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.
RELATED APPLICATION INFORMATION

This application claims the benefit of U.S. application Ser. No. 61/144,675 filed on Jan. 14, 2009, the contents of which are herein incorporated by reference.
TECHNICAL FIELD

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

Adult hemoglobin A (HbA) is a tetrameric protein of molecular weight 64.5 kD, composed of 2 α-globins and 2 β-globins (α2β2). The alpha α-subunit is composed of 141 amino acids (See, SEQ ID NO: 1). The β-subunit is composed of 146 amino acids (See, SEQ ID NO:2). Both α and β-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 α-globins and 2 δ-globins. The predominant fetal hemoglobin F consists of 2 α-globins and 2 γ-globins and is sometimes seen in neonates and adults.
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.
 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.
The determination of total hemoglobin concentration is also useful in assays reporting % hemoglobinAlc for monitoring blood glucose control.
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 μmol/L).
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, K₃Fe(CN)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.
 The method exemplified in the commercial MULTIGENT®Hemoglobin Alc Assay (Abbott Laboratories, List 02K96-20) converts digests hemoglobin with pepsin to give hematin which can be quantified at a wavelength of 604 nm.
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.
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.
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)).
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.
 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.
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

In one aspect, the present invention relates to a method of detecting hemoglobin in a test sample. The method comprises 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.
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.
In the above method, the basic solution is a solution having a pH of at least about 10.
 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:

wherein R¹ and R² are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and

wherein R³ through R¹⁵ 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^(⊕) is an anion.
Alternatively, the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II:

wherein R¹ is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and

wherein R³ through R¹⁵ 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^(⊕) is an anion.
In the above method, the indicator solution can further comprise at least one surfactant.
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.
In another aspect, the present invention relates to a method of detecting hemoglobin in a test sample. The method comprises 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.
 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.
In the above method, the basic solution is a solution having a pH of at least about 10.
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:

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.
In the above method, the test sample can be serum, plasma, whole blood, red blood cells and umbilical cord blood.
In the above method, the basic solution is a solution having a pH of at least about 10.
 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:

wherein R¹ and R² are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and

wherein R³ through R¹⁵ 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^(⊕) is an anion.
Alternatively, the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II:

wherein R¹ is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and

wherein R³ through R¹⁵ 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^(⊕) is an anion.
In the above method, the indicator solution can further comprise at least one surfactant.
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.
In the above method, the genetic disorder relating to hemoglobin metabolism can be anemia or β-thalassemia.
 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:

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.
In the above method, the test sample can be whole blood.
In the above method, the basic solution is a solution having a pH of at least about 10.
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:

wherein R¹ and R² are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and

wherein R³ through R¹⁵ 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^(⊕) is an anion.
 Alternatively, the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II:

wherein R¹ is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and

wherein R³ through R¹⁵ 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^(⊕) is an anion.
In the above method, the indicator solution can further comprise at least one surfactant.
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.
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:

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 blood sample is determined to be an older blood sample.
 In the above method, the blood sample is whole blood, red blood cells or umbilical cord blood.
In the above method, the basic solution is a solution having a pH of at least about 10.
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:

wherein R¹ and R² are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and

wherein R³ through R¹⁵ 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^(⊕) is an anion.
Alternatively, the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II:

wherein R¹ is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and

wherein R³ through R¹⁵ 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^(⊕) is an anion.
In the above method, the indicator solution can further comprise at least one surfactant.
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.
 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:

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 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.
In the above method, the basic solution is a solution having a pH of at least about 10.
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:

wherein R¹ and R² are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and

wherein R³ through R¹⁵ 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^(⊕) is an anion.
 Alternatively, the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II:

wherein R¹ is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and

wherein R³ through R¹⁵ 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^(⊕) is an anion.
In the above method, the indicator solution can further comprise at least one surfactant.
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.
In still yet another aspect, the present invention relates to a kit for detecting hemoglobin in a test sample. The kit comprises:

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.
In the above kit, the basic solution is a solution having a pH of at least about 10.
 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:

wherein R¹ and R² are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and

wherein R³ through R¹⁵ 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^(⊕) is an anion.
Alternatively, the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II:

wherein R¹ is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and

wherein R³ through R¹⁵ 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^(⊕) is an anion.
The indicator solution in the above kit can further comprise at least one surfactant.
In still yet another aspect, the present invention relates to a kit for detecting hemoglobin in a test sample. The kit comprises:

a. at least one basic solution;

b. at least one electrode; and

c. instructions for detecting hemoglobin in a test sample.
In the above kit, the basic solution is a solution having a pH of at least about 10.
 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:

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.
In the above kit, the basic solution is a solution having a pH of at least about 10.
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:

wherein R¹ and R² are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and

wherein R³ through R¹⁵ 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^(⊕) is an anion.
Alternatively, the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II:

wherein R¹ is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and

wherein R³ through R¹⁵ 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^(⊕) is an anion.
The indicator solution in the above kit can further comprise at least one surfactant.
 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:

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.
In the above kit, the basic solution is a solution having a pH of at least about 10.
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:

wherein R¹ and R² are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and

wherein R³ through R¹⁵ 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^(⊕) is an anion.
Alternatively, the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II:

wherein R¹ is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and

wherein R³ through R¹⁵ 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^(⊕) is an anion.
The indicator solution in the above kit can further comprise at least one surfactant.
 In still yet another embodiment, the present invention relates to a kit for determining the age of a stored blood sample. The kit comprises:

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.
In the above kit, the basic solution is a solution having a pH of at least about 10.
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:

wherein R¹ and R² are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and

wherein R³ through R¹⁵ 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^(⊕) is an anion.
Alternatively, the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II:

wherein R¹ is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and

wherein R³ through R¹⁵ 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^(⊕) is an anion.
The indicator solution in the above kit can further comprise at least one surfactant.
 In still yet another embodiment, the present invention relates to a kit for identifying a hemolyzed serum or plasma sample. The kit comprises:

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.
In the above kit, the basic solution is a solution having a pH of at least about 10.
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:

wherein R¹ and R² are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and

wherein R³ through R¹⁵ 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^(⊕) is an anion.
Alternatively, the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II:

wherein R¹ is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and

wherein R³ through R¹⁵ 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^(⊕) is an anion.
The indicator solution in the above kit can further comprise at least one surfactant.
BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the chemiluminescent detection reagent, 9-[[(3-Carboxypropyl)[(4-methylphenyl)sulfonyl]amino]-carbonyl]-10-(3-sulfopropyl)acridinium inner salt.
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.
 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.
FIG. 4 shows the peak chemiluminescent signal (RLUmax) for each whole blood dilution listed in Table 1 for each nominal hemoglobin concentration (μM) as described in Example 1.
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.
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.
FIG. 7 shows the chemiluminescence profile (RLU vs time) for each ferrous hemoglobin standard concentration as described in Example 4.
DETAILED DESCRIPTION

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

Section headings as used in this section and the entire disclosure herein are not intended to be limiting.
 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.
a) Acyl (and Other Chemical Structural Group Definitions)

As used herein, the term “acyl” refers to a —C(O)R_(a) group where R_(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.
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.
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.
As used herein, the term “alkyl radical” means any of a series of univalent groups of the general formula C_(n)H_(2n+1) derived from straight or branched chain hydrocarbons.
 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.
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.
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).
As used herein, the term “amino” means —NR_(b)R_(b), wherein R_(b) and R_(c) are independently selected from the group consisting of hydrogen, alkyl and alkylcarbonyl.
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.
 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.
As used herein, the term “carboxy” or “carboxyl” refers to —CO₂H or —CO₂.
As used herein, the term “carboxyalkyl” refers to a —(CH₂)_(n)CO₂H or —(CH₂)_(n)CO₂ ⁻ group where n is from 1 to 10.
As used herein, the term “cyano” means a —CN group.
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.
 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.
As used herein, the term “cycloalkylalkyl” means a —R_(d)R_(e) group where R_(d) is an alkylene group and R_(e) is cycloalkyl group. A representative example of a cycloalkylalkyl group is cyclohexylmethyl and the like.
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.
As used herein, the term “hydroxyl” means an —OH group.
As used herein, the term “nitro” means a —NO₂ group.
As used herein, the term “oxoalkyl” refers to —(CH₂)_(n)C(O)R_(a), where R_(a) is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl and where n is from 1 to 10.
As used herein, the term “phenylalkyl” means an alkyl group which is substituted by a phenyl group.
As used herein, the term “sulfo” means a —SO₃H group.
As used herein, the term “sulfoalkyl” refers to a —(CH₂)_(n)SO₃H or —(CH₂)_(n)SO₃ ⁻ group where n is from 1 to 10.
 b) Anion

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.
c) Pharmaceutical Composition

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.
d) Predetermined Level

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.
 e) Specific Binding Partner

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.
f) Subject

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.
 g) Test Sample

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®, Northfield Laboratories, Evanston, Ill.), polyethylene glycol-modified hemoglobin (e.g., Hemospan™, 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™, 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)).
 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 μL to about 100 μL.
B. METHODS OF THE PRESENT INVENTION

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.
 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.
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:

wherein R¹ and R² are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and

wherein R³ through R¹⁵ 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

optionally, if present, X^(⊕) is an anion.
 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).
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:

wherein R¹ is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and

wherein R³ through R¹⁵ 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^(⊕) is an anion.
 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).
