Methods and kits for detecting hemoglobin in test samples
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.
1. A method of detecting hemoglobin in a test sample, the method consisting essentially of 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 increase in light generated to detect the hemoglobin in the test sample, wherein the light generated is proportional to the amount of hemoglobin in the test sample; and wherein the at least one acridinium compound comprises an acridinium-9-carboxamide or an acridinium-9-carboxylate aryl ester,
wherein the acridiniurn-9-carboxamide has a structure according to formula I:
wherein R 1 and R 2 are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sutfoalkyl, carboxyalkyl and oxoalkyl, and;
wherein R 3 through R 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, carboxylalkyl and oxoalkyl; and optionally, if present, X ⊖ is an anion, and
wherein acridinium-9-carboxylate aryl ester has a structure according to formula II:
wherein R 1 is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and;
wherein R 3 through R 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, carboxylalkyl and oxoalkyl; and optionally, if present, X ⊖ is an anion.
2. The method of claim 1 , wherein the test sample is 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 indicator solution further comprises at least one surfactant.
5. 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.
6. The method of claim 5 , wherein the standard curve is generated from solutions of hemoglobin of a known concentration.
7. A method of diagnosing a subject suffering from a genetic disorder relating to hemoglobin metabolism, the method consisting essentially of 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 increased light generated to detect the hemoglobin in the test sample; and wherein the light generated is proportional to the amount of hemoglobin in the test sample; and
wherein the at least one acridinium compound comprises an acridinium-9-carboxamide or an acridinium-9-carboxylate aryl ester,
wherein the acridiniurn-9-carboxamide has a structure according to formula I:
wherein R 1 and R 2 are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and;
wherein R 3 through R 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, carboxylalkyl and oxoalkyl; and optionally, if present X ⊖ is an anion, and
wherein acridinium-9-carboxylate aryl ester has a structure according to formula II:
wherein R 1 is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and;
wherein R 3 through R 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, carboxylalkyl and oxoalkyl; and optionally, if present X ⊖ is an anion; 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.
8. The method of claim 7 , wherein the test sample is serum, plasma, whole blood, red blood cells or umbilical cord blood.
9. The method of claim 7 , wherein the basic solution is a solution having a pH of at least about 10.
10. The method of claim 7 , wherein the indicator solution further comprises at least one surfactant.
11. The method of claim 7 , 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.
12. The method of claim 11 , wherein the standard curve is generated from solutions of hemoglobin of a known concentration.
13. The method of claim 7 , wherein the genetic disorder relating to hemoglobin metabolism is anemia or β-thalassemia.
14. A method of determining the eligibility of a subject to be a blood donor, the method consisting essentially of 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 increased light generated to detect the hemoglobin in the test sample; wherein the light generated is proportional to the amount of hemoglobin in the test sample; and
wherein the at least one acridinium compound comprises an acridinium-9-carboxamide or an acridinium-9-carboxylate aryl ester,
wherein the acridiniurn-9-carboxamide has a structure according to formula I:
wherein R 1 and R 2 are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and;
wherein R 3 through R 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, carboxylalkyl and oxoalkyl; and optionally, if present, X ⊖ is an anion, and
wherein acridinium-9-carboxylate aryl ester has a structure according to formula II:
wherein R 1 is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and;
wherein R 3 through R 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, carboxylalkyl and oxoalkyl; and optionally, if present, X ⊖ is an anion; 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 not eligible to be a blood donor.
15. The method of claim 14 , wherein the test sample is whole blood.
16. The method of claim 14 , wherein the basic solution is a solution having a pH of at least about 10.
17. The method of claim 14 , wherein the indicator solution further comprises at least one surfactant.
18. The method of claim 14 , 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. A method of determining the age of a stored blood sample, the method consisting essentially of 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 increased light generated to detect the hemoglobin in the blood sample; wherein the light generated is proportional to the amount of hemoglobin in the test sample; and
wherein the at least one acridinium compound comprises an acridinium-9-carboxamide or an acridinium-9-carboxylate aryl ester,
wherein the acridiniurn-9-carboxamide has a structure according to formula I:
wherein R 1 and R 2 are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and;
wherein R 3 through R 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, carboxylalkyl and oxoalkyl; and optionally, if present, X ⊖ is an anion, and
wherein acridinium-9-carboxylate aryl ester has a structure according to formula II:
wherein R 1 is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and;
wherein R 3 through R 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, carboxylalkyl and oxoalkyl; and optionally, if present X ⊖ is an anion; and
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 than the predetermined level, then the plasma sample is determined to be an older blood sample.
21. The method of claim 20 , wherein the blood sample is whole blood, serum, plasma, platelets, red blood cells or umbilical cord blood.
22. The method of claim 20 , wherein the basic solution is a solution having a pH of at least about 10.
23. The method of claim 20 , wherein the indicator solution further comprises at least one surfactant.
24. The method of claim 20 , 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.
25. The method of claim 24 , wherein the standard curve is generated from solutions of hemoglobin of a known concentration.
26. A method of identifying a hemolyzed serum or plasma sample, the method consisting essentially of 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 increased light generated to detect the hemoglobin in the serum or plasma sample; wherein the light generated is proportional to the amount of hemoglobin in the test sample; and
wherein the at least one acridinium compound comprises an acridinium-9-carboxamide or an acridinium-9-carboxylate aryl ester,
wherein the acridiniurn-9-carboxamide has a structure according to formula I:
wherein R 1 and R 2 are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and;
wherein R 3 through R 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, carboxylalkyl and oxoalkyl; and optionally, if present, X ⊖ is an anion, and
wherein acridinium-9-carboxylate aryl ester has a structure according to formula II:
wherein R 1 is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and;
wherein R 3 through R 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, carboxylalkyl and oxoalkyl; and optionally, if present, X ⊖ is an anion; and
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 than 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 than the predetermined level, then the serum or plasma sample is determined not to be hemolyzed.
27. The method of claim 26 , wherein the basic solution is a solution having a pH of at least about 10.
28. The method of claim 26 , wherein the indicator solution further comprises at least one surfactant.
29. The method of claim 26 , 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.
30. The method of claim 29 , wherein the standard curve is generated from solutions of hemoglobin of a known concentration.