Chemistry: analytical and immunological testing – Hemoglobin – myoglobin – or occult blood – Glycosylated hemoglobin
Reexamination Certificate
1999-01-27
2001-10-30
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Hemoglobin, myoglobin, or occult blood
Glycosylated hemoglobin
C436S066000, C436S069000, C436S169000, C422S051000, C422S073000
Reexamination Certificate
active
06309887
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
(Not Applicable)
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the general area of remote site blood sample collection for medical diagnostic tests. More specifically, the invention relates to a improved filter paper for collecting a blood sample for higher precision analysis of hemoglobin or a hemoglobin fraction.
2. Description of Related Art
Filter paper has been used for more than 20 years as a means of gathering samples of blood, especially from newborns, for the purpose of identifying or measuring specific components in the blood in screening for congenital metabolic disorders, e.g., phenylketonuria (PKU). Although PKU screening has been one of the most common applications of blood collection on filter paper, many other applications have been in use, including the use of filter paper for collecting samples to test for blood components associated with thyroid dysfunction or in monitoring diabetic conditions. There are numerous papers in the scientific literature describing these testing methods. Although certain of these publications relate to characteristics of the filter paper itself, e.g., precision, lot-to-lot variations, and variability of results due to contamination of the filter paper used, most of the publications in the scientific literature focus on the methods employing a filter paper.
In 1984, a research paper was published on the use of filter paper for collecting blood samples for the purpose of measuring total glycohemoglobin. Eross, et al. (1984) Ann. Clin. Biochem. 21:477-483. Glycohemoglobin refers to the glycosylated state of hemoglobin resulting from glucose entering the red blood cell. Measurement of glycohemoglobin is extremely important in metabolic control of diabetics. Although blood glucose levels can fluctuate during the course of the day, relative to eating, medication, exercise, or other factors, glycohemoglobin levels are not so acutely affected. Thus, by measuring glycohemoglobin rather than glucose directly, a more accurate or more constant reading of blood glucose levels can be determined. Certain improvements to the total glycohemoglobin assay, which have been described in the scientific literature, have been achieved by modifying the filter paper used in the glycohemoglobin assay. Specifically, pretreatment of the filter paper with an enzyme, e.g., glucose oxidase, was used to reduce the glucose concentration in a blood sample being tested for total glycohemoglobin concentrations by Eross and by Little, et al. (1986) Clin. Chem. 32(5):869-871. The presence of glucose in the blood sample can cause an undesirable increase in glycohemoglobin concentrations compared to actual levels. A glycohemoglobin assay product using filter paper treated with glucose oxidase has been commercialized and was marketed under the trade name Self-Assure™, FlexSite Diagnostics, Palm City, Fla.
Although the use of a glucose oxidase-treated filter paper in measuring total glycohemoglobin has been described, such use has only been described in relation to affinity chromatography assays for total glycohemoglobin. Recently, methods have been introduced to measure a specific fraction of total glycohemoglobin, the so-called HbA
1c
fraction, Glucose glycates hemoglobin protein at a number of amino acid sites. About half of the glycation occurs at the N-terminal valine of the beta chain of hemoglobin. This glycation product is designated HbA
1c
. Antibody based methods for HbA
1c
have proved to be more specific than total glycohemoglobin methods. Thus, glucose present in the blood gives much larger errors in affinity chromatography methods for measuring total glycohemoglobin in dried blood spot than in an alternative method using an antibody assay, e.g., immunoprecipitation assays. Little, et al. (1996) Clin. Chem 42 (6): S193, showed that glucose present in a blood sample causes little interference in measuring the HbA
1c
fraction of glycohemoglobin using an antibody-based assay.
Two filter paper manufacturers, namely, Schleicher & Schuell (Keene, N.H., USA) and Whatman (Fairfield, N.J., USA) have devoted considerable commercial effort to refining certain of their filter paper products for application to dried blood spot methods, including glycohemoglobin testing. These refinements focus on consistency of the paper but not on treatment of the filter paper with various reagents. To date, it is not known to treat filter paper with a reagent other than glucose oxidase methods, according to Eross or Little, for use in a total glycohemoglobin assay.
In addition, it is not known that a treatment of the filter paper with a specific reagent can result in an improved assay for the HbA
1c
fraction of total glycohemoglobin by an antibody method. It is expected that when blood is applied to filter paper from a finger stick that coagulation will occur, thus binding the red blood cells into the clot matrix. It is further expected that when the blood is re-dissolved from the dried blood spot that coagulated blood will be more difficult to re-dissolve than un-coagulated blood, resulting in lower Hb concentrations in standard assays. Treatment with an anticoagulant of a filter paper used in an assay for measuring glycohemoglobin has not been heretofore described.
SUMMARY OF THE INVENTION
The subject invention concerns treatment of filter paper used for dried blood spot sample collection. More specifically, the subject invention concerns treatment of the filter paper with an anticoagulant, e.g., ethylenediamine tetra-acetic acid (EDTA), which results in easier dissolution of the dried blood spot compared to untreated filter paper, in the analysis of HbA
1c
and more direct comparison to EDTA anticoagulated venipuncture samples. The use of EDTA treated filter paper has the additional advantage of allowing exact comparison studies between freshly gathered finger stick blood applied to EDTA treated filter paper and larger quantities of veni-puncture blood collected in EDTA collection tubes. If EDTA were not used on the filter paper, the EDTA anti-coagulated veni-puncture samples might give different results in correlation studies with dried blood spots on untreated paper.
Thus, it is an object of the subject invention to provide an improved method and material for assaying components from dried blood samples collected on filter paper.
It is another object of the invention to provide an improved method and material for eluting dried blood samples collected on filter paper for purposes of assaying components in the blood sample.
It is yet another object of the invention to provide an improved material and method for assaying glycohemoglobin, or HbA
1c
, in a dried blood sample collected on filter paper. Specifically, it is an object of the invention to provide an improved assay for measuring the HbA
1c
function of glycohemoglobin in blood by collecting a blood sample to be assayed for that component onto a filter paper which has been pre-treated with an anticoagulant, e.g. EDTA.
These, and other objects of the invention achieved by an absorption material which comprises filter paper and a chelating agent. The filter is adapted to collect a liquid biological sample, and the chelating agent is contained within the filter paper to prevent the liquid biological sample from coagulating. The filter paper is preferably cellulose based and collects approximately 20-30 &mgr;l of the biological sample.
The liquid biological sample to be collected is preferably blood, and the component of interest is glycohemoglobin or a HbA
1c
fraction of total glycohemoglobin. When blood is collected, the preferable chelating agent is ethylenediamine tetra-acetic acid (EDTA) or possibly citric acid. If ethylenediamine tetra-acetic acid is the chelating agent, the EDTA is preferably present in a quantity of at least 5 &mgr;g/20 &mgr;l of blood collectable by the filter paper to prevent coagulation of the blood; however, most preferably the EDTA should be present in a quantity of at least 10 &mgr;g/20 &mgr;l of blo
Akerman & Senterfitt
FlexSite Diagnostics, Inc.
Wallenhorst Maureen M.
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