CN110596102A - Fluid control device and method for enriching cells and coloring cells - Google Patents

Abstract

The invention discloses a fluid control device and method for enriching cells and coloring cells. The device includes a filter chamber, a turntable, a sample chamber, a prefilled liquid chamber and a waste liquid chamber. The filter chamber is detachably installed on the turntable, and the sample The chamber and multiple pre-filled liquid chambers are evenly installed in the circumferential direction of the filter chamber, and the waste liquid chamber is installed at the bottom of the turntable. In the method of the present invention, a pressure difference is generated by pushing and pulling the piston, and the filter cavity is rotated to form a channel to flow the fluid into the filter cavity and perform filtration. After filtration, the cells are enriched on the microporous filter membrane, and the cells on the microporous filter membrane are colored. . The present invention adds liquid quantitatively during the coloring process, which reduces randomness and improves repeatability, and can also prevent operators from repeatedly contacting samples and causing risks. The invention relates to the technical field of medical sample processing.

Description

Fluid control device and method for enriching cells and coloring cells

technical field

The invention relates to the technical field of medical sample processing, in particular to a fluid control device and method for enriching and coloring cells.

Background technique

Making slices of prokaryotic or eukaryotic cells in body fluid samples and observing them under a microscope is a common method for diagnosing diseases in medical tests, and is also an important means in biological research. The cells in the sample are enriched, stained and other processed. Traditional cell enrichment methods include centrifugal enrichment method, biochemical capture method, membrane filtration enrichment method, etc. Although centrifugation method can enrich cells, the volume, shape and weight of cells vary, making some cells Discarded; biochemical capture methods usually use biomolecules or chemical molecules that can bind to cell surface molecules such as antibodies, ligands, polar or non-polar chemical molecules attached to a solid phase, and then capture cells by affinity to achieve enrichment However, the capture efficiency is affected by intermolecular affinity, molecular dosage, repeated cleaning, non-specific adsorption, etc., so losses will also occur; the membrane filtration enrichment method is to filter through a filter membrane with a smaller pore size than the cells to be captured, and the cells are completely Retained by the filter membrane on the surface of the membrane, it is an ideal enrichment method, but due to the difficulty in controlling the filter pressure, some cells are tightly adhered to the membrane and cannot be eluted, and some cells will also be lost.

Staining the cells allows medical staff and researchers to obtain the test results of the required cells from the microscopic examination. Commonly used staining methods for cells in body fluid samples include bacterial dye staining, fluorescent antibody combined with cell staining, etc., but the commonly used staining methods It is greatly affected by human factors; the staining process requires repeated washing of the slides, and some cells that are not firmly fixed will be washed away; the dripping of coloring solution and elution time depend on the experience of the operator, which is random and cannot be quantified. Difficult to repeat; open handling exposes operators to hazards.

Contents of the invention

The purpose of the present invention is to overcome the defects and deficiencies of the prior art, and provide a fluid control device for enriching cells in a sample and quantitatively staining the cells.

Another object of the present invention is to provide a cell staining method capable of standardizing the staining steps, the amount of staining solution, and the staining time.

The purpose of the present invention can be achieved through the following technical solutions: a fluid control device for enriching cells and coloring cells, including a filter chamber, a turntable, a sample chamber, a pre-filled liquid chamber and a waste liquid chamber, and the filter chamber is detachably installed on the turntable Above, the sample chamber and multiple prefilled liquid chambers are evenly installed in the circumferential direction of the filter cavity, and the waste liquid chamber is installed at the bottom of the turntable. The filter chamber is a hollow cylindrical structure, with a filter chamber rotating handle on the top, two symmetrical protrusions on the outer wall, a through filter chamber hole on the filter chamber wall, and a fixed filter chamber bottom. There is a microporous filter membrane, and the filter cavity is equipped with a piston push-pull rod that is suitable for the size of the cavity; the turntable is a hollow cylindrical structure, and the inner wall is provided with two symmetrical concave positions that are compatible with the convex positions; in the turntable The wall surface is provided with a turntable channel corresponding to the hole of the filter cavity, and the turntable channel runs through the top surface and the inner surface of the turntable; the sample chamber and the bottom of the pre-filled liquid chamber are provided with holes, and the opening position is on the same side as the turntable channel. The openings on the top surface are corresponding; the top of the waste liquid chamber is provided with a mesh structure.

As a preferred technical solution, the filter cavity body hole is arranged on one of the convex positions, and the turntable hole is arranged on a concave position corresponding to the convex position. Setting the filter cavity on the convex position can reduce the usage of the silica gel layer and reduce the cost.

As a preferred technical solution, a silica gel layer is provided at the connection between the filter cavity and the turntable, the connection between the turntable and the sample chamber, and the prefilled liquid chamber. The silicone layer can guarantee the airtightness inside the device.

As a preferred technical solution, sand chips are arranged on the mesh structure. When the waste liquid chamber is connected with the turntable, the sand chip is against the microporous membrane, which can better support the microporous membrane.

As a preferred technical solution, the microporous filter membrane is fixed between the silica gel sealing ring and the membrane-loading ring, and the silica gel sealing ring is connected to the bottom of the filter cavity. The microporous membrane is a track-etched polycarbonate membrane (PCTE membrane) or a track-etched polyethylene terephthalate membrane (PETTE membrane). This kind of microporous filter membrane is punched for the acceleration of heavy ions. The micropores pass through the membrane vertically. The pore size of each hole is fixed, which can ensure that all cells larger than the pore size are retained.

As a preferred technical solution, a sample injection port is provided on the top of the sample chamber, and a pre-filled liquid port is provided on the top of the pre-filled liquid chamber. A sealing film is provided on the sample loading port and the pre-loading liquid port, and a position mark is provided on the sealing film. marked.

Another object of the present invention is achieved through the following technical scheme: the method for enriching cells and coloring cells comprises the following steps: aligning the turntable channel with the hole in the sample chamber, opening the sealing film of the sample port, adding the sample to the sample chamber, Pull the piston push-pull rod upward, the sample flows into the filter chamber, rotate the filter chamber to the blank position, make the turntable hole and the sample chamber hole stagger, press down the piston push-pull rod, filter the sample; rotate the filter chamber to align the turntable hole with the first There are two pre-filled liquid chamber holes, pull the piston push-pull rod upward, the first pre-filled liquid flows into the filter cavity, rotate the filter cavity to the blank position, make the turntable hole and the bottom hole of the first pre-filled liquid chamber stagger, press down The piston pushes and pulls the rod to filter the first prefilled liquid; repeat to filter other prefilled liquids, and after all the prefilled liquids are filtered out, the coloring process is completed.

As a preferred technical solution, after the coloring is completed, the filter chamber is taken out, and the microporous filter membrane can be taken out for microscopic examination. Samples were processed blood, sputum, and spinal fluid.

The working principle of the fluid control device of the present invention is as follows: collect body fluid samples and add corresponding sample treatment liquid to decompose impurities or non-target cells in the samples for easy filtration; generate positive pressure to filter the samples through the action of the piston, and all target cells in the samples are eliminated. Retain on the top of the filter membrane; then add the coloring solution corresponding to the target cells to color, decolorize, and wash the cells on the filter membrane. After each step is completed, positive pressure is generated by the piston to drain the liquid. The cells were all stained; the filter was taken out for direct microscope observation and counting.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

1. The fluid control device of the present invention uses the movement of the piston as the driving force, and the pushing and pulling of the piston can control the inflow and filtration of the fluid; the microporous membrane is used to enrich the cells in the sample and color them in situ. The dosage of each component of liquid and coloring liquid is standardized to minimize the influence factors on the results, and the fully enclosed control has the advantages of safety, automation, easy operation and high detection rate.

2. The method of enriching cells and coloring cells of the present invention can enrich the target cells on a piece of filter membrane. During the coloring process, the cells are retained by the filter membrane and will not be washed away and lost, which can effectively improve the enrichment of cells. concentration and diagnostic sensitivity.

Description of drawings

Figure 1 is a front sectional view of a fluid control device in an embodiment of the present invention;

Fig. 2 is A-A sectional view of Fig. 1;

Fig. 3 is the B-B sectional view of Fig. 1;

Fig. 4 is a top view of the fluid control device in the embodiment of the present invention.

Among them: 1: Filter chamber, 2: Piston, 3: Push-pull rod, 4: Rotating handle of filter chamber, 5: Filter chamber hole, 6: Protrusion, 7: Silicone sealing ring, 8: Membrane ring, 9 : microporous filter membrane, 10: sand chip, 11: sample injection port, 12: sample chamber, 13: sample chamber hole, 14: pre-filled liquid port, 15: pre-filled liquid chamber, 15-1: pre-filled liquid 1 Chamber, 15-2: prefilled liquid chamber 2, 15-3: prefilled liquid chamber 3, 15-4: prefilled liquid chamber 4, 15-5: prefilled liquid chamber 5, 15-6: prefilled liquid chamber 6 Chamber, 15-7: prefilled liquid chamber 7, 15-8: prefilled liquid chamber 8, 15-9: prefilled liquid chamber 9, 16: prefilled liquid chamber hole, 17: turntable, 18: turntable channel, 19 : waste liquid chamber, 20: parafilm, 21: silica gel layer, 22: position mark.

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

A fluid control device for enriching and staining cells, including a filter chamber, a turntable, a sample chamber, a prefilled liquid chamber, and a waste liquid chamber.

The filter chamber is detachably installed on the turntable. The filter chamber is a hollow cylindrical structure with a filter chamber rotating handle on the top. There are two symmetrical strip protrusions on the outer wall surface, and the strip protrusions are pasted on the outer wall surface. One of the protruding positions is provided with a through filter cavity hole. The bottom of the filter chamber is fixed with a microporous filter membrane, the microporous filter membrane is fixed between the silica gel sealing ring and the film loading ring, and the silica gel seal ring is connected with the bottom of the filter chamber. The microporous filter membrane is in the shape of a disc with a diameter of 13 mm, and the pore size is 0.2 μm-8 μm. The microporous filter membrane can be track-etched polycarbonate membrane (PCTE membrane) or track-etched polyethylene terephthalate membrane (PETTE membrane). This kind of microporous filter membrane is punched for the acceleration of heavy ions. The micropores pass through the membrane vertically. The pore size of each hole is fixed, which can ensure that all cells larger than the pore size are retained. The filter cavity is provided with a piston and a push-pull rod suitable for the size of the cavity. The bottom of the push-pull rod is fixed with a piston, and the push-pull rod can drive the piston to move up and down.

The turntable is a hollow cylindrical structure, and the inner wall is provided with two symmetrical recesses corresponding to the convex positions; one of the recesses is provided with a turntable channel corresponding to the hole of the filter cavity, and the turntable channel presents an L shape, and the channel runs through Turntable top and inner sides.

The fluid control device is provided with 1 sample chamber and 9 pre-filled liquid chambers, and the sample chambers and pre-filled liquid chambers are evenly installed in the circumferential direction of the filter cavity. Holes are provided at the bottom of the sample chamber and the prefilled liquid chamber, and the opening positions correspond to the openings of the turntable channels on the top surface of the turntable. The top of the sample chamber is provided with a sample injection port, and the top of the prefilled liquid chamber is provided with a prefilled liquid port. The sample injection port and the prefilled liquid port are covered with parafilm. In order to precisely control the addition of prefilled liquid, there is a position mark on the sealing film. The mark 0 means that the hole of the turntable is aligned with the hole of the sample chamber. At this time, the sample can flow into the filter cavity. The mark □ means the empty position. The holes are staggered. Mark 1 indicates that the turntable channel is aligned with the bottom hole of pre-filled liquid chamber 1, and the adjacent □ indicates a vacant position. At this time, the turntable channel and the pre-filled liquid chamber hole are staggered. The meanings of other marks can be deduced in the same way.

The waste liquid chamber is installed at the bottom of the turntable, and the top is provided with a mesh structure, and the filtrate can flow into the waste liquid chamber from the mesh structure. In order to better support the microporous membrane, sand chips are also supported on the mesh structure. When the waste liquid chamber is connected with the turntable, the sand chip is against the bottom of the microporous membrane to support the microporous membrane.

In order to ensure the tightness of the entire fluid control device, a silica gel layer is provided at the connection between the filter cavity and the turntable, the connection between the turntable and the sample chamber, and the connection between the turntable and the prefilled liquid chamber.

The method for enriching cells and coloring cells using the fluid control device of the present invention comprises the following steps:

(1) The initial position of the device is that the opening of the turntable hole is aligned with the hole of the sample chamber, that is, the rotation handle of the filter chamber is rotated to 0 position.

(2) Open the sealing film of the sampling port of the device, and add 1-2 mL of sample to the sample chamber.

(3) Pull the push-pull rod upwards, the sample fluid enters the filter chamber, rotate the filter chamber to the blank position (□ position), at this time, the opening of the turntable hole is staggered from the hole of the sample chamber, press the push-pull rod down, and the sample is filtered .

(4) After the filtration is completed, rotate the rotating handle of the filter chamber so that the opening of the turntable hole is aligned with the hole (1 position) of the pre-installed liquid chamber 1, and pull the push-pull rod upward, and the pre-installed components in the pre-filled liquid chamber 1 The fluid enters the filter cavity, rotate the filter cavity to the blank position (□ position), at this time the opening of the turntable hole is staggered with the hole of the pre-filled liquid chamber 1, and the push-pull rod 3 is pressed down, and the pre-filled liquid 1 fluid is filtered out .

(5) After filtering, rotate the filter chamber so that the opening of the turntable hole is aligned with the hole (2 position) of the pre-filled liquid chamber 2, pull the push-pull rod upward, and the pre-installed component fluid in the pre-filled liquid chamber 2 enters the filter chamber Rotate the filter chamber to the blank position (□ position), at this time the opening of the turntable channel is staggered from the hole in the pre-filled liquid 2 chamber, press the push-pull rod down, and the pre-filled liquid 2 is filtered out.

(6) Repeat step (5). After all the pre-installed components have been used up, the coloring process is completed. At this time, the filter chamber can be taken out, and the microporous filter membrane can be removed and placed on a glass slide for microscopy. check.

Embodiment one:

In this embodiment, the fluid control device of the present invention is used to perform Gram staining to color bacteria. The microporous filter membrane used in the fluid control device is a track-etched polyethylene terephthalate (PETTE) membrane with a pore size of 0.45 μm. The components of the pre-filled solution 1-9 are pre-filled with the components of the Gram staining solution. The following are ammonium oxalate crystal violet solution, ultrapure water, iodine solution, ultrapure water, 95% alcohol, ultrapure water, ultrapure water, saffron red solution, and ultrapure water.

1. Sample collection and processing

(1) The patient coughs up about 5mL of sputum and spits it into a sterile 50mL centrifuge tube.

(2) Add 5 mL of 4% NaOH solution, shake for 1 minute, and let stand for 15 minutes.

(3) Add 0.067mol/L phosphate buffered saline (PBS) to 50mL, and centrifuge at 3000xg for 20 minutes.

(4) Pour off the supernatant, add 2mL to resuspend the precipitate.

2. Use the fluid control device of the present invention to enrich bacteria and carry out Gram staining on bacteria

(1) The initial position of the device is that the opening of the turntable channel is aligned with the hole of the sample chamber (position 0).

(2) Open the sealing film of the sampling port of the device, and add 2 mL of the above-mentioned sample to the sample chamber.

(3) Pull up the push-pull rod, the sample enters the filter chamber, rotate the filter chamber to the blank position (□ position), press the push-pull rod down, and the sample is filtered.

(4) After the filtration is completed, rotate the rotating handle of the filter chamber so that the opening of the turntable hole is aligned with the hole (1 position) of the pre-filled liquid chamber 1, pull the push-pull rod upward, and the ammonium oxalate crystal violet pre-installed in the pre-filled liquid 1 chamber The solution enters the filter chamber, rotate the filter chamber to the blank position (□ position), wait for 1 minute, press the push-pull rod down, and the ammonium oxalate crystal violet solution is filtered out.

(5) After the filtration is completed, rotate the rotating handle of the filter chamber so that the opening of the turntable hole is aligned with the hole (2 position) of the pre-filled liquid chamber 2, pull the push-pull rod upward, and the pre-installed ultrapure water in the pre-filled liquid chamber 2 enters Filter chamber, rotate the filter chamber to the blank position (□ position), press the push-pull rod down, and filter out the ultrapure water.

(6) After filtering, rotate the rotating handle of the filter chamber so that the opening of the turntable hole is aligned with the hole (3 position) of the pre-filled liquid chamber 3, pull the push-pull rod upward, and the pre-installed iodine solution in the pre-filled liquid chamber 3 enters the filter Chamber, rotate the filter chamber to the blank position (□ position), wait for 1 minute, press the push-pull rod down, and filter the iodine solution.

(7) After the filtration is completed, rotate the rotating handle of the filter chamber so that the opening of the turntable hole is aligned with the hole (4 position) of the pre-filled liquid chamber 4, pull the push-pull rod upward, and the pre-installed ultrapure water in the pre-filled liquid chamber 4 enters Filter chamber, rotate the filter chamber to the blank position (□ position), press the push-pull rod down, and filter out the ultrapure water.

(8) After filtering, rotate the rotating handle of the filter chamber so that the opening of the turntable hole is aligned with the hole (5 position) of the pre-filled liquid chamber 5, pull the push-pull rod upward, and the pre-installed 95% alcohol in the pre-filled liquid chamber 5 enters Filter chamber, rotate the filter chamber to the blank position (□ position), wait for 20 seconds, press the push-pull rod down, 95% alcohol is filtered out.

(9) After the filtration is completed, rotate the rotating handle of the filter chamber so that the opening of the turntable hole is aligned with the hole (6 position) of the pre-filled liquid chamber 6, pull the push-pull rod upward, and the pre-installed ultrapure water in the pre-filled liquid chamber 6 enters Filter chamber, rotate the filter chamber to the blank position (□ position), press the push-pull rod down, and filter out the ultrapure water.

(10) After the filtration is completed, rotate the rotating handle of the filter chamber so that the opening of the turntable hole is aligned with the hole (7 position) of the pre-filled liquid chamber 7, pull the push-pull rod upward, and the pre-installed ultrapure water in the pre-filled liquid chamber 7 enters Filter chamber, rotate the filter chamber to the blank position (□ position), press the push-pull rod down, and filter out the ultrapure water.

(11) After the filtration is completed, rotate the rotating handle of the filter chamber so that the opening of the turntable hole is aligned with the hole (8 position) of the pre-filled liquid chamber 8, pull the push-pull rod upward, and the saffron solution pre-installed in the pre-filled liquid chamber 8 enters Filter chamber, rotate the filter chamber to the blank position (□ position), wait for 1 minute, press the push-pull rod down, and filter out the saffron solution.

(12) After the filtration is completed, rotate the rotating handle of the filter chamber so that the opening of the turntable hole is aligned with the hole (position 9) of the pre-filled liquid chamber 9, pull the push-pull rod upward, and the pre-installed ultrapure water in the pre-filled liquid chamber 9 enters Filter chamber, rotate the filter chamber to the blank position (□ position), press the push-pull rod down, and filter out the ultrapure water.

(13) At this point, after all the pre-installed components have been used up, the coloring process is completed. At this time, the filter cavity can be taken out, and the microporous filter membrane is taken off and placed on a glass slide for microscope inspection. Gram Gram-positive bacteria are purple and Gram-negative bacteria are red.

Embodiment two:

In this embodiment, the fluid control device of the present invention is used to carry out the acid-fast staining method to color the acid-fast bacilli. The filter membrane used in the fluid control device is a track-etched polyethylene terephthalate (PETTE) membrane with a pore size of 0.45 μm. The components of the acid-fast staining solution are pre-filled in chambers 1-9 of the pre-filled solution, followed by stone Fuchsin carbonate solution, ultrapure water, ultrapure water, acidic alcohol, ultrapure water, ultrapure water, methylene blue solution, ultrapure water, ultrapure water.

1. Sample collection and processing

(1) The patient coughs up about 5mL of sputum and spits it into a sterile 50mL centrifuge tube.

(2) Add 5 mL of 4% NaOH solution, shake for 1 minute, and let stand for 15 minutes.

(3) Add 0.067mol/L phosphate buffered saline (PBS) to 50mL, and centrifuge at 3000xg for 20 minutes.

(4) Pour off the supernatant, add 2mL to resuspend the precipitate.

2. Use the fluid control device of the present invention to enrich acid-fast bacilli and color them

(1) The initial position of the device is that the opening of the turntable channel is aligned with the hole of the sample chamber (position 0).

(2) Open the sealing film of the sampling port of the device, and add 2 mL of the above-mentioned sample to the sample chamber.

(3) Pull up the push-pull rod, the sample enters the filter chamber, rotate the filter chamber to the blank position (□ position), press the push-pull rod down, and the sample is filtered.

(4) After the filtration is completed, rotate the filter chamber so that the opening of the turntable hole is aligned with the hole (1 position) of the pre-filled liquid chamber 1, pull the push-pull rod upwards, and the carbolic acid fuchsin solution pre-installed in the pre-filled liquid 1 chamber enters Filter chamber, rotate the filter chamber to the blank position (□ position), wait for 10 minutes, push the push-pull rod down, and filter out the carbolate fuchsin solution.

(5) After the filtration is completed, rotate the filter chamber so that the opening of the turntable hole is aligned with the hole (2 position) of the pre-filled liquid chamber 2, pull the push-pull rod upward, and the pre-installed ultrapure water in the pre-filled liquid chamber 2 enters the filter chamber body, rotate the filter chamber to the blank position (□ position), press the push-pull rod down, and filter out the ultrapure water.

(6) After filtering, rotate the filter chamber so that the opening of the turntable hole is aligned with the hole (3 position) of the pre-filled liquid chamber 3, pull the push-pull rod upward, and the pre-installed ultra-pure water in the pre-filled liquid chamber 3 enters the filter chamber body, rotate the filter chamber to the blank position (□ position), press the push-pull rod down, and filter out the ultrapure water.

(7) After filtering, rotate the filter chamber so that the opening of the turntable hole is aligned with the hole (4 position) of the pre-filled liquid chamber 4, pull the push-pull rod upward, and the pre-installed acid alcohol in the pre-filled liquid chamber 4 enters the filter chamber , rotate the filter chamber to the blank position (□ position), wait for 2 minutes, press the push-pull rod down, and filter out the acidic alcohol.

(8) After the filtration is completed, rotate the filter chamber so that the opening of the turntable hole is aligned with the hole (5 position) of the pre-filled liquid chamber 5, pull the push-pull rod upward, and the pre-installed ultrapure water in the pre-filled liquid chamber 5 enters the filter chamber body, rotate the filter chamber to the blank position (□ position), press the push-pull rod down, and filter out the ultrapure water.

(9) After filtering, rotate the filter chamber so that the opening of the turntable hole is aligned with the hole (6 position) of the pre-filled liquid chamber 6, pull the push-pull rod upward, and the pre-installed ultrapure water in the pre-filled liquid chamber 6 enters the filter chamber body, rotate the filter chamber to the blank position (□ position), press the push-pull rod down, and filter out the ultrapure water.

(10) After the filtration is completed, rotate the filter chamber so that the opening of the turntable hole is aligned with the hole (7 position) of the pre-filled liquid chamber 7, pull the push-pull rod upward, and the pre-installed methylene blue solution in the pre-filled liquid chamber 7 enters the filter chamber , rotate the filter chamber to the blank position (□ position), wait for 30 seconds, press the push-pull rod down, and filter out the methylene blue solution.

(11) After the filtration is completed, rotate the filter chamber so that the opening of the turntable hole is aligned with the hole (8 positions) of the pre-filled liquid chamber 8, pull the push-pull rod upward, and the pre-installed ultrapure water in the pre-filled liquid chamber 8 enters the filter chamber body, rotate the filter chamber to the blank position (□ position), press the push-pull rod down, and filter out the ultrapure water.

(12) After the filtration is completed, rotate the filter chamber so that the opening of the turntable hole is aligned with the hole (9 position) of the pre-filled liquid chamber 9, pull the push-pull rod upward, and the pre-installed ultrapure water in the pre-filled liquid chamber 9 enters the filter chamber body, rotate the filter chamber to the blank position (□ position), press the push-pull rod down, and filter out the ultrapure water.

(13) At this point, after all the pre-installed components have been used up, the coloring process is completed. At this time, the filter chamber can be taken out, and the microporous filter membrane can be removed and placed on a glass slide for microscope inspection. Anti-acid The bacillus is a red rod-shaped thallus, and the miscellaneous bacteria is a blue thallus.

Embodiment three:

In this embodiment, the fluid control device of the present invention is used to perform auramine O fluorescent staining to make acid-fast bacilli stain with fluorescence. The filter membrane used in the fluid control device is a track-etched polyethylene terephthalate (PETTE) membrane with a pore size of 0.45 μm. The components of the acid-fast staining solution are pre-filled in chambers 1-9 of the pre-filled solution, followed by gold Amine O dye solution, ultrapure water, ultrapure water, acid alcohol, ultrapure water, ultrapure water, potassium permanganate solution, ultrapure water, ultrapure water.

1. Sample collection and processing

(1) The patient coughs up about 5mL of sputum and spits it into a sterile 50mL centrifuge tube.

(2) Add 5 mL of 4% NaOH solution, shake for 1 minute, and let stand for 15 minutes.

(3) Add 0.067mol/L phosphate buffered saline (PBS) to 50mL, and centrifuge at 3000xg for 20 minutes.

(4) Pour off the supernatant, add 2mL to resuspend the precipitate.

2. Use the fluid control device of the present invention to enrich acid-fast bacilli and color them

(1) The initial position of the device is that the opening of the turntable channel is aligned with the hole of the sample chamber (position 0).

(2) Open the sealing film of the sampling port of the device, and add 2 mL of the above-mentioned sample to the sample chamber.

(3) Pull up the push-pull rod, the sample enters the filter chamber, rotate the filter chamber to the blank position (□ position), press the push-pull rod down, and the sample is filtered.

(4) After the filtration is completed, rotate the filter chamber so that the opening of the turntable hole is aligned with the hole (1 position) of the pre-filled liquid chamber 1, pull the push-pull rod upward, and the pre-installed auramine O dye solution in the pre-filled liquid chamber 1 enters Filter chamber, rotate the filter chamber to the blank position (□ position), wait for 10 minutes, press the push-pull rod down, and filter out the auramine O dye solution.

(5) After the filtration is completed, rotate the filter chamber so that the opening of the turntable hole is aligned with the hole (2 position) of the pre-filled liquid chamber 2, pull the push-pull rod upward, and the pre-installed ultrapure water in the pre-filled liquid chamber 2 enters the filter chamber body, rotate the filter chamber to the blank position (□ position), press the push-pull rod down, and filter out the ultrapure water.

(6) After filtering, rotate the filter chamber so that the opening of the turntable hole is aligned with the hole (3 position) of the pre-filled liquid chamber 3, pull the push-pull rod upward, and the pre-installed ultra-pure water in the pre-filled liquid chamber 3 enters the filter chamber body, rotate the filter chamber to the blank position (□ position), press the push-pull rod down, and filter out the ultrapure water.

(7) After filtering, rotate the filter chamber so that the opening of the turntable hole is aligned with the hole (4 position) of the pre-filled liquid chamber 4, pull the push-pull rod upward, and the pre-installed acid alcohol in the pre-filled liquid chamber 4 enters the filter chamber , rotate the filter chamber to the blank position (□ position), wait for 3 minutes, press the push-pull rod down, and the acidic alcohol is filtered out.

(8) After the filtration is completed, rotate the filter chamber so that the opening of the turntable hole is aligned with the hole (5 position) of the pre-filled liquid chamber 5, pull the push-pull rod upward, and the pre-installed ultrapure water in the pre-filled liquid chamber 5 enters the filter chamber body, rotate the filter chamber to the blank position (□ position), press the push-pull rod down, and filter out the ultrapure water.

(9) After filtering, rotate the filter chamber so that the opening of the turntable hole is aligned with the hole (6 position) of the pre-filled liquid chamber 6, pull the push-pull rod upward, and the pre-installed ultrapure water in the pre-filled liquid chamber 6 enters the filter chamber body, rotate the filter chamber to the blank position (□ position), press the push-pull rod down, and filter out the ultrapure water.

(10) After the filtration is completed, rotate the filter chamber so that the opening of the turntable hole is aligned with the hole (7 position) of the pre-filled liquid chamber 7, pull the push-pull rod upward, and the pre-installed potassium permanganate solution in the pre-filled liquid chamber 7 enters Filter chamber, rotate the filter chamber to the blank position (□ position), wait for 2 minutes, press the push-pull rod down, and filter out the potassium permanganate solution.

(11) After the filtration is completed, rotate the filter chamber so that the opening of the turntable hole is aligned with the hole (8 positions) of the pre-filled liquid chamber 8, pull the push-pull rod upward, and the pre-installed ultrapure water in the pre-filled liquid chamber 8 enters the filter chamber body, rotate the filter chamber to the blank position (□ position), press the push-pull rod down, and filter out the ultrapure water.

(12) After the filtration is completed, rotate the filter chamber so that the opening of the turntable hole is aligned with the hole (9 position) of the pre-filled liquid chamber 9, pull the push-pull rod upward, and the pre-installed ultrapure water in the pre-filled liquid chamber 9 enters the filter chamber body, rotate the filter chamber to the blank position (□ position), press the push-pull rod down, and filter out the ultrapure water.

(13) At this point, after all the pre-installed components have been used up, the coloring process is completed. At this time, the filter cavity can be taken out, the microporous filter membrane is taken off and placed on a glass slide for fluorescence microscope inspection. Under the excitation of ultraviolet light, the acid-fast bacilli are bright yellow or yellow-green rod-shaped bacteria.

The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (11)

1. A fluid control device for enriching cells and coloring cells, characterized in that it includes a filter chamber, a turntable, a sample chamber, a pre-filled liquid chamber and a waste liquid chamber, the filter chamber is detachably mounted on the turntable, the sample chamber and Multiple pre-filled liquid chambers are evenly installed in the circumferential direction of the filter cavity, and the waste liquid chamber is installed at the bottom of the turntable; The filter chamber is a hollow cylindrical structure, with a filter chamber rotating handle on the top, two symmetrical protrusions on the outer wall, a through filter chamber hole on the filter chamber wall, and a fixed filter chamber bottom. There is a microporous filter membrane, and the filter cavity is equipped with a piston push-pull rod suitable for the size of the cavity; The turntable is a hollow cylindrical structure, and the inner wall is provided with two symmetrical recesses corresponding to the convex positions; the inner wall of the turntable is provided with a turntable channel corresponding to the hole of the filter cavity, and the turntable channel runs through the top of the turntable face and inner face; Both the sample chamber and the bottom of the prefilled liquid chamber are provided with holes, and the opening positions correspond to the openings of the turntable channel on the top surface of the turntable; The top of the waste liquid chamber is provided with a mesh structure. 2 . The fluid control device according to claim 1 , wherein the filter cavity hole is arranged on one of the protrusions, and the turntable hole is arranged on a recess corresponding to the protrusion. 3 . 3 . The fluid control device according to claim 1 , wherein a silica gel layer is provided at the connection between the filter cavity and the turntable, the connection between the turntable and the sample chamber, and the prefilled liquid chamber. 4 . 4. The fluid control device according to claim 1, wherein sand chips are arranged on the mesh structure. 5 . The fluid control device according to claim 1 , wherein the microporous filter membrane is fixed between a silica gel sealing ring and a film-loading ring, and the silica gel sealing ring is connected to the bottom of the filter cavity. 6. The fluid control device according to claim 1 or 5, wherein the microporous filter membrane is a track-etched polycarbonate film or a track-etched polyethylene terephthalate film. 7 . The fluid control device according to claim 1 , wherein a sample injection port is provided on the top of the sample chamber, and a pre-filled liquid port is provided on the top of the pre-filled liquid chamber. 8. The fluid control device according to claim 7, wherein a sealing film is provided on the sample loading port and the preloading liquid port, and a position mark is provided on the sealing film, and the sample loading port and the preloading liquid port Between the two pre-installed liquid ports, there are blank position marks. 9. The method for enriching cells and coloring cells using the fluid control device according to any one of claims 1-8, characterized in that it comprises the following steps: Align the hole of the turntable with the hole of the sample chamber, open the sealing film of the sample inlet, add the sample into the sample chamber, pull the piston push-pull rod upward, the sample flows into the filter chamber, rotate the filter chamber to the blank position, and make the turntable hole and the sample chamber The holes are staggered, and the piston push-pull rod is pressed down to filter the sample; Rotate the filter chamber to align the hole of the turntable with the hole of the first pre-filled liquid chamber, pull the piston push-pull rod upward, the first pre-filled liquid flows into the filter cavity, rotate the filter cavity to the blank position, and align the hole of the turntable with the first Stagger the holes at the bottom of each pre-filled liquid chamber, press down the piston push-pull rod to filter the first pre-filled liquid; Repeat the filtering of other coloring liquids, and after all the coloring liquids are filtered out, the coloring process is completed. 10. The method for enriching cells and coloring cells according to claim 9, characterized in that, after the coloring is completed, the filter chamber is taken out, and the microporous filter membrane can be taken out for microscopic examination. 11. The method for enriching and staining cells according to claim 9, wherein the sample is processed blood, sputum, and spinal fluid.