Measurement method for glycosylated hemoglobin

Abstract

Provided is a measurement method for HbA1c that includes a step for calculating a peak area for unstable glycosylated hemoglobin E included in peaks for unstable glycosylated hemoglobin A1c or stable glycosylated hemoglobin A1c from a peak area for stable glycosylated hemoglobin E, a peak area for unstable glycosylated hemoglobin A1c, and a peak area for stable glycosylated hemoglobin A1c. The measurement method thereby makes it possible to accurately analyze HbA1c concentration even when the potential effect of unstable glycosylated hemoglobin E is high.

Description

Glycated hemoglobin measurement method

[0001] The present invention relates to a method for measuring glycated hemoglobin (HbA1c) based on hemoglobin fractions in blood.

[0002] Glycated proteins are produced by the non-enzymatic covalent bond formation between the aldehyde group of an aldose such as glucose and an amino group of a protein, resulting in an Amadori rearrangement. Examples of amino groups in proteins include the α-amino group at the amino terminus and the ε-amino group of the lysine residue side chain in the protein. A representative example of a glycated protein produced in vivo is hemoglobin A1c (HbA1c), which is glycated hemoglobin formed by glycation of hemoglobin in the blood. This is widely used clinically as an important blood glucose control marker for diagnosing and managing the symptoms of diabetes patients.

[0003] When hemoglobin is glycated, it is first bound to glucose via a reversible Schiff base bond, and this state is called unstable hemoglobin A1c (Labyrinthine A1c, LA1c), which gradually transitions to stable, irreversible hemoglobin A1c (Stable A1c, SA1c) through the Amadori transition.

[0004] For the quantification of HbA1c, an analytical method using hemoglobin fractionation based on liquid chromatography is widely used. Patent Document 1 discloses a technique for separating the above-mentioned LA1c and SA1c and measuring HbA1c with high accuracy.

[0005] Patent Document 2 reports that a type of modified hemoglobin, such as aldehyde hemoglobin or carbamylated hemoglobin, which is produced in the presence of acetaldehyde or sodium cyanate, co-elutes with LA1c.

[0006] It is known that in addition to hemoglobin A, there are abnormal hemoglobins (hemoglobin variants) in which part of the amino acid sequence of hemoglobin A is changed due to genetic mutations. Hemoglobin E is one of the hemoglobin variants that is widely found in Asia.

[0007] Patent Document 3 discloses a technique for separating the peak of stable glycated hemoglobin E (SE1c) and quantifying HbA1c.

[0008] The calculation of HbA1c when the SE1c peak is detected is based on empirical correction based on correlation with other methods, based on the knowledge that negative errors occur. While this simplifies the calculation, it has the problem that fluctuations in measurement conditions affect the stability of HbA1c measurement. Furthermore, in the presence of modified hemoglobin, not only hemoglobin A but also abnormal hemoglobin is thought to be affected by the modification, but this effect has not been avoided when calculating HbA1c.

[0009] Patent Document 4 also discloses a technique for separating the peak of stable glycated hemoglobin E (SE1c) and quantifying HbA1c by taking into consideration the influence of SE1c or SE1c and non-glycated hemoglobin E. Even when this technique is used, it is thought that in the presence of modified hemoglobin, not only hemoglobin A but also abnormal hemoglobin is affected by the modification, but this influence is not avoided when calculating HbA1c.

[0010] JP 2000-346848 JP 2000-146941 JP 2016-183871 JP 2012-215470

[0011] The present invention addresses the problem of providing a method for measuring HbA1c, which is capable of accurately analyzing the HbA1c concentration even when the potential influence of unstable glycated hemoglobin E is large when measuring a sample containing hemoglobin E.

[0012] As a result of extensive research, the present inventors have found that by including a step in the calculation of HbA1c concentration of calculating the peak area of ​​unstable glycated hemoglobin E contained in the peak of unstable glycated hemoglobin A1c or stable glycated hemoglobin A1c from the peak areas of stable glycated hemoglobin E, unstable glycated hemoglobin A1c, and stable glycated hemoglobin A1c, it is possible to accurately analyze HbA1c concentration even when the potential impact of unstable glycated hemoglobin E is large. Therefore, the present invention, which has been made to solve the above-mentioned problems, includes the following aspects: (1) A method for measuring HbA1c based on hemoglobin fractions in blood, which, when measuring a sample containing abnormal hemoglobin E, comprises: a step of separating at least unstable glycated hemoglobin A1c (LA1c), stable glycated hemoglobin A1c (SA1c), non-glycated hemoglobin A0 (A0), and stable glycated hemoglobin E (SE1c) and identifying the peaks of each component; and a calculation step represented by Equation 1, in which the peak area of ​​unstable glycated hemoglobin E (LE1c) contained in the peak of SA1c or LA1c is calculated from the areas of the peak identified as SE1c, the peak identified as SA1c, and the peak identified as LA1c by the hemoglobin fractionation. (wherein α represents the proportion of LE1c co-eluted with LA1c relative to the proportion of LE1c co-eluted with SA1c, and 0≦α≦1), and the formula for calculating the HbA1c concentration includes the area of ​​the LE1c peak. (2) A calculation step of calculating the HbA1c concentration represented by Formula 2, in which the HbA1c concentration is calculated as the ratio of the sum of the areas of the SA1c and SE1c peaks minus the area of ​​the LE1c peak to the area of ​​all peaks. (3) A calculation step of calculating the HbA1c concentration as a ratio of the area of ​​the SA1c peak minus the area of ​​the LE1c peak to the area of ​​the LE1c peak, the area of ​​the SE1c peak, and the area of ​​the non-glycated hemoglobin (E0) peak minus the area of ​​the SA1c peak, expressed by Equation 3. (4) A method for measuring HbA1c based on hemoglobin fractions in blood, comprising: a step of separating at least LA1c, SA1c, A0, and SE1c and identifying the peaks of each component when measuring a sample containing abnormal hemoglobin E; a calculation step represented by Equation 1 of calculating the area of ​​the peak of LE1c contained in the peak of SA1c or LA1c from the area of ​​the peak identified as SE1c, the area of ​​the peak identified as LA1c, and the area of ​​the peak identified as SA1c; (wherein α represents the proportion of LE1c co-eluted with LA1c relative to the proportion of LE1c co-eluted with SA1c, and 0≦α≦1), and a calculating step of calculating E0 contained in A0 from the peak area of ​​SE1c and the ratio of the peak area of ​​A0 to the peak area of ​​SA1c, wherein the formula for calculating the HbA1c concentration includes the peak areas of LE1c and E0. (5) The method for measuring HbA1c according to any one of (1) to (4), wherein hemoglobin fractionation in blood is performed by chromatography using a cation exchanger. (6) The method for measuring HbA1c according to (5), wherein the chromatography is HPLC.

[0013] The influence of stable glycated hemoglobin E and unstable glycated hemoglobin E can be avoided, and HbA1c can be accurately measured.

[0014] The technology of the present invention can accurately measure HbA1c concentration when a peak of stable glycated hemoglobin E is present, while avoiding the influence of unstable hemoglobin E or non-glycated hemoglobin E, but can also be applied when detecting peaks of unstable hemoglobin E or non-glycated hemoglobin E. Furthermore, the technology of the present invention can also be applied to abnormal hemoglobin species other than hemoglobin E that interfere with stable glycated hemoglobin A1c.

[0015] 1 is a configuration diagram of a measurement device according to an embodiment of the present disclosure; FIG. 2 is an example of a chromatogram of a normal sample measured by the measurement device of FIG. 1; FIG. 3 is a flowchart showing an example of an analysis process in the measurement device; FIG. 4 is an example of a chromatogram of an HbE sample (low LA1c concentration) measured by the measurement device of FIG. 1; and FIG. 5 is an example of a chromatogram of an HbE sample (high LA1c concentration) measured by the measurement device of FIG. 1.

[0016] The present invention will be described in more detail below using examples, but the aspects of the present invention are not limited to these.

[0017] [Method for Measuring HbA1c] Cation exchange liquid chromatography and capillary electrophoresis are widely used as methods for fractionating hemoglobin and quantifying HbA1c. These methods separate unstable hemoglobin A1c (LA1c), stable hemoglobin A1c (SA1c), and non-glycosylated hemoglobin A0 (A0) as the main components of hemoglobin, and can also separate hemoglobin A1a, hemoglobin A1b, fetal hemoglobin (hemoglobin F, HbF), and non-hemoglobin components such as the front peak as needed. Here, HbA1c and SA1c basically refer to the same measurement target, but SA1c is used to indicate the peak of stable hemoglobin A1c, and the peak that has undergone calculation and calibration for quantification is distinguished from HbA1c.

[0018] Furthermore, to deal with abnormal hemoglobin, peaks mainly for hemoglobin D, hemoglobin S, hemoglobin C, and hemoglobin E are separated.

[0019] Cation exchange liquid chromatography, a particularly preferred fractionation method in the present invention, will now be described. The cation exchange column is prepared using a packing material in which cation exchange groups have been introduced into non-porous cross-linked polymer particles. The synthesis method for the non-porous cross-linked polymer particles is not limited, and known methods such as suspension polymerization, emulsion polymerization, seed polymerization, and precipitation polymerization can be used. Furthermore, the present invention is not limited to non-porous cross-linked polymer particles; porous cross-linked polymers can also be used. The monomers and cross-linking agents used in the synthesis are not particularly limited, but hydrophilic methacrylic acid and acrylic esters are preferred. Examples of monomers include hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxypropyl acrylate, glycerin methacrylate, polyethylene glycol methacrylate, and polyethylene glycol acrylate. Examples of cross-linking agents include ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, and glycerin dimethacrylate. Preferably, cation exchange liquid chromatography is performed by HPLC.

[0020] When non-porous cross-linked polymer particles are used, there are no particular limitations on the method for introducing cation exchange groups onto the surface, and any known introduction method can be used. Examples of the cation exchange groups to be introduced include sulfopropyl groups, sulfoethyl groups, and carboxymethyl groups. Similar examples can be used when porous cross-linked polymers are used.

[0021] The eluent is not particularly limited, and may be a buffer solution containing an organic acid such as succinic acid or citric acid and its salt, a buffer solution containing an inorganic acid such as phosphoric acid and its salt, or a mixture of organic and inorganic acids. If necessary, other salts such as sodium chloride, sodium nitrate, sodium sulfate, etc. may be added to the buffer solution.

[0022] In the evaluations in the present examples, an automatic glycohemoglobin analyzer HLC-723GR01 (standard short mode) (manufactured by Tosoh Corporation) was used as the measurement device. This device is a liquid chromatography device based on the principle of cation exchange chromatography. Figure 1 shows the main system configuration. The analytical column (19) used was TSKgel GR01 (standard mode) (manufactured by Tosoh Corporation), and the eluents used were GR01 eluent 1 (10), GR01 eluent 2 (11), and GR01 eluent 3 (12) (manufactured by Tosoh Corporation). Under these measurement conditions, a measurement time of 30 seconds per sample was possible.

[0023] The eluents 10 to 12 are delivered by a delivery pump 14 and introduced into an analytical column 19 together with a measurement sample injected by an autosampler 18. The eluents are passed through a degasser 13 as needed, and different eluent mixture ratios are created by switching electromagnetic valves 15 to 17. The analytical column separates various components based on differences in salt concentration, etc., created by the mixture ratios, and the fractions are sequentially eluted. The sequentially eluted fractions are introduced into a detector 20, and the output values ​​thereof are continuously acquired.

[0024] The degassing device, solenoid valve, liquid pump, autosampler, column oven, detector, etc. are controlled by a control processing unit 22, such as a microcomputer or CPU, based on a control program and measurement conditions pre-stored in a storage unit 23. The detector signal obtained by the detector is converted by the control / processing unit into plot data consisting of, for example, time and detector signal. A chromatogram is then created based on this plot data. Furthermore, this chromatogram is subjected to waveform processing, and quantitative calculation of HbA1c is performed based on pre-stored calibration data, etc. The obtained quantitative calculation results, chromatogram, etc. can be displayed on a display unit 24, and this data can also be stored in a data storage unit 25.

[0025] In one embodiment of the present invention, a chromatogram is obtained using plot data in which the horizontal axis represents time (retention time) [t] and the vertical axis represents detector output (signal output) such as absorbance [A]. Alternatively, the horizontal axis may represent elution volume. The vertical axis may also represent a value converted from the detector signal. For example, noise reduction, smoothing, drift reduction, baseline correction, and other corrections may also be performed.

[0026] Affinity chromatography (AF) was used as a control for measuring HbA1c values. The measurement device used was an automatic glycohemoglobin analyzer HLC-723G8 (affinity mode) (manufactured by Tosoh Corporation). The analytical column used was TSKgel AF-GHb (manufactured by Tosoh Corporation), and the eluents used were TSKeluent AF-GHb A (S) and TSKeluent AF-GHb B (S) (manufactured by Tosoh Corporation).

[0027] [Method for Quantifying HbA1c in Normal Samples] An example of a chromatogram of a normal sample is shown in Figure 2. The peaks of A1a (100), A1b (101), F (102), LA1c (103), SA1c (104), and A0 (105) are fractionated on the chromatogram.

[0028] In normal specimens, no characteristic peaks are observed between SA1c and A0 or after A0 on the chromatogram.

[0029] Generally, HbA1c is calculated as the ratio of SA1c to the total amount of hemoglobin. That is, the SA1c peak area is divided by the total area (T.Area) to obtain [SA1c / T.Area], which is then adjusted based on the correction coefficient and calibration curve obtained by calibration performed on various instruments. Here, "total area (T.Area)" refers to the sum of the areas of all peaks actually detected on the chromatogram.

[0030] More precisely, HbA1c is quantified as the total amount of hemoglobin using the calculation formula [SA1c / (T.Area-F)], which removes the hemoglobin F peak area. Furthermore, non-hemoglobin components such as the front peak can be removed from the total amount of hemoglobin, but their presence is usually so small that their influence can be ignored. Therefore, when referring to "total area (T.Area)" in the present invention, particularly when referring to "total area (Total Area)" in the calculation step of formula (2) or formula (3), it may refer to either the sum of the areas of all peaks actually detected on the chromatogram, or the area obtained by subtracting the area of ​​the front peak from that sum.

[0031] Whether or not HbF is included in the total amount of hemoglobin depends on national and regional treatment guidelines. Calibration is also carried out in accordance with HbA1c units (NGSP units and IFCC units).

[0032] This flow chart is shown in FIG.

[0033] [Methods for detecting hemoglobin E] There are two methods for detecting abnormal hemoglobin E: one is to separate and detect non-glycated hemoglobin E (E0), which is present in a large proportion, after A0, and the other is to separate and detect stable glycated hemoglobin E (SE1c) between SA1c and A0. The former has the advantage of being able to estimate the relative amount of hemoglobin E, while the latter has the advantage of shortening the measurement time and being less likely to be mistaken for other abnormal hemoglobins.

[0034] In one embodiment of the present invention, the stable glycated hemoglobin E (SE1c) peak was detected separately between the SA1c and HbA0 peaks.

[0035] Figure 4 shows an example of a chromatogram of a sample containing hemoglobin E. The peaks indicated by dashed lines are hypothetical peaks that are not actually observed.

[0036] As mentioned above, LA1c is an unstable form of hemoglobin A1c in which a portion of hemoglobin A is bound to glucose via a reversible Schiff base bond. It was thought that unstable hemoglobin LE1c also forms in hemoglobin E, similar to hemoglobin A. It was also assumed that the frequency of this formation is similar to that of hemoglobin A, and that the ratio of LA1c to SA1c is the same as that of LE1c to SE1c. Furthermore, the peaks of A1a (100), A1b (101), F (102), LA1c (103), SA1c (104), and A0 (105) are fractionated on the chromatogram, and a peak of LE1c (200) can be observed between SA1c and A0. In this case, it was assumed that unstable glycated hemoglobin E (LE1c:200) is present in the LA1c (103) or SA1c (104) peak, and non-glycated hemoglobin E (E0:202) is present in the A0 peak. Because the area of ​​the LE1c peak may not be actually measured on a chromatogram, it can be calculated from the area of ​​the peaks of other components, assuming that it overlaps with the peaks of other components actually measured on a chromatogram, as shown in the above formula (1). When the HbA1c concentration was calculated based on the above assumptions, it was surprisingly found that the HbA1c concentration could be calculated with the same accuracy as affinity chromatography using a cation exchanger.

[0037] It is possible to determine that all peaks appearing between the SA1c and HbA0 peaks are SE1c peaks, but by setting a threshold, it is also possible to identify only peaks with a specific peak area or peak shape as SE1c peaks.

[0038] All documents mentioned herein are incorporated by reference in their entirety.

[0039] The following examples of the present invention are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. The present invention may be modified, for example, by adding, deleting, or substituting components of the present invention, provided that the modifications do not depart from the spirit of the present invention.

[0040] [Method for Quantifying HbA1c in Samples Containing Hemoglobin E] (Example 1) HbA1c in blood samples containing abnormal hemoglobin E was calculated as the ratio of glycated hemoglobin to the total amount of hemoglobin, rather than the ratio of SA1c to the total amount of hemoglobin.

[0041] That is, the calculation formula is [(SA1c + SE1c) / T. Area × 100], and more specifically, the calculation formula is [(SA1c + SE1c) / (T. Area - F) × 100]. Based on the assumption that LE1c co-elutes with SA1c from the peak position and also co-elutes with a portion of LA1c, the calculation formula is [(SA1c - LE1c + SE1c) / (T. Area - F) × 100]. This assumption is made when the elution position of SE1c approaches the elution position of SA1c due to a shortened measurement time, etc. The multiplication by 100 in the formula means converting from a ratio to a percentage, and the fraction, not the denominator, is multiplied by 100.

[0042] Therefore, the peak area of ​​LE1c contained in the SA1c peak is further calculated from the peak area of ​​SE1c and the peak area ratio of LA1c to SA1c using the formula [LE1c = SE1c × LA1c / (SA1c + α × EA1c)], where α is a constant determined based on the specifications of the measurement system, and takes the value 1 when all LE1c co-elutes with LA1c, and the value 0 when all LE1c co-elutes with SA1c, indicating the range of 0≦α≦1.

[0043] Therefore, from the HbA1c calculation formula [(SA1c-LE1c+SE1c) / (T.Area-F)×100], the calculation formula [{SA1c-SE1c×LA1c / (SA1c+α×SE1c)+SE1c} / (T.Area-F)×100] was derived.

[0044] In this example, α was set to 0.5, assuming that LE1c is evenly distributed between the LA1c and SA1c peaks. Therefore, the calculation formula used was [{SA1c - SE1c x LA1c / (SA1c + 0.5 x SE1c) + SE1c} / (T.Area - F) x 100]. This distribution is determined by the conditions of the measurement device, column, eluent, etc., and can be changed to suit the conditions.

[0045] Incidentally, values ​​obtained from the peaks are substituted into the terms in each equation, but in order to approach the true values ​​more precisely, it is also possible to create equations recursively and solve quadratic equations, etc.

[0046] That is, assuming SA1c'=SA1c-α×LE1c and LA1c'=LA1c-α×LE1c, it may be derived from {SA1c'-SE1c×LA1c' / (SA1c'+α×SE1c)+SE1c} / (T.Area-F)×100.

[0047] Example 2 HbA1c of a blood sample containing abnormal hemoglobin E is calculated from the ratio of SA1c to the total amount of hemoglobin A.

[0048] Here, it is assumed that LE1c co-elutes with SA1c from the peak position and also co-elutes with part of LA1c. This assumption is made by shortening the measurement time, etc., so that the elution position of SE1c approaches the elution position of SA1c. It is also assumed that E0 co-elutes with A0.

[0049] Therefore, a calculation formula [LE1c = SE1c × LA1c / (SA1c + α × EA1c)] was used to further determine the peak area of ​​LE1c contained in the SA1c peak from the peak area of ​​SE1c and the peak area ratio of LA1c to SA1c. Here, α is a constant, a value determined based on the specifications of the measurement system, and takes a value of 1 when all LE1c co-elutes with LA1c, and a value of 0 when all LE1c co-elutes with SA1c, indicating a range of 0≦α≦1. In addition, a calculation formula [E0 = SE1c × A0 / (SA1c + SE1c)] was used to calculate E0 contained in A0 from the SE1c peak and the ratio of A0 to SA1c.

[0050] Therefore, the calculation formula for HbA1c was used: [(SA1c-LE1c) / {T. Area-(LE1c+SE1c+E0)-F} x 100], that is, the calculation formula: [{SA1c-SE1c x LA1c / (SA1c+α x EA1c)} / {T. Area-(SE1c x LA1c / (SA1c+α x SE1c)+SE1c+SE1c x A0 / SA1c)-F} x 100].

[0051] In this example, α was set to 0.5, assuming that LE1c is evenly distributed between the LA1c peak and the SA1c peak. This distribution is determined by the conditions of the measuring device, the column, the eluent, etc.

[0052] Therefore, the calculation formula used is [{SA1c-SE1c x LA1c / (SA1c + 0.5 x EA1c)} / {T. Area-(SE1c x LA1c / (SA1c + 0.5 x SE1c) + SE1c + SE1c x A0 / SA1c)-F} x 100].

[0053] The values ​​obtained from the peaks are substituted into the terms in each equation, but in order to approach the true values ​​more precisely, it is also possible to create equations recursively and solve quadratic equations, etc.

[0054] That is, assuming SA1c' = SA1c - α x LE1c, LA1c' = LA1c - α x LE1c, and A0' = A0 - E0, it may be derived from {SA1c' - SE1c x LA1c' / (SA1c' + α x EA1c)} / {T. Area - (SE1c x LA1c' / (SA1c' + α x SE1c) + SE1c + SE1c x A0' / SA1c') - F} x 100.

[0055] Comparative Example 1 HbA1c of a blood sample containing abnormal hemoglobin E was calculated as the ratio of glycated hemoglobin to the total amount of hemoglobin, rather than the ratio of SA1c to the total amount of hemoglobin, using the calculation formula [(SA1c + SE1c) / (T.Area-F) x 100].

[0056] [Comparison of HbA1c Measurement Results of Hemoglobin E-Containing Samples] Sample 1 is a typical hemoglobin E sample with a low LA1c value. The measurement result by the AF method was 5.5%. The chromatogram of this sample is shown in Figure 4.

[0057] The HbA1c concentrations calculated using each calculation method are shown in Table 1, and no significant differences were observed between the calculation methods.

[0058] Sample 2 was a hemoglobin E sample with high LA1c. The measurement result by the AF method was 7.3%. The chromatogram of this sample is shown in Figure 5.

[0059] The HbA1c concentrations calculated by each calculation method are shown in Table 1. There was no large difference between Example 1, Example 2 and the AF method, but Comparative Example 1 showed a slight difference from the AF method.

[0060] [Comparison of the Effects of Glucose Loading on Hemoglobin E-Containing Samples] To further confirm the effects of the present invention, a glucose loading test was carried out and the HbA1c concentrations were compared between the calculation methods.

[0061] For the glucose tolerance test, a 10,000 mg / dL glucose solution was prepared and mixed with whole blood to achieve final concentrations of 0, 250, 500, 750, and 1,000 mg / dL. Each sample was left in a 37°C incubator for 1 hour, diluted with hemolysis and washing solution, and measured as a diluted sample. The HbA1c concentrations measured by the AF method and calculated by each calculation method are shown in Table 2. There was no significant difference between Examples 1 and 2 and the AF method, but Comparative Example 1 showed a deviation from the AF method as the glucose concentration increased.

[0062] [Comparison of the effects of modified hemoglobin in hemoglobin E-containing samples] To further confirm the effects of the present invention, an acetaldehyde challenge test (aldehyded hemoglobin) and a sodium cyanate challenge test (carbamylated hemoglobin) were performed, and the HbA1c concentrations were compared between the calculation methods.

[0063] For the acetaldehyde challenge test, a 250 mg / dL aqueous acetaldehyde solution was prepared and mixed with whole blood to achieve final concentrations of 0, 6.25, 12.5, 18.75, and 25 mg / dL. Each sample was left to stand in a 37°C incubator for 1 hour, diluted with hemolysis and washing solution, and measured as a diluted sample. The HbA1c concentrations measured by the AF method and calculated by each calculation method are shown in Table 3. There was no significant difference between Examples 1 and 2 and the AF method, but Comparative Example 1 showed a deviation from the AF method as the acetaldehyde concentration increased.

[0064] For the sodium cyanate challenge test, a 250 mg / dL sodium cyanate aqueous solution was prepared and mixed with whole blood to adjust final concentrations to 0, 6.25, 12.5, 18.75, and 25 mg / dL. Each sample was left to stand in a 37°C incubator for 1 hour, diluted with hemolysis and washing solution, and measured as a diluted sample. The HbA1c concentrations measured by the AF method and calculated by each calculation method are shown in Table 4. There was no significant difference between Examples 1 and 2 and the AF method, but in Comparative Example 1, the results deviated from the AF method as the sodium cyanate concentration increased.

[0065] 10 Eluent 1 11 Eluent 2 12 Eluent 3 13 Degasser 14 Liquid delivery pump 15, 16, 17 Solenoid valve 18 Autosampler 19 Analytical column 20 Detector 21 Column oven 22 Control / processing unit 23 Storage unit 24 Display unit (input unit) 25 Data storage unit 100 A1a peak 101 A1b peak 102 F peak 103 LA1c peak 104 SA1c peak 105 A0 peak 200 LE1c peak 201 SE1c peak 202 E0 peak

Claims

1. A method for measuring HbA1c based on hemoglobin fractions in blood, comprising, when measuring a sample containing abnormal hemoglobin E, a step of separating at least unstable glycated hemoglobin A1c (LA1c), stable glycated hemoglobin A1c (SA1c), non-glycated hemoglobin A0 (A0), and stable glycated hemoglobin E (SE1c) and identifying the peaks of each component; and a calculation step represented by Equation 1, in which the peak area of ​​unstable glycated hemoglobin E (LE1c) contained in the peak of SA1c or LA1c is calculated from the areas of the peak identified as SE1c, the peak identified as SA1c, and the peak identified as LA1c by the hemoglobin fractionation. (wherein α represents the proportion of LE1c co-eluted with LA1c relative to the proportion of LE1c co-eluted with SA1c, and 0≦α≦1), wherein the formula for calculating the HbA1c concentration includes the area of ​​the LE1c peak.

2. A calculation step of calculating the HbA1c concentration as the ratio of the sum of the areas of the SA1c and SE1c peaks minus the area of ​​the LE1c peak to the area of ​​all peaks, as expressed by Equation 2. The method for measuring HbA1c according to claim 1, comprising calculating 3. A calculation step of calculating the HbA1c concentration as expressed by Equation 3, which is the ratio of the area of ​​the SA1c peak minus the area of ​​the LE1c peak to the area of ​​the SA1c peak minus the area of ​​the LE1c peak, the area of ​​the SE1c peak, and the area of ​​the non-glycated hemoglobin E (E0) peak minus the area of ​​the SA1c peak. The method for measuring HbA1c according to claim 1, comprising calculating HbA1c by the following formula:

4. A method for measuring HbA1c based on hemoglobin fractions in blood, comprising: a step of separating at least LA1c, SA1c, A0, and SE1c and identifying the peaks of each component when measuring a sample containing abnormal hemoglobin E; and a calculation step represented by Equation 1, in which the area of ​​the peak identified as SE1c, the area of ​​the peak identified as LA1c, and the area of ​​the peak identified as SA1c are calculated from the area of ​​the peak identified as SE1c, LA1c, and SA1c. and calculating E0 contained in A0 from the ratio of the peak area of ​​A0 to the peak area of ​​SE1c and the peak area of ​​SA1c, wherein the peak area of ​​LE1c and the peak area of ​​E0 are included in a formula for calculating the HbA1c concentration.