CN113720669A - Immunohistochemical quality control product constructed based on animal organ or tissue - Google Patents
Immunohistochemical quality control product constructed based on animal organ or tissue Download PDFInfo
- Publication number
- CN113720669A CN113720669A CN202110995173.1A CN202110995173A CN113720669A CN 113720669 A CN113720669 A CN 113720669A CN 202110995173 A CN202110995173 A CN 202110995173A CN 113720669 A CN113720669 A CN 113720669A
- Authority
- CN
- China
- Prior art keywords
- quality control
- tissue
- pig
- immunohistochemical
- control product
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
- 238000003908 quality control method Methods 0.000 title claims abstract description 152
- 230000002055 immunohistochemical effect Effects 0.000 title claims abstract description 50
- 210000001557 animal structure Anatomy 0.000 title claims abstract description 7
- 210000000056 organ Anatomy 0.000 claims abstract description 71
- 238000000034 method Methods 0.000 claims abstract description 46
- 238000004519 manufacturing process Methods 0.000 claims abstract description 36
- 239000000463 material Substances 0.000 claims abstract description 26
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 20
- 230000018044 dehydration Effects 0.000 claims abstract description 17
- 238000006297 dehydration reaction Methods 0.000 claims abstract description 16
- 238000013115 immunohistochemical detection Methods 0.000 claims abstract description 12
- 230000007935 neutral effect Effects 0.000 claims abstract description 10
- 210000001519 tissue Anatomy 0.000 claims description 142
- 239000001993 wax Substances 0.000 claims description 31
- 239000012188 paraffin wax Substances 0.000 claims description 24
- 238000010186 staining Methods 0.000 claims description 20
- 241001465754 Metazoa Species 0.000 claims description 19
- 238000002360 preparation method Methods 0.000 claims description 18
- 238000004043 dyeing Methods 0.000 claims description 17
- 210000004185 liver Anatomy 0.000 claims description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 210000003734 kidney Anatomy 0.000 claims description 13
- 238000005520 cutting process Methods 0.000 claims description 12
- 238000003364 immunohistochemistry Methods 0.000 claims description 12
- 210000004556 brain Anatomy 0.000 claims description 11
- 210000002216 heart Anatomy 0.000 claims description 11
- 210000004072 lung Anatomy 0.000 claims description 10
- 210000000496 pancreas Anatomy 0.000 claims description 10
- 210000002826 placenta Anatomy 0.000 claims description 9
- 210000000952 spleen Anatomy 0.000 claims description 9
- 210000004291 uterus Anatomy 0.000 claims description 9
- 108060000903 Beta-catenin Proteins 0.000 claims description 8
- 102000015735 Beta-catenin Human genes 0.000 claims description 8
- 210000002429 large intestine Anatomy 0.000 claims description 8
- 210000001672 ovary Anatomy 0.000 claims description 8
- 102000007469 Actins Human genes 0.000 claims description 7
- 108010085238 Actins Proteins 0.000 claims description 7
- 210000003932 urinary bladder Anatomy 0.000 claims description 7
- 238000007598 dipping method Methods 0.000 claims description 6
- 235000015277 pork Nutrition 0.000 claims description 6
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 5
- 239000006285 cell suspension Substances 0.000 claims description 5
- 210000001165 lymph node Anatomy 0.000 claims description 5
- 238000007789 sealing Methods 0.000 claims description 5
- 102100034157 DNA mismatch repair protein Msh2 Human genes 0.000 claims description 4
- 101001134036 Homo sapiens DNA mismatch repair protein Msh2 Proteins 0.000 claims description 4
- 229910015837 MSH2 Inorganic materials 0.000 claims description 4
- 239000012189 cellwax Substances 0.000 claims description 4
- 101000642536 Apis mellifera Venom serine protease 34 Proteins 0.000 claims description 3
- 108010083123 CDX2 Transcription Factor Proteins 0.000 claims description 3
- 102000006277 CDX2 Transcription Factor Human genes 0.000 claims description 3
- 102000000905 Cadherin Human genes 0.000 claims description 3
- 108050007957 Cadherin Proteins 0.000 claims description 3
- 102100021147 DNA mismatch repair protein Msh6 Human genes 0.000 claims description 3
- 102100036912 Desmin Human genes 0.000 claims description 3
- 108010044052 Desmin Proteins 0.000 claims description 3
- 102100022183 E3 ubiquitin-protein ligase MIB1 Human genes 0.000 claims description 3
- 102100021260 Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1 Human genes 0.000 claims description 3
- 102100039289 Glial fibrillary acidic protein Human genes 0.000 claims description 3
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 claims description 3
- 102100027893 Homeobox protein Nkx-2.1 Human genes 0.000 claims description 3
- 101000968658 Homo sapiens DNA mismatch repair protein Msh6 Proteins 0.000 claims description 3
- 101000973503 Homo sapiens E3 ubiquitin-protein ligase MIB1 Proteins 0.000 claims description 3
- 101000894906 Homo sapiens Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1 Proteins 0.000 claims description 3
- 101000632178 Homo sapiens Homeobox protein Nkx-2.1 Proteins 0.000 claims description 3
- 101001015004 Homo sapiens Integrin beta-3 Proteins 0.000 claims description 3
- 101000994460 Homo sapiens Keratin, type I cytoskeletal 20 Proteins 0.000 claims description 3
- 101000934372 Homo sapiens Macrosialin Proteins 0.000 claims description 3
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 claims description 3
- 101000601724 Homo sapiens Paired box protein Pax-5 Proteins 0.000 claims description 3
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 claims description 3
- 101000874160 Homo sapiens Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial Proteins 0.000 claims description 3
- 101000845269 Homo sapiens Transcription termination factor 1 Proteins 0.000 claims description 3
- 206010062767 Hypophysitis Diseases 0.000 claims description 3
- 102100032999 Integrin beta-3 Human genes 0.000 claims description 3
- 102100032700 Keratin, type I cytoskeletal 20 Human genes 0.000 claims description 3
- 102100025136 Macrosialin Human genes 0.000 claims description 3
- 102000008071 Mismatch Repair Endonuclease PMS2 Human genes 0.000 claims description 3
- 108010074346 Mismatch Repair Endonuclease PMS2 Proteins 0.000 claims description 3
- 102000003729 Neprilysin Human genes 0.000 claims description 3
- 108090000028 Neprilysin Proteins 0.000 claims description 3
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 claims description 3
- 102100037504 Paired box protein Pax-5 Human genes 0.000 claims description 3
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 claims description 3
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 claims description 3
- 102100035726 Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial Human genes 0.000 claims description 3
- 210000005045 desmin Anatomy 0.000 claims description 3
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 claims description 3
- 229930192851 perforin Natural products 0.000 claims description 3
- 230000001817 pituitary effect Effects 0.000 claims description 3
- 210000003635 pituitary gland Anatomy 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 230000035945 sensitivity Effects 0.000 claims description 3
- 210000000813 small intestine Anatomy 0.000 claims description 3
- 101100381525 Mus musculus Bcl6 gene Proteins 0.000 claims description 2
- 238000000338 in vitro Methods 0.000 claims description 2
- 238000012216 screening Methods 0.000 claims description 2
- 238000002791 soaking Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims 1
- 208000005156 Dehydration Diseases 0.000 abstract description 12
- 230000001575 pathological effect Effects 0.000 abstract description 12
- 230000002349 favourable effect Effects 0.000 abstract description 7
- 239000013558 reference substance Substances 0.000 abstract description 5
- 241000282898 Sus scrofa Species 0.000 description 83
- 239000000047 product Substances 0.000 description 77
- 210000004027 cell Anatomy 0.000 description 18
- 238000001514 detection method Methods 0.000 description 13
- 230000000877 morphologic effect Effects 0.000 description 13
- 230000000694 effects Effects 0.000 description 11
- 206010028980 Neoplasm Diseases 0.000 description 10
- 210000001691 amnion Anatomy 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 7
- 230000009286 beneficial effect Effects 0.000 description 6
- 210000002808 connective tissue Anatomy 0.000 description 6
- 230000007170 pathology Effects 0.000 description 6
- 238000013461 design Methods 0.000 description 5
- 238000011532 immunohistochemical staining Methods 0.000 description 5
- 238000012545 processing Methods 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 210000000013 bile duct Anatomy 0.000 description 4
- 235000009508 confectionery Nutrition 0.000 description 4
- 210000005229 liver cell Anatomy 0.000 description 4
- 210000004379 membrane Anatomy 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 210000002460 smooth muscle Anatomy 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000282887 Suidae Species 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 210000001072 colon Anatomy 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000007762 localization of cell Effects 0.000 description 3
- 210000002751 lymph Anatomy 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 210000002381 plasma Anatomy 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000000834 fixative Substances 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 210000002220 organoid Anatomy 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 102100021631 B-cell lymphoma 6 protein Human genes 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 101150029707 ERBB2 gene Proteins 0.000 description 1
- 241000806990 Hala Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000971234 Homo sapiens B-cell lymphoma 6 protein Proteins 0.000 description 1
- 101000916173 Homo sapiens Catenin beta-1 Proteins 0.000 description 1
- 101000777314 Homo sapiens Choline kinase alpha Proteins 0.000 description 1
- 101000777313 Homo sapiens Choline/ethanolamine kinase Proteins 0.000 description 1
- 101000837845 Homo sapiens Transcription factor E3 Proteins 0.000 description 1
- 101001138544 Homo sapiens UMP-CMP kinase Proteins 0.000 description 1
- 102100025354 Macrophage mannose receptor 1 Human genes 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 102000043276 Oncogene Human genes 0.000 description 1
- 235000005311 Pandanus odoratissimus Nutrition 0.000 description 1
- 101001050928 Sus scrofa UMP-CMP kinase Proteins 0.000 description 1
- 102100028507 Transcription factor E3 Human genes 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000009534 blood test Methods 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 210000003701 histiocyte Anatomy 0.000 description 1
- 102000056097 human CTNNB1 Human genes 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 210000003963 intermediate filament Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000012758 nuclear staining Methods 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000010827 pathological analysis Methods 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 230000011218 segmentation Effects 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Images
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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
-
- 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
-
- 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
- G01N1/28—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
- G01N2001/366—Moulds; Demoulding
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to the field of pathological examination, in particular to an immunohistochemical quality control product constructed based on animal organs or tissues. In the scheme provided by the invention, the tissue and organ of the pig can effectively replace the organ and organ tissue of the human body as the source of the reference substance in the aspect of partial immunohistochemical detection. The material market is convenient to obtain, and the quality control product based on tissue morphology and immunohistochemical examination application is favorable for commercial production in the future. According to the basic requirements of histology, the quality control wax block can be manufactured by fixing with 10% neutral buffered formalin, an automatic tissue dehydration treatment instrument and the like. And the method is favorable for realizing normalized operation. No special method and change of the checking path are needed, and the method is simple and easy to implement.
Description
Technical Field
The invention relates to the field of pathological examination, in particular to an immunohistochemical quality control product constructed based on animal organs or tissues.
Background
Clinical immunohistochemistry (immunohistochemistry) can aid in diagnosis of benign and malignant tumors and guide medication. Known commercial antibodies are used to detect tumor antigens. Immunohistochemistry is used as a diagnostic tool which cannot be replaced by clinical pathology, and the detection result of the immunohistochemistry directly influences the accuracy of pathological diagnosis.
However, the quality of the antibody (with variable quality) cannot be uniform due to the secrecy of the sequence of the commercial antibody. Therefore, quality control of (anti-human) antibodies for clinical immunohistochemical use is required. Meanwhile, immunohistochemical detection (antigen-antibody reaction) is influenced by multiple factors (including temperature and humidity, concentration, batch difference, personnel operation and the like), and is easy to change, so that result deviation is caused. Therefore, quality control products are urgently needed to monitor the quality problem of the immunohistochemical detection process. Because antibody vendors do not provide quality control products and detection parties cannot provide the quality control products, the current immunohistochemical detection results of most laboratories only rely on the subjective interpretation of the result 'phenomenon' by an interpreter, and the results are often inconsistent.
Based on the above situation, the national pathology control center proposes the use of immunohistochemistry (negative and positive) controls. Currently, interventricular quality control often uses clinically confirmed pathological sections as a test for issuing photographs and explicitly identifies quality control usage rather than commercial transactions (anthropogenic, ethical constraints). However, the number is limited, and the detection results are variable and pragmatic and dispute each time only limited to a small number of laboratories.
Immunohistochemistry, like the result of a test (blood test), requires calibration of quality controls to ensure quality of detection. But the suppliers and the third parties can not provide quality control products due to ethical limitations.
The general principle requirements for immunohistochemical tissue quality control are as follows: (1) achieving the purpose of histological control: the system can help readers (pathologists) to confirm the authenticity (true and false negative and positive) of the immunohistochemical result in real time; (2) the concrete requirements are as follows: the cell shape positioning characteristics (nucleus, plasma and membrane) which are clear under a microscope can be used as a reference standard for interpretation of results; (3) the device has characteristic marks or forms which are convenient for readers to distinguish; (4) standardization can be realized, and mass production can be unified; (5) meets the ethical requirements.
At present, the commercial immunohistochemical tissue quality control product is lacking clinically, and the antibody manufacturer can not provide the quality control product required by the factory (due to the large demand and limited source). There are several major groups of immunohistochemical control reports (non-human-derived materials) that may be commercialized at home and abroad:
protein polypeptide: the polypeptide gel control (without the morphological structural characteristics of cells) is reported earlier in the literature to be dropped on the section to be detected in the form of different mass concentrations, and a brown light and dark signal can appear in the dyeing process. But no clear assessment based on cellular morphological localization can be given. The intended use of tissue quality control is not achieved.
Positive cell line: cell lines cultured by the supplier. However, the culture passage of the cell line is very affected by the variation, so that the method cannot be popularized in mass production. For example, in clinical breast cancer Her2(4B5) anti-human antibody tests, the supplier provided a control of cell lines at an early stage, but stopped the supply soon afterwards.
③ organoids: has cell components similar to human organs and connective tissue intermediate filament components, but has no regulation of in vivo environment and body fluid, and the application data and effect are unknown. At present, no marketable supply exists.
Fourthly, tumor transplantation: human tumor cells were implanted in nude mice to form tumor models. At present, the tumor still has great uncertainty and is not supplied by market. The commercial attempts of immunohistochemical controls, mentioned above, were unsuccessful.
Industry norms and quality management requirements (such as grade hospital reviews) of the pathology department prompt the internal production of quality control products of the pathology department. The laboratory can automatically make human-derived quality control products. Human Hala cells, carrying the relevant oncogene proteins. But cell replication of each organ cannot be achieved. There are no reports for clinical immunohistochemistry at present.
1. And (3) a laboratory human-derived cell suspension (human-derived attribute) is tried to be dripped on the sample immunohistochemical section to provide clue evidence for the reliable detection process and detection quality. However, due to underevaluation of manufacturing processes, ratios and effects, it is only self-made for use in individual laboratories. The main problems are that contrast signals are missing and the positioning is not accurate; lack of morphological recognition and structural features for reference. And ethics prohibits marketization.
2. Laboratory cell wax blocks. Collecting positive cases of human cancer cells, enriching, and making cell wax block according to clinical pathological tissue processing procedure. Cutting the wax block into positive pieces and pasting the positive pieces on one side of a sample to be detected. The above problems also exist. Lack of morphological structural features and negative and positive cell proportion distribution problems.
3. Tumor tissue chip set in laboratory. The Tissue chip (TMA) method was used. And pricking a dot tissue of about 2mm by using a handheld chip perforating needle to manufacture the quality control product chip wax block. Slicing in advance and mounting a positive tissue chip on the slice to be detected. The quality control chip tissue and the sample to be detected are dyed simultaneously, and real-time reference can be provided. The method is currently widely used for laboratory internal control and laboratory assessments. However, due to heterogeneity of tumor tissue, co-morbidities of negative or negative and positive often occur. Meanwhile, the pretreatment conditions of the pathological specimens in the laboratories are different, and the prepared pathological wax lump results are also inconsistent. More importantly, the ethical limitation is that the product cannot be commercially produced and can only be used internally. Therefore, the comparison method is not ideal in standard and yield performance, but is still used in a laboratory at present.
3. Closest to the prior art-the laboratory normal tissue set: reside in the principle of "normal tissue priority comparison"
(AIMM, 2015; 23:1-18), a tissue set custom-made in the laboratory called "amnion roll" and containing a plurality of normal tissues (Multi-tissue Block, MTB) (Lab Invest 1986; 55:244-8) (patent No. CN 207036856U). It has the characteristic of constant protein expression and definite morphological structure, and is favorable for the physician to interpret the result.
The preparation method comprises the following steps: when the detected material is human tumor pathological specimen, the pathologist takes the specimen and collects the rest tissues (normal tissues) beside the cancer of the specimen, and cuts the specimen into pieces with size of "matchstick" (2.5 x0.2x0.2cm)3) And then fixed in 10% neutral buffered formalin for 12 hours. Mixing organs such as liver, kidney, pancreas, spleen, lung, heart, brain, prostate, ovary and placenta to obtain tissue set, and wrapping with amnion (semitransparent biomembrane) on placenta surface to obtain Spring Roll shape. And (4) performing later-stage paraffin block preparation according to the preparation program of the pathological tissue paraffin block. And continuously slicing by a paraffin wheel rotation type slicing machine, cutting the wax block into 2-3 um positive pieces, and pasting the positive pieces on one side of the sample to be detected. The advantages are that: the result can be repeated due to the dyeing, and has in-situ morphological structural characteristics. The "amniotic membrane roll" is effective in providing negative and positive control references for immunohistochemistry, most of which are used in pathology laboratories approved by ISO 15189.
However, the material selected for the quality control product of the amniotic membrane roll is human organ tissue components, so that the quality control product related to ethics cannot be commercialized. And the difference of the manufacturing process in each laboratory is large, and the uniform manufacturing standard of the quality control reference substance cannot be formed. There are differences in the effects of actual use. Other control forms such as cell lines, transplanted tumors, organoids and the like are limited by the product itself, and no mature mass production and commercial supply exist at present.
The prior art adopts human organ and tissue to integrate the specific defects of the amniotic membrane roll:
1. human tissue and organs are not sufficiently sourced. In addition to the inability to collect vital organs (such as normal brain, heart, testis, etc.), amnion (i.e., placenta) is also lacking in many specialized hospitals (requiring the availability of materials from human placenta).
2. Ethical limitations. The manufacturing process involves ethical limitation of human organs, and mass production is prohibited.
3. The manufacturing process is not standard. At present, the manufacturing method is derived from reference documents, and manufacturing specifications and standards (the manufacturing process and the effect are to be evaluated) are not published in the industry; the quality control product is difficult to be a real quality control product due to the lack of uniform manufacturing procedures.
4. There is a lack of standardization. Most laboratories have a master role in thinking that: the preparation method of the amniotic membrane roll only comes from the literature (unpublished standard) and has no authoritative effect. The quality control product needs standard production, and can be repeatedly produced in large quantities. However, none of the above mentioned multiple tissue control sets can meet the above requirements. "
5. The implementation effect is not good. Most laboratories conflict with quality control products and feel troublesome. Even in the unit where the "amniotic membrane roll" quality control product is prepared, only 1 quality control product control stain is added to 29 pathological sections in one immunohistochemical detection. Control staining (which should be done at the same time as the same slide of sample) was not done for each case. Therefore, when any one of them shows a negative staining result, the accuracy of the result (whether it is true negative) still cannot be confirmed.
Therefore, it is of great practical significance to adopt a non-human source material to provide a practical approach that can be standardized, produce the same control results and beneficial technical effects, and be used for commercial production.
Disclosure of Invention
In view of the above, the present invention provides a manufacturing and production scheme of a commercialized immunohistochemical quality control product, which solves a part of the problems of reference products required for clinical immunohistochemical daily and quality control work, and achieves the purpose of controllable mass production and commercialization.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of organs or tissues of animals in preparing an immunohistochemical quality control product.
In some embodiments of the invention, the animal comprises a pig;
the organ or tissue includes, but is not limited to, one or more of the liver, lung, kidney, spleen, large intestine, small intestine, appendix, lymph node, bladder, pancreas, uterus, ovary, pituitary, placenta, heart or brain;
the immunohistochemical quality control product comprises a cell wax block or a cell suspension.
In some embodiments of the invention, the anti-human antibodies of the organ or group of animals comprise:
pig hearts: actin (HHF35) S100 Vim;
pork liver: CK (AE1/AE3)34 β E12 CK7(OVTL12/30) CK7(EPR1619Y) CK5/6(D5/16B4) SDHB;
pig lung: TTF1(8G7G3/1) TG CD117 TFE 3;
pig kidney E-Cadherin EMA WT-1(6FH 2);
pig spleen CD3 (polyclonal antibody) CD5 CD10 CD45 CD56 CD57 CD61 CD68 CD79a (HM57) PAX5(SP34) Perforin MyC MPO TdT BCL 6;
the pig large intestine: ki67(UMB107) Ki67(MIB1) CDX2(CDX-88) CgA (DAK-A3) MSH2 MSH6 PMS 2;
porcine bladder CK20(KS 20.8);
pig pancreas CK7 beta catenin;
porcine uterus ER P53P 63(4A4) Vim Desmin (D33) SMA (1A4) MHC;
pig ovary: inhibin-a (R1);
pig brain S100 NSE NF GFAP;
pig pituitary gland: GH PRL.
The preparation method of the immunohistochemical quality control product comprises the following steps:
step 1: obtaining an organ or tissue of an animal;
step 2: fixing;
and step 3: manufacturing a quality control wax block;
and 4, step 4: and (5) manufacturing a quality control wafer.
In some embodiments of the invention, the animal comprises a pig;
the organ or tissue includes one or more of the liver, lung, kidney, spleen, large intestine, bladder, pancreas, uterus, ovary, heart or brain.
In some embodiments of the invention, the immunohistochemical quality control product comprises one or more of a single type quality control product, a universal type quality control product, a porcine multi-organ mixed quality control product;
the preparation method of the single-type quality control product comprises the following steps:
step A, obtaining organs or tissues of the animal;
step B, fixing: taking organs or tissues of the animals, cutting the organs or tissues in vitro at the temperature of 2-8 ℃, and fixing the organs or tissues in 10% neutral buffered formalin for 24 hours;
step C, material taking: dissecting the organ with surgical knife according to the maximum diameter of the tissue, and selecting the tissue with the parenchymal part of 2 × 2 × 0.2cm3Carrying out tissue sampling;
step D, dehydration: putting the sampled tissue into 70-100% gradient alcohol for dehydration for 8 h; specifically, the dehydration temperature is set to 25 ℃ for 2h of 70% alcohol, 2h of 80% alcohol, 2h of 90% alcohol, 1h of 95% alcohol and 1h of 100% alcohol; this step can be done in an automated dehydration machine;
step E, transparency: the dehydrated tissue is transparent by adopting 3-channel dimethylbenzene, the transparent temperature is 25 ℃, and the time is 0.5h, 0.5h and 1h respectively; this step can be done in an automated dehydration machine;
step F, wax dipping: soaking the transparent tissue by using 3 paths of paraffin wax at the temperature of 60 ℃ for 0.5h, 0.5h and 1h respectively; this step can be done in an automated dehydration machine;
step G, embedding: the tissue that finishes the wax dipping is taken out from the automatic dehydrator, and the embedding of the mould is carried out to make quality control wax block: the paraffin block size was 3.5X 3X 0.8cm3To obtain the quality control wax block;
step H, manufacturing a quality control wafer: slicing the substrate according to the thickness of 2-3 mu m by using a paraffin wheel rotation type slicing machine to obtain the quality control sheet;
the preparation method of the universal quality control tablet or the pig multi-organ mixed quality control product comprises the following steps:
step I, obtaining organs or tissues of the animal;
step II, fixing: cutting organs or tissues of the animals at 2-8 ℃, and fixing in 10% neutral buffered formalin for 24 hours;
step III, material taking: taking the fixed organ or tissue according to 2 × 0.5 × 0.2cm3Cutting into strip-shaped tissue, cutting pig placenta into 5cm × 3cm, or coating the strip-shaped tissue of different organs or tissues into 3 × 1.5 × 0.5cm by using one or more of special microscope lens-wiping paper3Size, sealing both ends and/or knotting the center;
step IV, tissue dehydration, transparence, wax dipping, embedding and slicing steps are the same as the steps C to H, wherein C to F can be completed in an automatic dehydrator;
embedding (same as G) and flaking (same as H) were performed manually by personnel. The embedded paraffin block size was 3.5X 3X 0.8cm3Left and right; slicing according to the thickness of 2 mu m to obtain the quality control wafer.
More importantly, the invention also provides the immunohistochemical quality control product prepared by the preparation method.
Based on the above, the invention also provides the application of the immunohistochemical quality control product in the preparation of an immunohistochemical detection kit.
In addition, the invention also provides an evaluation method of the immunohistochemical quality control product, which comprises the following steps of by tissue immunohistochemical staining specificity: positioning; sensitivity: intensity and/or repeatability: and (3) evaluating the immunohistochemical quality control product by using a dyeing condition.
The invention also provides a screening method of the anti-human antibody, which is characterized in that the paraffin block of a sample to be detected is compared with the immunohistochemical quality control product, and if the paraffin block is obviously colored, the histological similarity is not lower than 60 percent.
The invention adopts a non-human-derived material, provides an implementation method of normalized operation, produces the same contrast result and beneficial technical effect, and can be used for commercial production.
Benefits include, but are not limited to:
the tissue and organ of the pig can effectively replace the organ and organ tissue of the human body as the source of the reference substance in the aspect of partial immunohistochemical detection. The material market is convenient to obtain, and the quality control product based on tissue morphology and immunohistochemical examination application is favorable for commercial production in the future.
According to the basic requirements of histology, the quality control wax block can be manufactured by fixing with 10% neutral buffered formalin, an automatic tissue dehydration treatment instrument and the like. And the method is favorable for realizing normalized operation. No special method and change of the checking path are needed, and the method is simple and easy to implement.
The general design idea solves the quality control problem of multiple antibodies (shown in table 1) in a one-to-many mode, and reduces the difficulty of implementation of contrast dyeing. The quality control product with tangible characteristics is prepared by adopting a multi-organization set 'sandwich roll' method and executing an operation instruction program according to fixed procedures. The identification between the reader and the sample to be detected is convenient, and the result interpretation and output are facilitated.
The application of the pig quality control product is realized, and more than 50 (anti-human) antibodies are tested at present. 1000 immunohistochemical control sections per hour were manually made from a single wax block. Such as automated production, can improve yield and efficiency.
On the basis of the same immunohistochemical route and mode (experimental conditions of human tissues are not changed), the control of the quality control tablet of the pig with the same tablet (On-Slide) is implemented, so that the quality monitoring of the detection antibody and the result is facilitated, and the beneficial clinical value of the clinical immunohistochemical examination result is improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows a flow chart of the procedure for preparing a multi-organ control set "sandwich roll" for a swine tissue immunohistochemical quality control product;
FIG. 2 is a diagram showing the simultaneous detection of the preparation of a plurality of tissue quality control slices of a pig and a sample to be detected; wherein, fig. 2(A) shows that the pig quality control wax block is made into slices; FIG. 2(B) shows the use of porcine quality control for human Actin antibody detection; FIG. 2(C) shows the use of porcine quality control for human CD117 antibody detection;
FIG. 3(A) shows porcine colon β catenin; FIG. 3(B) shows human intestine β catenin; the immunohistochemical staining is consistent in location through comparison verification;
fig. 4(a) shows porcine lymph Ki 67; fig. 4(B) shows human lymph Ki 67; the immunohistochemical staining is consistent in location through comparison verification;
FIG. 5(A) shows pig kidney CK; FIG. 5(B) shows a human kidney CK; the immunohistochemical staining is consistent in location through comparison verification;
FIG. 6 is a schematic diagram of another process for producing a multi-tissue control product of pork; FIG. 6(A) is a schematic diagram of a multi-tissue homogenate stirring process for pigs; FIG. 6(B) porcine tissue cell suspension control section; FIG. 6(C) porcine histiocyte suspension control staining effect CK.
Detailed Description
The invention discloses an immunohistochemical quality control product constructed based on animal organs or tissues, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention provides a commercial immunohistochemical quality control product constructed based on animal material tissues, which solves the problem of implementation of immunohistochemical control.
The material of the species provided by the invention is a general commercial histological material, namely tissue and organs of pigs, including liver, lung, kidney, large intestine, pancreas and precious organ tissues such as heart, brain and the like which are difficult to obtain. The organ acquisition channels are convenient and fast, and the requirements of users and market purchase can be met. And the qualified counting product of the paraffin block can be produced through the tissue processing procedure of the pathological specimen.
The morphological structure of the pig quality control product is almost the same as that of human tissues and organs through a morphological HE (hematoxylin-eosin) staining method. Due to species differences, not all anti-human antibodies reacted with porcine tissue (e.g., MLH1(ES05) without crossing). However, before the quality control product is prepared, the applicable application of the pig organ material can be given through the performance test of each immunohistochemical anti-human antibody (as shown in table 1). Of over one hundred clinically commercialized anti-human antibodies commonly used in the test, cross-reactivity was observed for nearly 50 antibodies. More antibodies to humans are also being tested on porcine material. The pigs tested are currently the most cross-reactive species.
TABLE 1 applicable application table of multiple tissue organ integration method for pig
The technical scheme provided by the invention comprises the following steps:
1. commercially available animal materials, porcine organ fractions, were used. The source of the pig organ material is convenient to obtain (commercial).
2. The material selection of the pig organ and the preparation method of the paraffin block are the same as the pathological tissue treatment scheme of the human. 10% neutral buffered formalin fixation was performed strictly for 24 hours.
3. Formalin-Fixed paraffin tissue wax blocks (FFPE) formed by adopting a pathological tissue processing scheme can be used for immunohistochemical detection.
4. It has almost no difference from the tissue morphology of human organs in the aspect of tissue morphology observation. Due to the morphological structure characteristics, the material is superior to a simple cell strain culture technology.
3. The beneficial effect of the pig tissue quality control product on the anti-human antibody which is commonly used in clinic is tested. The immunohistochemical staining result is the same as the result of the human tissue (similar to 90 percent or more). Has the characteristics of definite positioning (nucleus, plasma and membrane) of morphological attributes, and meets the specificity requirement of immunohistochemical control.
4. The pig quality control product has the strength characteristic based on the morphological basis and meets the sensitivity requirement of the immunohistochemical control. For example, CK (AE1/AE3) is strongly expressed in bile duct components of pig liver, and liver cell components are weakly expressed.
5. The repeatable characteristic of the quality control product is met. Because the normal organ tissues of the pig are regulated and controlled by the constant genes, the protein phenotype is constant, and the dyeing repeatability is 100 percent. The laboratory data alignment consistency including but not limited to the following antibodies was 100%. The results are the same through repeated experiments (more than or equal to 3 times). Antibodies that are consistent with complete staining of human tissue (including localization, intensity) such as:
firstly, Hept1 pig liver vs human liver cells, bile duct staining consistency 100% (including location, intensity).
② the dyeing consistency of the beta-catenin pig liver vs human liver cells and bile ducts is 100 percent (including positioning and strength).
③ CK7 pig liver vs human hepatobiliary duct dyeing consistency of 100% (including location and strength).
And fourthly, the dyeing consistency of the index of the human liver hepatocytes of Ki67 swine vs (including positioning and intensity) is 100%.
MMR protein such as MSH2 pig liver vs human liver cell, bile duct staining consistency 100% (including location and intensity).
Sixthly, the dyeing consistency of the protein pork and the blood vessels and connective tissues of the human meat is 100 percent (including positioning and strength).
Seventhly, the dyeing consistency of the MHC protein pork, human blood vessels and connective tissues is 100 percent (including positioning and strength).
The dyeing consistency of the porcine and human lymph follicles is 100% (including positioning and strength).
And ninthly, the staining consistency of the smooth muscle of the uterus of the pig uterus vs human is 100 percent (including positioning and strength).
6. Adopts a general design and a 'sandwich roll' manufacturing method. Comprises a plurality of different tissues and organs, and is made into a shape like a strip candy to form a tangible quality control product. After the quality control product is sliced, the cross section of the quality control product can be seen under a microscope, and the quality control product has definite morphological identification characteristics of tissue organ components. The different organ tissue components provide a staining presentation of the negative and positive cell localization (nuclear, plasma, membrane) that are complementary to each other. The quality control product for pig has the advantages of good appearance, simple process and easy application.
7. In the quality control comparison implementation process, the dyeing process and details are monitored in real time according to an on-slide detection scheme (the comparison tissue is pasted on the left side and the side close to a label of a sample) with a human sample to be detected.
8. The control quality control problem of more than or equal to 50 (still continuously tested) clinical anti-human antibodies can be solved by adopting a plurality of pig tissue sets and only needing one pair of photos. The workload can be simplified, and the trouble of selecting reference substances with different indexes is avoided. To date, this test alignment demonstrated that nearly 53 clinically useful antibodies (see table 1) were reactive with porcine tissue material.
9. The quality control product made of the pig tissue has the characteristic of mass production. Wax blocks with the thickness of 2mm can finish the production of 1000 quality control product slices (the slice thickness is 2 um/slice) within 1 hour. The quantity is rich, the available contrast section supply is sufficient, and the requirement of clinical practice can be met.
The solution provided by the present invention includes, but is not limited to, the following advantages: the tissue and organ of the pig can effectively replace the organ and organ tissue of the human body as the source of the reference substance in the aspect of partial immunohistochemical detection. The material market is convenient to obtain, and the quality control product based on tissue morphology and immunohistochemical examination application is favorable for commercial production in the future.
According to the basic requirements of histology, the quality control wax block can be manufactured by fixing with 10% neutral buffered formalin, an automatic tissue dehydration treatment instrument and the like. And the method is favorable for realizing normalized operation. No special method and change of the checking path are needed, and the method is simple and easy to implement.
The general design idea solves the quality control problem of multiple antibodies (shown in table 1) in a one-to-many mode, and reduces the difficulty of implementation of contrast dyeing. The quality control product with tangible characteristics is prepared by adopting a multi-organization set 'sandwich roll' method and executing an operation instruction program according to fixed procedures. The identification between the reader and the sample to be detected is convenient, and the result interpretation and output are facilitated.
The beneficial effects of the invention include: the application of the pig quality control product can test more than 50 (anti-human) antibodies at present. 1000 immunohistochemical control sections per hour were manually made from a single wax block. Such as automated production, can improve yield and efficiency.
The immunohistochemical application of the pig quality control product provided by the invention is not limited to:
more than 50 anti-human antibodies are commonly used in clinic to react with porcine organ tissues, for example:
pig hearts: actin (HHF35) S100 Vim
Pork liver: CK (AE1/AE3)34 beta E12 CK7(OVTL12/30) CK7(EPR1619Y) CK5/6(D5/16B4) SDHB
Pig lung: TTF1(8G7G3/1) TG CD117 TFE3
Pig kidney E-Cadherin EMA WT-1(6FH2)
Pig spleen CD3 (polyclonal antibody) CD5 CD10 CD45 CD56 CD57 CD61 CD68 CD79a (HM57) PAX5(SP34) Perforin MyC MPO TdT BCL6
The pig large intestine: ki67(UMB107) Ki67(MIB1) CDX2(CDX-88) CgA (DAK-A3) MSH2 MSH6 PMS2
Porcine bladder CK20(KS20.8)
Porcine pancreas CK7 beta catenin
Porcine uterus ER P53P 63(4A4) Vim Desmin (D33) SMA (1A4) MHC
Pig ovary: inhibin-a (R1)
Pig brain S100 NSE NF GFAP
Pig pituitary gland: GH PRL
On the basis of the same immunohistochemical route and mode (experimental conditions of human tissues are not changed), the control of the quality control tablet of the pig with the same tablet (On-Slide) is implemented, so that the quality monitoring of the detection antibody and the result is facilitated, and the beneficial clinical value of the clinical immunohistochemical examination result is improved.
In the immunohistochemical quality control product constructed based on animal organs or tissues, the used raw materials and reagents can be purchased from the market.
The invention is further illustrated by the following examples:
example 1 Single type quality control product design pig quality control wax block and quality control tablet
(1) Material source and acquisition: contacting with meat-processing factory to obtain healthy pig organ source.
(2) Fixing: the obtained fresh pig organs were segmented at low temperature and then immediately fixed in 10% neutral buffered formalin for 24 h. To ensure adequate fixation of the fixative, fixation was dissected every 1cm along the long axis of the organ, ensuring constant penetration of the fixative.
(3) Preparing a quality control wax block: single pig organ tissue at 2X2X0.2cm3After the segmentation, the tissue is placed in a tissue processor. The paraffin tissue block is prepared by an automatic tissue dehydration treatment procedure (70-100 percent gradient alcohol is dehydrated to 8 hours, transparent for 2 hours and wax is soaked for 2 hours), and then paraffin embedding is carried out. The specific step program is supplemented and added as required
(4) Quality control wafer: and (3) slicing by using a paraffin wheel rotation type slicer according to the thickness of 2-3 mu m. And mounting the tissue piece of the pig wax block on the side close to the label of the glass slide to obtain a quality control picture. For example, the pig liver quality control tablet can be used as a marker control of CK (AE1/AE3)34 beta E12 CK7(OVTL12/30) CK7(EPR1619Y) CK5/6(D5/16B4) and the like. The number of antibody clones is in parentheses.
Example 2 Universal design/pig multiple organ Mixed wax Block/"Sandwich roll" preparation method sheet
(1) Material taking and dividing: collecting the fixed tissue, and placing the tissues of liver, kidney, pancreas, spleen, lung, intestine, heart, brain, etc. of different organs of pig on the sampling board at 2x0.5x0.2cm3Cutting into cord-shaped tissue, selecting pig casing (commercial), cutting into 5x3cm pieces3Or the special lens wiping paper for the microscope adopts a sandwich roll mode to mix and wrap the cord-shaped tissues of different organs to be about 3x1.5x0.5cm3Size. The package is formed into a shape similar to a long strip candy.
Note that to avoid shape distortion during post-fabrication, a two-end stapler ties up the opening. If necessary, the center of the 'long strip candy' is knotted by a thread.
(2) Preparing a quality control wax block: and (3) putting the wrapped tissue set into a special tissue processing instrument for a pathology department to finish automatic dehydration (the procedure is the same as above, 70%, 80%, 90%, 95%, 100% gradient alcohol for 8h, transparent for 2h and paraffin for 2 h). After the special automatic dehydration program is finished, paraffin embedding is carried out. During embedding, the filter paper on the surface is removed, the 'strip-shaped candy' in the filter paper is stripped, the filter paper is cut according to the thickness of 2mm, the two ends of the cut surface are kept to be flat and aligned, and all tissues and organs of the pig are completely visible. Then embedding the tissue blocks of 2mm with paraffin to form the appearance size of 2x2x0.2cm3The shape of the quality control product.
(3) Slicing a quality control product: according to the requirement of the tissue thickness of 2 mu m, a pig quality control product wax block is cut on a paraffin wheel type slicer, the tissue is fished out from the end, close to the label, of the slide by using an adhesive slide, and after drying, the slide is placed in a 4-degree refrigerator for storage and standby application.
Example 3 dyeing of pig quality control
(1) Sample preparation: the human subject sample was mounted on the lower middle 1/3 area using a control slide with porcine tissue and organ components. The pig quality Control product and the sample to be detected are fished out On the same Slide to ensure the same quality Control (On-Slide Control).
(2) Immunohistochemical detection process: immunohistochemistry is the same industry standard procedure. The method mainly comprises the following steps: dewaxing, adding water, blocking by 3% hydrogen peroxide, antigen repairing, incubating, combining secondary antibody, developing DAB color, and dyeing and sealing.
(3) Evaluation of quality control chip: before observing the result of the specimen to be detected, observing whether the dyeing of the components of the tissue and the organ of the pig meets the signal requirement: if a marker staining smooth muscle connective tissue (e.g., MHC) is indicated, it is evident that porcine smooth muscle connective tissue exhibits a DAB tan signal, whereas non-smooth muscle connective tissue is completely absent. The control purpose of verifying the antibody and the staining condition is realized.
(4) And (4) outputting a result: under the reference prompt of the quality control sheet, objective positive and negative diagnoses are made on the human sample results under a microscope according to the specific principle of definite cell positioning.
(5) Repeatability: the three characteristics (same reagent, method and condition state) of simultaneous detection with the sample to be detected are realized by one-Slide Control (On-Slide Control) (figure 2), and the real-time quality monitoring effect is exerted. The experimental results are consistent when repeated for more than or equal to 3 times (figure 3).
Example 4
The test method comprises the following steps:
1. dewaxing pig single point quality control wafer or universal quality control wafer, adding water, sealing with 3% hydrogen peroxide at normal temperature for 15 min, and flushing with running water
PBS rinsing 3 times, adding one of the above 50 primary antibodies (such as β catenin, ki67.. actin.. s100.. vim.....) and incubating at 37 ℃ for 1 hour or overnight in a4 ℃ refrigerator,
washing with PBS for 3 times, adding secondary antibody (goat anti-human IgG-HRP), incubating at 37 deg.C for 20 min,
4, washing with PBS for 3 times, adding DAB to develop for 10 minutes,
5. after washing with water, hematoxylin was lining-stained for 30 seconds and turned blue. And (6) sealing and observing.
And (3) detecting data:
(1) beta catenin + of pig,
comparative example human beta catenin +,
the expression and location of the colon part are consistent according to the comparison data, and the colon part has the specificity and the strength of the staining location;
PK results: the morphology is similar, and the signal expression is consistent and consistent;
the quality control tablet of pig can completely replace the quality control tissue of human.
(2) Pig Ki67+,
comparative example human Ki67+,
the expression and location of the lymph node parts are consistent through comparison data, and the lymph node parts have nuclear staining location specificity and intensity;
PK results: the morphology is similar, and the signal expression is consistent and consistent;
the quality control tablet of pig can completely replace the quality control tissue of human.
(3) Pig CK +,
comparative example human CK +,
comparing data, expressing, positioning and describing kidney parts, and having staining positioning specificity and intensity;
PK results: 100% consistent;
the quality control tablet of pig can completely replace the quality control tissue of human.
(4) Pig Actin +,
comparative example human Actin +,
the contrast data expresses a location description, and has staining location specificity and intensity;
PK results: 100% consistent;
the quality control tablet of pig can completely replace the quality control tissue of human.
(5) The pig is S100+,
comparative example human S100+,
the contrast data expresses a location description, and has staining location specificity and intensity;
PK results: 100% consistent;
the quality control tablet of pig can completely replace the quality control tissue of human.
EXAMPLE 5 alternative Processes for making multiple texture controls
(1) The preparation method of the mixed liquid comprises the following steps:
as shown in fig. 6: a method for preparing a multi-tissue control product (pig) comprises the steps of adopting wax blocks of multiple tissue organs of a pig, continuously slicing, collecting sliced cells, mixing tissue slices (2-3 um) of different organs, dissolving the tissue slices with a xylene organic solvent, collecting residues in a vessel, and freezing at-20 ℃ overnight. Then stirring uniformly, adding PBS diluent for dilution, and preparing a suspension without precipitates. The slide was loaded in advance by a sample gun. 500 pieces can be dripped in an average time of 5 minutes, the efficiency is high, and automatic dripping can be realized. The cell suspension under the lens contains cell distribution of different organs, and can play a role in negative and positive display control. Immunohistochemical section quality control of 50 antibodies used in the above tests.
The effect is shown in comparison of pig and human materials as quality control products
TABLE 2 comparison of specificity of quality control for porcine and human immunohistochemistry
Description of data: the specific gray value values analyzed by the software were too large and were included in the table after log-log conversion.
The specificity of the tissue quality control has two characteristics: 1) positive tissue has definite cell staining location, 2) negative tissue has no location and staining signal at all.
The specificity of the quality control products of the pig and the human immunohistochemistry is compared by positive tissues and negative tissues as follows: as can be seen from the data in Table 2, the cell staining was localized through the comparative findings of the positive tissues: the morphologies of the cells are clearly positioned identically (such as cell nucleuses, cytoplasm and cell membranes) through microscopic observation, the positioned intensities are compared and analyzed, the human eyes observe the same result under the microscope (in situ cell nucleuses/cytoplasm/membranes show 1+, 2+ and 3+), and the eyes cannot see the difference; grey value analysis OD (mean ± standard deviation) versus cell localization intensity was determined to be consistent by ImagePro software. The comparative difference of the positioning gray values of the pig quality control product and the human quality control product is less than 10 percent through the example test, and the difference of the visual diagnosis is not caused. And (3) taking a human control as a standard, and determining that the gray value ratio of the staining positioning intensity of the pig control is more than 90 percent. The pig quality control product is presumed to have high consistency (100%) with human in the positioning applied to morphology.
As can be seen from the data in Table 2, the comparison of negative tissues shows that: the pig quality control product and the human quality control product in the negative tissue do not have any cell localization and staining signals, and the two are completely consistent (100%).
The pig quality control product meets the morphological requirements of the immunohistochemical detection kit on mesoscopic quality, and can replace a human quality control product.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. Application of animal organ or tissue in preparing immunohistochemical quality control product is provided.
2. The use of claim 1, wherein the animal comprises a pig;
the organ or tissue comprises one or more of liver, lung, kidney, spleen, large intestine, small intestine, appendix, lymph node, bladder, pancreas, uterus, ovary, pituitary, placenta, heart or brain;
the immunohistochemical quality control product comprises a cell wax block or a cell suspension.
3. The use of claim 1 or 2, wherein the anti-human antibody of the organ or group of animals comprises:
pig hearts: actin (HHF35) S100 Vim;
pork liver: CK (AE1/AE3)34 β E12 CK7(OVTL12/30) CK7(EPR1619Y) CK5/6(D5/16B4) SDHB;
pig lung: TTF1(8G7G3/1) TG CD117 TFE 3;
pig kidney E-Cadherin EMA WT-1(6FH 2);
pig spleen CD3 (polyclonal antibody) CD5 CD10 CD45 CD56 CD57 CD61 CD68 CD79a (HM57) PAX5(SP34) Perforin MyC MPO TdT BCL 6;
the pig large intestine: ki67(UMB107) Ki67(MIB1) CDX2(CDX-88) CgA (DAK-A3) MSH2 MSH6 PMS 2;
porcine bladder CK20(KS 20.8);
pig pancreas CK7 beta catenin;
porcine uterus ER P53P 63(4A4) Vim Desmin (D33) SMA (1A4) MHC;
pig ovary: inhibin-a (R1);
pig brain: s100 NSE NF GFAP;
pig pituitary gland: GH PRL.
4. The preparation method of the immunohistochemical quality control product is characterized by comprising the following steps:
step 1: obtaining an organ or tissue of an animal;
step 2: fixing;
and step 3: manufacturing a quality control wax block;
and 4, step 4: and (5) manufacturing a quality control wafer.
5. The method of claim 4, wherein the animal comprises a pig;
the organ or tissue includes one or more of the liver, lung, kidney, spleen, large intestine, small intestine, appendix, lymph node, bladder, pancreas, uterus, ovary, pituitary, placenta, heart or brain.
6. The method of claim 4 or 5, wherein the immunohistochemical quality control product comprises one or more of a single type quality control product, a universal type quality control product, a porcine multi-organ mixed quality control product;
the preparation method of the single-type quality control product comprises the following steps:
step A, obtaining organs or tissues of the animal;
step B, fixing: taking organs or tissues of the animals, cutting the organs or tissues in vitro at the temperature of 2-8 ℃, and fixing the organs or tissues in 10% neutral buffered formalin for 24 hours;
step C, material taking: dissecting the organ with surgical knife according to the maximum diameter of the tissue, and selecting the tissue with the parenchymal part of 2 × 2 × 0.2cm3Carrying out tissue sampling;
step D, dehydration: putting the sampled tissue into 70-100% gradient alcohol for dehydration for 8 h; specifically, the dehydration temperature is set to 25 ℃ for 2h of 70% alcohol, 2h of 80% alcohol, 2h of 90% alcohol, 1h of 95% alcohol and 1h of 100% alcohol;
step E, transparency: the dehydrated tissue is transparent by adopting 3-channel dimethylbenzene, the transparent temperature is 25 ℃, and the time is 0.5h, 0.5h and 1h respectively;
step F, wax dipping: soaking the transparent tissue by using 3 paths of paraffin wax at the temperature of 60 ℃ for 0.5h, 0.5h and 1h respectively;
step G, embedding: the tissue that finishes the wax dipping is taken out from the automatic dehydrator, and the embedding of the mould is carried out to make quality control wax block: the paraffin block size was 3.5X 3X 0.8cm3To obtain the quality control wax block;
step H, manufacturing a quality control wafer: slicing according to the thickness of 2-3 mu m to obtain the quality control wafer;
the preparation method of the universal quality control tablet or the pig multi-organ mixed quality control product comprises the following steps:
step I, obtaining organs or tissues of the animal;
step II, fixing: cutting organs or tissues of the animals at 2-8 ℃, and fixing in 10% neutral buffered formalin for 24 hours;
step III, material taking: taking the fixed organ or tissue according to 2 × 0.5 × 0.2cm3Cutting into rope-shaped tissue, cutting pig placenta into 5cm × 3cm or one or more of special microscope lens-wiping paper by using sandwich roll methodMixing and wrapping the cord-like tissue to 3 × 1.5 × 0.5cm3Size, sealing both ends and/or knotting the center;
step IV, tissue dehydration, transparence, wax dipping, embedding and slicing steps are the same as the steps C to H,
the embedded paraffin block size was 3.5X 3X 0.8cm3Left and right; slicing according to the thickness of 2 mu m to obtain the quality control wafer.
7. The immunohistochemical quality control product prepared by the preparation method according to any one of claims 4 to 6.
8. Use of the immunohistochemical quality control product of claim 7 in the preparation of an immunohistochemical detection kit.
9. The method of evaluating an immunohistochemical quality control substance according to claim 7, wherein the specific staining is performed by tissue immunohistochemistry: positioning; sensitivity: intensity and/or repeatability: and (3) evaluating the immunohistochemical quality control product by using a dyeing condition.
10. The method for screening anti-human antibody, wherein the paraffin block of the sample to be tested is compared with the immunohistochemical quality control product according to claim 7, and if the paraffin block is significantly colored, the histological similarity is not less than 60%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110995173.1A CN113720669A (en) | 2021-08-27 | 2021-08-27 | Immunohistochemical quality control product constructed based on animal organ or tissue |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110995173.1A CN113720669A (en) | 2021-08-27 | 2021-08-27 | Immunohistochemical quality control product constructed based on animal organ or tissue |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113720669A true CN113720669A (en) | 2021-11-30 |
Family
ID=78678479
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110995173.1A Pending CN113720669A (en) | 2021-08-27 | 2021-08-27 | Immunohistochemical quality control product constructed based on animal organ or tissue |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113720669A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115165511A (en) * | 2022-09-09 | 2022-10-11 | 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 (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002082026A (en) * | 2000-06-19 | 2002-03-22 | Chugai Pharmaceut Co Ltd | Method of producing sample |
| CN1439878A (en) * | 2003-01-17 | 2003-09-03 | 陕西超英生物医学研究开发有限公司 | Autoantibody detection tissue chip |
| KR20090011639A (en) * | 2007-07-27 | 2009-02-02 | 가톨릭대학교 산학협력단 | Slide for immunohistochemical staining and a method for manufacturing thereof |
| JP2010160018A (en) * | 2009-01-07 | 2010-07-22 | Kazuhisa Hasui | Cell analyzing method using immune tissue staining |
| CN104655475A (en) * | 2015-02-06 | 2015-05-27 | 丁伟 | Preparation method and application of paraffin embedding contrast suspension |
| CN107389417A (en) * | 2017-03-30 | 2017-11-24 | 贵州省人民医院 | A kind of detection method of hAECs DED organization engineering skins Proliferation, Differentiation vigor |
| CN207036856U (en) * | 2017-08-11 | 2018-02-23 | 湖北省肿瘤医院 | SABC positive control wax stone model |
| CN108106910A (en) * | 2017-12-18 | 2018-06-01 | 济南大学 | It is a kind of to detect antibody class drug and human body and the method for animal normal structure cross reaction |
| CN112198036A (en) * | 2020-09-16 | 2021-01-08 | 佛山科学技术学院 | Improved immunohistochemical staining method for paraffin section of pig brain tissue |
-
2021
- 2021-08-27 CN CN202110995173.1A patent/CN113720669A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002082026A (en) * | 2000-06-19 | 2002-03-22 | Chugai Pharmaceut Co Ltd | Method of producing sample |
| CN1439878A (en) * | 2003-01-17 | 2003-09-03 | 陕西超英生物医学研究开发有限公司 | Autoantibody detection tissue chip |
| KR20090011639A (en) * | 2007-07-27 | 2009-02-02 | 가톨릭대학교 산학협력단 | Slide for immunohistochemical staining and a method for manufacturing thereof |
| JP2010160018A (en) * | 2009-01-07 | 2010-07-22 | Kazuhisa Hasui | Cell analyzing method using immune tissue staining |
| CN104655475A (en) * | 2015-02-06 | 2015-05-27 | 丁伟 | Preparation method and application of paraffin embedding contrast suspension |
| CN107389417A (en) * | 2017-03-30 | 2017-11-24 | 贵州省人民医院 | A kind of detection method of hAECs DED organization engineering skins Proliferation, Differentiation vigor |
| CN207036856U (en) * | 2017-08-11 | 2018-02-23 | 湖北省肿瘤医院 | SABC positive control wax stone model |
| CN108106910A (en) * | 2017-12-18 | 2018-06-01 | 济南大学 | It is a kind of to detect antibody class drug and human body and the method for animal normal structure cross reaction |
| CN112198036A (en) * | 2020-09-16 | 2021-01-08 | 佛山科学技术学院 | Improved immunohistochemical staining method for paraffin section of pig brain tissue |
Non-Patent Citations (5)
| Title |
|---|
| ALBL ET AL.: "Tissue Sampling Guides for Porcine Biomedical Models", ORIGINAL ARTICLE, 16 February 2016 (2016-02-16) * |
| 丁会珍;刘丹丹;金贻铎;: "免疫组织化学检测阳性对照的应用现状与进展", 诊断病理学杂志, no. 07, 28 July 2020 (2020-07-28) * |
| 刘海霞;: "免疫组化病理技术质量控制问题分析与对策", 山西大同大学学报(自然科学版), no. 04, 28 August 2016 (2016-08-28) * |
| 王雪亮;肖艳群;王华梁;: "临床分子病理检测质控品的研究进展", 中华临床实验室管理电子杂志, no. 02, 28 May 2018 (2018-05-28) * |
| 高丽丽;刘春样;朱长乐;王景美;章宜芬;: "多组织羊膜卷蜡块在免疫组化染色阳性对照中的应用", 临床与实验病理学杂志, no. 09, 25 September 2018 (2018-09-25) * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115165511A (en) * | 2022-09-09 | 2022-10-11 | 迈杰转化医学研究(苏州)有限公司 | PD-L1 immunohistochemical detection quality control product and reference product |
| 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 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103347574B (en) | The cellular array quality control apparatus of pathology morphological analysis | |
| EP3514543B1 (en) | Quantitative controls and calibrators for cellular analytes | |
| CN103116029B (en) | Determining method for sensitivity and affinity of second antibody color appearance system for immunohistochemistry | |
| CN113720669A (en) | Immunohistochemical quality control product constructed based on animal organ or tissue | |
| CN113281516A (en) | Application of CUL9 as marker in colorectal cancer prognosis evaluation | |
| CN105606823A (en) | Detection method for PR genes of circulating tumor cells in peripheral blood of later-period breast cancer patient | |
| CN111207987A (en) | Immunofluorescence staining method for paraffin-embedded three-dimensional suspension cell mass | |
| CN110376036A (en) | A kind of preparation method and tumor cell line immunohistochemistry reference substance of tumor cell line immunohistochemistry reference substance | |
| CN116593262B (en) | Molecular marker detection product based on PDX/PDTX tumor living tissue biological sample and database and preparation method thereof | |
| EP2313754A1 (en) | Method for preparing cell standard | |
| WO2016123868A1 (en) | Method for preparing liquid-state dropping or coating pathological quality control product, and uses thereof | |
| CN113466458A (en) | Application of GPX4, NOX1 and ACSL4 in colorectal cancer prognosis evaluation | |
| CN112798322A (en) | 3D cell microsphere paraffin section method | |
| Zhang et al. | An efficient and user‐friendly method for cytohistological analysis of organoids | |
| CN108680424A (en) | It is multigroup to knit paraffin mass and preparation method thereof | |
| CN105784983A (en) | Kit for assessment of endometrial receptivity and use method thereof | |
| CN115508168A (en) | Preparation method and application of immunohistochemical quality control product | |
| CN109490056A (en) | A kind of cured embedding method of three-dimensional cell cultivation matrigel | |
| CN116773790B (en) | Preparation method and application of tumor tissue HER2 gradient detection product | |
| US20240167923A1 (en) | Bioengineered and standardized Human Tissue Reference Controls for Validation of IHC, FISH or CISH Cancer Test Results | |
| CA2607533A1 (en) | Xenograft tissue control for histology | |
| CN116425873B (en) | anti-CK 6 recombinant rabbit monoclonal antibody and application thereof | |
| CN112816295A (en) | Bone marrow tissue decalcification liquid and method for preparing bone marrow tissue paraffin section | |
| WO2016146616A1 (en) | Control slides for immunohistochemistry generated from three-dimensional cell line cultures | |
| CN108474025A (en) | Using more Phenotype typings of fluorescent quenching successively and the biological sample dyed again |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |