KR101525206B1 - Method for quantifying glycated hemoglobin using potentiometry without deviation depending hematocrit - Google Patents

Abstract

The present invention relates to a method for quantifying glycated hemoglobin without an error caused by hematocrit using a potentiometry, and more specifically, to a method for quantifying glycated hemoglobin using a potentiometry including a step of withdrawing a polynomial expression to correct difference of the measured displacement values according to the hematocrit using a standard sample with the matrix content of HbA1c with at least two red blood cell hematocrits different from each other, and applying the polynomial expression to the measurement of HbA1c of a unknown sample, to an apparatus for measuring glycated hemoglobin (HbA1c) using a potentiometry, a method for diagnosing diabetes mellitus using the apparatus, and a method for quantifying a target component of a blood sample by applying the principle same as the method.

Description

[0001] The present invention relates to a method for quantifying glycated hemoglobin using potentiometry without error according to the erythrocyte volume,

The present invention relates to a method for quantifying glycosylated hemoglobin using a potentiometric assay method without error according to the erythrocyte volume, and more particularly, to a method for quantifying glycated hemoglobin using a standard sample having a series of known HbA1c concentrations having two or more different erythrocyte volume values, A method of quantifying glycated hemoglobin using a potentiometric assay method, a method of quantitating glycated hemoglobin using a potentiometric assay, a method of measuring a glycated hemoglobin (HbA1c ), A method for diagnosing diabetes using the device, and a method for quantifying a target component in a blood sample by applying the same principle.

Diabetes is caused by improper carbohydrate metabolism that does not use the glucose absorbed in the body, and it can cause various complications due to excessive blood sugar in the blood. Type 1 diabetes is an insulin-dependent diabetes mellitus, which can be said to be a type that loses the ability to synthesize or secrete insulin by the autoimmune response of interest cells. Second, type 2 diabetes is non-insulin dependent diabetes, which is caused by insulin resistance to the insulin or inappropriate insulin secretion. There is also fetal diabetes that can occur during pregnancy. However, type 1 diabetes and fetal diabetes type of diabetes mellitus are not common, and most of the diabetes type 2 diabetes is known to account for 90 to 95% of advanced diabetes mellitus.

There are many ways to diagnose diabetes such as urinary glucose measurement and blood glucose measurement. However, urine glucose measurement is not reliable, and blood glucose measurement is inaccurate due to various factors such as eating and exercise. Among glycohemoglobin, especially HbA1c, which is a sugar-bound hemoglobin, HbA1c shows a good correlation with the blood glucose level when the HbA1c value is an average fasting time in the past 1 to 3 months, to be. In 1986, the American Diabetes Association began using glycated hemoglobin as a measure of diabetes mellitus as a relatively stable indicator of diabetes by proposing two glycated hemoglobin measurements per year to manage all forms of diabetes. In 1993, Direct Control and Complication (DCCT) Trial, Control of Diabetes and Complications), and started to use it in earnest, while reporting the relationship between glycated hemoglobin concentration and diabetic complications.

Regarding the setting of glycated hemoglobin reference, the American Diabetes Association (ADA), based on the reports of DCCT and UKPDS (United Kingdom Prospective Diabetes Study), controls the glycated hemoglobin level to within 7% And recommend reassessment and active treatment of diabetic management if glycated hemoglobin level is 8% or more. In 2001, the US Endocrinology Society presented a reference value of 6.5%, which refers to the results reported by the UKPDS that the incidence of diabetic retinopathy is increased even when it is 6.5% or more. In 1999, the International Diabetes Federation (IDF) also presented glycosylated hemoglobin 6.5% as a reference.

The glycated hemoglobin (HbA1c) value is expressed as a percentage (%) of the amount of HbA1c relative to the total amount of hemoglobin in the blood sample. Conventional HbA1c values were measured using high performance liquid chromatography (HPLC) or immunoassay. An immunoassay for measuring the HbA1c value is a latex immunoagglutination method in which HbA1c in the blood is adsorbed on the surface of latex particles and then reacted with the anti-HbA1c antibody, whereby the aggregation of latex caused by the antigen- Is used. In addition, ion exchange chromatography, affinity chromatography, electrophoresis, complex coloring, and the like are currently being applied commercially. However, these methods are difficult and complex to use and require skilled techniques.

When the HbA1c value is measured by such a method, a step of pretreating the test sample such as whole blood is required. For example, the collected blood is diluted 100-fold with hemolysis to prepare a specimen. The main reason for diluting the sample in this way is to measure the absorbance of the substance to be measured when it is analyzed by HPLC and the turbidity is measured by the immunoassay method. Therefore, the measurement value is adjusted within the detectable range because it is detected by optical method It is for this reason. That is, when the concentration of the sample is high, the measured absorbance and turbidity deviate from the range linearly proportional to the concentration of the substance to be measured, so accurate concentration measurement is impossible.

As described above, the HbA1c value is a percentage of the relative ratio of the amount of HbA1c to the total amount of hemoglobin. Therefore, when measuring the same sample, the value of HbA1c derived independently of the concentration of hemoglobin in the sample should be constant. However, when measured by an actual test apparatus, the HbA1c value measured depending on the concentration of hemoglobin in the specimen may be different. The measurement of the HbA1c value is usually carried out in a region where the hemoglobin concentration and the output of the detector (for example, the absorbance) exhibit a linear relationship, but an error may occur in the HbA1c value for a sample containing a high concentration of hemoglobin outside this region. For example, since the HbA1c value is derived by calculation, even if the HbA1c value is accurately measured, if there is an error in the measurement of the total hemoglobin amount, this error is also reflected in the HbA1c value derived therefrom. Therefore, there may be an error in the HbA1c value due to differences in the hemoglobin concentration in the sample, particularly hematocrit difference due to erythrocytes, due to the sex, age difference, anemia and the like of the subject.

In order to solve such a problem, when the concentration of hemoglobin in a sample is measured and the measured concentration exceeds a predetermined value, a dilution liquid is added to the sample to adjust the concentration of hemoglobin in the sample to fall within an allowable range Japanese Patent Application Laid-Open No. 4-70564). However, since the above-described method needs to additionally provide a valve, a pump, and a stirrer to adjust the concentration of hemoglobin in the sample, the cost of the device increases and the blood dilution treatment results are divided into two steps. There is a drawback that it takes time. In addition, there is a problem that the hemoglobin concentration can be adjusted only when the concentration is high and the hemoglobin concentration is low.

On the other hand, when latex immunoagglutination is used, a sample prepared by hemolysis dilution at a predetermined ratio is dispensed into a reagent solution, and latex particles adsorbed on various hemoglobin surfaces are reacted with an antibody that specifically binds to HbA1c, The turbidity of the reagent solution is optically detected using the phenomenon of agglutination by the HbA1c. Since the sample is diluted at a certain ratio without considering the difference in the concentration of hemoglobin in the sample, which may be caused by the sex, age difference, and anemia of the subject, the error is inevitably caused by the hemoglobin concentration. For example, when the concentration of hemoglobin in the sample is high, an HbA1c value may be generated due to the antigen antibody reaction proceeding even to the free HbA1c not adsorbed on the latex. On the other hand, when the hemoglobin concentration in the specimen is too low, an amount of hemoglobin sufficient to induce aggregation of the latex particles is not adsorbed on the latex, resulting in a lower value than the actual value. The latex agglutination immunization method in which a specimen subjected to dilution with a certain ratio of hemolysis is dispensed into a certain amount of reagent is required to have a separate control reagent for checking whether the HbA1c value is appropriate for the long-term control of diabetes , There is a disadvantage in that the inspection cost is increased due to a large amount of consumed reagent (especially, the antibody component in the reagent) by requiring more inspection process than necessary. Therefore, there is a need to find a detection method that can provide an accurate HbA1c value regardless of the total amount of hemoglobin without requiring a complicated pretreatment or assay procedure.

The present inventors have made intensive researches to find a method for providing a correct glycosylated hemoglobin value by excluding a measurement error which may be caused by hematocrit (Ht) in diagnosing diabetes using a potentiometric assay. As a result, A database is obtained by measuring the potential difference at two or more points using various standard samples having known hematocrit value and glycosylated hemoglobin (HbA1c), and a series of calculations are performed based on the measured potential difference to determine a constant HbA1c And the present invention has been completed.

A first aspect of the present invention is a method for quantifying glycosylated hemoglobin (HbA1c) of a blood sample separated by using potentiometric analysis, comprising the steps of: determining a first erythrocyte volume value selected within a normal erythrocyte volume range, A first step of setting a lower second hematocrit value and a third hematocyte volume value higher than the first hematocrit value; Preparing a first reference sample having a first hemocytic volume value and a known HbA1c value that are different from each other within a concentration range of 4 to 15% (ℓ is an integer of 3 or more), and calculating the second hemocyte volume value (M is an integer equal to or greater than 3) second standard samples having known HbA1c values different from each other within a concentration range of 4 to 15% and having a third hematocrit value of 4 to 15% A second step of preparing n third standard samples having known HbA1c values different from each other within a concentration range (n is an integer of 3 or more); Claim for measuring the potential difference of each of the standard samples in the first, second and third reference samples the first point in time from immediately after (t ₀₎ was added a hemolytic buffer solution each (t ₁₎ and the second point in time (t ₂₎ Step 3; The first standard sample ℓ dogs each HbA1c value of the base a first reference sample ℓ more of the potential difference measured value measured at a first point in time for each (x _ℓ value) or the potential difference measured value measured at a second time point (y _ℓ value ), And the known HbA1c value of each of the second standard samples m is compared with the measured value of the potential difference ( _xm value) measured at the first point of time or the potential difference measured at the second point of time ( _Ym value), and the known HbA1c value of each of the third standard samples n is compared with the measured value of the potential difference ( _xn value) measured at the first point of time or the second point of time To a measured potential difference value (y _n value); (X _l , y _l ) derived from the measured potential difference value (x _L value) measured at the first time point and the measured potential difference value (y _L value) measured at the second time point for each of the first standard sample _L , (X _m value) measured at a first time point and a measured potential difference value (x _m value) measured at a second time point for each of the second standard samples m, and deriving a first potential difference equation or chart for the first hemocyte volume value from the matrix, From the m (x _m , y _m ) matrix derived from the measured potential value (y _m value), a second potentiometric equation or diagram is derived for the second erythrocyte volume value, and for each of the n third standard samples From the n (x _n , y _n ) matrix derived from the potential difference measurement value (x _n value) measured at the first observation point and the potential difference measurement value (y _n value) measured at the second observation point in the case of the third red blood cell volume value A fifth step of deriving a three-potential inter-equation or diagram; (X, y) matrix of the reference sample obtained through the fifth step and the first to third relation or diagram derived therefrom, a potential difference value measured for the standard sample of the known concentration is substituted A sixth step of deriving an uncorrected HbA1c value; By using the first to third relationships derived for the first to third hematocrit values, a potential difference providing an arbitrarily selected concentration value within a 4 to 15% concentration range which is different from the standard sample is derived inversely, A seventh step of securing a pair; An eighth step of deriving first to third'quadratic polynomials for the HbA1c concentration and the potential difference values measured at the first time point t1 with respect to the first to third hemocyte volume values; And a ninth step of deriving an HbA1c value of a corrected unknown sample by applying one of the quadratic polynomial derived from the eighth step to a measured value of an unknown sample, wherein the first to third 'Is repeatedly performed until the quadratic polynomial is similarly derived within a deviation of 0 to 5%. The present invention also provides a method for quantifying HbA1c using the potentiometric assay.

A second aspect of the present invention is an apparatus for measuring glycated hemoglobin (HbA1c) using potentiometric analysis, comprising: a measurement unit having a reference electrode and a working electrode including a site capable of specifically binding to HbA1c on the current collector; A potential difference measurement circuit or potentiometric titration device for measuring a potential difference between a working electrode and a reference electrode by a potential difference analysis method; Control means for instruction from the (t ₀₎ shortly after addition of the hemolysis buffer, the unknown sample to be measured so as to measure the potential difference at a first time (t ₁₎ and the second point in time (t _2); For each of the l first standard samples (where l is an integer greater than or equal to 3) with known HbA1c values that are different from one another within a concentration range of 4 to 15%, with a first erythrocyte volume value selected within the normal red blood cell volume range, A database for a potential difference measured at a first point in time t ₁ and a potential difference measured at a second point in time t ₂ immediately after the addition of the hemolysis buffer solution (t ₀ ); (X _l , y) derived from the measured potential difference value (x _L value) measured at the first time point and the measured potential difference value (y _L value) measured at the second time point for each of the first standard sample As _ℓ) of a first hematocrit value and a lower second hematocrit value than the calculation means that in association with the HbA1c value of the base, the first hematocrit value from the matrix, 4 to 15% concentration range of different bases with each other in the m of the second reference samples the potential difference and the second point measured at the first point in time (t _1), immediately after (t ₀₎ by addition of hemolysis buffer for the (m being three or more integer) each having a HbA1c value (t ₂ A database for the potential difference measured in the database; (X _m , y) derived from the measured potential difference value (x _m value) measured at the first time point and the measured potential difference value y _m value measured at the second time point for each of the m second standard samples _m < / _RTI &_gt; matrix and a second HbA1c value that is greater than the first hemoglobin volume value and which has a third hematocrit value greater than the first hemoglobin volume value, of having a HbA1c value n of third reference samples (n is an integer greater than or equal to 3) for each of the potential difference and the second time measurement immediately after the addition of a hemolytic buffer solution from the (t ₀₎ at a first time (t ₁₎ (t ₂ ) a database of measured potential differences; (X _n , y) derived from the potential difference measurement value (x _n value) measured at the first time point and the potential difference measurement value (y _n value) measured at the second time point for each of the n second reference samples, _n ) matrix corresponding to a third red blood cell volume value and a known HbA1c value; The measured value of the potential difference measured at the first point of time t ₁ and the measured value of the potential difference measured at the second point of time t _{2 of the} unknown sample are introduced into the database or calculation means, Means for deriving an HbA1c value by deriving a Ht value and correcting for the Ht value of the derived unknown sample from the database or the calculation means of the reference sample; And a display unit for displaying the derived HbA1c value.

A third aspect of the present invention is a method for measuring HbA1c in a blood sample separated from a subject suspected of having diabetes using a device for measuring HbA1c according to the second aspect of the present invention, And a second step of determining that diabetes has occurred if the measured value is 6.5% or more.

In a fourth aspect of the present invention, there is provided a method of quantifying a target component in a blood sample separated by using potentiometry, wherein the sample has a hematocrit value of 3 or more known concentrations prior to the signal measurement on the unknown sample, A first step of preparing a standard sample containing a target component at a known concentration and measuring a signal to provide a relationship between a plurality of signals and a red blood cell volume value; A second step of measuring a signal for an unknown blood sample; And a third step of obtaining a corrected value by applying a measurement value for an unknown blood sample obtained from the second step to a relation provided from the first step.

Hereinafter, the present invention will be described in detail.

The present invention is based on the fact that the potential difference with respect to the glycated hemoglobin measured according to the hematocrit (Ht) differs from that of the glycated hemoglobin with the same concentration in diagnosing diabetes using the potential difference measurement method, This study was designed to solve the error in the measured glycosylated hemoglobin concentration and the diagnostic errors that may appear in the diagnosis of diabetes based on this.

The present invention is based on the use of various standard samples having a known hematocrit value and a glycosylated hemoglobin (HbA1c) value in order to find a method capable of providing an accurate glycosylated hemoglobin value by excluding the error due to the difference in the erythrocyte volume value of the sample. It is based on the first finding that a constant HbA1c value can be derived irrespective of the Ht value by measuring a potential difference at a time point or more, securing a database, and performing a series of calculations based thereon.

A method for quantifying glycosylated hemoglobin (HbA1c) of a blood sample separated by using the potentiometric assay of the present invention is characterized by measuring a first hemocytic blood volume value selected within a normal hemocytic blood volume range, a second hemocytic hematocrit value And setting a third erythrocyte volume value higher than the first erythrocyte volume value; Preparing a first reference sample having a first hemocytic volume value and a known HbA1c value different from each other within a concentration range of 4 to 15% (ℓ is an integer of 3 or more), and calculating the second hemocyte volume value (M is an integer equal to or greater than 3) second standard samples having known HbA1c values different from each other within a concentration range of 4 to 15% and having a third hematocrit value of 4 to 15% A second step of preparing n third standard samples having known HbA1c values different from each other within a concentration range (n is an integer of 3 or more); Claim for measuring the potential difference of each of the standard samples in the first, second and third reference samples the first point in time from immediately after (t ₀₎ was added a hemolytic buffer solution each (t ₁₎ and the second point in time (t ₂₎ Step 3; The first standard sample ℓ dogs each HbA1c value of the base a first reference sample ℓ more of the potential difference measured value measured at a first point in time for each (x _ℓ value) or the potential difference measured value measured at a second time point (y _ℓ value ), And the known HbA1c value of each of the second standard samples m is compared with the measured value of the potential difference ( _xm value) measured at the first point of time or the potential difference measured at the second point of time ( _Ym value), and the known HbA1c value of each of the third standard samples n is compared with the measured value of the potential difference ( _xn value) measured at the first point of time or the second point of time To a measured potential difference value (y _n value); (X _l , y _l ) derived from the measured potential difference value (x _L value) measured at the first time point and the measured potential difference value (y _L value) measured at the second time point for each of the first standard sample _L , (X _m value) measured at a first time point and a measured potential difference value (x _m value) measured at a second time point for each of the second standard samples m, and deriving a first potential difference equation or chart for the first hemocyte volume value from the matrix, From the m (x _m , y _m ) matrix derived from the measured potential value (y _m value), a second potentiometric equation or diagram is derived for the second erythrocyte volume value, and for each of the n third standard samples From the n (x _n , y _n ) matrix derived from the potential difference measurement value (x _n value) measured at the first observation point and the potential difference measurement value (y _n value) measured at the second observation point in the case of the third red blood cell volume value A fifth step of deriving a three-potential inter-equation or diagram; (X, y) matrix of the reference sample obtained through the fifth step and the first to third relation or diagram derived therefrom, a potential difference value measured for the standard sample of the known concentration is substituted A sixth step of deriving an uncorrected HbA1c value; By using the first to third relationships derived for the first to third hematocrit values, a potential difference providing an arbitrarily selected concentration value within a 4 to 15% concentration range which is different from the standard sample is derived inversely, A seventh step of securing a pair; An eighth step of deriving first to third'quadratic polynomials for the HbA1c concentration and the potential difference values measured at the first time point t1 with respect to the first to third hemocyte volume values; And a ninth step of deriving the corrected HbA1c value of the unknown sample by applying one of the quadratic polynomials derived from the eighth step to the measured value of the unknown sample. In this case, the sixth to eighth steps may be repeated until the derived first to third 'second order polynomials are similarly derived within a deviation of 0 to 5%.

Preferably, the equation derived in the fifth step may be a quadratic equation.

The term " hematocrit (Ht; HCT) "of the present invention is also referred to as PCV (packed cell volume) or EVF (erythrocyte volume fraction) as the volume ratio of red blood cells in blood. Approximately 45% of normal adult males and about 40% of normal adult females have individual differences, ranging from about 36% to 10% for normal adults, but about 45% to 68% for newborns , And about 28 to 42% in infancy to boyhood. The hematocrit value is performed as part of a general blood test and can be used for anemia, malnutrition, leukemia (blood cancer) and other medical conditions.

Preferably, the first erythrocyte volume value can be selected in the range of 35 to 50%.

On the other hand, preferably, the second hematocrit value and the third hematocrit value can be selected within the range of 20 to 35% and 50 to 60%, respectively, lower or higher than the hematocrit value of a normal adult.

In a specific example of the present invention, the first hemocyte volume value was 41%, which is a value found in a normal adult. In addition, 29% and 55%, respectively, were selected as the second and third erythrocyte volume values, respectively.

Further, the apparatus for measuring glycated hemoglobin (HbA1c) using the potentiometric analysis method of the present invention comprises: a measurement unit having a reference electrode and a working electrode including a site capable of specifically binding to HbA1c on the current collector; A potential difference measurement circuit or potentiometric titration device for measuring a potential difference between a working electrode and a reference electrode by a potential difference analysis method; Control means for instruction from the (t ₀₎ shortly after addition of the hemolysis buffer, the unknown sample to be measured so as to measure the potential difference at a first time (t ₁₎ and the second point in time (t _2); For each of the l first standard samples (where l is an integer greater than or equal to 3) with known HbA1c values that are different from one another within a concentration range of 4 to 15%, with a first erythrocyte volume value selected within the normal red blood cell volume range, A database for a potential difference measured at a first point in time t ₁ and a potential difference measured at a second point in time t ₂ immediately after the addition of the hemolysis buffer solution (t ₀ ); (X _l , y) derived from the measured potential difference value (x _L value) measured at the first time point and the measured potential difference value (y _L value) measured at the second time point for each of the first standard sample As _ℓ) of a first hematocrit value and a lower second hematocrit value than the calculation means that in association with the HbA1c value of the base, the first hematocrit value from the matrix, 4 to 15% concentration range of different bases with each other in the m of the second reference samples the potential difference and the second point measured at the first point in time (t _1), immediately after (t ₀₎ by addition of hemolysis buffer for the (m being three or more integer) each having a HbA1c value (t ₂ A database for the potential difference measured in the database; (X _m , y) derived from the measured potential difference value (x _m value) measured at the first time point and the measured potential difference value y _m value measured at the second time point for each of the m second standard samples _m < / _RTI &_gt; matrix and a second HbA1c value that is greater than the first hemoglobin volume value and which has a third hematocrit value greater than the first hemoglobin volume value, of having a HbA1c value n of third reference samples (n is an integer greater than or equal to 3) for each of the potential difference and the second time measurement immediately after the addition of a hemolytic buffer solution from the (t ₀₎ at a first time (t ₁₎ (t ₂ ) a database of measured potential differences; (X _n , y) derived from the potential difference measurement value (x _n value) measured at the first time point and the potential difference measurement value (y _n value) measured at the second time point for each of the n second reference samples, _n ) matrix corresponding to a third red blood cell volume value and a known HbA1c value; The measured value of the potential difference measured at the first point of time t ₁ and the measured value of the potential difference measured at the second point of time t _{2 of the} unknown sample are introduced into the database or calculation means, Means for deriving an HbA1c value by deriving a Ht value and correcting for the Ht value of the derived unknown sample from the database or the calculation means of the reference sample; And a display unit displaying the derived HbA1c value. Particularly, in the same manner as in the first aspect of the present invention, a database includes a measured potential difference value for a standard sample having a different red blood cell volume value, and a polynomial equation for correcting an error of a measured value according to the difference in red blood cell volume value derived therefrom And a calculation means for providing a corrected measurement value by using the calculation means.

Therefore, the apparatus for measuring HbA1c according to the present invention corrects the potential difference value measured differently according to the red blood cell volume value of the blood sample through the provided calculation means, so that even if the hemoglobin volume value is different for the same HbA1c value sample, the same HbA1c value Lt; / RTI > In other words, since the HbA1c value can be provided by correcting the error according to the red blood cell volume value, HbA1c value proportional to the HbA1c concentration in the blood sample can be obtained regardless of the erythrocyte volume value. One diabetes diagnosis is possible.

A first step of measuring the HbA1c level of blood isolated from a subject suspected of having diabetes using the apparatus for measuring HbA1c of the present invention; And a second step of determining that the diabetes has occurred if the measured value is 6.5% or more, to provide information for diagnosing diabetes.

The term "diagnosis" of the present invention means identifying the presence or characteristic of a pathological condition. In the present invention, the diagnosis can be interpreted as confirming the onset of diabetes.

The term "individual" of the present invention means all animals, including humans, who have developed or may have developed diabetes. The method of the present invention can be used to diagnose diabetes mellitus in a subject when a blood sample separated from an individual is put into an apparatus for measuring HbA1c of the present invention and the measured value is read and the value is 6.5% or more. In addition, even if the above value is less than 6.5, if the value is 5.7% or more, it can be diagnosed as a patient having a high risk of diabetes. Accordingly, the device for measuring HbA1c according to the present invention can be used to diagnose the incidence or risk of developing diabetes in a subject, and the result may lead to appropriate measures for the treatment or prevention of diabetes.

The principle and method for correcting the difference in the measured value of the potential difference signal according to the difference of the red blood cell volume value in the method of quantifying glycated hemoglobin (HbA1c) of the separated blood sample using the potentiometric analysis method according to the present invention are as follows: HbA1c It can also be applied to the analysis of other components. Specifically, a method for quantifying an objective component in a blood sample separated by using a potential difference analysis method, the method comprising the steps of: prior to signal measurement for an unknown sample, measuring a blood sample having a known red blood cell volume value of 3 or more, A first step of preparing a standard sample containing a component and measuring a potential difference signal to provide a relationship between a plurality of signals and a red blood cell volume value; A second step of measuring a potential difference signal with respect to an unknown blood sample; And a third step of obtaining a corrected value by applying a measurement value for an unknown blood sample obtained from the second step to a relation provided from the first step.

The target components that can be quantified using the method of the present invention include glucose, C-reactive protein (CRP), thyroid-stimulating hormone (TSH), free triiodothyronine (FT3) (HBsAg), hepatitis B core antigen (HBcAg), hepatitis C virus (HBsAg), thyroxine, human chorionic gonadotropin (hCG), hepatitis B surface antibody hepatitis C virus antibody (HCV Ab), TY antigen, antistreptotrypsin O (ASO), type 4 collagen, MMP-3 (matrix metalloproteinase-3) and PIVKA-II antagonist-II), alpha1 microbullin, beta1 microbuline, serum amyloid A (SAA), elastase 1, basic fetoprotein (BFP), Candida antigen ), Granulocyte elastase, digoxin in cervical mucus, The present invention relates to a pharmaceutical composition for the treatment and prophylaxis of cystatin C, transferrin (Tf), hyaluronic acid, fibrin monomer complex, von Willebrand factor (vWF), rheumatoid factor, immunoglobulin D (a1-acidglycoprotein),? 1 -antitrypsin (? 1-AT),? 2-macroglobulin (A1b), ceruloplasmin (Cp), haptoglobin (haptoglobin; Hp), prealbumin, retinol binding protein (RBP), β1C / β1A globulin (C3), β1E globulin (C4), immunoglobulin A (IgA), immunoglobulin G ; IgG), immunoglobulin M (IgM), beta-lipoprotein, apoprotein AI, apoprotein A-II, apoprotein B, apoprotein C-II, apoprotein C -III, apoprotein E, albumin, plasminogen (PLG), lactate, low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triacylglycerol and triacylglycerol (TG).

The method for quantifying the target component in the blood sample of the present invention is a method for measuring the change in the measured value for the objective component measured by the error of the measured value of the target component by the erythrocytes contained in the blood, When the erythrocyte volume value is high for the target component of concentration, when the measured potential difference value is low and the erythrocyte volume value is low, the measured potential difference value is high. Therefore, the erythrocyte volume value is corrected to eliminate the influence thereof, This can be achieved by applying a correction polynomial derived from a standard sample of the target component in order to provide the same measurement value for the sample. Therefore, if the potential difference value measured by the red blood cell volume value in blood is an object component to be affected, quantitative analysis can be performed by applying the method of the present invention without limitation.

A device for measuring glycosylated hemoglobin (HbA1c) of a blood sample using a potential difference analysis method including a step of correcting a red blood cell volume value of the present invention and a calculation means for achieving the same, It is possible to diagnose diabetes more precisely because it provides a constant HbA1c value regardless of the Ht value by excluding the variation of the measured HbA1c value according to the difference of the red blood cell volume (Ht) appearing. In addition, by applying the same principle, it is possible to correct the variation of the measured value by the red blood cell volume value in analyzing the target component in the blood other than the HbA1c value, so that more accurate and reproducible quantitative analysis of various blood components is also possible.

Figure 1 shows the measured values at 30 and 60 seconds for a series of standard samples (Ht; 29%, 41% and 55%, 4, 7, 9, 12 and 14% HbA1c for each Ht value) Potential difference and an optimization curve therefor.
Figure 2 shows the potential difference measured at 30 seconds for a series of standard samples (Ht; 29%, 41% and 55%, 4, 7, 9, 12 and 14% HbA1c for each Ht value) 0.0 > HbA1c < / RTI >
Figure 3 shows the results of a series of experiments in the range 0-5000 [mu] V for a series of standard samples (Ht; 29%, 41% and 55%, 4, 7, 9, 12 and 14% HbA1c for each Ht value) FIG. 5 is a graph showing HbA1c values derived from the respective optimization curves derived from the FIG. 1 with respect to the potential difference at 60 seconds set in units.
FIG. 4 shows the difference between the Ht value and the actual Ht value calculated from FIG. 3 using the potential difference measured at 60 seconds for a predetermined HbA1c concentration (6.5, 7.7, 11.5 and 13.3% Fig.
5 is a diagram showing a calculation method of the unmodified HbA1c value based on the potential difference measured at 60 seconds.
FIG. 6 is a graph showing HbA1c values measured for samples having different Ht values measured by a potential difference analysis method using an algorithm for Ht error correction according to the present invention.
7 is a graph showing HbA1c values measured for samples having different Ht values measured by potentiometric analysis without applying an algorithm for Ht error correction according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for describing the present invention more specifically, and the scope of the present invention is not limited by these examples.

Example  1: Red blood cell volume (Ht) Glycosylated hemoglobin  Comparative Analysis of Numbers

Standard samples of predetermined hematocrit (Ht) values (29%, 41% and 55% ± 2%) were prepared using venous blood (hereinafter referred to as Ht 29%, Ht 41% and Ht 55% ). Samples were prepared to contain HbA1c at predetermined concentrations (4, 7, 9, 12 and 14%) for each of the standard samples. Potential differences caused by the reaction of boric acid and HbA1c were measured on the above prepared samples using an electrochemical analyzer of BAS. In addition, the potential difference was measured using a biosensor (GBS KOREA) developed by the present inventor for measuring HbA1c. Potential difference measurement was performed at 30 seconds and 60 seconds with the moment when the buffer was added to the blood at 0 seconds. Each sample was repeatedly measured five times and the average value was calculated. The potential difference measured 30 seconds after the reaction and 60 seconds after the reaction for the series of HbA1c concentrations are plotted on the y-axis and the x-axis, respectively, and the results are shown in Fig. The experiment was carried out on each Ht value (open rhombus: Ht 29%, open squares: Ht 41% and open triangle: Ht 55%) and is shown together in FIG. The potential differences measured at 30 and 60 seconds for a series of varying concentrations of HbA1c (4, 7, 9, 12 and 14%) at each Ht concentration were optimized with a quadratic equation. The potential difference measured at the above two points showed the lowest value at Ht 55%, which is relatively high in Ht concentration, and increased sequentially from Ht 41% to Ht 29%. That is, for the same concentration of HbA1c, the measured potential difference decreases when the Ht value is high. In particular, the decline increased from 60 seconds to 30 seconds. This indicates that the potential difference measured is affected by the Ht value and the decrease width is increased when the time from the start of the reaction to the measurement is delayed. Therefore, we tried to compensate the variation of measured potential difference according to the Ht value from the difference of measured values at two points of view based on the behavior of the measured potential difference according to the concentration of Ht.

In addition, the potential difference measured at 30 seconds relative to the HbA1c concentration of each standard sample is shown in Fig. The series of data measured for each Ht concentration was also optimized with a quadratic equation. As shown in FIG. 2, when only one potential, that is, a potential difference at 30 seconds is shown, the concentration of HbA1c derived from the potential difference of the measured specific value differs depending on the Ht concentration. For example, when the measured potential difference is 5000 占,, the concentration of HbA1c derived therefrom is about 8% for Ht 29%, about 9.4% for Ht 41%, and about 11.5% for Ht 55% And a tendency to increase as the measured potential difference increases. Generally, when the value is less than or equal to 6.5%, the diagnosis result is normal, and when it is more than that, the diagnosis of diabetes is made. However, since different HbA1c concentrations measured at the same Ht concentration each exhibit a constant pattern, it can not be defined that either one value is correct or the other value is incorrect. Therefore, it was confirmed that a method of correcting the deviation of the HbA1c value induced by the difference of the measured value according to the Ht concentration is required.

Example  2: Ht  Error correction for the value

As shown in Fig. 1, a quadratic equation for the correlation of the potential difference was derived at 30 seconds and 60 seconds measured at the specific Ht value from the above-mentioned Example 1. [ The derived equations are as follows and the x and y values correspond to the potential difference values measured at 60 seconds and 30 seconds coinciding with the respective axes in Fig.

; R²=0.9971,About Ht 29%

; R ² = 0.9971,

; R²=0.9967, 및About Ht 41%

; R ² = 0.9967, and

; R²=0.9996.About Ht 55%

; R ² = 0.9996.

The values of y were calculated by assigning 0 to 5000 μV to each equation at 1000 μV intervals, and the results are shown in Table 1. That is, these values represent the expected potential difference at 30 seconds when the potential difference at 60 seconds is assumed to be 0 to 500 μV, respectively. This is shown in Fig.

60 seconds Potential difference (μV) 30 sec Potential difference (μV) Ht 29% Ht 41% Ht 55% 0 73.36 453.49 631.87 1000 1804.66 1716.19 1658.57 2000 3475.96 3038.89 2705.27 3000 5087.26 4421.59 3771.97 4000 6638.56 5864.29 4858.67 5000 8129.86 7366.99 5965.37

Based on the above Table 1, the error of Ht, that is, the difference between the calculated Ht value and the actual Ht value was obtained. Specifically, a sample containing HbA1c at various concentrations (6.5, 7.7, 11.5 and 13.3%) and having a known Ht value was prepared, and the potential difference was measured at 60 seconds after mixing with the buffer solution. Ht value was calculated inversely. Then, the difference between the calculated Ht value and the known Ht value of the sample is calculated and shown in FIG. As shown in FIG. 4, the HbA1c concentration showed an extremely low error value for samples in the range of 4 to 14%. Through this process, the Ht value can be converted more accurately within the HbA1c concentration range mentioned in Table 1 and FIG.

Example  3: Ht  Derivation and Application of Error Correction Algorithm for Value

Using the measurement results in Example 1, the measured potential difference for each HbA1c concentration of Ht 55% and the measured potential difference for each HbA1c concentration of Ht 21% were converged to the measured potential difference for the corresponding HbA1c concentration of Hb 41% The correction algorithm was derived using commercially available software, Microsoft Office Excel (R). The derivation process is as follows.

1. Since the normal range of Ht is 34.9 to 44.5 in adult female and 38.8 to 50.0 in adult male, the buffer is added to the sample having the intermediate value of 41% Ht in the above numerical value, and based on the measured potential difference after 60 seconds The relationship between the HbA1c concentration and the potential difference was created by a quadratic equation. The above equation is the unmodified HbA1c equation.

2. The HbA1c value was calculated based on the potential difference at 60 seconds measured in the first step. The HbA1c value was defined as the unmodified HbA1c value.

3. Using the potential difference measured at 30 seconds and the potential difference measured at 60 seconds, interpolation was performed from the Ht-potential difference as shown in Table 1, and the Ht value was deduced therefrom.

4. A quadratic equation optimized for low Ht (e.g., 29%), intermediate Ht (e.g., 41%) and high Ht (e.g., 55%) for the HbA1c value and unadjusted HbA1c values calculated from the second step To calculate the HbA1c value based on 11% unmodified HbA1c. From this, HbA1c value (Ht error HbA1c 11% value) with Ht error was obtained for Ht29%, Ht41% and Ht55% (11% for 41% Ht value when there is no error).

5. Using the 11% HbA1c value of the three Ht values calculated from the fourth step, a quadratic equation of Ht was calculated for 11% HbA1c with Ht error. This is called the correction equation of Ht.

6. The corrected HbA1c estimate based on Ht 41% was calculated using the uncorrected HbA1c value, the Ht estimate, and the Ht correction equation. The estimate is the last corrected HbA1c estimate.

As an example of the above process of performing the error correction for the Ht value, the potential difference measured at 60 seconds was plotted and the unmodified HbA1c value was obtained using the optimization curve derived therefrom. This is shown in FIG. The optimization curve derived for each Ht value is:

,About Ht 29%

,

, 및About Ht 41%

, And

.About Ht 55%

.

Therefore, x = 11 was substituted for each of the derived equations to obtain values of 3837.75 μV, 3776.07 μV and 3628.98 μV for 29% Ht, 41% Ht and 55% Ht, respectively. When these values are recalculated by adding them as data points, they converge to approximately the same values for the three Ht concentrations.

As described above, the concentration of HbA1c was calculated from the potential difference at two points in each standard sample in Example 1 using the above algorithm, and this is shown in FIG. FIG. 6 is a graph showing the relationship between the Ht value and the 41% Ht value by applying the algorithm derived through the above-described procedure so as to exclude the difference of the Ht value from the Ht value measured for the standard samples of various known concentrations The results are shown in Fig. In FIG. 6, the results for Ht 29% are open rhombus, the results for Ht 41% are shown as open squares, and the results for Ht 55% are shown as open triangles, all superimposed on points corresponding to the known concentrations. For comparison, the HbA1c concentration was calculated without such a correction procedure, that is, the step of correcting the Ht value with the measurement result at 60 seconds, and plotted against the known HbA1c concentration, and is shown in FIG. Specifically, the data was calculated from the equation shown in FIG. As shown in Fig. 7, when calculating the HbA1c value from the measured potential difference without the correction process for the Ht value, a different HbA1c value was provided depending on the Ht value. For example, HbA1c values were relatively high for samples with low Ht values and HbA1c values were low for samples with high Ht values. These data are proportional to the concentration of HbA1c for a sample with a single Ht value, but the results for a sample with the same HbA1c value are different depending on the Ht value, It is difficult to do. For example, in patients with low Ht values below normal (41%) according to age, sex, and physical condition of the patients to be collected, the measured HbA1c value may be higher than the actual concentration, Failure to classify patients as suspicious can lead to errors. On the contrary, in patients with high Ht values, the measured result shows a lower HbA1c than the actual value, and there is a risk that the patient suspected of diabetes is normal.

Therefore, correcting the measured signal with respect to an unknown sample by referring to the relationship between the amount of the target component and a plurality of signals therebetween enables a more accurate quantitative analysis and diagnosis through it, so that it is useful not only in research but also in clinical use .

Claims (9)

A method for quantifying glycosylated hemoglobin (HbA1c) of a blood sample separated by a potentiometric assay,
A first step of setting a first erythrocyte volume value selected within a normal erythrocyte volume range, a second erythrocyte volume value lower than the first erythrocyte volume value and a third erythrocyte volume value higher than the first erythrocyte volume value;
Preparing a first reference sample having a known hematocrit value and having known HbA1c values different from each other within a concentration range of 4 to 15% (ℓ is an integer of 3 or more)
Preparing m second standard samples (m is an integer of 3 or more) having the second hematocrit, having different known HbA1c values within the 4-15% concentration range,
A second step of preparing n third standard samples having the third hematocrit value and different known HbA1c values within a concentration range of 4 to 15% (n is an integer of 3 or more);
Claim for measuring the potential difference of each of the standard samples in the first, second and third reference samples the first point in time from immediately after (t ₀₎ was added a hemolytic buffer solution each (t ₁₎ and the second point in time (t ₂₎ Step 3;
The first standard sample ℓ dogs each HbA1c value of the base a first reference sample ℓ more of the potential difference measured value measured at a first point in time for each (x _ℓ value) or the potential difference measured value measured at a second time point (y _ℓ value ),
The second reference samples; m each HbA1c value of a known second reference samples the potential difference measurement value (x _m values) measured at a first time for the m pieces respectively, or the potential difference measured value measured at a second time point (y _m value ),
Third HbA1c value of a standard sample of n each base for the third standard sample n number of the potential difference measured value measured at a first point in time for each (x _n values) or the potential difference measured value measured at a second time point (y _n value ); And
(X _l , y _l ) derived from the measured potential difference value (x _L value) measured at the first time point and the measured potential difference value (y _L value) measured at the second time point for each of the first standard sample _L , Deriving from the matrix a first relationship or a first diagram showing a first potential difference relationship with respect to a first erythrocyte volume value at a measurement time point,
(X _m , y _m ) derived from the measured potential difference value (x _m value) measured at the first point in time and the measured potential difference value (y _m value) measured at the second point for each of the second standard samples, Deriving from the matrix a second relationship or a second diagram showing the relationship between the second potential differences with respect to the second erythrocyte volume value at the time of measurement,
(X _n , y _n ) derived from the potential difference measurement value (x _n value) measured at the first time point and the potential difference measurement value (y _n value) measured at the second time point, A fifth step of deriving a third relationship or a third diagram showing a relation between third potential differences according to a measurement time point of the third erythrocyte volume value from the matrix;
Based on the (x, y) matrix for the standard sample obtained through the fifth step and the first to third relation or diagram derived therefrom,
A sixth step of deriving an unmodified HbA1c value by substituting the measured potential difference value with the reference sample of the known concentration;
By using the first to third relationships derived for the first to third hematocrit values, a potential difference providing an arbitrarily selected concentration value within a 4 to 15% concentration range which is different from the standard sample is derived inversely, A seventh step of securing a pair;
The first to eighth steps of: deriving a first 'to 3' on the three quadratic polynomials for the potential difference values measured in the HbA1c concentration and the first point in time (t ₁₎ for the hematocrit value; And
And a ninth step of deriving an HbA1c value of a corrected unknown sample by applying one of the quadratic polynomials derived from the eighth step to a measured value of the unknown sample,
And the sixth to eighth steps are repeatedly performed until the derived first to third 'second order polynomials are similarly derived within a deviation of 0 to 5%. The method of quantifying HbA1c using potentiometric analysis.
The method according to claim 1,
Wherein the first hematocrit value is selected in the range of 35 to 50%.
The method according to claim 1,
Wherein the second hematocrit value and the third hematocrit value are selected within the range of 20 to 35% and 50 to 60%, respectively.
The method according to claim 1,
Wherein the first to third relationships derived in the fifth step are quadratic equations.
A device for measuring glycosylated hemoglobin (HbA1c) using a potentiometric assay,
A measurement unit having a reference electrode and a working electrode including a site capable of specifically binding to HbA1c on the current collector;
A potential difference measurement circuit or potentiometric titration device for measuring a potential difference between a working electrode and a reference electrode by a potential difference analysis method;
Control means for instruction from the (t ₀₎ shortly after addition of the hemolysis buffer, the unknown sample to be measured so as to measure the potential difference at a first time (t ₁₎ and the second point in time (t _2);
For each of the l first standard samples (where l is an integer greater than or equal to 3) with known HbA1c values that are different from one another within a concentration range of 4 to 15%, with a first erythrocyte volume value selected within the normal red blood cell volume range, A database for a potential difference measured at a first point in time t ₁ and a potential difference measured at a second point in time t ₂ immediately after the addition of the hemolysis buffer solution (t ₀ ); (X _l , y) derived from the measured potential difference value (x _L value) measured at the first time point and the measured potential difference value (y _L value) measured at the second time point for each of the first standard sample _ℓ ) computing means for mapping the first hemocyte volume value and the known HbA1c value from the matrix,
For each of the m second standard samples (m is an integer greater than or equal to 3) having a second hematocrit value lower than the first hematocrit, and having known HbA1c values different from each other within a 4-15% concentration range, A database for a potential difference measured at a first point in time t ₁ and a potential difference measured at a second point in time t ₂ immediately after the addition of the hemolysis buffer solution (t ₀ ); (X _m , y) derived from the measured potential difference value (x _m value) measured at the first time point and the measured potential difference value y _m value measured at the second time point for each of the m second standard samples _m ) means for associating a second hemocytic blood volume value and a known HbA1c value from the matrix, and
For each of the n third standard samples (where n is an integer of 3 or more) having a known hematocrit value higher than the first hematocrit, and having different known HbA1c values within the 4-15% concentration range, A database for a potential difference measured at a first point in time t ₁ and a potential difference measured at a second point in time t ₂ immediately after the addition of the hemolysis buffer solution (t ₀ ); (X _n , y) derived from the potential difference measurement value (x _n value) measured at the first time point and the potential difference measurement value (y _n value) measured at the second time point for each of the n second reference samples, _n ) matrix corresponding to a third red blood cell volume value and a known HbA1c value;
The measured value of the potential difference measured at the first point of time t ₁ and the measured value of the potential difference measured at the second point of time t _{2 of the} unknown sample are introduced into the database or calculation means, Means for deriving an erythrocyte volume value and deriving an HbA1c value by correcting the Ht value of the unknown sample derived from the database or the calculation means of the reference sample; And
And a display unit for displaying the derived HbA1c value.
A first step of measuring HbA1c levels of blood isolated from a subject suspected of having diabetes by using the apparatus for measuring HbA1c according to claim 5; And
And a second step of determining that diabetes has occurred if the measured value is 6.5% or more.
delete delete delete

Images