JP3516069B2 - How to measure glucose concentration - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring glucose concentration in blood. More specifically, using a glucose sensor, substantially only plasma is collected from a blood sample containing blood cells and plasma, and when measuring the glucose concentration, it is determined that blood cells are mixed in the collected plasma. The present invention relates to a method for measuring glucose concentration, which is characterized by being able to do so.

[0002]

2. Description of the Related Art In medical fields, medical research fields, etc.
Measurement of glucose concentration in blood (or plasma, and possibly serum) is required, for which various methods have been proposed and implemented. Among such methods, one widely used method is a method of measuring glucose concentration in blood using a biosensor. As is well known, this measurement method is generally called a glucose sensor method, in which glucose in a sample is decomposed using glucose oxidase (GOD), which is a glucose-degrading enzyme, and the decomposition product generated at that time is generated. The measurement principle is to electrochemically measure the amount of hydrogen peroxide as a substance and the amount of oxygen consumed, and obtain the glucose concentration in the sample from the measurement result.

Since such a glucose sensor method can measure glucose concentration with high sensitivity without the need for complicated pretreatment of a sample, it is widely used especially for the diagnosis and treatment of diabetes. A measuring device combining an immobilized membrane (glucose sensor) and a hydrogen peroxide electrode is often used in this method. For example, as a glucose concentration measuring device using this method, the GA-1140 device under the trade name is commercially available from Kyoto Daiichi Kagaku.

When the glucose concentration is measured by the glucose sensor method, blood is centrifuged to obtain a supernatant (usually plasma, and sometimes serum) from which blood cells are separated,
This is diluted with an appropriate buffer solution and the glucose decomposition method is used to measure the decomposition rate of glucose. When using such a glucose sensor method, it is necessary to take special care not to mix blood cells when sampling a supernatant (plasma or serum) from centrifuged blood. This is because blood cells have a significant amount of solids (usually 25-
45%), the dilution ratio of the sample with the buffer solution is different between the true ratio and the apparent ratio when blood samples are contained in the sample.

For example, consider a case where a predetermined amount A (for example, 20 μl) of a sample is diluted with a predetermined amount B (for example, 1.5 ml) of a buffer solution and the glucose concentration is measured by the glucose sensor method. In a proper measurement, A is blood plasma containing no blood cells, and therefore the dilution ratio is (A + B) /
A times. To convert the measured glucose concentration C (that is, the glucose concentration in the state diluted with a buffer solution) as raw data obtained by the measurement using the glucose sensor method into the glucose concentration in blood, C is (A + B)
It is necessary to multiply by / A.

However, when the solid content a is contained in A due to the mixing of a certain amount of blood cells in A, the true dilution ratio is (A−a + B) / (A−a) times. ,
Upon measurement, the apparent dilution ratio ((A +
Since only B) / A) is known, the measured glucose concentration C must be multiplied by (A + B) / A when converting the glucose concentration. To find the true glucose concentration,
Originally, the measured glucose concentration C was (A−a + B) / (A−
a) It is necessary to double. In addition, since the difference between the true dilution ratio and the apparent dilution ratio increases as the amount of blood cells mixed in increases, especially when many blood cells are mixed, the method for measuring the glucose concentration using the apparent dilution ratio is Not quite right.

As can be understood mathematically, apparent dilution ratio-true dilution ratio = (A + B) / A- (A-a + B) / (A-a) = (1 + B / A)-{1 + B / (A -A)} = B / A-B / (A-a) <0, the apparent dilution ratio is smaller than the true dilution ratio, and when blood cells are mixed in the sample, the apparent dilution ratio is used to calculate the glucose concentration. When calculated, the calculated glucose concentration becomes a value smaller than the true glucose concentration.

In recent years, also in the measurement of glucose concentration in blood, automation has progressed as in other fields. Especially, the sampling of the supernatant after centrifuging the collected blood sample is automated, and the glucose sensor thereafter. The measurement by is likewise automated. This sampling is usually performed by inserting a suction tube from above the separated sample and aspirating, but there are individual differences in the sample amount itself and also in the proportion of blood cells among individuals. Since the interface between the blood plasma portion and the lower blood cell portion is not always at a fixed position, when sampling the blood plasma portion, the tip of the suction tube is located inside the blood cell portion and the upper layer liquid containing the blood cell portion is aspirated. I have something to do.

In order to prevent such suction of blood cells, it has been proposed to detect the position of the interface in advance. Specifically, there is a method of using an electrode to detect the interface position by utilizing the difference in conductivity between the upper layer (plasma layer) and the lower layer (blood cell layer). In this method, two electrodes are used. It is necessary, and complete insulation is required for the conductivity measurement, which complicates the measurement system.

There is also a method of detecting the interface position by using the difference in light transmittance or reflectance between the upper layer and the lower layer by using CCD or LED. Usually, the collected blood is stored in test tubes, but these test tubes usually have an identification label attached, and there are cases where there is no part that reflects or transmits light near the interface. , In many cases, these methods cannot be applied. Furthermore, recently, there has been a tendency to reduce the amount of blood collected for the purpose of reducing the burden on the patient, and accordingly, it is desirable to further improve the accuracy of sampling and avoid mixing of blood cells. However, as described above, there is a practical problem in the method of detecting the position of the interface in advance in order to prevent the suction of blood cells.

[0011]

In view of the above-mentioned conventional techniques, it is not easy to prevent blood cells from being mixed in advance during automatic sampling of plasma in the automatic analysis of glucose concentration in blood. In fact, it is very difficult to avoid blood cell contamination with the current measurement technology. Therefore, even a blood cell-contaminated sample is automatically analyzed in the same manner as a blood cell-uncontaminated sample.Therefore, a measurement value for a sample without blood cells and a measurement value for a sample with blood cells mixed It is practically necessary to distinguish them.

[0012] Therefore, it is an object of the present invention to provide means for detecting the mixing of blood cells at the time of sampling plasma. If such a means is provided, it becomes possible to eliminate the measurement value of the sample in which the blood cell portion is mixed, and it is possible to prevent inappropriate judgment or treatment based on such measurement value. Become.

[0013]

According to the first aspect of the present invention, according to the present invention, after a liquid portion is sampled from a sample containing blood cells and a liquid component, the glucose concentration in the liquid portion of the sample is measured by a glucose sensor method. A method of measuring, using a glucose sensor method for a sample that is sampled, measuring the sensor output when glucose in the sample is decomposed by an enzyme, and based on this output, the glucose concentration in the sample by the equilibrium point method ( G1) and the glucose concentration (G2) in the sample by the first derivative method, respectively, and the glucose concentration (G1) and the glucose concentration (G
There is provided a method for measuring glucose concentration, which comprises detecting whether or not blood cells are mixed in a sampled sample by determining whether or not there is a significant difference between the sample and the sample.

In the present invention, blood cells mean at least one selected from red blood cells, white blood cells and platelets,
Practically, it is meant to consist mainly of red blood cells. The liquid component means a liquid existing outside blood cells, such as plasma and serum. Therefore, a sample containing blood cells and liquid components means, for example, a normal living body (organism, particularly human).
However, the blood is not limited to this, and may be a mixture of various liquid components and blood cell components of previously separated blood.

In the present invention, the glucose sensor method is a method of decomposing glucose dissolved in a liquid portion using a glucose-degrading enzyme such as glucose oxidase (which is usually immobilized on a suitable support) as an enzyme. Then, the amount of hydrogen peroxide decomposed and produced at that time and / or the amount of oxygen consumed is electrochemically measured by a hydrogen peroxide electrode and / or an oxygen electrode, and the glucose concentration in the liquid is determined from the measurement result. The measurement principle,
It means a method of measuring glucose concentration using a so-called biosensor as described in the description of the prior art. In the usual glucose concentration measurement, the glucose concentration is measured not by directly measuring the sample for which the glucose concentration is to be measured, but by diluting the sample with an appropriate liquid, particularly a buffer solution. Such glucose sensor methods are well known in the art, and it is particularly preferable to use a combination of glucose oxidase and hydrogen peroxide electrode in the present invention.

The present invention will be described below with reference to the case of using glucose oxidase and hydrogen peroxide electrodes, but the present invention can be similarly applied to the case of using other electrodes. Specifically, the glucose concentration is measured using a device as schematically shown in FIG. The glucose concentration measuring cell 1 has a hydrogen peroxide electrode 2 on which GOD is immobilized, and the liquid in the cell is sufficiently agitated by a stirrer 3 and a stirrer 4. The buffer solution is supplied into the cell 1 via the valve 6 by the pump 5, and the sample whose glucose concentration is to be measured is supplied into the cell 1 by the sampler 9, and when the measurement is completed, the sample is measured via the valve 8 by the pump 7. The liquid is drained. The measurement is performed by setting the time when the sample is supplied from the sampler 9 to 0 and obtaining the relationship between the output from the hydrogen peroxide electrode 2 and the elapsed time.

In the present invention, the equilibrium point method is one of glucose concentration measuring methods using the above-mentioned glucose sensor method.
That is, the measurement is continued until the output (current value) of the hydrogen peroxide electrode becomes substantially constant after starting the measurement for the standard solution with a known glucose concentration (that is, after injecting the sample into the cell). Then, the relationship between the constant output and the glucose concentration is obtained in advance as a calibration curve, and then the output of the hydrogen peroxide electrode is measured for a sample with an unknown glucose concentration, and similarly the output becomes a substantially constant value. It means a method of continuing the measurement up to and determining the glucose concentration from the constant value based on the calibration curve.

This equilibrium point method is based on the following matters: The rate of glucose decomposition by glucose oxidase is proportional to the glucose concentration in the buffer solution, but the amount of glucose decomposed by the enzyme itself is very small. Therefore, the glucose concentration hardly changes. Therefore, in a steady state, the production rate of hydrogen peroxide is constant. Specifically, when the measurement is performed on a sample having a certain glucose concentration, a relationship between the output of the electrode and the time after the injection of the sample as shown in FIG. 2 is obtained. It will be constant. There is a certain correlation between this constant output value and the glucose concentration in the buffer solution, and the equilibrium point method
This relationship is used as a calibration curve.

In such an equilibrium point method, there is sufficient time after the start of measurement until the output reaches a certain value, and when blood cells are mixed in the liquid sample to be measured, this time is It has been found that the glucose contained in the liquid inside is sufficient to diffuse into the buffer body against the resistance of the cell membrane of blood cells. Thus, the glucose that results in the measured glucose concentration (G1) as measured by the equilibrium method is both glucose dissolved in the liquid component and glucose dissolved in the liquid in the blood cells.

In the present invention, the first-order differential method is one of the glucose concentration measuring methods using the above-mentioned glucose sensor method, and the output of the hydrogen peroxide electrode after the start of measurement for a standard solution having a known glucose concentration ( The relationship between the maximum value of the temporal change in the output (that is, the differential value of the output with respect to the output, and thus the output speed) and the glucose concentration is calibrated by measuring the relationship between the current value) and the measurement time. Then, measure the temporal change in the output of the hydrogen peroxide electrode for a sample with an unknown glucose concentration, and similarly measure the maximum value of the temporal change amount, and based on the calibration curve from the maximum value. It means a method of obtaining glucose concentration.

For example, if the relationship between the output of an electrode and time as schematically shown in FIG. 2 is differentiated with respect to time, the amount of change over time of the output can be obtained. Specifically, a curve as shown in FIG. 3 is obtained. can get. There is a certain correlation between the maximum value of this curve and the glucose concentration in the buffer solution, and the first derivative method uses this relationship as a calibration curve. In such a first-order differential method, after the start of measurement, it takes only a few seconds (for example, 2-3
If the blood sample is mixed in the measurement sample, glucose contained in the liquid in the blood cell must pass through the cell membrane of the blood cell to diffuse into the buffer solution body. It turns out that it's not enough. Therefore, the measured glucose concentration (G2) measured by the first derivative method
The glucose that causes the is substantially only glucose that is dissolved in the liquid component.

Therefore, when measuring the glucose concentration in the liquid component, even if the glucose concentration in the liquid component is the same, if blood cells are contained in the sample to be measured, the glucose concentration by the equilibrium point method will be used. The measured value (G1) of (1) and the measured value of glucose concentration (G2) by the first derivative method are obviously different (G1> G2, of course). However, when blood cells are not contained in the sample for which the glucose concentration is to be measured, there is only the influence of glucose dissolved in plasma. Therefore, the glucose concentration measurement value (G1) by the equilibrium point method and the first derivative method are used. The glucose concentration measurement values (G2) according to the above are in agreement within the tolerance. Therefore, by comparing G1 and G2 to determine the presence or absence of a significant difference, it is possible to easily detect whether or not blood cells are contained in the sample being measured.

In any glucose concentration measurement, it is inevitable that an error will occur, and generally, an allowable error is set according to the measuring device, and the measured value (G1) and the measured value ( The criterion for determining whether or not there is a significant difference with G2) is whether or not the difference between G1 and G2 is within the tolerance, and this tolerance is affected by the measurement accuracy of the measuring device. . Therefore, in the present invention, 2
Since the scale for determining whether the measured values (for example, G1 and G2) of the glucose concentration measuring method of the species are different over the range of the tolerance is dependent on the measuring device used, how much G1 and G2 It is difficult to universally set a scale that it may be judged that there is a significant difference (or no significant difference) if there is a difference.

Considering a commercially available glucose concentration measuring device, in general, the ratio of the measured value (G2) is the measured value (G2).
1) up to about 80%, more preferably at least about 90%
% Or less, most preferably at least about 95% or less, it is often possible to determine that G1 and G2 differ by more than an acceptable margin of error, ie, are significantly different.
Further, recently, a device having a small tolerance is commercially available, and it may be determined that there is a significant difference between G1 and G2 even if G2 is 98% of G1, or 99% or more in some cases. . However, this ratio cannot theoretically exceed 100%.

[0025]

Therefore, the present invention can be advantageously applied to a method of centrifuging a collected blood and then sampling the obtained plasma to measure the glucose concentration therein. That is, in the second aspect, the present invention is a method of centrifuging the collected blood, sampling the obtained plasma, and measuring the glucose concentration therein.
The glucose sensor method is used for the sampled plasma, and the sensor output when glucose in the plasma is decomposed by glucose oxidase is measured. Based on this output, the glucose concentration (G
1) and the glucose concentration (G2) in plasma by the first derivative method are respectively obtained, and it is determined whether or not there is a significant difference between G1 and G2, thereby detecting the contamination of blood cells in the sampled plasma. A method for measuring glucose concentration is provided.

By such a glucose concentration measuring method, it is possible to automatically discard the inappropriately measured measured values and obtain only the really necessary measured values. Although such comparison and discarding of measured values can be basically performed manually, it is usually preferable that the comparison is automatically performed by a computer. In the first and second aspects of the present invention, it is possible to use the second derivative method instead of the equilibrium point method or the first derivative method.

Here, the second derivative method is one of the glucose concentration measuring methods using the above-mentioned glucose sensor method, and the output of the hydrogen peroxide electrode after the measurement is started with respect to a standard solution having a known glucose concentration ( Current value) and the measurement time is measured, and based on that, the relationship between the maximum value of the output acceleration (that is, the second derivative value according to the output time) and the glucose concentration is obtained in advance as a calibration curve, and then , A method of measuring the time change of the output of the hydrogen peroxide electrode for a sample with an unknown glucose concentration, measuring the maximum value of the output acceleration in the same way, and obtaining the glucose concentration from the maximum value based on the calibration curve. To do.

For example, if the relationship between the output of an electrode and time as schematically shown in FIG. 2 is second-order differentiated with respect to time (thus, similar to the case of differentiating again with time in FIG. 3), the output acceleration is obtained. Specifically, a curve as shown in FIG. 4 is obtained. There is a certain correlation between the maximum value of this curve and the glucose concentration in the buffer solution, and the second derivative method uses this relationship.

In such a second derivative method, after the start of measurement,
The time required for the output acceleration to reach the maximum value is shorter than the time required to reach the maximum value in the first derivative method, and it usually takes only a few seconds or less (for example, 2 seconds or less), and blood cells are mixed in the measurement sample. If this is the case, this time is not sufficient for glucose contained in the liquid in the blood cells to diffuse through the cell membrane of the blood cells into the buffer body, as in the case of the first derivative method. Is known. Therefore,
Measured glucose concentration (G
The glucose that provides 3) is essentially only the glucose dissolved in the liquid component.

Therefore, when the glucose concentration in the liquid component is measured, even if the glucose concentration in the liquid component is the same, when the blood cells are contained in the sample to be measured, the glucose concentration by the equilibrium point method is used. The measured value (G1) of G2 is different from the measured value of glucose concentration (G3) by the second derivative method. In this case, the determination as to whether or not the measured values are different is made in the case of comparing G1 and G3
The previously described measures of comparison of G1 and G2 can be applied as well.

In the first-order differential method and the second-order differential method, at least some glucose actually diffuses from the blood cells into the buffer solution through the cell membrane in the buffer solution at the time of measurement, but the extent of the diffusion reaches the maximum value. In the second derivative method, the time until is shorter than in the first derivative method, and therefore, the glucose concentration measured by the second derivative method is less affected by glucose present in the liquid in the blood cells.
Therefore, the glucose concentration by the first derivative method (G2) and the glucose concentration by the second derivative method (G3) are obviously different.

Therefore, even if the samples have the same liquid component,
When blood cells are included in the blood cells, G1, G2, and G3 are different from each other beyond the allowable error range (of course, G1>G2> G3). In the present invention, any two of these three measured values may be compared, and blood cell contamination can be determined.

For comparison of G2 and G3, see G2
Considering that is smaller than G1, in general,
If the percentage of the measured value (G3) is about 85% or less of the measured value (G2), more preferably at least about 90% or less, most preferably at least about 95% or less, there is a significant difference between G2 and G3. That is, in many cases, it may be determined that G2 and G3 are different by exceeding the allowable error range.
Most generally, it is the most preferable to determine whether or not the difference exceeds the allowable error range by statistically determining the allowable error as the characteristic of the measuring device to be used, and to make the determination based on that. Needless to say.

Similarly, instead of G1, G2 or G3, the third derivative method (the output is thirdly differentiated by time to obtain the relationship between the maximum value and the glucose concentration as in the second derivative method, and It is also possible in principle to apply the method of measuring the glucose concentration G4). However, since the time until the maximum value is reached is shortened, the influence of the mixing of the sample and the buffer solution and the influence of the tolerance become large, and therefore the application of the third derivative method is not so preferable mode. Considering both the difference in measured glucose concentration and the problem of measurement accuracy, it is most preferable to compare G1 and G2. For details of the equilibrium point method, the first derivative method, the second derivative method, and the like regarding the measurement of the glucose concentration as described above, refer to Japanese Patent Publication No. 7-37991.

In FIGS. 2, 3 and 4, broken lines and alternate long and short dash lines mean that the times correspond to each other. In addition, the method of the present invention further comprises the step of taking an appropriate treatment based on the determination when it is determined that the two types of measured values are substantially different and blood cells are mixed in the sampled sample. Good. Appropriate measures include, for example, informing the operator that blood cells have been mixed (and informing them to re-measure), issuing an alarm, and marking abnormal measurements when outputting measurement values. Be done.

In carrying out the above-described glucose concentration measuring method of the present invention, an existing glucose concentration measuring device can be used to obtain measured values such as the equilibrium point method, the first derivative method and the second derivative method. Processing of the measured values obtained, that is, the difference (G1
The calculation of -G2, G1-G3 and / or G2-G3) and the determination of whether or not the difference is significant can, of course, be performed manually, but can be easily performed by a computer. At this time, the scale for determining the presence or absence of a significant difference is set based on the characteristics such as the tolerance of the device used. Therefore, the above-described method of the present invention can be easily implemented as a softened circuit and can be combined with an existing glucose concentration measuring device.

Therefore, in the third summary, the present invention is
Provided is a glucose concentration measuring system for implementing the glucose concentration measuring method as described above, wherein the system incorporates a circuit for implementing the glucose concentration measuring method of the present invention as described above into an existing glucose concentration measuring device. This has the function of detecting blood cell contamination during sampling of the sample. The system may further include circuitry for performing an appropriate action following detection of blood cell contamination, such as the occurrence of a retest alarm as described above.

[0038]

【Example】

Example 1 A whole blood sample was centrifuged and the supernatant (plasma) 17
μl was diluted with 1.6 ml of buffer solution, and glucose concentration in plasma was measured by the equilibrium point method and the first derivative method.
Further, the glucose concentration was similarly measured for the sample containing 40% by volume of blood cells and the sample containing 10% by volume of the supernatant. For this measurement, GA-1140 manufactured by Kyoto Daiichi Kagaku Co., Ltd. was modified and used. The results are shown below: Supernatant Blood cell mixed sample Blood cell 10 volume% Blood cell 40 volume% Equilibrium point method 100 mg / dl 96 mg / dl 86 mg / dl First derivative 100 mg / dl 93.4 mg / dl 77 mg / dl

[Brief description of drawings]

FIG. 1 is a diagram schematically showing a glucose concentration measuring device.

FIG. 2 is a schematic graph showing the change over time in the output of a hydrogen peroxide electrode, showing the principle of measuring glucose concentration by the equilibrium point method.

FIG. 3 is a schematic graph showing the time derivative of the time change of the output of the hydrogen peroxide electrode, showing the principle of measuring the glucose concentration by the first derivative method.

FIG. 4 is a schematic graph when the output of the hydrogen peroxide electrode is second-order differentiated, showing the principle of measuring the glucose concentration by the second-order differentiation method.

[Explanation of symbols]

1 ... Glucose concentration measuring cell, 2 ... GOD-immobilized hydrogen peroxide electrode, 3 ... Stirrer, 4 ... Stirrer, 5 ... Pump, 6
... valve, 7 ... pump, 8 ... valve, 9 ... sampler.

Front page continuation (58) Fields surveyed (Int.Cl. ⁷ , DB name) G01N 27/46 G01N 27/26 371 G01N 27/30 G01N 33/49 G01N 33/66 A61K 35/14 A61M 1/02

Claims (5)

(57) [Claims] 1. A method for measuring a glucose concentration in a liquid part of a sample by a glucose sensor method after sampling a liquid part from a sample containing blood cells and a liquid component, wherein the glucose sensor method is used for the sampled sample. Then, the sensor output when the glucose in the sample is decomposed by the enzyme is measured, and the glucose concentration (G1) in the sample by the equilibrium point method and the glucose concentration (G2) in the sample by the first derivative method are measured based on this output. The glucose concentration (G1) and the glucose concentration (G2) are determined respectively to determine whether or not there is a significant difference between the glucose concentration (G1) and the glucose concentration (G2). Measuring method. 2. A method for centrifuging the collected blood and then sampling the obtained plasma to measure the glucose concentration therein, wherein glucose in the plasma is measured using a glucose sensor method. The sensor output at the time of decomposing by the enzyme was measured, and the glucose concentration in plasma (G1) by the equilibrium point method and the glucose concentration in plasma (G2) by the first derivative method were obtained based on this output, and G1 and G2 A method for measuring glucose concentration, which comprises detecting whether or not there is a blood cell contamination in the sampled plasma by determining whether or not there is a significant difference between the two. 3. The enzyme is glucose oxidase,
The sensor output is measured by a hydrogen peroxide electrode.
The method described. 4. The method according to claim 2, wherein the second derivative method is used instead of the first derivative method. 5. A glucose concentration measuring system including a circuit for carrying out the glucose concentration measuring method according to claim 2.