WO2021208262A1 - 干血斑定量采集装置及方法 - Google Patents

干血斑定量采集装置及方法 Download PDF

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WO2021208262A1
WO2021208262A1 PCT/CN2020/099852 CN2020099852W WO2021208262A1 WO 2021208262 A1 WO2021208262 A1 WO 2021208262A1 CN 2020099852 W CN2020099852 W CN 2020099852W WO 2021208262 A1 WO2021208262 A1 WO 2021208262A1
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tube
blood
quantitative
collection
sample
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PCT/CN2020/099852
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English (en)
French (fr)
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杭红
张云
李宇
张向辉
赵伟洁
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上海药明奥测医疗科技有限公司
苏州药明泽康生物科技有限公司
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Publication of WO2021208262A1 publication Critical patent/WO2021208262A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/10—Devices for withdrawing samples in the liquid or fluent state
    • G01N1/14—Suction devices, e.g. pumps; Ejector devices
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/15—Devices for taking samples of blood
    • A61B5/150007—Details
    • A61B5/150015—Source of blood
    • A61B5/150022—Source of blood for capillary blood or interstitial fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/15—Devices for taking samples of blood
    • A61B5/150007—Details
    • A61B5/150343—Collection vessels for collecting blood samples from the skin surface, e.g. test tubes, cuvettes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/15—Devices for taking samples of blood
    • A61B5/150007—Details
    • A61B5/150755—Blood sample preparation for further analysis, e.g. by separating blood components or by mixing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/15—Devices for taking samples of blood
    • A61B5/151—Devices specially adapted for taking samples of capillary blood, e.g. by lancets, needles or blades
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • 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
    • G01N1/14—Suction devices, e.g. pumps; Ejector devices
    • G01N2001/1472—Devices not actuated by pressure difference
    • G01N2001/149—Capillaries; Sponges
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/062—Preparation extracting sample from raw material

Definitions

  • the invention relates to the technical field of dry blood spot detection in biotechnology, and in particular to a dry blood spot quantitative collection device and method.
  • Dried Blood Spot (DBS) detection technology is a method in which a biological sample (usually whole blood) is dripped onto a filter paper and stored, and then extracted to detect analytes in the dried spot sample.
  • the technology is early Used for qualitative analysis and detection of nucleic acid genetic material. Because it has the advantages of convenient collection, small blood collection, and easy storage and transportation of samples, it has been used for newborn screening for phenylketonuria in the 1960s. At present, the most mature application of dried blood spot detection is still the screening of neonatal genetic and metabolic diseases. At the same time, its application is gradually expanding to the detection of endogenous biomarkers, therapeutic drug monitoring (TDM) and other fields.
  • TDM therapeutic drug monitoring
  • Dried blood spot detection is a set of systematic techniques, including: sample collection method; sample storage and transportation; establishment and verification of inspection methods; sample extraction processing; sample detection; analysis, processing and interpretation of test data.
  • dried blood spots have the following advantages as a sample collection technique:
  • Sampling is simple, not limited by time and space (can be extended to sampling in homes and remote areas). Dried blood spots are collected by puncturing the fingertips or heels with an automatic blood sampling needle. After the first drop of blood is wiped off, the remaining whole blood is directly dripped or transferred to the blood sampling card with a capillary tube. After proper training, users can realize self-service blood sampling. For some TDM drug monitoring, it is necessary to collect peak and trough concentrations at a specific time, and the use of dried blood spot detection can greatly reduce the difficulty of obtaining samples.
  • the blood collection process is minimally invasive and the blood collection volume is small.
  • An engineered automatic blood sampling needle is used to puncture the skin, and the puncture depth is generally about 2mm, which minimizes pain and trauma.
  • dried blood film testing can technically expand the application scenarios of in vitro diagnostics.
  • Dried blood spots can ensure the stability of most analytes, allowing samples to be stored at room temperature and long-distance transportation, greatly reducing the cost of storage and transportation, and making home sampling and third-party testing possible.
  • a study showed that the amino acids in dried blood spots were only slightly degraded after being stored at room temperature for 5 years. The literature also shows that most therapeutic drugs are relatively stable in dried blood spot samples.
  • the dried blood spot detection method also has some problems and shortcomings, which limit its further wide application. These problems and deficiencies include: the inability to review the blood source and sampling time during self-sampling; limited by the collection conditions and collection operations, although training and instructions have been given, many samples are still unqualified; patients are afraid of self-piercing the skin Psychological; due to the small sampling volume, effective detection of some low-concentration analytes cannot be carried out.
  • the most widely used blood collection card for dried blood spot technology is cotton filter paper, which is made of uniform cotton fiber, which can evenly distribute the absorbed whole blood.
  • the collection of blood spots is usually non-quantitative blood collection, direct fingertip blood to the blood collection card, or transfer with a microcapillary tube.
  • the traditional extraction procedure is to lay a fixed-size disc from the center of the dried blood spot, place it in a suitable container, and extract the analyte with a suitable extraction solution.
  • the detection instrument is usually a high-sensitivity liquid chromatography (HPLC) or chromatograph- Mass spectrometer (LC-MS).
  • the main influencing factors include the chromatographic effect of the filter paper (center-edge difference), blood volume effect, and hematocrit effect (also known as hematocrit, Hematocrit, HCT).
  • the chromatographic effect is due to the inconsistent diffusion rate of red blood cells and plasma in the filter paper in whole blood.
  • the analyte specifically binds to plasma or red blood cells, it may cause uneven distribution of the analyte on the blood spot card. This can usually be done by fixation. Punch in the center of the blood spot to solve it.
  • the volume effect is due to the difference in the volume of blood collected, which results in a different amount of blood contained in the filter paper per unit area. Usually, due to incorrect sampling, more than two drops of whole blood are dropped at the same position, and the volume effect is more obvious.
  • Hematocrit is the most important factor affecting the quantitative accuracy of dried blood spots. Changes in HCT will greatly affect blood viscosity, which in turn affects the diffusion characteristics of whole blood in the filter paper. Clinically, the HCT value of patient blood samples ranges from 20% to 60%. HCT will affect both the chromatographic effect and the volume effect. The research of Holub and Adam on prenatal screening found that due to the difference in the distribution of amino acids in plasma and red blood cells, as the HCT increases, the measured amino acid concentration will increase significantly. At low HCT, the amino acid concentration at the edge of the blood spot card will be significantly higher than the center. In addition, HCT can significantly affect the volume of whole blood per unit area.
  • Some products use microfluidics to achieve quantitative blood sampling. After transferring to the blood sampling card, all blood spots are extracted to avoid the volume and chromatography effects. However, because the filter paper is made of cotton fiber, the HCT has a major influence on the extraction rate. Interference factors, the test results will still be affected by HCT.
  • the current collection method of blood spot samples has a high probability of misoperation.
  • non-quantitative blood sampling is performed by dropping fingertip blood directly onto the blood sampling card, or transferring it with a microcapillary tube.
  • the volume of the blood drips varies greatly, excessive squeezing is required, and there is the possibility of repeated dripping.
  • the capillary is transferred, the slender capillary is easy to shake when held, causing difficulty in sampling, and the transfer process takes a long time, and the filter paper is easily scraped due to displacement at the contact with the filter paper, resulting in uneven blood diffusion.
  • the technical problem to be solved by the present invention is to provide a dry blood spot quantitative collection device, which can realize quantitative blood collection, complete sample recovery, improve the accuracy of quantitative detection results of blood spots, and enable sample collection, storage, extraction, and inspection
  • the operation is completed through the integration of the device.
  • the present invention provides a dried blood spot quantitative collection device, including: a sample collection tube, a sample collection filter paper, and a quantitative blood collection tube; wherein,
  • the sample collection tube includes a tube body and a tube cover; the tube body is a straight tube with opposite first and second ends; the first end is closed and is the bottom of the tube; the second end is An opening, which is a nozzle; the cap is used to close the nozzle;
  • the quantitative blood collection tube includes a quantitative capillary tube and a fixing element; the quantitative capillary tube is fixed to the fixing element, and the quantitative capillary tube has a blood sampling end for collecting blood and exposed outside the fixing element; the fixing element is engaged with At the mouth of the tube, the quantitative capillary tube extends into the tube body;
  • the sample collection filter paper is arranged in the tube body, and the blood collection end abuts against the sample collection filter paper.
  • the tube body is cylindrical
  • the tube opening is circular
  • the tube cover cooperates with the tube opening to form a seal.
  • the nozzle and the pipe cover are integrated or separated.
  • the nozzle has a first protrusion, which is clamped to the fixing member by the first protrusion.
  • the fixing member clamped to the nozzle plays a role of shielding and sealing the nozzle.
  • the bottom of the tube is U-shaped.
  • the tube body is made of plastic material.
  • the tube body is transparent.
  • the fixing member has a cavity, and the cavity is filled with a desiccant.
  • the fixing member has a second protrusion, which is clamped with the tank lid by the second protrusion. Therefore, the purpose of the tube cover closing the tube opening to form a closed space inside the tube body is achieved.
  • the fixing member is a pipe string
  • the pipe string is sleeved in the pipe body of the sample collection tube
  • the top end of the pipe string is open, and is engaged with the mouth of the pipe body
  • the bottom end of the pipe string is provided with a fixing hole
  • the quantitative capillary tube is inserted and fixed in the fixing hole.
  • the tube wall of the tube string, the bottom end of the tube string and the quantitative capillary form a circular columnar cavity surrounding the quantitative capillary tube, and the cavity can be used for placing a desiccant.
  • the pipe cover closes the pipe orifice, it is simultaneously pressed on the top of the pipe string, so that the position of the pipe string is fixed and the top of the pipe string is closed.
  • the fixing member is made of plastic material.
  • the fixing member is transparent.
  • the two ends of the quantitative capillary tube are respectively located on both sides of the bottom end of the pipe string; wherein, the end away from the top end of the pipe string is exposed outside the pipe string and is the blood sampling end; and is close to the pipe string
  • One end of the top end is located in the pipe string, and the height is lower than the top end of the pipe string.
  • the pipe string is cylindrical.
  • the fixing hole is located at the center of the cross section of the pipe string, and the quantitative capillary tube is inserted and fixed in the fixing hole along the axial direction of the pipe string.
  • the pipe string is made of plastic material.
  • the pipe string is transparent.
  • the quantitative capillary tube is made of glass.
  • the sample collection filter paper is spread on the bottom of the tube in a horizontal state
  • the quantitative blood collection tube is located above the sample collection filter paper in a vertical state, and is perpendicular to the sample collection filter paper, and the quantitative collection tube The blood collection end of the blood vessel abuts against the sample collection filter paper.
  • the sample collection filter paper has a circular sheet shape, and the blood sampling end abuts against the center of the circular sheet shape.
  • the tube body is cylindrical
  • the tube bottom is U-shaped
  • the sample collection filter paper is a round sheet
  • the sample collection filter paper is spread horizontally on the bottom of the tube
  • the sample collection A gap is formed between the filter paper and the U-shaped arc surface of the tube bottom.
  • the sample collection filter paper adopts glass fiber filter paper.
  • the present invention also provides a method for collecting dried blood spots using a quantitative collection device for dried blood spots, which includes the following steps:
  • Step S1 Open the tube cover, take out the quantitative blood collection tube, bring the blood collection end of the quantitative capillary in the quantitative blood collection tube close to and contact the blood, and wait for the capillary force to fill the entire quantitative capillary with blood;
  • Step S2 Insert the quantitative blood collection tube back into the sample collection tube, and ensure that the blood collection end of the quantitative capillary is in contact with the sample collection filter paper;
  • Step S3 Cover the tube cover, and rely on air pressure and filter paper suction to transfer all blood samples in the quantitative capillary to the sample collection filter paper.
  • the method further includes step S4: after step S3, the dried blood spot quantitative collection device is placed in a suitable environment for storage or transportation.
  • the method further includes step S5: after step S3, when the sample is analyzed, the tube cover is opened, the quantitative blood collection tube is taken out, the extraction liquid is directly added to the sample collection tube to extract the analyte, and the extracted analyte is sent for testing.
  • the method further includes step S6: in any of the foregoing steps, the sample source information is pasted on the outside of the sample collection tube.
  • the present invention provides a new dried blood spot sample collection device and method. Compared with the commonly used blood collection card collection and extraction methods, the main improvements of the present invention include:
  • the glass fiber filter paper is directly processed with the extraction solution, which is equivalent to the use of a pre-punched design, without the need for punching of dried blood spots. With quantitative blood sampling, all samples can be recovered.
  • special tools are required to lay a fixed area of blood slices in the sample center, which is time-consuming and labor-intensive, and there is a possibility of cross-contamination.
  • This method adopts pre-formed micro filter paper sheets to collect samples, saves the punching step, and directly adds the extraction liquid to the collection tube for extraction, which saves a lot of time and consumables.
  • the integrated blood sampling device design can realize the collection, drying, transportation, storage and extraction of samples in the same device, avoiding sample contamination.
  • the traditional blood collection card filter paper is exposed and needs to be exposed and dried after blood collection. There is a high risk of cross-contamination between different samples. In the process of storage, transfer, extraction, etc., due to improper holding and other reasons, samples are also susceptible to contamination from unknown sources.
  • the sample collection tube used in this method is closed from sampling to analysis, and contains desiccant so that the sample does not need to be exposed to dryness, and the probability of sample contamination is minimized.
  • Fig. 1 is a schematic diagram of a three-dimensional structure of a specific embodiment of the dried blood spot quantitative collection device of the present invention.
  • Fig. 2 is a cross-sectional view of Fig. 1.
  • Fig. 3 is an exploded schematic diagram of the structure of the dried blood spot quantitative collection device in Fig. 1.
  • Figure 4 is a flow chart of the collection and detection steps of dried blood spot samples.
  • Fig. 5 is a mass spectrum peak pattern diagram of some therapeutic drugs in a dried blood spot sample of the present invention.
  • Fig. 6 is a calibration curve diagram of a mass spectrum of a dried blood spot sample of the present invention.
  • Figure 7 shows the interference comparison of HCT to the blood concentration detection of imatinib.
  • Figure 8 shows the interference comparison of HCT on the detection of sirolimus blood concentration.
  • Figure 9 shows the interference comparison of HCT on the detection of valproic acid blood concentration.
  • Tube body 1a. Tube body
  • a quantitative collection device for dried blood spots includes a sample collection tube 1, a quantitative blood collection tube 2 and a sample collection filter paper 3.
  • the sample collection tube 1 is composed of two parts: a tube body 1a and a tube cover 1b.
  • the tube body 1a is a straight tube with opposite first and second ends, wherein the first end is a closed tube bottom 1a1, and the second end is an open nozzle 1a2.
  • the cap 1b is used to close the nozzle 1a2.
  • the quantitative blood collection tube 2 includes a fixing member 2a and a quantitative capillary tube 2b fixed on the fixing member 2a.
  • the fixing member 2a is engaged with the nozzle 1a2 of the sample collection tube 1, and the quantitative capillary tube 2b extends into the tube; the quantitative capillary tube 2b has a blood sampling end 2b1 for collecting blood, and the blood sampling end 2b1 is exposed outside the fixing element 2a.
  • the sample collection filter paper 3 is arranged in the tube body 1 a, and the blood collection end 2b1 abuts against the sample collection filter paper 3.
  • the dried blood spot quantitative collection device of the present invention can realize quantitative blood collection through the quantitative blood collection tube 2.
  • the collected blood samples are timely and all transferred to the sample collection filter paper 3, sealed in the sample collection tube 1, and sent to the sample collection tube during analysis. 1.
  • Adding the extraction liquid to extract all the samples which can realize the full recovery of the samples and improve the accuracy of the quantitative detection results of dried blood spots.
  • the overall design of the device enables the integration of sample collection, storage, extraction and inspection.
  • the dried blood spot quantitative collection device of the present invention is mainly used for the quantitative collection and storage of trace peripheral blood (such as fingertip blood), and can also be used to collect and store other clinical samples (such as venous blood, serum, plasma, urine). Wait).
  • the dry blood spot quantitative collection device of the present invention adopts loose glass fiber filter paper, which has a higher extraction recovery rate than cotton fiber, can realize all extraction and detection of collected samples, and is combined with quantitative blood collection means to avoid sample volume , Hematocrit (HCT), etc.
  • the dried blood spot quantitative collection device of the present invention has simpler sampling operation and low probability of misoperation.
  • the quantitative capillary can be operated by holding the fixing member. After the sample suction is completed, the quantitative blood collection tube is inserted into the sample collection tube, and the sample is completed by closing the lid. The sample is transferred to the filter paper by the suction force of the filter paper and air pressure.
  • blood is often directly dripped on the blood collection card, the sample volume varies greatly, excessive squeezing is required, and there is a possibility of repeated blood dripping.
  • the slender capillary is easy to shake when held, causing difficulty in sampling, and it is easy to scratch the filter paper during transfer and cause uneven spread of the sample.
  • the dry blood spot quantitative collection device of the present invention is closed from sampling to analysis, and contains a desiccant so that the sample does not need to be exposed to dryness, and the probability of sample contamination is minimized, and there is no risk of sample cross-contamination.
  • the traditional blood collection card filter paper is exposed to the outside and needs to be exposed and dried after blood collection. There is a high risk of cross-contamination between different samples. In the process of storage, transfer, extraction, etc., due to improper holding and other reasons, samples are also susceptible to contamination from unknown sources.
  • the dried blood spot quantitative collection device of the present invention directly adds all the samples on the sample collection filter paper 3 in the sample collection tube 1 for extracting liquid, without the need for the dried blood spot perforating operation, which saves a lot of time and consumables.
  • special tools are required to lay a fixed area of blood slices in the center of the dried blood spot sample, which is time-consuming and laborious, and there is a possibility of cross-contamination.
  • the size of the sample collection tube 1 is the same as that of a conventional 2 mL centrifuge tube, and the size can also be changed as needed.
  • the sample collection tube 1 is made of plastic material, which includes but is not limited to polypropylene, polyethylene, polyester, etc.
  • the main function of the sample collection tube 1 is to seal and preserve the sample collection filter paper 3 and serve as a container for sample extraction.
  • the tube body 1a is preferably transparent for easy visual operation.
  • the tube body 1a is cylindrical, the nozzle opening 1a2 is circular, and the tube cover 1b is used to shield the nozzle opening 1a2 so that the interior of the tube body 1a forms a closed space from the outside.
  • the tube opening 1a2 and the tube cover 1b are integrated or separated.
  • the tube cover 1b and the nozzle 1a2 are integrated, and there is a bendable connection between the tube cover 1b and the nozzle 1a2.
  • the fixing member 2a is clamped tightly with the nozzle 1a2 of the sample collection tube 1a. As shown in FIG.
  • the nozzle 1a2 of the pipe body 1a has a first protrusion 1a3, and the fixing member 2a extending into the pipe body 1a is clamped by the first protrusion 1a3 to keep the position of the fixing member 2a stable.
  • the fixing member 2a plays a role of shielding the nozzle 1a2.
  • the pipe cover 1b and the top end 2a1 of the fixing member 2a can be fixed by a snap structure to close the top end 2a1 of the fixing member 2a, and at the same time achieve the purpose of closing the nozzle 1a2.
  • the tube cover 1b and the nozzle 1a2 are separated, and the tube cover 1b and the top end 2a1 of the fixing member 2a can be fixed by a screw structure to close the top end 2a1 and the nozzle 1a2.
  • the pipe cover 1b can also completely cover or wrap the top end 2a1 of the fixing member 2a, and directly form a connection with the nozzle 1a2 so that both the top end 2a1 and the nozzle 1a2 are closed, such as the pipe cover 1b and the nozzle
  • the outer wall/inner wall of 1a2 forms a snap structure or a threaded structure.
  • the tube bottom 1a1 of the tube body 1a of the sample collection tube 1 is U-shaped, which is commonly referred to as a round bottom. .
  • the fixing member 2a also has a cavity for holding a desiccant 2c.
  • the desiccant 2c is beneficial to ensure that the internal environment of the sample collection tube 1 is dry and shorten the drying time after blood sample collection .
  • the device may also not contain the desiccant 2c, which will not affect the main function of the device.
  • the size of the fixing member 2a is larger than the diameter of the dosing capillary 2b, so as to facilitate holding and avoid shaking during sampling.
  • the fixing member 2a has a second protrusion 2a3.
  • the second protrusion 2a3 cooperates with the tube cover 1b to achieve the effect of closing the tube body 1a, that is, the tube cover 1b shields the top end 2a1 of the fixing member 2a.
  • the top end 2a1 of the fixing member 2a shields the nozzle 1a2, and the three are clamped to each other to form a closed space inside the pipe body 1a.
  • the fixing member 2a is made of plastic material.
  • the fixing member 2a is transparent.
  • the fixing member 2a is a pipe string, which is sleeved in the pipe body 1a of the sample collection tube 1 and clamped and fixed to the pipe mouth 1a2.
  • the two ends of the fixing member 2a are defined as a top end 2a1 and a bottom end 2a2, respectively.
  • the top end 2a1 of the pipe string is open, and the side surface of the top end 2a1 has a second protrusion 2a3, which is engaged with the pipe cover 1b through the second protrusion 2a3 to limit the position of the pipe string in the pipe body 1a.
  • the pipe The inner wall of the body 1a is provided with a recess that matches the protrusion 2a3; the bottom end 2a2 of the pipe string has a fixing hole 2a4, and the quantitative capillary tube 2b is inserted and fixed in the fixing hole 2a4.
  • the pipe wall of the pipe string, the bottom end 2a2 of the pipe string and the quantitative capillary tube 2b an annular cylindrical cavity surrounding the quantitative capillary tube 2b is formed, and the desiccant 2c can be placed in the cavity.
  • the pipe cover 1b When the pipe opening 1a2 is closed by the pipe cover 1b, the pipe cover 1b is pressed against the top end 2a1 of the pipe string at the same time, so that the position of the pipe string is fixed and the top end 2a1 is closed.
  • the diameter of the pipe column is larger than the quantitative capillary 2b, which is convenient for the user to hold and collect blood, and prevents shaking during sampling.
  • the pipe string is made of transparent plastic material, which is convenient for visual operation.
  • the two ends of the quantitative capillary tube 2b are respectively located on both sides of the bottom end 2a2 of the pipe string; one end is away from the top end 2a1 of the pipe string and is exposed on the pipe string The outside is the blood sampling end 2b1; and the end relatively close to the top end 2a1 of the pipe string is located inside the pipe string and is lower than the top end 2a1 of the pipe string.
  • the pipe string is cylindrical.
  • the fixing hole 2a4 is located at the center of the cross section of the pipe string (ie, at the center of the circle), and the quantitative capillary 2b is inserted and fixed in the fixing hole along the axial direction of the pipe string.
  • the quantitative capillary 2b is made of glass, and the liquid can fill the tube body by capillary force.
  • the glass material can also be replaced with other commonly used hydrophilic materials.
  • the sample collection filter paper 3 is spread on the bottom 1a1 of the tube in a horizontal state, and the quantitative blood collection tube 2b is vertically positioned directly above the sample collection filter paper 3, perpendicular to the sample collection filter paper 3, and the quantitative blood collection tube
  • the blood sampling end of 3 abuts the sample collection filter paper 3.
  • the sample collection filter paper 3 is in the shape of a circular sheet, and the blood sampling end 2b1 abuts against the center of the circular sheet.
  • the circular sheet-shaped sample collection filter paper 3 has a diameter of about 6 mm.
  • the material of the sample collection filter paper 3 is glass fiber filter paper, which can also be replaced by other porous loose materials.
  • the tube body 1a is cylindrical
  • the tube bottom 1a1 is U-shaped
  • the sample collection filter paper 3 is a round sheet
  • the sample collection filter paper 3 is spread horizontally on the tube bottom 1a1
  • the sample collection filter paper 3 is connected to the bottom of the tube.
  • a gap is formed between the U-shaped arc surfaces of 1a1. The sample flowing out from the blood collection end 2b1 is completely received by the sample collection filter paper 3.
  • the dried blood spot quantitative collection device of the present invention can complete the collection and detection of dried blood spot samples according to the following method steps:
  • Example 2 Collect fingertip blood for therapeutic drug monitoring (TDM)
  • This method can be used to collect blood from fingertips to make dried blood slice samples.
  • dried blood sheet samples have unique advantages such as less blood collection, stable samples, easy storage and transportation, and no time and space constraints for sampling.
  • TDM therapeutic drug monitoring
  • it is necessary to collect peak and trough concentrations at a specific time, or frequent sampling in a short period of time.
  • Dried peripheral blood samples can meet clinical needs.
  • This method is simple to operate and has high sample quality. After training, it can be extended to home self-sampling.
  • Extraction method 1 used to extract plasma protein-bound drugs
  • Extraction method 2 Used to extract blood cell-bound drugs
  • liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS/MS) is used for detection, which has high specificity and sensitivity.
  • the liquid-mass method is a mature method that can be retrieved in the literature.
  • Figure 5 shows the mass spectra of some therapeutic drugs detected by the device of the present invention, including imatinib, carbamazepine, valproic acid, mycophenolic acid, tacrolimus, cyclosporine A, and siro. Moss, showing that the relevant drug mass spectrum peak shape is excellent, there are no peaks and tailings, and it can be used for drug concentration detection.
  • the calibrator of the drug to be tested needs to be prepared.
  • the drug standard stock solution is added to the whole blood to prepare a calibrator with 6 concentration points.
  • the dried blood spot of the calibrator is made with a blood sampling device for dried blood tablets.
  • the dried blood spots are extracted and injected together to generate a calibration curve in the instrument, and quantitative detection can be realized.
  • Figure 6 shows the mass spectrometry calibration curves of dried blood spot samples of imatinib and sirolimus.
  • A is the calibration curve of imatinib, and the calibrator concentrations are: 50, 500, 1000, 2000, 3000 ng/mL, 5000 ng/mL
  • B is the calibration curve of sirolimus
  • the calibrator concentrations are respectively: 2, 4, 6, 12, 20, 30 ng/mL
  • the linear correlation coefficient r is all greater than 0.9990, indicating that the device of the present invention is effective
  • the integrated operation can improve the accuracy of the quantitative detection results of dried blood spots.
  • Example 3 Collecting human whole blood for nucleic acid detection
  • This method uses venous whole blood samples, which are loaded on a blood sample collection device, and used for gene mutation detection after nucleic acid extraction.
  • the sample can be tested for genetics.
  • the digital PCR method is used to detect the R140 locus of the IDH2 gene in the sample.
  • Digital PCR is a new method of nucleic acid detection and quantitative analysis, which can realize the absolute quantification of nucleic acid and the detection of rare alleles.
  • the following table is the quantitative and qualitative test results of this sample:
  • the sample is a simulated dried blood spot sample from normal human whole blood, and the test result is no R140 mutation (negative), and the results of the two are consistent. And the dried blood spot samples extracted this time can also meet the needs of subsequent genetic testing experiments. It shows that the device of the present invention can effectively collect and preserve dried blood spot samples, and is applied to clinical qualitative detection of molecular biology.
  • Example 4 Collect whole blood for qualitative detection of antibodies
  • This method can be used to collect whole blood for qualitative detection of antibodies.
  • dried blood sheet samples have unique advantages such as less blood collection, stable samples, easy storage and transportation, and no time and space constraints for sampling.
  • the method is simple to operate and the sample quality is high.
  • antibodies can be analyzed.
  • open the cover of the sample collection tube take out the quantitative blood collection tube, add 1 mL of sample diluent to the sample collection tube, shake at 600 rpm at 25°C for 3 hours, and perform the test according to the following experimental procedures.
  • Reading with microplate reader first use blank wells to zero, and then read the OD value of each well.
  • Example 5 Using the dry blood spot quantitative collection device of the present invention and the existing method hematocrit (HCT) interference comparison
  • HCT range 20% ⁇ 60%
  • the new method and the control method were used to detect and compare the bias of different HCT samples relative to 40% HCT samples, and the coefficient of variation (CV) of the sample population.
  • Test drug Imatinib
  • Test drug sirolimus
  • Test drug valproic acid

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Abstract

一种干血斑定量采集装置，包括：样本收集管（1）、样本收集滤纸（3）和定量采血管（2）；其中，样本收集管（1）包括管体（1a）和管盖（1b）；管体（1a）为直管，管盖（1b）用于封闭管口（1a2）；定量采血管（2）包括定量毛细管（2b）和固定件（2a）；定量毛细管（2b）固定于固定件（2a），定量毛细管（2b）具有用于采血且露出于所述固定件（2a）之外的采血端（2b1）；固定件（2a）卡合于管口（1a2），定量毛细管（2b）伸入管体（1a）内；样本收集滤纸（3）设置于管体（1a）内，采血端（2b1）相抵靠于样本收集滤纸（3）。另外，提供一种进行干血斑采集的方法。采用一体化采血装置设计，可在同一个装置实现样本的采集、干燥、运输保存和萃取，避免了样本的污染，提高干血斑定量检测结果的准确性。

Description

干血斑定量采集装置及方法 技术领域
本发明涉及生物技术中的干血斑检测技术领域，特别是涉及一种干血斑定量采集装置及方法。
背景技术
干血斑(Dried Blood Spot,DBS)检测技术，是将生物样本(通常为全血)滴加到滤纸片上干燥保存，再经过萃取后检测干斑样本中分析物的一种方法，该技术早期用于核酸类遗传物质的定性分析检测。因其具有采集方便，采血量少，样本易于保存运输的优点，上世纪六十年代开始被用于苯丙酮尿症的新生儿筛查。目前干血斑检测应用最成熟的领域依然为新生儿遗传代谢类疾病的筛查，同时其应用逐渐向内源性生物标志物检测，治疗药物监测(therapeutic drug monitoring,TDM)等领域扩展。
干血斑检测是一套系统的技术，包括：样本的采集方法；样本的保存运输；检验方法的建立及验证；样本的萃取处理；样本的检测；检测数据的分析处理及解释。其中干血斑作为一种样本采集技术具有以下一些优势：
1)采样简单，不受时间与空间限制(可拓展至家庭及偏远地区采样)。干血斑采集使用自动采血针刺破指尖或足跟，擦拭掉第一滴血后，将剩余全血直接滴到或用毛细管转移到采血卡上。用户经过适当培训后，能够实现自助采血。对于一些TDM药物监测，需要在特定的时间采集峰、谷浓度，采用干血斑检测能够大大降低样本获得的难度。
2)采血过程微创，采血体积少。采用工程化的自动采血针刺破皮肤，刺穿深度一般为2mm左右，最小化痛感及创伤。对于婴幼儿、凝血功能障碍病人、ICU病患以及偏远地区等采血存在困难的情形，干血片检测能够在技术上拓展体外诊断的应用场景。
3)干血斑能够保证绝大多数分析物的稳定性，使样本能够在常温保存及长距离运输，大大降低了保存及运输的成本，使居家采样、第三方检测成为可能。一项研究显示干血斑样本中的氨基酸在常温下保存5年后只有微小的降解。文献也显示大多数治疗药物在干血斑样本中都比较稳定。
另一方面，干血斑检测法同样存在着一些问题和不足，限制了其进一步的广泛应用。这些问题和不足包括：自助采样时无法复核血液来源及采样时间；受采集条件和采集操作的限制，虽然经过培训及说明书指导，但很多样本的采集仍然不合格；患者对自助刺破皮肤有恐 惧心理；由于采样体积少，对于一些低浓度分析物无法进行有效的检测。
目前干血斑技术使用最广泛的采血卡为棉质滤纸，材质为均匀的棉花纤维，能够使吸收的全血均匀分布。血斑的采集通常为非定量采血，直接将指尖血点到采血卡上，或者用微量毛细管转移。用于定量检测时，需要用相同的基质制备校准曲线卡。传统的萃取步骤为从干血斑中心打下一块固定大小的圆片，置于合适的容器中，用适当的萃取液萃取分析物，检测仪器常为高灵敏度的液相色谱(HPLC)或色谱-质谱联用仪(LC-MS)。
这种常用的方法相比静脉血液样本在定量准确度上会受到更多的干扰，这也是导致其应用局限性的主要原因。主要的影响因素包括滤纸的层析效应(中心边缘差异)，采血体积效应，及红细胞压积效应(又称红细胞比容,Hematocrit,HCT)。
层析效应是由于全血中红细胞和血浆在滤纸中的扩散速率不一致，当分析物与血浆或红细胞特异性结合时，可能导致分析物在血斑卡上的分布不均匀，这通常可以通过固定在血斑中心打卡来解决。体积效应是由于采血体积的差异，导致单位面积的滤纸含有的血量不同，通常由于错误采样，在同一位置滴加了两滴以上的全血，体积效应才较明显。
红细胞压积(HCT)是影响干血斑定量准确度的最主要因素。HCT变化会极大的影响血液黏度，进而影响全血在滤纸中的扩散特性，而临床上患者血液样本HCT值跨度从20％～60％不等。HCT会同时影响层析效应及体积效应，Holub和Adam对产前筛查的研究发现，由于氨基酸在血浆和红细胞中的分布差异，随着HCT升高，测得的氨基酸浓度会显著增高，而低HCT时，血斑卡边缘的氨基酸浓度会显著高于中心。此外HCT会显著影响单位面积全血体积，当HCT升高时，血斑的扩散面积减少，则打下相同面积的血斑含有的样本体积增多，导致检测结果受到显著干扰。如何消除HCT对干血斑样本定量结果的干扰，是干血斑检测应用方面的一大难题。
一些产品通过微流控实现定量采血，转移到采血卡后将血斑全部打下萃取，避开了体积和层析效应，但由于滤纸材质为棉质纤维，则HCT对萃取率的影响变成主要干扰因素，检测结果仍然会受到HCT影响。
目前血斑样本的采集方法误操作几率较大。通常为非定量采血，直接将指尖血滴到采血卡上，或者用微量毛细管转移。直接滴血时，血滴的体积差异较大，需过度挤压，且存在重复滴血的可能。毛细管转移时，细长的毛细管持握时容易抖动导致采样困难，转移过程耗时较长，与滤纸接触处容易因移位而刮破滤纸，导致血液扩散不均匀。现有的干血斑样本采集工具集成度低，耗材分散，滤纸常裸露在外，样本污染的几率非常大。现有采血卡干血斑样本的萃取过程繁琐，需要用专用工具在样本中心打下固定面积的血片，操作费时费力，且存 在交叉污染的可能。
发明内容
本发明所要解决的技术问题在于，提供一种干血斑定量采集装置，能够实现定量采血，样本全回收，提高血斑定量检测结果的准确性，并使样本的采集、保存、萃取和送检的操作通过该装置一体化完成。
为解决上述技术问题，本发明提供一种干血斑定量采集装置，包括：样本收集管、样本收集滤纸和定量采血管；其中，
所述样本收集管包括管体和管盖；所述管体为直管，具有相对的第一端和第二端；所述第一端呈封闭状，为管底；所述第二端呈开口状，为管口；所述管盖用于封闭所述管口；
所述定量采血管包括定量毛细管和固定件；所述定量毛细管固定于所述固定件，所述定量毛细管具有用于采血且露出于所述固定件之外的采血端；所述固定件卡合于所述管口，所述定量毛细管伸入所述管体内；
所述样本收集滤纸设置于所述管体内，所述采血端相抵靠于所述样本收集滤纸。
具体的，所述管体为圆筒形，所述管口为圆形，所述管盖与所述管口相配合形成密封。
具体的，所述管口与所述管盖为一体式的或分离式的。
具体的，所述管口具有第一凸起，通过所述第一凸起与所述固定件卡紧。卡紧于管口的固定件起到遮挡和封闭管口的作用。
具体的，所述管底呈U形。
优选的，所述管体为塑料材质。优选的，所述管体为透明的。
具体的，所述固定件具有腔体，所述腔体内装有干燥剂。
具体的，所述固定件具有第二凸起，通过所述第二凸起与所述罐盖卡紧。从而达到所述管盖封闭所述管口使所述管体内部形成密闭空间的目的。
具体的，所述固定件为一根管柱，所述管柱套在所述样本收集管的管体内；所述管柱的顶端为开口状，且与所述管体的管口卡合；所述管柱的底端具有一个固定孔，所述定量毛细管穿插于所述固定孔中并固定。所述管柱的管壁、管柱的底端以及定量毛细管之间形成一个围绕该定量毛细管的环形柱状的腔体，该腔体内可用于放置干燥剂。
具体的，所述管盖封闭所述管口时，同时压制于所述管柱的顶端，使所述管柱的位置固定并封闭所述管柱的顶端。
优选的，所述固定件为塑料材质。优选的，所述固定件为透明的。
优选的，所述定量毛细管的两端分别位于所述管柱的底端的两侧；其中，远离所述管柱的顶端的一端露出于所述管柱外，为采血端；靠近所述管柱的顶端的一端位于所述管柱内，高度低于所述管柱的顶端。
优选的，所述管柱为圆筒形。优选的，所述固定孔位于所述管柱横截面的中心，所述定量毛细管沿所述管柱的轴线方向穿插于所述固定孔中并固定。
优选的，所述管柱为塑料材质。优选的，所述管柱为透明的。
优选的，所述定量毛细管为玻璃材质。
具体的，所述样本收集滤纸呈水平状态铺展于所述管底，所述定量采血管呈竖直状态位于所述样本收集滤纸的上方，与所述样本收集滤纸呈垂直，且所述定量采血管的采血端抵靠于所述样本收集滤纸。
具体的，所述样本收集滤纸为圆形片状，所述采血端抵靠于所述圆形片状的圆心处。
具体的，所述管体为圆筒形，所述管底呈U形，所述样本收集滤纸为圆形片状，所述样本收集滤纸呈水平状态铺展于所述管底，所述样本收集滤纸与所述管底的U形弧面之间形成一间隙。
具体的，所述样本收集滤纸采用玻璃纤维滤纸。
另一方面，本发明还提供使用干血斑定量采集装置的进行干血斑采集的方法，包括以下步骤：
步骤S1：打开管盖，取出定量采血管，将定量采血管中定量毛细管的采血端靠近并接触血液，待毛细作用力将血液充满整个定量毛细管；
步骤S2：将定量采血管插回样本收集管，确保定量毛细管的采血端接触样本收集滤纸；
步骤S3：盖上管盖，依靠空气压力及滤纸吸力，将定量毛细管中全部血液样本转移至样本收集滤纸上。
具体的，所述方法还包括步骤S4：在步骤S3之后，将干血斑定量采集装置置于合适的环境下保存或运输。
具体的，所述方法还包括步骤S5：在步骤S3之后，分析样本时，打开管盖，将定量采血管取出，直接加入萃取液至样本收集管内以萃取分析物，将萃取分析物送检测。
具体的，所述方法还包括步骤S6：在前述任一步骤中，在样本收集管外贴上样本来源信息。
本发明提供了一种新的干血斑样本采集装置及方法，相比于常用的采血卡采集及萃取方法，本发明的主要改进包括：
1.采用定量毛细管进行定量采血，依靠毛细作用充满液体实现定量，无需借助外部吸力，玻璃毛细管的长度固定且均一，定量结果准确，采血体积可低至5μL，适合于微量样本采血分析。
2.采血载体更换为玻璃纤维滤纸，定量采血后全部转移至滤纸片上，分析时将所有样本全部萃取(不同于传统的部分打卡萃取)，相比棉花滤纸有更高的萃取回收率，能够大幅改善HCT对萃取回收率的影响，使干血斑定量检测结果更加准确，能够进一步应用于内源分析物检测或治疗药物监测(TDM)。
3.玻璃纤维滤纸直接使用萃取液处理，相当于是采用预打孔设计，无需再进行干血斑打孔操作，配合定量采血，可实现对样本的全部回收。传统采血卡样本萃取时需要用专用工具在样本中心打下固定面积的血片，操作费时费力，且存在交叉污染的可能。本方法采用预成型微型滤纸片收集样本，省去打孔步骤，直接将萃取液加在收集管内萃取，节省了大量时间及耗材。
4.一体化采血装置设计，可在同一个装置实现样本的采集、干燥、运输保存和萃取，避免了样本的污染。传统的采血卡滤纸曝露在外，采血后需裸露干燥，不同样本间交叉污染的风险很大。在保存、转移、萃取等过程中，由于持握不当等原因，样本也容易受到不明来源的污染。本方法采用的样本采集管，从采样到分析全程封闭式，内含干燥剂使样本无需曝露干燥，将样本污染的几率降到最低。
附图说明
图1为本发明的干血斑定量采集装置一具体实施例的立体结构示意图。
图2为图1的剖面图。
图3为图1中干血斑定量采集装置的结构分解示意图。
图4为干血斑样本采集及检测步骤流程图。
图5为本发明的干血斑样本中部分治疗药物质谱峰型图。
图6为本发明的干血斑样本质谱校准曲线图。
图7为HCT对伊马替尼血药浓度检测干扰比较。
图8为HCT对西罗莫司血药浓度检测干扰比较。
图9为HCT对丙戊酸血药浓度检测干扰比较。
附图中符号标记说明：
1、样本收集管；
1a、管体；
1a1、管底；
1a2、管口；
1a3、第一凸起；
1b、管盖；
2、定量采血管；
2a、固定件；
2a1、顶端；
2a2、底端；
2a3、第二凸起；
2a4、固定孔；
2b、定量毛细管；
2b1、采血端；
2c、干燥剂；
3、样本收集滤纸；
具体实施方式
下面将对本发明的技术方案进行清楚、完整的描述，显然，所描述的实施例是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
一种干血斑定量采集装置，如图1～3所示，包括：样本收集管1、定量采血管2和样本收集滤纸3。其中，该样本收集管1由管体1a和管盖1b两部分组成。管体1a为直管，具有相对的第一端和第二端，其中，第一端是呈封闭状的管底1a1，第二端是呈开口状的管口1a2。管盖1b用于封闭管口1a2。其中，该定量采血管2包括固定件2a和固定于该固定件2a上的定量毛细管2b。固定件2a卡合于样本收集管1的管口1a2，定量毛细管2b伸入该所述管体内；定量毛细管2b具有用于采血的采血端2b1，采血端2b1露出于固定件2a之外。其中，该样本收集滤纸3设置于管体1a内，采血端2b1相抵靠于该样本收集滤纸3。
本发明的干血斑定量采集装置，能够通过定量采血管2实现定量采血，采集的血液样本及时且全部转移至样本收集滤纸3上，密封于样本收集管1内，于分析时向样本收集管1内 加入萃取液将所有样本全部萃取，可实现样本全回收，提高干血斑定量检测结果的准确性，该装置的整体设计使样本的采集、保存、萃取和送检等操作一体化完成。本发明的干血斑定量采集装置，用途主要为对微量末梢血(如指尖血)的定量采集及保存，此外也可用于收集及保存其它临床样本(如静脉血、血清、血浆、尿液等)。
本发明的干血斑定量采集装置，采用疏松的玻璃纤维滤纸，相比棉花纤维具有更高的萃取回收率，能够实现对采集样本的全部萃取检测，与定量采血手段相结合，避免了样本体积、红细胞压积(HCT)等的影响。
本发明的干血斑定量采集装置，采样操作更加简单，误操作几率低。可通过握持固定件来操作定量毛细管，吸样完成后将定量采血管插回样本收集管，盖上盖子即完成采样，样本靠滤纸吸力及空气压力转移至滤纸上。而现有干血斑样本采集时，常将血直接滴在采血卡上，样本体积差异较大，需过度挤压，且存在重复滴血的可能。用毛细管转移时，细长的毛细管持握时容易抖动导致采样困难，转移时容易刮破滤纸导致样本扩散不均匀。
本发明的干血斑定量采集装置，从采样到分析全程封闭式，内含干燥剂使样本无需曝露干燥，将样本污染的几率降到最低，不存在样本交叉污染的风险。而传统的采血卡滤纸曝露在外，采血后需裸露干燥，不同样本间交叉污染的风险很大。在保存、转移、萃取等过程中，由于持握不当等原因，样本也容易受到不明来源的污染。
本发明的干血斑定量采集装置，直接加入萃取液萃取样本收集管1内的样本收集滤纸3上全部样本，无需干血斑打孔操作，节省了大量时间及耗材。而传统采血卡样本萃取时需要用专用工具在干血斑样本中心打下固定面积的血片，操作费时费力，且存在交叉污染的可能。
一种具体实施方式中，样本收集管1的尺寸与常规2mL离心管相同，也可根据需要变更尺寸。样本收集管1采用塑料材质，材质包括但不限于聚丙烯、聚乙烯、聚酯等。样本收集管1的主要功能为封闭保存样本收集滤纸3，及充当样本萃取的容器。管体1a优选为透明的，便于可视化操作。
如图1～3所示，管体1a为圆筒形，管口1a2为圆形，管盖1b用于遮挡管口1a2使管体1a内部形成与外界封闭的空间。本实用新型的干血斑定量采集装置中，管口1a2与管盖1b为一体式的或分离式的。如图1～3所示，管盖1b与管口1a2为一体式的，管盖1b与管口1a2之间具有一个可弯折的连接处。固定件2a与样本收集管1a的管口1a2卡紧。如图3所示，管体1a的管口1a2处具有第一凸起1a3，通过第一凸起1a3将伸入管体1a内的固定件2a卡紧，保持固定件2a的位置稳定。同时固定件2a起到遮挡管口1a2的作用。管盖1b与固定件2a的顶端2a1之间可通过卡扣结构固定以封闭固定件2a的顶端2a1，同时也达到封闭管口1a2 的目的。在其他的可替代实施方式中，管盖1b与管口1a2为分离式，管盖1b与固定件2a的顶端2a1之间可通过螺纹结构固定以封闭顶端2a1和管口1a2。在其他的可替代实施方式中，管盖1b也可以完全覆盖或包裹固定件2a的顶端2a1，并直接与管口1a2形成连接使顶端2a1和管口1a2都封闭，比如管盖1b与管口1a2的外壁/内壁形成卡扣结构或螺纹结构等。
如图1～3所示，该样本收集管1的管体1a的管底1a1呈U形，也就是通常所说的圆底。。
本发明的干血斑定量采集装置中，该固定件2a还具有用于装干燥剂2c的腔体，干燥剂2c有利于保证样本收集管1的内环境干燥，缩短血液样本采集后的干燥时间。该装置也可以不含有干燥剂2c，这不会影响到该装置的主要功能。固定件2a的尺寸比定量毛细管2b的直径更大，以便于握持，避免采样时抖动。
如图2～3所示，该固定件2a具有第二凸起2a3，通过第二凸起2a3与管盖1b相配合达到封闭管体1a的效果，即管盖1b遮挡固定件2a的顶端2a1，同时固定件2a的顶端2a1遮挡管口1a2，三者之间相互卡紧使管体1a内部形成封闭空间。本实施例中，该固定件2a采用塑料材质。本实施例中，该固定件2a为透明的。
如图1～3所示的一种具体实施方式中，固定件2a为一根管柱，该管柱套在样本收集管1的管体1a内，且与管口1a2卡紧固定。定义该固定件2a的两端分别为顶端2a1和底端2a2。该管柱的顶端2a1为开口状，顶端2a1的侧面具有第二凸起2a3，通过第二凸起2a3与管盖1b卡合，以限制该管柱在管体1a内的位置，通常该管体1a内壁设有与凸起2a3相配合的凹陷；该管柱的底端2a2具有一个固定孔2a4，定量毛细管2b穿插于该固定孔2a4中并固定。凭借该管柱的管壁、管柱的底端2a2以及定量毛细管2b之间形成一个围绕该定量毛细管2b的环形柱状的腔体，该腔体内可用于放置干燥剂2c。通过该管盖1b封闭管口1a2时，同时管盖1b压紧于管柱的顶端2a1，使该管柱的位置固定并封闭顶端2a1。管柱的直径大于定量毛细管2b，便于使用者握持和采血操作，防止采样时抖动。优选的实施方式中，该管柱采用透明的塑料材质，便于可视化操作。
如图1～3所示的一种具体实施方式中，该定量毛细管2b的两端分别位于该管柱的底端2a2的两侧；其中一端远离该管柱的顶端2a1且露出于该管柱外，即为采血端2b1；而相对的靠近该管柱的顶端2a1的一端是位于该管柱内，且高度低于该管柱的顶端2a1。
本实施例中，该管柱为圆筒形。固定孔2a4是位于该管柱横截面的中心(即圆心处)，而定量毛细管2b沿该管柱的轴线方向穿插于该固定孔中并固定。
本实施例中，所述定量毛细管2b为玻璃材质，依靠毛细作用力可以使液体充满管体。玻璃材质也可以用其他常用亲水材料替换。通过设置毛细管的内径及长度，可以调整毛细管的 采样量，即可实现对液体的定量采集，体积可低至5μL，典型的采集体积为10～20μL。
本实施例中，所述样本收集滤纸3呈水平状态铺展于管底1a1，定量采血管2b呈竖直状态位于该样本收集滤纸3的正上方，与样本收集滤纸3呈垂直，且定量采血管3的采血端抵靠于该样本收集滤纸3。如图1～3所示，该样本收集滤纸3为圆形片状，采血端2b1抵靠于圆形片状的圆心处。圆形片状的样本收集滤纸3直径为6mm左右。样本收集滤纸3的材质为玻璃纤维滤纸，也可以用其他的多孔疏松材料替代。
本实施例中，管体1a为圆筒形，管底1a1呈U形，样本收集滤纸3为圆形片状，样本收集滤纸3呈水平状态铺展于管底1a1，样本收集滤纸3与管底1a1的U形弧面之间形成一间隙。从采血端2b1流出的样本完全由样本收集滤纸3接收。
本发明的干血斑定量采集装置可按以下方法步骤完成干血斑样本采集及检测：
1)在样本收集管外贴上样本来源信息，打开样本收集管盖，将定量采血管取出备用。
2)选择血供充足的部位(如指尖内侧)作为采血处，酒精消毒后使用一次性自动采血针刺破皮肤，待血液自然流出，不得挤压，用干纱布擦拭掉第一滴血。
3)将定量采血管的毛细管端接触血液，待毛细作用力将血液充满整个毛细管。
4)将定量采血管插回样本收集管，确保毛细管端接触样本收集滤纸。盖上样本收集管盖，依靠空气压力及滤纸吸力，将毛细管中全部样本转移至滤纸上。待样本干燥后，将收集管置于合适的环境下保存或运输。
5)分析样本时，打开样本收集管盖，将定量采血管取出，直接加入萃取液至收集管内，按适当的程序萃取分析物后进行检测。
实施例2：采集指尖血用于治疗药物监测(TDM)
本方法可用于采集指尖末梢血制成干血片样本。相比静脉采样，干血片样本具有采血量少、样本稳定易于保存运输、采样不受时间空间限制等独特优势。对于一些治疗药物监测(TDM)，需要在特定的时间采集峰、谷浓度，或在短时间内频繁采样，末梢血干血片样本能够满足临床需求。本方法操作简单，样本质量高，经过培训后可拓展至家庭自助采样。
如图4所示，用于采集指尖血时，操作步骤如下：
1)在样本收集管外贴上样本来源信息，打开样本收集管盖，将定量采血管取出备用。
2)选择血供充足的部位(如指尖内侧)作为采血处，酒精消毒后使用一次性自动采血针刺破皮肤，待血液自然流出，不得挤压，用干纱布擦拭掉第一滴血。
3)将定量采血管的毛细管端接触血液，待毛细作用力将血液充满整个毛细管。
4)将定量采血管插回样本收集管，确保毛细管端接触样本收集滤纸。盖上样本收集管盖，依靠空气压力及滤纸吸力，将毛细管中全部样本转移至滤纸上。待样本干燥后，将收集管置于合适的环境下保存或运输。
对采集到的干血斑样本进行简单萃取后，即可分析药物浓度等信息。萃取样本时，打开样本收集管盖，将定量采血管取出，将萃取液加入样本收集管，按优化的程序萃取。
萃取方法①：用于萃取血浆蛋白结合药物
a)向80％甲醇水溶液中加入适量化合物内标，配制成含内标萃取液；
b)在双空白样品中加入300μL的80％甲醇水溶液，在其它样品中加入300μL含内标萃取液，涡旋振荡约15min；
c)4000rpm，4℃条件下离心10min；
d)转移上清液100μL至新的96孔板中，4000rpm，4℃条件下离心5min。
e)进样分析。
萃取方法②：用于萃取血细胞结合药物
f)用纯甲醇配制含内标萃取液；
g)在所有样品中加入150μL纯水，振荡5min；
h)在双空白样品中加入300μL甲醇，其它样品中加入300μL含内标萃取液，振荡混合15min；
i)4000rpm，4℃条件下离心10min；
j)转移上清液100μL至新的96孔板中，4000rpm，4℃条件下离心5min。
k)进样分析。
对于常见的小分子药物，采用液相色谱-串联三重四级杆质谱(LC-MS/MS)进行检测，具有较高的特异性和灵敏度，液质方法为文献中可检索到的成熟方法。
图5所示为采用本发明的装置检测的部分治疗药物质谱峰型图，包括伊马替尼、卡马西平、丙戊酸、霉酚酸、他克莫司、环抱霉素A、西罗莫司，显示相关药物质谱峰型优良，无杂峰及拖尾，可用于药物浓度检测。
用于定量检测时需配制待测药物的校准品，将药物标准品储备液加入全血中，配制成6个浓度点的校准品，采用干血片采血装置制成校准品干血斑，同样本干血斑一同萃取进样，在仪器中生成校准曲线，及可实现定量检测。图6所示为伊马替尼、西罗莫司两种药物的干血斑样本质谱校准曲线，其中A为伊马替尼校准曲线，校准品浓度分别为：50、500、1000、2000、3000、5000ng/mL，其中B为西罗莫司校准曲线，校准品浓度分别为：2、4、6、12、 20、30ng/mL，线性相关系数r均大于0.9990，说明本发明的装置的一体化操作可提高干血斑定量检测结果的准确性。
实施例3：采集人全血用于核酸检测
本方法采用静脉全血样本，加载到血样采集装置上，进行核酸提取后用于基因突变检测。
A.样本采集
(1).在样本收集管外贴上样本来源信息，打开样本收集管盖，将定量采血管取出备用；
(2).遵循静脉采血标准操作采集血样。
(3).使用移液器将静脉血加样至干血采集管的滤纸上，直至血样完全干燥。于适当温度条件下运输或保存。
B.核酸提取
在采集到的干血斑样本后，采用QIAgene公司的QIAamp DNA mini核酸提取试剂盒(货号51304)和QIAcube核酸自动抽提仪对样本进行核酸抽提。抽提步骤如下：
(a)从干血斑采集管中取出5个干血斑，放入1.5ml离心管中，添加180μL ATL，85°C孵育10min，短离；
(b)加入20μL蛋白酶K，混匀，56℃孵育1h，短离；
(c)将液体转移到新的1.5mL管中，作为初始样本待用；
(d)将QIAcube所需试剂、耗材准备、安装完毕；
(e)打开QIAcube电源，设置程序为DNA→QIAamp DNA mini→Tissue→Elution volume:200μL；
(f)放置好处理好的初始样本，开始QIAcube程序；
(g)待QIAcube正常运行结束后，取出含有抽提完成的200μL DNA 1.5mL管，-20℃保存。
(h)采用Qubit 3.0核酸定量荧光计对抽提后的核酸样本定量，定量结果如下表：
Figure PCTCN2020099852-appb-000001
C.基因突变检测
核酸抽提完成后，即可对样本进行基因检测。本实施例采用数字PCR法对样本中IDH2基因的R140位点进行检测。数字PCR是一种核酸检测和定量分析的新方法，可实现对核酸 的绝对定量及稀有等位基因的检测。下表分别为本次样本的定量和定性检测结果：
数字PCR定量结果
Figure PCTCN2020099852-appb-000002
突变检测定性结果
Figure PCTCN2020099852-appb-000003
样本为正常人全血模拟干血斑样本，检测结果无R140突变(阴性)，两者结果一致。且本次抽提的干血斑样本也均能满足后续基因检测实验需求。说明本发明的装置能有效采集、保存干血斑样本，并应用于分子生物学临床定性检测中。
实施例4：采集全血用于抗体定性检测
本方法可用于采集全血用于抗体定性检测。相比采血管采血，干血片样本具有采血量少、样本稳定易于保存运输、采样不受时间空间限制等独特优势。本方法操作简单，样本质量高。
操作步骤如下：
取500uL全血(血液来源：猪全血、兔全血、人全血都可)，添加抗体。将定量采血管的毛细管端接触血液，待毛细作用力将血液充满整个毛细管。将定量采血管插回样本收集管，确保毛细管端接触样本收集滤纸。盖上样本收集管盖，依靠空气压力及滤纸吸力，将毛细管中全部样本转移至滤纸上。待样本干燥后，将收集管置于合适的环境下保存或运输。
对采集到的干血斑样本进行简单处理后，即可分析抗体。处理样本时，打开样本收集管盖，将定量采血管取出，将1mL样本稀释液加入样本收集管，25℃600rpm振荡3小时，按如下实验步骤进行测试。
1)每轮实验均需设置空白对照2孔，阴性对照2孔；
2)每孔加经稀释的样本100uL(阴性对照除外)，空白对照每孔加样本稀释液100uL；
3)阴性对照加100uL于相应孔，振荡混匀，封板，37℃孵育30min；
4)洗板：弃去孔内液体，各孔注满洗涤液，静置5秒，甩干，重复以上5次拍干；
5)每孔加入100uL酶结合物，封板，37℃孵育30min；
6)洗板：重复步骤4；
7)每孔加入显色剂A，显色剂B各50uL，振荡混匀，封板，37℃孵育10min；
8)每孔加入终止液50uL，混匀；
9)酶标仪读数；先用空白孔校零，然后读取各孔OD值。
10)阳性判断：OD≥阴性对照0D均值+0.25
实验结果如下：
Figure PCTCN2020099852-appb-000004
血斑测定与非血斑测定的结果判读一致，该结果说明本发明的装置的一体化操作可进行抗体定性检测。
实施例5：使用本发明的干血斑定量采集装置与现有方法红细胞压积(HCT)干扰比较
HCT范围：20％～60％
样本制备方法：
1)调整全血HCT，制备5份HCT值分别为20％、30％、40％、50％、60％的全血基质。
2)分别向基质中加入等量的标准品储备液，得到5份浓度相同但HCT值不同的样本。低浓度区间和高浓度区间各制备一套。
3)用40％HCT的全血配制校准品。
4)用新方法及对照方法，将所有样本及校准品分别制备成相应的干血斑样本。
实验方法：
分别用新方法和对照方法检测，比较不同HCT样本相对40％HCT样本的偏倚，及样本总体的变异系数(CV)。
实验一：
检测药物：伊马替尼；
对照：Whatman 903卡，10μL定量采血，将血斑全部打下；
实验结果：如图7中B、C所示，在低、高浓度样本中，新方法相比903卡对照方法曲线更加平滑，显示HCT效应对检测结果的影响大幅改善，A、D显示新方法在HCT 20％～60％范围内检测变异系数CV值(5.17％，6.24％)明显小于903卡对照(11.46％，15.62％)。
实验二：
检测药物：西罗莫司；
对照：Whatman DMPK-A卡，非定量采血，3mm中心打孔；
实验结果：如图8中B、C所示，在低、高浓度样本中，新方法相比DMPK-A卡对照方法在HCT20％～60％范围内曲线更加平滑，显示HCT效应对检测结果的影响大幅改善，A、D显示新方法在HCT 20％～60％范围内检测变异系数CV值(5.27％，6.48％)明显小于DMPK-A卡对照(11.79％，14.65％)。
实验三：
检测药物：丙戊酸；
对照：Whatman DMPK-A卡，非定量采血，3mm中心打孔；
实验结果：如图9中B、C所示，在低、高浓度样本中，新方法相比DMPK-A卡对照方法在HCT20％～60％范围内曲线更加平滑，显示HCT效应对检测结果的影响大幅改善，A、D显示新方法在HCT 20％～60％范围内检测变异系数CV值(5.76％，8.63％)明显小于DMPK-A卡对照(10.82％，12.05％)。
以上实验说明本发明的装置的一体化操作可提高干血斑定量检测结果的准确性。
综上所述，上述各实施例仅为本发明的较佳实施例而已，并不用以限定本发明的保护范围，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，皆应包含在本发明的保护范围内。

Claims (16)

  1. 一种干血斑定量采集装置，其特征在于，包括：样本收集管、样本收集滤纸和定量采血管；其中，
    所述样本收集管包括管体和管盖；所述管体为直管，具有相对的第一端和第二端；所述第一端呈封闭状，为管底；所述第二端呈开口状，为管口；所述管盖用于封闭所述管口；
    所述定量采血管包括定量毛细管和固定件；所述定量毛细管固定于所述固定件，所述定量毛细管具有用于采血且露出于所述固定件之外的采血端；所述固定件卡合于所述管口，所述定量毛细管伸入所述管体内；
    所述样本收集滤纸设置于所述管体内，所述采血端相抵靠于所述样本收集滤纸。
  2. 如权利要求1所述的干血斑定量采集装置，其特征在于，所述管体为圆筒形，所述管口为圆形，所述管底呈U形。
  3. 如权利要求1所述的干血斑定量采集装置，其特征在于，所述管口与所述管盖为一体式的或分离式的。
  4. 如权利要求1所述的干血斑定量采集装置，其特征在于，所述管体为透明的塑料材质。
  5. 如权利要求1所述的干血斑定量采集装置，其特征在于，所述固定件具有腔体，所述腔体内装有干燥剂。
  6. 如权利要求5所述的干血斑定量采集装置，其特征在于，所述固定件具有第二凸起，通过所述第二凸起与所述罐盖卡紧。
  7. 如权利要求6所述的干血斑定量采集装置，其特征在于，所述固定件为一根管柱，所述管柱套在所述样本收集管的管体内；所述管柱的顶端为开口状，且与所述管体的管口卡合；所述管柱的底端具有一个固定孔，所述定量毛细管穿插于所述固定孔中并固定。
  8. 如权利要求7所述的干血斑定量采集装置，其特征在于，所述管柱为圆筒形；所述固定孔位于所述管柱横截面的中心，所述定量毛细管沿所述管柱的轴线方向穿插于所述固定孔中并固定。
  9. 如权利要求8所述的干血斑定量采集装置，其特征在于，所述管柱为透明的塑料材质。
  10. 如权利要求1所述的干血斑定量采集装置，其特征在于，所述样本收集滤纸呈水平状态铺展于所述管底，所述定量采血管呈竖直状态位于所述样本收集滤纸的上方，与所述样本收集滤纸呈垂直，且所述定量采血管的采血端抵靠于所述样本收集滤纸。
  11. 如权利要求10所述的干血斑定量采集装置，其特征在于，所述样本收集滤纸为圆形片状，所述采血端抵靠于所述圆形片状的圆心处。
  12. 如权利要求11所述的干血斑定量采集装置，其特征在于，所述样本收集滤纸采用玻 璃纤维滤纸。
  13. 一种使用如权利要求1-12任一项所述的干血斑定量采集装置的进行干血斑采集的方法，包括以下步骤：
    步骤S1：打开管盖，取出定量采血管，将定量采血管中定量毛细管的采血端靠近并接触血液，待毛细作用力将血液充满整个定量毛细管；
    步骤S2：将定量采血管插回样本收集管，确保定量毛细管的采血端接触样本收集滤纸；
    步骤S3：盖上管盖，依靠空气压力及滤纸吸力，将定量毛细管中全部血液样本转移至样本收集滤纸上。
  14. 如权利要求13所述的方法，其特征在于，所述方法还包括步骤S4：在步骤S3之后，将干血斑定量采集装置置于合适的环境下保存或运输。
  15. 如权利要求13所述的方法，其特征在于，所述方法还包括步骤S5：在步骤S3之后，分析样本时，打开管盖，将定量采血管取出，直接加入萃取液至样本收集管内以萃取分析物，将萃取分析物送检测。
  16. 如权利要求13所述的方法，其特征在于，所述方法还包括步骤S6：在前述任一步骤中，在样本收集管外贴上样本来源信息。
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