guidance for laboratories performing molecular pathology for cancer patients

Molecular testing is becoming an important part of the diagnosis of any cancer patient.
The challenge for the laboratory is to use reliable methods and processes to meet this need to ensure that patients receive timely and accurate reports as a basis for their treatment.
The purpose of this paper is to provide minimum requirements for the management of molecular pathology laboratories.
For different tumor types and tests, this general guidance should be enhanced with specific guidance.
Consideration before analysis is important, and it is necessary to carefully consider the way samples are obtained and arrived at the laboratory.
Sample Reception and Processing follow standard operating procedures, but some changes may need to be made if molecular testing is to be carried out, such as controlling tissue fixation.
DNA and RNA extraction can be standardized and the quality and quantity of the output should be checked regularly.
Selection of Analytical Methods (s)
Depending on the clinical needs, the desired turnaround time and the expertise available.
Internal quality control, regular internal audits of the entire testing process, laboratory recognition and continuous participation in external quality assessment programs are prerequisites for providing reliable services.
Molecular pathology reports should accurately convey the information that clinicians need to treat patients with sufficient information in order to properly interpret the results.
Rapid development of molecular pathology, further detailed evidence
Many of the topics covered here need to be based on suggestions.
Introduction The management of individual cancer patients is increasingly affected by specific features of the tumor, which is often due to specific genetic changes.
The concept of personalized medicine for cancer is not new and is called personalized, layered or precise medicine.
From estrogen receptor and estrogen receptor 2 (HER2)
Breast cancer was amplified as an mutation in the receptor or ALK in lung cancer, a Kristin mutation in malignant melanoma, a RAS mutation in colorectal cancer, and a B2C/ABL1 in Philadelphia-positive chronic leukemia.
Personalized approaches to tumor status include immune tissue chemistry and molecular pathology, which is often considered to refer to the analysis of nucleic acids in tissues, cells, or liquids.
Therefore, molecular testing is becoming an important part of this work --
For many patients with solid and blood tumors, if not most.
As a result, most hospitals with active oncology practices need to enter laboratories that provide the necessary information about gene formation
Tumor on biopsy material
This trend may strengthen as new drugs are available, and drugs on the market begin to be used in combination or in sequence to overcome the resistance mechanism.
The challenge for pathologists is to go beyond diagnosis and classification to produce the information needed to accurately guide the treatment and do so in the shortest possible time.
The easiest way to consider the requirements of the laboratory that provides these services is to consider the path from the patient to the outcome, and the requirements for each stage (figure 1).
Health care systems and laboratories inevitably differ in how they organize their work.
The purpose of this framework is to help molecular pathology laboratories provide the best services for patients.
Clear responsibility for requiring molecular analysis, pre-analysis sample processing, nucleic acid extraction and analysis, and results reporting is a prerequisite for safe and efficient service operation.
Download figureOpen in the new tabDownload powerpoint figure 1 workflow for the Laboratory of Molecular Pathology for cancer patients from formaldehydefixed paraffin-embedded (FFPE)tissue samples.
A frequently asked question is the number of samples that should be processed by any laboratory in order to be considered reliable.
Currently, there is no data to answer this question, but we know from other settings (eg, ERBB2 (HER2)testing3 ,4)
It is unwise to consider providing services to a small number of patients, in fact, it is difficult to achieve cost-
Effective solution for a small number of samples.
Similarly, few mutations occur in some genes, and with the lab adopting group testing as the standard, even the largest lab may find that they rarely report some mutations.
We believe that the key to this issue is the number of samples submitted for testing, as well as the clinical and logistics requirements of the service.
Therefore, the practicality of testing and clinical needs may make the decision of most tests simple.
Given that the number of patients that need to be tested may increase, most accredited laboratories may provide reliable services if they comply with the advice covered in subsequent chapters, although national agencies are advised to monitor this.
It should be noted that there are separate guidelines for each step in this path and for each test requirement.
A good example, based on a systematic review, is a recently published guide by the American College of Pathologists (CAP)
International Association for the Study of Lung Cancer and Association for Molecular Pathology (AMP)
It is used for ALK and TNF tests for lung cancer.
5 This paper is intended to outline the situation in which the laboratory is guided to run reliable services: we have adopted many of their suggestions as well as those in other related publications s2, 6-10 and ISO15189 guidelines.
Multiple medical disciplines (
Surgery, oncology, or pathology)
Molecular analysis may be required to define treatment strategies for individual patients, although in practice this is often an oncologist.
In many cases, a multi-disciplinary team (tumour board)
I will decide to request a test.
The request process should ensure that the request is made appropriately and that the test is provided to each patient who needs to be tested in a timely manner.
This is one of the most difficult aspects of operating a molecular pathology laboratory: it is important to ensure that all patients in need are tested, but again, unnecessary tests are not performed.
Although costs are falling at the moment, molecular testing is expensive and oncologists or multi-disciplinary teams may think they need to manage the needs, especially if they manage the budget.
However, automatic (reflex)
The pathologist may be more effective in testing based on the diagnosis and tissue availability of the pathology department, especially if more than 10% of specific diagnostic patients need testing (
Cree, unpublished).
The decision to implement a reflex test should be based on a business plan, taking into account the time and monetary cost of extracting specimens from the pathology file when clinical results are required.
6 In addition, the amount of tissue available and the amount of tissue required for Molecular Pathology should be included in the consideration of the sampling strategy: This is a special problem in lung cancer patients.
Flexibility is also needed: technology and needs are changing rapidly and it is critical to re-evaluate clinical needs frequently.
Good communication is required between laboratories, oncologists and surgeons to ensure that necessary tests are requested in a timely manner.
Pre-treatment (
Chemotherapy)
Gene expression and mutation status can be changed, 12-14, and should be recorded on the application form.
It is important to discuss with surgeons and pathologists any changes to the current protocol and they need to clearly determine the scope of the resection, but also need to be subject to the requirements of molecular analysis.
Again, the interpretation of molecular analysis requires an understanding of the diagnosis and the treatment strategies available.
The pathology laboratory must be able to handle multiple sample types.
This raises some challenges when the need for molecular analysis is included in the diagnostic pathway, although many problems are common in any diagnostic test.
At each stage, samples are likely to be incorrectly identified and this danger should be carefully noted.
It is the responsibility of the person taking the specimen to identify the patient, the sample required, to ensure that the sample is correctly marked and fixed with formaldehyde (If necessary)
And/or sent to the lab.
However, when samples are taken in specific circumstances, for example in the operating room, the primary responsibility of the operating room staff is the patient, not the sample, and the need for implementation and operational failure should be considered
Ensure a safe procedure for the correct handling of samples to ensure reliable and accurate diagnostic and test results are obtained.
The SPIDIA project has recently studied the pre-analysis process in detail (
The results we understand will be included in the ISO guidelines (ISO 15189).
Tissue: Most diagnostic biopsies are small and can be fixed quickly when placed in neutral buffer formaldehyde (4% formaldehyde)
However, large surgical specimens need to be fixed under control.
15, 16 it may be best to control the fixation process by the pathology laboratory.
This may involve transporting fresh specimens to the pathology laboratory, and if it takes more than an hour, it is better to be in a vacuum (see below).
Molecular analysis of fresh tumor samples can be collected (
Or organization Bank)
In the operating room, but preferably after receiving it in the pathology laboratory according to the defined protocol.
It is best to open the intestinal specimens and (where possible)
Cut a larger mass to allow penetration of the fixer.
Formaldehyde only penetrates the tissue around 1 µmm/h, and fixing will only begin when penetration occurs.
After dilution to 4% w/v, formaldehyde should only be used within 24 hours to reduce the polymerization effect and ensure a stable 4% concentration.
Cold ischemia occurs between the resection tissue and the fixed tissue, which is a special problem for large surgical specimens.
It changes the level of gene expression (
At RNA and protein levels)
Is the main factor in molecular pathology.
17 control of fixed time and temperature is recommended for molecular analysis of proteins such as db2 2 (HER2)
Or for gene expression analysis.
Organize vacuum packaging and transfer to the laboratory at 4 °c (Tissue Safe)
It is an option to attract attention and is very useful for locations far away from hospital labs.
Cooling should be as fast as possible, cold fixation can also preserve RNA well despite cold shock
Some biomarkers may consider induced changes.
19 standard operating procedures (SOP)
Should be designed to include the organizational handling of the aspects listed in Box 1.
Box 1 organizes the standard operating procedures required for processing, including staff responsibility for each stage and sample identification using four identifiers (
Name, date of birth, hospital identification code, for example).
Specimen type and origin. Container type—
Charts and lists in theaters help ensure proper use.
Transportation requirements-
Time and temperature
Fixed requirements, including open gut specimens and open large chunks, while respecting the removed edges.
Shedding cell samples should also be processed according to the defined standard operating procedures.
In some centers, pathologists are involved in performing fine needle aspiration, which helps to process samples.
Fixation is usually in an alcohol fixer so that nucleic acids can be preserved relatively completely.
This makes this sample suitable for most molecular analyses, although some issues have been noted in human hpv (HPV)and non-
Small cell lung cancer (NSCLC)testing.
20, 21, 22, 23 liquid biopsy: nucleic acid extraction by Decell (buffy coat)and plasma.
The white blood cell section provides off-the-shelf sources of seed DNA and has long been used to generate molecular data for blood tumors.
Viruses and bacteria can be isolated and identified from Buffalo coated preparations by molecular methods. Cell-
Free DNA can be used for mutation analysis, and although this method has not yet entered into routine practice, they show considerable prospects.
13, 24, 25 preferred plasma from edta4 blood sent directly to the molecular pathology laboratory or the blood science laboratory.
The serum contains DNA of white blood cells, which is not suitable.
Conventional hematology measurements using the collected samples are feasible, but since lithium is a PCR inhibitor, the use of lithium heparin tubes should be avoided.
RNA should be taken care of, because continuous production in the cells and continuous degradation of RNAse activity after the sample is collected will affect the measurement of the level.
Preventive measures should be taken to minimize the degradation of RNA, but in most hospitals, it is not a standard procedure to quickly transport samples to the laboratory on ice, when collecting samples, it can be difficult to establish a cold chain or add inhibitors to the sample.
There are specialized blood vessels to preserve RNA (
For example, PAXgene, PreAnalytix/Kaijie;
Valencia, California, USA)
But their clinical use requires validation of the relevant tests.
In clinical laboratories, standard operating procedures are required for all processes.
It is important to use and adhere to work instructions and lists when necessary.
There should be a dedicated sample receiving process for sample receiving and processing, which should be equipped with appropriate staff and allow the sample to be assigned to join numbers when entering the laboratory (
According to ISO 15189 and ISO 9001).
Most laboratory management information systems (LIMS)
This is allowed, but few recognize the need to integrate molecular pathology needs.
It is a good practice to bar code the samples and it is worth encouraging.
All samples should be tracked from patient to report.
The tissue, blood and liquid treatment in the laboratory shall be carried out in accordance with the prescribed standard operating procedures (see ISO 15189).
When most tissue biopsy samples are fixed, fresh tissue can also be sent to the laboratory for in-operative frozen sections or molecular analysis.
This requires separate standard operating procedures and specialized handling, care and laboratory staff.
Tissue samples are inspected and cut at macro level
On stage, when the building blocks are taken down and placed in the bar --
Coding and/or numbered plastic boxes for processing.
Reserve tissue in formaldehyde can be retained at this stage.
Vacuum or microwave
Processor-based temperature control devices ensure good penetration of graded alcohol and PX alternatives, allowing paraffin to be embedded.
The temperature of different machines and protocols may vary greatly, which may affect nucleic acid recovery (
Cree, unpublished).
The use of previously frozen fixed tissues is not recommended, and de-mining will reduce the recovery of DNA, especially RNA.
It is found that neither of them produces or is not an ideal result.
26, 27, 28 it is recommended that the clinical team and the pathology laboratory work closely together, and the key is to communicate the changes in the processing to the molecular pathology laboratory and make adjustments if necessary.
Pathological diagnosis is performed according to the minimum data set requirements, stained sections cut from paraffin blocks of 4-6 μm (
For molecular testing, it is critical to take strict precautions to prevent cross-cutting
Contamination between samples.
It is best to change the knife (blades)regularly (
It\'s best before every new formaldehyde. fixed paraffin-embedded (FFPE)
Cut tissue blocks).
In addition, parts should be transferred to slides using disposable plastic products (
If part is used).
By using a drop of PCR-quality water on the slide, it is possible to avoid the use of a water bath to stretch the slices.
It should be noted that the purification procedure does not reduce the level of DNA or RNA required for analysis when the slices are cut.
For example, the use of DNA zap wipes on the slicer blade immediately before cutting can greatly reduce DNA levels and should be avoided.
The use of substances that may inhibit PCR should be avoided.
29. if used, it is recommended that pathologists mark on H & E slides at the time of diagnosis of macro or micro-anatomy parts of the region containing the tumor.
Manual microanatomy may require labeling multiple regions of the tumor to account for heterogeneity of tumor cell content in large tumors.
As an alternative to microanatomy, 1mm punches in the marked area can be used.
Laser capture microcutting is largely a research tool that is not necessary for conventional molecular pathology.
Tissue pathologists should estimate the percentage of tumor cells in the sample (
Or some samples)
Selected for DNA/RNA extraction.
This estimation may be important to determine the success or failure of subsequent tests, as it may define the lower limit of detection, and the minimum percentage of tumor cells present can be applied to many tests.
31 if there is necrosis, this should be noted, but it is best to avoid this in the sample for molecular analysis.
The widely dispersed tumor cells or lymph duct inflammatory cancers in the sample are notorious examples in which a low proportion of tumor cells produce false negative results.
If the pathologist is directly responsible for requesting molecular analysis, this \"reflection\" test allows for a quick turnaround time and prevents the need to retrieve the block later, reducing the test turnaround time, and therefore, reduce the time and cost of employees.
This needs to be balanced against unnecessary testing in patients who do not need further treatment, or in the future it may be necessary to conduct unnecessary testing in patients who need treatment when testing alternative gene mutations.
A clear assessment of tumor cell content and responsibility for macroscopic or microscopic anatomy is required: this is an important role for tissue pathologists.
The estimated percentage of tumor cells present in tissues used for DNA/RNA extraction should be mentioned in the pathology report. Blood samples (EDTA)
It is usually rotated at low speed to precipitate red blood cells and recover Buffalo coating and plasma fraction.
Should start processing as soon as possible (
Ideally within 30 minutes)after sampling.
The collection time should be indicated on the test tube and the elapsed time between sampling and processing should be recorded.
Cells are usually removed from plasma by centrifugal 10 min at 1000-2000g.
Centrifuge at 2000g for 15 min will also exhaust platelets in plasma samples.
Similarly, an agreement should be strictly observed.
It is feasible to obtain white blood cells, foreign bodies and plasma from the same sample using density centrifuge.
Molecular analysis can be performed by tagging 32 samples on many LIMS, and reflection testing may be feasible.
Aliquoted plasma samples can be stored at-80 °c.
Standard operating procedures shall be established to ensure the safety of tissue and blood samples and their parts and, where possible, control the collection, processing and storage (
Including extracted nucleic acid)(
See ISO 15189 and any country guide).
Few laboratories preserve tissue blocks and slides in a temperature-controlled environment.
Similarly, blood samples are usually discarded within days of receipt.
Blood and Blood
Derivatives are considered biological hazards and should be treated accordingly.
Fresh organizations have similar risks, but FFPE organizations are considered safe, although the chemicals involved in the processing of the organization need to be carefully stored and processed.
DNA and RNA extraction nucleic acid extraction for clinical use requires a quality control reagent, ideally ivd ce-marked.
Many companies produce kits, although rarely sold to clinical use, it is the responsibility of the laboratory to verify and validate any method in accordance with existing standards.
Some multi-center validation data have been released, but this is less direct.
Consider which method to use should consider throughput, the quality and quantity of DNA or RNA, and the expected analytical method.
Manual methods are commonly used.
Precipitation is mostly used in manual methods-
Wash steps with a centrifugal rotating column or filter.
Training and considerable skills are required even for the kit, but excellent results can be provided.
Special attention should be paid to avoiding sample error identification and contamination.
It is important to follow the standard operating procedures carefully --
It can be helpful to use the checklist and careful staff training.
Automated methods are useful because they can save staff time and help prevent sample misidentification.
They tend to use magnetic bead extraction methods suitable for robotic systems.
It is important to try out these systems before implementation, because not everyone is good at blood and FFPE block extraction: several machines may be needed to handle all sample types and different throughput.
DNA and RNA can be quantified by luminosity, fluorescence or PCR.
Different methods advocate different machines: Nano-drops, quantum-bit fluorescence meters, or Agilent bio-analyzers are widely used.
DNA degradation in FFPE samples reduces the accuracy of the spectrometer measurement.
However, quantitative reality
Time PCR can be used to establish the quantity, size and amplification capacity of DNA and RNA with considerable accuracy and limited sample consumption.
Standard operating procedures should indicate which validation methods are preferred for each test performed in the laboratory and provide appropriate training to the staff.
Continuous audit of pre-analysis performance through internal quality control is important so that changes in sample processing and nucleic acid extraction can be safely implemented.
Care should be taken to control the storage of DNA and RNA.
Adding logs or barcode vials can be used to prevent sample misidentification.
Temperature records should be maintained.
Generally speaking, storing extracted DNA and RNA samples at-20 °c or-80 °c is a good practice to preserve PCR products in separate refrigerators at 20 °c or 80 °c, respectively, save the sequencing library in a separate refrigerator at 20 °c or 80 °c
For different analytical methods, there is not enough data on the effect of storage length on tissue and extracted DNA or RNA: this is a consideration that may become more and more important, because patients may need to be tested many years after sampling.
DNA (or cDNA) is more stable than RNA and may survive for many years under given conditions.
The selection test requirements for analytical methods are defined by clinical requirements, which in turn are defined by the availability of drugs that have identified operational mutations (table 1).
This list is rapidly expanding and each Laboratory needs to coordinate their testing on a regular basis according to the needs of oncologists.
34 in some cancers, mutations are mainly located in several exons (‘hotspots’)
Rarely found elsewhere in genes
Since the goal is to identify viable mutations, the laboratory should screen all mutations with evidence of clinical efficacy.
For example, in the epidermal growth factor receptor gene, even if mutations account for less than 5% of the identified changes, mutations in Gene 18 should be systematically screened.
The minimum requirements can also be stated in the national guidelines (
For example, Germany requires testing of biomarkers with an incidence of 1% or higher).
View this table: View the inline View pop-up table 1 Examples of actionable genes for solid tumors in many laboratories where people have moved away from a single
Gene analysis for panel testing and reflecting this change in practice.
External Quality Assessment (EQA)
The plan is a tumor, or has been transferred to the tumor-
Specific protocols, distribution of tumor samples used to detect multiple genes, according to individual Laboratory Practices (
This protocol is expected to test the current relevant actionable mutations, but will also assess the accuracy of typing for other genetic tests where appropriate.
Other considerations when selecting an analytical method are the number of samples that need to be tested, the number of genes tested, and the percentage of mutations tested in each gene.
Most pathology labs now have
Fully capable of running a good time PCR machine
Kit for actionable mutations covering 95% of specific genes was established.
Sanger and Coke sequencing methods are also widely used.
In high throughput units, use next-
Sequencing (NGS)
Platform, its advantage is to cover more and more interested genes more widely.
The number of organizations available is often a limiting factor in the scope of testing (
Fine needle aspiration, endoscopic or needle biopsy, for example)
It may affect which technology to choose.
Consideration should also be given to the level of staff expertise, equipment and infrastructure required.
Many workflows in molecular pathology need to be separated in different compartments (ideally rooms)
Prevent contamination of DNA and RNA
Pre-SeparationPCR and post-
PCR steps are particularly important.
ISO15189 needs to be in-
Housing testing compared to CE
In vitro diagnostic markers (CE-IVD)tests.
However, since most tests are only part of the whole process, the complete process including the pre-analysis, pre-analysis, and post-analysis stages should be in-depth.
These considerations often determine the choice of technology.
In practice, these options are based on real
Time PCR or sequencing solutions.
Companies that offer this option have independently evaluated the receptor (
And Jiashi mutation detection (
But the data of many methods is limited.
The speed of technical change is quite large, except for the instructions that instruments and kits should generally be ivd ce-
Mark and analyze validation according to existing performance specifications.
Validation and validation of tests is important, whether in a consortium of companies, academic and hospital laboratories, or in individual laboratories that provide testing for clinical use. Batch-to-
For complex reagents, batch change is a special problem. in recent years, the performance of several mutation detection kits has fluctuated.
ISO15189 requires the inspection of new batches of reagents without strict inspection.
Panel testing may become common in a few years.
In fact, some laboratories are already turning to arrays or NGS methods, and the advantage of these methods is that they can provide information about multiple gene mutations without a large amount of DNA or RNA. The NCCD (
A panel test of non-small cell lung cancer was recently recommended, indicating that it is generally accepted.
It should be noted that the NGS pair is low-
In the PCR steps in this process, genetic changes at the level, as well as incorporating bioinformatics rules into data analysis, are necessary to allow correct and appropriate interpretation.
For example, formaldehyde induced C> T artifacts in DNA that may mimic mutations that need to be identified by bioinformatics methods. The performance of molecular testing is critical for monitoring the performance of all tests in the pathology laboratory and the parameters remain within an acceptable range, as defined in the test validation report.
In most cases, these are statistically defined based on known changes.
Using simple statistics, CIs with correct analysis behavior can be established (
Rules 36, 37).
For example, kit manufacturers often (
Not always though)
Provide internal control or reference genes, and the level of these genes should not be changed for the same DNA input.
While differences between samples are common, this should not exceed certain parameters, which can be defined from standard operating procedures. In-
Use Control from external sources as much as possible.
The percentage of individual tumor type mutations detected may vary according to the population tested, but results outside the national confidence range should always be questioned.
Again, the experience from her-2 detection is very appropriate and it should be noted that the knowledge in this area comes from
Terminology Database project to collect data from many laboratories participating in the EQA program.
The percentage of patients with specific mutations may reflect ethnic combinations, environmental factors, and age distribution.
These may be very different from demographics in specific drug clinical trials and lead to a huge difference in treatment in the population.
Health care managers often use turnaround times to Judge services, which can be a blunt tool.
Timeliness of reporting is critical to patient care and depends on the patient path in the practice of each center.
For example, lung cancer is typically less than 5 business days, up to 10 business days, 5 business days, but for high-
The risk of developing melanoma or colorectal cancer, when results enter the patient\'s record, it may be enough to obtain results within a month if they need treatment.
Acceptability of longer turnaround times may allow for greater control of the test (
Requirement Management)
Or ingredients the sample to improve the cost-effectiveness of the test.
For all laboratories, regular participation in external quality assessments is required to verify and improve the quality of testing as described below.
Continuity planning is another problem: disruption of service should not be allowed to solve problems with equipment due to failure or limited staff.
Samples may need to be sent to another laboratory, but make sure there are alternative equipment or staff that can prevent the service from stopping and patients from having to wait for treatment decisions.
The referral laboratory shall check the service standards of the referral laboratory.
The pathology laboratory must be able to handle multiple sample types.
This raises some challenges when the need for molecular analysis is included in the diagnostic pathway, although many problems are common in any diagnostic test.
At each stage, samples are likely to be incorrectly identified and this danger should be carefully noted.
It is the responsibility of the person taking the specimen to identify the patient, the sample required, to ensure that the sample is correctly marked and fixed with formaldehyde (If necessary)
And/or sent to the lab.
However, when samples are taken in specific circumstances, for example in the operating room, the primary responsibility of the operating room staff is the patient, not the sample, and the need for implementation and operational failure should be considered
Ensure a safe procedure for the correct handling of samples to ensure reliable and accurate diagnostic and test results are obtained.
The SPIDIA project has recently studied the pre-analysis process in detail (
The results we understand will be included in the ISO guidelines (ISO 15189).
Tissue: Most diagnostic biopsies are small and can be fixed quickly when placed in neutral buffer formaldehyde (4% formaldehyde)
However, large surgical specimens need to be fixed under control.
15, 16 it may be best to control the fixation process by the pathology laboratory.
This may involve transporting fresh specimens to the pathology laboratory, and if it takes more than an hour, it is better to be in a vacuum (see below).
Molecular analysis of fresh tumor samples can be collected (
Or organization Bank)
In the operating room, but preferably after receiving it in the pathology laboratory according to the defined protocol.
It is best to open the intestinal specimens and (where possible)
Cut a larger mass to allow penetration of the fixer.
Formaldehyde only penetrates the tissue around 1 µmm/h, and fixing will only begin when penetration occurs.
After dilution to 4% w/v, formaldehyde should only be used within 24 hours to reduce the polymerization effect and ensure a stable 4% concentration.
Cold ischemia occurs between the resection tissue and the fixed tissue, which is a special problem for large surgical specimens.
It changes the level of gene expression (
At RNA and protein levels)
Is the main factor in molecular pathology.
17 control of fixed time and temperature is recommended for molecular analysis of proteins such as db2 2 (HER2)
Or for gene expression analysis.
Organize vacuum packaging and transfer to the laboratory at 4 °c (Tissue Safe)
It is an option to attract attention and is very useful for locations far away from hospital labs.
Cooling should be as fast as possible, cold fixation can also preserve RNA well despite cold shock
Some biomarkers may consider induced changes.
19 standard operating procedures (SOP)
Should be designed to include the organizational handling of the aspects listed in Box 1.
Box 1 organizes the standard operating procedures required for processing, including staff responsibility for each stage and sample identification using four identifiers (
Name, date of birth, hospital identification code, for example).
Specimen type and origin. Container type—
Charts and lists in theaters help ensure proper use.
Transportation requirements-
Time and temperature
Fixed requirements, including open gut specimens and open large chunks, while respecting the removed edges.
Shedding cell samples should also be processed according to the defined standard operating procedures.
In some centers, pathologists are involved in performing fine needle aspiration, which helps to process samples.
Fixation is usually in an alcohol fixer so that nucleic acids can be preserved relatively completely.
This makes this sample suitable for most molecular analyses, although some issues have been noted in human hpv (HPV)and non-
Small cell lung cancer (NSCLC)testing.
20, 21, 22, 23 liquid biopsy: nucleic acid extraction by Decell (buffy coat)and plasma.
The white blood cell section provides off-the-shelf sources of seed DNA and has long been used to generate molecular data for blood tumors.
Viruses and bacteria can be isolated and identified from Buffalo coated preparations by molecular methods. Cell-
Free DNA can be used for mutation analysis, and although this method has not yet entered into routine practice, they show considerable prospects.
13, 24, 25 preferred plasma from edta4 blood sent directly to the molecular pathology laboratory or the blood science laboratory.
The serum contains DNA of white blood cells, which is not suitable.
Conventional hematology measurements using the collected samples are feasible, but since lithium is a PCR inhibitor, the use of lithium heparin tubes should be avoided.
RNA should be taken care of, because continuous production in the cells and continuous degradation of RNAse activity after the sample is collected will affect the measurement of the level.
Preventive measures should be taken to minimize the degradation of RNA, but in most hospitals, it is not a standard procedure to quickly transport samples to the laboratory on ice, when collecting samples, it can be difficult to establish a cold chain or add inhibitors to the sample.
There are specialized blood vessels to preserve RNA (
For example, PAXgene, PreAnalytix/Kaijie;
Valencia, California, USA)
But their clinical use requires validation of the relevant tests.
In clinical laboratories, standard operating procedures are required for all processes.
It is important to use and adhere to work instructions and lists when necessary.
There should be a dedicated sample receiving process for sample receiving and processing, which should be equipped with appropriate staff and allow the sample to be assigned to join numbers when entering the laboratory (
According to ISO 15189 and ISO 9001).
Most laboratory management information systems (LIMS)
This is allowed, but few recognize the need to integrate molecular pathology needs.
It is a good practice to bar code the samples and it is worth encouraging.
All samples should be tracked from patient to report.
The tissue, blood and liquid treatment in the laboratory shall be carried out in accordance with the prescribed standard operating procedures (see ISO 15189).
When most tissue biopsy samples are fixed, fresh tissue can also be sent to the laboratory for in-operative frozen sections or molecular analysis.
This requires separate standard operating procedures and specialized handling, care and laboratory staff.
Tissue samples are inspected and cut at macro level
On stage, when the building blocks are taken down and placed in the bar --
Coding and/or numbered plastic boxes for processing.
Reserve tissue in formaldehyde can be retained at this stage.
Vacuum or microwave
Processor-based temperature control devices ensure good penetration of graded alcohol and PX alternatives, allowing paraffin to be embedded.
The temperature of different machines and protocols may vary greatly, which may affect nucleic acid recovery (
Cree, unpublished).
The use of previously frozen fixed tissues is not recommended, and de-mining will reduce the recovery of DNA, especially RNA.
It is found that neither of them produces or is not an ideal result.
26, 27, 28 it is recommended that the clinical team and the pathology laboratory work closely together, and the key is to communicate the changes in the processing to the molecular pathology laboratory and make adjustments if necessary.
Pathological diagnosis is performed according to the minimum data set requirements, stained sections cut from paraffin blocks of 4-6 μm (
For molecular testing, it is critical to take strict precautions to prevent cross-cutting
Contamination between samples.
It is best to change the knife (blades)regularly (
It\'s best before every new formaldehyde. fixed paraffin-embedded (FFPE)
Cut tissue blocks).
In addition, parts should be transferred to slides using disposable plastic products (
If part is used).
By using a drop of PCR-quality water on the slide, it is possible to avoid the use of a water bath to stretch the slices.
It should be noted that the purification procedure does not reduce the level of DNA or RNA required for analysis when the slices are cut.
For example, the use of DNA zap wipes on the slicer blade immediately before cutting can greatly reduce DNA levels and should be avoided.
The use of substances that may inhibit PCR should be avoided.
29. if used, it is recommended that pathologists mark on H & E slides at the time of diagnosis of macro or micro-anatomy parts of the region containing the tumor.
Manual microanatomy may require labeling multiple regions of the tumor to account for heterogeneity of tumor cell content in large tumors.
As an alternative to microanatomy, 1mm punches in the marked area can be used.
Laser capture microcutting is largely a research tool that is not necessary for conventional molecular pathology.
Tissue pathologists should estimate the percentage of tumor cells in the sample (
Or some samples)
Selected for DNA/RNA extraction.
This estimation may be important to determine the success or failure of subsequent tests, as it may define the lower limit of detection, and the minimum percentage of tumor cells present can be applied to many tests.
31 if there is necrosis, this should be noted, but it is best to avoid this in the sample for molecular analysis.
The widely dispersed tumor cells or lymph duct inflammatory cancers in the sample are notorious examples in which a low proportion of tumor cells produce false negative results.
If the pathologist is directly responsible for requesting molecular analysis, this \"reflection\" test allows for a quick turnaround time and prevents the need to retrieve the block later, reducing the test turnaround time, and therefore, reduce the time and cost of employees.
This needs to be balanced against unnecessary testing in patients who do not need further treatment, or in the future it may be necessary to conduct unnecessary testing in patients who need treatment when testing alternative gene mutations.
A clear assessment of tumor cell content and responsibility for macroscopic or microscopic anatomy is required: this is an important role for tissue pathologists.
The estimated percentage of tumor cells present in tissues used for DNA/RNA extraction should be mentioned in the pathology report. Blood samples (EDTA)
It is usually rotated at low speed to precipitate red blood cells and recover Buffalo coating and plasma fraction.
Should start processing as soon as possible (
Ideally within 30 minutes)after sampling.
The collection time should be indicated on the test tube and the elapsed time between sampling and processing should be recorded.
Cells are usually removed from plasma by centrifugal 10 min at 1000-2000g.
Centrifuge at 2000g for 15 min will also exhaust platelets in plasma samples.
Similarly, an agreement should be strictly observed.
It is feasible to obtain white blood cells, foreign bodies and plasma from the same sample using density centrifuge.
Molecular analysis can be performed by tagging 32 samples on many LIMS, and reflection testing may be feasible.
Aliquoted plasma samples can be stored at-80 °c.
Standard operating procedures shall be established to ensure the safety of tissue and blood samples and their parts and, where possible, control the collection, processing and storage (
Including extracted nucleic acid)(
See ISO 15189 and any country guide).
Few laboratories preserve tissue blocks and slides in a temperature-controlled environment.
Similarly, blood samples are usually discarded within days of receipt.
Blood and Blood
Derivatives are considered biological hazards and should be treated accordingly.
Fresh organizations have similar risks, but FFPE organizations are considered safe, although the chemicals involved in the processing of the organization need to be carefully stored and processed.
DNA and RNA extraction nucleic acid extraction for clinical use requires a quality control reagent, ideally ivd ce-marked.
Many companies produce kits, although rarely sold to clinical use, it is the responsibility of the laboratory to verify and validate any method in accordance with existing standards.
Some multi-center validation data have been released, but this is less direct.
Consider which method to use should consider throughput, the quality and quantity of DNA or RNA, and the expected analytical method.
Manual methods are commonly used.
Precipitation is mostly used in manual methods-
Wash steps with a centrifugal rotating column or filter.
Training and considerable skills are required even for the kit, but excellent results can be provided.
Special attention should be paid to avoiding sample error identification and contamination.
It is important to follow the standard operating procedures carefully --
It can be helpful to use the checklist and careful staff training.
Automated methods are useful because they can save staff time and help prevent sample misidentification.
They tend to use magnetic bead extraction methods suitable for robotic systems.
It is important to try out these systems before implementation, because not everyone is good at blood and FFPE block extraction: several machines may be needed to handle all sample types and different throughput.
DNA and RNA can be quantified by luminosity, fluorescence or PCR.
Different methods advocate different machines: Nano-drops, quantum-bit fluorescence meters, or Agilent bio-analyzers are widely used.
DNA degradation in FFPE samples reduces the accuracy of the spectrometer measurement.
However, quantitative reality
Time PCR can be used to establish the quantity, size and amplification capacity of DNA and RNA with considerable accuracy and limited sample consumption.
Standard operating procedures should indicate which validation methods are preferred for each test performed in the laboratory and provide appropriate training to the staff.
Continuous audit of pre-analysis performance through internal quality control is important so that changes in sample processing and nucleic acid extraction can be safely implemented.
Care should be taken to control the storage of DNA and RNA.
Adding logs or barcode vials can be used to prevent sample misidentification.
Temperature records should be maintained.
Generally speaking, storing extracted DNA and RNA samples at-20 °c or-80 °c is a good practice to preserve PCR products in separate refrigerators at 20 °c or 80 °c, respectively, save the sequencing library in a separate refrigerator at 20 °c or 80 °c
For different analytical methods, there is not enough data on the effect of storage length on tissue and extracted DNA or RNA: this is a consideration that may become more and more important, because patients may need to be tested many years after sampling.
DNA (or cDNA) is more stable than RNA and may survive for many years under given conditions.
The selection test requirements for analytical methods are defined by clinical requirements, which in turn are defined by the availability of drugs that have identified operational mutations (table 1).
This list is rapidly expanding and each Laboratory needs to coordinate their testing on a regular basis according to the needs of oncologists.
34 in some cancers, mutations are mainly located in several exons (‘hotspots’)
Rarely found elsewhere in genes
Since the goal is to identify viable mutations, the laboratory should screen all mutations with evidence of clinical efficacy.
For example, in the epidermal growth factor receptor gene, even if mutations account for less than 5% of the identified changes, mutations in Gene 18 should be systematically screened.
The minimum requirements can also be stated in the national guidelines (
For example, Germany requires testing of biomarkers with an incidence of 1% or higher).
View this table: View the inline View pop-up table 1 Examples of actionable genes for solid tumors in many laboratories where people have moved away from a single
Gene analysis for panel testing and reflecting this change in practice.
External Quality Assessment (EQA)
The plan is a tumor, or has been transferred to the tumor-
Specific protocols, distribution of tumor samples used to detect multiple genes, according to individual Laboratory Practices (
This protocol is expected to test the current relevant actionable mutations, but will also assess the accuracy of typing for other genetic tests where appropriate.
Other considerations when selecting an analytical method are the number of samples that need to be tested, the number of genes tested, and the percentage of mutations tested in each gene.
Most pathology labs now have
Fully capable of running a good time PCR machine
Kit for actionable mutations covering 95% of specific genes was established.
Sanger and Coke sequencing methods are also widely used.
In high throughput units, use next-
Sequencing (NGS)
Platform, its advantage is to cover more and more interested genes more widely.
The number of organizations available is often a limiting factor in the scope of testing (
Fine needle aspiration, endoscopic or needle biopsy, for example)
It may affect which technology to choose.
Consideration should also be given to the level of staff expertise, equipment and infrastructure required.
Many workflows in molecular pathology need to be separated in different compartments (ideally rooms)
Prevent contamination of DNA and RNA
Pre-SeparationPCR and post-
PCR steps are particularly important.
ISO15189 needs to be in-
Housing testing compared to CE
In vitro diagnostic markers (CE-IVD)tests.
However, since most tests are only part of the whole process, the complete process including the pre-analysis, pre-analysis, and post-analysis stages should be in-depth.
These considerations often determine the choice of technology.
In practice, these options are based on real
Time PCR or sequencing solutions.
Companies that offer this option have independently evaluated the receptor (
And Jiashi mutation detection (
But the data of many methods is limited.
The speed of technical change is quite large, except for the instructions that instruments and kits should generally be ivd ce-
Mark and analyze validation according to existing performance specifications.
Validation and validation of tests is important, whether in a consortium of companies, academic and hospital laboratories, or in individual laboratories that provide testing for clinical use. Batch-to-
For complex reagents, batch change is a special problem. in recent years, the performance of several mutation detection kits has fluctuated.
ISO15189 requires the inspection of new batches of reagents without strict inspection.
Panel testing may become common in a few years.
In fact, some laboratories are already turning to arrays or NGS methods, and the advantage of these methods is that they can provide information about multiple gene mutations without a large amount of DNA or RNA. The NCCD (
A panel test of non-small cell lung cancer was recently recommended, indicating that it is generally accepted.
It should be noted that the NGS pair is low-
In the PCR steps in this process, genetic changes at the level, as well as incorporating bioinformatics rules into data analysis, are necessary to allow correct and appropriate interpretation.
For example, formaldehyde induced C> T artifacts in DNA that may mimic mutations that need to be identified by bioinformatics methods. The performance of molecular testing is critical for monitoring the performance of all tests in the pathology laboratory and the parameters remain within an acceptable range, as defined in the test validation report.
In most cases, these are statistically defined based on known changes.
Using simple statistics, CIs with correct analysis behavior can be established (
Rules 36, 37).
For example, kit manufacturers often (
Not always though)
Provide internal control or reference genes, and the level of these genes should not be changed for the same DNA input.
While differences between samples are common, this should not exceed certain parameters, which can be defined from standard operating procedures. In-
Use Control from external sources as much as possible.
The percentage of individual tumor type mutations detected may vary according to the population tested, but results outside the national confidence range should always be questioned.
Again, the experience from her-2 detection is very appropriate and it should be noted that the knowledge in this area comes from
Terminology Database project to collect data from many laboratories participating in the EQA program.
The percentage of patients with specific mutations may reflect ethnic combinations, environmental factors, and age distribution.
These may be very different from demographics in specific drug clinical trials and lead to a huge difference in treatment in the population.
Health care managers often use turnaround times to Judge services, which can be a blunt tool.
Timeliness of reporting is critical to patient care and depends on the patient path in the practice of each center.
For example, lung cancer is typically less than 5 business days, up to 10 business days, 5 business days, but for high-
The risk of developing melanoma or colorectal cancer, when results enter the patient\'s record, it may be enough to obtain results within a month if they need treatment.
Acceptability of longer turnaround times may allow for greater control of the test (
Requirement Management)
Or ingredients the sample to improve the cost-effectiveness of the test.
For all laboratories, regular participation in external quality assessments is required to verify and improve the quality of testing as described below.
Continuity planning is another problem: disruption of service should not be allowed to solve problems with equipment due to failure or limited staff.
Samples may need to be sent to another laboratory, but make sure there are alternative equipment or staff that can prevent the service from stopping and patients from having to wait for treatment decisions.
The referral laboratory shall check the service standards of the referral laboratory.
Reporting molecular pathology tests for cancer patients sa Pathology reports should accurately communicate the information needed for clinicians to treat patients who are tested and provide sufficient information to properly interpret the results.
Therefore, accurate communication is the key.
Given the different groups of clinicians involved and the variable capabilities of the laboratory information management system, this is often a challenge.
There is an urgent need for enhanced communication between scientists, pathologists, oncologists and surgeons so that all groups are educated and tested, results and related treatments are correctly used and interpreted.
38, 39 different bodies have defined what should appear in the molecular pathology report.
Cancer Committee of American Society of SurgeonsACS-CoC)
Along with CAP, a checklist of scientific validation data elements containing all cancer sample reports was developed.
41-44 The External Quality Assessment Program for Molecular Pathology throughout Europe scores the report and test results and helps guide and educate what should be included in the laboratory report.
The European Pathology Society provides guidance for these programs, defining how to label the report, which will drive the response of the laboratory.
Similarly, laboratories across Europe often need to be certified.
In the UK, for example, this is certified by the UK service (UKAS)
ISO 15189 or equivalent, the inspectors pay close attention to the reporting and reporting standards.
In Germany, the national laboratory of cyclic testing conducted through QuiP (
Pathological quality, joint quality assurance program of the German Society of pathology and the German Association of Pathologists)
This makes it possible for clinicians and patients to check their reliability.
The Italian medical oncology association has similar policies (AIOM)
And the Italian Society of Pathology (SIAPEC)
Organize their external quality assessment program.
Finally, many hospitals and laboratories have internal considerations, often due to the preferences of clinicians or pathologists.
The suggested format is displayed in (
The following are three key areas that should be presented in the report.
Patient identification patients must be correctly identified-the laboratory requires at least two unique patient identifiers plus a unique sample identifier on the application form and report.
These include the patient\'s name (family name)
Date of birth, hospital and national identification number.
The ward or service, date of biopsy and name of the recommended clinician should also be stated. 2.
Rarely read the report in full style and long content, the length is important;
One page, or better yet, one page
Screen reports are preferred if they are legible.
If the length of the report is more than one page, then in order to link to the correct patient, each page should have the appropriate patient identifier and the page should be numbered, that is, the first page of 3, so the reader of the report knows if any page is missing (
See ISO 15189 for guidelines).
Clear presentation results, testing (s)
Any restrictions on testing (
Are all possible mutations detected or are they just options for common mutations? ).
For some tests, the percentage of samples occupied by the test target (
Tumor cells)is important.
Ideally, the report should contain basic information about fixing (
Fixed objects, fixed time, etc. )
For quality control, although this may be kept in department records, not reported.
The name and contact details of the individual responsible for testing are important and have some form of authorization for the date.
Due to the laws and regulations of some European countries, the final responsibility is to combine the \"form-
Molecular reports must be legible.
Spell checker is very useful for preventing spelling and printing errors. 3.
The interpretation must properly report the results of the test and provide an explanation, especially in cases involving treatment decisions.
Genetic typing results should be given according to HGVS nomenclature (
Provide appropriate reference sequences at both DNA and protein levels.
The external quality assurance program requires the laboratory to provide an accurate explanation of the results (s)
Obtain and include any relevant suggestions.
Attention should be paid to ensuring that this recommendation is as up-to-date as possible, and if conflicting data is released, then this should be included in the report.
In view of the growth in the accompanying diagnostic tests in molecular pathology, pathologists may increasingly be required to include clinical interpretation and treatment protocol advice.
The type and scope of molecular analysis used should be clearly described so that oncologists request further testing where clinical conditions permit. 4.
The comprehensive report broadly recognizes the need for comprehensive reporting results, and as the introduction of gene group tests becomes more widespread, the results of different genetic tests should be reported in one report.
In addition, the results of several pathology specialties in individual patients need to be integrated into the same report.
For example, patients with enlarged lymph nodes can perform biopsy and blood samples to provide reports of tissue pathology, microbiology, immunology, molecular pathology, hematology, and biochemistry.
Clinicians who treat patients must combine these with radiation and clinical investigations to make the best decision.
While it is helpful to obtain all pathological results in one report, not all countries provide comprehensive pathological services.
It is also difficult to do this with different reporting times and systems.
The best solution seems to be a joint reporting system, or an electronic medical record that allows clinicians or multi-disciplinary teams to determine if they have all the information available to come to a reasonable conclusion.
Certification and quality assurance
Internally and externally, we believe that all laboratories that provide molecular pathology services should obtain laboratory certification in accordance with ISO 15189 or their national equivalent standards.
Certification provides evidence of laboratory capabilities for patients, staff, service users and specialists.
The standards include the provision of adequate facilities, trained staff and documentation.
We suggest that as part of the certification, the laboratory should ensure that their supporting diagnostic tests cover all relevant genes that appear in the product feature summary (SmPC)
Published for drugs.
Laboratories that want to be certified should contact their national certification authority (NAB).
47. most NABs have formal application documents to fill in, and preliminary documentation of the laboratory itself and the existing quality management system is required (QMS).
The certification body will then appoint a chief assessor and a technical assessor (s)
Who is an expert in this field?
The panel of assessors will conduct an audit (external audit)
They will formally report the results of the assessment to the NAB in the laboratory.
In the case of minors
After compliance, the assessor will check whether the corrective action is sufficient through the submitted documentation.
In case of major non
Compliance, a new
In order to assess the effectiveness of corrective actions, an on-site assessment may be required.
Certification for up to 3 or 5 years is available, but NABs will conduct monitoring visits to ensure that the laboratory continues to meet the requirements normally every 1-2 years.
Procedures for extending certification are often very similar to those for new applicants, but the laboratory may consider expanding the scope of certification by adding new tests.
All laboratories that conduct molecular testing for cancer patients should be part of the EQA program (eg, (((((
In fact, in most countries, ongoing certification requires EQA and regular audits of test performance.
49 The European provider of solid tumor molecular pathology EQA program has published minimum acceptable standards for such EQA programs and signed agreements that meet them.
While there are still differences in practice in each programme, these principles have now been agreed.
There are fewer articles on the necessity of internal quality assessment.
Where technology allows, it is recommended to use control materials in each run.
Results of internal control tests should be monitored to ensure final resultsto-
The final performance of the test.
50 internal controls should be carried out for all batches.
Internal active control can be prepared from samples with sufficient remaining materials, and can also be purchased from commercial sources.
Standardized products should be used to ensure the stability of the results and external validation.
In some countries, national reference centres may provide control materials, which may be helpful as an inspection of control materials generated internally, although the quantity is often limited.
Errors found by EQA are divided into several categories.
Error recognition at all stages of the testing process is a problem that causes mutations or wild-
Type results, ultimately, incorrect treatment of the patient.
Typing errors do occur, usually due to human errors, not following standard operating procedures, and the method of execution is not ideal.
Careful internal verification of the test is essential, recording batches and batch numbers can save weeks of investigation with the manufacturer.
Interpretation errors are less common and if the data presented does not match the conclusions, the clinical team may find an interpretation error.
Therefore, the results of the tests and explanations must be stated in the report.
Those involved in the EQA program must read the report detailing the mistakes made by all participants and act on the suggestions made and ensure that their own mistakes are promptly corrected.
Laboratories with poor performance should stop service immediately, conduct root cause analysis, and review cases that may be affected by system errors.
Before determining that their tests are safe, they should send the samples to another provider.
While in some countries, E