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GENETIC TESTING BIOTECHNOLOGY

Advice to Scientists

Advice to Scientists Incorporating Authentication into Everyday Culture Practice

1) Planning a project or grant application

a) Identify a validated source of cell lines you wish to use, e.g., a cell bank that will provide evidence to guarantee authenticity and absence of microbial contamination including mycoplasma.

b) If such a source is not available:

i) Do a PubMed and Google search using the name of the cell line.

ii) Identify the originator of the cell line – only if cells are not available from the originator is it necessary to approach a secondary user of the cells.

iii) Check that cell line is not on ICLAC list of misidentified cell lines:http://standards.atcc.org/kwspub/home/the_international_cell_line_authentication_committee-iclac_/Cross_Contaminations_v7_1.pdf

iv) Confirm that source laboratory has some evidence of authenticity.

v) Confirm that cell line designation is unique and cannot be confused with a different cell line.

2) Initiating work on a new project

a) Obtain cell lines from a validated source if possible (as in 1a above).

b) Expand and freeze your own working stocks (as in 4 below).

c) Take additional precautions while preparing stocks if a validated source is not available:

i) Handle under quarantine.

ii) Freeze token stock (2-3 vials to guard against future loss).

iii) Confirm freedom from mycoplasma and other microorganisms.

iv) Confirm identity by performing authentication testing (as in 5 below).

v) If clean and authentic, release from quarantine and freeze additional stocks (as in 4 below).

3) Initiating new cell lines

a) Retain sample of patient's blood and/or biopsy in liquid nitrogen for DNA extraction and STR profiling.

b) When cell line is subcultured:

i) Freeze token stock (2-3 vials to guard against future loss).

ii) Check medium supernatant for mycoplasma contamination.

c) When cell line is established as a finite or continuous cell line (and before distributing as the originator of the new cell line):

i) Confirm identity with reference to donor DNA from blood or biopsy (as in 5 below).

ii) Confirm freedom from mycoplasma and other microorganisms.

iii) If clean and authentic, freeze additional stocks (as in 4 below).

iv) Keep records for all stored vials including total number of passages, date, and test results.

v) Deposit cell line at an international cell bank or distribute to others with appropriate data on validation.

4) Freezing down stocks

Laboratories needing a small number of vials, and who receive those vials from a validated source,may choose to prepare a single working stock (~20 vials). Stocks should be replenished if they reach the last 5 vials from the original stock.

Cell banks or laboratories needing a larger number of vials will prepare stocks at several passages:

A small stock (12-20 vials), also known as "seed stock" or a "master bank", is prepared first and used only to replenish stocks later on.

A single vial from that stock is expanded to give a large number of vials (up to 100 vials), also known as "distribution stock" or a "working bank". Vials from that stock are suitable to release to members of that laboratory for experimental work. Cell banks have permission from the originator to use those stocks for wider distribution.

A lab member receiving such a vial will prepare a working stock (~20 or more vials) before commencing experimental work using that cell line. Lab members should return to those stocks periodically to avoid overpassaging. Working stocks should not be distributed to other users,even within the same laboratory. Other users should obtain cells from the validated distributionstock.

When freezing down stocks for future use, always:

Keep records for all stored vials including total number of passages, date, test results, any unique distinguishing growth behavior, and any known genetic features (e.g., mutations).

Confirm identity of stocks by authentication testing.

Store frozen stocks across at least 2 independent storage systems, to reduce the risk of losing all stocks in case of equipment malfunction.

5) Authentication testing

Authentication testing aims to compare the DNA profile for that cell line to other samples from the same donor to confirm the cell line is not cross-contaminated or otherwise misidentified. If donor tissue or cell lines are not available, the comparison is made with reference to online databases containing DNA profiles from a large number of widely used cell lines.

For human cell lines, it is recommended that cell lines are tested using short tandem repeat (STR) profiling in accordance with the standard ANSI/ATCC ASN-0002-2011 Authentication of Human Cell Lines: Standardization of STR Profiling. Recommendations from the standard should be followed, including the use of at least eight core STR loci and application of match criteria (80% match threshold) to allow for a small amount of genetic drift in some cell lines.

For non-human cell lines, best practice will vary with the species being tested. As a minimum, it is recommended that non-human cell lines are tested for species-specificity and absence of human and rodent cell lines. Appropriate test methods include karyotyping, isoenzyme analysis, and mitochondrial DNA typing (DNA barcoding). Test methods, including STR profiling, are available commercially at a modest cost from various suppliers, including some of the cell repositories.

6) More Information

For additional information on good cell culture practice or testing for contamination, see:

Freshney RI. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (6th edition). Wiley-Blackwell 2010.

Cree IA (editor). Cancer Cell Culture: Methods and Protocols (2nd edition). Methods Mol Biol 2011; 731. Humana Press.

For more on contaminated cell lines or authentication requirements, and procedures, see:

ICLAC web page

http://standards.atcc.org/kwspub/home/the_international_cell_line_authentication_committee-iclac_/

List of cross-contaminated or misidentified cell lines

http://standards.atcc.org/kwspub/home/the_international_cell_line_authentication_committee-iclac_/Cross_Contaminations_v7_1.pdf

ANSI/ATCC ASN-0002-2011. Authentication of human cell lines: Standardization of STRprofiling. The Standard can be purchased at the ANSI eStandard store:

http://webstore.ansi.org/RecordDetail.shtmlx?sku=ANSI%2fATCC+ASN-0002-2011

Guide to human cell line authentication (Authentication SOP) See ICLAC web page

Guide

Guide to Human Cell Line Authentication

This protocol sets out the essential steps to follow when authenticating human cell lines.

SUMMARY

1. Send cells or DNA to an experienced supplier of STR profile testing, working according to the human cell line authentication Standard published by ANSI (1).

2. Compare STR profile to donor and all other known cell line STR profiles, interpret as below

3. Check the cell line name against the database below of misidentified cell lines

4. If in doubt, ask ICLAC

5. If the cell line is false, throw it out

More detailed information and caveats are included below.

A. Check the database of misidentified or cross-contaminated cell lines

Using the database, check the name of the cell line you wish to work with before you start work and before you test it. The latest version of the database is at:

http://standards.atcc.org/kwspub/home/the_international_cell_line_authentication_committee-iclac_/Database_of_Cross_Contaminated_or_Misidentified_Cell_Lines.pdf

This step will alert you to possible problems with the cell line and may save you considerable time andeffort. However, it should be noted that if a positive match is found, it does not necessarily mean thatyour sample is cross-contaminated. There may be multiple stocks of cell lines with the same name from many sources, only some of which may be cross-contaminated. However, the likelihood is that the cells are not as claimed. Paradoxically, the lack of any match again does not give 100% confidence of authentication as there are many name changes and false cell lines yet to be identified. Consequently any new cell line entering a laboratory stillmust be profiled using short tandem repeat (STR) genotyping.

For more background on the database, see Reference 2.

B. Prepare starting material for STR profiling

Starting material should include:

1. DNA from the donor of the cell line if established within your laboratory

Snap freeze a few milligrams of tissue in liquid nitrogen (e.g., part of the biopsy, blood cells, or buccal smear) from the donor. DNA from this sample can be used later to provide unequivocal evidence (STR profiling) that the cell line is derived from the donor.

2. DNA from the cell line with which you wish to work

Where possible, it is advisable to purchase a cell line from a reputable supplier that provides characterisation and an STR profile. This STR profile can then be used for comparison when testing your own stock.

At minimum, testing should be performed at the beginning and end of experimental work. Test each batch of frozen vials (cell bank) that you prepare for that cell line.

Some scientists prefer to check the STR profile of the cells before they are banked, others after they are banked. Either way, it is essential that all batches of cells are STR profiled at the time they are frozen down and before they are used or distributed. Confidence is then assured for the length of time the sample is available that the cells have the determined STR profile. Any other method of maintaining the cell stock (e.g., continuous culture or multiple freeze/thaw cycles) is poor tissue culture practice.

For more information on cryopreservation of cell line stocks, see Reference 3.

C. Perform STR profiling

STR profiling can be performed in-house or outsourced. STR profiling should only be done in-house if it is routinely used and conforms to the criteria set out in the human cell line authentication Standard (1). For various reasons, the International Cell Line Authentication Committee (ICLAC) is not able to specify suppliers. Suppliers can be found online (e.g., by entering "cell line str profiling" or "cell line authentication" into your search engine).

It is essential that the supplier chosen provides a rapid and reliable service with appropriate accreditation and quality assurance. The supplier should provide an STR profile and quality control data conforming to the Standard (1).

Some suppliers ask for DNA, while others accept either cells or DNA. Suppliers may express a preference for samples spotted onto FTA paper, allowing shipping and storage at room temperature. A single T25 flask will provide more than enough cells or DNA to obtain an STR profile. If the laboratory cannot produce DNA that meets the quality required by the Standard (1), cells should be supplied.

D. What to do with the result when it arrives: STR profile comparison

An STR profile is obtained by PCR amplification of a set of STR loci. The profile consists of a series of numbers corresponding to the number of repeats seen at each allele for that locus – for example, 8 STR loci will give a maximum of 16 numbers (corresponding to 16 alleles) if all are heterozygous. A minimum of eight core STR loci plus amelogenin for sex determination (X or XY) are required for cell line authentication. Incidentally, cells can lose some of the Y chromosome in culture and therefore sex determination can only be regarded as indicative.

The next step is to compare the STR profile to an appropriate reference sample – for example, DNA from the donor of that cell line. Guidelines on interpretation of results are given in the human cell line authentication Standard (1). A worksheet is available from ICLAC to summarise the essential concepts when comparing a test sample to a second reference sample (see References and Resources section).

The result should also be compared to every other STR profile available using an online interactive database of human cell line STR profiles. Some STR genotyping service providers offer this service along with the profile. The cell bank databases currently available for comparison include:

http://www.atcc.org/CulturesandProducts/CellBiology/STRProfileDatabase/tabid/174/Default.shtmlx

http://www.dsmz.de/services/services-human-and-animal-cell-lines/online-str-analysis.html

Additionally, a resource for human cell line STR profiles is currently under development by the National Center for Biotechnology Information (NCBI) as part of the BioSample database at

http://www.ncbi.nlm.nih.gov/biosample/.

The NCBI BioSample database is now accepting human cell line STR profiles from the community. Scientists and cell banks interested in submitting STR profiles to the database are invited to contact curation staff at biosamplehelp@ncbi.nlm.nih.gov for more information.

Points to be aware of when comparing cell line samples:

1) To be completely certain that a cell line is authentic, you need to compare it to another sample from the same donor. For many cell lines, another donor sample is not accessible. In those cases, comparison to a database with STR profiles from many different cell lines will give you a high degree of confidence that your sample is not misidentified.

2) STR profiling does not allow you to distinguish between cell lines arising from the same donor. All cell lines from that donor will have the same, or highly similar, STR profiles.

3) A small amount of STR profile variation may be seen between cultures derived from the same donor. This can be caused by genetic drift with passage, particularly in cell lines with microsatellite instability. Variation may also relate to laboratory differences in test methods or interpretation. It is important to apply match criteria (80 % match threshold) as recommended by the Standard (1) to allow for a small amount of variation in some cultured samples.

4) Increasing the number of STR loci included in the analysis will increase the probability that the STR profile is correctly authenticated as arising from that donor. Eight STR loci plus amelogenin are recommended as a minimum (1) and are used in the databases listed above.

To use an STR profile database:

1. You may be required to register the first time you use such a database. If so, have your log in and password information along with the STR profile you wish to compare, and log in to the system as your first step.

2. Enter your STR profile as set out in the search form. STR profiles include results for different loci (e.g., D5S818, TH01, amelogenin). For each locus on the search form, find the corresponding locus in your sample and enter its results. Results are given in the format "A,B" where A and B represent two different alleles present at that locus. You may not be required to enter all of the loci from your result. The online STR profile databases use a minimum set of loci; many of the current STR kits will amplify a greater number of loci.

3. Search the database to find the closest matches to your sample. Samples are compared using a match algorithm; search results will usually list the closest matches first.

4. To interpret your results, it helps to look at the percent match or EV value. Samples from the same donor generally yield a result in the 80-100 % match range (EV 0.8-1.0), while samples from different donors generally lie in the 0-55 % match range (EV 0-0.55).

5. It also helps to know the provenance of your cell line. Was it derived from another cell line, or are there other cell lines known to be established from the same donor? If so, these cell lines would legitimately have the same STR profile. A misidentified cell line is one where the STR profile fails to correspond to the expected donor, or where the STR profile unexpectedly corresponds to an unrelated cell line.

E. What to do with misidentified cell lines

If your testing shows that a cell line is misidentified, stocks of it should be discarded and any colleagues who have other stocks of that cell line should be informed.

If you discover a novel cross-contamination, you may choose to publish your finding. If the cell line is used widely and you believe that no authentic stocks exist, publication is an important step to alert others in the scientific community.

Please also contact ICLAC at standards@atcc.org so the cell line can be added to the database of misidentified cell lines. If you wish to add a new misidentified cell line or edit entries within the database, we will ask for more information to help us review your findings and decide on the best course of action.

References and Resources

References cited in this document:

1) ANSI/ATCC ASN-0002-2011. Authentication of human cell lines: Standardization of STR profiling. The Standard can be purchased at the ANSI eStandard store:

http://webstore.ansi.org/RecordDetail.shtmlx?sku=ANSI%2fATCC+ASN-0002-2011.

2) Capes-Davis et al. (2010) Check your cultures! A list of cross-contaminated or misidentified cell lines. Int J Cancer 127 (1): 1-8.

3) Stacey N and Masters JRW (2008) Cryopreservation and banking of mammalian cell lines. Nature Protocols 3: 1981-1989.

For additional resources relating to contaminated cell lines and authentication testing, see:

ICLAC web page

http://standards.atcc.org/kwspub/home/the_international_cell_line_authentication_committee-iclac_/

List of cross-contaminated or misidentified cell lines

http://standards.atcc.org/kwspub/home/the_international_cell_line_authentication_committee-iclac_/Database_of_Cross_Contaminated_or_Misidentified_Cell_Lines.pdf

Worksheet – match criteria for human cell line authentication Word file available under "References" at:

http://standards.atcc.org/kwspub/home/the_international_cell_line_authentication_committee-iclac_/

Advice to scientists – incorporating authentication into everyday culture practice See ICLAC web page

Terms of Reference

What Do We Mean by Cell Line Misidentification?

A cell line is misidentified if its DNA profile is no longer consistent with the donor from whom it was first established. Such a cell line can be described as a misidentified cell line, or as a false or imposter cell line. Investigators who are unaware that they are working with a false cell line may use it in their work, without realising that it comes from an entirely different cell type or tissue – perhaps even a different species – and is likely to be unfit for their application. This lack of awareness leads to unreliable research findings, and the use of those unreliable findings by other scientists in turn.

Although there are many causes of false cell lines, including mislabelling of culture samples, the problem is often caused by cross-contamination (see the Definitions later in this document). Many cell lines have been cross-contaminated during establishment and so all subsequent work based on the false cell line has used the contaminant rather than the correct species, tissue or cell type that was originally present in that culture. Once cross-contaminated, cell lines are often handed from laboratory to laboratory without anyone being aware that their stocks may be unfit for use. This practice can only lead to confusing results and represents a waste of precious resources.

Why Do We Focus on Cell Line Authentication?

Authentication testing is an effective way to combat the use of false cell lines. Authentication testing aims to compare a test sample to other reference samples from that donor, or to a database of reference samples if donor material is not available, to see whether samples correspond. The test method should distinguish between different species and different individuals within that species, although this will depend on the technology available to the field of authentication testing. When multiple cell lines have been established from a single individual, identification rests on additional test methods and characteristics such as tissue-specific markers, phenotype and morphology. While all such testing falls within the realm of authentication, the latter methods are unreliable as identity tests and therefore these aspects of authentication are outside the scope of this document.

In 2011, the American Type Culture Collections Standard Development Organization (ATCC SDO) published a standard on authentication testing of human cell lines (ANSI/ATCC ASN-0002-2011). A new group, the International Cell Line Authentication Committee (ICLAC), was formed after publication of the standard to provide guidance and an ongoing focus for improvement in this area.

ICLAC aims to make the use of false cell lines more visible and to promote awareness and authentication testing as effective ways to combat the problem.

Goal 1: Make Cell Line Misidentification More Visible

A number of laboratories and cell line repositories have uncovered false cell lines, in publications dating from the 1960s. More information will come from public databases such as the NCBI cell line database, currently in preparation as part of the standard. These online, interactive databases give cell banks and laboratories an effective way to compare samples and share authentication test results. But it is important that the older reports are not lost and that any incorrect or inaccurate information added to the literature, or to online databases, should be addressed.

To make this information more visible and informative, ICLAC aims to:

1. Review reports of misidentified or cross-contaminated cell lines

2. Gather information to provide a written response, where needed, to the scientific community

3. Manage a master list of misidentified cell lines for the research community and for feedback to the NCBI cell line database

As a resource for the research community in this area, members of the group have previously developed a single list of cross-contaminated or misidentified cell lines. The list will be used as an initial template for this goal, with ongoing updates and online release of updated information. It can then be used as a tool to help ensure that reports of cell line misidentification are recorded, made accessible to the research community, and any inaccuracies are corrected.

Goal 2: Promote Authentication Testing

The standard referred to above is an important resource for authenticating human cell lines. However, there is a need for education and resources to help laboratories apply the standard in their own situations.

How this is done will depend on the resources available to the group. Possibilities include:

Provide advice to scientists planning a project or grant application, starting new work or initiating new cell lines

Guidelines and protocols on application of the standard and recommended test measures

Shared policies on difficult issues

Shared data to provide more effective datasets for shared online databases

Approaches to journals and funding bodies to encourage mandatory authentication testing, with assistance if needed to make such testing recommendations easier to make.

Goal 3: Harmonization of Guidelines and Standards

In addition to the standard referred to above, other standards and guidelines are relevant to authentication testing or – more broadly – to good cell culture practice.

Use of standards and guidelines may vary from country to country because of different regulatory frameworks. Use may also vary from one application to another, depending on the type of cell culture being performed. However, it is important to maintain consistency across all reference documents wherever possible, so that laboratories are not faced with conflicting requirements. ICLAC aims to assist in harmonization of relevant guidelines and standards by providing an independent forum for communication and discussion of such reference documents as they arise.

Ground Rules

ICLAC members have been invited to join the group based on expertise in cell line misidentification, authentication testing, or database applications. Members act in a voluntary capacity and their individual contributions and commitment to addressing the problem of cell line misidentification are respectfully acknowledged.

Contributions from members are subject to the policies of their individual institutions. All shared policies and sharing of data must be approved by the contributing organisation.

The committee meets every three months by teleconference. Correspondence and other business are carried out by email as far as possible, to allow members from different time zones to contribute.

A new finding of cell line misidentification can be reported by any member. New reports will be distributed to the members with an opportunity to give feedback and contribute further data where available. If members accept or endorse the initial report, the curator of the list of cross-contaminated or misidentified cell lines will add that entry to the list. If any member challenges the initial report, the cell line will be reviewed by the members and a decision made on its status based on available data.A lack of a response from a member will be taken as agreement to the cell line being added to the list.

A change in status for cell lines already entered on the list can be reported using a similar process. For example, if authentic stock is found, the cell line can be moved from Table 1 (no known authentic stock) to Table 2 (authentic stock known).

Duration and Terms of Reference

No duration has been set for the committee. To promote effective use of resources, the committee will review its purpose and goals on an annual basis via teleconference. The Terms of Reference can be modified by the group following teleconference discussion. All changes must be reviewed by the members via email before being adopted.

Definitions

Some words are used in many different ICLAC documents and resources. They include:

Authentication. The aim of authentication is to confirm or verify the identity of a cell line, ensuring that it is derived from the correct species and donor. Testing involves comparison of a test sample to other reference samples from that donor, or to a database of reference samples if donor material is not available, to see whether samples correspond. Ideally, the test method should distinguish between different species and different individuals within that species, although this will depend on the technology available to the field of authentication testing. Not all currently used test methods have the power of discrimination of STR profiling or SNP testing; therefore authentication may not in all cases lead to unambiguous identification of cells to a specific donor or donor tissue. Where unambiguous identification is not possible, species verification is used as the best alternative currently available.

Cross-contamination. The term contamination refers to introduction of foreign material into a cell culture. Cross-contamination occurs when that foreign material consists of cells from another culture. Cross-contamination initially results in a mixed culture, containing cells from the authentic culture and the contaminant. If the contaminant has a survival advantage – for example, if it proliferates more rapidly – it will overgrow and replace the authentic cells within the culture. A contaminant usually comes from a different donor or species and so can be detected by authentication testing.

Misidentification. A misidentified cell line no longer corresponds to the donor or species from which it was originally established. Misidentification may arise due to cross-contamination. It may also arise from a variety of errors, including mislabelling of samples. If it happens early – for example, during cell line establishment – there will be no authentic material retained, and the cell line will be considered to be a false cell line. If misidentification happens late – for example, after the cell line is established and distributed to other locations – then authentic material may still exist and only some stocks may be false.

Misidentification does not refer to problems with the technical procedure of authenticating cell lines. It also does not typically extend to other characteristics such as tissue type, cell type or disease state.If the tissue type, cell type or disease state of a cell line is incorrectly attributed, the cell line is considered to be misclassified.

Members

Information on what the ICLAC members do can be found in the Ground Rules section.

A full list of committee members and their affiliations can be found on the ICLAC website.

Partner Organizations

Organizations have provided support to ICLAC since inception in a number of ways, including administrative support; website resources; sourcing of cell line samples; and sample testing. Organizations have also enabled staff to contribute their time as ICLAC members. We wish to acknowledge all of the organizations who have supported the work of the committee in different ways.

A full list of partner organizations and funding sources can be found on the ICLAC website.

Authentication

International Cell Line Authentication Committee

Goal 1: Make Cell Line Misidentification More Visible

A number of laboratories and cell line repositories have uncovered misidentified cell lines, in publications dating from the 1960s. More information will come from public databases such as the NCBI cell line database, currently in preparation as part of the standard. These online, interactive databases give cell banks and laboratories an effective way to compare samples and share authentication test results. But it is important that the older reports are not lost and that any incorrect or inaccurate information added to the literature, or to online databases, should be addressed.

To make this information more visible and informative, ICLAC aims to:

1. Review reports of misidentified or cross-contaminated cell lines

2. Gather information to provide a written response, where needed, to the scientific community

3. Manage a master list of misidentified cell lines for the research community and for feedback to the NCBI cell line database

As a resource for the research community in this area, members of the group have previously developed a single list of cross-contaminated or misidentified cell lines. The list will be used as an initial template for this goal, with ongoing updates and online release of updated information. It can then be used as a tool to help ensure that reports of cell line misidentification are recorded, accessible to the research community and any inaccuracies corrected.

Goal 2: Promote Authentication Testing

The standard referred to above is an important resource for authenticating cell lines. However, there is a need for education and resources to help laboratories apply the standard in their own situations.How this is done will depend on the resources available to the group. Possibilities include:

Provide advice to scientists planning a project or grant application, starting new work or initiating new cell lines

Guidelines on application of the standard and recommended test measures

Shared policies on difficult issues

Shared data to provide more effective datasets for shared online databases

Approaches to journals and funding bodies to encourage mandatory authentication testing, with assistance if needed to make such testing recommendations easier to make.

Ground Rules

ICLAC members have been invited to join the group based on expertise in cell line misidentification, authentication testing, or database applications. Members act in a voluntary capacity and their individual contributions and commitment to addressing the problem of cell line misidentification are respectfully acknowledged. Administrative support and website resources have been made available to the group from the ATCC SDO as part of an ongoing commitment to authentication testing.

Contributions from members are subject to the policies of their individual institutions. All shared policies and sharing of data must be approved by the contributing organisation.

The committee meets every three months by teleconference. Correspondence and other business are carried out by email as far as possible, to allow members from different time zones to contribute.

A new finding of cell line misidentification can be reported by any member. New reports will be distributed to the members with an opportunity to give feedback and contribute further data where available. If members accept or endorse the initial report, the curator of the list of cross-contaminated or misidentified cell lines will add that entry to the list. If any member challenges the initial report, the cell line will be reviewed by the members and a decision made on its status based on available data. No response from the members will be taken as agreement to the cell line being added to the list.

A change in status for cell lines already entered on the list can be reported using a similar process.For example, if authentic stock is found, the cell line can be moved from Table 1 (no known authentic stock) to Table 2 (authentic stock known).

Duration and Terms of Reference

No duration has been set for the committee. To promote effective use of resources, the committee will review its purpose and goals on an annual basis via teleconference. The Terms of Reference can be modified by the group following teleconference discussion. All changes must be reviewed by the members via email before being adopted.

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