WO2017006963A1 - Blood test kit and analysis method using same - Google Patents
Blood test kit and analysis method using same Download PDFInfo
- Publication number
- WO2017006963A1 WO2017006963A1 PCT/JP2016/070008 JP2016070008W WO2017006963A1 WO 2017006963 A1 WO2017006963 A1 WO 2017006963A1 JP 2016070008 W JP2016070008 W JP 2016070008W WO 2017006963 A1 WO2017006963 A1 WO 2017006963A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- blood
- test kit
- capillary
- sample
- blood sample
- Prior art date
Links
Images
Classifications
-
- 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
- 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
- G01N1/12—Dippers; Dredgers
-
- 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
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
-
- 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 test kit for analyzing a target component in a blood sample and a blood analysis method using the same.
- 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 5 discloses a hemolytic measurement method characterized by the above.
- 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-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.
- 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.
- Patent Documents 1 to 4 are not sufficient for performing analysis when a very small amount of blood sample is used with high accuracy.
- blood collection is an invasive action that damages the skin, and it may be uncomfortable to stare at the red color of the blood. It is common to want to stop the blood. For these reasons, the amount of blood collected is not always constant and often varies. If the variation in the amount of blood collected is large, the accuracy of the dilution rate will be reduced, so visualization and stabilization of the amount of blood collected is desired.
- the problem to be solved by the present invention is to provide a blood test kit and a blood analysis method capable of performing a blood test with a small amount of blood with high accuracy by visualizing and stabilizing the blood collection volume.
- the present inventors accommodate a blood collection device for collecting a blood sample, a diluent for diluting the collected blood sample, and a diluted blood sample.
- a blood collection device for collecting a blood sample; A diluent for diluting the collected blood sample; A storage device for storing a dilution of a blood sample; A blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is constantly present in blood, wherein the blood collection device is a capillary .
- a blood collection device for collecting a blood sample; A diluent for diluting the collected blood sample; A storage device for storing a dilution of a blood sample; A blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is constantly present in blood, The diluent contains standard components that are not present in the blood, A blood test kit, wherein the blood collection device is a capillary.
- a blood collection device for collecting a blood sample; A diluent for diluting a collected blood sample, which contains a standard component not present in the blood; A storage device for storing a dilution of a blood sample; A blood test kit comprising: A blood test kit, wherein the blood collection device is a capillary.
- the blood test kit includes a separation instrument for separating and collecting plasma from a diluted blood sample.
- the scale is attached to a position indicating the lower limit of the volume range of a blood sample to be collected, and the capillary has a stopper, 7.
- the blood test kit according to 10 wherein the inner wall of the capillary is subjected to hydrophilic treatment.
- the blood test kit according to 10 or 11 wherein an end of the capillary on the side for sucking a blood sample has a taper.
- At least one position indicating the volume range of the blood sample to be collected is marked on the capillary, and the inner diameter of the capillary is increased from the portion beyond the position of the scale.
- a scale is attached to at least one position of the capillary indicating the volume range of the blood sample to be collected, and an inner diameter of a portion including the position where the scale is attached of the capillary is smaller than other portions.
- the blood test kit according to any one of 10 to 12.
- the blood test kit according to 1 or 2 wherein the standard component that is constantly present in blood is sodium ion or chloride ion.
- the blood test kit according to 1 or 2 wherein the standard component that is constantly present in the blood is a standard component that is constantly present in sodium and chloride ions and still another blood.
- the blood test kit according to 17, wherein the another standard component is total protein or albumin.
- the blood test kit according to 2 wherein the standard component not present in the blood is lithium ion or glycerol triphosphate.
- the blood test kit according to 3 wherein the standard component not present in the blood is lithium ion or glycerol triphosphate.
- a method for analyzing the concentration of a target component in a blood sample excluding medical practice), characterized by using the blood test kit according to any one of 1 to 21.
- a blood test with a very small amount of blood can be performed with high accuracy by visualizing and stabilizing the amount of blood collected.
- FIG. 1 shows an example of the configuration of a storage device for storing a diluted blood sample.
- FIG. 2 shows a cross-sectional view of an example of a capillary.
- FIG. 3 shows an example of the shape of the capillary and the position of the scale.
- FIG. 4 shows an example of the shape of the capillary and the position of the scale.
- the range indicated by X to Y includes the upper limit X and the lower limit Y.
- a standard component that is constantly present in blood may be referred to as an external standard substance or an external standard.
- a standard component that does not exist in blood may be referred to as an internal standard substance or an internal standard.
- Patent Document 2 discloses a method using a porous material having high blood retention instead of filter paper.
- blood components absorbed in the material are extracted and measured with a buffer solution or the like, so that sodium ions, chloride ions, calcium ions, proteins, which are external standard substances that are constantly present in blood, are measured.
- sodium ions, chloride ions, calcium ions, proteins, which are external standard substances that are constantly present in blood are measured.
- the amount of blood collected varies, and when the dilution rate of the collected blood increases, the subsequent analysis accuracy decreases and the results vary, and the blood is agglomerated and solidified. As a result, the guarantee of the stability of the components may be insufficient.
- a buffer solution for extracting a biological component from a dried sample it is necessary to use a buffer solution to which NaOH, NaCl, or HCl is added for pH adjustment or biological component stabilization. For this reason, the concentration of other diluted biological components is corrected using the concentration of sodium ions and chloride ions, which are present at a relatively high concentration of sample components, have homeostasis, and have small differences between individuals as an external standard. There was a problem that it could not be used to do.
- Patent Document 1 a method described in Patent Document 1 is disclosed as a method for diluting a collected trace blood with a buffer solution containing an internal standard and quantifying the amount of a component having an unknown amount in diluted plasma from the dilution factor of the internal standard substance.
- N- (2-Hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline sodium salt (HSDA) or Acid Blue 9 (Brilliant Blue FCF) is used as an internal standard substance to stabilize blood. Buffering agents and preservatives are used for holding.
- Such a prescription achieved the stability of its components by not coagulating the blood, but when the blood collection volume is also variable and the collection volume is small, the dilution rate of the internal standard substance after dilution This is a problem that the reliability of the test accuracy is lowered due to the decrease in the amount of blood and the amount of the blood component itself.
- the method of diluting with a buffer solution is a biological component stored in a buffer solution with physiological conditions of pH 7.4 and is excellent in stability during transportation. There was a problem that measurement errors were likely to occur when the dilution rate was small and a small amount of sample was added.
- phosphate buffered saline is used as the buffer solution to be extracted because it is excellent in the stable retention of biological components.
- phosphate buffered saline contains sodium ions and chloride. Contain ions.
- sodium ions and chloride ions cannot be used as external standards, and calcium ions, proteins, and the like are used. Therefore, in order to perform a blood test with a small amount of blood with high accuracy, the use of an external standard substance that corrects the dilution rate as in the prior art or the use of a buffer solution containing a conventionally proposed internal standard substance Insufficient inspection accuracy.
- Patent Document 1 has a description regarding an internal standard, but there is no description regarding the combined use with an external standard. Therefore, there is no description regarding the contamination of the external standard, and no specific means for preventing the contamination has been proposed.
- Patent Document 5 collects blood with a capillary, but is diluted with a buffer containing NaCl, and there is no description about using an external standard.
- the present invention relates to a blood test kit and a kit for analyzing the concentration of a target component by more accurately determining the dilution factor in a method for analyzing the concentration of the target component by diluting a trace amount of blood with a buffer solution. It was made for the purpose of providing a blood analysis method. As a means for solving this problem, it is adopted that blood is collected using a capillary so that the blood collection amount is a constant value (at least equal to or greater than the allowable minimum blood collection amount). In a preferred embodiment, not only an external standard is used but also an internal standard is used.
- the blood sample in the method of analyzing a target component in a blood sample, even if the patient collects blood himself, the blood sample can be easily set to a constant value, and at least the minimum volume range of the desired blood sample It can be more than the amount.
- the influence of the external standard substance eluted in the buffer solution from the members constituting the kit on the calculation of the dilution ratio can be made constant, and the component to be analyzed can be accurately quantified.
- a graduated capillary is used as an embodiment, but since the blood sampler can grasp the volume of the collected blood, it becomes possible to predict the dilution rate relatively accurately, and the dilution rate based on the measurement result It is possible to accurately estimate whether or not the calculated value is valid. For blood collectors, it is possible to visually confirm that the volume of the collected blood sample is within the allowable range and that at least the minimum amount is satisfied, so that the blood sample can be safely transported to the laboratory. There are also advantages. Further, by using a graduated capillary, the volume of the collected blood sample can be grasped, and information on the grasped volume can be provided to the testing institution together with the blood sample. The provided information can be used for checking that the calculated dilution factor is appropriate in the inspection organization.
- the blood test kit of the present invention includes a blood collection device for collecting a blood sample, a diluent for diluting the collected blood sample, and a housing for housing a diluted blood sample.
- Analyzing the concentration of the target component in the 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. Including determination of whether or not, the form of analysis is not particularly limited.
- the blood test kit of the present invention is for collecting a blood sample for analysis of a target component in the blood sample. Collection of blood using the blood test kit of the present invention may be performed by the subject himself or by a qualified person such as a doctor.
- the patient himself / herself collects 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.
- one of the preferred embodiments of the present invention is a blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is constantly present in blood.
- using means determining the dilution factor for analyzing the concentration of the target component based on the standard value (constant value) of the standard component. Therefore, analyzing the concentration of a target component in a blood sample using a standard component that is constantly present in blood is based on the constant value (standard value) of the standard component that is constantly present in blood. Is to analyze the concentration of the target component.
- 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 / L (average value: 142 mmol / L), and the chloride ion concentration is 97 to 107 mmol / L (average) Value: 102 mmol / L), potassium ion concentration is 3.2 to 4.8 mmol / L (average value: 4.0 mmol / L), and magnesium ion concentration is 0.75 to 1.0 mmol / L (average value: 0.9 mmol / L), calcium ion concentration is 4.2 to 5.1 mmol / L (average value: 4.65 mmol / L), and total protein concentration is 6.7 to 8.3 g / 100 ml (average value: 7.5 g / 100 mL), and the albumin concentration
- 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, in order to detect the above-mentioned standard component with sufficient accuracy when a very small amount of blood component is diluted at a high dilution rate, it is preferable to measure a standard component present at a high concentration in a very small amount of blood.
- sodium ions Na +
- chloride ions Cl ⁇
- sodium ion having the highest amount in the blood among the standard components that are constantly present in the blood.
- the average value of sodium ion represents a standard value (median value of the reference range), and the value is 142 mmol / L, which accounts for 90 mol% or more of the total cations in plasma.
- One preferred embodiment of the present invention is a blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is not present in blood.
- a test kit may be used for using a standard component that does not exist in blood together with a standard component that exists constantly in blood, and does not use a standard component that exists constantly in blood.
- a standard component that does not exist in blood may be used alone.
- standard components that are not present in the blood can be used by adding them to a diluting solution described later so as to have a predetermined concentration.
- a substance that is not contained at all in the blood sample or is contained in a trace amount even if it is contained can be used.
- Standard components that are not present in blood include substances that do not interfere with the measurement of target components in blood samples, substances that do not degrade due to the action of biological enzymes in blood samples, substances that are stable in dilution, and blood cell membranes. It is preferable to use a substance that does not permeate and is not contained in blood cells, a substance that does not adsorb to a buffer storage container, and a substance that can use a detection system that can measure with high accuracy.
- a substance that is stable even if stored for a long time in a state of being added to a diluent is preferable.
- standard components not present in blood include glycerol triphosphate, alkali metals Li, Rb, Cs, or Fr, and alkaline earth metals Sr, Ba, or Ra, and Li and glycerol tris. Phosphoric acid is preferred.
- These standard components that are not present in blood can be colored by adding a second reagent during concentration measurement after blood dilution, and the concentration in the diluted blood can be determined from the color density.
- concentration in the diluted blood can be determined from the color density.
- the measurement of lithium ions added to the diluting solution is performed by biochemistry using a chelate colorimetric method (halogenated porphyrin chelate method: perfluoro-5,10,15,20-tetraphenyl-21H, 23H-porphyrin).
- a chelate colorimetric method halogenated porphyrin chelate method: perfluoro-5,10,15,20-tetraphenyl-21H, 23H-porphyrin.
- the blood test kit of the present invention includes a diluent for diluting the collected blood sample.
- Diluent does not contain standard components that are permanently present in the blood if the kit is used to analyze the concentration of the component of interest in the blood sample using standard components that are permanently present in the blood .
- “Not contained” means “not substantially contained”.
- substantially does not contain means that it does not contain a substance having homeostasis used at the time of determining the dilution factor, or even if it is contained, the homeostasis of the diluted solution after diluting the blood sample It means a case where it is contained at a very small concentration that does not affect the measurement of a toxic substance.
- sodium ions or chloride ions are used as a standard component that is constantly present in blood, a diluent that does not substantially contain sodium ions or chloride ions is used as the diluent.
- the diluted solution is pH 6.5 to pH 8.0 in order to prevent decomposition and denaturation of the target component.
- the buffer solution has a buffering action in the pH range of pH 7.0 to pH 7.5, more preferably pH 7.3 to pH 7.4, and the diluent contains a buffer component that suppresses fluctuations in pH. It is preferable to use a buffer solution.
- buffer types include acetate buffer (Na), phosphate buffer (Na), citrate buffer (Na), borate buffer (Na), tartrate buffer (Na), Tris (Tris).
- (Hydroxymethyl) aminoethane) buffer (Cl) Hepes ([2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid]) buffer, phosphate buffered saline (Na), etc.
- 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 of 8.08, so that it has a buffer capacity around pH 7.0 to pH 8.0.
- the buffer solution used does not contain sodium ions or chloride ions (included)
- a buffer 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] ethane also referred to as HEPES, which is a Good's buffer (Good buffer) and has a pKa of around 7.4.
- BES N, N-bis (2-hydroxyethyl)
- a diluent containing a buffer selected from the group consisting of -2-aminoethanesulfonic acid (pKa 7.15), among which 2-amino-2-methyl-1-propanol (AMP) and HEPES, A combination with TES, MOPS or BES is preferable, and a combination of 2-amino-2-methyl-1-propanol (AMP) and HEPES is most preferable, where pKa represents an acid dissociation constant.
- 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.
- a chelating agent include ethylenediaminetetraacetic acid (EDTA) salt, 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 an antimicrobial substance.
- 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 buffer also contains a standard component that is not present in blood in a kit for analyzing a target component using a standard component that is not present in blood. It is also important not to include an internal standard substance, which will be described later, and not to interfere with the blood analysis measurement system.
- the osmotic pressure of the buffer solution is equivalent to that of blood (285 mOsm / kg (mOsm / kg is the osmotic pressure of 1 kg of water in the solution and represents the number of millimolar ions)) or more By doing so, hemolysis of blood cells can be prevented.
- 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 osmotic pressure of the buffer solution can be measured with an osmometer.
- ALT alanine transaminase
- AST anaspartate aminotransferase
- ⁇ -GTP ⁇ glutamyl transpeptidase
- ALP alkaline phosphatase
- total bilirubin The concentration in the blood of several or more substances such as total protein and albumin is measured.
- a certain amount of diluted blood is required in consideration of the possibility of remeasurement. Therefore, it is important to secure a certain amount of the diluent for diluting the collected blood. Considering that blood is collected with low invasiveness as much as possible, it is desirable to avoid a blood collection volume of 100 ⁇ L or more, and therefore the dilution factor is about 7 times or more.
- the kit of the present invention employs a capillary as a blood collection device.
- a capillary as a blood collection device.
- a sufficient amount of blood sample specifically, If about 100 ⁇ L, the dilution factor can be calculated with high accuracy.
- the dilution ratio when calculating the dilution ratio using an external standard substance, if the blood volume is small, the dilution ratio will be high, and it will be easily affected by contamination of the external standard substance that is eluted from the kit components during dilution. The measurement accuracy is reduced.
- the present inventors have analyzed a concentration of a component by diluting a trace amount of blood sample with a diluent, and, for example, an external standard substance that has a blood collection amount not exceeding 100 ⁇ L and elutes from a member into the diluent.
- a diluent for example, an external standard substance that has a blood collection amount not exceeding 100 ⁇ L and elutes from a member into the diluent.
- FIG. 2 is a cross-sectional view of an embodiment of a capillary according to the present invention.
- the capillary is usually a tube composed of a thin cylindrical body 201 having a substantially constant inner diameter.
- a capillary phenomenon a phenomenon in which liquid permeates into the capillary against gravity.
- the other end of the capillary blood sampling device is sealed with a finger or the like, and suction is stopped.
- the capillary holding the blood sample with the end held with a finger is carried to a storage device that stores a diluent for diluting the blood sample. Then, after holding one end of the capillary in the storage device, the finger holding the end is released, and the blood sample is stored in the storage device.
- the capillaries may be sealed using a sealer such as a cap, a silicon putty or a paraffin resin, clay, or the like, instead of being pressed with a finger and sealed.
- the inner diameter of the capillary is preferably 0.5 to 2.0 mm in consideration of the size of blood cells and the occurrence of capillary action.
- the length of the capillary is preferably 5 to 15 cm in consideration of ease of handling and the like, and is preferably 5 to 10 cm because it is desirable to be compact in consideration of constituting a blood test kit. More preferred.
- the volume of blood that can be collected with a capillary for example, when the inner diameter is 1.1 mm to 1.2 mm and the length is 7.5 cm, 70 ⁇ L of blood can be collected.
- FIG. 3 shows an example of a capillary with a scale for confirming the volume of the collected blood sample.
- the maximum volume of a blood sample that can be collected by a capillary is theoretically the amount that fills the inside of the capillary, but the capillary indicates the position and upper limit that indicate the lower limit of the volume range of the collected blood sample.
- the position can be provided with a lower limit scale 204 and an upper limit scale 205, whereby it can be determined whether or not the volume of the collected blood sample is appropriate. For example, when it is preferable to collect a blood sample at about 65 ⁇ L, it is preferable to calibrate 55 ⁇ L and 75 ⁇ L, which are ⁇ 10 ⁇ L.
- the volume range of the blood sample to be collected is the volume range of the blood sample that is allowed to accurately analyze the target component contained in the blood sample. In the above example, the volume range is 55 to 75 ⁇ L.
- the inner diameter of the portion including the position where the capillaries are marked may be smaller than other portions. This is because the volume can be collected more accurately if there is a scale in a portion having a small inner diameter, that is, a portion where the tube is thin. Moreover, since the rising speed of the blood in the capillary is increased by reducing the inner diameter, it is easy to take the sealing timing.
- the scale 4 attached to the position indicating the lower limit affects the inspection accuracy if it is less than this, it is preferable that the scale line is thick and easy to see.
- the blood sampler can also reduce the risk that the blood contact end 212 for contacting the blood in the capillary will be mistaken for the sealing end 213 which is the other end.
- blood collection exceeding the upper limit tends to suppress the influence of contamination of the external standard substance from the member, but the variation tends to increase as compared with the case where the blood collection amount is close to the lower limit.
- a stopper with a hole is provided inside the position indicating the upper limit of the capillary (for example, the upper limit scale 205 in FIG. 3), and blood reaches this stopper.
- the suction may be stopped by sealing the upper end of the capillary with a finger or the like.
- the inner diameter of the end 212 on the side of the capillary that comes into contact with blood may be made smaller than that of other portions. As a result, capillary action can easily occur.
- the end portion 207 on the side of the capillary that comes into contact with blood may be a tapered type whose inner diameter becomes smaller toward the end.
- the capillary can be shaped to collect a blood sample of a defined volume.
- An example of such a capillary is designed to have a defined capacity when the interior is filled with a blood sample.
- the shape cannot be collected any more.
- the inner diameter is changed from a certain position of the capillary. The size is rapidly increased (for example, the inner diameter exceeds 2 mm), and the capillary phenomenon hardly occurs when the position is exceeded.
- the change in capacity with respect to the scale deviation is reduced, and the measurement accuracy can be increased.
- the capillary having such a shape may be graduated at a position indicating the upper limit or the lower limit of the blood volume to be collected.
- the position for changing the inner diameter can be a position with a scale or a position beyond the scale, but the latter is preferable when the inner diameter is increased. This is because if there is a scale in the narrow part of the tube before the inner diameter increases, the capacity can be judged accurately.
- the position beyond the scale means a position showing a volume increased by 1 ⁇ L to 7 ⁇ L, preferably 3 ⁇ L to 5 ⁇ L from the volume when the scale is collected.
- the capillary used in the present invention preferably has an anticoagulant inside when blood obtained by puncturing with a lancet or the like is directly collected by the capillary.
- An example of such a capillary is one in which an anticoagulant such as heparin or EDTA salt is applied to the inner surface of the capillary.
- Various anticoagulants can be used, but when an external standard is used in the analysis, one that does not substantially contain the external standard is selected.
- the capillary used in the present invention may be made of glass or synthetic resin. In addition, other materials may be coated with a synthetic resin. In this specification, plastic is used synonymously with synthetic resin.
- the capillaries are preferably made of plastic from the viewpoint of safety that damage is less likely to occur than glass. For example, polyethylene, polypropylene, polyvinyl chloride, polyvinylidene chloride, polystyrene, polyvinyl acetate, polyurethane, acrylonitrile butadiene styrene resin (ABS resin), acrylonitrile styrene resin (AS resin), acrylic resin (PMMA), polycarbonate, silicone resin, etc. Is mentioned.
- the water contact angle of the glass is 10 degrees or less, so that the inner wall of the capillary is sufficiently hydrophilic even if it is intact, and the capillary action of the capillary is sufficiently exhibited and blood can be collected quickly.
- the capillary is made of plastic, for example, the water contact angle of polycarbonate is 85 degrees, and the water contact angle of acrylic is 70 degrees.
- the inner wall of the capillary is preferably treated to be hydrophilic.
- hydrophilic treatment for example, hydrophilic treatment by plasma treatment is possible.
- the capillary for collecting blood used by medical professionals and researchers is transparent, but one of the preferred embodiments of the capillary used in the present invention is colored so that the red color of blood is not noticeable. It is a thing. If the capillary is made of plastic, coloring is easy. Alternatively, although the capillary is transparent, glasses or the like using a transmission medium capable of shielding the absorption wavelength region of hemoglobin in the blood so that the collected blood looks a color other than red is one of the components of the blood test kit. It's okay.
- hemoglobin combined with oxygen looks red, but it is difficult to absorb sharply from the wavelength 600 nm toward the long wavelength side. Therefore, for example, by using a filter that absorbs wavelengths longer than 680 nm, the red color of blood can be made inconspicuous (looks like an orange color).
- kits for separating and collecting plasma from diluted blood samples A blood sample collected by the kit of the present invention may elapse for a long time in a diluted state until analysis is performed. In the meantime, for example, when hemolysis of erythrocytes occurs, substances or enzymes present in the blood cells elute into the plasma or serum and affect the test results, or the absorption of the eluted hemoglobin causes the optical properties of the analyte to be analyzed. This may have an impact on the measurement of the amount of analysis target components using optical information such as typical absorption. Therefore, it is preferable to prevent hemolysis. Therefore, it is preferable that the kit includes a separation device for separating and collecting plasma from a diluted blood sample.
- a preferred example of the separation device is a separation membrane.
- the separation membrane can be used, for example, by applying pressure to a diluted blood sample to capture blood cell components with the separation membrane, allowing plasma components to pass through, separating blood cells, and collecting plasma components.
- an anticoagulant it is preferable to use an anticoagulant.
- the plasma that has passed through the separation membrane does not flow back to the blood cell side.
- a backflow prevention means described in JP-A-2003-270239 is used. It can be a component of a kit.
- the kit of the present invention makes it possible to realize a method capable of analyzing a component to be analyzed with high measurement accuracy even with a blood collection volume of 100 ⁇ L or less, and to accurately measure a patient with a small blood collection volume of 100 ⁇ L or less. It is preferable that the kit includes an instruction manual in which information such as the possibility of blood collection and to which position of the capillary the blood should be collected is described.
- the kit is not only a capillary but also a diluent, a first storage device containing the diluent (also a storage device for storing a diluted blood sample), and diluted with the diluent.
- Separation device for separating and recovering plasma from the collected blood sample holding device for holding the separation device, second storage device for storing the collected plasma, and the stored plasma in the second storage device Including a sealing device for maintaining
- FIG. 1 of Japanese Patent No. 3597827 is incorporated as FIG. 1 of the present application.
- the blood separation device 1 includes a blood collection container 2 (a storage device in which a diluent is stored, or a first storage device. It is also a storage device for storing a diluted blood sample) and a blood collection container 2.
- a cylindrical body 3 that can be inserted (second storage device for storing the collected plasma), a cap piston 4 that can be attached to the cylindrical body 3, and a sealing lid 5 provided at the lower end of the cap piston 4 ( Before use, the upper end opening of the blood collection container 2 is sealed with a cap 6 via a packing 7 before use.
- the storage device 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 number of storage devices 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 operation of separating and collecting plasma from a diluted blood sample is performed as follows. After the blood collected by the capillary is put into the blood collection container 2 containing the dilution liquid, the blood and the dilution liquid are sufficiently shaken and mixed while taking care not to foam by holding the upper part of the blood collection container 2. Next, the cylindrical body 3 holding the filtration membrane 21 (preventing liquid leakage due to sneaking into the side surface of the cylinder when separating blood plasma and blood cells) is inserted into the blood collection container 2 so that the filtration membrane is below, and slowly and constantly The filtration membrane is pushed down to the bottom of the blood collection container 2 at a speed.
- 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 material of the members other than the capillary included in the blood test kit of the present invention is preferably a synthetic resin from the viewpoint of resistance to breakage, hygiene, and cost.
- a synthetic resin for example, 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 and the like.
- the blood test kit of the present invention stores a capillary, a diluent for diluting a blood sample, a storage device for storing the diluted blood sample, and optional elements described above if desired. It can provide as a form stored in a storage container.
- the present invention also provides a blood analysis method using the kit having the configuration described in [1] above.
- the blood analysis method includes a mode that is a medical act on a human (an act performed by a doctor) and a mode that is not a medical act on a human (for example, a mode in which a blood sampler is a patient himself and an analyst is a person other than a doctor, Embodiments for human animals, etc.).
- 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. Good.
- 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 biological sample to be analyzed is blood
- blood is a concept including serum or plasma.
- plasma or serum obtained by collecting a small amount of blood from a subject, diluting with a buffer solution, and then separating blood cells by a filter or centrifugation can be used.
- the component of the blood sample is preferably a plasma component separated from the blood sample by the separation means.
- the origin of the blood sample is not limited to humans, but may be mammals, birds, fishes, etc., which are non-human animals (non-human animals). Examples of animals other than humans include horses, cows, pigs, sheep, goats, dogs, cats, mice, bears, pandas, and the like.
- the source of the biological sample is human.
- the concentration of a target component is analyzed using a standard component that is constantly present in a blood sample.
- a standard component that is constantly present in a blood sample.
- the description in [1] applies here as it is.
- the occupation ratio of plasma components in the blood of the subject is about 55% in terms of volume, but varies depending on changes in the amount of salt intake of the subject. Therefore, in the present invention, the dilution ratio of plasma is calculated using the standard value of the standard component that is constantly present in plasma, and the concentration of the target component in plasma in the blood sample is calculated using the calculated dilution ratio. analyse.
- the dilution factor can be obtained by calculating the dilution factor (Y / X) of the plasma component in the blood sample from the known concentration value (concentration Y; 142 mmol / L in the case of sodium ion) of ions, etc. . Using this dilution factor, the measurement value (concentration Z) of the target component in the plasma dilution is measured, and this measurement value is multiplied by the dilution factor, so that the analyte actually contained in the plasma of the blood sample The component concentration [Z ⁇ (Y / X)] can be measured.
- the concentration of sodium ion or the like can be measured by, for example, flame photometry, glass electrode method, titration method, ion selective electrode method, enzyme activity method and the like.
- the measurement of sodium ion utilizes the fact that ⁇ -galactosidase is activated by sodium ion, and uses the proportional relationship between sodium ion concentration and galactosidase activity in a sample diluted with a buffer solution. Measurement method is adopted.
- a dilution factor independently from the other standard components and confirm that the value matches the dilution factor obtained above.
- the coincidence means that in two measured values (a, b), the ratio of their difference to their average value, that is,
- a standard component that is constantly present in plasma other than sodium ions or chloride ions it is preferably selected from total protein or albumin, and more preferably total protein.
- Methods for measuring total protein include known methods such as the Burette method, the ultraviolet absorption method, the Breadford method, the Raleigh method, the bicinchoninic acid (BCA) method, and the fluorescence method.
- a method to be used as appropriate can be selected according to the amount and the like.
- the concentration of the target component is analyzed using standard components that are not present in the blood.
- a blood test kit containing a diluent containing standard components not present in blood is used.
- the concentration of the target component is analyzed using a standard component that is constantly present in blood and a standard component that is not present in blood.
- the dilution ratio of the blood sample is determined by using sodium ion as a standard component that is constantly present in the blood and lithium ion as a standard component that is not present in the blood.
- the measurement of sodium ion is proportional to ⁇ -galactosidase activity.
- it can be calculated by any one of the following formulas 1 to 4.
- A, B, C, D, B ′ and X are defined as follows.
- the blood sample component is calculated by calculating with Equation 5 using the root-mean-square method, and multiplying the concentration of the analysis target component in the diluted solution by the dilution rate calculated with Equation 5.
- An embodiment in which the concentration of the target component is analyzed is also preferable.
- the concentration of the target component in the blood sample component can be calculated from the concentration of the target component in the diluent based on the dilution factor.
- the analysis target component is not limited, and any substance contained in a biological sample 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. Further, 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.
- Dilution liquid 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 ions, and the enzyme activity method was used that utilized a proportional relationship between the sodium ion concentration in each dilution 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.
- the temperature 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 lithium ion measuring reagent having the following composition was prepared.
- Reference Example 2 80 ⁇ L of blood prepared in Reference Example 1 was weighed with a capillary, mixed with a diluting solution, and a blood plasma component was separated with a filter, and then the total protein concentration was measured by the method described below.
- the average value of the dilution rate determined from the total protein concentration was the same as the average dilution rate determined from the sodium ion concentration prepared in Reference Example 1. Thereby, it turned out that it can verify that the measurement of the dilution rate calculated
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Hydrology & Water Resources (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention addresses the problem of providing a blood test kit, by which blood collection amount is visualized and uniformized so that a blood test can be performed at a high accuracy using a minor amount of blood, and a blood analysis method. Provided is a blood test kit for analyzing the concentration of a target component in a blood specimen using a standard component constantly existing in blood, said blood test kit comprising a blood collecting device for collecting a blood specimen, a diluent for diluting the collected blood specimen, and a housing device for housing the diluted blood specimen, wherein the blood collecting device is a capillary.
Description
本発明は、血液検体中の対象成分を分析するための血液検査キット、及びそれを用いた血液分析方法に関する。
The present invention relates to a blood test kit for analyzing a target component in a blood sample and a blood analysis method using the same.
一般に、採血には、医師等一定の有資格者が注射器を用いて静脈から血液を採取する一般採血と、検査対象者が、自分の手の指等に採血針を刺して血液を採取する自己採血とがある。
In general, for blood collection, 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.
一般採血により採取された血液は、採取容器に密閉された状態で医療機関又は検査機関に搬送され、そこで検査が行われている。血液を血球と血漿とに分離せずに搬送する場合には、医療機関又は検査機関にて遠心分離機により血液を血球と血漿とに分離した後に検査が行われる。また、検査対象者が行う自己採血では、採血後の血液は分離膜により血球と血漿とに分離され、この分離された状態で検査場所に輸送され、そこで検査が行われる。
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. In the case where blood is transported without being separated into blood cells and plasma, a test is performed after blood is separated into blood cells and plasma by a centrifuge at a medical institution or inspection institution. In the self-collection performed by the subject to be examined, 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.
特許文献1には、自己採血により採取された血液検体の検査方法が記載されている。具体的には、1)容量を定量することなしに採取した定量すべき成分を含有する未知容量の生体試料と一定量の指示物質を含有する一定量の水性溶液とからなる定量用試料を調製する工程、2)一定量の指示物質を含有する一定量の水性溶液中の指示物質の濃度(C1)と定量用試料中の指示物質の濃度(C2)とから生体試料の希釈倍率(a)を求める工程、3)定量用試料中の定量すべき成分の濃度(Y)を求める工程、4)上記2)で求めた生体試料希釈倍率(a)と上記3)で求めた定量用試料中の定量すべき物質の濃度(Y)とから生体試料中の定量すべき成分を決定する工程を含む生体試料中の定量すべき成分の定量方法が記載されている。
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. 2 ) The biological sample dilution rate (C 1 ) in a fixed amount of aqueous solution containing a specified amount of indicator substance and the concentration (C 2 ) of the indicator substance in the sample for quantification Step for obtaining a), 3) Step for obtaining the concentration (Y) of the component to be quantified in the sample for quantification, 4) Biological sample dilution ratio obtained in 2) above and for quantification obtained in 3) above A method for quantifying a component to be quantified in a biological sample is described which includes a step of determining a component to be quantified in a biological sample from the concentration (Y) of a substance to be quantified in the sample.
特許文献2には、検体中の分析対象成分量を測定し、さらに、これ以外の恒常的に検体中に元来存在する標準成分の量を測定し、この標準成分の量と、検体中での標準成分の既知濃度とから検体の量を決定し、この検体量と、分析対象成分量とから、検体中の分析対象成分の濃度を決定する定量分析法が記載されている。
In 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.
また、特許文献3には、血液希釈定量器具を用いてヒトや動物から微量血液を採取しそのまま又は希釈したのち一定量を他の機器や容器又は直接試薬に供給することが記載されている。さらに特許文献4には、希釈用水溶液中の指示物質の吸光度を利用して、生物学的試料中の定量すべき成分の濃度を定量する方法が記載されている。
Further, 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. Further, 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.
さらに、微量採血管を用いて採血した血液を、溶血性希釈液で希釈して血球成分を溶血させ、しかる後該希釈溶血試料を免疫学的測定系に添加し、抗原又は抗体を測定することを特徴とする溶血式測定方法が特許文献5に記載されている。
Furthermore, blood collected using a micro blood collection tube is diluted with a hemolytic diluent to hemolyze blood cell components, and then the diluted hemolyzed sample is added to an immunological measurement system to measure antigens or antibodies. Patent Document 5 discloses a hemolytic measurement method characterized by the above.
一方、血液検体を検査対象者が採取する際には、小型ナイフが具備されたランセットにて採取し、血液中の任意成分の濃度の定量に使用されているが、通常100μL以上の血液検体を採取することが必要とされている。
On the other hand, when a test subject collects a blood sample, it is collected by a lancet equipped with a small knife and used for quantifying the concentration of an arbitrary component in blood. It is necessary to collect.
特許文献1に記載の方法においては、血液検体が微量である場合には、血液検体量に対する希釈液の割合を高くすることが必要になる。しかし、この場合、血液検体を希釈する前後での希釈液の体積変化率が非常に小さくなるため、内部標準物質の濃度の変化率が小さくなり、測定値の繰り返し再現性が低下するという問題がある。
In the method described in Patent Document 1, when the amount of blood sample is very small, it is necessary to increase the ratio of the diluent to the amount of blood sample. However, in this case, the volume change rate of the diluted solution before and after diluting the blood sample becomes very small, so that the rate of change of the concentration of the internal standard substance becomes small and the reproducibility of the measured value decreases. is there.
特許文献2には、健常者全血約100μLを多孔質膜に滴下し、血球を分離して血清を展開した後に、150μLの生食等張PBS(Phosphate-buffered saline: pH7.4)を添加して得た液を遠心分離して得られた上清を分析試料として分析しているが、100μL未満の採血については記載されていない。
In Patent Document 2, about 100 μL of whole blood of healthy subjects is dropped on a porous membrane, blood cells are separated and serum is developed, and then 150 μL of saline-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 μL.
特許文献3の方法では、10μLの血液量をマイクロピペットで正確に採取して分析しているが、採血に不慣れな患者が採取する場合には一定量を正確に採取することは難しく、誤差を含む採血で検査をした場合には測定値に誤差が含まれる結果となる。
In the method of Patent Document 3, a blood volume of 10 μL is accurately collected and analyzed with a micropipette. However, when a patient unaccustomed to blood collection collects a certain amount, it is difficult to accurately collect a certain amount of error. When the test is performed by including blood sampling, the measurement value includes an error.
特許文献4に記載の測定方法は希釈倍率が10倍程度の測定であり、希釈倍率をさらに高めて希釈血液量を十分に確保する場合には、特許文献1と同様に、測定値の繰り返し再現性が低下するという問題がある。
The measurement method described in Patent Document 4 is a measurement with a dilution factor of about 10 times. When the dilution factor is further increased to ensure a sufficient amount of diluted blood, the measurement value is repeatedly reproduced as in Patent Document 1. There is a problem that the performance is lowered.
上記の通り、微量の血液検体を使用する場合の分析を高精度で行うためには、特許文献1~4の方法では充分ではなかった。一方、採血者にとっては、血液採取は皮膚を傷つける侵襲的な行為であり、また血液の赤色を凝視することに不快を感じる場合もあることから、できるだけ早く採血して、傷部から流出している血液を止めたいと考えることが一般的である。このような事情から、採血量は必ずしも常に一定量であるとは限らず、ばらつく場合が多い。血液採取量のばらつきが大きいと希釈倍率の精度低下を招くことから、採血量の可視化及び一定化が望まれている。
As described above, the methods described in Patent Documents 1 to 4 are not sufficient for performing analysis when a very small amount of blood sample is used with high accuracy. On the other hand, for blood collectors, blood collection is an invasive action that damages the skin, and it may be uncomfortable to stare at the red color of the blood. It is common to want to stop the blood. For these reasons, the amount of blood collected is not always constant and often varies. If the variation in the amount of blood collected is large, the accuracy of the dilution rate will be reduced, so visualization and stabilization of the amount of blood collected is desired.
本発明が解決しようとする課題は、採血量の可視化及び一定化を図ることによって微量血液での血液検査を高精度で行うことができる、血液検査キット及び血液分析方法を提供することである。
The problem to be solved by the present invention is to provide a blood test kit and a blood analysis method capable of performing a blood test with a small amount of blood with high accuracy by visualizing and stabilizing the blood collection volume.
本発明者らは上記課題を解決するために鋭意検討した結果、血液検体を採取するための血液採取器具と、採取した血液検体を希釈するための希釈液と、血液検体の希釈物を収容するための収容器具と、を含む、血液中に恒常的に存在する標準成分を用いて血液検体中の対象成分の濃度を分析するための、血液検査キットにおいて、上記血液採取器具をキャピラリー状のものとすることにより、上記課題を解決できることを見出し、本発明を完成するに至った。すなわち、本発明によれば以下の発明が提供される。
As a result of intensive studies to solve the above-mentioned problems, the present inventors accommodate a blood collection device for collecting a blood sample, a diluent for diluting the collected blood sample, and a diluted blood sample. A blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is continually present in the blood, including a storage device for the blood sample. As a result, the present inventors have found that the above problems can be solved, and have completed the present invention. That is, according to the present invention, the following inventions are provided.
[1] 血液検体を採取するための血液採取器具と、
採取した血液検体を希釈するための希釈液と、
血液検体の希釈物を収容するための収容器具と、
を含み、血液中に恒常的に存在する標準成分を用いて血液検体中の対象成分の濃度を分析するための、血液検査キットであって、上記血液採取器具が、キャピラリーである、血液検査キット。
[2] 血液検体を採取するための血液採取器具と、
採取した血液検体を希釈するための希釈液と、
血液検体の希釈物を収容するための収容器具と、
を含む、血液中に恒常的に存在する標準成分を用いて血液検体中の対象成分の濃度を分析するための、血液検査キットであって、
上記希釈液が、血液中に存在しない標準成分を含有し、
上記血液採取器具が、キャピラリーである、血液検査キット。
[3] 血液検体を採取するための血液採取器具と、
採取した血液検体を希釈するための希釈液であって、血液中に存在しない標準成分を含有する希釈液と、
血液検体の希釈物を収容するための収容器具と、
を含む、血液検査キットであって、
上記血液採取器具が、キャピラリーである、血液検査キット。
[4] 上記希釈液が、血液中に恒常的に存在する標準成分を含まない、1から3のいずれかに記載の血液検査キット。
[5] 上記血液検査キットが、血液検体の希釈物から血漿を分離回収するための分離器具を含む、1から4のいずれかに記載の血液検査キット。
[6] 上記キャピラリーに、採取した血液検体の容量を確認するための目盛が付されている、1から5のいずれかに記載の血液検査キット。
[7] 上記目盛が、採取する血液検体の容量範囲の下限を示す位置に付されており、かつ
上記キャピラリーが、ストッパーを有し、
上記ストッパーが、採取した血液検体の容量が、採取する血液検体の容量範囲の上限を超えないためのものである、1から6のいずれかに記載の血液検査キット。
[8] 上記キャピラリーが、定められた容量の血液検体を採取するための形状を有する、1から7のいずれかに記載の血液検査キット。
[9] 上記キャピラリーが、内部に抗凝固剤を有する、1から8のいずれかに記載の血液検査キット。
[10] 上記キャピラリーが、合成樹脂からなる、1から9のいずれかに記載の血液検査キット。
[11] 上記キャピラリーの内壁が、親水性処理されたものである、10に記載の血液検査キット。
[12] 上記キャピラリーの血液検体を吸引する側の端部が、テーパーを有する、10又は11に記載の血液検査キット。
[13] 上記キャピラリーの、採取すべき血液検体の容量範囲を示す少なくとも1つの位置に目盛が付されており、上記キャピラリーの内径が、上記目盛の位置を越えた部位から大きくなる、10から12のいずれかに記載の血液検査キット。
[14] 上記キャピラリーの、採取すべき血液検体の容量範囲を示す少なくとも1つの位置に目盛が付されており、上記キャピラリーの上記目盛が付された位置を含む部分の内径が他の部分より小さい、10から12のいずれかに記載の血液検査キット。
[15] 上記キャピラリーを構成する材が、600nm以上の領域の波長の光の少なくとも一部を吸収する成分を含有する、10から14のいずれかに記載の血液検査キット。
[16] 血液中に恒常的に存在する上記標準成分が、ナトリウムイオン又は塩化物イオンである、1又は2に記載の血液検査キット。
[17] 血液中に恒常的に存在する上記標準成分が、ナトリウムイオン又は塩化物イオンと、さらに別の血液中に恒常的に存在する標準成分である、1又は2に記載の血液検査キット。
[18] 上記別の標準成分が、総タンパク又はアルブミンである、17に記載の血液検査キット。
[19] さらに別の血液中に恒常的に存在する標準成分を用いて、上記血液検体中の対象成分の濃度の分析を検証する、17又は18に記載の血液検査キット。
[20] 上記血液中に存在しない標準成分が、リチウムイオン又はグリセロール三リン酸である、2に記載の血液検査キット。
[21] 上記血液中に存在しない標準成分が、リチウムイオン又はグリセロール三リン酸である、3に記載の血液検査キット。
[22] 1から21のいずれかに記載の血液検査キットを用いることを特徴とする、血液検体中の対象成分の濃度を分析する方法(医療行為を除く。)。 [1] A blood collection device for collecting a blood sample;
A diluent for diluting the collected blood sample;
A storage device for storing a dilution of a blood sample;
A blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is constantly present in blood, wherein the blood collection device is a capillary .
[2] a blood collection device for collecting a blood sample;
A diluent for diluting the collected blood sample;
A storage device for storing a dilution of a blood sample;
A blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is constantly present in blood,
The diluent contains standard components that are not present in the blood,
A blood test kit, wherein the blood collection device is a capillary.
[3] a blood collection device for collecting a blood sample;
A diluent for diluting a collected blood sample, which contains a standard component not present in the blood;
A storage device for storing a dilution of a blood sample;
A blood test kit comprising:
A blood test kit, wherein the blood collection device is a capillary.
[4] The blood test kit according to any one of 1 to 3, wherein the diluent does not contain a standard component that is constantly present in blood.
[5] The blood test kit according to any one of 1 to 4, wherein the blood test kit includes a separation instrument for separating and collecting plasma from a diluted blood sample.
[6] The blood test kit according to any one of 1 to 5, wherein the capillary is provided with a scale for confirming the volume of the collected blood sample.
[7] The scale is attached to a position indicating the lower limit of the volume range of a blood sample to be collected, and the capillary has a stopper,
7. The blood test kit according to any one of 1 to 6, wherein the stopper is for the volume of the collected blood sample not to exceed the upper limit of the volume range of the collected blood sample.
[8] The blood test kit according to any one of 1 to 7, wherein the capillary has a shape for collecting a blood sample having a predetermined volume.
[9] The blood test kit according to any one of 1 to 8, wherein the capillary has an anticoagulant inside.
[10] The blood test kit according to any one of 1 to 9, wherein the capillary is made of a synthetic resin.
[11] The blood test kit according to 10, wherein the inner wall of the capillary is subjected to hydrophilic treatment.
[12] The blood test kit according to 10 or 11, wherein an end of the capillary on the side for sucking a blood sample has a taper.
[13] At least one position indicating the volume range of the blood sample to be collected is marked on the capillary, and the inner diameter of the capillary is increased from the portion beyond the position of the scale. The blood test kit according to any one of the above.
[14] A scale is attached to at least one position of the capillary indicating the volume range of the blood sample to be collected, and an inner diameter of a portion including the position where the scale is attached of the capillary is smaller than other portions. The blood test kit according to any one of 10 to 12.
[15] The blood test kit according to any one of 10 to 14, wherein the material constituting the capillary contains a component that absorbs at least part of light having a wavelength of 600 nm or more.
[16] The blood test kit according to 1 or 2, wherein the standard component that is constantly present in blood is sodium ion or chloride ion.
[17] The blood test kit according to 1 or 2, wherein the standard component that is constantly present in the blood is a standard component that is constantly present in sodium and chloride ions and still another blood.
[18] The blood test kit according to 17, wherein the another standard component is total protein or albumin.
[19] The blood test kit according to 17 or 18, wherein the analysis of the concentration of the target component in the blood sample is verified using a standard component that is constantly present in another blood.
[20] The blood test kit according to 2, wherein the standard component not present in the blood is lithium ion or glycerol triphosphate.
[21] The blood test kit according to 3, wherein the standard component not present in the blood is lithium ion or glycerol triphosphate.
[22] A method for analyzing the concentration of a target component in a blood sample (excluding medical practice), characterized by using the blood test kit according to any one of 1 to 21.
採取した血液検体を希釈するための希釈液と、
血液検体の希釈物を収容するための収容器具と、
を含み、血液中に恒常的に存在する標準成分を用いて血液検体中の対象成分の濃度を分析するための、血液検査キットであって、上記血液採取器具が、キャピラリーである、血液検査キット。
[2] 血液検体を採取するための血液採取器具と、
採取した血液検体を希釈するための希釈液と、
血液検体の希釈物を収容するための収容器具と、
を含む、血液中に恒常的に存在する標準成分を用いて血液検体中の対象成分の濃度を分析するための、血液検査キットであって、
上記希釈液が、血液中に存在しない標準成分を含有し、
上記血液採取器具が、キャピラリーである、血液検査キット。
[3] 血液検体を採取するための血液採取器具と、
採取した血液検体を希釈するための希釈液であって、血液中に存在しない標準成分を含有する希釈液と、
血液検体の希釈物を収容するための収容器具と、
を含む、血液検査キットであって、
上記血液採取器具が、キャピラリーである、血液検査キット。
[4] 上記希釈液が、血液中に恒常的に存在する標準成分を含まない、1から3のいずれかに記載の血液検査キット。
[5] 上記血液検査キットが、血液検体の希釈物から血漿を分離回収するための分離器具を含む、1から4のいずれかに記載の血液検査キット。
[6] 上記キャピラリーに、採取した血液検体の容量を確認するための目盛が付されている、1から5のいずれかに記載の血液検査キット。
[7] 上記目盛が、採取する血液検体の容量範囲の下限を示す位置に付されており、かつ
上記キャピラリーが、ストッパーを有し、
上記ストッパーが、採取した血液検体の容量が、採取する血液検体の容量範囲の上限を超えないためのものである、1から6のいずれかに記載の血液検査キット。
[8] 上記キャピラリーが、定められた容量の血液検体を採取するための形状を有する、1から7のいずれかに記載の血液検査キット。
[9] 上記キャピラリーが、内部に抗凝固剤を有する、1から8のいずれかに記載の血液検査キット。
[10] 上記キャピラリーが、合成樹脂からなる、1から9のいずれかに記載の血液検査キット。
[11] 上記キャピラリーの内壁が、親水性処理されたものである、10に記載の血液検査キット。
[12] 上記キャピラリーの血液検体を吸引する側の端部が、テーパーを有する、10又は11に記載の血液検査キット。
[13] 上記キャピラリーの、採取すべき血液検体の容量範囲を示す少なくとも1つの位置に目盛が付されており、上記キャピラリーの内径が、上記目盛の位置を越えた部位から大きくなる、10から12のいずれかに記載の血液検査キット。
[14] 上記キャピラリーの、採取すべき血液検体の容量範囲を示す少なくとも1つの位置に目盛が付されており、上記キャピラリーの上記目盛が付された位置を含む部分の内径が他の部分より小さい、10から12のいずれかに記載の血液検査キット。
[15] 上記キャピラリーを構成する材が、600nm以上の領域の波長の光の少なくとも一部を吸収する成分を含有する、10から14のいずれかに記載の血液検査キット。
[16] 血液中に恒常的に存在する上記標準成分が、ナトリウムイオン又は塩化物イオンである、1又は2に記載の血液検査キット。
[17] 血液中に恒常的に存在する上記標準成分が、ナトリウムイオン又は塩化物イオンと、さらに別の血液中に恒常的に存在する標準成分である、1又は2に記載の血液検査キット。
[18] 上記別の標準成分が、総タンパク又はアルブミンである、17に記載の血液検査キット。
[19] さらに別の血液中に恒常的に存在する標準成分を用いて、上記血液検体中の対象成分の濃度の分析を検証する、17又は18に記載の血液検査キット。
[20] 上記血液中に存在しない標準成分が、リチウムイオン又はグリセロール三リン酸である、2に記載の血液検査キット。
[21] 上記血液中に存在しない標準成分が、リチウムイオン又はグリセロール三リン酸である、3に記載の血液検査キット。
[22] 1から21のいずれかに記載の血液検査キットを用いることを特徴とする、血液検体中の対象成分の濃度を分析する方法(医療行為を除く。)。 [1] A blood collection device for collecting a blood sample;
A diluent for diluting the collected blood sample;
A storage device for storing a dilution of a blood sample;
A blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is constantly present in blood, wherein the blood collection device is a capillary .
[2] a blood collection device for collecting a blood sample;
A diluent for diluting the collected blood sample;
A storage device for storing a dilution of a blood sample;
A blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is constantly present in blood,
The diluent contains standard components that are not present in the blood,
A blood test kit, wherein the blood collection device is a capillary.
[3] a blood collection device for collecting a blood sample;
A diluent for diluting a collected blood sample, which contains a standard component not present in the blood;
A storage device for storing a dilution of a blood sample;
A blood test kit comprising:
A blood test kit, wherein the blood collection device is a capillary.
[4] The blood test kit according to any one of 1 to 3, wherein the diluent does not contain a standard component that is constantly present in blood.
[5] The blood test kit according to any one of 1 to 4, wherein the blood test kit includes a separation instrument for separating and collecting plasma from a diluted blood sample.
[6] The blood test kit according to any one of 1 to 5, wherein the capillary is provided with a scale for confirming the volume of the collected blood sample.
[7] The scale is attached to a position indicating the lower limit of the volume range of a blood sample to be collected, and the capillary has a stopper,
7. The blood test kit according to any one of 1 to 6, wherein the stopper is for the volume of the collected blood sample not to exceed the upper limit of the volume range of the collected blood sample.
[8] The blood test kit according to any one of 1 to 7, wherein the capillary has a shape for collecting a blood sample having a predetermined volume.
[9] The blood test kit according to any one of 1 to 8, wherein the capillary has an anticoagulant inside.
[10] The blood test kit according to any one of 1 to 9, wherein the capillary is made of a synthetic resin.
[11] The blood test kit according to 10, wherein the inner wall of the capillary is subjected to hydrophilic treatment.
[12] The blood test kit according to 10 or 11, wherein an end of the capillary on the side for sucking a blood sample has a taper.
[13] At least one position indicating the volume range of the blood sample to be collected is marked on the capillary, and the inner diameter of the capillary is increased from the portion beyond the position of the scale. The blood test kit according to any one of the above.
[14] A scale is attached to at least one position of the capillary indicating the volume range of the blood sample to be collected, and an inner diameter of a portion including the position where the scale is attached of the capillary is smaller than other portions. The blood test kit according to any one of 10 to 12.
[15] The blood test kit according to any one of 10 to 14, wherein the material constituting the capillary contains a component that absorbs at least part of light having a wavelength of 600 nm or more.
[16] The blood test kit according to 1 or 2, wherein the standard component that is constantly present in blood is sodium ion or chloride ion.
[17] The blood test kit according to 1 or 2, wherein the standard component that is constantly present in the blood is a standard component that is constantly present in sodium and chloride ions and still another blood.
[18] The blood test kit according to 17, wherein the another standard component is total protein or albumin.
[19] The blood test kit according to 17 or 18, wherein the analysis of the concentration of the target component in the blood sample is verified using a standard component that is constantly present in another blood.
[20] The blood test kit according to 2, wherein the standard component not present in the blood is lithium ion or glycerol triphosphate.
[21] The blood test kit according to 3, wherein the standard component not present in the blood is lithium ion or glycerol triphosphate.
[22] A method for analyzing the concentration of a target component in a blood sample (excluding medical practice), characterized by using the blood test kit according to any one of 1 to 21.
本発明の血液検査キット及び血液分析方法によれば、採血量の可視化及び一定化を図ることによって微量血液での血液検査を高精度で行うことができる。
According to the blood test kit and the blood analysis method of the present invention, a blood test with a very small amount of blood can be performed with high accuracy by visualizing and stabilizing the amount of blood collected.
以下、本発明の実施の形態を説明する。なお、X~Yで示される範囲は、上限Xと下限Yの値を含む。血液中に恒常的に存在する標準成分のことを、外部標準物質又は外部標準ということがある。また、血液中に存在しない標準成分のことを、内部標準物質又は内部標準ということがある。
Hereinafter, embodiments of the present invention will be described. The range indicated by X to Y includes the upper limit X and the lower limit Y. A standard component that is constantly present in blood may be referred to as an external standard substance or an external standard. In addition, a standard component that does not exist in blood may be referred to as an internal standard substance or an internal standard.
微量の血液を採取する方法として濾紙を用いて血液分析を行う方法が特開平10-104226号公報に開示されている。また濾紙の代わりに血液保持性の高い多孔質材を用いる方法が特許文献2に開示されている。これらの方法では、素材に吸収された血液成分を緩衝液などで抽出して測定することになるため、血液に恒常的に存在する外部標準物質であるナトリウムイオン、塩化物イオン、カルシウムイオン、蛋白などを、血液を溶出して再溶解した場合の緩衝液による希釈率を見積もる基準物質として用いることが記載されている。しかしながらこれらの方法では、血液の採取量はさまざまで、採取した血液の希釈率が高くなってしまうとその後の分析精度が下がって結果がばらつき、一旦血液を凝集させて固化するために分析対象となる成分の安定性の保証が不十分になることがあった。また、乾燥した試料から生体成分を抽出する緩衝液は、pH調整や生体成分安定化のためにNaOHやNaCl、HClを添加した緩衝液を利用する必要がある。このため、試料の成分で比較的高い濃度で存在し、恒常性があり、個体間差が少ないナトリウムイオンや塩化物イオンの濃度を外部標準として、希釈された元の他の生体成分濃度の補正をすることに利用できないという問題があった。
As a method of collecting a very small amount of blood, a method of performing blood analysis using a filter paper is disclosed in JP-A-10-104226. Further, Patent Document 2 discloses a method using a porous material having high blood retention instead of filter paper. In these methods, blood components absorbed in the material are extracted and measured with a buffer solution or the like, so that sodium ions, chloride ions, calcium ions, proteins, which are external standard substances that are constantly present in blood, are measured. Are used as reference substances for estimating the dilution rate with a buffer solution when blood is eluted and redissolved. However, with these methods, the amount of blood collected varies, and when the dilution rate of the collected blood increases, the subsequent analysis accuracy decreases and the results vary, and the blood is agglomerated and solidified. As a result, the guarantee of the stability of the components may be insufficient. Further, as a buffer solution for extracting a biological component from a dried sample, it is necessary to use a buffer solution to which NaOH, NaCl, or HCl is added for pH adjustment or biological component stabilization. For this reason, the concentration of other diluted biological components is corrected using the concentration of sodium ions and chloride ions, which are present at a relatively high concentration of sample components, have homeostasis, and have small differences between individuals as an external standard. There was a problem that it could not be used to do.
一方、採取した微量血液を内部標準入り緩衝液で希釈し、内部標準物質の希釈倍数から希釈血漿中の未知量の存在する成分量を定量する方法として特許文献1に記載の方法が開示されている。内部標準物質としてはN-(2-ヒドロキシ-3-スルホプロピル)-3,5-ジメトキシアニリン・ナトリウム塩(HSDA)、あるいはアシッドブルー9(ブリリアントブルーFCF)が利用されており、血液を安定に保持するための緩衝剤や防腐剤が用いられている。このような処方は、血液を凝固させないことでその成分の安定性を維持することを実現したが、やはり血液採取量がばらつき、採取量が少ない場合には、希釈後の内部標準物質の希釈率が小さくなることや、血液成分量そのものが低下する為に、検査精度の信頼性が低下する問題であった。また、緩衝液で希釈する方法は生体成分がpH7.4の生理的条件の緩衝液で保存され、輸送中の安定性にも優れているが、内部標準添加緩衝液への試料による内部標準の希釈率が小さく、少ない試料添加では測定誤差が生じ易い問題があった。
On the other hand, a method described in Patent Document 1 is disclosed as a method for diluting a collected trace blood with a buffer solution containing an internal standard and quantifying the amount of a component having an unknown amount in diluted plasma from the dilution factor of the internal standard substance. Yes. N- (2-Hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline sodium salt (HSDA) or Acid Blue 9 (Brilliant Blue FCF) is used as an internal standard substance to stabilize blood. Buffering agents and preservatives are used for holding. Such a prescription achieved the stability of its components by not coagulating the blood, but when the blood collection volume is also variable and the collection volume is small, the dilution rate of the internal standard substance after dilution This is a problem that the reliability of the test accuracy is lowered due to the decrease in the amount of blood and the amount of the blood component itself. In addition, the method of diluting with a buffer solution is a biological component stored in a buffer solution with physiological conditions of pH 7.4 and is excellent in stability during transportation. There was a problem that measurement errors were likely to occur when the dilution rate was small and a small amount of sample was added.
さらにこれら先行技術の実施例では、抽出する緩衝液にはリン酸緩衝生理食塩水が生体成分の安定保持に優れているので利用されているが、リン酸緩衝生理食塩水にはナトリウムイオンや塩化物イオンが含まれている。そのためナトリウムイオンや塩化物イオンは外部標準として利用できず、カルシウムイオン、蛋白等が利用されている。従って、微量血液での血液検査を精度高く行う為には、従来技術にあるように希釈率を補正する外部標準物質の使用や、従来提案されている内部標準物質を含有する緩衝液の使用では検査精度の確保が充分でなかった。
Furthermore, in these prior art examples, phosphate buffered saline is used as the buffer solution to be extracted because it is excellent in the stable retention of biological components. However, phosphate buffered saline contains sodium ions and chloride. Contain ions. For this reason, sodium ions and chloride ions cannot be used as external standards, and calcium ions, proteins, and the like are used. Therefore, in order to perform a blood test with a small amount of blood with high accuracy, the use of an external standard substance that corrects the dilution rate as in the prior art or the use of a buffer solution containing a conventionally proposed internal standard substance Insufficient inspection accuracy.
また、血液中の恒常性物質とはいえ、例えば、ナトリウムイオンにおいては、正常値134~146mmol/Lの分布の幅を有するため、より正確な希釈倍率を算出することが必要となる。希釈倍率の精度が低下すると検査精度に悪影響を与えて、検査の信頼性を損なってしまうリスクが高まってしまう。特に、キットを構成する部材から緩衝液中に溶出する外部標準物質による汚染(いわゆるコンタミ)が多少なりともあった場合に、血液採取量が多かったり少なかったりすると、コンタミの希釈倍率算出に与える影響度が異なってしまう。特許文献2には、このようなキットを構成する部材から緩衝液中に溶出する外部標準物質のコンタミの希釈倍率算出に与える影響度を一定にすることに関しては、一切言及していない。
In addition, although it is a homeostatic substance in blood, for example, sodium ions have a distribution range of normal values of 134 to 146 mmol / L, so it is necessary to calculate a more accurate dilution factor. Decreasing the accuracy of the dilution factor adversely affects inspection accuracy and increases the risk of impairing inspection reliability. In particular, if there is some contamination (so-called contamination) due to an external standard substance that elutes in the buffer solution from the components that make up the kit, the effect of contamination on the dilution rate calculation if the amount of blood collected is large or small The degree will be different. Patent Document 2 makes no mention of making the influence on the dilution ratio calculation of the contamination of the external standard substance eluted from the members constituting such a kit constant.
また、特許文献1には、内部標準に関する記載はあるが、外部標準と併用することに関する記載はない。従って、外部標準のコンタミに関する記載はなく、コンタミを防止するための具体的な手段は一切提案されていない。さらに特許文献5にはキャピラリで採血するが、NaClを含む緩衝液で希釈されており、外部標準を用いることに関する記載はない。
In addition, Patent Document 1 has a description regarding an internal standard, but there is no description regarding the combined use with an external standard. Therefore, there is no description regarding the contamination of the external standard, and no specific means for preventing the contamination has been proposed. Furthermore, Patent Document 5 collects blood with a capillary, but is diluted with a buffer containing NaCl, and there is no description about using an external standard.
本発明は、微量血液を緩衝液で希釈して対象成分の濃度を分析する方法において、より正確に希釈倍率を決定して対象成分の濃度の分析を行うための血液検査キット、及びキットを用いた血液分析方法を提供することを目的としてなされたものである。そのための解決手段として、キャピラリーを用いて血液を採取することにより、血液採取量を一定値(少なくとも許容最低採血量以上である)とすることを採用している。また、好ましい態様においては、外部標準を用いるのみならず、内部標準を用いることとしている。
The present invention relates to a blood test kit and a kit for analyzing the concentration of a target component by more accurately determining the dilution factor in a method for analyzing the concentration of the target component by diluting a trace amount of blood with a buffer solution. It was made for the purpose of providing a blood analysis method. As a means for solving this problem, it is adopted that blood is collected using a capillary so that the blood collection amount is a constant value (at least equal to or greater than the allowable minimum blood collection amount). In a preferred embodiment, not only an external standard is used but also an internal standard is used.
また、従来のファイバーロッド(繊維)を用いる血液採取は、採血者にとって、採血量が分かりにくく、かつファイバーロッド内部に染み込んだ血液が緩衝液中に完全に放出されないことによる検体ロス、すなわち少ない採血量による検査精度を低下させる要因を含んでしまう。これに対し、本発明は、キャピラリーを用いて血液を採取することで、採取した血液検体の容量が目視で確認でき、また検体ロスを防止することができる。採血した血液検体の容量が目視で確認できることは、採血者にとっても安心である。
In addition, blood sampling using conventional fiber rods (fibers) is difficult for blood collectors to understand the amount of blood collected, and the sample loss due to the fact that blood soaked inside the fiber rod is not completely released into the buffer solution, that is, low blood sampling The factor which reduces the inspection accuracy by quantity will be included. In contrast, according to the present invention, by collecting blood using a capillary, the volume of the collected blood sample can be confirmed visually, and sample loss can be prevented. The fact that the volume of the collected blood sample can be visually confirmed is also safe for the blood sampler.
本発明により、血液検体中の対象成分を分析する方法において、患者自らが血液を採取する場合であっても、血液採取量を容易に一定値とでき、かつ少なくとも望ましい血液検体の容量範囲の最低量以上とすることができる。またキットを構成する部材から緩衝液中に溶出する外部標準物質の希釈倍率算出に与える影響を一定にして、分析対象成分の定量を精度よく行うことが可能となる。
According to the present invention, in the method of analyzing a target component in a blood sample, even if the patient collects blood himself, the blood sample can be easily set to a constant value, and at least the minimum volume range of the desired blood sample It can be more than the amount. In addition, the influence of the external standard substance eluted in the buffer solution from the members constituting the kit on the calculation of the dilution ratio can be made constant, and the component to be analyzed can be accurately quantified.
さらに本発明では、実施態様として、目盛付きのキャピラリーを用いるが、採取した血液の容量を採血者が把握できるので、希釈倍率を比較的正確に予想することが可能となり、測定結果に基づく希釈倍率の算出値が妥当であるか否かを、精度よく見積もることが可能となる。採血者にとっては、採取された血液検体の容量が許容範囲内であること、及び少なくとも最低量は満たしていることを目視で確認できるため、安心して検査機関に血液検体を搬送することができるという利点もある。また、目盛付きキャピラリーを用いることにより、採取した血液検体の容量を把握することができ、その把握した容量の情報を、血液検体とともに検査機関に提供することができる。提供された情報は、検査機関においては算出した希釈倍率が適切であることのチェックに用いることができる。
Furthermore, in the present invention, a graduated capillary is used as an embodiment, but since the blood sampler can grasp the volume of the collected blood, it becomes possible to predict the dilution rate relatively accurately, and the dilution rate based on the measurement result It is possible to accurately estimate whether or not the calculated value is valid. For blood collectors, it is possible to visually confirm that the volume of the collected blood sample is within the allowable range and that at least the minimum amount is satisfied, so that the blood sample can be safely transported to the laboratory. There are also advantages. Further, by using a graduated capillary, the volume of the collected blood sample can be grasped, and information on the grasped volume can be provided to the testing institution together with the blood sample. The provided information can be used for checking that the calculated dilution factor is appropriate in the inspection organization.
[1]血液検査キット
本発明の血液検査キットは、血液検体を採取するための血液採取器具と、採取した血液検体を希釈するための希釈液と、血液検体の希釈物を収容するための収容器具と、を含み、血液中に恒常的に存在する標準成分を用いて血液検体中の対象成分の濃度を分析するための、血液検査キットであって、上記血液採取器具が、キャピラリーであることを特徴とする。 [1] Blood test kit The blood test kit of the present invention includes a blood collection device for collecting a blood sample, a diluent for diluting the collected blood sample, and a housing for housing a diluted blood sample. A blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is constantly present in blood, and the blood sampling device is a capillary It is characterized by.
本発明の血液検査キットは、血液検体を採取するための血液採取器具と、採取した血液検体を希釈するための希釈液と、血液検体の希釈物を収容するための収容器具と、を含み、血液中に恒常的に存在する標準成分を用いて血液検体中の対象成分の濃度を分析するための、血液検査キットであって、上記血液採取器具が、キャピラリーであることを特徴とする。 [1] Blood test kit The blood test kit of the present invention includes a blood collection device for collecting a blood sample, a diluent for diluting the collected blood sample, and a housing for housing a diluted blood sample. A blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is constantly present in blood, and the blood sampling device is a capillary It is characterized by.
血液検体中の対象成分の濃度を分析するとは、対象成分の濃度を決定すること(すなわち、対象成分を定量すること)、又は対象成分の濃度が所定の基準値以上であるか所定の基準値以下であるかを決定することなどを包含し、分析の形態は特に限定されない。
Analyzing the concentration of the target component in the 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. Including determination of whether or not, the form of analysis is not particularly limited.
[採血方法と量]
本発明の血液検査キットは、血液検体中の対象成分の分析のために血液検体を採取するためのものである。本発明の血液検査キットによる血液の採取は、対象者自身が行ってもよく、医師等の有資格者が行ってもよい。 [Blood collection method and volume]
The blood test kit of the present invention is for collecting a blood sample for analysis of a target component in the blood sample. Collection of blood using the blood test kit of the present invention may be performed by the subject himself or by a qualified person such as a doctor.
本発明の血液検査キットは、血液検体中の対象成分の分析のために血液検体を採取するためのものである。本発明の血液検査キットによる血液の採取は、対象者自身が行ってもよく、医師等の有資格者が行ってもよい。 [Blood collection method and volume]
The blood test kit of the present invention is for collecting a blood sample for analysis of a target component in the blood sample. Collection of blood using the blood test kit of the present invention may be performed by the subject himself or by a qualified person such as a doctor.
好ましい態様では、患者本人が、ランセットなどのナイフ付の器具を用いて指先などを傷つけて皮膚外にでた血液を採取する。患者の負担を減らすために、侵襲性低く血液を採取することが好ましく血液を採取するときに無痛、あるいは、痛みが非常に少ない状態で採血できることが望ましく、その場合、傷の深さ、大きさは小さいことが望ましく、採取できる血液も非常に少なくなる。従って、本発明の血液検査キットで採取される検体の容量(すなわち、採血量)は100μL以下であることが好ましい。このように少ない採血量でも、本発明では、血液採取量を一定値とすることが容易であり、算出される希釈倍率のばらつきを抑えることができる 。
In a preferred embodiment, the patient himself / herself collects 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. In order to reduce the burden on the patient, it is preferable to collect blood with low invasiveness. It is desirable that blood can be collected with no pain or very little pain when collecting blood, in which case the depth and size of the wound Is preferably small, and very little blood can be collected. Therefore, the volume of the sample collected by the blood test kit of the present invention (that is, the amount of blood collected) is preferably 100 μL or less. Even with such a small amount of blood collection, in the present invention, it is easy to set the blood collection amount to a constant value, and variations in the calculated dilution rate can be suppressed.
[血液中に恒常的に存在する標準成分]
上記のように、血漿成分の希釈倍率の高い希釈血漿の希釈後の対象成分について、希釈前の血液の血漿中に存在する濃度を正確に分析するためには、希釈液中にあらかじめ存在する物質の濃度の変化率から求める方法では、濃度の変化の割合が非常に小さくなるために測定誤差が大きく、また、測定の再現性が悪化する弊害がある。従って本発明の好ましい態様の一つは、血液中に恒常的に存在する標準成分を用いて血液検体中の対象成分の濃度を分析するための、血液検査キットである。 [Standard components that are permanently present in the blood]
As described above, in order to accurately analyze the concentration of the target component after dilution of the diluted plasma with a high dilution ratio of the plasma component, the concentration of the plasma component in the blood before dilution is determined in advance. In the method of obtaining from the density change rate, the density change rate is very small, so that the measurement error is large and the reproducibility of the measurement deteriorates. Accordingly, one of the preferred embodiments of the present invention is a blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is constantly present in blood.
上記のように、血漿成分の希釈倍率の高い希釈血漿の希釈後の対象成分について、希釈前の血液の血漿中に存在する濃度を正確に分析するためには、希釈液中にあらかじめ存在する物質の濃度の変化率から求める方法では、濃度の変化の割合が非常に小さくなるために測定誤差が大きく、また、測定の再現性が悪化する弊害がある。従って本発明の好ましい態様の一つは、血液中に恒常的に存在する標準成分を用いて血液検体中の対象成分の濃度を分析するための、血液検査キットである。 [Standard components that are permanently present in the blood]
As described above, in order to accurately analyze the concentration of the target component after dilution of the diluted plasma with a high dilution ratio of the plasma component, the concentration of the plasma component in the blood before dilution is determined in advance. In the method of obtaining from the density change rate, the density change rate is very small, so that the measurement error is large and the reproducibility of the measurement deteriorates. Accordingly, one of the preferred embodiments of the present invention is a blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is constantly present in blood.
ここで、標準成分を「用いて」とは、標準成分についての標準値(恒常値)に基づき、対象成分の濃度を分析するための希釈倍率を決定する意である。したがって、血液中に恒常的に存在する標準成分を用いて血液検体中の対象成分の濃度を分析することは、血液中に恒常的に存在する標準成分の恒常値(標準値)に基づき希釈倍率を決定し、対象成分の濃度を分析することでもある。
Here, “using” the standard component means determining the dilution factor for analyzing the concentration of the target component based on the standard value (constant value) of the standard component. Therefore, analyzing the concentration of a target component in a blood sample using a standard component that is constantly present in blood is based on the constant value (standard value) of the standard component that is constantly present in blood. Is to analyze the concentration of the target component.
血液中に恒常的に存在する標準成分は、例えば、ナトリウムイオン、塩化物イオン、カリウムイオン、マグネシウムイオン、カルシウムイオン、総タンパク、及びアルブミン等が挙げられる。血液検体の血清及び血漿中に含まれるこれらの標準成分の濃度は、ナトリウムイオン濃度は、134~146mmol/L(平均値:142mmol/L)、塩化物イオン濃度は、97~107mmol/L(平均値:102mmol/L)、カリウムイオン濃度は、3.2~4.8mmol/L(平均値:4.0mmol/L)、マグネシウムイオン濃度は、0.75~1.0mmol/L(平均値:0.9mmol/L)、カルシウムイオン濃度は、4.2~5.1mmol/L(平均値:4.65mmol/L)、総タンパク濃度は、6.7~8.3g/100ml(平均値:7.5g/100mL)、アルブミン濃度は、4.1~5.1g/100mL(平均値:4.6g/100mL)である。本発明では、患者の痛みを和らげるために採血する血液量が非常に少ない場合における対象成分の測定を可能にするためのものであり、微量の血液を希釈液で希釈した際に、希釈液中に存在する「血液中に恒常的に存在する標準成分」の濃度を精度よく測定する必要がある。希釈倍率が高くなると、もともと血液中に存在する成分の希釈液中の濃度が低下し、希釈倍率によっては濃度測定時に、測定誤差を含む可能性がある。従って、微量な血液成分を希釈倍率高く希釈したときに、上記標準成分を十分に精度高く検出するためには、微量な血液中に高い濃度で存在する標準成分を測定することが好ましい。本発明では、血液検体中に恒常的に存在する成分の中でも高濃度に存在する、ナトリウムイオン(Na+)又は塩化物イオン(Cl-)を用いることが好ましい。更には、上述の血液中に恒常的に存在する標準成分の中でも血液中に存在する量が一番高いナトリウムイオンを測定することが最も好ましい。ナトリウムイオンは、平均値が標準値(基準範囲の中央値)を表し、その値は、142mmol/Lであり、血漿中の総陽イオンの90モル%以上を占める。
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 / L (average value: 142 mmol / L), and the chloride ion concentration is 97 to 107 mmol / L (average) Value: 102 mmol / L), potassium ion concentration is 3.2 to 4.8 mmol / L (average value: 4.0 mmol / L), and magnesium ion concentration is 0.75 to 1.0 mmol / L (average value: 0.9 mmol / L), calcium ion concentration is 4.2 to 5.1 mmol / L (average value: 4.65 mmol / L), and total protein concentration is 6.7 to 8.3 g / 100 ml (average value: 7.5 g / 100 mL), and the albumin concentration is 4.1 to 5.1 g / 100 mL (average value: 4.6 g / 100 mL). 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. When 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”. When the dilution rate is increased, 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, in order to detect the above-mentioned standard component with sufficient accuracy when a very small amount of blood component is diluted at a high dilution rate, it is preferable to measure a standard component present at a high concentration in a very small amount of blood. In the present invention, it is preferable to use sodium ions (Na + ) or chloride ions (Cl − ) that are present in a high concentration among the components that are constantly present in the blood sample. Furthermore, it is most preferable to measure sodium ion having the highest amount in the blood among the standard components that are constantly present in the blood. The average value of sodium ion represents a standard value (median value of the reference range), and the value is 142 mmol / L, which accounts for 90 mol% or more of the total cations in plasma.
[血液中に存在しない標準成分]
本発明の好ましい態様の一つは、血液中に存在しない標準成分を用いて血液検体中の対象成分の濃度を分析するための、血液検査キットである。このような検査キットは、血液中に恒常的に存在する標準成分とともに、血液中に存在しない標準成分を用いるためのものであってもよく、血液中に恒常的に存在する標準成分を用いずに、血液中に存在しない標準成分を単独で用いるためのものであってもよい。 [Standard components not present in blood]
One preferred embodiment of the present invention is a blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is not present in blood. Such a test kit may be used for using a standard component that does not exist in blood together with a standard component that exists constantly in blood, and does not use a standard component that exists constantly in blood. In addition, a standard component that does not exist in blood may be used alone.
本発明の好ましい態様の一つは、血液中に存在しない標準成分を用いて血液検体中の対象成分の濃度を分析するための、血液検査キットである。このような検査キットは、血液中に恒常的に存在する標準成分とともに、血液中に存在しない標準成分を用いるためのものであってもよく、血液中に恒常的に存在する標準成分を用いずに、血液中に存在しない標準成分を単独で用いるためのものであってもよい。 [Standard components not present in blood]
One preferred embodiment of the present invention is a blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is not present in blood. Such a test kit may be used for using a standard component that does not exist in blood together with a standard component that exists constantly in blood, and does not use a standard component that exists constantly in blood. In addition, a standard component that does not exist in blood may be used alone.
いずれの場合も、血液中に存在しない標準成分は、後述する希釈液に所定の濃度になるように添加して用いることができる。血液中に存在しない標準成分としては、血液検体中に全く含まれないか、若しくは含まれていたとしても極微量である物質を使用することができる。血液中に存在しない標準成分としては、血液検体中の対象成分の測定に干渉を与えない物質、血液検体中の生体酵素の作用を受けて分解しない物質、希釈中で安定な物質、血球膜を透過せず血球中に含まれない物質、緩衝液の保存容器に吸着しない物質、精度良く測定できる検出系が利用できる物質を用いることが好ましい。
In any case, standard components that are not present in the blood can be used by adding them to a diluting solution described later so as to have a predetermined concentration. As the standard component that does not exist in the blood, a substance that is not contained at all in the blood sample or is contained in a trace amount even if it is contained can be used. Standard components that are not present in blood include substances that do not interfere with the measurement of target components in blood samples, substances that do not degrade due to the action of biological enzymes in blood samples, substances that are stable in dilution, and blood cell membranes. It is preferable to use a substance that does not permeate and is not contained in blood cells, a substance that does not adsorb to a buffer storage container, and a substance that can use a detection system that can measure with high accuracy.
血液中に存在しない標準成分としては、希釈液に添加した状態で長期間保管しても安定な物質が好ましい。血液中に存在しない標準成分の例としては、グリセロール三リン酸、アルカリ金属としてLi、Rb、Cs、又はFr、そしてアルカリ土類金属としてはSr、Ba、又はRaが挙げられ、Li及びグリセロール三リン酸が好ましい。
As a standard component that does not exist in blood, a substance that is stable even if stored for a long time in a state of being added to a diluent is preferable. Examples of standard components not present in blood include glycerol triphosphate, alkali metals Li, Rb, Cs, or Fr, and alkaline earth metals Sr, Ba, or Ra, and Li and glycerol tris. Phosphoric acid is preferred.
これらの血液中に存在しない標準成分は、血液希釈後の濃度測定時に第二の試薬を添加することで発色させ、その発色濃度から希釈血液中の濃度を求めることができる。例えば、希釈液に添加したリチウムイオンの測定は、キレート比色法(ハロゲン化ポルフィリンキレート法:パーフルオロ-5,10,15,20-テトラフェニル-21H,23H-ポルフィリン)を利用して生化学自動分析装置で大量試料を微量の試料で容易に測定できる。
These standard components that are not present in blood can be colored by adding a second reagent during concentration measurement after blood dilution, and the concentration in the diluted blood can be determined from the color density. For example, the measurement of lithium ions added to the diluting solution is performed by biochemistry using a chelate colorimetric method (halogenated porphyrin chelate method: perfluoro-5,10,15,20-tetraphenyl-21H, 23H-porphyrin). A large amount of sample can be easily measured with a small amount of sample by an automatic analyzer.
[希釈液]
本発明の血液検査キットは、採取した血液検体を希釈するための希釈液を含む。希釈液は、キットが血液中に恒常的に存在する標準成分を用いて 血液検体中の対象成分の濃度を分析するためのものである場合、血液中に恒常的に存在する標準成分を含有しない。「含有しない」とは、「実質的に含有しない」ことを意味する。ここで、「実質的に含有しない」とは、希釈倍率を求める時に使用する恒常性のある物質をまったく含まないか、あるいは含まれていたとしても、血液検体を希釈した後の希釈液の恒常性のある物質の測定に影響を及ぼさない程度の極微量の濃度で含まれる場合を意味する。血液中に恒常的に存在する標準成分として、ナトリウムイオン又は塩化物イオンを用いる場合には、希釈液としては、ナトリウムイオン又は塩化物イオンを実質的に含有しない希釈液を使用する。 [Diluted solution]
The blood test kit of the present invention includes a diluent for diluting the collected blood sample. Diluent does not contain standard components that are permanently present in the blood if the kit is used to analyze the concentration of the component of interest in the blood sample using standard components that are permanently present in the blood . “Not contained” means “not substantially contained”. Here, “substantially does not contain” means that it does not contain a substance having homeostasis used at the time of determining the dilution factor, or even if it is contained, the homeostasis of the diluted solution after diluting the blood sample It means a case where it is contained at a very small concentration that does not affect the measurement of a toxic substance. When sodium ions or chloride ions are used as a standard component that is constantly present in blood, a diluent that does not substantially contain sodium ions or chloride ions is used as the diluent.
本発明の血液検査キットは、採取した血液検体を希釈するための希釈液を含む。希釈液は、キットが血液中に恒常的に存在する標準成分を用いて 血液検体中の対象成分の濃度を分析するためのものである場合、血液中に恒常的に存在する標準成分を含有しない。「含有しない」とは、「実質的に含有しない」ことを意味する。ここで、「実質的に含有しない」とは、希釈倍率を求める時に使用する恒常性のある物質をまったく含まないか、あるいは含まれていたとしても、血液検体を希釈した後の希釈液の恒常性のある物質の測定に影響を及ぼさない程度の極微量の濃度で含まれる場合を意味する。血液中に恒常的に存在する標準成分として、ナトリウムイオン又は塩化物イオンを用いる場合には、希釈液としては、ナトリウムイオン又は塩化物イオンを実質的に含有しない希釈液を使用する。 [Diluted solution]
The blood test kit of the present invention includes a diluent for diluting the collected blood sample. Diluent does not contain standard components that are permanently present in the blood if the kit is used to analyze the concentration of the component of interest in the blood sample using standard components that are permanently present in the blood . “Not contained” means “not substantially contained”. Here, “substantially does not contain” means that it does not contain a substance having homeostasis used at the time of determining the dilution factor, or even if it is contained, the homeostasis of the diluted solution after diluting the blood sample It means a case where it is contained at a very small concentration that does not affect the measurement of a toxic substance. When sodium ions or chloride ions are used as a standard component that is constantly present in blood, a diluent that does not substantially contain sodium ions or chloride ions is used as the diluent.
血液のpHは、健常者では通常pH7.30~7.40程度で一定に保たれていることから、対象成分の分解や変性を防止するために、希釈液は、pH6.5~pH8.0、好ましくはpH7.0~pH7.5、更に好ましくはpH7.3~pH7.4のpH域で緩衝作用を有する緩衝液であることが好ましく、希釈液は、pHの変動を抑える緩衝成分を含有する緩衝液であることが好ましい。
Since the pH of blood is usually kept constant at about pH 7.30 to 7.40 in healthy individuals, the diluted solution is pH 6.5 to pH 8.0 in order to prevent decomposition and denaturation of the target component. Preferably, the buffer solution has a buffering action in the pH range of pH 7.0 to pH 7.5, more preferably pH 7.3 to pH 7.4, and the diluent contains a buffer component that suppresses fluctuations in pH. It is preferable to use a buffer solution.
従来、緩衝液の種類としては、酢酸緩衝液(Na)、リン酸緩衝液(Na)、クエン酸緩衝液(Na)、ホウ酸緩衝液(Na)、酒石酸緩衝液(Na)、Tris(トリス(ヒドロキシメチル)アミノエタン)緩衝液(Cl)、Hepes([2-[4-(2-ヒドロキシエチル)-1-ピペラジニル]エタンスルホン酸])緩衝液、リン酸緩衝生理食塩水(Na)等が知られている。これらの中でpH7.0~pH8.0付近の緩衝液としては、リン酸緩衝液、Tris緩衝液、Hepes緩衝液が代表的なものである。しかしながら、リン酸緩衝液はリン酸のナトリウム塩が含まれていること、Tris緩衝液は、解離pKaは8.08であるため、pH7.0~pH8.0付近で緩衝能を持たせるためには通常は塩酸と組み合わせて使用されること、Hepesのスルホン酸の解離のpKaは7.55であるが、イオン強度一定での緩衝溶液を調整するため、通常は水酸化ナトリウムと塩化ナトリウムとHEPESの混合物が用いられることから、これらはpHを一定に保つ作用を有する緩衝液としては有用であるが、本発明において外部標準物質として用いることが好ましい物質であるナトリウムイオンあるいは塩化物イオンを含有するため、キットが血液中に恒常的に存在する標準成分を用いて血液検体中の対象成分の濃度を分析するためのものである場合、適用は好ましくない。
Conventional buffer types include acetate buffer (Na), phosphate buffer (Na), citrate buffer (Na), borate buffer (Na), tartrate buffer (Na), Tris (Tris). (Hydroxymethyl) aminoethane) buffer (Cl), Hepes ([2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid]) buffer, phosphate buffered saline (Na), etc. Are known. Among these, phosphate buffers, Tris buffers, and Hepes buffers are representative examples of buffers around pH 7.0 to pH 8.0. However, the phosphate buffer contains a sodium salt of phosphate, and the Tris buffer has a dissociated pKa of 8.08, so that it has a buffer capacity around pH 7.0 to pH 8.0. Is usually used in combination with hydrochloric acid, and the pKa of dissociation of Hepes sulfonic acid is 7.55, but in order to adjust the buffer solution with constant ionic strength, usually sodium hydroxide, sodium chloride and HEPES These are useful as buffers having the action of keeping the pH constant, but contain sodium ions or chloride ions that are preferably used as external standard substances in the present invention. Therefore, the kit is for analyzing the concentration of the target component in the blood sample using the standard component that is constantly present in the blood. If the application is not preferable.
キットが血液中に恒常的に存在する標準成分を用いて 血液検体中の対象成分の濃度を分析するためのものである場合、用いる緩衝液としては、ナトリウムイオン又は塩化物イオンを含有しない(含有しないの意味は、すでに述べたとおりである。)ことが好ましい。このような緩衝液は好ましくは、2-アミノ-2-メチル-1-プロパノール(AMP)、2-エチルアミノエタノール、N-メチル-D-グルカミン、ジエタノールアミン、及びトリエタノールアミンからなる群から選択される少なくとも1種のアミノアルコール化合物、並びにGood’s緩衝液(グッドバッファー)でpKaが7.4付近の緩衝剤であるHEPESとも称する2-[4-(2-ヒドロキシエチル-1-ピペラジニル]エタンスルホン酸)(pKa=7.55)、TESとも称するN-トリス(ヒドロキシメチル)メチル-2-アミノエタンスルホン酸(pKa=7.50)、MOPSとも称する3-モルホリノプロパンスルホン酸(pKa=7.20)、及びBESとも称する(N,N-ビス(2-ヒドロキシエチル)-2-アミノエタンスルホン酸(pKa=7.15)からなる群から選択される緩衝剤を含む希釈液である。上記の中でも、2-アミノー2-メチル-1-プロパノール(AMP)とHEPES、TES、MOPS又はBESとの組み合わせが好ましく、さらに、2-アミノー2-メチル-1-プロパノール(AMP)とHEPESとの組み合わせが最も好ましい。なおpKaは、酸解離定数を表す。
When the kit is used for analyzing the concentration of a target component in a blood sample using a standard component that is constantly present in blood, the buffer solution used does not contain sodium ions or chloride ions (included) The meaning of not doing is as already mentioned.) Such a buffer is preferably selected from the group consisting of 2-amino-2-methyl-1-propanol (AMP), 2-ethylaminoethanol, N-methyl-D-glucamine, diethanolamine, and triethanolamine. 2- [4- (2-hydroxyethyl-1-piperazinyl] ethane, also referred to as HEPES, which is a Good's buffer (Good buffer) and has a pKa of around 7.4. Sulfonic acid) (pKa = 7.55), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (pKa = 7.50), also referred to as TES, 3-morpholinopropanesulfonic acid (pKa = 7), also referred to as MOPS .20), also referred to as BES (N, N-bis (2-hydroxyethyl) A diluent containing a buffer selected from the group consisting of -2-aminoethanesulfonic acid (pKa = 7.15), among which 2-amino-2-methyl-1-propanol (AMP) and HEPES, A combination with TES, MOPS or BES is preferable, and a combination of 2-amino-2-methyl-1-propanol (AMP) and HEPES is most preferable, where pKa represents an acid dissociation constant.
上記緩衝液を調製するためには、アミノアルコールとGood‘s緩衝液を1:2~2:1、好ましくは1:1.5~1.5:1、更に好ましくは1:1の濃度比で混合すればよい。緩衝液の濃度は限定されないが、アミノアルコール又はGood‘s緩衝液の濃度は、0.1~1000mmol/L、好ましくは、1~500mmol/L、さらに好ましくは10~100mmol/Lである。
In order to prepare the above buffer solution, 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. Can be mixed. 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.
緩衝液中には、分析対象成分を安定に保つことを目的にキレート剤、界面活性剤、抗菌剤、防腐剤、補酵素、糖類等が含有されていてもよい。キレート剤としては、エチレンジアミン四酢酸(EDTA)塩、クエン酸塩、シュウ酸塩等が挙げられる。界面活性剤としては、例えば、陽イオン界面活性剤、陰イオン界面活性剤、両性界面活性剤又は非イオン界面活性剤が挙げられる。防腐剤としては、例えば、アジ化ナトリウムや抗性物質等が挙げられる。補酵素としては、ピリドキサールリン酸、マグネシウム、亜鉛等が挙げられる。赤血球安定化剤の糖類としては、マンニトール、デキストロース、オリゴ糖等が挙げられる。特に、抗生物質の添加により、手指採血時に手指表面から一部混入する細菌の増殖を抑えることができ、細菌による生体成分の分解を抑制し、生体成分の安定化を図ることができる。
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. Examples of the chelating agent include ethylenediaminetetraacetic acid (EDTA) salt, citrate, and oxalate. Examples of the surfactant include a cationic surfactant, an anionic surfactant, an amphoteric surfactant, and a nonionic surfactant. Examples of the preservative include sodium azide and an antimicrobial substance. Examples of the coenzyme include pyridoxal phosphate, magnesium, zinc and the like. Examples of the saccharide of the erythrocyte stabilizer include mannitol, dextrose, oligosaccharide and the like. In particular, 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.
緩衝液はまた、血液中に存在しない標準成分を用いて対象成分を分析するためのキットにおいては、この血液中に存在しない標準成分を含む。後述する内部標準物質を含まず、血液分析の測定系に干渉を与えないことも重要である。
The buffer also contains a standard component that is not present in blood in a kit for analyzing a target component using a standard component that is not present in blood. It is also important not to include an internal standard substance, which will be described later, and not to interfere with the blood analysis measurement system.
全血を希釈するとの観点からは、緩衝液の浸透圧を血液と同等(285mOsm/kg(mOsm/kgは、溶液の水1kgが持つ浸透圧で、イオンのミリモル数をあらわす))又はそれ以上とすることにより血球の溶血を防止することができる。浸透圧は、対象成分の測定、及び血液中に恒常的に存在する標準成分の測定に影響しない塩類、糖類又は緩衝剤等により、等張に調整することができる。緩衝液の浸透圧は、浸透圧計により測定することができる。
From the standpoint of diluting whole blood, the osmotic pressure of the buffer solution is equivalent to that of blood (285 mOsm / kg (mOsm / kg is the osmotic pressure of 1 kg of water in the solution and represents the number of millimolar ions)) or more By doing so, hemolysis of blood cells can be prevented. 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 osmotic pressure of the buffer solution can be measured with an osmometer.
[希釈液の量、希釈倍率]
血液検査として、肝機能、腎機能、メタボリズムなど、特定の臓器、特定の疾患を検査する場合には、臓器や疾患に特有の複数の測定対象成分の情報を入手して、臓器の状態、生活習慣の予測などを行うために、一般的には、複数の測定対象成分の分析が同時に行われる。例えば、肝臓の状態を検査するためには、一般的には、ALT(アラニントランスアミナーゼ)、AST(アスパラギン酸アミノトランスフェラーゼ)、γ―GTP(γグルタミルトランスペプチダーゼ)、ALP(アルカリホスファターゼ)、総ビリルビン、総タンパク、アルブミン等、数種類以上もの物質の血液中の濃度が測定される。このように、複数の対象成分をいつの血液検体から測定するためには、再測定の可能性も考慮して、希釈された血液の量はある程度必要となる。従って、採取した血液を希釈する希釈液は、ある程度の量を確保することが重要である。侵襲性を少しでも低く血液を採取することを考慮すると、採血量100μL以上は避けたいので、従って、希釈倍率は7倍程度以上となる。 [Amount of dilution, dilution ratio]
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 or the like, generally, a plurality of components to be measured are analyzed simultaneously. For example, in order to examine the condition of the liver, generally, ALT (alanine transaminase), AST (aspartate aminotransferase), γ-GTP (γ glutamyl transpeptidase), ALP (alkaline phosphatase), total bilirubin, The concentration in the blood of several or more substances such as total protein and albumin is measured. Thus, in order to measure a plurality of target components from any blood sample, a certain amount of diluted blood is required in consideration of the possibility of remeasurement. Therefore, it is important to secure a certain amount of the diluent for diluting the collected blood. Considering that blood is collected with low invasiveness as much as possible, it is desirable to avoid a blood collection volume of 100 μL or more, and therefore the dilution factor is about 7 times or more.
血液検査として、肝機能、腎機能、メタボリズムなど、特定の臓器、特定の疾患を検査する場合には、臓器や疾患に特有の複数の測定対象成分の情報を入手して、臓器の状態、生活習慣の予測などを行うために、一般的には、複数の測定対象成分の分析が同時に行われる。例えば、肝臓の状態を検査するためには、一般的には、ALT(アラニントランスアミナーゼ)、AST(アスパラギン酸アミノトランスフェラーゼ)、γ―GTP(γグルタミルトランスペプチダーゼ)、ALP(アルカリホスファターゼ)、総ビリルビン、総タンパク、アルブミン等、数種類以上もの物質の血液中の濃度が測定される。このように、複数の対象成分をいつの血液検体から測定するためには、再測定の可能性も考慮して、希釈された血液の量はある程度必要となる。従って、採取した血液を希釈する希釈液は、ある程度の量を確保することが重要である。侵襲性を少しでも低く血液を採取することを考慮すると、採血量100μL以上は避けたいので、従って、希釈倍率は7倍程度以上となる。 [Amount of dilution, dilution ratio]
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 or the like, generally, a plurality of components to be measured are analyzed simultaneously. For example, in order to examine the condition of the liver, generally, ALT (alanine transaminase), AST (aspartate aminotransferase), γ-GTP (γ glutamyl transpeptidase), ALP (alkaline phosphatase), total bilirubin, The concentration in the blood of several or more substances such as total protein and albumin is measured. Thus, in order to measure a plurality of target components from any blood sample, a certain amount of diluted blood is required in consideration of the possibility of remeasurement. Therefore, it is important to secure a certain amount of the diluent for diluting the collected blood. Considering that blood is collected with low invasiveness as much as possible, it is desirable to avoid a blood collection volume of 100 μL or more, and therefore the dilution factor is about 7 times or more.
[キャピラリー]
本発明のキットは、血液採取器具として、キャピラリーを採用する。微量の血液検体を希釈液で希釈して成分を定量分析する方法において、希釈液中の内部標準物質として、例えばグリセロール-3-リン酸を使用する場合は、血液検体が十分量、具体的には100μL程度あれば、精度高く希釈倍率を算出できる。また外部標準物質を使って希釈倍率を算出する場合にも、血液量が少なければ希釈倍率が高くなり、キットの部材から希釈中に溶出する外部標準物質のコンタミの影響を受け易くなり、定量分析の測定精度が落ちる。本発明者らは、微量の血液検体を希釈液で希釈して成分の濃度を分析する上で、例えば100μLを超えない血液採取量であって、しかも部材から希釈液中に溶出する外部標準物質のコンタミの影響力を抑え、精度の高い検査結果を得るための手段を鋭意検討した。その結果、血液採取器具として、キャピラリーを用いることで、血液採取量を一定値採取することが最適であることを見出した。 [Capillary]
The kit of the present invention employs a capillary as a blood collection device. In a method for quantitative analysis of components by diluting a trace amount of blood sample with a diluent, when glycerol-3-phosphate is used as an internal standard substance in the diluent, a sufficient amount of blood sample, specifically, If about 100 μL, the dilution factor can be calculated with high accuracy. In addition, when calculating the dilution ratio using an external standard substance, if the blood volume is small, the dilution ratio will be high, and it will be easily affected by contamination of the external standard substance that is eluted from the kit components during dilution. The measurement accuracy is reduced. The present inventors have analyzed a concentration of a component by diluting a trace amount of blood sample with a diluent, and, for example, an external standard substance that has a blood collection amount not exceeding 100 μL and elutes from a member into the diluent. We have intensively studied ways to reduce the influence of contamination and obtain highly accurate test results. As a result, it has been found that it is optimal to collect a constant amount of blood by using a capillary as a blood collection device.
本発明のキットは、血液採取器具として、キャピラリーを採用する。微量の血液検体を希釈液で希釈して成分を定量分析する方法において、希釈液中の内部標準物質として、例えばグリセロール-3-リン酸を使用する場合は、血液検体が十分量、具体的には100μL程度あれば、精度高く希釈倍率を算出できる。また外部標準物質を使って希釈倍率を算出する場合にも、血液量が少なければ希釈倍率が高くなり、キットの部材から希釈中に溶出する外部標準物質のコンタミの影響を受け易くなり、定量分析の測定精度が落ちる。本発明者らは、微量の血液検体を希釈液で希釈して成分の濃度を分析する上で、例えば100μLを超えない血液採取量であって、しかも部材から希釈液中に溶出する外部標準物質のコンタミの影響力を抑え、精度の高い検査結果を得るための手段を鋭意検討した。その結果、血液採取器具として、キャピラリーを用いることで、血液採取量を一定値採取することが最適であることを見出した。 [Capillary]
The kit of the present invention employs a capillary as a blood collection device. In a method for quantitative analysis of components by diluting a trace amount of blood sample with a diluent, when glycerol-3-phosphate is used as an internal standard substance in the diluent, a sufficient amount of blood sample, specifically, If about 100 μL, the dilution factor can be calculated with high accuracy. In addition, when calculating the dilution ratio using an external standard substance, if the blood volume is small, the dilution ratio will be high, and it will be easily affected by contamination of the external standard substance that is eluted from the kit components during dilution. The measurement accuracy is reduced. The present inventors have analyzed a concentration of a component by diluting a trace amount of blood sample with a diluent, and, for example, an external standard substance that has a blood collection amount not exceeding 100 μL and elutes from a member into the diluent. We have intensively studied ways to reduce the influence of contamination and obtain highly accurate test results. As a result, it has been found that it is optimal to collect a constant amount of blood by using a capillary as a blood collection device.
以下、図面に示した実施例を参照しつつ、本発明に係るキャピラリーの実施の形態について説明する。
Hereinafter, an embodiment of a capillary according to the present invention will be described with reference to the examples shown in the drawings.
〔キャピラリーの構造等〕
図2は、本発明に係るキャピラリーの一実施例の断面図である。キャピラリーは、通常、内径が略一定の細い筒状本体201からなる管である。その両端202及び203の一方を血液に接触させると、毛細管現象(重力に逆らって液体がキャピラリー内部に浸透していく現象)により、血液をキャピラリー内部に採取することができる。そして適当な量の血液を吸い上げたときに、キャピラリー採血器具の他方の端を指で押さえる等して封止し、吸引を停止する。次いで、端を指で押さえたままの状態として内部に血液検体を保持したキャピラリーを、血液検体を希釈するための希釈液が収容された収容器具まで運ぶ。そしてキャピラリーの一端を収容器具内に保持した後、端を押さえていた指を離すことで、血液検体を収容器具内に収容する。キャピラリーは、指で押さえて封止する代わりに、キャップ、シリコンパテ、パラフィン樹脂等のシーラー、粘土等を使用して封止してもよい。 [Capillary structure, etc.]
FIG. 2 is a cross-sectional view of an embodiment of a capillary according to the present invention. The capillary is usually a tube composed of a thincylindrical body 201 having a substantially constant inner diameter. When one of the ends 202 and 203 is brought into contact with blood, blood can be collected inside the capillary by a capillary phenomenon (a phenomenon in which liquid permeates into the capillary against gravity). When an appropriate amount of blood is sucked up, the other end of the capillary blood sampling device is sealed with a finger or the like, and suction is stopped. Next, the capillary holding the blood sample with the end held with a finger is carried to a storage device that stores a diluent for diluting the blood sample. Then, after holding one end of the capillary in the storage device, the finger holding the end is released, and the blood sample is stored in the storage device. The capillaries may be sealed using a sealer such as a cap, a silicon putty or a paraffin resin, clay, or the like, instead of being pressed with a finger and sealed.
図2は、本発明に係るキャピラリーの一実施例の断面図である。キャピラリーは、通常、内径が略一定の細い筒状本体201からなる管である。その両端202及び203の一方を血液に接触させると、毛細管現象(重力に逆らって液体がキャピラリー内部に浸透していく現象)により、血液をキャピラリー内部に採取することができる。そして適当な量の血液を吸い上げたときに、キャピラリー採血器具の他方の端を指で押さえる等して封止し、吸引を停止する。次いで、端を指で押さえたままの状態として内部に血液検体を保持したキャピラリーを、血液検体を希釈するための希釈液が収容された収容器具まで運ぶ。そしてキャピラリーの一端を収容器具内に保持した後、端を押さえていた指を離すことで、血液検体を収容器具内に収容する。キャピラリーは、指で押さえて封止する代わりに、キャップ、シリコンパテ、パラフィン樹脂等のシーラー、粘土等を使用して封止してもよい。 [Capillary structure, etc.]
FIG. 2 is a cross-sectional view of an embodiment of a capillary according to the present invention. The capillary is usually a tube composed of a thin
キャピラリーの内径は、血球の大きさや毛細管現象が生じること等を考慮して0.5~2.0mmであることが好ましい。キャピラリーの長さは、扱いやすさ等を考慮して、5~15cmであることが好ましく、血液検査キットを構成することを考慮するとコンパクトであることが望ましいことから、5~10cmであることがより好ましい。キャピラリーで採取することができる血液の容量は、例えば内径が1.1mm~1.2mmである場合、長さが7.5cmあれば、70μLの血液採取が可能である。
The inner diameter of the capillary is preferably 0.5 to 2.0 mm in consideration of the size of blood cells and the occurrence of capillary action. The length of the capillary is preferably 5 to 15 cm in consideration of ease of handling and the like, and is preferably 5 to 10 cm because it is desirable to be compact in consideration of constituting a blood test kit. More preferred. As for the volume of blood that can be collected with a capillary, for example, when the inner diameter is 1.1 mm to 1.2 mm and the length is 7.5 cm, 70 μL of blood can be collected.
〔目盛、ストッパーを有するキャピラリー〕
図3は、採取した血液検体の容量を確認するための目盛が付されているキャピラリーの一実施例である。キャピラリーにより採取することができる血液検体の最大容量は、理論的にはキャピラリーの内部を一杯に満たす量であるが、キャピラリーには、採取する血液検体の容量範囲の下限を示す位置と上限を示す位置とに、下限目盛204及び上限目盛205を付すことができ、それにより採取した血液検体の容量が適切か否かを判断することができる。例えば、65μL程度で血液検体が採取されることが好ましい場合、±10μLである55μLと75μLに目盛をつけるとよい。もちろん、中心値の65μLにも目盛(中心目盛206)をつけてもよいが、下限又は上限を示す目盛と見誤らないように、外観が異なる目盛をつけることが好ましい。採取する血液検体の容量範囲とは、血液検体に含まれる対象成分の分析を精度よく行うために許容される血液検体の容量範囲であり、上記の例では、55~75μLである。 [Scale, capillary with stopper]
FIG. 3 shows an example of a capillary with a scale for confirming the volume of the collected blood sample. The maximum volume of a blood sample that can be collected by a capillary is theoretically the amount that fills the inside of the capillary, but the capillary indicates the position and upper limit that indicate the lower limit of the volume range of the collected blood sample. The position can be provided with alower limit scale 204 and an upper limit scale 205, whereby it can be determined whether or not the volume of the collected blood sample is appropriate. For example, when it is preferable to collect a blood sample at about 65 μL, it is preferable to calibrate 55 μL and 75 μL, which are ± 10 μL. Of course, a scale (center scale 206) may be attached to the center value of 65 μL. However, it is preferable to attach a scale having a different appearance so as not to misunderstand the lower limit or the upper limit. The volume range of the blood sample to be collected is the volume range of the blood sample that is allowed to accurately analyze the target component contained in the blood sample. In the above example, the volume range is 55 to 75 μL.
図3は、採取した血液検体の容量を確認するための目盛が付されているキャピラリーの一実施例である。キャピラリーにより採取することができる血液検体の最大容量は、理論的にはキャピラリーの内部を一杯に満たす量であるが、キャピラリーには、採取する血液検体の容量範囲の下限を示す位置と上限を示す位置とに、下限目盛204及び上限目盛205を付すことができ、それにより採取した血液検体の容量が適切か否かを判断することができる。例えば、65μL程度で血液検体が採取されることが好ましい場合、±10μLである55μLと75μLに目盛をつけるとよい。もちろん、中心値の65μLにも目盛(中心目盛206)をつけてもよいが、下限又は上限を示す目盛と見誤らないように、外観が異なる目盛をつけることが好ましい。採取する血液検体の容量範囲とは、血液検体に含まれる対象成分の分析を精度よく行うために許容される血液検体の容量範囲であり、上記の例では、55~75μLである。 [Scale, capillary with stopper]
FIG. 3 shows an example of a capillary with a scale for confirming the volume of the collected blood sample. The maximum volume of a blood sample that can be collected by a capillary is theoretically the amount that fills the inside of the capillary, but the capillary indicates the position and upper limit that indicate the lower limit of the volume range of the collected blood sample. The position can be provided with a
キャピラリーの目盛を付した位置を含む部分の内径は、他の部分より小さくしてもよい。内径が小さい部分、すなわち管が細い部分に目盛りがあると、容量をより正確に採取することができるからである。また内径を小さくすることによりキャピラリー内の血液の上昇スピードが速くなるため、封止するタイミングがとりやすいからである。
The inner diameter of the portion including the position where the capillaries are marked may be smaller than other portions. This is because the volume can be collected more accurately if there is a scale in a portion having a small inner diameter, that is, a portion where the tube is thin. Moreover, since the rising speed of the blood in the capillary is increased by reducing the inner diameter, it is easy to take the sealing timing.
下限を示す位置に付す目盛4は、これより少ないと検査精度に影響するため、目盛り線が太く見易いことが好ましい。このようにキャピラリーの上下で非対称としておくことで、採血者はキャピラリーの血液に接触させるための血液接触端212を、他方の端である封止端213と取り違えてしまうリスクを減じることもできる。一方、上限を超えての血液採取は、部材からの外部標準物質のコンタミによる影響をより抑える方向ではあるが、血液採取量が下限に近い場合と比べると、ばらつきは増える傾向にある。血液採取量をより確実に上限以下とするためには、例えば、キャピラリーの上限を示す位置(例えば図3の上限目盛205)の内部に、穴の開いたストッパーを設け、このストッパーに血液が達したときにキャピラリーの上端を指等で封止して吸引を停止することとしてもよい。ストッパーを用いることで、たとえ指等で封止するタイミングが少しずれたとしても、血液の吸引速度が抑えられ、より確実に上限以下の容量の血液を採取することができる。また、複数の目盛を付すと採血を行う者が混乱する原因となり得るので、キャピラリーには目盛を付さず、穴あきストッパーだけを設けることも好ましい。
Since the scale 4 attached to the position indicating the lower limit affects the inspection accuracy if it is less than this, it is preferable that the scale line is thick and easy to see. In this way, by making the upper and lower capillaries asymmetrical, the blood sampler can also reduce the risk that the blood contact end 212 for contacting the blood in the capillary will be mistaken for the sealing end 213 which is the other end. On the other hand, blood collection exceeding the upper limit tends to suppress the influence of contamination of the external standard substance from the member, but the variation tends to increase as compared with the case where the blood collection amount is close to the lower limit. In order to make the amount of blood collected more reliably below the upper limit, for example, a stopper with a hole is provided inside the position indicating the upper limit of the capillary (for example, the upper limit scale 205 in FIG. 3), and blood reaches this stopper. In this case, the suction may be stopped by sealing the upper end of the capillary with a finger or the like. By using the stopper, even if the timing of sealing with a finger or the like is slightly shifted, the blood suction speed can be suppressed, and blood with a volume below the upper limit can be collected more reliably. Moreover, since attaching a plurality of scales may cause confusion for a person who collects blood, it is also preferable to provide only a perforated stopper without providing a scale on the capillary.
またキャピラリーの血液に接触させる側の端212の内径を他の部分より小さくしてもよい。これにより毛細管現象を起こりやすくすることができる。このとき、キャピラリーの血液に接触させる側の端部207は、端に行くほど内径が小さくなるテーパー型としてもよい。
Also, the inner diameter of the end 212 on the side of the capillary that comes into contact with blood may be made smaller than that of other portions. As a result, capillary action can easily occur. At this time, the end portion 207 on the side of the capillary that comes into contact with blood may be a tapered type whose inner diameter becomes smaller toward the end.
〔キャピラリーの形状の例〕
さらにキャピラリーは、定められた容量の血液検体を採取するための形状とすることができる。このようなキャピラリーの例は、内部が血液検体で一杯に満たされると、定められた容量となるように設計されたものである。他の例は、定められた容量の血液検体を採取すると、それ以上採取できないような形状であるものであり、具体的には、例えば図4に示したように、キャピラリーのある位置から内径を急激に大きくし(例えば内径を2mmを超えるようにし)、その位置を超えると毛細管現象が生じ難くなるようにしたものである。あるいは、キャピラリーのある位置から内径を極小さくすることで、目盛りのズレに対する容量の変化が小さくなり、計量精度を高めることが可能となる。このような形状のキャピラリーには、採取する血液容量の上限を示す位置又は下限を示す位置に目盛を付してもよい。内径を変える位置は、目盛を付した位置又は目盛を超えた位置とすることができるが、内径を大きくする場合は、後者が好ましい。内径が大きくなる手前の、管が細い部分に目盛りがあると、容量を正確に判断しやすいからである。なお、目盛を超えた位置とは、目盛まで採取した場合の容量より1μLから7μL分、好ましくは3μLから5μL分、増加した容量を示す位置をいう。 [Example of capillary shape]
Furthermore, the capillary can be shaped to collect a blood sample of a defined volume. An example of such a capillary is designed to have a defined capacity when the interior is filled with a blood sample. In another example, when a blood sample of a predetermined volume is collected, the shape cannot be collected any more. Specifically, as shown in FIG. 4, for example, the inner diameter is changed from a certain position of the capillary. The size is rapidly increased (for example, the inner diameter exceeds 2 mm), and the capillary phenomenon hardly occurs when the position is exceeded. Alternatively, by reducing the inner diameter from a certain position of the capillary, the change in capacity with respect to the scale deviation is reduced, and the measurement accuracy can be increased. The capillary having such a shape may be graduated at a position indicating the upper limit or the lower limit of the blood volume to be collected. The position for changing the inner diameter can be a position with a scale or a position beyond the scale, but the latter is preferable when the inner diameter is increased. This is because if there is a scale in the narrow part of the tube before the inner diameter increases, the capacity can be judged accurately. In addition, the position beyond the scale means a position showing a volume increased by 1 μL to 7 μL, preferably 3 μL to 5 μL from the volume when the scale is collected.
さらにキャピラリーは、定められた容量の血液検体を採取するための形状とすることができる。このようなキャピラリーの例は、内部が血液検体で一杯に満たされると、定められた容量となるように設計されたものである。他の例は、定められた容量の血液検体を採取すると、それ以上採取できないような形状であるものであり、具体的には、例えば図4に示したように、キャピラリーのある位置から内径を急激に大きくし(例えば内径を2mmを超えるようにし)、その位置を超えると毛細管現象が生じ難くなるようにしたものである。あるいは、キャピラリーのある位置から内径を極小さくすることで、目盛りのズレに対する容量の変化が小さくなり、計量精度を高めることが可能となる。このような形状のキャピラリーには、採取する血液容量の上限を示す位置又は下限を示す位置に目盛を付してもよい。内径を変える位置は、目盛を付した位置又は目盛を超えた位置とすることができるが、内径を大きくする場合は、後者が好ましい。内径が大きくなる手前の、管が細い部分に目盛りがあると、容量を正確に判断しやすいからである。なお、目盛を超えた位置とは、目盛まで採取した場合の容量より1μLから7μL分、好ましくは3μLから5μL分、増加した容量を示す位置をいう。 [Example of capillary shape]
Furthermore, the capillary can be shaped to collect a blood sample of a defined volume. An example of such a capillary is designed to have a defined capacity when the interior is filled with a blood sample. In another example, when a blood sample of a predetermined volume is collected, the shape cannot be collected any more. Specifically, as shown in FIG. 4, for example, the inner diameter is changed from a certain position of the capillary. The size is rapidly increased (for example, the inner diameter exceeds 2 mm), and the capillary phenomenon hardly occurs when the position is exceeded. Alternatively, by reducing the inner diameter from a certain position of the capillary, the change in capacity with respect to the scale deviation is reduced, and the measurement accuracy can be increased. The capillary having such a shape may be graduated at a position indicating the upper limit or the lower limit of the blood volume to be collected. The position for changing the inner diameter can be a position with a scale or a position beyond the scale, but the latter is preferable when the inner diameter is increased. This is because if there is a scale in the narrow part of the tube before the inner diameter increases, the capacity can be judged accurately. In addition, the position beyond the scale means a position showing a volume increased by 1 μL to 7 μL, preferably 3 μL to 5 μL from the volume when the scale is collected.
〔抗凝固剤含有キャピラリー〕
本発明に用いられるキャピラリーは、ランセット等で穿刺して得られる血液を直接キャピラリーで採取する場合には、内部に抗凝固剤を有していることが好ましい。このようなキャピラリーの例は、例えばヘパリン又はEDTA塩等の抗凝固剤が、キャピラリー内面に塗布されているものである。抗凝固剤としては、種々のものを用いることができるが、分析の際に外部標準を用いる場合は、その外部標準を実質的に含まないものが選択される。 [Anti-coagulant-containing capillary]
The capillary used in the present invention preferably has an anticoagulant inside when blood obtained by puncturing with a lancet or the like is directly collected by the capillary. An example of such a capillary is one in which an anticoagulant such as heparin or EDTA salt is applied to the inner surface of the capillary. Various anticoagulants can be used, but when an external standard is used in the analysis, one that does not substantially contain the external standard is selected.
本発明に用いられるキャピラリーは、ランセット等で穿刺して得られる血液を直接キャピラリーで採取する場合には、内部に抗凝固剤を有していることが好ましい。このようなキャピラリーの例は、例えばヘパリン又はEDTA塩等の抗凝固剤が、キャピラリー内面に塗布されているものである。抗凝固剤としては、種々のものを用いることができるが、分析の際に外部標準を用いる場合は、その外部標準を実質的に含まないものが選択される。 [Anti-coagulant-containing capillary]
The capillary used in the present invention preferably has an anticoagulant inside when blood obtained by puncturing with a lancet or the like is directly collected by the capillary. An example of such a capillary is one in which an anticoagulant such as heparin or EDTA salt is applied to the inner surface of the capillary. Various anticoagulants can be used, but when an external standard is used in the analysis, one that does not substantially contain the external standard is selected.
〔合成樹脂製キャピラリー〕
本発明に用いられるキャピラリーは、ガラス製であっても合成樹脂製であってもよい。また他の素材の上に合成樹脂でコーティングされていてもよい。なお本明細書では、プラスチックを合成樹脂と同義に用いている。キャピラリーは、ガラス製よりも破損が起きにくいとの安全上の観点から、プラスチック製であることが好ましい。例えば、ポリエチレン、ポリプロピレン、ポリ塩化ビニル、ポリ塩化ビニリデン、ポリスチレン、ポリ酢酸ビニル、ポリウレタン、アクリロニトリルブタジエンスチレン樹脂(ABS樹脂)、アクリロニトリルスチレン樹脂(AS樹脂)、アクリル樹脂 (PMMA)、ポリカーボネート、シリコーン樹脂等が挙げられる。 [Synthetic resin capillaries]
The capillary used in the present invention may be made of glass or synthetic resin. In addition, other materials may be coated with a synthetic resin. In this specification, plastic is used synonymously with synthetic resin. The capillaries are preferably made of plastic from the viewpoint of safety that damage is less likely to occur than glass. For example, polyethylene, polypropylene, polyvinyl chloride, polyvinylidene chloride, polystyrene, polyvinyl acetate, polyurethane, acrylonitrile butadiene styrene resin (ABS resin), acrylonitrile styrene resin (AS resin), acrylic resin (PMMA), polycarbonate, silicone resin, etc. Is mentioned.
本発明に用いられるキャピラリーは、ガラス製であっても合成樹脂製であってもよい。また他の素材の上に合成樹脂でコーティングされていてもよい。なお本明細書では、プラスチックを合成樹脂と同義に用いている。キャピラリーは、ガラス製よりも破損が起きにくいとの安全上の観点から、プラスチック製であることが好ましい。例えば、ポリエチレン、ポリプロピレン、ポリ塩化ビニル、ポリ塩化ビニリデン、ポリスチレン、ポリ酢酸ビニル、ポリウレタン、アクリロニトリルブタジエンスチレン樹脂(ABS樹脂)、アクリロニトリルスチレン樹脂(AS樹脂)、アクリル樹脂 (PMMA)、ポリカーボネート、シリコーン樹脂等が挙げられる。 [Synthetic resin capillaries]
The capillary used in the present invention may be made of glass or synthetic resin. In addition, other materials may be coated with a synthetic resin. In this specification, plastic is used synonymously with synthetic resin. The capillaries are preferably made of plastic from the viewpoint of safety that damage is less likely to occur than glass. For example, polyethylene, polypropylene, polyvinyl chloride, polyvinylidene chloride, polystyrene, polyvinyl acetate, polyurethane, acrylonitrile butadiene styrene resin (ABS resin), acrylonitrile styrene resin (AS resin), acrylic resin (PMMA), polycarbonate, silicone resin, etc. Is mentioned.
キャピラリーがガラス製の場合、ガラスの水接触角は、は10度以下であるためにキャピラリーの内壁はそのままでも十分な親水性があり、キャピラリーの毛細管現象が十分に発揮され、すばやく血液を採取できる。一方、キャピラリーがプラスチック製である場合、例えばポリカーボネートの水接触角は、85度であり、アクリルの水接触角は70度である。血液をすばやく採取するためには、キャピラリー内壁を親水性に処理されていることが好ましい。親水性処理として、例えば、プラズマ処理による親水化が可能である。
When the capillary is made of glass, the water contact angle of the glass is 10 degrees or less, so that the inner wall of the capillary is sufficiently hydrophilic even if it is intact, and the capillary action of the capillary is sufficiently exhibited and blood can be collected quickly. . On the other hand, when the capillary is made of plastic, for example, the water contact angle of polycarbonate is 85 degrees, and the water contact angle of acrylic is 70 degrees. In order to quickly collect blood, the inner wall of the capillary is preferably treated to be hydrophilic. As the hydrophilic treatment, for example, hydrophilic treatment by plasma treatment is possible.
プラスチック製であることは、上述したように血液に接触させる端部をテーパー型にする加工や、キャピラリーの内径を変える加工が容易であるとの観点からも好ましい。
It is preferable that it is made of plastic from the viewpoint that it is easy to process the tapered end of the end contacting the blood or to change the inner diameter of the capillary as described above.
〔色つきキャピラリー等〕
自己採血でキャピラリーを使用する場合、血液の赤色を凝視することに抵抗がある人もいる。一般的に医療従事者や研究者が使用する血液を採取するためのキャピラリーは透明であるが、本発明で使用するキャピラリーの好ましい態様の一つは、血液の赤色が目立たないように色を付けたものである。キャピラリーがプラスチック製であれば、着色は容易である。あるいは、キャピラリーは透明であるが、採取した血液が赤色以外の色に見えるように、血液中のヘモグロビンの吸収波長領域を遮蔽できる透過媒体を用いた眼鏡等を、血液検査キットの構成要素の一つとしてもよい。 [Colored capillaries, etc.]
When using a capillary for self-collection, some people are resistant to staring at the red color of the blood. Generally, the capillary for collecting blood used by medical professionals and researchers is transparent, but one of the preferred embodiments of the capillary used in the present invention is colored so that the red color of blood is not noticeable. It is a thing. If the capillary is made of plastic, coloring is easy. Alternatively, although the capillary is transparent, glasses or the like using a transmission medium capable of shielding the absorption wavelength region of hemoglobin in the blood so that the collected blood looks a color other than red is one of the components of the blood test kit. It's okay.
自己採血でキャピラリーを使用する場合、血液の赤色を凝視することに抵抗がある人もいる。一般的に医療従事者や研究者が使用する血液を採取するためのキャピラリーは透明であるが、本発明で使用するキャピラリーの好ましい態様の一つは、血液の赤色が目立たないように色を付けたものである。キャピラリーがプラスチック製であれば、着色は容易である。あるいは、キャピラリーは透明であるが、採取した血液が赤色以外の色に見えるように、血液中のヘモグロビンの吸収波長領域を遮蔽できる透過媒体を用いた眼鏡等を、血液検査キットの構成要素の一つとしてもよい。 [Colored capillaries, etc.]
When using a capillary for self-collection, some people are resistant to staring at the red color of the blood. Generally, the capillary for collecting blood used by medical professionals and researchers is transparent, but one of the preferred embodiments of the capillary used in the present invention is colored so that the red color of blood is not noticeable. It is a thing. If the capillary is made of plastic, coloring is easy. Alternatively, although the capillary is transparent, glasses or the like using a transmission medium capable of shielding the absorption wavelength region of hemoglobin in the blood so that the collected blood looks a color other than red is one of the components of the blood test kit. It's okay.
一方、酸素と結びついたヘモグロビンは赤く見えるが、波長600nmから長波長側に向けて急峻に吸収し難くなる。従って、例えば、680nmより長い波長を吸収するフィルターを用いることで、血液の赤色を目立たなくする(オレンジ色のように見える)ことができる。
On the other hand, hemoglobin combined with oxygen looks red, but it is difficult to absorb sharply from the wavelength 600 nm toward the long wavelength side. Therefore, for example, by using a filter that absorbs wavelengths longer than 680 nm, the red color of blood can be made inconspicuous (looks like an orange color).
[血液検体の希釈物から血漿を分離回収するための分離器具]
本発明のキットにより採取された血液検体は、分析が行われるまで、希釈された状態で長時間経過する可能性がある。その間に、例えば赤血球の溶血が起こると、血球内に存在する物質や酵素などが血漿あるいは血清中に溶出して検査結果に影響を与えたり、溶出したヘモグロビンが有する吸収により、分析対象成分の光学的な吸収などの光情報で分析対象成分量を測定する場合に影響を及ぼす可能性がある。従って、溶血を防止することが好ましい。そのため、血液検体の希釈物から血漿を分離回収するための分離器具をキットに含む態様が好ましい。分離器具の好ましい例は、分離膜である。分離膜は、例えば血液検体の希釈物に圧力を加えることによって、血球成分は分離膜で捕獲し、血漿成分を通過させて、血球を分離して血漿成分を回収するように用いることができる。この場合、抗凝固剤を用いることが好ましい。また、測定の精度を確保するために、分離膜を通過した血漿が血球側へ逆流しないことが好ましく、そのためには具体的には、特開2003-270239号公報に記載の、逆流防止手段をキットの構成要素とすることができる。 [Separation device for separating and collecting plasma from diluted blood samples]
A blood sample collected by the kit of the present invention may elapse for a long time in a diluted state until analysis is performed. In the meantime, for example, when hemolysis of erythrocytes occurs, substances or enzymes present in the blood cells elute into the plasma or serum and affect the test results, or the absorption of the eluted hemoglobin causes the optical properties of the analyte to be analyzed. This may have an impact on the measurement of the amount of analysis target components using optical information such as typical absorption. Therefore, it is preferable to prevent hemolysis. Therefore, it is preferable that the kit includes a separation device for separating and collecting plasma from a diluted blood sample. A preferred example of the separation device is a separation membrane. The separation membrane can be used, for example, by applying pressure to a diluted blood sample to capture blood cell components with the separation membrane, allowing plasma components to pass through, separating blood cells, and collecting plasma components. In this case, it is preferable to use an anticoagulant. In order to ensure measurement accuracy, it is preferable that the plasma that has passed through the separation membrane does not flow back to the blood cell side. Specifically, for this purpose, a backflow prevention means described in JP-A-2003-270239 is used. It can be a component of a kit.
本発明のキットにより採取された血液検体は、分析が行われるまで、希釈された状態で長時間経過する可能性がある。その間に、例えば赤血球の溶血が起こると、血球内に存在する物質や酵素などが血漿あるいは血清中に溶出して検査結果に影響を与えたり、溶出したヘモグロビンが有する吸収により、分析対象成分の光学的な吸収などの光情報で分析対象成分量を測定する場合に影響を及ぼす可能性がある。従って、溶血を防止することが好ましい。そのため、血液検体の希釈物から血漿を分離回収するための分離器具をキットに含む態様が好ましい。分離器具の好ましい例は、分離膜である。分離膜は、例えば血液検体の希釈物に圧力を加えることによって、血球成分は分離膜で捕獲し、血漿成分を通過させて、血球を分離して血漿成分を回収するように用いることができる。この場合、抗凝固剤を用いることが好ましい。また、測定の精度を確保するために、分離膜を通過した血漿が血球側へ逆流しないことが好ましく、そのためには具体的には、特開2003-270239号公報に記載の、逆流防止手段をキットの構成要素とすることができる。 [Separation device for separating and collecting plasma from diluted blood samples]
A blood sample collected by the kit of the present invention may elapse for a long time in a diluted state until analysis is performed. In the meantime, for example, when hemolysis of erythrocytes occurs, substances or enzymes present in the blood cells elute into the plasma or serum and affect the test results, or the absorption of the eluted hemoglobin causes the optical properties of the analyte to be analyzed. This may have an impact on the measurement of the amount of analysis target components using optical information such as typical absorption. Therefore, it is preferable to prevent hemolysis. Therefore, it is preferable that the kit includes a separation device for separating and collecting plasma from a diluted blood sample. A preferred example of the separation device is a separation membrane. The separation membrane can be used, for example, by applying pressure to a diluted blood sample to capture blood cell components with the separation membrane, allowing plasma components to pass through, separating blood cells, and collecting plasma components. In this case, it is preferable to use an anticoagulant. In order to ensure measurement accuracy, it is preferable that the plasma that has passed through the separation membrane does not flow back to the blood cell side. Specifically, for this purpose, a backflow prevention means described in JP-A-2003-270239 is used. It can be a component of a kit.
本発明のキットは、100μL以下の採血量であっても、測定精度よく分析対象成分を分析できる方法を実現可能とするものであり、患者に、100μL以下の少ない採血量でも精度よく測定することが可能であることや、キャピラリーのどの位置まで血液を採取すべきか等の情報が記載された取り扱い説明書を含むキットであることが好ましい。
The kit of the present invention makes it possible to realize a method capable of analyzing a component to be analyzed with high measurement accuracy even with a blood collection volume of 100 μL or less, and to accurately measure a patient with a small blood collection volume of 100 μL or less. It is preferable that the kit includes an instruction manual in which information such as the possibility of blood collection and to which position of the capillary the blood should be collected is described.
[キットの具体例]
好ましい態様の一つにおいて、キットは、キャピラリーのほか、希釈液、希釈液が収容された第一の収容器具(血液検体の希釈物を収容するための収容器具でもある。)、希釈液で希釈された血液検体から血漿を分離回収するための分離器具、分離器具を保持するための保持器具、回収した血漿を収容するための第二の収容器具、及び収容した血漿を第二の収容器具内に維持するための封止器具を含む。キャピラリー以外の具体的な器具としては、例えば、特許第3597827号公報の図1から図13に記載された器具を使用することができる。特許第3597827号公報の図1を、本願の図1として援用する。 [Specific examples of kit]
In one of the preferred embodiments, the kit is not only a capillary but also a diluent, a first storage device containing the diluent (also a storage device for storing a diluted blood sample), and diluted with the diluent. Separation device for separating and recovering plasma from the collected blood sample, holding device for holding the separation device, second storage device for storing the collected plasma, and the stored plasma in the second storage device Including a sealing device for maintaining As a specific instrument other than the capillary, for example, the instrument described 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.
好ましい態様の一つにおいて、キットは、キャピラリーのほか、希釈液、希釈液が収容された第一の収容器具(血液検体の希釈物を収容するための収容器具でもある。)、希釈液で希釈された血液検体から血漿を分離回収するための分離器具、分離器具を保持するための保持器具、回収した血漿を収容するための第二の収容器具、及び収容した血漿を第二の収容器具内に維持するための封止器具を含む。キャピラリー以外の具体的な器具としては、例えば、特許第3597827号公報の図1から図13に記載された器具を使用することができる。特許第3597827号公報の図1を、本願の図1として援用する。 [Specific examples of kit]
In one of the preferred embodiments, the kit is not only a capillary but also a diluent, a first storage device containing the diluent (also a storage device for storing a diluted blood sample), and diluted with the diluent. Separation device for separating and recovering plasma from the collected blood sample, holding device for holding the separation device, second storage device for storing the collected plasma, and the stored plasma in the second storage device Including a sealing device for maintaining As a specific instrument other than the capillary, for example, the instrument described 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.
血液分離器具1は採血容器2(希釈液が収容された収容器具、第一の収容器具ということもある。血液検体の希釈物を収容するための収容器具でもある。)と、採血容器2に嵌挿可能な筒体3(回収した血漿を収容するための第二の収容器具)と、筒体3に冠着可能なキャップピストン4と、キャップピストン4の下端に設けられた密閉蓋5(封止器具)とを備え、使用前は、図1に示すように、採血容器2の上端開口部はキャップ6によりパッキン7を介して密閉されている。本発明における希釈された血液検体を収容するための収容器具は、図1の構成においては、採血容器2と筒体3の組み合わせに対応する。すなわち、希釈された血液検体を収容するための収容器具は1個でも2個以上の組み合わせでもよい。
The blood separation device 1 includes a blood collection container 2 (a storage device in which a diluent is stored, or a first storage device. It is also a storage device for storing a diluted blood sample) and a blood collection container 2. A cylindrical body 3 that can be inserted (second storage device for storing the collected plasma), a cap piston 4 that can be attached to the cylindrical body 3, and a sealing lid 5 provided at the lower end of the cap piston 4 ( Before use, the upper end opening of the blood collection container 2 is sealed with a cap 6 via a packing 7 before use. The storage device 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 number of storage devices for storing the diluted blood sample may be one or a combination of two or more.
採血容器2は透明な材質製で円筒状を成し、その上端部には、外面に螺子部8が形成され、内面に係止部9が突設されている。また、採血容器2の下端部には、逆円錐状の底部10が形成され、底部10の周囲に円筒状の脚部11が形成されている。脚部11は、血液の分析検査時に使用するサンプルカップと同一外径を有しており、好ましくは、その下端の対向する位置にそれぞれ鉛直方向にスリット溝12が形成されている。さらに、採血容器2内には、図1に示されているように、所要量、例えば、500mm3の希釈液13が予め入れられていてもよい。
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. In addition, 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.
筒体3は透明な材質製で円筒状を成し、その上端部には拡径部14が形成されている。拡径部14は薄肉部15を介して本体部16と接続されている。筒体3の下端部には、縮径部18が形成され、縮径部18の内面には係止突起部19が形成されている。さらに、縮径部18の下端部には外鍔部20(保持器具)が形成され、外鍔部20の下端開口部は濾過膜21(分離器具)により覆われ、濾過膜21は血液中の血漿の通過を許容し、血球の通過を阻止するようになっている。
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. Furthermore, 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.
縮径部18の外周にはシリコンゴム製のカバー22が装着されている(図1)。
A silicon rubber cover 22 is attached to the outer periphery of the reduced diameter portion 18 (FIG. 1).
キャップピストン4は、略円筒状の摘み部26と、摘み部26と同心で下方に延びる心棒部27とで構成されている。摘み部26の内側上端部には筒体3の拡径部14が嵌合可能な円筒状の空間28が形成され、また、その下方は螺刻され、螺子に螺合可能となっている。心棒部27はその下端部29がピン状に形成され、下端部29に密閉蓋5が着脱可能に設けられている(図1参照)。密閉蓋5はシリコンゴム製である。
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.
血液検体の希釈物からの血漿の分離回収操作は、具体的には、次のように行う。希釈液を収容する採血容器2に、キャピラリーで採取した血液を投入した後、採血容器2の上部を持って泡立てないように注意しながら血液と希釈液とを十分に振り混ぜる。次に、濾過膜21を保持する筒体3(血漿、血球分離時のシリンダ側面への回りこみによる液漏れを防止)を濾過膜が下になるように採血容器2に差し込み、ゆっくりと一定のスピードで採血容器2の底面まで濾過膜を押し下げる。このとき、筒体3の濾過膜を通って血漿が上部に上がり、血球は採血容器2の下部に残る。その後、キャップピストン4を筒体3にゆっくりと差し込んで、密閉栓5により逆流による血漿と血球の混合を防止する。
Specifically, the operation of separating and collecting plasma from a diluted blood sample is performed as follows. After the blood collected by the capillary is put into the blood collection container 2 containing the dilution liquid, the blood and the dilution liquid are sufficiently shaken and mixed while taking care not to foam by holding the upper part of the blood collection container 2. Next, the cylindrical body 3 holding the filtration membrane 21 (preventing liquid leakage due to sneaking into the side surface of the cylinder when separating blood plasma and blood cells) is inserted into the blood collection container 2 so that the filtration membrane is below, and slowly and constantly The filtration membrane is pushed down to the bottom of the blood collection container 2 at a speed. At this time, plasma goes up through the filtration membrane of the cylinder 3, and blood cells remain in the lower part of the blood collection container 2. Thereafter, the cap piston 4 is slowly inserted into the cylinder 3, and the sealing plug 5 prevents plasma and blood cells from being mixed by backflow.
上記した器具による血液分離方法の詳細は、特許第3597827号公報の段落番号0023~0026並びに図12及び図13に記載されており、その内容は本明細書に引用される。
The details of the blood separation method using the above-mentioned instrument are described in Japanese Patent No. 3597827, paragraph numbers 0023 to 0026, and FIGS. 12 and 13, the contents of which are cited in this specification.
本発明の血液検査キットに含まれる各々の要素の個数は特に限定されず、各々1個でもよいし、2個以上の複数でもよい。
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.
本発明の血液検査キットに含まれる、キャピラリー以外の部材の材料は、破損しにくさ、衛生面、価格等の観点から、合成樹脂であることが好ましい。例えば、ポリエチレン、ポリプロピレン、ポリ塩化ビニル、ポリ塩化ビニリデン、ポリスチレン、ポリ酢酸ビニル、ポリウレタン、ポリエチレンテレフタレート、ポリ乳酸、アクリロニトリルブタジエンスチレン樹脂(ABS樹脂)、アクリロニトリルスチレン樹脂(AS樹脂)、アクリル樹脂(PMMA)、ポリカーボネート、シリコーン樹脂等が挙げられる。
The material of the members other than the capillary included in the blood test kit of the present invention is preferably a synthetic resin from the viewpoint of resistance to breakage, hygiene, and cost. For example, 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 and the like.
本発明の血液検査キットは、キャピラリーと、血液検体を希釈するための希釈液と、希釈された血液検体を収容するための収容器具と、さらに所望により上記した任意の要素とを、これらを収納する収納容器に収納した形態として提供することができる。
The blood test kit of the present invention stores a capillary, a diluent for diluting a blood sample, a storage device for storing the diluted blood sample, and optional elements described above if desired. It can provide as a form stored in a storage container.
[2]血液分析方法
本発明はまた、本明細書の上記[1]で説明した構成のキットを用いた血液分析方法を提供する。血液分析方法は、ヒトに対する医療行為(医師が行う行為)である態様とヒトに対する医療行為ではない態様(例えば、採血者が患者自身であり、かつ分析者が医師以外の者である態様、非ヒト動物に対する態様、等)が含まれる。本発明の血液分析方法は、対象者自身が血液を採取する自己採血で実施してもよいし、医師等の有資格者が注射器を使用して血液を採取する一般採血においても実施してもよい。好ましい態様としては、患者本人が、ランセットなどのナイフ付の器具を用いて指先などを傷つけて皮膚外にでた血液を採取する。 [2] Blood analysis method The present invention also provides a blood analysis method using the kit having the configuration described in [1] above. The blood analysis method includes a mode that is a medical act on a human (an act performed by a doctor) and a mode that is not a medical act on a human (for example, a mode in which a blood sampler is a patient himself and an analyst is a person other than a doctor, Embodiments for human animals, etc.). 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. Good. As a preferred embodiment, 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.
本発明はまた、本明細書の上記[1]で説明した構成のキットを用いた血液分析方法を提供する。血液分析方法は、ヒトに対する医療行為(医師が行う行為)である態様とヒトに対する医療行為ではない態様(例えば、採血者が患者自身であり、かつ分析者が医師以外の者である態様、非ヒト動物に対する態様、等)が含まれる。本発明の血液分析方法は、対象者自身が血液を採取する自己採血で実施してもよいし、医師等の有資格者が注射器を使用して血液を採取する一般採血においても実施してもよい。好ましい態様としては、患者本人が、ランセットなどのナイフ付の器具を用いて指先などを傷つけて皮膚外にでた血液を採取する。 [2] Blood analysis method The present invention also provides a blood analysis method using the kit having the configuration described in [1] above. The blood analysis method includes a mode that is a medical act on a human (an act performed by a doctor) and a mode that is not a medical act on a human (for example, a mode in which a blood sampler is a patient himself and an analyst is a person other than a doctor, Embodiments for human animals, etc.). 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. Good. As a preferred embodiment, 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.
本発明において、分析対象となる生体試料は血液であり、血液とは、血清又は血漿を含む概念である。好ましくは、被験者より微量の血液を採取し、緩衝液で希釈した後、フィルタや遠心分離により血球を分離することにより得られた血漿又は血清を用いることができる。 血液検体の成分としては、分離手段により血液検体から分離された血漿成分であることが好ましい。
In the present invention, the biological sample to be analyzed is blood, and blood is a concept including serum or plasma. Preferably, plasma or serum obtained by collecting a small amount of blood from a subject, diluting with a buffer solution, and then separating blood cells by a filter or centrifugation can be used. The component of the blood sample is preferably a plasma component separated from the blood sample by the separation means.
血液検体の起源はヒトに限定されず、ヒト以外の動物(非ヒト動物)である哺乳類、鳥類、魚類等であっても良い。ヒト以外の動物としては、例えば、ウマ、ウシ、ブタ、ヒツジ、ヤギ、イヌ、ネコ、マウス、クマ、パンダ等が挙げられる。好ましくは、生体試料の起源はヒトである。
The origin of the blood sample is not limited to humans, but may be mammals, birds, fishes, etc., which are non-human animals (non-human animals). Examples of animals other than humans include horses, cows, pigs, sheep, goats, dogs, cats, mice, bears, pandas, and the like. Preferably, the source of the biological sample is human.
本発明の血液分析方法の第一の態様としては、血液検体中に恒常的に存在する標準成分を用いて、対象成分の濃度の分析を行う。血液検体中に恒常的に存在する標準成分については、[1]での説明が、ここでもそのまま当てはまる。
As a first aspect of the blood analysis method of the present invention, the concentration of a target component is analyzed using a standard component that is constantly present in a blood sample. For the standard components that are permanently present in the blood sample, the description in [1] applies here as it is.
被検者の血液中における血漿成分の占有率は、容積の比率で約55%であるが、被検者の塩分摂取量の変化などで変動する。そのため、本発明においては、血漿中に恒常的に存在する標準成分の標準値を用いて血漿の希釈倍率を算出し、算出した希釈倍率を用いて血液検体中の血漿中の対象成分の濃度を分析する。希釈倍率を算出する方法としては、血漿の希釈液中の外部標準物質(例えば、ナトリウムイオンなど)の測定値(濃度X)と、血液検体の血漿中に含まれる上記外部標準物質(例えば、ナトリウムイオンなど)の既知濃度値(濃度Y;ナトリウムイオンの場合には142mmol/L)とから、血液検体中の血漿成分の希釈倍率(Y/X)を算出することにより希釈倍率を求めることができる。この希釈倍率を用いて、血漿の希釈液中の対象成分の測定値(濃度Z)を測定し、この測定値に希釈倍率を掛け合わせることにより、実際に血液検体の血漿中に含まれる分析対象成分の濃度[Z×(Y/X)]を測定することが可能となる。
The occupation ratio of plasma components in the blood of the subject is about 55% in terms of volume, but varies depending on changes in the amount of salt intake of the subject. Therefore, in the present invention, the dilution ratio of plasma is calculated using the standard value of the standard component that is constantly present in plasma, and the concentration of the target component in plasma in the blood sample is calculated using the calculated dilution ratio. analyse. As a method for calculating the dilution rate, a measured value (concentration X) of an external standard substance (for example, sodium ion) in a diluted plasma solution and the external standard substance (for example, sodium) contained in the blood sample plasma. The dilution factor can be obtained by calculating the dilution factor (Y / X) of the plasma component in the blood sample from the known concentration value (concentration Y; 142 mmol / L in the case of sodium ion) of ions, etc. . Using this dilution factor, the measurement value (concentration Z) of the target component in the plasma dilution is measured, and this measurement value is multiplied by the dilution factor, so that the analyte actually contained in the plasma of the blood sample The component concentration [Z × (Y / X)] can be measured.
ナトリウムイオンなどの濃度は、例えば、炎光光度法、ガラス電極法、滴定法、イオン選択電極法、酵素活性法等により測定することができる。特に好ましい態様において、ナトリウムイオンの測定はβ-ガラクトシダーゼがナトリウムイオンで活性化することを利用し、緩衝液で希釈された試料のナトリウムイオン濃度とガラクトシダーゼ活性が比例関係にあることを利用した酵素的測定法が採用される。
The concentration of sodium ion or the like can be measured by, for example, flame photometry, glass electrode method, titration method, ion selective electrode method, enzyme activity method and the like. In a particularly preferred embodiment, the measurement of sodium ion utilizes the fact that β-galactosidase is activated by sodium ion, and uses the proportional relationship between sodium ion concentration and galactosidase activity in a sample diluted with a buffer solution. Measurement method is adopted.
また、部材に由来する標準成分の量を規定した血液検査キットが実際に使用されているか、また、血液の希釈と血漿の回収の方法が正常に行われているか確証するためには、血漿中の別の標準成分から独立に希釈倍率を追加的に求めて、その値が上で求めた希釈倍率と一致することを確認することが好ましい。一致するとは、2つの測定値(a,b)において、それらの差のそれらの平均値に対する割合、すなわち|a-b|/{(a+b)/2}×100が、20%以下であることであり、好ましくは10%以下であることであり、より好ましくは5%以下であることである。これにより、血液検体中の対象成分の濃度の分析が正常に行われていることの検証が可能となる。ここで、ナトリウムイオン又は塩化物イオン以外の血漿中に恒常的に存在する標準成分の例としては、総タンパク又はアルブミンから選択されることが好ましく、総タンパクであることがより好ましい。総タンパクの測定法は、ビューレット法や、紫外吸収法、ブレッドフォード法、ローリー法、ビシンコニン酸(Bicinchoninic Acid:BCA)法、蛍光法など公知の方法があり、測定試料の特性や感度、試料量などに応じて適宜使用する方法を選択することができる。
In addition, in order to confirm that blood test kits that specify the amount of standard components derived from components are actually used, and that blood dilution and plasma recovery methods are performed normally, It is preferable to additionally obtain a dilution factor independently from the other standard components and confirm that the value matches the dilution factor obtained above. The coincidence means that in two measured values (a, b), the ratio of their difference to their average value, that is, | a−b | / {(a + b) / 2} × 100 is 20% or less. Preferably, it is 10% or less, more preferably 5% or less. Thereby, it is possible to verify that the analysis of the concentration of the target component in the blood sample is performed normally. Here, as an example of a standard component that is constantly present in plasma other than sodium ions or chloride ions, it is preferably selected from total protein or albumin, and more preferably total protein. Methods for measuring total protein include known methods such as the Burette method, the ultraviolet absorption method, the Breadford method, the Raleigh method, the bicinchoninic acid (BCA) method, and the fluorescence method. A method to be used as appropriate can be selected according to the amount and the like.
本発明の血液分析方法の第二の態様としては、血液中に存在しない標準成分を用いて、対象成分の濃度の分析を行う。この場合、血液中に存在しない標準成分を含む希釈液を含む血液検査キットを用いる。
As a second aspect of the blood analysis method of the present invention, the concentration of the target component is analyzed using standard components that are not present in the blood. In this case, a blood test kit containing a diluent containing standard components not present in blood is used.
本発明の血液分析方法の第三の態様としては、血液中に恒常的に存在する標準成分、及び血液中に存在しない標準成分を用いて、対象成分の濃度の分析を行う。2つの標準成分を併用することで、より信頼性の高い分析方法とすることができる。
As a third aspect of the blood analysis method of the present invention, the concentration of the target component is analyzed using a standard component that is constantly present in blood and a standard component that is not present in blood. By using two standard components in combination, a more reliable analysis method can be obtained.
このとき、血液検体の希釈倍率は、血液中に恒常的に存在する標準成分としてナトリウムイオンを、血液中に存在しない標準成分としてリチウムイオンを用い、ナトリウムイオンの測定をβ-ガラクトシダーゼ活性が比例関係にあることを利用した酵素活性法(後述)で行い、リチウムイオンの測定をキレート比色法(後述)で行う場合には、下記式1から4のいずれかの式で算出することができる。
At this time, the dilution ratio of the blood sample is determined by using sodium ion as a standard component that is constantly present in the blood and lithium ion as a standard component that is not present in the blood. The measurement of sodium ion is proportional to β-galactosidase activity. In the case of performing the enzyme activity method (described later) utilizing the facts described above and measuring the lithium ion by the chelate colorimetric method (described later), it can be calculated by any one of the following formulas 1 to 4.
上記式において、A、B、C、D、B’及びXは、以下のように定義される。
A : 緩衝液を発色させた際の吸光度
B : 血漿添加後の吸光度変化量
C : 血漿ナトリウム中央値142 mmol/Lの吸光度
D : 血漿希釈後のナトリウムイオン濃度における吸光度
B’: 血漿ナトリウムの吸光度から算出した希釈倍率による、希釈血漿中
の血液中に存在しない標準成分の吸光度の補正値
X : 血漿希釈倍数 In the above formula, A, B, C, D, B ′ and X are defined as follows.
A: Absorbance when the buffer solution is developed B: Absorbance change after plasma addition C: Absorbance of plasma sodium median 142 mmol / L D: Absorbance at sodium ion concentration after plasma dilution B ': Absorbance of plasma sodium Correction value X of the absorbance of the standard component that is not present in the blood in the diluted plasma, based on the dilution factor calculated from X: dilution factor of plasma
A : 緩衝液を発色させた際の吸光度
B : 血漿添加後の吸光度変化量
C : 血漿ナトリウム中央値142 mmol/Lの吸光度
D : 血漿希釈後のナトリウムイオン濃度における吸光度
B’: 血漿ナトリウムの吸光度から算出した希釈倍率による、希釈血漿中
の血液中に存在しない標準成分の吸光度の補正値
X : 血漿希釈倍数 In the above formula, A, B, C, D, B ′ and X are defined as follows.
A: Absorbance when the buffer solution is developed B: Absorbance change after plasma addition C: Absorbance of plasma sodium median 142 mmol / L D: Absorbance at sodium ion concentration after plasma dilution B ': Absorbance of plasma sodium Correction value X of the absorbance of the standard component that is not present in the blood in the diluted plasma, based on the dilution factor calculated from X: dilution factor of plasma
希釈率を求める際のもう一つの算出方法として、二乗平均法を用いた式5で算出し、希釈液中の分析対象成分の濃度に、式5で算出した希釈率を乗じて血液検体の成分中の対象成分の濃度を分析する態様も好ましい。
As another calculation method for obtaining the dilution rate, the blood sample component is calculated by calculating with Equation 5 using the root-mean-square method, and multiplying the concentration of the analysis target component in the diluted solution by the dilution rate calculated with Equation 5. An embodiment in which the concentration of the target component is analyzed is also preferable.
血液検体の成分中の対象成分の濃度は、希釈液中の対象成分の濃度から、上記希釈倍率に基づいて算出できる。
The concentration of the target component in the blood sample component can be calculated from the concentration of the target component in the diluent based on the dilution factor.
分析の対象成分は限定されず、生体試料中に含まれるあらゆる物質が対象となる。例えば臨床診断に用いられる血液中の生化学検査項目、腫瘍マーカーや肝炎のマーカー等各種疾患のマーカー等が挙げられ、タンパク質、糖、脂質、低分子化合物等が挙げられる。また、測定は物質濃度だけでなく、酵素等の活性を有する物質の活性も対象となる。各対象成分の測定は、公知の方法で行うことができる。
The analysis target component is not limited, and any substance contained in a biological sample 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. Further, 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.
ナトリウムイオンの測定ではナトリウムイオンにより酵素ガラクトシダーゼは酵素活性が活性化することから、緩衝液で希釈された非常に低濃度ナトリウムイオン(24 mmol/L以下)試料を数μLで測定する酵素的測定法が使用できる。この方法は生化学・免疫自動分析装置に適応でき、ナトリウムイオン測定のために別の測定機器を必要としない点で効率性が高く経済的である。
In the measurement of sodium ion, the enzyme activity of the enzyme galactosidase is activated by sodium ion. Therefore, an enzymatic measurement method that measures a very low concentration sodium ion (24 mmol / L or less) sample diluted in a buffer solution in several μL. Can be used. This method can be applied to a biochemical / immunological automatic analyzer and is highly efficient and economical in that it does not require a separate measuring instrument for measuring sodium ions.
以下の実施例により本発明を説明するが、本発明は実施例によって限定されない。
The following examples explain the present invention, but the present invention is not limited to the examples.
(参考例1)
1.微量血液試料を希釈した希釈液の調製
ボランティアの患者から、インフォームドコンセントを行った後に静脈から注射器で採取した7mLを採血管に得た。この採血した血液から、80μL、60μLをそれぞれ10回ずつ、内径1mmのキャピラリーで秤量した。また、血液吸引器の先端のファイバーロットで10回ずつ、ファーバーロット全体が赤く染まるまで血液を吸引させて、下記のように調製した希釈液-1の360μLにそれぞれ混合し、フィルターで血球成分を分離して、希釈血漿を試料として生化学自動分析装置を用いて、生体成分の各濃度を測定した。 (Reference Example 1)
1. Preparation of Diluted Solution by Diluting a Trace Blood Sample From a volunteer patient, 7 mL obtained from a vein through a syringe after informed consent was obtained in a blood collection tube. From this collected blood, 80 μL and 60 μL were weighed 10 times each with a capillary having an inner diameter of 1 mm. In addition, aspirate the blood 10 times with the fiber lot at the tip of the blood aspirator until the entire fiber lot is stained red, mix with 360 μL of Diluent-1 prepared as follows, and filter the blood cell components with the filter. Separately, each concentration of the biological component was measured using a biochemical automatic analyzer using diluted plasma as a sample.
1.微量血液試料を希釈した希釈液の調製
ボランティアの患者から、インフォームドコンセントを行った後に静脈から注射器で採取した7mLを採血管に得た。この採血した血液から、80μL、60μLをそれぞれ10回ずつ、内径1mmのキャピラリーで秤量した。また、血液吸引器の先端のファイバーロットで10回ずつ、ファーバーロット全体が赤く染まるまで血液を吸引させて、下記のように調製した希釈液-1の360μLにそれぞれ混合し、フィルターで血球成分を分離して、希釈血漿を試料として生化学自動分析装置を用いて、生体成分の各濃度を測定した。 (Reference Example 1)
1. Preparation of Diluted Solution by Diluting a Trace Blood Sample From a volunteer patient, 7 mL obtained from a vein through a syringe after informed consent was obtained in a blood collection tube. From this collected blood, 80 μL and 60 μL were weighed 10 times each with a capillary having an inner diameter of 1 mm. In addition, aspirate the blood 10 times with the fiber lot at the tip of the blood aspirator until the entire fiber lot is stained red, mix with 360 μL of Diluent-1 prepared as follows, and filter the blood cell components with the filter. Separately, each concentration of the biological component was measured using a biochemical automatic analyzer using diluted plasma as a sample.
(希釈液組成)
希釈液を以下の組成で調製した。浸透圧は、OSMOATAT OM-6040(アークレイ(株)社製)を用いて測定した値を表示した。浸透圧の単位は、溶液の水1kgが持つ浸透圧で、イオンのミリモル数をあらわす。
HEPES 50mmol/L
2-アミノー2-メチル-1-プロパノール(AMP) 50mmol/L
D-マンニトール 284mmol/L
塩化リチウム 1mmol/L
EDTA-2K 0.8mmol/L
PALP(ピリドキサールリン酸) 0.05mmol/L
チアベンダゾール 0.0001質量%
アミカシン硫酸塩 0.0003質量%
硫酸カナマイシン 0.0005質量%
メロペネム三水和物 0.0005質量%
浸透圧 355mOsm/kg
pH 7.4 (Diluted solution composition)
Dilution liquid was prepared with the following composition. For the 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
希釈液を以下の組成で調製した。浸透圧は、OSMOATAT OM-6040(アークレイ(株)社製)を用いて測定した値を表示した。浸透圧の単位は、溶液の水1kgが持つ浸透圧で、イオンのミリモル数をあらわす。
HEPES 50mmol/L
2-アミノー2-メチル-1-プロパノール(AMP) 50mmol/L
D-マンニトール 284mmol/L
塩化リチウム 1mmol/L
EDTA-2K 0.8mmol/L
PALP(ピリドキサールリン酸) 0.05mmol/L
チアベンダゾール 0.0001質量%
アミカシン硫酸塩 0.0003質量%
硫酸カナマイシン 0.0005質量%
メロペネム三水和物 0.0005質量%
浸透圧 355mOsm/kg
pH 7.4 (Diluted solution composition)
Dilution liquid was prepared with the following composition. For the 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
2.ナトリウムイオン濃度の測定
1.で調製したそれぞれの希釈液について、ナトリウムイオン濃度の測定を行った。測定には、β-ガラクトシダーゼがナトリウムイオンで活性化することを利用し、それぞれの希釈液中のナトリウムイオン濃度とβ-ガラクトシダーゼ活性が比例関係にあることを利用した酵素活性法により測定した。具体的には、ナトリウムイオンを含まない精製水で血液の希釈液を5倍希釈した後、3μLを秤量し、下記のように調製した第一試薬52μLを加えて、37℃で5分間加温し、下記のように調製した第二試薬を26μL加え、1分間の吸光度の変化をJCA-BM6050型生化学自動分析装置(日本電子(株)社製)を用いて主波長410nm、副波長658nmで吸光度を測定することにより求めた。あらかじめ作成した検量線から、ナトリウムイオンの濃度を測定した。 2. Measurement of sodium ion concentration The sodium ion concentration was measured for each diluted solution prepared in (1). For the measurement, β-galactosidase was activated by sodium ions, and the enzyme activity method was used that utilized a proportional relationship between the sodium ion concentration in each dilution and β-galactosidase activity. Specifically, after diluting the dilutedblood solution 5 times with purified water not containing sodium ions, 3 μL was weighed, 52 μL of the first reagent prepared as follows was added, and the mixture was heated at 37 ° C. for 5 minutes. Then, 26 μL of the second reagent prepared as described below was added, and the change in absorbance for 1 minute was measured using a JCA-BM6050 type biochemical automatic analyzer (manufactured by JEOL Ltd.) with a main wavelength of 410 nm and a subwavelength of 658 nm. It was determined by measuring the absorbance at. The concentration of sodium ions was measured from a calibration curve prepared in advance.
1.で調製したそれぞれの希釈液について、ナトリウムイオン濃度の測定を行った。測定には、β-ガラクトシダーゼがナトリウムイオンで活性化することを利用し、それぞれの希釈液中のナトリウムイオン濃度とβ-ガラクトシダーゼ活性が比例関係にあることを利用した酵素活性法により測定した。具体的には、ナトリウムイオンを含まない精製水で血液の希釈液を5倍希釈した後、3μLを秤量し、下記のように調製した第一試薬52μLを加えて、37℃で5分間加温し、下記のように調製した第二試薬を26μL加え、1分間の吸光度の変化をJCA-BM6050型生化学自動分析装置(日本電子(株)社製)を用いて主波長410nm、副波長658nmで吸光度を測定することにより求めた。あらかじめ作成した検量線から、ナトリウムイオンの濃度を測定した。 2. Measurement of sodium ion concentration The sodium ion concentration was measured for each diluted solution prepared in (1). For the measurement, β-galactosidase was activated by sodium ions, and the enzyme activity method was used that utilized a proportional relationship between the sodium ion concentration in each dilution and β-galactosidase activity. Specifically, after diluting the diluted
(ナトリウムイオン測定試薬の調製)
以下の組成のナトリウムイオン測定試薬を調製した。
第一試薬
HPEPS・LiOH(pH8.0) 100mmol/L
D-マンニトール 60mmol/L
N-アセチルシステイン 30mmol/L
硫酸マグネシウム 1.52mmol/L
β-ガラクトシダーゼ 1.1kU/L
TritonX-100 0.05質量%
第二試薬
HPEPS・LiOH(pH8.0) 100mmol/L
o-Nitrophenyl-β-D-Galactpyranoside
15mmol/L (Preparation of sodium ion measurement reagent)
A sodium ion measuring reagent having the following composition was prepared.
First reagent HPEPS · LiOH (pH 8.0) 100 mmol / L
D-mannitol 60mmol / L
N-acetylcysteine 30mmol / L
Magnesium sulfate 1.52mmol / L
β-galactosidase 1.1 kU / L
TritonX-100 0.05% by mass
Second reagent HPEPS · LiOH (pH 8.0) 100 mmol / L
o-Nitrophenyl-β-D-Galactopyranoside
15 mmol / L
以下の組成のナトリウムイオン測定試薬を調製した。
第一試薬
HPEPS・LiOH(pH8.0) 100mmol/L
D-マンニトール 60mmol/L
N-アセチルシステイン 30mmol/L
硫酸マグネシウム 1.52mmol/L
β-ガラクトシダーゼ 1.1kU/L
TritonX-100 0.05質量%
第二試薬
HPEPS・LiOH(pH8.0) 100mmol/L
o-Nitrophenyl-β-D-Galactpyranoside
15mmol/L (Preparation of sodium ion measurement reagent)
A sodium ion measuring reagent having the following composition was prepared.
First reagent HPEPS · LiOH (pH 8.0) 100 mmol / L
D-mannitol 60mmol / L
N-acetylcysteine 30mmol / L
Magnesium sulfate 1.52mmol / L
β-galactosidase 1.1 kU / L
TritonX-100 0.05% by mass
Second reagent HPEPS · LiOH (pH 8.0) 100 mmol / L
o-Nitrophenyl-β-D-Galactopyranoside
15 mmol / L
3.ナトリウムイオン濃度による血漿の希釈倍率の測定
上記のように求めた希釈液中のナトリウムイオン濃度(X)と、血液の血漿中のナトリウムイオン濃度の標準値(Y)とからそれぞれの希釈液の希釈率(Y/X)を求め、採血した血液から、キャピラリーで80μL、60μLそれぞれ、10ずつ作製した試料、およびファイバーロットで10ずつ作製した試料の希釈率の平均値と希釈倍率のばらつきの尺度である変動係数であるCV(coefficient of variation)(%)をもとめた。結果を表1に示した。 3. Measurement of dilution ratio of plasma by sodium ion concentration Dilution of each diluted solution from the sodium ion concentration (X) in the diluted solution obtained as described above and the standard value (Y) of sodium ion concentration in blood plasma. The ratio (Y / X) was obtained, and the average of the dilution rate and the scale of the variation of the dilution rate of the samples prepared 10 by 80 μL and 60 μL each from the collected blood and 10 samples prepared by the fiber lot. CV (coefficient of variation) (%) which is a certain coefficient of variation was obtained. The results are shown in Table 1.
上記のように求めた希釈液中のナトリウムイオン濃度(X)と、血液の血漿中のナトリウムイオン濃度の標準値(Y)とからそれぞれの希釈液の希釈率(Y/X)を求め、採血した血液から、キャピラリーで80μL、60μLそれぞれ、10ずつ作製した試料、およびファイバーロットで10ずつ作製した試料の希釈率の平均値と希釈倍率のばらつきの尺度である変動係数であるCV(coefficient of variation)(%)をもとめた。結果を表1に示した。 3. Measurement of dilution ratio of plasma by sodium ion concentration Dilution of each diluted solution from the sodium ion concentration (X) in the diluted solution obtained as described above and the standard value (Y) of sodium ion concentration in blood plasma. The ratio (Y / X) was obtained, and the average of the dilution rate and the scale of the variation of the dilution rate of the samples prepared 10 by 80 μL and 60 μL each from the collected blood and 10 samples prepared by the fiber lot. CV (coefficient of variation) (%) which is a certain coefficient of variation was obtained. The results are shown in Table 1.
4.ナトリウムイオン濃度とリチウムイオン濃度による血漿の希釈倍率の測定
(希釈液中のリチウムイオンの測定)
上記の希釈液組成になるように調製した希釈液に添加したリチウムイオン濃度の測定は、キレート比色法(ハロゲン化ポリフィリンキレート法:perfluoro-5,10,15,20-tetraphenyl-21H,23H,-porphyrin)により行った。具体的には、リチウムイオンを含まない精製水を用いて血液の希釈液を4.5倍に希釈した後、5μLを秤量し下記に示した組成となるように調製した第三試薬55μLを加えて、37℃で10分間加温し、JCA-BM6050型生化学自動分析装置(日本電子(株)社製)を使用して、主波長545nm、副波長596nmで吸光度を測定することにより求めた。あらかじめ作成した検量線から、リチウムイオンの濃度を測定した。 4). Measurement of dilution ratio of plasma by sodium ion concentration and lithium ion concentration (measurement of lithium ion in diluted solution)
The lithium ion concentration added to the diluent prepared to have the above-mentioned diluent composition 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 μL was weighed and 55 μL of the third reagent prepared to have the composition shown below was added. The temperature 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.
(希釈液中のリチウムイオンの測定)
上記の希釈液組成になるように調製した希釈液に添加したリチウムイオン濃度の測定は、キレート比色法(ハロゲン化ポリフィリンキレート法:perfluoro-5,10,15,20-tetraphenyl-21H,23H,-porphyrin)により行った。具体的には、リチウムイオンを含まない精製水を用いて血液の希釈液を4.5倍に希釈した後、5μLを秤量し下記に示した組成となるように調製した第三試薬55μLを加えて、37℃で10分間加温し、JCA-BM6050型生化学自動分析装置(日本電子(株)社製)を使用して、主波長545nm、副波長596nmで吸光度を測定することにより求めた。あらかじめ作成した検量線から、リチウムイオンの濃度を測定した。 4). Measurement of dilution ratio of plasma by sodium ion concentration and lithium ion concentration (measurement of lithium ion in diluted solution)
The lithium ion concentration added to the diluent prepared to have the above-mentioned diluent composition 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 μL was weighed and 55 μL of the third reagent prepared to have the composition shown below was added. The temperature 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.
(リチウムイオン用測定試薬の調製)
以下の組成のリチウムイオン測定試薬を調製した。
第三試薬
Perfluoro-5,10,15,20-tetraphenyl-21H,23H-porphyrin 0.05質量%
ジメチルスルホキシド 5質量%
トリエタノールアミン 2質量%
ポリエチレングリコール-t-オクチルフェニルエーテル 2質量%
ドデシル硫酸ナトリウム 2質量% (Preparation of measurement reagent for lithium ion)
A lithium ion measuring reagent having the following composition was prepared.
Third reagent Perfluoro-5,10,15,20-tetraphenyl-21H, 23H-porphyrin 0.05 mass%
Dimethyl sulfoxide 5% by mass
Triethanolamine 2% by mass
Polyethylene glycol-t-octylphenyl ether 2% by mass
Sodium dodecyl sulfate 2% by mass
以下の組成のリチウムイオン測定試薬を調製した。
第三試薬
Perfluoro-5,10,15,20-tetraphenyl-21H,23H-porphyrin 0.05質量%
ジメチルスルホキシド 5質量%
トリエタノールアミン 2質量%
ポリエチレングリコール-t-オクチルフェニルエーテル 2質量%
ドデシル硫酸ナトリウム 2質量% (Preparation of measurement reagent for lithium ion)
A lithium ion measuring reagent having the following composition was prepared.
Third reagent Perfluoro-5,10,15,20-tetraphenyl-21H, 23H-porphyrin 0.05 mass%
Dimethyl sulfoxide 5% by mass
Triethanolamine 2% by mass
Polyethylene glycol-t-octylphenyl ether 2% by mass
Sodium dodecyl sulfate 2% by mass
(希釈倍率の測定)
先に求めたナトリウムイオン濃度の結果と、上記の希釈液組成になるように調製した希釈液、及び、上記で調製した微量血液試料を希釈した希釈血漿に対して、血液血漿の希釈倍率を求める方法と計算式を以下に示す。
A:リチウムイオン濃度測定時の希釈液を発色させた際の吸光度
B:希釈血漿中のリチウムイオン濃度測定時に測定した吸光度の変化量
C:血漿ナトリウム中央値142 mmol/Lの吸光度
D:血漿を希釈液で希釈した後のナトリウムイオン濃度における吸光度
X:血漿希釈倍数
血漿の緩衝液による希釈倍数は下記の(1)式の関係を用いた (Measurement of dilution factor)
Obtain the dilution ratio of blood plasma with respect to the result of the previously obtained sodium ion concentration, the diluted solution prepared to have the above diluted solution composition, and the diluted plasma obtained by diluting the micro blood sample prepared above. The method and formula are shown below.
A: Absorbance when coloring the diluted solution at the time of measuring the lithium ion concentration B: Absorbance change measured when measuring the lithium ion concentration in the diluted plasma C: Absorbance D of plasma sodium median 142 mmol / L D: Plasma Absorbance at sodium ion concentration after dilution with diluent X: Plasma dilution factor The dilution factor of the plasma with the buffer used the relationship of the following formula (1):
先に求めたナトリウムイオン濃度の結果と、上記の希釈液組成になるように調製した希釈液、及び、上記で調製した微量血液試料を希釈した希釈血漿に対して、血液血漿の希釈倍率を求める方法と計算式を以下に示す。
A:リチウムイオン濃度測定時の希釈液を発色させた際の吸光度
B:希釈血漿中のリチウムイオン濃度測定時に測定した吸光度の変化量
C:血漿ナトリウム中央値142 mmol/Lの吸光度
D:血漿を希釈液で希釈した後のナトリウムイオン濃度における吸光度
X:血漿希釈倍数
血漿の緩衝液による希釈倍数は下記の(1)式の関係を用いた (Measurement of dilution factor)
Obtain the dilution ratio of blood plasma with respect to the result of the previously obtained sodium ion concentration, the diluted solution prepared to have the above diluted solution composition, and the diluted plasma obtained by diluting the micro blood sample prepared above. The method and formula are shown below.
A: Absorbance when coloring the diluted solution at the time of measuring the lithium ion concentration B: Absorbance change measured when measuring the lithium ion concentration in the diluted plasma C: Absorbance D of plasma sodium median 142 mmol / L D: Plasma Absorbance at sodium ion concentration after dilution with diluent X: Plasma dilution factor The dilution factor of the plasma with the buffer used the relationship of the following formula (1):
上記の式(1)を用いて、1.で調製した血漿の希釈液それぞれについて、希釈率の平均値と希釈倍率のばらつきの尺度である変動係数CV(%)を求めた。結果を表2に示した。
Using the above equation (1), 1. The coefficient of variation CV (%), which is a measure of the variation of the average value of the dilution rate and the dilution ratio, was determined for each of the diluted plasma solutions prepared in (1). The results are shown in Table 2.
5.リチウムイオン濃度による血漿の希釈倍率の測定
上記の希釈液組成になるように調製した希釈液、及び、上記で調製した微量血液試料を希釈した希釈血漿に対して、4.で求めた血液検体を希釈した希釈後の希釈液中のリチウムイオン濃度(A)と、血液を希釈する前の希釈液中のリチウムイオン濃度(B)とから、それぞれの希釈液の希釈率[B/(B-A)]を求め、試料の希釈率の平均値と希釈倍率のばらつきの尺度である変動係数CV(%)をもとめた。結果を表3に示した。 5). 3. Measurement of dilution ratio of plasma based on lithium ion concentration 4. Diluted solution prepared to have the above-described diluted solution composition and diluted plasma obtained by diluting the micro blood sample prepared above. From the lithium ion concentration (A) in the diluted diluted solution obtained by diluting the blood sample obtained in step 1 and the lithium ion concentration (B) in the diluted solution before diluting the blood, the dilution rate of each diluted solution [ B / (BA)] was determined, and the coefficient of variation CV (%), which is a measure of the variation of the dilution ratio and the average value of the dilution ratio of the sample, was obtained. The results are shown in Table 3.
上記の希釈液組成になるように調製した希釈液、及び、上記で調製した微量血液試料を希釈した希釈血漿に対して、4.で求めた血液検体を希釈した希釈後の希釈液中のリチウムイオン濃度(A)と、血液を希釈する前の希釈液中のリチウムイオン濃度(B)とから、それぞれの希釈液の希釈率[B/(B-A)]を求め、試料の希釈率の平均値と希釈倍率のばらつきの尺度である変動係数CV(%)をもとめた。結果を表3に示した。 5). 3. Measurement of dilution ratio of plasma based on lithium ion concentration 4. Diluted solution prepared to have the above-described diluted solution composition and diluted plasma obtained by diluting the micro blood sample prepared above. From the lithium ion concentration (A) in the diluted diluted solution obtained by diluting the blood sample obtained in step 1 and the lithium ion concentration (B) in the diluted solution before diluting the blood, the dilution rate of each diluted solution [ B / (BA)] was determined, and the coefficient of variation CV (%), which is a measure of the variation of the dilution ratio and the average value of the dilution ratio of the sample, was obtained. The results are shown in Table 3.
表1、表2及び表3の結果から、血液中に存在する恒常性のある成分であるナトリウムイオンを標準物質に用いた場合において、ファイバーロットを用いて採血する場合に対して、目盛付きキャピラリーを用いることで、希釈倍率の測定値の繰り返し再現性はファイバーロットに対して非常に良く、また、内部標準物質であるリチウムイオンを用いた場合でも、繰り返し再現性が非常に良いことがわかった。従って、血液検査キットに用いる手軽さ、低いコストを考えるとキャピラリーが優れていることが判った。更に、血液中に存在する恒常性のある成分であるナトリウムイオンを標準物質として用い、加えて、ナトリウムイオンを実質的に含まない希釈液に含まれるリチウムイオンを標準物質として併用することで、更に一段と希釈倍率の測定値の繰り返し再現性は非常に良い結果が得られ、同様の結論を得た。この結論から、血液検体中の対象成分の濃度を精度よく計算して求めることが可能であることがわかった。
From the results of Table 1, Table 2 and Table 3, when using sodium ion, which is a homeostatic component present in blood, as a standard substance, a capillary with a scale is used for collecting blood using a fiber lot. It was found that the repetition reproducibility of the measured value of the dilution factor was very good for the fiber lot, and that the reproducibility was very good even when using lithium ion which is an internal standard substance. . Therefore, it was found that the capillaries are excellent considering the ease of use for blood test kits and the low cost. Furthermore, by using sodium ion, which is a homeostatic component present in blood, as a standard substance, and additionally using lithium ion contained in a diluent substantially free of sodium ions as a standard substance, The reproducibility of the measured values of the dilution ratio was very good, and the same conclusion was obtained. From this conclusion, it was found that the concentration of the target component in the blood sample can be accurately calculated.
(参考例2)
参考例1で調製した80μLの血液をキャピラリーで秤量し、希釈液と混合しフィルターで血球成分を分離した希釈血漿試料に対して、下記に示す方法により、総タンパクの濃度を測定した。 (Reference Example 2)
80 μL of blood prepared in Reference Example 1 was weighed with a capillary, mixed with a diluting solution, and a blood plasma component was separated with a filter, and then the total protein concentration was measured by the method described below.
参考例1で調製した80μLの血液をキャピラリーで秤量し、希釈液と混合しフィルターで血球成分を分離した希釈血漿試料に対して、下記に示す方法により、総タンパクの濃度を測定した。 (Reference Example 2)
80 μL of blood prepared in Reference Example 1 was weighed with a capillary, mixed with a diluting solution, and a blood plasma component was separated with a filter, and then the total protein concentration was measured by the method described below.
(希釈血漿試料中の総タンパクの濃度の測定)
ビューレット法を測定原理とする測定を行った。ビウレット試薬:3.0mmol/L、硫酸銅 400μL、酒石酸カリウムナトリウム 21.3mmol/L、NaOH 0.75mol/Lを準備し、希釈血漿と混合した。混合後、37℃で10分間放置して、アルカリ性下で血漿中のタンパクと銅イオンによる540~560nmの青紫色を呈する錯体が形成されるまで待ち、545nmで吸光度を測定し、標準溶液の吸光度から得た検量線を用いて血球分離後の希釈血漿中の総タンパク濃度を定量した。 (Measurement of total protein concentration in diluted plasma sample)
Measurements were performed using the burette method as the measurement principle. Biuret reagent: 3.0 mmol / L, copper sulfate 400 μL, potassium sodium tartrate 21.3 mmol / L, NaOH 0.75 mol / L were prepared and mixed with diluted plasma. After mixing, leave at 37 ° C. for 10 minutes, wait until a complex of 540 to 560 nm of blue-violet color is formed by plasma proteins and copper ions under alkalinity, measure the absorbance at 545 nm, and measure the absorbance of the standard solution Was used to quantitate the total protein concentration in the diluted plasma after separation of blood cells.
ビューレット法を測定原理とする測定を行った。ビウレット試薬:3.0mmol/L、硫酸銅 400μL、酒石酸カリウムナトリウム 21.3mmol/L、NaOH 0.75mol/Lを準備し、希釈血漿と混合した。混合後、37℃で10分間放置して、アルカリ性下で血漿中のタンパクと銅イオンによる540~560nmの青紫色を呈する錯体が形成されるまで待ち、545nmで吸光度を測定し、標準溶液の吸光度から得た検量線を用いて血球分離後の希釈血漿中の総タンパク濃度を定量した。 (Measurement of total protein concentration in diluted plasma sample)
Measurements were performed using the burette method as the measurement principle. Biuret reagent: 3.0 mmol / L, copper sulfate 400 μL, potassium sodium tartrate 21.3 mmol / L, NaOH 0.75 mol / L were prepared and mixed with diluted plasma. After mixing, leave at 37 ° C. for 10 minutes, wait until a complex of 540 to 560 nm of blue-violet color is formed by plasma proteins and copper ions under alkalinity, measure the absorbance at 545 nm, and measure the absorbance of the standard solution Was used to quantitate the total protein concentration in the diluted plasma after separation of blood cells.
総タンパクの濃度から求めた希釈倍率の平均値は、参考例1で調製した、ナトリウムイオン濃度から求めた希釈倍率の平均値と一致した値が得られた。これにより、ナトリウムイオン濃度から求めた希釈倍率の測定が正常に行われていることの検証が可能であることが分かった。
The average value of the dilution rate determined from the total protein concentration was the same as the average dilution rate determined from the sodium ion concentration prepared in Reference Example 1. Thereby, it turned out that it can verify that the measurement of the dilution rate calculated | required from the sodium ion concentration is performed normally.
1 血液分離器具
2 採血容器
3 筒体
4 キャップピストン
5 密閉蓋
6 キャップ
7 パッキン
8 螺子部
9 係止部
10 底部
11 脚部
12 スリット溝
13 希釈液
14 拡径部
15 薄肉部
16 本体部
18 縮径部
19 係止突起部
20 外鍔部
21 濾過膜
22 カバー
26 摘み部
27 心棒部
28 空間
29 下端部
31 段差部
33 上端部
34 頂部
201 筒状本体
202 端
203 端
204 下限目盛
205 上限目盛
206 中心目盛
207 端部
212 血液接触端
213 封止端 DESCRIPTION OF SYMBOLS 1 Blood separation instrument 2 Blood collection container 3 Cylindrical body 4Cap piston 5 Sealing lid 6 Cap 7 Packing 8 Screw part 9 Locking part 10 Bottom part 11 Leg part 12 Slit groove 13 Diluted solution 14 Wide diameter part 15 Thin part 16 Main body part 18 Shrinkage Diameter portion 19 Locking projection portion 20 Outer flange portion 21 Filtration membrane 22 Cover 26 Picking portion 27 Mandrel portion 28 Space 29 Lower end portion 31 Step portion 33 Upper end portion 34 Top portion 201 Cylindrical body 202 End 203 End 204 Lower limit scale 205 Upper limit scale 206 Center scale 207 End 212 Blood contact end 213 Sealed end
2 採血容器
3 筒体
4 キャップピストン
5 密閉蓋
6 キャップ
7 パッキン
8 螺子部
9 係止部
10 底部
11 脚部
12 スリット溝
13 希釈液
14 拡径部
15 薄肉部
16 本体部
18 縮径部
19 係止突起部
20 外鍔部
21 濾過膜
22 カバー
26 摘み部
27 心棒部
28 空間
29 下端部
31 段差部
33 上端部
34 頂部
201 筒状本体
202 端
203 端
204 下限目盛
205 上限目盛
206 中心目盛
207 端部
212 血液接触端
213 封止端 DESCRIPTION OF SYMBOLS 1 Blood separation instrument 2 Blood collection container 3 Cylindrical body 4
Claims (22)
- 血液検体を採取するための血液採取器具と、
採取した血液検体を希釈するための希釈液と、
血液検体の希釈物を収容するための収容器具と、
を含み、血液中に恒常的に存在する標準成分を用いて血液検体中の対象成分の濃度を分析するための、血液検査キットであって、
前記血液採取器具が、キャピラリーである、血液検査キット。 A blood collection device for collecting a blood sample;
A diluent for diluting the collected blood sample;
A storage device for storing a dilution of a blood sample;
A blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is constantly present in blood,
A blood test kit, wherein the blood collection device is a capillary. - 血液検体を採取するための血液採取器具と、
採取した血液検体を希釈するための希釈液と、
血液検体の希釈物を収容するための収容器具と、
を含む、血液中に恒常的に存在する標準成分を用いて血液検体中の対象成分の濃度を分析するための、血液検査キットであって、
前記希釈液が、血液中に存在しない標準成分を含有し、
前記血液採取器具が、キャピラリーである、血液検査キット。 A blood collection device for collecting a blood sample;
A diluent for diluting the collected blood sample;
A storage device for storing a dilution of a blood sample;
A blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is constantly present in blood,
The diluent contains standard components not present in the blood,
A blood test kit, wherein the blood collection device is a capillary. - 血液検体を採取するための血液採取器具と、
採取した血液検体を希釈するための希釈液であって、血液中に存在しない標準成分を含有する希釈液と、
血液検体の希釈物を収容するための収容器具と、
を含む、血液検査キットであって、
前記血液採取器具が、キャピラリーである、血液検査キット。 A blood collection device for collecting a blood sample;
A diluent for diluting a collected blood sample, which contains a standard component not present in the blood;
A storage device for storing a dilution of a blood sample;
A blood test kit comprising:
A blood test kit, wherein the blood collection device is a capillary. - 前記希釈液が、血液中に恒常的に存在する標準成分を含まない、請求項1から3のいずれか1項に記載の血液検査キット。 The blood test kit according to any one of claims 1 to 3, wherein the diluent does not contain a standard component that is constantly present in blood.
- 前記血液検査キットが、血液検体の希釈物から血漿を分離回収するための分離器具を含む、請求項1から4のいずれか1項に記載の血液検査キット。 The blood test kit according to any one of claims 1 to 4, wherein the blood test kit includes a separation instrument for separating and collecting plasma from a diluted blood sample.
- 前記キャピラリーに、採取した血液検体の容量を確認するための目盛が付されている、請求項1から5のいずれか1項に記載の血液検査キット。 The blood test kit according to any one of claims 1 to 5, wherein the capillary is provided with a scale for confirming the volume of the collected blood sample.
- 前記目盛が、採取する血液検体の容量範囲の下限を示す位置に付されており、かつ
前記キャピラリーが、ストッパーを有し、
前記ストッパーが、採取した血液検体の容量が、採取する血液検体の容量範囲の上限を超えないためのものである、請求項1から6のいずれか1項に記載の血液検査キット。 The scale is attached to a position indicating the lower limit of the volume range of the blood sample to be collected, and the capillary has a stopper,
The blood test kit according to any one of claims 1 to 6, wherein the stopper is for the volume of the collected blood sample not to exceed the upper limit of the volume range of the collected blood sample. - 前記キャピラリーが、定められた容量の血液検体を採取するための形状を有する、請求項1から7のいずれか1項に記載の血液検査キット。 The blood test kit according to any one of claims 1 to 7, wherein the capillary has a shape for collecting a blood sample having a predetermined volume.
- 前記キャピラリーが、内部に抗凝固剤を有する、請求項1から8のいずれか1項に記載の血液検査キット。 The blood test kit according to any one of claims 1 to 8, wherein the capillary has an anticoagulant inside.
- 前記キャピラリーが、合成樹脂からなる、請求項1から9のいずれか1項に記載の血液検査キット。 The blood test kit according to any one of claims 1 to 9, wherein the capillary is made of a synthetic resin.
- 前記キャピラリーの内壁が、親水性処理されたものである、請求項10に記載の血液検査キット。 The blood test kit according to claim 10, wherein the inner wall of the capillary is subjected to a hydrophilic treatment.
- 前記キャピラリーの血液検体を吸引する側の端部が、テーパーを有する、請求項10又は11に記載の血液検査キット。 The blood test kit according to claim 10 or 11, wherein an end of the capillary on the side for sucking a blood sample has a taper.
- 前記キャピラリーの、採取すべき血液検体の容量範囲を示す少なくとも1つの位置に目盛が付されており、前記キャピラリーの内径が、前記目盛の位置を越えた部位から大きくなる、請求項10から12のいずれか1項に記載の血液検査キット。 The scale is attached to at least one position indicating the volume range of the blood sample to be collected of the capillary, and the inner diameter of the capillary increases from a portion beyond the position of the scale. The blood test kit according to any one of the above.
- 前記キャピラリーの、採取すべき血液検体の容量範囲を示す少なくとも1つの位置に目盛が付されており、前記キャピラリーの前記目盛が付された位置を含む部分の内径が他の部分より小さい、請求項10から12のいずれか1項に記載の血液検査キット。 A scale is attached to at least one position indicating a volume range of a blood sample to be collected of the capillary, and an inner diameter of a portion including the position where the scale is attached of the capillary is smaller than other portions. The blood test kit according to any one of 10 to 12.
- 前記キャピラリーを構成する材が、600nm以上の領域の波長の光の少なくとも一部を吸収する成分を含有する、請求項10から14のいずれか1項に記載の血液検査キット。 The blood test kit according to any one of claims 10 to 14, wherein the material constituting the capillary contains a component that absorbs at least part of light having a wavelength of 600 nm or more.
- 血液中に恒常的に存在する前記標準成分が、ナトリウムイオン又は塩化物イオンである、請求項1又は2に記載の血液検査キット。 The blood test kit according to claim 1 or 2, wherein the standard component that is constantly present in blood is sodium ion or chloride ion.
- 血液中に恒常的に存在する前記標準成分が、ナトリウムイオン又は塩化物イオンと、さらに別の血液中に恒常的に存在する標準成分である、請求項1又は2に記載の血液検査キット。 The blood test kit according to claim 1 or 2, wherein the standard component that is constantly present in the blood is sodium ion or chloride ion and a standard component that is constantly present in another blood.
- 前記別の標準成分が、総タンパク又はアルブミンである、請求項17に記載の血液検査キット。 The blood test kit according to claim 17, wherein the another standard component is total protein or albumin.
- さらに別の標準成分を用いて、前記血液検体中の対象成分の濃度の分析を検証する、請求項17又は18に記載の血液検査キット。 The blood test kit according to claim 17 or 18, wherein the analysis of the concentration of the target component in the blood sample is verified using another standard component.
- 前記血液中に存在しない標準成分が、リチウムイオン又はグリセロール三リン酸である、請求項2に記載の血液検査キット。 The blood test kit according to claim 2, wherein the standard component not present in the blood is lithium ion or glycerol triphosphate.
- 前記血液中に存在しない標準成分が、リチウムイオン又はグリセロール三リン酸である、請求項3に記載の血液検査キット。 The blood test kit according to claim 3, wherein the standard component not present in the blood is lithium ion or glycerol triphosphate.
- 請求項1から21のいずれか1項に記載の血液検査キットを用いることを特徴とする、血液検体中の対象成分の濃度を分析する方法(医療行為を除く。)。 A method for analyzing the concentration of a target component in a blood sample (excluding medical practice), characterized by using the blood test kit according to any one of claims 1 to 21.
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020187000469A KR20180016513A (en) | 2015-07-06 | 2016-07-06 | Blood test kit and analyzing method using the same |
EP16821432.8A EP3321677B1 (en) | 2015-07-06 | 2016-07-06 | Blood test kit and analyzing method using the same |
CN201680039443.9A CN107864669A (en) | 2015-07-06 | 2016-07-06 | Blood test kit and the analysis method using blood test kit |
US15/861,238 US10697870B2 (en) | 2015-07-06 | 2018-01-03 | Blood test kit and analyzing method using the same |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2015-135066 | 2015-07-06 | ||
JP2015135066 | 2015-07-06 | ||
JP2016133959A JP6522556B2 (en) | 2015-07-06 | 2016-07-06 | Blood test kit and analysis method using the same |
JP2016-133959 | 2016-07-06 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/861,238 Continuation US10697870B2 (en) | 2015-07-06 | 2018-01-03 | Blood test kit and analyzing method using the same |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2017006963A1 true WO2017006963A1 (en) | 2017-01-12 |
Family
ID=57685045
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2016/070008 WO2017006963A1 (en) | 2015-07-06 | 2016-07-06 | Blood test kit and analysis method using same |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2017006963A1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107760673A (en) * | 2017-08-25 | 2018-03-06 | 南通普惠精准医疗科技有限公司 | The stabilizer of dissociative DNA and the heparin tube for dissociative DNA detection |
WO2018216607A1 (en) * | 2017-05-22 | 2018-11-29 | Okinawa Institute Of Science And Technology School Corporation | Integrated system for sampling and processing a liquid suspension |
WO2019220938A1 (en) * | 2018-05-15 | 2019-11-21 | 富士フイルム株式会社 | Blood specimen guide instrument and blood examination kit |
US20210010998A1 (en) * | 2018-03-28 | 2021-01-14 | Fujifilm Corporation | Sample acquisition information management device, sample acquisition information management system, and sample acquisition information management method |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS59180674U (en) * | 1983-05-20 | 1984-12-03 | テルモ株式会社 | vacuum blood collection tube |
JPH10221334A (en) * | 1997-02-07 | 1998-08-21 | Shima Kenkyusho:Kk | Hemolytic measuring method and microblood collecting tube and reagent kit used therefor |
JP2000232972A (en) * | 1999-02-15 | 2000-08-29 | Daikin Ind Ltd | Sample spot-sticking instrument |
JP2001330603A (en) * | 2000-05-18 | 2001-11-30 | Arkray Inc | Quantitative analysis method |
JP3093189U (en) * | 2002-10-02 | 2003-04-18 | 有限会社生理科学研究所 | Micro blood collection and dispenser |
JP2011112451A (en) * | 2009-11-25 | 2011-06-09 | Physical Screening Inc | Method for analyzing biological sample component |
-
2016
- 2016-07-06 WO PCT/JP2016/070008 patent/WO2017006963A1/en active Application Filing
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS59180674U (en) * | 1983-05-20 | 1984-12-03 | テルモ株式会社 | vacuum blood collection tube |
JPH10221334A (en) * | 1997-02-07 | 1998-08-21 | Shima Kenkyusho:Kk | Hemolytic measuring method and microblood collecting tube and reagent kit used therefor |
JP2000232972A (en) * | 1999-02-15 | 2000-08-29 | Daikin Ind Ltd | Sample spot-sticking instrument |
JP2001330603A (en) * | 2000-05-18 | 2001-11-30 | Arkray Inc | Quantitative analysis method |
JP3093189U (en) * | 2002-10-02 | 2003-04-18 | 有限会社生理科学研究所 | Micro blood collection and dispenser |
JP2011112451A (en) * | 2009-11-25 | 2011-06-09 | Physical Screening Inc | Method for analyzing biological sample component |
Non-Patent Citations (1)
Title |
---|
MASATOSHI HORITA ET AL.: "Establishment of Mail Medical Examination System Using Immediate Plasma Separating Device by the Self-Collection Blood : The Method of Dilution Ratio Calculation by Using Internal Standard for the Sample with Different Amount of Collecting Blood", THE JAPANESE JOURNAL OF CLINICAL PATHOLOGY, vol. 56, no. 7, 25 July 2008 (2008-07-25), pages 577 - 583, XP009503400 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018216607A1 (en) * | 2017-05-22 | 2018-11-29 | Okinawa Institute Of Science And Technology School Corporation | Integrated system for sampling and processing a liquid suspension |
CN107760673A (en) * | 2017-08-25 | 2018-03-06 | 南通普惠精准医疗科技有限公司 | The stabilizer of dissociative DNA and the heparin tube for dissociative DNA detection |
CN107760673B (en) * | 2017-08-25 | 2020-08-07 | 南通普惠精准医疗科技有限公司 | Stabilizer for free DNA and blood collection tube for detecting free DNA |
US20210010998A1 (en) * | 2018-03-28 | 2021-01-14 | Fujifilm Corporation | Sample acquisition information management device, sample acquisition information management system, and sample acquisition information management method |
WO2019220938A1 (en) * | 2018-05-15 | 2019-11-21 | 富士フイルム株式会社 | Blood specimen guide instrument and blood examination kit |
JP2019200085A (en) * | 2018-05-15 | 2019-11-21 | 富士フイルム株式会社 | Blood specimen guide tool, and blood inspection kit |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10697870B2 (en) | Blood test kit and analyzing method using the same | |
EP3321676B1 (en) | Blood test kit and blood analysis method | |
WO2017006963A1 (en) | Blood test kit and analysis method using same | |
WO2017006962A1 (en) | Blood test kit and blood analysis method | |
WO2018124275A1 (en) | Blood test kit and blood analysis method | |
US10788478B2 (en) | Blood test kit, member thereof, and method for manufacturing the same | |
US10634661B2 (en) | Blood analysis method and blood test kit | |
US10739361B2 (en) | Blood analysis method and blood test kit | |
JP6789108B2 (en) | Blood analysis method and blood test kit | |
US11179081B2 (en) | Blood analysis method and blood test kit | |
WO2018124273A1 (en) | Blood analysis method and blood test kit | |
WO2017006965A1 (en) | Blood analysis method and blood testing kit | |
WO2017006964A1 (en) | Blood test kit, members thereof, and production method for same |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 16821432 Country of ref document: EP Kind code of ref document: A1 |
|
DPE1 | Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101) | ||
ENP | Entry into the national phase |
Ref document number: 20187000469 Country of ref document: KR Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2016821432 Country of ref document: EP |