How to measure hemoglobin F

Abstract

To accurately measure HbF from a chromatograph obtained by subjecting a blood specimen to liquid chromatography.SOLUTION: A measuring method of hemoglobin F includes: calculating a ratio of a peak value of an HbF peak to a peak value of the whole hemoglobin peak from a chromatogram obtained by subjecting a blood specimen to liquid chromatography; and calculating a correction value of the HbF peak to the whole hemoglobin peak by multiplying the ratio by a prescribed coefficient.SELECTED DRAWING: Figure 12

Description

[Technical Field]

[0001] The present invention relates to a method for measuring hemoglobin F in a blood sample. [Background technology]

[0002] Various hemoglobins in blood samples can be measured by separation and fractionation methods such as high-performance liquid chromatography and capillary electrophoresis. Measurements of hemoglobin F (HbF), a type of hemoglobin, are required to be highly accurate because they are used as diagnostic materials for hemoglobinopathies and thalassemias. HbF is often measured by capillary electrophoresis, and the HbF measured by capillary electrophoresis is often used as diagnostic material. An example of a method for measuring hemoglobin F using liquid chromatography is the method disclosed in Patent Document 1 listed below. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2014-235023 Summary of the Invention [Problem to be solved by the invention]

[0004] The component peaks seen in the hemoglobin chromatogram obtained by liquid chromatography generally correspond in ratio to the component peaks seen in the hemoglobin analysis chart obtained by capillary electrophoresis, but there is a discrepancy between the two, only for the HbF peak. Specifically, the ratio of the HbF peak in liquid chromatography is smaller than that in capillary electrophoresis.

[0005] Therefore, an object of an embodiment of the present disclosure is to more accurately measure HbF from a chromatogram obtained by subjecting a blood sample to liquid chromatography. [Means for solving the problem]

[0006] In one embodiment of the method for measuring HbF, a blood sample is subjected to liquid chromatography to obtain a chromatogram, and the ratio of the HbF peak to all hemoglobin peaks is calculated from the chromatogram. The ratio is then multiplied by a predetermined coefficient to calculate a corrected value of the HbF peak to all hemoglobin peaks. [Effects of the Invention]

[0007] According to an embodiment of the present invention, it becomes possible to more accurately measure HbF from a chromatogram obtained by subjecting a blood sample to liquid chromatography. [Brief explanation of the drawings]

[0008] [Figure 1] 1 shows a schematic example of a hemoglobin chromatograph obtained by subjecting a blood sample to high performance liquid chromatography. [Figure 2] Schematic diagram showing the relationship between the HbF peak and the composite peak in a normal sample. [Figure 3] Schematic diagram showing the relationship between the HbF peak and the composite peak in a high HbF sample. [Figure 4] The hemoglobin analysis chart obtained by subjecting a normal sample to capillary electrophoresis is shown schematically below. [Figure 5] Schematic diagram of a hemoglobin analysis chart obtained by subjecting a high HbF sample to capillary electrophoresis. [Figure 6] An example of a chromatogram obtained using the apparatus 1 in the first mode is shown. [Figure 7] An example of a chromatogram obtained using the apparatus 1 in the second mode is shown. [Figure 8] An example of a chromatogram of a blood sample obtained using device 2 is shown. [Figure 9] 1 shows an example of a chromatogram of a blood sample obtained using the device 3 in the first mode. [Figure 10]1 shows an example of a chromatogram of a blood sample obtained using the device 3 in the second mode. [Figure 11] 1 shows an example of an analysis chart of a blood sample obtained using a capillary electrophoresis device. [Figure 12] 1 is a graph showing the correlation between HbF peak values ​​obtained by liquid chromatography and HbF peak values ​​obtained by capillary electrophoresis. DETAILED DESCRIPTION OF THE INVENTION

[0009] Hereinafter, embodiments of the present disclosure will be described with reference to the drawings. Common reference numerals in the various drawings indicate the same parts unless otherwise specified. Note that, in the present disclosure, a "peak value" refers to the height or area of ​​each peak observed in a chromatogram, and a relative value or an absolute value can be used. This relative value may be a ratio to the total area of ​​the chromatogram, a ratio to the total area of ​​hemoglobin-related peaks in the chromatogram, or a ratio to the area of ​​a specific peak (e.g., the HbA0 peak).

[0010] When a blood sample is subjected to liquid chromatography, a hemoglobin chromatogram such as that shown schematically in Figure 1 is obtained. In this chromatogram, the following peaks are observed in descending order of elution rate from the column: a composite peak 10 including an HbA1a peak 11 and an HbA1b peak 12 (see Figures 2 and 3), an HbF peak 20, an unstable HbA1c peak 30, an HbA1c peak 40, an HbA0 peak 50, and an HbA2 peak 60 (see Figure 8).

[0011] A chromatograph of a normal blood sample with an HbF peak value of less than 1% is shown schematically in Figure 2, and a chromatograph of a blood sample from a patient with hyperHbFemia is shown schematically in Figure 3. As can be seen by comparing the HbA1c peak 30, which is approximately the same size in both figures, the HbF peak 20 in the chromatograph of Figure 3 is higher than the HbF peak 20 in the chromatograph of Figure 2. At this time, the composite peak 10 in the chromatograph of Figure 3 is also higher than the composite peak 10 in the chromatograph of Figure 2.

[0012] Here, composite peak 10 contains HbA1a peak 11 and HbA1b peak 12 of HbA1, which is a glycated product of HbA, as shown in Figure 2. Furthermore, as shown in Figure 3, an increase in HbF peak 20 leads to an increase in composite peak 10. Because composite peak 10 contains HbA1a peak 11 and HbA1b peak 12, which are glycated products, the increase in composite peak 10 is thought to be due to an increase in modified HbF peak 13, which is a modification of HbF, as shown in Figure 3. On the other hand, in the normal sample shown in Figure 2, modified HbF peak 13 is very small, and it is thought that the majority of composite peak 10 is occupied by HbA1a peak 11 and HbA1b peak 12.

[0013] Next, Figure 4 shows a schematic diagram of an analytical chart obtained by capillary electrophoresis of a normal blood sample with an HbF peak value of less than 1%, and Figure 5 shows a schematic diagram of an analytical chart obtained by capillary electrophoresis of a blood sample from a patient with hyperHbFemia. As shown in both figures, peaks appear on the analytical chart in the following order: HbA peak 100, HbF peak 120, and HbA2 peak 160. Comparing the two figures, it can be seen that the HbF peak 120 in the chromatograph of Figure 5 is higher than the HbF peak 120 in the chromatograph of Figure 4. However, the magnitudes of the HbA peak 100 and HbA2 peak 160 in Figure 4 are almost the same as those of the HbA peak 100 and HbA2 peak 160 in Figure 5. This suggests that the HbF peak 120 in the capillary electrophoresis also contains a modified HbF peak value.

[0014] Based on the above observations, the method for measuring HbF according to an embodiment of the present disclosure calculates the ratio of the hemoglobin F peak to all hemoglobin peaks from a chromatogram obtained by subjecting a blood sample to liquid chromatography, and then multiplies this ratio by a predetermined coefficient to calculate a corrected value of the hemoglobin F peak to all hemoglobin peaks. Specifically, this predetermined coefficient is 1.15 or greater but less than 1.25, preferably 1.2. The calculation of this corrected value requires that the ratio of the hemoglobin F peak to all hemoglobin peaks is ultimately multiplied by the predetermined coefficient. For example, the corrected value of the hemoglobin F peak to all hemoglobin peaks can be calculated by multiplying the hemoglobin F peak value by the predetermined coefficient to obtain a corrected value, and then calculating the ratio of the corrected value to all hemoglobin peaks. [Example]

[0015] (1) Liquid chromatography equipment As liquid chromatography devices, the following cation exchange chromatography devices, Device 1, Device 2, Device 3 and Device 4, were used.

[0016] (1-1) Device 1 Apparatus 1 was an ADAMS HA-8180V (sold by Arkray). Apparatus 1 used Column Unit 80 (sold by Arkray), a column packed with 0.35 ml of a hydrophilic polymer consisting of a methacrylate ester copolymer as the packing material. The reagents used to elute hemoglobin from the column were 80A (sold by Arkray) as eluent A, 80B (sold by Arkray) as eluent B, and 80CV (sold by Arkray) as eluent C, respectively. 80H (sold by Arkray) was used as the hemolysis and washing solution. The compositions and pH of 80A, 80B, 80CV, and 80H are shown in Table 1 below. These reagents were passed through the column at a flow rate of 1.7 ml / min.

[0017] (1-2) Device 2 Apparatus 2 was an ADAMS HA-8180T (sold by Arkray). Apparatus 2 used a column unit 80T (sold by Arkray), a column packed with 0.45 ml of a hydrophilic polymer consisting of a methacrylic acid ester copolymer as the packing material. The reagents used for eluting hemoglobin from the column were 80A as eluent A, 80B as eluent B, and 80CT (sold by Arkray) as eluent C, respectively, and 80H as the hemolysis and washing solution. The compositions and pH of 80A, 80B, 80CT, and 80H are shown in Table 1 below. These reagents were passed through the column at a flow rate of 1.7 ml / min.

[0018] (1-3) Device 3 The ADAMS HA-8190V (sold by Arkray) was used as Apparatus 3. This Apparatus 3 used Column Unit 90 (sold by Arkray), a column packed with 0.25 ml of a hydrophilic polymer consisting of a methacrylate ester copolymer as the packing material. The reagents used to elute hemoglobin from the column were 90A (sold by Arkray) as eluent A, 90B (sold by Arkray) as eluent B, and 90CV (sold by Arkray) as eluent C, respectively. 90H (sold by Arkray) was used as the hemolysis and washing solution. The compositions and pH of 90A, 90B, 90CV, and 90H are shown in Table 1 below. These reagents were passed through the column at a flow rate of 4.0 ml / min.

[0019] [Table 1]

[0020] Among eluents A to C, eluent A had the smallest elution power for hemoglobin, and eluent B had the largest elution power.

[0021] (2) Liquid Chromatography Measurement Method Measurement of HbF using Apparatus 1, Apparatus 2, and Apparatus 3 was carried out in accordance with the attached documents from Arkray, Inc. For Apparatus 1 and Apparatus 3, measurements were carried out in both the first and second modes described below.

[0022] (2-1) First mode Eluent A was passed through the column to elute HbF and HbA1c, and then eluent B was passed through the column to elute all remaining hemoglobin.

[0023] (2-2) Second mode Eluent A was passed through the column to elute HbF and HbA1c, then eluent C was passed through the column to elute HbS, HbC, HbE, and HbD, and then eluent B was passed through the column to elute all remaining hemoglobin.

[0024] (3) Capillary electrophoresis device The capillary electrophoresis apparatus used was Capillarys 2 Flexpiercing (Sebia). HbF measurement using the capillary electrophoresis apparatus was performed according to the attached document provided by Sebia.

[0025] (4) Measurement results Using the above-mentioned Apparatus 1 to Apparatus 3 and a capillary electrophoresis apparatus, HbF peak values ​​were measured for 51 whole blood samples.

[0026] As liquid chromatography devices, an example of measurement using Device 1 in the first mode is shown in Figure 6, an example of measurement using Device 1 in the second mode is shown in Figure 7, an example of measurement using Device 2 is shown in Figure 8, an example of measurement using Device 3 in the first mode is shown in Figure 9, and an example of measurement using Device 3 in the second mode is shown in Figure 10. It was shown that with all devices, it was possible to separate the HbF peak 20 from the composite peak 10 of the HbA1a and HbA1b peaks.

[0027] An example of measurement using a capillary electrophoresis system is shown in Figure 11. The analysis chart in Figure 11 shows that the ratio of HbF peak 120 to the total hemoglobin peaks is clearly greater than the ratio of HbF peak 20 to the total hemoglobin peaks measured using a liquid chromatography system.

[0028] Figure 12 is a graph showing the correlation between the HbF peak values ​​obtained by measuring the same sample using each liquid chromatography system and the HbF peak values ​​obtained by measuring the same sample using a capillary electrophoresis system. The horizontal axis of the graph shows the HbF peak values ​​obtained using the capillary electrophoresis system, and the vertical axis of the graph shows the HbF peak values ​​obtained using the liquid chromatography system. The dashed line in the graph indicates the correlation between the horizontal axis (x) and the vertical axis (y). y=x This graph shows that the HbF peak value obtained by the capillary electrophoresis apparatus is greater than that obtained by the liquid chromatography apparatus.

[0029] The ratio of the HbF peak value obtained with the capillary electrophoresis system to the HbF peak value obtained with each liquid chromatography system is shown in Table 2. The HbF peak value obtained with the capillary electrophoresis system was found to be 1.18 to 1.23 times the HbF peak value obtained with the liquid chromatography system.

[0030] [Table 2]

[0031] From the above, it was demonstrated that by multiplying the HbF peak value obtained by a capillary electrophoresis apparatus by a coefficient of 1.15 or more and less than 1.25, preferably by a coefficient of 1.18 or more and 1.23 or less, and more preferably by a coefficient of 1.2 times, it is possible to measure a more accurate HbF peak value, such as that obtained by a capillary electrophoresis apparatus. [Industrial Applicability]

[0032] The present invention can be used to measure HbF in a blood sample using liquid chromatography. [Explanation of symbols]

[0033] 10 Composite Peak 11 HbA1a peak 12 HbA1b peak 13 Modified HbF peak 20 HbF peak 30 Unstable HbA1c peak 40 HbA1c peak 50 HbA0 peak 60 HbA2 peak 100 HbA peak (capillary electrophoresis) 120 HbF peak (capillary electrophoresis) 160 HbA2 peak (capillary electrophoresis)

Claims

1. calculating a ratio of the peak value of the hemoglobin F peak to the peak value of all hemoglobin peaks from a chromatogram obtained by subjecting the blood sample to liquid chromatography; multiplying the ratio by a predetermined coefficient to calculate a correction value of the hemoglobin F peak relative to the total hemoglobin peak; a hemoglobin F measuring method, wherein the predetermined coefficient is a value obtained in advance as a ratio of the peak value of hemoglobin F obtained by a capillary electrophoresis device to the peak value of hemoglobin F obtained by a liquid chromatography device for the same blood sample.

2. The method for measuring hemoglobin F according to claim 1, wherein the coefficient is 1.15 or greater but less than 1.

25.

3. The method for measuring hemoglobin F according to claim 2, wherein the coefficient is 1.

2.

4. The method for measuring hemoglobin F according to any one of claims 1 to 3, wherein the liquid chromatography is cation exchange chromatography.

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