U.S. patent application number 13/532905 was filed with the patent office on 2012-10-25 for methods and kits for detecting hemoglobin in test samples.
This patent application is currently assigned to ABBOTT LABORATORIES. Invention is credited to Maciej Adamczyk, Roy J. Brashear, Phillip G. Mattingly.
Application Number | 20120270258 13/532905 |
Document ID | / |
Family ID | 42319346 |
Filed Date | 2012-10-25 |
United States Patent
Application |
20120270258 |
Kind Code |
A1 |
Adamczyk; Maciej ; et
al. |
October 25, 2012 |
METHODS AND KITS FOR DETECTING HEMOGLOBIN IN TEST SAMPLES
Abstract
The present invention relates to methods of detecting hemoglobin
in a test sample. These methods can be used to diagnose a subject
suffering from a genetic disorder relating to hemoglobin
metabolism, to determine the eligibility of a subject to be a blood
donor, to determine the age of a stored blood sample or to identify
a hemolyzed plasma sample. The present invention also relates to
kits for use in the above described methods.
Inventors: |
Adamczyk; Maciej; (Gurnee,
IL) ; Brashear; Roy J.; (Mundelein, IL) ;
Mattingly; Phillip G.; (Third Lake, IL) |
Assignee: |
ABBOTT LABORATORIES
Abbott Park
IL
|
Family ID: |
42319346 |
Appl. No.: |
13/532905 |
Filed: |
June 26, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12362331 |
Jan 29, 2009 |
8241915 |
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13532905 |
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Current U.S.
Class: |
435/29 ;
436/66 |
Current CPC
Class: |
G01N 33/721 20130101;
G01N 21/76 20130101 |
Class at
Publication: |
435/29 ;
436/66 |
International
Class: |
G01N 21/76 20060101
G01N021/76 |
Claims
1. A method of detecting hemoglobin in a test sample, the method
comprising the steps of: a) adding at least one basic solution to a
test sample; b) adding an indicator solution to the test sample to
generate a light signal, wherein the indicator solution comprises
at least one acridinium compound; wherein steps a) and b) can be
performed in any order; and c) measuring the light generated to
detect the hemoglobin in the test sample.
2. The method of claim 1, wherein the test sample is a
non-biological forensic sample, stool, whole blood, serum, plasma,
interstitial fluid, saliva, ocular lens fluid, cerebral spinal
fluid, sweat, urine, ascites fluid, mucous, nasal fluid, sputum,
synovial fluid, peritoneal fluid, vaginal fluid, menses, amniotic
fluid, semen, soil or a blood substitute.
3. The method of claim 1, wherein the basic solution is a solution
having a pH of at least about 10.
4. The method of claim 1, wherein the acridinium compound is an
acridinium-9-carboxamide having a structure according to formula I:
##STR00041## wherein R.sup.1 and R.sup.2 are each independently
selected from the group consisting of: alkyl, alkenyl, alkynyl,
aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein
R.sup.3 through R.sup.15 are each independently selected from the
group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or
aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen,
halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl;
and optionally, if present, X.sup..crclbar. is an anion.
5. The method of claim 1, wherein the acridinium compound is an
acridinium-9-carboxylate aryl ester having a structure according to
formula II: ##STR00042## wherein R.sup.1 is an alkyl, alkenyl,
alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl;
and wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and optionally, if present,
X.sup..crclbar. is an anion.
6. The method of claim 1, wherein the indicator solution further
comprises at least one surfactant.
7. The method of claim 1, further comprising measuring the amount
of hemoglobin in the test sample by relating the amount of light
generated in the test sample by comparison to a standard curve for
hemoglobin or to a reference standard.
8. The method of claim 7, wherein the standard curve is generated
from solutions of hemoglobin of a known concentration.
9. A method of detecting hemoglobin in a test sample, the method
comprising the steps of: a) adding at least one basic solution to a
test sample; and b) measuring the current generated at at least one
electrode to detect the hemoglobin in the test sample.
10. The method of claim 9, wherein the test sample is a
non-biological forensic sample, stool, whole blood, serum, plasma,
interstitial fluid, saliva, ocular lens fluid, cerebral spinal
fluid, sweat, urine, ascites fluid, mucous, nasal fluid, sputum,
synovial fluid, peritoneal fluid, vaginal fluid, menses, amniotic
fluid, semen, soil or a blood substitute.
11. The method of claim 9, wherein the basic solution is a solution
having a pH of at least about 10.
12. A method of diagnosing a subject suffering from a genetic
disorder relating to hemoglobin metabolism, the method comprising
the steps of: a) adding at least one basic solution to a test
sample obtained from a subject suspected of suffering from a
genetic disorder relating to hemoglobin metabolism; b) adding an
indicator solution to the test sample to generate a light signal,
wherein the indicator solution comprises at least one acridinium
compound, wherein steps a) and b) can be performed in any order; c)
quantifying the light generated to detect the hemoglobin in the
test sample; and d) determining the concentration of hemoglobin in
the test sample based on the amount of light quantified in step c);
and e) comparing the concentration of hemoglobin in step (d) with a
predetermined level, wherein if the concentration of hemoglobin
determined in step (d) is lower or higher than the predetermined
level, then a determination is made that the subject is suffering
from a genetic disorder relating to hemoglobin metabolism.
13. The method of claim 12, wherein the test sample is serum,
plasma, whole blood, red blood cells and umbilical cord blood.
14. The method of claim 12, wherein the basic solution is a
solution having a pH of at least about 10.
15. The method of claim 12, wherein the acridinium compound is an
acridinium-9-carboxamide having a structure according to formula I:
##STR00043## wherein R.sup.1 and R.sup.2 are each independently
selected from the group consisting of: alkyl, alkenyl, alkynyl,
aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein
R.sup.3 through R.sup.15 are each independently selected from the
group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or
aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen,
halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl;
and optionally, if present, X.sup..crclbar. is an anion.
16. The method of claim 12, wherein the acridinium compound is an
acridinium-9-carboxylate aryl ester having a structure according to
formula II: ##STR00044## wherein R.sup.1 is an alkyl, alkenyl,
alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl;
and wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and optionally, if present,
X.sup..crclbar. is an anion.
17. The method of claim 12, wherein the indicator solution further
comprises at least one surfactant.
18. The method of claim 12, further comprising measuring the amount
of hemoglobin in the test sample by quantifying the amount of light
generated in the test sample by comparison to a standard curve for
hemoglobin or to a reference standard.
19. The method of claim 18, wherein the standard curve is generated
from solutions of hemoglobin of a known concentration.
20. The method of claim 12, wherein the genetic disorder relating
to hemoglobin metabolism is anemia or .beta.-thalassemia.
21. A method of determining the eligibility of a subject to be a
blood donor, the method comprising the steps of: a) adding at least
one basic solution to a test sample obtained from a subject; b)
adding an indicator solution to the test sample to generate a light
signal, wherein the indicator solution comprises at least one
acridinium compound, wherein steps a) and b) can be performed in
any order; c) quantifying the light generated to detect the
hemoglobin in the test sample; d) determining the concentration of
hemoglobin in the test sample based on the amount of light
quantified in step c); and e) comparing the concentration of
hemoglobin in step (d) with a predetermined level, wherein if the
concentration of hemoglobin determined in step (d) is lower or
higher than the predetermined level, then a determination is made
that the subject is not eligible to be a blood donor.
22. The method of claim 21, wherein the test sample is whole
blood.
23. The method of claim 21, wherein the basic solution is a
solution having a pH of at least about 10.
24. The method of claim 21, wherein the acridinium compound is an
acridinium-9-carboxamide having a structure according to formula I:
##STR00045## wherein R.sup.1 and R.sup.2 are each independently
selected from the group consisting of: alkyl, alkenyl, alkynyl,
aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein
R.sup.3 through R.sup.15 are each independently selected from the
group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or
aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen,
halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl;
and optionally, if present, X.sup..crclbar. is an anion.
25. The method of claim 21, wherein the acridinium compound is an
acridinium-9-carboxylate aryl ester having a structure according to
formula II: ##STR00046## wherein R.sup.1 is an alkyl, alkenyl,
alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl;
and wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and optionally, if present,
X.sup..crclbar. is an anion.
26. The method of claim 21, wherein the indicator solution further
comprises at least one surfactant.
27. The method of claim 21, further comprising measuring the amount
of hemoglobin in the test sample by quantifying the amount of light
generated in the test sample by comparison to a standard curve for
hemoglobin or to a reference standard.
28. The method of claim 27, wherein the standard curve is generated
from solutions of hemoglobin of a known concentration.
29. A method of determining the age of a stored blood sample, the
method comprising the steps of: a) adding at least one basic
solution to a blood sample; b) adding an indicator solution to the
blood sample to generate a light signal, wherein the indicator
solution comprises at least one acridinium compound, wherein steps
a) and b) can be performed in any order; c) quantifying the light
generated to detect the hemoglobin in the blood sample; d)
determining the concentration of hemoglobin in the blood sample
based on the amount of light quantified in step c); and e)
comparing the concentration of hemoglobin in step (d) with at least
one predetermined level, wherein if the concentration of hemoglobin
determined in step (d) is lower then the predetermined level, then
the plasma sample is determined to be an older blood sample.
30. The method of claim 29, wherein the blood sample is whole
blood, serum, plasma, platelets, red blood cells or umbilical cord
blood.
31. The method of claim 29, wherein the basic solution is a
solution having a pH of at least about 10.
32. The method of claim 29, wherein the acridinium compound is an
acridinium-9-carboxamide having a structure according to formula I:
##STR00047## wherein R.sup.1 and R.sup.2 are each independently
selected from the group consisting of: alkyl, alkenyl, alkynyl,
aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein
R.sup.3 through R.sup.15 are each independently selected from the
group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or
aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen,
halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl;
and optionally, if present, X.sup..crclbar. is an anion.
33. The method of claim 29, wherein the acridinium compound is an
acridinium-9-carboxylate aryl ester having a structure according to
formula II: ##STR00048## wherein R.sup.1 is an alkyl, alkenyl,
alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl;
and wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and optionally, if present,
X.sup..crclbar. is an anion.
34. The method of claim 29, wherein the indicator solution further
comprises at least one surfactant.
35. The method of claim 29, further comprising measuring the amount
of hemoglobin in the blood sample by quantifying the amount of
light generated in the blood sample by comparison to a standard
curve for hemoglobin or to a reference standard.
36. The method of claim 29, wherein the standard curve is generated
from solutions of hemoglobin of a known concentration.
37. A method of identifying a hemolyzed serum or plasma sample, the
method comprising the steps of: a) adding at least one basic
solution to a serum or plasma sample; b) adding an indicator
solution to the serum or plasma sample to generate a light signal,
wherein the indicator solution comprises at least one acridinium
compound, wherein steps a) and b) can be performed in any order; c)
quantifying the light generated to detect the hemoglobin in the
serum or plasma sample; d) determining the concentration of
hemoglobin in the serum or plasma sample based on the amount of
light quantified in step c); and e) comparing the concentration of
hemoglobin in step (d) with a predetermined level, wherein if the
concentration of hemoglobin determined in step (d) is the same as
or higher then the predetermined level, then the serum or plasma
sample is determined to be hemolyzed and further wherein if the
concentration of hemoglobin determined in step (d) is lower then
the predetermined level, then the serum or plasma sample is
determined not to be hemolyzed.
38. The method of claim 37, wherein the basic solution is a
solution having a pH of at least about 10.
39. The method of claim 37, wherein the acridinium compound is an
acridinium-9-carboxamide having a structure according to formula I:
##STR00049## wherein R.sup.1 and R.sup.2 are each independently
selected from the group consisting of: alkyl, alkenyl, alkynyl,
aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein
R.sup.3 through R.sup.15 are each independently selected from the
group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or
aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen,
halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl;
and optionally, if present, X.sup..crclbar. is an anion.
40. The method of claim 37, wherein the acridinium compound is an
acridinium-9-carboxylate aryl ester having a structure according to
formula II: ##STR00050## wherein R.sup.1 is an alkyl, alkenyl,
alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl;
and wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and optionally, if present,
X.sup..crclbar. is an anion.
41. The method of claim 37, wherein the indicator solution further
comprises at least one surfactant.
42. The method of claim 37, further comprising measuring the amount
of hemoglobin in the serum or plasma sample by quantifying the
amount of light generated in the serum or plasma sample by
comparison to a standard curve for hemoglobin or to a reference
standard.
43. The method of claim 42, wherein the standard curve is generated
from solutions of hemoglobin of a known concentration.
44. A kit for detecting hemoglobin in a test sample, the kit
comprising: a. at least one basic solution; b. at least one
indicator solution, wherein the indicator solution comprises at
least one acridinium compound; and c. instructions for detecting
hemoglobin in a test sample.
45. The kit of claim 44, wherein the basic solution is a solution
having a pH of at least about 10.
46. The kit of claim 44, wherein the acridinium compound is an
acridinium-9-carboxamide having a structure according to formula I:
##STR00051## wherein R.sup.1 and R.sup.2 are each independently
selected from the group consisting of: alkyl, alkenyl, alkynyl,
aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein
R.sup.3 through R.sup.15 are each independently selected from the
group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or
aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen,
halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl;
and optionally, if present, X.sup..crclbar. is an anion.
47. The kit of claim 44, wherein the acridinium compound is an
acridinium-9-carboxylate aryl ester having a structure according to
formula II: ##STR00052## wherein R.sup.1 is an alkyl, alkenyl,
alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl;
and wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and optionally, if present,
X.sup..crclbar. is an anion.
48. The kit of claim 44, wherein the indicator solution further
comprises at least one surfactant.
49. A kit for detecting hemoglobin in a test sample, the kit
comprising: a. at least one basic solution; b. at least one
electrode; and c. instructions for detecting hemoglobin in a test
sample.
50. The kit of claim 49, wherein the basic solution is a solution
having a pH of at least about 10.
51. A kit for diagnosing a subject suffering from a genetic
disorder relating to hemoglobin metabolism, the kit comprising: a.
at least one basic solution; b. at least one indicator solution,
wherein the indicator solution comprises at least one acridinium
compound; and c. instructions for diagnosing a subject suffering
from a genetic disorder relating to hemoglobin metabolism.
52. The kit of claim 51, wherein the basic solution is a solution
having a pH of at least about 10.
53. The kit of claim 51, wherein the acridinium compound is an
acridinium-9-carboxamide having a structure according to formula I:
wherein R.sup.1 and R.sup.2 are each independently selected from
the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl,
sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R.sup.3 through
R.sup.15 are each independently selected from the group consisting
of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino,
amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro,
cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and
optionally, if present, X.sup..crclbar. is an anion.
54. The kit of claim 51, wherein the acridinium compound is an
acridinium-9-carboxylate aryl ester having a structure according to
formula II: ##STR00053## wherein R.sup.1 is an alkyl, alkenyl,
alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl;
and wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and optionally, if present,
X.sup..crclbar. is an anion.
55. The kit of claim 51, wherein the indicator solution further
comprises at least one surfactant.
56. A kit for determining the eligibility of a subject to be a
blood donor, the kit comprising: a. at least one basic solution; b.
at least one indicator solution, wherein the indicator solution
comprises at least one acridinium compound; and c. instructions for
determining the eligibility of a subject to be a blood donor.
57. The kit of claim 56, wherein the basic solution is a solution
having a pH of at least about 10.
58. The kit of claim 56, wherein the acridinium compound is an
acridinium-9-carboxamide having a structure according to formula I:
##STR00054## wherein R.sup.1 and R.sup.2 are each independently
selected from the group consisting of: alkyl, alkenyl, alkynyl,
aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein
R.sup.3 through R.sup.15 are each independently selected from the
group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or
aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen,
halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl;
and optionally, if present, X.sup..crclbar. is an anion.
59. The kit of claim 56, wherein the acridinium compound is an
acridinium-9-carboxylate aryl ester having a structure according to
formula II: ##STR00055## wherein R.sup.1 is an alkyl, alkenyl,
alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl;
and wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and optionally, if present,
X.sup..crclbar. is an anion.
60. The kit of claim 56, wherein the indicator solution further
comprises at least one surfactant.
61. A kit for determining the age of a stored blood sample, the kit
comprising: a. at least one basic solution; b. at least one
indicator solution, wherein the indicator solution comprises at
least one acridinium compound; and c. instructions for determining
the age of a blood sample.
62. The kit of claim 61, wherein the basic solution is a solution
having a pH of at least about 10.
63. The kit of claim 61, wherein the acridinium compound is an
acridinium-9-carboxamide having a structure according to formula I:
##STR00056## wherein R.sup.1 and R.sup.2 are each independently
selected from the group consisting of: alkyl, alkenyl, alkynyl,
aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein
R.sup.3 through R.sup.15 are each independently selected from the
group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or
aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen,
halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl;
and optionally, if present, X.sup..crclbar. is an anion.
64. The kit of claim 61, wherein the acridinium compound is an
acridinium-9-carboxylate aryl ester having a structure according to
formula II: ##STR00057## wherein R.sup.1 is an alkyl, alkenyl,
alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl;
and wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and optionally, if present,
X.sup..crclbar. is an anion.
65. The kit of claim 61, wherein the indicator solution further
comprises at least one surfactant.
66. A kit for identifying a hemolyzed serum or plasma sample, the
kit comprising: a. at least one basic solution; b. at least one
indicator solution, wherein the indicator solution comprises at
least one acridinium compound; and c. instructions for identifying
a hemolyzed serum or plasma sample.
67. The kit of claim 66, wherein the basic solution is a solution
having a pH of at least about 10.
68. The kit of claim 66, wherein the acridinium compound is an
acridinium-9-carboxamide having a structure according to formula I:
##STR00058## wherein R.sup.1 and R.sup.2 are each independently
selected from the group consisting of: alkyl, alkenyl, alkynyl,
aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein
R.sup.3 through R.sup.15 are each independently selected from the
group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or
aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen,
halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl;
and optionally, if present, X.sup..crclbar. is an anion.
69. The kit of claim 66, wherein the acridinium compound is an
acridinium-9-carboxylate aryl ester having a structure according to
formula II: ##STR00059## wherein R.sup.1 is an alkyl, alkenyl,
alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl;
and wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and optionally, if present,
X.sup..crclbar. is an anion.
70. The kit of claim 66, wherein the indicator solution further
comprises at least one surfactant.
Description
RELATED APPLICATION INFORMATION
[0001] The present application is a divisional of and claims
priority to allowed U.S. application Ser. No. 12/362,331, filed on
Jan. 29, 2009, hereby incorporated in its entirety by
reference.
TECHNICAL FIELD
[0002] The present invention relates to methods of detecting and/or
quantifying the amount of hemoglobin in a test sample. The methods
of the present invention can be used to diagnose a subject
suffering from a genetic disorder relating to hemoglobin
metabolism, to determine the eligibility of a subject to be a blood
donor, to determine the age of a stored blood sample and to
identify a hemolyzed plasma sample. The present invention further
relates to kits for use in the above described methods.
BACKGROUND
[0003] Adult hemoglobin A (HbA) is a tetrameric protein of
molecular weight 64.5 kD, composed of 2 .alpha.-globins and 2
.beta.-globins (.alpha.2.beta.2). The alpha .alpha.-subunit is
composed of 141 amino acids (See, SEQ ID NO:1). The .beta.-subunit
is composed of 146 amino acids (See, SEQ ID NO:2). Both .alpha. and
.beta.-subunits are arranged in 8 helical segments (referred to as
helix A-G). Each globin chain also contains a covalently bound heme
molecule, composed of a porphyrin ring and an iron (Fe (II)) ligand
located between helix E and F of the globin protein. Hemoglobin A
constitutes approximately 97% of adult hemoglobin. Hemoglobin A2 is
minor adult hemoglobin consisting of 2 .alpha.-globins and 2
.delta.-globins. The predominant fetal hemoglobin F consists of 2
.alpha.-globins and 2 .gamma.-globins and is sometimes seen in
neonates and adults.
[0004] Hemoglobin constitutes almost 90% of the dry weight of
mature erythrocytes (e.g., red blood cells) and is responsible for
the transport of oxygen and carbon dioxide between the lungs and
body tissue. The heme-bound iron must be in the ferrous oxidation
state, e.g., Fe(II), for hemoglobin to bind oxygen reversibly.
Oxyhemoglobin can undergo autooxidation to methemoglobin
(HbFe(III)) and higher oxidation states in the presence of other
oxidants. In vivo, the methemoglobin concentration is less than
1.5% that of ferrous hemoglobin. Sometimes the intracellular
mechanisms (e.g., cytochrome b5 methemoglobin reductase,
glutathione, or nicotinamide adenine dinucleotide phosphate flavin
reductase) fail to maintain hemoglobin in the ferrous state due to
genetic abnormalities, or the presence of toxins or drugs,
rendering the hemoglobin nonfunctional. Hemolytic anemia releases
hemoglobin from erythrocytes where the free hemoglobin in
circulation is subject to oxidative denaturation. Oxidation of
hemoglobin has been problematic in the production and storage of
hemoglobin-based blood substitutes.
[0005] Determination of hemoglobin concentration is an essential
part of the blood donation process as an aid in eliminating harm to
both anemic donors and potential transfusion recipients. Current
standards require that donors have a minimum hemoglobin
concentration of 12.5 g/dL (e.g., 0.0019 mol/L) corresponding to
hematocrit of 38% or greater.
[0006] The determination of total hemoglobin concentration is also
useful in assays reporting % hemoglobinA1c for monitoring blood
glucose control.
[0007] The determination of hemoglobin in plasma is a sensitive
measure of damage to the red blood cells during blood collection
for clinical analysis, use of cardiovascular or hemodialysis
medical devices, or during the processing of blood products (for
example, packed red blood cells, plasma). Normally the
concentration of hemoglobin in plasma is less than 10 mg/dL (1.6
.mu.mol/L).
[0008] Methods for measuring the concentration of hemoglobin have
been reviewed (See, Malinauskas, R. A. Artif. Organs, 21, 1255-67
(1997)). Briefly, methods may be classified as direct optical
techniques that measure the absorbance of undiluted oxyhemoglobin
at a wavelength of 577 nm (e.g., Cripps, Kahn, Porter, Shinowara
and first derivative methods); direct optical techniques that
measure the absorbance of diluted hemoglobin at a wavelength of 415
nm (Harboe and Fairbanks All methods); and chemical methods such as
Drabkin the method supported by international standards (See, Lewis
S. M., Kumari S., Guidelines on Standard Operating Procedures for
HAEMATOLOGY. Chapter 7--Haemoglobinometry. New Delhi World Health
Organization, 1999). The Drabkin method converts most forms of
hemoglobin to cyanomethemoglobin (HiCN) by treatment with buffered
potassium ferricyanide, K.sub.3Fe(CN).sub.6 and potassium cyanide.
To quantify the concentration of hemoglobin, the absorbance at a
wavelength of 540 nm is measured and compared to the International
HiCN standard.
[0009] The method exemplified in the commercial Multigent
Hemoglobin A1c Assay (Abbott Laboratories, List 02K96-20) converts
digests hemoglobin with pepsin to give hematin which can be
quantified at a wavelength of 604 nm.
[0010] Alternatively, assays for quantifying hemoglobin have been
reported which are based on the use of hemoglobin to act as a
catalyst for the oxidation of a chromogenic substrate in the
presence of added hydrogen peroxide. Suitable substrates include
for example, tetramethylbenzidene, o-toluidine, chlorpromazine,
dianisidine and leucomalachite green (See, Malinauskas, R. A.
Artif. Organs, 21, 1255-67 (1997)). The absorbance of the oxidized
substrate is proportional to the concentration of hemoglobin
present.
[0011] Similarly, chemiluminescent assays for hemoglobin rely on
the hemoglobin-catalyzed oxidation of luminol (See, Tatsu, Y.;
Yoshikawa, S. Anal Chem., 62, 2103-6 (1990)) or iso-luminol
(Olsson, T.; Bergstrom, K.; Thore, A. Clinica Chimica Acta, 122:125
(1982)) in the presence of added hydrogen peroxide to generate a
light signal proportional to the concentration of hemoglobin
present.
[0012] Weak chemiluminescence has been reported from hemoglobin and
methemoglogin upon reaction with hydrogen peroxide (See, Lissi, E.
A.; Escobar, J.; Pascual, C.; del Castillo, M.; Schmitt, T. H.; Di
Mascio, P. Photochem. Photobiol., 60:405-11 (1994); Nohl, H.;
Stolze, K. Free Radic Biol Med., 15, 257-63 (1993)). The mechanism
remains unresolved (See, Yoshiki, Y.; Iida, T.; Okubo, K.;
Kanazawa, T. Photochem. Photobiol., 73, 545-50 (2001)).
[0013] A chemiluminescent hemoglobin assay is described in WO
98/54578. Briefly, the hemoglobin content of a sample is determined
by chemiluminescence based on the ability of hemoglobin to absorb
radiation emitted by the chemiluminescent reaction of lucigenin and
hydrogen peroxide. The concentration of hemoglobin is inversely
related to the chemiluminescent signal.
[0014] A chemiluminescent assay for glycated hemoglobin fraction
(See, Adamczyk, M.; Chen, Y.-Y.; Johnson, D. D.; Mattingly, P. G.;
Moore, J. A.; Pan, Y.; Reddy, R. E. Bioorg. Med. Chem. Lett., 16,
1324-8 (2006)) consisted of i) the conversion of all hemoglobin
fractions to methemoglobin, ii) formation of an
acridinium-9-carboxamide boronate/glycated hemoglobin complex, iii)
initiating the chemiluminescent signal by the addition of excess
hydrogen peroxide and base. The concentration of the glycated
fraction of hemoglobin inversely related to the chemiluminescent
signal.
[0015] There is a need in the art for new methods for determining
the concentration of hemoglobin in test samples that do not employ
toxic chemicals (such as potassium cyanide and potassium
ferricyanide) and that exhibit improved sensitivity.
SUMMARY
[0016] In one aspect, the present invention relates to a method of
detecting hemoglobin in a test sample. The method comprises the
steps of:
[0017] a) adding at least one basic solution to a test sample;
[0018] b) adding an indicator solution to the test sample to
generate a light signal, wherein the indicator solution comprises
at least one acridinium compound,
[0019] wherein steps a) and b) can be performed in any order;
and
[0020] c) measuring the light generated to detect the hemoglobin in
the test sample.
[0021] In the above method, the test sample can be a non-biological
forensic sample, stool, whole blood, serum, plasma, interstitial
fluid, saliva, ocular lens fluid, cerebral spinal fluid, sweat,
urine, ascites fluid, mucous, nasal fluid, sputum, synovial fluid,
peritoneal fluid, vaginal fluid, menses, amniotic fluid, semen,
soil or a blood substitute.
[0022] In the above method, the basic solution is a solution having
a pH of at least about 10.
[0023] In the above method, any acridinium compound can be used.
For example, the acridinium compound can be an
acridinium-9-carboxamide having a structure according to formula
I:
##STR00001##
[0024] wherein R.sup.1 and R.sup.2 are each independently selected
from the group consisting of: alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R.sup.3
through R.sup.15 are each independently selected from the group
consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl,
amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide,
nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and
optionally, if present, X.sup..crclbar. is an anion.
[0025] Alternatively, the acridinium compound is an
acridinium-9-carboxylate aryl ester having a structure according to
formula II:
##STR00002##
[0026] wherein R.sup.1 is an alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and
[0027] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0028] optionally, if present, X.sup..crclbar. is an anion.
[0029] In the above method, the indicator solution can further
comprise at least one surfactant.
[0030] In the above method, the method can further comprise
measuring the amount of hemoglobin in the test sample by relating
the amount of light generated in the test sample by comparison to a
standard curve for hemoglobin or to a reference standard.
Optionally, the standard curve can be generated from solutions of
hemoglobin of a known concentration.
[0031] In another aspect, the present invention relates to a method
of detecting hemoglobin in a test sample. The method comprises the
steps of:
[0032] a) adding at least one basic solution to a test sample;
and
[0033] b) measuring the current generated at at least one electrode
to detect the hemoglobin in the test sample.
[0034] In the above method, the test sample is a non-biological
forensic sample, stool, whole blood, serum, plasma, interstitial
fluid, saliva, ocular lens fluid, cerebral spinal fluid, sweat,
urine, ascites fluid, mucous, nasal fluid, sputum, synovial fluid,
peritoneal fluid, vaginal fluid, menses, amniotic fluid, semen,
soil or a blood substitute.
[0035] In the above method, the basic solution is a solution having
a pH of at least about 10.
[0036] In another aspect, the present invention relates to method
of diagnosing a subject suffering from a genetic disorder relating
to hemoglobin metabolism. The method comprises the steps of:
[0037] a) adding at least one basic solution to a test sample
obtained from a subject suspected of suffering from a genetic
disorder relating to hemoglobin metabolism;
[0038] b) adding an indicator solution to the test sample to
generate a light signal, wherein the indicator solution comprises
at least one acridinium compound,
[0039] wherein steps a) and b) can be performed in any order;
[0040] c) quantifying the light generated to detect the hemoglobin
in the test sample; and
[0041] d) determining the concentration of hemoglobin in the test
sample based on the amount of light quantified in step c); and
[0042] e) comparing the concentration of hemoglobin in step (d)
with a predetermined level, wherein if the concentration of
hemoglobin determined in step (d) is lower or higher than the
predetermined level, then a determination is made that the subject
is suffering from a genetic disorder relating to hemoglobin
metabolism.
[0043] In the above method, the test sample can be serum, plasma,
whole blood, red blood cells and umbilical cord blood.
[0044] In the above method, the basic solution is a solution having
a pH of at least about 10.
[0045] In the above method, any acridinium compound can be used.
For example, the acridinium compound can be an
acridinium-9-carboxamide having a structure according to formula
I:
##STR00003##
[0046] wherein R.sup.1 and R.sup.2 are each independently selected
from the group consisting of: alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R.sup.3
through R.sup.15 are each independently selected from the group
consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl,
amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide,
nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and
optionally, if present, X.sup..crclbar. is an anion.
[0047] Alternatively, the acridinium compound is an
acridinium-9-carboxylate aryl ester having a structure according to
formula II:
##STR00004##
[0048] wherein R.sup.1 is an alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and
[0049] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0050] optionally, if present, X.sup..crclbar. is an anion.
[0051] In the above method, the indicator solution can further
comprise at least one surfactant.
[0052] In the above method, the method can further comprise
measuring the amount of hemoglobin in the test sample by relating
the amount of light generated in the test sample by comparison to a
standard curve for hemoglobin or to a reference standard.
Optionally, the standard curve can be generated from solutions of
hemoglobin of a known concentration.
[0053] In the above method, the genetic disorder relating to
hemoglobin metabolism can be anemia or .beta.-thalassemia.
[0054] In yet another aspect, the present invention relates to a
method of determining the eligibility of a subject to be a blood
donor. The method comprises the steps of:
[0055] a) adding at least one basic solution to a test sample
obtained from a subject;
[0056] b) adding an indicator solution to the test sample to
generate a light signal, wherein the indicator solution comprises
at least one acridinium compound,
[0057] wherein steps a) and b) can be performed in any order;
[0058] c) quantifying the light generated to detect the hemoglobin
in the test sample;
[0059] d) determining the concentration of hemoglobin in the test
sample based on the amount of light quantified in step c); and
[0060] e) comparing the concentration of hemoglobin in step (d)
with a predetermined level, wherein if the concentration of
hemoglobin determined in step (d) is lower or higher than the
predetermined level, then a determination is made that the subject
is not eligible to be a blood donor.
[0061] In the above method, the test sample can be whole blood.
[0062] In the above method, the basic solution is a solution having
a pH of at least about 10.
[0063] In the above method, any acridinium compound can be used.
For example, the acridinium compound can be an
acridinium-9-carboxamide having a structure according to formula
I:
##STR00005##
[0064] wherein R.sup.1 and R.sup.2 are each independently selected
from the group consisting of: alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R.sup.3
through R.sup.15 are each independently selected from the group
consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl,
amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide,
nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and
optionally, if present, X.sup..crclbar. is an anion.
[0065] Alternatively, the acridinium compound is an
acridinium-9-carboxylate aryl ester having a structure according to
formula II:
##STR00006##
[0066] wherein R.sup.1 is an alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and
[0067] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0068] optionally, if present, X.sup..crclbar. is an anion.
[0069] In the above method, the indicator solution can further
comprise at least one surfactant.
[0070] In the above method, the method can further comprise
measuring the amount of hemoglobin in the test sample by relating
the amount of light generated in the test sample by comparison to a
standard curve for hemoglobin or to a reference standard.
Optionally, the standard curve can be generated from solutions of
hemoglobin of a known concentration.
[0071] In still yet a further aspect, the present invention relates
to a method of determining the age of a stored blood sample. The
method comprises the steps of:
[0072] a) adding at least one basic solution to a blood sample;
[0073] b) adding an indicator solution to the blood sample to
generate a light signal, wherein the indicator solution comprises
at least one acridinium compound,
[0074] wherein steps a) and b) can be performed in any order;
[0075] c) quantifying the light generated to detect the hemoglobin
in the blood sample;
[0076] d) determining the concentration of hemoglobin in the blood
sample based on the amount of light quantified in step c); and
[0077] e) comparing the concentration of hemoglobin in step (d)
with at least one predetermined level, wherein if the concentration
of hemoglobin determined in step (d) is lower then the
predetermined level, then the blood sample is determined to be an
older blood sample.
[0078] In the above method, the blood sample is whole blood, red
blood cells or umbilical cord blood.
[0079] In the above method, the basic solution is a solution having
a pH of at least about 10.
[0080] In the above method, any acridinium compound can be used.
For example, the acridinium compound can be an
acridinium-9-carboxamide having a structure according to formula
I:
##STR00007##
[0081] wherein R.sup.1 and R.sup.2 are each independently selected
from the group consisting of: alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R.sup.3
through R.sup.15 are each independently selected from the group
consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl,
amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide,
nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and
optionally, if present, X.sup..crclbar. is an anion.
[0082] Alternatively, the acridinium compound is an
acridinium-9-carboxylate aryl ester having a structure according to
formula II:
##STR00008##
[0083] wherein R.sup.1 is an alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and
[0084] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0085] optionally, if present, X.sup..crclbar. is an anion.
[0086] In the above method, the indicator solution can further
comprise at least one surfactant.
[0087] In the above method, the method can further comprise
measuring the amount of hemoglobin in the blood sample by relating
the amount of light generated in the test sample by comparison to a
standard curve for hemoglobin or to a reference standard.
Optionally, the standard curve can be generated from solutions of
hemoglobin of a known concentration.
[0088] In still yet a further aspect, the present invention relates
to a method of identifying a hemolyzed serum or plasma sample. The
method comprises the steps of:
[0089] a) adding at least one basic solution to a serum or plasma
sample;
[0090] b) adding an indicator solution to the serum or plasma
sample to generate a light signal, wherein the indicator solution
comprises at least one acridinium compound,
[0091] wherein steps a) and b) can be performed in any order;
[0092] c) quantifying the light generated to detect the hemoglobin
in the serum or plasma sample;
[0093] d) determining the concentration of hemoglobin in the serum
or plasma sample based on the amount of light quantified in step
c); and
[0094] e) comparing the concentration of hemoglobin in step (d)
with a predetermined level, wherein if the concentration of
hemoglobin determined in step (d) is higher then the predetermined
level, then the serum or plasma sample is determined to be
hemolyzed and further wherein if the concentration of hemoglobin
determined in step (d) is lower then the predetermined level, then
the serum or plasma sample is determined not to be hemolyzed.
[0095] In the above method, the basic solution is a solution having
a pH of at least about 10.
[0096] In the above method, any acridinium compound can be used.
For example, the acridinium compound can be an
acridinium-9-carboxamide having a structure according to formula
I:
##STR00009##
[0097] wherein R.sup.1 and R.sup.2 are each independently selected
from the group consisting of: alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R.sup.3
through R.sup.15 are each independently selected from the group
consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl,
amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide,
nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and
optionally, if present, X.sup..crclbar. is an anion.
[0098] Alternatively, the acridinium compound is an
acridinium-9-carboxylate aryl ester having a structure according to
formula II:
##STR00010##
[0099] wherein R.sup.1 is an alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and
[0100] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0101] optionally, if present, X.sup..crclbar. is an anion.
[0102] In the above method, the indicator solution can further
comprise at least one surfactant.
[0103] In the above method, the method can further comprise
measuring the amount of hemoglobin in the serum or plasma by
relating the amount of light generated in the serum or plasma by
comparison to a standard curve for hemoglobin or to a reference
standard. Optionally, the standard curve can be generated from
solutions of hemoglobin of a known concentration.
[0104] In still yet another aspect, the present invention relates
to a kit for detecting hemoglobin in a test sample. The kit
comprises:
[0105] a. at least one basic solution;
[0106] b. at least one indicator solution, wherein the indicator
solution comprises at least one acridinium compound; and
[0107] c. instructions for detecting hemoglobin in a test
sample.
[0108] In the above kit, the basic solution is a solution having a
pH of at least about 10.
[0109] In the above kit, any acridinium compound can be used. For
example, the acridinium compound can be an acridinium-9-carboxamide
having a structure according to formula I:
##STR00011##
[0110] wherein R.sup.1 and R.sup.2 are each independently selected
from the group consisting of: alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R.sup.3
through R.sup.15 are each independently selected from the group
consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl,
amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide,
nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and
optionally, if present, X.sup..crclbar. is an anion.
[0111] Alternatively, the acridinium compound is an
acridinium-9-carboxylate aryl ester having a structure according to
formula II:
##STR00012##
[0112] wherein R.sup.1 is an alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and
[0113] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0114] Xoptionally, if present, X.sup..crclbar. is an anion.
[0115] The indicator solution in the above kit can further comprise
at least one surfactant.
[0116] In still yet another aspect, the present invention relates
to a kit for detecting hemoglobin in a test sample. The kit
comprises:
[0117] a. at least one basic solution;
[0118] b. at least one electrode; and
[0119] c. instructions for detecting hemoglobin in a test
sample.
[0120] In the above kit, the basic solution is a solution having a
pH of at least about 10.
[0121] In still yet another aspect, the present invention relates
to a kit for diagnosing a subject suffering from a genetic disorder
relating to hemoglobin metabolism. The kit comprises:
[0122] a. at least one basic solution;
[0123] b. at least one indicator solution, wherein the indicator
solution comprises at least one acridinium compound; and
[0124] c. instructions for diagnosing a subject suffering from a
genetic disorder relating to hemoglobin metabolism.
[0125] In the above kit, the basic solution is a solution having a
pH of at least about 10.
[0126] In the above kit, any acridinium compound can be used. For
example, the acridinium compound can be an acridinium-9-carboxamide
having a structure according to formula I:
##STR00013##
[0127] wherein R.sup.1 and R.sup.2 are each independently selected
from the group consisting of: alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R.sup.3
through R.sup.15 are each independently selected from the group
consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl,
amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide,
nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and
optionally, if present, X.sup..crclbar. is an anion.
[0128] Alternatively, the acridinium compound is an
acridinium-9-carboxylate aryl ester having a structure according to
formula II:
##STR00014##
[0129] wherein R.sup.1 is an alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and
[0130] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0131] optionally, if present, X.sup..crclbar. is an anion.
[0132] The indicator solution in the above kit can further comprise
at least one surfactant.
[0133] In still yet another aspect, the present invention relates
to a kit for determining the eligibility of a subject to be a blood
donor. The kit comprises:
[0134] a. at least one basic solution;
[0135] b. at least one indicator solution, wherein the indicator
solution comprises at least one acridinium compound; and
[0136] c. instructions for determining the eligibility of a subject
to be a blood donor.
[0137] In the above kit, the basic solution is a solution having a
pH of at least about 10.
[0138] In the above kit, any acridinium compound can be used. For
example, the acridinium compound can be an acridinium-9-carboxamide
having a structure according to formula I:
##STR00015##
[0139] wherein R.sup.1 and R.sup.2 are each independently selected
from the group consisting of: alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R.sup.3
through R.sup.15 are each independently selected from the group
consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl,
amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide,
nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and
optionally, if present, X.sup..crclbar. is an anion.
[0140] Alternatively, the acridinium compound is an
acridinium-9-carboxylate aryl ester having a structure according to
formula II:
##STR00016##
[0141] wherein R.sup.1 is an alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and
[0142] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0143] optionally, if present, X.sup..crclbar. is an anion.
[0144] The indicator solution in the above kit can further comprise
at least one surfactant.
[0145] In still yet another embodiment, the present invention
relates to a kit for determining the age of a stored blood sample.
The kit comprises:
[0146] a. at least one basic solution;
[0147] b. at least one indicator solution, wherein the indicator
solution comprises at least one acridinium compound; and
[0148] c. instructions for determining the age of a blood
sample.
[0149] In the above kit, the basic solution is a solution having a
pH of at least about 10.
[0150] In the above kit, any acridinium compound can be used. For
example, the acridinium compound can be an acridinium-9-carboxamide
having a structure according to formula I:
##STR00017##
[0151] wherein R.sup.1 and R.sup.2 are each independently selected
from the group consisting of: alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R.sup.3
through R.sup.15 are each independently selected from the group
consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl,
amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide,
nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and
optionally, if present, X.sup..crclbar. is an anion.
[0152] Alternatively, the acridinium compound is an
acridinium-9-carboxylate aryl ester having a structure according to
formula II:
##STR00018##
[0153] wherein R.sup.1 is an alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and
[0154] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and optionally, if present,
X.sup..crclbar. is an anion.
[0155] The indicator solution in the above kit can further comprise
at least one surfactant.
[0156] In still yet another embodiment, the present invention
relates to a kit for identifying a hemolyzed serum or plasma
sample. The kit comprises:
[0157] a. at least one basic solution;
[0158] b. at least one indicator solution, wherein the indicator
solution comprises at least one acridinium compound; and
[0159] c. instructions for identifying a hemolyzed serum or plasma
sample.
[0160] In the above kit, the basic solution is a solution having a
pH of at least about 10.
[0161] In the above kit, any acridinium compound can be used. For
example, the acridinium compound can be an acridinium-9-carboxamide
having a structure according to formula I:
##STR00019##
[0162] wherein R.sup.1 and R.sup.2 are each independently selected
from the group consisting of: alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R.sup.3
through R.sup.15 are each independently selected from the group
consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl,
amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide,
nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and
optionally, if present, X.sup..crclbar. is an anion.
[0163] Alternatively, the acridinium compound is an
acridinium-9-carboxylate aryl ester having a structure according to
formula II:
##STR00020##
[0164] wherein R.sup.1 is an alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and
[0165] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0166] optionally, if present, X.sup..crclbar. is an anion.
[0167] The indicator solution in the above kit can further comprise
at least one surfactant.
BRIEF DESCRIPTION OF THE FIGURES
[0168] FIG. 1 shows the chemiluminescent detection reagent,
9-[[(3-Carboxypropyl)[(4-methylphenyl)sulfonyl]amino]-carbonyl]-10-(3-sul-
fopropyl)acridinium inner salt.
[0169] FIG. 2 shows the peak chemiluminescent signal (RLUmax) for
each whole blood dilution listed in Table 1 for each reciprocal
dilution factor (1/DF) as described in Example 1.
[0170] FIG. 3 shows the peak chemiluminescent signal RLUmax) for
each whole blood dilution listed in Table 1 for each dilution
factor (DF) as described in Example 1.
[0171] FIG. 4 shows the peak chemiluminescent signal (RLUmax) for
each whole blood dilution listed in Table 1 for each nominal
hemoglobin concentration (.mu.M) as described in Example 1.
[0172] FIG. 5 shows a comparison of the chemiluminescent profile
from whole blood samples treated with base before and after
ultrafiltration as described in Example 3.
[0173] FIG. 6 shows the peak chemiluminescent signal (RLUmax) for
each concentration of the ferrous hemoglobin standards listed in
Table 2 as described in Example 4.
[0174] FIG. 7 shows the chemiluminescence profile (RLU vs time) for
each ferrous hemoglobin standard concentration as described in
Example 4.
DETAILED DESCRIPTION
[0175] The present invention relates to methods of (a) detecting
the presence of hemoglobin in a test sample; (b) quantifying the
amount of hemoglobin in a test sample; (c) diagnosing a subject
suffering from a genetic disorder relating to hemoglobin
metabolism; (d) determining the eligibility of a subject to be a
blood donor; (e) determining the age of a stored blood sample; and
(f) identifying a hemolyzed plasma sample. The methods of the
present invention do not employ toxic chemicals employed in the
prior art and exhibit improved sensitivity compared to other
methods known in the art. Further, the methods of the present
invention can optionally employ very small test sample volumes and
provide assay results rapidly (namely, from about 1 second to about
60 minutes), facilitating high-throughput automation. Additionally,
the present invention relates to kits for performing each of the
above methods.
A. DEFINITIONS
[0176] Section headings as used in this section and the entire
disclosure herein are not intended to be limiting.
[0177] As used herein, the singular forms "a," "an" and "the"
include plural referents unless the context clearly dictates
otherwise. For the recitation of numeric ranges herein, each
intervening number there between with the same degree of precision
is explicitly contemplated. For example, for the range 6-9, the
numbers 7 and 8 are contemplated in addition to 6 and 9, and for
the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,
6.7, 6.8, 6.9 and 7.0 are explicitly contemplated.
[0178] a) Acyl (and Other Chemical Structural Group
Definitions)
[0179] As used herein, the term "acyl" refers to a --C(O)R.sub.a
group where R.sub.a is hydrogen, alkyl, cycloalkyl,
cycloalkylalkyl, phenyl or phenylalkyl. Representative examples of
acyl include, but are not limited to, formyl, acetyl,
cylcohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl,
benzylcarbonyl and the like.
[0180] As used herein, the term "alkenyl" means a straight or
branched chain hydrocarbon containing from 2 to 10 carbons and
containing at least one carbon-carbon double bond formed by the
removal of two hydrogens. Representative examples of alkenyl
include, but are not limited to, ethenyl, 2-propenyl,
2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl,
2-methyl-1-heptenyl, and 3-decenyl.
[0181] As used herein, the term "alkyl" means a straight or
branched chain hydrocarbon containing from 1 to 10 carbon atoms.
Representative examples of alkyl include, but are not limited to,
methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,
tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl,
2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl,
and n-decyl.
[0182] As used herein, the term "alkyl radical" means any of a
series of univalent groups of the general formula C.sub.nH.sub.2n+1
derived from straight or branched chain hydrocarbons.
[0183] As used herein, the term "alkoxy" means an alkyl group, as
defined herein, appended to the parent molecular moiety through an
oxygen atom. Representative examples of alkoxy include, but are not
limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy,
tert-butoxy, pentyloxy, and hexyloxy.
[0184] As used herein, the term "alkynyl" means a straight or
branched chain hydrocarbon group containing from 2 to 10 carbon
atoms and containing at least one carbon-carbon triple bond.
Representative examples of alkynyl include, but are not limited, to
acetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, and
1-butynyl.
[0185] As used herein, the term "amido" refers to an amino group
attached to the parent molecular moiety through a carbonyl group
(wherein the term "carbonyl group" refers to a --C(O)-- group).
[0186] As used herein, the term "amino" means --NR.sub.bR.sub.c,
wherein R.sub.b and R.sub.c are independently selected from the
group consisting of hydrogen, alkyl and alkylcarbonyl.
[0187] As used herein, the term "aralkyl" means an aryl group
appended to the parent molecular moiety through an alkyl group, as
defined herein. Representative examples of arylalkyl include, but
are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, and
2-naphth-2-ylethyl.
[0188] As used herein, the term "aryl" means a phenyl group, or a
bicyclic or tricyclic fused ring system wherein one or more of the
fused rings is a phenyl group. Bicyclic fused ring systems are
exemplified by a phenyl group fused to a cycloalkenyl group, a
cycloalkyl group, or another phenyl group. Tricyclic fused ring
systems are exemplified by a bicyclic fused ring system fused to a
cycloalkenyl group, a cycloalkyl group, as defined herein or
another phenyl group. Representative examples of aryl include, but
are not limited to, anthracenyl, azulenyl, fluorenyl, indanyl,
indenyl, naphthyl, phenyl, and tetrahydronaphthyl. The aryl groups
of the present disclosure can be optionally substituted with one-,
two, three, four, or five substituents independently selected from
the group consisting of alkoxy, alkyl, carboxyl, halo, and
hydroxyl.
[0189] As used herein, the term "carboxy" or "carboxyl" refers to
--CO.sub.2H or --CO.sub.2.
[0190] As used herein, the term "carboxyalkyl" refers to a
--(CH.sub.2).sub.nCO.sub.2H or --(CH.sub.2).sub.nCO.sub.2 group
where n is from 1 to 10.
[0191] As used herein, the term "cyano" means a --CN group.
[0192] As used herein, the term "cycloalkenyl" refers to a
non-aromatic cyclic or bicyclic ring system having from three to
ten carbon atoms and one to three rings, wherein each five-membered
ring has one double bond, each six-membered ring has one or two
double bonds, each seven- and eight-membered ring has one to three
double bonds, and each nine- to ten-membered ring has one to four
double bonds. Representative examples of cycloalkenyl groups
include cyclohexenyl, octahydronaphthalenyl, norbornylenyl, and the
like. The cycloalkenyl groups can be optionally substituted with
one, two, three, four, or five substituents independently selected
from the group consisting of alkoxy, alkyl, carboxyl, halo, and
hydroxyl.
[0193] As used herein, the term "cycloalkyl" refers to a saturated
monocyclic, bicyclic, or tricyclic hydrocarbon ring system having
three to twelve carbon atoms. Representative examples of cycloalkyl
groups include cyclopropyl, cyclopentyl, bicyclo[3.1.1]heptyl,
adamantyl, and the like. The cycloalkyl groups of the present
disclosure can be optionally substituted with one, two, three,
four, or five substituents independently selected from the group
consisting of alkoxy, alkyl, carboxyl, halo, and hydroxyl.
[0194] As used herein, the term "cycloalkylalkyl" means a
--R.sub.dR.sub.e group where R.sub.d is an alkylene group and
R.sub.e is cycloalkyl group. A representative example of a
cycloalkylalkyl group is cyclohexylmethyl and the like.
[0195] As used herein, the term "halogen" means a --Cl, --Br, --I
or --F; the term "halide" means a binary compound, of which one
part is a halogen atom and the other part is an element or radical
that is less electronegative than the halogen, e.g., an alkyl
radical.
[0196] As used herein, the term "hydroxyl" means an --OH group.
[0197] As used herein, the term "nitro" means a --NO.sub.2
group.
[0198] As used herein, the term "oxoalkyl" refers to
--(CH.sub.2).sub.nC(O)R.sub.a, where R.sub.a is hydrogen, alkyl,
cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl and where n is
from 1 to 10.
[0199] As used herein, the term "phenylalkyl" means an alkyl group
which is substituted by a phenyl group.
[0200] As used herein, the term "sulfo" means a --SO.sub.3H group.
As used herein, the term "sulfoalkyl" refers to a
--(CH.sub.2).sub.nSO.sub.3H or --(CH.sub.2).sub.nSO.sub.3.sup.-
group where n is from 1 to 10.
[0201] b) Anion
[0202] As used herein, the term "anion" refers to an anion of an
inorganic or organic acid, such as, but not limited to,
hydrochloric acid, hydrobromic acid, sulfuric acid, methane
sulfonic acid, formic acid, acetic acid, oxalic acid, succinic
acid, tartaric acid, mandelic acid, fumaric acid, lactic acid,
citric acid, glutamic acid, aspartic acid, phosphate,
trifluoromethansulfonic acid, trifluoroacetic acid and
fluorosulfonic acid and any combinations thereof
[0203] c) Pharmaceutical Composition
[0204] As used herein, the term "pharmaceutical composition" refers
to any agent or drug, whether a small molecule (e.g., a drug
containing an active agent, typically a non-peptidic) or biologic
(e.g., a peptide or protein based drug, including any with
modifications, such as, but not limited to PEGylation) that can be
used to treat a subject suffering from a disease or condition that
requires treatment.
[0205] d) Predetermined Level
[0206] As used herein, the term "predetermined level" refers
generally at an assay cutoff value that is used to assess
diagnostic results by comparing the assay results against the
predetermined level, and where the predetermined level already that
has been linked or associated with various clinical parameters
(e.g., assessing risk, severity of disease,
progression/nonprogression/improvement, determining the age of a
test sample, determining whether a test sample (e.g., serum or
plasma) has hemolyzed, etc.). The present invention provides
exemplary predetermined levels, and describes the initial linkage
or association of such levels with clinical parameters for
exemplary assays as described herein. However, it is well known
that cutoff values may vary dependent on the nature of the assay.
It further is well within the ordinary skill of one in the art to
adapt the invention herein for other assays to obtain
assay-specific cutoff values for those other assays based on this
description.
[0207] e) Specific Binding Partner
[0208] As used herein, the phrase "specific binding partner," as
used herein, is a member of a specific binding pair. That is, two
different molecules where one of the molecules, through chemical or
physical means, specifically binds to the second molecule.
Therefore, in addition to antigen and antibody specific binding
pairs of common immunoassays, other specific binding pairs can
include biotin and avidin (or streptavidin), carbohydrates and
lectins, complementary nucleotide sequences, effector and receptor
molecules, cofactors and enzymes, enzyme inhibitors, and enzymes
and the like. Furthermore, specific binding pairs can include
members that are analogs of the original specific binding members,
for example, an analyte-analog. Immunoreactive specific binding
members include antigens, antigen fragments, antibodies and
antibody fragments, both monoclonal and polyclonal and complexes
thereof, including those formed by recombinant DNA molecules.
[0209] f) Subject
[0210] As used herein, the terms "subject" and "patient" are used
interchangeably irrespective of whether the subject has or is
currently undergoing any form of treatment. As used herein, the
terms "subject" and "subjects" refer to any vertebrate, including,
but not limited to, a mammal (e.g., cow, pig, camel, llama, horse,
goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and
mouse, a non-human primate (for example, a monkey, such as a
cynomolgous monkey, chimpanzee, etc) and a human). Preferably, the
subject is a human.
[0211] g) Test Sample
[0212] As used herein, the term "test sample" generally refers to a
material being tested for and/or suspected of containing
hemoglobin. For example, the material can be a non-biological
forensic sample, such as clothing (e.g, shirts, pants, skirts,
pajamas, socks, underwear, coats, gloves, hats, pantyhose, etc.),
toothbrushes, combs, carpeting, towels, sheets, drapes, bedding,
chairs, couches, seats from vehicles or boats, etc. Alternatively,
the material may be a blood substitute. A number of blood
substitutes are known in the art. Example of blood substitutes
include, but are not limited to: recombinant human hemoglobin,
crosslinked bovine polyhemoglobin (e.g., Hemopure, Biopure
Corporation, Cambridge, Mass.) crosslinked human polyhemoglobin
(e.g., PolyHeme.RTM., Northfield Laboratories, Evanston, Ill.),
polyethylene glycol-modified hemoglobin (e.g., Hemospan.TM. Sangart
Inc., San Diego, Calif.), polymerized polynitroxyl hemoglobin
(e.g., HemoZyme, SynZyme Technologies, LLC, Irvine, Calif.),
perfluorocarbon based blood substitutes (See, U.S. Pat. No.
5,374,624; Oxycyte.TM., Costa Mesa, Calif.), etc. Alternatively,
the material can be a biological material being tested for and/or
suspected of containing hemoglobin. Biological materials may be
derived from any biological source. Examples of biological
materials include, but are not limited to, stool, whole blood,
serum, plasma, red blood cells, platelets, interstitial fluid,
saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine,
ascites fluid, mucous, nasal fluid, sputum, synovial fluid,
peritoneal fluid, vaginal fluid, menses, amniotic fluid, semen,
soil, etc. The test sample may be used directly as obtained from
the biological source or following a pretreatment to modify the
character of the sample. For example, such pretreatment may include
preparing plasma from blood, diluting viscous fluids and so forth.
Methods of pretreatment may also involve filtration, precipitation,
dilution, distillation, mixing, concentration, inactivation of
interfering components, the addition of reagents, lysing, etc. If
such methods of pretreatment are employed with respect to the test
sample, such pretreatment methods are such that hemoglobin remains
in the test sample at a concentration proportional to that in an
untreated test sample (e.g., namely, a test sample that is not
subjected to any such pretreatment method(s)).
[0213] The methods of present invention can optionally employ small
volumes of test samples. If such small volumes are employed, the
volume of test samples employed is from about 0.001 .mu.L to about
100 .mu.L.
B. METHODS OF THE PRESENT INVENTION
[0214] In one aspect, the present invention involves a method of
detecting and/or quantifying hemoglobin in a test sample. The
method involves obtaining a test sample. The type and source of the
test sample used in the method of the present invention is not
critical. For example, the test sample can be a biological sample
obtained from a subject. Alternatively, the test sample can be a
non-biological sample obtained from any source or location, such
as, but not limited to, a dwelling (e.g., house, apartment, trailer
home, dorm room, hotel room, bungalow, etc), a school, a place of
business, a car, a boat, motor home, bus, a park, etc.
[0215] Once the test sample is obtained, at least one basic
solution (which serves as a trigger solution) and optionally, at
least one indicator solution can each added to the test sample. The
order in which the at least one basic solution and, optionally, the
at least one indicator solution are added is not critical. The
basic solution used in the method is a solution that contains at
least one base and that has a pH greater than or equal to 10,
preferably, greater than or equal to 12. Examples of basic
solutions include, but are not limited to, sodium hydroxide,
potassium hydroxide, calcium hydroxide, ammonium hydroxide,
magnesium hydroxide, sodium carbonate, sodium bicarbonate, calcium
hydroxide, calcium carbonate and calcium bicarbonate. The amount of
basic solution added to the test sample depends on the
concentration of the basic solution used in the assay. Based on the
concentration of the basic solution used, one skilled in the art
could easily determine the amount of basic solution to be used in
the method described herein.
[0216] If an indicator solution is added to the test sample, the
indicator solution comprises at least one acridinium compound.
Preferably, the acridinium compound is an acridinium-9-carboxamide.
Specifically, the acridinium-9-carboxamide has a structure
according to formula I:
##STR00021##
[0217] wherein R.sup.1 and R.sup.2 are each independently selected
from the group consisting of: alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and
[0218] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and further wherein any of the alkyl,
alkenyl, alkynyl, aryl or aralkyl may contain one or more
heteroatoms; and
[0219] optionally, if present, X.sup..crclbar. is an anion.
[0220] Methods for preparing acridinium 9-carboxamides are
described in Mattingly, P. G. J. Biolumin. Chemilumin., 6, 107-14;
(1991); Adamczyk, M.; Chen, Y.-Y., Mattingly, P. G.; Pan, Y. J.
Org. Chem., 63, 5636-5639 (1998); Adamczyk, M.; Chen, Y.-Y.;
Mattingly, P. G.; Moore, J. A.; Shreder, K. Tetrahedron, 55,
10899-10914 (1999); Adamczyk, M.; Mattingly, P. G.; Moore, J. A.;
Pan, Y. Org. Lett., 1, 779-781 (1999); Adamczyk, M.; Chen, Y.-Y.;
Fishpaugh, J. R.; Mattingly, P. G.; Pan, Y.; Shreder, K.; Yu, Z.
Bioconjugate Chem., 11, 714-724 (2000); Mattingly, P. G.; Adamczyk,
M. In Luminescence Biotechnology: Instruments and Applications;
Dyke, K. V. Ed.; CRC Press: Boca Raton, pp. 77-105 (2002);
Adamczyk, M.; Mattingly, P. G.; Moore, J. A.; Pan, Y. Org. Lett.,
5, 3779-3782 (2003); and U.S. Pat. Nos. 5,468,646, 5,543,524 and
5,783,699 (each incorporated herein by reference in their
entireties for their teachings regarding same).
[0221] Alternatively, the acridinium compound can be an
acridinium-9-carboxylate aryl ester; the acridinium-9-carboxylate
aryl ester can have a structure according to formula II:
##STR00022##
[0222] wherein R.sup.1 is an alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and
[0223] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0224] optionally, if present, X.sup..crclbar. is an anion.
[0225] Examples of acridinium-9-carboxylate aryl esters having the
above formula II that can be used in the present disclosure
include, but are not limited to,
10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (available
from Cayman Chemical, Ann Arbor, Mich.). Methods for preparing
acridinium 9-carboxylate aryl esters are described in McCapra, F.,
et al., Photochem. Photobiol., 4, 1111-21 (1965); Razavi, Z et al.,
Luminescence, 15:245-249 (2000); Razavi, Z et al., Luminescence,
15:239-244 (2000); and U.S. Pat. No. 5,241,070 (each incorporated
herein by reference in their entireties for their teachings
regarding same).
[0226] In addition to the at least one acridinium compound, the
indicator solution can also contain at least one surfactant. Any
surfactant that when dissolved in water, lowers the surface tension
of the water and increases the solubility of organic compounds, can
be used in the present invention. Examples of surfactants that can
be used is one or more non-ionic or ionic surfactants (e.g.,
anionic, cationic or zwitterionic surfactants). Examples of
non-ionic surfactants that can be used include, but are not limited
to, t-octylpheoxypolyethoxyethanol (TRITON X-100, Sigma Aldrich,
St. Louis, Mo.), polyoxyethylenesorbitan monolaurate (Tween 20),
nonylphenol polyoxyethylene ether (Nonidet P10),
decyldimethylphosphine oxide (APO-10),
Cyclohexyl-n-ethyl-.beta.-D-Maltoside,
Cyclohexyl-n-hexyl-.beta.-D-Maltoside,
Cyclohexyl-n-methyl-.beta.-D-Maltoside, n-Decanoylsucrose,
n-Decyl-.beta.-D-glucopyranoside, n-Decyl-.beta.-D-maltopyranoside,
n-Decyl-.beta.-D-thiomaltoside, Digitonin, n-Dodecanoyl sucrose,
n-Dodecyl-.beta.-D-glucopyranoside, n-Dodecyl-.beta.-D-maltoside,
polyoxyethylene (10) dodecyl ether (Genapol C-100), isotridecanol
polyglycol ether (Genapol X-80), isotridecanol polyglycol ether
(Genapol X-100), Heptane-1,2,3-triol,
n-Heptyl-.beta.-D-glucopyranoside,
n-Heptyl-.beta.-D-thioglucopyranoside and combinations thereof. An
example of a ionic surfactant that can be used include, sodium
cholate, chenodeoxycholic acid, cholic acid, dehydrocholic acid,
docusate sodium, docusate sodium salt, glycocholic acid hydrate,
glycodeoxycholic acid monohydrate, glycolithocholic acid ethyl
ester, N-lauroylsarcosine sodium salt, N-lauroylsarcosine, lithium
dodecyl sulfate, calcium propionate, 1-octanesulfonic acid sodium
salt, sodium 1-butanesulfonate, sodium chenodeoxycholate, sodium
cholate hydrate, sodium 1-decanesulfonate, sodium
1-decanesulfonate, sodium deoxycholate, sodium deoxycholate
monohydrate, sodium dodecylbenzenesulfonate, sodium dodecyl
sulfate, sodium glycochenodeoxycholate, sodium glycocholate
hydrate, sodium 1-heptanesulfonate, sodium hexanesulfonate, sodium
1-nonanesulfonate, sodium octyle sulfate, sodium pentanesulfonate,
sodium 1-propanesulfonate hydrate, sodium taurodeoxycholate
hydrate, sodium taurohyodeoxycholate hydrate, sodium
tauroursodeoxycholate, taurocholic acid sodium salt hydrate,
taurolithocholic acid 3-sulfate disodium salt, Triton.RTM. X-200,
Triton.RTM. QS-15, Triton.RTM. QS-44, Triton.RTM. XQS-20,
Trizma.RTM. dodecyl sulfate, ursodeoxycholic acid,
alkyltrimethylammonium bromide, amprolium hydrocholoride,
benzalkonium chloride, benzethonium hydroxide,
benzyldimethylhexadecylammonium chloride,
benzyldodecyldimethylammonium bromide, choline p-toluenesulfonate
salt, dimethyldioctadecylammonium bromide,
dodecylethyldimethylammonium bromide, dodecyltrimethylammonium
bromide, ethylhexadecyldimethylammonium bromide, Ggirard's reagent,
hexadecylpyridinium bromide, hexadecylpyridinium chloride
monohydrate, hexadecylpyridinium chloride monohydrate,
hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium
p-toluenesulfonate, hexadecyltrimethylammonium bromide,
hexadecyltrimethylammonium p-toluenesulfonate, Hyamine.RTM. 1622,
methylbenzethonium chloride, myristyltrimethylammonium bromide,
oxyphenonium bromide, N,N',N'-polyoxyethylene
(10)-N-tallow-1,3-diaminopropane, tetraheptylammonium bromide,
tetrakis(decyl)ammonium bromide, thonzonium bromide and
Luviquat.TM. FC370, Luviquat.TM. HM 552, Luviquat.TM. HOLD,
Luviquat.TM. MS 370, Luviquat.TM. PQ 11PN and combinations thereof
(all available from Sigma Aldrich, St. Louis, Mo.).
[0227] Optionally, the test sample may be treated prior to addition
of the at least one basic solution, the at least one indicator
solution or prior to the addition of both the at least one basic
solution and the at least one indicator solution. Such treatment
may include dilution, ultrafiltration, extraction, precipitation,
dialysis, chromatography and digestion. Such treatment may be in
addition to and separate from any pretreatment that the test sample
may receive or be subjected to as discussed previously herein.
Moreover, if such treatment methods are employed with respect to
the test sample, such treatment methods are such that hemoglobin
remains in the test sample at a concentration proportional to that
in an untreated test sample (e.g., namely, a test sample that is
not subjected to any such treatment method(s)).
[0228] As mentioned briefly previously herein, the time and order
in which the test sample, the at least one basic solution and
optionally, the at least one indicator solution are added to form a
mixture is not critical. Additionally, the mixture formed by the at
least one basic solution, the at least one indicator solution or
the at least one basic solution and at least one indicator
solution, can optionally be allowed to incubate for a period of
time. For example, mixture can be allowed to incubate for a period
of time of from about 1 second to about 60 minutes. Specifically,
the mixture can be allowed to incubate for a period of from about 1
second to about 18 minutes.
[0229] If at least one indicator solution is used and added to the
test sample, after the addition of the at least one indicator
solution and the at least one basic solution to the test sample, a
detectable signal, namely, a chemiluminescent signal, is generated.
The signal generated by the mixture is detected for a fixed
duration of time. Preferably, the mixture is formed and the signal
is detected concurrently. The duration of the detection may range
from about 0.01 to about 360 seconds, more preferably from about
0.1 to about 30 seconds, and most preferably from about 0.5 to
about 5 seconds. Chemiluminescent signals generated can be detected
using routine techniques known to those skilled in the art.
[0230] Thus, in the assays of the present invention when an
indicator solution is used and added to the test sample, the
chemiluminescent signal generated after the addition of the basic
solution and the indicator solution indicates the presence of
hemoglobin in the test sample which can be detected. The amount or
concentration of hemoglobin in the test sample can be quantified
based on the intensity of the signal generated. Specifically, the
amount of hemoglobin contained in a test sample is proportional to
the intensity of the signal generated. Specifically, the amount of
hemoglobin present can be quantified based on comparing the amount
of light generated to a standard curve for hemoglobin or by
comparison to a reference standard. The standard curve can be
generated using serial dilutions or solutions to hemoglobin of
known concentration, by mass spectroscopy, gravimetrically and by
other techniques known in the art.
[0231] Alternatively, if no indicator solution is used or added to
the test sample, the presence of hemoglobin in the test sample can
be detected using electrochemical detection, the techniques for
which are well known to those skilled in the art. Such
electrochemical detection often employs one or more electrodes
connected to a device that measures and records an electrical
current. Such techniques can be realized in a number of
commercially available devices, such as the I-STAT.RTM. (Abbott
Laboratories, Abbott Park, Ill.) system, which comprises a
hand-held electrochemical detection instrument and self-contained
assay-specific reagent cartridges. For example, in the present
invention, the basic trigger solution could be contained in the
self-contained hemoglobin reagent cartridge and upon addition of
the test sample, a current would be generated at at least one
electrode that is proportional to the amount of hemoglobin in the
test sample.
[0232] The methods described herein can be used to identify the
presence of hemoglobin, and hence blood, in a test sample. For
example, the method described herein can be used to detect the
presence of blood in test samples obtained by the police during the
course of a criminal or missing persons investigation.
[0233] In another aspect, the present invention relates to methods
of diagnosing subjects suffering from a genetic disorder relating
to hemoglobin metabolism. The method involves obtaining a test
sample from a subject. A subject from which a test sample can be
obtained is any vertebrate. Preferably, the vertebrate is a mammal,
especially a human. Examples of mammals include, but are not
limited to, dogs, cats, rabbits, mice, rats, goats, sheep, cows,
pigs, horses, non-human primates and humans. The subject may be
suspected of suffering from a genetic disorder relating to
hemoglobin metabolism. The test sample can be obtained from the
subject using routine techniques known to those skilled in the
art.
[0234] Once the test sample from a subject is obtained, at least
one basic solution (which serves as a trigger solution) and at
least one indicator solution are each added to the test sample. The
order in which the at least one basic solution and the at least one
indicator solution are added is not critical. The basic solution
used in this method is a solution that contains at least one base
and that has a pH greater than or equal to 10, preferably, greater
than or equal to 12. Examples of basic solutions include, but are
not limited to, sodium hydroxide, potassium hydroxide, calcium
hydroxide, ammonium hydroxide, magnesium hydroxide, sodium
carbonate, sodium bicarbonate, calcium hydroxide, calcium carbonate
and calcium bicarbonate. The amount of basic solution added to the
test sample depends on the concentration of the basic solution used
in the assay. Based on the concentration of the basic solution
used, one skilled in the art could easily determine the amount of
basic solution to be used in the method described herein.
[0235] The indicator solution comprises at least one acridinium
compound. Preferably, the acridinium compound is an
acridinium-9-carboxamide. Specifically, the
acridinium-9-carboxamide has a structure according to formula
I:
##STR00023##
[0236] wherein R.sup.1 and R.sup.2 are each independently selected
from the group consisting of: alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and
[0237] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and further wherein any of the alkyl,
alkenyl, alkynyl, aryl or aralkyl may contain one or more
heteroatoms; and
[0238] optionally, if present, X.sup..crclbar. is an anion.
[0239] As discussed previously herein, methods for preparing
acridinium 9-carboxamides are described in Mattingly, P. G. J.
Biolumin. Chemilumin., 6, 107-14; (1991); Adamczyk, M.; Chen,
Y.-Y., Mattingly, P. G.; Pan, Y. J. Org. Chem., 63, 5636-5639
(1998); Adamczyk, M.; Chen, Y.-Y.; Mattingly, P. G.; Moore, J. A.;
Shreder, K. Tetrahedron, 55, 10899-10914 (1999); Adamczyk, M.;
Mattingly, P. G.; Moore, J. A.; Pan, Y. Org. Lett., 1, 779-781
(1999); Adamczyk, M.; Chen, Y.-Y.; Fishpaugh, J. R.; Mattingly, P.
G.; Pan, Y.; Shreder, K.; Yu, Z. Bioconjugate Chem., 11, 714-724
(2000); Mattingly, P. G.; Adamczyk, M. In Luminescence
Biotechnology: Instruments and Applications; Dyke, K. V. Ed.; CRC
Press: Boca Raton, pp. 77-105 (2002); Adamczyk, M.; Mattingly, P.
G.; Moore, J. A.; Pan, Y. Org. Lett., 5, 3779-3782 (2003); and U.S.
Pat. Nos. 5,468,646, 5,543,524 and 5,783,699 (each incorporated
herein by reference in their entireties for their teachings
regarding same).
[0240] Alternatively, the acridinium compound can be an
acridinium-9-carboxylate aryl ester; the acridinium-9-carboxylate
aryl ester can have a structure according to formula II:
##STR00024##
[0241] wherein R.sup.1 is an alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and
[0242] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0243] optionally, if present, X.sup..crclbar. is an anion.
[0244] Examples of acridinium-9-carboxylate aryl esters having the
above formula II that can be used in the present disclosure
include, but are not limited to,
10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (available
from Cayman Chemical, Ann Arbor, Mich.). Methods for preparing
acridinium 9-carboxylate aryl esters are described in McCapra, F.,
et al., Photochem. Photobiol., 4, 1111-21 (1965); Razavi, Z et al.,
Luminescence, 15:245-249 (2000); Razavi, Z et al., Luminescence,
15:239-244 (2000); and U.S. Pat. No. 5,241,070 (each incorporated
herein by reference in their entireties for their teachings
regarding same).
[0245] In addition to the at least one acridinium compound, the
indicator solution can also contain at least one surfactant. Any
surfactant that when dissolved in water, lowers the surface tension
of the water and increases the solubility of organic compounds, can
be used in the present invention. Examples of surfactants that can
be used is one or more non-ionic or ionic surfactants (e.g.,
anionic, cationic or zwitterionic surfactants). Examples of
non-ionic surfactants that can be used include, but are not limited
to, t-octylpheoxypolyethoxyethanol (TRITON X-100, Sigma Aldrich,
St. Louis, Mo.), polyoxyethylenesorbitan monolaurate (Tween 20),
nonylphenol polyoxyethylene ether (Nonidet P10),
decyldimethylphosphine oxide (APO-10),
Cyclohexyl-n-ethyl-.beta.-D-Maltoside,
Cyclohexyl-n-hexyl-.beta.-D-Maltoside,
Cyclohexyl-n-methyl-.beta.-D-Maltoside, n-Decanoylsucrose,
n-Decyl-.beta.-D-glucopyranoside, n-Decyl-.beta.-D-maltopyranoside,
n-Decyl-.beta.-D-thiomaltoside, Digitonin, n-Dodecanoyl sucrose,
n-Dodecyl-.beta.-D-glucopyranoside, n-Dodecyl-.beta.-D-maltoside,
polyoxyethylene (10) dodecyl ether (Genapol C-100), isotridecanol
polyglycol ether (Genapol X-80), isotridecanol polyglycol ether
(Genapol X-100), Heptane-1,2,3-triol,
n-Heptyl-.beta.-D-glucopyranoside,
n-Heptyl-.beta.-D-thioglucopyranoside and combinations thereof. An
example of a ionic surfactant that can be used include, sodium
cholate, chenodeoxycholic acid, cholic acid, dehydrocholic acid,
docusate sodium, docusate sodium salt, glycocholic acid hydrate,
glycodeoxycholic acid monohydrate, glycolithocholic acid ethyl
ester, N-lauroylsarcosine sodium salt, N-lauroylsarcosine, lithium
dodecyl sulfate, calcium propionate, 1-octanesulfonic acid sodium
salt, sodium 1-butanesulfonate, sodium chenodeoxycholate, sodium
cholate hydrate, sodium 1-decanesulfonate, sodium
1-decanesulfonate, sodium deoxycholate, sodium deoxycholate
monohydrate, sodium dodecylbenzenesulfonate, sodium dodecyl
sulfate, sodium glycochenodeoxycholate, sodium glycocholate
hydrate, sodium 1-heptanesulfonate, sodium hexanesulfonate, sodium
1-nonanesulfonate, sodium octyle sulfate, sodium pentanesulfonate,
sodium 1-propanesulfonate hydrate, sodium taurodeoxycholate
hydrate, sodium taurohyodeoxycholate hydrate, sodium
tauroursodeoxycholate, taurocholic acid sodium salt hydrate,
taurolithocholic acid 3-sulfate disodium salt, Triton.RTM. X-200,
Triton.RTM. QS-15, Triton.RTM. QS-44, Triton.RTM. XQS-20,
Trizma.RTM. dodecyl sulfate, ursodeoxycholic acid,
alkyltrimethylammonium bromide, amprolium hydrocholoride,
benzalkonium chloride, benzethonium hydroxide,
benzyldimethylhexadecylammonium chloride,
benzyldodecyldimethylammonium bromide, choline p-toluenesulfonate
salt, dimethyldioctadecylammonium bromide,
dodecylethyldimethylammonium bromide, dodecyltrimethylammonium
bromide, ethylhexadecyldimethylammonium bromide, Ggirard's reagent,
hexadecylpyridinium bromide, hexadecylpyridinium chloride
monohydrate, hexadecylpyridinium chloride monohydrate,
hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium
p-toluenesulfonate, hexadecyltrimethylammonium bromide,
hexadecyltrimethylammonium p-toluenesulfonate, Hyamine.RTM. 1622,
methylbenzethonium chloride, myristyltrimethylammonium bromide,
oxyphenonium bromide, N,N',N'-polyoxyethylene
(10)-N-tallow-1,3-diaminopropane, tetraheptylammonium bromide,
tetrakis(decyl)ammonium bromide, thonzonium bromide and
Luviquat.TM. FC370, Luviquat.TM. HM 552, Luviquat.TM. HOLD,
Luviquat.TM. MS 370, Luviquat.TM. PQ 11PN and combinations thereof
(all available from Sigma Aldrich, St. Louis, Mo.).
[0246] Optionally, the test sample may be treated prior to addition
of the at least one basic solution, the at least one indicator
solution or prior to the addition of both the at least one basic
solution and the at least one indicator solution. Such treatment
may include dilution, ultrafiltration, extraction, precipitation,
dialysis, chromatography and digestion. Such treatment may be in
addition to and separate from any pretreatment that the test sample
may receive or be subjected to as discussed previously herein.
Moreover, if such treatment methods are employed with respect to
the test sample, such treatment methods are such that hemoglobin
remains in the test sample at a concentration proportional to that
in an untreated test sample (e.g., namely, a test sample that is
not subjected to any such treatment method(s)).
[0247] As mentioned briefly previously herein, the time and order
in which the test sample, the at least one basic solution and the
at least one indicator solution are added to form a mixture is not
critical. Additionally, the mixture formed by the at least one
basic solution or the at least one indicator solution, can
optionally be allowed to incubate for a period of time. For
example, mixture can be allowed to incubate for a period of time of
from about 1 second to about 60 minutes. Specifically, the mixture
can be allowed to incubate for a period of from about 1 second to
about 18 minutes.
[0248] After the addition of the at least one indicator solution
and the at least one basic solution to the test sample, a
detectable signal, namely, a chemiluminescent signal, is generated.
The signal generated by the mixture is detected for a fixed
duration of time. Preferably, the mixture is formed and the signal
is detected concurrently. The duration of the detection may range
from about 0.01 to about 360 seconds, more preferably from about
0.1 to about 30 seconds, and most preferably from about 0.5 to
about 5 seconds. Chemiluminescent signals generated can be detected
using routine techniques known to those skilled in the art.
[0249] As discussed previously herein, the chemiluminescent signal
generated after the addition of the basic solution and the
indicator solution indicates the presence of hemoglobin in the test
sample. The amount or concentration of hemoglobin in the test
sample can then be quantified based on the intensity of the signal
generated. Specifically, the amount of hemoglobin contained in a
test sample is proportional to the intensity of the signal
generated. Specifically, the amount or concentration of hemoglobin
present can be quantified based on comparing the amount of light
generated to a standard curve for hemoglobin or by comparison to a
reference standard. The standard curve can be generated using
serial dilutions or solutions to hemoglobin of known concentration,
by mass spectroscopy, gravimetrically and by other techniques known
in the art.
[0250] Once the amount or concentration of hemoglobin in the test
sample is determined, the amount or concentration of hemoglobin
determined in the test sample is compared with a predetermined
level. Specifically, if the concentration of the hemoglobin
determined in the test sample is lower or higher then the
predetermined level (e.g., 7 to 8 g/dL), then a determination is
made that the subject is suffering from a genetic disorder relating
to hemoglobin metabolism. For example, if the concentration of
hemoglobin in a test sample obtained from a subject is determined
to be lower then a predetermined level, then the subject may be
identified as suffering from a genetic disorder such as anemia or
.beta.-thalassemia. Once a determination is made that a subject is
suffering from such a genetic disorder, the subject can be started
on treatment with one or more pharmaceutical compositions.
[0251] In still yet another aspect, the present invention relates
to a method of determining the eligibility of a subject to be a
blood donor. The method involves obtaining a test sample from a
subject. A subject from which a test sample can be obtained is any
vertebrate. Preferably, the vertebrate is a mammal, especially a
human. The test sample can be obtained from the subject using
routine techniques known to those skilled in the art.
[0252] Once the test sample from a subject is obtained, at least
one basic solution (which serves as a trigger solution) and at
least one indicator solution are each added to the test sample. The
order in which the at least one basic solution and the at least one
indicator solution are added is not critical. The basic solution
used in this method is a solution that contains at least one base
and that has a pH greater than or equal to 10, preferably, greater
than or equal to 12. Examples of basic solutions include, but are
not limited to, sodium hydroxide, potassium hydroxide, calcium
hydroxide, ammonium hydroxide, magnesium hydroxide, sodium
carbonate, sodium bicarbonate, calcium hydroxide, calcium carbonate
and calcium bicarbonate. The amount of basic solution added to the
test sample depends on the concentration of the basic solution used
in the assay. Based on the concentration of the basic solution
used, one skilled in the art could easily determine the amount of
basic solution to be used in the method described herein.
[0253] The indicator solution comprises at least one acridinium
compound. Preferably, the acridinium compound is an
acridinium-9-carboxamide. Specifically, the
acridinium-9-carboxamide has a structure according to formula
I:
##STR00025##
[0254] wherein R.sup.1 and R.sup.2 are each independently selected
from the group consisting of: alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and
[0255] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and further wherein any of the alkyl,
alkenyl, alkynyl, aryl or aralkyl may contain one or more
heteroatoms; and
[0256] optionally, if present, X.sup..crclbar. is an anion.
[0257] As discussed previously herein, methods for preparing
acridinium 9-carboxamides are described in Mattingly, P. G. J.
Biolumin. Chemilumin., 6, 107-14; (1991); Adamczyk, M.; Chen,
Y.-Y., Mattingly, P. G.; Pan, Y. J. Org. Chem., 63, 5636-5639
(1998); Adamczyk, M.; Chen, Y.-Y.; Mattingly, P. G.; Moore, J. A.;
Shreder, K. Tetrahedron, 55, 10899-10914 (1999); Adamczyk, M.;
Mattingly, P. G.; Moore, J. A.; Pan, Y. Org. Lett., 1, 779-781
(1999); Adamczyk, M.; Chen, Y.-Y.; Fishpaugh, J. R.; Mattingly, P.
G.; Pan, Y.; Shreder, K.; Yu, Z. Bioconjugate Chem., 11, 714-724
(2000); Mattingly, P. G.; Adamczyk, M. In Luminescence
Biotechnology: Instruments and Applications; Dyke, K. V. Ed.; CRC
Press: Boca Raton, pp. 77-105 (2002); Adamczyk, M.; Mattingly, P.
G.; Moore, J. A.; Pan, Y. Org. Lett., 5, 3779-3782 (2003); and U.S.
Pat. Nos. 5,468,646, 5,543,524 and 5,783,699 (each incorporated
herein by reference in their entireties for their teachings
regarding same).
[0258] Alternatively, the acridinium compound can be an
acridinium-9-carboxylate aryl ester; the acridinium-9-carboxylate
aryl ester can have a structure according to formula II:
##STR00026##
[0259] wherein R.sup.1 is an alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and
[0260] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0261] optionally, if present, X.sup..crclbar. is an anion.
[0262] Examples of acridinium-9-carboxylate aryl esters having the
above formula II that can be used in the present disclosure
include, but are not limited to,
10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (available
from Cayman Chemical, Ann Arbor, Mich.). Methods for preparing
acridinium 9-carboxylate aryl esters are described in McCapra, F.,
et al., Photochem. Photobiol., 4, 1111-21 (1965); Razavi, Z et al.,
Luminescence, 15:245-249 (2000); Razavi, Z et al., Luminescence,
15:239-244 (2000); and U.S. Pat. No. 5,241,070 (each incorporated
herein by reference in their entireties for their teachings
regarding same).
[0263] In addition to the at least one acridinium compound, the
indicator solution can also contain at least one surfactant. Any
surfactant that when dissolved in water, lowers the surface tension
of the water and increases the solubility of organic compounds, can
be used in the present invention. Examples of surfactants that can
be used is one or more non-ionic or ionic surfactants (e.g.,
anionic, cationic or zwitterionic surfactants). Examples of
non-ionic surfactants that can be used include, but are not limited
to, t-octylpheoxypolyethoxyethanol (TRITON X-100, Sigma Aldrich,
St. Louis, Mo.), polyoxyethylenesorbitan monolaurate (Tween 20),
nonylphenol polyoxyethylene ether (Nonidet P10),
decyldimethylphosphine oxide (APO-10),
Cyclohexyl-n-ethyl-.beta.-D-Maltoside,
Cyclohexyl-n-hexyl-.beta.-D-Maltoside,
Cyclohexyl-n-methyl-.beta.-D-Maltoside, n-Decanoylsucrose,
n-Decyl-.beta.-D-glucopyranoside, n-Decyl-.beta.-D-maltopyranoside,
n-Decyl-.beta.-D-thiomaltoside, Digitonin, n-Dodecanoyl sucrose,
n-Dodecyl-.beta.-D-glucopyranoside, n-Dodecyl-.beta.-D-maltoside,
polyoxyethylene (10) dodecyl ether (Genapol C-100), isotridecanol
polyglycol ether (Genapol X-80), isotridecanol polyglycol ether
(Genapol X-100), Heptane-1,2,3-triol,
n-Heptyl-.beta.-D-glucopyranoside,
n-Heptyl-.beta.-D-thioglucopyranoside and combinations thereof. An
example of a ionic surfactant that can be used include, sodium
cholate, chenodeoxycholic acid, cholic acid, dehydrocholic acid,
docusate sodium, docusate sodium salt, glycocholic acid hydrate,
glycodeoxycholic acid monohydrate, glycolithocholic acid ethyl
ester, N-lauroylsarcosine sodium salt, N-lauroylsarcosine, lithium
dodecyl sulfate, calcium propionate, 1-octanesulfonic acid sodium
salt, sodium 1-butanesulfonate, sodium chenodeoxycholate, sodium
cholate hydrate, sodium 1-decanesulfonate, sodium
1-decanesulfonate, sodium deoxycholate, sodium deoxycholate
monohydrate, sodium dodecylbenzenesulfonate, sodium dodecyl
sulfate, sodium glycochenodeoxycholate, sodium glycocholate
hydrate, sodium 1-heptanesulfonate, sodium hexanesulfonate, sodium
1-nonanesulfonate, sodium octyle sulfate, sodium pentanesulfonate,
sodium 1-propanesulfonate hydrate, sodium taurodeoxycholate
hydrate, sodium taurohyodeoxycholate hydrate, sodium
tauroursodeoxycholate, taurocholic acid sodium salt hydrate,
taurolithocholic acid 3-sulfate disodium salt, Triton.RTM. X-200,
Triton.RTM. QS-15, Triton.RTM. QS-44, Triton.RTM. XQS-20,
Trizma.RTM. dodecyl sulfate, ursodeoxycholic acid,
alkyltrimethylammonium bromide, amprolium hydrocholoride,
benzalkonium chloride, benzethonium hydroxide,
benzyldimethylhexadecylammonium chloride,
benzyldodecyldimethylammonium bromide, choline p-toluenesulfonate
salt, dimethyldioctadecylammonium bromide,
dodecylethyldimethylammonium bromide, dodecyltrimethylammonium
bromide, ethylhexadecyldimethylammonium bromide, Ggirard's reagent,
hexadecylpyridinium bromide, hexadecylpyridinium chloride
monohydrate, hexadecylpyridinium chloride monohydrate,
hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium
p-toluenesulfonate, hexadecyltrimethylammonium bromide,
hexadecyltrimethylammonium p-toluenesulfonate, Hyamine.RTM. 1622,
methylbenzethonium chloride, myristyltrimethylammonium bromide,
oxyphenonium bromide, N,N',N'-polyoxyethylene
(10)-N-tallow-1,3-diaminopropane, tetraheptylammonium bromide,
tetrakis(decyl)ammonium bromide, thonzonium bromide and
Luviquat.TM. FC370, Luviquat.TM. HM 552, Luviquat.TM. HOLD,
Luviquat.TM. MS 370, Luviquat.TM. PQ 11PN and combinations thereof
(all available from Sigma Aldrich, St. Louis, Mo.).
[0264] Optionally, the test sample may be treated prior to addition
of the at least one basic solution, the at least one indicator
solution or prior to the addition of both the at least one basic
solution and the at least one indicator solution. Such treatment
may include dilution, ultrafiltration, extraction, precipitation,
dialysis, chromatography and digestion. Such treatment may be in
addition to and separate from any pretreatment that the test sample
may receive or be subjected to as discussed previously herein.
Moreover, if such treatment methods are employed with respect to
the test sample, such treatment methods are such that hemoglobin
remains in the test sample at a concentration proportional to that
in an untreated test sample (e.g., namely, a test sample that is
not subjected to any such treatment method(s)).
[0265] As mentioned briefly previously herein, the time and order
in which the test sample, the at least one basic solution and the
at least one indicator solution are added to form a mixture is not
critical. Additionally, the mixture formed by the at least one
basic solution or the at least one indicator solution, can
optionally be allowed to incubate for a period of time. For
example, mixture can be allowed to incubate for a period of time of
from about 1 second to about 60 minutes. Specifically, the mixture
can be allowed to incubate for a period of from about 1 second to
about 18 minutes.
[0266] After the addition of the at least one indicator solution
and the at least one basic solution to the test sample, a
detectable signal, namely, a chemiluminescent signal, is generated.
The signal generated by the mixture is detected for a fixed
duration of time. Preferably, the mixture is formed and the signal
is detected concurrently. The duration of the detection may range
from about 0.01 to about 360 seconds, more preferably from about
0.1 to about 30 seconds, and most preferably from about 0.5 to
about 5 seconds. Chemiluminescent signals generated can be detected
using routine techniques known to those skilled in the art.
[0267] As discussed previously herein, the chemiluminescent signal
generated after the addition of the basic solution and the
indicator solution indicates the presence of hemoglobin in the test
sample. The amount or concentration of hemoglobin in the test
sample can then be quantified based on the intensity of the signal
generated. Specifically, the amount of hemoglobin contained in a
test sample is proportional to the intensity of the signal
generated. Specifically, the amount or concentration of hemoglobin
present can be quantified based on comparing the amount of light
generated to a standard curve for hemoglobin or by comparison to a
reference standard. The standard curve can be generated using
serial dilutions or solutions to hemoglobin of known concentration,
by mass spectroscopy, gravimetrically and by other techniques known
in the art.
[0268] Once the amount or concentration of hemoglobin in the test
sample is determined, the amount or concentration of hemoglobin
determined in the test sample is compared with a predetermined
level. Specifically, if the concentration of the hemoglobin
determined in the test sample is lower or higher then the
predetermined level (e.g., 12.5 grams/dL) then a determination is
made that the subject is not eligible to a blood donor.
[0269] In still yet another aspect, the present invention relates
to a method of determining the age of a stored blood sample based
on the concentration of ferrous hemoglobin in the stored blood
sample. Regulatory procedures have been established to define the
shelf-life of donated blood and its components. Currently, RBC
concentrates in CDPA-1 (citrate, dextrose, phosphate, and adenine)
have a shelf-life of about 35 days at a temperature of from about 1
to about 6.degree. C., while RBCs packed in an additive solution
can be stored for about 42 days. These limitations are based on the
75% viability of the erythrocytes 24 hours after transfusion.
Recent studies have indicated that these criteria may not be
sufficient and that poor outcomes may result in patients transfused
with RBCs older than 14 days (Koch et al., N. Eng. J. Med. 358:
1229-1239 (2008)). Some researchers have suggested that the
increased risk of adverse outcomes is due to depletion of
oxygen-carrying chemicals, i.e., hemoglobin. Methods for assessing
the suitability of erythrocytes for transfusion by assessing the
level of free choline in the erythrocytes, and a kit for use in
such a method and use of the method or kit to screen potential
blood donors and assess the suitability of processed blood products
for transfusion prior to use has been disclosed in Adamczyk, et al,
U.S. patent application Ser. No. 12/106,670, incorporated herein by
reference.
[0270] The present method involves obtaining a test sample from a
blood sample that has been stored for at least 30 minutes, at least
1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at
least 5 hours, at least 6 hours, at least 7 hours, at least 8
hours, at least 9 hours, at least 10 hours, at least 11 hours, at
least 12 hours, at least 13 hours, at least 14 hours, at least 15
hours, at least 16 hours, at least 17 hours, at least 18 hours, at
least 19 hours, at least 20 hours, at least 21 hours, at least 22
hours at least 23 hours, at least 1 day, at least 2 days, at least
3 days, at least 4 days, at least 5 days, at least 6 days, at least
7 days, at least 14 days, at least 21 days, at least 30 days, at
least 60 days, at least 90 days, at least 120 days, at least 5
months, at least 6 months, at least 7 months, at least 8 months, at
least 9 months, at least 10 months, at least 11 months, at least 12
months, at least 13 months, at least 14 months, at least 15 months,
at least 16 months, at least 17 months, at least 18 months, at
least 19 months, at least 20 months, at least 21 months, at least
22 months, at least 23 months, at least 24 months, at least 3
years, at least 4 years, at least 5 years, at least 6 years, at
least 7 years, at least 8 years, at least 9 years, at least 10
years, etc. The stored blood sample can be whole blood, red blood
cells (which can be buffered with glycerol), or umbilical cord
blood. Techniques for storing blood samples is well known to those
skilled in the art (e.g., a blood sample can be stored under
refrigeration at a temperature of from about 1.degree. C. to about
6.degree. C., a blood sample can be frozen and stored at
temperatures ranging from -18.degree. C. to about -80.degree. C.,
alternatively, a blood sample can be stored at room temperature).
The stored blood sample can be obtained from any vertebrate.
Preferably, the vertebrate is a mammal, especially a human. The
stored blood sample can be obtained from any source, such as, but
not limited to, a blood bank (e.g., a private or public blood bank)
or a company providing umbilical blood storage services.
[0271] Once the test sample from a stored blood sample is obtained,
at least one basic solution (which serves as a trigger solution)
and at least one indicator solution are each added to the test
sample. The order in which the at least one basic solution and the
at least one indicator solution are added is not critical. The
basic solution used in this method is a solution that contains at
least one base and that has a pH greater than or equal to 10,
preferably, greater than or equal to 12. Examples of basic
solutions include, but are not limited to, sodium hydroxide,
potassium hydroxide, calcium hydroxide, ammonium hydroxide,
magnesium hydroxide, sodium carbonate, sodium bicarbonate, calcium
hydroxide, calcium carbonate and calcium bicarbonate. The amount of
basic solution added to the test sample depends on the
concentration of the basic solution used in the assay. Based on the
concentration of the basic solution used, one skilled in the art
could easily determine the amount of basic solution to be used in
the method described herein.
[0272] The indicator solution comprises at least one acridinium
compound. Preferably, the acridinium compound is an
acridinium-9-carboxamide. Specifically, the
acridinium-9-carboxamide has a structure according to formula
I:
##STR00027##
[0273] wherein R.sup.1 and R.sup.2 are each independently selected
from the group consisting of: alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and
[0274] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and further wherein any of the alkyl,
alkenyl, alkynyl, aryl or aralkyl may contain one or more
heteroatoms; and
[0275] optionally, if present, X.sup..crclbar. is an anion.
[0276] As discussed previously herein, methods for preparing
acridinium 9-carboxamides are described in Mattingly, P. G. J.
Biolumin. Chemilumin., 6, 107-14; (1991); Adamczyk, M.; Chen,
Y.-Y., Mattingly, P. G.; Pan, Y. J. Org. Chem., 63, 5636-5639
(1998); Adamczyk, M.; Chen, Y.-Y.; Mattingly, P. G.; Moore, J. A.;
Shreder, K. Tetrahedron, 55, 10899-10914 (1999); Adamczyk, M.;
Mattingly, P. G.; Moore, J. A.; Pan, Y. Org. Lett., 1, 779-781
(1999); Adamczyk, M.; Chen, Y.-Y.; Fishpaugh, J. R.; Mattingly, P.
G.; Pan, Y.; Shreder, K.; Yu, Z. Bioconjugate Chem., 11, 714-724
(2000); Mattingly, P. G.; Adamczyk, M. In Luminescence
Biotechnology: Instruments and Applications; Dyke, K. V. Ed.; CRC
Press: Boca Raton, pp. 77-105 (2002); Adamczyk, M.; Mattingly, P.
G.; Moore, J. A.; Pan, Y. Org. Lett., 5, 3779-3782 (2003); and U.S.
Pat. Nos. 5,468,646, 5,543,524 and 5,783,699 (each incorporated
herein by reference in their entireties for their teachings
regarding same).
[0277] Alternatively, the acridinium compound can be an
acridinium-9-carboxylate aryl ester; the acridinium-9-carboxylate
aryl ester can have a structure according to formula II:
##STR00028##
[0278] wherein R.sup.1 is an alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and
[0279] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0280] optionally, if present, X.sup..crclbar. is an anion.
[0281] Examples of acridinium-9-carboxylate aryl esters having the
above formula II that can be used in the present disclosure
include, but are not limited to,
10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (available
from Cayman Chemical, Ann Arbor, Mich.). Methods for preparing
acridinium 9-carboxylate aryl esters are described in McCapra, F.,
et al., Photochem. Photobiol., 4, 1111-21 (1965); Razavi, Z et al.,
Luminescence, 15:245-249 (2000); Razavi, Z et al., Luminescence,
15:239-244 (2000); and U.S. Pat. No. 5,241,070 (each incorporated
herein by reference in their entireties for their teachings
regarding same).
[0282] In addition to the at least one acridinium compound, the
indicator solution can also contain at least one surfactant. Any
surfactant that when dissolved in water, lowers the surface tension
of the water and increases the solubility of organic compounds, can
be used in the present invention. Examples of surfactants that can
be used is one or more non-ionic or ionic surfactants (e.g.,
anionic, cationic or zwitterionic surfactants). Examples of
non-ionic surfactants that can be used include, but are not limited
to, t-octylpheoxypolyethoxyethanol (TRITON X-100, Sigma Aldrich,
St. Louis, Mo.), polyoxyethylenesorbitan monolaurate (Tween 20),
nonylphenol polyoxyethylene ether (Nonidet P10),
decyldimethylphosphine oxide (APO-10),
Cyclohexyl-n-ethyl-.beta.-D-Maltoside,
Cyclohexyl-n-hexyl-.beta.-D-Maltoside,
Cyclohexyl-n-methyl-.beta.-D-Maltoside, n-Decanoylsucrose,
n-Decyl-.beta.-D-glucopyranoside, n-Decyl-.beta.-D-maltopyranoside,
n-Decyl-.beta.-D-thiomaltoside, Digitonin, n-Dodecanoyl sucrose,
n-Dodecyl-.beta.-D-glucopyranoside, n-Dodecyl-.beta.-D-maltoside,
polyoxyethylene (10) dodecyl ether (Genapol C-100), isotridecanol
polyglycol ether (Genapol X-80), isotridecanol polyglycol ether
(Genapol X-100), Heptane-1,2,3-triol,
n-Heptyl-.beta.-D-glucopyranoside,
n-Heptyl-.beta.-D-thioglucopyranoside and combinations thereof. An
example of a ionic surfactant that can be used include, sodium
cholate, chenodeoxycholic acid, cholic acid, dehydrocholic acid,
docusate sodium, docusate sodium salt, glycocholic acid hydrate,
glycodeoxycholic acid monohydrate, glycolithocholic acid ethyl
ester, N-lauroylsarcosine sodium salt, N-lauroylsarcosine, lithium
dodecyl sulfate, calcium propionate, 1-octanesulfonic acid sodium
salt, sodium 1-butanesulfonate, sodium chenodeoxycholate, sodium
cholate hydrate, sodium 1-decanesulfonate, sodium
1-decanesulfonate, sodium deoxycholate, sodium deoxycholate
monohydrate, sodium dodecylbenzenesulfonate, sodium dodecyl
sulfate, sodium glycochenodeoxycholate, sodium glycocholate
hydrate, sodium 1-heptanesulfonate, sodium hexanesulfonate, sodium
1-nonanesulfonate, sodium octyle sulfate, sodium pentanesulfonate,
sodium 1-propanesulfonate hydrate, sodium taurodeoxycholate
hydrate, sodium taurohyodeoxycholate hydrate, sodium
tauroursodeoxycholate, taurocholic acid sodium salt hydrate,
taurolithocholic acid 3-sulfate disodium salt, Triton.RTM. X-200,
Triton.RTM. QS-15, Triton.RTM. QS-44, Triton.RTM. XQS-20,
Trizma.RTM. dodecyl sulfate, ursodeoxycholic acid,
alkyltrimethylammonium bromide, amprolium hydrocholoride,
benzalkonium chloride, benzethonium hydroxide,
benzyldimethylhexadecylammonium chloride,
benzyldodecyldimethylammonium bromide, choline p-toluenesulfonate
salt, dimethyldioctadecylammonium bromide,
dodecylethyldimethylammonium bromide, dodecyltrimethylammonium
bromide, ethylhexadecyldimethylammonium bromide, Ggirard's reagent,
hexadecylpyridinium bromide, hexadecylpyridinium chloride
monohydrate, hexadecylpyridinium chloride monohydrate,
hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium
p-toluenesulfonate, hexadecyltrimethylammonium bromide,
hexadecyltrimethylammonium p-toluenesulfonate, Hyamine.RTM. 1622,
methylbenzethonium chloride, myristyltrimethylammonium bromide,
oxyphenonium bromide, N,N',N'-polyoxyethylene
(10)-N-tallow-1,3-diaminopropane, tetraheptylammonium bromide,
tetrakis(decyl)ammonium bromide, thonzonium bromide and
Luviquat.TM. FC370, Luviquat.TM. HM 552, Luviquat.TM. HOLD,
Luviquat.TM. MS 370, Luviquat.TM. PQ 11PN and combinations thereof
(all available from Sigma Aldrich, St. Louis, Mo.).
[0283] Optionally, the test sample may be treated prior to addition
of the at least one basic solution, the at least one indicator
solution or prior to the addition of both the at least one basic
solution and the at least one indicator solution. Such treatment
may include dilution, ultrafiltration, extraction, precipitation,
dialysis, chromatography and digestion. Such treatment may be in
addition to and separate from any pretreatment that the test sample
may receive or be subjected to as discussed previously herein.
Moreover, if such treatment methods are employed with respect to
the test sample, such treatment methods are such that hemoglobin
remains in the test sample at a concentration proportional to that
in an untreated test sample (e.g., namely, a test sample that is
not subjected to any such treatment method(s)).
[0284] As mentioned briefly previously herein, the time and order
in which the test sample, the at least one basic solution and the
at least one indicator solution are added to form a mixture is not
critical. Additionally, the mixture formed by the at least one
basic solution or the at least one indicator solution, can
optionally be allowed to incubate for a period of time. For
example, mixture can be allowed to incubate for a period of time of
from about 1 second to about 60 minutes. Specifically, the mixture
can be allowed to incubate for a period of from about 1 second to
about 18 minutes.
[0285] After the addition of the at least one indicator solution
and the at least one basic solution to the stored blood sample, a
detectable signal, namely, a chemiluminescent signal, is generated.
The signal generated by the mixture is detected for a fixed
duration of time. Preferably, the mixture is formed and the signal
is detected concurrently. The duration of the detection may range
from about 0.01 to about 360 seconds, more preferably from about
0.1 to about 30 seconds, and most preferably from about 0.5 to
about 5 seconds. Chemiluminescent signals generated can be detected
using routine techniques known to those skilled in the art.
[0286] As discussed previously herein, the chemiluminescent signal
generated after the addition of the basic solution and the
indicator solution indicates the presence of hemoglobin in the
stored blood sample. The amount or concentration of hemoglobin in
the stored blood sample can then be quantified based on the
intensity of the signal generated. Specifically, the amount of
hemoglobin contained in a stored blood sample is proportional to
the intensity of the signal generated. Specifically, the amount or
concentration of hemoglobin present can be quantified based on
comparing the amount of light generated to a standard curve for
hemoglobin or by comparison to a reference standard. The standard
curve can be generated using serial dilutions or solutions to
hemoglobin of known concentration, by mass spectroscopy,
gravimetrically and by other techniques known in the art.
[0287] Once the amount or concentration of hemoglobin in the stored
blood sample is determined, the amount or concentration of
hemoglobin determined in the test sample is compared with a
predetermined level. Specifically, if the concentration of the
hemoglobin determined in the stored blood sample is lower then the
predetermined level, then a determination is made that the stored
blood sample is an older blood sample. Since the rate of hemoglobin
degradation may be affected by the storage conditions of the
sample, the predetermined level is preferably determined in the
end-user setting. The steps to validate the predetermined level may
include, the periodic measurement (for example, hourly, daily,
weekly, monthly or yearly) of hemoglobin in one or more blood
samples, or pooled blood samples, stored under conditions
representative of the standard operating procedures used in the
end-user setting, and over a set duration of time following the
initial collection of the sample from the source subject and
extending until the hemoglobin concentration has decreased by
greater than about 50% or preferably about 75%, or more preferably,
about 95%. The assignment of the predetermined level may be aided
by plotting the periodic measurements of hemoglobin concentration
versus time, and analyzing the resulting curve.
[0288] In still yet another aspect, the present invention further
relates to a method of identifying a hemolyzed serum or plasma
sample. Hemolysis is the breakage of the membrane of at least one
red blood cell which cause the release of hemoglobin and other
internal components into the surrounding fluid. Hemolysis is a
common occurrence seen in serum and plasma samples and can
compromise a laboratory's test parameters. In vitro hemolysis may
be caused by improper specimen collection, specimen processing or
specimen transport. Hemolysis may cause certain analytes to be
increased due to leakage of red cell constituents or may cause
interference in a test method.
[0289] The method of identifying a hemolyzed serum or plasma sample
involves obtaining a serum or plasma sample. The serum or plasma
sample may be a stored blood sample as described previously herein.
The serum or plasma sample can be obtained is any vertebrate.
Preferably, the vertebrate is a mammal, especially a human. The
serum or plasma sample can be obtained using routine techniques
known to those skilled in the art.
[0290] Once the serum or plasma sample is obtained, at least one
basic solution (which serves as a trigger solution) and at least
one indicator solution are each added to the serum or plasma
sample. The order in which the at least one basic solution and the
at least one indicator solution are added is not critical. The
basic solution used in this method is a solution that contains at
least one base and that has a pH greater than or equal to 10,
preferably, greater than or equal to 12. Examples of basic
solutions include, but are not limited to, sodium hydroxide,
potassium hydroxide, calcium hydroxide, ammonium hydroxide,
magnesium hydroxide, sodium carbonate, sodium bicarbonate, calcium
hydroxide, calcium carbonate and calcium bicarbonate. The amount of
basic solution added to the serum or plasma sample depends on the
concentration of the basic solution used in the assay. Based on the
concentration of the basic solution used, one skilled in the art
could easily determine the amount of basic solution to be used in
the method described herein.
[0291] The indicator solution comprises at least one acridinium
compound. Preferably, the acridinium compound is an
acridinium-9-carboxamide. Specifically, the
acridinium-9-carboxamide has a structure according to formula
I:
##STR00029##
[0292] wherein R.sup.1 and R.sup.2 are each independently selected
from the group consisting of: alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and
[0293] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and further wherein any of the alkyl,
alkenyl, alkynyl, aryl or aralkyl may contain one or more
heteroatoms; and
[0294] optionally, if present, X.sup..crclbar. is an anion.
[0295] As discussed previously herein, methods for preparing
acridinium 9-carboxamides are described in Mattingly, P. G. J.
Biolumin. Chemilumin., 6, 107-14; (1991); Adamczyk, M.; Chen,
Y.-Y., Mattingly, P. G.; Pan, Y. J. Org. Chem., 63, 5636-5639
(1998); Adamczyk, M.; Chen, Y.-Y.; Mattingly, P. G.; Moore, J. A.;
Shreder, K. Tetrahedron, 55, 10899-10914 (1999); Adamczyk, M.;
Mattingly, P. G.; Moore, J. A.; Pan, Y. Org. Lett., 1, 779-781
(1999); Adamczyk, M.; Chen, Y.-Y.; Fishpaugh, J. R.; Mattingly, P.
G.; Pan, Y.; Shreder, K.; Yu, Z. Bioconjugate Chem., 11, 714-724
(2000); Mattingly, P. G.; Adamczyk, M. In Luminescence
Biotechnology: Instruments and Applications; Dyke, K. V. Ed.; CRC
Press: Boca Raton, pp. 77-105 (2002); Adamczyk, M.; Mattingly, P.
G.; Moore, J. A.; Pan, Y. Org. Lett., 5, 3779-3782 (2003); and U.S.
Pat. Nos. 5,468,646, 5,543,524 and 5,783,699 (each incorporated
herein by reference in their entireties for their teachings
regarding same).
[0296] Alternatively, the acridinium compound can be an
acridinium-9-carboxylate aryl ester; the acridinium-9-carboxylate
aryl ester can have a structure according to formula II:
##STR00030##
[0297] wherein R.sup.1 is an alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and
[0298] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0299] optionally, if present, X.sup..crclbar. is an anion.
[0300] Examples of acridinium-9-carboxylate aryl esters having the
above formula II that can be used in the present disclosure
include, but are not limited to,
10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (available
from Cayman Chemical, Ann Arbor, Mich.). Methods for preparing
acridinium 9-carboxylate aryl esters are described in McCapra, F.,
et al., Photochem. Photobiol., 4, 1111-21 (1965); Razavi, Z et al.,
Luminescence, 15:245-249 (2000); Razavi, Z et al., Luminescence,
15:239-244 (2000); and U.S. Pat. No. 5,241,070 (each incorporated
herein by reference in their entireties for their teachings
regarding same).
[0301] In addition to the at least one acridinium compound, the
indicator solution can also contain at least one surfactant. Any
surfactant that when dissolved in water, lowers the surface tension
of the water and increases the solubility of organic compounds, can
be used in the present invention. Examples of surfactants that can
be used is one or more non-ionic or ionic surfactants (e.g.,
anionic, cationic or zwitterionic surfactants). Examples of
non-ionic surfactants that can be used include, but are not limited
to, t-octylpheoxypolyethoxyethanol (TRITON X-100, Sigma Aldrich,
St. Louis, Mo.), polyoxyethylenesorbitan monolaurate (Tween 20),
nonylphenol polyoxyethylene ether (Nonidet P10),
decyldimethylphosphine oxide (APO-10),
Cyclohexyl-n-ethyl-.beta.-D-Maltoside,
Cyclohexyl-n-hexyl-.beta.-D-Maltoside,
Cyclohexyl-n-methyl-.beta.-D-Maltoside, n-Decanoylsucrose,
n-Decyl-.beta.-D-glucopyranoside, n-Decyl-.beta.-D-maltopyranoside,
n-Decyl-.beta.-D-thiomaltoside, Digitonin, n-Dodecanoyl sucrose,
n-Dodecyl-.beta.-D-glucopyranoside, n-Dodecyl-.beta.-D-maltoside,
polyoxyethylene (10) dodecyl ether (Genapol C-100), isotridecanol
polyglycol ether (Genapol X-80), isotridecanol polyglycol ether
(Genapol X-100), Heptane-1,2,3-triol,
n-Heptyl-.beta.-D-glucopyranoside,
n-Heptyl-.beta.-D-thioglucopyranoside and combinations thereof. An
example of a ionic surfactant that can be used include, sodium
cholate, chenodeoxycholic acid, cholic acid, dehydrocholic acid,
docusate sodium, docusate sodium salt, glycocholic acid hydrate,
glycodeoxycholic acid monohydrate, glycolithocholic acid ethyl
ester, N-lauroylsarcosine sodium salt, N-lauroylsarcosine, lithium
dodecyl sulfate, calcium propionate, 1-octanesulfonic acid sodium
salt, sodium 1-butanesulfonate, sodium chenodeoxycholate, sodium
cholate hydrate, sodium 1-decanesulfonate, sodium
1-decanesulfonate, sodium deoxycholate, sodium deoxycholate
monohydrate, sodium dodecylbenzenesulfonate, sodium dodecyl
sulfate, sodium glycochenodeoxycholate, sodium glycocholate
hydrate, sodium 1-heptanesulfonate, sodium hexanesulfonate, sodium
1-nonanesulfonate, sodium octyle sulfate, sodium pentanesulfonate,
sodium 1-propanesulfonate hydrate, sodium taurodeoxycholate
hydrate, sodium taurohyodeoxycholate hydrate, sodium
tauroursodeoxycholate, taurocholic acid sodium salt hydrate,
taurolithocholic acid 3-sulfate disodium salt, Triton.RTM. X-200,
Triton.RTM. QS-15, Triton.RTM. QS-44, Triton.RTM. XQS-20,
Trizma.RTM. dodecyl sulfate, ursodeoxycholic acid,
alkyltrimethylammonium bromide, amprolium hydrocholoride,
benzalkonium chloride, benzethonium hydroxide,
benzyldimethylhexadecylammonium chloride,
benzyldodecyldimethylammonium bromide, choline p-toluenesulfonate
salt, dimethyldioctadecylammonium bromide,
dodecylethyldimethylammonium bromide, dodecyltrimethylammonium
bromide, ethylhexadecyldimethylammonium bromide, Ggirard's reagent,
hexadecylpyridinium bromide, hexadecylpyridinium chloride
monohydrate, hexadecylpyridinium chloride monohydrate,
hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium
p-toluenesulfonate, hexadecyltrimethylammonium bromide,
hexadecyltrimethylammonium p-toluenesulfonate, Hyamine.RTM. 1622,
methylbenzethonium chloride, myristyltrimethylammonium bromide,
oxyphenonium bromide, N,N',N'-polyoxyethylene
(10)-N-tallow-1,3-diaminopropane, tetraheptylammonium bromide,
tetrakis(decyl)ammonium bromide, thonzonium bromide and
Luviquat.TM. FC370, Luviquat.TM. HM 552, Luviquat.TM. HOLD,
Luviquat.TM. MS 370, Luviquat.TM. PQ 11PN and combinations thereof
(all available from Sigma Aldrich, St. Louis, Mo.).
[0302] Optionally, the serum or plasma sample may be treated prior
to addition of the at least one basic solution, the at least one
indicator solution or prior to the addition of both the at least
one basic solution and the at least one indicator solution. Such
treatment may include dilution, ultrafiltration, extraction,
precipitation, dialysis, chromatography and digestion. Such
treatment may be in addition to and separate from any pretreatment
that the serum or plasma sample may receive or be subjected to as
discussed previously herein. Moreover, if such treatment methods
are employed with respect to the serum or plasma sample, such
treatment methods are such that hemoglobin remains in the serum or
plasma sample at a concentration proportional to that in an
untreated serum or plasma sample (e.g., namely, a serum or plasma
sample that is not subjected to any such treatment method(s)).
[0303] As mentioned briefly previously herein, the time and order
in which the serum or plasma sample, the at least one basic
solution and the at least one indicator solution are added to form
a mixture is not critical. Additionally, the mixture formed by the
at least one basic solution or the at least one indicator solution,
can optionally be allowed to incubate for a period of time. For
example, mixture can be allowed to incubate for a period of time of
from about 1 second to about 60 minutes. Specifically, the mixture
can be allowed to incubate for a period of from about 1 second to
about 18 minutes.
[0304] After the addition of the at least one indicator solution
and the at least one basic solution to the serum or plasma sample,
a detectable signal, namely, a chemiluminescent signal, is
generated. The signal generated by the mixture is detected for a
fixed duration of time. Preferably, the mixture is formed and the
signal is detected concurrently. The duration of the detection may
range from about 0.01 to about 360 seconds, more preferably from
about 0.1 to about 30 seconds, and most preferably from about 0.5
to about 5 seconds. Chemiluminescent signals generated can be
detected using routine techniques known to those skilled in the
art.
[0305] As discussed previously herein, the chemiluminescent signal
generated after the addition of the basic solution and the
indicator solution indicates the presence of hemoglobin in the
serum or plasma sample. The amount or concentration of hemoglobin
in the serum or plasma sample can then be quantified based on the
intensity of the signal generated. Specifically, the amount of
hemoglobin contained in a serum or plasma sample is proportional to
the intensity of the signal generated. Specifically, the amount or
concentration of hemoglobin present can be quantified based on
comparing the amount of light generated to a standard curve for
hemoglobin or by comparison to a reference standard. The standard
curve can be generated using serial dilutions or solutions to
hemoglobin of known concentration, by mass spectroscopy,
gravimetrically and by other techniques known in the art.
[0306] Once the amount or concentration of hemoglobin in the serum
or plasma sample is determined, the amount or concentration of
hemoglobin determined in the serum or plasma sample is compared
with a predetermined level (e.g., 10 mg/dL). Specifically, if the
concentration of the hemoglobin determined in the serum or plasma
sample is lower then the predetermined level, then the serum or
plasma sample is determined not to be hemolyzed. However, if the
concentration of hemoglobin determined in the serum or plasma
sample is the same as or higher the predetermined level, then the
serum or plasma sample is determined to be hemolyzed. Serum or
plasma samples determined to be hemolyzed pursuant to the method
described herein are rejected and the sample is redrawn.
C. KITS
[0307] In another aspect, the present invention relates to a kit
for detecting and/or quantifying the amount of hemoglobin in a test
sample. The kit can contain at least one basic solution. The basic
solution is a solution that contains at least one base and that has
a pH greater than or equal to 10, preferably, greater than or equal
to 12. Examples of basic solutions that can be included in the kit
include, but are not limited to, sodium hydroxide, potassium
hydroxide, calcium hydroxide, ammonium hydroxide, magnesium
hydroxide, sodium carbonate, sodium bicarbonate, calcium hydroxide,
calcium carbonate and calcium bicarbonate.
[0308] Additionally, the kit can optionally contain at least one
indicator solution containing at least one acridinium compound. The
acridinium compound may comprise at least one
acridinium-9-carboxamide, at least one acridinium-9-carboxylate
aryl ester or any combinations thereof. More specifically, the
acridinium-9-carboxamide that can be used has the structure
according to Formula I:
##STR00031##
[0309] wherein R.sup.1 and R.sup.2 are each independently selected
from the group consisting of: alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and
[0310] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0311] further wherein any of the alkyl, alkenyl, alkynyl, aryl or
aralkyl may contain one or more heteroatoms; and
[0312] optionally, if present, X.sup..crclbar. is an anion.
[0313] Additionally, the acridinium-9-carboxylate aryl ester that
can be used has a structure according to formula II:
##STR00032##
[0314] wherein R.sup.1 is an alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and
[0315] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0316] optionally, if present, X.sup..crclbar. is an anion.
[0317] In addition, the at least one indicator solution can also
contain at least one surfactant. Any surfactant that when dissolved
in water, lowers the surface tension of the water and increases the
solubility of organic compounds, can be included. Examples of
surfactants that can be used is one or more non-ionic or ionic
surfactants (e.g., anionic, cationic or zwitterionic surfactants).
Examples of non-ionic surfactants that can be used include, but are
not limited to, t-octylpheoxypolyethoxyethanol (TRITON X-100, Sigma
Aldrich, St. Louis, Mo.), polyoxyethylenesorbitan monolaurate
(Tween 20), nonylphenol polyoxyethylene ether (Nonidet P10),
decyldimethylphosphine oxide (APO-10),
Cyclohexyl-n-ethyl-.beta.-D-Maltoside,
Cyclohexyl-n-hexyl-.beta.-D-Maltoside,
Cyclohexyl-n-methyl-.beta.-D-Maltoside, n-Decanoylsucrose,
n-Decyl-.beta.-D-glucopyranoside, n-Decyl-.beta.-D-maltopyranoside,
n-Decyl-.beta.-D-thiomaltoside, Digitonin, n-Dodecanoyl sucrose,
n-Dodecyl-.beta.-D-glucopyranoside, n-Dodecyl-.beta.-D-maltoside,
polyoxyethylene (10) dodecyl ether (Genapol C-100), isotridecanol
polyglycol ether (Genapol X-80), isotridecanol polyglycol ether
(Genapol X-100), Heptane-1,2,3-triol,
n-Heptyl-.beta.-D-glucopyranoside,
n-Heptyl-.beta.-D-thioglucopyranoside and combinations thereof. An
example of a ionic surfactant that can be used include, sodium
cholate, chenodeoxycholic acid, cholic acid, dehydrocholic acid,
docusate sodium, docusate sodium salt, glycocholic acid hydrate,
glycodeoxycholic acid monohydrate, glycolithocholic acid ethyl
ester, N-lauroylsarcosine sodium salt, N-lauroylsarcosine, lithium
dodecyl sulfate, calcium propionate, 1-octanesulfonic acid sodium
salt, sodium 1-butanesulfonate, sodium chenodeoxycholate, sodium
cholate hydrate, sodium 1-decanesulfonate, sodium
1-decanesulfonate, sodium deoxycholate, sodium deoxycholate
monohydrate, sodium dodecylbenzenesulfonate, sodium dodecyl
sulfate, sodium glycochenodeoxycholate, sodium glycocholate
hydrate, sodium 1-heptanesulfonate, sodium hexanesulfonate, sodium
1-nonanesulfonate, sodium octyle sulfate, sodium pentanesulfonate,
sodium 1-propanesulfonate hydrate, sodium taurodeoxycholate
hydrate, sodium taurohyodeoxycholate hydrate, sodium
tauroursodeoxycholate, taurocholic acid sodium salt hydrate,
taurolithocholic acid 3-sulfate disodium salt, Triton.RTM. X-200,
Triton.RTM. QS-15, Triton.RTM. QS-44, Triton.RTM. XQS-20,
Trizma.RTM. dodecyl sulfate, ursodeoxycholic acid,
alkyltrimethylammonium bromide, amprolium hydrocholoride,
benzalkonium chloride, benzethonium hydroxide,
benzyldimethylhexadecylammonium chloride,
benzyldodecyldimethylammonium bromide, choline p-toluenesulfonate
salt, dimethyldioctadecylammonium bromide,
dodecylethyldimethylammonium bromide, dodecyltrimethylammonium
bromide, ethylhexadecyldimethylammonium bromide, Ggirard's reagent,
hexadecylpyridinium bromide, hexadecylpyridinium chloride
monohydrate, hexadecylpyridinium chloride monohydrate,
hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium
p-toluenesulfonate, hexadecyltrimethylammonium bromide,
hexadecyltrimethylammonium p-toluenesulfonate, Hyamine.RTM. 1622,
methylbenzethonium chloride, myristyltrimethylammonium bromide,
oxyphenonium bromide, N,N',N'-polyoxyethylene
(10)-N-tallow-1,3-diaminopropane, tetraheptylammonium bromide,
tetrakis(decyl)ammonium bromide, thonzonium bromide and
Luviquat.TM. FC370, Luviquat.TM. HM 552, Luviquat.TM. HOLD,
Luviquat.TM. MS 370, Luviquat.TM. PQ 11PN and combinations thereof
(all available from Sigma Aldrich, St. Louis, Mo.).
[0318] Optionally, if no indicator solution is included in the kit,
the kit can comprise at least one electrode.
[0319] Also, the kit can also contain one or more instructions for
detecting and/or quantifying the amount of hemoglobin in a test
sample. The kit can also contain instructions for generating a
standard curve for the purposes of quantifying the hemoglobin or a
reference standard for purposes of quantifying the hemoglobin in
the test sample. Such instructions optionally can be in printed
form or on CD, DVD, or other format of recorded media.
[0320] In another embodiment, the present invention relates to a
kit for diagnosing a subject suffering from a genetic disorder
relating to hemoglobin metabolism. The kit can contain at least one
basic solution. The basic solution is a solution that contains at
least one base and that has a pH greater than or equal to 10,
preferably, greater than or equal to 12. Examples of basic
solutions that can be included in the kit include, but are not
limited to, sodium hydroxide, potassium hydroxide, calcium
hydroxide, ammonium hydroxide, magnesium hydroxide, sodium
carbonate, sodium bicarbonate, calcium hydroxide, calcium carbonate
and calcium bicarbonate.
[0321] Additionally, the kit can contain at least one indicator
solution containing at least one acridinium compound. The
acridinium compound may comprise at least one
acridinium-9-carboxamide, at least one acridinium-9-carboxylate
aryl ester or any combinations thereof More specifically, the
acridinium-9-carboxamide that can be used has the structure
according to Formula I:
##STR00033##
[0322] wherein R.sup.1 and R.sup.2 are each independently selected
from the group consisting of: alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and
[0323] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0324] further wherein any of the alkyl, alkenyl, alkynyl, aryl or
aralkyl may contain one or more heteroatoms; and
[0325] optionally, if present, X.sup..crclbar. is an anion.
[0326] Additionally, the acridinium-9-carboxylate aryl ester that
can be used has a structure according to formula II:
##STR00034##
[0327] wherein R.sup.1 is an alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and
[0328] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0329] optionally, if present, X.sup..crclbar. is an anion.
[0330] In addition to the at least one acridinium compound, the
indicator solution can also contain at least one surfactant. Any
surfactant that when dissolved in water, lowers the surface tension
of the water and increases the solubility of organic compounds, can
be used included. Examples of surfactants that can be used is one
or more non-ionic or ionic surfactants (e.g., anionic, cationic or
zwitterionic surfactants). Examples of non-ionic surfactants that
can be used include, but are not limited to,
t-octylpheoxypolyethoxyethanol (TRITON X-100, Sigma Aldrich, St.
Louis, Mo.), polyoxyethylenesorbitan monolaurate (Tween 20),
nonylphenol polyoxyethylene ether (Nonidet P10),
decyldimethylphosphine oxide (APO-10),
Cyclohexyl-n-ethyl-.beta.-D-Maltoside,
Cyclohexyl-n-hexyl-.beta.-D-Maltoside,
Cyclohexyl-n-methyl-.beta.-D-Maltoside, n-Decanoylsucrose,
n-Decyl-.beta.-D-glucopyranoside, n-Decyl-.beta.-D-maltopyranoside,
n-Decyl-.beta.-D-thiomaltoside, Digitonin, n-Dodecanoyl sucrose,
n-Dodecyl-.beta.-D-glucopyranoside, n-Dodecyl-.beta.-D-maltoside,
polyoxyethylene (10) dodecyl ether (Genapol C-100), isotridecanol
polyglycol ether (Genapol X-80), isotridecanol polyglycol ether
(Genapol X-100), Heptane-1,2,3-triol,
n-Heptyl-.beta.-D-glucopyranoside,
n-Heptyl-.beta.-D-thioglucopyranoside and combinations thereof. An
example of a ionic surfactant that can be used include, sodium
cholate, chenodeoxycholic acid, cholic acid, dehydrocholic acid,
docusate sodium, docusate sodium salt, glycocholic acid hydrate,
glycodeoxycholic acid monohydrate, glycolithocholic acid ethyl
ester, N-lauroylsarcosine sodium salt, N-lauroylsarcosine, lithium
dodecyl sulfate, calcium propionate, 1-octanesulfonic acid sodium
salt, sodium 1-butanesulfonate, sodium chenodeoxycholate, sodium
cholate hydrate, sodium 1-decanesulfonate, sodium
1-decanesulfonate, sodium deoxycholate, sodium deoxycholate
monohydrate, sodium dodecylbenzenesulfonate, sodium dodecyl
sulfate, sodium glycochenodeoxycholate, sodium glycocholate
hydrate, sodium 1-heptanesulfonate, sodium hexanesulfonate, sodium
1-nonanesulfonate, sodium octyle sulfate, sodium pentanesulfonate,
sodium 1-propanesulfonate hydrate, sodium taurodeoxycholate
hydrate, sodium taurohyodeoxycholate hydrate, sodium
tauroursodeoxycholate, taurocholic acid sodium salt hydrate,
taurolithocholic acid 3-sulfate disodium salt, Triton.RTM. X-200,
Triton.RTM. QS-15, Triton.RTM. QS-44, Triton.RTM. XQS-20,
Trizma.RTM. dodecyl sulfate, ursodeoxycholic acid,
alkyltrimethylammonium bromide, amprolium hydrocholoride,
benzalkonium chloride, benzethonium hydroxide,
benzyldimethylhexadecylammonium chloride,
benzyldodecyldimethylammonium bromide, choline p-toluenesulfonate
salt, dimethyldioctadecylammonium bromide,
dodecylethyldimethylammonium bromide, dodecyltrimethylammonium
bromide, ethylhexadecyldimethylammonium bromide, Ggirard's reagent,
hexadecylpyridinium bromide, hexadecylpyridinium chloride
monohydrate, hexadecylpyridinium chloride monohydrate,
hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium
p-toluenesulfonate, hexadecyltrimethylammonium bromide,
hexadecyltrimethylammonium p-toluenesulfonate, Hyamine.RTM. 1622,
methylbenzethonium chloride, myristyltrimethylammonium bromide,
oxyphenonium bromide, N,N',N'-polyoxyethylene
(10)-N-tallow-1,3-diaminopropane, tetraheptylammonium bromide,
tetrakis(decyl)ammonium bromide, thonzonium bromide and
Luviquat.TM. FC370, Luviquat.TM. HM 552, Luviquat.TM. HOLD,
Luviquat.TM. MS 370, Luviquat.TM. PQ 11PN and combinations thereof
(all available from Sigma Aldrich, St. Louis, Mo.).
[0331] Also, the kit can also contain one or more instructions for
diagnosing whether a subject is suffering from a genetic disorder.
The kit can also contain instructions for generating a standard
curve for the purposes of quantifying the hemoglobin or a reference
standard for purposes of quantifying the hemoglobin in the test
sample obtained from a subject. Such instructions optionally can be
in printed form or on CD, DVD, or other format of recorded
media.
[0332] In still yet another aspect, the present invention relates
to a kit for determining the eligibility of a subject to be a blood
donor. The kit can contain at least one basic solution. The basic
solution is a solution that contains at least one base and that has
a pH greater than or equal to 10, preferably, greater than or equal
to 12. Examples of basic solutions that can be included in the kit
include, but are not limited to, sodium hydroxide, potassium
hydroxide, calcium hydroxide, ammonium hydroxide, magnesium
hydroxide, sodium carbonate, sodium bicarbonate, calcium hydroxide,
calcium carbonate and calcium bicarbonate.
[0333] Additionally, the kit can contain at least one indicator
solution containing at least one acridinium compound. The
acridinium compound may comprise at least one
acridinium-9-carboxamide, at least one acridinium-9-carboxylate
aryl ester or any combinations thereof. More specifically, the
acridinium-9-carboxamide that can be used has the structure
according to Formula I:
##STR00035##
[0334] wherein R.sup.1 and R.sup.2 are each independently selected
from the group consisting of: alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and
[0335] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0336] further wherein any of the alkyl, alkenyl, alkynyl, aryl or
aralkyl may contain one or more heteroatoms; and
[0337] optionally, if present, X.sup..crclbar. is an anion.
[0338] Additionally, the acridinium-9-carboxylate aryl ester that
can be used has a structure according to formula II:
##STR00036##
[0339] wherein R.sup.1 is an alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and
[0340] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0341] optionally, if present, X.sup..crclbar. is an anion.
[0342] In addition to the at least one acridinium compound, the
indicator solution can also contain at least one surfactant. Any
surfactant that when dissolved in water, lowers the surface tension
of the water and increases the solubility of organic compounds, can
be included. Examples of surfactants that can be used is one or
more non-ionic or ionic surfactants (e.g., anionic, cationic or
zwitterionic surfactants). Examples of non-ionic surfactants that
can be used include, but are not limited to,
t-octylpheoxypolyethoxyethanol (TRITON X-100, Sigma Aldrich, St.
Louis, Mo.), polyoxyethylenesorbitan monolaurate (Tween 20),
nonylphenol polyoxyethylene ether (Nonidet P10),
decyldimethylphosphine oxide (APO-10),
Cyclohexyl-n-ethyl-.beta.-D-Maltoside,
Cyclohexyl-n-hexyl-.beta.-D-Maltoside,
Cyclohexyl-n-methyl-.beta.-D-Maltoside, n-Decanoylsucrose,
n-Decyl-.beta.-D-glucopyranoside, n-Decyl-.beta.-D-maltopyranoside,
n-Decyl-.beta.-D-thiomaltoside, Digitonin, n-Dodecanoyl sucrose,
n-Dodecyl-.beta.-D-glucopyranoside, n-Dodecyl-.beta.-D-maltoside,
polyoxyethylene (10) dodecyl ether (Genapol C-100), isotridecanol
polyglycol ether (Genapol X-80), isotridecanol polyglycol ether
(Genapol X-100), Heptane-1,2,3-triol,
n-Heptyl-.beta.-D-glucopyranoside,
n-Heptyl-.beta.-D-thioglucopyranoside and combinations thereof. An
example of a ionic surfactant that can be used include, sodium
cholate, chenodeoxycholic acid, cholic acid, dehydrocholic acid,
docusate sodium, docusate sodium salt, glycocholic acid hydrate,
glycodeoxycholic acid monohydrate, glycolithocholic acid ethyl
ester, N-lauroylsarcosine sodium salt, N-lauroylsarcosine, lithium
dodecyl sulfate, calcium propionate, 1-octanesulfonic acid sodium
salt, sodium 1-butanesulfonate, sodium chenodeoxycholate, sodium
cholate hydrate, sodium 1-decanesulfonate, sodium
1-decanesulfonate, sodium deoxycholate, sodium deoxycholate
monohydrate, sodium dodecylbenzenesulfonate, sodium dodecyl
sulfate, sodium glycochenodeoxycholate, sodium glycocholate
hydrate, sodium 1-heptanesulfonate, sodium hexanesulfonate, sodium
1-nonanesulfonate, sodium octyle sulfate, sodium pentanesulfonate,
sodium 1-propanesulfonate hydrate, sodium taurodeoxycholate
hydrate, sodium taurohyodeoxycholate hydrate, sodium
tauroursodeoxycholate, taurocholic acid sodium salt hydrate,
taurolithocholic acid 3-sulfate disodium salt, Triton.RTM. X-200,
Triton.RTM. QS-15, Triton.RTM. QS-44, Triton.RTM. XQS-20,
Trizma.RTM. dodecyl sulfate, ursodeoxycholic acid,
alkyltrimethylammonium bromide, amprolium hydrocholoride,
benzalkonium chloride, benzethonium hydroxide,
benzyldimethylhexadecylammonium chloride,
benzyldodecyldimethylammonium bromide, choline p-toluenesulfonate
salt, dimethyldioctadecylammonium bromide,
dodecylethyldimethylammonium bromide, dodecyltrimethylammonium
bromide, ethylhexadecyldimethylammonium bromide, Ggirard's reagent,
hexadecylpyridinium bromide, hexadecylpyridinium chloride
monohydrate, hexadecylpyridinium chloride monohydrate,
hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium
p-toluenesulfonate, hexadecyltrimethylammonium bromide,
hexadecyltrimethylammonium p-toluenesulfonate, Hyamine.RTM. 1622,
methylbenzethonium chloride, myristyltrimethylammonium bromide,
oxyphenonium bromide, N,N',N'-polyoxyethylene
(10)-N-tallow-1,3-diaminopropane, tetraheptylammonium bromide,
tetrakis(decyl)ammonium bromide, thonzonium bromide and
Luviquat.TM. FC370, Luviquat.TM. HM 552, Luviquat.TM. HOLD,
Luviquat.TM. MS 370, Luviquat.TM. PQ 11PN and combinations thereof
(all available from Sigma Aldrich, St. Louis, Mo.).
[0343] Also, the kit can also contain one or more instructions for
determining the eligibility of a subject to be a blood donor. The
kit can also contain instructions for generating a standard curve
for the purposes of quantifying the hemoglobin or a reference
standard for purposes of quantifying the hemoglobin in the test
sample. Such instructions optionally can be in printed form or on
CD, DVD, or other format of recorded media.
[0344] In still yet a further aspect, the present invention relates
to a kit for determining the age of a stored blood sample (such as
whole blood, serum, plasma, platelets, red blood cells or umbilical
cord blood). The kit can contain at least one basic solution. The
basic solution is a solution that contains at least one base and
that has a pH greater than or equal to 10, preferably, greater than
or equal to 12. Examples of basic solutions that can be included in
the kit include, but are not limited to, sodium hydroxide,
potassium hydroxide, calcium hydroxide, ammonium hydroxide,
magnesium hydroxide, sodium carbonate, sodium bicarbonate, calcium
hydroxide, calcium carbonate and calcium bicarbonate.
[0345] Additionally, the kit can contain at least one indicator
solution containing at least one acridinium compound. The
acridinium compound may comprise at least one
acridinium-9-carboxamide, at least one acridinium-9-carboxylate
aryl ester or any combinations thereof. More specifically, the
acridinium-9-carboxamide that can be used has the structure
according to Formula I:
##STR00037##
[0346] wherein R.sup.1 and R.sup.2 are each independently selected
from the group consisting of: alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and
[0347] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0348] further wherein any of the alkyl, alkenyl, alkynyl, aryl or
aralkyl may contain one or more heteroatoms; and
[0349] optionally, if present, X.sup..crclbar. is an anion.
[0350] Additionally, the acridinium-9-carboxylate aryl ester that
can be used has a structure according to formula II:
##STR00038##
[0351] wherein R.sup.1 is an alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and
[0352] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0353] optionally, if present, X.sup..crclbar. is an anion.
[0354] In addition to the at least one acridinium compound, the
indicator solution can also contain at least one surfactant. Any
surfactant that when dissolved in water, lowers the surface tension
of the water and increases the solubility of organic compounds, can
be included. Examples of surfactants that can be used is one or
more non-ionic or ionic surfactants (e.g., anionic, cationic or
zwitterionic surfactants). Examples of non-ionic surfactants that
can be used include, but are not limited to,
t-octylpheoxypolyethoxyethanol (TRITON X-100, Sigma Aldrich, St.
Louis, Mo.), polyoxyethylenesorbitan monolaurate (Tween 20),
nonylphenol polyoxyethylene ether (Nonidet P10),
decyldimethylphosphine oxide (APO-10),
Cyclohexyl-n-ethyl-.beta.-D-Maltoside,
Cyclohexyl-n-hexyl-.beta.-D-Maltoside,
Cyclohexyl-n-methyl-.beta.-D-Maltoside, n-Decanoylsucrose,
n-Decyl-.beta.-D-glucopyranoside, n-Decyl-.beta.-D-maltopyranoside,
n-Decyl-.beta.-D-thiomaltoside, Digitonin, n-Dodecanoyl sucrose,
n-Dodecyl-.beta.-D-glucopyranoside, n-Dodecyl-.beta.-D-maltoside,
polyoxyethylene (10) dodecyl ether (Genapol C-100), isotridecanol
polyglycol ether (Genapol X-80), isotridecanol polyglycol ether
(Genapol X-100), Heptane-1,2,3-triol,
n-Heptyl-.beta.-D-glucopyranoside,
n-Heptyl-.beta.-D-thioglucopyranoside and combinations thereof. An
example of a ionic surfactant that can be used include, sodium
cholate, chenodeoxycholic acid, cholic acid, dehydrocholic acid,
docusate sodium, docusate sodium salt, glycocholic acid hydrate,
glycodeoxycholic acid monohydrate, glycolithocholic acid ethyl
ester, N-lauroylsarcosine sodium salt, N-lauroylsarcosine, lithium
dodecyl sulfate, calcium propionate, 1-octanesulfonic acid sodium
salt, sodium 1-butanesulfonate, sodium chenodeoxycholate, sodium
cholate hydrate, sodium 1-decanesulfonate, sodium
1-decanesulfonate, sodium deoxycholate, sodium deoxycholate
monohydrate, sodium dodecylbenzenesulfonate, sodium dodecyl
sulfate, sodium glycochenodeoxycholate, sodium glycocholate
hydrate, sodium 1-heptanesulfonate, sodium hexanesulfonate, sodium
1-nonanesulfonate, sodium octyle sulfate, sodium pentanesulfonate,
sodium 1-propanesulfonate hydrate, sodium taurodeoxycholate
hydrate, sodium taurohyodeoxycholate hydrate, sodium
tauroursodeoxycholate, taurocholic acid sodium salt hydrate,
taurolithocholic acid 3-sulfate disodium salt, Triton.RTM. X-200,
Triton.RTM. QS-15, Triton.RTM. QS-44, Triton.RTM. XQS-20,
Trizma.RTM. dodecyl sulfate, ursodeoxycholic acid,
alkyltrimethylammonium bromide, amprolium hydrocholoride,
benzalkonium chloride, benzethonium hydroxide,
benzyldimethylhexadecylammonium chloride,
benzyldodecyldimethylammonium bromide, choline p-toluenesulfonate
salt, dimethyldioctadecylammonium bromide,
dodecylethyldimethylammonium bromide, dodecyltrimethylammonium
bromide, ethylhexadecyldimethylammonium bromide, Ggirard's reagent,
hexadecylpyridinium bromide, hexadecylpyridinium chloride
monohydrate, hexadecylpyridinium chloride monohydrate,
hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium
p-toluenesulfonate, hexadecyltrimethylammonium bromide,
hexadecyltrimethylammonium p-toluenesulfonate, Hyamine.RTM. 1622,
methylbenzethonium chloride, myristyltrimethylammonium bromide,
oxyphenonium bromide, N,N',N'-polyoxyethylene
(10)-N-tallow-1,3-diaminopropane, tetraheptylammonium bromide,
tetrakis(decyl)ammonium bromide, thonzonium bromide and
Luviquat.TM. FC370, Luviquat.TM. HM 552, Luviquat.TM. HOLD,
Luviquat.TM. MS 370, Luviquat.TM. PQ 11PN and combinations thereof
(all available from Sigma Aldrich, St. Louis, Mo.).
[0355] Also, the kit can also contain one or more instructions for
determining the age of a stored blood sample. The kit can also
contain instructions for generating a standard curve for the
purposes of quantifying the hemoglobin or a reference standard for
purposes of quantifying the hemoglobin in the stored blood sample.
Such instructions optionally can be in printed form or on CD, DVD,
or other format of recorded media.
[0356] In still yet a further aspect, the present invention relates
to a kit for identifying a hemolyzed serum or plasma sample. The
kit can contain at least one basic solution. The basic solution is
a solution that contains at least one base and that has a pH
greater than or equal to 10, preferably, greater than or equal to
12. Examples of basic solutions that can be included in the kit
include, but are not limited to, sodium hydroxide, potassium
hydroxide, calcium hydroxide, ammonium hydroxide, magnesium
hydroxide, sodium carbonate, sodium bicarbonate, calcium hydroxide,
calcium carbonate and calcium bicarbonate.
[0357] Additionally, the kit can contain at least one indicator
solution containing at least one acridinium compound. The
acridinium compound may comprise at least one
acridinium-9-carboxamide, at least one acridinium-9-carboxylate
aryl ester or any combinations thereof. More specifically, the
acridinium-9-carboxamide that can be used has the structure
according to Formula I:
##STR00039##
[0358] wherein R.sup.1 and R.sup.2 are each independently selected
from the group consisting of: alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and
[0359] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0360] further wherein any of the alkyl, alkenyl, alkynyl, aryl or
aralkyl may contain one or more heteroatoms; and
[0361] optionally, if present, X.sup..crclbar. is an anion.
[0362] Additionally, the acridinium-9-carboxylate aryl ester that
can be used has a structure according to formula II:
##STR00040##
[0363] wherein R.sup.1 is an alkyl, alkenyl, alkynyl, aryl or
aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and
[0364] wherein R.sup.3 through R.sup.15 are each independently
selected from the group consisting of: hydrogen, alkyl, alkenyl,
alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl,
carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,
carboxyalkyl and oxoalkyl; and
[0365] optionally, if present, X.sup..crclbar. is an anion.
[0366] In addition to the at least one acridinium compound, the
indicator solution can also contain at least one surfactant. Any
surfactant that when dissolved in water, lowers the surface tension
of the water and increases the solubility of organic compounds, can
be included. Examples of surfactants that can be used is one or
more non-ionic or ionic surfactants (e.g., anionic, cationic or
zwitterionic surfactants). Examples of non-ionic surfactants that
can be used include, but are not limited to,
t-octylpheoxypolyethoxyethanol (TRITON X-100, Sigma Aldrich, St.
Louis, Mo.), polyoxyethylenesorbitan monolaurate (Tween 20),
nonylphenol polyoxyethylene ether (Nonidet P10),
decyldimethylphosphine oxide (APO-10),
Cyclohexyl-n-ethyl-.beta.-D-Maltoside,
Cyclohexyl-n-hexyl-.beta.-D-Maltoside,
Cyclohexyl-n-methyl-.beta.-D-Maltoside, n-Decanoylsucrose,
n-Decyl-.beta.-D-glucopyranoside, n-Decyl-.beta.-D-maltopyranoside,
n-Decyl-.beta.-D-thiomaltoside, Digitonin, n-Dodecanoyl sucrose,
n-Dodecyl-.beta.-D-glucopyranoside, n-Dodecyl-.beta.-D-maltoside,
polyoxyethylene (10) dodecyl ether (Genapol C-100), isotridecanol
polyglycol ether (Genapol X-80), isotridecanol polyglycol ether
(Genapol X-100), Heptane-1,2,3-triol,
n-Heptyl-.beta.-D-glucopyranoside,
n-Heptyl-.beta.-D-thioglucopyranoside and combinations thereof. An
example of a ionic surfactant that can be used include, sodium
cholate, chenodeoxycholic acid, cholic acid, dehydrocholic acid,
docusate sodium, docusate sodium salt, glycocholic acid hydrate,
glycodeoxycholic acid monohydrate, glycolithocholic acid ethyl
ester, N-lauroylsarcosine sodium salt, N-lauroylsarcosine, lithium
dodecyl sulfate, calcium propionate, 1-octanesulfonic acid sodium
salt, sodium 1-butanesulfonate, sodium chenodeoxycholate, sodium
cholate hydrate, sodium 1-decanesulfonate, sodium
1-decanesulfonate, sodium deoxycholate, sodium deoxycholate
monohydrate, sodium dodecylbenzenesulfonate, sodium dodecyl
sulfate, sodium glycochenodeoxycholate, sodium glycocholate
hydrate, sodium 1-heptanesulfonate, sodium hexanesulfonate, sodium
1-nonanesulfonate, sodium octyle sulfate, sodium pentanesulfonate,
sodium 1-propanesulfonate hydrate, sodium taurodeoxycholate
hydrate, sodium taurohyodeoxycholate hydrate, sodium
tauroursodeoxycholate, taurocholic acid sodium salt hydrate,
taurolithocholic acid 3-sulfate disodium salt, Triton.RTM. X-200,
Triton.RTM. QS-15, Triton.RTM. QS-44, Triton.RTM. XQS-20,
Trizma.RTM. dodecyl sulfate, ursodeoxycholic acid,
alkyltrimethylammonium bromide, amprolium hydrocholoride,
benzalkonium chloride, benzethonium hydroxide,
benzyldimethylhexadecylammonium chloride,
benzyldodecyldimethylammonium bromide, choline p-toluenesulfonate
salt, dimethyldioctadecylammonium bromide,
dodecylethyldimethylammonium bromide, dodecyltrimethylammonium
bromide, ethylhexadecyldimethylammonium bromide, Ggirard's reagent,
hexadecylpyridinium bromide, hexadecylpyridinium chloride
monohydrate, hexadecylpyridinium chloride monohydrate,
hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium
p-toluenesulfonate, hexadecyltrimethylammonium bromide,
hexadecyltrimethylammonium p-toluenesulfonate, Hyamine.RTM. 1622,
methylbenzethonium chloride, myristyltrimethylammonium bromide,
oxyphenonium bromide, N,N',N'-polyoxyethylene
(10)-N-tallow-1,3-diaminopropane, tetraheptylammonium bromide,
tetrakis(decyl)ammonium bromide, thonzonium bromide and
Luviquat.TM. FC370, Luviquat.TM. HM 552, Luviquat.TM. HOLD,
Luviquat.TM. MS 370, Luviquat.TM. PQ 11PN and combinations thereof
(all available from Sigma Aldrich, St. Louis, Mo.).
[0367] Also, the kit can also contain one or more instructions for
identifying a hemolyzed serum or plasma sample. The kit can also
contain instructions for generating a standard curve for the
purposes of quantifying the hemoglobin or a reference standard for
purposes of quantifying the hemoglobin in the serum or plasma
sample. Such instructions optionally can be in printed form or on
CD, DVD, or other format of recorded media.
D. ADAPTATIONS OF THE METHODS OF THE PRESENT INVENTION
[0368] The present invention as described herein also can be
adapted for use in a variety of automated and semi-automated
systems (including those wherein the solid phase comprises a
microparticle), as described, e.g., in U.S. Pat. Nos. 5,089,424 and
5,006,309, and as, e.g., commercially marketed by Abbott
Laboratories (Abbott Park, Ill.) including but not limited to
Abbott's ARCHITECT.RTM., AxSYM, IMX, PRISM, and Quantum II
instruments, as well as other platforms. Moreover, the disclosure
optionally is adaptable for the Abbott Laboratories commercial
Point of Care (I-STAT.RTM.) electrochemical assay system for
performing a variety of different types of assays (e.g.,
immunoassays, clinical chemistry assays, etc) Immunosensors, and
their methods of manufacture and operation in single-use test
devices are described, for example in, U.S. Pat. No. 5,063,081,
U.S. Patent Application 2003/0170881, U.S. Patent Application
2004/0018577, U.S. Patent Application 2005/0054078, and U.S. Patent
Application 2006/0160164, which are incorporated in their
entireties by reference for their teachings regarding same.
[0369] In particular, with regard to the adaptation of the present
assay to the I-STAT.RTM. system, the following example is provided.
Other adaptations and configurations are well within the skill of
those in the art. Specifically, a microfabricated silicon chip is
manufactured with a pair of gold amperometric working electrodes
and a silver-silver chloride reference electrode. This chip is
assembled into an I-STAT.RTM. cartridge with a fluidics format
suitable for the assay. Within the fluid pouch of the cartridge is
an aqueous basic reagent.
In operation, a sample suspected of containing hemoglobin is added
to the holding chamber of the hemoglobin test cartridge and the
cartridge is inserted into the I-STAT.RTM. reader. A pump element
within the cartridge forces the sample into a conduit containing
the chip. Fluid is forced out of the pouch and into the conduit to
mix with the sample. After applying the appropriate potential
across the indicator electrode surface, with respect to a reference
electrode, one or more electrochemical reactions takes place, all
of which result in the consumption of the electroactive species
generated upon reaction of hemoglobin and the basic aqueous reagent
with the production of a measurable current. Based on the measured
current, the reader is able to calculate the amount of hemoglobin
in the sample by means of an embedded algorithm and
factory-determined calibration curve.
[0370] It further goes without saying that the methods and kits as
described herein necessarily encompass other reagents and methods
for carrying out the assay. For instance, encompassed are various
buffers such as are known in the art and/or which can be readily
prepared or optimized to be employed, e.g., for washing, as a
conjugate diluent, and/or as a calibrator diluent. An exemplary
conjugate diluent is ARCHITECT.RTM. conjugate diluent employed in
certain kits (Abbott Laboratories, Abbott Park, Ill.) and
containing 2-(N-morpholino)ethanesulfonic acid (MES), other salt,
protein blockers, antimicrobial and surfactant. An exemplary
calibrator diluent is ARCHITECT.RTM. Human calibrator diluent
employed in certain kits (Abbott Laboratories, Abbott Park, Ill.),
which comprises a buffer containing MES, other salt, a protein
blocker and an antimicrobial.
[0371] By way of example, and not of limitation, examples of the
present disclosures shall now be given.
Example 1
Detection of Hemoglobin in Whole Blood
[0372] Chemiluminescent detection reagent.
9-[[(3-Carboxypropyl)[(4-methylphenyl)sulfonyl]amino]-carbonyl]-10-(3-sul-
fopropyl)acridinium inner salt (See, FIG. 1 and Adamczyk, M.; Chen,
Y.-Y., Mattingly, P. G.; Pan, Y. J. Org. Chem., 63, 5636-5639
(1998)) was dissolved in reagent grade water containing sodium
cholate (0.1% wt/vol) to give a concentration of 4 .mu.M.
[0373] Whole blood dilutions. A pooled whole blood sample (30
.mu.L) was serially 2-fold diluted with buffer across the first row
of a clear microplate to give 1:1, 2, 4, 8, 16, 32, 64, 128, 512,
1024, 2048 dilutions. By dilution 1:512 the solution was not
visibly colored when viewed against a white background. These
dilutions corresponded to nominal hemoglobin concentrations [Hb] of
2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625,
1.953125, 0.9765625 .mu.M.
[0374] Assay protocol. The whole blood dilutions (1 .mu.L) were
arrayed in replicates of six in a 96-well microplate (Costar part
no. 3972). The plate was loaded into a microplate luminometer
(Mithras LB-940, Berthold Technologies U.S.A. LLC, Oak Ridge,
Tenn.) at 28.degree. C. Well by well, the chemiluminescent
detection reagent (10 .mu.L) and aqueous base (0.25 N sodium
hydroxide, 30 .mu.L, VWR part no. 3469-1) were sequentially added
and the chemiluminescent signal (RLU, i.e, relative light units)
recorded for 2 s. The peak chemiluminescent signal (RLUmax) for
each whole blood dilution is listed below in Table 1 for each
dilution factor (DF) and graphically in FIGS. 2 to 4.
TABLE-US-00001 TABLE 1 Detection of Hemoglobin in Whole Blood
RLUmax DF 1/DF [Hb] .mu.M 2742134 1 1 2000 2732867 2 0.5 1000
1999592 4 0.25 500 1141733 8 0.125 250 418842 16 0.0625 125 144405
32 0.03125 62.5 41587 64 0.015625 31.25 14193 128 0.0078125 15.625
6313 256 0.00390625 7.8125 3547 512 0.001953125 3.90625 3210 1024
0.000976563 1.953125 3183 2048 0.000488281 0.9765625
Example 2
Effect of Ultrafiltration of Hemoglobin Concentration
[0375] An aliquot of whole blood (100 .mu.L) was filtered through a
Nanosep 10K spin-filter (Pall Corporation, East Hills, N.Y., part
no OD010C3J) using a TDx (Abbott Laboratories, Abbott Park, Ill.)
microcentrifuge. The filtrate was colorless when viewed against a
white background, indicating the removal of hemoglobin from the
test sample.
[0376] The filtrate was analyzed by the assay protocol of Example
1. The resulting peak chemiluminescent signal (RLUmax) of 2493,
indicated a residual hemoglobin concentration in the filtrate below
1 nM from the dose response curve generated in Example 1.
Example 3
Combined Effect of Aqueous Base Pretreatment and Ultrafiltration on
Hemoglobin Analysis
[0377] A whole blood sample (6 mL) was diluted with aqueous base
(0.25 N sodium hydroxide, 180 .mu.L, VWR part no. 3469-1) then
filtered through a Nanosep 10K spin-filter (Pall Corporation, East
Hills, N.Y., part no OD010C3J) using a TDx (Abbott Laboratories,
Abbott Park, Ill.) microcentrifuge. The filtrate (31 mL) was
arrayed in replicates of six (e.g. 6.times.31 mL) in a 96-well
microplate (Costar part no. 3972). The plate was loaded into a
microplate luminometer (Mithras LB-940, Berthold Technologies
U.S.A. LLC, Oak Ridge, Tenn.) at 28.degree. C. Well by well, the
chemiluminescent detection reagent (10 mL) was added and the
chemiluminescent signal (RLU, i.e, relative light units) recorded
for 2 seconds. The peak chemiluminescent signal (RLUmax) was
40940.
[0378] A comparison of the chemiluminescent profile from whole
blood samples treated with base before (-- Example 3) and after
(---- Example 2) ultrafiltration is shown in FIG. 5.
[0379] This demonstrates, among other things, that the order of
addition of the aqueous base and chemiluminescent detection reagent
may be reversed in the detection of hemoglobin.
Example 4
Analysis of Fresh Whole Blood
[0380] Ferrous hemoglobin stock solution. Ferrous hemoglobin (mwt
64,500, Sigma Aldrich, St. Louis, Mo., part no. H-0267, <10%
methemoglobin) was dissolved in phosphate buffer to give a 1 mM
stock solution.
[0381] Ferrous hemoglobin standard solutions. The stock solution
was diluted in phosphate buffer to give standard solutions of
100.00, 75.00, 50.00, 30.00, 20.00, 10.00, 5.00, 0.00 .mu.M
hemoglobin.
[0382] Standard dose-response curve. The ferrous hemoglobin
standards (1 .mu.L) were arrayed in replicates of six in a 96-well
microplate (Costar part no. 3972). The plate was loaded into a
microplate luminometer (Mithras LB-940, Berthold Technologies
U.S.A. LLC, Oak Ridge, Tenn.) at 28.degree. C. Well by well, the
chemiluminescent detection reagent (10 .mu.L) and aqueous base
(0.25 N sodium hydroxide, 30 .mu.L, VWR part no. 3469-1) were
sequentially added and the chemiluminescent signal (RLU, i.e,
relative light units) recorded for 2 s. The peak chemiluminescent
signal (RLUmax) for standard dilution are listed in Table 2 and
graphically in FIG. 6. The chemiluminescence profile (RLU vs time)
for each standard concentration is shown in FIG. 7.
[0383] Fresh whole blood assay. Fresh whole blood collected by
finger stick was rapidly lysed by freeze/thaw (dry ice/acetone, -78
C, 3.times.), then an aliquot was diluted 1:50 in phosphate
buffer.
[0384] The diluted test sample (1 .mu.L) was arrayed in replicates
of six in a 96-well microplate (Costar part no. 3972). The plate
was loaded into a microplate luminometer (Mithras LB-940, Berthold
Technologies U.S.A. LLC, Oak Ridge, Tenn.) at 28.degree. C. Well by
well, the chemiluminescent detection reagent (10 .mu.L) and aqueous
base (0.25 N sodium hydroxide, 30 .mu.L, VWR part no. 3469-1) were
sequentially added and the chemiluminescent signal (RLU, i.e,
relative light units) recorded for 2 seconds.
[0385] The peak chemiluminescent signal (RLUmax) was 565322, which
corresponded to a hemoglobin concentration of 2.77 mM in the
undiluted fresh whole blood sample.
TABLE-US-00002 TABLE 2 Concentration RLUmax 100.00 1280088 75.00
888918 50.00 468027 30.00 186545 20.00 110975 10.00 36548 5.00
14685 0.00 9480
Example 5
Effect of Sample Age on Hemoglobin Concentration
[0386] The blood sample of Example 4 was stored overnight at
-20.degree. C., and then re-analyzed using the same protocol as
Example 4 the next day. The recorded peak chemiluminescent signal
(RLUmax) was 391470 corresponding to a hemoglobin concentration of
2.23 mM, thus showing a decrease in the hemoglobin concentration in
the stored blood sample of 0.54 mM (19.5%).
[0387] A lysed whole blood sample that had been stored for greater
than one year, was analyzed using the same protocol as Example 4.
The recorded peak chemiluminescent signal (RLUmax) was 77892,
corresponding to a hemoglobin concentration of 0.75 mM.
[0388] This would indicate that hemoglobin concentration in this
sample decreased about 70% from that expected in a freshly
collected sample. This is consistent with what has been reported
for samples stored for a year or more under similar conditions
(See, Stratton, L. P.; Rudolph, A. S.; Knoll, W. K., Jr.; Bayne,
S.; Farmer, M. C. Hemoglobin, 12:353 (1988)).
[0389] One skilled in the art would readily appreciate that the
present disclosure is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The molecular complexes and the methods, procedures,
treatments, molecules, specific compounds described herein are
presently representative of preferred embodiments, are exemplary,
and are not intended as limitations on the scope of the invention.
It will be readily apparent to one skilled in the art that varying
substitutions and modifications may be made to the invention
disclosed herein without departing from the scope and spirit of the
invention.
[0390] All patents and publications mentioned in the specification
are indicative of the levels of those skilled in the art to which
the invention pertains. All patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
[0391] The invention illustratively described herein suitably may
be practiced in the absence of any element or elements, limitation
or limitations which is not specifically disclosed herein. Thus,
for example, in each instance herein any of the terms "comprising,"
"consisting essentially of" and "consisting of" may be replaced
with either of the other two terms. The terms and expressions which
have been employed are used as terms of description and not of
limitation, and there is no intention that in the use of such terms
and expressions of excluding any equivalents of the features shown
and described or portions thereof, but it is recognized that
various modifications are possible within the scope of the
invention claimed. Thus, it should be understood that although the
present disclosure has been specifically disclosed by preferred
embodiments and optional features, modification and variation of
the concepts herein disclosed may be resorted to by those skilled
in the art, and that such modifications and variations are
considered to be within the scope of this invention as defined by
the appended claims.
* * * * *