A collection device and method for quantitative collection of dried blood spots

Abstract

The application discloses a collecting device for quantitatively collecting dry blood spots, which comprises a sample collecting ring with both ends being permeable, a sample carrying sheet and a quantitative capillary for sampling, and the sample carrying sheet is detachably arranged in the sample collecting ring. The application also discloses a collecting method for collecting dry blood spots by using the collecting device. The application can quantitatively collect blood samples, and the sample can be directly sealed and sent without drying, punching and sampling, so as to reduce cross contamination, improve the accuracy of quantitative detection results of the dry blood spots, and make the quantitative collection, preservation, sample information input and sample sending operation of the sample be integrally completed by the device.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of dried blood spot detection, in particular to a dried blood spot quantitative collection device and a collection method. BACKGROUND

[0002] Dried blood spot (DBS) detection is a method for detecting analytes in dried blood spots by adding clinical biological samples to filter paper, drying and then detecting the analytes in the dried blood spots after solvent extraction. The technology uses filter paper as a carrier and is widely used for qualitative detection of nucleic acid genetic material, qualitative and quantitative detection of biomarkers, monitoring of drug concentration and the like. With the development of sequencing technology and mass spectrometry technology, the application of dried blood spots in clinical disease detection and screening projects is becoming more and more extensive. However, the current dried blood spot collection card needs to be dried for two to three hours in a cool place during blood spot preparation, which not only affects the sample preparation efficiency, but also is prone to cross contamination during the process. In the process of using the dried blood spot, manual punching is required, which is time-consuming and laborious and is prone to cross contamination and confusion between samples. In addition, due to the blood volume effect, hematocrit effect (HCT) and chromatographic effect, the components of the same blood spot are not uniform at the center and the edge, and the components are not uniform at different parts of the blood spot, which causes large detection error and poor reproducibility.

[0003] The patent specification with the publication number CN111474257A discloses a dried blood spot quantitative collection device, which comprises a sample collection tube, a sample collection filter paper and a quantitative blood collection tube. The sample collection tube comprises a tube body and a tube cover. The tube body is a straight tube, and the tube cover is used to close the tube opening. The quantitative blood collection tube comprises a quantitative capillary tube and a fixing piece. The quantitative capillary tube is fixed to the fixing piece, and the quantitative capillary tube has a blood collection end exposed outside the fixing piece. The fixing piece is clamped to the tube opening, and 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.

[0004] The patent specification with the publication number CN206192677U discloses a collection tube type dried blood spot sampling device, which comprises a collection tube main body with both ends being transparent. The top of the collection tube main body is provided with a top sealing film which can be broken by pressure. An information identification code is printed on the top sealing film. The bottom of the collection tube main body is provided with a matched filter paper.

[0005] The above two schemes have the problem of long blood spot drying time, which affects the sample preparation efficiency. SUMMARY

[0006] The application aims to provide a collection device for quantitatively collecting dry blood spots, which can quantitatively collect blood samples, directly seal and send the samples without drying, avoid punching and sampling, reduce cross contamination, improve the accuracy of quantitative detection results of dry blood spots, and integrate the quantitative collection, preservation, sample information input and sample sending operations of the sample through the device.

[0007] The application relates to a collection device for quantitatively collecting dry blood spots, which comprises a sample collection ring with both ends being permeable, a sample carrier and a quantitative capillary for sampling, wherein the sample carrier is detachably arranged in the sample collection ring.

[0008] In the application, the quantitative sampling is performed by using the quantitative capillary, and all the quantitatively collected blood samples are transferred to the sample carrier, and the whole sample carrier is put into an extraction tube for extracting analytes, so that the problem of large quantitative result fluctuation and low accuracy caused by uneven distribution of the sample on the sample carrier can be avoided.

[0009] Preferably, the sample carrier comprises a plurality of pieces arranged in the sample collection ring and arranged along the axial direction of the sample collection ring.

[0010] Preferably, a fixing clamp for fixing the sample carrier is arranged on the inner wall of the sample collection ring; the fixing clamp comprises a plurality of fixing clamps uniformly distributed along the circumferential direction of the inner wall of the sample collection ring, and at least one of the fixing clamps is retractable, so that the hard sample carrier can be conveniently put into the sample collection ring.

[0011] Further preferably, the end of the fixing clamp for fixing is provided with a bayonet for the sample carrier to extend into.

[0012] Preferably, a plurality of uniformly distributed hollows are arranged along the circumferential direction of the ring wall of the sample collection ring. Specifically, three hollows are arranged at the middle part of the sample collection ring, so that the blood spots on the sample carrier can be conveniently dried.

[0013] Preferably, the sample carrier is made of full cotton fiber filter paper, glass fiber filter paper or diatomite. Specifically, the waterman 903 filter paper can be used, and the sample carrier can also be a round piece pressed by other appropriate materials, such as a round piece pressed by inorganic powder; the diameter of the sample carrier is 3mm to 12mm, preferably 3mm to 8mm.

[0014] Preferably, the application further comprises a support, and a plurality of recess cavities for placing a plurality of sample collection rings are arranged on the support. Each sample collection ring is marked with a number, and the side surface of the support has an area for pasting a sample information barcode, which is used for pasting the sample information barcode. The material of the sample collection ring and the material of the support are the same plastic material, and the plastic material is preferably transparent.

[0015] As preferred, the support is further provided with a placement cavity for placing the quantitative capillary, one end of the placement cavity is closed, the other end is open, and the open end is provided with an end cover for closing. The placement cavity is horizontally placed at the front end of the support, and the length is slightly longer than the length of the quantitative capillary.

[0016] As preferred, a sealed bag for placing the support is further included, and a desiccant is arranged in the sealed bag. The sealed bag is provided with a concave-convex sealing card strip at the opening, which can be opened and sealed. The sealed bag can be transparent and made of PE material, and if the measured sample is sensitive to light, a light-shielding sealed bag can be selected.

[0017] Another object of the present application is to provide a collection method, which uses the above-mentioned collection device for dry blood spot collection, comprising the following steps:

[0018] S1: The blood sampling end of the quantitative capillary is close to and contacts the blood, and the capillary force fills the blood in the entire quantitative capillary or to the corresponding volume scale;

[0019] S2: After collecting the blood sample, the blood sampling end of the quantitative capillary is contacted with the sample loading sheet, and the blood in the quantitative capillary is completely transferred to the sample loading sheet in the sample collection ring by air pressure and the suction force of the sample loading sheet;

[0020] S3: The sample collection ring is sealed and stored.

[0021] Before packaging, the blood sample information barcode is attached to the support, and after sealing, the dry blood spot quantitative collection device is stored or transported in a suitable environment. For some samples sensitive to air oxidation, nitrogen can be filled in the sealed bag before sealing to improve the stability of the sample.

[0022] As preferred, when analyzing the sample, one or several sample loading sheets are taken out and placed in a sample extraction tube, an extraction liquid is directly added for extracting the analyte, and the extracted analyte is sent for inspection.

[0023] The present application has the following advantages:

[0024] (1) The quantitative capillary is used for quantitative sampling, and the blood sample collected quantitatively is completely transferred to the sample loading sheet, and the whole sample loading sheet is placed in an extraction tube for extracting the analyte, which can avoid the problem of large quantitative result fluctuation and low accuracy caused by uneven distribution of the sample on the sample loading sheet.

[0025] (2) The quantitative capillary is used for collecting the blood sample, so that the sample can be quantitatively transferred to the sample loading sheet, and the detection result is more accurate.

[0026] (3) After the blood sample is added to the sample loading sheet, it does not need to be dried, and can be directly sealed and stored or transported, which reduces the waiting time in the drying process, improves the effect, and also reduces the possibility of sample contamination in the drying process.

[0027] (4) The sample does not need to be punched before extraction, which reduces the work intensity, improves the efficiency, and reduces the cross contamination caused by punching.

[0028] (5) For samples with different polarities, different materials of the sample carrier can be selected, which can improve the extraction recovery and improve the detection sensitivity. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is a schematic view of the structure of the support of the application;

[0030] Figure 2 It is a schematic view of the structure of the sample collection ring;

[0031] Figure 3 It is Figure 2 It is a schematic view of the structure of the local part A in the middle;

[0032] Figure 4 It is a schematic view of the drying time of different sampling amounts in Examples 1-3;

[0033] Figure 5 It is a schematic view of the relationship between the concentration of the analyte and the sampling amount in Example 4;

[0034] Figure 6 It is a schematic view of the relationship between the number of sample carriers and the concentration of the analyte in Example 6;

[0035] Figure 7 It is a schematic view of the stability of the sample within 12 days in Example 7;

[0036] Figure 8 It is a typical chromatogram of the mass spectrometric detection of 25-hydroxyvitamin D. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the application will be described clearly and completely below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0038] As Figures 1-3 shown, a collection device for quantitatively collecting a dry blood spot includes a sealed bag, a support 1, a sample collection ring 2, a sample carrier 3, and a quantitative capillary. The support 1 is used to accommodate the sample collection ring 2, the sample carrier 3, and the quantitative capillary. After sampling, they are packed into the sealed bag for storage.

[0039] The sample sampling ring 2 is cylindrical and has two ends which are permeable, and a plurality of sample carriers 3 are arranged in the sample sampling ring 2 along the axial direction of the sample sampling ring 2, and the sample carriers 3 are arranged in parallel with the ports of the sample sampling ring 2.

[0040] In the embodiment, two sample carriers 3 are arranged near the quarter positions of the two ports of the sample sampling ring 2, and two groups of fixing clamps 21 are arranged on the inner wall of the sample sampling ring 2 for fixing the sample carriers 3. In the embodiment, each group of fixing clamps 21 is specifically provided with three fixing clamps 21 which are uniformly distributed along the circumferential direction of the inner wall of the sample sampling ring 2, and one of the fixing clamps 21 is retractable. The fixing clamp 21 is made of elastic material, and a bayonet 211 is arranged on one end of the fixing clamp 21 for fixing. The diameter of the sample carrier 3 is smaller than the inner diameter of the sample sampling ring 2, so that the sample carrier 3 can be easily put in. When the sample carrier 3 is placed, the retractable fixing clamp 21 is pressed, the edge of the sample carrier 3 is placed in the bayonet 211 of the fixing clamp 21, and the retractable fixing clamp 21 is reset to complete the fixing.

[0041] In the embodiment, three evenly distributed hollows 22 are arranged on the circumferential direction of the middle part of the ring wall of the sample sampling ring 2, so as to facilitate the air drying of the blood spots on the sample carrier 3. The sample carrier 3 can be made of all-cotton fiber filter paper, glass fiber filter paper or diatomite, or can be made of a circular piece of other appropriate materials with a diameter of 3-8 mm.

[0042] In the embodiment, the support 1 is provided with recesses 11 arranged in 2*3, each recess 11 corresponds to one sample sampling ring 2, and each sample sampling ring 2 is marked with a number. The side surface of the support 1 has an area for pasting a sample information barcode.

[0043] The front end of the support 1 is provided with a horizontal placement cavity 12, the length of which is slightly longer than the length of the quantitative capillary, and the opening end is provided with an end cover 13 for closing.

[0044] In the embodiment, a desiccant is arranged in the sealing bag, and a concave-convex sealing strip is arranged on the opening of the sealing bag, so that the sealing bag can be opened and sealed. The sealing bag can be transparent and made of PE material. If the measured sample is sensitive to light, a light-shielding sealing bag can be selected.

[0045] A collection method for collecting dry blood spots by using the above-mentioned collection device, comprising the following steps:

[0046] S1: opening the sealing bag, taking out the quantitative capillary and the sample sampling ring with the sample carrier from the support, and placing the blood sampling end of the quantitative capillary close to and in contact with the blood, until the capillary force fills the entire quantitative capillary with blood or reaches the corresponding volume scale;

[0047] S2: After collecting the blood sample, the blood collection end of the quantitative capillary is brought into contact with the center of the sample carrier. The blood sample is transferred to the sample carrier by air pressure and suction of the sample carrier.

[0048] S3: Attach the blood sample information barcode to the holder;

[0049] S4: After the quantitative capillary tube and sample sampling loop are stored, put them into a sealed bag containing desiccant, squeeze out the air from the bag, seal the bag, and store or transport the dried blood spot collection device in a suitable environment.

[0050] S5: When analyzing samples, open the sealed bag, take out the holder, scan the code to enter the sample information, take out one or more sample slides and put them into the extraction tube, add the extraction solution to extract the analyte, and send the extracted analyte for testing.

[0051] Examples 1-3

[0052] Using this quantitative collection device, 20, 30, and 40 μL of whole blood were collected, respectively. After collection, the dried blood spots were measured after being left at room temperature for 2 h, 4 h, 6 h, and 24 h. The results are as follows: Figure 4 As shown, the mass is the total weight of the dried blood spot and the filter paper.

[0053] from Figure 4 It can be seen that the weight of the dried blood spot basically no longer changes after 6 hours, indicating that the drying time of the collection device at room temperature is 6 hours or more.

[0054] Example 4

[0055] Using this quantitative collection device, 10, 20, 30, and 40 μL of whole blood were collected. After collection, the dried blood spots were left at room temperature overnight and the concentration of 25-hydroxyvitamin D in the dried blood spots of different blood volumes was measured.

[0056] The testing conditions are as follows:

[0057] 1. Sample processing

[0058] Remove the dried blood spot sample and place it in a 96-well plate;

[0059] Add 100 μL of reagent A to each well;

[0060] Add 200 μL of reagent B to each well;

[0061] Add 40 μL of 10 ng / mL VD-IS and shake continuously for at least 30 minutes.

[0062] Transfer the solution from each well to a 96-well SLE plate and let it stand for about 5 minutes.

[0063] Add 400 uL reagent C to each well, stand for about 5 min;

[0064] Add 400 uL reagent C to each well again, stand for about 5 min;

[0065] Nitrogen blowing dry;

[0066] Add 60 uL reagent D to each sample well, vortex for 2 min;

[0067] Derive at 50℃ for 30 min;

[0068] Add 40 uL reagent B to each well, vortex for 2 min;

[0069] Nitrogen blowing dry;

[0070] Add 100 uL reagent E to each sample well, vortex for 2 min;

[0071] Put the 96-well plate with silica gel cover plate into the autosampler, start the program for sample injection detection.

[0072] 2. Liquid phase conditions

[0073] Chromatographic column: BEH C18, 3.0*50mm (waters);

[0074] Mobile phase A: 0.1% formic acid aqueous solution;

[0075] Mobile phase B: 0.1% formic acid acetonitrile solution;

[0076] Injection volume: 5 uL;

[0077] Flow rate: 0.4 mL / min;

[0078] Column temperature: 45℃;

[0079] Sample injector temperature: 15℃;

[0080] Elution gradient:

[0081] Time (min) A(％) B(％) Time (min) A(％) B(％) 0.0 40 60 1.8 5 95 0.5 40 60 1.81 40 60 1.0 10 90 2.5 40 60

[0082] 3. Mass spectrometry conditions

[0083] Source parameters

[0084]

[0085] MRM ion channel

[0086] Tested / IS MRM Conical hole voltage (V) Collision energy (eV) VD2 570.4 / 298.2 10 8 VD2-IS 573.4 / 301.4 10 8 VD3 558.4 / 298.2 4 8 VD3-IS 564.3 / 298.4 4 8

[0087] The detection results of 25-hydroxyvitamin D in blood spots under different sample loading conditions are shown in Table 1 below. Typical chromatograms of 25-hydroxyvitamin D detected by liquid chromatography-tandem mass spectrometry are shown below. Figure 8 As shown.

[0088] Table 1. Detection results of 25-hydroxyvitamin D in dried blood spots with different sample loading amounts.

[0089]

[0090]

[0091] Unit: ng / mL

[0092] As shown in the table above, when the sampling volume is 10, 20, and 30 μL, the concentration is basically proportional to the sampling volume. However, when the sampling volume is 40 μL, the concentration is much lower than the theoretical concentration. By dividing the detection concentrations at sampling volumes of 20, 30, and 40 μL by the detection concentration at a sampling volume of 10 μL, and plotting this ratio on the y-axis and the theoretical ratio on the x-axis, we obtain the following results. Figure 5 .

[0093] Depend on Figure 5 It can be seen that the actual ratio of a 40μL sampling volume is much lower than the theoretical ratio (the inflection point in the figure appears at the fourth point). In conclusion, the sample volume of a single sample carrier in the sampling device described in this paper should preferably not exceed 30μL.

[0094] Example 5

[0095] In this example, the concentration of the analyte in whole blood samples (i.e., whole blood used to prepare dried blood spots) was detected simultaneously, and the difference in the concentration of the analyte between the two samples was compared, as shown in Table 2 below.

[0096] Table 2 Recovery rate of dried blood spot samples

[0097]

[0098] As can be seen from Table 2, the recovery rate is greater than 100%, indicating that the analyte in the dried blood spot can be completely extracted, and that the sampling device described in this invention does not produce a large adsorption of the analyte.

[0099] Example 6

[0100] This quantitative collection device can hold different numbers of sample slides (one, two, three, and four slides). 20 μL of whole blood is collected from each slide. After collection, the blood is left at room temperature overnight before measuring the concentration of 25-hydroxyvitamin D in the dried blood spots from different numbers of sample slides. The results are shown in Table 3 below.

[0101] Table 3. Results of 25-hydroxyvitamin D assay with different numbers of sample slides.

[0102]

[0103] Divide the detection concentrations for sample numbers 1, 2, 3, and 4 in the table by the detection concentration for sample number 1, and plot a scatter plot with this ratio on the y-axis and the theoretical ratio on the x-axis, as shown below. Figure 6 As shown.

[0104] From Table 3 and Figure 6 It can be seen that the number of sample slides is directly proportional to the concentration of the analyte, indicating that the sampling volume of this sampling device can be increased by increasing the number of sample slides.

[0105] Example 7

[0106] After sampling 20 μL using this quantitative collection device, the samples were left at room temperature for 1 hour, 6 hours, 1 day, 4 days, 7 days, and 12 days, and then processed uniformly to measure the stability of 25-hydroxyvitamin D in the dried blood spots. The stability of the samples was as follows: Figure 7 As shown.

[0107] from Figure 7 As can be seen, the samples are stable within 12 days, and we believe that the samples collected by this collection device can be stored at room temperature for 12 days.

[0108] Example 8

[0109] In this example, a sample slide made of diatomaceous earth was used instead of the original filter paper. The recovery rate of the sample when using diatomaceous earth is shown in Table 4 below.

[0110] Table 4 Recovery rate of dried blood spot samples

[0111]

[0112] As can be seen from Table 2, the recovery rate is between 89% and 104%, indicating that the analyte in the dried blood spot of the sample slide prepared using diatomaceous earth can be completely extracted, which shows that the sampling device of the present invention can also use diatomaceous earth as a sample slide.

[0113] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A collection device for the quantitative collection of a dried blood spot, characterized in that The sample collection ring is provided with a sample collection ring, a sample carrier sheet and a quantitative capillary for sampling, and the sample carrier sheet is detachably arranged in the sample collection ring. The sample carrier sheet is arranged in the sample collection ring and is arranged in an axial direction. The sample collection ring is provided with a plurality of fixing clamps on the inner wall of the sample collection ring for fixing the sample carrier sheet. The fixing clamps are arranged in a circumferential direction of the sample collection ring.

2. The collection device of claim 1, wherein, The sample collection ring is provided with a plurality of hollows arranged in a circumferential direction of the ring wall.

3. The collection device of claim 2, wherein, The bracket is provided with a plurality of recesses for placing a plurality of sample collection rings.

4. The collection device of claim 2, wherein, The bracket is also provided with a placement cavity for placing the quantitative capillary.

5. A method of acquisition, characterized by, The placement cavity is closed at one end and open at the other end, and the open end is provided with an end cover for closing. The bracket is also provided with a sealing bag for placing the bracket, and the sealing bag is provided with a drying agent. The collection device is used for collecting dry blood spots, and the collection device comprises the following steps: S1: The blood sampling end of the quantitative capillary is close to and contacts the blood, and the capillary force fills the blood in the entire quantitative capillary or to the corresponding volume scale; 6. The method of claim 5, wherein, S2: After collecting the blood sample, the blood sampling end of the quantitative capillary is contacted with the sample carrier sheet, and the blood in the quantitative capillary is transferred to the sample carrier sheet in the sample collection ring by air pressure and the suction force of the sample carrier sheet; S3: The sample collection ring is sealed and stored. When analyzing the sample, one or several sample carrier sheets are taken out and placed in a sample extraction tube, an extraction liquid is directly added for extracting the analyte, and the extracted analyte is sent for inspection.

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