All-in-one kit and secondary staining method

Abstract

In the integrated reagent kit and secondary staining method, the integrated reagent kit includes a reagent storage and mixed solution preparation area and a tiling area; the tiling area is used for holding and tiling the sample mixture to be observed in microscopy; the reagent storage and mixed solution preparation area is used for storing sample processing reagents and sample processing; in the first state, the reagent storage and mixed solution preparation area stores reagents for sample processing; in the second state, the reagent storage and mixed solution preparation area is used to receive samples input from the outside and process the samples in the reagent storage and mixed solution preparation area; in the third state, the processed sample mixture enters the tiling area from the reagent storage and mixed solution preparation area for tiling. The reagent kit has a simple and ingenious structure, is very convenient for efficient secondary staining of samples, is simple to operate and highly efficient, and is suitable for large-scale sample processing.

Description

Technical Field

[0001] The present application belongs to the field of integrated reagent devices, and in particular, relates to a reagent device and method for performing sample processing more than twice. Background Art

[0002] It is a routine operation in sample testing to prepare a sample mixture for observation. In some more complex sample processing processes, multiple reagents are required to process the sample step by step.

[0003] In the prior art, multiple reagents are usually stored separately in different containers, and operators are required to take out reagents of corresponding components from different containers at different locations for sample processing. The operation efficiency is low, and when the reagents are similar, the requirements for step management are relatively high.

[0004] Even if the sample mixture is prepared, it is still necessary to transfer the prepared sample mixture to a dedicated carrier such as a glass slide for microscopic observation. The transfer process also requires strict management of the transferred sample mixture volume and transfer time, which places high demands on the operator and related equipment. In addition, the entire process is cumbersome and inefficient, and cannot be operated and applied in batches.

[0005] In the cell detection of biological body fluids, in order to distinguish the types, morphologies and states of cells in body fluids or secretions, cell staining technology is usually used to distinguish cells of different types, different morphologies and different states. In order to have a better staining effect, a variety of reagents are required to perform the staining steps in a suitable sequence. And after the stained sample mixture is prepared, it needs to be used for observation as soon as possible. However, using the equipment and steps in the above-mentioned prior art, not only are the steps extremely cumbersome and time-consuming, but the operation of each step requires a well-trained professional operator to produce a qualified observation sample, and any problem in any link in the middle may affect the subsequent observation effect. The staining efficiency in the prior art is extremely low, and it is impossible to stain sample cells in large quantities. Summary of the invention

[0006] The technical problem to be solved by the present application is to avoid the above-mentioned deficiencies of the prior art, and propose a simple and efficient integrated reagent kit and secondary staining method, which is suitable for large-scale batch operation of sample processing including secondary staining of samples.

[0007] The technical solution of the present application to solve the above-mentioned problems is an integrated test kit, including a reagent storage and mixed liquid preparation area and a tiling area; the tiling area is used for storing and tiling the sample mixed liquid to be observed in microscopy; the reagent storage and mixed liquid preparation area is used for storing sample processing reagents and sample processing; in a first state, the reagent storage and mixed liquid preparation area stores reagents for sample processing; in a second state, the reagent storage and mixed liquid preparation area is used to receive samples input from the outside and process the samples in the reagent storage and mixed liquid preparation area; in a third state, the processed sample mixed liquid enters the tiling area from the reagent storage and mixed liquid preparation area for tiling.

[0008] The integrated reagent kit can not only be used to store reagents, but also to process and prepare sample mixtures using reagents, and can also lay out the prepared sample mixtures, integrating three functions in one. It greatly improves the efficiency of processing and preparing sample mixtures, avoids the steps of taking and weighing various reagents, and also avoids the management of the application sequence of various different reagents. The sample mixture can enter the observation area as soon as it is prepared, so that the observation area can obtain fresh sample mixtures for observation as soon as possible. It greatly improves the process efficiency and application efficiency of sample mixture preparation.

[0009] A sealing film is provided on one side of the reagent storage and mixed liquid preparation area; the sealing film is used to seal the reagent storage and mixed liquid preparation area; in the second state, the external sampling device breaks through the sealing film, allowing the sample to be processed to enter the reagent storage and mixed liquid preparation area for sample processing.

[0010] The sealing film allows the reagents to be stored in an airtight container, giving them the longest possible shelf life.

[0011] The integrated test kit also includes a sampling device, which includes a sampling device body and a sampling nozzle; the sampling nozzle is arranged at one end of the sampling device body, and the sampling nozzle is used to load the external sample into the sampling device; the sampling nozzle is also used to break through the sealing film and send the external sample obtained by sampling into the reagent storage and mixed liquid preparation area.

[0012] The sealing film and the sampling nozzle are arranged in cooperation with each other, so that the sampling device can be easily penetrated to prepare the sample mixture.

[0013] A positioning ring is provided on the outer wall of the sampling device body; in the second state, when the sampling device breaks through the sealing film, the positioning ring is used to locate the depth of the sampling device body entering the end accommodating space, and at the same time, the positioning ring is used to replace the sealing film to isolate the end accommodating space from the outside world.

[0014] The positioning ring can locate the depth of entry, ensuring that the space that the sampling device enters each time is predictable, avoiding the situation of not entering in place or exceeding the positioning, and ensuring the reliability of the sample mixture preparation process. The positioning ring also ensures the sealing during the sample mixture preparation process, making it quantitatively accurate and leak-free.

[0015] The reagent storage and mixed liquid preparation area includes a bottom accommodating space and an end accommodating space; the paving area and the bottom accommodating space are connected; and a partition is provided between the bottom accommodating space and the end accommodating space.

[0016] Different spaces are used to store reagents needed in different steps, simplifying the sample mixture preparation process.

[0017] In the first state, the bottom storage space is empty and the end storage space is used to store sample processing reagents; in the second state, the end storage space is used to receive samples input from the outside and process the samples in the end storage space; in the third state, the processed sample mixture enters the bottom storage space from the end storage space and enters the paving area through the bottom storage space.

[0018] The reserved bottom accommodating space can be used as a buffer space between the reagent storage and preparation area and the observation area. The buffer space is conducive to the uniform mixing of the sample mixture, and it enters the observation area more quickly, and can make the observation area stable for observation as soon as possible.

[0019] In the first state, the bottom accommodating space and the end accommodating space each independently stores reagents for sample processing; the end accommodating space is used to accommodate initial reagents, which are used for the first sample processing; the bottom accommodating space is used to accommodate final test reagents, which are used for the final sample processing; in the second state, the end accommodating space is used to receive samples input from the outside and perform the first processing on the samples in the end accommodating space; the sample mixture after the first processing enters the bottom accommodating space from the end accommodating space for the final processing of the samples; in the third state, the sample mixture after the final processing enters the paving area from the bottom accommodating space.

[0020] At least one intermediate accommodating space is also arranged between the bottom accommodating space and the end accommodating space, and the intermediate accommodating space is used to accommodate intermediate reagents, and the intermediate reagents are used for intermediate sample processing; a first partition is arranged between the intermediate accommodating space and the end accommodating space; a second partition is arranged between the intermediate accommodating space and the bottom accommodating space; in the first state, the integrated reagent box is placed vertically, and the bottom accommodating space, the intermediate accommodating space and the end accommodating space each independently stores reagents for sample processing; in the second state, the external sample breaks through the sealing film, the first partition and the second partition in turn, so that the sample enters the end accommodating space, the intermediate accommodating space and the bottom accommodating space in turn to complete the initial processing, intermediate secondary processing and final processing of the sample in turn.

[0021] Reagents are stored in each space, so that the types of reagents that can be stored can be increased, and the sample mixture preparation process that requires the participation of more reagents can be adapted.

[0022] The integrated test kit also includes a sampling device, which includes a sampling device body and a sampling nozzle; the sampling nozzle is arranged at one end of the sampling device body, and the sampling nozzle is used to load an external sample into the sampling device; the sampling nozzle also sends the sample obtained by sampling into the end accommodating space, the middle accommodating space and the bottom accommodating space; a first positioning ring, a second positioning ring and a third positioning ring are arranged on the outer wall of the sampling device body; in the second state, when the external sampling device breaks through the sealing film, the first positioning ring is used to locate the depth of the sampling device body entering the end accommodating space, and the first positioning ring is used to replace the role of the sealing film to isolate the end accommodating space from the outside; when the external sampling device breaks through the first partition, the second positioning ring is used to locate the depth of the sampling device body entering the middle accommodating space, and the second positioning ring is used to replace the role of the sealing film to isolate the end accommodating space from the outside; when the external sampling device breaks through the second partition, the third positioning ring is used to locate the depth of the sampling device body entering the bottom accommodating space, and the third positioning ring is used to replace the role of the sealing film to isolate the end accommodating space from the outside.

[0023] A sealing ring groove is also provided on the inner wall close to the sealing film on one side of the end accommodating space, and a sealing ring is embedded in the sealing ring groove. When the sampling device is pushed inward in sequence, any one of the first positioning ring, the second positioning ring or the third positioning ring and the end accommodating space are sealed by the sealing ring.

[0024] The sealing ring further ensures the sealing of the reagent storage and mixed liquid preparation area relative to the outside after the sampling device enters, ensuring the sealing of the reagent storage and preparation process.

[0025] The sampling device is a quantitative sampling device, and the capacity range of quantitative sampling is 5ul to 15ul.

[0026] The capacity range of the end accommodation space is 50ul to 3000ul.

[0027] The capacity range of the bottom accommodation space is 50ul to 1000ul.

[0028] The capacity range of the middle accommodating space is 50ul~1000ul.

[0029] The quantitative sampling device and the fixed-volume spaces cooperate with each other, and a fixed-volume reagent can be stored in the fixed-volume space, which eliminates the need to weigh the reagents separately in each step. Moreover, due to the clever setting of each space, each step can be completed in sequence by pushing the sampling device, which greatly improves the process efficiency of sample mixture preparation.

[0030] A plurality of intermediate accommodating spaces are also arranged between the bottom accommodating space and the end accommodating space; the plurality of intermediate accommodating spaces are arranged adjacent to each other, and the plurality of intermediate accommodating spaces are arranged as a whole between the end accommodating space and the bottom accommodating space, and the plurality of intermediate accommodating spaces are used to store a plurality of different processing reagents; a first partition is arranged between the end accommodating space and the intermediate accommodating space, a middle partition is arranged between each intermediate accommodating space, and a last partition is arranged between the intermediate accommodating space and the bottom accommodating space; in a first state, the integrated reagent kit is placed longitudinally; the bottom accommodating space, the plurality of intermediate accommodating spaces and the end accommodating space each independently stores reagents for sample processing; in a second state, the integrated reagent kit is placed longitudinally, and external samples break through the sealing film, the first partition, the middle partition and the last partition in turn, so that the samples complete the initial processing, multiple intermediate processing and the last processing of the samples in turn when entering the end accommodating space, the plurality of intermediate accommodating spaces and the bottom accommodating space in turn.

[0031] The integrated test kit also includes a sampling device, which includes a sampling device body and a sampling nozzle; the sampling nozzle is arranged at one end of the sampling device body, and the sampling nozzle is used to load an external sample into the sampling device; the sampling nozzle also sequentially sends the sample obtained by sampling into the end accommodating space, multiple intermediate accommodating spaces and the bottom accommodating space; a first positioning ring, multiple intermediate positioning rings and a final positioning ring are arranged on the outer wall of the sampling device body; in the second state, when the external sampling device breaks through the sealing film, the first positioning ring is used to locate the depth of the sampling device body entering the end accommodating space, and the first positioning ring is used to replace the role of the sealing film to isolate the end accommodating space from the outside; when the external sampling device breaks through the first partition, the intermediate positioning ring is used to locate the depth of the sampling device body entering the intermediate accommodating space, and the intermediate positioning ring is used to replace the role of the sealing film to isolate the end accommodating space from the outside; when the external sampling device breaks through the final partition, the final positioning ring is used to locate the depth of the sampling device body entering the bottom accommodating space, and the final positioning ring is used to replace the role of the sealing film to isolate the end accommodating space from the outside.

[0032] A sealing ring groove is also provided on the inner wall close to the sealing film on one side of the end accommodating space, and a sealing ring is embedded in the sealing ring groove. When the sampling device is pushed inward in sequence, any one of the first positioning ring, the middle positioning ring or the last positioning ring and the end accommodating space are sealed by the sealing ring.

[0033] The sampling device is a quantitative sampling device; the capacity of the end accommodating space, the capacity of the middle accommodating space and the capacity of the bottom accommodating space match the capacity of the quantitative sampling device.

[0034] When quantitatively diluting a quantitative sample, the end accommodating space, the middle accommodating space, and the bottom accommodating space may be filled with reagents or not; the end accommodating space, the middle accommodating space, and the bottom accommodating space should be sufficient to complete the processing of the quantitative sample in the quantitative sampling device. That is, the capacity of the end accommodating space, the middle accommodating space, and the bottom accommodating space should at least be greater than or equal to the minimum amount of reagent required to be stored.

[0035] The technical solution of the present application to solve the above-mentioned problem can also be a secondary staining method, which is used for secondary staining treatment of cells in biological samples and is based on the above-mentioned integrated reagent kit; the initial reagent in the end accommodating space is the first staining reagent used for the first sample staining treatment; the final reagent in the bottom accommodating space is the second staining reagent used for the second sample staining treatment; the cell secondary staining method includes the following steps: Step 1: Use a sampling device to perform quantitative sampling of the sample to be stained; Step 2: Use the sampling device in step one to send a quantitative sample to be stained into the end accommodating space for the first staining, and in the second state of the integrated reagent kit, the sample and the first staining reagent are mixed evenly, that is, the first staining is completed; Step 3: Use the sampling device in step 2 to send the mixed liquid after the first staining treatment into the bottom accommodating space, and in the second state of the integrated reagent kit, the mixed liquid after the first staining treatment is mixed evenly with the second staining reagent in the bottom accommodating space, that is, the second staining is completed.

[0036] The technical solution of the present application to solve the above-mentioned problem can also be a secondary staining method, which is used for secondary staining of cells in biological samples and is based on the above-mentioned integrated reagent kit; the initial reagent in the end accommodating space is a diluent used for the first sample dilution treatment; the intermediate reagent in the middle accommodating space is a first staining reagent used for the first sample staining treatment; the final reagent in the bottom accommodating space is a second staining reagent used for the second sample staining treatment; the cell secondary staining method comprises the following steps: step one: quantitatively sampling the sample to be stained by using a sampling device; step two: using the sampling device in step one, sending a quantitative sample to be stained into the end accommodating space for dilution treatment; in step one In the second state of the integrated reagent kit, the sample and the diluent are mixed evenly, that is, the dilution step is completed, and the diluted sample liquid is obtained; step three: using the sampling device in step two, the diluted sample liquid is sent to the middle accommodating space for the first staining. In the second state of the integrated reagent kit, the diluted sample liquid and the first staining reagent are mixed evenly, that is, the first staining is completed, and the mixed liquid after the first staining treatment is obtained; step four: using the sampling device in step three, the mixed liquid after the first staining treatment is sent to the bottom accommodating space for the second staining. In the second state of the integrated reagent kit, the mixed liquid after the first staining treatment and the second staining reagent are mixed evenly, that is, the second staining is completed.

[0037] Compared with the prior art, the beneficial effects of the present application are: 1. Ingenious integrated reagent kit structure design, the three functions of reagent storage, sample processing, and sample tiling are integrated together at the same time, and the operations of each step are concentrated in one reagent kit to complete, with a high degree of integration and high staining efficiency; 2. The quantitative sampling device and the fixed-volume reagent storage space eliminate the weighing process and improve the process efficiency; 3. The reagent storage space arranged in a certain order can very conveniently use the sampling device to perform a multi-step sample processing process step by step, further improving the process efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is an axonometric projection diagram showing the coordination relationship between the integrated test kit and the sampling device in a disassembled state;

[0039] Figure 2 It is a schematic diagram of the axonometric projection of the sampling device;

[0040] Figure 3 It is a schematic diagram of the top orthographic projection of the sampling device;

[0041] Figure 4 yes Figure 3 AA cross-sectional view of ;

[0042] Figure 5 is an axonometric projection schematic diagram of the integrated reagent box when the sampling device is inserted into the integrated reagent box;

[0043] Figure 6 It is a schematic diagram of the rear view orthographic projection of the integrated test kit and the sampling device in the combined state;

[0044] Figure 7 yes Figure 6 BB cross-sectional view;

[0045] Figure 8 It is the state in which the sampling device has not been inserted into the integrated reagent box, and the integrated reagent box is in a vertical upright state; usually after the integrated reagent box is packaged, it will be in a vertical placement state; of course, when the bottom accommodating space is empty at the beginning, that is, when no reagent is pre-buried therein, there is no requirement for the placement state of the integrated reagent box; when reagents are stored in the bottom accommodating space, but a one-way valve or other intercepting device for closing the pipeline is provided in the outflow hole and the reflux hole, there is no requirement for the placement state of the integrated reagent box; when reagents are stored in the bottom accommodating space, and no intercepting device for closing the pipeline is provided in the outflow hole and the reflux hole, there is a requirement for the placement state of the integrated reagent box, and it must be placed in the vertical position;

[0046] Fig. 9 The sampling device is inserted into the integrated reagent box, and the integrated reagent box is in a vertically upright state; and the sample and the reagent are mixed in this state;

[0047] Fig.10 It is the state where the sampling device is inserted into the integrated reagent box, and the integrated reagent box is in a horizontally flat state; in this state, the liquid in the bottom accommodating space will flow to the flat area. DETAILED DESCRIPTION

[0048] The implementation methods of the present application are further described in detail below in conjunction with the accompanying drawings.

[0049] like Figures 5 to 7 In the embodiment of the integrated reagent kit shown, a reagent storage and mixed solution preparation area 100 and a paving area 200 are provided together on a base 900. The paving area 200 is used for storing and paving the sample mixed solution to be observed in microscopic examination; the reagent storage and mixed solution preparation area 100 is used for storing sample processing reagents and sample processing; in the first state, the reagent storage and mixed solution preparation area 100 stores reagents for sample processing; in the second state, the reagent storage and mixed solution preparation area 100 is used to receive samples input from the outside and process the samples in the reagent storage and mixed solution preparation area; in the third state, the processed sample mixed solution enters the paving area 200 from the reagent storage and mixed solution preparation area 100.

[0050] like Figure 1In the embodiment of the integrated reagent kit shown, an intermediate accommodating space 120 is further provided between the bottom accommodating space 130 and the end accommodating space 110, and the intermediate accommodating space 120 is used to accommodate intermediate reagents, and the intermediate reagents are used for intermediate processing of samples; when only one intermediate accommodating space 120 is provided, a first partition 121 is provided between the intermediate accommodating space 120 and the end accommodating space 110; a second partition 131 is provided between the intermediate accommodating space 120 and the bottom accommodating space 130; in the first state, the integrated reagent kit is placed longitudinally; the bottom accommodating space 130, the intermediate accommodating space 120 and the end accommodating space 110 each independently store reagents for sample processing; in the second state, the integrated reagent kit is placed longitudinally, and the external sample breaks through the sealing film, the first partition 121 and the second partition 131 in turn, so that the sample enters the end accommodating space 110, the intermediate accommodating space 120 and the bottom accommodating space 130 in turn to complete the initial processing, intermediate secondary processing and final processing of the sample in turn. Figure 1 The middle number 160 is an upper cover covering the bottom accommodating space 130, the middle accommodating space 120 and the end accommodating space 110. The decomposition in the figure is to show the relationship between the various spaces, and it should not be understood that the various spaces are interconnected. Each space is an independent space during the storage of reagents. Only when the sample mixture is prepared, the reagents or liquids stored in each area are mixed by bringing in the sampling device. That is, the bottom accommodating space 130, the middle accommodating space 120 and the end accommodating space will only be interconnected when needed during the preparation process.

[0051] In some embodiments not shown in the drawings, the reagent storage and mixed solution preparation area may only include the bottom accommodating space 130 and the end accommodating space 110; the paving area 200 and the bottom accommodating space 130 are connected; and a partition is provided between the bottom accommodating space 130 and the end accommodating space 110. In the first state, the bottom accommodating space 130 is empty; in the second state, the end accommodating space 110 is used to receive samples input from the outside and process the samples in the end accommodating space 110; in the third state, the processed sample mixed solution enters the bottom accommodating space 130 from the end accommodating space 110, and the processed sample mixed solution enters the paving area 200 from the bottom accommodating space 130.

[0052] In some embodiments not shown in the accompanying drawings, when multiple intermediate accommodating spaces are provided between the end accommodating space and the bottom accommodating space, the multiple intermediate accommodating spaces are adjacent to each other, and the multiple intermediate accommodating spaces are provided as a whole between the end accommodating space and the bottom accommodating space, and the multiple intermediate accommodating spaces are used to store multiple different processing reagents; a first partition is provided between the end accommodating space and the intermediate accommodating space, a middle partition is provided between each intermediate accommodating space, and a second partition is provided between the intermediate accommodating space and the bottom accommodating space; in a first state, the integrated reagent kit is placed longitudinally; the bottom accommodating space, the multiple intermediate accommodating spaces and the end accommodating space each independently store reagents for sample processing; in a second state, the integrated reagent kit is placed longitudinally, and the external sample breaks through the sealing film, the first partition, the middle partition and the second partition in turn, so that the sample completes the initial processing, multiple intermediate processing and the final processing of the sample in turn when entering the end accommodating space, the multiple intermediate accommodating spaces and the bottom accommodating space in turn. In the first state, the bottom accommodating space and the end accommodating space each independently store reagents for sample processing; the bottom accommodating space is used to accommodate final test reagents, which are used for the final processing of the sample; the end accommodating space is used to accommodate initial reagents, which are used for the first processing of the sample; in the second state, the end accommodating space is used to receive samples input from the outside and perform the first processing on the samples in the end accommodating space; the sample mixture after the first processing enters the bottom accommodating space from the end accommodating space for the second processing; in the third state, the sample mixture after the second processing enters the paving area from the bottom accommodating space.

[0053] The multiple intermediate accommodating spaces enable the integrated test kit to be applicable to scenarios where multiple reagents need to be used for processing in steps, and the integrated test kit can be applicable to more complex applications.

[0054] like Figure 1 In the embodiment of the integrated reagent kit shown, a sealing film 111 is provided on one side of the reagent storage and mixed liquid preparation area; the sealing film 111 is fixed to the outer wall of the reagent storage and mixed liquid preparation area by a sealing film fixing ring 115; the sealing film 111 is used to seal the reagent storage and mixed liquid preparation area; in the second state, the external sampling device 300 breaks through the sealing film 111, allowing the sample to be processed to enter the reagent storage and mixed liquid preparation area for sample processing.

[0055] like Figure 1In the embodiment of the integrated reagent kit shown, the tiling area 200 includes a carrier body 210; a sinking space is provided on the carrier body 210, the sinking space is used to accommodate the sample mixture, and an observation area is provided in the sinking space; in the third state, the treated sample mixture flows from the reagent storage and mixture preparation area 100 into the observation area in the sinking space; the observation area is used to lay out the sample mixture to be observed, forming an observation field for microscopic examination. The carrier body 210 and the reagent storage and mixture preparation area 100 are fixed on the same mounting surface, i.e., the base 900; in the third state, the integrated reagent kit is placed horizontally; after being placed horizontally, the reagent or the sample mixture after multiple treatments automatically flows into the tiling area 200 under the action of gravity.

[0056] like Figures 2 to 7 In the embodiment of the integrated reagent kit shown, a sampling device 300 is also included. The sampling device 300 includes a sampling device body 320 and a sampling nozzle 310. The sampling nozzle 310 is arranged at one end of the sampling device body 320, and the sampling nozzle 310 is used to load an external sample into the sampling device 300. The sampling nozzle 310 is also used to break through the sealing film 111 and send the sample obtained by sampling into the reagent storage and mixed liquid preparation area 100. The sampling nozzle 310 also sends the sample obtained by sampling into the end accommodating space 110, the middle accommodating space 120 and the bottom accommodating space 130; a first positioning ring 321, a second positioning ring 322 and a third positioning ring 323 are arranged on the outer wall of the sampling device body 320; in the second state, when the external sampling device 300 breaks through the sealing film 111, the first positioning ring 321 is used to position the sampling device body 320 to enter the depth of the end accommodating space 110, and at the same time, the first positioning ring 321 is used to replace the function of the sealing film 111 to isolate the end accommodating space 110 from the outside world; when the external sampling device 300 breaks through the sealing film 111, the first positioning ring 321 is used to position the sampling device body 320 to enter the end accommodating space 110. When the external sampling device 300 breaks through the first partition 121, the second positioning ring 322 is used to position the sampling device body 320 to enter the depth of the middle accommodating space 120, and the second positioning ring 323 is used to replace the sealing film 111 to isolate the reagent storage and mixed liquid preparation area 100 from the outside; when the external sampling device 300 breaks through the second partition 131, the third positioning ring 323 is used to position the sampling device body 320 to enter the depth of the bottom accommodating space 130, and the third positioning ring 323 is used to replace the sealing film 111 to isolate the reagent storage and mixed liquid preparation area 100 from the outside.

[0057] like Figures 2 to 7In the embodiment of the integrated test kit shown, the sampling device 300 also includes a handle 325 and a limiting ring 324. The handle 325 is arranged at the end of the sampling device body 320 where the sampling nozzle 310 is not arranged; the handle is used to push or rotate the sampling device body 320; the limiting ring 324 is arranged between the handle 325 and the third positioning ring 323, and is used to limit the depth of the sampling device 300 as a whole entering the reagent storage and mixed liquid preparation area 100.

[0058] like Figures 2 to 7 In the embodiment of the integrated test kit shown, a sealing ring groove 180 is further provided on the inner wall close to the sealing film on one side of the end accommodating space 110, and a sealing ring 150 is embedded in the sealing ring groove 180. When the sampling device is pushed inward in sequence, any one of the first positioning ring 321, the second positioning ring 322 or the third positioning ring 323 and the end accommodating space 110 are sealed by the sealing ring 150.

[0059] In some embodiments not shown in the accompanying drawings, the sampling device is a quantitative sampling device, and the capacity range of quantitative sampling is 5ul (microliter) to 15ul. The capacity range of quantitative sampling can also be 7.5ul to 10ul. The capacity range of the end accommodating space 110 is 50ul to 3000ul. The capacity range of the bottom accommodating space 130 is 50ul to 1000ul. The capacity range of the middle accommodating space 120 is 50ul to 1000ul. The size of the three spaces of the end accommodating space 110, the middle accommodating space 120 and the bottom accommodating space 130 can be reasonably set according to the capacity of the reagents to be placed; the end accommodating space 110, the middle accommodating space 120 and the bottom accommodating space 130 can reserve a certain space and not be filled with reagents; of course, the end accommodating space 110, the middle accommodating space 120 and the bottom accommodating space 130 can also be filled with reagents; the size of each space will be greater than or equal to the volume of the reagent that must be accommodated. The volumes of the reagents contained in the end containing space 110 , the middle containing space 120 , and the bottom containing space 130 match the sampling volume of the quantitative sampling device.

[0060] like Figure 1 In the embodiment of the integrated reagent kit shown, the sinking space is a space with a square cross section; a cover sheet 290 is arranged above the sinking space; the shape and size of the cover sheet correspond to the shape and size of the sinking space, and can be embedded and covered on the sinking space. The cover sheet 290 is embedded above the sinking space, so that the sinking space 230 covered by the cover sheet 290 forms a sealed space for containing and spreading the sample mixture to be observed.

[0061] In an embodiment of a secondary staining method not shown in some drawings, it is based on the above-mentioned integrated reagent kit; the initial reagent in the end accommodating space is the first staining reagent used for the first sample staining treatment; the final reagent in the bottom accommodating space is the second staining reagent used for the second sample staining treatment; the cell secondary staining method includes the following steps: Step 1: Use a sampling device to perform quantitative sampling of the sample to be stained; Step 2: Use the sampling device in step one to send a quantitative sample to be stained into the end accommodating space for the first staining, and in the second state of the integrated reagent kit, the sample and the first staining reagent are mixed evenly, that is, the first staining is completed; Step 3: Use the sampling device in step 2 to send the mixed liquid after the first staining treatment into the bottom accommodating space, and in the second state of the integrated reagent kit, the mixed liquid after the first staining treatment is mixed evenly with the second staining reagent in the bottom accommodating space, that is, the second staining is completed.

[0062] In an embodiment of a secondary dyeing method not shown in some drawings, it is based on Figure 1 or Figure 7 The integrated reagent kit shown; the initial reagent in the end accommodating space 110 is a diluent for the first sample dilution treatment; the intermediate reagent in the middle accommodating space 120 is a first staining reagent for the first sample staining treatment; the final reagent in the bottom accommodating space 130 is a second staining reagent for the second sample staining treatment; the cell secondary staining method comprises the following steps: step one: using the sampling device 300 to perform quantitative sampling of the sample to be stained; step two: using the sampling device in step one, the quantitative sample to be stained is sent into the end accommodating space for dilution treatment; in the second state of the integrated reagent kit, the sample and the diluent are mixed evenly, that is, the dilution step is completed; step three: using the sampling device in step two, the diluted sample to be stained is sent into the middle accommodating space for the first staining, and in the second state of the integrated reagent kit, the diluted sample to be stained and the first staining reagent are mixed evenly, that is, the first staining is completed; step four: using the sampling device in step three, the mixed solution after the first staining treatment is sent into the bottom accommodating space for the second staining, and in the second state of the integrated reagent kit, the mixed solution after the first staining treatment and the second staining reagent are mixed evenly, that is, the second staining is completed.

[0063] In the present application, the cells in the sample to be stained complete the staining process in a liquid suspension, and the environment used for the staining solution and the diluent is relatively close to the physiological state of the cells. Therefore, during the staining process, the cell activity is better maintained. On the one hand, the Brownian motion of the cells and the dye molecules in the solution, and the electrostatic force can be utilized to carry out the staining reaction, which makes the staining speed faster and more efficient.

[0064] In the present application, 1. The ingenious integrated reagent kit structure design integrates the three functions of reagent storage, sample processing and sample tiling at the same time, and the operations of each step are concentrated in one reagent kit, with a high degree of integration and high staining efficiency; 2. The ingenious structural design enables the stained sample to flow quickly to the tiling area with the help of gravity; 3. The quantitative sampling device and the fixed-volume reagent storage space eliminate the weighing process and improve the process efficiency; the ratio between the sample staining reagent and the sample is reasonably set, and the ratio of various reagents can be completed by setting the fixed-volume reagent storage space, which is simple and efficient; 4. The reagent storage space arranged in sequence in a certain order can very conveniently use the sampling device to perform a multi-step sample processing process step by step, thereby improving efficiency.

[0065] The kit and secondary staining method provided by the present application are simple to operate, have low requirements on the operator, are uniform in staining, and are short in time, and can realize a fool-proof staining process; they avoid the shortcomings of the prior art in that the cell staining operation is cumbersome, has high professional requirements, is time-consuming, and is prone to uneven staining. The staining operation is simple and can realize a fool-proof staining operation, making it suitable for a variety of application scenarios. Scenarios such as instant testing are also suitable for various application scenarios such as first aid, bedside, battlefield, etc. that lack large-scale professional equipment, professionals, and complex testing environments.

[0066] The above descriptions are merely embodiments of the present application and are not intended to limit the patent scope of the present application. Any equivalent structure or equivalent process transformation made using the contents of the invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A secondary staining method for secondary staining of cells in a biological sample, characterized in that: It is based on an all-in-one kit; The integrated test kit includes a reagent storage and mixed solution preparation area and a tiling area; The paving area is used for holding and paving the sample mixture to be observed in microscopic examination; Reagent storage and mixed solution preparation area, used for storage of sample processing reagents and sample processing; In the first state, the reagent storage and mixed solution preparation area stores reagents used for sample processing; In the second state, the reagent storage and mixed solution preparation area is used to receive samples input from the outside and process the samples in the reagent storage and mixed solution preparation area; In the third state, the processed sample mixture enters the paving area from the reagent storage and mixture preparation area and is paved; The reagent storage and mixed solution preparation area includes a bottom accommodating space and an end accommodating space; The paving area and the bottom accommodating space are connected; A partition is provided between the bottom accommodating space and the end accommodating space; At least one intermediate accommodating space is also provided between the bottom accommodating space and the end accommodating space, and the intermediate accommodating space is used to accommodate an intermediate reagent, and the intermediate reagent is used for intermediate sample processing; A first partition is provided between the middle accommodating space and the end accommodating space; a second partition is provided between the middle accommodating space and the bottom accommodating space; In the first state, the integrated reagent box is placed vertically, and the bottom accommodating space, the middle accommodating space and the end accommodating space each independently stores reagents for sample processing; In the second state, the external sample breaks through the sealing film, the first partition and the second partition in sequence, so that the sample enters the end accommodating space, the middle accommodating space and the bottom accommodating space in sequence to complete the initial processing, the intermediate processing and the final processing of the sample in sequence; Also included is a sampling device, which includes a sampling device body and a sampling nozzle; The sampling nozzle is arranged at one end of the sampling device body, and the sampling nozzle is used to load the external sample into the sampling device; The sampling nozzle also delivers the sample obtained by sampling into the end accommodating space, the middle accommodating space and the bottom accommodating space; A first positioning ring, a second positioning ring and a third positioning ring are arranged on the outer wall of the sampling device body; In the second state, when the external sampling device breaks through the sealing film, the first positioning ring is used to position the depth of the sampling device body entering the end accommodation space, and the first positioning ring is used to replace the sealing film to isolate the end accommodation space from the outside; When the external sampling device breaks through the first partition, the second positioning ring is used to position the depth of the sampling device body entering the middle accommodating space, and the second positioning ring is used to replace the sealing film to isolate the end accommodating space from the outside; When the external sampling device breaks through the second partition, the third positioning ring is used to position the depth of the sampling device body entering the bottom accommodation space, and the third positioning ring is used to replace the sealing film to isolate the end accommodation space from the outside world; The initial reagent in the end accommodating space is a diluent used for the first sample dilution process; The intermediate reagent in the intermediate accommodating space is a first staining reagent used for staining the first sample; The final test reagent in the bottom accommodating space is a second staining reagent used for the second sample staining process.

2. The secondary dyeing method according to claim 1, characterized in that: A sealing film is provided on one side of the reagent storage and mixed solution preparation area; the sealing film is used to seal the reagent storage and mixed solution preparation area; In the second state, the external sampling device breaks through the sealing film, allowing the sample to be processed to enter the reagent storage and mixed liquid preparation area for sample processing.

3. The secondary dyeing method according to claim 2, characterized in that: Also included is a sampling device, which includes a sampling device body and a sampling nozzle; The sampling nozzle is arranged at one end of the sampling device body, and the sampling nozzle is used to load the external sample into the sampling device; The sampling nozzle is also used to break through the sealing film and send the external samples obtained by sampling into the reagent storage and mixed liquid preparation area.

4. The secondary dyeing method according to claim 3, characterized in that: A positioning ring is provided on the outer wall of the sampling device body; In the second state, when the sampling device breaks through the sealing film, the positioning ring is used to locate the depth of the sampling device body entering the end accommodating space. At the same time, the positioning ring is used to replace the sealing film to isolate the end accommodating space from the outside.

5. The secondary dyeing method according to claim 1, characterized in that: In the first state, the bottom accommodating space is empty, and the end accommodating space is used to accommodate sample processing reagents; In the second state, the end accommodating space is used to receive a sample input from the outside and process the sample in the end accommodating space; In the third state, the processed sample mixture enters the bottom accommodating space from the end accommodating space, and enters the flattening area through the bottom accommodating space.

6. The secondary dyeing method according to claim 1, characterized in that: In the first state, the bottom accommodating space and the end accommodating space each independently store reagents for sample processing; the end accommodating space is used to accommodate initial reagents, which are used for the first sample processing; The bottom accommodation space is used to accommodate the final test reagent, which is used for the final treatment of the sample; In the second state, the end accommodating space is used to receive a sample input from the outside and perform a first process on the sample in the end accommodating space; The sample mixture after the first treatment enters the bottom accommodation space from the end accommodation space for the final treatment of the sample; In the third state, the sample mixture after the last sample processing enters the flattening area from the bottom accommodating space.

7. The secondary dyeing method according to claim 1, characterized in that: A sealing ring groove is also provided on the inner wall close to the sealing film on one side of the end accommodating space, and a sealing ring is embedded in the sealing ring groove. When the sampling device is pushed inward in sequence, any one of the first positioning ring, the second positioning ring or the third positioning ring and the end accommodating space are sealed by the sealing ring.

8. The secondary dyeing method according to claim 1, characterized in that: The sampling device is a quantitative sampling device, and the capacity range of quantitative sampling is 5ul to 15ul.

9. The secondary dyeing method according to claim 8, characterized in that: The capacity range of the end accommodation space is 50ul to 3000ul.

10. The secondary dyeing method according to claim 8, characterized in that: The capacity range of the bottom accommodation space is 50ul to 1000ul.

11. The secondary dyeing method according to claim 8, characterized in that: The capacity range of the middle accommodating space is 50ul~1000ul.

12. The secondary dyeing method according to claim 1, characterized in that: A plurality of intermediate accommodating spaces are also provided between the bottom accommodating space and the end accommodating space; A plurality of intermediate accommodating spaces are arranged adjacent to each other, and the plurality of intermediate accommodating spaces are arranged as a whole between the end accommodating space and the bottom accommodating space, and the plurality of intermediate accommodating spaces are used to store a plurality of different treatment reagents; A first partition is provided between the end accommodating space and the middle accommodating space, a middle partition is provided between each middle accommodating space, and a last partition is provided between the middle accommodating space and the bottom accommodating space; In the first state, the integrated reagent box is placed vertically; the bottom accommodating space, the plurality of middle accommodating spaces and the end accommodating space each independently stores reagents for sample processing; In the second state, the integrated test kit is placed vertically, and the external samples break through the sealing film, the first partition, the middle partition and the last partition in sequence, so that the samples enter the end accommodating space, multiple intermediate accommodating spaces and the bottom accommodating space in sequence to complete the initial processing, multiple intermediate processing and the final processing of the samples.

13. The secondary dyeing method according to claim 12, characterized in that: Also included is a sampling device, which includes a sampling device body and a sampling nozzle; The sampling nozzle is arranged at one end of the sampling device body, and the sampling nozzle is used to load the external sample into the sampling device; The sampling nozzle also delivers the sample obtained by sampling into the end accommodating space, a plurality of middle accommodating spaces and the bottom accommodating space in sequence; A first positioning ring, a plurality of intermediate positioning rings and a final positioning ring are arranged on the outer wall of the sampling device body; In the second state, when the external sampling device breaks through the sealing film, the first positioning ring is used to position the depth of the sampling device body entering the end accommodation space, and the first positioning ring is used to replace the sealing film to isolate the end accommodation space from the outside; When the external sampling device breaks through the first partition, the middle positioning ring is used to position the depth of the sampling device body entering the middle accommodation space, and the middle positioning ring is used to replace the sealing film to isolate the end accommodation space from the outside world; When the external sampling device breaks through the last partition, the last positioning ring is used to locate the depth of the sampling device body entering the bottom accommodating space. At the same time, the last positioning ring is used to replace the sealing film to isolate the end accommodating space from the outside world.

14. The secondary dyeing method according to claim 13, characterized in that: A sealing ring groove is also provided on the inner wall close to the sealing film on one side of the end accommodating space, and a sealing ring is embedded in the sealing ring groove. When the sampling device is pushed inward in sequence, any one of the first positioning ring, the middle positioning ring or the last positioning ring and the end accommodating space are sealed by the sealing ring.

15. The secondary dyeing method according to claim 13, characterized in that: The sampling device is a quantitative sampling device; the capacity of the end accommodating space, the capacity of the middle accommodating space and the capacity of the bottom accommodating space match the capacity of the quantitative sampling device.

16. The secondary dyeing method according to claim 1, characterized in that: The cell secondary staining method comprises the following steps: Step 1: Use a sampling device to quantitatively sample the sample to be stained; Step 2: Using the sampling device in step 1, a quantitative amount of the sample to be stained is sent into the end receiving space for dilution treatment; in the second state of the integrated reagent kit, the sample and the diluent are mixed evenly, that is, the dilution step is completed to obtain a diluted sample solution; Step 3: Using the sampling device in step 2, the diluted sample solution is sent into the middle accommodation space for the first staining. In the second state of the integrated reagent kit, the diluted sample solution and the first staining reagent are mixed evenly, that is, the first staining is completed to obtain a mixed solution after the first staining treatment; Step 4: Use the sampling device in step 3 to send the mixed liquid after the first dyeing treatment into the bottom accommodating space for second dyeing. In the second state of the integrated reagent kit, the mixed liquid after the first dyeing treatment and the second dyeing reagent are evenly mixed to complete the second dyeing.

17. A secondary staining method for secondary staining of cells in a biological sample, characterized in that: It is based on an all-in-one kit; The integrated test kit includes a reagent storage and mixed solution preparation area and a tiling area; The paving area is used for holding and paving the sample mixture to be observed in microscopic examination; Reagent storage and mixed solution preparation area, used for storage of sample processing reagents and sample processing; In the first state, the reagent storage and mixed solution preparation area stores reagents used for sample processing; In the second state, the reagent storage and mixed solution preparation area is used to receive samples input from the outside and process the samples in the reagent storage and mixed solution preparation area; In the third state, the processed sample mixture enters the paving area from the reagent storage and mixture preparation area and is paved; The reagent storage and mixed solution preparation area includes a bottom accommodating space and an end accommodating space; The paving area and the bottom accommodating space are connected; A partition is provided between the bottom accommodating space and the end accommodating space; In the first state, the bottom accommodating space and the end accommodating space each independently store reagents for sample processing; the end accommodating space is used to accommodate initial reagents, which are used for the first sample processing; The bottom accommodation space is used to accommodate the final test reagent, which is used for the final treatment of the sample; In the second state, the end accommodating space is used to receive a sample input from the outside and perform a first process on the sample in the end accommodating space; The sample mixture after the first treatment enters the bottom accommodation space from the end accommodation space for the final treatment of the sample; In the third state, the sample mixture after the last sample treatment enters the paving area from the bottom accommodating space; The initial reagent in the end accommodating space is a first staining reagent used for the first sample staining process; The final test reagent in the bottom accommodating space is a second staining reagent used for the second sample staining process; The cell secondary staining method comprises the following steps: Step 1: Use a sampling device to quantitatively sample the sample to be stained; Step 2: using the sampling device in step 1, a quantitative amount of the sample to be stained is sent into the end accommodating space for the first staining, and in the second state of the integrated reagent box, the sample and the first staining reagent are mixed evenly, that is, the first staining is completed; Step three: Use the sampling device in step two to send the mixed liquid after the first dyeing treatment into the bottom accommodating space. In the second state of the integrated reagent kit, the mixed liquid after the first dyeing treatment is evenly mixed with the second dyeing reagent in the bottom accommodating space to complete the second dyeing.

Images