CN106645153A - Fast staining and detecting method for cell pathological sections - Google Patents
Fast staining and detecting method for cell pathological sections Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
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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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- 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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
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Abstract
The invention discloses a fast staining and detecting method for cell pathological sections. Fast staining treatment is performed on sections with a special dyeing method, cell nucleuses and cytoplasm can be stained with colors, and the color contrast is obvious; the color of cytoplasm is filtered through detection software conversion channel and nonlinear calibration, the DNA content of the cell nucleuses is accurately measured, a corresponding cell morphology picture of each cell nucleuses is collected by a high-resolution microscope and a digital color camera lens, and integral digital sections are synthesized; lesions are judged according to change of DNA content of single and cluster cell nucleuses in combination with characteristic change of cell morphology. The special dyeing method is adopted, the cell nucleuses and the cytoplasm can show clear colors in a short time, during DNA measurement, all single independent cell nucleuses in the sections can be accurately measured, cluster cell nucleuses can be measured and recorded, and the diagnosis sensitivity and specificity can be improved; scanning and digitalization of the whole sections can be completed during DNA measurement, and remote diagnosis is facilitated.
Description
Technical field
The present invention relates to biological cell, tissue staining technical field and cytology is qualitative and DNA quantitative detecting analysis methods,
Specifically cut into slices after specific stain, while carrying out qualitative and quantitative determination.
Background technology
, used as modern pathology component, because of its draw materials convenience, relative noninvasive, detection is quick and economical real for cell pathology
With increasingly receiving publicity.Traditional cell detection method refers to that tester passes through under the microscope observation of cell morphological feature
Change, so as to judge whether cell occurs pathology.In order to the effective observation of cell, generally need to enter cell before testing
Row dyeing, conventional colouring method has haematoxylin eosin staining procedures, Papanicolaou's vaginal smear technique, shore decoration method etc..These methods it is many according to
Acidity of Aikalinity according to different organelles is different, so as to reference to the dyestuff of different colours, to distinguish cell membrane, cell under the microscope
Some changes of slurry and nucleus and cell other structures.This morphological analysis are only observer, and to present some thin
Born of the same parents' feature（Such as there is situation in nucleus size, karyoplasmic ratio or nuclear particulate）.Due to individual otherness, cause cell
Can complex shape be changeable, accurately judge and understand metamorphosis whether real pathology actually, depends primarily on cell and studies medicine
Raw experience and qualification, therefore, the subjective repeatability of testing result is bad.Due to the restriction of naked eyes identification, also very
Hardly possible distinguishes the property of Junctional rhythm, or cannot find the atypical pathology of some morphological changes, can unavoidably cause
The case that certain amount is failed to pinpoint a disease in diagnosis.
Chromosome is a complete Encyclopedia of Life, and each species has the chromosome of a set of uniqueness（That is chromosome
Group）, for the presence of species, chromosome has decisive role.Meanwhile, fine mitosis mechanism also ensure that dyeing
During passage, quantity and structure remain stable to body.But carcinogen, rare inherited disorder and accidental
Mitosis mistake is destroyed may this stable state.Beginning of the century in 19 end of the century 20, David von Hansemann and
Theodor Boveri Von Hansemann have found that all cancer cells all contain abnormal chromosome.With entering for detection technique
Step, more and more as shown by data：Cancer cell and just all can run counter to " genome is stable " in the namely sick cell of canceration
This law of nature, by producing the cell of quantity abnormal chromosome tumour is caused.The abnormal quantity of chromosome makes total with thousand
The gene of meter and they encode protein be in imbalance state so that cell produce malignant phenotype, also for cell development into
Condition is created for more serious chromosome abnormality cell.Cancer cell numerical abnormalities of chromosomes degree is higher, chromosomal variation speed
Degree is faster, therefore the pathology situation of cell can be judged by the change detection to chromosome number in nucleus or DNA content,
This belongs to quantitative determination.DNA specific stains are first passed through, cell DNA is marked, because dyeing is chemical reaction, can be
Label sets up linear relationship and DNA content between, by detect label number, judge DNA content size.It is this
Method can be good in the change of pathology early detection inhereditary material, sensitiveness；Detection is carried out by equipment, with the experience of operating personnel or
Qualification is unrelated, it is to avoid the error that subjective factor is caused, reproducibility of results is good.But the colouring method used in this detection is made
Nucleus is can only see when detection, in the case of shortage is cytoplasmic, it is impossible to judge sick cell type.Additionally, checking
During nucleus, as without cytoplasm, but the core by dyeing is difficult to distinguish impurity in sample or real thin
Karyon, if impurity is mistaken for into diagnosis cell, will also result in mistaken diagnosis.The graphical analysis of DNA measurements is overlapped to multiple nucleus
The segmentation of i.e. agglomerating core is difficult point, typically directly skips this kind of cell, nonrecognition, and this can cause to fail to pinpoint a disease in diagnosis.
Therefore, the method dyeing time of cytomorphology qualitative analysis is short, and flow process is simple, but unstable, will to technician
Ask high, diagnosis is based only on the morphological feature of cell, the experience and skill of dependent cells scholar, misdiagnosis rate is very high；DNA is quantitative
, based on DNA special colouring method, dyeing time is long, and dyeing condition has high demands, and reagent is unstable for analysis method.DNA measurement according to
The nuclear features of Lai Yu, to individual cells nuclear energy measurement is split well, for multiple nucleus overlaps are dividing for agglomerating core
The difficult point that measurement is DNA quantitative analyses is cut, this kind of cell, nonrecognition is typically directly skipped, and can not provide any with regard to cell
Information in terms of morphology.
The content of the invention
The technical problem to be solved in the present invention is：Overcome the deficiencies in the prior art, there is provided a kind of cell pathology section is quick
Dyeing detection method, makes dyeing time short, and flow process is simple, and section after dyeing can simultaneously carry out cytomorphology analysis and nucleus
DNA quantitative determinations, improve the degree of accuracy of sick cell detection.
The technical solution adopted for the present invention to solve the technical problems is：A kind of cell pathology section rapid dyeing detection side
Method, comprises the following steps：
1st step：Dyeing process is carried out to section, makes nucleus and cytoplasm catch color, and both contaminate color contrast
Substantially, it is ensured that in the image for capturing, nucleus and cytoplasm can clearly be presented；
2nd step：By inspection software ALT-CH alternate channel and gamma correction, filtration cell matter color, accurately measure nucleus DNA and contain
Amount, while gathering the corresponding cytomorphology figure of each nucleus using high resolution microscope and the collection of digital color camera
Piece and synthesis whole number section；
3rd step：Sick cell is judged according to change of the single and packed cell nuclear dna content change with reference to cytomorphology.
Dyeing in above-mentioned 1st step is processed and included：
Step a：The sample for making is spontaneously dried；
Step b：Is put in dry section and fix in pretreatment fluid 10-30 minutes, then water displacement wash 4-5 time；
Step c：Acidifying 5-10 minutes in 50～55 DEG C of hydrochloric acid solution are put into through the sample of step b process, then water displacement wash
4-5 time；
Step d：Dyeing 5-15 minutes in 50～55 DEG C of specific stain liquid A are put into through the sample of step c acidifying, then flowing water
Washing 4-5 time；
Step e：5 minutes are embathed through the sample distilled water of step d dyeing；
Step f：The sample embathed through step d is sequentially placed into 50% alcohol and is dehydrated 2 minutes, dehydration 2 minutes in 75% alcohol,
Dehydration 3 minutes in 95% alcohol；
Step g：Sample after step f is dehydrated step by step is placed in dyeing 1-5 minutes in specific stain liquid B；
Step h：Through step g dyeing sample in soaked in absolute ethyl alcohol twice, 3 minutes every time；
Step i：Through the sample natural gum cover plate sealing of step h.
Reagent used in above-mentioned dyeing is processed includes pretreatment fluid, hydrochloric acid solution, specific stain liquid A and specific stain liquid B,
Wherein：
The pretreatment fluid is using any one in AF liquid, 4% paraformaldehyde, 10% neutral formalin, 95% ethanol, BS liquid or one
More than kind；
The hydrochloric acid solution includes by mass percentage 35%～45% concentrated hydrochloric acid, 55%～65% distilled water；
The specific stain liquid A by mass percentage comprising 0.10%～1% phenothiazines dyestuff, 1.8%～2.2% hydrochloric acid solution,
0.09%～0.12% Sodium Metabisulfite, residual mass percentage are supplied by distilled water, and the phenothiazines dyestuff is by methine
One or more compositions in blue, reddish black A, thionine；
Described specific stain liquid B includes by mass percentage 0.005%～0.1% acid dyes, 75%～96% alcohol reagent, remains
Remaining mass percent is supplied by distilled water, and the acid dyes is by Yihong, oil red O, Ponceaux 2R, biebrich Scarlet
Plant or more than one compositions, alcohol reagent is by one or more groups in ethanol, methyl alcohol, isopropanol, the tert-butyl alcohol, ethylene glycol
Into.
The volume of each reagent component of foregoing description and performance figure provide a kind of proportionate relationship, are not offered as this
Each reagent can only be with above-mentioned volume and quality in bright.
Preferably, the optimal process time in above-mentioned steps is：Step b fixes 15 minutes, and step c is acidified 7 minutes, step
Rapid d is dyeed 10 minutes, and step f is dyeed 3 minutes.
In 2nd step on inspection software by original green Channel-shifted be red passage, filtration cell matter color；Gamma correction mistake
Journey includes adjustment light-source brightness, image exposure value, gradation of image deviant, image segmentation threshold compensating parameter, Bayer pattern
（Bayer）The parameters such as model analyzing parameter, exponential model conversion, so as to accurately measure nuclear DNA content.
The process of gamma correction is specific as follows：
Step（1）：One class index model conversion regulation parameter, Bayer pattern are set（Bayer）Parsing regulation parameter and image point
Cut valve value compensation parameter；
Step（2）：Routinely microscope regulative mode adjusts DNA testing equipment illuminations, field stop, aperture diaphragm, adjusts and exposes
The light time, image histogram showed the bright dark state of image in unsaturation scope in specified range intervals；
Step（3）：Place a standard to cut into slices and arrange a scanning area, after the completion of scanning, the IOD of check criteria section（Product
Light splitting density）, diploid cell standard variance CV, to determine that DNA testing equipments resolution ratio is whether normal, calculate tetraploid IOD
Whether linear relationship is accorded with the ratio of dliploid IOD.The sweep parameter of above parameter and quality control system is contrasted, with Quality Control equipment
For standard, exponential model transformation parameter and Bayer analytic parameters are adjusted；
Step（4）：Repeat the above steps（1）-（3）, until sweep parameter index IOD of same standard section（Integral light is close
Degree）, diploid cell standard variance CV and tetraploid IOD and dliploid IOD linear relationship, DNA testing equipments with
In the range of allowable error, correction is completed parameter its difference that the scanning of standard quality control system is obtained.
Digital color camera collection cellular morphology figure is clapped in the case where high resolution microscope is set to 20 times in 2nd step
Take the photograph, testing staff can point out without the need for diagosis under mirror according to DNA content, reading section picture can make diagnosis, save diagosis people
Member's time, improve diagnosis efficiency.
Concrete steps in 3rd step include：
I：Choose the normal cell in same section and do internal reference cell, and contained all nucleus according to DNA by inspection software
The descending order arrangement of amount；
II：During DNA content quantitative analysis, corresponding nuclear full cellular morphology can be observed, determine whether real cell
Core and karyoplasmic ratio, metamorphosis, and cell type is judged；
III：During cytomorphology interpretation, inspection software presses the corresponding cellular morphology figure of the descending arrangement of nuclear DNA content
Piece, points out according to DNA content, and reading section picture can make diagnosis；
Ⅳ：Multiple nucleus overlaps are agglomerating core image segmentation and calculate the DNA content of these agglomerating cores by inspection software, then
By cytology qualitative analysis；
Ⅴ：According to II, III, IV step comprehensive analysis, judge to be detected cell as normal or improper.
The present invention is presented at short notice clear face as a result of special colouring method, nucleus and cell mass-energy
Color, flow process is simple, and efficiency high, applied range provides possibility for Frozen Section Diagnosis in art；By inspection software ALT-CH alternate channel
And gamma correction, filtration cell pulp color, accurately measure nuclear DNA content；Not only will own in section when DNA is measured
Single independent cell core accurate measurement, and packed cell core is measured and recorded, it is qualitative by cytology, improve diagnostic sensitivity
And specificity；The corresponding cytomorphology picture of each nucleus and the scanning of whole digital slices are gathered while DNA is measured,
Can digital slice transmission, remote medical consultation with specialists etc..
Description of the drawings
Fig. 1 is the flow chart of method for integrative detection of cells of the present invention；
Fig. 2 is effect diagram after Feulgen dyeing；
Fig. 3 is effect diagram after rapid dyeing of the invention；
Fig. 4 is according to the arranging cells morphology picture that nuclear DNA content is descending in the present invention；
Fig. 5 is the view in a visual field in nucleus DNA quantitative analysis core collection of illustrative plates in the present invention；
Fig. 6 is the corresponding nuclear full cellular morphology figure of arrow indication in Fig. 5；
Fig. 7 is the nucleus view of arrow indication cell in Fig. 5；
Fig. 8 is cytomorphology analysis report.
Specific embodiment
Below in conjunction with the accompanying drawings embodiment, is described further to the present invention：
Flow chart as shown in Figure 1, the 1st step：Dyeing process is carried out to section, makes nucleus and cytoplasm catch color, and
It is obvious that both contaminate color contrast, it is ensured that in the image for capturing, nucleus and cytoplasm can clearly be presented；
2nd step：By inspection software ALT-CH alternate channel and gamma correction, filtration cell matter color, accurately measure nucleus DNA and contain
Amount, while gathering the corresponding cytomorphology figure of each nucleus using high resolution microscope and the collection of digital color camera
Piece and synthesis whole number section；
3rd step：Sick cell is judged according to change of the single and packed cell nuclear dna content change with reference to cytomorphology.
Dyeing in above-mentioned 1st step is processed and included：
Step a：The sample for making is spontaneously dried；
Step b：Is put in dry section and fix in pretreatment fluid 10-30 minutes, then water displacement wash 4-5 time；
Step c：Acidifying 5-10 minutes in 50～55 DEG C of hydrochloric acid solution are put into through the sample of step b process, then water displacement wash
4-5 time；
Step d：Dyeing 5-15 minutes in 50～55 DEG C of specific stain liquid A are put into through the sample of step c acidifying, then flowing water
Washing 4-5 time；
Step e：5 minutes are embathed through the sample distilled water of step d dyeing；
Step f：The sample embathed through step d is sequentially placed into 50% alcohol and is dehydrated 2 minutes, dehydration 2 minutes in 75% alcohol,
Dehydration 3 minutes in 95% alcohol；
Step g：Sample after step f is dehydrated step by step is placed in dyeing 1-5 minutes in specific stain liquid B；
Step h：Through step g dyeing sample in soaked in absolute ethyl alcohol twice, 3 minutes every time；
Step i：Through the sample natural gum cover plate sealing of step h.
Reagent used in above-mentioned dyeing is processed includes pretreatment fluid, hydrochloric acid solution, specific stain liquid A and specific stain liquid B,
Wherein：
The pretreatment fluid is using any one in AF liquid, 4% paraformaldehyde, 10% neutral formalin, 95% ethanol, BS liquid or one
More than kind；
The hydrochloric acid solution includes by mass percentage 35%～45% concentrated hydrochloric acid, 55%～65% distilled water；
The specific stain liquid A by mass percentage comprising 0.10%～1% phenothiazines dyestuff, 1.8%～2.2% hydrochloric acid solution,
0.09%～0.12% Sodium Metabisulfite, residual mass percentage are supplied by distilled water, and the phenothiazines dyestuff is by methine
One or more compositions in blue, reddish black A, thionine；
Described specific stain liquid B includes by mass percentage 0.005%～0.1% acid dyes, 75%～96% alcohol reagent, remains
Remaining mass percent is supplied by distilled water, and the acid dyes is by Yihong, oil red O, Ponceaux 2R, biebrich Scarlet
Plant or more than one compositions, alcohol reagent is by one or more groups in ethanol, methyl alcohol, isopropanol, the tert-butyl alcohol, ethylene glycol
Into.
The volume of each reagent component of foregoing description and performance figure provide a kind of proportionate relationship, are not offered as this
Each reagent can only be with above-mentioned volume and quality in bright.
Preferably, the optimal process time in above-mentioned steps is：Step b fixes 15 minutes, and step c is acidified 7 minutes, step
Rapid d is dyeed 10 minutes, and step f is dyeed 3 minutes.
In 2nd step on inspection software by original green Channel-shifted be red passage, filtration cell matter color；Gamma correction mistake
Journey includes adjustment light-source brightness, image exposure value, gradation of image deviant, image segmentation threshold compensating parameter, Bayer pattern
（Bayer）The parameters such as model analyzing parameter, exponential model conversion, so as to accurately measure nuclear DNA content.
The process of gamma correction is specific as follows：
Step（1）：One class index model conversion regulation parameter, Bayer pattern are set（Bayer）Parsing regulation parameter and image point
Cut valve value compensation parameter；
Step（2）：Routinely microscope regulative mode adjusts DNA testing equipment illuminations, field stop, aperture diaphragm, adjusts and exposes
The light time, image histogram showed the bright dark state of image in unsaturation scope in specified range intervals；
Step（3）：Place a standard to cut into slices and arrange a scanning area, after the completion of scanning, the IOD of check criteria section（Product
Light splitting density）, diploid cell standard variance CV, to determine that DNA testing equipments resolution ratio is whether normal, calculate tetraploid IOD
Whether linear relationship is accorded with the ratio of dliploid IOD.The sweep parameter of above parameter and quality control system is contrasted, with Quality Control equipment
For standard, exponential model transformation parameter and Bayer analytic parameters are adjusted；
Step（4）：Repeat the above steps（1）-（3）, until sweep parameter index IOD of same standard section（Integral light is close
Degree）, diploid cell standard variance CV and tetraploid IOD and dliploid IOD linear relationship, DNA testing equipments with
In the range of allowable error, correction is completed parameter its difference that the scanning of standard quality control system is obtained.
Digital color camera collection cellular morphology figure is clapped in the case where high resolution microscope is set to 20 times in 2nd step
Take the photograph, testing staff can point out without the need for diagosis under mirror according to DNA content, reading section picture can make diagnosis, save diagosis people
Member's time, improve diagnosis efficiency.
Concrete steps in 3rd step include：
Ⅰ：Choose the normal cell in same section and do internal reference cell, and contained all nucleus according to DNA by inspection software
The descending order arrangement of amount；
II：During DNA content quantitative analysis, corresponding nuclear full cellular morphology can be observed, determine whether real cell
Core and karyoplasmic ratio, metamorphosis, and cell type is judged；
III：During cytomorphology interpretation, inspection software presses the corresponding cellular morphology figure of the descending arrangement of nuclear DNA content
Piece, points out according to DNA content, and reading section picture can make diagnosis；
Ⅳ：Multiple nucleus overlaps are agglomerating core image segmentation and calculate the DNA content of these agglomerating cores by inspection software, then
By cytology qualitative analysis；
Ⅴ：According to II, III, IV step comprehensive analysis, judge to be detected cell as normal or improper.
Through above-mentioned colouring method staining cell sample under high resolution microscope and the collection of digital color camera
View such as Fig. 3, Fig. 2 be Feulgen dyeing after effect diagram, both contrast understand the present invention dyeing after view cytoplasm
With nucleus significant difference, more morphologic informations can be provided；Fig. 4 is to be previously mentioned according to nucleus DNA in III in the 3rd step
The cytomorphology picture of the descending arrangement of content, microscope records each visual field, and by these visual field pictures according to thin
Born of the same parents' DNA content intensity of anomaly carries out auto-sequencing, when facilitating diagnosis digital slices is carried out according to cell abnormal degree
Check；Fig. 5-7 show the DNA content quantitative analysis of II in the 3rd step, corresponding nuclear full cellular morphology can be observed, often
Numeral below one nucleus is DNA content, and its corresponding cellular morphology can also be seen simultaneously；Fig. 8 is born of the same parents' morphology
Analysis report will embody final result.
The above, is only presently preferred embodiments of the present invention, is not the restriction for making other forms to the present invention, is appointed
What those skilled in the art changed possibly also with the technology contents of the disclosure above or be modified as equivalent variations etc.
Effect embodiment.But it is every without departing from technical solution of the present invention content, according to the technical spirit of the present invention to above example institute
Any simple modification, equivalent variations and the remodeling made, still falls within the protection domain of technical solution of the present invention.
Claims (6)
1. a kind of cell pathology is cut into slices rapid dyeing detection method, it is characterised in that comprised the following steps：
1st step：Dyeing process is carried out to section, makes nucleus and cytoplasm catch color, and both contaminate color contrast
Substantially, it is ensured that in the image for capturing, nucleus and cytoplasm can clearly be presented；
2nd step：By inspection software ALT-CH alternate channel and gamma correction, filtration cell matter color, accurately measure nucleus DNA and contain
Amount, while gathering the corresponding cytomorphology figure of each nucleus using high resolution microscope and the collection of digital color camera
Piece and synthesis whole number section；
3rd step：Sick cell is judged according to change of the single and packed cell nuclear dna content change with reference to cytomorphology；
Dyeing in above-mentioned 1st step is processed and included：
Step a：The sample for making is spontaneously dried；
Step b：Is put in dry section and fix in pretreatment fluid 10-30 minutes, then water displacement wash 4-5 time；
Step c：Acidifying 5-10 minutes in 50～55 DEG C of hydrochloric acid solution are put into through the sample of step b process, then water displacement wash
4-5 time；
Step d：Dyeing 5-15 minutes in 50～55 DEG C of specific stain liquid A are put into through the sample of step c acidifying, then flowing water
Washing 4-5 time；
Step e：5 minutes are embathed through the sample distilled water of step d dyeing；
Step f：The sample embathed through step d is sequentially placed into 50% alcohol and is dehydrated 2 minutes, dehydration 2 minutes in 75% alcohol,
Dehydration 3 minutes in 95% alcohol；
Step g：Sample after step f is dehydrated step by step is placed in dyeing 1-5 minutes in specific stain liquid B；
Step h：Through step g dyeing sample in soaked in absolute ethyl alcohol twice, 3 minutes every time；
Step i：Through the sample natural gum cover plate sealing of step h.
2. a kind of cell pathology according to claim 1 is cut into slices rapid dyeing detection method, it is characterised in that dyeing is processed
Used in reagent include pretreatment fluid, hydrochloric acid solution, specific stain liquid A and specific stain liquid B, wherein：
The pretreatment fluid is using any one in AF liquid, 4% paraformaldehyde, 10% neutral formalin, 95% ethanol, BS liquid or one
More than kind；
The hydrochloric acid solution includes by mass percentage 35%～45% concentrated hydrochloric acid, 55%～65% distilled water；
The specific stain liquid A by mass percentage comprising 0.10%～1% phenothiazines dyestuff, 1.8%～2.2% hydrochloric acid solution,
0.09%～0.12% Sodium Metabisulfite, residual mass percentage are supplied by distilled water, and the phenothiazines dyestuff is by methine
One or more compositions in blue, reddish black A, thionine；
Described specific stain liquid B includes by mass percentage 0.005%～0.1% acid dyes, 75%～96% alcohol reagent, remains
Remaining mass percent is supplied by distilled water, and the acid dyes is by Yihong, oil red O, Ponceaux 2R, biebrich Scarlet
Plant or more than one compositions, alcohol reagent is by one or more groups in ethanol, methyl alcohol, isopropanol, the tert-butyl alcohol, ethylene glycol
Into.
3. a kind of cell pathology according to claim 1 is cut into slices rapid dyeing detection method, it is characterised in that in step
Optimal process time is：Step b fixes 15 minutes, and step c is acidified 7 minutes, and step d is dyeed 10 minutes, and step f dyes 3 points
Clock.
4. a kind of cell pathology according to claim 1 is cut into slices rapid dyeing detection method, it is characterised in that in the 2nd step
On inspection software by original green Channel-shifted be red passage, filtration cell matter color；Gamma correction process includes adjustment light source
Brightness, image exposure value, gradation of image deviant, image segmentation threshold compensating parameter, Bayer pattern（Bayer）Model analyzing is joined
The parameters such as number, exponential model conversion, so as to accurately measure nuclear DNA content.
5. a kind of cell pathology according to claim 4 is cut into slices rapid dyeing detection method, it is characterised in that non-linear school
Positive process is specific as follows：
Step（1）：One class index model conversion regulation parameter, Bayer pattern are set（Bayer）Parsing regulation parameter and image point
Cut valve value compensation parameter；
Step（2）：Routinely microscope regulative mode adjusts DNA testing equipment illuminations, field stop, aperture diaphragm, adjusts and exposes
The light time, image histogram showed the bright dark state of image in unsaturation scope in specified range intervals；
Step（3）：Place a standard to cut into slices and arrange a scanning area, after the completion of scanning, the IOD of check criteria section（Product
Light splitting density）, diploid cell standard variance CV, to determine that DNA testing equipments resolution ratio is whether normal, calculate tetraploid IOD
Whether linear relationship is accorded with the ratio of dliploid IOD；The sweep parameter of above parameter and quality control system is contrasted, with Quality Control equipment
For standard, exponential model transformation parameter and Bayer analytic parameters are adjusted；
Step（4）：Repeat the above steps（1）-（3）, until sweep parameter index IOD of same standard section（Integral light is close
Degree）, diploid cell standard variance CV and tetraploid IOD and dliploid IOD linear relationship, DNA testing equipments with
In the range of allowable error, correction is completed parameter its difference that the scanning of standard quality control system is obtained.
6. a kind of cell pathology according to claim 1 is cut into slices rapid dyeing detection method, it is characterised in that in the 3rd step
Concrete steps include：
I：Choose the normal cell in same section and do internal reference cell, and contained all nucleus according to DNA by inspection software
The descending order arrangement of amount；
II：During DNA content quantitative analysis, corresponding nuclear full cellular morphology can be observed, determine whether real cell
Core and karyoplasmic ratio, metamorphosis, and cell type is judged；
III：During cytomorphology interpretation, inspection software presses the corresponding cellular morphology figure of the descending arrangement of nuclear DNA content
Piece, points out according to DNA content, and reading section picture can make diagnosis；
Ⅳ：Multiple nucleus overlaps are agglomerating core image segmentation and calculate the DNA content of these agglomerating cores by inspection software, then
By cytology qualitative analysis；
Ⅴ：According to II, III, IV step comprehensive analysis, judge to be detected cell as normal or improper.
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