Diagnostic assay

For blood or other physiological fluid sample collection kits that use filter paper to collect the sample, the performance of the kit and associated analytical method can be improved by using a material having properties which are superior to those of standard filter paper or modified filter paper routinely used in standard biological assays. Certain materials currently available for uses other than blood collection, storage, or transport have properties that are advantageous as employed in assays of biological fluids, including the use of specific cellulose blotting materials for collecting blood samples for hemoglobin or hemoglobin A1c monitoring.

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 medical diagnostic tests using filter paper to collect the
 blood sample.
 DISCUSSION OF THE RELEVANT ART
 Historically, blood samples have been drawn from a patient in a hospital or
 physician's office using an evacuated test-tube to collect blood from a
 venipuncture by a physician, nurse, or other medical professional.
 Alternatively, a method for sampling of blood at a site remote from the
 hospital, physician's office, or laboratory has developed over the years
 where a small amount of blood may be obtained from a fingerstick and the
 blood can be absorbed onto filter paper to collect the sample. This is
 known in the art as a "dry" technique, as opposed to a "wet" remote site
 sampling technique that involves collection of a blood sample in a
 capillary tube.
 Dry remote site blood spot sampling had its beginning in the 1960's with
 the use of filter paper to gather blood spot samples from neonates for use
 in the determination of the presence of phenylketones (Guthrie, et al.,
 [1963] Pediatrics 32(3): 338-343). Since that time, a number of products
 have been introduced that are designed to facilitate remote site sample
 collection and transport of the sample to a laboratory for analysis. The
 availability of remote site blood collection such that the collected
 sample can be transported to a laboratory for analysis has been very
 successful in monitoring blood components of diabetics.
 Diabetes is a chronic and serious disease which affects over 100 million
 individuals worldwide. In the United States, there are 8 million diagnosed
 cases of diabetes, with an estimated 8 million cases undiagnosed. Diabetes
 is the leading cause of blindness, stroke, kidney failure, and amputations
 in the U.S. Because a person with diabetes is ten (10) times more likely
 to require hospitalization than the general population, the direct and
 indirect medical costs for treating diabetes have been estimated to exceed
 $105 billion annually. This total equals 14.6% of total U.S. health care
 expenditures being consumed by 3% of the population.
 In about 1980, Schleicher and Schuell Corp. (S&S) began producing a filter
 paper attachable to a test request form (designed to order). In using the
 S&S system, a blood spot is placed in one or more designated areas of the
 filter paper, allowed to dry, and then mailed along with the test request
 form to the laboratory.
 Eross, et al. introduced the use of filter paper spots for the gathering of
 blood spot samples for the measurement of glycohemoglobin. Eross, et al.
 (1984) Ann. Clin. Biochem. 21: 477-483. In 1986 Little, et al. reported on
 the use of filter paper blood spotting for measuring glycohemoglobin by
 affinity chromatography (Little, et al., [1986] Clin. Chem. 32(5):
 869-871). Measurement of glycosylated hemoglobin (glycohemoglobin) or a
 component thereof, e.g., HbA1c, is extremely important in metabolic
 control of diabetics. Using the Little technology, Evalu-lab, a subsidiary
 of Awareness Technology, produced a product, Self-Assure.TM. (now owned by
 FlexSite Diagnostics), between 1987 and 1992 which gathered a blood spot
 sample on filter paper for glycohemoglobin or HbA1c determination. The
 blotting material used in these assays was a glucose oxidase-treated
 filter paper.
 The methods and materials in this art have been the subject of many
 patents, including U.S. Pat. No. 5,516,487, which describes the use of
 various antibiotics or preservatives in combination with a cotton fiber
 filter paper, as well as the use of multiple application zones on the
 filter paper which are isolated from each other by perforations in the
 filter paper; U.S. Pat. No. 5,508,200, which describes the use of S&S 903
 and S&S 470 filter papers in a complex integrated analytical system and
 measurement of chemical reactions on the filter paper matrix; U.S. Pat.
 No. 5,432,097, concerning digestion of the filter paper with cellulase so
 that recovery of intact cells can be achieved; U.S. Pat. No. 5,427,953,
 which concerns measurement of a heavy metal (e.g., lead) from blood
 samples collected on filter paper; U.S. Pat. No. 5,204,267, which
 describes preservation of blood samples collected on various filter
 matrices for glucose analysis; U.S. Pat. No. 4,816,224, which is directed
 to a multiple layer device for separating plasma or serum from a blood
 sample collected for glucose analysis; U.S. Pat. No. 4,299,812, pertaining
 to an improved thyroid function (T4) test; and U.S. Pat. No. 4,227,249,
 which primarily concerns a drying procedure and its effect on the results
 of an assay measuring somatomedin. The disclosures of these patents are
 hereby incorporated by reference.
 Other patents describing the use of certain blotting materials used in
 biological assay methods include U.S. Pat. Nos. 5,496,626; 5,460,057;
 5,415,758; 4,790,797; and 4,774,192. None of the above-referenced patents
 relate to the use of blotting materials used in association with a
 standard assay for hemoglobin (Hb) or hemoglobin A1c (HbA1c) or describe
 superior performance of the Hb or HbA1c assay by use of those materials.
 In about 1992, Bio-Rad introduced a 100 test kit (wet samples) to
 facilitate the collection of capillary samples for analysis on their
 Diamat.TM. HPLC (High Performance Liquid Chromatography) system. Eli Lilly
 subsequently packaged a one test version of this kit as a promotional tool
 for their insulin products.
 In Sweden about 1994 Boehringer Mannheim Corp. (BMC) introduced a mail-in,
 filter paper based ("dry") blood spot kit under the name of "Via Post.TM."
 for HbA1c measurement. This BMC kit was evaluated by Jeppsson, et al.
 (Diabetes Care 19(2): pp. 142-145). BMC also introduced a "wet" capillary
 HbA1c kit for analysis on a Bio-Rad HPLC in 1995, and in 1996 introduced a
 screen material for blood spot sampling for analysis with their
 TinaQuant.TM. method for Hemoglobin A1c (Niederau, et al. [1996] Clin.
 Chem. 42(6): 167). Little, et al. ([1996] Clin. Chem. 42(6): 193)
 evaluated the use of filter paper for HbA1c sample collection with
 analysis by HPLC and by the Roche Unimate.TM. method Voss, et al.
 evaluated capillary collection systems for HbA1c analyses. Diabetes Care
 (1992) 15: 700.
 Historically, the most commonly used material for these assays has been S&S
 903, a cotton linter paper. However, certain disadvantages have been
 associated with the S&S 903 paper and its equivalents available from other
 manufacturers, e.g., Whatman. Specifically, certain of these commercially
 available and commonly used materials lack characteristics which provide
 precision values and accuracy that is preferred for carrying out a
 commercially superior HbA1c assay.
 Measurement of HbA1c, unlike blood glucose monitoring which provides an
 individual with their glucose value at that instant, measures the degree
 of diabetes control over the last 2-3 month period. In doing so, HbA1c
 monitoring provides the health care professional and patient a critical
 tool to determine the effectiveness of the patient's therapy and/or the
 patient's overall compliance.
 Albeit recognized that HbA1c monitoring is beneficial, it is estimated that
 fewer than 16% of the 8 million individuals with diabetes have an A1c test
 annually, less than 10% have the test performed on a quarterly basis as
 recommended. The primary reasons for this is that a majority of patients
 with diabetes are treated by primary care physicians who are not current
 on the benefits of intensive therapy and HbA1c monitoring. Also, A1c
 testing has historically been an expensive laboratory test which required
 a blood draw by venipuncture which is poorly tolerated by both young and
 older patients.
 Thus, discovery of unexpectedly improved or superior Hb or HbA1c assay
 results using materials that are commercially available and presumed to
 provide only equivalent results adds significantly to this art by
 improving the accuracy of detection of the blood components of interest.
 Improved accuracy of detection can contribute to more precise monitoring
 and overall better health care and quality of life for the patients who
 may rely on these assays to monitor glycohemoglobin.
 SUMMARY OF THE INVENTION
 It is an object of the invention to provide an improved assay for measuring
 or detecting a component of interest in a biological fluid, e.g., blood.
 It is another object of the invention to provide an improved assay method
 or an article of manufacture for detecting or measuring hemoglobin A1c
 (HbA1c) in a biological fluid.
 It is a further object of the invention to provide a kit having particular
 unexpected advantages for measuring or detecting a component of interest
 in a biological sample, e.g., HbA1c in blood.
 The subject invention thus concerns a system which can provide an
 improvement, e.g., superior performance, in a standard assay for measuring
 HbA1c. Specifically, the improved performance results from the utilization
 as a blotting material of a particular membrane or filter product made
 from natural or synthetic materials or composites. The material selected
 for use in accordance with the subject invention, e.g., the cellulose
 filter paper, Ahlstrom 319, which is commercially available,
 advantageously exploits one or more properties that can give improved
 performance characteristics such as assay precision, sample stability,
 accuracy of quantitative or qualitative detection, ease of sample
 handling, and the like. Certain materials according to the subject
 invention can provide improved precision or other advantages as compared
 to the commonly used S&S 903 cellulose filter paper as a blotting
 material. Improved precision or other such performance characteristics can
 result in superior product performance. Improving the procedure of
 measuring or detecting HbA1c so that the precision of the HbA1c assay has
 a within-run precision coefficient of variation (CV) less than 2%, as
 provided by the subject invention, can be commercially significant and
 advantageous.
 DETAILED DESCRIPTION OF THE INVENTION
 The subject invention concerns an advantageous system for detecting or
 measuring a component of interest in a biological sample. For example, the
 system according to the instant invention includes the use of a particular
 blotting material for collection of a blood specimen for assaying the
 presence or amount of a component that is present in or absent from the
 blood of a patient being tested for that component. In a preferred
 embodiment, the invention concerns the use of a particular blotting or
 "blood spotting" material in a method article of manufacture, or kit for
 determining concentrations of hemoglobin A1c in the blood of a patient. In
 a most preferred embodiment, the blotting material comprises a single
 layer of material. A plurality of distinct layers of materials can also be
 used in accordance with the subject invention. For example, certain
 laminations can be included to achieve separation of certain components,
 e.g., red blood cells, in a blood sample.
 Certain properties or characteristics of the blotting materials have shown
 unexpected improvements in biological assays as compared to commonly used
 blotting materials, e.g. S&S 903. The advantageous properties include:
 ability to absorb blood quickly and neatly, release of protein readily
 during elution, reproducibility of protein elution, spot diameter, spot
 appearance, wet strength, and ability to be punched, or "punchability".
 Several categories of blotting materials for blood specimen collection are
 available. These include: cellulose (wood or cotton derived), glass fiber,
 glass fiber/cellulose composites, nylon, modified polyester,
 polypropylene, nitrocellulose, modified polyethersulfone, polyvinylidene
 fluoride, modified natural and synthetic fibers, laminated materials, and
 screens. From more than 100 materials evaluated with regard to their
 properties for blood spot collection and recovery, we have discovered that
 the use of certain membranes or filter materials as a blotting material
 for gathering, storing and/or transporting a physiological fluid, e.g.,
 blood, can facilitate or improve the capabilities of measuring hemoglobin
 (Hb) or hemoglobin A1c (HbA1c).
 One requirement for a material that can be used in accordance with the
 subject invention is its ability to absorb blood readily and quickly. Many
 of the materials examined do not. A second property of a material used in
 accordance with the subject invention is its ability to release the
 components of interest, e.g., hemoglobin and hemoglobin A1c, that has been
 absorbed into or onto the blotting material. In a preferred embodiment,
 the material used can release the component of interest efficiently and
 precisely. That is, the material itself should minimally affect
 measurement or detection of the component of interest. In general, the
 better the precision of the hemoglobin elution, the better the precision
 of the HbA1c measurement. Thus the most favorable materials provide high
 Hb recovery, low coefficient of variation (CV) for Hb precision, and low
 coefficient of variation (CV) for HbA1c precision.
 One preferred material is a cellulose (cotton fiber) filter paper, Ahlstrom
 319, which has a low precision characteristic and a low CV for the
 hemoglobin measurement. It also gives a spot with a good appearance and is
 easy to punch after drying. The hemoglobin recovery is relatively low
 compared with other cotton fiber filter papers, but this does not seem to
 hurt the performance in the critical area of precision of HbA1c
 measurement. Another preferred material is Ahlstrom 205. Its performance
 mirrors that of Ahlstrom 319 and can give slightly higher hemoglobin
 recovery than Ahlstrom 319. It would be understood by those of ordinary
 skill in the art that the blotting material can be treated, e.g., with
 enzymes, enzyme inhibitors, or other chemicals, to achieve certain results
 in accordance with the assay being conducted. Both of these filter
 materials, Ahlstrom 319 and Ahistrom 205 are commercially available from
 Ahlstrom Filtration, Inc., Mt. Holly Springs, Pa.
 In one embodiment of the invention, the blotting material can be included
 as part of a kit for remote site blood sampling. The kit can comprise a
 blotting material having at least one designated area for placing a blood
 drop, a sterile lancet commonly used for obtaining a finger prick blood
 sample, instructions for use of the kit, and a sealable container, e.g., a
 plastic bag suitable for containing a biological sample. The preferred
 embodiment of the kit of the subject invention can include Ahlstrom 319 or
 Ahlstrom 205 as the blotting material. The kit can further comprise a
 return mailer, with instructions for transmitting the sample to the
 laboratory for analysis. Other components, as would be readily recognized
 by those of ordinary skill in the art, can also be included as part of the
 kit.
 Following are examples which illustrate procedures for practicing the
 invention. These examples should not be construed as limiting. All
 percentages are by weight and all solvent mixture proportions are by
 volume unless otherwise noted.

Example 1--Precision of S&S 903 filter paper:
 Spot-to-spot precision of S&S 903 paper was determined by spotting five
 spots of EDTA antimicrobial-coagulated blood from three patients (low,
 medium, or high percent of HbA1c) on cards made of the blotting material
 of interest, drying overnight, placing the cards in plastic bags and
 storing at room temperature, eluting the spots on the day noted and
 assaying the eluate on the Roche Cobas Mira to determined HbA1c percentage
 in the blood of the patient. The results are shown below in Table 1.
 TABLE 1
 SPOT-TO-SPOT PRECISION OF PERCENTAGE OF HbA1c
 MEASUREMENTS USING S & S 903
 Sample Result 1 2 3 4 5 Mean s.d.
 CV %
 Low day 2 6.5 6.9 6.9 6.9 6.6 6.5 0.17
 2.6
 (5-7% HbA1c) day 4 6.7 6.4 6.5 6.4 6.6 6.5 0.12
 1.8
 day 6 6.6 6.4 6.7 6.4 6.3 6.5 0.15
 2.3
 day 9 6.5 6.4 6.4 6.4 6.9 6.5 0.19
 3.0
 day 11 6.6 6.4 6.5 6.4 6.6 6.5 0.09
 1.4
 Medium day 2 8.5 8.0 8.6 8.7 8.4 8.4 0.24
 2.9
 (7-9% HbA1c) day 4 8.3 8.0 8.3 8.2 8.8 8.3 0.26
 3.2
 day 6 8.0 7.8 7.9 8.4 8.0 8.0 0.20
 2.5
 day 9 8.0 8.6 8.5 8.0 8.6 8.3 0.37
 4.3
 day 11 8.1 8.6 8.2 8.7 8.3 8.4 0.23
 2.8
 High day 2 12.4 12.6 12.0 12.4 12.5 12.4 0.20
 1.7
 (10-15% HbA1c) day 4 12.4 12.9 12.7 12.7 12.4 12.6 0.19
 1.5
 day 6 12.1 12.7 12.8 13.0 12.6 12.6 0.30
 2.4
 day 9 13.2 12.8 13.3 12.6 13.0 13.0 0.26
 2.0
 day 11 13.3 12.9 13.8 13.2 13.6 13.6 0.31
 2.4
 The median coefficient of variation (CV), which is a measurement of
 precision of the assay, is at least about 2.5% for measurement of HbA1c
 using S&S 903 filter paper as a blotting material. However, the range of
 CV's is 1.4-4.5% more than 46% of all runs have a CV in excess of 2.5%.
 These precision values were measured at HbA1c levels in the normally
 encountered range (typically 5-15% HbA1c).
 Example 2--Properties of other blotting materials:
 While S&S 903 has been utilized rather extensively for many years as a
 means to gather and transport blood samples, we have discovered certain
 materials which offer better performance in one or more properties. The
 precision of the materials studied for better performance was also
 determined by measuring the spot-to-spot variability of five spots. The
 reproducibility of the performance of a given material was determined by
 repeating a precision study several times (typically 5 or more) over a
 period of several days. The other properties of interest were recorded
 during the precision studies. Several materials have been found which
 advantageously have precision coefficients of variation (CV), at most
 below about 2.5%, and advantageously consistently below 2%. Each of the
 materials has the ability to readily absorb blood from a fingerstick.
 Table 2, below, lists the materials with performance superior to that of
 S&S 903.
 TABLE 2
 CHARACTERISTICS OF PREFERRED MATERIALS
 Hb HbA1c Precision
 Punch-
 Mfr Material Composit'n Hb CV % CV % Appearance
 ability
 S & S 470 Cellulose 210 5 1.9, 2.2, 2.5, 2.6, 0.9 Good
 Good
 S & S 740E Cellulose 250 8 2.0, 0.0, 0.0, 1.9, 2.3 Good
 Good
 Whatman C/DE 30 Cellulose 210 4 2.1, 2.2, 1.2, 1.5, 2.0 Good
 Good
 Whatman 3MMchr Cellulose 235 4 1.5, 2.1, 1.5 Good
 Good
 Whatman C-5031 Cellulose 260 4 0.8, 1.5, 1.1, 2.1 Good
 Good
 Ahlstrom 205 Cellulose 200 5 0.9, 1.1, 1.4, 1.0 Good
 Good
 Ahlstrom 319 Cellulose 150 3 1.3, 1.4, 0.9, 1.9, 1.7 Good
 Good
 Pail Hemsep-L Modified 160 5 2.0, 1.3, 2.5, 2.8 Good
 Hard
 Polyester
 Pail Loprosorb Modified 320 8 2.1, 0.9, 1.1, 2.3 Irreg
 Hard
 Fiber
 The Hb and Hb CV values are approximate averages. Precision values are
 determined from five replicate spots of 20 microliters of blood applied to
 the material with a pipet.
 All of the materials listed in Table 2 give precision performance superior
 to that of S&S 903 in the experiments performed. However, in combination
 with the other properties certain of the materials are superior in overall
 performance. For example, a cellulose blotting material sold as Ahlstrom
 319 has a low precision characteristic and the lowest CV for hemoglobin
 measurement of the cellulose tested. These characteristics can provide a
 technical and commercial advantage for HbA1c determinations by maintaining
 an HbA1c precision value consistently at 2.0% or below.