JPH07120447A - Glycohemoglobin analyzer - Google Patents

Abstract

PURPOSE:To carry out an analysis at every blood collection by leading a pipe line between eluent and a pump through a constant temperature unit, heating the pipe line and arresting bubbles generated in the pipe line with a bubble trap. CONSTITUTION:A constant temperature unit 18 is provided for maintaining the temperature of a separation column 19 forming a separation and detection mechanism at a level higher than room temperature by 5 deg.C or more. Furthermore, constitution is so made that a pipe line 20 where eluent 14 (14a and 14b) flows, passes the unit 18 on the way to a gradient unit 16. Bubbles, when generated in the eluent 14 at a process of flowing through the unit 18, are caught by an air trap 21 (21a and 21b). As a result, the eluent 14 in transition from a low flowrate at a standby process to a steady flowrate at an analysis process can quickly give a stable flowrate condition. Thus, blood can be analyzed every time it is gathered from a patient.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus and method for biochemical analysis, and more particularly to an analyzer and apparatus for quantifying hemoglobin subfraction based on liquid chromatography.

[0002]

2. Description of the Related Art Conventionally, a glycohemoglobin analyzer based on liquid chromatography has a cation exchange column as a separation column and two or more different salt concentrations as eluents, as described in JP-A-3-255360. The hemoglobin subfraction was separated by stepwise or gradient elution using three types of buffers. This method requires a long time for equilibration and separation of the separation column, and the analysis time per sample was long. Therefore, the efficiency was poor unless the control was analyzed several times before the analysis of the unknown sample, and after the equilibration of the separation column was sufficiently secured, continuous analysis was performed.

[0003]

The above-mentioned conventional technique is effective in a facility where a considerable number of specimens can be secured by the time the analysis is started, but outpatients are irregular and intermittent like small hospitals. In facilities that come to Japan, it was necessary to take measures such as delaying the analysis until the number of samples collected. On the other hand, with the increase in the number of diabetic patients, specialized outpatient hospitals have appeared. At such hospitals, the number of specimens was at most several tens per day, and analysis was usually outsourced. However, in such a case, the treatment or life guidance of the patient by the doctor will be performed based on the analysis data at the previous visit (usually about one month before). Glycohemoglobin, by its nature, is said to reflect the blood glucose level of about 1 to 2 months ago, so when outsourced analysis is performed as before, it is 2
There was a problem because I was instructed based on my blood glucose level three months ago.

The present invention has been made under these circumstances, and an object of the present invention is to provide a glycohemoglobin analyzer capable of performing an analysis each time blood is collected from a patient.

[0005]

In order to achieve such an object, the present invention heats a pipe connecting an eluent and a pump through a column constant temperature unit, and a bubble trap causes bubbles in the pipe to flow into the pump. It was designed so that it would not enter.

Further, in order to analyze without waiting until the equilibrium state of the separation column is reached, the sample is continuously analyzed immediately after shifting from the standby state to the sample analysis, and the quantitative values of the nth time and the (n-1) th time are analyzed. The analysis of the sample is terminated when the absolute value of the difference becomes less than a specified constant value.

Further, the maximum number of repetitions n and n is set so as to be allowable even if there is a lot difference between columns and a difference between apparatuses.
The difference (absolute value) between the quantitative value at the 1st time and the quantitative value at the (n-1) th time can be rewritten.

[0008]

In the glycohemoglobin analyzer according to the present invention, the separation column is maintained at a temperature higher than room temperature, and the eluent is passed through the constant temperature unit of the column to make the dissolved air in the saturated state into bubbles, which are collected by the bubble trap. To do. As a result, a stable flow rate state can be promptly obtained when the low flow rate in the standby process is changed to the steady flow rate in the analysis process.

In addition, the analyzer according to the present invention continuously analyzes the same sample after shifting to the analysis step, and the difference (absolute position) between the quantitative values at the n-th time and the (n-1) -th time becomes less than a certain value. The analysis ends at this point. In a glycohemoglobin analyzer based on a liquid chromatograph, two to three kinds of eluents having different concentrations are sent at regular time intervals. Therefore, several cycles are required until the state of the separation column is equilibrated, and conventionally, the sample analysis was performed after equilibration before a series of analyses. In the present invention, since repeated analysis is performed until the quantitative value is stably output, an incorrect quantitative value before equilibration is not output.

[0010]

EXAMPLE A glycohemoglobin analyzer according to the present invention will be described with reference to FIG.

A nozzle 12 for sucking, discharging and injecting a sample,
A movable arm 8 for fixing the nozzle, a motor 9 for driving the movable arm 8, a dilution port 7 for diluting / hemolyzing a sample of whole blood, a syringe 10 used for aspirating the sample, a dilution / hemolysis 11, and a hemolyzed sample are injected. Sampling mechanism composed of an injection valve 13, a gradient unit 16 for generating a concentration gradient between eluents 14a and 14b, a liquid supply mechanism composed of a liquid supply pump 15, and a hemolysis sample subfractionation of hemoglobin Separation column 17 for separating into two, constant temperature unit 18 for keeping the separation column higher than room temperature, detector 19
It is composed of a separation / detection mechanism 3 and a data processing mechanism 4. The pipe 20 through which the eluent flows passes through the constant temperature unit 18 on the way to the gradient unit 16. If bubbles are generated in the eluent while passing through the constant temperature unit 18, they are collected by the bubble trap 21.

Next, the operation of this analyzer will be described with reference to FIG.

(1) Start When the power supply is turned on, all the parts constituting this analyzer are energized.

(2) Preparatory operation The pump starts the liquid transfer at a normal flow rate. Constant temperature unit 1
8. Confirm that the column pressure and the lamp strength of the detector 19 are stable. It operates until certain conditions such as detection signal noise, drift, and pressure are satisfied.

(3) Reference analysis When the conditions for completing the preparatory operation are cleared, the reference (hemolysis) contained in the dilution port 6 is analyzed, and glycohemoglobin (HbA _1c ), hemoglobin F, hemoglobin (H) is obtained.
Check the holding time of bA ₀ ).

(4) Standby The solution flow rate of the eluent is set to be lower than in the normal analysis, and the process waits until the analysis key is pressed.

(5) Analysis When the analysis key is pressed, the analysis process starts. The flow rate of the pump rises to a normal state, and the movable arm 8 slides forward to suck a fixed amount of the sample. Next, the sample is discharged to the dilution port 6 together with the diluted / lysed blood 11. The hemolyzed sample is sent from the injection port 7 to the injection valve 13. At this time, the flow path of the injection valve 13 is as shown by the broken line. Next, the injection valve 13 is switched to the flow path indicated by the solid line, and at the same time, the eluents 14 with different concentrations are
A and b are mixed to generate a gradient, and the hemolyzed sample is guided to a separation column and separated into a hemoglobin subfraction.
When the separation of the hemolyzed sample injected for the first time is completed, the hemolyzed sample remaining in the dilution port is injected again through the injection port 7 to start the separation. This analysis is repeated n times, but when the difference between the n-th quantitative value and the (n-1) -th quantitative value becomes a certain value or less, the analysis of this sample is terminated and waits. The number of repetitions n is an integer and can be set arbitrarily. If the difference between the quantified values at the n-th time and the (n-1) -th time is not less than a fixed value even if the analysis is performed by the same number as the set number of repetitions n, an error is displayed and the operation is stopped.

Next, an actual analysis example will be described.

The separation column used was one packed with a carboxymethyl group-introduced methacrylate polymer gel. The column has an inner diameter of 4.6 mm and a length of 35 mm. The eluent is 52.5 mmol / l phosphate buffer (pH
6.2) and 210 mMol / l phosphate buffer (pH 6.
1) was used. The flow rate during the analysis was 1.2 ml / min, the standby time was 0.2 ml / min, and the column temperature was 40 ° C.
0.1% surfactant (Triton X-1) for dilution and hemolysis
00) was used to dilute a whole blood sample about 200 times with hemolyzed blood to prepare a hemolyzed sample. This 10 μl was injected. A visible photometer with a wavelength of 415 nm was used to detect the hemoglobin subfraction. The chromatogram obtained under the above conditions is shown in FIG.

Table 1 shows the results of analysis using the three columns according to the flow shown in FIG. The blood samples of three diabetic patients were used as samples. Columns 1 and 2 were new, and column 3 was about 2000 samples analyzed. In the case of a new column, the first quantitative value tends to be higher, which is because hemoglobin A ₀ (HbA ₀ ) cannot be completely eluted and remains in the column in one analysis. Conceivable. As shown in Table 1, the repetition number n is set to 3,
Further, if the absolute value of the difference between the quantitative values is set to 0.1, it can be seen that the analysis is completed when columns 1 and 2 are analyzed three times and when column 3 is analyzed twice.

[0021]

[Table 1]

[0022]

As is clear from the above description, the preparation operation is made to wait after the completion of the preparatory operation, and the same sample is repeatedly analyzed at the time of analysis, and the operation is terminated when the absolute value of the difference between the quantitative values becomes a certain value or less. By setting a quantitative value at this time, it is possible to analyze the blood of an outpatient who visits intermittently.

[Brief description of drawings]

FIG. 1 is a diagram showing a flow path of a glycohemoglobin analyzer according to the present invention.

FIG. 2 is a diagram showing an analysis procedure of the present invention.

FIG. 3 is a chromatogram obtained by the glycohemoglobin analyzer shown in FIG.

[Explanation of Codes] 1 ... Sampling mechanism, 2 ... Liquid feeding mechanism, 3 ... Separation / detection mechanism, 4 ... Data processing mechanism, 5 ... Specimen, 8 ... Movable arm, 9 ... Drive motor, 10 ... Syringe, 13 ... Injection valve,
14 ... Eluent, 15 ... Liquid feed pump, 16 ... Gradient unit, 17 ... Separation column, 18 ... Constant temperature unit, 1
9 ... Detector.

Claims (3)

[Claims] 1. A sampling mechanism for aspirating / discharging a specimen, diluting / hemolyzing and injecting a fixed amount thereof, and 2 having different salt concentrations.
A liquid feeding mechanism that feeds more than one type of eluent, a hemolysis sample injected by the sampling mechanism is separated into hemoglobin subfractions, and a separation / detection mechanism that detects each fraction, and the separation / detection mechanism
In a glycohemoglobin analyzer including a data processing mechanism that processes a detection signal from the detection mechanism to perform a quantitative calculation, the separation column that constitutes the separation / detection mechanism is kept at room temperature from 5
A constant temperature unit is installed so that the temperature is kept higher than ℃, and the pipe connecting the eluent storage tank and the liquid sending pump passes through the constant temperature unit, and a bubble trap is provided in the middle of the pipe connecting the constant temperature unit and the liquid sending pump. A glycohemoglobin analyzer characterized by being provided. 2. The glycohemoglobin analyzer according to claim 1, wherein after the power is turned on, a preparatory operation step that operates until pump pressure, an output signal level, an output signal noise, etc. are satisfied, and an analysis after the preparatory operation is completed. Until the execution, the eluent flow rate is made lower than the preparatory operation or / and waiting during the analysis, and the same unknown sample is continuously analyzed twice or more times n times. Glycohemoglobin analyzer characterized by comprising an analysis procedure composed of a step of ending the analysis when the absolute value of the difference from the quantitative value at the second time becomes equal to or less than a specified constant value, and shifting to a standby step. . 3. The glycohemoglobin analyzer according to claim 2, wherein the absolute value of the difference between the quantitative values at the n-th time and the (n-1) -th time and the number of repetitions n are rewritable. Analyzer.