CN107831058A - The preparation method and cell block of cell block based on courageous and upright cell sample - Google Patents
The preparation method and cell block of cell block based on courageous and upright cell sample Download PDFInfo
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- CN107831058A CN107831058A CN201711341745.4A CN201711341745A CN107831058A CN 107831058 A CN107831058 A CN 107831058A CN 201711341745 A CN201711341745 A CN 201711341745A CN 107831058 A CN107831058 A CN 107831058A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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Abstract
The present invention relates to a kind of preparation method and cell block of the cell block based on courageous and upright cell sample, it is related to biological technical field.Preparation method includes：By courageous and upright cell sample after the first fixer liquid cleans, centrifuge for the first time, the precipitation sample of collection first, the second fixer of addition, after being well mixed, second of centrifugation, obtain the second precipitation, by the second precipitation dehydration, transparent, waxdip and embedding.Wherein, the first fixer is alcohol, acetic acid liquid, and alcohol, acetic acid liquid is 18 19 according to volume ratio with acetic acid by ethanol that volume fraction is 24 25.5%：1 is obtained by mixing, and its preparation efficiency is high, easy to operate, obtained cell block, its can persistence, cell component is more, and eucaryotic cell structure is complete, high power clear background, effectively improves the accuracy rate of diagnosis.
Description
Technical field
The present invention relates to biological technical field, and the preparation of more particularly to a kind of cell block based on courageous and upright cell sample
Method and cell block.
Background technology
Exfoliative cytology inspection is one of diagnosing tumor effective means, a small amount of chest and abdomen drawn in common smear manufacturing process
Water can not represent the situation of whole Pleural effusions, and make and finish remaining Pleural effusions often abandoned afterwards, and cell block can
To collect most cells in censorship Pleural effusions, to observe the form of most cells, and cell block is easy to preserve,
The Sensitivity and Specificity of cell pathology diagnosis is substantially increased, regular growth smear is compensate for and makes limited and not enough make more
The weak point of kind of dyeing, can further be studied and perspective study.
But the set time is grown during existing cell block makes, and cell loss is obvious, influences diagnostic result, meanwhile, it is existing
Courageous and upright cell sample in due to substantial amounts of red blood cell be present, the quality and diagnostic result of wax stone are influenceed, to some difficult diseases
Example can also influence the judgement to positive findings when needing to do cell immunohistochemical staining.
The content of the invention
It is an object of the invention to provide a kind of cell block, its can persistence, cell component is more, and eucaryotic cell structure is complete
It is whole, high power clear background, effectively improve the accuracy rate of diagnosis.
Another object of the present invention is to provide a kind of preparation method of the cell block based on courageous and upright cell sample, it is made
Standby efficiency high, it is easy to operate, effectively solve the above problems.
The present invention is solved its technical problem and realized using following technical scheme.
The present invention proposes a kind of preparation method of the cell block based on courageous and upright cell sample, and it includes：
By courageous and upright cell sample after the cleaning of the first fixer liquid, centrifuge for the first time, collect the first precipitation sample, add the
Two fixers, after being well mixed, second of centrifugation, the second precipitation is obtained, by the second precipitation dehydration, transparent, waxdip and embedding.
Wherein, the first fixer is alcohol, acetic acid liquid, and alcohol, acetic acid liquid is by ethanol and second that volume fraction is 24-25.5%
Acid is 18-19 according to volume ratio：1 is obtained by mixing.
The present invention proposes a kind of cell block as made from above-mentioned preparation method.
The preparation method of the cell block based on courageous and upright cell sample of the embodiment of the present invention and the beneficial effect of cell block
Fruit is：
By when courageous and upright cell sample is cleaned through the first fixer, on the one hand effectively destroying red blood cell, carrying on the back high power
Scape is clear, prevents the interference of red blood cell, and on the other hand effectively fixed required cell, such as tumour cell etc., prevents it certainly
It is molten.Improve the enrichment of wax stone cell.
By first time centrifuge, after removing most of red blood cell, collect first precipitation sample, by with the second fixer
Coordinate, further effectively improve the fixation of tangible required cell, broken cell is removed, further improve wax stone cell
Integrality.
The cell block as made from aforesaid way, its can persistence, cell component is more, and eucaryotic cell structure is complete, high power the back of the body
Scape is clear, effectively improves the accuracy rate of diagnosis.
Embodiment
, below will be in the embodiment of the present invention to make the purpose, technical scheme and advantage of the embodiment of the present invention clearer
Technical scheme be clearly and completely described.Unreceipted actual conditions person, builds according to normal condition or manufacturer in embodiment
The condition of view is carried out.Agents useful for same or the unreceipted production firm person of instrument, it is the conventional production that can be obtained by commercially available purchase
Product.
The preparation method and cell block of the cell block based on courageous and upright cell sample of the embodiment of the present invention are entered below
Row illustrates.
The present invention provides a kind of cell block, and its cell component is more, and eucaryotic cell structure is complete, high power clear background, effectively carries
The accuracy rate of height diagnosis, for applied to pathological detection research etc., it to be made by following methods：
S1. courageous and upright cell sample is centrifuged for the first time after the cleaning of the first fixer, collects the first precipitation sample.
Due to containing substantial amounts of red blood cell in courageous and upright cell sample, and the presence of red blood cell for wax stone quality and examine
Disconnected result, and the judgement to positive findings can be also influenceed when needing to do cell immunohistochemical staining to some intractable cases
Bad influence all be present.Therefore, destroyed using the first fixer for red blood cell so that the section of obtained wax stone
High power clear background, it is easy to accurately observe and diagnose.
Preferably, the first fixer is alcohol, acetic acid liquid.Wherein, ethanol handles existing fixation again to histiocytic
There is dehydration, ethanol borrows precipitation to make protein denaturation, acetic acid also known as glacial acetic acid, and it is unable to albumin, but can sink
Shallow lake nucleoprotein, permeability is strong, fixed fast, and red blood cell can be made to expand and crush, good with the use of effect with ethanol.Therefore, inciting somebody to action
When courageous and upright cell sample cleans through the first fixer, red blood cell is on the one hand effectively destroyed, makes high power clear background, on the other hand has
Cell required for effect is fixed, such as tumour cell etc., prevents its self-dissolving.
It when courageous and upright cell sample is cleaned through the first fixer, can be vibrated, it is red to effectively facilitate the destruction of alcohol, acetic acid liquid
Cell, at the same it is easy to operation, effectively save the time prepared and technique.
In preferred embodiments of the present invention, alcohol, acetic acid liquid by ethanol and acetic acid that volume fraction is 24-25.5% according to
Volume ratio is 18-19：1 is obtained by mixing.Alternatively, the volume ratio of alcohol, acetic acid liquid and courageous and upright cell sample should be 12-16:1,
Red blood cell is effectively destroyed, while is centrifuged through first time, broken red blood cell is removed with supernatant, improves last cell block
Accuracy.
Preferably, in preferred embodiments of the present invention, centrifugation for the first time is to be centrifuged under conditions of 2900-3100r/min
5-6min, effective cell effectively is precipitated, remove broken red blood cell etc., collect the first precipitation sample, meanwhile, in the centrifugation model
Enclosing interior effectively prevents required effective cell from rupturing.
Preferably, if precipitation is less, the supernatant after centrifuging for the first time is collected, is centrifuged under these conditions, will
The precipitation of centrifugation gained is incorporated to the first precipitation sample, but which collection efficiency is relatively low, it is preferable that after centrifuging for the first time, stands
After 5-10min, collect precipitation and apart from precipitation 0-1.5cm, i.e., distance precipitate 1.5cm in supernatant, break up, again from
The heart, abandons supernatant, collects the first precipitation sample, under which, effectively avoids the waste of cell, while effectively improve the first precipitation sample
This accumulation.
Alternatively, before being cleaned through the first fixer liquid, courageous and upright cell sample is stood, cell deposition is taken in bottom
Prepared by bottom sample, effectively improve the producing efficiency of the preparation method of the cell block based on courageous and upright cell sample.
S2. the second fixer is added in the first precipitation sample, after being well mixed, second of centrifugation, obtains the second precipitation.
Wherein, the second fixer is the neutral buffered formalin liquid that concentration is 3.5-4.3wt%.Because formaldehyde can be with protein
Amino combines, and makes protein coagulating, and then effectively fixed cell, prevents aqtocytolysis, influence the quality of cell block.Need
Bright, the first precipitation sample is well mixed with the second fixer, it is necessary to first gently break up the first precipitation sample.
The shrinkage factor of cell during in order to further reduce fixed, so as to get cell block in cell closer to reality
State, to prevent hypersystole, alternatively, the temperature of neutral buffered formalin liquid is 3-5 DEG C, i.e. precooling neutral buffered formalin in advance
Liquid.
But because neutral buffered formalin liquid has certain pollution for environment, it is preferred that the second fixer is also
It can be the ethanol solution that volume fraction is 93-96%.More preferably volume fraction is 95% ethanol solution, wherein, volume
The environmental sound while ethanol solution that fraction is 95% can effectively fix cell, while certain take off can be carried out to cell
Water.
Alternatively, centrifuge for the second time and 5-6min is centrifuged under conditions of 2800-3100r/min, effectively make cell precipitation, go
Except supernatant, the second precipitation sample is collected, meanwhile, effectively prevent cell rupture in the range of the centrifugation.
Wherein, the collection of the second precipitation sample, such as can be to sign to choose the second precipitation sample firmly using tip, use
Filter paper is wrapped.
S3. by the second precipitation dehydration, transparent, waxdip and embedding.
Wherein, dehydration includes：At 44-46 DEG C, 110- is dehydrated in the ethanol solution that volume fraction is 90-96%
125min, then it is dehydrated 1-2 times in absolute ethyl alcohol, is dehydrated 85-120min every time.It is fixed before dehydration, makes second to sink
Cell block is more readily formed in shallow lake.
It is transparent including：Under conditions of 45-47 DEG C, continuous dipping 1- is carried out to the 3rd precipitation after dehydration using dimethylbenzene
2 times, 20-30min is carried out every time.In time and numbers range, transparent processing effect is good, and eucaryotic cell structure is good, can effectively prevent
Cell becomes fragile caused by long soaking time, the problem of being unfavorable for the later stage and cut into slices.
In preferred embodiments of the present invention, the waxdip under conditions of 58-65 DEG C, the efficiency of waxdip can be effectively facilitated.
Waxdip includes：After the 3rd wax of the precipitation coated on melt surface shape after will be transparent, by the quick pressing of the 3rd precipitation
In the surface of wax, 20-25 DEG C is quickly cooled to, 5-20min is stood and toasts 4-6min after 60-65 DEG C.
Using above method waxdip, cell block is located at the top of wax stone, on the one hand easily section, while cell knot under light microscopic
Structure is more complete, and cell and clear background, thickness is uniform, and mesothelium, tumour and inflammatory cell bright, cell is clear, caryoplasm dyeing
Contrast is obvious, and cellular morphology is approximate with conventional organization making paraffin section HE, easily differentiates, is easy to observation analysis, on the other hand
Paraffin mass can preserve for a long time.
Waxdip can also be after 58-65 DEG C of first time waxdip 30-35min, after being incubated 10-20min, then in 58-65
DEG C of second waxdip carries out 110-130min.Effectively improve the efficiency of waxdip.
Alternatively, in addition to by the courageous and upright cell sample after embedding the thin slice that thickness is 3-6um is switched to, effectively solved because cutting
Not the problem of too thick caused eucaryotic cell structure of piece is not known, while also allow for subsequently carrying out routine or immunohistochemical staining, detection etc.,
It will not be described here.
Nearly all cell in courageous and upright cell sample is collected using the above method, effectively avoids wasting.
The feature and performance of the present invention are described in further detail with reference to embodiments.
Embodiment 1
A kind of cell block, it is made by following preparation method：
By courageous and upright cell sample after the cleaning of the first fixer, after centrifuging 5min for the first time under conditions of 3000r/min,
After standing 10min, precipitation and the supernatant apart from precipitation 1cm are collected, breaks up, is centrifuged again under conditions of 3000r/min
5min, supernatant is abandoned, collect the first precipitation sample, the second fixer is added, after being well mixed, second under conditions of 3000r/min
Secondary centrifugation 5min, obtains the second precipitation, the second precipitation is dehydrated, transparent, waxdip, embedding, the courageous and upright cell sample after embedding cut
To the thin slice that thickness is 4um.
Wherein, the first fixer is alcohol, acetic acid liquid, and alcohol, acetic acid liquid is pressed by the ethanol that volume fraction is 25% with acetic acid
It is 19 according to volume ratio：1 is obtained by mixing.
Second fixer is the neutral buffered formalin liquid that the concentration that temperature is 4 DEG C is 4wt%.
Dehydration includes：At 45 DEG C, 120min is dehydrated in the ethanol solution that volume fraction is 95%, then in absolute ethyl alcohol
Middle dehydration 120min.
It is transparent including：Under conditions of 45 DEG C, continuous dipping is carried out to the second precipitation after dehydration using dimethylbenzene
30min。
After waxdip is included in 65 DEG C of first time waxdip 30min, after being incubated 20min, then carried out in 65 DEG C of second of waxdips
110min。
Embodiment 2
A kind of cell block, it is made by following preparation method：
Courageous and upright cell sample is stood overnight, takes bottom 20ml courageous and upright cell sample after the cleaning of the first fixer,
5min is centrifuged under conditions of 3100r/min for the first time, the first precipitation sample is collected, adds the second fixer, after being well mixed,
Second of centrifugation 5min, obtains the second precipitation under conditions of 3100r/min, the second precipitation is dehydrated, transparent, waxdip, embedding, will wrapped
Courageous and upright cell sample after burying is switched to the thin slice that thickness is 5um.
Wherein, the first fixer is alcohol, acetic acid liquid, and alcohol, acetic acid liquid is by ethanol and acetic acid that volume fraction is 25.5%
It is 19 according to volume ratio：1 is obtained by mixing.
Second fixer is the neutral buffered formalin liquid that the concentration that temperature is 4.5 DEG C is 3.5wt%.
Dehydration includes：At 45 DEG C, 110min is dehydrated in the ethanol solution that volume fraction is 96%, then in absolute ethyl alcohol
Middle dehydration 85min.
It is transparent including：Under conditions of 47 DEG C, continuous dipping is carried out 2 times to the second precipitation after dehydration using dimethylbenzene,
20min is carried out every time.
After waxdip is included in 60 DEG C of first time waxdip 35min, after being incubated 10min, then carried out in 62 DEG C of second of waxdips
115min。
Embodiment 3
A kind of cell block, it is made by following preparation method：
Courageous and upright cell sample is stood overnight, takes bottom 30ml courageous and upright cell sample after the cleaning of the first fixer,
6min is centrifuged under conditions of 3100r/min for the first time, the first precipitation sample is collected, adds the second fixer, after being well mixed,
Second of centrifugation 6min, obtains the second precipitation under conditions of 2900r/min, the second precipitation is dehydrated, transparent, waxdip, embedding, will wrapped
Courageous and upright cell sample after burying is switched to the thin slice that thickness is 3um.
Wherein, the first fixer is alcohol, acetic acid liquid, and alcohol, acetic acid liquid is by ethanol and acetic acid that volume fraction is 25.5%
It is 18 according to volume ratio：1 is obtained by mixing.
Second fixer is the neutral buffered formalin liquid that the concentration that temperature is 3.5 DEG C is 4wt%.
Dehydration includes：At 45 DEG C, 120min is dehydrated in the ethanol solution that volume fraction is 95%, then in absolute ethyl alcohol
It is middle to be dehydrated 2 times, 85min is dehydrated every time.
It is transparent including：Under conditions of 45 DEG C, continuous dipping is carried out 2 times to the second precipitation after dehydration using dimethylbenzene,
20min is carried out every time.
After waxdip is included in 60 DEG C of first time waxdip 32min, after being incubated 15min, then carried out in 65 DEG C of second of waxdips
110min。
Embodiment 4
A kind of cell block, it is made by following preparation method：
Courageous and upright cell sample is stood overnight, takes bottom 25ml courageous and upright cell sample after the cleaning of the first fixer,
6min is centrifuged under conditions of 2900r/min for the first time, the first precipitation sample is collected, adds the second fixer, after being well mixed,
Second of centrifugation 6min, obtains the second precipitation under conditions of 2900r/min, the second precipitation is dehydrated, transparent, waxdip, embedding, will wrapped
Courageous and upright cell sample after burying is switched to the thin slice that thickness is 6um.
Wherein, the first fixer is alcohol, acetic acid liquid, and alcohol, acetic acid liquid is by ethanol and acetic acid that volume fraction is 25.5%
It is 18 according to volume ratio：1 is obtained by mixing.
Second fixer is the neutral buffered formalin liquid that the concentration that temperature is 4.5 DEG C is 4wt%.
Dehydration includes：At 44 DEG C, 125min is dehydrated in the ethanol solution that volume fraction is 90%, then in absolute ethyl alcohol
It is middle to be dehydrated 2 times, 100min is dehydrated every time.
It is transparent including：Under conditions of 46 DEG C, continuous dipping is carried out to the second precipitation after dehydration using dimethylbenzene
30min。
Waxdip is included under conditions of 60 DEG C, after the second wax of the precipitation coated on melt surface shape after transparent processing,
Second precipitation is quickly flattened in the surface of wax, is quickly cooled to 23 DEG C, 10min is stood and toasts 5min after 62 DEG C.
Embodiment 5
A kind of cell block, it is made by following preparation method：
By 20ml courageous and upright cell sample after the cleaning of the first fixer, centrifuged for the first time under conditions of 3000r/min
5min, the first precipitation sample is collected, adds the second fixer, after being well mixed, is centrifuged for the second time under conditions of 3000r/min
5min, the second precipitation is obtained, the second precipitation is dehydrated, transparent, waxdip, embedding, the courageous and upright cell sample after embedding is switched to thickness
For 6um thin slice.
Wherein, the first fixer is alcohol, acetic acid liquid, and alcohol, acetic acid liquid is pressed by the ethanol that volume fraction is 25% with acetic acid
It is 19 according to volume ratio：1 is obtained by mixing.
Second fixer is the neutral buffered formalin liquid that the concentration that temperature is 4 DEG C is 4wt%.
Dehydration includes：At 45 DEG C, 120min is dehydrated in the ethanol solution that volume fraction is 95%, then in absolute ethyl alcohol
Middle dehydration 120min.
It is transparent including：Under conditions of 45 DEG C, continuous dipping is carried out to the second precipitation after dehydration using dimethylbenzene
30min。
After waxdip is included in 60 DEG C of first time waxdip 30min, after being incubated 10min, then carried out in 60 DEG C of second of waxdips
120min。
To sum up, the preparation method of the cell block of the offer of the embodiment of the present invention, its is simple and convenient, and preparation efficiency is high,
Set time is shorter, the almost all of cell in effective fixed sample, prevents the waste of cell.It is thin as made from the above method
Born of the same parents' wax stone, its eucaryotic cell structure understand, improve the accuracy rate of pathological diagnosis.
Embodiments described above is part of the embodiment of the present invention, rather than whole embodiments.The reality of the present invention
The detailed description for applying example is not intended to limit the scope of claimed invention, but is merely representative of the selected implementation of the present invention
Example.Based on the embodiment in the present invention, what those of ordinary skill in the art were obtained under the premise of creative work is not made
Every other embodiment, belongs to the scope of protection of the invention.
Claims (10)
- A kind of 1. preparation method of the cell block based on courageous and upright cell sample, it is characterised in that including：By courageous and upright cell sample after the cleaning of the first fixer, centrifuge for the first time, collect the first precipitation sample, add second and fix Liquid, after being well mixed, second of centrifugation, the second precipitation is obtained, by the described second precipitation dehydration, transparent, waxdip and embedding；Wherein, first fixer is alcohol, acetic acid liquid, and the alcohol, acetic acid liquid is by ethanol that volume fraction is 24-25.5% According to volume ratio it is 18-19 with acetic acid：1 is obtained by mixing.
- 2. preparation method according to claim 1, it is characterised in that second fixer is that concentration is 3.5- 4.3wt% neutral buffered formalin liquid.
- 3. preparation method according to claim 2, it is characterised in that the temperature of the neutral buffered formalin liquid is 3-5 DEG C.
- 4. preparation method according to claim 1, it is characterised in that second fixer is that volume fraction is 93- 96% ethanol solution.
- 5. preparation method according to claim 1, it is characterised in that dehydration includes：At 44-46 DEG C, it is in volume fraction 110-125min is dehydrated in 90-96% ethanol solution, is then dehydrated 1-2 times in absolute ethyl alcohol, is dehydrated 85- every time 120min。
- 6. preparation method according to claim 3, it is characterised in that it is transparent including：Under conditions of 45-47 DEG C, use Dimethylbenzene carries out continuous dipping 1-2 times to the second precipitation after dehydration, carries out 20-30min every time.
- 7. preparation method according to claim 3, it is characterised in that the waxdip under conditions of 58-65 DEG C.
- 8. preparation method according to claim 7, it is characterised in that be coated on second precipitation after transparent processing After the wax of melt surface shape, the described second precipitation is quickly flattened in the surface of the wax, is quickly cooled to 20-25 DEG C, is stood 5-20min toasts 4-6min after 60-65 DEG C.
- 9. preparation method according to claim 1, it is characterised in that also include the courageous and upright cell sample after embedding being switched to Thickness is 3-5um thin slice.
- 10. cell block made from the preparation method as described in claim 1-9 any one.
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Cited By (1)
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| CN116296701A (en) * | 2023-03-27 | 2023-06-23 | 江苏凯基生物技术股份有限公司 | Cell fixing liquid and application thereof |
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Application publication date: 20180323 |