CN104215490B - 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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- CN104215490B CN104215490B CN201410497784.3A CN201410497784A CN104215490B CN 104215490 B CN104215490 B CN 104215490B CN 201410497784 A CN201410497784 A CN 201410497784A CN 104215490 B CN104215490 B CN 104215490B
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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 of nasal secretion
Treatment fluid and processing method thereof.
Background technology
The cytolgical examination of nasal secretion has critically important meaning to clinical diagnosis and treatment
Justice.Traditional nasal discharge cytolgical examination is secretions or mucosa to be scraped thing be coated directly on
Microscope slide center, is dried after fixing and directly carries out dyeing process, but, nasal discharge contains
Substantial amounts of impurity and stickum, cause the purpose cell in secretions can not be equal at microscope slide
Even dispersion, it is impossible to carry out immunohistochemical staining；Additionally, the impurity in secretions also can be to painting
The Color of sheet and the correct of result judge there is the biggest impact.
Summary of the invention
It is an object of the invention to provide treatment fluid and the process side thereof of a kind of nasal secretion
Method, to solve above-mentioned problem.
Provide the treatment fluid of a kind of nasal secretion in an embodiment of the present invention, including depending on
The cleanout fluid of secondary use, Digestive system and protection liquid；
Described cleanout fluid includes ethylenediaminetetraacetic acid, methanol, disodium hydrogen phosphate, biphosphate
Potassium, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, described ethylenediaminetetraacetic acid is heavy with described
The mass volume ratio steaming water is 0.2～0.4g/L, described methanol and the volume ratio of described redistilled water
For 1:0.5～1.5, the mass volume ratio of described disodium hydrogen phosphate and described redistilled water be 1.0～
1.5g/L, described potassium dihydrogen phosphate is 0.2～0.3 with the mass volume ratio of described redistilled water
G/L, described sodium chloride is 7～9g/L with the mass volume ratio of described redistilled water, described chlorination
Potassium is 0.1～0.3g/L with the mass volume ratio of described redistilled water, and described hydrochloric acid is by cleanout fluid
PH is adjusted to 7.2～7.4；
Described Digestive system includes dithiothreitol, DTT, disodium hydrogen phosphate, potassium dihydrogen phosphate, chlorination
The quality of sodium, potassium chloride, redistilled water and hydrochloric acid, described dithiothreitol, DTT and described redistilled water
Volume ratio is 90～110g/L, described disodium hydrogen phosphate and the mass volume ratio of described redistilled water
Being 1.0～1.5g/L, described potassium dihydrogen phosphate with the mass volume ratio of described redistilled water is
0.2～0.3g/L, described sodium chloride is 7～9g/L with the mass volume ratio of described redistilled water,
Described potassium chloride is 0.1～0.3g/L with the mass volume ratio of described redistilled water, described hydrochloric acid
Digestive system pH is adjusted to 7.2～7.4；
Described protection liquid includes tween, ethylenediaminetetraacetic acid, methanol, disodium hydrogen phosphate, phosphorus
Acid dihydride potassium, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, described tween and described heavy steaming
The volume ratio of water is 0.04～0.06:1, described ethylenediaminetetraacetic acid and the quality of described redistilled water
Volume ratio is 0.2～0.4g/L, the volume ratio of described methanol and described redistilled water be 1:0.5～
1.5, described disodium hydrogen phosphate is 1.0～1.5g/L with the mass volume ratio of described redistilled water,
Described potassium dihydrogen phosphate is 0.2～0.3g/L with the mass volume ratio of described redistilled water, described chlorine
The mass volume ratio changing sodium and described redistilled water is 7～9g/L, and described potassium chloride is heavy with described
Steam water mass volume ratio be 0.1～0.3g/L, described hydrochloric acid protection liquid pH is adjusted to 7.2～
7.4。
Preferably, described tween is polysorbas20, tween 21, polysorbate40, polysorbate60, tells
Any one or more mixing in temperature 61, Tween 80, sorbimacrogol oleate100 and polysorbate85.
Preferably, the treatment fluid of described nasal secretion also includes fixative I, described fixing
Liquid I is by paraformaldehyde, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, weight
Steam water and hydrochloric acid composition, the mass volume ratio of described paraformaldehyde and described redistilled water be 35～
45g/L, described disodium hydrogen phosphate is 1.0～1.5g/L with the mass volume ratio of described redistilled water,
Described potassium dihydrogen phosphate is 0.2～0.3g/L with the mass volume ratio of described redistilled water, described chlorine
The mass volume ratio changing sodium and described redistilled water is 7～9g/L, and described potassium chloride is heavy with described
The mass volume ratio steaming water is 0.1～0.3g/L, and the pH of fixative I is adjusted to by described hydrochloric acid
7.2～7.4.
Preferably, the treatment fluid of described nasal secretion also includes fixative II, described fixing
Liquid II by methanol and acetone with volume ratio as 1:0.5～1.5 formulated.
The processing method of nasal secretion comprises the following steps:
A () takes nasal secretion, the nasal secretion obtained is transferred to described cleanout fluid
In 20～40ml, add described Digestive system, the described Digestive system of addition and described cleanout fluid
Volume ratio is 1～5:100, agitator 400～600rpm, vibration mixing in 20～40 minutes；
B () 1500～2500rpm is centrifuged 5～15 minutes, supernatant discarded, and shake is scattered gently
The precipitation of bottom, adds and protects liquid described in 20～40ml and mix, be 30～200 with aperture
μm cell strainer filters, and collects filtrate；
(c) by the cell membrane filtration that described filtrate aperture is 3～8 μm, by cell
Cell on filter membrane is transferred on microscope slide, obtains cell smear；
D () carries out cell to described cell smear and fixes, dyeing processes, then by aobvious
Micro mirror observation of cell smear.
Preferably, in described step (c), described filtrate is made by cell slide machine
Cell smear.
Preferably, described cell slide machine make cell smear particularly as follows:
Described cell filter membrane is carried out Negative pressure, when covering 8～15% on cell filter membrane
Long-pending cell, vacuum suction stops, and the cell absorption in described filtrate is filtered at described cell
Film surface；
The side that described cell filter membrane is adsorbed with cell is attached on microscope slide, passes through malleation
Air-blowing and electrostatic adsorption, make absorption be transferred to microscope slide at the cell of cell filter membrane surface
On, obtain cell smear.
Preferably, in described (d), described cell is fixed and is used the fixing of present invention offer
Liquid I or fixative II is fixed.
Preferably, in described (b), the aperture of described cell strainer is 80～100 μm.
Preferably, in described (a), use sampler that nasal secretion is sampled.
The treatment fluid of the nasal secretion that the embodiment of the present invention provides and processing method thereof, use
Nasal secretion is suspended by cleanout fluid, simultaneously with Digestive system to gluing in nasal secretion
Property composition cracks, and is filtered by cell strainer and be centrifuged, and removes the removal of impurity and goo
Matter, is subsequently adding protection liquid, makes cell dispersed, by homodisperse in protection liquid
Cell is transferred to cell strainer, then is transferred on microscope slide by the cell on cell strainer, Gu
Fixed, obtain cell distribution uniform and thin, cell smear the most attractive in appearance, use microscope to see
When examining, cell distribution is uniform, and representative, not only cell is non-overlapping, and eliminates resistance
Hinder the mucus of observation and the impact of impurity, it is possible to directly carry out immunohistochemical staining.
Accompanying drawing explanation
Fig. 1 shows under 100 times of mirrors of microscope that the embodiment of the present invention 1 cell HE dyes
Observe figure；
Fig. 2 shows the microscope 100 that the embodiment of the present invention 1 cellular control unit HE dyes
Times Microscopic observation figure；
Fig. 3 shows under 100 times of mirrors of microscope that the embodiment of the present invention 2 cell HE dyes
Observe figure；
Fig. 4 shows the microscope 100 that the embodiment of the present invention 2 cellular control unit HE dyes
Times Microscopic observation figure；
Fig. 5 shows that in the embodiment of the present invention 3, MUC5AC dyes by microscope 400
Times sem observation figure；
Fig. 6 shows that in the embodiment of the present invention 3, MUC5AC dyes by microscope 1000
Times sem observation figure；
Fig. 7 shows that in the embodiment of the present invention 3, MUC2 dyes by 400 times of mirrors of microscope
Observe figure；
Fig. 8 shows that in the embodiment of the present invention 3, MUC2 dyes by microscope 1000 times
Sem observation figure；
Fig. 9 shows that in the embodiment of the present invention 4, tubulin dyes by 100 times of mirrors of microscope
Observe figure；
Figure 10 shows that in the embodiment of the present invention 4, tubulin dyes by microscope 1000 times
Sem observation figure.
Detailed description of the invention
Below by specific embodiment, the present invention is described in further detail.
The embodiment of the present invention provides treatment fluid and the process side thereof of a kind of nasal secretion
Method.
A kind for the treatment of fluid of nasal secretion, including the cleanout fluid used successively, Digestive system and
Protection liquid；
Described cleanout fluid includes ethylenediaminetetraacetic acid, methanol, disodium hydrogen phosphate, biphosphate
Potassium, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, described ethylenediaminetetraacetic acid is heavy with described
The mass volume ratio steaming water is 0.2～0.4g/L, described methanol and the volume ratio of described redistilled water
For 1:0.5～1.5, the mass volume ratio of described disodium hydrogen phosphate and described redistilled water be 1.0～
1.5g/L, described potassium dihydrogen phosphate is 0.2～0.3 with the mass volume ratio of described redistilled water
G/L, described sodium chloride is 7～9g/L with the mass volume ratio of described redistilled water, described chlorination
Potassium is 0.1～0.3g/L with the mass volume ratio of described redistilled water, and described hydrochloric acid is by cleanout fluid
PH is adjusted to 7.2～7.4；
Described Digestive system includes dithiothreitol, DTT, disodium hydrogen phosphate, potassium dihydrogen phosphate, chlorination
The quality of sodium, potassium chloride, redistilled water and hydrochloric acid, described dithiothreitol, DTT and described redistilled water
Volume ratio is 90～110g/L, described disodium hydrogen phosphate and the mass volume ratio of described redistilled water
Being 1.0～1.5g/L, described potassium dihydrogen phosphate with the mass volume ratio of described redistilled water is
0.2～0.3g/L, described sodium chloride is 7～9g/L with the mass volume ratio of described redistilled water,
Described potassium chloride is 0.1～0.3g/L with the mass volume ratio of described redistilled water, described hydrochloric acid
Digestive system pH is adjusted to 7.2～7.4；
Described protection liquid includes tween, ethylenediaminetetraacetic acid, methanol, disodium hydrogen phosphate, phosphorus
Acid dihydride potassium, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, described tween and described heavy steaming
The volume ratio of water is 0.04～0.06:1, described ethylenediaminetetraacetic acid and the quality of described redistilled water
Volume ratio is 0.2～0.4g/L, the volume ratio of described methanol and described redistilled water be 1:0.5～
1.5, described disodium hydrogen phosphate is 1.0～1.5g/L with the mass volume ratio of described redistilled water,
Described potassium dihydrogen phosphate is 0.2～0.3g/L with the mass volume ratio of described redistilled water, described chlorine
The mass volume ratio changing sodium and described redistilled water is 7～9g/L, and described potassium chloride is heavy with described
Steam water mass volume ratio be 0.1～0.3g/L, described hydrochloric acid protection liquid pH is adjusted to 7.2～
7.4。
The treatment fluid of the nasal secretion that the embodiment of the present invention provides and processing method thereof, use
Nasal secretion is suspended by cleanout fluid, according to how many addition digestion of mucus in secretions
Liquid, the viscosity component in nasal secretion is cracked, and passes through cell strainer by Digestive system
Filter and centrifugal, remove the removal of impurity and stickum, be subsequently adding protection liquid, make cell protect
Protect in liquid dispersed, homodisperse cell is transferred to cell strainer, then cell is filtered
Online cell is transferred on microscope slide, fixing, obtains cell distribution uniform and thin, clearly
Cell smear attractive in appearance, when using microscope to observe, cell distribution is uniform, representative,
Not only cell is non-overlapping, and eliminates and hinder the mucus and the impact of impurity observed, and can
Directly to carry out immunohistochemical staining.
Tween (or Polysorbate) is nonionic surfactant, has well protection
The effect of cell.Preferably, described tween is polysorbas20, tween 21, polysorbate40, tells
Any one or more in temperature 60, Tween61, Tween 80, sorbimacrogol oleate100 and polysorbate85 is mixed
Close.
In order to obtain more preferable fixed effect, it is preferable that the treatment fluid of described nasal secretion
Also including fixative I, described fixative I is by paraformaldehyde, disodium hydrogen phosphate, biphosphate
Potassium, sodium chloride, potassium chloride, redistilled water and hydrochloric acid composition, described paraformaldehyde is heavy with described
The mass volume ratio steaming water is 35～45g/L, described disodium hydrogen phosphate and described redistilled water
Mass volume ratio is 1.0～1.5g/L, described potassium dihydrogen phosphate and the mass body of described redistilled water
Long-pending ratio is 0.2～0.3g/L, the mass volume ratio of described sodium chloride and described redistilled water be 7～
9g/L, described potassium chloride is 0.1～0.3g/L with the mass volume ratio of described redistilled water, described
The pH of fixative I is adjusted to 7.2～7.4 by hydrochloric acid.Fixative I is used for regular growth morphology
Fixing of the cell observed, good fixing effect.
Preferably, the treatment fluid of described nasal secretion also includes fixative II, described fixing
Liquid II by methanol and acetone with volume ratio as 1:0.5～1.5 formulated.Fixative II is used for
Fixing the cell that nucleus is observed, good fixing effect.
The processing method of nasal secretion comprises the following steps:
A () takes nasal secretion, the nasal secretion obtained is transferred to described cleanout fluid
In 20～40ml, add described Digestive system, the described Digestive system of addition and described cleanout fluid
Volume ratio is 1～5:100, agitator 400～600rpm, vibration mixing in 20～40 minutes；
B () 1500～2500rpm is centrifuged 5～15 minutes, supernatant discarded, and shake is scattered gently
The precipitation of bottom, adds and protects liquid described in 20～40ml and mix, be 30～200 with aperture
μm cell strainer filters, and collects filtrate；
(c) by the cell membrane filtration that described filtrate aperture is 3～8 μm, by cell
Cell on filter membrane is transferred on microscope slide, obtains cell smear；
D () carries out cell to described cell smear and fixes, dyeing processes, then by aobvious
Micro mirror observation of cell smear.
The process method step of this nasal secretion is simple, and the cell smear obtained is thin
Born of the same parents are distributed uniform and thin, cell smear the most attractive in appearance, and when using microscope to observe, cell divides
Cloth is uniform, and representative, not only cell is non-overlapping, and eliminates and hinder that observes to glue
Liquid and the impact of impurity, it is possible to directly carry out immunohistochemical staining.
Wherein, the fixing middle cell smear immersion fixative 20 of cell～30 minutes, to reach more
Good fixed effect.In described step (b), centrifugal use 1500～2500rpm from
The heart 5～15 minutes, it is sufficient to cell dispersion, the most do not destroy cellular morphology.
Preferably, in described step (c), described filtrate is made by cell slide machine
Cell smear.Using cell slide machine to make cell smear, efficiency is high, saves manpower, and
The cell smear cell distribution obtained is uniform.
Preferably, described cell slide machine make cell smear particularly as follows:
Described cell filter membrane is carried out Negative pressure, when covering 8～15% on cell filter membrane
Long-pending cell, vacuum suction stops, and the cell absorption in described filtrate is filtered at described cell
Film surface；
The side that described cell filter membrane is adsorbed with cell is attached on microscope slide, passes through malleation
Air-blowing and electrostatic adsorption, make absorption be transferred to microscope slide at the cell of cell filter membrane surface
On, obtain cell smear.
Adopting and make cell smear in this way, the impurity content of cell is few, thin by controlling
The quantity covering cell on born of the same parents' filter membrane, can avoid the overlap of cell, and cell is carrying glass
Being distributed thin and uniform on sheet, the cell smear prepared is observed, and the most attractive in appearance, cell divides
Cloth is uniform, and representative, not only cell is non-overlapping, and eliminates and hinder that observes to glue
Liquid and the impact of impurity, it is possible to directly carry out immunohistochemical staining.
Fixative I is used for the fixing of the cell of regular growth morphological observation；Fixative II uses
In fixing the cell that nucleus is observed；The observation mesh that both fix according to different cells
And use, and both good fixing effect.Preferably, in described (d), described cell
Fixative I or fixative II that the fixing present invention of employing provides are fixed.
Cell dia is 10～20 μm, and cell strainer is will to be more than cell volume in liquid component
Impurity composition filter out.Preferably, in described (b), the aperture of described cell strainer
It is 80～100 μm.
Preferably, in described (a), use sampler that nasal secretion is sampled.
Nasal secretion sampler, including: brush, brush body and brush holder, described brush and brush
Handle lays respectively at the two ends of described brush body；
A length of the 3 of described brush～30mm, width is 1～5mm, and thickness is 2～10mm；
A length of the 50 of described brush body～80mm, a length of the 50 of described brush holder～80mm；
Described brush is cylinder, and the front end surface of described brush and its side are respectively arranged with brush
Hair.
Preferably, it is additionally provided with convergence part 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,
Described brush body and described brush holder lay respectively at the both sides of described convergence part, described brush holder and described
Angle between the extended line of brush body is 0～30 degree.
Preferably, the cross section of described bristle is circular or oval or cross or polygon.
Preferably, the material of described bristle is polyamide 6, the cross-sectional diameter of described bristle
It is 0.05～0.5mm.
Preferably, a length of the 2～5mm of described bristle.
Preferably, described bristle is at the edge 0.1～5mm away from described end face or described side
Place is arranged.
Preferably, described brush holder is strip, and the cross section of described brush holder is flat or circle
Column, its width or a diameter of 2～10mm.
Preferably, the width of described brush body is gradually reduced along the direction of described brush, described brush
Its width of position that body is connected with described convergence part is 4～6mm, described brush body and described brush
Its width of position connected is 2～3mm.
Preferably, the surface configuration of described brush holder has anti-slip tank.
A length of the 3 of brush～30mm, the width of brush is 1～5mm, and the thickness of brush is
2～10mm, a length of the 50 of brush body～80mm, a length of the 50 of brush holder～80mm, nasal cavity
The size of secretion sampler is fitted mutually with nostril；Brush is cylinder, the end face of brush and side
It is provided with bristle.By the bristle that arranges in brush side in nasal cavity swipe, collect nasal cavity
Secretions on sidewall；By the bristle arranged in brush front end, can be with precise positioning swipe
The secretions of appointment scope, the sample anchor point of collection accurately and more fully, additionally, adopt
After collecting secretions with bristle, bristle rinses the most in the solution, bristle not residual itself
Thing, and specimen will not be produced extra chaff interference.
Embodiment 1
Cleanout fluid include ethylenediaminetetraacetic acid, methanol, disodium hydrogen phosphate, potassium dihydrogen phosphate,
Sodium chloride, potassium chloride, redistilled water and hydrochloric acid, described ethylenediaminetetraacetic acid and described redistilled water
Mass volume ratio be 0.3g/L, the volume ratio of described methanol and described redistilled water is 1:1,
Described disodium hydrogen phosphate is 1.44g/L with the mass volume ratio of described redistilled water, described phosphoric acid
Potassium dihydrogen is 0.24g/L with the mass volume ratio of described redistilled water, and described sodium chloride is with described
The mass volume ratio of redistilled water is 8g/L, described potassium chloride and the quality volume of described redistilled water
Ratio is 0.2g/L, and cleanout fluid pH is adjusted to 7.3 by described hydrochloric acid；
Digestive system include dithiothreitol, DTT, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride,
The quality volume of potassium chloride, redistilled water and hydrochloric acid, described dithiothreitol, DTT and described redistilled water
It is 1.44 than the mass volume ratio for 100g/L, described disodium hydrogen phosphate and described redistilled water
G/L, described potassium dihydrogen phosphate is 0.24g/L with the mass volume ratio of described redistilled water, described
Sodium chloride is 8g/L with the mass volume ratio of described redistilled water, described potassium chloride and described heavy steaming
The mass volume ratio of water is 0.2g/L, and Digestive system pH is adjusted to 7.3 by described hydrochloric acid；
Protection liquid includes tween 20, ethylenediaminetetraacetic acid, methanol, disodium hydrogen phosphate, phosphoric acid
Potassium dihydrogen, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, described tween 20 is heavy with described
The volume ratio steaming water is 0.05:1, described ethylenediaminetetraacetic acid and the mass body of described redistilled water
Long-pending is 1:1 than the volume ratio for 0.3g/L, described methanol and described redistilled water, described phosphoric acid
The mass volume ratio of disodium hydrogen and described redistilled water is 1.44g/L, described potassium dihydrogen phosphate with
The mass volume ratio of described redistilled water is 0.24g/L, described sodium chloride and the matter of described redistilled water
Amount volume ratio is 8g/L, and described potassium chloride is 0.2 with the mass volume ratio of described redistilled water
G/L, protection liquid pH is adjusted to 7.3 by described hydrochloric acid.
Fixative I, by paraformaldehyde, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, chlorine
Change potassium, redistilled water and hydrochloric acid composition, described paraformaldehyde and the quality volume of described redistilled water
It is 1.44 than the mass volume ratio for 40g/L, described disodium hydrogen phosphate and described redistilled water
G/L, described potassium dihydrogen phosphate is 0.24g/L with the mass volume ratio of described redistilled water, described
Sodium chloride is 8g/L with the mass volume ratio of described redistilled water, described potassium chloride and described heavy steaming
The mass volume ratio of water is 0.2g/L, and the pH of fixative I is adjusted to 7.3 by described hydrochloric acid.
The processing method of nasal secretion, comprises the following steps:
A () uses sampler to take nasal secretion, be transferred to clearly by the nasal secretion obtained
In washing liquid 20ml, add 0.2ml Digestive system, use agitator 500rpm, 20min, shake
Swing mixing；
B (), by centrifuge, 2000rpm, 10min, supernatant discarded, is shaken gently and is dissipated
Open the precipitation of bottom, add 30ml protection liquid and mix, be 100 μm cell filters with aperture
Net filtration, collects filtrate；
(c) by the cell membrane filtration that filtrate aperture is 3 μm, by cell filter membrane
Cell be transferred on microscope slide, obtain cell smear；
D () uses fixative I that cell smear is carried out cell and fixes, cell is coated with by fixative
Cell on sheet is fixing is that cell smear is directly immersed in fixative 20min, for routine
Morphological observation, then carries out HE dyeing process, and HE dyeing is carried out according to a conventional method,
Then by 100 times of observation of cell smears of microscope, result is as shown in Figure 1；Matched group is adopted
Process nasal secretion by conventional mode and then carry out HE dyeing process, then by aobvious
100 times of observation of cell smears of micro mirror, result is as shown in Figure 2.It will be seen from figure 1 that it is thin
Born of the same parents' dispersion effect is good, and does not substantially have overlapping phenomenon, and in Fig. 2, cell overlap is serious, point
Dissipate property poor.
Embodiment 2
Cleanout fluid include ethylenediaminetetraacetic acid, methanol, disodium hydrogen phosphate, potassium dihydrogen phosphate,
Sodium chloride, potassium chloride, redistilled water and hydrochloric acid, described ethylenediaminetetraacetic acid and described redistilled water
Mass volume ratio be 0.2g/L, the volume ratio of described methanol and described redistilled water is 1:0.5,
Described disodium hydrogen phosphate is 1.0g/L with the mass volume ratio of described redistilled water, described di(2-ethylhexyl)phosphate
Hydrogen potassium is 0.2g/L with the mass volume ratio of described redistilled water, described sodium chloride and described heavy steaming
The mass volume ratio of water is 7g/L, and described potassium chloride with the mass volume ratio of described redistilled water is
0.1g/L, cleanout fluid pH is adjusted to 7.2 by described hydrochloric acid；
Digestive system include dithiothreitol, DTT, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride,
The quality volume of potassium chloride, redistilled water and hydrochloric acid, described dithiothreitol, DTT and described redistilled water
It is 1.0g/L than the mass volume ratio for 90g/L, described disodium hydrogen phosphate and described redistilled water,
Described potassium dihydrogen phosphate is 0.2g/L with the mass volume ratio of described redistilled water, described sodium chloride
It is 7g/L with the mass volume ratio of described redistilled water, described potassium chloride and the matter of described redistilled water
Amount volume ratio is 0.1g/L, and Digestive system pH is adjusted to 7.2 by described hydrochloric acid；
Protection liquid includes tween 80, ethylenediaminetetraacetic acid, methanol, disodium hydrogen phosphate, phosphoric acid
Potassium dihydrogen, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, described tween 80 is heavy with described
The volume ratio steaming water is 0.04:1, described ethylenediaminetetraacetic acid and the mass body of described redistilled water
Long-pending is 1:0.5 than the volume ratio for 0.2g/L, described methanol and described redistilled water, described phosphoric acid
Disodium hydrogen is 1.0g/L with the mass volume ratio of described redistilled water, described potassium dihydrogen phosphate and institute
The mass volume ratio stating redistilled water is 0.2g/L, described sodium chloride and the quality of described redistilled water
Volume ratio is 7g/L, and described potassium chloride is 0.1g/L with the mass volume ratio of described redistilled water,
Protection liquid pH is adjusted to 7.2 by described hydrochloric acid.
Fixative I, by paraformaldehyde, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride,
Potassium chloride, redistilled water and hydrochloric acid composition, described paraformaldehyde and the mass body of described redistilled water
Long-pending is 1.0 than the mass volume ratio for 35g/L, described disodium hydrogen phosphate and described redistilled water
G/L, described potassium dihydrogen phosphate is 0.2g/L with the mass volume ratio of described redistilled water, described chlorine
The mass volume ratio changing sodium and described redistilled water is 7g/L, described potassium chloride and described redistilled water
Mass volume ratio be 0.1g/L, the pH of fixative I is adjusted to 7.2 by described hydrochloric acid.
The processing method of nasal secretion, comprises the following steps:
A () uses sampler to take nasal secretion, be transferred to clearly by the nasal secretion obtained
In washing liquid 20ml, add 0.2ml Digestive system, use agitator 500rpm, 20min, shake
Swing mixing；
B (), by centrifuge, 2000rpm, 10min, supernatant discarded, is shaken gently and is dissipated
Open the precipitation of bottom, add 30ml protection liquid and mix, being 80 μm cells with aperture
Strainer filtering, collects filtrate；
(c) by the cell membrane filtration that filtrate aperture is 3 μm, by cell filter membrane
On cell be transferred on microscope slide, obtain cell smear；
D () uses fixative I that cell smear is carried out cell and fixes, cell is coated with by fixative
Cell on sheet is fixing is that cell smear is directly immersed in fixative 20min, for routine
Morphological observation, then carries out HE dyeing process, and HE dyeing is carried out according to a conventional method,
By 100 times of observation of cell smears of microscope, result is as shown in Figure 3；Matched group often uses
The mode of rule processes nasal secretion and then carries out HE dyeing process, then passes through microscope
100 times of observation of cell smears, result is as shown in Figure 4.From figure 3, it can be seen that cell divides
Dissipate effective, and substantially there is no overlapping phenomenon, and in Fig. 4, cell overlap is serious, dispersibility
Difference.
Embodiment 3
Cleanout fluid include ethylenediaminetetraacetic acid, methanol, disodium hydrogen phosphate, potassium dihydrogen phosphate,
Sodium chloride, potassium chloride, redistilled water and hydrochloric acid, described ethylenediaminetetraacetic acid and described redistilled water
Mass volume ratio be 0.4g/L, the volume ratio of described methanol and described redistilled water is 1:1.5,
Described disodium hydrogen phosphate is 1.5g/L with the mass volume ratio of described redistilled water, described di(2-ethylhexyl)phosphate
Hydrogen potassium is 0.3g/L with the mass volume ratio of described redistilled water, described sodium chloride and described heavy steaming
The mass volume ratio of water is 9g/L, and described potassium chloride with the mass volume ratio of described redistilled water is
0.3g/L, cleanout fluid pH is adjusted to 7.4 by described hydrochloric acid；
Digestive system include dithiothreitol, DTT, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride,
The quality volume of potassium chloride, redistilled water and hydrochloric acid, described dithiothreitol, DTT and described redistilled water
It is 1.5 than the mass volume ratio for 110g/L, described disodium hydrogen phosphate and described redistilled water
G/L, described potassium dihydrogen phosphate is 0.3g/L with the mass volume ratio of described redistilled water, described chlorine
The mass volume ratio changing sodium and described redistilled water is 9g/L, described potassium chloride and described redistilled water
Mass volume ratio be 0.3g/L, Digestive system pH is adjusted to 7.4 by described hydrochloric acid；
Protection liquid includes tween 20, ethylenediaminetetraacetic acid, methanol, disodium hydrogen phosphate, phosphoric acid
Potassium dihydrogen, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, described tween 20 is heavy with described
The volume ratio steaming water is 0.06:1, described ethylenediaminetetraacetic acid and the mass body of described redistilled water
Long-pending is 1:1.5 than the volume ratio for 0.4g/L, described methanol and described redistilled water, described phosphorus
The mass volume ratio of acid disodium hydrogen and described redistilled water is 1.5g/L, described potassium dihydrogen phosphate with
The mass volume ratio of described redistilled water is 0.3g/L, described sodium chloride and the matter of described redistilled water
Amount volume ratio is 9g/L, and described potassium chloride is 0.3 with the mass volume ratio of described redistilled water
G/L, protection liquid pH is adjusted to 7.4 by described hydrochloric acid.
Fixative II is formulated with volume ratio for 1:0.5 by methanol and acetone.
The processing method of nasal secretion, comprises the following steps:
A () uses sampler to take nasal secretion, be transferred to clearly by the nasal secretion obtained
In washing liquid 40ml, add 1ml Digestive system, use agitator 600rpm, 30min, vibration
Mixing；
B (), by centrifuge, 1500rpm, 15min, supernatant discarded, is shaken gently and is dissipated
Open the precipitation of bottom, add 40ml protection liquid and mix, be 200 μm cell filters with aperture
Net filtration, collects filtrate；
(c) by the cell membrane filtration that filtrate aperture is 8 μm, by cell filter membrane
Cell be transferred on microscope slide, obtain cell smear；
D () uses fixative II to be fixed, for nuclear observation, fixative
Fixing to the cell on cell smear is that cell smear is directly immersed in fixative 10min,
Taking out, then carry out immunohistochemical staining, MUC5AC dyeing and MUC2 dye, point
Not Tong Guo 400 times and 1000 times observation of cell smears of microscope, MUC5AC dyeing pass through
Microscope 400 is the most as shown in Figure 5；MUC5AC dyeing is by microscope 1000 times such as figure
Shown in 6；MUC2 dyes by microscope 400 the most as shown in Figure 7；MUC2 dyeing is logical
Cross microscope 1000 the most as shown in Figure 8.It can be seen that cell dyeing effect from Fig. 5～8
The best, and observation background is pure, disturbs without other impurity.
Embodiment 4
Cleanout fluid include ethylenediaminetetraacetic acid, methanol, disodium hydrogen phosphate, potassium dihydrogen phosphate,
Sodium chloride, potassium chloride, redistilled water and hydrochloric acid, described ethylenediaminetetraacetic acid and described redistilled water
Mass volume ratio be 0.2g/L, the volume ratio of described methanol and described redistilled water is 1:0.5,
Described disodium hydrogen phosphate is 1.0g/L with the mass volume ratio of described redistilled water, described di(2-ethylhexyl)phosphate
Hydrogen potassium is 0.2g/L with the mass volume ratio of described redistilled water, described sodium chloride and described heavy steaming
The mass volume ratio of water is 7g/L, and described potassium chloride with the mass volume ratio of described redistilled water is
0.1g/L, cleanout fluid pH is adjusted to 7.2 by described hydrochloric acid；
Digestive system include dithiothreitol, DTT, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride,
The quality volume of potassium chloride, redistilled water and hydrochloric acid, described dithiothreitol, DTT and described redistilled water
It is 1.0g/L than the mass volume ratio for 100g/L, described disodium hydrogen phosphate and described redistilled water,
Described potassium dihydrogen phosphate is 0.2g/L with the mass volume ratio of described redistilled water, described sodium chloride
It is 7g/L with the mass volume ratio of described redistilled water, described potassium chloride and the matter of described redistilled water
Amount volume ratio is 0.1g/L, and Digestive system pH is adjusted to 7.2 by described hydrochloric acid；
Protection liquid includes Tween-60, ethylenediaminetetraacetic acid, methanol, disodium hydrogen phosphate, phosphoric acid
Potassium dihydrogen, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, described Tween-60 is heavy with described
The volume ratio steaming water is 0.04:1, described ethylenediaminetetraacetic acid and the mass body of described redistilled water
Long-pending is 1:0.5 than the volume ratio for 0.2g/L, described methanol and described redistilled water, described phosphoric acid
Disodium hydrogen is 1.0g/L with the mass volume ratio of described redistilled water, described potassium dihydrogen phosphate and institute
The mass volume ratio stating redistilled water is 0.2g/L, described sodium chloride and the quality of described redistilled water
Volume ratio is 7g/L, and described potassium chloride is 0.1g/L with the mass volume ratio of described redistilled water,
Protection liquid pH is adjusted to 7.2 by described hydrochloric acid.
Fixative II is formulated with volume ratio for 1:1.5 by methanol and acetone.
The processing method of nasal secretion, comprises the following steps:
A () uses sampler to take nasal secretion, be transferred to clearly by the nasal secretion obtained
In washing liquid 30ml, add 0.3ml Digestive system, use agitator 400rpm, 40min, shake
Swing mixing；
B (), by centrifuge, 2500rpm, 5min, supernatant discarded, is shaken gently and is dissipated
Open the precipitation of bottom, add 20ml protection liquid and mix, be 30 μm cell filters with aperture
Net filtration, collects filtrate；
(c) by the cell membrane filtration that filtrate aperture is 5 μm, by cell filter membrane
Cell be transferred on microscope slide, obtain cell smear；
D () uses fixative II to be fixed, for nuclear observation, fixative
Fixing to the cell on cell smear is that cell smear is directly immersed in fixative 15min,
Take out, then carry out immunohistochemical staining, then carry out tubulin dyeing, respectively by aobvious
Micro mirror 100 times (Fig. 9) and 1000 times of (Figure 10) observation of cell smears.From Fig. 9 and Tu
It can be seen that cell dyeing effect is the best in 10, and observation background is pure, miscellaneous without other
Matter is disturbed.
The treatment fluid of the nasal secretion that the present invention provides and processing method thereof, not only inherit
All advantages of conventional inspecting method, there is cell and lose in more solution tradition secretions smear technique
Lose, can not all be colored, viscous liquid in secretions, impurity exists and cell can not be equal
The judgements correct on result such as even dispersion, overlap affect problem.Use the nose that the present invention provides
The treatment fluid of solinocrine thing and processing method thereof, make effective purpose cell in nasal discharge in
Monolayer, tile on microscope slide equably, make that Cell Component is abundant, clear background, shape
The ultra-thin cell smear that state is complete, be evenly distributed, it is possible to directly carry out immunohistochemical staining,
The accuracy judging result is greatly improved.
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.All
Within the spirit and principles in the present invention, any modification, equivalent substitution and improvement etc. made,
Should be included within the scope of the present invention.
Claims (10)
1. the treatment fluid of a nasal secretion, it is characterised in that include using successively is clear
Washing liquid, Digestive system and protection liquid；
Described cleanout fluid include ethylenediaminetetraacetic acid, methanol, disodium hydrogen phosphate, potassium dihydrogen phosphate,
Sodium chloride, potassium chloride, redistilled water and hydrochloric acid, described ethylenediaminetetraacetic acid and described redistilled water
Mass volume ratio is 0.2～0.4g/L, and described methanol is 1 with the volume ratio of described redistilled water:
0.5～1.5, described disodium hydrogen phosphate is 1.0～1.5 with the mass volume ratio of described redistilled water
G/L, described potassium dihydrogen phosphate is 0.2～0.3g/L with the mass volume ratio of described redistilled water, institute
The mass volume ratio stating sodium chloride and described redistilled water is 7～9g/L, and described potassium chloride is with described
The mass volume ratio of redistilled water is 0.1～0.3g/L, and cleanout fluid pH is adjusted to by described hydrochloric acid
7.2～7.4；
Described Digestive system include dithiothreitol, DTT, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride,
The mass volume ratio of potassium chloride, redistilled water and hydrochloric acid, described dithiothreitol, DTT and described redistilled water
Be 90～110g/L, the mass volume ratio of described disodium hydrogen phosphate and described redistilled water be 1.0～
1.5g/L, described potassium dihydrogen phosphate is 0.2～0.3g/L with the mass volume ratio of described redistilled water,
Described sodium chloride is 7～9g/L with the mass volume ratio of described redistilled water, described potassium chloride and institute
The mass volume ratio stating redistilled water is 0.1～0.3g/L, and Digestive system pH is adjusted to by described hydrochloric acid
7.2～7.4；
Described protection liquid includes tween, ethylenediaminetetraacetic acid, methanol, disodium hydrogen phosphate, phosphoric acid
Potassium dihydrogen, sodium chloride, potassium chloride, redistilled water and hydrochloric acid, described tween and described redistilled water
Volume ratio is 0.04～0.06:1, described ethylenediaminetetraacetic acid and the quality volume of described redistilled water
Ratio is 0.2～0.4g/L, and described methanol is 1:0.5～1.5 with the volume ratio of described redistilled water, institute
The mass volume ratio stating disodium hydrogen phosphate and described redistilled water is 1.0～1.5g/L, described phosphoric acid
Potassium dihydrogen is 0.2～0.3g/L with the mass volume ratio of described redistilled water, described sodium chloride and institute
The mass volume ratio stating redistilled water is 7～9g/L, described potassium chloride and the quality of described redistilled water
Volume ratio is 0.1～0.3g/L, and protection liquid pH is adjusted to 7.2～7.4 by described hydrochloric acid.
The treatment fluid of nasal secretion the most according to claim 1, it is characterised in that
Described tween be polysorbas20, tween 21, polysorbate40, polysorbate60, Tween61, Tween 80,
Any one or more mixing in sorbimacrogol oleate100 and polysorbate85.
The treatment fluid of nasal secretion the most according to claim 1, it is characterised in that
The treatment fluid of described nasal secretion also include fixative I, described fixative I by paraformaldehyde,
Disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, redistilled water and hydrochloric acid composition, institute
The mass volume ratio stating paraformaldehyde and described redistilled water is 35～45g/L, described phosphoric acid hydrogen two
Sodium is 1.0～1.5g/L with the mass volume ratio of described redistilled water, described potassium dihydrogen phosphate and institute
The mass volume ratio stating redistilled water is 0.2～0.3g/L, described sodium chloride and described redistilled water
Mass volume ratio is 7～9g/L, and described potassium chloride with the mass volume ratio of described redistilled water is
0.1～0.3g/L, the pH of fixative I is adjusted to 7.2～7.4 by described hydrochloric acid.
The treatment fluid of nasal secretion the most according to claim 1, it is characterised in that
The treatment fluid of described nasal secretion also includes fixative II, and described fixative II is by methanol and third
Ketone is with volume ratio as 1:0.5～1.5 formulated.
5. use the treatment fluid described in claim 1 or 2 to carry out the process side of nasal secretion
Method, it is characterised in that comprise the following steps:
A () takes nasal secretion, the nasal secretion obtained is transferred to described cleanout fluid
In 20～40ml, add described Digestive system, the described Digestive system of addition and the body of described cleanout fluid
Long-pending ratio is 1～5:100, agitator 400～600rpm, vibration mixing in 20～40 minutes；
B () 1500～2500rpm is centrifuged 5～15 minutes, abandoning supernatant, and shake dissipates gently
Open the precipitation of bottom, add and protect liquid described in 20～40ml and mix, be 30～200 with aperture
μm cell strainer filters, and collects filtrate；
C cell, by the cell membrane filtration that described filtrate aperture is 3～8 μm, is filtered by ()
Cell on film is transferred on microscope slide, obtains cell smear；
D () carries out cell to described cell smear and fixes, dyeing processes, then by micro-
Sem observation cell smear.
The processing method of nasal secretion the most according to claim 5, it is characterised in that
In described step (c), described filtrate makes cell smear by cell slide machine.
The processing method of nasal secretion the most according to claim 6, it is characterised in that
Described cell slide machine make cell smear particularly as follows:
Described cell filter membrane is carried out Negative pressure, when covering 8～15% on cell filter membrane
Long-pending cell, vacuum suction stops, and the cell in described filtrate adsorbs at described cell filter membrane
Surface；
The side that described cell filter membrane is adsorbed with cell is attached on microscope slide, by positive drum pressure
Gas and electrostatic adsorption, make absorption be transferred on microscope slide at the cell of cell filter membrane surface,
Obtain cell smear.
The processing method of nasal secretion the most according to claim 5, it is characterised in that
In described (d), described cell is fixed and is used the fixative I in claim 3 or right
Require that the fixative II in 4 is fixed.
The processing method of nasal secretion the most according to claim 5, it is characterised in that
In described (b), the aperture of described cell strainer is 80～100 μm.
The processing method of nasal secretion the most according to claim 5, it is characterised in that
In described (a), use sampler that nasal secretion is sampled.
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| CN102393318A (en) * | 2011-08-19 | 2012-03-28 | 张雪云 | Simple and quick preparation method of thin-layer liquid-based cytology cell smear |
| CN102435728A (en) * | 2011-09-07 | 2012-05-02 | 福州大学 | Preparation method for positive control substance for inspection and control of quality in immunohistochemical process |
| CN102762982A (en) * | 2010-02-05 | 2012-10-31 | 株式会社日冷生物科学 | Pretreatment solution for immunohistochemical staining and condensed solution thereof |
| CN103196729A (en) * | 2013-04-10 | 2013-07-10 | 吴鹏 | Reagent for processing mucus |
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| US20070054258A1 (en) * | 2003-10-07 | 2007-03-08 | Sysmex Corporation | Method of removing mucus and cell treatment solution and preservative solution used therefor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102762982A (en) * | 2010-02-05 | 2012-10-31 | 株式会社日冷生物科学 | Pretreatment solution for immunohistochemical staining and condensed solution thereof |
| CN102393318A (en) * | 2011-08-19 | 2012-03-28 | 张雪云 | Simple and quick preparation method of thin-layer liquid-based cytology cell smear |
| CN102435728A (en) * | 2011-09-07 | 2012-05-02 | 福州大学 | Preparation method for positive control substance for inspection and control of quality in immunohistochemical process |
| CN103196729A (en) * | 2013-04-10 | 2013-07-10 | 吴鹏 | Reagent for processing mucus |
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