Clonal chromosomal abnormalities are the features of tumors or hereditary diseases, including abnormalities in the number or structure of chromosome. These abnormalities can be detected by FISH. At present, FISH is widely applied in the department of pathology, hematology department, prenatal diagnosis center, and so on.
As a person in charge of FISH, you should focus on the following:
• Operation conditions: What conditions, instruments and equipment should be made available?
• Application: What can we do using these platforms?
• Process QC: What should we pay attention to at the detection process and QC link?
Part 1. Operation conditions
According to the 2016 Guidelines for the Construction of Molecular Pathology Diagnostic Laboratory (Trial), the department of pathology in third-grade class-A hospitals should set up its molecular pathology diagnostic laboratory, and other hospitals can also set up such laboratory if conditions permit.
1. Personnel
Technicians: Having the basic knowledge about pathology and molecular biology, college degree or above, and having received training or further study on relevant professional technical skills, and obtained the corresponding qualification certificate(s).
Authorized signatories: Having obtained the Certificate of Licensed Practicing Physician in clinical pathology and/or genetics, having intermediate or above professional titles, and engaged in their specialty as an on-the-job doctor or technician for their own employer.
Laboratory director: Having clinical medicine and pathology background, molecular biology related work experience, associate chief physician or above professional and technical titles, engaged in his/her own specialty as an on-the-job physician for his/her own employer.
2. Setup requirements
Laboratory area
Used for specimen pretreatment, digestion, denatured hybridization, washing, slide sealing, and so on. Detection with fluorescent labeled probes should ensure operation in a dark environment, including electromagnetic oven, water bath, centrifuge, pH meter, hybridizer, and refrigerator (-20~４℃).
Image collection and analysis area
Used for slide reading, image collection, analysis, report issuing and so on. Detection with fluorescent labeled probes should ensure operation in a dark environment, including fluorescence microscope, PC and image collection and analysis software.
3. Item admission
3.1 Workload: There should be a certain amount of workload, at least 1 molecular pathology detection item and in every year, at least 100 cases of detection.
4. Quality control
4.1 Detection capacity and detection system assessment: Before the commencement of new items, the laboratory detection capacity and detection system (instrument, reagent and consumables) must be evaluated. 1-2 laboratories that have conducted such items and obtained qualifications should be selected for comparison and verification and their result consistency should be more than 95%. Standard operating procedures have been established and complete detection records and technical files are available.
4.2 Pathological evaluation: Before the detection of any lesional tissue or cell, a pathologist is required to further make sure whether the lesional tissue is in line with the pathological diagnosis and assess whether there is bleeding and necrosis on the specimen and pretreatment unfavorable nucleic acid detection (e.g. treatment with decalcifying solution containing HCL), so as to avoid false negative results due to improper specimen treatment and evaluate the total number and proportion of pathological cells (e.g. tumor cells) meet the detection requirements.
4.3 Intra-laboratory and inter-laboratory QC evaluation: Intra-laboratory QC is performed on a regular basis, keeping the records and timely solving the problems. Participation in external QC activities should occur 1-2 times every year. The consistency of positive and negative results during external QC should reach more than 90%.
4.4 Equipment calibration: The analysis equipment, including sample loading, detection and temperature systems, should be calibrated. The PCR instrument, sample injector, thermometer and thermostatic equipment should be calibrated on a regular basis and the calibration records and reports should be generated. Some equipment, e.g. biosafety cabinet, microtome and microscope can be calibrated according to the manufacturers' calibration procedure in accordance with the detection objective and requirements.
5. Reagent management
5.1 Reagent selection: Reagents with CFDA certification should be selected. A complete set of SOP for reagent purchasing and quality inspection should be established and strictly implemented and recorded.
5.2 Use and record of reagents and consumables: The reagents and consumables should be used within their validity period. Records for check, receiving or rejection, storage and use of the reagents and consumables should be made available. The records for the use record of commercial reagents should include their expiration date and decapping date.
5.3 Inventory control system for reagents and consumables: The inventory control system should include the batch record, and records on laboratory receiving date and use date of all related reagents, controlled substances and calibrators.
5.4 Evaluation on the quality of reagents and consumables: The laboratory should evaluate the key reagents, consumables and service providers that affect the detection quality, maintain the evaluation records and list the directory of the above reagents, consumables and providers approved.
6. Report format
The molecular pathological diagnosis report should be made in a perfect format and content, including the basic information about patients, specimen information (part, type, fixing time and fixing method), usage, reagent source, control setting, pathological evaluation, detection result, conclusions and necessary interpretation.
7. Selection of filter blocks
Different manufacturers vary with their fluorescein. When selecting the filter lens parameters of fluorescence microscope, it is recommended to contact the supplier to obtain compatible parameters. LBP's FISH detection reagent parameters are recommended as follows:
8. Specimen types
Tissue specimen (FFPE): including excision tissue and puncture specimens;
Cell specimens: peripheral blood, bone marrow, washing liquid, urine, amniotic fluid and villi.
Part 2. Application
As a molecular detection means based on morphology, the FISH detection platform has been extensively applied in the detection of tumors or hereditary diseases, which is mainly reflected in the following aspects:
Medication guidance: With the development of tumor therapy, more and more targeted drugs are applied to tumor therapy. Many targeted drugs must be subjected to molecular detection to determine whether they are suitable for targeted therapy before use.
Molecular diagnosis: It has been found that some tumors with special molecular change mechanisms are similar in morphology to those without such molecular change, but their treatment or prognosis varies greatly, so they are named as a special classification. For example, renal cellcarcinoma in the MIT family is characterized by molecular changes such as TFE3 or TFEB gene rearrangement with 1p/19q changed glioma.
Prognosis management: In tumors of lymphatic hematopoietic system, the molecular change mechanism is complex, and different molecular changes often suggest different risk grades or prognosis; therefore, molecular detection results are needed to help clinicians manage the prognosis.
Auxiliary diagnosis: In soft tissue tumors or some other special tumors, pathologists will encounter similar morphology and non-specific protein expression when making diagnosis, so it is difficult to make a definite diagnosis. At this time, some characteristic molecular detection results are used to help them make an accurate diagnosis.
In addition to clinical diagnosis and treatment, the FISH detection platform can also help doctors in the related departments to conduct research and article study. From 2014 to date, LBP's probe users have published more than 70 papers in domestic and foreign journals.
Part 3. Process QC
It generally takes 2.5 days for FISH detection. It takes about 2-3 hours for the experiment before hybridization. The routine denaturation often lasts overnight, for about 10-18 hours; after hybridization, washing and reading consumes about 2-3 hours. In case of fast hybridization, the time can be shortened to 2-3 hours.
If the LBP-6612 Automatic Slide Processing System is used for FISH experiment, on the one hand, the quality of experimental process can be stably controlled; on the other hand, it can replace most manual processes to greatly improve the experimental efficiency.
Part 4. Terminology
DNA & gene & chromosome
• DNA molecule consists of sugar backbone and four kinds of nucleotide molecules (A, G, C and T).
• Gene is a long DNA fragment, also the most basic unit of inheritance; biological traits in the form of genes pass from parents to the next generation.
• Long chain DNA is entwined in histone skeletons to make up genes, many of which are contained in chromosomes separated by long segment DNA.
Chromosomal band: Used to indicate the approximate position of a certain chromosomal region on the chromosome. For example, the probe for FGFR2 gene will be written as 10q26, which generally indicates that FGFR2 is located in zone 2 and band 6 of long arm of chromosome 10.
DAPI: Namely 4,6-diamidino-2-phenylindole dihydrochloride, a fluorescent dye capable of binding with DNA, which is often used for fluorescence microscope observation. Since DAPI can penetrate a complete cell membrane, it can be used for staining of viable cells and anchor cells (blue cell nucleus).
Single channel: Refer to single-color channel filter block, which is divided into green channel, red channel and cyan channel. In this channel, you can observe the signal point of a single color.
Double channel: Refer to double-color channel filter block. In the double-color channel, you can observe the red and green signal points, which is often used for interpreting cleavage probe and fusion probe.
