Method for measuring glycated hemoglobin and device for measuring glycated hemoglobin

Abstract

A method for measuring a proportion of sA1c (%), which includes, when a peak derived from abnormal hemoglobin D, abnormal hemoglobin S or abnormal hemoglobin C is identified, calculation of the peak area, and measurement of the proportion of sA1c (%) corrected by using the calculation results. Results of measurement are obtained, by cation exchange chromatography, of sA1c (%) with a subject who provided a blood sample containing abnormal hemoglobin D, abnormal hemoglobin S or abnormal hemoglobin C by eliminating influences by such abnormal hemoglobin.

Claims

1. A method for measuring stable glycated hemoglobin sA1c, which comprises (1) a step of subjecting a blood sample after hemolysis to cation exchange chromatography to elute sA1c as separated from other hemoglobin components thereby to obtain a chromatogram showing elution of hemoglobin fractions, (2) a step of identifying a sA1c peak in the obtained chromatogram and calculating its peak area, (3) a step of identifying peaks of hemoglobin components of hemoglobin A eluted before and including non-glycated peak of hemoglobin A, A0, other than sA1c in the obtained chromatogram and calculating their peak areas, (4) a step of calculating a proportion (%) of the sA1c peak area obtained in the step 2 to a total hemoglobin peak area which is a sum of the peak areas obtained in the step 3 and sA1c peak area obtained in the step 2, (5) a step of identifying a non-glycated peak of abnormal hemoglobin D, abnormal hemoglobin S or abnormal hemoglobin C from peaks which appear after the A0, and (6) a step of estimating an area of a glycated peak of abnormal hemoglobin D, abnormal hemoglobin S or abnormal hemoglobin C based on a non-glycated peak area of the abnormal hemoglobin identified, wherein the proportion (%) of the sA1c peak area in the above step 4 is corrected using the estimated glycated peak area of the identified abnormal hemoglobin, wherein when a non-glycated peak of abnormal hemoglobin D or abnormal hemoglobin S is identified in the step 5, the proportion (%) of the sA1c peak area to the total peak area of hemoglobin A is corrected in accordance with the following formulae, on an assumption that the glycated peak of the abnormal hemoglobin D or abnormal hemoglobin S appears as overlapping the A0, and that a proportion of the sA1c peak area to the total peak area of hemoglobin A is equal to a proportion of the glycated peak area of the abnormal hemoglobin to a sum of the peak areas of the glycated abnormal hemoglobin, non-glycated abnormal hemoglobin, and other peaks that elute after the A0 and do not include the peak of glycated abnormal hemoglobin and the peak of non-glycated abnormal hemoglobin:
A1c %=100sA1c/(A0+α)=100X1c/(X0+β+X1c)
A′=A0+X1c
X1c=[(A′+α−sA1c)−√{(A′+α−sA1c).sup.2−4sA1c(X0+β)}]/2 wherein A′ is a sum of A0 and a glycated peak area of abnormal hemoglobin which coelutes with A0 and is an area of the peak observed as A0 on the chromatogram, sA1c and A0 are respectively glycated and non-glycated peak areas of hemoglobin A, X1c and X0 are respectively a glycated peak area of abnormal hemoglobin which coelutes with A0 and a non-glycated peak area of abnormal hemoglobin which appears after A0, α=A1a+A1b+LA1c+sA1c, and β is one obtained by subtracting X0 from a total area of peaks which appear after A0; and when a non-glycated peak of abnormal hemoglobin C is identified in the step 5, the proportion (%) of the sA1c peak area to the total peak area of hemoglobin A is corrected in accordance with the following formula, on an assumption that the glycated peak of the abnormal hemoglobin C appears after the non-glycated peak A0 of hemoglobin A:
A1c %=100sA1c/(A′+α) wherein A′ is a peak area observed as A0 on the chromatogram, sA1c and A0 are respectively glycated and non-glycated peak areas of hemoglobin A, and
α=A1a+A1b+LA1c+sA1c.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a diagram illustrating the correlation between measurement results by affinity chromatography and measurement results by cation exchange chromatography, with respect to blood samples containing abnormal hemoglobin D and blood samples containing no abnormal hemoglobin D.

(2) FIG. 2 is a diagram illustrating the correlation between measurement results by affinity chromatography and measurement results by cation exchange chromatography, with respect to blood samples containing abnormal hemoglobin S and blood samples containing no abnormal hemoglobin S.

(3) FIG. 3 is a diagram illustrating the correlation between measurement results by affinity chromatography and measurement results by cation exchange chromatography, with respect to blood samples containing abnormal hemoglobin C and blood samples containing no abnormal hemoglobin C.

(4) FIG. 4 is a chromatogram obtained by cation exchange chromatography with respect to a blood sample containing no abnormal hemoglobin D, abnormal hemoglobin S or abnormal hemoglobin C.

(5) FIG. 5 is a chromatogram obtained by cation exchange chromatography with respect to a blood sample containing abnormal hemoglobin D.

(6) FIG. 6 is a chromatogram obtained by cation exchange chromatography with respect to a blood sample containing abnormal hemoglobin S.

(7) FIG. 7 is a chromatogram obtained by cation exchange chromatography with respect to a blood sample containing abnormal hemoglobin C.

(8) FIG. 8 is a diagram illustrating the correlation between measurement results by affinity chromatography and measurement results obtained by the method of the present invention by correcting A1c % based on the area of a remarkable peak (H-V0) corresponding to abnormal hemoglobin D and other peak area information on a chromatogram, with respect to blood samples containing abnormal hemoglobin D and blood samples containing no abnormal hemoglobin D.

(9) FIG. 9 is a diagram illustrating the correlation between measurement results by affinity chromatography and measurement results obtained by the method of the present invention by correcting A1c % based on the area of a remarkable peak (H-V1) corresponding to abnormal hemoglobin S and other peak area information on a chromatogram, with respect to blood samples containing abnormal hemoglobin S and blood samples containing no abnormal hemoglobin S.

(10) FIG. 10 is a diagram illustrating the correlation between measurement results by affinity chromatography and measurement results obtained by the method of the present invention by correcting A1c % based on the area of a remarkable peak (H-V2) corresponding to abnormal hemoglobin C and other peak area information on a chromatogram, with respect to blood samples containing abnormal hemoglobin C and blood samples containing no abnormal hemoglobin C.

(11) FIG. 11 is a diagram illustrating the measurement device of the present invention.

(12) FIG. 12 is a diagram illustrating the measurement device of the present invention.

DESCRIPTION OF EMBODIMENTS

(13) FIGS. 1, 2 and 3 are diagrams illustrating the correlation between measurement results by affinity chromatography and measurement results by cation exchange chromatography, with respect to blood samples containing abnormal hemoglobin D, abnormal hemoglobin S or abnormal hemoglobin C (38, 33 or 53 samples respectively) and blood samples containing no such abnormal hemoglobin (22 samples, open circle).

(14) The former samples were confirmed to contain abnormal hemoglobin D, abnormal hemoglobin S or abnormal hemoglobin C by a commercial apparatus (Capillarys 2 manufactured by Sebia).

(15) Measurement by affinity chromatography is carried out by a commercial apparatus (Ultra.sup.2 manufactured by Trinity Biotech), and measurement by cation chromatography is carried out by cation exchange chromatography using an Automated Glycohemoglobin Analyzer HLC-723G8 (tradename) manufactured by TOSOH CORPORATION equipped with a non-porous cation exchange column.

(16) The vertical axis of FIGS. 1, 2 and 3 indicates measurement results by cation exchange chromatography, and the horizontal axis indicates measurement results by affinity chromatography, and all the results are the proportion (%) of sA1c to total hemoglobin, and all the results are obtained by plotting measurement results output from the device with dedicated reagents after calibration.

(17) With respect to blood samples containing no abnormal hemoglobin (22 samples, open circle), there is a good correlation between results by affinity chromatography and results by cation exchange chromatography, however, with respect to blood samples containing abnormal hemoglobin D, abnormal hemoglobin S or abnormal hemoglobin C (38, 33 or 53 samples, filled circle), the results by cation exchange chromatography show lower values than the results by affinity chromatography.

(18) FIG. 4 is an example of a chromatogram obtained by subjecting a blood sample of a healthy subject to cation exchange chromatography. With a blood sample from a healthy subject, no remarkable peak derived from other component appears after the hemoglobin A peak (A0). FIGS. 5, 6 and 7 are examples of chromatograms obtained by subjecting blood samples from subjects containing abnormal hemoglobin D, abnormal hemoglobin S or abnormal hemoglobin C, to cation exchange chromatography. As evident from FIGS. 5, 6 and 7, with the blood samples containing abnormal hemoglobin D, abnormal hemoglobin S or abnormal hemoglobin C, a remarkable peak (H-V0, H-V1 or H-V2) derived from such abnormal hemoglobin, which is not observed in FIG. 4, appears after the hemoglobin A peak (A0).

(19) Accordingly, peaks derived from abnormal hemoglobin D, abnormal hemoglobin S and abnormal hemoglobin C (respectively H-V0, H-V1 and H-V2) can be identified by comparison with a chromatogram of a blood sample containing no such abnormal hemoglobin. Accordingly, when elution times at which H-V0, H-V1 and H-V2 appear under the predetermined conditions (in the case of examples in Figs., 1.0 minute, 1.2 minutes and 1.3 minutes, respectively) are stored, peaks derived from abnormal hemoglobin D, abnormal hemoglobin S and abnormal hemoglobin C can be identified from the elution times, so long as sA1c is measured under the same conditions.

EXAMPLES

Example 1

(20) The method of the present invention was carried out such that in a chromatogram (FIG. 5) obtained by cation exchange chromatography with respect to a blood sample containing abnormal hemoglobin D, the total peak area (Total area) and the respective peak areas of A1a, A1b, HbF, LA1c, sA1c, A′ and H-V0 were calculated, the X1 c area was obtained from the formula (3), the A0 area was determined by the formula (2), and A1c % was calculated by the formula (1). The peak detection range for H-V0 was 1.00±0.07 minutes.

(21) (1) A′ Peak Area

(22) The A′ peak area was calculated to be 921.2 from the chromatogram.

(23) (2) α Peak Area

(24) The α peak area was calculated to be 8.0+6.7+38.1+63.9=116.7 from the chromatogram.

(25) (3) sA1c Peak Area

(26) The sA1c peak area was calculated to be 63.9 from the chromatogram.

(27) (4) X0 Peak Area

(28) The X0=H-V0 peak area was calculated to be 576.7 from the chromatogram.

(29) (5) β Peak Area

(30) The peak area can be calculated as Total area-HbF-α-A′-X0, from the Total area, the β peak area was calculated to be 1,645.2-12.6-116.7-921.2-576.7=18.0 from the chromatogram.

(31) (6) Calculation of X1c

(32) The above values were assigned to the formula (3) to calculate X1c=40.7.

(33) (7) Calculation of A0

(34) The above-obtained value of X1c was assigned to the formula (2) to calculate A0=880.5.

(35) (8) Calculation of A1c %

(36) The above-obtained values were assigned to the formula (1) to calculate A1c %=6.4%.

(37) (9) Conversion to NGSP Units (Conversion Factors: 1.1151, 0.6558)
NGSP converted value (%)=6.4×1.1151+0.6558=7.8(%)

Example 2

(38) The method of the present invention was carried out such that in a chromatogram (FIG. 6) obtained by cation exchange chromatography with respect to a blood sample containing abnormal hemoglobin S, the total peak area (Total area) and the respective peak areas of A1a, A1b, HbF, LA1c, sA1c, A′ and H-V1 were calculated, the X1 c area was obtained from the formula (3), the A0 area was determined by the formula (2), and A1c % was calculated by the formula (1). The peak detection range for H-V1 was 1.16±0.09 minutes.

(39) (1) A′ Peak Area

(40) The A′ peak area was calculated to be 899.0 from the chromatogram.

(41) (2) α Peak Area

(42) The α peak area was calculated to be 11.8+7.8+31.4+78.6=129.6 from the chromatogram.

(43) (3) sA1c Peak Area

(44) The sA1c peak area was calculated to be 78.6 from the chromatogram.

(45) (4) X0 Peak Area

(46) The X0=H-V1 peak area was calculated to be 573.6 from the chromatogram.

(47) (5) β Peak Area

(48) The peak area can be calculated as Total area-HbF-α-A′-X0, from the Total area, the β peak area was calculated to be 1,619.0-10.5-129.6-899.0-573.6=6.3 from the chromatogram.

(49) (6) Calculation of X1c

(50) The above values were assigned to the formula (3) to calculate X1c=50.7.

(51) (7) Calculation of A0

(52) The above-obtained value of X1c was assigned to the formula (2) to calculate A0=848.3.

(53) (8) Calculation of A1c %

(54) The above-obtained values were assigned to the formula (1) to calculate A1c %=8.0%.

(55) (9) Conversion to NGSP Units (Conversion Factors: 1.1151, 0.6558)
NGSP converted value (%)=8.0×1.1151+0.6558=9.6(%)

Example 3

(56) The method of the present invention was carried out such that in a chromatogram (FIG. 7) obtained by cation exchange chromatography with respect to a blood sample containing abnormal hemoglobin C, the respective peak areas of A1a, A1b, LA1c, sA1c and A0 were calculated, and A1c % was calculated by the formula (4). The peak detection range for H-V2 was 1.34±0.09 minutes.

(57) (1) A0 Peak Area

(58) The A0 peak area was calculated to be 1,140.3 from the chromatogram.

(59) (2) α Peak Area

(60) The α peak area was calculated to be 10.8+11.8+34.8+89.9=147.3 from the chromatogram.

(61) (3) sA1c Peak Area

(62) The sA1c peak area was calculated to be 89.9 from the chromatogram.

(63) (8) Calculation of A1c %

(64) The above-obtained values were assigned to the formula (4) to calculate A1c %=6.7%.

(65) (9) Conversion to NGSP Units (Conversion Factors: 1.1151, 0.6558)
NGSP converted value (%)=6.7×1.1151+0.6558=8.1(%)

Example 4

(66) The present invention shown in Examples 1, 2 and 3 were applied to the chromatograms obtained by subjecting the blood samples containing abnormal hemoglobin D, S and C shown in FIGS. 5, 6 and 7 to cation exchange chromatography, and the results are shown in FIGS. 8, 9 and 10, respectively. As shown in these Figs., as a result of application of the present invention, the differences with measurement results by affinity chromatography are improved, and the slopes in the correlation coefficients were improved from 0.8841 to 0.9731, from 0.8767 to 0.9732, and from 0.8919 to 1.018, respectively as compared with FIGS. 1, 2, and 3.

(67) The present invention was described in detail with reference to specific embodiments, however, it is obvious to those skilled in the art that various changes and modifications are possible without departing from the spirit and scope of the present invention.

(68) The entire disclosure of Japanese Patent Application No. 2016-095191 filed on May 11, 2016 including specification, claims, drawings and summary is incorporated herein by reference in its entirety.

REFERENCE SYMBOLS

(69) 1 Cation exchange chromatography device 11 A means of sample injection 12 A means of liquid transportation 13 A means of separation 14 A means of detection 15 A means of analysis 16 Baseline setting module 17 Peak identification module 18 Peak area calculation module 19 sA1c proportion calculation module 20 A means of output