JP2019219309A - Method for calculating similarity of chromatogram - Google Patents

Abstract

To provide a method for calculating similarity between a measurement sample and a standard sample based on a chromatogram that is acquired in a liquid chromatograph exercised on glycosylated hemoglobin as a measurement object.SOLUTION: Provided is a method for calculating similarity between a measurement sample and a standard sample based on a chromatogram that is acquired in a liquid chromatograph exercised on glycosylated hemoglobin as a measurement object. The method includes removing a drift in two or more sections, of the chromatograms of the measurement sample and standard sample, in which similarity calculation is performed, and thereafter calculating section similarity between the two in each section and obtaining the average value of weighted section similarities as final similarity between the two.SELECTED DRAWING: None

Description

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

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

  Amino acid substitution, omission or insertion in the amino acid sequence of α-chain globin or β-chain globin that constitutes a hemoglobin molecule, or those containing unusual components such as those containing a fused globin chain by gene fusion 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.

  In the field of spectroscopic analysis and the field of mass spectrometry, a technique using “similarity” is frequently used for the purpose of specifying / identifying a measurement sample. This is a method of comparing a spectrum to be compared with a spectrum obtained by measuring an unknown sample, and evaluating the similarity numerically. The wavelength or molecular weight is plotted on the x-axis, and the absorbance or intensity corresponding to the wavelength or molecular weight is plotted on the y-axis. It is desired that the x-axis of the data to be compared, that is, the wavelength or the molecular weight does not include uncertainty.

  Patent Document 1 is an example in which the idea of similarity is applied to the analysis of glycated hemoglobin by liquid chromatography, and the method is applied 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 serving as 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 having a high ratio greatly changes the value of the similarity and is not an accurate indicator. There are issues.

JP-A-9-251016

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

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

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

  In one aspect of the calculation method according to the present invention, the calculation of the section similarity is calculated by the following equation.

  Further, the present invention includes a method of determining whether the measurement sample is normal or abnormal based on whether or not the final similarity calculated by the above-described calculation method is equal to or greater than an arbitrary threshold.

  Further, by using a sample having a composition equivalent to that of the standard sample as the measurement sample, whether the final similarity calculated by the above-described calculation method is equal to or more than an arbitrary threshold value, The present invention also includes a method for determining the presence / absence of a chromatographic apparatus abnormality.

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

FIG. 4 is a diagram schematically illustrating an effect of drift removal of a chromatogram in the present invention. FIG. 5 is a diagram schematically illustrating time sections in similarity calculation according to the present invention. FIG. 2 is a diagram illustrating a system configuration used in an embodiment. FIG. 3 is a diagram illustrating an example of a chromatogram of a sample used for verification in Example 1. FIG. 4A is a diagram illustrating a chromatogram of a standard sample used in Example 1, and FIG. 4B is a diagram illustrating a target section of similarity calculation. FIG. 6 is a diagram illustrating a list of weighted average similarities of three sections according to the first embodiment. FIG. 5 is a diagram illustrating a list of simple average similarities in three sections according to the first embodiment. FIG. 6 is a diagram illustrating a list of similarities in which all sections in the first embodiment are regarded as one section. FIG. 14 is a diagram illustrating a list of weighted average similarities of three sections according to the second embodiment. FIG. 13 is a diagram illustrating a list of similarities in which all sections in the second embodiment are regarded as one section.

  The calculation method of the present invention, for the chromatograms of the measurement sample and the standard sample, after removing the drift of two or more sections for performing the similarity calculation, calculate the similarity between the two in each section as a section similarity, An average value obtained by weighting the section similarities 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 by liquid chromatography using glycated hemoglobin as a measurement target. Further, the chromatogram of the standard sample may be referred to as “reference chromatogram”, and the chromatogram of the measurement sample may be referred to as “comparative chromatogram”.

  As an example of the process of removing the drift in the section in which the similarity calculation is performed, a start point (x1, y1) and an end point (x2, y2) [x: time, y: output] of the section in 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 may be used. However, this step is not limited to this method. A method is also useful in which a polynomial is calculated based on a plurality of points at which peaks other than the start point and the end point are not eluted, and the y value of the polynomial is subtracted 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. However, the drift may be removed in the above-described procedure for each section, or the drift may be removed in a range including a plurality of sections. For example, it is convenient to specify the measurement start point as the start point and the measurement end point as the end point, which is preferable (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 includes an S-A1c component peak having an area ratio of about 5 to 6% in a healthy person and an A0 component peak having an area ratio of about 90%. Therefore, it is preferable to divide the similarity calculation into three sections: a section including the S-A1c component peak, a section including the A0 component peak, and a section including the component peak that appears specifically (FIG. 2). Further, the sections in which the similarity calculation is performed may be continuous or discontinuous.

  The method of calculating the “section similarity” is typically a method as shown in Expression 1, but is not particularly limited as long as it is an index of waveform similarity. The similarity calculated using Expression 1 is 1000 at the maximum, and the closer to 1000, the higher the identity.

  After calculating the section similarity in each section, the average value obtained by weighting the section similarity is used as the final similarity between the measurement sample and the standard sample. There is no problem even if the “simple average similarity” is set so that all the weighting coefficients are 1 or the same value. It is preferable to reduce the coefficient of, because the variation of the similarity due to the variation of the component having a low ratio can be seen largely.

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

  Whether the measurement sample is normal or abnormal can be determined based on whether or not the final similarity between the measurement sample and the standard sample obtained by the above-described method is equal to or greater than an arbitrary threshold. In addition, if a sample having a composition equivalent to that of the standard sample is used as the measurement sample, that is, if the similarity calculation is performed on the chromatograms of the standard samples, whether or not the threshold value is equal to or more than an arbitrary threshold is used for the measurement. It is also possible to determine the presence or absence of a liquid chromatography apparatus abnormality.

  Examples will be shown below, and the 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 in detail. Furthermore, the present invention is not limited to the above-described embodiment, and various changes can be made within the scope shown in the claims, and embodiments obtained by appropriately combining the disclosed technical means are also described. It is included in the technical scope of the invention. In addition, all of the documents described in this specification are incorporated by reference.

Actual measurement was performed using the system shown in FIG. As the glycohemoglobin analyzer, HLC-723G8 manufactured by Tosoh Corporation, a dedicated column, and an eluent were used. The measurement mode was a “variant mode” in which abnormal hemoglobin species could be easily identified.
By switching the three different eluents (1, 2, 3) at specified times and sending them to the analytical column (10), the injected sample is separated into each component by changing the dissolution power, The amount is measured by a visible detector (12). The sample injection device (8) is configured to automatically hemolyze / dilute and inject whole blood collected by a blood collection tube as it is, and to inject a diluted sample such as a standard sample or a control sample.

(Example 1)
In the present example, it was verified whether a normal specimen and an abnormal hemoglobin species could be distinguished from each other by judging the identity of chromatograms.
Normal samples (10 samples, one of which was used as a standard sample), HbAlc calibrator (Level 1, manufactured by Tosoh Corporation), HbAlc control (Level 1, manufactured by Tosoh Corporation), and a variant hemoglobin to the normal sample Evaluation was performed using samples (28 samples) to which two types of controls were added. As the variant hemoglobin control, a variant hemoglobin control AS (HC-104) and a variant hemoglobin control AC (HC-105) manufactured by Analytical Control Systems were used. The 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 the measurement end point.

FIG. 4 shows chromatograms of (a) a normal sample, (b) a sample added with variant hemoglobin control AS (HC-104), and (c) a sample added with variant hemoglobin control AC (HC-105).
In the present embodiment, the first section (0.0 minutes to 0.7 minutes), the second section (0.7 minutes to 1.0 minutes), and the The three sections (1.0 to 1.6 minutes) were divided into three sections, and the section similarity was calculated (see FIG. 5B). The first section is a time area including the S-A1c peak, the second section is a time area including the A0 peak, and the third section is a time area in which the component peak of the abnormal hemoglobin species is likely to appear. The weight coefficient was set to 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 section similarity by the weighting coefficients (1, 2, 3) for each section by the sum of the weighting coefficients (1 + 2 + 3) is defined as the weighted average similarity.

  FIG. 6 shows a list of the results of the weighted average similarity of three sections, and FIG. 7 shows a list of the results of the simple average similarity of three sections. In any of the calculation methods, there is a significant difference between the normal sample and the abnormal hemoglobin species, but the difference in the weighted average similarity is clearer. On the other hand, when the similarity calculation is performed with all sections as one section, as shown in FIG. 8, there are many places where it is not possible to discriminate between a normal specimen and an abnormal hemoglobin type. In the figure, “N” indicates a normal sample, “+ (1)” indicates a sample obtained by adding HC-104 to a normal sample, “+ (2)” indicates a sample obtained by adding HC-105 to a normal sample, and “U” "Indicates an abnormal hemoglobin specimen.

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

(Example 2)
In the present example, it was verified whether an abnormality (fluctuation in flow rate) of the measuring device could be detected by determining the identity of the chromatogram.
A normal sample (Sample_A) and a sample obtained by adding two types of variant hemoglobin controls to the normal sample (Sample_I, J) were also used as samples. As the variant hemoglobin control, Variant Hemoglobin Control AS (HC-104) and Variant Hemoglobin Control AC (HC-105) manufactured by Analytical Control Systems were used.
The flow rate factor (relative value of the flow rate) at the time of standard measurement is operated at 1.00, and when it is 0.98 to 1.02, it can be said that normal measurement can be performed. The measurement was performed with factors of 1.00, 1.02, 1.04, 1.06, 0.98, 0.96, and 0.94, and the threshold value was set as the similarity 850 for the presence or absence of the device abnormality. In the present embodiment, the result of the normal sample Sample_A measured at a factor of 1.00 was used as a reference chromatogram (standard sample), and the conditions such as the time division and the weighting factor for calculating the similarity were the same as in the first embodiment.

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

1. 1. First eluent 2. second eluent Third eluent4. Hemolysis washing solution5. Deaerator 6. Solenoid valve7. Liquid sending pump8. Sample injection mechanism 9. Prefilter 10. Analytical column Resistance tube12. 12. Visible absorption detector Thermostatic bath 14. Sample 15. Waste liquid

Claims (5)

In liquid chromatography using glycated hemoglobin as a measurement target,
Based on the obtained chromatogram, a method for calculating the similarity between the measurement sample and the standard sample,
For the chromatogram of the measurement sample and the standard sample,
After removing the drift in two or more sections for performing the similarity calculation,
The similarity between the two in each section is calculated as the section similarity,
The above method wherein an average value obtained by weighting the section similarities is used as a final similarity between the two.   The method according to claim 1, wherein the method of setting the weight to the section similarity is determined based on the intensity of a component peak of a chromatogram. The method according to claim 1, wherein the calculation of the section similarity is calculated by the following equation.

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

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