How to measure hemoglobin F

Abstract

To accurately measure HbF from a chromatogram obtained by subjecting a blood specimen to liquid chromatography.SOLUTION: A measuring method of hemoglobin F includes: determining in advance a first correlation equation being a correlation equation of an HbA1c peak value and a synthetic peak value including an HbA1a peak and an HbA1b peak from a chromatogram obtained by subjecting, to liquid chromatography, a first blood specimen containing HbA1c and whose HbF content to the total hemoglobin is known to be less than a prescribed content; calculating a modified HbF peak value by subtracting the synthetic peak value obtained by applying, to the first correlation equation, the HbA1c peak value of the chromatogram that is obtained by subjecting the measuring object blood specimen to the liquid chromatography, from the synthetic peak value including the HbA1a peak and the HbA1b peak of the blood specimen; and correcting the HbF peak value by adding the modified HbF peak value to the HbF peak value of the blood specimen.SELECTED DRAWING: Figure 8

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. Measurement values ​​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. 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] In the chromatogram obtained by subjecting a blood sample to cation exchange liquid chromatography, a composite peak appears on the side of the column with a faster elution rate than the HbF peak, which is a peak formed by overlapping the hemoglobin A1a (HbA1a) peak and the hemoglobin A1b (HbA1b) peak of hemoglobin A1 (HbA1), which is a glycated product of hemoglobin A (HbA).

[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] Just as HbA becomes HbA1c upon glycation, it is believed that HbF also undergoes modifications such as glycation to become modified HbF. Because modified HbF is also a type of HbF, measuring the peak value of modified HbF more accurately requires measuring the peak value of modified HbF. In the inventor's experiments, it was observed that the magnitude of this composite peak changes depending on the magnitude of the HbF peak. From these observations, it was inferred that this composite peak also contains modified HbF. Because the charges and magnitudes of HbA1a, HbA1b, and modified HbF contained in the composite peak are similar to each other, it is difficult to separate HbA1a, HbA1b, and modified HbF by cation exchange chromatography, and the peak value of modified HbF cannot be accurately measured. As a result, it is impossible to measure the total amount of HbF in a blood sample (in other words, the total concentration of unmodified HbF and modified HbF).

[0007] In one embodiment of the present disclosure, a method for measuring HbF involves: subjecting a first group of blood samples, which are known to contain HbA1c and have an HbF content less than a predetermined percentage relative to total hemoglobin, to liquid chromatography to obtain a chromatogram; determining in advance a first correlation equation that correlates the HbA1c peak value with a composite peak value including HbA1a and HbA1b peaks; applying the HbA1c peak value of the blood sample to be measured to the first correlation equation to obtain a composite peak value; subtracting the composite peak value obtained from the blood sample's composite peak value including HbA1a and HbA1b peaks to calculate a modified HbF peak value; and adding this modified HbF peak value to the HbF peak value of the blood sample to correct the HbF peak value. [Effects of the Invention]

[0008] 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]

[0009] [Figure 1] Schematic diagram of an example of a hemoglobin chromatogram 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] 1 is a chromatogram obtained by subjecting blood sample 1 to high performance liquid chromatography. [Figure 5] 1 is a chromatogram obtained by subjecting blood sample 2 to high performance liquid chromatography. [Figure 6] 1 is a chromatogram obtained by subjecting blood sample 3 to high performance liquid chromatography. [Figure 7] 1 is a chromatogram obtained by subjecting blood sample 4 to high performance liquid chromatography. [Figure 8] FIG. 1 is a scatter plot showing the correlation between the HbA1c peak value and the composite peak value including the HbA1a peak and the HbA1b peak in a chromatogram obtained by subjecting a group of blood samples from healthy individuals to high performance liquid chromatography. [Figure 9] FIG. 1 is a scatter plot showing the correlation between HbF peak values ​​and modified HbF peak values ​​in chromatograms obtained by subjecting a group of blood samples known to contain HbF to high performance liquid chromatography. DETAILED DESCRIPTION OF THE INVENTION

[0010] 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).

[0011] (1) First embodiment When a blood sample is subjected to cation exchange 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 containing 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 Figures 4 to 7).

[0012] A chromatogram of a normal blood sample with an HbF peak value of less than 1% is shown schematically in Figure 2, and a chromatogram of a blood sample from a patient with hyperHbFemia is shown schematically in Figure 3. As can be seen by comparing the HbA1c peak 40, which is approximately the same size in both figures, the HbF peak 20 in the chromatogram of Figure 3 is higher than the HbF peak 20 in the chromatogram of Figure 2. At this time, the composite peak 10 in the chromatogram of Figure 3 is also higher than the composite peak 10 in the chromatogram of Figure 2.

[0013] 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.

[0014] On the other hand, it is assumed that the ratio of the sum of the values ​​of HbA1a peak 11 and HbA1b peak 12 to HbA1c peak 40 is approximately constant, whether in a normal sample as shown in Figure 2 or a high HbF sample as shown in Figure 3. Under this assumption, if a correlation is found between the peak value of HbA1c peak 40 and the peak value of composite peak 10 (essentially the sum of the peak values ​​of HbA1a peak 11 and HbA1b peak 12) in a normal sample, this correlation can be applied to HbA1c peak 40 in a high HbF sample to calculate the sum of HbA1a peak 11 and HbA1b peak 12 in composite peak 10. The difference between the sum of the peak values ​​of HbA1a peak 11 and HbA1b peak 12 and the peak value of composite peak 10 is based on the value of modified HbF peak 13. It is believed that modified HbF peak 13 can be calculated by subtracting the calculated total from composite peak 10 in the chromatogram shown in Figure 3. As will be shown in the Examples below (see Figure 8), it was found that there is a high correlation between HbA1c peak 40 and composite peak 10 for multiple normal samples.

[0015] Based on this correlation, the HbF measurement method of the first embodiment of the present disclosure involves: determining a first correlation equation between the HbA1c peak value and a composite peak value including HbA1a and HbA1b peaks from a chromatogram obtained by subjecting a first group of blood samples known to contain HbA1c but not HbF to liquid chromatography; fitting the HbA1c peak value of the chromatogram obtained by subjecting the blood samples to liquid chromatography to the first correlation equation to obtain a composite peak value; subtracting the composite peak value obtained from the blood sample's HbA1a and HbA1b peaks to calculate a modified HbF peak value; and adding the modified HbF peak value to the HbF peak value of the blood sample to correct the HbF peak value. Because the HbF peak value correlates with the HbF concentration in the blood sample, the HbF concentration in the blood sample and the ratio of HbF to total hemoglobin can be determined based on the HbF peak value.

[0016] Specifically, for multiple blood samples known to contain HbA1c and whose HbF content relative to total hemoglobin is less than a predetermined percentage, a first correlation equation is determined from the correlation between the HbA1c peak value and the composite peak value (essentially the sum of the HbA1a peak value and the HbA1b peak value). The "predetermined content" referred to here can be an HbF peak value below the normal value (for example, 5%, preferably 3%, and more preferably 1% of the peak value of total hemoglobin). The "plurality of blood samples known to have an HbF content below the predetermined content relative to total hemoglobin" may be obtained as blood samples whose HbF peak values, obtained when hemoglobin contained in the blood samples is separated and analyzed by liquid chromatography, are below the predetermined content, or may be obtained as blood samples whose hemoglobin F content relative to total hemoglobin is shown to be below the predetermined content by analysis using a measurement principle other than liquid chromatography (for example, capillary electrophoresis). This first correlation equation is expressed as the following equation (1), where the HbA1c peak value is variable x and the composite peak value is variable y.

[0017] y=a1x+b1 (1) x: HbA1c peak value (variable) y: composite peak value (variable) a1: slope (constant) a2: Intercept (constant)

[0018] The HbA1c peak value is then determined from a chromatogram obtained from the blood sample to be measured, and this is substituted for variable x in equation (1) above to estimate the composite peak value as the calculated variable y. The modified HbF peak value contained in composite peak 10 is calculated by subtracting this calculated estimated composite peak value from the composite peak value obtained from composite peak 10 appearing in the chromatogram (i.e., measured using composite peak 10 appearing in the chromatogram). The calculated modified HbF peak value is then added to the peak value of HbF peak 20 obtained from the chromatogram, correcting the HbF peak value and estimating the true HbF peak value in the blood sample. The modified HbF peak value is expressed by equation (2) below. If the modified HbF peak value is w and the HbF peak value is variable z, the true HbF peak value (v) can be expressed by equation (3) below.

[0019] w=y-(a1x+b1) (2) v=z+w=z+(y-(a1x+b1)) ···(3) v: True HbF peak value (variable) w: peak modified HbF value (variable) x: HbA1c peak value (variable) y: composite peak value (variable) z: HbF peak value (variable) a1: slope (constant) b1: Intercept (constant)

[0020] (2) Second embodiment In the first embodiment described above, it is assumed that the blood sample to be measured contains HbA1c, but for blood samples that do not contain HbA1c or blood samples in which HbA1c is not detected, the HbA1c peak value cannot be fitted to the first correlation equation.

[0021] Therefore, in the HbF measurement method of the second embodiment, a first correlation equation is determined in advance as shown in the first embodiment above, and further, a second blood sample group known to contain HbA1c and HbF is subjected to liquid chromatography to obtain a chromatogram, and the HbA1c peak value is substituted into the first correlation equation to obtain a composite peak value. An estimated modified HbF peak value for the second blood sample group is obtained by subtracting the composite peak value from the composite peak value containing the HbA1a peak and the HbA1b peak of the second blood sample group. A second correlation equation, which is the correlation equation between the HbF peak value of the second blood sample group and the estimated modified HbF peak value, is determined in advance, and the HbF peak value of the chromatogram obtained by subjecting the blood sample to be measured to liquid chromatography is substituted into the second correlation equation to calculate the modified HbF peak value. The peak value of the modified HbF is then added to the HbF peak value of the blood sample to correct the HbF peak value.

[0022] Specifically, first, as described in the first embodiment, the first correlation equation expressed by the above equation (1) is determined.

[0023] Next, for chromatograms of multiple blood samples known to contain both HbA1c and HbF, the HbA1c peak value is first determined, and this is substituted for variable x in the above formula (1), to estimate the composite peak value as the calculated variable y. The calculated composite peak value is subtracted from the composite peak value obtained from composite peak 10 appearing in the chromatogram (i.e., measured using composite peak 10 appearing in the chromatogram), to calculate the modified HbF peak value contained in composite peak 10. Meanwhile, HbF peak values ​​are obtained from the same chromatogram, and a second correlation equation is determined from the correlation between these HbF peak values ​​and the calculated modified HbF peak values. This second correlation equation is expressed as the following formula (4), where the HbF peak value is variable z and the modified HbF peak value is variable w.

[0024] w=a2z+b2 (4) w: peak modified HbF value (variable) z: HbF peak value (variable) a2: slope (constant) b2: Intercept (constant)

[0025] The HbF peak value is then determined from the chromatogram obtained from the blood sample to be measured, and this value is substituted for variable z in equation (4) above to calculate the modified HbF peak value as variable w. This calculated modified HbF peak value is then added to the HbF peak value obtained from the chromatogram to correct the HbF peak value and estimate the true HbF peak value in the blood sample. The true HbF peak value (v) in the blood sample is expressed by equation (5) below.

[0026] v=z+w=z+(a2z+b2) (5) v: True HbF peak value (variable) w: peak modified HbF value (variable) z: HbF peak value (variable) a2: slope (constant) b2: Intercept (constant)

[0027] Instead of the second correlation equation, a third correlation equation may be determined from the correlation between the HbF peak value and the corrected HbF peak value (the sum of the HbF peak value and the modified HbF peak value). This third correlation equation is expressed as the following equation (6), where z is the HbF peak value and v is the corrected HbF peak value, which is the true HbF peak value.

[0028] v=a3z+b3 (6) v: True HbF peak value (variable) z: HbF peak value (variable) a3: slope (constant) b3: Intercept (constant)

[0029] The HbF peak value is then determined from the chromatogram obtained from the blood sample to be measured, and this is substituted for the variable z in the above equation (5) or (6). The corrected HbF peak value, i.e., the true HbF peak value in the blood sample, is estimated as the calculated variable v.

[0030] (3) Third embodiment The first embodiment described above is suitable for blood samples containing HbA1c. The second embodiment described above is suitable for blood samples not containing HbA1c. Therefore, it is desirable to selectively use the measurement method of the first embodiment described above or the measurement method of the second embodiment described above depending on whether or not HbA1c is contained in the chromatogram.

[0031] That is, in the HbF measurement method of the third embodiment, a first correlation equation is determined in advance as shown in the first embodiment, and a second correlation equation is determined in advance as shown in the second embodiment. Then, if a target chromatogram obtained by subjecting a blood sample to liquid chromatography has an HbA1c peak, the HbF peak value of the blood sample is corrected as shown in the first embodiment. On the other hand, if a target chromatogram obtained by subjecting a blood sample to liquid chromatography does not have an HbA1c peak, the HbF peak value of the blood sample is corrected as shown in the first embodiment. [Example]

[0032] (1) Liquid chromatography equipment A commercially available cation exchange chromatography column packed with a hydrophilic polymer consisting of a methacrylate ester copolymer was connected to a commercially available high-performance liquid chromatography system. An optical detector (specifically, an absorbance meter) was attached to a predetermined position in the flow path downstream of the cation exchange chromatography column to detect the concentration of hemoglobin flowing through the flow path.

[0033] (2) Eluent Three types of eluents were prepared as aqueous solutions with the compositions shown in Table 1 below.

[0034] [Table 1]

[0035] Eluent A was adjusted to pH 5.08, eluent B to pH 8.0, and eluent C to pH 6.82. Eluent A had the lowest elution ability for hemoglobin, while eluent B had the highest.

[0036] (3) Chromatogram Eluent A was passed through the liquid chromatography apparatus described above (1) to equilibrate the column. Then, a hemolyzed blood sample was introduced into the column. Eluent A was then passed through for 13 seconds to elute HbA1a, HbA1b, HbF, and HbA1c. Next, a 1:9 mixture of eluent A and eluent C was passed through for 5 seconds to elute HbA0. Next, eluent C was passed through for 5 seconds to elute HbA2. Then, eluent B was passed through for 2 seconds to elute all of the hemoglobin remaining in the column, and then eluent A was passed through for 5 seconds. During this elution, a chromatogram was created from the absorbance measured by an optical detector at a detection wavelength of 420 nm.

[0037] Examples of the obtained chromatograms are shown in Figures 4 to 7. As is clear from these figures, the area of ​​HbF peak 20 (HbF peak value) increased, as did the area of ​​composite peak 10 (composite peak value). Therefore, it was strongly speculated that composite peak 10, which is known to contain HbA1a peak 11 and HbA1b peak 12 (see Figure 3), also contains modified HbF peak 13 (see Figure 3).

[0038] (4) First correlation equation To determine the first correlation equation, blood samples from 66 healthy individuals (blood samples in which the HbF peak value was less than 1% of the total hemoglobin peak value) were prepared. Chromatograms were obtained from each of these blood samples, and the HbA1c peak value and composite peak value were measured for each. A correlation was found, as shown in the scatter plot in Figure 8. The horizontal axis of the graph indicates the HbA1c peak value, and the vertical axis indicates the composite peak value (i.e., the sum of the HbA1a peak value and the HbA1b peak value). The first correlation equation, represented by the dashed straight line in Figure 8, was expressed as follows: where x is the HbA1c peak value and y is the composite peak value. The correlation coefficient (r) of this first correlation equation was 0.8951.

[0039] y=0.1475x+0.8048 (7) x: HbA1c peak value (variable) y: composite peak value (variable)

[0040] The correlation between composite peak 10, which contains HbA1a and HbA1b, and HbA1c peak 40 is presumably due to the fact that HbA1a, HbA1b, and HbA1c are all glycated products of HbA0, and the respective ratios of HbA1a, HbA1b, and HbA1c produced by the glycation of HbA0 are approximately constant.

[0041] The HbA1c peak value is then measured from the chromatogram obtained from the blood sample to be measured, and this is substituted into the above formula (7) as the variable x to calculate the sum of the HbA1a and HbA1b peak values ​​(hereinafter referred to as the "AB value") as the variable y. The difference between the composite peak value measured from the chromatogram and this AB value is based on the modified HbF peak value. The modified HbF peak value is then calculated by subtracting this AB value from the composite peak value measured from the chromatogram. The value obtained by adding this calculated modified HbF peak value to the HbF peak value measured from the chromatogram is then estimated to be the correct HbF peak value for the blood sample.

[0042] (2) Second correlation equation To determine the second correlation equation, blood samples from 66 healthy subjects (blood samples showing HbF peak values ​​less than 1% of the total hemoglobin peak value) and 48 patients with high HbF were prepared. Chromatograms were obtained from each of these blood samples. HbA1c peak values ​​were measured from these chromatograms and substituted into the above equation (7) as variable x to calculate the AB value as variable y. Furthermore, this AB value was subtracted from the composite peak value measured from the chromatogram to calculate the modified HbF peak value. A correlation was found between the measured HbF peak values ​​from these chromatograms and the calculated modified HbF peak value, as shown in the scatter diagram in Figure 9. The horizontal axis of the graph represents the HbF peak value, and the vertical axis represents the modified HbF peak value. The second correlation equation, represented by the dashed line in Figure 9, was expressed as the following equation (8), where the HbF peak value is variable z and the modified HbF peak value is variable w. The correlation coefficient (r) of this first correlation equation was 0.9697.

[0043] w=0.2323z (8)

[0044] The HbF peak value is then measured from a chromatogram obtained from the blood sample to be measured, and this is substituted into the above equation (8) as variable z to calculate the modified HbF peak value as variable w. The value obtained by adding this calculated modified HbF peak value to the HbF peak value measured from the chromatogram is then estimated to be the correct HbF peak value for the blood sample. [Industrial Applicability]

[0045] The present invention can be used to measure HbF in a blood sample using liquid chromatography. [Explanation of symbols]

[0046] 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

Claims

1. a first correlation equation is determined in advance from a chromatogram obtained by subjecting a first group of blood samples, which are known to contain hemoglobin A1c and have a hemoglobin F content less than a predetermined percentage relative to total hemoglobin, to liquid chromatography, the first correlation equation being a correlation equation between a hemoglobin A1c peak value and a composite peak value including a hemoglobin A1a peak and a hemoglobin A1b peak; calculating a modified hemoglobin F peak value by applying the hemoglobin A1c peak value of a chromatogram obtained by subjecting a blood sample to liquid chromatography to the first correlation equation and subtracting the composite peak value obtained from the composite peak value including the hemoglobin A1a peak and the hemoglobin A1b peak of the blood sample; A method for measuring hemoglobin F, comprising adding the modified hemoglobin F peak value to the hemoglobin F peak value of the blood sample to correct the hemoglobin F peak value.

2. a first correlation equation is determined in advance from a chromatogram obtained by subjecting a first group of blood samples, which are known to contain hemoglobin A1c and have a hemoglobin F content less than a predetermined percentage relative to total hemoglobin, to liquid chromatography, the first correlation equation being a correlation equation between a hemoglobin A1c peak value and a composite peak value including a hemoglobin A1a peak and a hemoglobin A1b peak; a second blood sample group known to contain hemoglobin A1c and hemoglobin F is subjected to liquid chromatography to obtain a chromatogram, and a hemoglobin A1c peak value is fitted to the first correlation equation to obtain a composite peak value; a second blood sample group is subjected to liquid chromatography to obtain a modified hemoglobin F peak estimated value by subtracting the composite peak value from a composite peak value of the second blood sample group containing a hemoglobin A1a peak and a hemoglobin A1b peak; and a second correlation equation is determined in advance as a correlation equation between the hemoglobin F peak value and the modified hemoglobin F peak value, using the hemoglobin F peak value of the second blood sample group and the estimated modified hemoglobin F peak value; the hemoglobin F peak value of the chromatogram obtained by subjecting the blood sample to be measured to liquid chromatography is applied to the second correlation equation to calculate the modified hemoglobin F peak value; A method for measuring hemoglobin F, comprising adding the modified hemoglobin F peak value to the hemoglobin F peak value of the blood sample to correct the hemoglobin F peak value.

3. a first correlation equation is determined in advance from a chromatogram obtained by subjecting a first group of blood samples, which are known to contain hemoglobin A1c and have a hemoglobin F content less than a predetermined percentage relative to total hemoglobin, to liquid chromatography, the first correlation equation being a correlation equation between a hemoglobin A1c peak value and a composite peak value including a hemoglobin A1a peak and a hemoglobin A1b peak; a second blood sample group known to contain hemoglobin A1c and hemoglobin F is subjected to liquid chromatography to obtain a chromatogram, and a hemoglobin A1c peak value is fitted to the first correlation equation to obtain a composite peak value; a second blood sample group is subjected to liquid chromatography to obtain a modified hemoglobin F peak estimated value by subtracting the composite peak value from a composite peak value of the second blood sample group containing a hemoglobin A1a peak and a hemoglobin A1b peak; and a second correlation equation is determined in advance as a correlation equation between the hemoglobin F peak value and the modified hemoglobin F peak value, using the hemoglobin F peak value of the second blood sample group and the estimated modified hemoglobin F peak value; When a chromatogram obtained by subjecting a blood sample to be measured to liquid chromatography has a hemoglobin A1c peak, calculating a modified hemoglobin F peak value by subtracting a composite peak value obtained by applying the hemoglobin A1c peak value of the blood sample to the first correlation equation from a composite peak value including the hemoglobin A1a peak and the hemoglobin A1b peak of the blood sample; adding the modified hemoglobin F peak value to the hemoglobin F peak value of the blood sample to correct the hemoglobin F peak value; When the chromatogram obtained by subjecting the blood sample to be measured to liquid chromatography does not have a hemoglobin A1c peak, calculating a modified hemoglobin F peak value by applying the hemoglobin F peak value of the blood sample to the second correlation equation; A method for measuring hemoglobin F, comprising adding the modified hemoglobin F peak value to the hemoglobin F peak value of the blood sample to correct the hemoglobin F peak value.

4. determining in advance a third correlation equation, which is a correlation equation between the hemoglobin F peak value of the second blood sample group and the sum of the hemoglobin F peak value and the estimated modified hemoglobin F peak value, instead of the second correlation equation; 4. The method for measuring hemoglobin F according to claim 2, wherein the hemoglobin F peak value of a chromatogram obtained by subjecting a blood sample to liquid chromatography is corrected by applying the hemoglobin F peak value to the third correlation equation instead of the second correlation equation.

5. The method according to any one of claims 1 to 4, wherein the liquid chromatography is cation exchange chromatography.

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