WO2014114009A1 - Immunohistochemical quality control reference object and quality control method - Google Patents

Immunohistochemical quality control reference object and quality control method Download PDF

Info

Publication number
WO2014114009A1
WO2014114009A1 PCT/CN2013/071117 CN2013071117W WO2014114009A1 WO 2014114009 A1 WO2014114009 A1 WO 2014114009A1 CN 2013071117 W CN2013071117 W CN 2013071117W WO 2014114009 A1 WO2014114009 A1 WO 2014114009A1
Authority
WO
WIPO (PCT)
Prior art keywords
quality control
control
antigen
paraffin
dextran
Prior art date
Application number
PCT/CN2013/071117
Other languages
French (fr)
Chinese (zh)
Inventor
王小亚
Original Assignee
Wang Xiaoya
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wang Xiaoya filed Critical Wang Xiaoya
Publication of WO2014114009A1 publication Critical patent/WO2014114009A1/en

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
    • G01N1/06—Devices for withdrawing samples in the solid state, e.g. by cutting providing a thin slice, e.g. microtome
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N2001/2893—Preparing calibration standards
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples
    • G01N2001/368—Mounting multiple samples in one block, e.g. TMA [Tissue Microarrays]

Definitions

  • the present invention relates to the field of biological pathology detection, and more particularly to an immunohistochemical quality control reference material and a method for quality control using the same. Background technique
  • Immunohistochemical quality control refers to the use of continuously optimized measures and techniques to develop laboratory systems and routine work, standardize and improve the laboratory immunohistochemical staining procedures and procedures to ensure that the quality of immunohistochemical staining achieves the best results, and An accurate diagnosis can be made.
  • Immunohistochemical staining is an important technique and means in clinical pathological diagnosis and morphological studies.
  • the application of immunohistochemistry to pathological diagnosis began in the 1970s and has been widely used in the global pathology community. It has become an indispensable part of the routine work of pathologists.
  • Immunohistochemical technology has the advantages of specificity, sensitivity and ease of operation, which has made the immunohistochemical technology widely popularized and applied in the field of disease diagnosis. Immunohistochemical technology not only improves the accuracy of pathological diagnosis, but also penetrates into clinical and basic disciplines. It plays an invaluable role in exploring the etiology, pathogenesis and research of diseases.
  • Immunohistochemical technology has the advantages of simple operation, high sensitivity and strong specificity. It is widely used in clinical pathology diagnosis and research in hospitals at various levels, but it is immune due to differences in experimental conditions and other technical factors. The results of histochemical staining vary greatly, which affects the results of pathological diagnosis. At present, the need to improve the level of immunohistochemical staining technology is more and more urgent, and there is still a long way to go.
  • IQC internal quality control
  • EQA external quality assurance
  • the primary step is to establish a reference standard sample (ie, reference). Only the establishment of positive and negative controls can ensure the accuracy of the immunohistochemical staining process, the effectiveness of antibodies and related reagents, confirm the authenticity of the staining results, and detect the presence of false positive or false negative results.
  • Control settings include reagent controls, tissue controls, and internal controls. Since immunohistochemical staining may have false positive and false negative results, both positive and negative controls must be established for each experiment to achieve the best quality control.
  • the current routine immunohistochemistry scan is based on a single section containing tissue that is positive or negative for the antibody to be tested.
  • the present invention provides an immunohistochemical quality control reference material and a method for quality control using the same.
  • the reference is a tissue section control with a stable antigen content to be tested.
  • the present invention provides an immunohistochemical quality control reference and methods for quality control using the same.
  • a reference for quality control of immunohistochemical techniques comprising three controls: a quality controlled positive control, a quality controlled negative control, and an antigen repair control.
  • the quality control positive control is prepared by treating the antigen with a dextran material, curing it, and then embedding it in paraffin.
  • the quality control negative control is prepared by: collecting glucose without antigen The sugar material is embedded in paraffin.
  • the antigen retrieval control is prepared by treating the antigen with a dextran material and solidifying it, and then immersing it in paraffin after being soaked in formalin.
  • the prepared quality control positive control, the quality control negative control and the antigen retrieval control are taken out from the paraffin block, embedded in the same paraffin block in sequence, and sliced and adhered on the microtome.
  • the slide was used as a reference for immunohistochemical quality control.
  • the invention also provides a method for quality control of immunohistochemistry, which simulates a conventional paraffin tissue section by using a material embedded with an antigen to be tested, and cuts into a thin slice on a slicer and adheres to the slide and the tissue to be detected.
  • the immunohistochemical staining is performed together for the purpose of quality control; the material in which the target antigen to be tested is embedded is the above reference substance.
  • the material embedded with the antigen to be tested refers to a solid particle formed by solidification after mixing the dextran with the target antigen to be tested.
  • the antigen to be tested refers to the same protein as the antigen to be detected in the pathological tissue or a protein having the same antigenic determinant.
  • the negative control of the quality control means that the material without the target antigen is prepared by conventional paraffin sectioning, and is attached to the glass slide to be stained together with the tissue to be detected;
  • the positive control of the quality control means that the material in which the target antigen is embedded is prepared by conventional paraffin section, but is not fixed by formalin, so that the antigenic determinant of the target antigen is not blocked;
  • the antigen retrieval control means that the material in which the antigen of interest is embedded is prepared by conventional paraffin sectioning and fixed by formalin so that the antigenic determinant of the target antigen is blocked.
  • the method of the invention utilizes a protein obtained in vitro having the same protein or the same antigenic determinant as the antigen to be detected in the tissue as a control, embedded in the polysaccharide material and solidified to form solid particles, dehydrated, transparent, and immersed in wax. And the embedding and other procedures are made into paraffin blocks, sliced in a tissue microtome and attached to the same slide glass with the tissue to be tested, and immunohistochemical staining is performed together with the tissue to be tested.
  • the method of the present invention designed three controls (ie, reference materials) for quality control of immunohistochemistry, including negative control, positive control, and antigen retrieval control, and directly judged the success or failure of staining by the result of staining of the reference substance, and the staining failure.
  • the reason behind. details as follows: (1) Negative results in negative results for the photos, positive positive results for the photos, and positive results for the antigen retrieval controls. It indicates that the dyeing process is successful and the staining results can be used for pathological diagnosis.
  • Negative control, positive control, and antigen-repair control are all negative results, indicating that the staining process failed.
  • the cause of the failure may be the failure of the experimental reagent or the operation process.
  • Negative controls, positive controls, and antigen-repair controls were all positive. It indicates that the dyeing process failed, that is, non-specific staining was produced.
  • the reference material provided by the present invention is a non-biological tissue material which has high stability and provides a reliable reference for quality control of immunohistochemistry.
  • the traditional quality control method is to provide positive tumor reference photos, but the source of tumor tissue is limited, and the staining results of different tissues are quite different, which cannot provide a stable quality control reference marker for the immunohistochemistry process.
  • the reference material provided by the present invention is low in cost and has long-term stability.
  • the reference photos prepared by the method have the same concentration of the markers, and also have the characteristics of reliable, stable and consistent quality control signals.
  • Figure 1 is a schematic diagram showing the results of correct staining of photos by slides
  • Fig. 2 is a schematic diagram showing the result of incomplete staining of the antigen in the immunohistochemical process
  • Fig. 3 is a schematic diagram showing the results of the anti-staining failure in the immunohistochemical process
  • Figure 4 is a schematic diagram showing the results of anti-staining failure in the immunohistochemistry process. detailed description
  • the preparation steps of the negative control material in the present invention are as follows: 0.2 g of skim milk powder is weighed, dissolved in 10 mL of double distilled water, and thoroughly stirred and dissolved. Weighing 0.2 g of dextran into the above solution, adding 2 ( ⁇ L glacial acetic acid after stirring, stirring constantly to completely dissolve the dextran, and removing the bubbles in the dextran solution at 4 ° C. The needle is used to solidify the dextran solution into a sodium hydroxide solution to form white solid particles. The obtained solid particles are subjected to pathology.
  • the preparation steps of the positive control material in the present invention are as follows: 10 mL of double distilled water is weighed, and an antigen protein having a final concentration ranging from 0.1 g/L to 10 mg/L is added and fully dissolved. Weigh 0.2 g of skim milk powder, dissolve it in the above antigen solution, and stir well. Weighed 0.2 g of dextran into the above solution, and after stirring, 2 (?L glacial acetic acid was added, stirring was continued to completely dissolve the dextran, and the bubbles in the dextran solution were removed at 4 ° C. The needle solidifies the dextran solution into sodium hydroxide solution to form white solid particles.
  • the obtained solid particles are dehydrated by gradient alcohol and dehydrated by gradient alcohol (dehydration step: 80%, 90%, 95%, 100% various The concentration of ethanol was dehydrated for 2 hours), the xylene was transparent, the wax was soaked, and the paraffin was embedded in paraffin.
  • the preparation steps of the antigen retrieval control material in the present invention are as follows: 10 mL of double distilled water is weighed, and an antigen protein in a concentration range of 0.1 g/L to 10 mg/L is added and fully dissolved. 0.2 g of skim milk powder was weighed, dissolved in the above antigen solution, and thoroughly stirred and dissolved. Weighing 0.2 g of dextran into the above solution, stirring, adding 2 ( ⁇ L glacial acetic acid, stirring constantly to completely dissolve the dextran, and removing the bubbles in the dextran solution at 4 ° C. The needle is used to solidify the dextran solution into sodium hydroxide solution to form white solid particles.
  • the obtained solid particles are fixed by formalin (35-40% formaldehyde and 10-15% aqueous methanol solution), and the gradient alcohol is dehydrated through a gradient.
  • Alcohol dehydration dehydration step: 80%, 90%, 95%, 100% ethanol dehydration for 2 hours
  • xylene transparent paraffin immersion
  • paraffin embedding into paraffin specimens paraffin specimens.
  • the obtained solid particles are subjected to the same procedure as the pathological tissue, and subjected to dehydration by gradient alcohol (step of dehydration: 80%, 90%, 95%, 100%)
  • steps of dehydration: 80%, 90%, 95%, 100% Various concentrations of ethanol were dehydrated for 2 hours), transparent with xylene, paraffin immersed, and finally paraffin embedded to form paraffin specimens.
  • the obtained solid particles are dehydrated by gradient alcohol and dehydrated by gradient alcohol (dehydration step: 80%, 90%, 95%, 100% various The concentration of ethanol was dehydrated for 2 hours), the xylene was transparent, the paraffin was soaked, and the paraffin was embedded in paraffin.
  • the obtained solid particles are fixed by formalin (35-40% formaldehyde and 10-15% aqueous methanol solution), and the gradient alcohol is dehydrated through a gradient.
  • Alcohol dehydration dehydration step: 80%, 90%, 95%, 100% ethanol dehydration for 2 hours
  • xylene transparent paraffin immersion
  • paraffin embedding into paraffin specimens paraffin specimens.
  • the obtained solid particles are dehydrated by gradient alcohol and dehydrated by gradient alcohol (dehydration step: 80%, 90%, 95%, 100% various The concentration of ethanol was dehydrated for 2 hours), the xylene was transparent, the paraffin was soaked, and the paraffin was embedded in paraffin.
  • the obtained solid particles are dehydrated by gradient alcohol and dehydrated by gradient alcohol (dehydration step: 80%, 90%, 95%, 100% various The concentration of ethanol was dehydrated for 2 hours), the xylene was transparent, the paraffin was soaked, and the paraffin was embedded in paraffin.
  • the obtained solid particles are fixed by formalin (35-40% formaldehyde and 10-15% aqueous methanol solution), and the gradient alcohol is dehydrated through a gradient.
  • Alcohol dehydration dehydration step: 80%, 90%, 95%, 100% ethanol dehydration for 2 hours
  • xylene transparent paraffin immersion
  • paraffin embedding into paraffin specimens paraffin specimens.
  • the normal sheep serum working solution was blocked, and the mixture was washed at 37 ° C for 10 min. Incubate with anti-ER primary antibody at 4 °C overnight, rinse with PBS for 3 X 5 min ; add biotin-labeled secondary antibody, incubate at 37 °C for 30 min, rinse with PBS for 3 X 5 min; add horseradish peroxidase-labeled Streptomycin avidin working solution, incubated at 37 ° C for 30 min, washed with PBS for 3 X 5 min; DAB / H 2 0 2 reaction staining, tap water after thorough washing, hematoxylin counterstaining, conventional dehydration, transparent, dry, and mounted.
  • Figure 1 is a schematic diagram of the correct staining of the photos by the slides. If the staining result is as shown in Figure 1, the negative photo is negative, positive. Positive results were obtained for the photos and positive results were obtained for the antigen retrieval controls. It indicates that the dyeing process is successful and the staining results can be used for pathological diagnosis.
  • Fig. 2 is a schematic diagram showing the results of staining the photos in the immunohistochemical process.
  • Fig. 2 The staining results of the control control tablets prepared by the patented technique are as shown in Fig. 2, that is, the negative control shows a negative result, the positive control shows a positive result, and the antigen is repaired.
  • the blocked antigenic determinants were not completely exposed, and new repair methods should be selected or the repair strength should be improved.
  • the credibility of the staining results of the pathological tablets decreased to some extent.
  • Fig. 3 is a schematic diagram showing the results of anti-staining failure in the immunohistochemical process.
  • the staining results of the control control tablets prepared by the patented technique are as shown in Fig.
  • Fig. 4 is a schematic diagram showing the results of anti-staining failure in the immunohistochemical process.
  • the staining results of the control control tablets prepared by the patented technique are positive as shown in Fig. 4, that is, the negative control, the positive control and the antigen retrieval control. This indicates that the staining process failed, ie, non-specific staining was produced.
  • the pathological staining results are not reliable and the staining operation should be repeated.
  • the negative control spots showed negative results
  • the positive control spots showed positive results
  • the antigen retrieval control spots showed positive results
  • the staining process was successful, and the pathological sections were used for further diagnosis.
  • the pathological section is a positive result, and if no brown color appears, the pathological section is negative.

Abstract

Provided in the present invention are an immunohistochemical quality control reference object and a method for quality control using the reference object. In the present invention, particles solidified by a glucan solution are used to simulate biological tissue, and target antigen proteins are uniformly embedded in the particles. The solid glucan particles in which the target antigens are embedded are formed into a paraffin block after being dehydrated, transparentized, wax-dipped and paraffin-embedded, and the paraffin block is stuck to a glass slide after being sliced on a slicer, so as to be dyed together with tissue to be detected. In the method of the present invention, negative control, positive control and antigen retrieval control are designed according to different materials, and the purpose of quality control is achieved through a dyeing result of a reference object.

Description

免疫组化质量控制参照物及质量控制方法 技术领域- 本发明属于生物病理检测领域， 具体的， 本发明涉及一种免疫组 化质量控制参照物及利用该参照物进行质量控制的方法。 背景技术  BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the field of biological pathology detection, and more particularly to an immunohistochemical quality control reference material and a method for quality control using the same. Background technique
为了促进免疫组织化学实验室的标准化、检测免疫组织化学染色 结果的可靠性， 免疫组化过程应该进行有效的质量控制。免疫组织化 学质量控制是指，采取不断优化的措施和技术制定实验室制度及常规 工作， 规范和完善实验室的免疫组织化学染色操作流程与步骤， 保证 免疫组织化学染色质量达到最佳结果， 并可以做出准确的诊断。  In order to promote the standardization of immunohistochemistry laboratories and to test the reliability of immunohistochemical staining results, the immunohistochemical process should be subjected to effective quality control. Immunohistochemical quality control refers to the use of continuously optimized measures and techniques to develop laboratory systems and routine work, standardize and improve the laboratory immunohistochemical staining procedures and procedures to ensure that the quality of immunohistochemical staining achieves the best results, and An accurate diagnosis can be made.
在临床病理诊断和形态学研究中，免疫组织化学染色是一种很重 要的技术和手段。免疫组化技术应用于病理诊断始于上世纪 70年代， 目前在全球病理界已经得到广泛应用，它已成为病理医生常规工作中 不可缺少的部分。免疫组化技术拥有特异性、敏感性强及操作简便等 优点， 使得免疫组化技术在疾病诊断领域得到了广泛的推广和应用。 免疫组化技术不仅能提高了病理诊断的准确性，同时也渗透到了临床 和基础学科， 在探讨疾病的病因学、发病机制及科研工作中起到了不 可估量的作用。  Immunohistochemical staining is an important technique and means in clinical pathological diagnosis and morphological studies. The application of immunohistochemistry to pathological diagnosis began in the 1970s and has been widely used in the global pathology community. It has become an indispensable part of the routine work of pathologists. Immunohistochemical technology has the advantages of specificity, sensitivity and ease of operation, which has made the immunohistochemical technology widely popularized and applied in the field of disease diagnosis. Immunohistochemical technology not only improves the accuracy of pathological diagnosis, but also penetrates into clinical and basic disciplines. It plays an invaluable role in exploring the etiology, pathogenesis and research of diseases.
免疫组化技术具有操作简单， 灵敏度高、 特异性强等优点， 在各 级医院的临床病理诊断、研究等相关学科中得到广泛应用， 但是由于 实验条件的差别及其他技术因素的影响，使得免疫组化染色结果差别 很大， 从而影响到病理诊断的结果， 目前提高免疫组化染色技术水平 的需要越来越迫切， 而且任重道远。  Immunohistochemical technology has the advantages of simple operation, high sensitivity and strong specificity. It is widely used in clinical pathology diagnosis and research in hospitals at various levels, but it is immune due to differences in experimental conditions and other technical factors. The results of histochemical staining vary greatly, which affects the results of pathological diagnosis. At present, the need to improve the level of immunohistochemical staining technology is more and more urgent, and there is still a long way to go.
质量控制由室内质控 (internal quality control， IQC) 和室间质控 (external quality assurance, EQA) 两部分组成。 IQC 是指实验室内部 自己制定免疫组织化学实验的质控方法和程序; EQA是由某个外部 机构或组织，对各个实验室之间的不同免疫组织化学染色结果进行检 测和评估， 是在实验室之间进行的。 IQC 和 EQA对于实现免疫组织 化学染色的标准化方面起到了非常重要的作用，并推动了我国免疫组 织化学在病理诊断中的应用与发展。 Quality control consists of two parts: internal quality control (IQC) and external quality assurance (EQA). IQC refers to the quality control methods and procedures for the development of immunohistochemistry experiments in the laboratory; EQA is the detection and evaluation of different immunohistochemical staining results between laboratories by an external organization or organization. Between the rooms. IQC and EQA for achieving immune organization The standardization of chemical staining plays a very important role, and promotes the application and development of immunohistochemistry in pathological diagnosis in China.
对于免疫组织化学染色的内部质控程序来说，主要的步骤便是对照 标准样品 （即参照物） 的设立。 只有设立阳性对照和阴性对照， 才能 确保免疫组织化学染色过程的准确性、 抗体及相关试剂的有效性，确 认染色结果的真实性， 检测是否存在假阳性或假阴性结果。对照的设 立包括试剂对照、组织对照以及内部对照等。 由于免疫组织化学染色 有可能出现假阳性和假阴性结果，因此每次实验都必须同时设立阳性 对照和阴性对照， 以达到质控的最佳效果。 目前免疫组织化学常规对 照的设立，是由一张单独的切片上包含有待测抗体阳性或阴性的组织 组成的。在日常免疫组织化学染色中， 每一种抗体都需设立一张单独 的阳性或阴性组织对照片， 以确认抗体及其他试剂是否有效， 染色过 程是否运行正常。 目前采用的阳性、 阴性对照组织方法， 虽然具有一 定的效果， 但是由于每张组织片都有一定差异， 因此很难为检测组织 提供一个标准含量的参考。且目前市场上没有利用非生物组织的材料 作为免疫组化质量控制的对照材料。基于此， 本发明提供了一种免疫 组化质量控制参照物及利用该参照物进行质量控制的方法。该参照物 是一种待测抗原含量稳定的组织切片对照物。 发明内容  For internal quality control procedures for immunohistochemical staining, the primary step is to establish a reference standard sample (ie, reference). Only the establishment of positive and negative controls can ensure the accuracy of the immunohistochemical staining process, the effectiveness of antibodies and related reagents, confirm the authenticity of the staining results, and detect the presence of false positive or false negative results. Control settings include reagent controls, tissue controls, and internal controls. Since immunohistochemical staining may have false positive and false negative results, both positive and negative controls must be established for each experiment to achieve the best quality control. The current routine immunohistochemistry scan is based on a single section containing tissue that is positive or negative for the antibody to be tested. In daily immunohistochemical staining, a separate positive or negative tissue pair photo is required for each antibody to confirm that the antibody and other reagents are effective and that the staining process is functioning properly. The positive and negative control tissue methods currently used, although having certain effects, are difficult to provide a standard reference for the test tissue because of differences in each tissue piece. There are currently no materials on the market that use non-biological tissue as a control material for immunohistochemical quality control. Based on this, the present invention provides an immunohistochemical quality control reference material and a method for quality control using the same. The reference is a tissue section control with a stable antigen content to be tested. Summary of the invention
为了在免疫组化质控过程中提供稳定的易于获得的参照物，本发 明提供了提供一种免疫组化质量控制参照物及利用该参照物进行质 量控制的方法。  In order to provide a stable, readily available reference during immunohistochemical quality control, the present invention provides an immunohistochemical quality control reference and methods for quality control using the same.
为实现上述目的， 本发明提供的技术方案如下：  To achieve the above object, the technical solution provided by the present invention is as follows:
一种用于免疫组织化学技术质量控质的参照物，所述参照物包括 三个对照物： 质量控制的阳性对照物、质量控制的阴性对照物以及抗 原修复对照物。  A reference for quality control of immunohistochemical techniques, the reference comprising three controls: a quality controlled positive control, a quality controlled negative control, and an antigen repair control.
所述质量控制的阳性对照物的制备方法为： 将抗原经过葡聚糖材 料处理、 固化、 然后包埋于石蜡中。  The quality control positive control is prepared by treating the antigen with a dextran material, curing it, and then embedding it in paraffin.
所述质量控制的阴性对照物的制备方法为：将不含有抗原的葡聚 糖材料包埋于石蜡中。 The quality control negative control is prepared by: collecting glucose without antigen The sugar material is embedded in paraffin.
所述抗原修复对照物的制备方法为：将抗原经过葡聚糖材料处理 并固化， 再经福尔马林浸泡后包埋于石蜡中。  The antigen retrieval control is prepared by treating the antigen with a dextran material and solidifying it, and then immersing it in paraffin after being soaked in formalin.
本发明将制备好的质量控制的阳性对照物、质量控制的阴性对照物以 及抗原修复对照物从石蜡块中取出， 按顺序包埋在同一个石蜡块中， 在切片机上实现切片并黏贴于载玻片上作为免疫组化质量控质的参 照物。 In the present invention, the prepared quality control positive control, the quality control negative control and the antigen retrieval control are taken out from the paraffin block, embedded in the same paraffin block in sequence, and sliced and adhered on the microtome. The slide was used as a reference for immunohistochemical quality control.
本发明还提供了一种免疫组化质量控制的方法， 该方法是利用包 埋有待测目标抗原的材料模拟常规石蜡组织切片，在切片机上切成薄 片黏贴于载玻片上与待检测组织一同进行免疫组化染色，达到质量控 制的目的； 所述包埋有待测目标抗原的材料即上述的参照物。  The invention also provides a method for quality control of immunohistochemistry, which simulates a conventional paraffin tissue section by using a material embedded with an antigen to be tested, and cuts into a thin slice on a slicer and adheres to the slide and the tissue to be detected. The immunohistochemical staining is performed together for the purpose of quality control; the material in which the target antigen to be tested is embedded is the above reference substance.
所述的包埋有待测目标抗原的材料，是指利用葡聚糖与待测目标 抗原混合后经过固化形成的固体颗粒。  The material embedded with the antigen to be tested refers to a solid particle formed by solidification after mixing the dextran with the target antigen to be tested.
所述的待测目标抗原， 是指与病理组织中待检测抗原相同的蛋白 或者具有相同的抗原决定簇的蛋白质。  The antigen to be tested refers to the same protein as the antigen to be detected in the pathological tissue or a protein having the same antigenic determinant.
所述质量控制的阴性对照，是指没有包埋目标抗原的材料经过常 规石蜡切片制作， 贴于载玻片上与待检测组织一同染色；  The negative control of the quality control means that the material without the target antigen is prepared by conventional paraffin sectioning, and is attached to the glass slide to be stained together with the tissue to be detected;
所述质量控制的阳性对照，是指包埋了目标抗原的材料经过常规 石蜡切片制作， 但是不经过福尔马林固定， 使得目标抗原的抗原决定 簇不被封闭；  The positive control of the quality control means that the material in which the target antigen is embedded is prepared by conventional paraffin section, but is not fixed by formalin, so that the antigenic determinant of the target antigen is not blocked;
所述抗原修复对照，是指是包埋了目标抗原的材料经过常规石蜡 切片制作，并经过福尔马林固定，使得目标抗原的抗原决定簇被封闭。  The antigen retrieval control means that the material in which the antigen of interest is embedded is prepared by conventional paraffin sectioning and fixed by formalin so that the antigenic determinant of the target antigen is blocked.
本发明方法利用体外获得的与组织中待检测抗原具有相同的蛋 白或具有相同的抗原决定簇的蛋白质作为对照物，包埋于多糖材料中 并经过固化形成固体颗粒， 经过脱水、 透明、 浸蜡和包埋等程序制作 成石蜡块， 在组织切片机实现切片并与待测组织贴于同一张载玻片 上， 与待测组织一同进行免疫组化染色。  The method of the invention utilizes a protein obtained in vitro having the same protein or the same antigenic determinant as the antigen to be detected in the tissue as a control, embedded in the polysaccharide material and solidified to form solid particles, dehydrated, transparent, and immersed in wax. And the embedding and other procedures are made into paraffin blocks, sliced in a tissue microtome and attached to the same slide glass with the tissue to be tested, and immunohistochemical staining is performed together with the tissue to be tested.
本发明方法设计了三个对照（即参照物）用于免疫组化的质量控 制， 包括阴性对照、 阳性对照和抗原修复对照， 通过参照物染色的结 果直接判断染色的成功与否， 以及染色失败后的原因。 具体如下： ( 1 ) 阴性对照片呈现阴性结果， 阳性对照片呈现阳性结果， 抗 原修复对照呈现阳性结果。说明染色过程成功， 染色结果可以用于病 理诊断。 The method of the present invention designed three controls (ie, reference materials) for quality control of immunohistochemistry, including negative control, positive control, and antigen retrieval control, and directly judged the success or failure of staining by the result of staining of the reference substance, and the staining failure. The reason behind. details as follows: (1) Negative results in negative results for the photos, positive positive results for the photos, and positive results for the antigen retrieval controls. It indicates that the dyeing process is successful and the staining results can be used for pathological diagnosis.
(2 ) 阴性对照呈现阴性结果， 阳性对照呈现阳性结果， 抗原修 复对照呈现阴性结果。说明染色过程失败，而且失败原因是抗原修复， 被封闭的抗原决定簇没有暴露出来，应当选择新的修复方法或提高修 复强度。  (2) The negative control showed a negative result, the positive control showed a positive result, and the antigen repair control showed a negative result. This indicates that the staining process failed, and the cause of the failure was antigen retrieval. The blocked antigenic determinants were not exposed, and new repair methods should be selected or the repair strength should be improved.
(3 ) 阴性对照、 阳性对照、 抗原修复对照都为阴性结果， 说明 染色过程失败， 失败的原因可能是实验试剂或操作过程的失误。  (3) Negative control, positive control, and antigen-repair control are all negative results, indicating that the staining process failed. The cause of the failure may be the failure of the experimental reagent or the operation process.
(4) 阴性对照、 阳性对照和抗原修复对照都呈现阳性。 说明染 色过程失败， 即产生了非特异性染色。  (4) Negative controls, positive controls, and antigen-repair controls were all positive. It indicates that the dyeing process failed, that is, non-specific staining was produced.
本发明的显著优点： 本发明提供的参照物为非生物组织材料，该 材料稳定性高， 为免疫组化技术的质量控制提供了一个可靠的参照 物。传统的质控方法是提供阳性肿瘤参照片， 但是肿瘤组织的来源有 限， 不同组织的染色结果差异较大， 无法为免疫组化过程提供一个稳 定的质控参考标志物。本发明提供的参照物成本低廉， 具有长久的稳 定性。通过该方法制备的参照片， 其标志物浓度一致， 同时还具有来 源可靠、 稳定、 质控信号一致的特点。 附图说明  Significant advantages of the present invention: The reference material provided by the present invention is a non-biological tissue material which has high stability and provides a reliable reference for quality control of immunohistochemistry. The traditional quality control method is to provide positive tumor reference photos, but the source of tumor tissue is limited, and the staining results of different tissues are quite different, which cannot provide a stable quality control reference marker for the immunohistochemistry process. The reference material provided by the present invention is low in cost and has long-term stability. The reference photos prepared by the method have the same concentration of the markers, and also have the characteristics of reliable, stable and consistent quality control signals. DRAWINGS
图 1为载玻片对照片正确染色结果示意图； Figure 1 is a schematic diagram showing the results of correct staining of photos by slides;
图 2为免疫组化过程抗原修复不彻底对照片染色结果示意图； 图 3为免疫组化过程抗染色失败结果示意图； Fig. 2 is a schematic diagram showing the result of incomplete staining of the antigen in the immunohistochemical process; Fig. 3 is a schematic diagram showing the results of the anti-staining failure in the immunohistochemical process;
图 4为免疫组化过程抗染色失败结果示意图。 具体实施方式 Figure 4 is a schematic diagram showing the results of anti-staining failure in the immunohistochemistry process. detailed description
本发明中阴性对照材料的制备步骤如下： 称取 0.2g脱脂奶粉， 溶 解于 10mL双蒸水中，充分搅拌溶解。再称取 0.2g葡聚糖加入到上述溶 液中， 搅拌后加入 2(^L冰乙酸， 不断搅拌使葡聚糖彻底溶解， 4°C放 置除去葡聚糖溶液中的气泡。用一次性注射针头将葡聚糖溶液打到氢 氧化钠溶液中固化， 形成白色固体颗粒。获得的固体颗粒进行与病理 组织同样的程序，经过梯度酒精脱水（脱水的步骤： 80%、 90%、 95%、 100%各种浓度的乙醇分别脱水 2小时）， 用二甲苯透明， 石蜡浸泡， 最后石蜡包埋制作成石蜡标本。按照与病理组织一样的程序切片和贴 片。 The preparation steps of the negative control material in the present invention are as follows: 0.2 g of skim milk powder is weighed, dissolved in 10 mL of double distilled water, and thoroughly stirred and dissolved. Weighing 0.2 g of dextran into the above solution, adding 2 (^L glacial acetic acid after stirring, stirring constantly to completely dissolve the dextran, and removing the bubbles in the dextran solution at 4 ° C. The needle is used to solidify the dextran solution into a sodium hydroxide solution to form white solid particles. The obtained solid particles are subjected to pathology. Organize the same procedure, after gradient alcohol dehydration (dehydration step: 80%, 90%, 95%, 100% ethanol dehydration for 2 hours), transparent with xylene, paraffin soaked, and finally paraffin embedded Paraffin specimens. Slice and patch according to the same procedure as pathology.
本发明中阳性对照材料的制备步骤如下： 量取 10mL双蒸水， 加入 终浓度为 0.1 g/L到 10mg/L浓度范围的抗原蛋白并充分溶解。称取 0.2g 脱脂奶粉， 溶解于上述抗原溶液中， 充分搅拌溶解。 再称取 0.2g葡聚 糖加入到上述溶液中，搅拌后加入 2(^L冰乙酸，不断搅拌使葡聚糖彻 底溶解， 4°C放置除去葡聚糖溶液中的气泡。 用一次性注射针头将葡 聚糖溶液打到氢氧化钠溶液中固化， 形成白色固体颗粒。获得的固体 颗粒经过梯度酒精脱水经过梯度酒精脱水（脱水的步骤： 80%、 90%、 95%、 100%各种浓度的乙醇分别脱水 2小时），二甲苯透明，石蜡浸泡， 石蜡包埋制作成石蜡标本。  The preparation steps of the positive control material in the present invention are as follows: 10 mL of double distilled water is weighed, and an antigen protein having a final concentration ranging from 0.1 g/L to 10 mg/L is added and fully dissolved. Weigh 0.2 g of skim milk powder, dissolve it in the above antigen solution, and stir well. Weighed 0.2 g of dextran into the above solution, and after stirring, 2 (?L glacial acetic acid was added, stirring was continued to completely dissolve the dextran, and the bubbles in the dextran solution were removed at 4 ° C. The needle solidifies the dextran solution into sodium hydroxide solution to form white solid particles. The obtained solid particles are dehydrated by gradient alcohol and dehydrated by gradient alcohol (dehydration step: 80%, 90%, 95%, 100% various The concentration of ethanol was dehydrated for 2 hours), the xylene was transparent, the wax was soaked, and the paraffin was embedded in paraffin.
本发明中抗原修复对照材料的制备步骤如下： 量取 10mL双蒸水， 加入终浓度 0.1 g/L到 10mg/L浓度范围的抗原蛋白并充分溶解。 称取 0.2g脱脂奶粉， 溶解于上述抗原溶液中， 充分搅拌溶解。 再称取 0.2g 葡聚糖加入到上述溶液中，搅拌后加入 2(^L冰乙酸，不断搅拌使葡聚 糖彻底溶解， 4°C放置除去葡聚糖溶液中的气泡。 用一次性注射针头 将葡聚糖溶液打到氢氧化钠溶液中固化， 形成白色固体颗粒。获得的 固体颗粒经过福尔马林 （35-40%甲醛和 10-15%甲醇水溶液） 固定， 梯度酒精脱水经过梯度酒精脱水 （脱水的步骤： 80%、 90%、 95%、 100%各种浓度的乙醇分别脱水 2小时）， 二甲苯透明， 石蜡浸泡， 石 蜡包埋制作成石蜡标本。  The preparation steps of the antigen retrieval control material in the present invention are as follows: 10 mL of double distilled water is weighed, and an antigen protein in a concentration range of 0.1 g/L to 10 mg/L is added and fully dissolved. 0.2 g of skim milk powder was weighed, dissolved in the above antigen solution, and thoroughly stirred and dissolved. Weighing 0.2 g of dextran into the above solution, stirring, adding 2 (^L glacial acetic acid, stirring constantly to completely dissolve the dextran, and removing the bubbles in the dextran solution at 4 ° C. The needle is used to solidify the dextran solution into sodium hydroxide solution to form white solid particles. The obtained solid particles are fixed by formalin (35-40% formaldehyde and 10-15% aqueous methanol solution), and the gradient alcohol is dehydrated through a gradient. Alcohol dehydration (dehydration step: 80%, 90%, 95%, 100% ethanol dehydration for 2 hours), xylene transparent, paraffin immersion, paraffin embedding into paraffin specimens.
下面通过具体实施示例对本发明的技术方案做进一步说明，但是 不能以此限制本发明的范围。以下所用生化试剂除特别标明外均购自 生工生物工程（上海）有限公司， 抗体和临床组织石蜡切片为申请人 (福州迈新生物技术开发有限公司）提供， 操作步骤若无说明均为常 规操作步骤。  The technical solutions of the present invention are further illustrated by the specific embodiments, but the scope of the present invention is not limited thereto. The following biochemical reagents were purchased from Bioengineering (Shanghai) Co., Ltd., and the antibody and clinical tissue paraffin sections were provided by the applicant (Fuzhou Maixin Biotechnology Development Co., Ltd.), unless otherwise stated. step.
实施例 1、 阴性对照材料的制备 Example 1. Preparation of negative control material
称取 0.2g脱脂奶粉， 溶解于 10mL双蒸水中， 充分搅拌溶解。再称 取 0.2g葡聚糖加入到上述溶液中， 搅拌后加入 2(^L冰乙酸， 不断搅拌 使葡聚糖彻底溶解， 4°C放置除去葡聚糖溶液中的气泡。 用一次性注 射针头将葡聚糖溶液打到氢氧化钠溶液中固化， 形成白色固体颗粒。 获得的固体颗粒进行与病理组织同样的程序， 经过梯度酒精脱水（脱 水的步骤： 80%、 90%、 95%、 100%各种浓度的乙醇分别脱水 2小时）， 用二甲苯透明， 石蜡浸泡， 最后石蜡包埋制作成石蜡标本。 按照与病 理组织一样的程序切片和贴片。 Weigh 0.2 g of skimmed milk powder, dissolve it in 10 mL of double distilled water, and stir well. Again Add 0.2 g of dextran to the above solution, add 2 (^L glacial acetic acid after stirring, stir constantly to completely dissolve the dextran, and remove the bubbles in the dextran solution at 4 ° C. Use a disposable injection needle The dextran solution is solidified in a sodium hydroxide solution to form white solid particles. The obtained solid particles are subjected to the same procedure as the pathological tissue, and subjected to dehydration by gradient alcohol (step of dehydration: 80%, 90%, 95%, 100%) Various concentrations of ethanol were dehydrated for 2 hours), transparent with xylene, paraffin immersed, and finally paraffin embedded to form paraffin specimens. Slice and patch according to the same procedure as pathological tissue.
实施例 2、 阳性对照材料的制备 Example 2. Preparation of positive control material
量取 10mL双蒸水， 加入终浓度为 O.l g/L的抗原蛋白并充分溶解。 称取 0.2g脱脂奶粉， 溶解于上述抗原溶液中， 充分搅拌溶解。 再称取 0.2g葡聚糖加入到上述溶液中， 搅拌后加入 2(^L冰乙酸， 不断搅拌使 葡聚糖彻底溶解， 4°C放置除去葡聚糖溶液中的气泡。 用一次性注射 针头将葡聚糖溶液打到氢氧化钠溶液中固化， 形成白色固体颗粒。获 得的固体颗粒经过梯度酒精脱水经过梯度酒精脱水 （脱水的步骤： 80%、 90%、 95%、 100%各种浓度的乙醇分别脱水 2小时）， 二甲苯透 明， 石蜡浸泡， 石蜡包埋制作成石蜡标本。  10 mL of double distilled water was weighed, and an antigen protein of a final concentration of 0.1 g/L was added and fully dissolved. 0.2 g of skim milk powder was weighed, dissolved in the above antigen solution, and thoroughly stirred and dissolved. Weighed 0.2 g of dextran into the above solution, and after stirring, 2 (?L glacial acetic acid was added, stirring was continued to completely dissolve the dextran, and the bubbles in the dextran solution were removed at 4 ° C. The needle solidifies the dextran solution into sodium hydroxide solution to form white solid particles. The obtained solid particles are dehydrated by gradient alcohol and dehydrated by gradient alcohol (dehydration step: 80%, 90%, 95%, 100% various The concentration of ethanol was dehydrated for 2 hours), the xylene was transparent, the paraffin was soaked, and the paraffin was embedded in paraffin.
实施例 3、 抗原修复对照材料的制备 Example 3, Preparation of antigen retrieval control material
量取 10mL双蒸水， 加入终浓度为 lmg/L的抗原蛋白并充分溶解。 称取 0.2g脱脂奶粉， 溶解于上述抗原溶液中， 充分搅拌溶解。 再称取 0.2g葡聚糖加入到上述溶液中， 搅拌后加入 2(^L冰乙酸， 不断搅拌使 葡聚糖彻底溶解， 4°C放置除去葡聚糖溶液中的气泡。 用一次性注射 针头将葡聚糖溶液打到氢氧化钠溶液中固化， 形成白色固体颗粒。获 得的固体颗粒经过福尔马林 （35-40%甲醛和 10-15%甲醇水溶液） 固 定，梯度酒精脱水经过梯度酒精脱水（脱水的步骤： 80%、 90%、 95%、 100%各种浓度的乙醇分别脱水 2小时）， 二甲苯透明， 石蜡浸泡， 石 蜡包埋制作成石蜡标本。  10 mL of double distilled water was weighed, and an antigen protein of a final concentration of 1 mg/L was added and fully dissolved. 0.2 g of skim milk powder was weighed, dissolved in the above antigen solution, and thoroughly stirred and dissolved. Weighed 0.2 g of dextran into the above solution, and after stirring, 2 (?L glacial acetic acid was added, stirring was continued to completely dissolve the dextran, and the bubbles in the dextran solution were removed at 4 ° C. The needle is used to solidify the dextran solution into sodium hydroxide solution to form white solid particles. The obtained solid particles are fixed by formalin (35-40% formaldehyde and 10-15% aqueous methanol solution), and the gradient alcohol is dehydrated through a gradient. Alcohol dehydration (dehydration step: 80%, 90%, 95%, 100% ethanol dehydration for 2 hours), xylene transparent, paraffin immersion, paraffin embedding into paraffin specimens.
实施例 4、 质量控质参照物的制作 Example 4: Production of quality control reference materials
获得的阴性对照、 阳性对照和抗原修复对照材料， 从石蜡块中取 出， 按顺序包埋在同一个石蜡块中， 经过组织切片机切片贴于载玻片 上， 作为免疫组化质控参照物。 实施例 5、 阳性对照材料的制备 The obtained negative control, positive control and antigen retrieval control materials were taken out from the paraffin block, embedded in the same paraffin block in sequence, and sliced on a glass slide through a tissue microtome to serve as an immunohistochemical quality control reference. Example 5, Preparation of Positive Control Materials
量取 10mL双蒸水， 加入终浓度为 10mg/L的抗原蛋白并充分溶解。 称取 0.2g脱脂奶粉， 溶解于上述抗原溶液中， 充分搅拌溶解。 再称取 0.2g葡聚糖加入到上述溶液中， 搅拌后加入 2(^L冰乙酸， 不断搅拌使 葡聚糖彻底溶解， 4°C放置除去葡聚糖溶液中的气泡。 用一次性注射 针头将葡聚糖溶液打到氢氧化钠溶液中固化， 形成白色固体颗粒。获 得的固体颗粒经过梯度酒精脱水经过梯度酒精脱水 （脱水的步骤： 80%、 90%、 95%、 100%各种浓度的乙醇分别脱水 2小时）， 二甲苯透 明， 石蜡浸泡， 石蜡包埋制作成石蜡标本。  10 mL of double distilled water was weighed, and an antigen protein of a final concentration of 10 mg/L was added and fully dissolved. 0.2 g of skim milk powder was weighed, dissolved in the above antigen solution, and thoroughly stirred and dissolved. Weighed 0.2 g of dextran into the above solution, and after stirring, 2 (?L glacial acetic acid was added, stirring was continued to completely dissolve the dextran, and the bubbles in the dextran solution were removed at 4 ° C. The needle solidifies the dextran solution into sodium hydroxide solution to form white solid particles. The obtained solid particles are dehydrated by gradient alcohol and dehydrated by gradient alcohol (dehydration step: 80%, 90%, 95%, 100% various The concentration of ethanol was dehydrated for 2 hours), the xylene was transparent, the paraffin was soaked, and the paraffin was embedded in paraffin.
实施例 6、 阳性对照材料的制备 Example 6. Preparation of positive control material
量取 10mL双蒸水， 加入终浓度为 lmg/L的抗原蛋白并充分溶解。 称取 0.2g脱脂奶粉， 溶解于上述抗原溶液中， 充分搅拌溶解。 再称取 0.2g葡聚糖加入到上述溶液中， 搅拌后加入 2(^L冰乙酸， 不断搅拌使 葡聚糖彻底溶解， 4°C放置除去葡聚糖溶液中的气泡。 用一次性注射 针头将葡聚糖溶液打到氢氧化钠溶液中固化， 形成白色固体颗粒。获 得的固体颗粒经过梯度酒精脱水经过梯度酒精脱水 （脱水的步骤： 80%、 90%、 95%、 100%各种浓度的乙醇分别脱水 2小时）， 二甲苯透 明， 石蜡浸泡， 石蜡包埋制作成石蜡标本。  10 mL of double distilled water was weighed, and an antigen protein of a final concentration of 1 mg/L was added and fully dissolved. 0.2 g of skim milk powder was weighed, dissolved in the above antigen solution, and thoroughly stirred and dissolved. Weighed 0.2 g of dextran into the above solution, and after stirring, 2 (?L glacial acetic acid was added, stirring was continued to completely dissolve the dextran, and the bubbles in the dextran solution were removed at 4 ° C. The needle solidifies the dextran solution into sodium hydroxide solution to form white solid particles. The obtained solid particles are dehydrated by gradient alcohol and dehydrated by gradient alcohol (dehydration step: 80%, 90%, 95%, 100% various The concentration of ethanol was dehydrated for 2 hours), the xylene was transparent, the paraffin was soaked, and the paraffin was embedded in paraffin.
实施例 7、 抗原修复对照材料的制备 Example 7. Preparation of antigen retrieval control material
量取 10mL双蒸水， 加入终浓度为 O.^g/L的抗原蛋白并充分溶解。 称取 0.2g脱脂奶粉， 溶解于上述抗原溶液中， 充分搅拌溶解。 再称取 0.2g葡聚糖加入到上述溶液中， 搅拌后加入 2(^L冰乙酸， 不断搅拌使 葡聚糖彻底溶解， 4°C放置除去葡聚糖溶液中的气泡。 用一次性注射 针头将葡聚糖溶液打到氢氧化钠溶液中固化， 形成白色固体颗粒。获 得的固体颗粒经过福尔马林 （35-40%甲醛和 10-15%甲醇水溶液） 固 定，梯度酒精脱水经过梯度酒精脱水（脱水的步骤： 80%、 90%、 95%、 100%各种浓度的乙醇分别脱水 2小时）， 二甲苯透明， 石蜡浸泡， 石 蜡包埋制作成石蜡标本。  10 mL of double distilled water was weighed, and an antigen protein of a final concentration of O.g/L was added and fully dissolved. 0.2 g of skim milk powder was weighed, dissolved in the above antigen solution, and thoroughly stirred and dissolved. Weighed 0.2 g of dextran into the above solution, and after stirring, 2 (?L glacial acetic acid was added, stirring was continued to completely dissolve the dextran, and the bubbles in the dextran solution were removed at 4 ° C. The needle is used to solidify the dextran solution into sodium hydroxide solution to form white solid particles. The obtained solid particles are fixed by formalin (35-40% formaldehyde and 10-15% aqueous methanol solution), and the gradient alcohol is dehydrated through a gradient. Alcohol dehydration (dehydration step: 80%, 90%, 95%, 100% ethanol dehydration for 2 hours), xylene transparent, paraffin immersion, paraffin embedding into paraffin specimens.
实施例 8、 抗原修复对照材料的制备 Example 8. Preparation of antigen retrieval control material
量取 10mL双蒸水，加入终浓度为 10mg/L的抗原蛋白并充分溶解。 称取 0.2g脱脂奶粉， 溶解于上述抗原溶液中， 充分搅拌溶解。 再称取 0.2g葡聚糖加入到上述溶液中， 搅拌后加入 2(^L冰乙酸， 不断搅拌使 葡聚糖彻底溶解， 4°C放置除去葡聚糖溶液中的气泡。 用一次性注射 针头将葡聚糖溶液打到氢氧化钠溶液中固化， 形成白色固体颗粒。获 得的固体颗粒经过福尔马林 （35-40%甲醛和 10-15%甲醇水溶液） 固 定，梯度酒精脱水经过梯度酒精脱水（脱水的步骤： 80%、 90%、 95%、 100%各种浓度的乙醇分别脱水 2小时）， 二甲苯透明， 石蜡浸泡， 石 蜡包埋制作成石蜡标本。 10 mL of double distilled water was weighed, and an antigen protein of a final concentration of 10 mg/L was added and fully dissolved. Weigh 0.2 g of skim milk powder, dissolve it in the above antigen solution, and stir well. Weighed 0.2 g of dextran into the above solution, and after stirring, 2 (?L glacial acetic acid was added, stirring was continued to completely dissolve the dextran, and the bubbles in the dextran solution were removed at 4 ° C. The needle is used to solidify the dextran solution into sodium hydroxide solution to form white solid particles. The obtained solid particles are fixed by formalin (35-40% formaldehyde and 10-15% aqueous methanol solution), and the gradient alcohol is dehydrated through a gradient. Alcohol dehydration (dehydration step: 80%, 90%, 95%, 100% ethanol dehydration for 2 hours), xylene transparent, paraffin immersion, paraffin embedding into paraffin specimens.
实施例 9、 具体应用 Example 9, specific application
分别取一块乳腺癌病理组织蜡块和本专利技术制作的蜡块（即含 参照物的蜡块）。然后按照公认的标准的程序各组织取一片进行切片、 载玻片贴片。 烤片， 68°C， 20分钟， 公认的二甲苯脱蜡， 梯度酒精脱 水 （二甲苯 I 20min=>二甲苯 II 20min=> 100%酒精 10min=> 100%酒精 10min=>95%酒精 5min=>80%酒精 5min=>70%酒精 5min)， 阻断灭活内 源性过氧化物酶： 3%H20237°C孵育 10min， PBS冲洗 3 X 5min; 取出 后进行抗原修复： 置 0.01M枸橼酸缓冲液 （PH6.0) 中用煮沸 （95°C， 15-20min), 自然冷却 20min以上， 再用冷水冲洗缸子， 加快冷却至室 温， PBS冲洗 3 X 5min。 正常羊血清工作液封闭， 37°C 10min， 倾去勿 洗。滴加抗 ER的一抗 4 °C冰箱孵育过夜， PBS冲洗 3 X 5min; 滴加生物 素标记二抗， 37°C孵育 30min， PBS冲洗 3 X 5min; 滴加辣根过氧化物 酶标记的链霉素卵白素工作液， 37°C孵育 30min， PBS冲洗 3 X 5min; DAB/H202反应染色， 自来水充分冲洗后， 苏木素复染， 常规脱水， 透明， 干燥， 封片。 Take a piece of breast cancer pathology wax block and a wax block made by this patent technology (ie, a wax block containing reference material). Then, one piece of each tissue was taken for slicing and slide patch according to the accepted standard procedure. Baked slices, 68 ° C, 20 minutes, recognized dewaxing of xylene, gradient alcohol dehydration (xylene I 20min => xylene II 20min => 100% alcohol 10min => 100% alcohol 10min => 95% alcohol 5min = >80% alcohol 5min=>70% alcohol 5min), block inactivation of endogenous peroxidase: 3%H 2 0 2 37 °C for 10min, PBS rinse for 3 X 5min; remove antigenic repair: In 0.01 M citrate buffer (pH 6.0), boil (95 ° C, 15-20 min), naturally cool for more than 20 min, then rinse the tank with cold water, accelerate to cool to room temperature, rinse with PBS for 3 X 5 min. The normal sheep serum working solution was blocked, and the mixture was washed at 37 ° C for 10 min. Incubate with anti-ER primary antibody at 4 °C overnight, rinse with PBS for 3 X 5 min ; add biotin-labeled secondary antibody, incubate at 37 °C for 30 min, rinse with PBS for 3 X 5 min; add horseradish peroxidase-labeled Streptomycin avidin working solution, incubated at 37 ° C for 30 min, washed with PBS for 3 X 5 min; DAB / H 2 0 2 reaction staining, tap water after thorough washing, hematoxylin counterstaining, conventional dehydration, transparent, dry, and mounted.
在进行病理组织结果判断之前， 首先确定试剂及其整个处理中试 剂的有效性及处理过程是否正确。 根据对照片的结果进行判断。 （具 体判定方法为： 图 1-4中的圆形斑点中， 深色斑点为阳性， 无色斑点 为阴性）： 图 1 是载玻片对照片正确染色结果示意图本专利技术制备的对 照质控片染色结果若如图 1所示， 即阴性对照片呈现阴性结果， 阳性 对照片呈现阳性结果， 抗原修复对照呈现阳性结果。则说明染色过程 成功， 染色结果可以用于病理诊断。 图 2 是免疫组化过程抗原修复不彻底对照片染色结果示意图，本 专利技术制备的对照质控片染色结果若如图 2所示， 即阴性对照呈现 阴性结果， 阳性对照呈现阳性结果， 抗原修复对照呈现阴性结果。则 说明染色过程失败， 而且失败原因是抗原修复， 被封闭的抗原决定簇 没有完全暴露出来， 应当选择新的修复方法或提高修复强度。病理片 的染色结果可信度有一定程度的下降。 图 3 是免疫组化过程抗染色失败结果示意图，本专利技术制备的 对照质控片染色结果若如图 3所示， 即阴性对照、 阳性对照、 抗原修 复对照都为阴性结果， 说明染色过程失败， 失败的原因可能是试剂或 操作过程的失误。 病理染色结果不可信， 应重新进行染色操作。 图 4 是免疫组化过程抗染色失败结果示意图，本专利技术制备的 对照质控片染色结果若如图 4所示， 即阴性对照、 阳性对照和抗原修 复对照都呈现阳性。 说明染色过程失败， 即产生了非特异性染色。病 理染色结果不可信， 应重新进行染色操作。 Before performing the pathological results judgment, first determine the validity of the reagents and the reagents in the entire treatment and whether the treatment process is correct. Judging based on the results of the photos. (The specific determination method is: in the circular spots in Figure 1-4, the dark spots are positive, and the colorless spots are negative): Figure 1 is a schematic diagram of the correct staining of the photos by the slides. If the staining result is as shown in Figure 1, the negative photo is negative, positive. Positive results were obtained for the photos and positive results were obtained for the antigen retrieval controls. It indicates that the dyeing process is successful and the staining results can be used for pathological diagnosis. Fig. 2 is a schematic diagram showing the results of staining the photos in the immunohistochemical process. The staining results of the control control tablets prepared by the patented technique are as shown in Fig. 2, that is, the negative control shows a negative result, the positive control shows a positive result, and the antigen is repaired. The control presented a negative result. This indicates that the staining process failed, and the cause of the failure was antigen retrieval. The blocked antigenic determinants were not completely exposed, and new repair methods should be selected or the repair strength should be improved. The credibility of the staining results of the pathological tablets decreased to some extent. Fig. 3 is a schematic diagram showing the results of anti-staining failure in the immunohistochemical process. The staining results of the control control tablets prepared by the patented technique are as shown in Fig. 3, that is, the negative control, the positive control, and the antigen-repair control are all negative results, indicating that the staining process fails. The cause of the failure may be a mistake in the reagent or operation process. The pathological staining results are not reliable and the staining operation should be repeated. Fig. 4 is a schematic diagram showing the results of anti-staining failure in the immunohistochemical process. The staining results of the control control tablets prepared by the patented technique are positive as shown in Fig. 4, that is, the negative control, the positive control and the antigen retrieval control. This indicates that the staining process failed, ie, non-specific staining was produced. The pathological staining results are not reliable and the staining operation should be repeated.
如图 1所示， 即阴性对照斑点呈现阴性结果， 阳性对照斑点呈现 阳性结果， 抗原修复对照斑点呈现阳性结果， 染色过程成功， 则病理 切片可用于进一步的诊断。 在显微镜下观察， 如果呈现棕色结果，则 病理切片为阳性结果， 如果无棕色颜色出现， 则病理切片的诊断结果 为阴性。  As shown in Fig. 1, the negative control spots showed negative results, the positive control spots showed positive results, the antigen retrieval control spots showed positive results, and the staining process was successful, and the pathological sections were used for further diagnosis. Under the microscope, if the brown result is present, the pathological section is a positive result, and if no brown color appears, the pathological section is negative.

Claims

权利要求书 claims
1、 一种用于免疫组织化学技术质量控质的参照物， 其特征在于： 所 述参照物包括三个对照物： 质量控制的阳性对照物、质量控制的阴性 对照物以及抗原修复对照物。 1. A reference substance used for quality control of immunohistochemistry technology, characterized in that: the reference substance includes three controls: a positive control substance for quality control, a negative control substance for quality control, and an antigen retrieval control substance.
2、根据权利要求 1所述的用于免疫组织化学技术质量控质的参照物， 其特征在于： 所述质量控制的阳性对照物的制备方法为： 将抗原经过 葡聚糖材料处理后， 然后包埋于石蜡中。 2. The reference material for quality control of immunohistochemistry technology according to claim 1, characterized in that: the preparation method of the positive control material for quality control is: after treating the antigen with dextran material, and then Embedded in paraffin.
3、根据权利要求 1所述的用于免疫组织化学技术质量控质的参照物， 其特征在于： 所述质量控制的阴性对照物的制备方法为： 将不含有抗 原的葡聚糖材料包埋于石蜡中。 3. The reference material for quality control of immunohistochemistry technology according to claim 1, characterized in that: the preparation method of the negative control material for quality control is: embedding dextran material that does not contain antigen in paraffin.
4、根据权利要求 1所述的用于免疫组织化学技术质量控质的参照物， 其特征在于： 所述抗原修复对照物的制备方法为： 将抗原经过葡聚糖 材料处理并固化， 再经福尔马林浸泡后包埋于石蜡中。 4. The reference material for quality control of immunohistochemistry technology according to claim 1, characterized in that: the preparation method of the antigen repair reference material is: treating the antigen with dextran material and solidifying it, and then Soaked in formalin and embedded in paraffin.
5、 一种如权利要求 1所述的参照物的应用， 其特征在于： 所述应用 是将制备好的质量控制的阳性对照物、质量控制的阴性对照物以及抗 原修复对照物从石蜡块中取出， 按顺序包埋在同一个石蜡块中， 在切 片机上实现切片并黏贴于载玻片上作为免疫组化质量控质的参照物。 5. An application of the reference material according to claim 1, characterized in that: the application is to remove the prepared positive control material for quality control, the negative control material for quality control and the antigen retrieval control material from the paraffin block. Take them out, embed them in the same paraffin block in sequence, slice them on a microtome and stick them on a glass slide as a reference for immunohistochemistry quality control.
6、 一种免疫组化质量控制的方法， 其特征在于： 该方法是利用包埋 有待测目标抗原的材料模拟常规石蜡组织切片，在切片机上切成薄片 黏贴于载玻片上与待检测组织一同进行免疫组化染色，达到质量控制 的目的；所述包埋有待测目标抗原的材料即为如权利要求 1中所述的 6. A method for immunohistochemistry quality control, characterized by: This method uses materials embedded with the target antigen to be detected to simulate conventional paraffin tissue sections, and cuts thin slices on a microtome and sticks them on a glass slide with the target antigen to be detected. The tissue is subjected to immunohistochemical staining together to achieve the purpose of quality control; the material embedded with the target antigen to be tested is as described in claim 1
7、 根据权利要求 6所述的免疫组化质量控制的方法， 其特征在于： 所述的包埋有待测目标抗原的材料，是指利用葡聚糖与待测目标抗原 混合后经过固化形成的固体颗粒。 7. The immunohistochemistry quality control method according to claim 6, characterized in that: the material embedded with the target antigen to be tested refers to the material formed by mixing dextran and the target antigen to be tested and then solidifying of solid particles.
8、 根据权利要求 6所述的免疫组化质量控制的方法， 其特征在于： 所述的待测目标抗原，是指与病理组织中待检测抗原相同的蛋白或者 具有相同的抗原决定簇的蛋白质。 8. The immunohistochemical quality control method according to claim 6, characterized in that: the target antigen to be detected refers to the same protein or the same antigenic determinant as the antigen to be detected in the pathological tissue. .
PCT/CN2013/071117 2013-01-25 2013-01-30 Immunohistochemical quality control reference object and quality control method WO2014114009A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201310029327.7 2013-01-25
CN201310029327.7A CN103116018B (en) 2013-01-25 2013-01-25 Immunohistochemical quality control reference object and quality control method

Publications (1)

Publication Number Publication Date
WO2014114009A1 true WO2014114009A1 (en) 2014-07-31

Family

ID=48414445

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2013/071117 WO2014114009A1 (en) 2013-01-25 2013-01-30 Immunohistochemical quality control reference object and quality control method

Country Status (2)

Country Link
CN (1) CN103116018B (en)
WO (1) WO2014114009A1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106353164A (en) * 2016-07-21 2017-01-25 深圳市美雅洁技术股份有限公司 Ultrasonic rapid wax dipping system for pathological tissues and usage method
EP3639031A4 (en) * 2017-06-15 2021-05-05 Sunstone Scientific Limited. Method for ihc antigen imaging scale extrapolation
CN114689407A (en) * 2022-04-14 2022-07-01 四川大学华西医院 Method for manufacturing paraffin section of animal microtissue

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104655475A (en) 2015-02-06 2015-05-27 丁伟 Preparation method and application of paraffin embedding contrast suspension
US11662564B2 (en) 2017-06-15 2023-05-30 Shenzhen Prs Limited Paraffin shield coating for microscope slide
WO2018228575A1 (en) * 2017-06-15 2018-12-20 Sunstone Scientific Limited. Process record slide for immunohistochemical staining
US10921223B2 (en) * 2018-10-12 2021-02-16 Shenzhen Prs Limited Process record slide for staining and method of using the same
CN110132693A (en) * 2019-05-27 2019-08-16 福州迈新生物技术开发有限公司 A kind of coloration result method for early warning of full-automatic pathological staining system
CN110376036A (en) * 2019-07-30 2019-10-25 河南赛诺特生物技术有限公司 A kind of preparation method and tumor cell line immunohistochemistry reference substance of tumor cell line immunohistochemistry reference substance
CN111426532A (en) * 2020-04-09 2020-07-17 北京大学深圳医院 Bacterial wax block preparation method and method for positive quality control of bacterial wax block as acid-fast staining
CN117295946A (en) * 2021-06-07 2023-12-26 株式会社日立高新技术 Control slide for pathological examination and method for manufacturing same
CN114188039A (en) * 2021-11-02 2022-03-15 宁波市科技园区明天医网科技有限公司 Digital molecular pathology laboratory and pathology department service interaction system and method
CN115165511B (en) * 2022-09-09 2023-01-06 迈杰转化医学研究(苏州)有限公司 PD-L1 immunohistochemical detection quality control product and reference product

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060035292A1 (en) * 2001-01-12 2006-02-16 Mount Sinai School Of Medicine Of New York University Differential diagnosis of cancer and other conditions based on expression of p63
CN102183651A (en) * 2011-02-22 2011-09-14 沈阳药科大学 Caspase-3 proenzyme protein immunohistochemical diagnosis kit and application
CN102435728A (en) * 2011-09-07 2012-05-02 福州大学 Preparation method for positive control substance for inspection and control of quality in immunohistochemical process

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1108384C (en) * 2000-10-12 2003-05-14 陈学银 A tissue array and its preparation method
CN102292113B (en) * 2009-01-22 2014-06-25 株式会社奎真生物技术 Beta-glucan-based scaffold for biological tissue engineering using radiation fusion technology, and production method therefor
DE102009030845A1 (en) * 2009-06-26 2010-12-30 Siemens Aktiengesellschaft In vitro method for e.g. prognosis of allergic reaction to contrast agent used in imaging techniques in blood sample comprises contacting sample with the agent and stimulant, incubating, determining marker substance content and comparing
CN101957282B (en) * 2010-10-20 2013-06-05 杭州市中医院 Preparation method and application of single cell or tissue particle suspension
WO2012124763A1 (en) * 2011-03-16 2012-09-20 コニカミノルタエムジー株式会社 Tissue evaluation method
CN102507284A (en) * 2011-11-11 2012-06-20 福州迈新生物技术开发有限公司 Tissue paraffin removing liquid

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060035292A1 (en) * 2001-01-12 2006-02-16 Mount Sinai School Of Medicine Of New York University Differential diagnosis of cancer and other conditions based on expression of p63
CN102183651A (en) * 2011-02-22 2011-09-14 沈阳药科大学 Caspase-3 proenzyme protein immunohistochemical diagnosis kit and application
CN102435728A (en) * 2011-09-07 2012-05-02 福州大学 Preparation method for positive control substance for inspection and control of quality in immunohistochemical process

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106353164A (en) * 2016-07-21 2017-01-25 深圳市美雅洁技术股份有限公司 Ultrasonic rapid wax dipping system for pathological tissues and usage method
EP3639031A4 (en) * 2017-06-15 2021-05-05 Sunstone Scientific Limited. Method for ihc antigen imaging scale extrapolation
CN114689407A (en) * 2022-04-14 2022-07-01 四川大学华西医院 Method for manufacturing paraffin section of animal microtissue
CN114689407B (en) * 2022-04-14 2023-05-19 四川大学华西医院 Method for manufacturing animal micro tissue paraffin section

Also Published As

Publication number Publication date
CN103116018A (en) 2013-05-22
CN103116018B (en) 2015-04-15

Similar Documents

Publication Publication Date Title
WO2014114009A1 (en) Immunohistochemical quality control reference object and quality control method
CN103940658A (en) Method for manufacturing paraffin-embedded tissue cell specimen
KR20140019833A (en) Biomarker for breast cancer
CN103116029B (en) Determining method for sensitivity and affinity of second antibody color appearance system for immunohistochemistry
WO2014100220A2 (en) Antibody cocktail systems and methods for classification of histologic subtypes in lung cancer
CN104359746B (en) Cell mass collector and the method for collecting cell in humoral specimen
CN116593262B (en) Molecular marker detection product based on PDX/PDTX tumor living tissue biological sample and database and preparation method thereof
CN111190001B (en) Special diluent for immunohistochemical antibody and preparation method thereof
Renshaw Immunochemical staining techniques
JP3723204B1 (en) Impermeable tissue quick fixative
US20220365081A1 (en) Sample clarification and reduction of background fluorescence for fluorescent detection of analytes
CN106198996A (en) Early diagnosis biomarker, test kit and the application thereof of minimal change nephropathy
JP2003520600A (en) A system for internal qualitative and quantitative validation of marker indices
Zheng et al. CD45 immunohistochemistry in mouse kidney
JPWO2003104806A1 (en) Detection kit, measurement plate used therefor, detection method, evaluation method, frog vitellogenin polyclonal antibody and method for producing the same
CN111474358B (en) 3D (three-dimensional) three-dimensional immunofluorescence staining kit and application thereof
Al-Refu General methods in preparation of skin biopsies for haematoxylin and eosin stain and immunohistochemistry
CN115125331A (en) EB virus detection method based on in-situ hybridization method
Miller et al. Effects of delayed or prolonged fixation on immunohistochemical detection of bovine viral diarrhea virus type I in skin of two persistently infected calves
CN103076448B (en) Preparation method and device for novel labeling technique of cervix cancer cells
CN111393524A (en) In-situ detection method of Alzheimer's disease brain tissue A β protein based on metal marker
CN116659987A (en) Preparation method and application of paraffin section for cytopathology detection
CN105585627A (en) Antigen polypeptide and application of anti-GBM nephritis model built from same
Dakhil et al. Detection of HER-2 receptor over expression in Breast Cancer by using of Immunohistochemistry procedure
WO2022211084A1 (en) Method for treating biological sample

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13873059

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205N DATED 06.11.2015)

122 Ep: pct application non-entry in european phase

Ref document number: 13873059

Country of ref document: EP

Kind code of ref document: A1