CN104215490A - Nasal cavity secretion treating liquid and treating method thereof - Google Patents
Nasal cavity secretion treating liquid and treating method thereof Download PDFInfo
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Abstract
The invention relates to the field of clinical medicine, particular to nasal cavity secretion treating liquid and a treating method thereof. The nasal cavity secretion treating liquid comprises cleaning fluid, digestive fluid and protective solution which are used sequentially. The treating method includes the steps of suspension of nasal cavity secretion with the cleaning liquid, splitting decomposition of viscous components of the nasal cavity secretion with the digestive fluid, filtration and centrifugation with a cell strainer to remove impurities and viscous substances, addition of the protective solution, uniformly dispersion of cells in the preservative solution, transference of uniformly-dispersed cells to the cell strainer, transference of cells on the cell strainer to a glass slide, and fixing to obtain a clear and beautiful cell smear of thin and uniformly-distributed cells. The uniformly-distributed cells can be observed through a microscope, the cells are representative and are not overlapped, the influence of mucus and impurities on the observation of the cells is removed, and the immunohistochemical staining can be directly performed.
Description
Technical field
The present invention relates to clinical medicine domain, in particular to a kind for the treatment of fluid and disposal route thereof of nasal secretion.
Background technology
The cytolgical examination of nasal secretion has very important significance to clinical Diagnosis and Treat tool.Traditional nasal discharge cytolgical examination secretion or mucous membrane is scraped effluent be directly coated in microslide center, dyeing process is directly carried out after drying is fixing, but, nasal discharge contains a large amount of impurity and stickum, cause the object cell in secretion can not be dispersed at microslide, can not immunohistochemical staining be carried out; In addition, the impurity in secretion also can have very large impact to the correct judgement of the Color of smear and result.
Summary of the invention
The object of the present invention is to provide a kind for the treatment of fluid and disposal route thereof of nasal secretion, to solve the above problems.
Provide a kind for the treatment of fluid of nasal secretion in an embodiment of the present invention, comprise the cleaning fluid, digestive juice and the protection liquid that use successively;
Described cleaning fluid comprises ethylenediamine tetraacetic acid, methyl alcohol, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the mass volume ratio of described ethylenediamine tetraacetic acid and described redistilled water is 0.2 ~ 0.4g/L, the volume ratio of described methyl alcohol and described redistilled water is 1:0.5 ~ 1.5, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.0 ~ 1.5g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.2 ~ 0.3g/L, the mass volume ratio of described sodium chloride and described redistilled water is 7 ~ 9g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.1 ~ 0.3g/L, cleaning fluid pH is adjusted to 7.2 ~ 7.4 by described hydrochloric acid,
Described digestive juice comprises dithiothreitol (DTT), sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the mass volume ratio of described dithiothreitol (DTT) and described redistilled water is 90 ~ 110g/L, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.0 ~ 1.5g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.2 ~ 0.3g/L, the mass volume ratio of described sodium chloride and described redistilled water is 7 ~ 9g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.1 ~ 0.3g/L, digestive juice pH is adjusted to 7.2 ~ 7.4 by described hydrochloric acid,
Described protection liquid comprises tween, ethylenediamine tetraacetic acid, methyl alcohol, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the volume ratio of described tween and described redistilled water is 0.04 ~ 0.06:1, the mass volume ratio of described ethylenediamine tetraacetic acid and described redistilled water is 0.2 ~ 0.4g/L, the volume ratio of described methyl alcohol and described redistilled water is 1:0.5 ~ 1.5, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.0 ~ 1.5g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.2 ~ 0.3g/L, the mass volume ratio of described sodium chloride and described redistilled water is 7 ~ 9g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.1 ~ 0.3g/L, protection liquid pH is adjusted to 7.2 ~ 7.4 by described hydrochloric acid.
Preferably, described tween is any one or more mixing in polysorbas20, tween 21, polysorbate40, polysorbate60, Tween61, Tween 80, sorbimacrogol oleate100 and polysorbate85.
Preferably, the treating fluid of described nasal secretion also comprises immobile liquid I, described immobile liquid I is by paraformaldehyde, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid composition, the mass volume ratio of described paraformaldehyde and described redistilled water is 35 ~ 45g/L, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.0 ~ 1.5g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.2 ~ 0.3g/L, the mass volume ratio of described sodium chloride and described redistilled water is 7 ~ 9g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.1 ~ 0.3g/L, the pH of immobile liquid I is adjusted to 7.2 ~ 7.4 by described hydrochloric acid.
Preferably, the treating fluid of described nasal secretion also comprises immobile liquid II, and described immobile liquid II is that 1:0.5 ~ 1.5 are formulated by methyl alcohol and acetone with volume ratio.
The disposal route of nasal secretion comprises the following steps:
A () gets nasal secretion, be transferred in described cleaning fluid 20 ~ 40ml by the nasal secretion obtained, add described digestive juice, the described digestive juice added and the volume ratio of described cleaning fluid are 1 ~ 5:100, oscillator 400 ~ 600rpm, vibration mixing in 20 ~ 40 minutes;
Centrifugal 5 ~ 15 minutes of (b) 1500 ~ 2500rpm, supernatant discarded, shake is scattered the precipitation of bottom gently, adds and protects liquid described in 20 ~ 40ml and mix, be that 30 ~ 200 μm of cell strainer filter, collect filtered fluid with aperture;
C described filtered fluid aperture is the cell membrane filtration of 3 ~ 8 μm by (), be transferred on microslide, obtain cell smear by the cell on cell filter membrane;
D () is carried out cell to described cell smear and is fixed, dyeing process, then by microscope observing cell smear.
Preferably, in described step (c), described filtered fluid makes cell smear by cell slide machine.
Preferably, described cell slide machine making cell smear is specially:
Described cell filter membrane is carried out Negative pressure, and when cell filter membrane covering the cell of 8 ~ 15% areas, vacuum suction stops, and the cell adsorption in described filtered fluid is at described cell filter membrane surface;
The side that described cell filter membrane is adsorbed with cell is attached on microslide, by malleation air-blowing and electrostatic adsorption, makes the cell being adsorbed on cell filter membrane surface be transferred on microslide, obtain cell smear.
Preferably, in described (d), the fixing immobile liquid I provided by the invention or immobile liquid II that adopts of described cell is fixed.
Preferably, in described (b), the aperture of described cell strainer is 80 ~ 100 μm.
Preferably, in described (a), sampler is adopted to sample nasal secretion.
The treating fluid of the nasal secretion that the embodiment of the present invention provides and disposal route thereof, cleaning fluid is adopted to suspend to nasal secretion, with digestive juice, cracking is carried out to the viscosity component in nasal secretion simultaneously, and by cell strainer filter and centrifugal, remove impurity and stickum, then protection liquid is added, make cell dispersed in protection liquid, homodisperse cell is transferred to cell strainer, again the cell on cell strainer is transferred on microslide, fixing, obtain cell distribution thin and even, clear cell smear attractive in appearance, during employing microscopic examination, cell distribution is even, representative, not only cell zero lap, and eliminate the impact hindering mucus and the impurity observed, and directly can carry out immunohistochemical staining.
Accompanying drawing explanation
Fig. 1 shows microscope 100 times of Microscopic observation figure that the embodiment of the present invention 1 cell HE dyes;
Fig. 2 shows microscope 100 times of Microscopic observation figure that the embodiment of the present invention 1 cellular control unit HE dyes;
Fig. 3 shows microscope 100 times of Microscopic observation figure that the embodiment of the present invention 2 cell HE dyes;
Fig. 4 shows microscope 100 times of Microscopic observation figure that the embodiment of the present invention 2 cellular control unit HE dyes;
Fig. 5 shows MUC5AC in the embodiment of the present invention 3 and dyes by microscope 400 times of sem observation figure;
Fig. 6 shows MUC5AC in the embodiment of the present invention 3 and dyes by microscope 1000 times of sem observation figure;
Fig. 7 shows MUC2 in the embodiment of the present invention 3 and dyes by microscope 400 times of sem observation figure;
Fig. 8 shows MUC2 in the embodiment of the present invention 3 and dyes by microscope 1000 times of sem observation figure;
Fig. 9 shows tubulin in the embodiment of the present invention 4 and dyes by microscope 100 times of sem observation figure;
Figure 10 shows tubulin in the embodiment of the present invention 4 and dyes by microscope 1000 times of sem observation figure.
Embodiment
Below by specific embodiment, the present invention is described in further detail.
A kind for the treatment of fluid and disposal route thereof of nasal secretion is provided in the embodiment of the present invention.
A treating fluid for nasal secretion, comprises the cleaning fluid, digestive juice and the protection liquid that use successively;
Described cleaning fluid comprises ethylenediamine tetraacetic acid, methyl alcohol, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the mass volume ratio of described ethylenediamine tetraacetic acid and described redistilled water is 0.2 ~ 0.4g/L, the volume ratio of described methyl alcohol and described redistilled water is 1:0.5 ~ 1.5, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.0 ~ 1.5g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.2 ~ 0.3g/L, the mass volume ratio of described sodium chloride and described redistilled water is 7 ~ 9g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.1 ~ 0.3g/L, cleaning fluid pH is adjusted to 7.2 ~ 7.4 by described hydrochloric acid,
Described digestive juice comprises dithiothreitol (DTT), sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the mass volume ratio of described dithiothreitol (DTT) and described redistilled water is 90 ~ 110g/L, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.0 ~ 1.5g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.2 ~ 0.3g/L, the mass volume ratio of described sodium chloride and described redistilled water is 7 ~ 9g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.1 ~ 0.3g/L, digestive juice pH is adjusted to 7.2 ~ 7.4 by described hydrochloric acid,
Described protection liquid comprises tween, ethylenediamine tetraacetic acid, methyl alcohol, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the volume ratio of described tween and described redistilled water is 0.04 ~ 0.06:1, the mass volume ratio of described ethylenediamine tetraacetic acid and described redistilled water is 0.2 ~ 0.4g/L, the volume ratio of described methyl alcohol and described redistilled water is 1:0.5 ~ 1.5, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.0 ~ 1.5g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.2 ~ 0.3g/L, the mass volume ratio of described sodium chloride and described redistilled water is 7 ~ 9g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.1 ~ 0.3g/L, protection liquid pH is adjusted to 7.2 ~ 7.4 by described hydrochloric acid.
The treating fluid of the nasal secretion that the embodiment of the present invention provides and disposal route thereof, cleaning fluid is adopted to suspend to nasal secretion, number according to mucus in secretion adds digestive juice, digestive juice carries out cracking to the viscosity component in nasal secretion, and by cell strainer filter and centrifugal, remove impurity and stickum, then protection liquid is added, make cell dispersed in protection liquid, homodisperse cell is transferred to cell strainer, again the cell on cell strainer is transferred on microslide, fixing, obtain cell distribution thin and even, clear cell smear attractive in appearance, during employing microscopic examination, cell distribution is even, representative, not only cell zero lap, and eliminate the impact hindering mucus and the impurity observed, and directly can carry out immunohistochemical staining.
Tween (or polysorbate) is non-ionics, has the effect of good Cell protection.Preferably, described tween is any one or more mixing in polysorbas20, tween 21, polysorbate40, polysorbate60, Tween61, Tween 80, sorbimacrogol oleate100 and polysorbate85.
In order to obtain better fixed effect, preferably, the treating fluid of described nasal secretion also comprises immobile liquid I, described immobile liquid I is by paraformaldehyde, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid composition, the mass volume ratio of described paraformaldehyde and described redistilled water is 35 ~ 45g/L, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.0 ~ 1.5g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.2 ~ 0.3g/L, the mass volume ratio of described sodium chloride and described redistilled water is 7 ~ 9g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.1 ~ 0.3g/L, the pH of immobile liquid I is adjusted to 7.2 ~ 7.4 by described hydrochloric acid.Immobile liquid I is used for the fixing of the cell of regular growth morphological observation, good fixing effect.
Preferably, the treating fluid of described nasal secretion also comprises immobile liquid II, and described immobile liquid II is that 1:0.5 ~ 1.5 are formulated by methyl alcohol and acetone with volume ratio.Immobile liquid II is used for fixing the cell that nucleus is observed, good fixing effect.
The disposal route of nasal secretion comprises the following steps:
A () gets nasal secretion, be transferred in described cleaning fluid 20 ~ 40ml by the nasal secretion obtained, add described digestive juice, the described digestive juice added and the volume ratio of described cleaning fluid are 1 ~ 5:100, oscillator 400 ~ 600rpm, vibration mixing in 20 ~ 40 minutes;
Centrifugal 5 ~ 15 minutes of (b) 1500 ~ 2500rpm, supernatant discarded, shake is scattered the precipitation of bottom gently, adds and protects liquid described in 20 ~ 40ml and mix, be that 30 ~ 200 μm of cell strainer filter, collect filtered fluid with aperture;
C described filtered fluid aperture is the cell membrane filtration of 3 ~ 8 μm by (), be transferred on microslide, obtain cell smear by the cell on cell filter membrane;
D () is carried out cell to described cell smear and is fixed, dyeing process, then by microscope observing cell smear.
The process method step of this nasal secretion is simple, and the cell smear cell distribution obtained is thin and even, clear cell smear attractive in appearance, during employing microscopic examination, cell distribution is even, representative, not only cell zero lap, and eliminate the impact hindering mucus and the impurity observed, and directly can carry out immunohistochemical staining.
Wherein, cell smear immersion immobile liquid 20 ~ 30 minutes during cell is fixing, to reach better fixed effect.In described step (b), centrifugal 5 ~ 15 minutes of centrifugal employing 1500 ~ 2500rpm, is enough to cell dispersion, does not destroy cellular morphology again.
Preferably, in described step (c), described filtered fluid makes cell smear by cell slide machine.Use cell slide machine to make cell smear, efficiency is high, save manpower, and the cell smear cell distribution obtained is even.
Preferably, described cell slide machine making cell smear is specially:
Described cell filter membrane is carried out Negative pressure, and when cell filter membrane covering the cell of 8 ~ 15% areas, vacuum suction stops, and the cell adsorption in described filtered fluid is at described cell filter membrane surface;
The side that described cell filter membrane is adsorbed with cell is attached on microslide, by malleation air-blowing and electrostatic adsorption, makes the cell being adsorbed on cell filter membrane surface be transferred on microslide, obtain cell smear.
Adopt and make cell smear in this way, the impurity content of cell is few, by controlling the quantity of the covering cell on cell filter membrane, can avoid the overlap of cell, and cell distributes thin and evenly on microslide, obtained cell smear is observed, and clear attractive in appearance, cell distribution is even, representative, not only cell zero lap, and eliminate the impact hindering mucus and the impurity observed, and directly can carry out immunohistochemical staining.
Immobile liquid I is used for the fixing of the cell of regular growth morphological observation; Immobile liquid II is used for fixing the cell that nucleus is observed; Both use according to the fixing observation object of different cells, and both good fixing effect.Preferably, in described (d), the fixing immobile liquid I provided by the invention or immobile liquid II that adopts of described cell is fixed.
Cell dia is 10 ~ 20 μm, and cell strainer is filtered out by the impurity composition being greater than cell volume in liquid component.Preferably, in described (b), the aperture of described cell strainer is 80 ~ 100 μm.
Preferably, in described (a), sampler is adopted to sample nasal secretion.
Nasal secretion sampler, comprising: brush, brush body and brush holder, and described brush and brush holder lay respectively at the two ends of described brush body;
The length of described brush is 3 ~ 30mm, and width is 1 ~ 5mm, and thickness is 2 ~ 10mm; The length of described brush body is 50 ~ 80mm, and the length of described brush holder is 50 ~ 80mm;
Described brush is cylinder, and front end surface and its side of described brush are respectively arranged with bristle.
Preferably, also convergence part is provided with between described brush body and described brush holder;
One end of described convergence part is connected with described brush body, and the other end is connected with described brush holder, and described brush body and described brush holder lay respectively at the both sides of described convergence part, and the angle between the extended line of described brush holder and described brush body is 0 ~ 30 degree.
Preferably, the xsect of described bristle is circular or oval or cruciform or polygon.
Preferably, the material of described bristle is polyamide 6, and the cross-sectional diameter of described bristle is 0.05 ~ 0.5mm.
Preferably, the length of described bristle is 2 ~ 5mm.
Preferably, described bristle is arranged at the 0.1 ~ 5mm place, edge apart from described end face or described side.
Preferably, described brush holder is strip, and the xsect of described brush holder is flat or cylindric, and its width or diameter are 2 ~ 10mm.
Preferably, the width of described brush body reduces gradually along the direction of described brush, and its width of position that described brush body is connected with described convergence part is 4 ~ 6mm, and its width of position that described brush body is connected with described brush is 2 ~ 3mm.
Preferably, the surface of described brush holder is provided with anti-slip tank.
The length of brush is 3 ~ 30mm, and the width of brush is 1 ~ 5mm, and the thickness of brush is 2 ~ 10mm, and the length of brush body is 50 ~ 80mm, and the length of brush holder is 50 ~ 80mm, and the size of nasal secretion sampler is fitted mutually with nostril; Brush is cylinder, and end face and the side of brush are provided with bristle.Got at Brush for cleaning nasal cavity by the bristle arranged in brush side, collect the secretion on nasal wall; By the bristle arranged in brush front end, the secretion of specified scope can be got by precise positioning brush, the sample anchor point collected accurately and more comprehensive, in addition, after adopting bristle to collect secretion, bristle is directly rinsed in the solution and is washed, and bristle itself does not have residue, and can not produce extra chaff interference to sample.
Embodiment 1
Cleaning fluid comprises ethylenediamine tetraacetic acid, methyl alcohol, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the mass volume ratio of described ethylenediamine tetraacetic acid and described redistilled water is 0.3g/L, the volume ratio of described methyl alcohol and described redistilled water is 1:1, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.44g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.24g/L, the mass volume ratio of described sodium chloride and described redistilled water is 8g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.2g/L, cleaning fluid pH is adjusted to 7.3 by described hydrochloric acid,
Digestive juice comprises dithiothreitol (DTT), sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the mass volume ratio of described dithiothreitol (DTT) and described redistilled water is 100g/L, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.44g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.24g/L, the mass volume ratio of described sodium chloride and described redistilled water is 8g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.2g/L, and digestive juice pH is adjusted to 7.3 by described hydrochloric acid;
Protection liquid comprises Tween-20, ethylenediamine tetraacetic acid, methyl alcohol, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the volume ratio of described Tween-20 and described redistilled water is 0.05:1, the mass volume ratio of described ethylenediamine tetraacetic acid and described redistilled water is 0.3g/L, the volume ratio of described methyl alcohol and described redistilled water is 1:1, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.44g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.24g/L, the mass volume ratio of described sodium chloride and described redistilled water is 8g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.2g/L, protection liquid pH is adjusted to 7.3 by described hydrochloric acid.
Immobile liquid I, be made up of paraformaldehyde, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the mass volume ratio of described paraformaldehyde and described redistilled water is 40g/L, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.44g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.24g/L, the mass volume ratio of described sodium chloride and described redistilled water is 8g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.2g/L, and the pH of immobile liquid I is adjusted to 7.3 by described hydrochloric acid.
The disposal route of nasal secretion, comprises the following steps:
A () adopts sampler to get nasal secretion, be transferred in cleaning fluid 20ml by the nasal secretion obtained, add 0.2ml digestive juice, use oscillator 500rpm, 20min, vibration mixing;
B () uses centrifuge, 2000rpm, 10min, supernatant discarded, shakes the precipitation of bottom of scattering gently, adds 30ml and protects liquid and mix, and is that 100 μm of cell strainer filter with aperture, collection filtered fluid;
C filtered fluid aperture is the cell membrane filtration of 3 μm by (), be transferred on microslide, obtain cell smear by the cell on cell filter membrane;
D () adopts immobile liquid I to carry out cell to cell smear and fixes, immobile liquid is fixedly that cell smear is directly immersed in immobile liquid 20min to the cell on cell smear, for regular growth morphological observation, then HE dyeing process is carried out, HE dyeing is carried out according to a conventional method, then by microscope 100 times of observation of cell smears, result as shown in Figure 1; Control group adopts conventional mode process nasal secretion then to carry out HE dyeing process, and then by microscope 100 times of observation of cell smears, result as shown in Figure 2.As can be seen from Figure 1, cell dispersal is effective, and does not substantially have overlapping phenomenon, and in Fig. 2, cell overlap is serious, bad dispersibility.
Embodiment 2
Cleaning fluid comprises ethylenediamine tetraacetic acid, methyl alcohol, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the mass volume ratio of described ethylenediamine tetraacetic acid and described redistilled water is 0.2g/L, the volume ratio of described methyl alcohol and described redistilled water is 1:0.5, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.0g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.2g/L, the mass volume ratio of described sodium chloride and described redistilled water is 7g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.1g/L, cleaning fluid pH is adjusted to 7.2 by described hydrochloric acid,
Digestive juice comprises dithiothreitol (DTT), sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the mass volume ratio of described dithiothreitol (DTT) and described redistilled water is 90g/L, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.0g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.2g/L, the mass volume ratio of described sodium chloride and described redistilled water is 7g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.1g/L, and digestive juice pH is adjusted to 7.2 by described hydrochloric acid;
Protection liquid comprises Tween-80, ethylenediamine tetraacetic acid, methyl alcohol, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the volume ratio of described Tween-80 and described redistilled water is 0.04:1, the mass volume ratio of described ethylenediamine tetraacetic acid and described redistilled water is 0.2g/L, the volume ratio of described methyl alcohol and described redistilled water is 1:0.5, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.0g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.2g/L, the mass volume ratio of described sodium chloride and described redistilled water is 7g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.1g/L, protection liquid pH is adjusted to 7.2 by described hydrochloric acid.
Immobile liquid I, be made up of paraformaldehyde, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the mass volume ratio of described paraformaldehyde and described redistilled water is 35g/L, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.0g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.2g/L, the mass volume ratio of described sodium chloride and described redistilled water is 7g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.1g/L, and the pH of immobile liquid I is adjusted to 7.2 by described hydrochloric acid.
The disposal route of nasal secretion, comprises the following steps:
A () adopts sampler to get nasal secretion, be transferred in cleaning fluid 20ml by the nasal secretion obtained, add 0.2ml digestive juice, use oscillator 500rpm, 20min, vibration mixing;
B () uses centrifuge, 2000rpm, 10min, supernatant discarded, shakes the precipitation of bottom of scattering gently, adds 30ml and protects liquid and mix, and is that 80 μm of cell strainer filter with aperture, collection filtered fluid;
C filtered fluid aperture is the cell membrane filtration of 3 μm by (), be transferred on microslide, obtain cell smear by the cell on cell filter membrane;
D () adopts immobile liquid I to carry out cell to cell smear and fixes, immobile liquid is fixedly that cell smear is directly immersed in immobile liquid 20min to the cell on cell smear, for regular growth morphological observation, then HE dyeing process is carried out, HE dyeing is carried out according to a conventional method, by microscope 100 times of observation of cell smears, result as shown in Figure 3; Control group adopts conventional mode process nasal secretion then to carry out HE dyeing process, and then by microscope 100 times of observation of cell smears, result as shown in Figure 4.As can be seen from Figure 3, cell dispersal is effective, and does not substantially have overlapping phenomenon, and in Fig. 4, cell overlap is serious, bad dispersibility.
Embodiment 3
Cleaning fluid comprises ethylenediamine tetraacetic acid, methyl alcohol, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the mass volume ratio of described ethylenediamine tetraacetic acid and described redistilled water is 0.4g/L, the volume ratio of described methyl alcohol and described redistilled water is 1:1.5, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.5g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.3g/L, the mass volume ratio of described sodium chloride and described redistilled water is 9g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.3g/L, cleaning fluid pH is adjusted to 7.4 by described hydrochloric acid,
Digestive juice comprises dithiothreitol (DTT), sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the mass volume ratio of described dithiothreitol (DTT) and described redistilled water is 110g/L, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.5g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.3g/L, the mass volume ratio of described sodium chloride and described redistilled water is 9g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.3g/L, and digestive juice pH is adjusted to 7.4 by described hydrochloric acid;
Protection liquid comprises Tween-20, ethylenediamine tetraacetic acid, methyl alcohol, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the volume ratio of described Tween-20 and described redistilled water is 0.06:1, the mass volume ratio of described ethylenediamine tetraacetic acid and described redistilled water is 0.4g/L, the volume ratio of described methyl alcohol and described redistilled water is 1:1.5, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.5g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.3g/L, the mass volume ratio of described sodium chloride and described redistilled water is 9g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.3g/L, protection liquid pH is adjusted to 7.4 by described hydrochloric acid.
Immobile liquid II is that 1:0.5 is formulated by methyl alcohol and acetone with volume ratio.
The disposal route of nasal secretion, comprises the following steps:
A () adopts sampler to get nasal secretion, be transferred in cleaning fluid 40ml by the nasal secretion obtained, add 1ml digestive juice, use oscillator 600rpm, 30min, vibration mixing;
B () uses centrifuge, 1500rpm, 15min, supernatant discarded, shakes the precipitation of bottom of scattering gently, adds 40ml and protects liquid and mix, and is that 200 μm of cell strainer filter with aperture, collection filtered fluid;
C filtered fluid aperture is the cell membrane filtration of 8 μm by (), be transferred on microslide, obtain cell smear by the cell on cell filter membrane;
D () adopts immobile liquid II to be fixed, for to nuclear observation, immobile liquid is fixedly that cell smear is directly immersed in immobile liquid 10min to the cell on cell smear, take out, then immunohistochemical staining is carried out, MUC5AC dyeing and MUC2 dyeing, respectively by 400 times, microscope and 1000 times of observation of cell smears, MUC5AC dyeing is by microscope 400 doubly as shown in Figure 5; MUC5AC dyeing by microscope 1000 doubly as shown in Figure 6; MUC2 dyeing by microscope 400 doubly as shown in Figure 7; MUC2 dyeing by microscope 1000 doubly as shown in Figure 8.As can be seen from Fig. 5 ~ 8, cell dyeing effect is very good, and observation background is pure, disturbs without other impurity.
Embodiment 4
Cleaning fluid comprises ethylenediamine tetraacetic acid, methyl alcohol, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the mass volume ratio of described ethylenediamine tetraacetic acid and described redistilled water is 0.2g/L, the volume ratio of described methyl alcohol and described redistilled water is 1:0.5, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.0g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.2g/L, the mass volume ratio of described sodium chloride and described redistilled water is 7g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.1g/L, cleaning fluid pH is adjusted to 7.2 by described hydrochloric acid,
Digestive juice comprises dithiothreitol (DTT), sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the mass volume ratio of described dithiothreitol (DTT) and described redistilled water is 100g/L, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.0g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.2g/L, the mass volume ratio of described sodium chloride and described redistilled water is 7g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.1g/L, and digestive juice pH is adjusted to 7.2 by described hydrochloric acid;
Protection liquid comprises Tween-60, ethylenediamine tetraacetic acid, methyl alcohol, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the volume ratio of described Tween-60 and described redistilled water is 0.04:1, the mass volume ratio of described ethylenediamine tetraacetic acid and described redistilled water is 0.2g/L, the volume ratio of described methyl alcohol and described redistilled water is 1:0.5, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.0g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.2g/L, the mass volume ratio of described sodium chloride and described redistilled water is 7g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.1g/L, protection liquid pH is adjusted to 7.2 by described hydrochloric acid.
Immobile liquid II is that 1:1.5 is formulated by methyl alcohol and acetone with volume ratio.
The disposal route of nasal secretion, comprises the following steps:
A () adopts sampler to get nasal secretion, be transferred in cleaning fluid 30ml by the nasal secretion obtained, add 0.3ml digestive juice, use oscillator 400rpm, 40min, vibration mixing;
B () uses centrifuge, 2500rpm, 5min, supernatant discarded, shakes the precipitation of bottom of scattering gently, adds 20ml and protects liquid and mix, and is that 30 μm of cell strainer filter with aperture, collection filtered fluid;
C filtered fluid aperture is the cell membrane filtration of 5 μm by (), be transferred on microslide, obtain cell smear by the cell on cell filter membrane;
D () adopts immobile liquid II to be fixed, for to nuclear observation, immobile liquid is fixedly that cell smear is directly immersed in immobile liquid 15min to the cell on cell smear, take out, then immunohistochemical staining is carried out, then tubulin dyeing is carried out, respectively by microscope 100 times (Fig. 9) and 1000 times of (Figure 10) observation of cell smears.As can be seen from Fig. 9 and Figure 10, cell dyeing effect is very good, and observation background is pure, disturbs without other impurity.
The treating fluid of nasal secretion provided by the invention and disposal route thereof, not only inherit all advantages of conventional inspecting method, more solve traditional secretion smear technique and there is cell loss, can not all be colored, viscous liquid in secretion, impurity exists and cell does not can be uniformly dispersed, overlapping etc. result is correctly judged affect problem.Adopt treating fluid and the disposal route thereof of nasal secretion provided by the invention, the effective object cell in nasal discharge is made to be individual layer, to tile on microslide equably, make the ultra-thin cell smear that Cell Component enriches, clear background, form are complete, be evenly distributed, and directly can carry out immunohistochemical staining, the accuracy that result judges is improved greatly.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a treating fluid for nasal secretion, is characterized in that, comprises the cleaning fluid, digestive juice and the protection liquid that use successively;
Described cleaning fluid comprises ethylenediamine tetraacetic acid, methyl alcohol, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the mass volume ratio of described ethylenediamine tetraacetic acid and described redistilled water is 0.2 ~ 0.4g/L, the volume ratio of described methyl alcohol and described redistilled water is 1:0.5 ~ 1.5, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.0 ~ 1.5g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.2 ~ 0.3g/L, the mass volume ratio of described sodium chloride and described redistilled water is 7 ~ 9g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.1 ~ 0.3g/L, cleaning fluid pH is adjusted to 7.2 ~ 7.4 by described hydrochloric acid,
Described digestive juice comprises dithiothreitol (DTT), sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the mass volume ratio of described dithiothreitol (DTT) and described redistilled water is 90 ~ 110g/L, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.0 ~ 1.5g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.2 ~ 0.3g/L, the mass volume ratio of described sodium chloride and described redistilled water is 7 ~ 9g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.1 ~ 0.3g/L, digestive juice pH is adjusted to 7.2 ~ 7.4 by described hydrochloric acid,
Described protection liquid comprises tween, ethylenediamine tetraacetic acid, methyl alcohol, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, the volume ratio of described tween and described redistilled water is 0.04 ~ 0.06:1, the mass volume ratio of described ethylenediamine tetraacetic acid and described redistilled water is 0.2 ~ 0.4g/L, the volume ratio of described methyl alcohol and described redistilled water is 1:0.5 ~ 1.5, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.0 ~ 1.5g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.2 ~ 0.3g/L, the mass volume ratio of described sodium chloride and described redistilled water is 7 ~ 9g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.1 ~ 0.3g/L, protection liquid pH is adjusted to 7.2 ~ 7.4 by described hydrochloric acid.
2. the treating fluid of nasal secretion according to claim 1, is characterized in that, described tween is any one or more mixing in polysorbas20, tween 21, polysorbate40, polysorbate60, Tween61, Tween 80, sorbimacrogol oleate100 and polysorbate85.
3. the treating fluid of nasal secretion according to claim 1, it is characterized in that, the treating fluid of described nasal secretion also comprises immobile liquid I, described immobile liquid I is by paraformaldehyde, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid composition, the mass volume ratio of described paraformaldehyde and described redistilled water is 35 ~ 45g/L, the mass volume ratio of described sodium hydrogen phosphate and described redistilled water is 1.0 ~ 1.5g/L, the mass volume ratio of described potassium dihydrogen phosphate and described redistilled water is 0.2 ~ 0.3g/L, the mass volume ratio of described sodium chloride and described redistilled water is 7 ~ 9g/L, the mass volume ratio of described potassium chloride and described redistilled water is 0.1 ~ 0.3g/L, the pH of immobile liquid I is adjusted to 7.2 ~ 7.4 by described hydrochloric acid.
4. the treating fluid of nasal secretion according to claim 1, is characterized in that, the treating fluid of described nasal secretion also comprises immobile liquid II, and described immobile liquid II is that 1:0.5 ~ 1.5 are formulated by methyl alcohol and acetone with volume ratio.
5. adopt the treating fluid described in claim 1 or 2 to carry out the disposal route of nasal secretion, it is characterized in that, comprise the following steps:
A () gets nasal secretion, be transferred in described cleaning fluid 20 ~ 40ml by the nasal secretion obtained, add described digestive juice, the described digestive juice added and the volume ratio of described cleaning fluid are 1 ~ 5:100, oscillator 400 ~ 600rpm, vibration mixing in 20 ~ 40 minutes;
Centrifugal 5 ~ 15 minutes of (b) 1500 ~ 2500rpm, supernatant discarded, shake is scattered the precipitation of bottom gently, adds and protects liquid described in 20 ~ 40ml and mix, be that 30 ~ 200 μm of cell strainer filter, collect filtered fluid with aperture;
C described filtered fluid aperture is the cell membrane filtration of 3 ~ 8 μm by (), be transferred on microslide, obtain cell smear by the cell on cell filter membrane;
D () is carried out cell to described cell smear and is fixed, dyeing process, then by microscope observing cell smear.
6. the disposal route of nasal secretion according to claim 5, is characterized in that, in described step (c), described filtered fluid makes cell smear by cell slide machine.
7. the disposal route of nasal secretion according to claim 6, is characterized in that, described cell slide machine makes cell smear and is specially:
Described cell filter membrane is carried out Negative pressure, and when cell filter membrane covering the cell of 8 ~ 15% areas, vacuum suction stops, and the cell adsorption in described filtered fluid is at described cell filter membrane surface;
The side that described cell filter membrane is adsorbed with cell is attached on microslide, by malleation air-blowing and electrostatic adsorption, makes the cell being adsorbed on cell filter membrane surface be transferred on microslide, obtain cell smear.
8. the disposal route of nasal secretion according to claim 5, is characterized in that, in described (d), described cell is fixed and adopted the immobile liquid I in the claim 3 or immobile liquid II in claim 4 to be fixed.
9. the disposal route of nasal secretion according to claim 5, is characterized in that, in described (b), the aperture of described cell strainer is 80 ~ 100 μm.
10. the disposal route of nasal secretion according to claim 5, is characterized in that, in described (a), adopts sampler to sample nasal secretion.
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