Breast cancer auxiliary diagnosis detection sample kit

Abstract

The invention relates to the related technical field of breast cancer auxiliary diagnosis and detection, in particular to a sample kit for breast cancer auxiliary diagnosis and detection, which can quickly and accurately diagnose breast cancer, greatly improve the early discovery rate of breast cancer and improve the detection efficiency of breast cancer. The kit provides better treatment selection and survival rate for breast cancer patients, realizes accurate detection of breast cancer markers, contains specific antibodies and primers, the antibodies and primers can specifically recognize and combine specific molecules released by breast cancer cells, and by detecting the molecules, the specific molecules released by the breast cancer cells can be specifically recognized and combined with the specific antibodies and primers. Whether a patient suffers from breast cancer can be accurately judged, and the progress degree and type of the breast cancer can be preliminarily evaluated.

Description

Technical Field

[0001] The present invention relates to the technical field related to breast cancer auxiliary diagnosis and detection, and in particular to a breast cancer auxiliary diagnosis and detection sample kit. Background Art

[0002] Breast cancer screening is to screen asymptomatic women through effective, simple and economical breast examination measures in order to detect, diagnose and treat the disease early. Its ultimate goal is to reduce the mortality rate of breast cancer in the population. With the continuous advancement of medical technology, early diagnosis of breast cancer has become the key to improving patients' survival rate and quality of life. However, traditional breast cancer screening methods often have problems such as complex operation, high cost or insufficient diagnostic accuracy, which are difficult to meet the needs of the majority of women. Therefore, there is a special need for a breast cancer auxiliary diagnosis test sample kit.

[0003] Because existing breast cancer auxiliary diagnosis test sample kits often require cumbersome operating steps during use, or require high professional skills of the operator, this has to some extent limited its popularity and use among the general female population. Summary of the invention

[0004] The purpose of the present invention is to provide a breast cancer auxiliary diagnosis detection sample kit to solve the problem that the existing breast cancer auxiliary diagnosis detection sample kit proposed in the above background technology often requires cumbersome operating steps during use, or requires high professional skills of the operator, which to a certain extent limits its popularization and use among the general female population.

[0005] To achieve the above object, the present invention provides a breast cancer auxiliary diagnosis and detection sample kit, which is carried out according to the following steps:

[0006] S1. Sample addition: Add the sample to the bottom of the ELISA plate well, try not to touch the well wall, and gently shake to mix;

[0007] S2, incubation: seal the plate with a sealing film and incubate at 37°C for 30 minutes;

[0008] S3, liquid preparation: dilute 30 (20 times of 48T) times of concentrated washing liquid with 30 (20 times of 48T) times of distilled water for later use;

[0009] S4, washing: carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times, and pat dry;

[0010] S5, enzyme addition: add 50 μl of enzyme labeling reagent to each well, except for the blank well;

[0011] S6, incubation: seal the plate with a sealing film and incubate at 37°C for 30 minutes;

[0012] S7, washing: carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times, and pat dry;

[0013] S8, color development: first add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake to mix, and color development at 37°C in the dark for 15 minutes;

[0014] S9, stop: add 50 μl of stop solution to each well to stop the reaction (the blue color immediately turns yellow);

[0015] S10. Determination: Use blank as zero and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The determination should be carried out within 15 minutes after adding the stop solution.

[0016] Preferably, the standard in S1 is set with 10 wells at 5 concentration points, and parallel wells are set for each concentration, and 50 μl of standards of different concentrations are added. The blank well is set with 1 well and 50 μl of distilled water is added (the blank control well does not contain sample and enzyme-labeled reagent, and the other steps are the same), and the sample well to be tested is set with 40 μl of sample diluent first added to the sample well to be tested on the enzyme-labeled coated plate, and then 10 μl of the sample to be tested is added (the final dilution of the sample is 5 times).

[0017] Preferably, in S3, the required reagents and standard curve are prepared according to the requirements of the kit. The reagents usually consist of an ELISA plate and a washing buffer solution. The standard curve is a reference standard for determining samples of unknown concentration.

[0018] Preferably, in S5, the coated antibody or antigen is first added to the wells of the ELISA plate, and then incubated at room temperature for a period of time, which will form a layer of antigen or antibody in the wells for subsequent reactions.

[0019] Preferably, in S1, samples and standards are added to the wells according to the requirements of the kit, and several wells are reserved as blank controls. Note that samples and standards are added to the correct wells to avoid cross contamination.

[0020] Preferably, in S5, an enzyme-labeled secondary antibody (usually horseradish peroxidase against human IgG) is added to each well of the ELISA plate to allow it to bind to the target substance.

[0021] Preferably, in S7, the ELISA plate is placed back into the incubator for incubation, and then washed again to remove unbound substances.

[0022] Preferably, in S8, a substrate is added to each well to react with the enzyme to produce a color change, and the reaction time is controlled according to the requirements of the kit.

[0023] Preferably, a reaction stopping solution is added to S9 to stop the enzyme reaction and prevent further color change. The specific reaction stopping method should be carried out according to the requirements of the kit.

[0024] Preferably, in S10, a microplate reader or a photometer is used to measure the color intensity in each well, and the color intensity is compared with the standard value on the standard curve to determine the concentration of the target substance in the sample.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: the breast cancer auxiliary diagnosis detection sample kit can quickly and accurately diagnose breast cancer, greatly improve the early detection rate of breast cancer, provide better treatment options and survival rates for breast cancer patients, and realize accurate detection of breast cancer markers. The kit contains specific antibodies and primers, which can specifically recognize and bind to specific molecules released by breast cancer cells. By detecting these molecules, we can accurately determine whether the patient has breast cancer, and can make a preliminary assessment of the degree of progression and type of breast cancer. In addition, the present invention also has the advantages of high sensitivity and high specificity, which can effectively avoid misdiagnosis and missed diagnosis. At the same time, the use of the kit is simple and convenient, and does not require complex equipment and technical support. It is suitable for routine detection work in a large number of medical institutions and laboratories. First of all, the design of the kit fully considers the complexity and diversity of breast cancer. By selecting and optimizing antibodies and primers in a targeted manner, it can realize the simultaneous detection of multiple breast cancer markers, so as to have a more comprehensive understanding of the patient's condition. This comprehensive The detection method helps to more accurately assess the risk and progression of breast cancer, and provides strong support for the development of personalized treatment plans. Secondly, this kit uses advanced biotechnology and nanomaterials to improve the sensitivity and specificity of detection. Through the enhancement effect of nanomaterials, the antibodies and primers bind to the target molecules more tightly and stably, thereby improving the accuracy of detection. At the same time, this technology can also effectively reduce the interference of background signals and reduce the incidence of false positives and false negatives. This kit also has good stability and reliability. After strict quality control and test verification, the activity and stability of the antibodies and primers in the kit are ensured, which means that even during long-term storage or transportation, the performance of the kit will not be significantly affected, ensuring the stability and reliability of the test results. With the continuous increase in the incidence of breast cancer, early detection and accurate diagnosis are crucial to improving patient survival rates and quality of life. The rapidity, accuracy, sensitivity and specificity of this kit can meet the needs of medical institutions and laboratories for auxiliary diagnosis of breast cancer, and provide strong support for the prevention, control and treatment of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic diagram of the steps of using the breast cancer auxiliary diagnosis and detection sample kit of the present invention;

[0027] Figure 2 This is a schematic diagram of the structure of HER2 detection using a validated IHC method in the present invention;

[0028] Figure 3 The present invention uses a validated dual-probe in situ hybridization method for HER2 detection. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the implementation regulations described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] See also Figure 1-3 , the present invention provides a breast cancer auxiliary diagnosis and detection sample kit: according to the following steps;

[0031] S1. Sample addition: Add the sample to the bottom of the ELISA plate well, try not to touch the well wall, and gently shake to mix;

[0032] S2, incubation: seal the plate with a sealing film and incubate at 37°C for 30 minutes;

[0033] S3, liquid preparation: dilute 30 (20 times of 48T) times of concentrated washing liquid with 30 (20 times of 48T) times of distilled water for later use;

[0034] S4, washing: carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times, and pat dry;

[0035] S5, enzyme addition: add 50 μl of enzyme labeling reagent to each well, except for the blank well;

[0036] S6, incubation: seal the plate with a sealing film and incubate at 37°C for 30 minutes;

[0037] S7, washing: carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times, and pat dry;

[0038] S8, color development: first add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake to mix, and color development at 37°C in the dark for 15 minutes;

[0039] S9, stop: add 50 μl of stop solution to each well to stop the reaction (the blue color immediately turns yellow);

[0040] S10. Determination: Use blank as zero and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The determination should be carried out within 15 minutes after adding the stop solution.

[0041] Through the above steps, the HER2 detection is mainly performed using the validated IHC method and the validated dual-probe in situ hybridization method for HER2 detection for breast cancer detection. The breast cancer HER2 detection guidelines recommend the use of immunohistochemistry (IHC) to detect the expression level of the patient's HER2 protein, and the use of in situ hybridization to detect the amplification level of the patient's HER2 gene. Among them, the in situ hybridization method includes two detection methods, namely fluorescence in situ hybridization (FISH) and bright field in situ hybridization. In order to ensure the accuracy of the test results, it is recommended to combine the two detection methods of IHC and in situ hybridization to detect HER2 in patients with breast cancer. Immunohistochemistry (IHC), IHC 0: HER2 negative infiltrating cancer cell membrane is not stained or no more than 10% of the infiltrating cancer cells show incomplete and weak cell membrane staining; IHC 1+: HER2 negative: more than 10% of the infiltrating cancer cells show incomplete and weak cell membrane staining; IHC 2+: HER2 uncertain:

[0042] ① More than 10% of the infiltrating cancer cells showed weak to moderate intensity of intact cell membrane staining;

[0043] ② No more than 10% of the infiltrating cancer cells showed strong and complete cell membrane staining;

[0044] Note: For IHC 2+ cases, further in situ hybridization testing is required on the patient's breast cancer specimen to determine the patient's HER2 gene amplification status. At the same time, different tissue samples of the patient can be selected for re-testing or sent to other laboratories for testing. IHC 3+: More than 10% of the HER2-positive infiltrating cancer cells show strong, complete and uniform cell membrane staining.

[0045] Fluorescence in situ hybridization (FISH) HER2 / CEP17 ratio ≥ 2.0

[0046] FISH positive

[0047] ① Average HER2 copy number / cell ≥4.0;

[0048] ②Numerical HER2 signals are connected into clusters.

[0049] FISH negative

[0050] The average HER2 copy number / cell was <4.0, and the result remained unchanged after increasing the number of cells counted.

[0051] HER2 / CEP17 ratio <2.0

[0052] FISH positive

[0053] ① The average HER2 copy number / cell is ≥6.0, and the result remains unchanged after increasing the number of counted cells;

[0054] ② The average HER2 copy number / cell is ≥4.0 and <6.0, and the result remains unchanged after recounting the signals in at least 20 cell nuclei, and the IHC result is 3+.

[0055] FISH negative

[0056] ① Average HER2 copy number / cell <4.0;

[0057] ② The average HER2 copy number / cell is ≥4.0 and <6.0, and the result remains unchanged after recounting the signals in at least 20 cell nuclei, and the IHC result is 0, 1+ or 2+.

[0058] Note: If the result changes after recounting the signals in at least 20 nuclei, it is necessary to comprehensively judge and analyze the results twice.

[0059] If a patient's initial HER2 test result in a primary breast cancer biopsy specimen is negative, HER2 testing should be repeated on the patient's surgical resection specimen if one of the following is observed:

[0060] ① The tumor histological grade is grade 3;

[0061] ②The number of invasive tumors in the biopsy specimen is very small;

[0062] ③ The resection specimen contains high-grade cancer, and its morphology is different from that of the puncture biopsy specimen;

[0063] ④After ISH and IHC testing, the HER2 results of the biopsy specimen are unclear;

[0064] ⑤ There is doubt about the handling of the biopsy tissue specimen (such as long ischemia, short fixation time, different fixatives) or the pathologist doubts the test results

[0065] Chromogenic in situ hybridization (CISH)

[0066] CISH negative

[0067] ① The average HER2 copy number of tumor cells / cell is <4.0;

[0068] ② The average HER2 copy number / cell is ≥4.0 and <6.0, and the result remains unchanged after recounting the signals in at least 20 cell nuclei, and the IHC result is 0, 1+ or 2+.

[0069] CISH positive

[0070] ① The average HER2 copy number of tumor cells / cell ≥ 6.0;

[0071] ② The average HER2 copy number / cell is ≥4.0 and <6.0, and the result remains unchanged after recounting the signals in at least 20 cell nuclei, and the IHC result is 3+.

[0072] Note: If the result changes after recounting the signals in at least 20 nuclei, it is necessary to comprehensively judge and analyze the results twice.

[0073] (3) Silver-enhanced in situ hybridization (SISH)

[0074] The interpretation of the results of the double-probe silver-enhanced in situ hybridization method can refer to FISH, and the interpretation of the results of the single-probe silver-enhanced in situ hybridization method is the same as CISH.

[0075] Furthermore, in S1, 10 wells with 5 concentration points of the standard were set, and parallel wells were set for each concentration, and 50 μl of standard of different concentrations were added. The blank well was set to 1 well and 50 μl of distilled water was added (the blank control well was not added with sample and enzyme-labeled reagent, and the other steps were the same), and the wells for the samples to be tested were set to add 40 μl of sample diluent to the wells for the samples to be tested on the enzyme-labeled plate, and then add 10 μl of the sample to be tested (the final dilution of the sample was 5 times).

[0076] Further, according to the requirements of the kit, the required reagents and standard curve are prepared. The reagents usually consist of an ELISA plate and a washing buffer solution. The standard curve is a reference standard for determining samples of unknown concentration.

[0077] Further, the coated antibody or antigen is first added to the wells of the ELISA plate, which is then incubated at room temperature for a period of time, which will form a layer of antigen or antibody in the wells for subsequent reactions.

[0078] Furthermore, according to the requirements of the kit, samples and standards are added to the wells, while several wells are left as blank controls. Note that samples and standards are added to the correct wells to avoid cross contamination.

[0079] Furthermore, an enzyme-labeled secondary antibody (usually horseradish peroxidase against human IgG) is added to each well of the ELISA plate to allow it to bind to the target substance.

[0080] Furthermore, the ELISA plate is returned to the incubator for incubation, and then washed again to remove unbound substances.

[0081] Further, a substrate is added to each well to react with the enzyme to produce a color change, and the reaction time is controlled according to the requirements of the kit.

[0082] Furthermore, a reaction stopping solution is added to stop the enzyme reaction and prevent further color change. The specific reaction stopping method should be carried out according to the requirements of the kit.

[0083] Further, the intensity of the color in each well is measured using a microplate reader or a photometer and compared with the standard value on the standard curve to determine the concentration of the target substance in the sample.

[0084] Types of specimens: 1. Surgical resection specimens;

[0085] 2. Core needle biopsy specimen;

[0086] 3. Mammotome biopsy specimen;

[0087] 4. Cytological specimens with more than 100 cancer cells.

[0088] Fixation of specimens:

[0089] 1. All breast cancer specimens should be fixed as soon as possible (within 1 hour) after being removed from the body.

[0090] 2. When fixing, the specimen should be cut every 5-10 mm, and gauze or filter paper can be embedded between the tissues.

[0091] 3. The ratio of the amount of fixative to the tissue to be immersed should be sufficient.

[0092] 4. The fixed time should be 6-72 hours.

[0093] 5. Fixative type: 3.7% neutral (phosphate buffered) formaldehyde fixative.

[0094] Tissue sections

[0095] 1. Unstained sections should not be kept at room temperature for more than 6 weeks to prevent antigen loss.

[0096] 2. The thickness of the slices used for IHC staining should be 3-5 μm, and that for in situ hybridization should be 4-5 μm.

[0097] 3. For the slices that have completed the test, IHC and bright field in situ hybridization can be stored for a long time as usual. The FISH results should be photographed and archived immediately and stored for a long time. It is recommended that the FISH slices be stored at -20℃ for at least 3 months for future reference.

[0098] 4. All detection methods should be compared with HE-stained sections. Quality Control

[0099] Including specimen preparation, antibody selection, antigen retrieval methods, staining and other related laboratory techniques, all should be carried out strictly according to SOP.

[0100] IHC automated staining systems are easier to standardize, but rigorous comparison tests and program optimization should also be performed.

[0101] IHC staining requires the establishment of positive and negative controls, and tissue chips with different staining intensities are the best external controls. Normal breast epithelial cells adjacent to the cancer in the tested slices are good negative internal controls. The use of computer head image analysis or artificial intelligence is conducive to the accuracy and repeatability of judgments, but the results must be confirmed by a pathologist, and the equipment must be calibrated before use.

[0102] FISH detection FISH technology uses fluorescently labeled DNA probes to hybridize with DNA target sequences in the cell nucleus. The probe signals hybridized to the DNA target sequence in the cell nucleus are observed and analyzed under a fluorescence microscope to obtain information on the gene status of chromosomes (or chromosome fragments) in the cell nucleus.

[0103] Currently, most probes used to monitor the status of the HER2 gene are double probes that contain both the HER2 gene and the centromere (CEP17) sequence of chromosome 17 where the gene is located. A single probe that only contains the HER2 gene can also be used.

[0104] The observation procedure should first observe the entire section under a low-power microscope to determine whether the staining is satisfactory and whether there is heterogeneity in HER2 expression. Normal breast epithelium should not show strong cell membrane staining. Only the staining of invasive carcinoma is assessed, and the staining of carcinoma in situ cannot be used as an assessment object. If there are too few micro-invasive lesions in the IHC section and it is difficult to assess the HER2 status, a note can be made. Observe the proportion of invasive cancer cells with cell membrane staining and the staining intensity. If cytoplasm or nuclear staining occurs, indicating that the IHC staining effect is not ideal or the tissue is not well processed, it is recommended to adjust the staining conditions or replace the tissue before staining. When interpreting, avoid cancer tissues at the edge of the tissue and those with poor tissue processing (such as obvious squeezing).

[0105] Interpretation criteria: 10% of the infiltrating cancer cells showed incomplete and weak cell membrane staining;

[0106] >10% of the infiltrating cancer cells showed weak to moderate staining of intact cell membranes;

[0107] 2+≤10% of infiltrating cancer cells showed strong and complete cell membrane staining;

[0108] 3+>10% of infiltrating cancer cells showed strong, complete and uniform cell membrane staining.

[0109] Average HER2 copy number / cell ≥4.0: This situation is judged as FISH positive. If many HER2 signals are connected into clusters, it can be directly judged as FISH positive. The ratio is greater than or equal to 2.0 and the average HER2 copy number / cell is <4.0; it is recommended to increase the cell count in this case. If the result remains unchanged, it is judged as FISH negative. It is recommended to note in the report: In the existing clinical trial data, there is a lack of sufficient evidence to show that this part of the patients can benefit from anti-HER2 targeted therapy. More evidence-based medicine needs to be accumulated for this special group of people. It is recommended to add positive and negative controls during each staining process to confirm the quality of the reagents and the function of the instrument. If possible, it is recommended to set a low amplification control.

[0110] Number of chromosome 17

[0111] The purpose of adding the CEP17 probe to the FISH dual-probe test is to detect the number of chromosome 17 while detecting the HER2 gene, thereby distinguishing aneuploidy of chromosome 17 from simple HER2 gene amplification (especially low-level amplification).

[0112] However, studies in recent years have shown that polysomy of the entire chromosome 17 is rare, and the increase in CEP17 does not represent polysomy of the entire chromosome 17.

[0113] In some breast cancers, there is co-amplification of the HER2 gene and the centromere on chromosome 17.

[0114] More and more scholars believe that compared with the HER2 / CEP17 ratio, the HER2 copy number is more important for determining HER2 gene amplification.

[0115] Therefore, in addition to reporting the HER2 / CEP17 ratio in the FISH test results of HER2, the HER2 copy number and CEP17 values ​​should also be reported separately.

[0116] There are literature reports that multiple other gene probes on chromosome 17 can be used to replace CEP17 for FISH testing to solve the problem of interpreting difficult cases with special FISH results. However, this test often obtains multiple different results due to the use of multiple different probes, and there is a lack of basis for how to make a comprehensive judgment in the end. Therefore, it is not recommended for routine use at present.

[0117] Regarding HER2 gene heterogeneity: HER2 expression or amplification may be heterogeneous in invasive breast cancer. Although the clinical significance of HER2 gene heterogeneity is still unclear, it can lead to inconsistent test results between IHC and in situ hybridization, primary lesions and metastatic lesions, and puncture specimens and surgical resection specimens.

[0118] Before in situ hybridization counting, the entire slide should be observed or IHC slides should be used to determine the possible HER2 amplification areas. It should be emphasized that even if heterogeneity exists, as long as the amplified cells are continuous, homogeneous, and account for more than 10% of the invasive carcinoma, they should be clearly reported as in situ hybridization positive.

[0119] The count values ​​of different cell populations (>10%) can be supplemented (including the total number of cells counted, HER2 copy number, CEP17 value, HER2 / CEP17 ratio), and the proportion of the amplified cell population to all infiltrating cancer cells can be reported.

[0120] SISH testing

[0121] The most widely used method in SISH is dual-color in situ hybridization.

[0122] In this test, HER2 is detected by a probe labeled with dinitrobenzene (DNP), and silver staining in situ hybridization DNP staining solution is used for color development. CEP17 is detected by a probe labeled with digoxigenin, and digoxigenin red staining solution is used. The DISH results can be observed under a light microscope, where HER2 appears as a black signal in the nucleus of tumor cells and CEP17 as a red signal. A single probe SISH targeting only the HER2 gene can also be used.

[0123] Observation Procedure

[0124] Combined with HE staining, target areas containing invasive breast cancer are selected for observation. Most cells in the selected area should show both black and red signals, and these signals should not be covered by nonspecific background staining.

[0125] Scan the entire slide under a 4x objective to observe whether there is heterogeneity in HER2 amplification and the quality of the specimen. Then observe the results under a high-power microscope (40x or 60x objective) and perform signal counting and ratio calculation.

[0126] It is recommended to count ≥20 invasive cancer cells in at least 2 representative areas.

[0127] Interpretation of results

[0128] 1. Select cells with uniform nuclei, intact nuclear borders, no overlapping nuclei, and clear red and black signals. Randomly count at least 20 dual-color signals in the nuclei of infiltrating cancer cells.

[0129] 2. When HER2 signal clusters are present, the copy number can be estimated based on the size of a single copy. The interpretation criteria for dual-probe SISH are as for dual-probe FISH, and the interpretation criteria for single-probe SISH are the same as for CISH.

[0130] Quality Control

[0131] Internal control: HER2 signal and CEP17 signal of normal cells in breast tissue (such as fibroblasts, vascular endothelial cells, lymphocytes, normal breast epithelial cells) can be used as internal control. External control:

[0132] It is recommended that positive and negative controls be included in each staining run to confirm reagent quality and instrument functionality.

[0133] CISH Testing

[0134] CISH Testing Principle

[0135] In CISH detection, digoxigenin-labeled probes are often used to perform in situ hybridization on paraffin sections, and then mouse anti-digoxigenin antibodies and horseradish peroxidase-anti-mouse antibodies are used for immunoconjugation. After diaminobenzidine color development, the HER2 gene signal is observed under an ordinary microscope. There are also reports on dual-probe CISH.

[0136] CISH testing can simultaneously display genetic status and tissue morphology, and the test sections can be preserved for a long time.

[0137] Observation Procedure

[0138] Combined with HE staining, the target area containing invasive breast cancer is selected for observation. Most of the cells in the area must have brown signals, and these signals should not be covered by nonspecific background staining.

[0139] The entire slide was observed under a low-power microscope to determine the specimen quality and whether there was heterogeneity in HER2 amplification.

[0140] Then, count the signals under a high-power microscope (40x or 60x objective lens). It is recommended to count ≥20 infiltrating cancer cells in at least 2 representative areas.

[0141] The results should be interpreted by selecting cells with uniform nuclei, complete nuclear borders, no overlapping nuclei, and clear signals. Randomly count the HER2 signals in the nuclei of at least 20 infiltrating cancer cells.

[0142] Interpretation criteria: Quality control:

[0143] Internal control:

[0144] The HER2 signal of normal cells in breast tissue (such as fibroblasts, vascular endothelial cells, lymphocytes, and normal breast epithelial cells) can be used as an internal control.

[0145] External control:

[0146] It is recommended to add positive and negative controls (the quality control photos provided by the kit can be used) during each staining process to confirm the quality of the reagents and the function of the instrument.

[0147] IHC and ISH are recommended for HER2 testing in breast cancer.

[0148] ISH is divided into 3 methods, FISH, SISH and CISH. Currently, there is no data on the consistency rate of FISH, SISH and CISH in breast cancer. Compared with gastric cancer, ISH detection of HER2 in breast cancer pays more attention to the HER2 copy number, so in addition to examining the HER2 / CEP ratio, it is also necessary to examine the average copy number of HER2. HER2 / CEP17 ratio ≥ 2.0, and average HER2 copy 1. The average HER2 copy number of tumor cells / cell ≥ 6.0 is positive CISH positive number / cell ≥ 4.0, positive HER2 / CEP17 ratio < 2.0, average HER2 copy number 1. The average HER2 copy number of tumor cells / cell < 4.0 is CISH negative / cell < 4.0, the interpretation criteria of negative double-probe SISH see double-probe FISH, and the interpretation criteria of single-probe SISH are the same as CISH.

[0149] 1. HER2 / CEP17 ratio <2.0, average HER2 copy number / cell ≥4.0 and <6.0, increase the count of cells, if the result remains unchanged, the IHC result is 3+, and the HER2 status is judged as positive. If the HC result is 0, 1+ or 2+, the HER2 status should be judged as negative1. The average HER2 copy number / cell is ≥4.0 and <6.0,2. HER2 / CEP17 ratio <2.0, average HER2 copy number / increase the count of cells, if the result remains unchanged, the IHC result is 3+, and the HER2 status is judged as positive. If the IHC is 0, 1+ or 2+, the HER2 status is judged as negativeResample and test cells ≥6.0: increase the count of cells, if the result remains unchanged, then FISH is positiveHER2 / CEP17 ratio ≥2.0, average HER2 copy number / 3. Cells <4.0: increase the count of cells, if the result remains unchanged, then FISH is negative.

[0150] Working principle: When performing in situ hybridization detection of HER2 gene, it is crucial to ensure the accuracy and reliability of experimental results. This involves precise control of the experimental process, strict management of experimental conditions, and careful analysis of experimental results. First of all, for the control of the experimental process, we should follow the standard operating procedures to ensure that each step is carried out according to the predetermined procedures. When preparing slices and performing hybridization reactions, special attention should be paid to avoid contamination and cross-contamination to ensure the specificity of the results. At the same time, the selection and use of reagents should also be strictly followed in accordance with the instructions to ensure the effectiveness of the experiment. Secondly, for the management of experimental conditions, we should keep the laboratory clean and quiet to ensure the normal operation of the experimental equipment. When performing hybridization reactions, the temperature and time should be controlled to avoid the influence of too high or too low temperature on the experimental results. In addition, for the reagents used in the experiment, their validity period and storage conditions should be checked regularly to ensure the quality of the reagents. Finally, for the careful analysis of the experimental results, we should combine the HE staining results to accurately count and calculate the ratio of the in situ hybridization signals. When interpreting the results, it is important to distinguish between normal HER2 expression and abnormal HER2 amplification to avoid confusing the two. At the same time, any abnormalities in the results, such as signal clusters and uneven signals, should be carefully analyzed and judged to determine whether HER2 gene amplification exists.

[0151] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the implementation rules without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A breast cancer auxiliary diagnosis and detection sample kit, which is carried out according to the following steps; S1. Sample addition: Add the sample to the bottom of the ELISA plate well, try not to touch the well wall, and gently shake to mix; S2, incubation: seal the plate with a sealing film and incubate at 37°C for 30 minutes; S3, liquid preparation: dilute 30 (20 times of 48T) times of concentrated washing liquid with 30 (20 times of 48T) times of distilled water for later use; S4, washing: carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times, and pat dry; S5, enzyme addition: add 50 μl of enzyme labeling reagent to each well, except for the blank well; S6, incubation: seal the plate with a sealing film and incubate at 37°C for 30 minutes; S7, washing: carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times, and pat dry; S8, color development: first add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake to mix, and color development at 37°C in the dark for 15 minutes; S9, stop: add 50 μl of stop solution to each well to stop the reaction (the blue color immediately turns yellow); S10. Determination: Use blank as zero and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The determination should be carried out within 15 minutes after adding the stop solution.

2. A breast cancer auxiliary diagnosis detection sample kit according to claim 1, characterized in that: The standard in S1 is set with 10 wells at 5 concentration points, and parallel wells are set for each concentration. 50 μl of standard of different concentrations are added. The blank well is set with 1 well and 50 μl of distilled water is added (the blank control well is not added with sample and enzyme labeling reagent, and the other steps are the same). For the wells of the samples to be tested, 40 μl of sample diluent is first added to the wells of the samples to be tested on the enzyme labeling coated plate, and then 10 μl of the sample to be tested is added (the final dilution of the sample is 5 times).

3. A breast cancer auxiliary diagnosis detection sample kit according to claim 1, characterized in that: In S3, the required reagents and standard curve are prepared according to the requirements of the kit. The reagents are usually composed of an ELISA plate and a washing buffer solution. The standard curve is a reference standard for determining samples of unknown concentration.

4. A breast cancer auxiliary diagnosis detection sample kit according to claim 1, characterized in that: In the S5, the coated antibody or antigen is first added to the wells of the ELISA plate, and then incubated at room temperature for a period of time, which will form a layer of antigen or antibody in the wells for subsequent reactions.

5. A breast cancer auxiliary diagnosis detection sample kit according to claim 1, characterized in that: In S1, samples and standards are added to the wells according to the requirements of the kit, and several wells are reserved as blank controls. Note that samples and standards are added to the correct wells to avoid cross contamination.

6. A breast cancer auxiliary diagnosis detection sample kit according to claim 1, characterized in that: In the S5, an enzyme-labeled secondary antibody (usually horseradish peroxidase against human IgG) is added to each well of the ELISA plate to allow it to bind to the target substance.

7. A breast cancer auxiliary diagnosis detection sample kit according to claim 1, characterized in that: In S7, the ELISA plate is placed back into the incubator for incubation, and then washed again to remove unbound substances.

8. A breast cancer auxiliary diagnosis detection sample kit according to claim 1, characterized in that: In S8, a substrate is added to each well to react with the enzyme to produce a color change. The reaction time is controlled according to the requirements of the kit.

9. A breast cancer auxiliary diagnosis detection sample kit according to claim 1, characterized in that: A reaction stopping solution is added to the S9 to stop the enzyme reaction and prevent further color change. The specific reaction stopping method should be carried out according to the requirements of the kit.

10. A breast cancer auxiliary diagnosis detection sample kit according to claim 1, characterized in that: In the S10, a microplate reader or a photometer is used to measure the intensity of the color in each well, and the intensity is compared with the standard value on the standard curve to determine the concentration of the target substance in the sample.

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