CN112485426A - Circulating tumor cell IHC antigen repairing method based on ISET principle - Google Patents
Circulating tumor cell IHC antigen repairing method based on ISET principle Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
Abstract
The invention belongs to the field of biomedicine, and particularly relates to a circulating tumor cell IHC antigen repairing method based on the ISET principle. The method comprises the following steps: adding 0.01mol/L sodium citrate buffer solution into a water bath, heating to 95 ℃, putting a circulating tumor cell slide fixed by a magnet, and heating for 15 minutes; after repair was complete, slides were removed and washed 2min x 3 times with PBS. After the antigen provided by the invention is subjected to thermal restoration, the IHC staining effect on circulating tumor cells is good, and false positive caused by antigen restoration can be reduced; the invention further perfects the circulating tumor cell detection and identification technical system.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a circulating tumor cell IHC antigen repairing method based on the ISET principle.
Background
Since paraffin section specimens are mostly fixed by formaldehyde, aldehyde bonds, carboxymethyl groups and the like formed by formaldehyde in tissues block part of antigenic determinants, or the antigenic determinants are hidden due to cross-linking among protein molecules. Therefore, before staining, some antigens need to be repaired or exposed to make the antigen and antibody in the tissues fully combined, and the ideal staining result is achieved. And the antigen needing to be repaired needs to be determined when a staining program is established. Two methods commonly used are heat antigen retrieval and enzymatic digestion. A large number of experiments prove that the heating antigen retrieval method is superior to the enzyme digestion method, the mechanism is still unclear at present, and the heating probably opens the cross-linking of antigenic determinants of the tissue antigen caused by formaldehyde fixation, so that the sensitivity of immunohistochemical staining is greatly improved. The paraffin-embedded tissue is subjected to light staining due to loss of protein antigen activity in the fixing process, so that false negative of a detection result is caused, the staining result is also influenced by the type of a fixing agent and the like, and antigen restoration is used for compensating the loss of the antigen activity in the fixing process, but the restoration of the antigen also causes non-specific staining to a certain extent, so that false positive of the detection result is caused. In the prior art, no record exists about repairing circulating tumor cells.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a circulating tumor cell IHC antigen repairing method based on the ISET principle.
The technical scheme adopted by the invention for realizing the purpose is as follows:
the invention provides a circulating tumor cell IHC antigen repairing method based on an ISET principle, which comprises the following steps:
(1) adding 0.01mol/L sodium citrate buffer solution into a water bath, heating to 95 ℃, putting a circulating tumor cell slide fixed by a magnet, and heating for 15 minutes;
(2) after repair was complete, slides were removed and washed 2min x 3 times with PBS.
Further, the pH value of the sodium citrate buffer solution is 6.0.
The invention also provides an immunohistochemical detection method for the circulating tumor cells repaired by the antigen repairing method, which comprises the following steps:
1) dripping 100 μ l of 0.1% Triton X-100 into the repaired circulating tumor cell slide, incubating at room temperature for 15min, and washing with DI water for 2min × 3 times;
2) 100 μ l of 0.3% H was added dropwise2O2Incubating at room temperature for 10min, washing with PBS for 2min × 3 times;
3) dripping 100 μ l of primary antibody working solution, incubating at room temperature for 2h, washing with PBS for 2min × 3 times;
4) dripping 100 μ l goat anti-rabbit/mouse IgG/HRP, incubating at room temperature for 15-30min, washing with PBS for 2min × 3 times;
5) dripping 100 mul of DAB color development solution, and incubating for 3-10 min at room temperature;
6) after the color development is finished, discarding DAB color development liquid, flushing with running water for 5min, and dyeing with hematoxylin for 15 min;
7) rinsing with water for 1 second, differentiating with hydrochloric acid and ethanol for 3-8 seconds, and bluing with tap water for 15 min;
8) dehydrating with 75% ethanol (1 min), 95% ethanol (1 min), 100% ethanol (1 min) by gradient ethanol, air drying, and sealing with neutral resin.
9) And (5) performing microscopic examination by using an optical microscope.
Further, in the step 3), the primary anti-working solution is CD45+ CD 31.
The preparation process of the primary anti-working solution comprises the following steps: adding 100 mu lCD45 primary antibody working solution into 1 mu lCD31 primary antibody concentrated solution, and mixing.
Further, the specific dehydration process of the gradient ethanol comprises the following steps: dehydrating with 75% ethanol for 1min, dehydrating with 95% ethanol for 1min, and dehydrating with 100% ethanol for 1 min.
The specific experimental route of the invention is shown in figure 1.
The invention has the beneficial effects that:
(1) after the antigen provided by the invention is subjected to thermal restoration, the IHC staining effect on circulating tumor cells is good, and false positive caused by antigen restoration can be reduced;
(2) the invention further perfects the circulating tumor cell detection and identification technical system.
Drawings
FIG. 1 is a comparative experiment for IHC antigen repair of circulating tumor cells based on the ISET principle.
FIG. 2 is a graph of immunohistochemical effect without machine antigen retrieval.
FIG. 3 is a graph of immunohistochemical effect following machine antigen retrieval.
FIG. 4 is a report of the effect on immunohistochemistry after antigen retrieval.
Detailed Description
The technical solution of the present invention is further explained and illustrated by the following specific examples.
(I) CTCs routine detection experiment consumable material and reagent
TABLE 1
(II) antigen retrieval special equipment and consumable
TABLE 2
Example 1
1. Blood sampling
1) The collection of the peripheral blood samples needs to be carried out by professional medical personnel, and BD Vacutainer K is used2E (EDTA) anticoagulation vacuum blood collection tube (other products need to be confirmed by the company);
2) BD Vacutainer K2E (EDTA) 10.8mg 10.0 mL 5mL peripheral blood was collected intravenously, 2 tubes;
3) immediately after the sample is collected, the mixture is gently turned upside down and mixed for 8 times to avoid blood coagulation(very critical)。
2. Filter activation
1) Opening the upper plug and the lower plug of the filter and properly placing the upper plug and the lower plug of the filter;
2) placing the filter on a filter rinsing rack, and adding about 2ml of 75% medical alcohol into the filter by using a Pasteur tube until the alcohol is naturally filtered from the lower opening of the filter;
3) adding normal saline into the filter, repeatedly rinsing (4 ml × 3 times), and removing residual alcohol in the filter;
4) filtering out normal saline naturally, reserving proper amount of normal saline to ensure the filter membrane is wet, and installing upper and lower filter plugs.
Note: if the alcohol can not be naturally filtered out during rinsing, the alcohol is immediately discarded.
3. A10 blood sample pretreatment
1) Checking the quality of the sample, and recording the information of the sample (filling a patient information statistical table);
2) taking 15ml centrifuge tubes and marking, wherein each sample corresponds to one centrifuge tube;
3) adding 10ml of normal saline into a 15ml centrifuge tube, adding 375 mu l of fixing agent (8% PFA), uniformly mixing, adding 5ml of blood sample into the centrifuge tube by using a Pasteur pipette, and uniformly mixing by reversing the upper part and the lower part for 8 times;
4) fixing at room temperature for 10 min;
5) and (5) detecting.
Note:
in the step 3, the mixture is directly inverted and uniformly mixed in the blood sampling tube, so that the possibility of falling off of blood clots agglutinated on the tube wall is prevented. If blood coagulation, hemolysis, apparent lack of volume or other abnormalities are found, they should be discarded immediately.
And 3, after the blood sample is uniformly mixed, taking back the redundant blood into the corresponding blood sampling tube.
And (4) avoiding generating air bubbles during sample adding in the step 4, and preventing the blood sample from polluting the opening of the filter and the upper end pipe wall.
4. Isolation of A10 CTC
1) Checking the waste liquid barrel, and opening a machine switch;
2) putting the pretreated filter into a filter bin, and ensuring that a sample feeding needle penetrates through a lower plug;
3) taking down the upper plug of the filter, and transferring 8ml of diluted sample to the filter;
4) selecting an automatic/manual mode, clicking a 'start' button, and starting filtering;
5) when 1ml of sample still exists in the filter, transferring the rest sample to the filter until all samples are filtered;
6) the 4ml PBS was rinsed three times to complete the separation and the waste was collected.
5. Giemsa Richardson staining
1) Taking down the filter, adding 500 μ l methanol, and fixing at room temperature for 1 min;
2) removing methanol, taking the membrane, sticking the membrane with the front side facing upwards to the center of the glass slide, and airing at room temperature for 2 min;
3) drawing a closed circle slightly larger than the filter membrane around the filter membrane by using a grouping pen, and waiting for 2min for airing;
4) adding 300 mu l A dye solution by using a pipette, dyeing for 1min, then adding 300 mu l PBS to dilute the A dye solution, and discarding the diluted dye solution;
5) adding 300 mu l B dye solution, dyeing for 1 minute and 30 seconds, and discarding the dye solution B;
6) adding DI, cleaning the B dyeing residual liquid on the filter membrane to ensure that no obvious dyeing liquid color residue exists on the filter membrane, and drying for 5min in a drying oven at 50 ℃;
7) using a pipette to take 2.5 mul of the patch agent onto a clean glass slide, and uniformly coating the patch agent into the area of the size of a filter membrane;
8) transferring the dyed filter membrane onto a glass slide coated with a sticking tablet, and placing the glass slide in a drying oven at 50 ℃ for 30 min;
9) dripping neutral resin and sealing;
10) and (5) reading the film and counting the detection rate of CTC.
Note:
1) the dye solutions used above were all from the D100 matched dye solution kit.
2) If the filter is not completely covered by 300. mu.l of the staining solution, the amount of the staining solution used can be increased.
3) If the filter membrane floats during the dyeing process, the filter membrane can be pressed below the liquid level by using tweezers.
4) When the dye solution is added, the solution is slowly dropped onto the filter membrane.
5) When the residual liquid is discarded, a pipette can be used to suck out the residual liquid from the edge of the filter membrane.
6. Decolorization after microscopic examination
1) The slide is placed in PBS or DI water until the coverslip is naturally removed.
2) Put into a dyeing jar with DI water, rinsed for 5 minutes up and down, and thoroughly washed with glycerin.
3) And (3) sequentially placing the glass slide into 100% ethanol (1 min), 95% ethanol (1 min) and 75% ethanol for decolorization for more than 20 minutes, placing the glass slide into a DI dyeing tank after the red dye is sufficiently faded, rinsing the glass slide up and down for 5 minutes, and drying the glass slide. (Observation under microscope whether the dye completely faded)
7. Antigen retrieval
Adding 0.01mol/L sodium citrate buffer solution (pH 6.0) into a water bath, heating to about 95 ℃, putting a circulating tumor cell slide fixed by a magnet, and heating for 15 minutes (the longest time is not more than 15 minutes), wherein the other slide is not treated;
after repair was complete, slides were removed and washed 2min x 3 times with PBS.
8. Immunohistochemistry
1) Mu.l of 0.1% Triton X-100 was added dropwise, incubated at room temperature for 15min, and washed with DI water for 2min X3 times.
2) 100 μ l of 0.3% H was added dropwise2O2Incubation was carried out at room temperature (18-26 ℃) for 10min, and washed with PBS 2min × 3 times.
3) 100 mu l (CD 45+ CD 31) of primary antibody working solution (100 mu lCD45 of primary antibody working solution is added with 1 mu lCD31 of primary antibody concentrated solution to be mixed evenly) is dripped in, incubated for 2h at room temperature (18-26 ℃) (or incubated overnight at 4 ℃, rewarming for 30 min), and washed for 2min multiplied by 3 times by PBS.
4) 100 mul goat anti-rabbit/mouse IgG/HRP is dripped, incubated for 15-30min at room temperature (18-26 ℃) and washed with PBS for 2min multiplied by 3 times.
5) Dripping 100 mul DAB color development solution, incubating at room temperature (18-26 ℃) and observing the color development condition under a microscope at any time (generally 3-10 min, the time can not exceed 10 min). The DAB color developing solution is prepared according to the specification.
6) After the color development is finished, discarding DAB color development liquid, flushing with running water for 5min, and dyeing with hematoxylin for 15 min;
7) rinsing with water for 1 second, differentiating with hydrochloric acid and ethanol for 3-8 seconds, and returning blue with tap water for 15min (flushing with running water);
8) dehydrating with 75% ethanol (1 min), 95% ethanol (1 min), 100% ethanol (1 min) by gradient ethanol, air drying, and sealing with neutral resin.
9) And (5) performing microscopic examination by using an optical microscope.
Results and discussion
The Tianjin tumor hospital provides a method for repairing machine antigens when operating by using Roche immunohistochemical equipment, and compared with immunohistochemical effects through a subject group, as shown in figures 2 and 3, compared with the conventional detection method, the number of positive blood cells after antigen repair is more (the cell nucleus is blue, the cell membrane is brown yellow), the number of negative CTC (the cell nucleus is blue, and the cell membrane is not brown yellow), and the expression of immunohistochemical signals is stronger. Therefore, the subject group proposes the experiment in combination with the research foundation, and aims to better express the immunohistochemical effect and perfect the CTC detection and identification technical system by a water bath antigen repair method.
(II) the subject
Selecting 12 samples of the prostataceae of Shandong tumor hospital, and collecting 2 extravascular peripheral blood samples of each sample; CEC samples were provided in 12 cases, each sample collecting 2 tubes of peripheral blood.
And analyzing and evaluating the histochemical staining effect of the two slides, and judging whether the CTC slide subjected to antigen retrieval has good staining effect or not according to the staining result, wherein the specific result is shown in Table 3.
TABLE 3
(III) calculation of test example
The invention mainly compares the influence of tumor patients and normal people on immunohistochemistry after thermal restoration; two groups of samples with the same number, N1= N2=0.5, alpha =0.05, 1-beta =0.9, are required, the detected immunohistochemical effective rate after thermal repair is 100% and the immunohistochemical effective rate after thermal repair is 50% for normal people by comparing samples of Tianjin tumor patients, and the obtained result report is shown in figure 4 by calculating with pass11 software.
Claims (6)
1. A circulating tumor cell IHC antigen repairing method based on an ISET principle is characterized by comprising the following steps:
(1) adding 0.01mol/L sodium citrate buffer solution into a water bath, heating to 95 ℃, putting a circulating tumor cell slide fixed by a magnet, and heating for 15 minutes;
(2) after repair was complete, slides were removed and washed 2min x 3 times with PBS.
2. The method for IHC antigen retrieval from circulating tumor cells based on the ISET principle of claim 1, wherein the pH value of the sodium citrate buffer solution is 6.0.
3. An immunohistochemical detection method of circulating tumor cells after the repair by the antigen retrieval method according to claim 1 or 2, comprising the steps of:
1) dripping 100 μ l of 0.1% Triton X-100 into the repaired circulating tumor cell slide, incubating at room temperature for 15min, and washing with DI water for 2min × 3 times;
2) 100 μ l of 0.3% H was added dropwise2O2Incubating at room temperature for 10min, washing with PBS for 2min × 3 times;
3) dripping 100 μ l of primary antibody working solution, incubating at room temperature for 2h, washing with PBS for 2min × 3 times;
4) dripping 100 μ l goat anti-rabbit/mouse IgG/HRP, incubating at room temperature for 15-30min, washing with PBS for 2min × 3 times;
5) dripping 100 mul of DAB color development solution, and incubating for 3-10 min at room temperature;
6) after the color development is finished, discarding DAB color development liquid, flushing with running water for 5min, and dyeing with hematoxylin for 15 min;
7) rinsing with water for 1 second, differentiating with hydrochloric acid and ethanol for 3-8 seconds, and bluing with tap water for 15 min;
8) dehydrating with 75% ethanol (1 min), 95% ethanol (1 min), 100% ethanol (1 min) by gradient ethanol, air drying, and sealing with neutral resin.
9) And (5) performing microscopic examination by using an optical microscope.
4. The immunohistochemical detection method according to claim 3, wherein in step 3), the primary anti-cancer fluid is CD45+ CD 31.
5. The immunohistochemical detection method according to claim 4, wherein the primary antibody working solution is prepared by the following specific steps: adding 100 mu lCD45 primary antibody working solution into 1 mu lCD31 primary antibody concentrated solution, and mixing.
6. The immunohistochemical detection method according to claim 3, wherein the gradient ethanol specifically dehydrates in the following steps: dehydrating with 75% ethanol for 1min, dehydrating with 95% ethanol for 1min, and dehydrating with 100% ethanol for 1 min.
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| US20140212881A1 (en) * | 2013-01-26 | 2014-07-31 | Denovo Sciences, Inc. | System and method for capturing and analyzing cells |
| US20190107542A1 (en) * | 2013-02-02 | 2019-04-11 | Duke University | Method of isolating circulating tumor cells |
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| CN105973681A (en) * | 2016-05-19 | 2016-09-28 | 四川金域医学检验中心有限公司 | Immunohistochemical operation method |
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