BLOOD SAMPLE ASSAY METHOD
20170349933 · 2017-12-07
Assignee
Inventors
- Arne Ludvig Faaren (Oslo, NO)
- Frank Frantzen (Oslo, NO)
- Arne Kristian Nordhei (Oslo, NO)
- Erling Sundrehagen (Oslo, NO)
- Lars Ørning (Oslo, NO)
Cpc classification
International classification
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.
Claims
1. An enzymatic method for measuring the concentration of at least one analyte in the plasma portion of a blood-cell containing sample, wherein the sample contains at least a non-HDL and a HDL, where said HDL interferes with the measurement of said non-HDL, if said HDL is present and unblocked, said method comprising the steps of i) contacting said blood cell containing sample with a reagent mixture that dilutes the sample; ii) substantially removing blood cells to provide a substantially cell free sample; iii) contacting said sample with at least one reagent which serves to temporarily prevent reaction of said HDL, whereby to generate a blocked HDL; iv) contacting said sample with at least one reagent mixture comprising at least one converting enzyme for at least one constituent of each analyte of said at least one analyte, whereby to cause the selective reaction of a constituent of the or each analyte to directly or indirectly generate detectable reaction products, wherein the, or one of, said analytes is said non-HDL; v) monitoring said detectable reaction product or products; vi) relating an amount of said detectable product or products and/or a rate of formation of said detectable product or products to the concentration of the or each of said at least one analyte in said blood sample, wherein the concentration of at least said first component is related to a corresponding detectable reaction product by means of estimating an un-measurable (fictive) endpoint; wherein step iii) may be carried out at any stage up to and including step iv) but before steps v) or vi) and wherein the reagent of step iii) may be applied to the sample separately or may be included in at least one reagent mixture of steps i) or iv).
2. A method as claimed in claim 1 wherein said non-HDL and said HDL are lipoprotein components having a lipid constituent in common but having protein constituents which are different.
3. A method as claimed in claim 1 wherein said converting enzyme for at least one constituent is a converting enzyme for a lipid constituent of said at least one analyte, such as a converting enzyme for cholesterol, triglyceride or phospholipid.
4. A method as claimed in claim 1 wherein said at least one analyte(s) are from the group of cholesterol, triglycerides or phospholipid, in total and/or associated with a specific lipoprotein from the group consisting of VLDL, IDL, LDL, HDL and non-HDL.
5. A method as claimed in claim 4 wherein said analyte is cholesterol associated with non-HDL.
6. A method as claimed in claim 5 wherein said non-HDL is LDL cholesterol.
7. A method as claimed in claim 1 wherein the blood cell containing sample is a whole blood sample.
8. The method as claimed in claim 1 wherein steps i) and iii) are carried out before step ii).
9. The method of claim 8 wherein steps i) and iii) are carried out simultaneously.
10. The method of claim 9 wherein said reagent mixture that dilutes the sample in step i) comprises said at least one reagent which serves to temporarily prevent reaction of said HDL in step iii).
11. The method of claim 1 comprising an enzymatic method for measuring the concentrations of at least three different analytes in the plasma portion of a blood-cell containing sample, wherein the sample contains a HDL and non-HDL, where said HDL interferes with the measurement of said non-HDL if said HDL is present and unblocked, said method comprising the steps of i) contacting said blood cell containing sample with a reagent mixture that dilutes the sample; ii) substantially removing blood cells to provide a substantially cell free sample; iii) contacting said sample with at least one reagent which serves to temporarily prevent reaction of said HDL, whereby to generate a blocked HDL; iv) dividing said cell free sample into at least three portions and contacting each portion with at least one of at least three reagent mixtures comprising lipid converting enzymes, whereby to cause the selective reactions of each of the at least three different analytes to (directly or indirectly) generate respective detectable reaction products, wherein one of said analytes is said first component; v) monitoring said detectable reaction products; vi) relating an amount of said detectable products and/or a rate of formation (or consumption) of said detectable products to the concentration of said at least three different analytes in said blood sample, wherein the concentration of at least said first component is related to a corresponding detectable reaction product by means of estimating an unmeasurable (fictive) endpoint; wherein step iii) may be carried out at any stage up to and including step iv) but before steps v) or vi) and wherein the reagent of step iii) may be applied to the sample separately or may be included in at least one reagent mixture of steps i) or iv).
12. A method as claimed in claim 1 wherein the method comprises determining a lipid profile comprising the concentrations of; cholesterol associated with at least one specific lipoprotein, as said non-HDL and a first analyte; total cholesterol as a second analyte; and total triglycerides as a third analyte, in the plasma portion of said blood cell containing sample.
13. A method as claimed in claim 12 wherein step iv) comprises; iv) contacting said cell free sample or a portion thereof with at least one reagent mixture comprising lipid converting enzymes, wherein said at least one reagent mixture reacts in the presence of said blocked HDL to cause the at least partially selective reaction of cholesterol associated with a specific lipoprotein or specific group of lipoproteins, such as LDL or non-HDL.
14. A method as claimed in claim 12 wherein step iv) comprises; iv) contacting a first portion of said cell free sample with at least one reagent mixture comprising lipid converting enzymes, wherein said at least one reaction mixture reacts in the presence of said blocked HDL to cause the at least partially selective reaction of cholesterol associated with a specific lipoprotein or specific group of lipoproteins, such as LDL or non-HDL; contacting a second portion of said cell free sample with at least one reagent mixture comprising lipid converting enzymes, wherein said at least one reaction mixture reacts with the total cholesterol in said second portion; and contacting a third portion of said cell free sample with at least one reagent mixture comprising lipid converting enzymes, wherein said at least one reaction mixture reacts with the total triglycerides in said third portion; and concomitantly generating detectable reaction products corresponding to each of said non-HDL (such as non-HDL cholesterol or LDL-cholesterol), total cholesterol and total triglycerides.
15. The method as claimed in claim 1 wherein said non-HDL is a lipoprotein having a lipid constituent (e.g. cholesterol) and a protein constituent and said HDL comprises the same lipid constituent associated with proteins other than said protein constituent of said non-HDL.
16. The method as claimed in claim 1 wherein the presence or generation of said blocked HDL in step iii) does not interfere with the reaction of any of said analytes or analyte constituents in step iv).
17. A method as claimed in claim 1 wherein said detectable products are detectable photometrically.
18. A method as claimed in claim 1 wherein the dilution of the blood sample is at least 10-fold.
19. A method as claimed in claim 1 wherein said substantially cell-free sample contains less than 5% blood cells.
20. A method as claimed in claim 1 wherein step ii) is carried out by filtration.
21. A method as claimed in claim 1 wherein step ii) is carried out without substantially causing cell lysis, such as by causing lysis of no more than 5% of blood cells.
22. A method as claimed in claim 1 wherein step ii) is carried out without substantially affecting plasma lipid components, such as by decreasing any of said components by not more than 5%.
23. A method as claimed in claim 1 wherein said at least one reagent which serves to temporarily prevent reaction of said HDL comprises at least one antibody specific for said HDL over said non-HDL.
24. A method as claimed in claim 1 wherein said at least one reagent which serves to temporarily prevent reaction of said HDL comprises at least one specific binder, such as at least one antibody, which is specific for a lipoprotein constituent of said HDL.
25. A method as claimed in claim 1 requiring no more than 20 L of blood.
26. A method as claimed in claim 1 wherein a suitable algorithm is used to determine the un-measurable end-point of at least said first component.
27. A method as claimed in claim 26 wherein said algorithm is a first order rate equation or logistic function.
28. A method as claimed in claim 1 wherein said un-measurable endpoint cannot be measured because of an actual or potential unblocking of said blocked HDL.
29. A method as claimed in claim 1 for the measurement of a plurality of analytes.
30. A method as claimed in claim 29 wherein the concentration of each analyte is related to the formation or consumption of a corresponding detectable product by means of estimating an endpoint, and wherein at least one of the endpoints is an unmeasurable (fictive) endpoint.
31. A kit for use in determining the concentration of at least three different analytes in the plasma portion of a blood-cell containing sample, wherein the sample contains a HDL and non-HDL, where said HDL interferes with the measurement of said non-HDL if said HDL is present and unblocked, said kit comprising; a) a first reagent mixture formulated to dilute said sample; b) a cell separation unit; c) a second reagent which serves to temporarily and/or competitively prevent reaction of (block) said HDL, whereby to generate a blocked HDL; d) at least three further reagent mixtures formulated to cause the selective conversion of said at least three different analytes, whereby to generate detectable indirect products corresponding to each analyte.
32. A kit as claimed in claim 31 wherein reagent c) is comprised within reagent mixture a).
33. A kit as claimed in claim 31 wherein said at least three analytes comprise total cholesterol, total triglycerides and non-HDL associated cholesterol.
Description
DETAILED DESCRIPTION OF THE INVENTION
[0064] The present invention provides for an assay method in which the concentration of at least one analyte in a blood-cell containing sample is measured.
[0065] As used herein, the term analyte is used to indicate a component or group of components whose measurement is desired. Analytes may thus be a single component, such as HDL cholesterol, or may be a set of components. Typically such a set will have a common feature, such as the set of all cholesterol containing lipoproteins.
[0066] Among the most preferred analytes for use in the present invention are lipid components, which is to say the total amount of a particular type of lipid (e.g. TG or CH) in the plasma portion of the sample or that part of a particular type of lipid present as a particular lipoprotein (e.g. HDL CH) or a particular set of lipoproteins (e.g. non-HDL CH).
[0067] The use of end-point estimation is useful in assays for a single component, where the end-point of the conversion of a particular constituent of that component (e.g. conversion of the lipid constituent of HDL) will be estimated by the methods indicated herein. The technique is, however, also highly valuable in parallel assays for multiple analytes (often termed panel assays). This applies particularly at the point-of-care. In such cases, two, three, four, five or more analytes may be measured in the assay. Furthermore, the instrument may be able to calculate the level of other analytes from the measured data and thus may be able to report on more components than are measured directly (see the discussions herein for example). In the panel assay embodiment of the invention it is preferable that at least three analytes are measured.
[0068] Where at least two (e.g. at least three) analytes are measured, end-point estimation methods will be used for analysis of one or more of such analytes. Where at least one analyte is a first component which cannot conveniently/reliably be measured without blocking of another component then this first component will be analysed by estimation of an immeasurable (fictive) endpoint. In combination with this method, however, other components that could be measured by end-point methods may also be measured by end-point estimation in order to reduce the time required for the total assay. Thus in one embodiment all analytes are measured by end-point estimation methods with at least one analyte being measured by estimation of an unmeasurable (fictive) endpoint.
[0069] As indicated above, preferred analytes are lipid components of the blood sample. Typical analytes will thus be cholesterol, triglycerides or phospholipid, in total and/or associated with a specific lipoprotein from the group consisting of VLDL, IDL, LDL and HDL. Cholesterol, triglycerides and/or phospholipid associated with groups of lipoproteins (e.g. non-HDL CH) may also be measured and form further preferred analytes.
[0070] The first component referred to herein will typically be a particular lipid associated with a particular lipoprotein (or group of lipoproteins) such as HDL_CH or non-HDL-CH. Thus, typically said first component is one analyte having a lipid constituent selected from cholesterol, triglycerides or phospholipid associated with at least one lipoprotein constituent selected from VLDL, IDL, LDL and HDL, such as cholesterol associated with HDL.
[0071] The interference caused between the second component and the measurement of the first component as indicated herein will generally be due to the need to distinguish the reaction of the lipid constituent of the first component from that of the second. Typically, therefore, the first and second components will comprise the same lipid constituent(s). In a typical example, it may be desired to measure HDL-CH (first component and analyte) in the presence of non-HDL-CH (second component). This cannot easily be done without blocking of the second component because the lipid constituents are both CH and thus if both are allowed to react then it is not possible to distinguish between them. In such situations the second component is blocked so that only or primarily the first component is available for reaction.
[0072] There are many methods by which components (especially the second component as discussed herein) may be blocked. Many of these methods are discussed herein and others are known to the skilled worker. If such a blocking is or can be made permanent (e.g. by some covalent attachment of a blocking moiety) then end-point estimation is not necessary (although it may still be an advantage in order to speed the assay method). However, techniques currently know and/or widely used are temporary blocking methods, which is to say the second component is rendered fully or largely uncreative but re-gains this reactivity either independently or in the presence of the enzymatic conversion reagents and/or surfactants employed in step iv) of the processes described herein. As used herein, a reagent that serves to temporarily prevent reaction of a component will prevent significant reaction of that component under appropriate conditions (e.g. the conditions of step iv)) for at least 60 seconds, preferably at least 240 seconds and most preferably at least 360 seconds. By significant reaction is indicated reaction of more than 10%, preferably more than 5% and most preferably reaction of more than 3% of any constituent (e.g. the lipid part) of that component.
[0073] Correspondingly, a blocked component will not undergo reaction of its lipid constituent to an extent of more than 10%, preferably 5%, more preferably 3% over a period of at least 60 seconds, preferably at least 240 seconds, more preferably at least 360 seconds when subjected to reactive conditions, such as the conditions of step iv) as described in any embodiment herein.
[0074] In preferred embodiments, the conditions of steps i), ii) and/or iii) are chosen such that the cells of the blood sample remain intact, or substantially intact (e.g. less than 10% of the cells, preferably less than 5%, and most preferably less than 3% (e.g. 0.1 to 3%) of the cells suffer lysis during steps i)-iii). Factors including ionic strength and surfactant type/concentration are particularly important in this respect.
[0075] Optionally, reaction accelerators such as disclosed in U.S. Pat. No. 6,818,414 may be included in the reagent solution, especially at step iv) to increase reaction rate and thus decrease assay time.
[0076] In the present invention, the method and other aspects relate to the measurement of at least one analyte. Such analytes will typically be lipid analytes and each independently may the total amount of a certain lipid component such as triglyceride, cholesterol, etc or may be a lipid component associated with a specific lipoprotein or a group of specific lipoproteins. Non-exhaustive examples of these include all combinations of lipid components cholesterol, triglycerides or phospholipid with any of lipoproteins VLDL, IDL, LDL and HDL, as well as the corresponding parts of each total lipid component not associated with such lipoproteing. Thus, for example, analytes include HDL cholesterol and non-HDL cholesterol, the latter being that part of the total cholesterol not bound to the HDL lipoprotein. Particularly preferred analytes include total TG, total CH, nonHDL, LDL, small dense LDL and HDL cholesterol.
[0077] In order to measure the parts of certain lipid components associated with specific lipoproteins, it is necessary to block or render uncreative either that component or all other lipoproteins having the same lipid component. This allows measurement of one component in the presence of another which would normally interfere.
[0078] Although this blocking is in itself known and many examples are described herein below, the known methods for achieving this do not result in a permanent blockage or do not hold the lipid component permanently. Rather, after a certain period and/or if the concentration of the lipid component elsewhere in the sample falls too low then the blockage will begin to fail and the lipid component will become reactive. Thus, in such situations, a reaction consuming the same lipid constituent as is constituted in a blocked component cannot be run to its endpoint because the blockage would become ineffective before that point was reached. Such endpoints are described herein as unreachable, unmeasureable or fictive to reflect the fact that they can never in fact be reached.
[0079] In view of the above, has been assumed in the art that endpoint type assays cannot be used with blocked lipid components of this type because the endpoint is unmeasureable. This has resulted in the need to use calibration or more commonly dried reagents in such assays with all of the disadvantages associated with those (as known in the art and as described herein).
[0080] However, the present inventors have now established that end-point estimation techniques can be effectively used even on such unmeasurable endpoints and so that unreachable or unmeasureable (fictive) endpoint can still be calculated even if it can never be reached in practice.
[0081] With regard to the selective reaction of specific class of lipid or specific class of lipoprotein, and the conversion of the lipid constituent of a lipoprotein to detectable secondary analyte, there are a number of methods which are well known in the art and any of these are suitable for use in the present invention. All of the homogeneous methods described above are suitable and included within the scope of the invention. Further details arc supplied below and in the referenced citations.
[0082] Two methods arc currently used routinely for quantification of CH. Both methods are enzymatic and suitable for use in the method of the present invention. In the first method, cholesterol esterase converts cholesterol esters into CH. Cholesterol oxidase then converts CH to choleste-4-ene-3-one and hydrogen peroxide. Finally hydrogen peroxidase uses the hydrogen peroxide formed to convert 4-aminoantipyrin in the presence of phenol to generate a coloured quinoneimine compound. The quinoneimine is monitored by photometry at 500-600 nm wavelength. Other well known colour producing substrates (e.g. TMB which product is blue and monitored at 650 nm) or fluorescent or chemiluminescent substrates may be substituted for the 4-aminoantipyrin/phenol.
[0083] The other method substitutes cholesterol dehydrogenase for cholesterol oxidase and determines the amount of CH in the sample on the basis of the amount of the produced NADH or NADPH.
[0084] With regard to the detection of TG, this is determined routinely in a four step enzymatic reaction, in which lipoprotein lipase hydrolyzes TG to unesterified glycerol and free fatty acids. The glycerol is then phosphorylated by glycerokinase and oxidized by glycerol-3-phosphate oxidase to di-hydroxy-acetone-phosphate and hydrogen peroxide. In a final colour forming step hydrogen peroxidase uses the hydrogen peroxide formed to convert 4-aminoantipyrin in the presence of phenol into a coloured quinoneimine. The quinoneimine is monitored spectrophotometrically at 500 nm wavelength. By selecting the appropriate substrates and phenols the formed coloured products may be monitored at wavelengths from 450 to 850 nm. Likewise fluorescent or chemiluminescent substrates may be substituted for the 4-aminoantipyrin. Evidently, the final steps in both peroxide-based methods are equivalent and interchangable.
[0085] Thus, the enzyme reactions converting the specific plasma lipid component into a detectable chemical product preferably may be performed using the above described enzyme systems.
[0086] For CH:
[0087] (i) cholesterol esterase+cholesterol oxidase+peroxidase, or
[0088] (ii) cholesterol esterase+cholesterol dehydrogenase.
[0089] For TG:
[0090] (iii) lipase+glycerol kinase+glycerol-3-phosphate oxidase+peroxidase.
[0091] As enzymes, commercially available enzymes derived from animals, micro-organisms or plants may be used. Enzymes from specific sources may display selectivity for certain lipoprotein classes, such as lipoprotein lipase and cholesterol esterase from Chromobacterium viscosum or Pseudomonas which react preferentially with the lipoprotein class VLDL. Such enzymes may be used to assay for a specific component, either in isolation or in combination with the other selection methods described herein. The enzymes may be chemically modified so as to change their specificity and stability, e.g conjugation of cholesterol oxidase and cholesterol esterase with PEG in order to make the enzymes less reactive with LDL associated CH (U.S. Pat. No. 5,807,696). Enzymes are typically used at concentrations from 100-100,000 U/L.
[0092] Measurement of lipid components of specific lipoprotein classes may be performed by several methods that exploit different chemical and physical characteristics of the different lipoprotein classes. In general, these methods rely on the specificity of the enzyme, and/or allow the reaction of the desired component after separating, converting or rendering inactive those other components which might interfere.
[0093] Non-ionic, anionic, cationic, and zwitterionic surfactants may be included in order to increase the selectivity of the enzymatic reactions or to increase the rate of reactions. Any suitable surfactant may be used that allows maintaining the intact status of the cells during the course of the reactions. One property of non-ionic surfactants of particular importance for the status of cells, is the hydrophil-lipophil balance (HLB). Surfactants with HLB values below 10 and above 13 are in particular suitable for use in the presence of intact cells. However, surfactants with HLB values between 10 and 13 may also be compatible with intact cells depending on the concentrations used, and depending on the construction and formulation of the assay, such as reaction times and temperatures used, ion strength, pH and types of salts used in the assay mixture, and the presence of stabilizing substances such as serum albumin. Examples of suitable surfactants are polyoxyethylene alkyl ethers (Brij 35 and 78), polyoxyethylene alkyl aryl ethers (Triton X45 and X305, Igepal 210 and 272), polyoxyethylene sorbitan monolaurate monolaurate (Tween 80), polyoxyethylene-cooxypropylene block copolymer (Pluronic F68 and L64), telomere B monoether with polyethylene glycol (Zonyl FSN 100), ethylenediamine alkoxalate block copolymer (Tetronic 1307), 2,4,7,9-tetramethyl-5-decyne-4,7-diol ethoxylate (Surfonyl 465 and 485), polydimethylsiloxane methylethoxylate (Silwet L7600), polyethoxylated oleyl alcohol (Rhodasurf ON-870), polyethoxylated castor oil (Cremophor EL), p-isononylphenoxy-poly(glycidol) (Surfactant 10G), and a polyether sulfonate (Triton X200). Surfactants are used for this purpose typically at concentrations of 0.001-10%, preferably 0.01 to 1%.
[0094] The following methods are among those suitable for selectively reacting the particular lipoprotein components as described. In all cases, the referenced published material is incorporated herein by reference.
[0095] In homogenous methods to measure the HDL associated CH fraction, the non-HDL particles have been blocked from reaction by different methods and rendered inaccessible to the CH metabolizing enzymes. The most recent development has been specific surfactants that selectively dissolve HDL. These include:
[0096] (i) In the PEG/cyclodextrin method (U.S. Pat. No. 5,691,159) sulfated alpha-cyclodextrins in the presence of Mg.sup.2+ forms soluble complexes with non-HDL which thereby become refractory to breakdown by PEG modified enzymes.
[0097] (ii) The polyanion method (U.S. Pat. No. 5,773,304) uses a synthetic polymer together with a polyanion, or a surfactant (U.S. Pat. No. 7,851,174) to block non-HDL and make them refractory to solubilization with specific detergents and enzymatic measurement.
[0098] (iii) The immunologic method (U.S. Pat. No. 6,162,607) exploits the presence of apolipoprotein B in all non-HDL and its absence in HDL. Antibodies to apoB block non-HDL for reaction with the cholesterol enzymes.
[0099] (iv) In the clearance method (U.S. Pat. No. 6,479,249) non-HDL is first consumed in a reaction not generating colour. A specific detergent is then added allowing the enzymes to react with HDL in a reaction generating colour.
[0100] (v) The accelerator/detergent method (U.S. Pat. No. 6,818,414) degrades unesterified cholesterol of non-HDL using an accelerator to speed up the reaction, and removes the formed H2O2 in a process not generating colour. In a second step HDL cholesterol is degraded using an HDL-specific detergent in a colour forming process.
[0101] Any of these methods, either individually or in combination may be applied to the measurement of HDL associated CH in the method of the present invention. LDL associated CH has been measured by homogenous methods which did not require a physical separation of non-LDL lipoproteins to measure the LDL CH fraction. Such methods include:
[0102] (i) U.S. Pat. No. 5,588,8827 describes a method whereby non-LDL is masked by surfactants and cyclodextrins in the presence of Mg.sup.2+ and becomes refractory to breakdown by PEG modified enzymes.
[0103] (ii) U.S. Pat. No. 5,925,534 describes a method using polyanions and surfactants to protect LDL in a sample and allow non-LDL to be enzymatically eliminated whereupon addition of a deprotecting reagent allows the enzymatic determination of LDL associated CH.
[0104] (iii) U.S. Pat. No. 6,057,118 and U.S. Pat. No. 6,194,164 describe two different methods utilizing specific surfactants to selectively eliminate non-LDL associated CH in an enzymatic reaction before determining LDL associated CH.
[0105] During recent years homogenous methods for determining VLDL associated CH have been developed. These include:
[0106] (i) U.S. Pat. No. 6,986,998 describes a method using albumin and calixarene to block HDL and LDL, respectively, allowing the selective decomposition of VLDL in an enzymatic reaction using VLDL selective enzymes from Chromobacterium viscosum.
[0107] (ii) U.S. Pat. No. 7,208,287 describes a method using specific surfactants to selectively decompose VLDL in an enzymatic reaction.
[0108] Cholesterol associated with IDL (also called VLDL remnants or remnant-like particles) is commonly determined using ultracentrifugation, high performance liquid chromatography or electrophoresis. Two homogenous methods were recently developed (U.S. Pat. No. 7,272,047 and US 2007/0161068) that use specific surfactants to further the selective enzymatic decomposition of IDL associated cholesterol. Any of these methods, either individually or in combination may be applied to the measurement of LDL associated CH in the method of the present invention.
[0109] Measurement of TG of specific lipoprotein classes may be performed by methods that exploit different chemical and physical characteristics of the different lipoprotein classes. These methods are thus analogous to those described above for cholesterol, but utilising enzymatic detection of TG, such as by those methods described herein above. Such methods include:
[0110] (i) U.S. Pat. No. 6,811,994 describes using selective surfactants and polyethylene glycol modified enzymes to block lipoproteins other than the particular lipoprotein.
[0111] (ii) WO2004/087945 and US2009/0226944 describe using selective surfactants to remove TG from nonLDL in a reaction not producing detectable products then converting TG associated with LDL into detectable products.
[0112] (iii) WO2000/06112 describes using selective surfactants to remove TG from lipoprotein other than VLDL and/or IDL in a reaction not producing detectable products then converting TG associated with VLDL and/or IDL into detectable products.
[0113] Any of these methods, either individually or in combination may be applied to the measurement of TG associated with specific lipoproteins in the method of the present invention.
[0114] The detection method used in the assay methods of the present invention is typically photometric, and the indirect product is generally chosen such that it is detectable photometrically (e.g. by its absorbance, fluorescence or chemiluminescence at one or more pre-identified wavelengths).
[0115] A highly preferred method for signal generation is via hydrogen peroxide, which serves as a substrate for the enzymatic oxidation of a colour-producing substance. The oxidizable color producing reagent or reagents that react with formed hydrogen peroxide to produce the detectable chemical product may be any molecule known in the art, the oxidized product of which can be measured by ultraviolet, visual, or infra-red spectroscopy, or by fluorescence or luminescence.
[0116] Examples of suitable chromogenic reagents are Trinder reagents, which in the presence of H.sub.2O.sub.2 react with a coupler to form colored products. Preferred examples of couplers are 4-aminoantipyrin (4AA), 3-methyl-2-benzolinone hydrazone (MBTH), N-methyl-N-phenyl-4-aminoaniline (NCP-04), N-methyl-N-(3-methylphenyl)-4-aminoaniline (NCP-05), N-methyl-N-(3-methoxyphenyl)-4-aminoaniline (NCP-06), N-methyl-N-(3-methoxyphenyl)-3-methoxy-4-aminoaniline (NCP-07). Preferred examples of Trinder reagents are those forming products that can be colorimetrically determined at wavelengths at or above 600 nm: N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethyloxyaniline (DAOS), N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxy-4-fluoroaniline (FDAOS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N,N-bis(4-sulfobutyl)-3,5-dimethylaniline (MADB), N-ethyl-N-(2-hydroxy-3-sulfoprpyl)-3,5-dimethylaniline MAOS). Preferred examples that are not Trinder reagents are: 3,3,5,5-Tetramethylbenzidine (TMB), N-(3-sulfopropyl)-3,3,5,5-tetramethylbenzidine (TMBZ-PS), N,N-bis(2-hydroxy-3-sulfopropyl)tolidine (SAT Blue), N-(carboxymethyl-aminocarbonyl)-4,4-bis(dimethylamino)-biphenyl amine (DA64), 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenotiazine (DA67). The concentration of the chromogen is preferably 0.01-10 g/L, and is limited by solubility.
[0117] Examples of suitable fluorescent substrates are dihydrocalceins, dihydroethidium, dihydrofluoresceins, dihydrophenoxazine (Amplex red;10-acetyl-3,7-dihydroxyphenoxazine), and dihydrorhodamines.
[0118] Examples of suitable chemiluminescent substrates are luminol (3-aminotriphenylene complexes), Lumigen PS-2 and Lumigen PS-atto.
[0119] Non-ionic, anionic, cationic, and zwitterionic surfactants may be used for the purpose of cell lysis prior to measurement of colored indirect analyte. Any suitable surfactant may be used that does not affect the measurement, typically at concentrations between 0.01-10%, but preferably between 0.1-2%.
[0120] Examples of suitable surfactants are the anionic surfactants amine aklylbenzene sulfonate (Ninate 411), sodium dioctylsulfosuccinate (Aerosol OT), sodium N-oleyl-N-methyltaurate (Geropon T-77), sodium olefin sulfonate (Bioterge AS-40), sodium polyoxyethylene lauryl sulphate (Standapol ES-1), and the non-ionic surfactants polyoxyethylene alkyl aryl ether (Triton X100 and X114) and polyoxyethylene lauryl alcohol (Chemal LA-9).
[0121] Where cell lysis has occurred, there will evidently be a corresponding release of haemoglobin and other cell products into the reaction medium. In order to gain maximum advantage from the lysis and reduce the background signal as far as possible, it is thus preferred in such cases to select a product (secondary analyte) for which detection is not inhibited by these released products. Secondary analytes detectable at wavelengths above 450 nm, preferably above 500 nm and most preferably 600 nm or higher (e.g. 500 to 1400 nm or 600 to 1200 nm) are thus preferred where cell lysis is used.
[0122] Physical separation of blood cells in the sample may be performed by any suitable method including flow past a specific binder for the whole cells, but will most commonly be by filtration. It is notable that filtration to remove cells following reaction step (i) has considerable advantages over filtration of the original blood sample. In particular, the reaction step can be carried out with a small volume of blood sample from a finger-stick. After one or more reagents have been added, the volume of the reaction mixture is much greater and separation of the cells can be conducted without the dead-volume being significant. The cells are also more dilute, whereby the cells upon filtration will spread more evenly over the filter surface and the filtration becomes more effective.
[0123] Filtration as described herein is thus a preferred technique for use in setp ii) of the methods of the present invention and corresponding filters etc are suitable for supply with the kits of the invention. Some preferred embodiments of such filtration are provided below, any of which may be applied individually or in any combination to the methods and kits of the invention.
[0124] In filtering, the sample is passed through a porous interface (filter, membrane or sieve) of small pore size, in which the cells are trapped. The porous interface may be solid (e.g. sintered glass) or fibrous (e.g. made of cellulose or glass), and the flow may be transversely through or laterally. The sample may be passed through the filter by gravity, centrifugation, capillary forces, pressure or suction. The filtering material may additionally contain reagents (e.g. lectins, antibodies) that capture the blood cells. Other methods use electrostatic attractions.
[0125] Suitable materials for transverse filtering are given in Table 2. Suitable materials for lateral flow filtering are Hemasep L (Pall Corp) and LF1 and MF1 (Whatman).
TABLE-US-00001 TABLE 2 Filtration recovery of lipids and colored product TG CH HDL Filter type (%) (%) (%) No filtration 100 100 100 PAD 901 99 95 97 PAD Whatman 98 92 94 QA Pall 102 96 97 Whatman grade 103 99 97 CF/CM30 Gelman ITLC SG 103 96 99 Gelman /E 103 98 98 Munktell filter 101 97 98 paper
[0126] Mathematical treatment of enzyme progress curves based on absorbance readings in order to calculate enzyme activities is well-known. Such mathematical procedures, algorithms are based on the integrated Michelis-Menten equation (London J W (1977) Analytical Chemistry 48:1716-9). However, the purpose of such algorithms is to determine enzyme activities by extrapolating the rate to zero time, they are not applicable to the determination of product end-points.
[0127] Recently, one group reported that using a combination of fixed time and kinetic measurements allowed the reading of the progress curve to be terminated when it had reached 90-95% of the predicted end-point, rather than following it to the end. This allowed for a substantially shortened assay time (Kvam (2009) Point of Care 8:16-20).
[0128] What the inventors have now surprisingly found is that in reactions without a measureable end-point it is still possible to estimate a fictive end-point which is a direct representative of the true, non-measurable end-point (
[0129] The algorithm used to predict the unmeasurable or fictive end-point in the various aspects of the present invention can be any mathematical equation that adequately describes the reaction progress, and might be used to describe the curve formed when such progress is plotted. Many such equations will be known to those of skill in the art. Usually the algorithm will be one based upon a first order rate equation (e.g. Y=Y.sub.0*e(kX)) or a logistic function (e.g. Y=1/(1+e(X))) (Table 3). Which algorithm will be most preferable for a particular situation will depend on many factors such as how the measurement is performed (transmission or absorbance), reaction conditions (e.g. reaction temperature), as well as measurement interval and other factors. It will, however, be a routine matter to fit the curves from each suitable equation to the experimental data and thereby determine the closest fit in each case. Different instrument platforms may thus favour different curve fitting algorithms but a suitable algorithm in each case will be rapidly established. In many cases more than one algorithm will give acceptable results.
TABLE-US-00002 TABLE 3 Some Examples of reaction-curve fitting algorithms Type Equation 1st order Y = Y.sub.max + (Y.sub.0 Y.sub.max)*e{circumflex over ()}(kX) (Exponential association/ dissociation) 1st order-2 Y = Y.sub.max Y.sub.max*e{circumflex over ()}(k(X X.sub.0)) 1st order-3 Y = Y.sub.max + (Y.sub.0 Y.sub.max)*e{circumflex over ()}(kX) + X*C Logistic-1 Y = Y.sub.0 + (Y.sub.max Y.sub.0)/(1 + 10{circumflex over ()}(logX.sub.50 logX)*B (Sigmoidal) Logistic-2 Y = Y.sub.max + (Y.sub.0 Y.sub.max)/(1 + (X/X.sub.50).sup.B (4 parameter logistic) Logistic-3 Y = Y.sub.0 + (Y.sub.max Y.sub.0)/(1 + (X/X.sub.50).sup.B Logistic-4 Y = Y.sub.max + (Y.sub.0 Y.sub.max)/(1 + (X/X.sub.50).sup.B + X*C Y.sub.0, Y at X = 0; Y.sub.max, Y at X = infinite; k, rate constant; X.sub.50, X at half maximal Y; B, slope factor, steepness of slope; C, slope factor of parallel reaction..
[0130] When estimating an unmeasurable end-point, the measurement interval should preferably be chosen such that a minimum of parallel reactions are occurring. However, it is not a strict requirement that parallel reactions cannot be present. The influence of such parallel reactions may be corrected for in a post-measurement analysis. This is particuarly true where at least two analytes are measured in the sample and a known (absolute or approximate) relationship is present between at least two of said analytes.
[0131] For instance, in the HDL assay the estimated unmeasureable end-point may be influenced by a concomitant conversion of nonHDL, also when nonHDL has been rendered temporarily unreactive by blocking reagents depending on different factors such as (i) the measurement interval, (ii) reaction temperature, (iii) HDL/nonHDL ratio. In a lipid-panel assay this may be corrected for in an iterative process involving HDL the simultaneously measured total cholesterol and a calibration curve for nonHDL, determined in advance and placed in the information (e.g. barcode) supplied for the reagent, in the same way as done with the HDL calibration curve. An example of how a post-analysis correction may be carried out is described in Example 6.
[0132] It would also be possible to take into account parallel reactions occurring in the algorithm used to estimate the unmeasurable end-point. For instance the reaction conditions may be formulated such that parallel reactions are essentially linear during the measurement interval, and can be represented in the algorithm by a linear term (Table 3).
[0133] One of the considerable advantages of the present assay method is the resultant decrease in the assay time. It is thus preferable that the time from adding the reagent in step i) to end of the the monitoring of detectable reaction products in step v) is no more than 10 minutes, more preferably no greater than 8 minutes, and most preferably in the range 5 to 7 minutes.
[0134] It is a further advantage of the present invention that it may be carried out on a minimal volume of blood. It is preferred, therefore that the volume of whole blood sample in step i) of the assay method is no greater than 40 L, more preferably no greater than 20 L and most preferably in the range of 10 to 15 L.
[0135] The present inventors have additionally established that further reaction time may be saved by the early contact of the sample with the blocking reagent (the reagent which serves to temporarily prevent reaction of said second component). This blocking reagent (many of which are described herein) typically takes some time (several minutes) to react with the second component. Thus, a typical point-of-care assay in which this blocking reagent was added at or around step iv) of the method would then be delayed by several minutes to allow reaction of this blocking reagent.
[0136] The present inventors have now established that it is possible to add a suitable blocking reagent to the cell-containing blood sample (e.g. whole blood sample) without significantly lysing the cells such that this reagent can be reacting with the second component of the sample during the cell separation steps(s). Evidently, this reduces the need to wait for the blocking reaction and can reduce the assay time by around 30 seconds, preferably by around 1 minute. In this embodiment, the blocking reagent will typically be included in the dilution mixture added at step i), or may alternatively be added after dilution, either before cell separation or after that cell separation process. In any case, if the blocking reagent can be added earlier than previously then assay time can be saved.
[0137] It is important in multi-analyte (panel) assays that where the blocking reagent is added before the sample is divided into portions for specific reaction, the blocking reagent should not interfere with detection of the various analytes. Where then analytes are HDL-CH, total-CH and total-TG, for example, the blocking reagent may be added at any of steps i) to iii) without intereference because the reagent causes blocking of non-HDL-CH (the second component) and thus allows specific reaction of HDL-CH. With regard to the total-CH and total-TG, although the blocking reagent will be present, the reagent mixture for specific reaction of these need only contain a suitable surfactant to release the total CH or total TG in spite of the block. Such surfactants will be known and easily selected by the skilled worker and so no interference with total-CH or total TG need take place.
[0138] In the kits of the present invention, the first reagent mixture is preferably formulated together with the first reagent mixture of the HDL reaction as a single reagent mixture. This reduces the number of steps, and thus reduces the assay time, and furthermore reduces the reagent storage and handling demands on the assay equipment, allowing the method to be practiced with less sophisticated automatic analysers.
[0139] The kit may additionally contain a lysing agent to cause cell lysis allowing determining the hematocrit.
[0140] The invention will now be further illustrated by the following non-limiting examples, and the attached Figures, in which:
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EXAMPLES
Example 1
Estimation of a Fictive End Measurement of HDL Associated Cholesterol in the Presence of Cholesterol Associated With Non-HDL
[0149] A sample containing 66 mg/dL HDL, 255 mg/dL LDL and 300 mg/dL TG was analyzed on a Cobas Mira plus instrument (ABX Diagnostics) using Wako HDL-C L-type reagents and protocol (Table 4). The absorbance was monitored at 600 nm (
Y=Ymax(1e.sup.(-K(X-X0))),
which coincided with the true end-point of a calibrator containing only HDL of almost the same concentration, 64 mg/dL versus 66 mg/dL. Other well known algorithms for following the kinetics of enzymatic reactions could be used in an analogous way.
TABLE-US-00003 TABLE 4 Wako HDL-C L-type reagent and protocol. R1 Pretreatment 5 minutes 37 C. R2 Incubation 5 minutes 37 C. R1 pretreatment Good's buffer, pH 7.0 30 mmol/L 4-aminoantipyrine 0.9 mmol/L peroxidase 2400 U/L ascorbate oxidase 2700 U/L anti-human -lipoprotein antibody R2 enzyme reagent Good's buffer, pH 7.0 30 mmol/L cholesterol esterase 4000 U/L cholesterol oxidase 20000 U/L F-DAOS 0.8 mmol/L
Example 2
Comparison of End-Point, Fix-Point and Estimated End-Point Measurements in the TG Assay
[0150] Roche TG reagent (Table 5) was stored at 25 C. and samples were taken out at different time points and used to measure a plasma sample containing 186 mg/dL triglyceride. The plasma sample was stored in aliquots frozen at 40 C. and thawed prior to measurement. The measurements were performed on a LabSystems Multiscan RC plate reader at ambient temperature (20-22 C.). The progress curves were followed until reaction completion and the end-points determined. In addition, the end-points were estimated based upon the first 300 s of the progress curves using a 4 parameter logistic function. The obtained estimated end-points were compared to true end-points and to the absorbance at the end of the time interval used for the estimation, 300 s. As seen in
TABLE-US-00004 TABLE 5 Roche triglyceride GPO-PAP reagent buffer Pipes 50 mmol/L, pH 6.8, containing 40 mmol/L Mg2+, sodium cholate 0.20 mmol/L, and 1 mol/L potassium hexacyanoferrate (II) ATP 1.4 mmol/L 4-aminoantipyrine 0.13 mmol/L 4-chlorophenol 4.7 mmol/L fatty alcohol 0.65% polyglycol ether lipoprotein lipase 5000 U/L (from Pseudomonas spec.) glycerokinase 190 U/L (from Bacillus stearothermoohilus) glycerol phosphate 2500 U/L (from E. coli) oxidase peroxidase 100 U/L (from horseradish)
Example 3
End-Point Estimation of the Roche HDL Assay
[0151] The HDL level of 8 different serum samples was determined on a Cobas Mira plus instrument from ABX technologies using HDLC3 reagent from Roche (Table 6). Using the initial 100 seconds of the progress curves, fictive end-points were estimated using a 4-parameter logistic function. As shown in
TABLE-US-00005 TABLE 6 Roche Direct HDLC3 reagents. R1 Pretreatment 5 minutes 37 C. R2 Incubation 5 minutes 37 C. R1 pretreatment Hepes, pH 7.4 10 mmol/L CHES, pH 7.4 97 mmol/L dextran sulfate 1.5 g/L magnesium nitratehexahydrate 12 mmol/L HSDA 1 mmol/L Ascorbate oxidase 16.7 kat/L (Eupenicillium sp) R2 enzyme reagent Hepes, pH 7.0 10 mmol/L PEG-cholesterol esterase 3.33 kact/L (Pseudomonas spec.) PEG-cholesterol oxidase 127 kat/L (Streptomyces spec.) peroxidase 333 kat/L (horseradish) 4-aminoantipyrine 2.5 mmol/L
[0152] Example 4.
Example 4
The Afinion HDL Assay (Point-of-Care)Comparison to Large Clinical Instrument Assay Method
[0153] The HDL levels of 49 scrum samples were determined on an Advia instrument and reagents from Siemens, the reference method. The samples also were measured by the estimated end-point method using Wako HDL-C L-type reagents (Table 4) on an Afinion point-of-care apparatus. Progress curves were monitored for 160 seconds and end-points estimated using two different algorithms: a 1.sup.st order equation (Table 3, type 1) and a logistic equation (Table 3, type log-transformed). The estimated end-points were converted to concentrations using a calibration curves established on Afinion in a separate experiment using calibrators from Trina Bionostics, whose HDL levels had been determined on Cobas Mira plus using HDL direct CP reagents from ABX Pentra (Table 7).
TABLE-US-00006 TABLE 7 ABX HDL Direct CP reagents. R1 Pretreatment 5 minutes 37 C. R2 Incubation 5 minutes 37 C. R1 pretreatment Good's buffer, pH 7.0 cholesterol oxidase 1000 U/L peroxidase 1300 ppg U/L DSBmt 1 mmol/L accelerator 1 mmol/L R2 enzyme reagent Good's buffer, pH 7.0 cholesterol esterase 1500 U/L 4-aminoantipyrine 1 mmol/L ascorbic acid oxidase 3000 U/L detergent 2% restrainer 0.15%
Example 5
The Afinion HDL AssayComparison to Point of Care Assay Method
[0154] The HDL level of 42 serum samples were determined on a point-of-care assay system from Cholestech, LDX system Lipid profile Glu. The samples also were measured and HDL values computed by the Afinion HDL method, essentially as described in Example 4.
Example 6
Correcting the Estimated End-Point of HDL in a Multi-Component Assay Method for Influence of Parallel Conversion of NonHDL
[0155] To exemplify the possibility of correcting the HDL assay for an influence of simultaneously converted nonHDL by postanalysis computations based upon concomitant measurement of totoal CH in the sample, the following experiment was performed:
[0156] (i) a calibration curve for HDL was constructed from a preparation of pure HDL (Wako High Unit HDL-C) using a Cobas Mira plus instrument and Wako HDL-C L-type reagents and protocol (Table 4). Estimated end-point absorbance from the first 300 s of the progress curves and curve fitting using a 1.sup.st order function were used in the construction of the calibration curve:
HDL=1.2+136.3ABS(1)
[0157] (ii) a calibration curve for the influence on the measured HDL value by a parallel conversion of nonHDL was constructed from mixtures of pure HDL and pure LDL (Wako High Unit LDL-C), two different concentrations of HDL and 4 different concentrations of LDL. The increase in the HDL measured was plotted as a function of the nonHDL concentration. The increase was exponentially correlated to the nonHDL concentration
HDL increase=2.433 exp0.008499nonHDL(2)
[0158] The concentration of nonHDL was calculated from the difference between CH (total cholesterol measured) and HDL:
nonHDL=CHHDL(3)
[0159] A serum sample containing 111 mg/dL HDL was mixed at two different concentrations with different amounts of pure LDL. The CH and HDL levels were measured on a Cobas Mira plus instrument with ABX Pentra Cholesterol CP and the Wako HDL-C L-type reagents and protocol, respectively.
[0160] Measured values were on average 48% overestimated. After iterative correction using the equations (1) to (3) above the mean overestimation was decreased to 5% (Table 8).
TABLE-US-00007 TABLE 8 Influence of nonHDL on HDL measurement and correction thereof. HDL HDL HDL postanalysis Sample CH true measured corr 36-1 117 36 41 36 36-2 231 36 52 40 36-3 336 36 68 38 62-1 204 62 70 62 62-2 314 62 84 64 62-3 428 62 120 69 Grand mean 49 72.5 51.5 Samples were run in 2-3 replicates.
Example 7
Elimination of Cell Lysis by Blocking Reagent
[0161] 20 L of whole blood was added to 400 L of HDL-C L-type R1 (Wako) added 0, 60, 90 or 120 mmol/L of NaCl. After mixing the absorbance of the samples were measured at 660 nm. At this wavelength there is no absorption from hemoglobin but strong interference (through scattering of light) from intact cells. While R1 added no NaCl caused almost complete lysis, R1 added 120 mmol/L NaCl caused insignificant cell lysis.
TABLE-US-00008 NaCl added Absorption at 660 nm Hemolysis (mmol/L) (AU) (%) 0 0.062 100 60 1.306 23 90 1.569 7 120 1.615 4
[0162] As can be seen, reduction of cell lysis to below 5% is achievable by appropriate choice of ionic strength in reagent R1.
Example 8
Use of Blocking Reagent Comprised Within Non-Lytic Dilution Buffer
[0163] Effect of using HDL reagent R1 as general dilution buffer on measurement of triglycerides.
[0164] The TO levels of 46 serum samples were determined on Afinion point-of-care instrument using HDL reagent R1 (Table) as the general dilution buffer and Roche TO reagent (Table 5).
[0165] 15 L sample was diluted into 280 L of HDL reagent R1 and 58 L was transferred to 100 L of TG reagent. Reaction end-points were determined and converted to concentrations using a calibration curve established on Afinion in a separate experiment using calibrators that had been previously quantified at a CRMLN laboratory (Seattle, USA). In a parallel experiment the same samples were determined in a separate laboratory (Frst Medical Laboratory, Oslo, Norway) using an Advia instrument and Siemens reagents. The samples compared very well between the two methods and there was no indication of a significant interference from the HDL R1 dilution buffer on the Afinion TG results (
[0166] Table Dilution buffer
[0167] Dilution buffer Good's buffer, pH 7.0 [0168] NaCl 175 mmol/L [0169] 4-aminoantipyrine 0.9 mmol/L [0170] peroxidase 2400 U/L [0171] Ascorbate oxidase 2700 U/L [0172] Anti-human apo--lipoprotein antibody
Example 9
Dilution of Whole Blood in Isotonic HDL R1 and Filtration to Obtain an Essentially Cell Free Filtrate
[0173] The applicability of HDL R1 (non-HDL blocking) reagent as a general dilution and filtration buffer for whole blood was investigated in an Afinion instrument supplied with filtration capability by placing a filter pad at the bottom of well nr.2. The dilution/filtration buffer was HDL-C L-type reagent 1 (R1) (Table 4) made nonlytic by adding NaCl to 175 mM.
[0174] Whole blood was drawn into a 15 L sample device by capillary forces and inserted into the Multiwell cartridge which was placed in the instrument. The sample was emptied and mixed into 250 L of the modified HDL R1, and the entire volume transferred into the well containing the filter pad. The membrane tube of the Multiwell cartridge was then positioned tightly over the filter pad. When below ambient pressure was applied to the open end of the membrane tube the diluted whole blood flowed into the filter pad and whereas blood cells were trapped in the filter, the diluted plasma flowed through the filter and into the membrane tube. When pressure started rising (sucking air) the membrane tube was removed from the filter, lowered into an empty well and forced to release the filtered plasma by applying an above ambient pressure. Blood contamination was measured after converting contaminating haemoglobin to methhemoglobin using NaNO.sub.2 and measuring the absorbance at a wavelength of 410 nm. The absorbance was converted to %Hb by interpolation from a calibration curve constructed from known concentrations of methhemoglobin. Results given in Table 9 are the compounded results of hemolysis caused by dilution into the R1 reagent, hemolysis caused by filtration, and contamination by blood cells, not trapped in the filter pad.
TABLE-US-00009 TABLE 9 Total haemoglobin contamination after dilution into R1 and filtration. Whole blood % Filtrate % Hb Filtertype hematocrit contamination Whatman 37 0.9 Whatman 61 1.2 Millipore AP25 37 0.5 Millipore AP25 61 1.5 Millipore 2 37 0.8 Millipore 2 61 1.2 Mean of 5-6 replicates
Example 10
Estimation of a Fictive End Measurement of NonHDL Associated Cholesterol in the Presence of Cholesterol Associated with HDL. Comparison to Certified Laboratory Method
[0175] A method for the direct determination of nonHDL was constructed by using a HDL specific block polymer that protects HDL while cholesterol associated with all other lipoproteins (nonHDL) are converted to a detectable product (HDL-X; Wako, Japan). As source for the HDL specific block polymer was used Reagent 1 from the Wako HDL-C L-M/2-PM reagent (Wako, Japan). The exact composition of Reagent 1 is not available, but it is disclosed to contain Good's buffer pH 7.0, cholesterol esterase, cholesterol oxidase, HMMPS, catalase, ascorbate oxidase and HDL block-polymer. By supplementing Reagent 1 with peroxidise (6000 U/L), 4-aminoantipurin (0.8 mmol/L) and sodium azide (0.025%) a reagent was formulated that converted nonHDL into a detectable product while HDL was temporarily protected.
[0176] 2 L of calibrator or plasma sample was mixed with 150 L of supplemented Reagent 1. The reaction was performed at 37C and monitored at 600 nm using a Cobas Mira plus instrument. End-point absorbance was estimated from the first 300 s of the progress curve using a 4 parameter logistic function (Table 3).
[0177] Calibrators and samples had been previously quantified at a CRMLN laboratory (Seattle, USA) with respect to CH, HDL and TG. From these values nonHDL values were calculated according to
nonHDL=CHHDL.
[0178] The nonHDL values of the 9 serum samples determined by the method of the invention correlated very well with the nonHDL values computed from the CRMLN values (
Example 11
[0179] Whole Blood and Plasma Lipid Profiles Determined on an Afinion Point-of-Care Analyzer
[0180] The plasma levels of CH, TG and HDL were determined on 15 L of fresh whole blood obtained from 60 healthy volunteers, using an Afinion analyzer. From these data the LDL levels were computed in mg/dL using the Friedewald equation, LDL=CH(HDL+TG/5). The plasma fraction of the same samples were also obtained (by centrifuging at 1000 g for 10 minutes) and measured on the Minion for the same analytes. The reagents used were those described in Tables 4 (HDL), 5 (TG) and 10 (CH). Reaction end-points were determined for TG and CH. The unmeasurable end-point of the HDL reaction was estimated using the first 100 s of the progress curve and curve fitting using a 1.sup.st order function (Table 3). Whole blood hematocrit was determined as described in Example 9 and whole blood results were converted to plasma results by dividing each result with (1-hematocrit) using the hematocrit value obtained for each specific whole blood sample.
TABLE-US-00010 TABLE 10 Roche Chol2 reagent buffer Pipes 225 mmol/L, pH 6.8, containing 10 mmol/L Mg2+ sodium cholate 0.6 mmol/L 4-aminoantipyrine 0.45 mmol/L phenol 12.6 mmol/L fatty alchol polyglycol ether 3% cholesterol esterase 1500 U/L (Pseudomonas spec.) cholesterol oxidase 450 U/L (E. coli) peroxidase 750 U/L (horseradish)