CN116593262B - Molecular marker detection product based on PDX/PDTX tumor living tissue biological sample and database and preparation method thereof - Google Patents
Molecular marker detection product based on PDX/PDTX tumor living tissue biological sample and database and preparation method thereof Download PDFInfo
- Publication number
- CN116593262B CN116593262B CN202310861191.XA CN202310861191A CN116593262B CN 116593262 B CN116593262 B CN 116593262B CN 202310861191 A CN202310861191 A CN 202310861191A CN 116593262 B CN116593262 B CN 116593262B
- Authority
- CN
- China
- Prior art keywords
- tissue
- tumor
- pdtx
- pdx
- molecular marker
- 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.)
- Active
Links
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 113
- 239000003147 molecular marker Substances 0.000 title claims abstract description 54
- 238000001514 detection method Methods 0.000 title claims abstract description 53
- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 239000012472 biological sample Substances 0.000 title claims abstract description 13
- 239000000523 sample Substances 0.000 claims abstract description 70
- 238000001574 biopsy Methods 0.000 claims abstract description 40
- 238000000034 method Methods 0.000 claims abstract description 24
- 238000012216 screening Methods 0.000 claims abstract description 13
- 238000003908 quality control method Methods 0.000 claims abstract description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 118
- 239000001993 wax Substances 0.000 claims description 57
- 239000012188 paraffin wax Substances 0.000 claims description 35
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 31
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 29
- 238000011532 immunohistochemical staining Methods 0.000 claims description 21
- 238000004043 dyeing Methods 0.000 claims description 20
- 210000004881 tumor cell Anatomy 0.000 claims description 19
- 238000002509 fluorescent in situ hybridization Methods 0.000 claims description 18
- 239000011521 glass Substances 0.000 claims description 16
- 230000001575 pathological effect Effects 0.000 claims description 14
- 238000003892 spreading Methods 0.000 claims description 11
- 230000007480 spreading Effects 0.000 claims description 11
- 238000009966 trimming Methods 0.000 claims description 10
- 239000008096 xylene Substances 0.000 claims description 10
- 241000699670 Mus sp. Species 0.000 claims description 9
- 230000003321 amplification Effects 0.000 claims description 9
- 230000018044 dehydration Effects 0.000 claims description 9
- 238000006297 dehydration reaction Methods 0.000 claims description 9
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 9
- 238000013102 re-test Methods 0.000 claims description 9
- 230000017074 necrotic cell death Effects 0.000 claims description 8
- 238000005520 cutting process Methods 0.000 claims description 7
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 230000007935 neutral effect Effects 0.000 claims description 6
- 229910001220 stainless steel Inorganic materials 0.000 claims description 6
- 239000010935 stainless steel Substances 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 5
- 230000012010 growth Effects 0.000 claims description 5
- 238000010008 shearing Methods 0.000 claims description 5
- 238000003559 RNA-seq method Methods 0.000 claims description 4
- 238000009826 distribution Methods 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 238000005070 sampling Methods 0.000 claims description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 3
- 241001465754 Metazoa Species 0.000 claims description 3
- 239000008098 formaldehyde solution Substances 0.000 claims description 3
- 238000007654 immersion Methods 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 238000004018 waxing Methods 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 210000001519 tissue Anatomy 0.000 description 147
- 239000000047 product Substances 0.000 description 36
- 235000019441 ethanol Nutrition 0.000 description 31
- 238000010186 staining Methods 0.000 description 22
- 210000004027 cell Anatomy 0.000 description 15
- 239000000243 solution Substances 0.000 description 13
- 239000008367 deionised water Substances 0.000 description 12
- 229910021641 deionized water Inorganic materials 0.000 description 12
- 230000008569 process Effects 0.000 description 11
- 239000003153 chemical reaction reagent Substances 0.000 description 10
- 230000002055 immunohistochemical effect Effects 0.000 description 9
- 239000000872 buffer Substances 0.000 description 8
- 238000011282 treatment Methods 0.000 description 8
- 108010067770 Endopeptidase K Proteins 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 210000002536 stromal cell Anatomy 0.000 description 7
- 239000013068 control sample Substances 0.000 description 6
- 238000009396 hybridization Methods 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 210000000170 cell membrane Anatomy 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 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 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000003517 fume Substances 0.000 description 4
- 238000007901 in situ hybridization Methods 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 210000003855 cell nucleus Anatomy 0.000 description 3
- 239000006059 cover glass Substances 0.000 description 3
- 230000007170 pathology Effects 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000036571 hydration Effects 0.000 description 2
- 238000006703 hydration reaction Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 230000001338 necrotic effect Effects 0.000 description 2
- 239000013610 patient sample Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000007306 turnover Effects 0.000 description 2
- 235000012431 wafers Nutrition 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- 101100314454 Caenorhabditis elegans tra-1 gene Proteins 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 238000000116 DAPI staining Methods 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000003416 augmentation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000005243 fluidization Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 238000011221 initial treatment Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000004882 non-tumor cell Anatomy 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000010827 pathological analysis Methods 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 238000012123 point-of-care testing Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 239000012128 staining reagent Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 150000003738 xylenes Chemical class 0.000 description 1
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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
- Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The method for preparing the molecular marker detection product based on PDX/PDTX tumor biopsy biological samples and a database comprises the steps of (1) screening PDX/PDTX tumor biopsy samples; (2) PDX/PDTX tumor biopsy sample detection; (3) PDX/PDTX modeling resuscitation and expansion of tumor tissue; (4) retesting PDX/PDTX tumor biopsy samples; (5) preparation of molecular marker detection products. The kit/quality control sheet can assist in accurately judging and reading the molecular marker expression result, reduce subjective interpretation errors among different hospitals and doctors, and further improve the specificity and accuracy of molecular marker identification and judgment.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a molecular marker detection product based on a PDX/PDTX tumor living tissue biological sample and a database and a preparation method thereof.
Background
With the rapid development of basic research and clinical medicine, more and more molecular markers involved in tumorigenesis, progression and influencing prognosis are successively discovered, and diagnostic molecular markers can be used to detect or confirm disease states, or to identify molecular markers of different disease subtypes. The enrichment research design of the predictive molecular marker can accurately screen out patient groups potentially benefiting to develop clinical tests.
Protein receptor expression is one of molecular markers, and an Immunohistochemical (IHC) method is a common method for detecting protein expression, which uses a specific antibody to bind to a target protein, and then detects the expression level of the protein by staining or fluorescence or the like. IHC plays a very important role in pathological diagnosis, but the immunohistochemical staining process is complex, and factors influencing the staining result are also many, so that the setting of the immunohistochemical control is particularly important. Archiving and diagnosing human tissues (pathological wax) and diagnosing residual human tissues are the most widely used control tissue sources in clinic at present, but most tissue treatment conditions are inconsistent, the pathological wax is patient data which must be stored in hospitals, the control is very limited, and providing enough suitable control tissues is one of the difficulties in meeting the current clinical application demands. CNAS-GL048 of China: 2021 approved guidelines for the field of histopathological examination in medical laboratories, 5.6.2.2, specifies that immunohistochemical staining or fluorescent staining, and specific staining should be followed by appropriate positive and negative controls (external controls are preferred). Researchers have been working on finding new control materials, including cell lines, transplanted tumor tissue, and the like.
The flow control of the molecular marker detection technology for liquid samples at home and abroad is basically mature, and the molecular marker detection technology comprises a chemical reaction mechanism, sample comparison setting, result interpretation standards and the like. The detection method for the molecular marker in clinic mainly depends on products such as a gene detection (PCR quantitative) kit, an enzyme-linked immunosorbent assay (ELISA) kit, a point of care (POCT) kit and the like, and the requirements of the products on a sample to be detected in the detection flow are liquid (such as blood, urine, tissue fluid and the like) samples or the sample to be detected is subjected to fluidization (cell tissue disruption and separation) treatment. The detection method is not suitable for direct detection of solid tissues, cannot truly show the distribution condition of molecular markers in tissue structures, lacks technical standards related to solid tissue detection, cannot perform quality control record on a sample dyeing process, and cannot screen out the reasons of abnormal detection results.
The existing molecular marker substance control chip mostly adopts a cell line as a control sample, mainly comprises cell clusters with different expression levels of molecular markers, but the control sample has unstable biological characteristic consistency, limited sample sources, and is generally difficult to trace the sample sources and realize sustainable supply. The control sample from the cell line is only tumor cells and non-tumor tissues, so that the microenvironment of the tumor tissues is absent, and the true tissue structure cannot be represented. More importantly, the samples to be tested for clinical immunohistochemical staining are tumor tissues, not tumor cells, and the non-specific staining cannot be distinguished by using a control derived from a cell line. The control sample and the sample to be detected of the existing molecular marker are respectively attached to the surfaces of different slides, the immunohistochemical staining conditions and the process are asynchronous, the slide and the microscope need to be replaced for refocusing in the contrast reading process, the operation is complicated, the staining result is read subjectively by a pathologist, the judgment scale of the immunohistochemical staining intensity of different pathologists exists, the subjective heterogeneity exists, if the immunohistochemical staining intensity caused by experimental system errors is increased or decreased, and the clinical pathologist is not identified from the reading.
Disclosure of Invention
The technical problems to be solved are as follows: aiming at the problems that the prior molecular marker substance control chip adopts a cell line as a control sample to cause unstable consistency of biological characteristics of the control sample and limited sample sources, and is difficult to trace back the sample sources and realize sustainable supply, the invention provides a molecular marker detection product based on a PDX/PDTX tumor living tissue biological sample and a database and a preparation method thereof.
The technical scheme is as follows: the preparation method of the molecular marker detection product based on the PDX/PDTX tumor living tissue biological sample and the database comprises the following steps: (1) PDX/PDTX tumor biopsy sample screening:
1.1 PDX/PDTX tumor biopsy sample primary screening: setting positive samples and negative samples according to the CNV value and the RNA-seq TPM value corresponding to the molecular marker expression, and then grouping, wherein the positive samples are as follows: CNV is more than or equal to 10 and TPM is more than or equal to 50, and the negative samples are: CNV is less than or equal to 4 and TPM is less than or equal to 10; performing sample preliminary screening through a sample CNV value and an RNA-seqTPM value stored in a PDX/PDTX tumor biopsy database, screening PDX/PDTX tumor biopsy samples meeting positive or negative requirements, and numbering;
1.2 PDX/PDTX swellingTumor biopsy sample library selection: according to the number and grouping condition of the screened samples, at least 5 samples of each group are distributed, samples are extracted from a PDX/PDTX tumor living tissue biological sample library, and the sampling volume of each sample is not less than 8mm 3 ；
(2) PDX/PDTX tumor biopsy sample detection:
2.1 PDX/PDTX tumor biopsied wax block preparation: performing tissue fixation and wax block preparation on PDX/PDTX tumor living tissue stored in the sample library obtained in the step 1.2;
2.2 And (3) pathological detection: performing HE staining on the prepared PDX/PDTX tumor biopsy wax block, removing samples with the tumor cell proportion less than 30% and the necrosis proportion more than 20%, performing IHC staining, and observing whether the expression level of the molecular marker of the PDX/PDTX tumor biopsy sample meets the 1.1 grouping requirement under a microscope;
2.3 FISH detection determines the optimal target sample: performing FISH detection on PDX/PDTX tumor living tissue wax blocks which pass through pathological detection and meet the 1.1 grouping requirement, and determining an optimal target sample according to the FISH information characteristics corresponding to the molecular marker expression;
(3) Modeling resuscitation and expansion of tumor tissue PDX/PDTX: carrying out modeling resuscitation and amplification on tumor tissues on the target sample determined in the step 2.3;
(4) PDX/PDTX tumor biopsy samples retest:
4.1 PDX/PDTX tumor biopsied wax block preparation: preparing the revived and amplified PDX/PDTX tumor living tissue into wax blocks;
4.2 Pathological retesting: HE staining is carried out on the prepared PDX/PDTX tumor living tissue wax block, samples with the tumor cell proportion more than or equal to 30% and the necrosis proportion less than or equal to 20% are screened out, IHC staining is carried out, and whether the expression level of the molecular marker of the PDX/PDTX tumor living tissue sample meets the grouping requirement is observed under a microscope;
4.3 FISH retest: performing FISH retest on PDX/PDTX tumor living tissue wax blocks which pass through pathological detection and meet the requirements, and determining an optimal target sample according to FISH information characteristic retest corresponding to molecular marker expression;
(5) Preparation of molecular marker detection products: and taking out a qualified tissue chip by using a tissue chip sampler according to the IHC staining effect under a microscope by using an optimal target sample determined by retesting, arranging and combining the qualified tissue chip on the same wax block, and slicing the wax block to prepare a molecular marker detection finished product.
The specific steps of the tissue fixation and wax block preparation are as follows: (1) tissue fixation: adding PDX/PDTX tumor living tissue into 10 times volume of 10% neutral formaldehyde solution, and fixing at room temperature for 12-24h; (2) tissue material selection: cutting the fixed tissue into tissue blocks with the thickness less than or equal to 1.5cm multiplied by 0.5cm, placing the tissue blocks into an embedding box and placing the embedding box into a tissue dehydration basket; (3) tissue dehydration: sequentially immersing the tissues into ethanol with different concentration gradients to dehydrate the tissues: 70% ethanol 1h, 80% ethanol 2h, 85% ethanol 70min, 95% ethanol 1h, 95% ethanol 50min, absolute ethanol 1h, absolute ethanol 50min; (4) tissue transparency: sequentially immersing the tissue subjected to gradient ethanol dehydration into xylene for transparency: 30min of dimethylbenzene and 25min of dimethylbenzene; (5) tissue waxing: immersing the tissue subjected to tissue transparency into melted paraffin for tissue paraffin immersion: paraffin 1 h- & gt paraffin 1h; (6) tissue embedding: the tissue embedding is carried out in a tissue embedding machine, melted paraffin is dripped into a stainless steel embedding mould, after the paraffin at the bottom is solidified, the tissue is placed at the bottom of the mould, the position of the tissue is adjusted to enable the tissue to be on the same plane, an embedding box is placed on the mould, the mould is continuously filled with paraffin liquid, the mould is transferred to a refrigerating table which is pre-cooled in advance for 30min, the paraffin block is taken down, and redundant paraffin is trimmed.
The specific method for resuscitation and amplification comprises the following steps: shearing the PDX/PDTX tumor living tissue meeting the requirements into 2X 2mm 3 Inoculating the strain into the back subcutaneous of NCG mice; after inoculation, the animal status was observed daily, tumor volume was measured 1 time a week and tumor-bearing mice were weighed and tumor volume V (mm) was calculated 3 ）= (a×b 2 ) 2, a is a tumor long diameter, b is a tumor short diameter; after the growth volume of the P0 generation transplanted tumor is accumulated to 800-2000 mm 3 Dissecting, taking out and shearing to randomly inoculate P1 generation to the back of 5 NCG mice for subcutaneously carrying out passage amplification; tumor growth accumulation for P1 generation transplantationThe product is up to 800-2000 mm 3 Samples were dissected out and paraffin embedded and section prepared as alternative control areas.
The specific preparation steps of the molecular marker detection product are as follows: (1) preparing a tissue chip: drawing a positive region or a negative region with uniform molecular marker expression under a microscope according to IHC dyeing results, respectively taking out PDX/PDTX tumor biopsy sample columns with the diameters of 1mm by utilizing a tissue chip sampler to contrast the drawn region, putting the PDX/PDTX tumor biopsy sample columns into a pre-cast wax block mold according to a specific sequence, and determining specific distribution of a contrast region sample in a tissue chip according to the characteristics of the molecular markers; placing the wax block with the tissue into a stainless steel bottom die with the section facing downwards, placing into a 70 ℃ oven for 30-60min, and taking out the wax block after the wax block is completely transparent; placing the tissue embedding box above the wax block, continuously injecting wax until the bottom of the box is completely immersed, and placing and cooling; taking out and then carrying out corner cutting marking; (2) tissue section: slicing the wax block on a slicing machine, and repairing the wax block firstly: in the trimming mode, the thickness of a trimming slice is adjusted to 15-20 mu m, after trimming wax blocks until all tissues are exposed, the thickness of the slice is adjusted to 3 mu m, and then the slice is carried out; (3) spreading: moving the slices to the water surface in the spreading machine by using tweezers to spread; (4) fishing out the slices: and sticking the unfolded slice on a glass slide, tilting the glass slide to 45 degrees to enable excessive water on the slice to flow down, and airing to obtain the finished product.
The molecular marker detection product prepared by the preparation method is prepared.
Such products include, but are not limited to, quality control wafers or kits.
The beneficial effects are that: the invention is based on PDX/PDTX tumor living tissue biological sample library and database, and utilizes molecular techniques such as gene copy number variation (Copy number variations, CNV) and transcriptome sequencing technology (RNA-seq), and pathological techniques such as HE staining, IHC staining, fluorescence in situ hybridization (Fluorescence in situ hybridization, FISH), and the like to prepare a standardized and stable and reliable quality molecular marker detection kit/quality control sheet which can be continuously supplied and takes PDX/PDTX tumor living tissue samples as a contrast, fills the blank of relevant fields at home and abroad, can assist in accurately judging the expression result of the molecular marker, reduces subjective interpretation errors between different hospitals and doctors, and can further improve the specificity and accuracy of molecular marker identification and judgment.
Drawings
FIG. 1 is a chart of the histomorphology consistency of PDX/PDTX tumor biopsies versus primary tumor tissue;
FIG. 2 is a molecular marker profile consistency control of PDX/PDTX tumor biopsies versus primary tumor tissue;
FIG. 3 is a schematic illustration of a technical route;
FIG. 4 is a flow chart of PDX/PDTX tumor biopsy modeling resuscitation and amplification;
fig. 5 is a diagram of sample 011 versus sample 012 detection;
FIG. 6 is a chart comparing the staining pattern of the product with other brands;
fig. 7 is a schematic diagram of a product chip size study, 1, 3mm diameter, magnification 50×: poor uniformity, much necrosis and difficult control. 2, diameter 2mm magnification 50×inthe figure: the necrosis is more and is not easy to control; 3, diameter 1mm magnification 100×inthe figure: relatively uniform.
Detailed Description
The invention is described in detail below by way of examples, which are given in detail embodiments and specific operating steps, for further illustration of the invention, and are not to be construed as limiting the scope of the invention.
PDX/PDTX tumor living tissue biological sample library and construction of database
1. Criteria for inclusion
Fresh tissue (including tumor tissue and paracancerous tissue) and blood samples taken in a reasonable manner, and the preservation amount standard of each sample is respectively as follows: tumor tissue samples are not smaller than 1cm at a time 3 The method comprises the steps of carrying out a first treatment on the surface of the The tissue sample beside cancer is not smaller than 1cm each time 3 The method comprises the steps of carrying out a first treatment on the surface of the The blood sample was 3 mL/tube.
2. Exclusion criteria
Incorrect sampling patterns lead to necrotic or contaminated tissue and blood samples.
3. Reject criteria
Tissue and blood samples that are incorrectly stored or handled resulting in necrosis or contamination.
4. Preservation conditions
After receiving the tissue samples, the primary treatment is carried out under aseptic condition, the obtained primary fresh tumor tissue is amplified into tumor living tissue meeting the quality control requirement by utilizing the PDTX technology, and the tumor living tissue is stored at low temperature (-80 ℃/-196 ℃) and is subjected to subculture and paraffin embedding treatment (room temperature storage).
Example 1
Step 1: PDX/PDTX tumor biopsy sample prescreening
According to the actual condition of clinical pathology detection of the molecular marker A, the control samples are divided into positive and negative groups, wherein the requirements of the positive samples are as follows: CNV is more than or equal to 10 and TPM is more than or equal to 50, and the requirements of negative samples are as follows: CNV is less than or equal to 4 and TPM is less than or equal to 10. According to the requirements, sample preliminary screening is carried out through sample CNV information and RNAseq-TPM information stored in a PDX/PDTX tumor living tissue database, 5 positive samples and 5 negative samples (see table 1 in detail) of the PDX/PDTX tumor living tissue meeting the requirements simultaneously are screened out, and then the operation is carried out according to the steps 2-5 respectively.
Samples between the samples CNV 4-10 and the TPM 10-50 are in gray area, so that the samples are abandoned.
The results of sample 011 and sample 012 are shown in table 2 and fig. 5:
step 2: tissue fixation and wax block preparation
(1) Tissue fixation: PDX/PDTX tumor biopsies were added to 10 volumes of 10% neutral formaldehyde solution and fixed at room temperature for 12-24h.
(2) Tissue material selection: cutting the fixed tissue into tissue blocks smaller than or equal to 1.5cm multiplied by 0.5cm, placing the tissue blocks into an embedding box with printed labels, and placing the embedding box into a tissue dehydration basket.
(3) Tissue dehydration: sequentially immersing the tissues into ethanol with different concentration gradients to dehydrate the tissues: 70% ethanol 1h, 80% ethanol 2h, 85% ethanol 70min, 95% ethanol 1h, 95% ethanol 50min, absolute ethanol 1h, absolute ethanol 50min.
(4) Tissue transparency: sequentially immersing the tissue subjected to gradient ethanol dehydration into xylene for transparency: 30min of xylene I to 25min of xylene II.
(5) Tissue waxing: immersing the tissue subjected to tissue transparency into melted paraffin for tissue paraffin immersion: paraffin I1h → paraffin II1h → paraffin III1h.
(6) Tissue embedding: tissue embedding is performed in a tissue embedding machine. Dropping a little melted paraffin into a preheated stainless steel embedding mould, placing the tissue at the bottom of the mould after the paraffin at the bottom is slightly solidified, adjusting the position of the tissue, slightly pressing the tissue to enable the tissue to be on the same plane, placing the embedding box on the mould, continuously filling with paraffin liquid, transferring to a pre-cooled freezing table, cooling for 30min, taking down the paraffin block, and repairing redundant paraffin.
Step 3: HE staining
(1) And (5) placing the wax block into a clamping groove of a slicing machine to slice. The 3 μm white slices were cut out and moved to a 45 ℃ water bath until the slices were fully spread.
(2) After deployment, the tissue was adhered to a glass slide.
(3) The slices can be baked after slightly drying, and the slices are baked for 15-30min by a slice baking machine at the temperature of about 62 ℃, so that the slice baking time can be prolonged by special samples.
(4) And after the baking sheet is finished, putting the baked sheet into a Leica ST5010 automatic dyeing machine for dyeing.
(5) The reagent sequence and staining time for the Leica ST5010 autostainer were as follows:
(6) The stained sections were transferred to a Leica CV5030 sealer for sealing.
Step 4: immunohistochemical (IHC) staining
(1) Tissue section: the wax block is placed on a cold table for 30min, and then is placed on a slicing machine for slicing. Firstly, repairing the wax block: in the trimming mode, the thickness of the trimming slice can be adjusted to 10 mu m, after the wax block is trimmed until the tissue is completely exposed, the thickness of the slice is adjusted to be about 3 mu m, and then the slice is cut, so that the slice is required to be complete and uniform, and has no knife mark, no fold and no crack.
(2) Spreading: the slices are gently picked up by a brush pen, moved into the water surface in the tablet spreading machine by tweezers and spread (the temperature is 45 ℃ and the slices can rotate but cannot turn over in the process of transferring the slices to the water surface of the tablet spreading machine).
(3) Fishing: the unfolded slice is attached to a glass slide, and the glass slide sanding area is held by the right hand during the glass slide dragging, so that bubbles are prevented from being generated between the slice and the glass slide as much as possible. Tilting the slide to 45 deg. allowed excess water flow over the slice.
(4) Baking slices: the slide glass is placed on a slide baking machine and baked at 62 ℃ for 60min to prepare the tissue paraffin section.
(5) Dewaxing: after the baking sheet is finished, taking out the paraffin section, and rapidly transferring the paraffin section into dimethylbenzene, wherein the dimethylbenzene is from 5min to 5min of dimethylbenzene II to 5min of dimethylbenzene III.
(6) Hydration: taking out slices from dimethylbenzene, draining, transferring into ethanol with different concentration gradients for hydration, and washing with water after the completion of the steps of absolute ethanol I for 5min, absolute ethanol II for 5min, 95% ethanol for 5min and 75% ethanol for 5min.
(7) Antigen retrieval: adding proper amount of antigen retrieval liquid into a staining jar for thermal retrieval, boiling for 10min, naturally cooling, and washing with PBS buffer solution for 3 times and 3min each time.
(8) Closing: the sections were thrown clean of excess PBS buffer, endogenous peroxidase blocking reagent was added, 100. Mu.L was added dropwise to the tissue, incubated for 10min at 37℃and washed 3 times with PBS buffer for 3min each.
(9) Incubation resistance: the sections were thrown clean of excess PBS buffer, 100. Mu.L of diluted molecular marker A antibody was added, incubated for 1h at 37℃and washed 3 times with PBS buffer for 3min each.
(10) Secondary antibody incubation: the sections were thrown clean of excess PBS buffer, 100. Mu.L of secondary antibody was added and incubated at room temperature for 20min, and the PBS buffer was washed 3 times for 3min each.
(11) DAB color development: the sections were thrown clean of excess PBS buffer, incubated with 100. Mu. LDAB cocktail (solution A: solution B=1:1) for 5-8min, and washed with water to terminate color development.
(12) Counterstaining and checking: the slices are dried to remove redundant liquid, 100 mu L of hematoxylin dye solution is added, the dyeing time can be adjusted according to the actual process for 20-60s, and the slices are washed for 5min by running water.
(13) Differentiation (whether or not needed according to hematoxylin property selection): the sections were immersed in the differentiation solution for 2-5s, and the sections were rapidly removed and washed with tap water for 5min.
(14) Dehydrating and transparentizing: the water from which the sections were rinsed was spin-dried, gradient ethanol dehydrated, 75% ethanol in the following order: 5 min- > 95% ethanol: 5 min- & gt absolute ethanol: 5min, sections taken from absolute ethanol were put into xylene I:5min→xylene II: and (3) carrying out transparent treatment on the mixture for 5min and dimethylbenzene III for 5min. (the process is performed in a fume hood)
(15) And (3) sealing the neutral resin sheet: the sections were removed and placed in a fume hood, neutral gum was added dropwise before the xylene volatilized, and covered with a cover slip. (the process is performed in a fume hood)
Step 5: fluorescence In Situ Hybridization (FISH)
The step adopts a gene amplification detection kit (fluorescence in situ hybridization method) to carry out relevant detection.
1. Preparation of related reagent
(1) 20 XSSC (sodium citrate buffer), pH 5.3.+ -. 0
The above reagents were dissolved with 800mL of deionized water, the pH was adjusted to 5.3.+ -. 0.2 at room temperature, and the volume was fixed to 1L with deionized water. Sterilizing with high pressure steam, preserving at 2-8deg.C, and keeping for 6 months, and if the reagent is turbid or contaminated, it can not be used.
(2) 2 XSSC, pH 7.0.+ -. 0.2
100mL of the 20 XSSC is taken, diluted and uniformly mixed with 800mL of deionized water, the pH value is regulated to 7.0+/-0.2 at room temperature, the deionized water is used for constant volume to 1L, the preservation is carried out at 2-8 ℃, the shelf life is 6 months, and the reagent cannot be used if turbidity or pollution occurs.
(3) Ethanol solution: 70% ethanol, 85% ethanol
700mL and 850mL of absolute ethyl alcohol are respectively diluted to 1L by deionized water, the shelf life is 6 months, and the reagent cannot be used if turbidity or pollution occurs.
(4) Proteinase K
Proteinase K stock (20 mg/mL): 0.1g of proteinase K dry powder was weighed and dissolved in 5ml of 2 XSSC (pH 7.0) solution and gently shaken until completely dissolved, stored at-20℃and shelf-life for 6 months.
Proteinase K working solution (200. Mu.g/mL): 0.8mL of proteinase K stock solution was dissolved in 80mL of 2 XSSC (pH 7.0) solution and mixed well for ready use.
(5) 0.3% NP-40/0.4 XSSC solution, pH 7.0-7.5
150mL of deionized water is added, the mixture is uniformly mixed, the pH value is regulated to 7.0-7.5 at room temperature, deionized water is used for fixing the volume to 200mL, the mixture is preserved at 2-8 ℃, the shelf life is 6 months, and the mixture cannot be used if the reagent is turbid or polluted.
(6) 0.1% NP-40/2 XSSC solution, pH 7.0.+ -. 0.2
150mL of deionized water is added, the mixture is uniformly mixed, the pH value is regulated to 7.0+/-0.2 at room temperature, deionized water is used for constant volume to 200mL, the mixture is preserved at 2-8 ℃, the shelf life is 6 months, and the mixture cannot be used if the reagent is turbid or polluted.
(7) Diamidinophenyl indole (DAPI) counterstain, commercial DAPI counterstain with anti-quencher was chosen.
(8) Dewaxing and other steps are used, and commercial xylenes are selected.
2. Hybridization pretreatment
It is recommended to select specimen pieces positive and negative for amplification of known molecular marker genes as external controls.
(1) Slicing and manufacturing: the neutral formalin-fixed paraffin-embedded tissue sections were placed on a clean slide.
(2) Baking slices: the tissue sections were placed on a slice roaster overnight at 65℃for roasting and aging the sections.
(3) Dewaxing: tissue sections were immersed in a xylene-containing dye vat for 10min for dewaxing, repeated once, and immediately immersed in 100% ethanol for 5min for washing.
(4) And (3) rehydrating: at room temperature, placing the tissue slices in 100% ethanol, 85% ethanol and 70% ethanol for 2 min each, immersing in deionized water for 3min, taking out the slices, and sucking off excessive water around the tissues with a non-velvet tissue.
(5) And (3) water treatment: and immersing the tissue slices in deionized water for 30-40 min at the temperature of 95 ℃ in a water bath (the deionized water is preheated in the water bath).
(6) Washing: the tissue sections were rinsed twice in 2 XSSC solution for 5min at room temperature.
(7) Proteinase K treatment: and immersing the tissue slices in proteinase K working solution, and treating at 37 ℃ for 5-30 min.
(8) Washing: the tissue sections were rinsed twice in 2 XSSC solution for 5min at room temperature.
(9) Dehydrating: the tissue slices are sequentially placed in 70 percent ethanol, 85 percent ethanol and 100 percent ethanol for 2 minutes each, taken out and naturally dried.
3. Denaturing hybridization
The following operations are performed in a dark room.
(1) Taking out the bicolor probe, uniformly mixing, centrifuging briefly, taking 10 mu L of the bicolor probe, dripping the bicolor probe on a tissue slice hybridization area, immediately covering a cover glass with the thickness of 22mm multiplied by 22mm, uniformly spreading the probe under the cover glass, and sealing the edge by rubber glue.
(2) The sections were placed on a hybridization apparatus, co-denatured at 83℃for 5min (hybridization apparatus should be preheated to 83℃in advance), and hybridized at 42℃for 2-16 h.
4. Washing
The following operations are performed in a dark room.
(1) Taking out the hybridized slice, removing the cover glass, immediately placing the slice in a 67 ℃ 0.3% NP-40/0.4 XSSC solution, oscillating for 1-3 s, and soaking for 1-2 min.
(2) At room temperature, placing the slices into 0.1% NP-40/2 XSSC solution, oscillating for 1-3 s, and soaking for 1-2 min.
(3) placing the slices in 70% ethanol at room temperature, and soaking for 1-3 min; the sections were dried naturally in the dark.
5. Counterstain
The following operations are performed in a dark room.
And (3) dripping 10-15 mu L of DAPI compound agent in the hybridization area, immediately covering the dark place of the slice, standing for 10-20 min, and then selecting a proper optical filter under a fluorescence microscope to observe the slice.
Fish results observations
The section after counterstaining is placed under a fluorescence microscope, and the cell area in the PDX/PDTX tumor living tissue is confirmed under a low power objective lens (10X); turning to a position under a 40 multiplied by objective lens, and finding a position where cells are uniformly distributed; cells with consistent cell nucleus size, complete nucleus boundary, uniform DAPI staining, no overlapping cell nuclei and clear amplified signal of the molecular marker A gene should be selected under a high power objective lens (60X and 100X), at least 20 tumor cells are randomly selected, and color signals in the cell nuclei are counted.
Step 6: PDX/PDTX tumor biopsy sample pathology detection screening
In order to ensure the detection effectiveness of the molecular marker A of the PDX/PDTX tumor biopsy sample in the control area, the appropriate PDX/PDTX tumor biopsy sample is screened out according to the biological characteristics of the molecular marker A and the actual conditions of HE staining, IHC staining and FISH detection (see Table 7 for details), and then resuscitated and amplified according to the step 7.
Step 7: PDX/PDTX modeling resuscitation and augmentation
(1) Shearing the PDX/PDTX tumor living tissue meeting the requirements after screening in the step 2-5 into 2X 2mm 3 Inoculated subcutaneously into the back of NCG mice.
(2) After inoculation, the animal status was observed daily, tumor volume was measured 1 time a week and tumor-bearing mice were weighed and tumor volume V (mm) was calculated 3 ）= (a×b 2 ) And 2, a is the long diameter of the tumor, and b is the short diameter of the tumor.
(3) After the growth volume of the P0 generation transplanted tumor is accumulated to 800-2000 mm 3 The back of 5 NCG mice inoculated randomly (P1 generation) was dissected out and sheared for subcutane passaged expansion.
(4) After the growth volume of the P1 generation transplanted tumor is accumulated to 800-2000 mm 3 Samples were dissected out and paraffin embedded and section prepared as alternative control areas.
Step 8: pathological retest screening of PDX/PDTX tumor biopsy samples
In order to ensure the detection effectiveness of the molecular marker A of the PDX/PDTX tumor biopsy sample in the control area, pathological retesting is carried out on the tumor biopsy after PDX/PDTX modeling resuscitation and amplification, meanwhile, the optimal PDX/PDTX tumor biopsy sample is screened out according to the biological characteristics of the molecular marker A (see table 8 for details), and then the preparation of the molecular marker A detection kit/quality control sheet is carried out according to the step 9.
Step 9: preparation of molecular marker A detection kit/quality control wafer
(1) Preparing a tissue chip: and observing IHC staining results under a microscope, drawing a tissue region meeting requirements, taking out PDX/PDTX tumor biopsy sample columns with the diameters of 1mm and with positive expression and negative expression of a molecular marker A respectively by using a tissue chip sampler, putting the sample columns into a precast wax block, and determining the specific distribution of a control region sample in a tissue chip according to the characteristics of the molecular marker A. Placing the wax block with the structure into a stainless steel bottom die, and paying attention to the direction: cutting into sections, placing into oven at 70deg.C for 30-60min, and taking out when the wax block is completely transparent. The tissue embedding box is arranged above the wax block, and the wax is continuously injected until the bottom of the box is completely immersed, and the tissue embedding box is placed for cooling. And taking out and then carrying out corner cutting marking.
Product chip size study, namely embedding, slicing and staining tissues with diameters of 1mm, 2mm and 3mm respectively, wherein the staining results are shown in figure 7: conclusion: the diameter is 1mm, the tissue uniformity is good, and the necrosis proportion and the tumor cell content are easy to control.
a. The diameter is 1mm, the tissue area just occupies 100X of visual field, and the observation is easy;
b. the tissue area is small, and the consumed staining reagent is also small;
c. the tissue area is small, the uniformity is easy to control, and the necrotic area and the tumor tissue content are easy to control. The uniformity and quality of the product are improved.
d. The diameter is small, 2-4 tissue chips can be combined in a wax block grinding tool, and after slicing, a sufficient area is arranged on a glass slide to be attached to the tissue of a patient.
(2) Tissue section: the wax block is placed on a cold table for 30min, and then is placed on a slicing machine for slicing. Firstly, repairing the wax block: in the trimming mode, the thickness of the trimming slice can be adjusted to 10 mu m, after the wax block is trimmed until the tissue is completely exposed, the thickness of the slice is adjusted to be about 3 mu m, and then the slice is cut, so that the slice is required to be complete and uniform, and has no knife mark, no fold and no crack.
(3) Spreading: the slices are gently picked up by a brush pen, moved into the water surface in the tablet spreading machine by tweezers and spread (the temperature is 45 ℃ and the slices can rotate but cannot turn over in the process of transferring the slices to the water surface of the tablet spreading machine).
(4) Fishing: the unfolded slice is attached to a glass slide, and the glass slide sanding area is held by the right hand during the glass slide dragging, so that bubbles are prevented from being generated between the slice and the glass slide as much as possible. The slide glass is inclined to 45 degrees to enable redundant water flow on the slice to be downwards, and the slice is dried.
(5) Quality inspection: according to the sampling principle, the first, the middle and the last of each wax block serial slice are selected for immunohistochemical staining, and the slice staining result must meet the inspection requirement.
Step 10: clinical sample detection and pathological reading sheet
(1) Pretreatment: when the product is used, a customer cuts the wax blocks of a patient and sticks the wax blocks to the corresponding area of the product, and then roast and dewax the slices together;
(2) Dyeing: antigen retrieval and staining were performed according to immunohistochemical staining procedures after dewaxing.
(3) Sealing piece: after dyeing, the dyed sheet is sealed (in a fume hood).
(4) Reading: the pathologist reads the staining results. The product has a staining control area containing positive and negative controls. When a customer reads a film, the dyeing condition of the control area is checked, the positive control is positively developed, the negative control is not positively developed, and the dyeing process can be confirmed. If the positive control area does not show positive color development, false negative conditions may exist, and the reasons need to be searched, and the clinical sample dyeing negative result at the time is not credible. If the negative control area shows positive color development, false positive conditions may exist, and the reasons need to be searched, so that the positive dyeing result of the clinical sample is not credible.
(5) The product has the advantages that: the result of immunohistochemical staining is affected by antibody reagents, the staining ability of the inspector, and subjective judgment when the pathologist reads the film. The product can be used for monitoring the dyeing process and the quality of the dyed antibodies, and eliminating the problem of the dyeing quality of more than 90% before reading. Meanwhile, the product is screened by CNV, TPM, FISH, IHC, and the corresponding positive and negative expression levels and dyeing intensity are confirmed. According to the dyeing intensity, a pathologist can make more accurate dyeing intensity judgment on the patient section, the accuracy and consistency of the dyeing judgment can be improved, and the difference of reading among different doctors and different hospitals is greatly reduced. According to related research, the problem of insufficient consistency in the current clinical interpretation is solved, the interpretation difference between the dyeing scores 0 and 1+ is the largest, and the gradient dyeing product provided by the company can solve the problem and is improved by 20% on the basis of the existing interpretation consistency.
(6) Comparing similar products: the product is compared with the similar or analogous products on the market at present. IHC staining pattern is shown in FIG. 6.
Referring to Table 9, a single cell line smear with different staining gradients was used for a commercial product (HER-2 four-in-one pathology quality control patch). Tumor cells distributed in a scattered way are arranged on the slice, and the slice has no tissue structure and no stromal cells. The product parameters are IHC and FISH detection results.
The product adopts tissue chip embedding tablets with different staining gradients. The section is a tissue section, has a complete tissue structure, has tumor cells and stromal cells, and the positively expressed tumor cells show positive staining with different gradients, and the stromal cells are stained negative. Product parameters included CNV values for WES sequencing, TPM values for RNA-seq sequencing, FISH detection results (HER 2/CEP 17).
In the IHC staining chart (FIG. 6), both our product and some company product contained 4 staining levels: 0. 1+, 2+, 3+.
Our product is an organizational chip. Complete tissue structure, tumor cells, stromal cells, etc. can be seen after IHC staining. The positive staining sites were cell membranes. Cell membranes of tumor cells at level 0 are not stained; the cell membranes of tumor cells at the 1+, 2+, 3+ level are positively colored at different intensities.
A product of a certain company is a tumor cell strain. Only scattered cells can be seen after IHC staining, and no whole tissue structure, stromal cells and the like are present. Cell membranes of tumor cells at level 0 are not stained; the cell membranes of tumor cells at the 1+, 2+, 3+ level are positively colored at different intensities. Cell strain products cannot embody the structure of human tissues, only have tumor cells, and have no non-tumor cell contrast such as stromal cells (the cells are negative in staining), so that the cell strain products are unfavorable for comparison between clinicians and clinical patient samples. The clinical patient sample is an intact tissue and contains tumor cells, stromal cells, blood vessels and other components.
Claims (5)
1. The preparation method of the molecular marker detection product based on the PDX/PDTX tumor living tissue biological sample and the database is characterized by comprising the following steps: (1) PDX/PDTX tumor biopsy sample screening: 1.1 PDX/PDTX tumor biopsy sample primary screening: setting positive samples and negative samples according to the CNV value and the RNA-seq TPM value corresponding to the molecular marker expression, and then grouping, wherein the positive samples are as follows: CNV is more than or equal to 10 and TPM is more than or equal to 50, and the negative samples are: CNV is less than or equal to 4 and TPM is less than or equal to 10; performing sample preliminary screening through a sample CNV value and an RNA-seqTPM value stored in a PDX/PDTX tumor biopsy database, screening PDX/PDTX tumor biopsy samples meeting positive or negative requirements, and numbering; 1.2 PDX/PDTX tumor biopsy sample pool selection: according to the number and grouping condition of the screened samples, at least 5 samples of each group are distributed, samples are extracted from a PDX/PDTX tumor living tissue biological sample library, and the sampling volume of each sample is not less than 8mm 3 ；
(2) PDX/PDTX tumor biopsy sample detection: 2.1 PDX/PDTX tumor biopsied wax block preparation: performing tissue fixation and wax block preparation on PDX/PDTX tumor living tissue stored in the sample library obtained in the previous step; 2.2 And (3) pathological detection: performing HE staining on the prepared PDX/PDTX tumor biopsy wax block, removing samples with the tumor cell proportion less than 30% and the necrosis proportion more than 20%, performing IHC staining, and observing whether the expression level of the molecular marker of the PDX/PDTX tumor biopsy sample meets the 1.1 grouping requirement under a microscope; 2.3 FISH detection determines the optimal target sample: performing FISH detection on PDX/PDTX tumor living tissue wax blocks which pass through pathological detection and meet the 1.1 grouping requirement, and determining an optimal target sample according to the FISH information characteristics corresponding to the molecular marker expression;
(3) Modeling resuscitation and expansion of tumor tissue PDX/PDTX: carrying out modeling resuscitation and amplification on tumor tissues on the target sample determined in the step 2.3;
(4) PDX/PDTX tumor biopsy samples retest: 4.1 PDX/PDTX tumor biopsied wax block preparation: preparing the revived and amplified PDX/PDTX tumor living tissue into wax blocks; 4.2 Pathological retesting: HE staining is carried out on the prepared PDX/PDTX tumor living tissue wax block, samples with the tumor cell proportion more than or equal to 30% and the necrosis proportion less than or equal to 20% are screened out, IHC staining is carried out, and whether the expression level of the molecular marker of the PDX/PDTX tumor living tissue sample meets the grouping requirement is observed under a microscope; 4.3 FISH retest: performing FISH retest on PDX/PDTX tumor living tissue wax blocks which pass through pathological detection and meet grouping requirements, and determining an optimal target sample according to FISH information characteristic retest corresponding to molecular marker expression;
(5) Preparation of molecular marker detection products: 1) Preparing a tissue chip: drawing a positive region or a negative region with uniform molecular marker expression under a microscope according to IHC dyeing results, respectively taking out PDX/PDTX tumor biopsy sample columns with the diameters of 1mm by utilizing a tissue chip sampler to contrast the drawn region, putting the PDX/PDTX tumor biopsy sample columns into a pre-cast wax block mold according to a specific sequence, and determining specific distribution of a contrast region sample in a tissue chip according to the characteristics of the molecular markers; placing the wax block with the tissue into a stainless steel bottom die with the section facing downwards, placing into a 70 ℃ oven for 30-60min, and taking out the wax block after the wax block is completely transparent; placing the tissue embedding box above the wax block, continuously injecting wax until the bottom of the box is completely immersed, and placing and cooling; taking out and then carrying out corner cutting marking; 2) Tissue section: slicing the wax block on a slicing machine, and repairing the wax block firstly: in the trimming mode, the thickness of a trimming slice is adjusted to 15-20 mu m, after trimming wax blocks until all tissues are exposed, the thickness of the slice is adjusted to 3 mu m, and then the slice is carried out; 3) Spreading: moving the slices to the water surface in the spreading machine by using tweezers to spread; 4) Fishing: and sticking the unfolded slice on a glass slide, tilting the glass slide to 45 degrees to enable excessive water on the slice to flow down, and airing to obtain the finished product.
2. The method for preparing a molecular marker detection product based on a PDX/PDTX tumor biopsy biological sample and a database according to claim 1, wherein the specific steps of tissue fixation and wax block preparation are as follows: (1) tissue fixation: adding PDX/PDTX tumor living tissue into 10 times volume of 10% neutral formaldehyde solution, and fixing at room temperature for 12-24h; (2) tissue material selection: cutting the fixed tissue into tissue blocks with the thickness less than or equal to 1.5cm multiplied by 0.5cm, placing the tissue blocks into an embedding box and placing the embedding box into a tissue dehydration basket; (3) tissue dehydration: sequentially immersing the tissues into ethanol with different concentration gradients to dehydrate the tissues: 70% ethanol 1h, 80% ethanol 2h, 85% ethanol 70min, 95% ethanol 1h, 95% ethanol 50min, absolute ethanol 1h, absolute ethanol 50min; (4) tissue transparency: sequentially immersing the tissue subjected to gradient ethanol dehydration into xylene for transparency: 30min of dimethylbenzene and 25min of dimethylbenzene; (5) tissue waxing: immersing the tissue subjected to tissue transparency into melted paraffin for tissue paraffin immersion: paraffin 1 h- & gt paraffin 1h; (6) tissue embedding: the tissue embedding is carried out in a tissue embedding machine, melted paraffin is dripped into a stainless steel embedding mould, after the paraffin at the bottom is solidified, the tissue is placed at the bottom of the mould, the position of the tissue is adjusted to enable the tissue to be on the same plane, an embedding box is placed on the mould, the mould is continuously filled with paraffin liquid, the mould is transferred to a refrigerating table which is pre-cooled in advance for 30min, the paraffin block is taken down, and redundant paraffin is trimmed.
3. The method for preparing the molecular marker detection product based on the PDX/PDTX tumor living tissue biological sample and the database according to claim 1, wherein the specific method for resuscitation and amplification is as follows: shearing PDX/PDTX tumor living tissue meeting grouping requirements into 2X 2mm 3 Inoculating the strain to the back of NCG mice to obtain the generation P0 subcutaneously; after inoculation, the animal status was observed daily, tumor volume was measured 1 time a week and tumor-bearing mice were weighed and tumor volume V (mm) was calculated 3 ）= (a×b 2 ) 2, a is a tumor long diameter, b is a tumor short diameter; after the growth volume of the P0 generation transplanted tumor is accumulated to 800-2000 mm 3 Dissecting, taking out, shearing, randomly inoculating 5 NCG mice, and carrying out subcutaneously to carry out passage amplification to obtain the generation P1; after the growth volume of the P1 generation transplanted tumor is accumulated to 800-2000 mm 3 Samples were dissected out and paraffin embedded and section prepared as alternative control areas.
4. A molecular marker detection product produced by the production method of any one of claims 1 to 3.
5. The molecular marker detection product of claim 4, wherein the product comprises a quality control sheet or a kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310861191.XA CN116593262B (en) | 2023-07-13 | 2023-07-13 | Molecular marker detection product based on PDX/PDTX tumor living tissue biological sample and database and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310861191.XA CN116593262B (en) | 2023-07-13 | 2023-07-13 | Molecular marker detection product based on PDX/PDTX tumor living tissue biological sample and database and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116593262A CN116593262A (en) | 2023-08-15 |
| CN116593262B true CN116593262B (en) | 2023-09-26 |
Family
ID=87601183
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310861191.XA Active CN116593262B (en) | 2023-07-13 | 2023-07-13 | Molecular marker detection product based on PDX/PDTX tumor living tissue biological sample and database and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116593262B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116773790B (en) * | 2023-08-18 | 2023-11-28 | 南京普恩瑞生物科技有限公司 | Preparation method and application of tumor tissue HER2 gradient detection product |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999044063A2 (en) * | 1998-02-25 | 1999-09-02 | The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Tumor tissue microarrays for rapid molecular profiling |
| CN103602720A (en) * | 2013-06-24 | 2014-02-26 | 复旦大学附属肿瘤医院 | Application and method of prostatic cancer genetic marker in marking relapse and metastasis of prostatic cancer |
| CN112326961A (en) * | 2020-10-30 | 2021-02-05 | 福州迈新生物技术开发有限公司 | Analysis method and storage device for proportion of PD-L1 positive tumor cells in non-small cell lung cancer |
| CN113142135A (en) * | 2021-04-09 | 2021-07-23 | 山东第一医科大学附属省立医院(山东省立医院) | Construction method of digestive tract tumor PDX model and standardized model library |
| CN115881311A (en) * | 2022-12-23 | 2023-03-31 | 南京普恩瑞生物科技有限公司 | Method for screening antibody coupling drug indications by using tumor biopsy simulation clinical test |
-
2023
- 2023-07-13 CN CN202310861191.XA patent/CN116593262B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999044063A2 (en) * | 1998-02-25 | 1999-09-02 | The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Tumor tissue microarrays for rapid molecular profiling |
| CN103602720A (en) * | 2013-06-24 | 2014-02-26 | 复旦大学附属肿瘤医院 | Application and method of prostatic cancer genetic marker in marking relapse and metastasis of prostatic cancer |
| CN105861692A (en) * | 2013-06-24 | 2016-08-17 | 复旦大学附属肿瘤医院 | Method for studying on prostate cancer reoccurrence and metastasis |
| CN112326961A (en) * | 2020-10-30 | 2021-02-05 | 福州迈新生物技术开发有限公司 | Analysis method and storage device for proportion of PD-L1 positive tumor cells in non-small cell lung cancer |
| CN113142135A (en) * | 2021-04-09 | 2021-07-23 | 山东第一医科大学附属省立医院(山东省立医院) | Construction method of digestive tract tumor PDX model and standardized model library |
| CN115881311A (en) * | 2022-12-23 | 2023-03-31 | 南京普恩瑞生物科技有限公司 | Method for screening antibody coupling drug indications by using tumor biopsy simulation clinical test |
Non-Patent Citations (2)
| Title |
|---|
| Maria Rivera等.Patient-derived xenograft (PDX) models of colorectal carcinoma (CRC) as a platform for chemosensitivity and biomarker analysis in personalized medicine .《Neoplasia》.2021,第21-35页. * |
| 马双平等.ＰDＸ模型在肿瘤医学中的应用进展.《中国实验动物学报》.2023,第245-250页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116593262A (en) | 2023-08-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1195585C (en) | Apparatus and methods for efficient processing of biological samples on slides | |
| CN104007257B (en) | Method for detecting non-humoral rare karyotes, and kit thereof | |
| Kolostova et al. | Isolation, primary culture, morphological and molecular characterization of circulating tumor cells in gynecological cancers | |
| CN105588943B (en) | A kind of detection method of the genes of peripheral blood from patients with gastric cancer circulating tumor cell Her 2 | |
| CN116593262B (en) | Molecular marker detection product based on PDX/PDTX tumor living tissue biological sample and database and preparation method thereof | |
| CN103620058B (en) | Automated circulating tumor cell detection | |
| CN104569397B (en) | A kind of breast cancer detection quality-control product and preparation method thereof | |
| EP0248840A1 (en) | An apparatus and method for analyses of biological specimens. | |
| CN105510600B (en) | The detection method of advanced breast cancer patient Peripheral Circulation tumour cell ER genes | |
| WO2014114009A1 (en) | Immunohistochemical quality control reference object and quality control method | |
| CN103116029B (en) | Determining method for sensitivity and affinity of second antibody color appearance system for immunohistochemistry | |
| CN113281516A (en) | Application of CUL9 as marker in colorectal cancer prognosis evaluation | |
| CN111929447B (en) | PD-L1 immunohistochemical reference product and preparation method and application thereof | |
| CN105606823A (en) | Detection method for PR genes of circulating tumor cells in peripheral blood of later-period breast cancer patient | |
| CN113834941B (en) | Marker for prognosis diagnosis of colon cancer based on B cell expression and application thereof | |
| WO2016123868A1 (en) | Method for preparing liquid-state dropping or coating pathological quality control product, and uses thereof | |
| CN104359746B (en) | Cell mass collector and the method for collecting cell in humoral specimen | |
| CN109946278A (en) | Fluorescent dye DAPI carries out cell DNA to dye quantitative screening for cancer and diagnostic method | |
| CN113466458A (en) | Application of GPX4, NOX1 and ACSL4 in colorectal cancer prognosis evaluation | |
| CN209555255U (en) | A kind of kit early sieved for three-dimensional noninvasive tumour | |
| CN116773790B (en) | Preparation method and application of tumor tissue HER2 gradient detection product | |
| CN220983302U (en) | Tumor tissue HER2 gradient detection product | |
| CN105717104A (en) | Peripheral blood GPC3 detection method for hepatocellular carcinoma patient | |
| CN105606824B (en) | The detection method of the genes of advanced breast cancer patient Peripheral Circulation tumour cell Her 2 | |
| CN105699657B (en) | A kind of patients with renal cell carcinoma peripheral blood Vimentin detection methods |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |