JPH08233824A - Continuous measuring method for saccharified hemoglobin by chromatography without column regeneration - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method enabling continuous measurements for the content of saccharified hemoglobin in blood without regenerating an ion exchange column between a previous measuring sample and a coming measuring sample. SOLUTION: A boronate-affinitive absorption column 23 is utilized as an analyzer column. A series of hemolytic samples 11 is injected in a carrier liquid at 12, 13 every predetermined time interval. An absorbance is measured by a spectrophotometer 21 with the light of a wavelength absorbed by the hemoglobin before and after a liquid flow 14 including the samples pass through the column. Two measuring values are compared with each other, thereby to calculate the content of saccharified hemoglobin. Accordingly, 100 or more samples can be analyzed without detaching/regenerating the column.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention belongs to a technical field to which glycated hemoglobin contained in mammalian blood and its equivalent are related.

[0002]

BACKGROUND OF THE INVENTION Diabetes mellitus is a disease that affects more than 5% of the world's population. The disease is characterized by an insulin deficiency that reduces the body's ability to metabolize glucose and other nutrients, increasing the body's susceptibility to vascular complications. Examples of the treatment of diabetes include a method of externally administering insulin to the human body in an amount and frequency as needed. In that case, the need is usually assessed by monitoring the glucose level in the blood and a dosing regimen is aimed at keeping the glucose in the blood as close to normal levels as possible.

One of the methods for measuring the glucose content in blood is to measure blood glucose only once after fasting. However, it is recognized that such a single measurement is only indicative of the patient's immediate previous condition and does not necessarily represent the true condition of glucose in the blood. A more reliable index is the mean glucose concentration, which can be accurately measured by measuring the level of glycated hemoglobin in the blood.

When hemoglobin is saccharified, a bare 1,2-cis-diol group is added to the hemoglobin molecule. These groups are used as a means for separating glycated hemoglobin from non-glycated hemoglobin, and an ion exchange method and an affinity method have been developed based on this separation principle. One particularly effective separation technique is boronate affinity chromatography. In this chromatography, the geminal hydroxyl group bonded to the boronate functional group on the separation medium of the solid phase forms a reversible pair of condensed forms and bonds with the diol group on the glycated hemoglobin. Retention of the saccharified fraction on the chromatography column occurs under basic conditions, during which the non-saccharified fraction is washed out of the column. The saccharified fraction is then eluted by running a competitive cis-diol form or by acidifying the pH. Then, both the glycated hemoglobin fraction and the non-glycated hemoglobin fraction are individually quantified by spectrophotometric analysis.

This analysis lends itself to automation, and an automated continuous analyzer named "PRIMUS" is commercially available for clinical laboratories from Primus Corporation of Kansas City, Missouri, USA. Another such analyzer is the Variant Total GHb Program (VARI Program, available from Bio-Rad Laboratories, Inc. of Hercules, Calif., USA.
ANT Total GHb Program) ”.

As with any continuous analyzer, it is desirable to have frequent sample injections, but this sample injection interval is limited by the time required for column re-equilibration between subsequent injections. It In order to quantify the two peaks emanating from the column (the non-saccharified fraction is followed by the saccharified fraction), the period of the analysis is such that these two peaks are measured separately and accurately. Sufficient time must be taken to ensure that the peaks do not overlap and that the back portion of the peak is completely finished before the next injection. Also, changes in the buffer solution often reduce the degree of mixing, which hinders sharp transitions and increases the time required for re-equilibration between samples.

[0007]

The fact that the inventors have discovered here is that when a large number of samples are analyzed for glycated hemoglobin in a boronate affinity column, the re-equilibration of that column between samples is required. This means that analysis can be carried out without performing and without losing accuracy. Since detection is performed on both the inlet and outlet sides of the column as well as the outlet side of the column, absorbance measurements on the sample are made both before and after passing through the separating medium. This measurement is performed under conditions such that the saccharification fraction is retained by the medium while the sample is passing through the medium. Only the second of these measurements is done at the elution peak. The difference between the two measurements indicates the glycated hemoglobin retained by the column and correlates exactly with the amount of glycated hemoglobin in the sample in ratio to total hemoglobin.

Typical sample volumes used in automated continuous analyzers are on the order of 1-100 microliters of lysed sample, which lysed the blood sample to about 100 times or more hemolytic reagents. Prepared by diluting to the degree of dilution. A typical column has a length of about 1 cm to about 10 cm, an inner diameter of about 2 to about 10 mm, and is packed with a fixed bed (a packed bed for catalytic reaction). The present invention is based on the idea that such sample and column size should be used for analysis while performing re-equilibration between injections in order to clean the column in which all glycated hemoglobin is retained. However, it was attributed to the finding that the analytical results obtained by using the same size column and sample without re-equilibration were as accurate and reproducible as those obtained by re-equilibration. . Therefore, it is not necessary to change the buffer solution between samples, and continuous injection can be performed frequently with continuous absorption buffer solution flow.

These and other features of the invention,
And the advantages are explained in more detail below.

[0010]

DETAILED DESCRIPTION OF THE INVENTION Blood samples suitable for analysis according to the invention are generally mammalian blood samples in which blood cells have been lysed and all hemoglobin has been released into solution. Conventional hemolysis methods and reagents can be utilized and the method itself is not critical to the invention. In one of the conventional methods, a blood sample is at least about 1: 1 in deionized water.
The osmotic pressure is used to hemolyse by diluting at a dilution of 00, preferably at a dilution of at least about 1: 200.

The hemolysate is then analyzed according to the invention with other hemolysate samples. This analysis is done by continuously passing each hemolysate through the separation medium and performing a spectral analysis for each sample at both upstream and downstream adjacencies of the separation medium. The sample injection can be done manually, but in the present invention it is preferably done by an automated system. In this automated system, each sample is automatically and periodically injected in equal volumes into a continuously moving stream of carrier liquid. Note that the carrier liquid flows through the separating medium and either the two fluid cuvettes of the single detector or the two detectors.

As the separation medium, various forms and arrangements known to be usable in performing boronate affinity chromatography can be used. In the preferred form, the medium is a fixed bed of solid phase material in a high performance liquid phase chromatography (HPLC) column. The solid phase material is provided with a dihydroxyboryl group as a functional group, and suitable boron compounds having such a group for attachment to a supporting material are alkaneboronic acid, phenylboronic acid, and phenyl ring. It is a phenylboronic acid having various substituents. Examples of the phenylboronic acid having a substituent on the phenyl ring include alkylphenylboronic acid, nitrophenylboronic acid, and aminophenylboronic acid. In general, specific examples of suitable dihydroxyboryl compounds include ethaneboronic acid, 1-propaneboronic acid, 3-methyl-1-butaneboronic acid, phenylboronic acid, and meta-aminophenylborolide. Nick Acid. The last boronic acid is particularly preferred.

The solid support to which these functional groups are attached is substantially inert to the reagents used in the sample and the analytical and column preparation, and the derivative is formed by attaching the dihydroxyboryl ligand. It is possible to employ a natural or synthetic polymer capable of Hydrophilic materials are preferred, and examples thereof include polyacrylamide, polyvinyl alcohol, agarose, polyacrylamide-agarose copolymers, cellulose and cellulose derivatives such as cellulose acetate, starch, dextran, cross-linked dextran, and dextran-acrylamide. Polymers, methacrylates such as cross-linked hydroxyethyl methacrylate, polyethylene terephthalate, and polystyrene such as chloromethylated polystyrene are included. The support is preferably in the form of microscopic beads or other small particle form with a high surface area per unit volume. Functionalization of the support is accomplished by conventional chemistry well known in the art of preparing boronate affinity media.

The container holding the separating medium is preferably a column, most preferably of the cartridge type, ie the separating medium is pre-packed and the cartridge is easily incorporated into the flow path of the carrier liquid. It is a built-in, encapsulated column with an end mounting structure configured to allow. The size of the column is not critical and can vary widely, but the present invention has an inner diameter of about 3 mm to about 10 mm and a length (ie, the length of the bed contained inside the column) of about 0. .5c
It works best in columns that are in the range of m to about 50 cm, preferably about 1 cm to about 10 cm. The column can be made of glass, stainless steel, or other inert, preferably hydrophilic material.

The carrier liquid into which the sample is injected may be any liquid as long as it dissolves the sample and promotes the condensation reaction in which the glycated hemoglobin is retained by the dihydroxyboryl group on the separation medium. This liquid is preferably an aqueous buffer solution at basic pH, about 8.
More preferred is a pH of 0 or greater, and most preferred is a pH between about 8.0 and about 9.0. Examples of suitable buffers are ammonium salts such as ammonium acetate, phosphate buffers, glycine, morpholine, taurine, and HEP.
ES (N-2-hydroxyethylpiperazine-N-2-
Ethanesulfonic acid). Like the ionic strength, the pH of the buffer solution has a secondary interaction between the separation medium and the sample components, that is, an interaction other than the condensation reaction between the diol and the dihydroxyboryl ligand of glycated hemoglobin. Selected to minimize.

The detection of hemoglobin in the carrier liquid, which is carried out both before and after the liquid has passed through the separation medium, can be detected online if the method is capable of displaying the concentration of hemoglobin in the fluid. , Any method can be used. A common technique for making this type of detection is spectrophotometry, which measures the absorbance of electromagnetic radiation at the wavelength absorbed by hemoglobin. 405nm ~ 4
The 20 nm wavelength Soret region is particularly suitable for hemoglobin and is suitable for the purposes of the present invention. Conventional spectrophotometric techniques already used in hemoglobin analyzers can also be used here.

The detector signals are either recorded on a single common recording medium or read by a single common peak integrator as appropriate. In the preferred instrument configuration, the detection result for each sample exhibits two peaks, the first peak corresponds to the sample before passing through the column and the second peak is the glycated hemoglobin removed by the column. Corresponding to the peaks coming out of the column. The flow rate of the carrier liquid, the length of the column, the sample size, and other parameters of the system are such that these peaks appear in close proximity and in sequence, but there is no overlap between them. , Or selected to minimize. 1 representing the total hemoglobin amount
2nd peak and non-glycated hemoglobin 2
Saccharification by subtracting the peak area of unglycated hemoglobin from the peak area of total hemoglobin using the second peak and dividing by the peak area of total hemoglobin (multiply this by 100 to give a percentage). The ratio of hemoglobin released is easily measured, with appropriate comparison to calibration parameters if necessary. The system also includes a system calibrator to standardize the relative response of the flow cell or detector before and after the cartridge, or to standardize the response between the reagents and the instrument system, or both. I have it.

System parameters other than those mentioned above which are used in the practice of the invention can be varied considerably, but the best results, ie the ones which are most advantageous from an economic point of view, are often within a certain range. It is obtained by performing the measurement of. One such parameter is the ratio of the volume of aliquots of the original blood sample that is injected into the analyzer to the volume of the fixed bed of separation medium. In this case, the amount of aliquot is expressed by the volume excluding any diluent such as hemolytic reagent or carrier liquid. In a preferred embodiment of this invention, this ratio is about 0.1.
Is in the range of about 10 × 10 ⁻⁶ to about 100 × 10 ⁻⁶ , and in the most preferred embodiment, the range is about 0.3 × 10 6.
^-6 to about 30 x 10 ^-6 . Another such parameter is the velocity of the carrier liquid continuously flowing through the separation medium, which is expressed as a multiple of the carrier liquid fixed bed volume per minute. In the preferred embodiment, the rate is at least about 0.03, and in the most preferred embodiment the rate is at least about 0.1.

The present invention allows for the continuous injection of samples at a fairly high frequency compared to an automated continuous analyzer which separately analyzes both the fraction of glycated hemoglobin and the fraction of unglycated hemoglobin for each sample. To In the analyzer carrying out the invention, once every 10 minutes or less, once every 3 minutes or less, and in the preferred method once every 1 minute or less The sample can be injected.

As mentioned above, a very large number of samples can be run without regenerating the column to desorb glycated hemoglobin and without compromising accuracy, ie reproducibility from one sample to the next. It can be analyzed by passing through a column. The number of samples that can be analyzed without the elimination of interfering glycated hemoglobin depends to some extent on some of the system parameters mentioned above, in particular on the size of the column and the sample size. However, in general, at least 10 samples can be analyzed without interfering desorption. And in many cases 30, 50, 100
Individual or more samples can be analyzed. If you need to regenerate a column,
It may be purged with a releasing liquid for reversing the condensation reaction that resulted in the fixation of glycated hemoglobin with the dihydroxyboryl group. For desorption liquids, conventional automated systems, i.e. desorption at the end of every run,
That is, the same type of elution buffer used for desorption of glycated hemoglobin can be used in the conventional automated system performed as part of the analysis. For example, acidic pH buffers, or competitive ligands containing cis-diol groups such as 0.1 M sorbitol are effective elimination liquids. One example is pH
Is about 6.0 or less, eg, 5.3, sodium citrate buffer.

A schematic flow diagram of an analyzer illustrating the present invention is shown in FIG. A plurality of hemolyzed samples are placed in individual containers and placed in the automatic sampler 11. The automatic sampler 11 communicates the containers, one by one, to a sampling probe (not specifically shown, but part of the sampler), which directs the sample into the sample loop 12 of the injection valve 13. To be At appropriate intervals, the injection valve 13 is activated and the contents of the sample loop 12 are injected into the moving stream 14 of carrier liquid (basic pH). This liquid flow 14 is supplied from a reservoir 15 through a pump 16 and a valve 17. The carrier liquid flow contains the sample and passes through one channel 21 of the two-channel flow cell provided in the optical path 22 of the spectrophotometric detector. The liquid stream emerging from this spectrophotometric detector is then absorbed by the absorption column 23.
Pass through. The liquid stream emerging from the column 23 passes through the second channel 24 of the detector flow cell. The liquid flow emerging from this second channel is guided to the discharge path 25. The extinction coefficient due to the combination of the contents of the first and second flow channels 21, 24 is continuously measured by the photocell 26 in the detector. From the photocell 26, the absorptance signal is sent to a strip chart recorder (not shown) and an integrator (not shown).

The arrangement of the detector shown in FIG. 1 can be modified and modified in various ways. As an example, two separate one-channel detectors can be used, one upstream of the absorption column and another downstream. As a second example, a single 1-channel detector could be used in combination with a switching valve. In that case, the valve first directs the sample through the detector before it enters the absorption column. The valve is then switched so that the liquid flow through the detector is at the output of the column, such that at no point does any component of the sample bypass the detector. Done in. The third example also uses a single one-channel detector on the sample input side of the column, but the direction of flow through the column can be reversed immediately after the sample has passed through the column. Thus, there are various fluid flow paths arranged in combination with one or more switching valves. Therefore, the sample first passed through the detector on its way to the column, then once the sample had passed through the column, it contained all the components of the sample that were not adsorbed on that column and exited the column. The exiting carrier liquid is redirected backwards through the column and detector, leaving the absorption fraction on the column.

Referring again to the flow schematic shown in FIG. Once a large number of samples have passed through the absorption column 23, the flow of carrier liquid is interrupted by stopping the pump 16, closing the valve 17, or both, and purging or dissociating liquid. The flow of (liquid with acidic pH or liquid containing competitive cis-diol groups) begins. This purging liquid is pump 3
2 and the valve 33 to supply from the reservoir 31.
For automatic analysis, the operation timings of the sampler 11, the injection valve 13, the transfer liquid pump 16 and the purging liquid pump 32, and the valves 17 and 33 associated therewith are controlled by a program controller (not shown). )
Controlled by.

In the presently preferred embodiment of this invention, the system parameters are as follows:

Hemolytic Reagent: Deionized Water Dilution of Blood Blood Sample into Hemolytic Reagent: 1: 1600,
Optimal dilution is 1: 200 Sample loop volume: 20 μL, optimal volume is 2 μL Column dimensions: 4.6 mm diameter, 10 mm length Column packing: meta-aminophenylboronic acid retained on an inert support. Acid Transport (absorption) liquid: Glycylglycine buffer, pH
8.3 Removal liquid: Aqueous sodium citrate buffer containing sorbitol, pH 5.5 Injection interval: 2.5 minutes, optimal interval is 0.7-1.0 minutes Carrier fluid flow rate: 1 mL / min Detection: 415 nm Light absorbance at

[0026]

EXAMPLE A preferred analyzer with the parameters listed above was compared with several analyzers and manual methods currently available in clinical laboratories. here,
The analyzer of the present invention was the only one capable of continuous analysis without regenerating the column. For each analyzer, the same set of samples consisting of 86 samples with a percentage range of glycated hemoglobin ranging from about 5% to about 20% was used with a suitable calibrator. Comparative controls are shown in Figures 2-7, where the horizontal axis represents the data obtained by the analyzer of the present invention,
Data obtained by other analyzers listed below are shown for samples with the same vertical axis.

FIG. 2 vertical axis: VARIANT total G
Hb Program (Bio-Rad Laboratories, Inc., Hercules, CA, USA) Figure 3 Vertical axis: Glycotest II (Pierce Chemical Company, Rockford, IL, USA) Figure 4 Vertical axis: Glyc-Affin GHb (United States) Isolab Inc., Akron, Ohio) Figure 5 Vertical axis: Glyco-Tek (Helena Chemical, West Helena, Arkansas, USA)
Company) Figure 6 Vertical axis: Primus GHb (Primus Corporation, Kansas City, MO, USA) Figure 7 Vertical axis: DIAMAT A1c (Bio-Rad Laboratories, Inc., Hercules, CA, USA) These plots are correlated. It shows that the relationships are very close, which confirms the accuracy and reproducibility of the analyzer of the invention.

The above is provided primarily for purposes of illustration. It is to be understood that the analyzer operating unit arrangements, materials, operating conditions, process steps, and other parameters in the systems described herein may be altered or replaced in various ways without departing from the spirit and scope of the invention. It will be readily apparent to the trader.

[Brief description of drawings]

FIG. 1 is a block diagram illustrating the operation of an analyzer according to the present invention.

FIG. 2 is a plot graph showing a correlation between test data obtained for a series of samples using the analyzer of the present invention and test data obtained for the same sample using a conventional analyzer.

FIG. 3 is a plot graph showing the same correlation as shown in FIG. 2 using another conventional analyzer.

FIG. 4 is a plot graph showing the same correlation as shown in FIG. 2 using a third conventional analyzer.

FIG. 5 is a plot graph showing the same correlation as shown in FIG. 2 using a fourth conventional analyzer.

FIG. 6 is a plot graph showing the same correlation as shown in FIG. 2 using a fifth conventional analyzer.

FIG. 7 is a plot graph showing a correlation similar to that shown in FIG. 2 using a sixth conventional analyzer.

[Explanation of symbols]

11 ... Automatic sampler, 12 ... Sample loop, 13 ... Injection valve, 14 ... Carrier flow, 15 ... Reservoir, 16 ... Pump, 17 ... Valve, 21 ... Channel, 22 ... Optical path,
23 ... Absorption column, 24 ... Channel, 25 ... Discharge path, 26 ... Photocell, 31 ... Reservoir, 32 ... Pump,
33 ... Valve.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication location G01N 33/50 G01N 33/50 U (72) Inventor Sergey Vlasenko United States California 94510 Benicia Southampton Road 1300 Number 191

Claims (10)

[Claims] 1. A method of analyzing a plurality of mammalian blood samples, wherein the ratio of glycated hemoglobin to total hemoglobin in individual samples of a plurality of mammalian blood samples is continuously measured, comprising: (a) Dissolving one of the samples in a solution to liberate the hemoglobin contained in the sample; (b) this dissolved sample; (i) a first detection pass of the absorbance detector (ii) Fixed bed of dihydroxyboryl affinity medium (iii) The first detection in the flow of the carrier liquid having a pH of about 8.0 or more that continuously flows in this order through the second detection path of the absorbance detector. Injecting into the upstream side of the path, and (c) measuring the absorbance of light by the flow of the carrier liquid in the first and second detection paths at a wavelength absorbed by hemoglobin. And (d) as a means for measuring the ratio of glycated hemoglobin to total hemoglobin in the sample, a step of comparing the absorbance measured in the first detection path and the absorbance measured in the second detection path, (E) The steps (a) to (d) are successively repeated for the remaining samples of the plurality of samples without desorbing glycated hemoglobin from the fixed layer. And the analysis method. 2. The method for analyzing a plurality of blood samples according to claim 1, wherein the ratio of the volume of the sample injected in the step (b) to the volume of the dihydroxyboryl affinity medium in the fixed layer is about 0. 1 x 10 ^-6 to approximately 100 x 10 ^-6
An analysis method characterized by being. 3. The method for analyzing a plurality of blood samples according to claim 1, wherein the ratio of the volume of the sample injected in the step (b) to the volume of the dihydroxyboryl affinity medium in the fixed layer is about 0. .3 × 10 ⁻⁶ to about 30 × 10 ⁻⁶ . 4. The method for analyzing a plurality of blood samples according to claim 1, wherein the flow of the carrier liquid is at least about 0.03 times the volume of the dihydroxyboryl affinity medium in the fixed bed per minute. Analytical method characterized by continuous flow in 5. The method for analyzing a plurality of blood samples according to claim 1, wherein the steps (a) to (d) are repeated at least 10 times without desorbing glycated hemoglobin from the fixed layer. Analytical method characterized by. 6. The method for analyzing a plurality of blood samples according to claim 1, wherein the flow of the carrier liquid is at least about 0.03 times the volume of the dihydroxyboryl affinity medium in the fixed bed per minute. Analytical method, characterized in that the sample is injected continuously at a frequency of at least about 1 sample per 10 minutes, and the step (e) is continuously flowing. 7. The method of analyzing a plurality of blood samples according to claim 1, wherein the flow rate of the carrier liquid is at least about 0.1 times the volume of the dihydroxyboryl affinity medium in the fixed bed per minute. Analytical method, characterized in that the sample is injected continuously at a frequency of at least about 1 sample per 3 minutes, and the step (e) is continuously flowing. 8. The method of analyzing a plurality of blood samples according to claim 1, further comprising: (f) the carrier flowing through the fixed layer after a predetermined number of sample injections exceeding 10 times. Analytical method, characterized in that it comprises the steps of interrupting the flow of liquid and purging the fixed bed with a releasing liquid in order to dissociate the glycated hemoglobin from the dihydroxyboryl affinity medium. 9. The method for analyzing a plurality of blood samples according to claim 1, wherein the dihydroxyboryl affinity medium is alkaneboronic acid, phenylboronic acid, alkylphenylboronic acid, nitrophenylboronic acid, And a solid support functionalized with a boronic acid selected from the group consisting of and aminophenylboronic acid. 10. The method for analyzing a plurality of blood samples according to claim 1, wherein the dihydroxyboryl affinity medium is a solid support functionalized with meta-aminophenylboronic acid. Analysis method.