JP7070137B2 - How to calculate the similarity of chromatograms - Google Patents

Description

The present invention relates to a method for calculating the similarity of chromatograms.

The value of hemoglobin A1c (HbA1c) in which glucose is bound to hemoglobin is used as an index for evaluating the blood glucose state in the diagnosis and treatment of diabetes. HbA1c reflects the average blood glucose level in the past 1 to 2 months and is useful as a blood glucose control marker in diabetic patients.

Amino acid sequences of α-chain globin or β-chain globin that make up the hemoglobin molecule have amino acid substitutions, omissions, or insertions, or those containing fusion globin chains due to gene fusion, whose constituents are different from normal. Is called abnormal hemoglobin and is known to be rarely present. It is known that when such a sample is measured by liquid chromatography, it shows a separation pattern different from that of a normal sample.

Further, in the fields of spectroscopic analysis and mass spectrometry, a method using "similarity" is frequently used for the purpose of identifying / identifying a measurement sample. This is a method of comparing the spectrum to be compared with the spectrum obtained by measuring an unknown sample and evaluating the similarity numerically. The wavelength or molecular weight is taken on the x-axis, and the corresponding absorbance or intensity is taken as the y-axis, and both are compared and used for similarity calculation. It is desirable that the x-axis of the data to be compared, that is, the wavelength or molecular weight, contains no uncertainty.

Patent Document 1 is an example of applying the idea of similarity to the analysis of glycohemoglobin by liquid chromatography and applying it to a method of determining whether a chromatogram is normal or abnormal. In the separation of glycohemoglobin, the area ratio of the S-A1c peak, which is an index, is about 5% of the whole, and about 90% is occupied by the A0 peak. When the similarity is calculated for such an uneven chromatogram, a slight change in the shape of the A0 peak with a high ratio will greatly change the similarity value, and it will not be an accurate index. There are challenges.

Japanese Unexamined Patent Publication No. 9-251016

An object of the present invention is to provide a method for calculating the degree of similarity between a measurement sample and a standard sample based on the obtained chromatogram in liquid chromatography using glycohemoglobin as a measurement target.

In the liquid chromatography of glycohemoglobin according to the present invention, the method of calculating the similarity between the measurement sample and the standard sample based on the obtained chromatogram is based on the chromatogram of the measurement sample and the standard sample. After removing the drift of two or more sections for which similarity calculation is performed, the similarity between the two in each section is calculated as the section similarity, and the average value obtained by weighting the section similarity is the final value of both. It is characterized by having a degree of similarity.

Further, in one aspect of the calculation method according to the present invention, the method for setting the weight to the interval similarity is determined based on the intensity of the component peak of the chromatogram.

Further, in one aspect of the calculation method according to the present invention, the calculation of the section similarity is calculated by the following formula.

Further, the present invention includes a method for determining whether the measured sample is normal or abnormal depending on whether or not the final similarity between the two calculated by the above calculation method is equal to or higher than an arbitrary threshold value.

Further, using a sample having the same composition as the standard sample as the measurement sample, the liquid is determined by whether or not the final similarity between the two calculated by the above calculation method is equal to or higher than an arbitrary threshold value. The present invention also includes a method for determining the presence or absence of a chromatographic device abnormality.

According to the present invention, even if the chromatogram is uneven, the average value obtained by weighting the similarity for each section is used as the final similarity between the measurement sample and the standard sample, so that an accurate index can be used. Obtainable.

It is a figure which showed the effect of the drift removal of the chromatogram in this invention schematically. It is a figure which showed schematically the time division in the similarity calculation in this invention. It is a figure which showed the system configuration used in an Example. It is a figure which showed an example of the chromatogram of the sample used for the verification of Example 1. FIG. It is a figure which showed (a) the chromatogram of the standard sample used in Example 1, and (b) the target section of the similarity calculation. It is a figure which showed the list of the weight average similarity of 3 sections in Example 1. FIG. It is a figure which showed the list of the simple average similarity of 3 sections in Example 1. FIG. It is a figure which showed the list of the degree of similarity which made all the sections in Example 1 into one section. It is a figure which showed the list of the weight average similarity of 3 sections in Example 2. FIG. It is a figure which showed the list of the degree of similarity which made all the sections in Example 2 into one section.

In the calculation method of the present invention, after removing the drift of two or more sections for which similarity calculation is performed with respect to the chromatograms of the measurement sample and the standard sample, the similarity between the two in each section is calculated as the section similarity. The average value obtained by weighting the interval similarity is used as the final similarity between the two. Hereinafter, an embodiment of the present invention will be described.

The "chromatogram" in the present invention refers to a chromatogram obtained in liquid chromatography using glycohemoglobin as a measurement target. Further, the chromatogram of the standard sample may be referred to as a "reference chromatogram", and the chromatogram of the measurement sample may be referred to as a "comparison target chromatogram".

As an example of the process of removing the drift of the section for which the similarity calculation is performed, the start point (x1, y1) and the end point (x2, y2) [x: time, y: output] of the section for which the similarity calculation is performed are determined. Then, a method of calculating a linear equation (y = ax + b) and subtracting the y value of the linear equation from each data point (time, output) of the chromatogram can be mentioned. However, this step is not limited to this method. It is also useful to calculate a polynomial based on a plurality of points where peaks other than the start point and the end point are not eluted, and subtract the y value of the polynomial from each data point (time, output) of the chromatogram. ..

In the present invention, the number of sections for which the similarity calculation is performed is two or more, but the drift may be removed by the procedure as described above for each section, or the drift may be removed in a range including a plurality of sections. For example, it is preferable to specify the measurement start point as the start point and the measurement end point as the end point because it is convenient (see FIG. 1).

After removing the drift, the similarity between the two in each section is calculated as the section similarity. The chromatogram of glycohemoglobin contains an S—A1c component peak having an area ratio of about 5 to 6% and an A0 component peak having an area ratio of about 90%. Therefore, it is preferable to divide the section for calculating the similarity into three sections: a section including the S—A1c component peak, a section including the A0 component peak, and a section including the specifically appearing component peak (Fig.). 2). Further, the interval for which the similarity calculation is performed may be continuous or discontinuous.

The method for calculating the "interval similarity" is typically a method as described in Equation 1, but is not particularly limited as long as it can be used as an index of waveform similarity. The maximum similarity calculated using Equation 1 is 1000, and the closer it is to 1000, the higher the identity.

After calculating the interval similarity in each interval, the average value obtained by weighting the interval similarity is taken as the final similarity between the measurement sample and the standard sample. There is no problem even if the weighting coefficients are all 1 or the same value as "simple average similarity", but the weighting coefficient for the section similarity is the section where the ratio of the component peaks is small and the ratio of the component peaks is high. By reducing the coefficient of, it is preferable because the fluctuation of the similarity due to the fluctuation of the component having a low ratio can be seen large.

As a method of setting the weight to the interval similarity, the measurer may set an arbitrary weight coefficient for the purpose of clearly reflecting the change in the chromatogram. Further, based on the intensity of the component peaks of the chromatogram, that is, the weight of the section including the high-intensity peak may be reduced, and the weight of the section including the low-intensity peak may be increased. Further, it is also possible to determine the weight of the section according to the inverse of the average area ratio for each section in various samples (for example, first section area ratio: 20%, second section area ratio: 70%, first. When the three-section area ratio: 10%, the first section weight: 3.5, the second section weight: 1.0, and the third section weight: 7.0).

It is possible to judge whether the measurement sample is normal or abnormal depending on whether or not the final similarity between the measurement sample obtained by the above method and the standard sample is equal to or higher than an arbitrary threshold value. In addition, if a sample having the same composition as the standard sample is used as the measurement sample, that is, if the similarity is calculated for the chromatograms of the standard samples, it is used for the measurement depending on whether or not it exceeds an arbitrary threshold value. It is also possible to determine the presence or absence of an abnormality in the liquid chromatography device.

Examples are shown below, and embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible for details. Furthermore, the present invention is not limited to the above-described embodiment, and various modifications can be made within the scope of the claims, and the present invention also relates to an embodiment obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention. In addition, all of the documents described in this specification are incorporated by reference.

The actual measurement was performed using the system shown in FIG. The glycohemoglobin analyzer used HLC-723G8 manufactured by Tosoh Corporation, a dedicated column, and an eluent. The measurement mode was "variant mode", which makes it easy to identify abnormal hemoglobin species.
By switching between three different eluents (1, 2, 3) at a specified time and sending the solution to the analysis column (10), the injected sample is separated into each component by changing the dissolution output. The amount is measured by the visible detector (12). The sample injection device (8) is configured to automatically hemolyze / dilute and inject the whole blood collected in the blood collection tube as it is, and to inject a diluted sample such as a standard sample or a control sample.

(Example 1)
In this example, it was verified whether a normal sample and an abnormal hemoglobin species could be distinguished by determining the identity of the chromatogram.
Normal sample (10 samples, 1 of which was used as the standard sample), HbAlc calibrator (Level1, 2 Tosoh Co., Ltd.), HbAlc control (Level1, 2 Tosoh Co., Ltd.), and variant hemoglobin to the normal sample. Evaluation was performed using a sample (28 samples) to which 2 types of controls were added. As the variant hemoglobin control, Variant Hemoglobin Control AS (HC-104) and Variant Hemoglobin Control AC (HC-105) manufactured by Analytical Control Systems, Inc. were used. In addition, chromatograms of clinically measured abnormal hemoglobin samples (29 samples) were also evaluated.
The above samples were measured, chromatograms were obtained, and drift removal processing was performed at the measurement start point and measurement end point.

FIG. 4 shows chromatograms of (a) normal sample, (b) variant hemoglobin control AS (HC-104) -added sample, and (c) variant hemoglobin control AC (HC-105) -added sample.
In this example, the first section (0.0 minutes to 0.7 minutes), the second section (0.7 minutes to 1.0 minutes), and the first section, where changes in the characteristics of the chromatogram are expected to appear significantly. The section similarity was calculated by dividing into three sections (1.0 to 1.6 minutes) (see FIG. 5 (b)). The first section is a time domain including the S—A1c peak, the second section is the time domain including the A0 peak, and the third section is the time domain in which the component peak of the abnormal hemoglobin species is likely to appear. The weighting coefficient was 2 for the first section, 1 for the second section, and 3 for the third section. That is, the value obtained by dividing the sum of the values obtained by multiplying the interval similarity by the weighting coefficient (1, 2, 3) for each interval by the sum of the weighting coefficients (1 + 2 + 3) was defined as the weight average similarity.

FIG. 6 shows a list of the results of the weight average similarity of the three sections, and FIG. 7 shows a list of the results of the simple average similarity of the three sections. In both calculation methods, there is a dominant difference between the normal sample and the abnormal hemoglobin species, but the difference is clearer in the results with the weight average similarity. On the other hand, when the similarity calculation is performed with all the sections as one section, there are many places where it is not possible to distinguish between the normal sample and the abnormal hemoglobin species as shown in FIG. In the figure, "N" is a normal sample, "+ (1)" is a sample obtained by adding HC-104 to a normal sample, and "+ (2)" is a sample obtained by adding HC-105 to a normal sample, "U". "Indicates an abnormal hemoglobin sample.

In the case of simple average similarity calculation, the threshold of similarity is set to about 900, and in the case of calculation of weighted average similarity, even if the threshold of similarity is set to about 850, which is lower, the normal sample and abnormal hemoglobin species are clearly defined. The chromatogram of the sample can be distinguished.

(Example 2)
In this example, it was verified whether or not an abnormality (fluctuation in flow rate) of the measuring device could be detected by judging the identity of the chromatogram.
As a sample, a normal sample (Sample_A) and a sample (Sample_I, J) in which two variants of hemoglobin control were added to the normal sample were also used. As the variant hemoglobin control, Variant Hemoglobin Control AS (HC-104) and Variant Hemoglobin Control AC (HC-105) manufactured by Analytical Control Systems, Inc. were used.
The flow rate factor (relative value of the flow rate) at the time of standard measurement is operated at 1.00, and it can be said that normal measurement can be performed if it is 0.98 to 1.02. Measurements were performed with factors of 1.00, 1.02, 1.04, 1.06, 0.98, 0.96, and 0.94, and a threshold value of similarity 850 was set for the presence or absence of device abnormality. In this example, the result of the normal sample Sample_A whose factor was measured at 1.00 was used as a reference chromatogram (standard sample), and the conditions such as the time division for calculating the similarity and the weighting coefficient were the same as in Example 1.

FIG. 9 shows a list of results of weight average similarity of three sections, and FIG. 10 shows a list of results of similarity with all sections as one section.
As can be seen from FIG. 9, in the three-interval weight average similarity, it can be seen that there are two groups of three types of Sample_A, I, and J measured at a flow rate factor of 1.00. When the measurement was performed with the flow rate factors of 0.98, 1.00, and 1.02, the similarity of Sample_A was 850 or more, but when the measurement was performed with other flow rate factors, the similarity of Sample_A was similar. The degree was less than 850, and it was confirmed that the determination of the device abnormality was performed normally.
On the other hand, as can be seen from FIG. 10, in the similarity when all the sections are calculated as one section, for example, there is almost no difference between the three types of Sample_A, I, and J measured at a flow rate factor of 1.00. .. Then, when the measurement was performed with the flow rate factor of 1.02, the similarity of Sample_A was less than 850, and it was determined that the device was abnormal.

1. 1. First eluent 2. Second eluent 3. Third eluent 4. Hemolytic cleaning solution 5. Deaerator 6. Solenoid valve 7. Liquid feed pump 8. Sample injection mechanism 9. Pre-filter 10. Analytical column 11. Resistance tube 12. Visible absorption detector 13. Constant temperature bath 14. Specimen 15. Waste liquid

Claims (5)

In liquid chromatography using glycohemoglobin as a measurement target,
It is a method of calculating the similarity between the measurement sample and the standard sample based on the obtained chromatogram.
For the chromatograms of the measurement sample and the standard sample
After removing the drift of two or more sections for similarity calculation,
The similarity between the two in each section is calculated as the interval similarity, and
The method in which the average value obtained by weighting the interval similarity is used as the final similarity between the two. The method according to claim 1, wherein the method for setting the weight to the interval similarity is determined based on the intensity of the component peak of the chromatogram. The method according to claim 1 or 2, wherein the calculation of the interval similarity is calculated by the following formula.

A method for determining whether the measurement sample is normal or abnormal depending on whether or not the final similarity between the two calculated by the method according to any one of claims 1 to 3 is equal to or higher than an arbitrary threshold value. Whether the final similarity between the two calculated by the method according to any one of claims 1 to 3 using a sample having the same composition as the standard sample as the measurement sample is equal to or higher than an arbitrary threshold value. A method for determining the presence or absence of an abnormality in the liquid chromatography apparatus.

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