One Reading: The Gold Standard of Tumor Diagnosis---Pathological Diagnosis

Cancer patients and their family members should be familiar with pathology reports, but it is not clear how the pathology reports are produced and what they are used for
.

Today, the editor will give you an in-depth talk about pathological diagnosis
.

Pathological diagnosis refers to obtaining human tissues or cells through surgical resection, endoscopic biopsy, fine needle aspiration, etc.
, and using microscopes and other tools to process and observe the samples to study the etiology, pathogenesis, morphology, structure, function, and metabolism of the disease.
Medical science, which reveals the law of occurrence and development of diseases and clarifies the nature of diseases, is the "gold standard" for the diagnosis of most diseases, especially tumor diseases
.

The pathological diagnosis process mainly includes four steps: sampling, film making, diagnosis, and reporting
.

According to the sample type, it can be divided into two types: histological examination and cytological examination
.

The sampling methods of the two are different.
Histological samples are generally obtained through open surgery, endoscopy or percutaneous biopsy, while cytological samples are generally obtained through body fluids, netting, fine needle aspiration, and exfoliated cells
.

Histological examination and cytological examination are the two basic methods of pathological diagnosis, usually called histopathology and cytopathology, and the main thing is morphological observation
.

By combining with immunodiagnosis and molecular diagnosis respectively, pathological diagnosis has formed two other important branches: immunopathology and molecular pathology.
The pathological diagnosis extends from the morphological observation at the tissue and cell level to the protein and molecular level
.

1.
Histopathological examination can generally be divided into two methods: paraffin section and intraoperative frozen section.
The application scenarios of the two are different and each has its own advantages and disadvantages
.

Paraffin sections are the most widely used, but the preparation process is cumbersome and takes a long time.
It usually takes 3-5 days to produce results, but the clarity is high and can be stored for a long time.
Frozen sections are generally used for rapid diagnosis during surgery.
The tissue is rapidly cooled and hardened by low temperature, and the diagnosis result can be issued in only about half an hour, but the quality of the slice is not as good as the paraffin section, and there is a certain misdiagnosis rate, which requires high requirements for the pathologist
.

According to the requirements of the 2009 "Guidelines for the Construction and Management of Pathology Departments (Trial"), physicians who issue general pathological diagnosis reports must have the qualifications for professional and technical positions in pathology or above, and have undergone pathological diagnosis professional knowledge training or specialist training for 1-3 years.
Rapid pathological diagnosis physicians should have intermediate or above professional qualifications in pathology, and have more than 5 years of pathological diagnosis experience
.

HE staining is the most common and basic method of section staining, and hematoxylin staining solution is alkaline.
It mainly makes the chromatin in the nucleus and the nucleic acid in the cytoplasm colored purple-blue; eosin is an acid dye, which mainly makes the components in the cytoplasm and extracellular matrix colored red
.

2.
Cytopathology At present, cytopathology commonly used techniques for film production It is a Pap smear and liquid-based thin-layer cytology technique
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The Pap smear method uses a scraper to scrape the exfoliated cells from the cervix, and then directly smear it on the glass slide to complete the fixation and staining
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Because of the Pap smear There are problems such as cell loss and poor production quality, which are gradually replaced by liquid-based thin-layer cytology production methods; liquid-based thin-layer cytology production can be divided into membrane production (TCT) and sedimentation production ( LCT/LBP) are two types, which are generally collectively referred to as TCT, and no specific distinction is made
.

TCT: Wash the cervical exfoliated cells into the cell preservation solution bottle, stir in the vial with the scraper brush, and filter through the high-precision filter membrane.
The impurities in the specimen are separated, and the filtered epithelial cells are made into a thin layer of cells with a diameter of 20 mm on a glass slide, and then fixed, stained, and mounted
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LCT/LBP: Using two centrifugation and independent staining, it has great advantages in automation and standardization, and can achieve good control of the quality of pathological diagnosis.
It has become the mainstream method of cytology in China
.

In addition to being mainly used in cervical cytology testing, it has advantages in the testing of non-gynecological samples such as sputum, pleural and ascites, urine, and alveolar lavage fluid
.

3.
Immunohistochemistry Pathology Immunohistochemistry (Immunohistochemistry, IHC) refers to the use of the specific binding principle between antigen and antibody and the color reagents (enzymes, fluorescein, isotopes, metal ions, etc.
) labeled on the antibody to affect the tissue Specific antigen or antibody for localization, qualitative or quantitative detection
.

IHC has the characteristics of strong specificity, high sensitivity, accurate positioning, and the combination of form and function, which is conducive to in-depth research in the field of pathology and plays an important role in modern pathological diagnosis
.

Related terms also include immunocytochemistry (Immunocytochemistry, ICC) and immunofluorescence (Immunofluorescence, IF).
IHC and ICC refer to color development by labeling enzymes, which are generally collectively referred to as IHC in clinical practice, without strict distinction; IF uses luciferin instead of enzymes To perform color development, sometimes referred to as fluorescence IHC
.

IF is the earliest established immunohistological technique
.

Label the known antibody with fluorescein and use it as a probe to check the corresponding antigen in the cell or tissue
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When the fluorescein in the antigen-antibody complex is irradiated by the excitation light, it will emit fluorescence of a certain wavelength, so as to locate or quantify the antigen in the tissue, and it needs to be observed with a fluorescence microscope
.

IHC/ICC technology appeared after IF and is currently the most widely used clinically
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IHC/ICC first acts on tissues or cells with enzyme-labeled antibodies, and then adds substrates to generate colored insoluble substances under the catalysis of enzymes, thereby positioning the intracellular antigens, which can be observed by ordinary optical microscopes
.

Immunohistochemistry is widely used in pathological diagnosis and can provide objective evidence at the level of protein expression.
It can be used to judge benign and malignant tumors, determine the source of tumor cells, differentially diagnose tumor types or subtypes, tumor differentiation direction, tumor grade, prognostic judgment, and target It has been widely used in the direction of treatment, discovery and determination of micrometastases
.

For infectious diseases, immunohistochemistry technology is used to identify specific bacteria, viruses and other microorganisms to improve the accuracy of pathological diagnosis of infectious diseases
.

In addition, immunohistochemistry can also be used for the determination of tumor targets of targeted drugs, the prediction of tumor chemical drug treatment response, and the comprehensive evaluation of tumor prognosis to achieve and realize the significance of companion diagnosis
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Immunohistochemical pathology: staining process 1.
Baked slice: stick the waxed tissue section on the glass slide to prevent the slice from falling off during the staining process; 2.
Dewaxing and hydration: Dewaxing with xylene, then Use descending gradient ethanol to replace xylene in the tissue; 3.
Antigen retrieval: Part of the antigen peptide chain is distorted after the previous steps and cannot be displayed in the immunohistochemical staining process, so it will be blocked by chemical reagents or heat.
Re-exposure of antigen; 4.
Cell membrane perforation: Use fat-soluble reagents to perforate cell membrane to increase the permeability of cell membrane to antibodies (this step can be omitted when detecting cell membrane antigen); 5.
Inactivate endogenous enzymes and block endogenous Biotin: prevent the decomposition of substrate H202 and DAB precipitation; 6.
Non-immune serum blocking: use the same non-immune serum as the secondary antibody to block the charge to prevent the primary antibody from binding to it, inhibit non-specific background coloring, and prevent the antibody from being organized The charged collagen and connective tissue components in the slices are adsorbed; 7.
Primary antibody incubation: Make a gradient dilution according to the recommended concentration to determine the optimal concentration; 8.
Secondary antibody incubation: Add a secondary antibody that matches the species of the primary antibody for incubation; 9.
Color development: add a color developer, generally DAB or AEC; 10.
Counterstain: use common dyes such as hematoxylin to counterstain the slices to make the slices clearly show the tissue structure and facilitate accurate positioning; 11.
Mount the slides ; Immunohistochemical pathology: The antibodies used in the immunohistochemical staining of the detection and chromogenic system are mainly divided into primary antibodies and secondary antibodies
.

The primary antibody is an antibody that specifically binds to an antigen
.

The types of primary antibodies include monoclonal antibodies and polyclonal antibodies, whose main function is to identify the substance to be detected in the test; the secondary antibody can specifically bind to the primary antibody, that is, the antibody of the antibody
.

All antibodies produced by the secondary antibody against a specific species are specific, and the secondary antibody is usually labeled with enzymes, fluorescent groups and other substances for subsequent color development or luminescence
.

The role of the secondary antibody is to detect the presence of the primary antibody and amplify the signal of the primary antibody
.

A variety of different detection systems can be formed according to whether the secondary antibody is selected and the type of the secondary antibody marker.
At present, the biotin method and the enzyme-labeled polymer method are widely used
.

Color rendering system: also includes many types, of which HRP-DAB is the most widely used
.

Immunohistochemical pathology: clinical application (take NSCLC as an example) immunohistochemistry technology is widely used in the diagnosis of various tumors.
Taking non-small cell lung cancer (NSCLC) as an example, NSCLC accounts for 80% of all lung cancers and can be divided into different The histological types mainly include adenocarcinoma (≥40%) and squamous cell carcinoma (30%), as well as the relatively rare large cell carcinoma (LCC) (10%)
.

With the increasingly different individualized treatment options for lung adenocarcinoma and squamous cell carcinoma, there is a strong clinical demand for a clear diagnosis of NSCLC with different histological types
.

4.
Molecular pathology Molecular diagnosis is the application of molecular biology methods to make diagnosis by detecting changes in the structure or content of the subject's genetic material or the genetic material carrying viruses or pathogens.
It is widely used in infectious diseases and blood screening.
Diagnostics, genetic diseases, tumor companion diagnosis and other fields
.

Molecular pathology is the application of molecular diagnostic technology to pathological diagnosis, using histological/cytological specimens as carriers to detect molecular genetic changes in cells and tissues at the genetic level to assist in pathological diagnosis and typing, and to guide targeted therapy , Predict treatment response and judge prognosis
.

The current molecular pathology technical routes mainly include polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH) and high-throughput sequencing (NGS).
Different technical routes have their own advantages and disadvantages.
Among them, PCR and FISH technology platforms are relatively mature.

.

Molecular pathology: technical route-PCR polymerase chain reaction (PCR) refers to in vitro replication under the catalysis of DNA polymerase, using parent strand DNA as a template and specific primers as the starting point for extension, through denaturation, annealing, extension and other steps The process of extracting the daughter strand DNA complementary to the template DNA of the parent strand
.

Since its advent in the 1980s, PCR technology has been iterated to the third generation: the first generation of qualitative PCR: PCR products are analyzed by agarose gel electrophoresis, the analysis only stays at the qualitative or semi-quantitative level, and the detection time is long, There is a risk of cross infection
.

Second-generation qPCR (real-time fluorescent quantitative PCR): Add a fluorescent group that can specifically bind to the DNA product in the PCR reaction system, use the accumulation of fluorescent signals to monitor the entire PCR process in real time, and realize quantitative analysis through the standard curve.
Data collection can be The PCR amplification process is completed
.

The third generation of dPCR (digital PCR): Based on chip-type droplet microfluidic technology, a sample is divided into tens of thousands to millions of copies and distributed to different reaction units, each unit contains one or more copies of the target molecule (DNA template), the target molecule is amplified separately in each reaction unit, and after the amplification is completed, the fluorescent signal of each reaction unit is statistically analyzed, which can realize absolute quantitative detection
.

Molecular pathology: qPCR technology extension-Mutation Amplification Block System (ARMS) ARMS technology: Add "mutant" primers for the detection target in the reaction system, and use wild-type nucleic acid template can not be used with the 3'end of the "mutant" primer The principle that bases are complementary paired and cannot be extended by Taq enzyme realizes the blockade of wild-type nucleic acid template amplification
.

The mutant nucleic acid template can be complementary to the 3'end base of the “mutant” primer, and the “mutation” signal is amplified by the PCR reaction and finally detected by the qPCR instrument
.

Using ARMS technology to combine fluorescent probes to detect one or more genetic mutations in sample DNA on a real-time fluorescent PCR platform is currently the most mature and clinically applied technology platform
.

The advantages of this technology include: (1) High detection sensitivity, which can detect mutant genes with a mutation ratio of 1% or even lower in tumor cells; (2) It is suitable for fragmented DNA and is conducive to molecular pathological diagnosis (due to the fixation of formaldehyde, Most of the DNA of the paraffin-embedded specimen tissue is fragmented); (3) Combining qPCR to achieve closed-tube operation can reduce the possibility of contamination
.

Molecular pathology: technical route-FISH fluorescence in situ hybridization (FISH) is based on the principle of base complementation, using nucleic acid probes directly or indirectly labeled with fluorescein to detect interphase nuclear staining on tissue sections, cell smears, and chromosome spreads Qualitative and relative quantitative analysis and detection technology are carried out for qualitative, quantitative and structural changes
.

Since DNA molecules are linearly arranged along the longitudinal axis on the chromosome, probes can be used to directly hybridize with the chromosome to locate a specific gene on the chromosome
.

By using hapten-labeled DNA or RNA probes to complement the target sequence, detecting the hapten by the antibody with a fluorescent group, or directly labeling the probe with a fluorescent group and binding to the target sequence, and finally using fluorescence The microscope directly observes the distribution of the target sequence in the nucleus, chromosome or slice tissue
.

Advantages: A variety of fluorescent labels can be used to display the relative position and direction between DNA fragments and genes, and the spatial positioning is accurate; sensitive and specific, it can analyze multiple cells in the division and interphase at the same time, and perform quantification; can detect Occult or tiny chromosomal aberrations and complex karyotypes
.

Disadvantages: 100% hybridization cannot be achieved, and efficiency is significantly reduced when using shorter cDNA probes; higher requirements for operation and interpretation techniques; high cost and low throughput
.

Molecular pathology: technical route-NGS high-throughput sequencing (NGS) is a revolutionary advancement following Sanger sequencing.
It refers to the chemical modification of template DNA molecules, anchoring them on nanopores or microcarrier chips, and using base complementation Pairing principle, in the process of DNA polymerase chain reaction or DNA ligase reaction, by collecting fluorescent label signal or chemical reaction signal to realize the interpretation of base sequence
.

NGS can perform massively parallel sequencing, and can sequence millions of fragments at the same time in each run, which improves the speed and accuracy while greatly reducing the cost of sequencing
.

The above is the introduction and application of the basics of pathological diagnosis technology
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Content from: Medical World