Blood sample assay method
Inventors
Faaren, Arne Ludvig • Frantzen, Frank • Nordhei, Arne Kristian • Sundrehagen, Erling • Ørning, Lars
Assignees
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Publication Number
US-11085066-B2
Publication Date
2021-08-10
Expiration Date
Abstract
The invention provides an enzymatic method for measuring the concentration of one or more analytes in the plasma portion of a blood derived sample, containing a first and a second component, where said second component interferes with the measurement of said first component. The method includes: i) diluting the sample with a reagent mixture; ii) substantially removing blood cells; iii) using a reagent which serves to temporarily prevent reaction of the second component, to generate a blocked second component; iv) causing the selective reaction of a constituent of each analyte to directly or indirectly generate detectable reaction products, where one of the analytes is the first component; v) monitoring the detectable reaction product or products; vi) relating an amount of the detectable product or products and/or a rate of formation of the detectable product or products to the concentration of each analyte, where the concentration of at least the first component is related to a corresponding detectable reaction product by means of estimating an un-measurable (fictive) endpoint. Step iii) may be carried out at any stage up to and including step iv) but before steps v) or vi). The reagent of step iii) may be applied to the sample separately or may be included in a reagent mixture during steps i) or iv). A corresponding kit is also provided.
Core Innovation
The invention provides an enzymatic point-of-care assay kit for determining concentrations of at least three different analytes in a sample comprising HDL and non-HDL, where HDL interferes with the measurement of non-HDL if HDL is present and unblocked. The kit includes a first reagent mixture formulated to dilute the sample and a cell separation unit to generate a substantially cell free sample. A first blocking reagent temporarily and/or competitively prevents reaction of HDL, thereby producing blocked HDL so that non-HDL measurement is not compromised.
The kit further comprises a second reagent mixture including a first converting enzyme that converts a first analyte to a first detectable product, a third reagent mixture including a second converting enzyme that converts a second analyte to a second detectable product, and a fourth reagent mixture including a third converting enzyme that converts a third analyte to a third detectable product. Detectable products are generated in parallel as the converting enzymes convert their respective analytes, enabling determination of concentrations for at least three analytes from the sample while using temporary or competitive HDL blocking to mitigate interference.
The described approach includes estimating an unmeasurable or fictive enzymatic end-point via curve-fitting algorithms, such as first-order and logistic models, based on monitored product formation. It also supports multi-analyte panel correction using iterative or post-measurement computation to account for parallel conversion, and emphasizes reduced assay time with liquid reagents without calibrators, including an optional early addition of the blocking reagent during dilution.
Claims Coverage
Independent claim clm-00001 defines a multiplex kit for at least three analytes where HDL interferes with non-HDL measurement unless HDL is temporarily or competitively blocked, and where three analytes are converted by respective converting enzymes into detectable products. Dependent claims refine the kit by specifying sample types and additional computational features, including predicting an unmeasurable or fictive enzymatic end-point.
Multiplex HDL/non-HDL interference-avoiding kit
A kit for use in determining the concentrations of at least three different analytes in a sample comprising HDL and non-HDL, wherein said HDL interferes with the measurement of said non-HDL if said HDL is present and unblocked.
Dilution with cell separation to produce a substantially cell free sample
A first reagent mixture formulated to dilute said sample and a cell separation unit to generate a substantially cell free sample.
Temporary and/or competitive HDL blocking reagent
A first blocking reagent that temporarily and/or competitively prevents reaction of HDL in said sample, thereby producing blocked HDL.
Parallel converting enzymes for first, second, and third analytes
A second reagent mixture comprising a first converting enzyme that converts a first analyte to a first detectable product, a third reagent mixture comprising a second converting enzyme that converts a second analyte to a second detectable product, and a fourth reagent mixture comprising a third converting enzyme that converts a third analyte to a third detectable product.
Photometric detectability of indirect products
The kit is configured for use such that the indirect products are detectable photometrically.
Predicting an unmeasurable or fictive enzymatic end-point
The kit further includes computer-readable media with instructions for running an algorithm to predict an unmeasurable or fictive end-point of an enzymatic reaction.
Across the independent claim and its highlighted dependents, the core claim coverage centers on diluting and cell-separating a blood-containing sample, temporarily or competitively blocking HDL to prevent interference with non-HDL measurement, and converting at least three analytes in parallel using distinct converting enzymes to form detectable products. Additional claim coverage includes photometric detectability of products and computer-readable media for algorithmic prediction of an unmeasurable or fictive enzymatic end-point.
Stated Advantages
Enables determination of at least three analytes in a sample comprising HDL and non-HDL where HDL interferes with non-HDL measurement if HDL is present and unblocked, by producing blocked HDL.
Allows estimation of an unmeasurable or fictive enzymatic end-point using curve fitting algorithms and monitored product formation.
Supports multi-analyte panel correction using iterative or post-measurement computation to account for parallel conversion.
Claims extensive point-of-care assay context with reduced assay time, including a target assay time of less than 10 minutes.
Documented Applications
Enzymatic point-of-care assay for plasma analytes in whole blood using HDL blocking to enable measurement of non-HDL, including HDL and non-HDL associated cholesterol, with multi-analyte panels.
Use cases include cholesterol and lipoprotein classes and lipid and lipoprotein analyte measurements, including HDL and non-HDL associated cholesterol and related lipid analytes described in the document context.
Demonstrated use with a point-of-care apparatus and analysis on an Afinion analyzer.
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