CN108956242A - A kind of fixing process method of mammary cancer armpit lymph gland in vitro tissue sample - Google Patents
A kind of fixing process method of mammary cancer armpit lymph gland in vitro tissue sample Download PDFInfo
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- PVNIQBQSYATKKL-UHFFFAOYSA-N Glycerol trihexadecanoate Natural products CCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCC PVNIQBQSYATKKL-UHFFFAOYSA-N 0.000 description 22
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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
- 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
- G01N2001/305—Fixative compositions
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
This application involves a kind of fixing process methods of mammary cancer armpit lymph gland in vitro tissue sample, belong to test sample preparation technical field.In vitro mammary cancer armpit lymph gland to be processed is cleaned tissue specimen to be soaked in fixer 2-24 hours, after flushing, 30-120min is impregnated with hydrochloric acid solution, is rinsed, with dipping by lye 30-120min, it is transferred in digestive juice, the digestive juice contains the EDTA of 0.1-2mg/ml Proteinase K, 1-100mg/ml trypsase and 0.1-2mg/ml and cuts lymph node after water bath with thermostatic control is handled 1-10 hours, processing work is completed in conventional dehydration, embedding, slice, dyeing.Lymph no in vitro tissue sample is in the above way handled, not only effectively softening dissolved fat tissue, guarantee lymph node are sufficiently fixed in time, also effectively improve lymph nodes number, accurate lymphatic metastasis positive rate.
Description
Technical field
This application involves a kind of fixing process methods of mammary cancer armpit lymph gland in vitro tissue sample, belong to test sample
Product preparation technical field.
Background technique
Existing fresh in vitro sample is usually to impregnate in formalin fixer, this can be for a long time mainly due to formaldehyde
And tectology structure feature is saved well, while there is good economic utility again.Though such sample processing method
It so can be very good the form of fixing organization, but the sample containing adipose tissue also made to be hardened, especially drenched in pathology department's materials
Difficulty is considerably increased when fawning on materials.For in the Radical resections sample such as gastric cancer, intestinal cancer, breast cancer, thoroughly lymph node
Materials to diagnosing tumor, by stages, formulate therapeutic scheme and judging prognosis is most important, and conventional fixer and existing sample
Fixing means is all unable to satisfy the materials speed and recall rate that lymph node is improved while quick fixed, so that pathologist exists
Hand touches knife cut type and finds lymph node in long-term isolated adipose tissue, this method has insufficient, operating time length etc. of drawing materials
Many drawbacks, and finally influence the therapeutic scheme of patient, prognosis and lapse to.Skill is handled to solve existing tumor radical cure tissue specimen
The problem of art, be badly in need of in clinical practice work softening or sufficiently exposed and fixed lymph node while dissolved fat,
Keep lymph node tissue structure and the complete method of cellular morphology.
Based on this, the application is made.
Summary of the invention
Drawbacks described above present in lymph node materials technology is cleaned for existing tumour, the application provides a kind of completely new cream
The processing method of gland cancer lymph node dissection flesh tissue sample, fixed lymph node, guarantees while softening emulsifies dissolved fat
The complete and optimum dyeing effect of lymph node structure.
To achieve the above object, the technical solution that the application takes is as follows:
A kind of fixing process method of mammary cancer armpit lymph gland in vitro tissue sample, by vitro mammary cancer armpit to be processed
Lymph node dissection tissue specimen is soaked in fixer 2-24 hours, after flushing, is impregnated 30-120min with hydrochloric acid solution, is rinsed,
It with dipping by lye 30-120min, is transferred in digestive juice, the digestive juice contains 0.1-2mg/ml Proteinase K, 1-100mg/ml pancreas
The EDTA of protease and 0.1-2mg/ml cuts lymph node, is routinely dehydrated, embeds, cuts after water bath with thermostatic control is handled 1-10 hours
Piece, dyeing complete processing work.
Further, as preferred:
The fixer includes formaldehyde, lecithin, dehydrated alcohol and acetone, concentration difference of each component in fixer
Are as follows: 50~150ml/L of formaldehyde, 30~70g/L of lecithin, 200~300ml/L of dehydrated alcohol, 200~300ml/L of acetone.
The fixer further includes having deoxycholic aicd, methanol and PBS solution, and concentration of the deoxycholic aicd in fixer is 30
~70g/L, concentration of the methanol in fixer are 200-300ml/L, and the PBS solution is added to its concentration in fixer and is
100-200ml/L.It is furthermore preferred that the fixer is made of each component of following concentration ratio: formaldehyde 100ml/L, lecithin
50g/L, dehydrated alcohol 250ml/L, acetone 250ml/L, deoxycholic aicd 47.5g/L, methanol 250ml/L, PBS solution 1 ×.
The fixer further includes having deoxycholic aicd, methanol, PBS solution and methylene blue, and deoxycholic aicd is dense in fixer
Degree is 30~70g/L, and concentration of the methanol in fixer is 200-300ml/L, and PBS solution is added to its concentration in fixer and is
100-200ml/L, concentration of the methylene blue in fixer are 0.05-0.15g/L.It is furthermore preferred that the fixer is by following dense
The each component for spending ratio is constituted: formaldehyde 100ml/L, lecithin 50g/L, dehydrated alcohol 250ml/L, acetone 250ml/L, deoxycholic aicd
47.5g/L, methanol 250ml/L, PBS solution 1 ×, methylene blue 0.1g/L.The hydrochloric acid solution is 0.05-0.2NHCl/PBS solution.
The lye is 0.05-0.2N NaOH/PBS solution.
Sample after digestion process is transferred to soaking at room temperature in fixer, takes out when to be processed, and carries out cutting lymph node, often
Processing work is completed in rule dehydration, embedding, slice, dyeing.
Above-mentioned palmitin fixer is handled into sample, advantage major embodiment are as follows:
(1) the fixing process method of operation excision mammary cancer armpit lymph gland in vitro tissue sample provided herein,
Palmitin is first carried out with fixer, after buffer rinses, then with HCl/PBS solution room temperature immersion treatment, through the secondary punching of buffer
It after washing, with NaOH/PBS solution soaking at room temperature, then is placed in digestive juice and carries out digestion process, Proteinase K and pancreas egg are selected in digestion
Pre-processing is completed in white enzyme-EDTA water bath with thermostatic control processing.In this scenario, palmitin is carried out first, lymph node is determined, so
Fat cell membrane permeability is enhanced with high concentration soda acid alternate treatment again afterwards, and lymph node is because of surface compact connective tissue quilt
The protection of film, cell composition component cells film is unaffected or influences the change for slightly being not enough to cause cell and institutional framework.
(2) soft with the fixer progress for softening the multiple actions such as chyle fat, fixed lymph node after chemical method processing
Rouge processing can make the adipose tissue softening for wrapping up lymph node and lymph node periphery, increase leaching when finally being handled using digestive juice
Fawn on the hardness difference with adipose tissue, it is easier to and lymph node is facilitated to draw materials, improve working efficiency and materials quality.
(3) the application is using palmitin fixer to the fixed effect of lymph node and Various Tissues sample and conventional formalin
The effect for save fixation is suitable, improves the detection number of lymph node, accurate lymphatic metastasis positive rate decreases disease
The dissection of natural sciences doctor is drawn materials the time, possibility is provided for the outdoor quality control of later period pathology subject, in clinic after commercialization
Effect in wide popularization and application is especially prominent.
Detailed description of the invention
Fig. 1 is the process flow diagram of the application；
Fig. 2 is the adipose tissue of palmitin fixer provided herein before and after the processing, wherein A is the fresh rouge before processing
Fat tissue, B are fatty for treated；C is that metastatic carcinoma amplifies 40 times, and D is that small metastatic carcinoma amplifies 200 times；
Fig. 3 is breast tissue using palmitin fixer (uplink) provided herein and uses conventional neutral formalin
The effect comparison figure of (downlink) processing, wherein being followed successively by conventional H E dyeing and CD10, P63 and PR immunohistochemistry dye from left to right
Color, × 200.
Specific embodiment
Embodiment 1: differently composed palmitin fixer
The central role ingredient of palmitin fixer provided herein is mainly formaldehyde, lecithin, dehydrated alcohol and third
Ketone can also additionally increase any one of deoxycholic aicd, methanol, PBS solution and methylene blue or more under different implementation environments
Kind, specific ingredient selection mode can be found in shown in table 1.
The palmitin fixer of the different compositions of table 1
We respectively survey the palmitin fixer of core constituent (serial number 1 i.e. in table 1) and complementary element
Examination, available above-mentioned 16 kinds of situations, same composition do parallel test to same sample using same concentrations, the results showed that
The palmitin fixer of core constituent is whether to adipose tissue or organ-tissue (such as parathyroid tissue, breast tissue, lung
Tissue) or colonic tissue, all there is good fixed effect, lymph node is well exposed；But the effect of complementary element
Can not be ignored, as using serial number 2 in table 1 scheme is provided when, under the cooperation of deoxycholic aicd and core constituent, fatty disappears
It is preferable to change effect, the trend is in serial number 1 and serial number 2, serial number 3 and serial number 6, serial number 8 and serial number 12 and serial number 14 and serial number 16
Comparison in have same embodiment；In scheme provided by serial number 3, methanol plays the role of cooperateing with dehydrated alcohol, cooperates other
Core constituent, dissolution efficiency is higher, the trend in serial number 1 and serial number 3, serial number 4 and serial number 8, serial number 6 and serial number 7 and
There is same embodiment in the comparison of serial number 15 and serial number 16；In scheme provided by serial number 4, PBS solution and core constituent
Under cooperation, fatty dissolution and bating effect are more uniform, and the trend is in serial number 1 and serial number 4, serial number 13 and serial number 16；Serial number 5
In provided scheme, under the cooperation with core constituent, can more quickly it develop the color in subsequent dyeing processing, the spy
Property also have same embodiment in the comparison of serial number 1 and serial number 5, serial number 6 and serial number 13, serial number 12 and serial number 16.
Therefore, based on different fixed purpose and processing requirement, can choose above-mentioned differently composed palmitin fixer into
Row palmitin (fat melting) processing.
We also (remove core constituent in fixer (formaldehyde, lecithin, dehydrated alcohol, acetone) with complementary element
Oxycholic acid, methanol, PBS solution, methylene blue) concentration tested, the results showed that in palmitin fixer, the concentration of formaldehyde is suitable
The suitable control of concentration preferably controlled in 50-150ml/L, lecithin exists in the suitable control of concentration of 30-70g/L, dehydrated alcohol
200-300ml/L, acetone concentration be suitable for control in 200-300ml/L, in this case, four kinds of core action components can be with
It is good to play collaboration and mating reaction, achieve the effect that dissolution or softening fat, being lower than or exceed above range then can be right
Fat melting effect causes certain interference and adverse effect；When carrying out the addition of complementary element, above-mentioned four kinds of cores can be kept to make
With ingredient additive amount it is constant or within the above range appropriate adjustment, the addition of complementary element should not be too large, be otherwise easy to make
It is unbalance at fixer, softening and solute effect are influenced, therefore, concentration of the deoxycholic aicd in fixer is 30~70g/L, methanol
Concentration in fixer is 200-300ml/L, and concentration of the PBS solution in fixer is 100-200ml/L, and methylene blue is in fixation
Concentration in liquid is 0.05-0.15g/L, and these four complementary elements can add simultaneously, also may be selected any or appoints several points
It does not add.
Embodiment 2: the palmitin fixer of same composition various concentration
This gives the full palmitins for constituting and (containing whole core constituent and whole complementary elements)
Fixer composition and concentration, and representative several groups are enumerated as shown in table 2:
The palmitin fixer of 2 various concentration of table
Pretreated operation is handled using palmitin fixer corresponding to each serial number of table 2 to cut off nethike embrane sample 4 hours, then
Lymph node materials, dehydration, embedding, slice, dyeing, immunohistochemistry are carried out, under the microscope, the results showed that above-mentioned each concentration
Different degrees of realizing to sample is dissolved, and the soft liquefaction of fat, lymph node is effectively exposed, and lymph node cells form has saved
Whole, no deformation, immunohistochemistry works well, and especially with serial number 9-12 in table 2, (serial number 10,11,12 is followed successively by original offer
Embodiment 3,2,1) when provided scheme carries out palmitin experiment, fat melting significant effect, good fixing effect, fatty softening agent
Change, lymph node exposure is obvious, and lymph node cells form saves completely, and no deformation, immunohistochemistry works well.
The cleaning of kinds of tumor lymph node flesh tissue sample compares
Individually below using palmitin fixer provided by serial number 12 in table 2 as representing, it is solid that palmitin is carried out to different specimens
Change, and is compared with conventional neutral formalin (10% formalin, pH7.2) treatment effect, specific as follows:
(1) adipose tissue
Operation excision fresh fat tissue fritter and lymph node are taken, is impregnated 4 hours with palmitin fixer, is rushed through PBS first
After washing, with 0.1N HCl/PBS soaking at room temperature 1 hour, treatment fluid is abandoned, after PBS is rinsed, with 0.1N NaOH/PBS soaking at room temperature 1
Hour, treatment fluid is abandoned, is soaked with 0.5mg/ml Proteinase K -25mg/ml trypsase -1mg/ml EDTA in 37 DEG C of constant water bath box
Bubble 4 hours.In next step, sample is taken out, conventional materials, dehydration, embedding, slice, dyeing can be observed, such as Fig. 2 institute
Show, this is former adipose tissue compared with degreasing fixer is handled after 2h, it is seen that after using the application fixing process, adipose tissue
Obviously soften thinning, lymph node structure is complete, and metastatic carcinoma tissue and small metastasis cancer cell group are high-visible.
(2) breast tissue
It takes operation to cut off fresh mammary gland glandular tissue, is impregnated 4 hours with palmitin fixer first, after PBS is rinsed, use 0.1N
HCl/PBS soaking at room temperature 1 hour, treatment fluid is abandoned, after PBS is rinsed, with 0.1N NaOH/PBS soaking at room temperature 1 hour, abandoning was handled
Liquid is impregnated 4 hours with 0.5mg/ml Proteinase K -25mg/ml trypsase -1mg/ml EDTA in 37 DEG C of constant water bath box.Under
One step, sample is taken out, and conventional materials, dehydration, embedding, slice, dyeing can be observed, as a result as shown in figure 3, result
Show: it is fixed compared with sample with conventional formalin, the processing of this law fixer to the general morphology of breast tissue sample,
The equal indifference of immunohistochemical staining.
Wherein, 0.1N HCl used in above scheme is prepared by the following steps: the hydrochloric acid that mass fraction is 36.5%
Drip the HCl solution being configured in distilled water.
Fixer is prepared by the following steps: it weighs PBS powder and 15ml distilled water is uniformly mixed, obtain 7 × PBS solution,
Then dehydrated alcohol, methanol, acetone, formaldehyde, lecithin, deoxycholic aicd, methylene blue are sequentially added, 100ml is settled to, is stirred to mixed
Close uniformly (in fixer PBS solution concentration be 1 ×, it is excessive or too small can all cause cytomorphosis or structural damage).
Claims (9)
1. a kind of fixing process method of mammary cancer armpit lymph gland in vitro tissue sample, it is characterised in that: will be to be processed in vitro
Mammary cancer armpit lymph gland cleans tissue specimen and is soaked in fixer 2-24 hours, after flushing, impregnates 30- with hydrochloric acid solution
120min, rinse, with dipping by lye 30-120min, be transferred in digestive juice, the digestive juice contain 0.1-2mg/ml Proteinase K,
The EDTA of 1-100mg/ml trypsase and 0.1-2mg/ml cuts lymph node after water bath with thermostatic control is handled 1-10 hours, conventional
Processing work is completed in dehydration, embedding, slice, dyeing.
2. a kind of fixing process method of mammary cancer armpit lymph gland in vitro tissue sample as described in claim 1, feature
Be: the sample after digestion process is transferred to soaking at room temperature in fixer, takes out when to be processed, and carries out cutting lymph node, conventional
Processing work is completed in dehydration, embedding, slice, dyeing.
3. a kind of fixing process method of mammary cancer armpit lymph gland in vitro tissue sample as described in claim 1, feature
It is, the fixer includes formaldehyde, lecithin, dehydrated alcohol and acetone, and concentration of each component in fixer is respectively as follows: first
50 ~ 150ml/L of aldehyde, 30 ~ 70g/L of lecithin, dehydrated alcohol 200 ~ 300 ml/L, 200 ~ 300ml/L of acetone.
4. a kind of fixing process method of mammary cancer armpit lymph gland in vitro tissue sample as claimed in claim 3, feature
It is: further includes having deoxycholic aicd, methanol and PBS solution, concentration of the deoxycholic aicd in fixer is 30 ~ 70g/L, and methanol exists
Concentration in fixer is 200-300ml/L, and it is 100-200ml/L that the PBS solution, which is added to its concentration in fixer,.
5. a kind of fixing process method of mammary cancer armpit lymph gland in vitro tissue sample as claimed in claim 4, feature
It is, the fixer is made of each component of following concentration ratio: formaldehyde 100ml/L, lecithin 50g/L, dehydrated alcohol 250
Ml/L, acetone 250ml/L, deoxycholic aicd 47.5g/L, methanol 250ml/L, PBS solution 1 ×.
6. a kind of fixing process method of mammary cancer armpit lymph gland in vitro tissue sample as claimed in claim 3, feature
It is: further includes having deoxycholic aicd, methanol, PBS solution and methylene blue, concentration of the deoxycholic aicd in fixer is 30 ~ 70g/L,
Concentration of the methanol in fixer is 200-300ml/L, and it is 100-200ml/L, beauty that PBS solution, which is added to its concentration in fixer,
Concentration of the orchid in fixer is 0.05-0.15g/L.
7. a kind of fixing process method of mammary cancer armpit lymph gland in vitro tissue sample as claimed in claim 6, feature
It is, the fixer is made of each component of following concentration ratio: formaldehyde 100ml/L, lecithin 50g/L, dehydrated alcohol 250
Ml/L, acetone 250ml/L, deoxycholic aicd 47.5g/L, methanol 250ml/L, PBS solution 1 ×, methylene blue 0.1g/L.
8. a kind of fixing process method of mammary cancer armpit lymph gland in vitro tissue sample as described in claim 1, feature
Be: the hydrochloric acid solution is 0.05-0.2NHCl/PBS solution.
9. a kind of fixing process method of mammary cancer armpit lymph gland in vitro tissue sample as described in claim 1, feature
Be: the lye is 0.05-0.2N NaOH/PBS solution.
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