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WO2017006965A1 - Procédé d'analyse du sang, et kit d'examen du sang - Google Patents

Procédé d'analyse du sang, et kit d'examen du sang Download PDF

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Publication number
WO2017006965A1
WO2017006965A1 PCT/JP2016/070010 JP2016070010W WO2017006965A1 WO 2017006965 A1 WO2017006965 A1 WO 2017006965A1 JP 2016070010 W JP2016070010 W JP 2016070010W WO 2017006965 A1 WO2017006965 A1 WO 2017006965A1
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WIPO (PCT)
Prior art keywords
blood
diluent
analysis method
sample
component
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PCT/JP2016/070010
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English (en)
Japanese (ja)
Inventor
達也 石坂
中津川　晴康
進 大澤
晋哉 杉本
Original Assignee
富士フイルム株式会社
株式会社リージャー
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by 富士フイルム株式会社, 株式会社リージャー filed Critical 富士フイルム株式会社
Priority to EP16821434.4A priority Critical patent/EP3321679B1/fr
Priority to KR1020207005609A priority patent/KR102111903B1/ko
Priority to CN201680039333.2A priority patent/CN107949789B/zh
Priority to KR1020187000464A priority patent/KR102084410B1/ko
Priority claimed from JP2016133960A external-priority patent/JP6417367B2/ja
Publication of WO2017006965A1 publication Critical patent/WO2017006965A1/fr
Priority to US15/861,209 priority patent/US10634661B2/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/10—Devices for withdrawing samples in the liquid or fluent state
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard

Definitions

  • the present invention relates to a blood analysis method and a blood test kit for analyzing a target component in a very small amount of blood sample.
  • a general qualified blood sample is collected by a doctor or other qualified person using a syringe to collect blood from the vein, and the subject is self-collected by inserting a blood collection needle into his / her finger or the like. There is blood sampling.
  • Blood collected by general blood collection is transported to a medical institution or inspection in a state of being sealed in a collection container, where it is inspected.
  • a test is performed after blood is separated into blood cells and plasma by a centrifuge at a medical institution or inspection institution.
  • the collected blood is separated into blood cells and plasma by a separation membrane and transported to the examination site in this separated state, where the examination is performed.
  • Patent Document 1 describes a method for examining a blood sample collected by self blood collection. Specifically, 1) Prepare a sample for quantification consisting of an unknown volume of a biological sample containing a component to be quantified collected without quantifying the volume and a fixed amount of an aqueous solution containing a fixed amount of an indicator substance.
  • Patent Document 2 the amount of a component to be analyzed in a sample is measured, and further, the amount of a standard component originally present in the sample other than the above is measured.
  • a quantitative analysis method is described in which the amount of a sample is determined from the known concentrations of the standard components, and the concentration of the analysis target component in the sample is determined from the sample amount and the analysis target component amount.
  • Patent Document 3 describes that a small amount of blood is collected from a human or animal using a blood dilution quantification instrument and is supplied as it is or after dilution to supply a constant amount to another device, container or reagent.
  • Patent Document 4 describes a method of quantifying the concentration of a component to be quantified in a biological sample using the absorbance of an indicator substance in an aqueous solution for dilution.
  • Patent Document 2 about 100 ⁇ L of whole blood of healthy subjects is dropped on a porous membrane, blood cells are separated and serum is developed, and then 150 ⁇ L of saline-isotonic PBS (Phosphate-buffered saline: pH 7.4) is added. The supernatant obtained by centrifuging the obtained liquid is analyzed as an analysis sample, but there is no description of blood collection of less than 100 ⁇ L.
  • saline-isotonic PBS Phosphate-buffered saline: pH 7.4
  • the measurement method described in Patent Document 4 is a measurement with a dilution factor of about 10 times.
  • the measurement value is repeatedly reproduced as in Patent Document 1. There is a problem that the performance is lowered.
  • An object of the present invention is to provide a blood analysis method having high reproducibility of measurement values in a blood sample of 50 ⁇ L or less, and a blood test kit for use in the blood analysis method.
  • the present inventors diluted a collected blood sample with a diluent, and determined the dilution ratio using the standard values of standard components that are constantly present in the blood.
  • the volume of the blood sample is 50 ⁇ L or less
  • the dilution factor of the plasma component in the blood sample is 14 times or more
  • the blood sample is constantly in the blood as a diluent.
  • the present invention has been completed by finding that the above problem can be solved by using a diluent that does not contain the above-described standard component. That is, according to the present invention, the following inventions are provided.
  • the blood analysis method according to (1) comprising: Blood analysis wherein the volume of the blood sample is 50 ⁇ L or less, the dilution factor of the plasma component in the blood sample is 14 times or more, and the diluent is a diluent that does not contain a standard component that is constantly present in the blood Method.
  • the blood analysis method according to (1) comprising a step of recovering a plasma component-containing sample from the diluted blood sample after the step of diluting the collected blood sample with a diluent.
  • the blood analysis method according to (2) wherein the collecting step is a step using a separation membrane.
  • the blood analysis method according to (2) or (3) comprising a step of transporting the plasma component-containing sample after the step of recovering the plasma component-containing sample from the diluted blood sample.
  • the blood analysis method according to any one of (1) to (4) wherein the standard component that is constantly present in the blood is sodium ion or chloride ion.
  • the blood analysis method according to any one of (1) to (5), wherein the standard components that are constantly present in the blood are sodium ions or chloride ions and at least one standard component. .
  • the blood analysis method according to (6), wherein the at least one standard component is a standard component selected from total protein or albumin.
  • the blood analysis method according to (5), wherein the diluent is a diluent not containing sodium ions or chloride ions.
  • the blood analysis method according to (5), wherein the standard component that is constantly present in the blood is sodium ion.
  • an amino alcohol compound wherein the diluent is selected from the group consisting of 2-amino-2-methyl-1-propanol, 2-ethylaminoethanol, N-methyl-D-glucamine, diethanolamine, and triethanolamine; 2- [4- (2-hydroxyethyl-1-piperazinyl) ethanesulfonic acid), also referred to as HEPES, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid, also referred to as TES, 3-morpholinopropane, also referred to as MOPS (1) to (11), which is a diluent containing a buffer selected from the group consisting of sulfonic acid and BES (N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid)
  • a buffer selected from the group consisting of sulfonic acid and BES (N, N-bis (2-hydroxyethyl) -2-aminoethan
  • the method according to any one of (1) to (12), further including a step of verifying an analysis of the concentration of the target component based on a dilution factor obtained using a standard value of a standard component different from the standard component The blood analysis method according to 1.
  • (14) For use in the blood analysis method according to any one of (1) to (13), including a diluent for diluting a blood sample and a container for containing the diluted blood sample Blood test kit.
  • the blood analysis method and the blood test kit of the present invention it is possible to perform analysis with high reproducibility of measured values even if the amount of blood sample is small.
  • FIG. 1 shows an example of the configuration of a container for accommodating a diluted blood sample.
  • Patent Document 2 a blood volume of about 100 ⁇ L is collected.
  • the wound on the skin such as a fingertip needs to be enlarged, and the patient is painful, Some people feel it as a strong pain. There is also a concern that hemostasis will be delayed because the wound is deep.
  • dilution is performed using magnesium ion having a central homeostasis value in blood of 0.9 mmol / L, 4.65 mmol / L calcium ion, and 7.5 g / 100 mL total protein. The magnification is measured.
  • the concentration of the homeostatic component in the diluent decreases, resulting in a measurement error that cannot be ignored when measuring the homeostatic component.
  • the measurement value of the dilution factor also includes an error, and the measurement reliability is impaired.
  • Patent Document 4 discloses a technique for improving the measurement accuracy by correcting the influence of milk using two wavelengths of light, but it is a measurement with a dilution factor of about 10 times.
  • the method of Patent Document 4 is effective for analysis with a diluted blood volume of 100 ⁇ L or less, but there are few components to be analyzed, information on many various components to be analyzed is obtained, and the state of organs and lifestyle habits are obtained. It cannot handle inspections for the purpose of prediction. In this case, when the dilution rate is further increased to sufficiently secure the diluted blood volume, the same problem as in Patent Document 1 is encountered.
  • the present invention has been studied in consideration of the above-mentioned problems.
  • the dilution rate is increased.
  • a sufficient amount of the diluent to be analyzed is secured, a dilution ratio with good reproducibility can be realized, and the target component can be analyzed with high accuracy.
  • the amount of blood to be collected is very small and the dilution factor is high, use a diluent that does not contain standard components that are permanently present in the blood as the diluent, and use the standard values for the standard components to determine the dilution factor. Being able to perform analysis with high reproducibility of measured values by determining is a completely unexpected effect that cannot be predicted from the prior art documents.
  • the blood analysis method of the present invention comprises: Diluting the collected blood sample with a diluent, Determining the dilution factor using standard values of standard components that are permanently present in the blood; Analyzing the concentration of the target component in the blood sample; A blood analysis method comprising: A method in which the volume of the blood sample is 50 ⁇ L or less, the dilution factor of the plasma component in the blood sample is 14 times or more, and the diluent is a diluent that does not contain the above-mentioned standard component that is constantly present in the blood. is there.
  • a blood sample is collected and the target component in the blood sample is analyzed.
  • the blood analysis method of the present invention may be performed by self-collection in which the subject himself collects blood, or may be performed in general blood collection in which a qualified person such as a doctor collects blood using a syringe. .
  • the patient himself / herself collects the blood that has come out of the skin by damaging a fingertip or the like using an instrument with a knife such as a lancet.
  • the volume of the blood sample used in the blood analysis method of the present invention (that is, the amount of blood collected) is 50 ⁇ L or less, preferably 40 ⁇ L or less, more preferably 30 ⁇ L or less, further preferably 20 ⁇ L or less, and the lower limit is not particularly limited.
  • the necessary blood volume for performing blood analysis is preferably 5 ⁇ L or more.
  • the target component can be analyzed with high measurement accuracy even when the amount of blood sample is very small.
  • the collected blood sample is diluted with a diluent.
  • a diluent When testing specific organs and specific diseases such as liver function, renal function, and metabolism as a blood test, obtain information on multiple components to be measured that are specific to the organ and the disease to determine the state of the organ, In order to predict habits and the like, generally, a plurality of target components are analyzed simultaneously. For example, in order to examine the state of the liver, generally, ALT (alanine transaminase), AST (aspartate aminotransferase), ⁇ -GTP ( ⁇ glutamyl transpeptidase), ALP (alkaline phosphatase), total bilirubin, The concentration in the blood of several or more components such as total protein and albumin is measured.
  • the amount of the diluent to be used is preferably 250 ⁇ L or more, more preferably 300 ⁇ L or more, even more preferably 350 ⁇ L or more, and 400 ⁇ L or more in order to measure a plurality of target components.
  • the upper limit of the amount of the diluent is not particularly limited, but is generally preferably 1000 ⁇ L or less in order to realize a dilution rate effective for measurement.
  • the blood to be collected contains a plasma component and a blood cell component.
  • a plasma component obtained by removing the blood cell component from the blood and a diluent.
  • the blood cell component may be separated from the blood in advance and then mixed with the diluted solution, or the collected blood may be mixed with the diluted solution, and then the blood cell component may be separated using a separation membrane or the like.
  • dilution can be performed after performing a step of separating blood cells from blood collected by a patient and collecting plasma.
  • a plasma component-containing sample is a sample containing a plasma component and a diluent. In this case, the plasma component-containing sample collected as described above can be transported to, for example, an examination place.
  • the method for separating blood cells from blood and collecting plasma there is no particular limitation on the method for separating blood cells from blood and collecting plasma, and the method for separating blood cells from diluted blood and collecting a plasma component-containing sample.
  • the blood After blood collection with an anticoagulant blood collection tube, the blood may be separated into blood cell components and plasma components by centrifugation, or the blood components are passed through a separation membrane such as a filtration membrane by applying pressure to the blood components. May be captured by a separation membrane to separate blood cell components from blood.
  • an anticoagulant may be used.
  • the step of collecting the plasma component is preferably a step using a separation membrane.
  • a biological sample separation instrument or the like having means can be used.
  • the present invention it is preferable to collect blood with low invasiveness, and it is important to prepare a diluted blood solution from a very small amount of blood sample, and the plasma in the blood sample when the collected blood is diluted with the diluted solution It is required that the dilution ratio of the components is high.
  • the dilution rate here is the dilution rate of the plasma component obtained by removing the blood cell component from the blood component, and the dilution rate in the present invention is 14 times or more, preferably 17 times or more, more preferably 21 times or more, It is more preferably 25 times or more, particularly preferably 30 times or more, most preferably 40 times or more, and the upper limit is not particularly limited, but in order to enable high-accuracy measurement, it is generally preferably 100 times or less.
  • the reproducibility of the dilution factor measurement when diluted with a diluent is good even at a high magnification from 6 times or more, and the patient collects blood.
  • the dilution ratio of blood is preferably 8 times or more, more preferably 10 times or more, still more preferably 13 times or more, most preferably 18 times or more,
  • the upper limit is not particularly limited, but is generally preferably 50 times or less in order to enable highly accurate measurement.
  • the concentration of the plasma component in the blood before dilution is determined in advance.
  • the density change rate is very small, so that the measurement error is large and the reproducibility of the measurement deteriorates. Therefore, in the present invention, in order to increase the measurement accuracy, the dilution rate is determined using the standard values of standard components that are constantly present in blood.
  • the standard component that is constantly present in the blood is also referred to as an external standard substance.
  • Examples of standard components that are constantly present in blood include sodium ions, chloride ions, potassium ions, magnesium ions, calcium ions, total protein, and albumin.
  • the concentration of these standard components contained in the serum and plasma of the blood sample is such that the sodium ion concentration is 134 to 146 mmol / liter (average value: 142 mmol / liter), and the chloride ion concentration is 97 to 107 mmol / liter (average) Value: 102 mmol / liter), potassium ion concentration is 3.2 to 4.8 mmol / liter (average value: 4.0 mmol / liter), and magnesium ion concentration is 0.75 to 1.0 mmol / liter (average value: 0.9 mmol / liter), calcium ion concentration is 4.2 to 5.1 mmol / liter (average value: 4.65 mmol / liter), and total protein concentration is 6.7 to 8.3 g / 100 mL (average value: 7.5 g /
  • the present invention is intended to enable measurement of a target component when the amount of blood collected to relieve pain of a patient is very small.
  • a very small amount of blood is diluted with a diluent, It is necessary to accurately measure the concentration of “a standard component that is continually present in blood”.
  • the concentration of components originally present in the blood decreases in the diluted solution, and depending on the dilution rate, there is a possibility that a measurement error is included in the concentration measurement. Therefore, among these “standard components that are continually present in the blood”, when a very small amount of blood component is diluted at a high dilution ratio, the above-mentioned standard component can be detected with sufficient accuracy. It is preferable to measure standard components present at high concentrations.
  • sodium ions Na +
  • chloride ions Cl ⁇
  • the average value of sodium ions represents a standard value (the median value of the reference range), which is 142 mmol / liter, which accounts for 90% or more of the total cations in plasma.
  • the percentage of the plasma component in the blood of the patient who is a patient is about 55% in volume ratio, but varies depending on the change in the amount of salt intake of the subject, and also varies depending on the subject. . Therefore, in the present invention, the dilution rate is determined using the standard value of the standard component that is constantly present in the blood, and the concentration of the target component in the blood sample is analyzed using the determined dilution rate.
  • concentration X concentration of an external standard substance (for example, sodium ion) in a diluted solution of plasma and the known external standard substance (for example, sodium ion) in plasma are known.
  • the dilution factor can be determined by calculating the dilution factor (Y / X) of the plasma component in the blood sample from the concentration value (concentration Y; 142 mmol / liter in the case of sodium ion). Using this dilution factor, the measurement value (concentration Z) of the target component in the diluted plasma solution is measured, and this measurement value is multiplied by the dilution factor, so that the actual analysis target component contained in the blood sample is obtained. The density [Z ⁇ (Y / X)] can be measured.
  • the concentration analysis of the analysis target component in the blood is performed normally, two or more different components that are constantly present in the blood are used as standard components, and each is independently contained in the blood sample. It is preferable to determine the dilution rate of the plasma component of the blood and confirm that the values match.
  • the coincidence means that in two measured values (a, b), the ratio of their difference to their average value, that is,
  • Chloride ion measurement methods include an electrode method using an ion-selective electrode (Ion Selective Electrode: ISE), an enzyme method using an enzyme such as amylase, a silver nitrate titration method, and the like. A method to be used as appropriate can be selected in accordance with the above.
  • total protein or albumin As an example of standard components that are constantly present in plasma other than sodium ions and chloride ions, it is preferable to select from total protein or albumin, more preferably from total protein, There are known methods such as the Burette method, the ultraviolet absorption method, the Breadford method, the Raleigh method, the bicinchoninic acid (BCA) method, the fluorescence method, and the characteristics and sensitivity of the measurement sample and the sample amount.
  • BCA bicinchoninic acid
  • the sodium ion concentration and chloride ion concentration can be measured by, for example, flame photometry, glass electrode method, titration method, ion selective electrode method, enzyme activity method and the like.
  • to analyze the concentration of a target component in a blood sample is to determine the concentration of the target component (that is, to quantify the target component), or whether the concentration of the target component is equal to or higher than a predetermined reference value.
  • concentration of the target component that is, to quantify the target component
  • concentration of the target component is equal to or higher than a predetermined reference value.
  • the form of analysis is not particularly limited, including determining whether it is below a predetermined reference value, performing qualitative detection that includes a certain level of concentration, and the like.
  • a standard component that is constantly present in blood (hereinafter also referred to as a homeostatic substance) is measured after dilution with a diluent, and the dilution factor is determined as described above to determine the target component in the blood sample.
  • the concentration can be analyzed.
  • a diluent for diluting a blood sample is a diluent that does not contain “a standard component that is constantly present in blood” used to determine the dilution factor.
  • “does not contain” means “does not contain substantially”.
  • substantially free means that the homeostatic substance used when determining the dilution factor is not included at all, or even if it is included, the homeostatic substance in the diluted solution after the blood sample is diluted It means the case where it is contained at a very small concentration that does not affect the measurement of the above.
  • a diluent that does not substantially contain sodium ions or chloride ions is used as the diluent.
  • the diluting solution is pH 6.5 to pH 8.0, preferably pH 7.0 to pH 7.5, more preferably pH 7.3 to pH 7.4.
  • a buffer solution having a buffering action is preferable, and the diluent is preferably a buffer solution containing a buffer component that suppresses fluctuations in pH.
  • the types of buffer include acetate buffer (Na), phosphate buffer (Na), citrate buffer (Na), borate buffer (Na), tartrate buffer (Na), Tris (Tris (hydroxy Methyl) aminoethane) buffer (Cl), HEPES ([2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid]) buffer, phosphate buffered saline (Na), etc. are known ing.
  • phosphate buffers, Tris buffers, and HEPES buffers are representative examples of buffers around pH 7.0 to pH 8.0.
  • the phosphate buffer contains a sodium salt of phosphate
  • the Tris buffer has a dissociated pKa (Ka is an acid dissociation constant) of 8.08
  • the pH is around 7.0 to 8.0.
  • the pKa of HEPES sulfonic acid dissociation is 7.55, but in order to adjust the buffer solution with constant ionic strength, water is usually used. Since a mixture of sodium oxide, sodium chloride and HEPES is used, these are useful as a buffer having an action of keeping the pH constant, but sodium ions or chloride ions which are preferably used as an external standard substance Therefore, application to the present invention is not preferable.
  • a buffer solution that does not contain sodium ions or chloride ions.
  • the diluent used in the present invention is preferably selected from the group consisting of 2-amino-2-methyl-1-propanol (AMP), 2-ethylaminoethanol, N-methyl-D-glucamine, diethanolamine, and triethanolamine.
  • AMP 2-amino-2-methyl-1-propanol
  • 2-ethylaminoethanol N-methyl-D-glucamine
  • diethanolamine diethanolamine
  • triethanolamine triethanolamine.
  • 2- [4- (2-hydroxyethyl-1-piperazinyl]] also referred to as HEPES, which is a buffer with a Good's buffer (Good buffer) and a pKa of around 7.4.
  • the concentration ratio of amino alcohol and Good's buffer solution is 1: 2 to 2: 1, preferably 1: 1.5 to 1.5: 1, more preferably 1: 1.
  • the concentration of the buffer is not limited, but the concentration of amino alcohol or Good's buffer is 0.1 to 1000 mmol / L, preferably 1 to 500 mmol / L, more preferably 10 to 100 mmol / L.
  • the buffer solution may contain a chelating agent, a surfactant, an antibacterial agent, a preservative, a coenzyme, a saccharide and the like for the purpose of keeping the analysis target component stable.
  • chelating agents include ethylenediaminetetraacetate (EDTA), citrate, and oxalate.
  • the surfactant include a cationic surfactant, an anionic surfactant, an amphoteric surfactant, and a nonionic surfactant.
  • the preservative include sodium azide and antibiotics.
  • the coenzyme include pyridoxal phosphate, magnesium, zinc and the like.
  • saccharide of the erythrocyte stabilizer examples include mannitol, dextrose, oligosaccharide and the like.
  • by adding antibiotics it is possible to suppress the growth of bacteria partially mixed from the finger surface at the time of hand blood collection, to suppress the degradation of the biological components by bacteria, and to stabilize the biological components.
  • the osmotic pressure of the buffer solution is equivalent to that of blood (285 mOsm / kg (mOsm / kg is 1 kg of water of the solution).
  • the osmotic pressure represents the number of milligrams of ions)) or more to prevent hemolysis of blood cells.
  • the osmotic pressure can be adjusted to be isotonic with salts, sugars, buffers or the like that do not affect the measurement of the target component and the measurement of the standard component that is constantly present in the blood.
  • the component to be analyzed in the present invention is not limited, and any substance contained in blood is targeted.
  • examples include biochemical test items in blood used for clinical diagnosis, markers for various diseases such as tumor markers and hepatitis markers, and include proteins, sugars, lipids, low molecular weight compounds, and the like.
  • the measurement includes not only the substance concentration but also the activity of substances having an activity such as an enzyme.
  • Each target component can be measured by a known method.
  • the blood test kit of the present invention is used in the blood analysis method of the present invention described above, including a diluent for diluting a blood sample and a container for containing the diluted blood sample.
  • a diluent for diluting a blood sample and a container for containing the diluted blood sample.
  • This is a blood test kit.
  • the material of the container is preferably a synthetic resin from the viewpoint of resistance to breakage, hygiene, and price.
  • polyethylene polypropylene, polyvinyl chloride, polyvinylidene chloride, polystyrene, polyvinyl acetate, polyurethane, polyethylene terephthalate, polylactic acid, acrylonitrile butadiene styrene resin (ABS resin), acrylonitrile styrene resin (AS resin), acrylic resin (PMMA) , Polycarbonate, silicone resin, silicone rubber and the like.
  • ABS resin acrylonitrile butadiene styrene resin
  • AS resin acrylonitrile styrene resin
  • PMMA acrylic resin
  • Polycarbonate silicone resin, silicone rubber and the like.
  • a diluting solution for diluting a blood sample a first containing device containing the diluting solution, and a separation for separating and collecting plasma from the blood sample diluted with the diluting solution
  • a device a holding device for holding the separating device, a second containing device for containing the collected plasma, a sealing device for maintaining the contained plasma in the second containing device, and damaging the skin
  • It is equipped with a needle that bleeds blood outside the skin, a lancet, an adhesive bandage or disinfecting member (for example, a non-woven fabric impregnated with isopropanol (70% by mass isopropanol) or ethanol), an instruction manual, etc. be able to.
  • the diluent described above in this specification can be used.
  • the first storage device and the second storage device may be used as a first storage device and a second storage device, or may be provided with separate devices.
  • the first storage device and the second storage device The instrument is preferably made of a transparent material.
  • transparent as used in the present invention is not limited as long as the observer can confirm the amount of liquid inside, and is a concept including translucency.
  • the separation instrument for separating plasma is preferably a separation membrane, and more preferably a filter having pores capable of separating blood cell components. It is preferable that the holding device for holding the separation device is a gasket.
  • the sealing device when the storage device is a tube-shaped device, a cap that can cover the opening, a cover having a spiral groove, or a rubber plug is used. can do.
  • the present invention makes it possible to realize a method capable of analyzing an analysis target component with a high measurement accuracy even with a small amount of blood of 50 ⁇ L or less, and allows a patient to accurately analyze even with a small blood sample of 50 ⁇ L or less.
  • the kit includes an instruction manual in which information indicating that measurement is possible is described.
  • a first storage device containing a diluent containing a blood sample diluted with the diluent
  • a holding device for holding the separation device for holding the separation device
  • a first device for containing the collected plasma are described, for example, in FIGS. 1 to 13 of Japanese Patent No. 3597827. Can be used.
  • FIG. 1 of Japanese Patent No. 3597827 is incorporated as FIG. 1 of the present application.
  • the blood separation instrument 1 includes a blood collection container 2 (first accommodation instrument in which a diluent is accommodated), a cylinder 3 (second accommodation instrument for accommodating the collected plasma) that can be inserted into the blood collection container 2, and
  • the cap piston 4 that can be attached to the cylindrical body 3 and a sealing lid 5 (sealing device) provided at the lower end of the cap piston 4, and before use, as shown in FIG.
  • the upper end opening is sealed with a cap 6 via a packing 7.
  • the container for storing the diluted blood sample in the present invention corresponds to the combination of the blood collection container 2 and the cylinder 3 in the configuration of FIG. That is, the container for storing the diluted blood sample may be one or a combination of two or more.
  • the blood collection container 2 is made of a transparent material and has a cylindrical shape.
  • a screw portion 8 is formed on the outer surface of the blood collection container 2 and an engaging portion 9 is projected on the inner surface.
  • an inverted conical bottom portion 10 is formed at the lower end portion of the blood collection container 2, and a cylindrical leg portion 11 is formed around the bottom portion 10.
  • the legs 11 have the same outer diameter as the sample cup used at the time of blood analysis and testing, and preferably, slit grooves 12 are formed in the vertical direction at positions opposite to the lower ends thereof. Further, as shown in FIG. 1, a required amount, for example, a diluted solution 13 of 500 mm 3 may be placed in the blood collection container 2 in advance.
  • the cylindrical body 3 is made of a transparent material and has a cylindrical shape, and an enlarged diameter portion 14 is formed at an upper end portion thereof.
  • the enlarged diameter portion 14 is connected to the main body portion 16 through a thin portion 15.
  • a reduced diameter portion 18 is formed at the lower end of the cylindrical body 3, and a locking projection 19 is formed on the inner surface of the reduced diameter portion 18.
  • the outer flange portion 20 (holding device) is formed at the lower end portion of the reduced diameter portion 18, the lower end opening portion of the outer flange portion 20 is covered with a filtration membrane 21 (separation device), and the filtration membrane 21 is in the blood. It allows passage of plasma and prevents passage of blood cells.
  • a silicon rubber cover 22 is attached to the outer periphery of the reduced diameter portion 18 (FIG. 1).
  • the cap piston 4 includes a substantially cylindrical knob 26 and a mandrel 27 that is concentric with the knob 26 and extends downward.
  • a cylindrical space 28 into which the enlarged diameter portion 14 of the cylindrical body 3 can be fitted is formed at the inner upper end portion of the knob portion 26, and the lower portion thereof is screwed and can be screwed into the screw.
  • the lower end portion 29 of the mandrel portion 27 is formed in a pin shape, and the sealing lid 5 is detachably provided on the lower end portion 29 (see FIG. 1).
  • the sealing lid 5 is made of silicon rubber.
  • the number of each element included in the blood test kit of the present invention is not particularly limited, and may be one each or two or more.
  • the blood test kit of the present invention comprises a diluent for diluting a blood sample, a container for containing the diluted blood sample, and optional elements described above in a storage container for storing them. It can be provided as a stored form.
  • composition of Diluent-1 Diluent-1 was prepared with the following composition.
  • osmotic pressure a value measured using OSMOATAT OM-6040 (manufactured by ARKRAY, Inc.) was displayed.
  • the unit of osmotic pressure is the osmotic pressure of 1 kg of solution water, and represents the number of millimoles of ions.
  • HEPES 50mmol / L 2-Amino-2-methyl-1-propanol (AMP) 50mmol / L D-mannitol 284 mmol / L Lithium chloride 1mmol / L EDTA-2K 0.8mmol / L PALP (pyridoxal phosphate) 0.05mmol / L Thiabendazole 0.0001 mass% Amikacin sulfate 0.0003 mass% Kanamycin sulfate 0.0005% by mass Meropenem trihydrate 0.0005% by mass Osmotic pressure 355mOsm / kg pH 7.4
  • the sodium ion concentration was measured for each diluted solution prepared in (1).
  • ⁇ -galactosidase was activated by sodium, and the measurement was performed by an enzyme activity method utilizing the proportional relationship between the sodium ion concentration in each diluted solution and ⁇ -galactosidase activity. Specifically, after diluting the diluted blood solution 5 times with purified water not containing sodium ions, 3 ⁇ L was weighed, 52 ⁇ L of the first reagent prepared as follows was added, and the mixture was heated at 37 ° C. for 5 minutes. did.
  • the lithium ion added to the diluted solution was measured by a chelate colorimetric method (halogenated polyphyrin chelate method: perfluoro-5,10,15,20-tetraphenyl-21H, 23H, -porphyrin). Specifically, after diluting the diluted blood solution 4.5 times with purified water not containing lithium ions, 5 ⁇ L was weighed and 55 ⁇ L of the third reagent prepared as follows was added. Warmed for minutes.
  • the change in absorbance per minute was determined by measuring the absorbance at a main wavelength of 545 nm and a sub wavelength of 596 nm using a JCA-BM6050 type biochemical automatic analyzer (manufactured by JEOL Ltd.). .
  • the concentration of lithium ions was measured from a calibration curve prepared in advance.
  • a measurement reagent for lithium ions having the following composition was prepared.
  • Example 1 Measurement of ALT (Alanine Transaminase) and AST (Aspartate Aminotransferase) Immediately after blood was collected from a vein using a syringe in Reference Example 1, blood was squeezed from the fingertip of the same patient who collected the blood outside the skin of the fingertip.
  • ALT Alanine Transaminase
  • AST Aspartate Aminotransferase
  • the patient sucks blood using a sponge capable of absorbing about 20 ⁇ L to 40 ⁇ L of liquid, and soaks the sponge having absorbed blood in 360 ⁇ L of a diluent having the same composition as the diluent used in Reference Example 1,
  • the blood was sufficiently extracted from the sponge into the diluted solution and filtered through a filter to separate blood cell components, thereby obtaining a diluted solution of plasma components of the blood sample. Seal the diluted solution and transport it to another facility where inspection is possible. Then, remove the diluted solution and dilute in the same manner as the method for measuring the dilution factor using sodium ions in blood in Reference Example 1. When the magnification was measured, it was 22.3 times.
  • ALT and AST concentration of ALT and AST in the diluted sample was measured using a commercially available measurement kit (Transaminase CII-Test Wako: Wako Pure Chemical Industries, Ltd.). The results were almost the same as the measured values of ALT and AST, which were analyzed based on the dilution rate measured using sodium ion concentration.
  • Example 2 In Example 1, the concentration of chloride ions was measured by the method described below using a diluted solution obtained by measuring the dilution rate of plasma components in a blood sample using sodium ions.
  • Chloride ions were measured using an ion selective electrode (ISE). A biological sample was passed between an ion selective electrode that selectively responds to chloride ions and a reference electrode, and the chloride ion concentration was calculated from the electromotive voltage generated between the electrodes.
  • ISE ion selective electrode

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Abstract

L'invention a pour objet de fournit un procédé d'analyse du sang qui présente une reproductibilité répétée élevée d'une valeur de mesure pour un échantillon de sang de 50μＬ ou moins, et un kit d'examen du sang destiné à être mis en œuvre dans ledit procédé d'analyse du sang. Le procédé d'analyse du sang de l'invention inclut : une étape au cours de laquelle l'échantillon de sang prélevé est dilué à l'aide d'une solution de dilution; une étape au cours de laquelle le degré de dilution est déterminé à l'aide d'une valeur de référence d'un composant de référence présent de manière constante dans le sang; et une étape au cours de laquelle la concentration d'un composant objet de l'analyse dans l'échantillon de sang, est analysée. La quantité d'échantillon de sang est de 50μＬ ou moins, le degré de dilution d'un composant plasma dans l'échantillon de sang, est de 14 fois ou plus, et la solution de dilution ne comprend pas le composant de référence présent de manière constante dans le sang.
PCT/JP2016/070010 2015-07-06 2016-07-06 Procédé d'analyse du sang, et kit d'examen du sang WO2017006965A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP16821434.4A EP3321679B1 (fr) 2015-07-06 2016-07-06 Procédé d'analyse du sang
KR1020207005609A KR102111903B1 (ko) 2015-07-06 2016-07-06 혈액 분석 방법 및 혈액 검사 키트
CN201680039333.2A CN107949789B (zh) 2015-07-06 2016-07-06 血液分析方法及血液检查试剂盒
KR1020187000464A KR102084410B1 (ko) 2015-07-06 2016-07-06 혈액 분석 방법 및 혈액 검사 키트
US15/861,209 US10634661B2 (en) 2015-07-06 2018-01-03 Blood analysis method and blood test kit

Applications Claiming Priority (4)

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JP2015-135064 2015-07-06
JP2015135064 2015-07-06
JP2016133960A JP6417367B2 (ja) 2015-07-06 2016-07-06 血液分析方法及び血液検査キット
JP2016-133960 2016-07-06

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
JP2020512107A (ja) * 2017-04-04 2020-04-23 ジェネウェル シーオー．，エルティーディー． 外科手術後の切開部位の痛み軽減又は治療のためのキット

Citations (2)

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JP2001330603A (ja) * 2000-05-18 2001-11-30 Arkray Inc 定量分析法
JP2011112451A (ja) * 2009-11-25 2011-06-09 Physical Screening Inc 生体試料成分の分析方法

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JP2001330603A (ja) * 2000-05-18 2001-11-30 Arkray Inc 定量分析法
JP2011112451A (ja) * 2009-11-25 2011-06-09 Physical Screening Inc 生体試料成分の分析方法

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SUSUMU OSAWA ET AL.: "Revolution of medical services at home using a small amount of blood collected from the fingertip", CLINICAL TESTING, vol. 59, no. 5, 15 May 2015 (2015-05-15), pages 397 - 404, XP009503398 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020512107A (ja) * 2017-04-04 2020-04-23 ジェネウェル シーオー．，エルティーディー． 外科手術後の切開部位の痛み軽減又は治療のためのキット

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