CN111474257A

CN111474257A - Dry blood spot quantitative collection device and method

Info

Publication number: CN111474257A
Application number: CN202010305434.8A
Authority: CN
Inventors: 杭红; 张云; 李宇; 张向辉; 赵伟洁
Original assignee: Suzhou Yaoming Zekang Biotechnology Co ltd; Shanghai Youming Autometric Medical Technology Co ltd
Current assignee: Suzhou Yaoming Zekang Biotechnology Co ltd; Shanghai Youming Autometric Medical Technology Co ltd
Priority date: 2020-04-17
Filing date: 2020-04-17
Publication date: 2020-07-31
Also published as: WO2021208262A1

Abstract

The invention provides a dry blood spot quantitative collecting device, comprising: the device comprises a sample collecting pipe, sample collecting filter paper and a quantitative blood collecting pipe; the sample collection tube comprises a tube body and a tube cover; the pipe body is a straight pipe, and the pipe cover is used for sealing the pipe orifice; the quantitative blood collection tube comprises a quantitative capillary tube and a fixing piece; the quantitative capillary tube is fixed on the fixing piece and provided with a blood sampling end which is used for sampling blood and is exposed out of the fixing piece; the fixing piece is clamped on the pipe orifice, and the quantitative capillary tube extends into the pipe body; the sample collection filter paper is arranged in the tube body, and the blood sampling end is abutted against the sample collection filter paper. The invention also provides a method for collecting the dried blood spots. The invention adopts the design of an integrated blood sampling device, can realize the collection, drying, transportation, storage and extraction of samples in the same device, avoids the pollution of the samples and improves the accuracy of the quantitative detection result of the dried blood spots.

Description

Dry blood spot quantitative collection device and method

Technical Field

The invention relates to the technical field of dry blood spot detection in biotechnology, in particular to a dry blood spot quantitative collection device and a dry blood spot quantitative collection method.

Background

The Dry Blood Spot (DBS) detection technology is a method for dropping a biological sample (generally whole Blood) onto a filter paper sheet for drying and storage, and detecting an analyte in the dry Spot sample after extraction. The reagent kit has the advantages of convenient collection, less blood collection amount and easy storage and transportation of samples, and is used for screening the newborn phenylketonuria in the sixties of the last century. At present, the most mature field of dried blood spot detection is still the screening of newborn genetic metabolic diseases, and the application of the dried blood spot detection is gradually expanded to the fields of endogenous biomarker detection, Therapeutic Drug Monitoring (TDM) and the like.

Dry blood spot detection is a systematic technique, comprising: a method of collecting a sample; storing and transporting the sample; establishing and verifying a detection method; extracting and processing a sample; detecting a sample; and analyzing, processing and interpreting the detection data. Among them, dried blood spots as a sample collection technique have some advantages:

1) the sampling is simple, and is not limited by time and space (the sampling can be expanded to families and remote areas). The collection of the dry blood spots is realized by using an automatic blood taking needle to puncture the finger tip or the heel, wiping off the first drop of blood, and then directly dropping the residual whole blood or transferring the residual whole blood onto a blood taking card by using a capillary tube. After the user is trained properly, self-service blood sampling can be realized. For some TDM drug monitoring, peak and valley concentrations need to be collected at specific time, and the difficulty of obtaining samples can be greatly reduced by adopting dry blood spot detection.

2) The blood sampling process is minimally invasive, and the blood sampling volume is small. The skin is punctured by adopting an engineered automatic blood taking needle, the puncturing depth is about 2 mm generally, and pain and trauma are minimized. For the situations of difficult blood sampling of infants, patients with blood coagulation dysfunction, ICU patients, remote areas and the like, the application scene of in-vitro diagnosis can be technically expanded by dry blood slice detection.

3) The stability of most analytes can be guaranteed to the dried blood spot, so that the sample can be stored at normal temperature and transported at a long distance, the storage and transportation cost is greatly reduced, and home sampling and third-party detection are possible. One study showed that amino acids in dried blood spot samples were only slightly degraded after 5 years of storage at ambient temperature. The literature also shows that most therapeutic drugs are relatively stable in dry blood spot samples.

On the other hand, the dry blood spot detection method also has some problems and disadvantages, which limit the further wide application thereof. These problems and deficiencies include: the blood source and the sampling time cannot be rechecked during self-service sampling; limited by collection conditions and collection operation, although the collection of a plurality of samples is still unqualified under the guidance of training and specifications; the patient has fear to puncture the skin by oneself; due to the small sample volume, effective detection cannot be performed for some low concentration analytes.

The current technique of dry blood spots uses the most widely used blood collection card as cotton filter paper, which is made of uniform cotton fibers and can uniformly distribute absorbed whole blood, the collection of blood spots is usually non-quantitative blood collection, fingertip blood is directly spotted on the blood collection card, or micro capillary transfer is used, when the method is used for quantitative detection, a calibration curve card is prepared by using the same substrate, the traditional extraction step is to punch a wafer with fixed size from the center of the dry blood spots, place the wafer in a proper container, extract analytes by using proper extraction liquid, and the detection instrument is usually a high-sensitivity liquid chromatography (HP L C) or a chromatography-mass spectrometer (L C-MS).

This commonly used method is more disturbed in the accuracy of the quantification than the venous blood sample, which is also a major cause of its limited applicability. The main influencing factors include the chromatographic effect (central edge difference) of filter paper, the blood sampling volume effect, and the Hematocrit effect (also known as Hematocrit, HCT).

The chromatographic effect is due to the inconsistent diffusion rates of red blood cells and plasma in whole blood in filter paper, which when the analyte binds specifically to plasma or red blood cells, may result in an uneven distribution of the analyte on the blood spot card, which can be usually solved by fixing the card in the center of the blood spot. The volume effect is caused by the difference of blood sampling volume, so that the blood volume contained in the filter paper in unit area is different, and usually, more than two drops of whole blood are dripped at the same position due to wrong sampling, so that the volume effect is obvious.

Hematocrit (HCT) is the most important factor affecting the accuracy of quantification of dried blood spots. HCT change can greatly influence blood viscosity, and further influence the diffusion characteristic of whole blood in filter paper, and clinical HCT value span of patient blood samples is different from 20% -60%. HCT affects both chromatographic and volume effects, and studies by Holub and Adam on prenatal screening found that the measured amino acid concentration increased significantly with increasing HCT, while at low HCT the amino acid concentration at the periphery of the plaque was significantly higher than at the center, due to differences in the distribution of amino acids in plasma and erythrocytes. In addition, HCT can significantly affect the whole blood volume per unit area, when HCT is increased, the diffusion area of blood spots is reduced, and the volume of a sample contained in the blood spots with the same area is increased, so that the detection result is significantly interfered. How to eliminate the interference of HCT on the quantitative result of the dry blood spot sample is a big problem in the aspect of dry blood spot detection application.

Some products realize quantitative blood sampling through micro-fluidic, take down the extraction with blood spot totally after shifting to the blood sampling card, avoided volume and chromatography effect, but because the filter paper material is cotton fiber, HCT becomes main interference factor to the influence of extraction rate, and the testing result still can receive HCT influence.

The current blood spot sample collection method has a high misoperation probability. Generally, non-quantitative blood collection is carried out by directly dripping fingertip blood on a blood collection card or transferring the fingertip blood by using a trace capillary. When directly dripping blood, the volume difference of the blood drops is large, excessive extrusion is needed, and the possibility of repeated blood dripping exists. When the capillary is transferred, the slender capillary is easy to shake when being held, so that the sampling is difficult, the time consumption of the transfer process is long, and the filter paper is easy to be scraped at the contact part of the capillary and the filter paper due to the displacement, so that the blood is not uniformly diffused. The existing dry blood spot sample collecting tool is low in integration level, consumables are scattered, filter paper is always exposed outside, and the probability of sample pollution is very high. The extraction process of the existing blood sampling card dry blood spot sample is complicated, a blood sheet with a fixed area needs to be punched at the center of the sample by a special tool, the operation is time-consuming and labor-consuming, and the possibility of cross contamination exists.

Disclosure of Invention

The invention aims to solve the technical problem of providing a quantitative dry blood spot collecting device, which can realize quantitative blood collection and full sample recovery, improve the accuracy of a quantitative blood spot detection result and integrally complete the operations of collecting, storing, extracting and submitting a sample.

In order to solve the above technical problems, the present invention provides a quantitative collection device for dried blood spots, comprising: the device comprises a sample collecting pipe, sample collecting filter paper and a quantitative blood collecting pipe; wherein the content of the first and second substances,

the sample collecting pipe comprises a pipe body and a pipe cover; the pipe body is a straight pipe and is provided with a first end and a second end which are opposite; the first end is closed and is a tube bottom; the second end is open and is a pipe orifice; the tube cover is used for closing the tube orifice;

the quantitative blood collection tube comprises a quantitative capillary tube and a fixing piece; the quantitative capillary tube is fixed on the fixing piece and provided with a blood sampling end which is used for sampling blood and is exposed out of the fixing piece; the fixing piece is clamped on the pipe orifice, and the quantitative capillary tube extends into the pipe body;

the sample collection filter paper is arranged in the tube body, and the blood sampling end is abutted against the sample collection filter paper.

Specifically, the body is the cylinder, the mouth of pipe is circular, the tube cap with the mouth of pipe cooperatees and forms sealedly.

Specifically, the nozzle and the tube cover are integrated or separated.

Specifically, the pipe orifice is provided with a first bulge, and the pipe orifice is clamped with the fixing piece through the first bulge. The fixing piece clamped on the pipe orifice plays a role in shielding and closing the pipe orifice.

Specifically, the tube bottom is U-shaped.

Preferably, the tube body is made of plastic. Preferably, the tube is transparent.

Specifically, the fixing piece is provided with a cavity, and a drying agent is filled in the cavity.

Specifically, the fixing part is provided with a second bulge, and the second bulge is clamped with the tank cover. Thereby achieving the purpose that the pipe cover seals the pipe orifice to form a closed space inside the pipe body.

Specifically, the fixing piece is a pipe column which is sleeved in a pipe body of the sample collecting pipe; the top end of the pipe column is in an opening shape and is clamped with the pipe orifice of the pipe body; the bottom end of the pipe column is provided with a fixing hole, and the quantitative capillary is inserted into the fixing hole and fixed. An annular columnar cavity surrounding the quantitative capillary tube is formed among the tube wall of the tube column, the bottom end of the tube column and the quantitative capillary tube, and a drying agent can be placed in the cavity.

Specifically, when the pipe orifice is closed by the pipe cover, the pipe cover is pressed on the top end of the pipe column at the same time, so that the position of the pipe column is fixed and the top end of the pipe column is closed.

Preferably, the fixing piece is made of plastic. Preferably, the fixing member is transparent.

Preferably, two ends of the quantitative capillary are respectively positioned at two sides of the bottom end of the tubular column; one end far away from the top end of the pipe column is exposed out of the pipe column and is a blood sampling end; one end close to the top end of the pipe column is positioned in the pipe column and is lower than the top end of the pipe column.

Preferably, the column is cylindrical. Preferably, the fixing hole is located at the center of the cross section of the column, and the quantitative capillary is inserted into the fixing hole along the axial direction of the column and fixed.

Preferably, the pipe column is made of plastic. Preferably, the tubing string is transparent.

Preferably, the quantitative capillary is made of glass.

Specifically, the sample collection filter paper is the horizontality and spreads in the tube bottom, the ration blood sampling pipe is vertical state and is located the top of sample collection filter paper, with the sample collection filter paper is perpendicular, just the blood sampling end of ration blood sampling pipe support lean on in the sample collection filter paper.

Specifically, the sample collection filter paper is in a circular sheet shape, and the blood sampling end abuts against the circle center of the circular sheet shape.

Specifically, the tube body is cylindrical, the tube bottom is U-shaped, the sample collecting filter paper is circular and flaky, the sample collecting filter paper is horizontally spread at the tube bottom, and a gap is formed between the sample collecting filter paper and the U-shaped cambered surface of the tube bottom.

Specifically, the sample collection filter paper is glass fiber filter paper.

In another aspect, the present invention further provides a method for collecting dried blood spots using a dried blood spot quantitative collecting device, comprising the steps of:

step S1: opening the tube cover, taking out the quantitative blood collection tube, enabling the blood collection end of the quantitative capillary tube in the quantitative blood collection tube to be close to and contact with blood, and filling the whole quantitative capillary tube with the blood by capillary force;

step S2: inserting the quantitative blood collection tube back into the sample collection tube to ensure that the blood collection end of the quantitative capillary tube is contacted with the sample collection filter paper;

step S3: and (4) covering the tube cover, and transferring the whole blood sample in the quantitative capillary tube to the sample collection filter paper by virtue of air pressure and filter paper suction.

Specifically, the method further includes step S4: after step S3, the quantitative dry blood spot collecting device is placed in a suitable environment for storage or transportation.

Specifically, the method further includes step S5: after step S3, when analyzing the sample, the tube cap is opened, the quantitative blood collection tube is taken out, the extraction liquid is directly added into the sample collection tube to extract the analyte, and the extracted analyte is sent to the test.

Specifically, the method further includes step S6: in any of the preceding steps, sample source information is attached to the outside of the sample collection tube.

Compared with the common blood sampling card acquisition and extraction method, the invention provides a novel dry blood spot sample acquisition device and method, and the main improvements of the invention comprise:

1. the quantitative capillary tube is adopted for quantitative blood collection, the liquid is filled by virtue of capillary action to realize quantification, external suction is not needed, the length of the glass capillary tube is fixed and uniform, the quantitative result is accurate, the blood collection volume can be as low as 5 mu L, and the quantitative capillary tube is suitable for micro sample blood collection analysis.

2. The blood sampling carrier is replaced by glass fiber filter paper, all samples are transferred to the filter paper sheet after quantitative blood sampling, all samples are extracted (different from traditional partial card punching extraction) during analysis, compared with cotton filter paper, the extraction recovery rate is higher, the influence of HCT on the extraction recovery rate can be greatly improved, the dry blood spot quantitative detection result is more accurate, and the method can be further applied to endogenous analyte detection or Therapeutic Drug Monitoring (TDM).

3. The glass fiber filter paper is directly treated by the extraction liquid, so that the pre-punching design is adopted, the punching operation of the dried blood spots is not needed, and the whole recovery of the sample can be realized by matching with quantitative blood sampling. When a traditional blood sampling card is used for sample extraction, a special tool is required to punch down a blood slice with a fixed area in the center of a sample, the operation is time-consuming and labor-consuming, and the possibility of cross contamination exists. The method adopts the preformed micro filter paper sheet to collect the sample, omits the step of punching, directly adds the extract liquid into the collecting tube for extraction, and saves a large amount of time and material consumption.

4. The design of the integrated blood sampling device can realize the collection, drying, transportation, storage and extraction of samples in the same device, thereby avoiding the pollution of the samples. The traditional blood sampling card filter paper is exposed outside, and needs to be exposed and dried after blood sampling, so that the risk of cross contamination among different samples is great. Samples are also susceptible to contamination from unknown sources during storage, transfer, extraction, etc., due to improper handling, etc. The sample collecting tube adopted by the method is closed in the whole process from sampling to analysis, and contains the drying agent, so that the sample does not need to be exposed and dried, and the probability of sample pollution is reduced to the minimum.

Drawings

Fig. 1 is a schematic perspective view of a quantitative collection device for dried blood spots according to an embodiment of the present invention.

Fig. 2 is a cross-sectional view of fig. 1.

Fig. 3 is an exploded view of the quantitative collection device for dried blood spots in fig. 1.

FIG. 4 is a flow chart of the dry blood spot sample collection and detection steps.

FIG. 5 is a mass spectrum peak pattern of a portion of the therapeutic agent in a dried blood spot sample of the present invention.

FIG. 6 is a calibration graph of mass spectra of dried blood spot samples according to the present invention.

FIG. 7 is a comparison of HCT interference with blood imatinib concentration detection.

FIG. 8 is a comparison of HCT interference with blood sirolimus level detection.

FIG. 9 shows the interference of HCT on the detection of plasma valproic acid concentration.

Description of the symbols in the drawings:

1. a sample collection tube;

1a, a pipe body;

1a1, tube bottom;

1a2, mouthpiece;

1a3, a first protrusion;

1b, a tube cover;

2. a quantitative blood collection tube;

2a, a fixing piece;

2a1, top;

2a2, bottom end;

2a3, second projection;

2a4, fixation holes;

2b, quantitative capillary;

2b1, a blood sampling end;

2c, a drying agent;

3. sample collection filter paper;

Detailed Description

The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Example 1

A quantitative collection device for dry blood spots, as shown in figures 1-3, comprises: 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 a tube body 1a and a tube cover 1 b. The tube body 1a is a straight tube and has a first end and a second end opposite to each other, wherein the first end is a closed tube bottom 1a1, and the second end is an open tube mouth 1a 2. The tube cover 1b is used to close the spout 1a 2. The quantitative blood collection tube 2 includes a fixing member 2a and a quantitative capillary tube 2b fixed to the fixing member 2 a. The fixing piece 2a is clamped at the pipe orifice 1a2 of the sample collection pipe 1, and the quantitative capillary 2b extends into the pipe body; the quantitative capillary 2b has a blood collection end 2b1 for collecting blood, and the blood collection end 2b1 is exposed outside the holder 2 a. Wherein, the sample collection filter paper 3 is arranged in the tube body 1a, and the blood sampling end 2b1 is abutted against the sample collection filter paper 3.

The quantitative collection device for the dried blood spots can realize quantitative blood collection through the quantitative blood collection tube 2, collected blood samples are timely and completely transferred to the sample collection filter paper 3 and sealed in the sample collection tube 1, all samples are completely extracted by adding the extraction liquid into the sample collection tube 1 during analysis, the samples can be completely recovered, the accuracy of the quantitative detection result of the dried blood spots is improved, and the operations of collection, storage, extraction, inspection and the like of the samples are integrally completed due to the integral design of the device. The quantitative collection device for the dry blood spots is mainly used for quantitatively collecting and storing trace peripheral blood (such as fingertip blood), and can also be used for collecting and storing other clinical samples (such as venous blood, serum, plasma, urine and the like).

The dry blood spot quantitative collecting device provided by the invention adopts loose glass fiber filter paper, has a higher extraction recovery rate compared with cotton fiber, can realize all extraction detection on collected samples, is combined with a quantitative blood collecting means, and avoids the influences of sample volume, Hematocrit (HCT) and the like.

The dry blood spot quantitative collecting device is simpler in sampling operation and low in misoperation probability. The accessible is held the mounting and is operated the ration capillary, inhales kind and inserts the sample collecting pipe back with the ration heparin tube after accomplishing, covers the lid and accomplishes the sampling promptly, and the sample leans on filter paper suction and air pressure transfer to filter paper on. When the existing dry blood spot sample is collected, blood is often directly dripped on the blood sampling card, the sample volume difference is large, excessive extrusion is needed, and the possibility of repeated blood dripping exists. When the capillary tube is used for transferring, the slender capillary tube is easy to shake when being held, so that the sampling is difficult, and the filter paper is easy to scratch during transferring, so that the sample is not uniformly diffused.

The dry blood spot quantitative collecting device is closed in the whole process from sampling to analysis, contains the drying agent, enables the sample not to be exposed and dried, reduces the probability of sample pollution to the minimum, and does not have the risk of sample cross contamination. The traditional blood sampling card filter paper is exposed outside, and needs to be exposed and dried after blood sampling, so that the risk of cross contamination among different samples is great. Samples are also susceptible to contamination from unknown sources during storage, transfer, extraction, etc., due to improper handling, etc.

According to the quantitative collection device for the dried blood spots, the extraction liquid is directly added to extract all samples on the sample collection filter paper 3 in the sample collection pipe 1, the dried blood spots are not required to be punched, and a large amount of time and consumables are saved. And when the traditional blood sampling card is used for extracting a sample, a blood slice with a fixed area needs to be punched at the center of a dry blood spot sample by using a special tool, the operation is time-consuming and labor-consuming, and the possibility of cross contamination exists.

In one embodiment, the sample collection tube 1 is the same size as a conventional 2m L centrifuge tube, and the size can be changed as required, the sample collection tube 1 is made of plastic material including but not limited to polypropylene, polyethylene, polyester, etc. the main function of the sample collection tube 1 is to seal and store the sample collection filter paper 3 and serve as a container for sample extraction, and the tube body 1a is preferably transparent for visual operation.

As shown in fig. 1 to 3, the tube body 1a is cylindrical, the spout 1a2 is circular, and the tube cover 1b is used to shield the spout 1a2 to form a space enclosed with the outside inside of the tube body 1 a. In the device for quantitatively collecting dry blood spots of the present invention, the nozzle 1a2 and the cap 1b are integrated or separated. As shown in fig. 1 to 3, the cap 1b and the spout 1a2 are integrated, and a bendable joint is formed between the cap 1b and the spout 1a 2. The fixing member 2a is engaged with the nozzle 1a2 of the sample collection tube 1 a. As shown in fig. 3, the nozzle 1a2 of the tube body 1a has a first protrusion 1a3, and the first protrusion 1a3 clamps the fixing member 2a extending into the tube body 1a, so as to keep the position of the fixing member 2a stable. While the fixing member 2a functions to shield the spout 1a 2. The tube cover 1b and the top end 2a1 of the fastener 2a can be fixed by a snap structure to close the top end 2a1 of the fastener 2a, and simultaneously achieve the purpose of closing the nozzle 1a 2. In other alternative embodiments, the cap 1b is separate from the nozzle 1a2, and the cap 1b and the top end 2a1 of the fastener 2a can be fastened together by a screw structure to close the top end 2a1 and the nozzle 1a 2. In other alternative embodiments, the tube cover 1b can completely cover or wrap the top end 2a1 of the fixing member 2a and directly form a connection with the nozzle 1a2 to close both the top end 2a1 and the nozzle 1a2, such as the tube cover 1b forms a snap structure or a thread structure with the outer/inner wall of the nozzle 1a 2.

As shown in FIGS. 1 to 3, the bottom 1a1 of the tube body 1a of the sample collection tube 1 has a U-shape, that is, a so-called round bottom. .

In the device for quantitatively collecting the dry blood spots, the fixing piece 2a is also provided with a cavity for containing a drying agent 2c, and the drying agent 2c is favorable for ensuring the drying of the internal environment of the sample collecting pipe 1 and shortening the drying time after the blood sample is collected. The device may also be free of desiccant 2c, which does not affect the main function of the device. The size of the fixing member 2a is larger than the diameter of the quantitative capillary 2b so as to be easily held and prevent shaking during sampling.

As shown in fig. 2 to 3, the fixing member 2a has a second protrusion 2a3, and the second protrusion 2a3 cooperates with the tube cap 1b to achieve the effect of closing the tube body 1a, i.e. the tube cap 1b shields the top end 2a1 of the fixing member 2a, and simultaneously the top end 2a1 of the fixing member 2a shields the tube opening 1a2, and the three are mutually clamped to form a closed space inside the tube body 1 a. In this embodiment, the fixing member 2a is made of plastic. In this embodiment, the fixing member 2a is transparent.

In one embodiment shown in fig. 1 to 3, the fixing member 2a is a tubular column, which is sleeved in the tubular body 1a of the sample collection tube 1 and tightly fixed with the tube opening 1a 2. The two ends of the fixing member 2a are defined as a top end 2a1 and a bottom end 2a 2. The top end 2a1 of the column is open, the side of the top end 2a1 is provided with a second protrusion 2a3, the position of the column in the tube body 1a is limited by the engagement of the second protrusion 2a3 and the tube cover 1b, usually, the inner wall of the tube body 1a is provided with a recess matching with the protrusion 2a 3; the lower end 2a2 of the column has a fixing hole 2a4, and the quantitative capillary 2b is inserted into the fixing hole 2a4 and fixed. By virtue of the annular cylindrical cavity surrounding the quantitative capillary 2b formed between the tube wall of the tube column, the bottom end 2a2 of the tube column and the quantitative capillary 2b, the desiccant 2c can be placed in the cavity. When the nozzle 1a2 is closed by the cap 1b, the cap 1b presses against the top end 2a1 of the column, thereby fixing the position of the column and closing the top end 2a 1. The diameter of the pipe column is larger than that of the quantitative capillary 2b, so that a user can conveniently hold and take blood, and the shaking during sampling is prevented. In a preferred embodiment, the tubular column is made of transparent plastic materials, so that visual operation is facilitated.

In one embodiment shown in fig. 1-3, the two ends of the quantitative capillary 2b are located at two sides of the bottom end 2a2 of the column respectively; one end of the blood sampling tube is far away from the top end 2a1 of the tube column and is exposed outside the tube column, namely a blood sampling end 2b 1; the opposite end, near the top end 2a1 of the column, is located within the column at a lower elevation than the top end 2a1 of the column.

In this embodiment, the column is cylindrical. The fixed hole 2a4 is located at the center (i.e., the center of the circle) of the cross section of the column, and the quantitative capillary 2b is inserted into the fixed hole and fixed along the axial direction of the column.

In the embodiment, the quantitative capillary 2b is made of glass, liquid can be filled in the tube body by virtue of capillary force, the glass material can also be replaced by other common hydrophilic materials, the sampling amount of the capillary can be adjusted by setting the inner diameter and the length of the capillary, so that the quantitative collection of the liquid can be realized, the volume can be as low as 5 mu L, and the typical collection volume is 10-20 mu L.

In this embodiment, the filter paper 3 is spread at the tube bottom 1a1 in a horizontal state, the quantitative blood collection tube 2b is located right above the filter paper 3 in a vertical state, and is perpendicular to the filter paper 3, and the blood collection end of the quantitative blood collection tube 3 abuts against the filter paper 3. As shown in FIGS. 1 to 3, the sample collection filter paper 3 has a circular sheet shape, and the blood collection end 2b1 abuts against the center of the circular sheet. The round sheet-like specimen 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, and other porous loose materials can be used for replacing the glass fiber filter paper.

In this embodiment, the tube body 1a is cylindrical, the tube bottom 1a1 is U-shaped, the sample collection filter paper 3 is circular sheet-shaped, the sample collection filter paper 3 is horizontally spread over the tube bottom 1a1, and a gap is formed between the sample collection filter paper 3 and the U-shaped arc surface of the tube bottom 1a 1. The specimen flowing out of the drawing end 2b1 is completely received by the specimen collection filter paper 3.

The dry blood spot quantitative collecting device can complete the collection and detection of the dry blood spot sample according to the following method steps:

1) attaching sample source information to the outside of the sample collecting tube, opening the cover of the sample collecting tube, and taking out the quantitative blood collecting tube for later use.

2) Selecting the part with sufficient blood supply (such as inner side of fingertip) as blood sampling part, disinfecting with alcohol, puncturing skin with disposable automatic blood sampling needle, and wiping off the first drop of blood with dry gauze when blood naturally flows out without squeezing.

3) Contacting the capillary end of the quantitative blood collection tube with blood until the capillary force fills the whole capillary.

4) The quantitative blood collection tube was inserted back into the sample collection tube, ensuring that the capillary end contacted the sample collection filter paper. The sample collection tube was capped and the entire sample in the capillary tube was transferred to the filter paper by air pressure and filter paper suction. After the sample is dried, the collection tube is placed in a suitable environment for storage or transport.

5) When the sample is analyzed, the cover of the sample collecting tube is opened, the quantitative blood collecting tube is taken out, the extraction liquid is directly added into the collecting tube, and the analyte is extracted according to a proper program and then is detected.

Example 2: collecting fingertip blood for Therapeutic Drug Monitoring (TDM)

The method can be used for collecting fingertip peripheral blood to prepare a dried blood slice sample. Compared with vein sampling, the dried blood slice sample has the unique advantages of less blood collection amount, stable sample, easy storage and transportation, no time and space limitation on sampling and the like. For some Therapeutic Drug Monitoring (TDM), peak and trough concentrations need to be collected at specific times, or samples are frequently taken over short periods of time, and peripheral blood stem and blood slice samples can meet clinical requirements. The method is simple to operate, high in sample quality and capable of being expanded to home self-service sampling after training.

As shown in fig. 4, when the method is used for collecting blood from fingertips, the operation steps are as follows:

After the collected dry blood spot sample is simply extracted, the information such as the drug concentration and the like can be analyzed. And when the sample is extracted, opening the cover of the sample collecting tube, taking out the quantitative blood collecting tube, adding the extract into the sample collecting tube, and extracting according to an optimized program.

Extraction method ① for extracting plasma protein-bound drug

a) Adding a proper amount of compound internal standard into 80% methanol aqueous solution to prepare internal standard-containing extract liquor;

b) adding 300 mu L of 80% methanol aqueous solution into the double blank sample, adding 300 mu L of internal standard-containing extract into other samples, and carrying out vortex oscillation for about 15 min;

c) centrifuging at 4000rpm and 4 deg.C for 10 min;

d) transfer the supernatant 100. mu. L to a new 96-well plate and centrifuge at 4000rpm for 5min at 4 ℃.

e) And (6) sample injection analysis.

Extraction method ② for extracting blood cell-bound medicine

f) Preparing an internal standard-containing extract by using pure methanol;

g) adding 150 mu L pure water into all samples, and oscillating for 5 min;

h) adding 300 mu L methanol into the double blank sample, adding 300 mu L internal standard-containing extract into other samples, and mixing for 15min by oscillation;

i) centrifuging at 4000rpm and 4 deg.C for 10 min;

j) transfer the supernatant 100. mu. L to a new 96-well plate and centrifuge at 4000rpm for 5min at 4 ℃.

k) And (6) sample injection analysis.

For common small molecule drugs, the detection is carried out by adopting liquid chromatography-tandem triple quadrupole mass spectrometry (L C-MS/MS), the specificity and the sensitivity are higher, and the liquid chromatography method is a mature method which can be retrieved in the literature.

Fig. 5 shows a mass spectrum peak pattern diagram of a part of therapeutic drugs detected by the device of the present invention, including imatinib, carbamazepine, valproic acid, mycophenolic acid, tacrolimus, cyclosporin a, and sirolimus, showing that the mass spectrum peak pattern of the related drugs is excellent, has no miscellaneous peaks and tails, and can be used for drug concentration detection.

The calibration curve is shown in figure 6, the calibration curve is dry blood spot sample mass spectrum calibration curves of two medicines of imatinib and sirolimus, wherein A is an imatinib calibration curve, the concentrations of the calibrators are respectively 50, 500, 1000, 2000, 3000 and 5000ng/m L, B is a sirolimus calibration curve, the concentrations of the calibrators are respectively 2, 4, 6, 12, 20 and 30ng/m L, and linear correlation coefficients r are all more than 0.9990, which indicates that the accuracy of the dry blood spot quantitative detection result can be improved by the integrated operation of the device.

Example 3: collection of human whole blood for nucleic acid detection

The method adopts a vein whole blood sample, loads the vein whole blood sample on a blood sample collecting device, and is used for detecting gene mutation after nucleic acid extraction.

A. Sample collection

(1) Attaching sample source information to the outside of the sample collection tube, opening the cover of the sample collection tube, and taking out the quantitative blood collection tube for later use;

(2) blood samples were collected following the standard venous blood collection procedure.

(3) Venous blood was loaded onto the filter paper of the dry blood collection tube using a pipette until the blood sample was completely dry. Transporting or storing under proper temperature condition.

B. Nucleic acid extraction

After the collected dry blood spot sample, nucleic acid extraction was performed on the sample using QIAamp DNA mini nucleic acid extraction kit (cat # 51304) from QIAgene and QIAcube nucleic acid auto-extractor. The extraction steps are as follows:

(a) taking 5 dried blood spots from a dried blood spot collecting tube, putting the dried blood spots into a 1.5ml centrifuge tube, adding 180 mu L AT L, incubating for 10min AT 85 ℃, and performing short-cut separation;

(b) adding 20 mu L proteinase K, mixing uniformly, incubating for 1h at 56 ℃, and performing short-cut separation;

(c) the liquid was transferred to a new 1.5m L tube as an initial sample for use;

(d) preparing and installing reagents and consumables required by QIAcube;

(e) turning on QIAcube power, the program was DNA → QIAamp DNA mini → Tissue → Elutionvolume: 200. mu. L;

(f) placing the processed initial sample, and starting a QIAcube program;

(g) after the completion of the normal operation of QIAcube, the 1.5m L tube containing the extracted 200. mu. L DNA was removed and stored at-20 ℃.

(h) The extracted nucleic acid samples were quantified using a Qubit 3.0 nucleic acid quantification fluorometer, the quantification results are given in the following table:

C. gene mutation detection

After the nucleic acid extraction is completed, the gene detection can be carried out on the sample. This example uses digital PCR to detect the R140 site of IDH2 gene in samples. Digital PCR is a new method for nucleic acid detection and quantitative analysis, and can realize absolute quantification of nucleic acid and detection of rare allele. The following table respectively shows the quantitative and qualitative detection results of the sample:

digital PCR quantitative results

Qualitative results of mutation detection

The sample is a normal human whole blood simulated dry blood spot sample, the detection result has no R140 mutation (negative), and the results of the two are consistent. And the extracted dry blood spot sample can meet the requirements of subsequent gene detection experiments. The device of the invention can effectively collect and store the dry blood spot sample and is applied to the clinical qualitative detection of molecular biology.

Example 4: collection of whole blood for qualitative detection of antibody

The method can be used for collecting whole blood for antibody qualitative detection. Compared with blood sampling by a blood sampling tube, the dried blood slice sample has the unique advantages of small blood sampling amount, stable sample, easy storage and transportation, no time and space limitation on sampling and the like. The method is simple to operate and high in sample quality.

The operation steps are as follows:

taking 500u L whole blood (blood source: pig whole blood, rabbit whole blood, human whole blood all can), adding antibody, contacting the capillary end of the quantitative blood collection tube with blood, filling the whole capillary tube with blood by capillary force, inserting the quantitative blood collection tube back into the sample collection tube, ensuring that the capillary end contacts sample collection filter paper, covering the sample collection tube cover, transferring all samples in the capillary tube onto the filter paper by air pressure and filter paper suction, and storing or transporting the collection tube in a proper environment after the samples are dried.

When the sample is processed, the cover of the sample collecting tube is opened, the quantitative blood collecting tube is taken out, 1m L sample diluent is added into the sample collecting tube, the sample collecting tube is oscillated at the temperature of 25 ℃ and the rpm of 600rpm for 3 hours, and the test is carried out according to the following experimental steps.

1) Setting 2 blank control holes and 2 negative control holes in each round of experiment;

2) diluted sample 100u L was added to each well (except negative control), blank control was added with sample diluent 100u L to each well;

3) adding 100u L into the negative control, shaking, mixing, sealing, and incubating at 37 deg.C for 30 min;

4) washing the plate: discarding liquid in the holes, filling washing liquid in each hole, standing for 5 seconds, spin-drying, and repeating the above steps for 5 times to dry;

5) adding 100u L enzyme conjugate into each well, sealing, and incubating at 37 deg.C for 30 min;

6) washing the plate: repeating the step 4;

7) adding 50u L of color-developing agent A and B into each well, shaking, mixing, sealing, and incubating at 37 deg.C for 10 min;

8) adding the stop solution 50u L into each hole, and uniformly mixing;

9) reading by an enzyme-linked immunosorbent assay; blank wells were first zeroed and the OD read from each well.

10) And (4) positive judgment: OD is not less than negative control 0D mean value +0.25

The experimental results are as follows:

the results of the blood spot assay and the non-blood spot assay were interpreted in agreement, which demonstrates that the integrated operation of the device of the invention allows for the qualitative detection of antibodies.

Example 5: the quantitative collection device for the dried blood spots is used for comparing the interference of the Hematocrit (HCT) with the prior method

HCT range: 20 to 60 percent

The sample preparation method comprises the following steps:

1) whole blood HCT was adjusted to prepare 5 portions of whole blood matrices having HCT values of 20%, 30%, 40%, 50%, and 60%, respectively.

2) Equal amounts of stock solutions of standards were added to the matrices to obtain 5 samples of the same concentration but different HCT values. The low concentration interval and the high concentration interval are respectively prepared in one set.

3) Calibrators were formulated with whole blood at 40% HCT.

4) All samples and calibrators are prepared into corresponding dry blood spot samples respectively by using a new method and a comparison method.

The experimental method comprises the following steps:

the bias of different HCT samples against 40% HCT samples, and the Coefficient of Variation (CV) of the sample population were compared, as measured by the new and control methods, respectively.

Experiment one:

and (3) detecting the medicine: imatinib;

the control is Whatman 903 card, 10 mu L quantitative blood sampling, and the blood spots are all removed;

the experimental results are as follows: as shown in B, C in fig. 7, in the low and high concentration samples, the new method has a smoother curve compared to the 903 ka control method, showing that the effect of HCT on the detection result is greatly improved, A, D shows that the CV values of the coefficient of variation (5.17%, 6.24%) of the new method in the range of HCT 20% to 60% are significantly less than those of the 903 ka control (11.46%, 15.62%).

Experiment two:

and (3) detecting the medicine: sirolimus;

comparison: whatman DMPK-A card, collecting blood non-quantitatively, and punching a hole in the center of 3 mm;

the experimental results are as follows: as shown in B, C in fig. 8, in the low and high concentration samples, the curve of the new method is smoother in the HCT 20% -60% range than that of the DMPK-a card control method, showing that the effect of HCT on the detection result is greatly improved, and A, D shows that the CV values (5.27%, 6.48%) of the coefficient of variation detected by the new method in the HCT 20% -60% range are significantly smaller than those of the DMPK-a card control (11.79%, 14.65%).

Experiment three:

and (3) detecting the medicine: valproic acid;

the experimental results are as follows: as shown in B, C in fig. 9, in the low and high concentration samples, the curve of the new method is smoother in the HCT 20% -60% range than that of the DMPK-a card control method, showing that the effect of HCT on the detection result is greatly improved, and A, D shows that the CV values (5.76%, 8.63%) of the coefficient of variation detected by the new method in the HCT 20% -60% range are significantly smaller than those of the DMPK-a card control (10.82%, 12.05%).

The above experiments demonstrate that the integrated operation of the device of the present invention can improve the accuracy of the quantitative detection result of dry blood spots.

In summary, the above embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. A dry blood spot quantitative collection device, characterized by comprising: the device comprises a sample collecting pipe, sample collecting filter paper and a quantitative blood collecting pipe; wherein the content of the first and second substances,

2. The dry blood spot quantitative collection device according to claim 1, wherein said tube body is cylindrical, said nozzle is circular, and said tube bottom is U-shaped.

3. The dry blood spot quantitative collection device according to claim 1, wherein the nozzle is integrated with or separated from the tube cover.

4. The device according to claim 1, wherein the tube is made of a transparent plastic material.

5. The device according to claim 1, wherein the mounting member has a cavity, and wherein a desiccant is disposed in the cavity.

6. The device according to claim 5, wherein the fixing member has a second protrusion, and the second protrusion is engaged with the cap.

7. The quantitative dry blood spot collection device according to claim 6, wherein the fixing member is a tubular column which is sleeved in the tubular body of the sample collection tube; the top end of the pipe column is in an opening shape and is clamped with the pipe orifice of the pipe body; the bottom end of the pipe column is provided with a fixing hole, and the quantitative capillary is inserted into the fixing hole and fixed.

8. The dry blood spot quantitative collection device according to claim 7, wherein said column is cylindrical; the fixed hole is positioned in the center of the cross section of the pipe column, and the quantitative capillary pipe is inserted into the fixed hole along the axial direction of the pipe column and is fixed.

9. The device according to claim 8, wherein the column is made of transparent plastic.

10. The dry blood spot quantitative collection device according to claim 1, wherein the sample collection filter paper is horizontally spread on the bottom of the tube, the quantitative blood collection tube is vertically positioned above the sample collection filter paper and is perpendicular to the sample collection filter paper, and the blood collection end of the quantitative blood collection tube abuts against the sample collection filter paper.

11. The dry blood spot quantitative collection device according to claim 10, wherein the sample collection filter paper has a circular sheet shape, and the blood collection end abuts against the center of the circular sheet shape.

12. The dry blood spot quantitative collection device according to claim 11, wherein the sample collection filter paper is a glass fiber filter paper.

13. A method of dry blood spot collection using the dry blood spot quantitative collection device according to any one of claims 1 to 12, comprising the steps of:

14. The method of claim 13, wherein the method further comprises step S4: after step S3, the quantitative dry blood spot collecting device is placed in a suitable environment for storage or transportation.

15. The method of claim 13, wherein the method further comprises step S5: after step S3, when analyzing the sample, the tube cap is opened, the quantitative blood collection tube is taken out, the extraction liquid is directly added into the sample collection tube to extract the analyte, and the extracted analyte is sent to the test.

16. The method of claim 13, wherein the method further comprises step S6: in any of the preceding steps, sample source information is attached to the outside of the sample collection tube.