A preparation method of a quality control product for intraoperative rapid frozen immunohistochemistry, a matching reagent and a rapid frozen immunohistochemistry method

Abstract

The application relates to a preparation method of a quality control product for intraoperative rapid frozen immunohistochemistry, a matching reagent and a rapid frozen immunohistochemistry method, and belongs to the technical field of pathological detection. The preparation method comprises the following steps: ① a positive cell line containing specific antigen expression is preserved by using a preservation solution to obtain a preservation solution containing the positive cell line; the preservation solution is composed of KCl, NaH2PO4, Na2HPO4, low-molecular alcohol, a polyhydroxy component and water; ② the preservation solution containing the positive cell line obtained in step 1 is added dropwise on a slide and is dried to be semi-dry; ③ a fixing solution is added dropwise on the position of the positive cell line on the slide in step 2 for fixing, the fixing solution is removed, and the slide is dried; the fixing solution is composed of formaldehyde, dimethyl sulfoxide and water. The preparation method of the quality control product for intraoperative rapid frozen immunohistochemistry does not need to carry out antigen repair before the quality control product is used, the operation is simple, and the product has good stability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the pathological detection technical field, and particularly relates to a preparation method of a quality control product for intraoperative rapid frozen immunohistochemistry, a matching reagent, and a rapid frozen immunohistochemistry method. BACKGROUND

[0002] At present, the main method of intraoperative biopsy is intraoperative frozen section HE staining. The frozen section is also called intraoperative rapid frozen section. The advantage of the frozen section is that the examination is rapid. Pathologists rapidly freeze the lesion tissue removed by a doctor in a frozen section machine to prepare a tissue section, the tissue section is dyed by hematoxylin-eosin, and the pathologist observes under a microscope in real time to make a judgment in a short time. The whole process is generally completed within half an hour. However, since the HE staining judgment only relies on morphological evidence, the tissue inevitably forms crystals in the freezing process, the cell morphology performance quality is poor, the cells are deformed, artificial artifacts are generated, and the limited material of the frozen section further increases the difficulty of diagnosis, and there is a possibility of missed diagnosis and misdiagnosis.

[0003] The intraoperative frozen immunohistochemical diagnosis can well make up for the defects of the intraoperative HE staining which only relies on morphological judgment. For some diseases which are difficult to judge the benignity and malignancy from morphology, specific antigens can be marked by immunohistochemical staining in a short time, immunological evidence is increased, diagnosis is made according to the immunological results, the defects of the intraoperative HE staining which is difficult to diagnose are filled, the accuracy of diagnosis is improved, micro lesions are helped to be found, and the false negative rate is reduced. Due to the particularity of the purpose of use, the accuracy of the result is particularly important. The quality control system of the frozen immunohistochemistry as a new detection means is still in the exploratory stage. Under the condition that the system is not perfect, the positive control is particularly important.

[0004] The limited quality control products on the market are mainly paraffin-embedded tissue wax blocks and cell line wax blocks. The quality control products are mainly for conventional immunohistochemistry, and antigen repair is required before use. The operation is complicated, inconvenient to use, and the price is high, so it is not suitable for the quality control of intraoperative frozen immunohistochemistry. The frozen immunohistochemistry is used for pathological diagnosis of intraoperative patients. Due to the unpredictability of the use, the pretreated quality control product is not suitable for the quality control of intraoperative immunohistochemistry. SUMMARY

[0005] The purpose of the present application is to provide a preparation method of a quality control product for intraoperative rapid frozen immunohistochemistry. The prepared quality control product does not need to be subjected to antigen repair before use, the operation is simple, and the product stability is good.

[0006] The second purpose of the present application is to provide a matching reagent of a quality control product for intraoperative rapid frozen immunohistochemistry.

[0007] The third purpose of the present application is to provide an intraoperative rapid frozen immunohistochemistry method.

[0008] To achieve the above object, the technical scheme adopted by the present application is:

[0009] A preparation method of a quality control product for intraoperative rapid immunohistochemistry, comprising the following steps:

[0010] (1) A positive cell line containing a specific antigen expression is preserved using a preservation solution to obtain a preservation solution containing the positive cell line; the preservation solution is composed of the following components: 0.5wt%-3wt% KCl, 0.5wt%-3wt% NaH2PO4, 0.1wt%-2wt% Na2HPO4, 15vol%-35vol% low molecular alcohol, 2wt%-6wt% polyhydroxy component, and water as the solvent; the number of hydroxyl functional groups in the low molecular alcohol is not more than 2, and the number of hydroxyl functional groups in the polyhydroxy component is more than 3;

[0011] (2) The preservation solution containing the positive cell line obtained in step (1) is added dropwise on a glass slide and dried to semi-dry;

[0012] (3) The position of the positive cell line on the glass slide in step (2) is added dropwise with a fixing solution to fix it, the fixing solution is removed, and it is dried; the fixing solution is composed of the following components by volume fraction: 5%-15% formalin, 15%-25% dimethyl sulfoxide, and the balance is water.

[0013] The preparation method of the quality control product for intraoperative rapid immunohistochemistry of the present application uses a preservation solution and a fixing solution with specific components, which can interact with each other, so that the quality control product prepared by the present application has the following advantages: no antigen repair is required before use, direct synchronization with frozen section staining is achieved, and operation is convenient; good stability, can be stored in cold storage and used at room temperature like paraffin sections; lower cost, conducive to popularization and use.

[0014] The mass fraction of formaldehyde in formalin is 35%-40%. Formalin can play a fixing role, maintain the original morphology of the cells, and make the antigenicity of the cells well maintained, and dimethyl sulfoxide (DMSO) can act as a moisturizing agent and a transdermal agent.

[0015] Further, the positive cell line is SKBR-3, MCF-7, hela, hTERT-HME1 or hTCEpi (corneal epithelial cell line).

[0016] Further, the cell amount of the positive cell line is 3×10 5 ~ 8×10 5 / cm 2 .

[0017] Further, the low molecular alcohol is one or any combination of ethylene glycol, methanol, and ethanol. The polyhydroxy component is one or any combination of mannitol, trehalose, and sucrose.

[0018] Further, the air-drying to semi-dry in step (2) is performed at room temperature for 30 seconds to 2 minutes. Air-drying to semi-dry is air-drying to slightly wet, not completely dry. The time of 30 seconds to 2 minutes can achieve better staining effect, making it easier to read.

[0019] Further, the drying in step (3) is air-drying at room temperature for 30 seconds to 2 minutes. Air-drying is air-drying to no flowing water on the smear, which can be adjusted according to the ambient temperature and humidity.

[0020] Further, the dropwise volume of the preservation solution is 0.5 to 5 μL. The dropwise volume of the fixing solution is 60 to 100 μL. Further, the dropwise volume of the preservation solution is 0.5 to 2 μL.

[0021] Further, the fixing time is 3 to 10 minutes. Further, the fixing time is 3 to 8 minutes. The time of 3 to 10 minutes can ensure that the fixing solution has a good fixing effect on the positive cell line, and at the same time, it will not be over-fixed, which will interfere with the subsequent staining effect.

[0022] Further, the preservation solution is composed of the following components: 0.5 wt% to 2 wt% KCl, 1 wt% to 3 wt% NaH2PO4, 0.1 wt% to 1 wt% Na2HPO4, 15 vol% to 35 vol% low molecular alcohol, and 3 wt% to 5 wt% polyhydroxy component, and the solvent is water. The fixing solution is composed of the following components by volume fraction: 10% to 15% formaldehyde and 15% to 25% dimethyl sulfoxide, and the balance is water.

[0023] Further, the preparation method of the quality control product for intraoperative rapid immunohistochemistry further comprises the following steps: storing the slide after air-drying in step (3) at 4°C or under -20°C refrigeration conditions.

[0024] Further, before using the preservation solution to preserve the positive cell line containing specific antigen expression, the positive cell line culture solution is centrifuged to obtain the precipitate, the positive cell line is washed with PBS, and then the excess PBS is removed. Further, the centrifugal speed is 1000 to 1500 r / min, and the centrifugal time is 4 to 8 minutes.

[0025] Further, before adding the preservation solution containing the positive cell line containing specific antigen expression on the slide, an area is circled on the slide using an immunohistochemical oil pen, and the size is about 0.5 to 1 cm 2 The preservation solution is added to the circled area.

[0026] The quality control material of the present invention for intraoperative rapid frozen immunohistochemistry can be stored for more than 6 months under refrigeration and more than 6 days at room temperature; it has strong anti-interference ability. In the intraoperative rapid frozen immunohistochemistry method, frozen tissue sections need to be fixed after freezing. Whether or not the quality control material undergoes a secondary fixation process, it does not affect the staining effect; after staining, the quality control material is diffusely positive, which is relatively easy to interpret.

[0027] The technical solution adopted for the supporting reagents of the quality control materials for intraoperative rapid frozen immunohistochemistry of the present invention is as follows:

[0028] A quality control reagent for intraoperative rapid frozen immunohistochemistry includes a preservation solution and a fixative. The preservation solution consists of the following components: 0.5wt%–3wt% KCl, 0.5wt%–3wt% NaH2PO4, 0.1wt%–2wt% Na2HPO4, 15vol%–35vol% low molecular weight alcohol, and 2wt%–6wt% polyhydroxy component, with water as the solvent. The low molecular weight alcohol contains no more than two hydroxyl functional groups, and the polyhydroxy component contains three or more hydroxyl functional groups. The fixative consists of the following components in volume fractions: 5%–15% formalin, 15%–25% dimethyl sulfoxide, with the balance being water.

[0029] The accompanying reagents for quality control products used in intraoperative rapid frozen immunohistochemistry of the present invention combine preservation solutions and fixatives of specific components to prepare quality control products for intraoperative rapid frozen immunohistochemistry. Through the synergistic effect between the components, the quality control products have good stability and can be used directly without antigen retrieval.

[0030] Furthermore, the preservation solution in the accompanying reagent consists of the following components: 0.5wt%–2wt% KCl, 1wt%–3wt% NaH2PO4, 0.1wt%–1wt% Na2HPO4, 15vol%–35vol% low molecular weight alcohol, 3wt%–5wt% polyhydroxy components, and water as the solvent.

[0031] Furthermore, the fixative in the accompanying reagent consists of the following components in volume fractions: 10%–15% formalin, 15%–25% dimethyl sulfoxide, and the balance being water.

[0032] The technical solution adopted by the intraoperative rapid frozen immunohistochemistry method of the present invention is as follows:

[0033] A rapid intraoperative frozen immunohistochemistry method includes the following steps: thawing the quality control sample obtained by the above method after refrigeration or freezing, attaching the tissue to be tested to the non-positive cell line position on the quality control sample, fixing it with fixative, washing it with water after fixation, and performing immunohistochemical staining.

[0034] In the intraoperative rapid frozen immunohistochemistry method, the lesion tissue is rapidly frozen in a cryostat to prepare sections. After the quality control material is thawed, the frozen sections can adhere better to the glass slide due to the temperature difference between the tissue sections and the quality control material.

[0035] The intraoperative rapid frozen immunohistochemistry method of the present invention uses the quality control material of the present invention for intraoperative rapid frozen immunohistochemistry as a positive control. It can be used for re-examination of frozen immunohistochemistry reagent samples, monitoring of the staining process, and evaluation of immediate staining results, ensuring the accuracy of intraoperative immunohistochemistry staining results, providing pathologists with more accurate evidence, and thus better and more accurately guiding surgical plans.

[0036] The intraoperative rapid frozen immunohistochemistry method of the present invention fills the gap in the field of intraoperative frozen immunohistochemistry quality control, solves the problem of the lack of positive controls in intraoperative frozen immunohistochemistry, and is conducive to promoting the establishment of an intraoperative frozen immunohistochemistry quality control system.

[0037] Furthermore, in the intraoperative rapid frozen immunohistochemistry method, the fixation time of the slide in the fixative is 30 seconds to 5 minutes. The washing time is 30 seconds to 5 minutes. Even further, in the intraoperative rapid frozen immunohistochemistry method, the fixation time of the slide in the fixative is 30 seconds to 2 minutes. The washing time is 1 minute. Attached Figure Description

[0038] Figure 1 This is a staining result image after testing the quality control material used for intraoperative rapid frozen immunohistochemistry in Example 15 of the present invention;

[0039] Figure 2 This is a staining result image after testing the quality control material used for intraoperative rapid frozen immunohistochemistry in Example 16 of the present invention;

[0040] Figure 3 This is a staining result image after testing the quality control material used for intraoperative rapid frozen immunohistochemistry in Example 17 of the present invention;

[0041] Figure 4 The staining results of the quality control material (numbered p63-A) in Example 4 of this invention after being refrigerated at 4°C for 1, 2, 3, 4, 5, and 6 months, respectively, were obtained after being used for intraoperative rapid frozen immunohistochemistry.

[0042] Figure 5 The staining results of the quality control material (numbered p63-B) in Comparative Example 1 of this invention after being refrigerated at 4°C for 1, 2, 3, 4, 5, and 6 months, respectively, were obtained after being tested by intraoperative rapid frozen immunohistochemistry.

[0043] Figure 6 The staining result chart of the control sample (CK Guang-A) in Example 5 of the present application after detection by the control sample for intraoperative rapid frozen immunohistochemistry after being stored at room temperature for 1, 2, 3, 4, 5, and 6 days;

[0044] Figure 7 The staining result chart of the control sample (CK Guang-B) in Comparative Example 2 of the present application after detection by the control sample for intraoperative rapid frozen immunohistochemistry after being stored at room temperature for 1, 2, 3, 4, 5, and 6 days;

[0045] Figure 8 The staining result chart of the control sample (CK Guang-A) in Example 5 of the present application after detection by the control sample for intraoperative rapid frozen immunohistochemistry after being stored at room temperature for 1, 2, 3, 4, 5, and 6 days;

[0046] Figure 9 The staining result chart of the control sample (CK Guang-A) in Example 5 of the present application after detection by the control sample for intraoperative rapid frozen immunohistochemistry after being stored at room temperature for 1, 2, 3, 4, 5, and 6 days;

[0047] Figure 10 The staining result chart of the control sample in Example 1 of the present application after detection of breast sentinel lymph nodes by the first group;

[0048] Figure 11 The staining result chart of the control sample in Example 2 of the present application after detection of breast punch biopsy samples by the second group. DETAILED DESCRIPTION

[0049] The present application is further described below in conjunction with examples.

[0050] I. Examples of the preparation method of the control sample for intraoperative rapid frozen immunohistochemistry

[0051] Example 1

[0052] The preparation method of the control sample for intraoperative rapid frozen immunohistochemistry in the present example comprises the following steps:

[0053] (1) Take the cell line SKBR-3 expressing CK broad spectrum, culture, and harvest, centrifuge at a speed of 1200 r / min for 5 minutes, wash with PBS once, add an appropriate amount of preservative, and adjust the cell amount to about 5×10 5 cells / cm 2The preservative solution is composed of 0.5wt% KCl, 2.5wt% NaH2PO4, 1wt% Na2HPO4, 5vol% ethylene glycol, 15vol% methanol, 5wt% mannitol, 80vol% pure water;

[0054] (2) Circle the area under the glass slide with an immunohistochemical oil pen, about 1cm in size 2 ;

[0055] (3) Mix the cells in the preservative solution gently, and drop 1μL of the preservative solution into the area circled by the oil pen, and slightly dry at room temperature for 1min;

[0056] (4) Add 100μL of the fixing solution into the area circled by the oil pen, and fix for 3min, the fixing solution is composed of 10vol% formaldehyde + 15vol% DMSO in water;

[0057] (5) Remove the excess fixing solution, dry at room temperature for 1min, and store in the refrigerator at 4℃.

[0058] The quality control product in this embodiment is coated under the glass slide, and needs to be fixed together with the tissue to be tested, and is fixed twice by the fixing solution.

[0059] Example 2

[0060] The preparation method of the quality control product for intraoperative rapid frozen immunohistochemistry in this embodiment comprises the following steps:

[0061] (1) Culture and harvest the p63-expressing cell line hTERT-HME1, centrifuge at 1500r / min for 4min, wash with PBS once, add an appropriate amount of preservative solution, and adjust the cell amount to about 3×10 5 cells / cm 2 The preservative solution is composed of 2wt% KCl, 1wt% KH2PO4, 0.5wt% Na2HPO4, 10vol% ethylene glycol, 25vol% ethanol, 3wt% trehalose, and 65vol% pure water;

[0062] (2) Circle the area above the glass slide with an immunohistochemical oil pen, about 0.6cm in size 2 ;

[0063] (3) Mix the cells in the preservative solution gently, and drop 2μL of the preservative solution into the area circled by the oil pen, and slightly dry at room temperature for 2min;

[0064] (4) Add 80μL of the fixing solution into the area circled by the oil pen, and fix for 5min, the fixing solution is composed of 10vol% formaldehyde + 20vol% DMSO in water;

[0065] (5) remove the excess fixative, air dry at room temperature for 1.5 min, and store in a refrigerator at 4°C.

[0066] The quality control sample in this embodiment is coated on the glass slide, and does not need to be fixed together with the tissue to be tested, and is not subjected to secondary fixation with a fixative.

[0067] Example 3

[0068] The preparation method of the quality control sample for intraoperative rapid frozen immunohistochemistry in this embodiment includes the following steps:

[0069] (1) Take the CK5 / 6-expressing cell line hTCEpi, culture, harvest, centrifuge at 1000 r / min for 8 minutes, wash with PBS once, add an appropriate amount of preservation solution, and adjust the cell amount to about 8x10 5 / cm 2 ; the preservation solution is composed of the following components: 1wt% KCl, 3wt% NaH2PO4, 0.1wt% Na2HPO4, 5vol% ethylene glycol, 10vol% methanol, 3wt% mannitol, 2wt% sucrose, 85vol% pure water;

[0070] (2) Circle the area on the glass slide with an immunohistochemical oil pen, with a size of about 0.5cm 2 ;

[0071] (3) Gently beat and mix the cells in the preservation solution, take 0.5μL and drop it into the oil pen circled area, and air dry at room temperature for 30s;

[0072] (4) Take 60μL of fixative, add it to the oil pen circled area, and fix for 8min, the fixative is composed of 10vol% formaldehyde + 25vol% DMSO in water;

[0073] (5) remove the excess fixative, air dry at room temperature for 1min, and store in a refrigerator at -20°C.

[0074] The quality control sample in this embodiment is coated on the glass slide, and does not need to be fixed together with the tissue to be tested, and is not subjected to secondary fixation with a fixative.

[0075] Example 4

[0076] The preparation method of the quality control sample for intraoperative rapid frozen immunohistochemistry in this embodiment includes the following steps:

[0077] (1) Take the p63-expressing cell line hTERT-HME1, culture, harvest, centrifuge at 1200 r / min for 5 minutes, wash with PBS once, add an appropriate amount of preservation solution, and adjust the cell amount to about 5x10 5 / cm 2; the preservative solution is composed of the following components: 1wt% KCl, 1wt% NaH2PO4, 1wt% Na2HPO4, 5vol% ethylene glycol, 20vol% ethanol, 3wt% trehalose, 75vol% pure water;

[0078] (2) Circle the area on the slide with an immunohistochemical oil pen, about 0.6cm in size 2 ;

[0079] (3) Gently mix the cells in the preservative solution, and drop 2μL of the preservative solution into the area circled by the oil pen, and slightly air dry at room temperature for 2min;

[0080] (4) Take 80μL of the fixing solution, and add it into the area circled by the oil pen, and fix for 5min; the fixing solution is composed of 10vol% formaldehyde + 20vol% DMSO in water;

[0081] (5) Remove the excess fixing solution, and air dry at room temperature for 2min; according to the above steps, a total of 7 groups of samples are prepared, wherein 1 group is directly used for intraoperative rapid frozen immunohistochemical detection in Example 18 after completion, and the remaining 6 groups (numbered p63-A) are placed in the refrigerator and stored at 4°C for 1, 2, 3, 4, 5, and 6 months respectively.

[0082] Example 5

[0083] The preparation method of the quality control product for intraoperative rapid frozen immunohistochemistry in this embodiment comprises the following steps:

[0084] (1) Take the CK broad-spectrum expressing cell line SKBR-3, culture, and harvest, centrifuge at 1000r / min for 5min, wash with PBS once, add an appropriate amount of preservative solution, and adjust the cell amount to about 6×10 5 / cm 2 ; the preservative solution is composed of the following components: 1wt% KCl, 1wt% NaH2PO4, 1wt% Na2HPO4, 8vol% ethylene glycol, 25vol% methanol, 3wt% trehalose, 67vol% pure water;

[0085] (2) Circle the area below the slide with an immunohistochemical oil pen, about 1cm in size 2 ;

[0086] (3) Gently mix the cells in the preservative solution, and drop 1μL of the preservative solution into the area circled by the oil pen, and slightly air dry at room temperature for 1min;

[0087] (4) Take 100μL of the fixing solution, and add it into the area circled by the oil pen, and fix for 3min; the fixing solution is composed of 10vol% formaldehyde + 20vol% DMSO in water;

[0088] (5) remove the excess fixative, air dry at room temperature for 1 min, and prepare 7 groups of samples according to the above steps, wherein one group is directly used for intraoperative rapid frozen immunohistochemical detection in Example 19 after completion, and the remaining 6 groups (numbered CK broad-A) are stored at room temperature for 1, 2, 3, 4, 5, and 6 days.

[0089] Example 6

[0090] The preparation method of the quality control for intraoperative rapid frozen immunohistochemistry in this example comprises the following steps:

[0091] (1) Take the CK5 / 6 expressing cell line hTCEpi, culture and harvest, centrifuge at a speed of 1000 r / min for 5 minutes, wash with PBS once, add an appropriate amount of preservation solution, and adjust the cell amount to about 4x10 5 / cm 2 ; the preservation solution is composed of the following components: 0.5wt% KCl, 1.5wt% NaH2PO4, 1wt% Na2HPO4, 10vol% ethylene glycol, 20vol% methanol, 5wt% mannitol, and 70vol% pure water;

[0092] (2) Circle the area on the slide with an immunohistochemical oil pen, about 0.5cm 2 in size;

[0093] (3) Gently mix the cells in the preservation solution, take 0.5μL and drop it into the oil pen circled area, and air dry at room temperature for 30s;

[0094] (4) Take 60μL of fixative, add it to the oil pen circled area, and fix for 3min, the fixative is composed of 10vol% formaldehyde + 20vol% DMSO in water;

[0095] (5) remove the excess fixative, air dry at room temperature for 30s, and store in the refrigerator at -20℃.

[0096] Example 7

[0097] The preparation method of the quality control for intraoperative rapid frozen immunohistochemistry in this example comprises the following steps:

[0098] (1) Take the CK broad-spectrum expressing cell line SKBR-3, culture and harvest, centrifuge at a speed of 1000 r / min for 5 minutes, wash with PBS once, add an appropriate amount of preservation solution, and adjust the cell amount to about 7x10 5 / cm 2 ; the preservation solution is composed of the following components: 0.5wt% KCl, 1.5wt% NaH2PO4, 1wt% Na2HPO4, 10vol% ethylene glycol, 20vol% methanol, 5wt% mannitol, and 70vol% pure water;

[0099] (2) Circle the area on the slide with an immunohistochemical oil pen, about 0.7 cm in size 2 ;

[0100] (3) Gently beat the cells in the preservation solution to mix, take 1 μL and drop in the oil pen circle area, slightly dry at room temperature for 1 min;

[0101] (4) Take 60 μL of the fixing solution, add to the oil pen circle area, fix for 3 min, the fixing solution is composed of 10 vol% formaldehyde + 20 vol% DMSO in water;

[0102] (5) Remove the excess fixing solution, dry at room temperature for 1 min, and store in the refrigerator at -20℃.

[0103] II. Examples of the matched reagents for the quality control product for intraoperative rapid frozen immunohistochemistry

[0104] Example 8

[0105] The matched reagents for the quality control product for intraoperative rapid frozen immunohistochemistry of the present example are the preservation solution and the fixing solution in Example 1.

[0106] Example 9

[0107] The matched reagents for the quality control product for intraoperative rapid frozen immunohistochemistry of the present example are the preservation solution and the fixing solution in Example 2.

[0108] Example 10

[0109] The matched reagents for the quality control product for intraoperative rapid frozen immunohistochemistry of the present example are the preservation solution and the fixing solution in Example 3.

[0110] Example 11

[0111] The matched reagents for the quality control product for intraoperative rapid frozen immunohistochemistry of the present example are the preservation solution and the fixing solution in Example 4.

[0112] Example 12

[0113] The matched reagents for the quality control product for intraoperative rapid frozen immunohistochemistry of the present example are the preservation solution and the fixing solution in Example 5.

[0114] Example 13

[0115] The matched reagents for the quality control product for intraoperative rapid frozen immunohistochemistry of the present example are the preservation solution and the fixing solution in Example 6.

[0116] Example 14

[0117] The reagent kit for the quality control product for intraoperative rapid frozen immunohistochemistry of the embodiment is the preservative solution and the fixing solution in Embodiment 7.

[0118] III. Embodiment of intraoperative rapid frozen immunohistochemistry method

[0119] Embodiment 15

[0120] The intraoperative rapid frozen immunohistochemistry method of the embodiment uses the quality control product for intraoperative rapid frozen immunohistochemistry prepared in Embodiment 1, and includes the following steps:

[0121] (1) The quality control product is taken out of the refrigerator, and the test tissue is attached to the slide coated with the cell line after rewarming for 1 min;

[0122] (2) The slide is quickly placed in the fixing solution for fixation for 1 min, and then washed with water for 1 min;

[0123] (3) The test tissue is circled by an immunohistochemical oil pen, and the rapid frozen immunohistochemical staining is completed together with the quality control product. The staining result is shown in Figure 1 , and it can be known from Figure 1 that the staining effect of the quality control product in the embodiment is good.

[0124] Embodiment 16

[0125] The intraoperative rapid frozen immunohistochemistry method of the embodiment uses the quality control product for intraoperative rapid frozen immunohistochemistry prepared in Embodiment 2, and includes the following steps:

[0126] (1) The quality control product is taken out of the refrigerator, and the test tissue is attached to the slide coated with the cell line after rewarming for 1 min;

[0127] (2) The slide is quickly placed in the fixing solution for fixation for 30 s, and then washed with water for 1 min;

[0128] (3) The test tissue is circled by an immunohistochemical oil pen, and the rapid frozen immunohistochemical staining is completed together with the quality control product. The staining result is shown in Figure 2 , and it can be known from Figure 2 that the staining effect of the quality control product in the embodiment is good.

[0129] Embodiment 17

[0130] The intraoperative rapid frozen immunohistochemistry method of the embodiment uses the quality control product for intraoperative rapid frozen immunohistochemistry prepared in Embodiment 3, and includes the following steps:

[0131] (1) The quality control product is taken out of the refrigerator, and the test tissue is attached to the slide coated with the cell line after rewarming for 2 min;

[0132] (2) The slide is quickly placed in the fixing solution for fixation for 2 min, and then washed with water for 1 min;

[0133] (3) The test tissue was circled by an immunohistochemical oil pen, and the rapid frozen immunohistochemical staining was completed together with the quality control sample. Figure 3 As shown in the staining results, Figure 3 it can be seen that the staining effect of the quality control sample in this embodiment is good.

[0134] Example 18

[0135] The intraoperative rapid frozen immunohistochemical method of this embodiment uses the quality control sample for intraoperative rapid frozen immunohistochemistry prepared in Example 4, and includes the following steps:

[0136] (1) The quality control sample was taken out of the refrigerator, and after rewarming for 2 min, the test tissue was attached to the glass slide coated with the cell line;

[0137] (2) The glass slide was quickly placed in the fixing solution for 2 min, and washed with water for 1 min;

[0138] (3) The test tissue was circled by an immunohistochemical oil pen, and the rapid frozen immunohistochemical staining was completed together with the quality control sample.

[0139] Example 19

[0140] The intraoperative rapid frozen immunohistochemical method of this embodiment uses the quality control sample for intraoperative rapid frozen immunohistochemistry prepared in Example 5, and includes the following steps:

[0141] (1) The quality control sample was taken out of the refrigerator, and after rewarming for 2 min, the test tissue was attached to the glass slide coated with the cell line;

[0142] (2) The glass slide was quickly placed in the fixing solution for 2 min, and washed with water for 1 min;

[0143] (3) The test tissue was circled by an immunohistochemical oil pen, and the rapid frozen immunohistochemical staining was completed together with the quality control sample.

[0144] Example 20

[0145] The intraoperative rapid frozen immunohistochemical method of this embodiment uses the quality control sample for intraoperative rapid frozen immunohistochemistry prepared in Example 6, and includes the following steps:

[0146] (1) The quality control sample was taken out of the refrigerator, and after rewarming for 2 min, the test tissue was attached to the glass slide coated with the cell line;

[0147] (2) The glass slide was quickly placed in the fixing solution for 2 min, and washed with water for 1 min;

[0148] (3) The test tissue was circled by an immunohistochemical oil pen, and the rapid frozen immunohistochemical staining was completed together with the quality control sample.

[0149] Example 21

[0150] The intraoperative rapid frozen immunohistochemical method of this example uses the quality control product for intraoperative rapid frozen immunohistochemistry prepared in Example 7, and includes the following steps:

[0151] (1) Take the quality control product out of the refrigerator, and after rewarming for 1 min, attach the tissue to be tested to the slide coated with the cell line;

[0152] (2) Quickly place the slide in the fixing solution and fix for 1 min, and then wash with water for 1 min;

[0153] (3) Circle the tissue to be tested using an immunohistochemical oil pen, and complete the rapid frozen immunohistochemical detection together with the quality control product.

[0154] The subsequent rapid frozen immunohistochemical detection can be performed according to the existing method, or according to the following staining method:

[0155] ① After washing the slide with pure water, circle the tissue area to be tested on the slide using an immunohistochemical oil pen, and add 50-100 μL of blocking agent to the circled tissue area to be tested, incubate for 30 s, and then wash the slide with washing buffer for 30 s;

[0156] ② Remove the excess washing buffer, and add 50-100 μL of antibody reagent or blank control reagent to the tissue area to be tested (if there are bubbles on the tissue, gently flush away all the bubbles), incubate for 2-4 min; wash with washing buffer for 1 min;

[0157] ③ Remove the washing buffer, and add 50-100 μL of anti-mouse / rabbit polymer to the tissue area to be tested, incubate for 2-3 min, and then wash with washing buffer for 1 min;

[0158] ④ Remove the excess washing buffer, and add 50-100 μL of freshly prepared DAB staining solution to the tissue area to be tested, incubate at room temperature for 2 min;

[0159] ⑤ Wash the slide with purified water, add 50-100 μL of hematoxylin to the tissue area to be tested and incubate for 10-15 s, and then wash with purified water to return the slide to blue;

[0160] ⑥ Dehydrate the tissue to be tested with gradient ethanol, and then transparentize the tissue to be tested with xylene, and finally mount the slide with neutral balsam and a cover glass;

[0161] ⑦ The pathologist reviews the slide, and interprets the results by comparing with HE staining.

[0162] Example 21

[0163] Comparative Example 1

[0164] The in-operation quick frozen immunohistochemical method of the present comparative example is only different from that of Example 18 in that the preservative solution in the quality control sample for in-operation quick frozen immunohistochemistry used in the present comparative example omits trehalose, and the specific implementation is as follows:

[0165] Example 18 uses the quality control sample in Example 4, and the preservative solution of the quality control sample consists of 1wt% KCl, 1wt% NaH2PO4, 1wt% Na2HPO4, 5vol% ethylene glycol, 20vol% ethanol, 3wt% trehalose, and 75vol% pure water; the preservative solution of the present comparative example consists of 1wt% KCl, 1wt% NaH2PO4, 1wt% Na2HPO4, 5vol% ethylene glycol, 20vol% ethanol, and 75vol% pure water.

[0166] The steps not mentioned refer to those of Example 18, and a total of 7 groups of samples are prepared, of which 1 group is directly used for in-operation quick frozen immunohistochemical detection in the present comparative example, and the remaining 6 groups (numbered as p63-B) are placed in a refrigerator and stored at 4°C for 1, 2, 3, 4, 5, and 6 months, respectively.

[0167] Comparative Example 2

[0168] The in-operation quick frozen immunohistochemical method of the present comparative example is only different from that of Example 19 in that the formula of the fixing solution in the quality control sample for in-operation quick frozen immunohistochemistry used in the present comparative example is 80vol% methanol + 20vol% DMSO, and the specific implementation is as follows:

[0169] Example 19 uses the quality control sample in Example 5, and the fixing solution of the quality control sample consists of an aqueous solution of 10vol% formalin + 20vol% DMSO; the fixing solution of the present comparative example consists of 80vol% methanol + 20vol% DMSO.

[0170] The steps not mentioned refer to those of Example 19. A total of 7 groups of samples are prepared, of which 1 group is directly used for in-operation quick frozen immunohistochemical detection in the present comparative example, and the remaining 6 groups (numbered as CK broad-B) are stored at room temperature for 1, 2, 3, 4, 5, and 6 days, respectively.

[0171] Comparative Example 3

[0172] The in-operation quick frozen immunohistochemical method of the present comparative example is only different from that of Example 20 in that the formula of the fixing solution in the quality control sample for in-operation quick frozen immunohistochemistry used in the present comparative example is 90vol% ethanol + 10vol% glacial acetic acid, and the specific implementation is as follows:

[0173] Example 20 uses the quality control sample from Example 6. The stationary phase of the quality control sample consists of an aqueous solution of 10 vol% formalin and 20 vol% DMSO. The stationary phase of this comparative example consists of 90 vol% ethanol and 10 vol% glacial acetic acid.

[0174] For any content not mentioned, refer to the steps in Example 20.

[0175] Comparative Example 4

[0176] The intraoperative rapid frozen immunohistochemistry method in this comparative example differs from that in Example 21 only in that the drying time in step (3) of the quality control material used for intraoperative rapid frozen immunohistochemistry is 5 min.

[0177] For any content not mentioned, refer to the steps in Example 21.

[0178] V. Experimental Examples

[0179] Comparison of preservation solution formulations in Experiment Example 1

[0180] The remaining 6 groups (p63-A) from Example 4 and the remaining 6 groups (p63-B) from Comparative Example 1 were subjected to rapid frozen immunohistochemical staining after the storage time was completed (the immunohistochemical method was performed according to the steps in Example 18). The staining results are as follows. Figure 4 , Figure 5 As shown in Table 1.

[0181] Table 1. Stability of the quality control samples in Example 4 and Comparative Example 1 at 4°C

[0182]

[0183] Depend on Figure 4 , Figure 5 As shown in Table 1, the quality control sample in Example 4 is stable and can still be used normally after being stored at 4°C for 6 months. The quality control sample has good thermal stability. However, the preservation solution in Comparative Example 1 did not contain trehalose, so its preservation stability was poor. The antigen began to be lost after being stored at 4°C for two months, and was completely lost after being stored for 4 months, so it could not be used as a positive reference.

[0184] Comparison of Fixative Formulations in Experiment Example 2

[0185] 2.1 Comparison when methanol is used as the stationary phase

[0186] The remaining 6 groups (numbered CK-A) from Example 5 and the remaining 6 groups (numbered CK-B) from Comparative Example 2 were used for rapid frozen immunohistochemical detection after the storage time was completed (the immunohistochemical method was performed according to the steps in Example 19). The staining results are as follows. Figure 6 , Figure 7 As shown in Table 2.

[0187] Table 2 Quality control room temperature storage stability (25°C) of Example 5 and Comparative Example 2

[0188]

[0189]

[0190] From Figure 6 , Figure 7 and Table 2, the quality control in Example 5 is stable in properties, and can still be used normally after being placed at room temperature for 6 days, while the formaldehyde in the fixing solution of Comparative Example 2 is replaced by methanol, and the storage stability is poor, the antigen starts to lose after being stored at room temperature for 1 day, and the antigen is completely lost after being stored for 3 days, and cannot be used as a positive reference.

[0191] 2.2 Comparison of using ethanol and glacial acetic acid in fixing solution

[0192] The staining results of the quality control in Example 20 and Comparative Example 3 are shown in CK5 / 6-A group and CK5 / 6-B group in Table 2, and it can be seen that compared with the well-stained CK5 / 6-A group, the quality control in CK5 / 6-B group replaces the formula of the fixing solution with ethanol + glacial acetic acid, which causes the cell membrane to rupture, the cytoplasm to melt out, the antigen to lose, and cannot be used as a positive reference. Figure 8

[0193] Experimental Example 3 Air-drying time comparison

[0194] The staining results of the quality control in Example 21 and Comparative Example 4 are shown in CK wide-A group and CK wide-B group in Table 2, and it can be seen that the staining effect of CK wide-A group is better, which is obviously stronger than that of CK wide-B group, so after adding the preservation solution on the slide, it only needs to be slightly air-dried, and cannot be air-dried for too long. Figure 9 Experimental Example 4

[0195] 4.1 Breast sentinel lymph node detection

[0196] In this experimental example, the quality control (CK wide quality control) in Example 1 is used to detect the breast sentinel lymph node, and the specific steps are as follows:

[0197] (1) Take the breast sentinel lymph node and place it on the frozen head with a little OCT embedding agent, freeze to the appropriate hardness, prepare 3 frozen sections with a thickness of about 4 μm, and one of which is pasted on the slide with CK wide quality control;

[0198] (2) Fix the slide in the fixing solution for 1 min, 2 sections are subjected to HE staining, and 1 section is subjected to CK wide rapid immunohistochemical staining;

[0199]

[0200] ​​(3) After staining, dehydrate and mount the slides, observe the slides, and record the staining results of the quality control samples and the tissue to be tested. Repeat the above steps three times. The results are shown in Table 3. Figure 10 (As shown in Group ① of Table 3).

[0201] Table 3. Results of CK quality control sample and breast sentinel lymph node staining.

[0202]

[0203]

[0204] From Table 3 and Figure 10 It can be seen that in this experimental case, the CK quality control sample was positive, and the test tissue showed positive small cell clusters. Therefore, this case is a case of micrometastasis of the sentinel lymph nodes in the breast, and it is necessary to expand the surgical wound and perform breast lymph node dissection.

[0205] 4.2 Detection of breast tissue excision samples

[0206] This experimental example uses the quality control material (p63 quality control material) from Example 2 to test breast tissue excised samples. The specific steps are as follows:

[0207] a. Take breast tissue, place it on a cryoprobe with a small amount of OCT embedding agent, freeze to a suitable hardness, and prepare 3 frozen sections with a thickness of about 4 μm. One of them is attached to a glass slide with p63 quality control material.

[0208] b. Fix in fixative for 1 min, perform HE staining on 2 sections, and perform p63 rapid immunohistochemical staining on 1 section;

[0209] c. After staining, dehydrate and mount the slides, observe the sections, and record the staining results of the quality control samples and the tissue to be tested. Repeat the above steps three times. The results are shown in Table 4. Figure 11 (As shown in Group ② of Table 4).

[0210] Table 4. Results of staining of p63 quality control material and breast tissue excise.

[0211] Group p63 quality control Tissue lesion to be tested Result reliability Result interpretation ① + + Reliable Non-invasive carcinoma ② + - Reliable Invasive carcinoma ③ - + Unreliable Need to retest ④ - - Unreliable Need to retest

[0212] From Table 4 and Figure 11 It can be seen that in this experimental case, the p63 quality control was positive, while the p63 staining of the lesion site in the test tissue was negative. Based on HE morphological judgment, this case is invasive tubular carcinoma of the breast, which requires expanding the surgical scope and performing surgical margin detection.

Claims

1. A method for preparing a quality control product for intraoperative rapid frozen immunohistochemistry, characterized in that, The method comprises the following steps: (1) using a preservation solution to preserve a positive cell line containing specific antigen expression, to obtain a preservation solution containing the positive cell line; the preservation solution is composed of the following components: 0.5wt%-3wt% KCl, 0.5wt%-3wt% NaH2PO4, 0.1wt%-2wt% Na2HPO4, 15vol%-35vol% low-molecular alcohol, 2wt%-6wt% polyhydroxy component, and water as the solvent; the low-molecular alcohol has no more than 2 hydroxyl functional groups, and the polyhydroxy component is one or any combination of mannitol, trehalose, and sucrose; (2) dropping the preservation solution containing the positive cell line obtained in step (1) on a glass slide and air-drying to semi-dryness; (3) dropping a fixing solution on the position of the positive cell line on the glass slide in step (2) for fixation, removing the fixing solution, and drying; the fixing solution is composed of the following components in volume fraction: 5%-15% formalin, 15%-25% dimethyl sulfoxide, and the balance of water.

2. The method for preparing a quality control for intraoperative rapid frozen immunohistochemistry according to claim 1, characterized in that, The low-molecular alcohol is one or any combination of ethylene glycol, methanol, and ethanol.

3. The method for preparing quality control for intraoperative rapid frozen immunohistochemistry according to claim 1, wherein, The air-drying to semi-dryness in step (2) is performed at room temperature for 30s-2min.

4. The method for preparing a quality control for intraoperative rapid frozen immunohistochemistry according to claim 1, characterized in that, The dropping volume of the preservation solution is 0.5-5μL; and the dropping volume of the fixing solution is 60-100μL.

5. The method for preparing quality control for intraoperative rapid frozen immunohistochemistry according to claim 1, wherein, The fixation time is 3-10min.

6. The method for preparing quality control for intraoperative rapid frozen immunohistochemistry according to claim 1, wherein, The preservation solution is composed of the following components: 0.5wt%-2wt% KCl, 1wt%-3wt% NaH2PO4, 0.1wt%-1wt% Na2HPO4, 15vol%-35vol% low-molecular alcohol, 3wt%-5wt% polyhydroxy component, and water as the solvent; and the fixing solution is composed of the following components in volume fraction: 10%-15% formalin, 15%-25% dimethyl sulfoxide, and the balance of water.

7. The method for preparing a quality control product for intraoperative rapid frozen immunohistochemistry according to any one of claims 1 to 6, characterized in that, The method further comprises the following steps: The glass slide dried in step (3) is stored under refrigeration at 4℃ or freezing at-20℃.

8. A kit reagent for quality control of intraoperative rapid frozen immunohistochemistry, characterized by, The method comprises the following steps:

9. An intraoperative rapid frozen immunohistochemical method, characterized by, The method comprises the following steps: The refrigerated or frozen quality control product obtained by the method of any one of claims 1-7 is warmed, a tissue to be tested is attached to a non-positive cell line position on the quality control product, and then a fixing solution is used for fixation, and the quality control product is washed with water after fixation, and then immunohistochemical staining is performed.

10. The intraoperative rapid frozen immunohistochemical method according to claim 9, characterized in that, The fixation time is 30s-5min, and the water washing time is 30s-5min.

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