DIASYS HBA1C PDF

Kajikinos The method according to claim 1, wherein the at least one stabiliser is hhba1c zwitterionic detergent of the general formula III wherein R is selected from an alkyl residue of a chain length in the range of C8 to C Just 19 of those 39 families can be associated with the so-called neutral zinc metalloprotease. The fructosyl amino acid or fructosyl peptide is oxidised by the activity of the enzyme fructosyl amino acid oxidase FAOX or the enzyme fructosyl peptide oxidase FPOXwherein a result of that oxidation step is the production of hydrogen peroxide H 2 O 2. Further examples of leuco dyes suitable for the present invention are the diphenylamine derivatives described in EP, EP and EP The method hbaa1c to claim 8, wherein the at least one thio compound is selected from thiodiglycol, thiomalic acid, thionicontinomide, thio-NAD and mixtures thereof. In certain embodiments of the invention the pH-value is in the range of 2 to 3. Those reagent 2 variations with respectively different haemolysis solution and reagent 1 were measured on a BM c.

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The method according to claim 1, wherein the at least one stabiliser is a phosphatidylcholine of the general formula I wherein R1 and R2 are selected from completely unsaturated or singly or multiply unsaturated straight-chain or branched-chain fatty acid residues of a chain length in the range of C8 to C The method according to claim 1, wherein the at least one stabiliser is a zwitterionic detergent of the general formula III wherein R is selected from an alkyl residue of a chain length in the range of C8 to C The method according to claim 1, wherein the stabiliser used is a mixture of two or more chemical compounds of the above-indicated kind.

The method according to claim 1, wherein the stabiliser is used with a concentration in the range of 0. The method according to claim 1, wherein quantification of the glycated haemoglobin degradation products is effected in method step c by oxidation thereof by means of fructosyl peptide oxidase or fructosyl amino acid oxidase with the production of hydrogen peroxide and by determining the resulting amount of hydrogen peroxide, wherein the amount of hydrogen peroxide is quantified on the basis of the colour reaction of a leuco dye in the presence of a peroxidase, and wherein the leuco dye is produced in a solution which for stabilisation of the dye contains a compound of the general formula IV : wherein P stands for a phosphorus atom and wherein X1, X2 and X3 are selected independently of each other from substituted or unsubstituted straight-chain or branched-chain C1C8-alkyl residues, substituted or unsubstituted cyclohexyl residues and substituted or unsubstituted phenyl residues.

The method according to claim 7, wherein the solution containing the leuco dye for stabilisation of the dye contains at least one thio compound.

The method according to claim 8, wherein the at least one thio compound is selected from thiodiglycol, thiomalic acid, thionicontinomide, thio-NAD and mixtures thereof. The method according to claim 1, wherein an operation for determining the total haemoglobin concentration is carried out during or between method steps a -c. The reagent kit according to claim 11, wherein the solution R2 for stabilisation of the dye contains at least one compound of the general formula IV : wherein P stands for a phosphorus atom and wherein X1, X2 and X3 are selected independently of each other from substituted or unsubstituted straight-chain or branched-chain C1C8-alkyl residues, substituted or unsubstituted cyclohexyl residues and substituted or unsubstituted phenyl residues.

The reagent kit according to claim 11, wherein the solution R2 for stabilising the leuco dye contains at least one thio compound, wherein the at least one thio compound is preferably selected from thiodiglycol, thiomalic acid, thionicotinamide, thio-NAD and mixtures thereof.

An enzymatic method has also been available for some time, in which a reaction of glycated haemoglobin with a fructosyl amino acid oxidoreductase FAOD is quantified. In the enzymatic investigation—as moreover is also the case with all other HbA1c methods—the first step is to haemolytically rupture the erythrocytes in the blood sample to release the HbA1c contained therein.

The released glycated haemoglobin is then brought into contact with a proteolytic agent to produce glycated haemoglobin degradation products. Those proteolytically produced degradation products include the fructosyl valine Fru-Val and fructosyl valine histidine Fru-Val-His or even longer-chain functosyl peptides, which are cleaved from the amino-terminal end of the beta chain of glycated haemoglobin. The fructosyl amino acid or fructosyl peptide is oxidised by the activity of the enzyme fructosyl amino acid oxidase FAOX or the enzyme fructosyl peptide oxidase FPOX , wherein a result of that oxidation step is the production of hydrogen peroxide H2O2.

The amount of hydrogen peroxide produced in the above-mentioned oxidation step correlates with the amount of fructosylated amino acid or peptide. Accordingly the amount of hydrogen peroxide produced in this step is a measurement in respect of the amount of HbA1c in the sample. Therefore, determination of the amount of HbA1c can ultimately be effected by quantifying the amount of hydrogen peroxide, for example on the basis of a colour reaction which is to be evaluated photometrically and which stoichiometrically correlates with the amount of hydrogen peroxide.

In principle however it is also possible to correspondingly use any other analysis method for quantifying the amount of hydrogen peroxide in a sample. In a given method of quantifying hydrogen peroxide, a reduced leuco dye is oxidised with hydrogen peroxide.

That method however entails the difficulty that autooxidation of the leuco dye causes a non-specific blank value signal and an increase in the spectral background which causes difficulty in precise photometric measurement of the analyte signal. On the other hand leuco dyes have the advantage over other dyes that they usually have higher molecular absorption coefficients. In addition for the major part leuco dyes have such high absorption maxima that optical influencing by interaction with constituents of the blood like for example bilirubin and haemoglobin can generally be disregarded.

It will be appreciated that this also applies to the same extent as for the above-discussed leuco dyes, for the enzymes and other reagents used in HbA1c tests.

At the same time however it is also of not immaterial importance for the stability of the starting material which is to be to be investigated as well as that of the intermediate and end products of the reaction to be ensured to achieve reliably reproducible results. Therefore the inventors of the present application set themselves the object of providing a method of determining the amount of glycated haemoglobin HbA1c in a sample and reagents which can be used in that respect, in which the chemical compounds essential to the reaction are present in sufficiently stable form.

Preferably the aim of the method according to the invention and the reagents according to the invention is to permit total haemoglobin determination at the same time as determining the amount of HbA1c.

DESCRIPTION OF THE INVENTION In accordance with the invention the above-described object is attained by various aspects which are described in detail hereinafter and which usually have the common denominator that they comprise a method of determining the amount of HbA1c in a sample, in which the following method steps are performed: a haemolysis of the erythrocytes in the sample to release the haemoglobin, including HbA1c, contained therein, b bringing the haemoglobin, including HbA1c, released in method step a into contact with a proteolytically acting agent for producing glycated haemoglobin degradation products, and c determining the amount of HbA1c by quantification of the glycated haemoglobin degradation products produced in method step b.

In most cases the sample will be a sample of fresh whole blood. The present invention however also embraces such samples like for example blood preserves, purified blood, whole blood lyophilisate, erythrocytes concentrate, pre-haemolysed blood samples, haemoglobin standard solutions, HbA1c standard solutions and standard solutions which contain synthetic haemoglobin degradation products like for example synthetic HbA1c degradation products. Insofar as the HbA1c is already present free in the sample to be analysed method step a is not required.

In the case of standard solutions which contain synthetic haemoglobin or HbA1c degradation products method step b is in addition also not required. Insofar as the haemoglobin or the HbA1c inherently contained therein or degradation products thereof are not already present in solution in the sample to be analysed they are put into a usually aqueous solution prior to or during step a. Haemolysis of the erythrocytes can basically be effected with all mechanical, chemical or osmotic haemolysis means or methods, of which the man skilled in the art knows that they lead to complete haemolysis of the erythrocytes.

One means or method in accordance with the present invention is deemed to be haemolytically acting when it leads to dissolution of the erythrocytes by destruction of the cell membrane and transfer of the haemoglobin contained in the erythrocytes into the ambient medium.

Examples of haemolytically acting means or methods which are known to the man skilled in the art are ultrasound treatment or the addition of haemolytic detergents or strongly hypotonic salt solutions. In the embodiments of the invention in which haemolytically acting detergents are used they can be selected from non-ionic, anionic, cationic and zwitterionic detergents, wherein the term detergent is used here to mean that this embraces substances which reduce the surface tension of a liquid or the interfacial tension between two phases.

Detergents are organic compounds which are made up of a nonpolar and a polar part, wherein the nonpolar part is at least an alkyl group or an alkylbenzene group and the polar part is selected at least from an alcohol, ether, alcohol-ether, carboxyl, sulphonyl, sulphatyl or quaternary ammonium group.

In the embodiments in which haemolytically acting detergents are used they are preferably stored and used in the form of a haemolysis solution. In principle all proteolytically acting means known to the man skilled in the art fall to be considered like for example proteases, wherein a means in accordance with the present invention has a proteolytic effect when it leads to cleaving of proteins by hydrolysis of the peptide bonds.

In the embodiments in which the proteolytic agent is a protease, it in principle can be selected from all proteases obtained recombinantly from eukaryotes or prokaryotes or obtained endogenously from organisms or organism constituents from serine, threonine, cysteine, asparagine, metal or unknown type, like for example acrosin, aminopeptidase B, bromelain, calpain I, carboxypeptidase A, cathepsin A, cathepsin B, cathepsin D, cathepsin E, cathepsin K, chymotrypsin, collagenase, dipeptidyl peptidase 4, dispase, elastase, factor IIa, factor Xa, ficin, gpr-endopeptidase, HIV-protease, kallikrein, MBTPS1, bromelain, papain, pepsin, plasmin, prepilin type IV peptidase, prolyl-oligopeptidase, proteinase K, proteasom, renin, seccretases alpha-, beta- and gamma-secretase , thermolysin EC 3,4,24,27 , thrombin, trypsin, urokinase, protease N from Bacillus sp.

In certain embodiments of the invention there can be a related advantage in using proteases which specifically cleave HbA1c. In general the present invention however does not require any specificity of the protease in regard to differentiation between glycated and non-glycated haemoglobin.

In that respect therefore it is also possible to use such proteases which do not specifically distinguish between HbA1c and non-glycated haemoglobin. In many embodiments it may even be explicitly desired for a protease to be used, which does not act specifically in a corresponding fashion. In many aspects of the present invention there is no need for the protease used to lead to given degradation products.

In many embodiments of the invention however a protease is specifically used, whose proteolytic activity leads to the release of fructosyl valine histidine or fructosyl valine from the amino-terminal end of the beta chain of glycated haemoglobin.

Determining the amount of HbA1c can basically be effected by all quantification procedures, known to the man skilled in the art, for the glycated haemoglobin degradation products produced in method step b , like for example by an HPLC analysis or enzymatic determination, as was described hereinbefore.

Besides the above-described method the present invention also proposes reagent kits for use in a method of determining the amount of HbA1c in a sample, which are characterised in that they comprise at least two different solutions in separate containers, wherein the at least two different solutions are respectively of such a composition that the various aspects of the invention described in detail hereinafter are implemented.

Aspect 1—Stabilisation of the Protease In accordance with a first aspect the above-described object of the invention is attained in that there is proposed a method of determining the amount of HbA1c in a sample, in which—insofar as required—method steps a to c are performed. In a specific embodiment of the invention the pH-value of the solution is in the range of 4. At the present time 54 metalloprotease families are divided into 15 clans, wherein outstanding significance is attributed to the clan MA with 39 families.

Just 19 of those 39 families can be associated with the so-called neutral zinc metalloprotease. The other metalloprotease are manganese- or cobalt-dependent. Inter alia the following metalloproteases belong to the clan MA: membrane alanyl aminopeptidase, peptidyl-dipeptidase A, thimet-oligopeptidase, oligopeptidase F Lactococcus , mycolysin, Immune-Inhibitor A Bacillus , no neutral streptomyces-protease, leishmanolysin, microbial collagenase, collagenase colA, matrix-metallopeptidase 1, serralysin, fragilysin, autolysin Chlamydomonas , astacin, reprolysin, neprilysin, IgA-specific metalloendopeptidase, t entoxilysin, t hermolysin, neutral staphylococcus-protease, carboxypeptidase t aq, lethal anthrax-factor, deuterolysin, fungalysin, cell cleaving protein ftsH, cytophagalysin, pappalysin 1, Steendopeptidase Saccharomyces , HtpX-endopeptidase E.

If proteases are in their active form over a prolonged period there is the risk that significant proportions of the enzyme are destroyed by self-digestion. In that respect temporary inactivation of the enzymatic activity of proteases is frequently wanted if self-digestion of the enzyme is to be avoided.

The problem of self-digestion of proteases is already known per se in the state of the art. To resolve that problem the state of the art proposed for example for the protease thermolysin removing zinc from the theremolysin by chelators, in particular SH group-containing reagents, or by a simple excess of EDTA.

As calcium or magnesium ions are of essential significance for the stability of the protein structure of the protease the use of an excess of chelator consequently causes destabilisation of the protease protein. Examples of chelators for divalent metal ions, that are suitable in accordance with the present invention, are acetylacetone, nitrilotriacetic acid NTA , ethylenediamine, ethylenediamine tetraacetate EDTA , N- 2-hydroxyethyl -ethylenediamine-N.

The chelator concentration can be freely selected within the above-specified range in dependence on the amount of protease. In an embodiment the solution contains 0. In another embodiment the solution contains 0. The amount of calcium or magnesium ions can also be freely selected in the above-mentioned ranges of concentration. The metal ions can be used in the reagent solution employed in any suitable salt form as long as the selected salt form affords the required amount of dissolved metal ion in the batch like for example chlorides, nitrates, sulphates, formiates and acetates.

In other embodiments the ratio is inn the range of to The inventors of the present application found that this specific amount of the respective divalent metal ions is sufficient to reoccupy the sites in the protein structure of the protease, that are required for activation of the protease, in spite of the simultaneously present significant amounts of chelator and calcium or magnesium ions. The metal ion content of the activation solution can be freely selected in the specified range depending on the respective embodiment involved.

More specifically one of the objects of the present invention is also that of combining together as many as possible of the reagents used in the HbA1c determination operation into as few as possible combined reagent solutions.

The above-mentioned reagent kit can be used in a method of HbA1c determination in the following manner. Firstly—insofar as is required—sample preparation is effected, in which whole blood is mixed with the haemolysing solution. The first reagent solution R1 is then added to the haemolysate resulting therefrom, the result of this being that the FPOX contained in the first reagent solution already breaks down endogenous fructosyle peptides possibly present, but not the terminal fructosylated peptides which are relevant to HbA1c determination as they are not yet released.

After the reaction is substantially concluded photometric determination of the total haemoglobin content of the pre-incubated haemolysate is effected.

Actual incubation is then effected, in which the second reagent solution R2 is added to the pre-incubated haemolysate. The protease apo enzyme contained therein for example thermolysin apo enzyme is activated by the additional divalent metal ions for example zinc ions already contained in the composition by way of the haemolysis solution and cleaves inter alia N-terminal glycated peptide from the beta chain of the haemoglobin.

The cleaved glycated peptide is then reacted by the FPOX, wherein hydrogen peroxide is produced upon cleaving of the glycated peptide into peptide and glucosone. The peroxidase already introduced into the composition by way of the first reagent solution R1 , in the presence of the resulting hydrogen peroxide, causes oxidation of the leuco dye towards its coloured oxidation form. The actual HbA1c determination operation can then be effected by photometric measurement for example very quickly for example 10 to 30 seconds, depending on the nature of the measuring instrument after addition of the second reagent solution R2 , that is to say immediately before the FPOX-induced reaction occurs, and once again at a later time for example 2 to 15 minutes, depending on the respective nature of the measuring instrument after the addition of R2, that is to say after conclusion of the hydrogen peroxide-induced oxidation of the leuco dye.

Ultimately, determination of the HbA1c content is effected in consideration of the difference between the two measurements and by formation of the quotient from the contents of HbA1c and the previously determined total haemoglobin. The measurement intervals however are very heavily dependent on the respectively employed analyser, photometer and so forth. Accordingly only one direct measurement after 2 to 15 minutes would also be possible. Aspect 2—Stabilisation of the Unfolded Hemoglobin Besides stabilisation of the protease the inventors of the present invention also set themselves the object of being able to unfold the haemoglobin contained in a sample, including HbA1c, as greatly as possible, and stabilise it in that unfolded form in order for example to permit digestion of the utmost efficiency of the haemoglobin by a protease and to put the haemoglobin into a measurable photometrically stable form.

It is known from the state of the art that haemoglobin is unfolded to a certain degree by the reduction in pH-value in the haemolycate. In the conventional methods of determining the amount of HbA1c unfolding is effected at a pH-value of about 5. A greater reduction in the HbA1c value however has the disadvantage that the haemoglobin treated in that way is severely denatured and agglutinated and precipitates in that form so that it is no longer available in a suitable form for digestion with a protease.

The inventors of the present invention however found a way in which, with a very low pH-value, extremely rapid and efficient unfolding and subsequent stabilisation of the unfolded haemoglobin can be achieved. The term phosphatidylcholine is used here to denote a compound of the general formula I : wherein R1 and R2 are selected from completely saturated or singly or multiply unsaturated straight-chain or branched-chain fatty acid residues.

In the embodiments with multiply unsaturated fatty acid residues they are preferably doubly, trebly or quadruply unsaturated and in certain embodiments independently of the degree of saturation the fatty acid residues are selected from those with a chain length in the range of C8 to C22 or those with a chain length in the range of C16 to C22 for example 1, 2-dioleoyl-sn-glycerophosphocholine.

Preferably the zwitterionic detergent has exactly two functional groups of opposite charges so that the molecule overall is electrically neutral. Preferably the zwitterionic detergent is selected from at least one chemical compound which is covered by the following general formula III wherein R is selected from an alkyl residue of a chain length in the range C8 to C Preferably R is selected from an alkyl residue of a chain length in the range of C8 to C The stabiliser used according to the invention can be a single chemical compound of the above-indicated kind a compound which is covered by one of formulae I , II or III or a mixture of two or more chemical compounds of the above-indicated kind.

Preferably the stabiliser is used with a concentration in the range of 0. In the cases in which the stabilizer includes a zwitterionic detergent which has a haemolytic action or comprises one or more haemolytically acting zwitterionic detergents various variants are possible: a In the haemolysis operation a haemolytically acting zwitterionic detergent is additionally used as a stabiliser, from which all further haemolytic detergents which are used differ in their chemical constitution so that in this variant a distinction is to be drawn between haemolytically acting stabiliser and a haemolytic detergent which does not have a stabilising action.

After the addition of the haemolysis solution the pH-value of the haemolysate corresponds to that of the haemolysis solution. That is achieved by suitable buffering of the haemolysis solution. The constantly low pH-value of the haemolysate in conjunction with the stabilising detergents has the result that the unfolded haemoglobin can be quickly and efficiently broken down.

The reduction of the pH-value into the range of 1 to 3 leads to very rapid and strong unfolding of the haemoglobin.

AASHTO T321 PDF

DIASYS HBA1C PDF

The method according to claim 1, wherein the at least one stabiliser is a phosphatidylcholine of the general formula I wherein R1 and R2 are selected from completely unsaturated or singly or multiply unsaturated straight-chain or branched-chain fatty acid residues of a chain length in the range of C8 to C The method according to claim 1, wherein the at least one stabiliser is a zwitterionic detergent of the general formula III wherein R is selected from an alkyl residue of a chain length in the range of C8 to C The method according to claim 1, wherein the stabiliser used is a mixture of two or more chemical compounds of the above-indicated kind. The method according to claim 1, wherein the stabiliser is used with a concentration in the range of 0. The method according to claim 1, wherein quantification of the glycated haemoglobin degradation products is effected in method step c by oxidation thereof by means of fructosyl peptide oxidase or fructosyl amino acid oxidase with the production of hydrogen peroxide and by determining the resulting amount of hydrogen peroxide, wherein the amount of hydrogen peroxide is quantified on the basis of the colour reaction of a leuco dye in the presence of a peroxidase, and wherein the leuco dye is produced in a solution which for stabilisation of the dye contains a compound of the general formula IV : wherein P stands for a phosphorus atom and wherein X1, X2 and X3 are selected independently of each other from substituted or unsubstituted straight-chain or branched-chain C1C8-alkyl residues, substituted or unsubstituted cyclohexyl residues and substituted or unsubstituted phenyl residues. The method according to claim 7, wherein the solution containing the leuco dye for stabilisation of the dye contains at least one thio compound.

HIPERTENSION INTRACRANEANA PDF

Kits for respons®: one HbA1c FS

Aranos It will be hga1c that this also applies to the same extent as for the above-discussed leuco dyes, for the enzymes and other reagents used in HbA1c tests. The metal ion content of the activation solution can be freely selected in the specified range depending on the respective embodiment involved. However, as was already mentioned hereinbefore, there is the risk that the haemoglobin is denatured, agglutinated and precipates, as can also be effected for example by adding trichloroacetic acid. In many embodiments of the invention however a protease is specifically used, whose proteolytic activity leads to the release of fructosyl valine histidine or fructosyl valine from the amino-terminal end of the beta chain of glycated haemoglobin. In an embodiment of the present invention the various reagents used in HbA1c determination are brought together in the form of the following solutions which are provided in separate containers: Table 2 C shows the AmE loss of the individual calibrators under different storage conditions. A plurality of thio compounds were checked in respect of their dye-stabilising action.

CHARLES EISENSTEIN ASCENT OF HUMANITY PDF

Diagnostic Reagents and System Solutions of Outstanding Quality

Goltilmaran In that respect therefore it is also possible to use such proteases which do not specifically distinguish between HbA1c and non-glycated haemoglobin. To improve the stability of the leuco dye in a reagent matrix various water-soluble stable substances were checked in regard hba1f their leuco dye-stabilising action. Preferably the zwitterionic detergent is selected from at least one chemical compound which is covered by the following general formula III. In the embodiments of the invention diazys which haemolytically acting detergents are used they can be selected from non-ionic, anionic, cationic and zwitterionic detergents, wherein the term detergent is used here to mean that this embraces substances which reduce the surface tension of a liquid or the interfacial tension between two phases. Alternatively for stabilising the leuco dye it is also possible to add thio compounds, more especially single ones or a plurality of thioalcohols, thioethers, thioketones or mixtures thereof. In principle all proteolytically acting means known to the man skilled in the art fall to be considered like for example proteases, wherein a means in accordance with the present invention has a proteolytic effect when it leads to cleaving of proteins by hydrolysis of the peptide bonds. A markedly greater amount of the protease thermolysin is evidently visible in the bands 4 and 5 in comparison with the other bands of the gel.

6ES7 134-4JB50-0AB0 PDF

HBA1C ANALYZER / POCT DEVICE

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