation to occur or for aggregates to
otherwise form. Alternatively, the sample can be passed through a
substrate such as a glass membrane which contains the particles and
other reagents (e.g., polyethylene glycol 8000 and dextran 10,000
MW) helpful to promote the aggregation reaction. Where the sample
contains ligand, aggregates and other moieties will be released
from the substrate. The released aggregates and other moieties also
constitute test mixtures according to this invention.
[0086] In certain embodiments, a reaction enhancer solution is
added to the sample/particles mixture to promote the aggregation
reaction. In one embodiment, the reaction enhancer solution has a
pH of 7.2. In a specific embodiment, the reaction enhancer solution
comprises polyethylene glycol, sodium chloride, and glycine. In a
more specific embodiment, the reaction enhancer solution comprises
polyethylene glycol 8000 in the range of from about 5% w/v to about
15% w/v, preferably from about 8% w/v to about 12% w/v. In another
more specific embodiment, the reaction enhancer solution comprises
either no sodium chloride (i.e., about 0.0%) or up to about 1% w/v,
preferably about 0.1% w/v. In yet another more specific embodiment,
the reaction enhancer solution comprises glycine in the range of
from about 0.01 molar to about 0.2 molar, preferably from about
0.02 molar to about 0.1 molar.
[0087] The test mixture is then exposed to a filter having
apertures which are larger than the particles but generally smaller
than the clumps of ligand/particle aggregates which might have
formed. In one embodiment, the filter comprises a controlled pore
membrane. In a specific embodiment, the filter comprises a
controlled pore polycarbonate membrane.
[0088] The filter should have a defined pore size which is about 5
to about 15 times larger than the latex particle diameter,
preferably about 10 to about 12 times larger, more preferably about
3 micrometers in diameter. It will be appreciated that there may be
some small variance in the diameters of the pores. Preferably, the
pore diameters will not vary from the nominal diameter by more than
30%, preferably not by more than 15%, 10%, or 5%.
[0089] The pore size of the filter is chosen to retain
heparin/platelet factor 4 antibodies/particle aggregates yet permit
the passage of any relatively small aggregates which may be formed
by non-specific aggregation. It will be appreciated that
non-specific aggregation is the aggregation of particles in the
absence of heparin/platelet factor 4 antibodies. The sensitivity of
the assay should be adjusted to produce aggregates larger than the
pore size, roughly 10 to 15 particles in diameter. Preferably, the
filter will be an absolute channel membrane having pores of
controlled diameter.
[0090] The filter separates a majority of any specific aggregates
from any non-specifically aggregated particles and/or any
unaggregated particles. In certain embodiments, the filter
separates about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%,
or all specific aggregates from any non-specifically aggregated
particles and/or any unaggregated particles. In a preferred
embodiment, the filter separates more than 90% of specific
aggregates from any non-specifically aggregated particles and/or
any unaggregated particles. The specific aggregates may be
separated from the non-specifically aggregated particles and
unaggregated particles based on, for example, size and/or weight,
and the level of separation can be determined and/or confirmed by
separation methods known to one skilled in the art (e.g., mass
spectroscopy, chromatography, etc.).
[0091] Preferred controlled pore membranes are which comprise
polycarbonate, such as those commercially available from the
Poretics Corporation (Livermore, Calif.).
[0092] After passing through the filter, the filtrate is passed
through a wicking membrane for further separation. In one
embodiment, the wicking membrane comprises a polymeric material. In
a specific embodiment, the wicking membrane comprises non-woven
fibers of glass or synthetic polymeric material. In a preferred
embodiment, the wicking membrane comprises polyester.
[0093] The wicking membrane separates a majority of any
unaggregated particles from any non-specifically aggregated
particles and/or any specific aggregates. In certain embodiments,
the wicking membrane separates about 50%, 60%, 70%, 80%, 90%, 95%,
96%, 97%, 98%, 99%, or all unaggregated particles from any
non-specifically aggregated particles and/or any specific
aggregates. In a specific embodiment, the wicking membrane
separates more than 90% of unaggregated particles from any
non-specifically aggregated particles and/or any specific
aggregates. The unaggregated aggregates may be separated from the
non-specifically aggregated particles and specific aggregates based
on, for example, size and/or weight, and the level of separation
can be determined and/or confirmed by separation methods known to
one skilled in the art (e.g., mass spectroscopy, chromatography,
etc.).
[0094] The unaggregated particles migrate faster through the
wicking membrane than non-specifically aggregated particles, and
the non-specifically aggregated particles migrate faster through
the wicking membrane than the specific aggregates. In one
embodiment, the unaggregated particles migrate horizontally through
the wicking membrane at a rate faster than the specific aggregates.
In a specific embodiment, the unaggregated particles migrate
horizontally through the wicking membrane at a rate 1,000 times,
500 times, 100 times, 50 times, 30 times, 20 times, 15 times, 10
times, 5 times, 4 times, 3 times, 2 times faster than the
non-specifically aggregated particles. In another specific
embodiment, the unaggregated particles migrate horizontally through
the wicking membrane at a rate 1,000 times, 500 times, 100 times,
50 times, 30 times, 20 times, 15 times, 10 times, 5 times, 4 times,
3 times, 2 times faster than the specific aggregates. In yet
another specific embodiment, the non-specifically aggregated
particles migrate horizontally through the wicking membrane at a
rate 1,000 times, 500 times, 100 times, 50 times, 30 times, 20
times, 15 times, 10 times, 5 times, 4 times, 3 times, 2 times
faster than the specific aggregates.
[0095] Once the mixture is filtered, the filtrate produced thereby
is analyzed for the presence of particles which are unaggregated.
While it will be appreciated that such analysis may be performed by
any of the appropriate physical and/or chemical methods known in
the art, such as centrifugation or particle counting, analysis of
the filtrate is preferably performed by visually inspecting the
filtrate to determine the presence therein of a recognizable color
corresponding to the particles. Thus, where the proportions of
particles and heparin/platelet factor 4 antibodies have been
carefully selected, a qualitative system is established wherein the
presence in the filtrate of a color corresponding to the particles
indicates the absence of heparin/platelet factor 4 antibodies in
the sample, and the absence of such color in the filtrate indicates
the presence of heparin/platelet factor 4 antibodies in the sample.
It is, of course, also possible to determine the quantity of
heparin/platelet factor 4 antibodies present in a sample in
accordance with the present invention. A suitable quantitative
system may be established by comparing the filtrate with one or
more visual standards corresponding to known concentrations of
colored particles in the filtrate. Such visual standards will be
prepared from samples having known concentrations of
heparin/platelet factor 4 antibodies.
[0096] In certain other embodiments, the color of the filtrate is
compared optically, and the amount of heparin/platelet factor 4
antibodies in the filtrate is determined by instruments known to
one skilled in the art.
[0097] 5.2 Kits
[0098] The present invention further provides kits suitable for
implementing the described methods. In general, such kits comprise:
a reaction cell comprising isolated PF4 complexed to particles or
beads. The kits additionally contain components such as an assay
plate suitable for performance of the filter method and analysis
method discussed above.
[0099] In a preferred embodiment, the kit comprises two components:
a container, such as a plastic pipette, containing the reagents.
The reagents are contained in crushable ampoules sealed inside the
pipette and comprise PF4-coated particles and other agents helpful
to promote rapid antigen/antibody binding.
[0100] In one embodiment, the reaction cell comprises a breakable
vessel that contains PF4-complexed particles. Optionally, the
reaction cell comprises a solution that enhances the aggregation
reaction. Optionally, the reaction cell comprises a solution that
biologically inactivates any infectious agents in the sample.
[0101] The reaction cells can be used for mixing and/or incubating
a sample with PF4-complexed particles.
[0102] Assay plates according to this invention comprise a top
member, a filter means, a wicking means, and a bottom member. In a
specific embodiment, the top member comprises a filter well and an
observation well that is at a fixed distance from said filter well.
In a specific embodiment, the filter means is adjacent the top
member and extending across the filter well. In a specific
embodiment, the wicking means is adjacent and in fluid
communication with the filter means and extending the length of the
filter well and the observation well. In a specific embodiment, the
bottom member is adjacent the wicking means. In preferred
embodiments, the top member, filter means, wicking means, and
bottom member are held in position with an appropriately applied
adhesive.
[0103] In one embodiment, the top member comprises a material that
is substantially impermeable to aqueous solutions such as those
associated with the human body. In a preferred embodiment, the top
member comprises polystyrene. In certain embodiments, the top
member receives the sample/particles mixture.
[0104] In one embodiment, the filter means comprises a controlled
pore membrane. In a specific embodiment, the filter means comprises
a controlled pore polycarbonate membrane. In preferred embodiments,
the filter means has apertures which are larger than the particles
but smaller than the aggregates.
[0105] In certain embodiments, the filter means has apertures which
are 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times,
9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15
times, 20 times, 30 times, 50 times, 100 times, or larger than the
particles but smaller than the aggregates. In a specific
embodiment, the apertures are from about 5 to about 15 larger than
the particles. In another specific embodiment, the apertures are
from about 10 to about 12 larger than the particles. In yet another
specific embodiment, the apertures are from about 2 micrometers to
about 12 micrometers, preferably about 3 micrometers.
[0106] The filter means is capable of separating a majority of any
specific aggregates from any non-specifically aggregated particles
and/or any unaggregated particles. In certain embodiments, the
filter means separates about 50%, 60%, 70%, 80%, 90%, 95%, 96%,
97%, 98%, 99%, or all specific aggregates from any non-specifically
aggregated particles and/or any unaggregated particles. In a more
preferred embodiment, the filter means separates more than 90% of
specific aggregates from any non-specifically aggregated particles
and/or any unaggregated particles.
[0107] In one embodiment, the wicking means comprises a polymeric
material. In a specific embodiment, the wicking membrane comprises
non-woven fibers of glass or synthetic polymeric material. In a
preferred embodiment, the wicking membrane comprises polyester. In
preferred embodiments, the wicking means separates a majority of
any unaggregated particles from any non-specifically aggregated
particles and/or any specific aggregates. In a specific embodiment,
the wicking means separates more than 90% of any unaggregated
particles from any non-specifically aggregated particles and/or any
specific aggregates.
[0108] In one embodiment, the bottom member comprises a relatively
non-rigid material. In a specific embodiment, the bottom member
comprises a vinyl polymer.
[0109] In one embodiment, the assay plate further comprises a
substrate positioned between the top member and the filter means
and extending across the filter well. In a preferred embodiment,
the substrate is a glass substrate that contains PF4-complexed
particles and one or more reagents that promote the aggregation
reaction.
[0110] In one embodiment, the assay plate further comprises a
barrier positioned between the wicking means and the bottom member
and is as long as the wicking means. In a preferred embodiment, the
barrier prevents the wicking means from coming into direct contact
with the bottom member.
[0111] Examples of preferred assay plate (1) that can be used in
the kits of the present invention are shown in FIGS. 1 through 7,
which are taken from U.S. Pat. No. 5,565,366, which is incorporated
herein by reference in its entirety. The assay plates useful for
the methods and kits of this invention generally comprise: a
substantially flat top member (10) of predetermined dimensions
having a filter well (12) and an observation well (14); filter
means (20) adjacent the top member and extending across the filter
well; wicking means (30) adjacent the filter means and extending
the length and width of the filter well and the observation well;
and a substantially flat bottom member (50) having the approximate
dimensions of the top member, adjacent the wicking means. It will
be appreciated that analysis means comprises elements of the assay
plates other than the filter means.
[0112] The top member preferably comprises a material which is
substantially impermeable to aqueous solutions such as those
associated with the human body. The top member preferably is cut or
stamped from a rigid material and, thus, is able to impart some
degree of support to the assay plate. It is preferred that the top
member comprise polystyrene and have a length of about 100
millimeters, a width of about 20 millimeters, and a thickness of
about 1.0 millimeters.
[0113] The top member should be cut, stamped, or otherwise
fabricated to have a filter well (12) and an observation well (14)
extending though the entire thickness of the top member.
Preferably, the filter well and the observation well are circular,
but other shapes are possible. It is also preferred that the filter
well and the observation well be a predetermined distance (X) from
one another. Since there exists the possibility that some
aggregates might not form clumps of sufficient diameter to be
retained by the filter, the predetermined distance (X) is selected
such that any aggregates which pass through the filter do not reach
the observation window. Thus, the predetermined distance (X) will
vary with the specific particle and wicking means employed. It will
generally be the case that the distance (X) varies in an inverse
fashion with the capacity of the wicking means to retain
aggregates.
[0114] The filter means (20) is preferably a filter as described
above having apertures (22) which are larger than the particles but
generally smaller than the clumps of aggregates. It is preferred
that the filter means be a controlled pore polycarbonate membrane.
While the filter means need only extend across the filter well,
where the filter means is transparent or nearly transparent, such
as where the filter means is a controlled pore polycarbonate
membrane, the filter means preferably also extends across the
observation well, as in FIG. 3.
[0115] Adjacent the filter means is the wicking means (30). The
wicking means is preferably positioned in close physical contact
with the filter means such that filtrate flows vertically into the
wicking means and migrates horizontally from a position beneath the
filter well to a position beneath the observation well. While the
filter means need only extend the length of the filter well and the
observation well, the wicking means is preferably somewhat longer,
as in FIG. 3. The filter means and the wicking means are preferably
attached to one another with a porous adhesive, such as the
adhesive available from Adhesive Research Company (Glen Rock, Pa.)
under the trade name ARcare Porous. It is preferred that the
wicking means comprise non-woven fibers of glass or natural or
synthetic polymeric materials, preferably polyester. The
composition and arrangement of the fibers in the wicking means are
selected such that the aggregates and the particles migrate thereon
at different rates. Preferably the particles migrate faster. It is
also preferred that the wicking means have an embossed or otherwise
formed visually recognizable pattern, such as a cross-hatch pattern
(32), to facilitate the visual detection of color at the
observation well.
[0116] The bottom member (50) is adjacent the wicking means and
preferably comprises a material which is substantially impermeable
to aqueous solutions. The bottom member preferably is cut or
stamped to have the approximate width and length of the top member.
The top member and/or the bottom member should serve to support the
assay plate. Thus, where the top member provides adequate support,
the bottom member may comprise a relatively non-rigid material,
such as a vinyl polymer. The bottom member is preferably physically
attached to the other components of the assay plate with an
adhesive. Since many suitable adhesives impair the wicking
properties of the wicking means, preferred assay plates have a
barrier (40) such as a thin polyethylene film at least as long as
the wicking means and positioned between the wicking means and the
adhesive-bearing bottom member.
[0117] The assay plates useful for the methods and kits of this
invention optionally also comprise a substrate (60), such as a
glass membrane, containing PF4-complexed particles and other
reagents necessary to promote the aggregation reaction. Such a
substrate is to be employed where the sample is to be applied
directly into the filter well, rather than pre-mixed with a
solution containing the receptor-bearing particles. The substrate
should be positioned between the top member and the filter means
and should extend across the filter well, as shown in FIG. 4.
[0118] A preferred type of reaction cell (70) is depicted in FIG.
5. One element of reaction cells according to this invention is a
container (80) in which PF4-complexed particles may be contacted
with a sample suspected to contain a ligand. Such containers may
have a variety of shapes. However, a preferably-shaped container is
a pipette, such as shown in FIG. 5. It will be appreciated that
containers having an open end preferably further comprise a cap
(72) for containing the sample and PF4-complexed particles.
Preferred containers are disposable and comprise any of the
relatively inexpensive, substantially transparent synthetic
polymers known in the art. Suitable transparent pipette-shaped
containers are available from Franklin, Inc. (Franklin, N.J.).
[0119] Within preferred reaction cells are breakable vessels (90)
containing the receptor-bearing particles (92). The breakable
vessel may comprise glass or some synthetic polymer, so long as the
material employed has sufficient structural integrity to contain
the particles securely until the particles are to be contacted with
the sample, at which time the vessel is broken or ruptured by
applied force. Where a reaction cell contains a breakable vessel,
it is necessary that the container comprise a relatively supple
material through which such rupturing force may be applied to the
breakable vessel.
[0120] Preferred reaction cells further comprise a kill solution.
It is intended that the term "kill solution" denote any solution
having the capacity to biologically inactivate any infectious
agents in the patient samples employed in performing the assay.
Solutions comprising ethanol, formaldehyde, glutaraldehyde,
iodophors, or oxidizing bleaches provide examples of kill solutions
according to this invention. It is preferred that kill solutions
comprise an oxidizing bleach such as sodium hypochlorite. The kill
solution is preferably contained in a compartment (100) at one end
of the container and separated therefrom by a rupturable membrane
(102). Alternatively, the kill solution is contained in a breakable
vessel (110) located within the container. The membrane or vessel
comprises a material which has sufficient structural integrity to
contain the kill solution securely until broken or ruptured by
applied force. The kill solution is then released and contacted
with any biologically active substances located in the container or
on the assay plate, usually upon the completion of an assay.
[0121] A preferred embodiment of an apparatus for detecting
heparin/platelet factor 4 antibodies in a fluid sample is shown in
exploded views in FIGS. 8 and 9. Apparatus 800 comprises front side
870 and a tower 805 comprising a sample well 803, latches 807 on
two sides, foot 810, and block channel foot 815. Tower 805 engages
into opening 820 in cover 825 such that foot 810 crushes ampoule
830 placed in slot 840 in ampoule support 835. Although FIGS. 8 and
9 only show slot 840 with spaces for containing two ampoules 830,
more than two ampoules can be used in the apparatus 800 and
additional feet may be included in tower 805. The cover 825 also
has latches 808 inside such that when tower 805 is depressed, the
latches 807 of tower 805 engages with the latches 808 of the cover
825 such that the tower 805 can only be raised to the position 809.
Block channel foot 815 of tower 805 blocks the flow of fluid to
reagent well 845 in ampoule support 835 following crushing of
ampoule 830 by foot 810 of tower 805 when tower 805 is engaged with
and pressed into opening 820 in cover 825. Cover 825 is placed over
ampoule support 835.
[0122] Preferably, cover 825 and ampoule support 835 can snap
together with bottom plate 850 holding test strip 855 in place.
Test strip 855, which contains a particulate immunofiltration assay
as described above, has a test window 847 and a control window 848,
and is held in place by indentation 865. In alternative
embodiments, different structures may be employed to hold and
orient test strip 855. Test strip 855 receives one or more reagents
from reagent well 845 via channel 875, typically when reagent is
released from ampoule 830 by depressing tower 805 and then
withdrawing it to position 809 to unblock channel 875 blocked by
block channel foot 815 of tower 805. Preferably, the reagent
comprises particles complexed to platelet factor 4 (PF4). Tower 805
further comprises spur 817 for crushing another ampoule to allow
mixing two (or more) reagents prior to their flowing to reagent
well 845. In alternative embodiments, spur 817 may not be adjacent
to block channel foot 815.
[0123] FIG. 9 shows reagent well underside 846 and channel
underside 876. As shown by FIGS. 8 and 9, the indentations guide
and preferably engage different parts such that bottom plate 860,
test strip 855, ampoule support 835, and cover 825 are engaged to
form test device 800 operable by depressing and withdrawing tower
805.
[0124] In one embodiment, the apparatus 800 is used in the
following manner:
[0125] First, the tower 805 is pushed all the way down to engage
into opening 820 in cover 825. Second, a patient sample is injected
into the sample well 803 of the tower 805. Third, the apparatus 800
is shook side to side along the longitudinal axis of the test strip
855 for five seconds. Fourth, the apparatus 800 is rested on a flat
surface for one minute when the patient sample and reagent(s) in
the ampoule(s) will form a mixture in the channel 875. Preferably,
the reagent comprises particles complexed to platelet factor 4
(PF4). Fifth, the tower 805 is pulled up to a stop position 809 and
the apparatus 800 is tilted forward at a forty-five degrees angle
and lightly tapped on the side opposing front side 870 such that
the mixture will flow into the reagent well 845 where it undergoes
reaction in a particulate immunofiltration assay as described
above. Finally, the apparatus 800 is laid flat again and rested for
ten minutes or until a color is observed in the control window
848.
[0126] In certain other embodiments, the kit comprises two
components: a container, such as a plastic pipette, containing the
reagents. The reagents are contained in crushable ampoules sealed
inside the pipette and comprise PF4-coated particles and other
agents helpful to promote rapid antigen/antibody binding.
[0127] In one embodiment, the reaction cell comprises a breakable
vessel that contains PF4-complexed particles. Optionally, the
reaction cell comprises a solution that enhances the aggregation
reaction. Optionally, the reaction cell comprises a solution that
biologically inactivates any infectious agents in the sample.
[0128] The reaction cells can be used for mixing and/or incubating
a sample with PF4-complexed particles.
[0129] Additional objects, advantages, and novel features of this
invention will become apparent to those skilled in the art upon
examination of the following examples thereof, which are not
intended to be limiting, wherein parts and percents are by weight
to volume unless otherwise indicated.
6. EXAMPLES
[0130] 6.1 HealthTEST.TM. Heparin/Platelet Factor 4 Antibody
Assay
[0131] The HealthTEST.TM. Heparin/Platelet Factor 4 Antibody Assay
(Akers Biosciences, Inc.) is a qualitative in vitro diagnostic
device, based on PIFA.RTM. technology, designed for the detection
of heparin/platelet factor 4 antibodies. The device is supplied in
a kit comprising a MiniReactor device containing a membrane
filtration system and a results window, and a push button reagent
dispensing system containing microparticle-based reaction
reagents.
[0132] The MiniReactor contains a reaction well that allows the
sample to react with the reagents. The sample is added to the
reaction wall followed by the reagents contained in the reagents
dispenser. The reagents contain microparticles coated with isolated
or purified PF4 protein as well as additional enhancing agents
(e.g., polyethylene glycol 8000 and dextran 10,000 MW) designed to
promote rapid aggregate formation of the particles in the presence
of specific antibodies in the test sample.
[0133] Once the reagents have reacted with the sample in the
reaction well, the reaction mixture automatically collects over the
membrane filtration system. This system acts to filter aggregated
particles, while allowing non-aggregated particles to pass through.
Thus, an aggregated, reactive sample will be trapped within the
membrane. Since the dyed particles are trapped on this filter, no
particles and hence no color, are able to migrate past the
positive/negative line on the results window. Conversely, a
non-aggregated, non-reactive sample will pass through the membrane
filter and into the wicking layers, and color will migrate past the
positive/negative line.
[0134] The test is a rapid manual assay and can be easily performed
when immediate results are required. The test determines the
presence of heparin/platelet factor 4 antibodies in serum or plasma
through a visual determination of color in a disposable test
device. The assay takes less than 5 minutes to perform with a
minimal number of steps.
[0135] Dyed microparticles coated with heparin/platelet factor 4
antigen provide the visual signal for the results of the assay. The
ability of aggregated or non-aggregated particles to move through a
filter medium is the measure of the reactivity/non-reactivity of
the test sample.
[0136] 6.1.1 Materials and Methods
[0137] Two studies were performed to evaluate the performance of
the HealthTEST.TM. Heparin/Platelet Factor 4 Antibody Assay
compared to commercially available standard laboratory methods
using fresh samples originating from field sources.
[0138] These studies measured the specificity and sensitivity of
the test. The results of the comparison studies are summarized in
Section 6.1.2.
[0139] 6.1.2 Results
[0140] Specificity and Sensitivity TABLE-US-00001 ELISA Positive
Negative Study # 1 Plasma HealthTEST .TM. Positive 21 15 Negative 2
137 23 152 Specificity = 90.1% (or 137/152) Sensitivity = 91.3% (or
21/23) Overall Agreement = 90.3% (or 158/173) Study # 2 Serum
HealthTEST .TM. Positive 21 3 Negative 2 153 23 156 Specificity =
98.1% (or 153/156) Sensitivity = 91.3% (or 21/23) Overall Agreement
= 97.2% (or 174/179)
[0141] Reproducibility
[0142] A study, which measured the reproducibility of the test, was
performed using 5 replicates each of positive and negative patient
controls which were tested with the HealthTEST.TM. Heparin/Platelet
Factor 4 Antibody Assay daily for 4 consecutive days.
[0143] The results show the test is 100% reproducible for both
serum and plasma.
[0144] 6.1.3 Discussion
[0145] The HealthTEST.TM. Heparin/Platelet Factor 4 Antibody Assay
is designed to identify patients at risk for developing
heparin-induced thrombocytopenia (HIT), a severe allergic-like side
effect associated with the use of the anticoagulant heparin. The
presence of heparin/platelet factor 4 antibodies is associated with
patients at risk for HIT, and is rapidly becoming a standard of
care in hematology and cardiology. The determination of the
presence of heparin/platelet factor 4 antibodies may therefore
contribute significantly in the risk assessment of vascular access
thrombosis. The test is intended for use, preferably in a health
care facility such as a hospital, where heparin is administered
during surgical and other medical procedures. The test was
evaluated for performance characteristics in comprehensive studies.
These studies have demonstrated that the test is safe and effective
for intended use.
[0146] Also, these studies show that the HealthTEST.TM.m
Heparin/Platelet Factor 4 Antibody Assay is as sensitive and
specific as current laboratory procedures, such as enzyme
immunoassays (ELISA), yet do not require instrumentation and can be
performed in as little as 5 min. These advantages will enable the
rapid diagnostic assay system to take advantage of market forces
and trends shaping the alternate-site health-care industry.
7. EQUIVALENTS
[0147] The present invention is not to be limited in scope by the
specific embodiments described which are intended as single
illustrations of individual aspects of the invention, and
functionally equivalent methods and components are within the scope
of the invention. Indeed, various modifications of the invention,
in addition to those shown and described herein, will become
apparent to those skilled in the art from the foregoing description
and accompanying drawings using no more than routine
experimentation. Such modifications and equivalents are intended to
fall within the scope of the appended claims.
[0148] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference into the
specification to the same extent as if each individual publication,
patent or patent application was specifically and individually
indicated to be incorporated herein by reference.
[0149] Citation or discussion of a reference herein shall not be
construed as an admission that such is prior art to the present
invention.
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