JP2016183871A - Method for quantifying stable glycated hemoglobin A1c - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method capable of measuring easily in a short time, HbA1c% in a blood sample containing HbE, by using HPLC.SOLUTION: In analysis of stable type glycosylated hemoglobin A1c using cation exchange liquid chromatography, when a peak appearing between a stable type glycosylated hemoglobin A1c peak and an adult type hemoglobin A0 peak is separated and detected, a stable type glycosylated hemoglobin A1c value is corrected by being multiplied by a coefficient in the range of 1.1-1.4.SELECTED DRAWING: Figure 6

Description

  The present invention relates to a method for measuring glycated hemoglobin in a blood sample by liquid chromatography. More specifically, the present invention relates to measurement of glycated hemoglobin, which is an index for diabetes diagnosis, in a blood sample group in which blood samples containing abnormal hemoglobin may be mixed.

  Analysis of hemoglobins using a cation chromatography method by HPLC is widely used as a method for diagnosing hemoglobin disorders such as diabetes, congenital hemolytic anemia, abnormal hemoglobin disease and thalassemia.

  In diabetes diagnosis using the HPLC method, in order to determine the glycation rate of hemoglobin, the amount of total hemoglobin in blood and the amount of stable glycated hemoglobin A1c (hereinafter referred to as HbA1c) are quantified, and the presence of stable glycated hemoglobin A1c is determined from the ratio. The rate (hereinafter referred to as HbA1c%) is obtained. More specifically, HbA1c% is calculated from the ratio of the total peak area obtained by adding the peak areas of all detected peaks to the peak area of the HbA1c peak. In healthy individuals, 95% or more of hemoglobin in blood is adult hemoglobin (HbA0). Therefore, in order to obtain HbA1c%, it is only necessary to determine the total amount of hemoglobin in the sample and the amount of stable glycated hemoglobin derived from HbA0.

  HbA0 consists of two types of globulin chains, an α chain and a β chain. However, patients with abnormal hemoglobin disease, which is a congenital genetic disease, have abnormal hemoglobin composed of abnormal globin chains due to mutations in addition to HbA0 due to the congenital abnormality of globulin chain synthesis. Hemoglobin S (hereinafter referred to as HbS), which is known as a typical abnormal hemoglobin species, is obtained by replacing the 6th residue Glu of β chain with Val, and hemoglobin C (hereinafter referred to as HbC) is β chain 6 Residue Glu is replaced with Lys, and abnormal hemoglobin D (hereinafter referred to as HbD) is obtained by replacing β-chain 121st residue Glu with Gln.

  As a method of measuring HbA1c% of patients with abnormal hemoglobin disease by HPLC method, a method of quantifying abnormal hemoglobin in a sample and correcting by subtracting the abnormal hemoglobin amount from the total hemoglobin amount, and identifying glycated hemoglobin components derived from abnormal hemoglobin are also identified -A method of measuring the quantity and correcting the measured value is used.

  HbD, HbS, HbC and their saccharified components can be easily separated by HPLC because their interaction with the column packing used in cation chromatography is significantly different from HbA0 or HbA1c, and each component can be quantified. Therefore, it is relatively easy to perform the correction.

  However, hemoglobin E (hereinafter referred to as HbE), which is an abnormal hemoglobin often found in Southeast Asia and India, is an abnormal hemoglobin in which the β-chain 26th residue Glu is replaced with Lys, and its interaction with the cation exchanger. Is close to HbA0, it was difficult to easily separate and quantify the components as in HbD, HbS, and HbC. Therefore, in order to obtain HbA1c% of the specimen containing HbE, the analysis time is lengthened to separate HbA0 and HbE, or from the state where HbA0 and HbE are not sufficiently separated, It was necessary to quantify HbE.

  As a prior art of the HPLC method, using a peak peculiar to a blood sample containing HbE, a method of calculating HbA1c% by correcting the peak area of HbA1c or the peak area of all hemoglobin based on this peak area is also reported. (Patent Document 1). However, with this method, it is necessary to separate the peaks peculiar to the HbE-containing blood sample and to calculate the peak area accurately, so the analysis time can be shortened while maintaining the accuracy of clinical diagnosis. It was difficult to do.

JP 2012-215470 A JP-A-3-255360

  When a blood sample to be measured for HbA1c% contains HbE, it has been necessary to separate a peak peculiar to the blood sample containing HbE and accurately calculate the peak area in the conventional HPLC method. . However, it has been difficult and difficult to reduce the analysis time while maintaining the accuracy of clinical diagnosis. Furthermore, as compared with immunization methods (hereinafter sometimes referred to as AIA methods) and the like, the cation chromatography method has a problem that it is difficult to specifically separate HbE in a short time.

  Therefore, in the present invention, when HbA1c% in a blood sample containing HbE is measured by the HPLC method, HbE is separated from HbA0, which has been an indispensable element, and hemoglobin is quantified, or is specific to a blood sample containing HbE. Thus, the present inventors have completed the present invention by finding a method for measuring HbA1c% accurately in a short time and simply without accurately quantifying a simple peak.

That is, the present invention
[1] In the analysis of stable glycated hemoglobin A1c using cation exchange liquid chromatography, cation exchange is performed when a peak appearing between the stable glycated hemoglobin A1c peak and the adult hemoglobin A0 peak is separated and detected. A method for quantifying stable glycated hemoglobin A1c, which comprises correcting the stable glycated hemoglobin A1c value obtained by liquid chromatography by a coefficient in the range of 1.1 to 1.4.
[2] The method for quantifying stable glycated hemoglobin A1c according to [1], wherein the peak appearing between the stable glycated hemoglobin A1c peak and the adult hemoglobin A0 peak is abnormal hemoglobin E.
It is about.

  When there is a peak between the stable glycated hemoglobin A1c peak and the adult hemoglobin A0 peak as in the present invention, it is possible to correct abnormal hemoglobin continuously in a short time by multiplying HbA1c% by a coefficient. It has become possible to provide a hemoglobin analyzer that randomly measures samples that contain or do not contain. Therefore, HbA1c% of hemoglobin E holders can be measured in the same manner as other samples in a short time.

It is a figure which shows the chromatogram example of a healthy subject sample. (Example 1) which shows the example of a chromatogram of abnormal hemoglobinosis (hemoglobin AE). (Example 2) which shows the example of a chromatogram of abnormal hemoglobinosis (hemoglobin AE). It is a figure which shows the example of a chromatogram of abnormal hemoglobinosis (hemoglobin AS). It is a figure which shows the difference of the measured value of the HbE sample in the reference example 1, and the measured value of an immunity method It is a figure which shows the difference of the measured value of the HbE sample in Example 1, and the measured value of an immunity method. It is a figure which shows the correlation with the measured value of the immunity method in the sample group containing abnormal hemoglobinosis in the comparative example 1. It is a figure which shows the correlation with the measured value of the immunity method in the sample group containing abnormal hemoglobin disease in Example 2.

  The present invention is described in detail below. The cation exchange liquid chromatography in this specification will be described. The cation exchange column is prepared from a filler in which cation exchange groups are introduced into non-porous crosslinked polymer particles. However, the synthesis method of the non-porous crosslinked polymer particles is not limited, and known suspension polymerization methods and emulsion polymerization methods. , Seed polymerization method, precipitation polymerization method and the like can be used. In the present invention, not only non-porous crosslinked polymer particles but also a porous crosslinked polymer can be used.

  The monomer and crosslinking agent used in the synthesis are not particularly limited, but hydrophilic methacrylic acid and acrylic esters are desirable. Examples of the monomer include hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxypropyl acrylate, glycerin methacrylate, polyethylene glycol methacrylate, and polyethylene glycol acrylate.

  Examples of the crosslinking agent include ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, and glycerin dimethacrylate.

  When non-porous crosslinked polymer particles are used, the method for introducing a cation exchange group onto the surface is not particularly limited, and a known introduction method can be used. Examples of the cation exchange group to be introduced include a sulfopropyl group, a sulfoethyl group, and a carboxylmethyl group. Similar examples can be given when using a porous crosslinked polymer.

  Moreover, although the HPLC method to be used is not limited, the HPLC method which can identify a component derived from HbE in a short analysis time is desirable. The eluent is not particularly limited, and a buffer solution composed of an organic acid such as succinic acid or citric acid and a salt thereof and a buffer solution composed of an inorganic acid such as phosphoric acid or a salt thereof, or both an organic acid and an inorganic acid are confused. Can be used. If necessary, other salts such as sodium chloride, sodium nitrate, sodium sulfate and the like can be added to the buffer.

  HbA1c is used for diabetes diagnosis, and the glycation rate of hemoglobin is determined by quantifying the total hemoglobin amount and HbA1c amount in blood.

  Adult hemoglobin A0 is known to have congenital mutants such as HbD, HbE, HbS, and HbC, and is generally eluted later than HbA1c in cation exchange chromatography. . In the embodiment of the present invention, when at least one of HbD, HbS, and HbC is detected in one sample in addition to HbE in the same sample, the HbA1c value is corrected simultaneously using the method of the present invention. Or before and after that, you may correct | amend considering HbD, HbS, and HbC by a well-known method.

  As means for correcting the stable glycated hemoglobin A1c value by multiplying by a coefficient in the range of 1.1 to 1.4, the preferable coefficient range is 1.1 to 1.4, more preferably 1.2 to 1.3. Value, more preferably 1.25. In addition, as a result of the present inventors diligently studying to achieve the above-mentioned problem, the range of the coefficient is as follows. The specimen having HbE and HbA0 (HbAE) has a ratio of HbE of 20-30%, which is almost 25%. Things that can be seen (Seama Rao et.al., Indian J Med Re, November 2010, 132, p.513-519, Martin H. Steinberg, Hemoglobinopathy, p.453-473., Etc.) and HbE This is a range derived from finding that HbA1c% of the specimen to be tested is about 25% lower than that of immunization.

  EXAMPLES Next, although an Example and a reference example demonstrate this invention further in detail, this invention is not limited to these.

Reference example 1
A specimen in which the presence of HbE was confirmed by genetic analysis was determined for HbA1c% by a typical HPLC method and immunization method. The HPLC method uses a Tosoh automated glycohemoglobin analyzer HLC-723G8 equipped with a non-porous cation exchanger column, and the immunization method is Roche capable of measuring HbA1c% without being affected by abnormal hemoglobin including HbE. A manufactured analyzer was used. In any measurement method, the HbA1c value was not corrected.

FIG. 1 shows an example of a chromatogram of a healthy person, and FIGS. 2 and 3 show examples of a chromatogram of a patient specimen having HbE. There is a peak that appears only in the HbE holder but not in the healthy person at the position of the arrow in FIGS.
FIG. 4 shows an example of a chromatogram of another patient with abnormal hemoglobinosis (HbS). Here, an HbS peak appears at a position far from the HbA0 peak, but there is no peak at the position of the arrow in FIGS. 2 and 3, and the peak indicated by the arrow in FIGS. It can be determined that the peak is specific to HbE. By detecting this peak (the arrow in FIGS. 2 and 3), the specimen containing HbE can be easily identified and can be separated from other specimens.
As shown in FIG. 5, the HPLC method showed a low value in all samples compared to the immunization method.

Example 1
HbA1c% was calculated from the corrected HbA1c value by multiplying HbA1c% in the HPLC method of Comparative Example 1 described later by a correction factor of 1.25. The results are shown in FIG.
Regardless of the value of HbA1c%, the difference between the HPLC method and the immunization method was within 0.1% for all samples.

Comparative Example 1
A sample group consisting of a sample containing HbE and a sample not containing HbE was measured by the HPLC method and the immunization method.
The measurement conditions are the same as in Reference Example 1. FIG. 7 shows a correlation diagram of HbA1c% comparing the two. It was divided into a group showing a good correlation (white circle in the figure) and a group showing a low HPLC method (black circle in the figure). There is a discrepancy between the two in the sample group containing HbE.

Example 2
The same as in Comparative Example 1 in which an HPLC specific Tohso glycohemoglobin analyzer HLC-723G8 detects a HbE-specific peak appearing between the HbA1c peak and the HbA0 peak, and automatically detects HbA1c% when detected. Sample groups were measured. The correction conditions were the same as in Example 1.
FIG. 8 shows the correlation results. All of the sample groups (black circles in FIG. 7) that were different in Comparative Example 1 recognized HbE-containing samples and showed almost the same value as the immunization method by automatic correction.

  Separation is easier than conventional separation of HbE and HbA0, the presence of HbE can be detected in a short period of time, a sample in which a peak is detected is treated as an HbE sample, and HbE is not included by correcting by the method of the present invention A specimen containing HbE can be corrected without affecting the specimen.

Claims (2)

In the analysis of stable glycated hemoglobin A1c using cation exchange liquid chromatography, when a peak appearing between stable glycated hemoglobin A1c peak and adult hemoglobin A0 peak is separated and detected, cation exchange liquid chromatography A method for quantifying stable glycated hemoglobin A1c, wherein the value is corrected by multiplying the stable glycated hemoglobin A1c value obtained by the above by a coefficient in the range of 1.1 to 1.4. The method for quantifying stable glycated hemoglobin A1c according to claim 1, wherein the peak that appears between the stable glycated hemoglobin A1c peak and the adult hemoglobin A0 peak is abnormal hemoglobin E.

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