Chemistry: analytical and immunological testing – Hemoglobin – myoglobin – or occult blood – Glycosylated hemoglobin
Reexamination Certificate
1999-09-17
2001-11-13
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Hemoglobin, myoglobin, or occult blood
Glycosylated hemoglobin
C436S066000, C436S164000, C436S172000, C436S805000, C436S824000, C436S518000, C436S524000, C435S007100, C435S960000
Reexamination Certificate
active
06316265
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for detecting the presence or amount of glycated hemoglobin (GHb) in a blood sample, and more particularly, to an improved, highly accurate, one-read method for assessing percentage glycated hemoglobin, that is, a method which does not require a measurement of total hemoglobin (THb).
BACKGROUND OF THE INVENTION
Glycated hemoglobin (GHb) refers to a series of minor hemoglobin components that are formed via the attachment of various sugars (most commonly glucose) to the hemoglobin molecule. The human erythrocyte is freely permeable to glucose. Within each erythrocyte, GHb is formed at a rate that is directly proportional to the ambient glucose concentration. The reaction of glucose with hemoglobin is nonenzymatic, irreversible and slow, so that only a fraction of the total hemoglobin is glycated during the life span of an erythrocyte (120 days). As a result, the measurement of GHb provides a weighted “moving” average of blood glucose levels that can be used to monitor long-term blood glucose levels, providing an accurate index of the mean blood glucose concentration over the preceding 2 to 3 months. The most important clinical application of this is in the assessment of glycemic control in a diabetic patient.
Hemoglobin A1c (HbA1c) is one specific type of glycated hemoglobin and is the most important hemoglobin species with respect to diabetes. The amount of total hemoglobin that is HbA1c is approximately 3 to 6% in nondiabetics, and 20% or greater in diabetes that is poorly controlled (Goldstein DE, et al, Clin Chem 32: B64-B70 (1986)). In HbA1c, glucose is attached to the amino terminal valine residue of one or both of the hemoglobin A beta chains. HbA1c (as well as other glycated Hemoglobin A1 species) can be separated from nonglycated hemoglobins by methods that separate molecules based on differences in their electrical charges. Glycation of hemoglobin also occurs at other sites on the hemoglobin molecule, but these species cannot be separated from nonglycated hemoglobins based on charge differences, so all of these species of hemoglobin are termed HbA0. Methods that measure all forms of glycated hemoglobin are said to measure total GHb. Since glycation at one site appears to be proportional to glycation at any other site, there is a linear relationship between GHb and HbA1c. The Diabetes Control and Complications Trial (DCCT) Research Group reported that a 1% change in GHb (%HbA1c) represents an average change of 300 mg/L in blood glucose levels over the preceding 120 days.
Traditional methods of assessing blood glucose control in diabetes, including urine and blood glucose levels, have a limited value since they can fluctuate, do not provide information on glucose levels over time, and are influenced dramatically by diet. However, measurement of GHb is an accurate index of a person's mean blood glucose level over the preceding 2 to 3 months and can provide a diabetic patient an overview of their success in meeting long-term goals for controlling their blood glucose levels. Since GHb levels can be used to monitor a patient's glycemic control over time, a high degree of long-term assay precision and standardization across different methodology is essential. In response to these clinical requirements, the American Association of Clinical Chemistry (AACC) formed a subcommittee on GHb standardization in 1993. The GHb Standardization Subcommittee recommended that within-laboratory, between-run CVs be maintained at 5% or lower for all GHb assays, and that standardization be based on correlation to the DCCT for fresh samples. All manufactured assays must meet these requirements to receive certification.
Clinical assay methods separate GHb from total hemoglobin based on either charge differences or structural characteristics. Methods based on charge differences include cation exchange chromatography and electrophoresis, and separate HbA1 or HbA1c from HbA0 based on the difference in their charges. Ion exchange chromatography can be performed either in large columns, mini or micro columns, or by high pressure liquid chromatography (HPLC). Large column methods are impractical for routine use in a clinical laboratory, but simplified mini or micro columns are available. However these methods show poor reproducibility and are very sensitive to variations in temperature, pH and ionic strength. While electrophoretic methods are not as sensitive to temperature, pH or ionic strength, they have other drawbacks which are also seen with ion exchange methods, namely, interference by a labile GHb intermediate, which must be removed prior to GHb testing, problems if a hemoglobinopathy is present, sensitivity to sample storage conditions and interference from extraneous clinical factors, such as aspirin therapy, ethanol levels and uremia. Also, HPLC and electrophoresis require specialized equipment.
Methods based on structural characteristics include affinity binding or chromatography and immunoassays. These methods are less sensitive to small variations in temperature, pH or ionic strength, and generally are not affected by labile GHb intermediates, hemoglobinopathies or sample storage conditions or the extraneous clinical factors mentioned above. However these methods either involve separation of GHb from nonglycated components or require 2 separate determinations—one for total hemoglobin and a second for GHb or HbA1c—to calculate %GHb. Use of a boronate ligand coupled to a solid phase matrix can be used in affinity binding assays due to the affinity of boronate for GHb. Ratios of bound (glycated) to nonbound (nonglycated) hemoglobin can then be quantified. Immunoassays measure HbA1c using HbA1c specific antibodies, but require 2 separate determinations, one for total hemoglobin and the other for HbA1c, in order to calculate %GHb. Alternatively an immunoassay may bind all hemoglobin species using passive adsorption, then detect HbA1c with a specific antibody conjugate; however this method may be adversely affected by hemoglobin variants.
What is therefore needed in the art is an improved, highly accurate method of detecting the presence or amount of glycated hemoglobin in a blood sample which does not require a determination of the total hemoglobin content as well.
SUMMARY OF THE INVENTION
The unique and novel approach described herein measures %GHb in whole blood samples and provides a %GHb result following only a single measurement. This differs from the methods described above and currently available in the art which require separate total and GHb measurements to calculate a %GHb result as, for example, a ratio of GHb/THb×100. The GHb assay described herein uses a simple procedure for the determination of DCCT Standardized %GHb in whole blood samples. First, a lysed whole blood sample is incubated with a solid phase that is coupled with boronic, phenylboronic or boric acid or related boronate compound through covalent linkage chemistries known in the art. This solid phase is novel since it specifically captures GHb in direct proportion to the %GHb in the sample. Next, a labeled component that recognizes human hemoglobin is added and the resulting signal is directly proportional to the %GHb in the sample. The advantages of measuring %GHb using a single determination include high precision (less than about 5% CVs) and, since the assay is easily automatable, high throughput (100 to 200 tests/hour). With automation, this assay can also be consolidated with other testing on one analyzer.
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Adamczyk Janina
Christensen Melissa A.
Lee Evelyn Mok
Westerberg David A.
Yao Haiou H.
Abbott Laboratories
Anderson Regina M.
Wallenhorst Maureen M.
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