This invention relates to a method for measuring the presence and/or an amount of analyte in a sample of blood with improved accuracy and precision by reducing the effect of hematocrit on the measurement. The invention further relates to a test article useful in performing the method, and test kits comprising the test article.
It has become increasingly important to medical science to be able to quantify the chemical and biochemical components in whole blood. Such ability is important in testing for exposure to hazardous materials, intoxicants, and therapeutic drugs, and in diagnostics. In some applications, it is important for a lay person to be able to perform the test outside a laboratory environment, with rapid and accurate results. For example, diabetics must test samples of their own blood for glucose several times a day to moderate their diet and medication. The test results must be both rapidly available and accurate. Assays for glucose in either plasma or whole blood can employ either oxidative or reductive chemistries that employ either colorimetric or electrochemical detection systems. Other analytes of interest in blood include cholesterol, triglycerides, ethanol, lactic acid, beta-hydroxy butyrate, ketone bodies, and fructosamine.
Test kits for the determination of glucose and other analytes in blood are well known in the art. Such test kits usually involve a test article such as a test strip or microfluidic device impregnated, coated, deposited, or printed with one or more chemicals that react in the presence of glucose to cause a dose-dependent response that may be measured by electrochemical or optical methods, or any combination thereof. Optical measurements can include transmittance, absorbance, and reflectance methods. Electrochemical measurements can include amperometric or coulometric methods.
It is well known that variations in hematocrit between whole blood samples used in diagnostic tests can interfere with accurate measurement of an analyte. Whole blood hematocrit (abbreviated hct) is a measure of the percentage of whole blood volume occupied by the red blood cells. It is also referred to as the packed cell volume, or the proportion of red blood cells to plasma. The interference caused by the hematocrit variation can arise from at least three factors: 1) interference with the detection of an optical signal used in analyte measurement by reflectance, absorbance or scatter of light; 2) interference with the rate of the chemical reaction by obstructing the diffusion of analyte within a whole blood sample; and 3) interference by reducing the amount of fluid available in a sample for adequate rehydration of a dried reagent on a test strip or other test article. Thus some test kits of the prior art require the user to dilute the sample, or require that the red blood cells be filtered out of the sample or lysed prior to applying the sample to a test device, or are designed such that these functions are carried out by the device itself without user intervention.
One series of patents relating to reducing hematocrit interference in the colorimetric determination of glucose in blood includes U.S. Pat. Nos. 4,935,346, 5,049,487, 5,049,394, 5,179,005, and 5,304,468, all assigned to Lifescan, Inc. of Mountain View, Calif., and all incorporated herein by reference in their entireties. The method disclosed therein involves taking a reflectance reading from one surface of an inert two-sided porous matrix. The matrix is impregnated with a reagent that will interact with the analyte to produce a light-absorbing reaction product when the fluid being analyzed is applied to the first surface and migrates through the matrix to the second surface. Reflectance measurements of the second surface are made at two separate wavelengths in order to eliminate interferences, and a timing circuit is triggered by an initial decrease in reflectance by the wetting of the second surface by the fluid which passes through the inert matrix. The method does not require the separation of red blood cells from serum or plasma.
U.S. Pat. No. 5,789,255, also assigned to Lifescan, Inc. and incorporated herein by reference, is entitled “Blood Glucose Strip Having Reduced Sensitivity to Hematocrit.” This patent discloses a reagent strip comprising an anisotropic membrane having a sample side with relatively large pores and a testing side with relatively small pores, such that a test sample is applied to the sample side and passes through the membrane toward the testing side, while the relatively large red blood cells are filtered out of the blood sample. The membrane is impregnated with a testing agent comprising a component that reacts with glucose and oxygen to create hydrogen peroxide, a color indicator that reacts with the hydrogen peroxide, and an acrylic acid polymer that reduces the effect of the red blood cells on the glucose concentration measurement.
International Publication Number WO 01/57239 A2 entitled “Reagent test strip for analyte determination” and assigned to Lifescan, Inc. describes the use of hemolyzing agents to lyse the red blood cells so that separation of the cells from plasma is not necessary. As stated therein, “The reagent test strips of the subject invention are characterized by having at least one of the following components: a porous matrix, one or more members of an analyte oxidation signal producing system; and at least one hemolyzing agent.”
Japanese Kokai Patent Application No. HEI 1[1989]-262470, entitled “Dry Type Whole-Blood Analytical Element,” related to Japanese Abstract JP 01262470 entitled “Dry process total blood analytical element—used esp. for detecting a high concn. of glucose in total blood,” assigned to Fuji Photo Film Co. Ltd., discloses an element for determining a given component in blood containing erythrocytes. The element comprises a reagent layer and a porous developing layer, both layers being water permeable. One of the layers contains a substance capable of forming an optically detectable substance in the presence of an analyte. The porous developing layer develops additional liquid from red blood cells to wet the other layers of the test system. The porous developing layer contains at least 5 g/m2 of either NaCl or KCl.
A journal article by Pegg, D. E., “Red Cell Volume in glycerol/sodium chloride/water mixtures, Cryobiology, (1984), 21 (2), 234-9, discloses methods for maintaining red blood cell volume in relation to cryopreservation of cells.
It is thus one object of the invention to provide a method of analyzing a sample of whole blood for an analyte that does not require separation of the red blood cells from the whole blood sample, and a test kit and an article useful in the method.
It is yet another object of the invention to provide a method for analyzing a sample of whole blood for an analyte that reduces interferences caused by variations in hematocrit among the various blood samples analyzed, and a test kit and an article useful in the method.
It is another object of the invention to provide a method for analyzing a sample of whole blood for an analyte that does not require blood to be lysed to reduce interference by variations in hematocrit, and a test kit and an article useful in the method.
It is another object of the invention to provide a method for analyzing a sample of whole blood for an analyte that does not require blood to be diluted to reduce interference by variations in hematocrit, and a test kit and an article useful in the method.