JP6938964B2 - Biological sample pretreatment equipment - Google Patents

Description

The present invention relates to a pretreatment device for removing contaminants such as bilirubin that cause measurement errors when quantifying hemoglobin in a biological sample.

Glycated hemoglobin (hereinafter, may be abbreviated as HbA1c), which is one of the diagnostic items for diabetes, is hemoglobin (hereinafter, sometimes abbreviated as Hb), which is responsible for transporting oxygen in the blood, and sugar (glucose) is added to the hemoglobin. Among the bound glycated hemoglobin, the valine residue located on the N-terminal side of the hemoglobin β chain refers to a glycated substance, and the HbA1c concentration with respect to the total amount of hemoglobin reflects the average blood glucose level over the past 1 to 2 months. , Used to observe the long-term course of diabetes.

In recent years, the word POCT has been attracting attention in the medical field. POCT is an abbreviation for Point Of Care Testing, and refers to a clinical test performed by a healthcare professional near a subject. Unlike clinical tests performed in a central laboratory of a large-scale hospital, POCT is becoming widespread in diabetes diagnosis because test results can be obtained instantly on the spot.

In POCT for the purpose of measuring HbA1c concentration, a technique using an immunochromatography method has been proposed. The immunochromatography method is an immunoassay method that utilizes capillarity, and is an analytical technique used in pregnancy test agents and influenza test agents that use the immunochromatography method, and is widely used worldwide. In the conventional immunochromatography method, visual judgment (qualitative evaluation) is common, but in recent years, a technique for quantifying the concentration of a substance contained in a sample by using an analyzer such as a chromatographic reader has been developed.

However, reducing substances such as bilirubin and ascorbic acid are present in blood, and the presence of these substances greatly affects the measured values of hemoglobin and glycated hemoglobin, which may cause an error in the measured values. In addition, since bilirubin and the like also act as dyes, they may cause an error depending on the measurement wavelength.

As a method of avoiding the influence of bilirubin, a method of adding an amphoteric tenside agent to a measurement reagent is known. For example, Patent Document 1 describes that an amphoteric surfactant is added to the first reagent for the purpose of avoiding the influence of bilirubin. Further, in Patent Document 2, in a method for measuring a biological component in which hydrogen peroxide produced by an enzymatic reaction is detected by a peroxidase and an oxidizable color former, both the first reagent or the first reagent and the second reagent are amphoteric. A method for measuring a biological component in which a surfactant and a ferrocyanide compound are present is described.

Japanese Unexamined Patent Publication No. 7-039394 Japanese Unexamined Patent Publication No. 7-155196

The present invention provides a biological sample pretreatment device that can selectively and efficiently remove measurement-inhibiting substances such as bilirubin that cause measurement errors when quantitatively measuring the hemoglobin concentration in a biological sample. The challenge is to provide.

As a result of diligent studies to solve the above problems, the present inventor can selectively and instantly remove contaminants in a biological sample by treating the biological sample with a pretreatment instrument having a specific configuration. The present invention was completed.

That is, the present invention has the following configuration.
(1) A pretreatment device for removing bilirubin from the biological sample when quantitatively analyzing hemoglobin in the biological sample.
The instrument is a housing case having an inlet and an outlet for the biological sample, and the housing case is filled with a bilirubin removing material.
The removal material is granular activated carbon or activated carbon fiber.
A pretreatment instrument characterized in that the total pore volume of the removal material is 0.25 to 1.00 cc / g.

According to the present invention, by bringing a biological sample into contact with a pretreatment instrument having a specific configuration, contaminants in the biological sample can be selectively, efficiently and promptly removed, so that the biological sample is not affected by the contaminants. Hemoglobin concentration can be measured quantitatively. In addition, since the influence of contaminants is reduced, it is possible to measure hemoglobin in the maximum absorption wavelength range, and it is possible to measure with high accuracy even when the hemoglobin concentration in the biological sample is low.

The schematic which shows an example (syringe filter type) of embodiment of the pretreatment instrument of this invention. The schematic which shows another example (dropper type) of embodiment of the pretreatment instrument of this invention.

In the present invention, the biological sample means a body fluid containing hemoglobin such as blood, plasma, serum, stool, and urine, and among them, hemoglobin is quantitatively analyzed, and in particular, the absorptiometry is used. It contains various contaminants (measurement-inhibiting substances) that give a measurement error when analyzing (measuring).

The present invention is an instrument for removing contaminants in a biological sample before subjecting the biological sample to a detection device (immunochromatographic test piece) or the like, and the instrument is a housing having an inlet and an outlet for the biological sample. The case and the housing case are filled with a material for removing contaminants. Hereinafter, the details of the pretreatment instrument of the present invention will be described with reference to FIG.

[Embodiment 1]
FIG. 1 shows a cross-sectional view of a syringe filter type pretreatment instrument. In FIG. 1, reference numeral 1 denotes a sample inlet for introducing a biological sample. In addition, 2 is a material for removing contaminants. Reference numeral 3 denotes a membrane filter for preventing contamination of the removal material itself and the eluate from the removal material on the treated biological sample. Further, reference numeral 4 denotes a sample outlet for taking out the processed biological sample. Note that 3 may or may not be present depending on the purpose and application. Further, other substances may be substituted as long as they can achieve contamination prevention.

The sample inlet 1 may have a structure that allows the syringe to be connected in a liquid-tight manner, and may have a female luar lock structure if necessary. On the other hand, it is preferable to adjust the shape and the like of the sample outlet 4 so that an appropriate amount of the sample can be directly dropped onto the immunochromatographic test piece. In the measurement using an immunochromatographic test piece, the shape may be such that about 100 μL to 500 μL can be dropped, although it depends on the size.

In the present invention, the material for removing contaminants is activated carbon, activated carbon fiber (hereinafter, the activated carbon and activated carbon fiber may be collectively referred to as activated carbon), titanium oxide, divinylbenzene / glycidyl methacrylate copolymer, and hydrophobicity. It is preferably one or more selected from the group consisting of silica. Of these, activated carbon is preferable in terms of availability and cost. Further, among the activated carbons, those for water treatment are more preferable.

In the present invention, the material for removing contaminants is preferably in one or more forms selected from the group consisting of fibers, woven fabrics, non-woven fabrics, particles and powders. Since the pretreatment instrument can be made compact, it is more preferable that it is in the form of a non-woven fabric, particles, or powder. Here, titanium oxide and hydrophobic silica are available in the form of particles or powder, and other than that, fibrous, non-woven fabric, particulate or powder can be obtained and prepared.

The raw material (precursor) of the activated charcoal is not particularly limited, and for example, phenol, cellulose, rayon, polyacrylonitrile-based, pitch-based, aramid, polyimide, polyamide, polyamideimide, polyphenylene benzobisoxazole, polyvinyl alcohol, and the like. Examples thereof include polyethersulfone, polysulfone, and polyphenylene oxide. These materials can be produced by molding into fibers, woven fabrics, non-woven fabrics, particles, and powders, and then carbonizing, activating, and purifying if necessary.

In the present invention, when a porous body is used as the material for removing contaminants, the total pore volume thereof is preferably in the range of 0.25 to 1.00 cc / g. If the total pore volume is too small, contaminants in the biological sample may not be sufficiently removed, and if it is too large, the strength of the single fiber may be significantly reduced and the morphology may not be maintained. The total pore volume can be measured using, for example, a specific surface area / pore distribution measuring device Gemini2375 (manufactured by Micromeritics).

In the present invention, the specific surface area of the material for removing contaminants is preferably in the range of 5 to ^{2500 m 2 / g.} If the specific surface area is too small, contaminants in the biological sample may not be sufficiently removed, or the pretreatment instrument needs to be made larger than necessary. Further, if the specific surface area is too large, the strength of the single fiber is remarkably lowered, and the morphology may not be maintained. The specific surface area means the specific surface area obtained by the BET method using a nitrogen gas adsorption isotherm at a liquid nitrogen temperature, and can be measured using, for example, a specific surface area / pore distribution measuring device Gemini2375 (manufactured by Micromeritics). ..

In the present invention, when a porous body is used as the material for removing contaminants, it is preferable that the micropore volume having a pore diameter of 10 Å or less is 95% or more of the total micropore volume. If the micropore volume with a pore diameter of 10 Å or less is less than 95% of the total micropore volume, the pores may become too large and contaminants may not be sufficiently removed. The ratio of the micropore volume having a pore diameter of 10 Å or less can be measured using, for example, a specific surface area / pore distribution measuring device Gemini2375 (manufactured by Micromeritics).

In the present invention, when the material for removing contaminants has a fiber shape, the single fiber diameter is preferably 5 to 30 μm. If the single fiber diameter is too small, it may not be possible to secure effective pores for removing contaminants. On the other hand, if the single fiber diameter is too large, the compactness of the pretreatment instrument may be impaired. When the material for removing contaminants is in the form of particles or powder, the average particle size is preferably 1 nm to 1 mm. If the average particle size is too small, it may not be possible to secure effective pores for removing contaminants. On the other hand, if the average particle size is too large, the compactness of the pretreatment instrument may be impaired.

In the present invention, it is not clear why the pretreatment instrument constructed using the above-mentioned material does not remove hemoglobin, but exhibits the selectivity of removing contaminants such as bilirubin and milky lotion. However, as will be described later, even if the solution in which hemoglobin is dissolved is treated with a pretreatment instrument, the absorbance does not decrease almost before and after the treatment, but when the solution in which bilirubin is dissolved is treated with a pretreatment instrument, the absorbance greatly decreases. It is clear that bilirubin is selectively removed.

In the first embodiment, for example, after sucking up a required amount of a biological sample diluted with a diluent into a syringe, the pretreatment instrument of the present invention is attached to the tip of the syringe, and the plunger of the syringe is pushed into the biological sample or diluted. By extruding the biological sample from the sample outlet of the pretreatment instrument, measurement-inhibiting substances such as bilirubin can be easily removed.

[Embodiment 2]
The first embodiment is different from the pretreatment instrument in that the pretreatment instrument is separate from the sampling instrument, whereas the second embodiment is different in that the sampling instrument is integrated with the pretreatment instrument (see FIG. 2). ).

(Measurement of specific surface area)
Approximately 30 mg of the contaminant removing material was sampled, vacuum dried at 120 ° C. for 12 hours, weighed, and measured using a specific surface area / pore distribution measuring device Gemini2375 (manufactured by Micromeritics). The adsorption amount of nitrogen gas at the boiling point (-195.8 ° C.) of liquid nitrogen was measured in the range of relative pressure of 0.02 to 0.95, and an adsorption isotherm of the material was prepared. Based on the results in the range of relative pressure 0.02 to 0.15, the specific surface area per weight (unit: m ² / g) was determined by the BET method.

(Measurement of total pore volume)
Approximately 30 mg of the contaminant removing material was sampled, vacuum dried at 120 ° C. for 12 hours, weighed, and measured using a specific surface area / pore distribution measuring device Gemini2375 (manufactured by Micromeritics). The adsorption amount of nitrogen gas at the boiling point (-195.8 ° C.) of liquid nitrogen was measured in the range of relative pressure of 0.02 to 0.95, and an adsorption isotherm of the material was prepared. The total pore volume (unit: cc / g) was calculated from the result at a relative pressure of 0.95.

(Total micropore volume (A))
Approximately 30 mg of the contaminant removing material was sampled, vacuum dried at 120 ° C. for 12 hours, weighed, and measured using a specific surface area / pore distribution measuring device Gemini2375 (manufactured by Micromeritics). The adsorption amount of nitrogen gas at the boiling point (-195.8 ° C.) of liquid nitrogen was measured in the range of relative pressure of 0.02 to 0.95, and an adsorption isotherm of the material was prepared. The analysis range of this result is 0 to 20 Å by the MP method, and the t-determination formula H. The analysis was performed under the condition of J, and the total micropore volume (A) (unit: cc / g) was calculated from the result of the micropore diameter distribution number table at the time of adsorption.

(Micropore volume (B) with a pore diameter of 10 Å or less)
Approximately 30 mg of the contaminant removing material was sampled, vacuum dried at 120 ° C. for 12 hours, weighed, and measured using a specific surface area / pore distribution measuring device Gemini2375 (manufactured by Micromeritics). The adsorption amount of nitrogen gas at the boiling point (-195.8 ° C.) of liquid nitrogen was measured in the range of relative pressure of 0.02 to 0.95, and an adsorption isotherm of the material was prepared. This result was analyzed by the MP method in an analysis range of 0 to 2 nm, and the t-determination formula H. Analysis under the condition of J, and subtract the micropore volume of 10.03 Å or more from the total micropore volume (A) from the result of the micropore diameter distribution number table at the time of adsorption, and micropores with a pore diameter of 10 Å or less. Volume B (unit: cc / g) was calculated.

(Measurement of absorbance)
For each measurement sample, the absorbance at 420 nm was measured using a spectrophotometer UV-2450 (manufactured by Shimadzu Corporation). As the measurement cell, a 1 mL disposable cell was used, and the measurement was performed with an optical path length of 10 mm.

(Preparation of pretreatment jig 1)
^{Activated carbon fibers having a specific surface area of 973 m 2} / g and a total pore volume of 0.42 cc / g obtained by carbonizing and activating phenol fibers in a polypropylene filter holder having an inlet and an outlet as shown in FIG. A pretreatment instrument 1 was prepared by filling with 34 mg.

(Preparation of pretreatment jig 2)
Pretreatment device 2 in the same manner as pretreatment device 1 except that it was filled with 34 mg of activated carbon fiber having a specific surface area of 1962 m ^{2 / g and a total pore volume of 0.85 cc / g obtained by carbonizing and activating phenol fibers.} Was produced.

(Preparation of pretreatment jig 3)
Pretreatment equipment similar to pretreatment equipment 1 except that it was filled with 34 mg of activated carbon fiber having a specific surface area of 850 m ^{2 / g and a total pore volume of 0.35 cc / g obtained by carbonizing and activating pitch fibers.} 3 was prepared.

(Preparation of pretreatment jig 4)
Pretreatment device 4 in the same manner as pretreatment device 1 except that it was filled with 34 mg of activated carbon fiber having a specific surface area of 1520 m ^{2 / g and a total pore volume of 0.65 cc / g obtained by carbonizing and activating phenol fibers.} Was produced.

(Preparation of pretreatment jig 5)
The pretreatment device 5 is the same as the pretreatment device 1 except that it is filled with 34 mg of activated carbon fiber ^{having a specific surface area of 1700 m 2} / g and a total pore volume of 0.80 cc / g obtained by carbonizing and activating the pitch fibers. Was produced.

(Preparation of pretreatment jig 6)
Pretreatment instrument 6 in the same manner as pretreatment instrument 1 except that it was filled with 34 mg of granular activated carbon (KD-GW-A manufactured by US Corporation, specific surface area 1329 m ^{2 / g, total pore volume 0.44 cc / g).} Was produced.

[Reference example 1]
Interference Check A Plus (Cysmex) hemolyzed hemoglobin was dissolved in distilled water to 135 g / L, and then diluted with a diluent (1% Triton X-100, 1% Tween-20, 50 mM PBS, pH 7.1). The sample 1 for measurement was obtained by diluting 500 times. After sucking up 1 mL of the measurement sample 1 into a 1 mL syringe, a pretreatment instrument 1 was attached to the tip of the syringe, and the measurement sample 1 was extruded into a cell. The cell was set in a spectrophotometer and the absorbance at 420 nm was measured. The results are summarized in Table 1.

[Reference example 2]
Bilirubin F of Interference Check A Plus (Sysmex Corporation) was dissolved in distilled water to a concentration of 200 mg / dL, and then diluted 500-fold with the same diluent as in Reference Example 1 to obtain Sample 2 for measurement. After sucking up 1 mL of the measurement sample 2 into a 1 mL syringe, the pretreatment instrument 1 at the tip of the syringe was attached, and the measurement sample 2 was pushed out into the cell. The cell was set in a spectrophotometer and the absorbance at 420 nm was measured. The results are summarized in Table 1.

As is clear from the results in Table 1, when the solution in which hemoglobin was dissolved was brought into contact with the pretreatment instrument, the absorbance did not change much before and after the contact, but the solution in which bilirubin was dissolved was brought into contact with the pretreatment instrument. In that case, the absorbance was greatly reduced. This indicates that the pretreatment device of the present invention does not remove hemoglobin in the biological sample, but removes contaminants such as bilirubin.

[Example 1]
After sucking up 1 mL of the measurement sample 1 into a 1 mL syringe, a pretreatment instrument 1 was attached to the tip of the syringe, and the measurement sample 1 was extruded into a cell. The cell was set in a spectrophotometer and the absorbance at 420 nm was measured. Further, the measurement sample 2 was also subjected to the same treatment to measure the absorbance. The results are summarized in Table 2.

[Example 2]
After sucking up 1 mL of the same measurement sample as in Example 1 into a 1 mL syringe, the pretreatment instrument 2 was attached to the tip of the syringe, and the measurement sample was pushed out into the cell. The cell was set in a spectrophotometer and the absorbance at 420 nm was measured. The results are summarized in Table 2.

[Example 3]
After sucking up 1 mL of the same measurement sample as in Example 1 into a 1 mL syringe, the pretreatment instrument 3 was attached to the tip of the syringe, and the measurement sample was pushed out into the cell. The cell was set in a spectrophotometer and the absorbance at 420 nm was measured. The results are summarized in Table 2.

[Example 4]
After sucking up 1 mL of the same measurement sample as in Example 1 into a 1 mL syringe, the pretreatment instrument 4 was attached to the tip of the syringe, and the measurement sample was pushed out into the cell. The cell was set in a spectrophotometer and the absorbance at 420 nm was measured. The results are summarized in Table 2.

[Example 5]
After sucking up 1 mL of the same measurement sample as in Example 1 into a 1 mL syringe, the pretreatment instrument 5 was attached to the tip of the syringe, and the measurement sample was pushed out into the cell. The cell was set in a spectrophotometer and the absorbance at 420 nm was measured. The results are summarized in Table 2.

[Example 6]
After sucking up 1 mL of the same measurement sample as in Example 1 into a 1 mL syringe, the pretreatment instrument 6 was attached to the tip of the syringe, and the measurement sample was pushed out into the cell. The cell was set in a spectrophotometer and the absorbance at 420 nm was measured. The results are summarized in Table 2.

[Comparative Example 1]
After sucking up 1 mL of the same measurement sample as in Example 1 into a 1 mL syringe, the measurement sample was extruded into the cell without attaching a pretreatment instrument to the tip of the syringe. The cell was set in a spectrophotometer and the absorbance at 420 nm was measured. The results are summarized in Table 2.

As is clear from the results in Table 2, in Examples 1-6 using the pretreatment instrument 1-6, the absorbance at a wavelength of 420 nm is significantly reduced as compared with Comparative Example 1. Hemoglobin A1c (%) is an index for diagnosing diabetes obtained by hemoglobin A1c concentration / hemoglobin concentration × 100, and accurate diagnosis cannot be made if the measured value of hemoglobin concentration contains an error. According to the present invention, the hemoglobin concentration can be measured more accurately.

According to the present invention, the hemoglobin concentration can be quantitatively measured without being affected by contaminants. In addition, since the influence of contaminants is reduced, it is possible to measure hemoglobin in the maximum absorption wavelength range, and even when the hemoglobin concentration in the biological sample is low, it can be measured with high accuracy, which is extremely important in diabetes diagnosis. Suitable.

1. 1. Sample inlet 2. Material for removing contaminants 3. Membrane filter 4. Sample outlet

Claims (1)

A pretreatment device for removing bilirubin from the biological sample when quantitatively analyzing hemoglobin in the biological sample.
The instrument is a housing case having an inlet and an outlet for the biological sample, and the housing case is filled with a bilirubin removing material.
The removal material is granular activated carbon or activated carbon fiber.
A pretreatment instrument characterized in that the total pore volume of the removal material is 0.25 to 1.00 cc / g.

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