RELATED APPLICATION INFORMATION
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.
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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.
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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 % hemoglobinA1c 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 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.
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.
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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: