Turbidity normalization algorithm and method for reducing intralipid / lipidemia interference in hemoglobin A1c determination

Abstract

A method for spectroscopically measuring the % glycated hemoglobin or the glycated hemoglobin:total hemoglobin ratio in a biological sample is disclosed. The method comprises normalizing the total hemoglobin concentration calculated from the spectral measurement using a turbidity normalization algorithm to substantially remove any turbidity interference therefrom. Turbidity normalization eliminates the negative bias observed with intralipid / lipemia and thus provides an glycated hemoglobin measurement that is not significantly interfered with by intralipid / lipemia.

Description

[0001] This application is a divisional application of an invention patent application filed on November 15, 2019, with application number 201980087342.2 and invention name “Turbidity normalization algorithm and method for reducing intralipid / lipemia interference in hemoglobin A1c determination”. Technical Field

[0002] The present invention relates to the field of biological sample assays, and in particular to a method for reducing intralipid / lipidemia interference in hemoglobin A1c assays. Background Art

[0003] Hemoglobin present in red blood cells can be glycated by non-enzymatically adding glucose molecules to the amino termini of the β-chains of hemoglobin. Once a hemoglobin molecule is glycated, it remains glycated, and the accumulation of glycated hemoglobin within red blood cells reflects the average glucose level to which the cells have been exposed during their life cycle. Therefore, the level of glycated hemoglobin present in an individual's blood is directly proportional to the glucose level in the blood and is an indicator of the individual's average daily blood glucose concentration over the previous four to three months. Therefore, the ratio of glycated hemoglobin to total hemoglobin in a whole blood sample is quite useful in the diagnosis and monitoring of patients with diabetes.

[0004] Precise control of blood glucose can improve many of the morbidity and mortality associated with diabetes. Therefore, many different hemoglobin assays have been developed based on the physical and chemical properties of hemoglobin or on epitopes recognized by specific antibodies. Clinical studies have shown that HbA1c results improve decision-making, patient compliance, and prognosis (Thaler et al. (1999) Diabetes Care, 22:1415-1421; and Miller et al. (2003) Diabetes Care, 26:1158-1163).

[0005] Glycated hemoglobin (HbA1c) is formed by non-enzymatic glycation of the N-terminus of the β-chain of hemoglobin A. Measurement of HbA1c is used as an aid in diagnosing and monitoring long-term blood glucose control in patients with diabetes, and as an aid in identifying patients at risk of developing diabetes. HbA1c levels reflect the average glucose concentration over a previous period (approximately 8-12 weeks, depending on the individual) and provide a better indicator of long-term blood glucose control than blood and urine glucose measurements. Studies have shown that long-term control of HbA1c levels can reduce the risk of developing and progressing chronic complications caused by diabetes.

[0006] Immunoassays are the most common type of hemoglobin measurement method currently used in clinical laboratory settings. These immunoassays utilize antibodies that recognize hemoglobin epitopes, and in specific cases, epitopes of glycated hemoglobin (HbA1c), such as (but not limited to) at least a portion of its N-terminal glycated amino acids. For example, the turbidimetric inhibition immunoassay (TINIA) for the analyte HbA1c utilizes anti-HbA1c antibodies and polyhapten agglutinating agents (i.e., synthetic molecules containing multiple HbA1c epitopes to induce agglutination with free antibodies). However, incompletely dispersed polyhapten reagents are a major interference in this assay because they essentially mimic insoluble antibody-polyhapten complexes; incompletely dispersed polyhapten reagents cause light scattering, which is then measured turbidimetrically and, due to the inverse relationship between absorbance and analyte concentration, translates into erroneously low HbA1c values.

[0007] Spectroscopic methods are also used for hemoglobin determination; however, the presence of lipids within biological samples can cause interference at one or more wavelengths utilized in these methods. Therefore, there is a need for new and improved hemoglobin A1c assays that utilize spectroscopic methods and in which no significant interference from intralipid / lipidemia is observed. Summary of the Invention

[0008] Before explaining at least one embodiment of the present disclosure in detail by way of exemplary language and results, it should be understood that the present disclosure is not limited in its application to the details of construction and arrangement of components set forth in the following description. The present disclosure is capable of other embodiments or of being practiced or implemented in various ways. Therefore, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary—not exhaustive. Furthermore, it should be understood that the phraseology and terminology employed herein are for descriptive purposes and should not be considered as limiting.

[0009] Unless otherwise defined herein, the scientific and technical terms used in conjunction with the present disclosure should have the meaning commonly understood by those of ordinary skill in the art. Further, unless the context requires otherwise, singular terms should include plural, and plural terms should include singular. Generally, the terms and techniques associated with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization as described herein are well-known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reaction and purification techniques are carried out according to the manufacturer's instructions or as generally completed in the art or as described herein. The aforementioned techniques and procedures are generally implemented according to conventional methods well-known in the art and as described in the various general and more specific references cited and discussed in this specification sheet. See, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)) and Coligan et al. (Current Protocols in Immunology, Wiley Interscience (1994)). The nomenclature utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly employed in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0010] All patents, published patent applications, and non-patent publications mentioned in this specification are indicative of the levels of skill of those skilled in the art to which the present disclosure pertains. All patents, published patent applications, and non-patent publications cited in any part of this application are expressly incorporated herein by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0011] In view of the present disclosure, all compositions, kits and / or methods disclosed herein can be prepared and performed without undue experimentation. Although the compositions, kits and / or methods have been described in terms of specific embodiments, it will be apparent to those skilled in the art that variations can be applied to the compositions, kits and / or methods, as well as to the steps or sequence of steps in the methods described herein, without departing from the concept, spirit and scope of the present disclosure. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope and concept of the present disclosure as defined in the appended claims.

[0012] As utilized in accordance with this disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0013] When used in conjunction with the term "comprising" in the claims and / or specification, the use of the term "a" or "an" can mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." Thus, the terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, a reference to "a compound" can refer to one or more compounds, 2 or more compounds, 3 or more compounds, 4 or more compounds, or a greater number of compounds. The term "plurality" means "two or more."

[0014] The use of the term "at least one" will be understood to include one and any number more than one, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" can extend to as many as 100 or 1000 or more, depending on the term to which it is attached; furthermore, the number of 100 / 1000 is not considered limiting, as higher limits may also produce satisfactory results. Furthermore, the use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) is used solely for the purpose of distinguishing two or more items and is not meant to imply any order or priority or importance of one item relative to another, or any order of addition, for example.

[0015] When used in the claims, the term "or" is intended to mean the inclusive "and / or" unless explicitly stated to refer to only alternatives or unless the alternatives are mutually exclusive. For example, any of the following satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0016] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. For example, the phrases "in some embodiments" or "an example" appearing in various places in the specification are not necessarily all referring to the same embodiment. Furthermore, all references to one or more embodiments or examples should be construed as non-limiting to the claims.

[0017] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error of the composition / instrument / device, method used to determine the value, or the variation that exists between study subjects. For example, but not limitation, when the term "about" is utilized, the specified value can vary from the recited value by plus or minus 20%, or 15%, or 12%, or 11%, or 10%, or 9%, or 8%, or 7%, or 6%, or 5%, or 4%, or 3%, or 2%, or 1%, as such variations are applicable to performing the disclosed methods and are understood by one of ordinary skill in the art.

[0018] As used in this specification and in one or more claims, the terms “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0019] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed before the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB if the order is important in the particular context. Continuing with this example, specifically included are combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so on. The skilled artisan will understand that, unless otherwise apparent from the context, there is generally no limit on the number of items or terms in any combination.

[0020] As used herein, the term "substantially" means that the subsequently described event or circumstance occurs completely or that the subsequently described event or circumstance occurs to a large extent or degree. For example, when related to specific events or circumstances, the term "substantially" means that the subsequently described event or circumstance occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. The term "substantially adjacent" can mean that two items are 100% adjacent to each other, or that the two items are in close proximity to each other but not 100% adjacent to each other, or that a portion of one of the two items is not 100% adjacent to the other item, but is in close proximity to the other item.

[0021] As used herein, the term "sample" will be understood to include any type of biological sample that can be used in accordance with the present disclosure. Examples of fluid biological samples that can be used include, but are not limited to, whole blood or any portion thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, stool, pleural fluid, nasopharyngeal fluid, combinations thereof, and the like.

[0022] As used herein, the term "reaction cuvette" includes any device capable of performing at least one diagnostic assay as described herein. The reaction cuvette can perform the diagnostic assay manually, but in most cases, the reaction cuvette will be inserted into a system that automatically performs the diagnostic assay. In a non-limiting embodiment, the reaction cuvette includes a reaction cuvette for an automatic diagnostic assay, which is performed by, for example, but not limited to, the Atellica clinical chemistry system commercially available from Siemens Healthineers (Tarrytown, NY). However, it should be understood that the reaction cuvette can be any commercially available product or cuvette described herein or otherwise contemplated that can perform one or more diagnostic assays according to the present disclosure.

[0023] Turning now to the inventive concepts disclosed and / or claimed herein, certain non-limiting embodiments of the present disclosure are generally directed to methods for reducing interference in a hemoglobin A1c assay, wherein the interference (for example, but not by way of limitation) is caused by Intralipid / lipemia, and wherein the interference is reduced by using a turbidity normalization algorithm. Specifically, certain non-limiting embodiments of the present disclosure are directed to a glycated hemoglobin assay from which any turbidity interference has been substantially removed; for example, but not by way of limitation, any turbidity interference can be substantially reduced to less than + / - about 5% at Intralipid concentrations up to about 1000 mg / dL. In this manner, any negative bias observed with Intralipid / lipemia is removed from the assay.

[0024] Certain non-limiting embodiments of the present disclosure relate to methods of measuring % glycated hemoglobin or the glycated hemoglobin:total hemoglobin ratio in a biological sample. The method comprises the following steps: (i) measuring absorbance at a first wavelength and a second wavelength in a biological sample containing lysed red blood cells, and determining a total hemoglobin concentration (tHb) based on the measurements obtained at the two wavelengths, wherein the first wavelength is 478 nm and the second wavelength is in the range of about 689 nm to about 699 nm; (ii) measuring absorbance at a third wavelength and a fourth wavelength in the biological sample containing lysed red blood cells, and determining a glycated hemoglobin concentration (A1c) based on the measurements obtained at the two wavelengths, wherein the third wavelength is 658 nm and the fourth wavelength is in the range of about 785 nm to about 825 nm; and (iii) normalizing the total hemoglobin concentration calculated in (i) using the absorbance (cHb) measured at the fourth wavelength in step (ii) and a turbidity normalization algorithm to substantially remove any turbidity interference from the wavelength measurements of (i), wherein the turbidity normalization algorithm is used to normalize ... to substantially remove any turbidity interference from the wavelength measurements of (i), wherein the turbidity normalization algorithm is used to normalize the total hemoglobin concentration calculated in (i) using the turbidity normalization algorithm to normalize the total hemoglobin concentration calculated in (i) using the turbidity normalization algorithm is used to normalize the total hemoglobin concentration calculated in (i) using the turbidity normalization algorithm At a concentration of 100 mg / dL of Intralipid, any turbidity interference is substantially reduced to less than + / - about 5%, and wherein the turbidity normalization algorithm is: Normalized tHb = and (iv) calculating % glycated hemoglobin or glycated hemoglobin:total hemoglobin ratio based on the concentrations calculated in (ii) and (iii).

[0025] In certain non-limiting embodiments of the above methods, the biological sample is a lysed whole blood sample.

[0026] In certain non-limiting embodiments of the above methods, the steps are performed in a single reaction cuvette.

[0027] The second wavelength can be any integer in the range of about 689 nm to about 699 nm, including about 689 nm, about 690 nm, about 691 nm, about 692 nm, about 693 nm, about 694 nm, about 695 nm, about 696 nm, about 697 nm, about 698 nm, and about 699 nm. In a specific (but non-limiting) embodiment, the second wavelength is 694 nm.

[0028] The fourth wavelength can be any integer in the range of about 785 nm to about 825 nm, including about 785 nm, about 786 nm, about 787 nm, about 788 nm, about 789 nm, about 790 nm, about 791 nm, about 792 nm, about 793 nm, about 794 nm, about 795 nm, about 796 nm, about 797 nm, about 798 nm, about 799 nm, about 800 nm, about 801 nm, about 802 nm, about 803 nm, about 804 nm, about 805 nm, about 806 nm, about 807 nm, about 808 nm, about 809 nm, about 810 nm, about 811 nm, about 812 nm, about 813 nm, about 814 nm, about 815 nm, about 816 nm, about 817 nm, about 818 nm, about 819 nm, about 820 nm, about 821 nm, about 822 nm In one embodiment, the fourth wavelength is about 805 nm.

[0029] Certain non-limiting embodiments of the present disclosure relate to methods of measuring % glycated hemoglobin or the glycated hemoglobin:total hemoglobin ratio in a biological sample. The method comprises the following steps: (a) lysing red blood cells present in the biological sample; (b) reacting the lysed red blood cells with a reagent to oxidize hemoglobin to methemoglobin; (c) cleaving an N-terminal fructosyl dipeptide fragment from the hemoglobin beta chain with a protease; (d) converting the methemoglobin to azide-methemoglobin; (e) measuring absorbance at a first wavelength and a second wavelength, and determining a total hemoglobin concentration (tHb) based on the measurements obtained at the two wavelengths, wherein the first wavelength is 478 nm and the second wavelength is in the range of about 689 nm to about 699 nm; (f) reacting the N-terminal fructosyl peptide fragment with a reagent to generate hydrogen peroxide; (g) measuring absorbance at a third wavelength and a fourth wavelength, and determining a glycated hemoglobin concentration (A1c) based on the measurements obtained at the two wavelengths, wherein the third wavelength is 658 nm and the fourth wavelength is in the range of about 785 nm to about 825 nm. (h) normalizing the total hemoglobin concentration calculated in step (e) using the absorbance (cHb) measured at the fourth wavelength in step (g) and a turbidity normalization algorithm to substantially remove any turbidity interference from the wavelength measurement in step (e); and (i) calculating % glycated hemoglobin or a glycated hemoglobin:total hemoglobin ratio based on the concentrations calculated in (g) and (h).

[0030] In certain non-limiting embodiments, the turbidity normalization algorithm used in step (h) is:

[0031] Normalized tHb = .

[0032] In certain non-limiting embodiments, any turbidity interference is substantially reduced to less than + / - about 5% at concentrations of Intralipid up to about 1000 mg / dL.

[0033] In certain non-limiting embodiments, all steps (a)-(g) of the method are performed in a single reaction cuvette.

[0034] The second wavelength can be any integer in the range of about 689 nm to about 699 nm, including about 689 nm, about 690 nm, about 691 nm, about 692 nm, about 693 nm, about 694 nm, about 695 nm, about 696 nm, about 697 nm, about 698 nm, and about 699 nm. In a specific (but non-limiting) embodiment, the second wavelength is 694 nm.

[0035] The fourth wavelength can be any integer in the range of about 785 nm to about 825 nm, including about 785 nm, about 786 nm, about 787 nm, about 788 nm, about 789 nm, about 790 nm, about 791 nm, about 792 nm, about 793 nm, about 794 nm, about 795 nm, about 796 nm, about 797 nm, about 798 nm, about 799 nm, about 800 nm, about 801 nm, about 802 nm, about 803 nm, about 804 nm, about 805 nm, about 806 nm, about 807 nm, about 808 nm, about 809 nm, about 810 nm, about 811 nm, about 812 nm, about 813 nm, about 814 nm, about 815 nm, about 816 nm, about 817 nm, about 818 nm, about 819 nm, about 820 nm, about 821 nm, about 822 nm In one embodiment, the fourth wavelength is about 805 nm.

[0036] In certain non-limiting embodiments, the biological sample is a whole blood sample.

[0037] In accordance with the present disclosure, any lysing reagent known in the art or otherwise contemplated herein can be used to lyse red blood cells present in a biological sample. Lysing reagents are well known in the art and widely commercially available, and therefore further discussion thereof is deemed unnecessary.

[0038] According to the present disclosure, any reagent known in the art that is capable of oxidizing hemoglobin to methemoglobin in the methods disclosed herein can be utilized. In certain non-limiting embodiments, the reagent used to oxidize hemoglobin to methemoglobin in step (b) is sodium nitrite.

[0039] According to the present disclosure, any azide that converts methemoglobin to azide-methemoglobin in step (d) of the methods disclosed herein can be utilized. In certain non-limiting embodiments, methemoglobin is converted to azide-methemoglobin in the presence of sodium azide.

[0040] According to the present disclosure, any reagent that can generate hydrogen peroxide after reaction with the N-terminal fructosyl peptide fragment of the hemoglobin beta chain in step (f) of the method disclosed herein can be utilized. In certain non-limiting embodiments, the reagent used in step (f) is fructosyl peptide oxidase.

[0041] Certain non-limiting embodiments of the present disclosure relate to methods of measuring % glycated hemoglobin or the glycated hemoglobin:total hemoglobin ratio in a biological sample. The method comprises the following steps: (a) lysing red blood cells present in the biological sample; (b) reacting the lysed red blood cells with sodium nitrite to oxidize hemoglobin to methemoglobin; (c) cleaving an N-terminal fructosyl dipeptide fragment from the hemoglobin beta chain with a protease; (d) converting the methemoglobin to azide-methemoglobin in the presence of sodium azide; (e) measuring absorbance at a first wavelength and a second wavelength, and determining a total hemoglobin concentration (tHb) based on the measurements obtained at the two wavelengths, wherein the first wavelength is 478 nm and the second wavelength is in the range of about 689 nm to about 699 nm; (f) reacting the N-terminal fructosyl peptide fragment with a reagent to generate hydrogen peroxide; (g) measuring absorbance at a third wavelength and a fourth wavelength, and determining a glycated hemoglobin concentration (A1c) based on the measurements obtained at the two wavelengths, wherein the third wavelength is 658 nm and the fourth wavelength is in the range of about 785 nm to about 825 nm. (h) normalizing the total hemoglobin concentration calculated in step (e) using the absorbance (cHb) measured at the fourth wavelength in step (g) and a turbidity normalization algorithm to substantially remove any turbidity interference from the wavelength measurement of (e), wherein any turbidity interference is substantially reduced to less than + / - about 5% at an intralipid concentration of up to about 1000 mg / dL, and wherein the turbidity normalization algorithm is: Normalized tHb = ; and (i) calculating % glycated hemoglobin or glycated hemoglobin:total hemoglobin ratio based on the concentrations calculated in (g) and (h).

[0042] In certain non-limiting embodiments of the above methods, the biological sample is a whole blood sample.

[0043] In certain non-limiting embodiments of the above methods, steps (a)-(g) are performed in a single reaction cuvette.

[0044] The second wavelength can be any integer in the range of about 689 nm to about 699 nm, including about 689 nm, about 690 nm, about 691 nm, about 692 nm, about 693 nm, about 694 nm, about 695 nm, about 696 nm, about 697 nm, about 698 nm, and about 699 nm. In a specific (but non-limiting) embodiment, the second wavelength is 694 nm.

[0045] The fourth wavelength can be any integer in the range of about 785 nm to about 825 nm, including about 785 nm, about 786 nm, about 787 nm, about 788 nm, about 789 nm, about 790 nm, about 791 nm, about 792 nm, about 793 nm, about 794 nm, about 795 nm, about 796 nm, about 797 nm, about 798 nm, about 799 nm, about 800 nm, about 801 nm, about 802 nm, about 803 nm, about 804 nm, about 805 nm, about 806 nm, about 807 nm, about 808 nm, about 809 nm, about 810 nm, about 811 nm, about 812 nm, about 813 nm, about 814 nm, about 815 nm, about 816 nm, about 817 nm, about 818 nm, about 819 nm, about 820 nm, about 821 nm, about 822 nm In one embodiment, the fourth wavelength is about 805 nm. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The graph illustrates the results of % glycated hemoglobin (% A1c) versus Intralipid concentration (mg / dL) obtained on an Atellica CH clinical chemistry analyzer (Siemens Healthineers, Tarrytown, NY) using whole blood samples spiked with Intralipid.

[0047] Figure 2 The graph illustrates the results obtained using whole blood spiked with Intralipid (6.5% HbA1c) compared to Intralipid concentration (mg / dL).

[0048] Figure 3The graph illustrates the results obtained using whole blood spiked with Intralipid (8.0% HbA1c) compared to Intralipid concentration (mg / dL).

[0049] Figure 4 Graph illustrating the results of A1c (µmol / L) versus Intralipid concentration (mg / dL) obtained using Intralipid-spiked whole blood (6.5% and 8% HbA1c).

[0050] Figure 5 The graph illustrates the results of total hemoglobin (tHb; µmol / L) obtained using intralipid-spiked whole blood (6.5% and 8% HbA1c) compared to intralipid concentration (mg / dL).

[0051] Figure 6 The graph illustrates the effect of increasing Intralipid on tHb (%) compared to the absence of Intralipid.

[0052] Figure 7 Describe the dilution scheme for samples spiked with Intralipid.

[0053] Figure 8 Graph illustrating delta tHb (µmol / L) with increasing amounts of Intralipid compared to absorbance at 805 nm in whole blood (6.5% HbA1c) spiked with Intralipid.

[0054] Figure 9 Describe the dilution scheme used for normal samples.

[0055] Figure 10 Graph illustrating absorbance at 805 nm compared to tHb (μmol) in normal samples diluted with saline.

[0056] Figure 11 Describes the dilution scheme used for Atellica CH-Intralipid Interference. Specific implementation plan

[0057] Examples are provided below. However, it should be understood that the present disclosure and its application are not limited to the specific experiments, results and laboratory procedures disclosed herein. On the contrary, the examples are provided as one of various embodiments only and are intended to be illustrative, not exhaustive.

[0058] The currently used A1c assay (developed for the Advia clinical chemistry analyzer; Siemens Healthineers, Tarrytown, NY) consists of two separate measurements: A1c (A1c_E) and total hemoglobin (tHb_E). These two measurements are used to determine %HbA1c (NGSP units) or the hemoglobin A1c_E / tHb_E ratio, expressed in mmol / mol (IFCC units).

[0059] Anticoagulated whole blood samples are lysed on the system for automated assays or can be manually lysed using pretreatment solutions to obtain hemolyzed lysates for manual assays.

[0060] This glycated hemoglobin assay is an enzymatic method that specifically measures the N-terminal fructosyl dipeptide on the β-chain of HbA1c. In a pretreatment step, red blood cells are lysed and hemoglobin is oxidized to methemoglobin by reacting with sodium nitrite. In the first step of the reaction, a protease is used to cleave the N-terminal fructosyl dipeptide fragment from the hemoglobin β chain. Simultaneously, in the presence of sodium azide, methemoglobin is converted to stable azide-methemoglobin, and the total hemoglobin concentration is determined by measuring the absorbance at 478 / 805 nm. In the second step of the reaction, fructosyl peptide oxidase (FPOX) is added to react with the fructosyl dipeptide to generate hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide reacts with a chromogen to produce a color that is measured at 658 / 805 nm.

[0061] This glycated hemoglobin assay also incorporates a turbidity normalization mechanism measured at 884 nm (cHb_E) to effectively remove any sample turbidity that may affect the tHb_E measurement.

[0062] However, other clinical chemistry analyzers do not have a filter at the wavelength (884 nm) used for the turbidity normalization component of the assay described above; therefore, other normalization methods must be developed to allow these clinical chemistry analyzers to perform an A1c assay that includes a turbidity normalization component. As described below, the A1c assay described herein has been found to remove any significant interference from Intralipid / lipidemia.

[0063] Turning now to the glycated hemoglobin assay of the present disclosure, this assay is also an enzymatic endpoint assay in which the results are measured as a percentage of two parameters: A1c, measured at 658 / 805 nm, and total hemoglobin (tHb), measured at 478 / 694 nm. The assay further includes the incorporation of a third sub-parameter, cHb, to normalize for falsely elevated tHb values in samples with intralipid interference and / or high turbidity.

[0064] Compared to the assay developed for the Advia clinical chemistry analyzer described above, in the present glycated hemoglobin assay, cHb is measured at 805 nm during the same reading cycle as tHb, and the absorbance at 805 nm is not associated with interference from Intralipid or increased turbidity. The following normalization equation is used in the glycated hemoglobin assay disclosed herein:

[0065] Normalized tHb_E = .

[0066] Samples with either Intralipid or increased turbidity were tested on two different clinical chemistry analyzers (Atellica CH and Advia modules, Siemens Healthineers, Tarrytown, NY) and found to perform similarly on both platforms. Without turbidity normalization, these samples exhibited falsely elevated tHb values and, therefore, falsely decreased HbA1c (%) values ( Figure 1 ).

[0067] When spiked with Intralipid, the whole blood samples did not meet the |+ / -5%| deviation specification. When calculated to target |+ / -5%| deviation, the samples that passed had only approximately 300 mg / dL of Intralipid, which is well below the target concentration of 1000 mg / dL Intralipid ( Figure 2 and Figure 3 and Table I).

[0068] Table I: Atellica CH - Samples spiked with Intralipid without normalization

[0069]

[0070] Similar to the previous assay platform, the influence of intralipid interference and sample turbidity on the glycosylated hemoglobin assay in this paper is manifested in the tHb portion. Therefore, this platform also requires a turbidity normalization equation ( Figure 4 、 Figure 5 、 Figure 6 and Table II).

[0071] Table II: Effect of increasing Intralipid compared to zero Intralipid on A1c (%) and tHb (%)

[0072]

[0073] Not all clinical chemistry analyzers on which the HbA1c assay is performed have a filter at 884 nm; therefore, the HbA1c assay described herein includes recalculating the tHb and cHb test wavelengths along with the tHb normalization constant to allow execution on other analyzers (such as, but not limited to, the Atellica CH module, Siemens Healthineers, Tarrytown, NY). The A1c test wavelengths used in the HbA1c assay remain the same, at 658 / 805 nm. The tHb test wavelengths used in the assay herein are 478 / 694 nm, and the cHb test wavelength used in the assay herein is 805 nm.

[0074] The following protocol describes the process for optimizing the parameters of the Glycated Hemoglobin assay of the present disclosure.

[0075] Whole blood Medical Decision Pools with HbA1c values of 5.0, 6.5, 8.0, and 12.0% were used in multiple feasibility studies. These samples were prepared as follows. Fresh field samples were collected and spiked with high BBI material (hemoglobin A1c HbA1c, SKU P186-0B; BBI Solutions, Crumlin, UK) to achieve the target %HbA1c values mentioned above. Samples were assigned values on a Tosoh G8 HPLC analyzer (Tosoh Bioscience, Inc., South San Francisco, CA; SN 13523607). Samples were pipetted into 0.5 mL aliquots and frozen.

[0076] Linear regression was used to evaluate the effect of Intralipid on tHb concentration at the new wavelength. A 2 mL frozen whole blood MDP (Medical Decision Point) sample (value of 6.5% HbA1c) was thawed and gently inverted for approximately one hour. 50 μL of 20,000 mg / dL Intralipid was spiked into 950 μL of thawed MDP for a total of one (1) mL whole blood sample, with a final Intralipid concentration of 1000 mg / dL. The mixture was inverted for approximately 30 minutes to ensure homogeneity. Then, the mixture was inverted using Figure 7 Using the dilution scheme described in [ 1 ], five unique whole blood samples with increasing concentrations of Intralipid (0 mg / dL, 250 mg / dL, 500 mg / dL, 750 mg / dL, and 1000 mg / dL) were created. Each level was then tested in triplicate on the Atellica CH module. The data was then plotted and the slope and y-intercept were found via linear regression analysis.

[0077] like Figure 8 As shown in , the equation y = 0.7899x – 4.7676 was established via linear regression, and its slope was used to develop a new turbidity normalization equation.

[0078] Linear regression was used to assess the relationship between tHb concentration and absorbance at 805 nm. A total of 10 mL of human whole blood was drawn from the donor site and a hemolytic lysate was prepared. The EDTA tubes were allowed to sit refrigerated for >4 hours and the cells and plasma separated naturally. The plasma was removed from each tube and transferred to separate containers. The red blood cells were then centrifuged at 2000 g for 10 minutes to pack the cells, and the supernatant was discarded. The packed cells were washed with saline and centrifuged again 3 times, discarding the supernatant between each wash. The washed cells were frozen at -70°C overnight and thawed the next day for testing. Follow Figure 9 The dilution diagram shown in the , produces equidistant dilutions of plasma and concentrated hemolyzed lysate (≥ 250 μmol / L tHb), for a total of six unique samples with increasing tHb concentrations. The Atellica dilution probe was unable to aspirate the fifth and highest level (≥ 250 μmol / L tHb), which prompted the viscosity sample delta flag; therefore, levels 4 and 5 were used to make a 4.5 level. Each level was then tested in triplicate on the Atellica CH analyzer. The data was then plotted and the slope and y-intercept were found.

[0079] like Figure 10 As shown in , the equation y = 0.0326x + 1.2168 was established via linear regression, and its slope was used to develop a new turbidity normalization equation.

[0080] Using the slopes of 0.7899 and 0.0326 from the two experiments, the normalization equation is formulated as follows:

[0081] Normalized tHb_E is calculated according to equation [1]:

[0082] [1] tHb_E norm = tHb_E not-norm – tHb_E 浊度

[0083] where tHb_E norm is the normalized total hemoglobin (μmol / L); tHb_E not-norm is the total hemoglobin before normalization (μmol / L); and tHb_E 浊度 is the turbidity contribution to tHb_E (μmol / L).

[0084] tHb_E 浊度Calculated according to equation [2a]. In equation [2a], the absorbance at 805 nm (cHb) is used to determine the equivalent tHb contribution due to turbidity at the assay reading wavelength (478 nm). The second part of the equation (tHb_E not-norm x 0.0326) is responsible for the true hemoglobin contribution at 884 nm and must be subtracted to determine the true turbidity contribution to tHb_E.

[0085] The factors 0.7899 and 0.0326 are derived empirically. 0.7899 is the absorbance at 805 nm (Abs 805nm ), total hemoglobin (tHb_E) measured at 478 nm in samples of increasing turbidity (whole blood spiked with Intralipid to simulate lipemic sample conditions) notnorm 0.0326 is the slope of the linear regression of the absorbance at 884 nm versus increasing hemoglobin concentration.

[0086] [2a] tHb_E 浊度 =

[0087] Equation [2a] is simplified to equations [2b] and [2c].

[0088] [2b] tHb_E 浊度 =

[0089] [2c] tHb_E 浊度 =

[0090] Substituting equation [2c] back into equation [1] yields equation [3a]:

[0091] [3a] tHb_E norm =

[0092] Equation [3a] then simplifies to [3b] and [3c]:

[0093] [3b] tHb_E norm =

[0094] [3c] tHb_E norm =

[0095] The final normalized equation:

[0096] Normalized tHb_E =

[0097] To evaluate the wavelength, a 2 mL frozen whole blood MDP sample with a HbA1c value of 6.5% was thawed and gently inverted for approximately one hour. 50 μL of 20,000 mg / dL Intralipid was spiked into 950 μL of the thawed MDP for a total of one (1) mL whole blood sample, with a final Intralipid concentration of 1000 mg / dL. The mixture was inverted for approximately 30 minutes to ensure homogeneity. The control sample (MDP without Intralipid) and the test sample (MDP spiked with Intralipid) were tested in triplicate on the analyzer. The interference (Dobs) and % bias were calculated and evaluated for the following wavelength combinations: (1) A1c - 658 / 805 nm and tHb - 478 / 805 nm, as shown in Table III; (2) A1c - 658 / 694 nm and tHb - 478 / 694 nm, as shown in Table IV; (3) A1c - 658 / 694 nm and tHb - 478 / 805 nm, as shown in Table V; and (4) A1c - 658 / 805 nm and tHb - 478 / 694 nm, as shown in Table VI. The test wavelengths of 658 / 805 nm for A1c and 478 / 694 nm for tHb (Table VI) showed the best performance and were therefore determined to be the new test wavelengths.

[0098] Table III: Intralipid-spiked samples measured at 658 / 805 nm (A1c) and 478 / 805 nm (tHb)

[0099]

[0100] Table IV: Intralipid spiked samples measured at 658 / 694 nm (A1c) and 478 / 694 nm (tHb)

[0101]

[0102] Table V: Intralipid-spiked samples measured at 658 / 694 nm (A1c) and 478 / 805 nm (tHb)

[0103]

[0104] Table VI: Intralipid spiked samples measured at 658 / 805 nm (A1c) and 478 / 694 nm (tHb)

[0105]

[0106] All the following feasibility data were analyzed using the new normalization equation.

[0107] Thaw 2 mL frozen whole blood MDP samples with HbA1c values of 6.5% and 8.0% and gently invert for approximately one hour. Spike 50 μL of 20,000 mg / dL of Intralipid into 950 μL of thawed 6.5% MDP to achieve a final concentration of 1000 mg / dL of Intralipid in one (1) mL of whole blood. Spike 50 μL of 20,000 mg / dL of Intralipid into 950 μL of thawed 8.0% MDP to achieve a final concentration of 1000 mg / dL of Intralipid in one (1) mL of whole blood. Invert the mixture for approximately 30 minutes to ensure homogeneity. Then use Figure 11 Five unique whole blood samples with increasing concentrations of Intralipid (0 mg / dL, 250 mg / dL, 500 mg / dL, 750 mg / dL, and 1000 mg / dL) were created for two levels of MDP using the dilution scheme described in

[0015] . Each level was then tested in triplicate on the Atellica CH analyzer, and the mean and % deviation were calculated as shown in Tables VII and VIII.

[0108] Table VII: Interference and dose response of Intralipid without normalization

[0109]

[0110] Table VIII: Normalized Intralipid Interference and Dose Response

[0111]

[0112] As can be seen, Intralipid interference had no effect in the novel HbA1c assay of the present disclosure, and the resulting % Deviation was below the specified target |+ / - 5%| when measured at the target Intralipid concentration (1000 mg / dL).

[0113] Thus, according to the present disclosure, there have been provided compositions and methods of making and using the same that fully satisfy the objects and advantages described above. Although the present disclosure has been described in conjunction with the specific figures, experiments, results, and language set forth above, it is apparent that many alternatives, modifications, and variations will be apparent to those skilled in the art. Therefore, it is intended to encompass all such alternatives, modifications, and variations that fall within the spirit and broad scope of the present disclosure.

Claims

1. A method for measuring % glycated hemoglobin or the ratio of glycated hemoglobin to total hemoglobin in a biological sample, the method comprising the following steps: (i) measuring absorbance at a first wavelength and a second wavelength in a biological sample containing lysed red blood cells, and determining a total hemoglobin concentration, tHb, based on the measurements obtained at the two wavelengths, wherein the first wavelength is 478 nm and wherein the second wavelength is 694 nm; (ii) measuring absorbance at a third wavelength and a fourth wavelength in a biological sample containing lysed red blood cells, and determining a glycated hemoglobin concentration A1c based on the measurement values obtained at the two wavelengths, wherein the third wavelength is 658 nm and wherein the fourth wavelength is 805 nm; (iii) normalizing the total hemoglobin concentration calculated in (i) using the absorbance cHb measured at the fourth wavelength in step (ii) and a turbidity normalization algorithm to remove any turbidity interference from the wavelength measurement of (i), wherein any turbidity interference is reduced to less than + / - 5% at an Intralipid concentration of up to 1000 mg / dL, and wherein the turbidity normalization algorithm is: Normalized tHb = 1.03 X tHb (μmol / L) – cHb X 0.7899; and (iv) Calculate the % glycated hemoglobin or the glycated hemoglobin:total hemoglobin ratio based on the concentrations calculated in (ii) and (iii).

2. The method of claim 1, wherein the biological sample is a lysed whole blood sample.

3. The method of claim 1 or 2, wherein the steps are performed in a single reaction cuvette.

4. A method for measuring % glycated hemoglobin or the ratio of glycated hemoglobin to total hemoglobin in a biological sample, the method comprising the steps of: (a) lysing red blood cells present in the biological sample; (b) reacting the lysed red blood cells with sodium nitrite to oxidize hemoglobin to methemoglobin; (c) cleaving the N-terminal fructosyl dipeptide fragment from the hemoglobin β chain using a protease; (d) converting methemoglobin to azide-methemoglobin in the presence of sodium azide; (e) measuring absorbance at a first wavelength and a second wavelength, and determining the total hemoglobin concentration tHb based on the measurements obtained at the two wavelengths, wherein the first wavelength is 478 nm and wherein the second wavelength is 694 nm; (f) reacting the N-terminal fructosyl peptide fragment with fructosyl peptide oxidase to generate hydrogen peroxide; (g) measuring absorbance at a third wavelength and a fourth wavelength, and determining a glycated hemoglobin concentration A1c based on the measurement values obtained at the two wavelengths, wherein the third wavelength is 658 nm and wherein the fourth wavelength is 805 nm; (h) normalizing the total hemoglobin concentration calculated in step (e) using the absorbance cHb measured at the fourth wavelength in step (g) and a turbidity normalization algorithm to remove any turbidity interference from the wavelength measurement in step (e), wherein any turbidity interference is reduced to less than + / - 5% at an Intralipid concentration of up to 1000 mg / dL, and wherein the turbidity normalization algorithm is: Normalized tHb = 1.03 X tHb (μmol / L) – cHb X 0.7899; and (i) Calculating % glycated hemoglobin or glycated hemoglobin:total hemoglobin ratio based on the concentrations calculated in steps (g) and (h).

5. The method of claim 4, wherein the biological sample is a whole blood sample.

6. The method of claim 4 or 5, wherein steps (a) to (g) are performed in a single reaction cuvette.

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