EP2235543A1 - Procédé pour déterminer des lipides - Google Patents

Procédé pour déterminer des lipides

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Publication number
EP2235543A1
EP2235543A1 EP08864896A EP08864896A EP2235543A1 EP 2235543 A1 EP2235543 A1 EP 2235543A1 EP 08864896 A EP08864896 A EP 08864896A EP 08864896 A EP08864896 A EP 08864896A EP 2235543 A1 EP2235543 A1 EP 2235543A1
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EP
European Patent Office
Prior art keywords
specific class
lipoprotein
reaction
lipid
plasma
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EP08864896A
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German (de)
English (en)
Inventor
Arne Ludvig Faaren
Frank Frantzen
Arne Kristian Nordhei
Erling Sundrehagen
Lars ÖRNING
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Axis Shield ASA
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Axis Shield ASA
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Publication of EP2235543A1 publication Critical patent/EP2235543A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

  • the present invention relates to the measurement of specific classes of lipid and lipoprotein in blood samples. Specifically, the invention relates to the measurement of plasma cholesterol and triglycerides associated with certain classes of lipoprotein, particularly by means of enzymatic assays. Most particularly, the invention relates to diagnostic and prognostic assays for use in automated methods, including those conducted on laboratory or "point-of-care" apparatus.
  • the measurement of components in body samples is a common feature of clinical assessments. Many diagnoses can be made or confirmed, or the risk of many conditions can be assessed, from the concentration of a particular analyte in a body sample, or from the profile of concentrations of several components.
  • concentration of a particular analyte in a body sample or from the profile of concentrations of several components.
  • the increasing number of known correlations between disease states and the concentrations of one or more analytes makes sample analysis an increasingly valuable tool, and also increases pressure on clinical laboratories to analyse ever larger numbers of samples increasingly quickly. To satisfy this, there is a need for assays which are quicker, higher throughput, simpler and more completely automated.
  • Point of care assays are exceptionally demanding in terms of the need for simple manipulation and rapid results. If an assay takes more than a few minutes then much of the advantage of conducting it at the point-of-care is lost. Furthermore, although the staff conducting such assays are likely to be healthcare professionals, they are not analytical specialists and will not have access to multiple instruments. It is therefore necessary that such assays be designed to rely on the minimum of sample handling.
  • the most common clinical samples taken for assay are fluids, particularly blood and urine, since these are relatively easy to take and to manipulate. In blood, it is typically the content of the fluid plasma which is analysed. Some of the most common and clinically important measurements made on blood-derived samples relate to the plasma lipid content.
  • the predominant lipids present in blood plasma are phospholipids, triglycerides (TG), and cholesterol (CH). Of these TG and CH are of particular diagnostic interest because of their association with cardiovascular disease, which in turn is one of the most prevalent diseases in the developed world.
  • Lipids are by their very nature water insoluble and in blood are transported in complex with apolipoproteins, which render them soluble. These complexes, the lipoproteins are classified into five groups, based on their size and lipid-to-protein ratio: chylomicrons, very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), and high density lipoproteins (HDL).
  • Chylomicrons are basically droplets of fat, and consist of TG to about 90%. Chylomicrons function as vehicles for the transport of dietary lipids from the ileum to adipose tissue and liver and are present in the general circulation for only a short period after a meal.
  • VLDL, IDL, and LDL are responsible for transporting lipids from the liver to the tissues
  • HDL is engaged in the reverse transport of superfluous lipids from peripheral tissues back to the liver for further hepatobiliary secretion.
  • VLDL and IDL have short half-lives and deliver mainly TG to the tissues.
  • LDL and HDL have longer half-lives and are the major participants in blood cholesterol homeostasis.
  • LDL and HDL combined carry about 95% of the cholesterol present in blood, with LDL carrying about 70% and HDL about 25%.
  • Apolipoprotein A Apolipoprotein A
  • B B, C, D, and E
  • Apolipoprotein B Apolipoprotein B
  • HDL HDL is devoid of apoB and its principal protein is apo-Al.
  • TG TG-derived neurotrophic factor
  • pathophysiological disorders such as diabetes, cardiovascular disease, hyperlipidaemia, hyperglyceridaemia types I and IV, and nephritic syndromes.
  • Low concentrations are found in hepatic infection and malnutrition.
  • CH associated with chylomicrons, VLDL, IDL, and LDL is a major risk factor for cardiovascular disease (CVD), with increasing concentrations correlating with increased risk of CVD.
  • the CH associated with LDL particles is considered the main risk factor.
  • CH associated with HDL on the other hand is inversely correlated with risk for cardiovascular disease. The lower the concentration of HDL, the higher the risk for cardiovascular disease. Therefore it is common practice to determine CH associated with LDL and HDL to diagnose and predict cardiovascular disease, as well as in formulating the risk of CVD, potentially in combination with other factors.
  • Two methods are currently used routinely for quantification of CH. Both methods are enzymatic. In the first method utilises an enzyme chain beginning with cholesterol esterase and cholesterol oxidase to generate a coloured or fluorescent signal by the generation of hydrogen peroxide. The other method substitutes cholesterol dehydrogenase in place of cholesterol oxidase and determines the amount of CH in the sample on the basis of the amount of the produced NADH or NADPH.
  • HDL associated CH may determined by separating this class of lipoprotein from non-HDL lipoproteins. After making the non-HDL unavailable, HDL associated CH is measured using the enzymatic methods of total CH.
  • the precipitated non-HDL is then removed by centrifugation.
  • the latter method is still recommended by the Cholesterol Reference Method Laboratory Network as reference method for measurement of HDL associated CH (Kimberly et al (1999) Clin Chem 45:1803-12).
  • NEP National Cholesterol Education Program
  • ATPIII Adult Treatment Panel III
  • LDL associated CH was measured using ultracentrifugation (Havel RJ et al, J Clin Invest 1955;34: 1345-53). This is still the most used reference method, but evidently requires sample pre-treatment. Homogenous methods were later developed which did not require a physical separation of non-LDL lipoproteins to measure the LDL CH fraction.
  • VLDL associated CH was originally measured using ultracentrifugation. This remains a preferred reference method, but during recent years homogenous methods for determining VLDL associated CH have been developed. These include methods from US6986998 and US7208287.
  • CH 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 (US7272047 and US 2007/0161068) that use specific surfactants to further the selective enzymatic decomposition of IDL associated cholesterol.
  • TG 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 (glycerokinase) and oxidized (glycerol-3 -phosphate oxidase) to di-hydroxy-acetone-phosphate and hydrogen peroxide, which is used to generate a coloured, fluorescent or chemiluminescent signal.
  • measurement of TG of specific lipoprotein classes may be performed by several methods that exploit different chemical and physical characteristics of the different lipoprotein classes.
  • a pre-treatment step is used, and blood cell plasma separation is accomplished by centrifugation.
  • blood sample is transported from its site of collection to the clinical laboratory where centrifugation and sample analysis is performed.
  • the steps of blood collection, transport to a clinical laboratory and centrifugation before performing the intended analyses constitute severe limitations both with respect to labour and time resources required and to risks of infection / contamination. It is an established fact that health-care workers experience an increased risk of acquiring in particular hepatic virus infections.
  • Assays for detecting certain antigens and antibodies, which are practicable on whole-blood have been proposed in US6143510; US2004/0048397.
  • Some assays for use at the point-of-care allow for whole-blood to be added to the test device. These devices, however, then incorporate separation steps, such as passage through membranes to separate the plasma from the intact blood cells. This is effective, but considerably increases the complexity of the assay device (e.g. cartridge) and therefore the cost and waste associated with the assay. Furthermore, since the separation is conducted directly on the whole blood sample added to the device, and there is a considerable dead-volume involved in such a separation method, the volume of sample required is considerably increased. Ideally, a point- of-care test should use a minimal volume of blood (e.g. less than 20 ⁇ l), so that this can be obtained with least discomfort for the patient.
  • a minimal volume of blood e.g. less than 20 ⁇ l
  • In-device separation also requires complex assay devices also stand a greater chance of blocking or generating unreliable results, especially where the separation is conducted at high concentrations of cells. If the discomfort or cost of a point-of-care assay becomes too great or the reliability too low, then the clinician, patient or medical insurer will elect for the assays to be carried out at a specialist laboratory rather than at the point of care.
  • this method should be simpler, quicker, more reliable, require a smaller sample and/or require less instrumentation than currently available methods.
  • the present inventors have now surprisingly established that the perceived need for assays measuring plasma lipid components to be conducted in separated plasma, rather than whole blood, is not in fact an essential requirement of a successful assay, providing that the assay is suitably formulated.
  • they have established that the lipid content of the blood cell component does not interfere with enzyme assays for lipoprotein associated lipids providing that the assay is conducted under conditions maintaining cell integrity.
  • They have furthermore established that although scattering by whole blood cells increases assay background signals, this does not prevent effective measurement of lipid components by suitable methods.
  • the interference caused by haemoglobin in the sample can be tolerated.
  • the present invention thus provides an enzymatic method for determining the concentration of at least one specific class of lipid or specific class of lipoprotein in the plasma portion of a blood sample comprising plasma and intact blood cells, said method comprising;
  • reaction mixture causes the selective reaction of said specific class of lipid or said specific class of lipoprotein and concomitantly generates a reaction product or consumes a reaction substrate;
  • step i) is conducted under conditions which substantially maintain said blood cells in an intact state.
  • step i The conditions of the specific reaction step (step i) should be such as to substantially maintain the blood cells in an intact state. This requires control of the chemical environment of the assay in a way that has not been provided in any previous assay for lipids or lipoproteins. Specifically, the reagents added during step i (or during step i and any additional steps carried out simultaneously) should control the osmotic pressure and surfactant content of the assay so as to substantially avoid cell lysis. Other factors such as the presence of solvents and the reaction pH will have lesser effects. After osmotic strength, which should preferably be approximately isotonic, the use of detergents and/or surfactants will have the next most significant effect upon cell lysis in the system.
  • the present invention additionally provides a kit for use in determining the concentration of at least one specific class of lipid or specific class of lipoprotein in the plasma portion of a blood sample comprising plasma and intact blood cells, said kit comprising;
  • step i) are chosen such that the cells of the blood sample remain intact, or substantially intact (e.g. less than 20% of the cells, preferably less than 10%, and most preferably less than 5% (e.g. 0.1 to 5%) of the cells suffer lysis during step i).
  • Factors including ionic strength and surfactant type/concentration are particularly important in this respect.
  • HLB hydrophil-lipophil balance
  • surfactants with HLB values below 10 and above 13 are in particular suitable for use in the presence of intact cells. Such surfactants are thus preferred for use in the method of the invention (e.g. surfactants with HLB from 1 to 10 or from 13 to 20).
  • surfactants with HLB values between 10 and 13 may also be compatible with maintaining intact cells depending on the concentrations used, and depending on the construction and formulation of the assay, such as reaction times and temperatures used, ionic strength, pH and types of salts used in the assay mixture, and the presence of stabilizing substances such as serum albumin.
  • the method of the invention is preferably carried out with the reagent mixtures of steps i and ii formulated as a single reagent mixture.
  • steps i) and ii) of the method are conducted simultaneously. This reduces the number of individual reagent mixtures which must be loaded into the instrument and accommodated therein, and furthermore allows a faster assay, since steps i and ii are conducted simultaneously.
  • reaction accelerators such as disclosed in US6818414 may be included in the reagent solution, especially at step i) to increase reaction rate and thus decrease assay time, as long as the included accelerator allows maintaining blood cells in an intact state.
  • accelerators which act specifically on the cholesterol reaction are: (i) membrane intercalators, which increase release of cholesterol from the phospholipid environment of the outer shell of lipoproteins.
  • membrane intercalators which increase release of cholesterol from the phospholipid environment of the outer shell of lipoproteins.
  • Examples are amphiphatic alcohols (octanol (see Example 11), hexadecanol; 0.1-100 mM), ceramids (N-hexanoyl-D-erythrosphingosine; 1-1000 ⁇ M), diglycerides (1,2- dioctanoyl-s,n-glycerol, hexadecyl glycerol, dipalmitoyl glycerol; 1-1000 ⁇ M), and the steroid based antibiotic, fusidic acid;
  • triglyceride or lipoprotein lipase see Example 1 1
  • substances that stabilize released cholesterol in the solvent phase such as serum albumin, polyethylen glycol 6000, cyclodextrins, and dextran; (compounds are available from Sigma-Aldrich, Fluka, and Molecular Probes (Eugene, OR)).
  • reactions may be accelerated by increasing the reaction temperature.
  • the time needed for the measurement of HDL cholesterol was 180s (3 min) at 37°C. This time (i.e. the time needed to reach the same amount of colored product formed) could be reduced to 105s when the reaction temperature was increased to 42 0 C and further reduced to 75s when the reaction temperature was increased to 47°C.
  • Two methods are currently used routinely for quantification of CH. Both methods are enzymatic and suitable for use in the method of the present invention.
  • cholesterol esterase converts cholesterol esters into CH.
  • Cholesterol oxidase then converts CH to choleste-4-ene-3-one and hydrogen peroxide.
  • 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
  • fluorescent or chemiluminescent substrates may be substituted for the 4-aminoantipyrin/phenol .
  • 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.
  • 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.
  • 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.
  • the formed coloured products may be monitored at wavelengths from 450 to 850 nm.
  • fluorescent or chemiluminescent substrates may be substituted for the 4-aminoantipyrin.
  • the final steps in both peroxide-based methods are equivalent and interchangable.
  • the enzyme reactions converting the specific plasma lipid component into a detectable chemical product preferably may be performed using the above described enzyme systems.
  • 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 (US5807696). Enzymes are typically used at concentrations from 100 - 100,000 U/L.
  • 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.
  • 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.
  • HLB hydrophil-lipophil balance
  • 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.
  • 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),
  • Surfactants are used for this purpose typically at concentrations of 0.001-10%, preferably 0.01 to 1%. 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.
  • the polyanion method (ii) uses a synthetic polymer together with a polyanion to block non-HDL and make them refractory to solubilization with specific detergents and enzymatic measurement.
  • the immunologic method (iii) 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.
  • the accelerator/detergent method 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.
  • HDL cholesterol is degraded using an HDL-specific detergent in a colour forming process.
  • 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:
  • US5888827 describes a method whereby non-LDL is masked by surfactants and cyclodextrins in the presence OfMg 2+ and becomes refractory to breakdown by PEG modified enzymes.
  • US5925534 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.
  • US6986998 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.
  • 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 (US7272047 and US 2007/0161068) that use specific surfactants to further the selective enzymatic decomposition of IDL associated cholesterol.
  • 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.
  • 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).
  • the presence of intact blood cells in such an assay tends to increase the non-specific background signal, especially for absorbance methods, but does not alter the change or rate of change in signal, which is used to assess the concentrations of the analytes in the sample. Since the signal is detectable without removal of the blood cells at any stage, in one preferred embodiment, the blood cells remain intact or substantially intact in the sample throughout the assay method (e.g. for steps i to iv).
  • 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.
  • chromogenic reagents are Trinder reagents, which in the presence OfH 2 O 2 react with a coupler to form colored products.
  • 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).
  • Trinder reagents are those forming products that can be colorimetrically determined at wavelengths 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 l -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).
  • concentration of the chromogen is preferably 0.01-10 g/L, and is limited by solubility.
  • fluorescent substrates examples include dihydrocalceins, dihydroethidium, dihydrofluoresceins, dihydrophenoxazine (Amplex red;10-acetyl-3,7- dihydroxyphenoxazine), and dihydrorhodamines.
  • chemiluminescent substrates examples include luminol (3-aminotriphenylene complexes), Lumigen PS-2 and Lumigen PS-atto.
  • the present invention therefore provides that the concentration of intact blood cells is reduced after step i but before the detection step iii.
  • concentration is used herein to indicate concentration relative to the concentration of the other components (especially the signal-generating secondary analyte or the reaction product or reaction substrate referred to in step (i)).
  • step i) of the method an inhibitor is added to inhibit further reaction (e.g. to inhibit further reaction of the lipid converting enzyme or further generation of the indirect product) and the intact blood cells in the sample are reduced by means of cell lysis.
  • an inhibitor is added to inhibit further reaction (e.g. to inhibit further reaction of the lipid converting enzyme or further generation of the indirect product) and the intact blood cells in the sample are reduced by means of cell lysis.
  • a suitable inhibitor of any step in the pathway from the lipid to the indirect product may be used, such as an inhibitor of the lipid converting enzyme.
  • cell lysis may be achieved by different means, such as by lowering the osmotic pressure causing the cells to take up water, swell and disrupt, or by using detergents that dissolve the cell membrane. Such methods are known in the art.
  • 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%.
  • 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-I), and the non-ionic surfactants polyoxyethylene alkyl aryl ether (Triton XlOO and Xl 14) and polyoxyethylene lauryl alcohol (Chemal LA-9).
  • anionic surfactants amine aklylbenzene sulfonate (Ninate 411), sodium dioctylsulfosuccinate (Aerosol OT), sodium N-oleyl- N-methyltaurate (Geropon T-77), sodium olefin sulfonate (Bioter
  • inhibition of peroxidase is a suitable method to avoid generating further signal after cell lysis.
  • the common peroxidase inhibitor, Methimazole (2-mercapto- 1 - methylimidazole) is particularly suitable.
  • inhibitors of peroxidase reaction are: alphatocopherol, benzhydroxamic acid, diethyldithiocarbamate, dithiothreitol, glutathione, hydroquinone, hydroxylamine, phenyl hydrazine, potassium cyanide, PTU (6-n-propyl-thiouracil), salts of metals (Al 2+ , Hg 2+ , Mn + ), sodium azide, Tiron (dihydrobenzene disulfonic acid).
  • Trinder coupler e.g. 4-aminoantipyrine
  • Inhibitors of other enzymes used in the conversion of specific lipid to detectable chemical product may also be used, in combination or alone.
  • these include:
  • Cholesterol oxidase inhibitors Fenipropimorph, l-fluoro-2,4-dinitrobenzene, N- bromosuccinimide, salts of metals (Ag + , Fe 2+ , Hg 2+ ), sarcosyl, SDS, trinitrobenzene sulfonate, Triton X-100 (>0.2%).
  • Cholesterol esterase inhibitors cetyltriethylammonium bromide, diethyldicarbonate, diisopropyl fluorophosphates, dithiothreitol, 1-hexadecanesulfonyl chloride, 4- hydroxymercury benzoate, N-bromosuccinimide, p-chloromercury benzoate, phenyl methyl sulfonyl fluoride, potassium cyanide, protamine, salts of metals (Ag + , Ca 2+ , Cd 2+ , Cu 2+ , Fe 2+ , Fe 3+ , Hg 2+ ), Triton X-IOO (>1%).
  • Glycerol-3-phosphate dehydrogenase inhibitors ATP, N-ethyl maleimide, p-chloro mercury benzoate, 1,10-phenantroline, salts of metals (Cu 2+ , Mn 2+ , Ni 2+ , Zn 2+ ).
  • Hepatic lipase inhibitors cetyltri ethyl ammonium bromide, p-chloromercury benzoate , diethyl 4-nitrophenyl phosphate, EDTA, 1 -hexandecansulfonyl chloride, 4-hydroxymercury benzoate, 2-mercaptoethanol, N-bromosuccinimide, 1,10- phenantroline, protamine, salts of metals (Ag + , Ca 2+ , Co 2+ , Cu 2+ , Fe 2+ , Hg 2+ , Mn 2+ , Ni 2+ , Zn 2+ ), SDS, Triton XlOO (>1%).
  • Glycerol-3-phosphate oxidase inhibitors Acriflavone, atebin, benzoyl formic acid, glyoxylic acid, methylglyoxidde, sodium azide.
  • the second method by which the intact blood cells in the sample may be reduced is by means of physical separation. This may be 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.
  • 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, however, 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 and the filtration thus more effective.
  • 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.
  • Suitable materials for transverse filtering are given in Table 1.
  • Suitable materials for lateral flow filtering are Hemasep® L (Pall Corp) and LFl and MFl (Whatman).
  • the intact blood cells may be separated by filtration prior to monitoring formed colored product.
  • the intact blood cells may be separated before or after the enzymatic reactions have been completed, but where this takes place before, then a controlled reaction period is generally necessary.
  • Table 1 lists different filters and their effect on the recovery of colored product and lipids.
  • the concentration of cells is preferably reduced by at least 50% (e.g. 50 to 90%), preferably by at least 70% (e.g. 70 to 95%), and most preferably by at least 90% (e.g. between 90% and substantially 100%) .
  • the relevant "concentration” is preferably not absolute concentration as might be reduced by dilution, but is rather concentration relative to the sample as a whole, and in particular relative to the concentration of the reaction product or reaction substrate referred to in step (i), or to the secondary analyte.
  • the at the time from adding the reagent in step i) to the detection of the secondary analyte in step iii) is no more than 7 minutes, preferably no more than 5 minutes, and more preferably in the range 3 to 5 minutes.
  • the volume of whole blood sample in step i) of the assay method is no greater than 40 ⁇ L, preferably no greater than 20 ⁇ L and most preferably no greater than 12 ⁇ L (e.g. 12 to 25 ⁇ L).
  • the first reagent mixture and said second reagent mixture are preferably formulated together 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.
  • the kit preferably comprises an inhibitor to inhibit further reaction of said specific class of lipid or said specific class of lipoprotein, or to inhibit the generation of further secondary analyte.
  • Suitable inhibitors are disclosed herein are those named above are particularly preferred both individually and in combination.
  • the kit may additionally contain a lysing agent to cause cell lysis.
  • a lysing agent to cause cell lysis.
  • This is preferably in combination with an inhibitor and suitable lysing agents are again disclosed herein and those mentioned herein (including in the examples attached hereto) are preferred.
  • Surfactants of HLB of 10 to 13 are particularly preferred, as are tonicity adjusters.
  • Reference samples of at least one lipoprotein selected from the group consisting of very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) may also be incorporated into the kits of the invention.
  • Fig.1. illustrates the effect of intact blood cells on the measurement of plasma TG.
  • the upper graph depicts the strong effect of intact cells on the signal.
  • the middle graph depicts the same results normalized to give the same initial absorption level.
  • the intact cells have no or only a very small effect on the development of the curve, i.e. on the conversion of TG to colored product.
  • intact blood cells do not contribute lipid to the determination of plasma TG.
  • the lower graph shows that lysed cells on the other hand do contribute lipid to the determination of plasma TG.
  • Fig.2. Absorption profiles of intact and lysed blood cells.
  • Fig 4. illustrates the effect of intact blood cells on the measurement of plasma total CH.
  • the upper graph depicts the strong effect of intact cells on the signal.
  • the middle graph depicts the same results normalized to give the same initial absorption level.
  • the intact cells have no or only a very small effect on the development of the curve, i.e. on the conversion of plasma CH to colored product.
  • intact blood cells do not contribute lipid to the determination of plasma CH.
  • the lower graph shows that lysed cells on the other hand do contribute lipid to the determination of plasma CH.
  • Fig.5. illustrates the effect of intact blood cells on the measurement of HDL associated CH.
  • the upper graph depicts the strong effect of intact cells on the signal. The more intact cells are present, the higher the signal.
  • the lower graph depicts the same results normalized to give the same initial absorption level.
  • the inset shows the slope of the curves (i.e. rate of product formation) as a function of cell concentration.
  • the intact cells have a nonsignificant effect on the development of the curve, i.e. on the conversion of HDL associated CH to colored product. Thus, intact blood cells do not contribute lipid to the determination of HDL associated CH.
  • Example 1 Evaluation of the effect of intact blood cells on the measurement of plasma TG.
  • Plasma samples were drawn from healthy volunteers into EDTA-plasma tubes. Plasma and cells were separated by centrifugation at 200Og for 15 min. All reactions were performed at 37 0 C.
  • the ABX Pentra Triglyceride CP reagent was reformulated so as to maintain cell integrity. 6 ⁇ L of plasma with or without 1.5 ⁇ L of blood cells or 1.5 ⁇ L of blood cells in buffer was added to 500 ⁇ L of the reformulated assay reagent, containing lipoprotein lipase (108 kU/L), glycerokinase (505 U/L), glycerol-3 -phosphate oxidase (4150 U/L), peroxidase (495 U/L), ATP (3.14 mmol/L), p-chlorophenol (2.69 mmol/L), 4-aminoantipyrin (0.31 mmol/L), and magnesium chloride (15 mmol/L) in Good's buffer (50 mmol/L), pH 7.4.
  • lipoprotein lipase 108 kU/L
  • glycerokinase 505 U/L
  • glycerol-3 -phosphate oxidase 4
  • the non-specific increase in background is due to the light scattering effect of the intact blood cells present in the pathway of the light used to measure the amount of colored product formed. This interference may be reduced by cell lysis prior to measurement, in particular if measurement is performed using wavelengths at 600 nm or higher (Fig. 2)
  • intact blood cells do not contribute lipid to the measurement of plasma TG, but the intact cells give rise to high background.
  • Cell lysis lowers the background, but lysed cells contribute lipid to the measurement of plasma TG.
  • performing the conversion of plasma TG to colored product in the presence of intact blood cells, and then performing cell lysis prior to determination of amount of product formed, in the presence of enzyme inhibitors which block the further conversion of lipid released from lysed cells into colored product will allow direct determination of plasma lipids in the presence of blood cells, i.e. in whole blood.
  • intact blood cells do not contribute lipid to the measurement of plasma CH, but the intact cells give rise to high background.
  • Cell lysis lowers the background, but lysed cells contribute lipid to the measurement of plasma CH.
  • performing the conversion of plasma CH to colored product in the presence of intact blood cells, and then performing cell lysis prior to determination of amount of product formed, in the presence of enzyme inhibitors which block the further conversion of lipid released from lysed cells into colored product will allow direct determination of plasma lipids in the presence of blood cells, i.e. in whole blood.
  • 5 ⁇ L of human EDTA plasma was mixed with blood cells in different portions to mimic different degrees of cell removal (from 0 to 100% cell removal) and added to 810 ⁇ L of Wako HDL-C L-type Reagent 1, containing anti-human apoB antibody, peroxidase (2700 U/L), ascorbate oxidase (2700 U/L), and 4-aminoantipyrin (0.9 mmol/L) in Good's buffer, pH 7.0, and adjusted with sodium chloride to isotonicity to avoid cell lysis.
  • Fig 2 shows the absorption spectrum between 300 nm and 700 nm of blood plasma in the presence of intact blood cells or lysed blood cells. Cell lysis clearly reduces background substantially and above 600 nm eliminates it almost completely.
  • Figs 1 and 4 show cell lysis renders cell associated CH and TG available for the enzyme systems used to measure plasma CH and TG, leading to an overestimation of these analytes.
  • This problem is solved by including inhibitors of the enzymes converting the lipids to colored product.
  • Fig.3 shows the dose dependent decrease in the formation of colored product by hydrogen peroxidase by the common peroxidase inhibitor, Methimazole (2-mercapto-l-methylimidazole).
  • TG is determined in blood plasma in the presence of intact blood cells.
  • the TG in the sample is measured using two reagents, Rl and R2 (Table 4).
  • lO ⁇ L of fingerprick blood is aspirated from a patient.
  • the Sample Device is inserted into the Cartridge and placed in the Afinion instrument.
  • Reaction vessel (RV)-I of the Cartridge is filled with 300 ⁇ L PBS, RV-2 with 100 ⁇ L enzyme/substrate solution (Rl), and RV-3 with 300 ⁇ L lysis/inhibition solution (R2) (Table 4).
  • the sample is automatically transferred into RV-I of the Cartridge, which contains 300 ⁇ L of PBS.
  • 100 ⁇ L of diluted sample is transferred from RV-I to RV-2 containing lOO ⁇ L Rl .
  • the reaction converting plasma TG to colored product is allowed to proceed for 3 min.
  • 100 ⁇ L R2 is transferred from RV-3 to RV-2, and the amount of colored product is determined by measuring the absorbance at 630 nm.
  • the hematocrit is determined by transferring 50 ⁇ L diluted sample from RV-I to RV-3 and measuring absorbance at 530 nm.
  • the absorbance values obtained for hematocrit and amount of product formed are converted to % and mmol/L, respectively by intrapolating from standard curves constructed using calibrators of known hematocrit and TG concentration.
  • Example 5 Determination of plasma total TG in the presence of blood cells in a point-of-care instrument
  • Determination of total TG in plasma in the presence of intact blood cells is performed with the instrument and reagent 1 of Example 4, but filtration is used instead of cell lysis to reduce the concentration of intact blood cells, and reflectance is measured instead of absorption to quantify formed colored product.
  • HDL associated CH is determined in blood plasma in the presence of intact blood cells.
  • the HDL associated CH in the sample is measured using three reagents, Rl, R2, and R3 (Table 6).
  • Rl, R2, and R3 three reagents, Rl, R2, and R3 (Table 6).
  • Rl, R2, and R3 three reagents, Rl, R2, and R3 (Table 6).
  • Rl, R2, and R3 Table 6
  • Reaction vessel (RV)-I of the Cartridge is filled with 300 ⁇ L PBS, RV-2 with 100 ⁇ L Rl, RV-3 with 150 ⁇ L R2, and RV-4 with 250 ⁇ L R3.
  • RV-I of the Cartridge is filled with 300 ⁇ L PBS, RV-2 with 100 ⁇ L Rl, RV-3 with 150 ⁇ L R2, and RV-4 with 250 ⁇ L R3.
  • the sample is automatically transferred into RV-I of the Cartridge and diluted in PBS.
  • 50 ⁇ L of diluted sample is transferred from RV-I to RV-2 and mixed with Rl . After 3 min 50 ⁇ L of R2 is transferred from RV-3 to RV-2 and the reaction continued for 3 min. Finally, 50 ⁇ L of R3 is transferred from RV-4 to RV-2, whereupon cell lysis and enzyme inhibition occurs.
  • the amount of colored product is determined by measuring the absorbance at 630 nm.
  • the hematocrit is determined by transferring 50 ⁇ L of diluted sample from RV-I to RV-4 and measuring absorbance at 530 nm. The absorbance values obtained for hematocrit and amount of product formed are converted to % and mmol/L, respectively by intrapolating from standard curves constructed using calibrators of known hematocrit and CH concentration. Table 6.
  • N-bromosuccinimide 50 ⁇ mol/L Methimazole 500 mmol/L Triton XlOO 2 %
  • LDL associated CH is determined in blood plasma in the presence of intact blood cells.
  • the LDL associated CH in the sample is measured using three reagents, Rl, R2, and R3 (Table 7).
  • lO ⁇ L of f ⁇ ngerprick blood is aspirated from a patient.
  • the Sample Device is inserted into the Cartridge and placed in the Afinion instrument.
  • Reaction vessel (RV)-I of the Cartridge is filled with 300 ⁇ L PBS, RV-2 with 100 ⁇ L Rl, RV-3 with 150 ⁇ L R2, and RV-4 with 250 ⁇ L R3.
  • the sample is automatically transferred into RV-I of the Cartridge and diluted in PBS. 50 ⁇ L of diluted sample is transferred from RV-I to RV-2 and mixed with Rl. After 3 min 50 ⁇ L of R2 is transferred from RV-3 to RV-2 and the reaction continued for 3 min.
  • R3 50 ⁇ L of R3 is transferred from RV-4 to RV-2, whereupon cell lysis and enzyme inhibition occurs.
  • the amount of colored product is determined by measuring the absorbance at 630 run.
  • the hematocrit is determined by transferring 50 ⁇ L of diluted sample from RV-I to RV-4 and measuring absorbance at 530 nm.
  • the absorbance values obtained for hematocrit and amount of product formed are converted to % and mmol/L, respectively by intrapolating from standard curves constructed using calibrators of known hematocrit and CH concentration.
  • lO ⁇ L of fingerprick blood is aspirated from a patient.
  • the sample is automatically transferred into reaction vessel 1 (RV-I) of the Cartridge, which contains 190 ⁇ L of Rl .
  • RV-I reaction vessel 1
  • the hematocrit is determined by photometric measurement at 850 nm.
  • 60 ⁇ L of R2 is transferred from RV-2 to RV-I and the reaction continued for 5 min.
  • 50 ⁇ L of R3 is transferred from RV-3 to RV-I, whereupon cell lysis and enzyme inhibition occurs.
  • the amount of colored product is determined by measuring the absorbance at 630 nm.
  • the absorbance values obtained for hematocrit and amount of product formed are converted to % and mmol/L, respectively by intrapolating from standard curves constructed using calibrators of known hematocrit and VLDL concentration.
  • a manual prototype assay for the determination of plasma triglyceride concentration utilizing whole blood was constructed for the point-of-care platform Af ⁇ nion.
  • Blood samples were drawn from 10 healthy volunteers, two samples from each person. One sample was centrifuged to separate plasma and cells. The triglyceride concentration of the samples was analyzed using the prototype assay as well as three reference methods: two point-of-care methods, Colestech Lipid Profile (main reference method) and Callegari Triglycerides, and one automatic laboratory method, ABX Pentra Triglycerides CP.
  • the ABX method uses plasma, Cholestech performs in-device filtration of whole blood and thus does the measurement on plasma, and Callegari uses lyzed whole blood.
  • TG assay reagent (306 iiL):
  • Quench reagent (33 ⁇ L): 4-amino antipyrine 0.9 mol/L Triton X-100 1 %
  • Example 1 A manual prototype assay for total plasma cholesterol in whole blood
  • Blood samples were drawn from 10 healthy volunteers, two samples from each person. One sample was centrifuged to separate plasma and cells. The plasma was used for those tests requiring plasma. Whole blood samples were used for tests utilizing whole blood. The cholesterol concentration of the samples was then analyzed using the prototype assay as well as three reference methods: two point-of- care methods, Colestech Lipid Profile (main reference method) and Callegari Cholesterol, and one automatic laboratory method, ABX Pentra Cholesterol CP. The ABX method uses plasma, Cholestech performes in-device filtration of whole blood and thus measures on plasma, and Callegari ues lyzed whole blood.
  • CH assay reagent Cholesterol oxidase (rec. microbial in E CoIi) 1000 U/L
  • Example 12 A manual prototype assay for plasma HDL in whole blood
  • a manual prototype assay for the determination of plasma HDL concentration using whole blood was constructed for the point-of-care platform Afinion. Blood samples were drawn from 10 healthy volunteers. The samples were centrifuged to separate plasma and cells. The plasma was used for those tests requiring plasma. Whole blood samples were constructed by mixing whole blood and packed cells (from the same donor) 1 :1, to obtain identical hematocrit, 50%, for all samples. The HDL concentration was then analyzed in each sample using the prototype assay as well as three reference methods: two point-of-care instruments, Colestech (main reference method) using capillary blood and Callegari using plasma, and one automatic laboratory instrument, Cobas Mira with WAKO HDL-C M-reagent utilizing plasma. Cholestech performs in-device filtration of the capillary blood sample and thus performs the measurement on plasma. HDL assay reagent 1 TRl) (270 uU:
  • HDL assay reagent 2 (R2) (9OuL): WAKO HDL reagent 2
  • the LDL levels were computed from the measured values for total cholesterol, triglycerides, and HDL cholesterol by using the Friedewald formula (values in mmol/L):
  • LDL Total cholesterol - (HDL + Triglycerides/2.2)
  • Table 13 Summary of averages of Tables 9-12 and method bias compared to Cholestech.

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Abstract

L'invention concerne un procédé enzymatique pour déterminer la concentration plasmatique d'au moins une classe spécifique de lipide et une classe spécifique de lipoprotéine dans un échantillon de sang. L'invention concerne également des trousses utilisées dans le même procédé.
EP08864896A 2007-12-21 2008-12-19 Procédé pour déterminer des lipides Withdrawn EP2235543A1 (fr)

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