JPH09178719A - Automatic measuring method for glycohemoglobin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure stabilized HbAlc accurately with high reproducibility by analyzing glycohemoglobin quickly through fast liquid chromatography using an instable HbAlc removing agent. SOLUTION: A large number of sets of rack 7 holding a plurality of sample cases are mounted at a sample holding section 1. At a sampling section 2, a sample suction pump P2 is driven at first to suck a specified quantity of sample through a first nozzle 9. A sampling nozzle mechanism 11 is then shifted above a dilution/dispensation tank 12 where a hemolysis-cleaning liquid is delivered through a second nozzle 10 thus cleaning the outer wall of first nozzle 9. Subsequently, pumps P1 , P2 are driven to deliver the sample into the dilution/ dispensation tank 12 along with a specified quantity of hemolysis-cleaning liquid and a mixed liquid 15 is fed to a sample introduction valve 18 at an analyzing section 4 where it is heated. It is then pushed out by an eluate being fed from a bottle unit section 3 and injected into a column 19.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an improved method for automatically measuring glycated hemoglobin based on high performance liquid chromatography.

[0002]

BACKGROUND OF THE INVENTION glycated hemoglobin to hemoglobin in sugar is bound (HbA ₁₎ can be found in many diabetic patients, have been especially HbA _1c is an important measurement items as an indicator of long-term control of health screening and diabetes medical checkup, etc. ing. This is because HbA _1c is the most abundant in glycated hemoglobin (HbA ₁ ), and the increase in diabetes is significantly higher than that of other components, and the value of HbA _1c is 1
By showing good correlation with mean fasting blood glucose levels for ~ 3 months.

By the way, glycated hemoglobin contains HbA.
_In addition to _1c , there are HbA _1a , HbA _{1b and the} like, which are fractionated and measured by a colorimetric method, an electrophoresis method, a mini-column method, high performance liquid chromatography and the like. In the field of internal clinical examination, high performance liquid chromatography (HPLC method) has been widely used recently from the viewpoint of required time and separation performance.

However, it is said that the stable type of HbA _1c has a good correlation with the blood glucose level in the past, and there is an unstable type of HbA _1c . It is said that the ratio is about 10 to 15% in the whole fasted HbA _1c in a healthy person. The unstable HbA _1c is a β-chain N terminal of hemoglobin and a reducing terminal of glucose reversibly bound by a Shiff base, and is produced and decomposed within a relatively short time depending on the blood glucose concentration. Therefore, the number of diabetic patients is higher than that of healthy people, and it may reach 10 to 20% of the total HbA _1c . In addition, the amount after meals is larger than that on an empty stomach, and is greatly affected by the state at the time of blood collection.

On the other hand, the stable HbA _1c is an unstable HbA _1c.
Slowly, persistently and irreversibly produced from, well reflecting past long-term blood glucose levels. Therefore, it is desirable to measure only the stable type separately. However, both of them are structurally similar and separation by liquid chromatography is quite difficult.

As one method of coping with this, a high-resolution column having a long length is used to improve the separation performance. This method has an advantage that it is less likely to cause denaturation of glycohemoglobin by a chemical treatment, but it requires a dozen minutes or more of analysis time for one sample to achieve good separation. Therefore, there is a drawback that the number of samples to be measured increases and the measurement in an emergency cannot be sufficiently dealt with. In addition, this type has a long column and therefore requires a large apparatus and is expensive.

On the other hand, as another method for separating and measuring stable HbA _1c , there is a method of chemically decomposing and removing unstable HbA _1c by pretreatment. This is unstable HbA _1c
It is focused on that it is easily decomposed because it is temporarily bound to glucose (Shiff base bond). For example, there is a method in which washed red blood cells are incubated with an isotonic phosphate buffer (37 ° C., 4 hours) or physiological saline (room temperature, 14 hours) to separate glucose from unstable HbA _1c .

Alternatively, there is a method of adding a hemolytic reagent to whole blood and incubating at 35 ° C. for more than 10 hours. This is to reduce the concentration of unstable HbA _1c by hemolyzing and diluting the sample. Especially, the effect is large in the acidic region of pH 6 or less, the reaction is fast, and the effect is large when the temperature is raised. Furthermore,
It is more effective to add a commercially available unstable HbA _1c removing reagent containing boric acid, which has been used in the conventional mini-column method.

However, these pretreatments are time-consuming and, along with the degradation of unstable HbA _1c , the degradation and denaturation of other glycohemoglobin and pure hemoglobin (HbA ₀ ) also proceed at the same time.

Further, the hemolytic type has a drawback that the amount of stable HbA _1c also varies depending on the elapsed time after hemolysis and the elapsed temperature until the measurement. In particular, when automatically measuring a large number of specimens, diluting a blood sample with hemolyzed blood containing a decomposing reagent increases the elapsed time of the specimens to be measured later among the waiting specimens, resulting in excessive decomposition reactions. However, there was a drawback that the specimen proceeded to denature and the sample was denatured.

[0011]

DISCLOSURE OF THE INVENTION The present invention is to perform rapid analysis of glycohemoglobin using a labile HbA _1c removing reagent (decomposition reagent) by high performance liquid chromatography and to decompose glycohemoglobin other than labile HbA _1c. Stable H with high accuracy and good reproducibility
It is an object of the present invention to provide a method for measuring bA _1c . Another object of the present invention is to provide a glycated hemoglobin analysis method in which the structure of the device is relatively simple and the handling and preparation of the sample is easy.

A further object of the present invention is to provide a sample introduction valve which is optimal for controlling the temperature of a mixed liquid of a sample (not limited to a blood sample) and a reaction reagent in a high performance liquid chromatograph.

[0013]

SUMMARY OF THE INVENTION The above object is to make a large number of blood samples stand by as whole blood or blood cell layers, and to sample rapidly when the turn of measurement comes, and then to rapidly produce an unstable H type.
This can be achieved by diluting and mixing with hemolyzed blood containing a bA _1c removal reagent, and injecting into a column of a high-performance liquid chromatograph apparatus after a lapse of a predetermined time from the start of mixing. In the following, "sample" refers to a whole blood sample (or blood cell layer) collected in a sample container, and "specimen" refers to a mixed solution obtained by diluting this sample with hemolyzed blood.

Furthermore, the reaction can be promoted by heating the mixed solution (sample) before it is injected into the column, and more rapid measurement can be performed. When this heating is performed in the sample loop portion of the sample introduction valve, it is not necessary to provide another heating zone or to heat an extra sample, and space saving and energy saving can be achieved.

Further, it is achieved by omitting the labor of manually mixing the reagents and preventing an error due to the manual mixing and the denaturation of the sample before the measurement after the mixing.

Next, the procedure of the method of the present invention and the configuration of the measuring device will be described. First, a blood sample collected from each patient or subject is put in a sample container such as a blood collection tube or a sampling cup, and kept in the sample holding unit as whole blood or a blood cell layer. Since whole blood or a blood cell layer obtained by centrifuging it is used, pretreatment is not necessary and it is simple. It should be noted that the use of the blood cell layer is assumed to effectively use the rest of the plasma used for other tests, but if whole blood is kept waiting for a long time, the blood cells will be on the bottom. The ingredients settle. To deal with these, sampling should be performed from a portion near the bottom of the sample container. Even if the proportion of red blood cells in the sampled blood sample varies, the proportion of HbA _1c relative to HbA ₀ and other HbA ₁ is constant for each sample.
No problem. Anticoagulants are usually added to blood samples. As the anticoagulant, commercially available ones such as heparin and EDTA-2Na can be used.

Each sample container includes a rack, a snake chain,
A large number of holding means such as a turntable are held, and they are sequentially sent to the sampling position.

In the sampling section, a predetermined amount (about 1 to several μl) of the sample is sucked from the sampling nozzle by the suction action of the pump, diluted with the hemolyzed blood containing the unstable HbA _1c removing reagent supplied separately, and mixed. To be done. The dilution ratio is several tens to several hundreds, especially about 100 to 400 times.

The sampling and dilution procedures may have various configurations and modifications depending on the structures and combinations of various pumps, but the point is to remove the blood sample sampled within a few tens of seconds to a few minutes immediately before the measurement and remove the reagent. Dilute with hemolysis / washing solution containing
Any structure may be used as long as it can be injected into the column through the sample introduction valve after a lapse of a certain time after the start of mixing. These operations can be performed automatically and continuously,
It is necessary that measures have been taken to prevent pollution.

As the labile HbA _1c removing reagent, boric acid, phosphoric acid compounds, or commercially available reagents containing these are used. The pH should be acidic. A commercially available hemolytic agent is used.

As the column, for example, a column for high performance liquid chromatography packed with a spherical ion exchange gel (cation, anion) is used, and the processing time of the sample is regulated by this separation ability.

The progress of the reaction after hemolysis is naturally dependent on the performance of the unstable HbA _1c removing reagent, but it is also a function of the elapsed time and the elapsed temperature after the start of the reaction. The time required for measurement is the sum of the time required for operations such as sampling, dilution / mixing, liquid feeding, and washing and the heating time, and the shorter the measurement time is, the better for processing a large number of samples. Therefore, if the reagent has a low ability to decompose unstable HbA _1c , it is possible to shorten the reaction time by heating the sample (mixed solution) feeding path within a range that does not cause inconvenience, for example, deterioration of the sample. In this case, in consideration of the analysis time and the decomposition ability of the reagent, the temperature of the whole or part of the feeding path including the sample introduction valve portion is controlled so that the time until the measurement becomes constant. The heating temperature is set to about 30 to 65 ° C, preferably about 40 to 55 ° C in consideration of the decomposition ability of the reagent and the heating time. Above 65 ° C, protein denaturation occurs, which is not preferable. If the temperature is 30 ° C or lower, cooling may be required in the summer.

Next, the cleaning liquid will be described. The cleaning liquid is for cleaning the sampling nozzle and each flow path in order to prevent contamination after measuring the previous sample. However, in the present invention, since the sample that has been sampled is diluted and mixed with the hemolyzed blood, it is essentially necessary to provide the two liquid feeding systems for the washing liquid and the hemolyzed blood. Of course, this configuration may be used, but if both are used together as a hemolysis / washing solution, there is an advantage that the number of pumps for feeding is reduced by one and piping and a feeding sequence are simplified.

Further, in the present invention, after a lapse of a certain time from the start of mixing the blood sample and the hemolyzed blood, the mixed solution (specimen) is injected into the column. Therefore, if for some reason, for example, the movement of the rack did not go smoothly, or the timing of interrupt measurement did not match, the backward calculation from the next injection timing and the start of mixing did not meet the schedule It is desirable to perform an empty measurement operation without performing injection or injection. This difference in the mixing timing does not pose a problem when one type of eluent is used. However, when two or more kinds of eluents having different concentrations and pH are used, the imbalance in the column will be lost if the injection timing is incorrect.

[0025]

BEST MODE FOR CARRYING OUT THE INVENTION Next, the method of the present invention will be described in detail with reference to an example of a glycohemoglobin measuring apparatus embodying the method of the present invention. In this example, the hemolyzed blood and the washing liquid are commonly used, and the sample is heated by the sample loop of the sample introduction valve.

FIG. 1 is a flow diagram showing an example of a glycohemoglobin automatic measuring apparatus in which the method of the present invention is embodied, FIG. 2 is a flow diagram of the sampling section, and FIG. 3 is a schematic perspective view of a sample introduction valve. is there.

This apparatus comprises a sample holder 1 for holding and pooling a plurality of sample containers containing whole blood samples or blood cell layers.
And a sampling unit 2 for sampling a sample and diluting and mixing with hemolysate, a bottle unit 3 for accommodating each container of eluent, hemolyzing / washing liquid, and waste liquid, an analyzing unit including a sample introduction valve, column, photometric means, and the like. 4, a storage / control unit that controls the operation of the entire device based on programs and input commands, stores and calculates measured values, and outputs the results to a display device such as a printer together with the patient number, measurement date and time, etc. , An operation keyboard for inputting operation commands and the like. The storage / control unit uses, for example, the microcomputer 5, and the display device also uses a digital display.

Next, the configuration and operation of each section will be described. First, the sample holder 1 includes a rack 7 holding a plurality of sample containers 6.
A plurality of sets are mounted, and each rack 7 is driven in the direction of the arrow so that the sample containers 6 are sequentially placed at the sampling positions. An interrupt measurement port 8 may be provided for emergency measurement.

The sampling unit 2 has two nozzles 9,
A sampling nozzle mechanism 11 provided with 10, a hemolysis / washing liquid pump (P ₁ ), a sample suction pump (P ₂ ), a sample introduction pump (P ₃ ), and a dilution / dispensing tank 12 are provided. The sample suction pump (P ₂ ) has a capacity of 1 to several μl, and the washing liquid pump (P ₁ ) and the sample introduction pump (P ₃ ) have a capacity of several hundred μl. The sampling nozzle mechanism 11 moves up and down with rotation. The hemolyzing / washing solution 13 is the above-mentioned unstable HbA _1c decomposing reagent or hemolyzing agent dissolved in a diluting solution of the sample. In this example, this solution is used in combination with each pump, nozzle, and other channels for washing. There is. Further, in the present invention, sample 1
Reference numeral 4 refers to a whole blood sample or blood cell layer collected in the sample container 6, and specimen 15 refers to the sample 14 diluted with the hemolysis / washing solution 13 by several tens to several hundreds of times.

In the sampling unit 2, first, the sample suction pump (P ₂ ) is driven to suck a predetermined amount of sample 14 from the first nozzle 9. Next, the sampling nozzle mechanism 11 is moved onto the diluting / dispensing tank 12, the hemolyzing / cleaning liquid 13 is discharged from the second nozzle 10, and the outer wall of the first nozzle 9 is cleaned. For this purpose, the tip of the second nozzle 10 is bent towards the first nozzle 9. Waste liquid is the waste liquid valve 1
It is guided to the drain bottle 17 of the bottle unit section 3 through 6. Next, the pumps (P ₁ ) and (P ₂ ) are driven to discharge the sample 14 sampled in the dilution and dispensing tank 12 and a predetermined amount of hemolyzing / washing liquid 13. Both 13 by this discharge
・ 14 is agitated. Furthermore, the first nozzle 9 may be repeatedly sucked and discharged to sufficiently stir and mix.
The mixed sample 15 is sucked from the first nozzle 9 by the suction action of the sample introduction pump (P ₃ ) and sent to the sample introduction valve 18 of the analysis unit 4 (states of FIGS. 1 and 2).

Thereafter, the dilution / dispensing tank 12, the pipe, the outer wall of the first nozzle 9 and the like are washed with the hemolyzing / washing liquid 13, and the waste liquid is discarded to prepare for the next sample suction.

On the other hand, the sample loop 1 of the sample introduction valve 18
The sample 15 sent to 8a is held in the sample loop 18a and heated. As shown in FIG. 3, the sample loop 18a is surrounded by a loop warmer 18b, and a heat insulating cover 18c covers the loop warmer 18b and the main part of the sample introduction valve 18. In the figure, reference numeral 18d is a sample inlet / outlet tube, 18e is an eluent inlet / outlet tube, 18f is a motor, and 18g.
Is a gear box, and 18h is a heat insulating block. The heat insulating cover 18c is screwed to the block 18h.

The heating by this system is performed by the sample loop 18a.
Since only the sample inside is heated reliably, there is no waste of energy, and there is no risk of injecting into the column 19 a part that is not heated, that is, a sample in which unstable HbA _1c is not sufficiently separated and removed.

Next, the motor 18f of the sample introduction valve 18
Rotates to the state shown in FIG. 4, and the sample loop 18a
The sample 15 therein is pushed out by the eluent sent from the bottle unit 3 and injected into the column 19. Each component of the specimen 15, that is, HbA _1a , HbA _1b , HbA _1c , H
bA _{0 and the} like are separated in the column 19 and are sequentially placed in the photometer 20.
The light is metered in and discarded in the drain container 21. The photometric result is sent to the microcomputer 5, where each fractionation pattern, peak elution time, content% of each component, etc. are calculated and visualized by the printer 22. In addition, a filter 24 for removing impurities is arranged in front of the column 19, and the whole is kept in a constant temperature bath 25.
Stable measurement can be performed by storing it inside.

Based on FIG. 2, the pump (P
₁ ) ・ (P ₂ ) ・ (P ₃ ) operation and hemolysis / washing solution 13
Table 1 shows a summary of the flow path states of the flow path switching valves 38 and 39. In addition, ↓ of each pump is suction, ↑ is discharge, and blank indicates stop.

[Table 1]

In the above operation, if the sampling amount of the sample 14 is 1.5 μl and the amount of the hemolyzing / washing solution 13 for diluting the sample 14 is 450 μl, the dilution ratio becomes 300 times. The heating time and temperature of the sample 15 depend on the decomposing ability of the unstable HbA _1c removing reagent. However, the unstable HbA _1c removing reagent of the phosphate compound type sold by the applicant under the name “21H” is used. When using the hemolyzed blood containing the hemolyzate, the optimal dilution ratio is 2 minutes 40 seconds at 48 ° C. still,
Degradation of unstable HbA _1c is almost completely completed in 2 minutes at 60 ° C., 3 minutes at 40 ° C., and 4 minutes at 33 ° C.

By the way, in this example, three kinds of eluents are used. Each of the eluents (A), (B), and (C) passes through the heating coil 26, the cooling coil 27, and the defoaming devices 28, 29, and 30 based on the liquid feeding sequence, and the switching valve 31 respectively.
-Sequentially fed to the manifold 34 by 32 and 33. Each eluent that became one flow path in the manifold 34
The liquid is pumped by the liquid feed pump 35 to the sample introduction valve 18, then injected into the column 19, and while passing the sample 15, the sample 15 passes through the photometer 20 and reaches the drain container 21. In the figure, reference numeral 36 is a pressure detector, and 37 is a damper.

In FIGS. 1 and 2, reference numerals 38 and 39 are flow path switching valves for the hemolyzing / washing solution 13, 40 is a liquid shortage sensor for the hemolyzing / washing solution 13, and 41 is a suction air pump for the drain bottle 17. , 42 is a display, and 43 is an operation keyboard.

The above description is for the case of measuring samples arranged in series, and in this case, if the operation is always performed in a fixed sequence, the time from dilution to injection becomes constant. However, in the case where the sample container is discontinuously placed on the rack 7 or the like due to time variation of blood collection or the like, extra irregular time is required to search for the next sample. Even when a sample container is set in the interrupt port for emergency measurement, the blood sample suction timing may be shifted. Therefore, it is advisable to search for the next sample at the time when the injection of the sample 15 into the column 19 is finished, and calculate backward from the time of the next injection to start the dilution at the necessary timing.

Regarding the start of dilution, if it is already known that it will not be in time for the next injection, by stopping mixing and injecting the sample into the column at that time, and continuing the liquid feeding casing for one measurement, Even when using three kinds of eluents having different concentrations and pH, stable measurement can be continued without breaking the equilibrium conditions of the column.

Comparison with the conventional method (Comparison with the blood cell washing method with isotonic phosphate buffer) The results are shown in Table 2, and the unstable HbA _1c by the conventional method is shown.
An effect equivalent to the removal of the fraction was obtained. Blood cell washing and 21
When H was also used in combination, it was compared, but no further decrease in unstable HbA _1c fraction or increase in A ₁ a + b component due to denaturation was observed. All values were high when neither pretreatment nor unstable HbA _1c removal reagent was used (21 L). Difference _{L-A 1 c + S-} A 1 in the c 21L and other processing can be inferred that labile HbA _1c. In this example, unstable HbA _1c accounts for about 0.7% of the total hemoglobin.
It is.

[Table 2]

The measurement was performed under the following conditions.・ 21L: Hemolysis that does not contain unstable HbA _1c removal reagent
Cleaning solution. Nonionic surfactant 1 g / l Potassium dihydrogen phosphate 0.1 g / l Potassium dihydrogen phosphate 0.3 g / l pH 7.5 ・ 21H: Hemolysis / washing solution containing labile HbA _1c removal reagent Nonionic surfactant 1 g / l phosphoric acid compound 0.1 g / l KOH 0.3 g / l pH6 “S” +21 L: Incubated by blood cell washing method (37 ° C.) for 6 hours. S indicates Saline. Washed red blood cells (1 volume) added to isotonic phosphate buffer (14 volumes):
Unstable HbA _1c is removed by incubation at 37 ° C. for 6 hours with continuous rotary mixing. Then, the blood cell layer was collected, diluted with 21 L to 300 times and measured. -"S" + 21H: 21H was loaded on the saline.
As in S + 21L, the blood cell layer treated with isotonic phosphate buffer was collected and diluted 300 times with 21H for measurement. -Sample: healthy person. Experiment immediately after blood collection. -Measurement conditions: All are based on the method of the example. Heating temperature 4
8 ° C, heating time 2 minutes 40 seconds, measurement time 4 minutes. n = 5.

As described in detail above, the method of the present invention relates to an improvement in the automatic measurement method of glycated hemoglobin based on the principle of high performance liquid chromatography, and a large number of blood samples collected from each patient are sampled as whole blood or blood cell layers. Hold it in the holding part, sample just before the measurement (several tens of seconds to several minutes), mix with the hemolyzing / washing solution containing the unstable HbA _1c removal reagent, and mix it with the high-speed liquid after a certain time has elapsed from the start of mixing. It is designed to be injected into the column of a chromatograph. Further, if necessary, the mixture is heated after being diluted and before being injected into the column.

[0044]

As a result, the measurement can be performed much more rapidly than the conventional method using a high performance long column, and
It is possible to obtain the same degree of accuracy. Eliminates the need to manually mix reagents and enables accurate measurement without error due to manual dilution. No pretreatment is necessary because whole blood or a blood cell layer obtained by centrifugation of whole blood is used.
Since it is not diluted with a hemolytic reagent in advance, the sample does not denature until the measurement, and stable HbA _1c can be accurately measured by suppressing the decomposition and denaturation of glycohemoglobin other than unstable HbA _1c . Since the measured value is not affected by unstable HbA _1c , there is no time limitation for blood collection and no burden on the patient. Furthermore, since the sample is hemolyzed immediately before the measurement and always injected into the column at a constant timing, stable analysis is possible. In particular, even if the sample is set discontinuously, the equilibration condition of the column is not disturbed, so that the chromatographic pattern is stable. It has many excellent effects.

On the other hand, the sample introduction valve of the present invention heats the sample solution and the reaction reagent to a predetermined temperature for a certain period of time.
Since only the sample in the sample loop can be heated reliably, there is no waste of energy, and there is no risk of injecting the unheated portion into the column, and accurate control is possible. Further, the conventional sample introduction valve can be obtained simply by making a simple modification, and since it is not necessary to provide any other heating device, there is an advantage that no extra space is required.

[Brief description of the drawings]

FIG. 1 is a flow diagram showing an example of an automatic glycated hemoglobin measurement apparatus in which the method of the present invention is implemented.

FIG. 2 is a flow diagram of a sampling unit similar to FIG.

FIG. 3 is a schematic perspective view of a sample introduction valve.

FIG. 4 is a plan view showing a state of a sample introduction valve at the time of injecting a sample into a column.

[Explanation of symbols]

1 sample holding part 2 sampling part 4 analysis part 6 sample container 9, 10 nozzle 11 sampling nozzle mechanism 12 dilution dispensing tank 13 hemolysis / washing liquid 14 sample 15 specimen 18 sample introduction valve 19 column P ₁ hemolysis / washing liquid pump P ₂ sample suction Pump P ₃ Sample introduction pump

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location G01N 33/72 G01N 33/72 A 35/10 35/06 F

Claims (2)

[Claims] 1. A labile H in the case of measuring a fraction of glycated hemoglobin by high performance chromatography.
An automatic measuring method for glycohemoglobin, characterized in that hemolyzed blood containing a bA _1c removing reagent is used as a washing liquid for washing each flow path including a sample loop and a sampling nozzle portion. 2. A large number of blood samples collected in a sample container are allowed to stand by as whole blood or a blood cell layer, the sample containers are sequentially sent to a sampling unit, and the blood sample sucked from a sampling nozzle contains an unstable HbA _1c removal reagent. A method of mixing and diluting with hemolyzed blood, introducing a part of the mixed solution into a sample loop of a sample introduction valve, and injecting it into a column after a lapse of a certain time from the start of mixing to measure with unstable HbA _1c removed or reduced The method for automatically measuring glycated hemoglobin according to claim 1.