Managing the Cold Chain to Minimize Pre-Analytical Variability
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The presentation discusses the critical importance of maintaining the integrity of biological samples throughout the cold chain process to minimize pre-analytical variability. It provides guidelines for proper inventorying, transport, and storage at various temperature ranges to avoid freezing and thawing, which can damage samples. The document emphasizes the need for controlled methods during sample transfer and re-freezing to ensure quality and reliability in clinical diagnostics and research.
Managing the Cold Chain to Minimize Pre-Analytical Variability
- 1. Managing the Cold Chain to Minimize Pre-Analytical Variability Dr. Alex Esmon Senior Product Manager Thermo Fisher Scientific
- 2. 2 Today’s agenda • We will discuss three scenarios • Importance of maintaining sample conditions • Concerns during Inventorying • Sample Transport • Unit transfer • Content transfer
- 3. 3 Why is this important? • The integrity of samples must be maintained at all times otherwise there will be variation brought about that can skew results and analysis • For clinical diagnostic development, therapeutics, epidemiological studies, and “-omics” studies, the ability for biobanks to "control" pre- analytic variation throughout the biosample journey from collection and processing through storage and transport is going to be the foundation for the development of precision medicine.
- 4. How Do I Maintain Sample Integrity? Product Management Team Thermo Fisher Scientific
- 5. 5 Inventorying, Shipping and Maintaining Sample Integrity • Ensuring sample integrity is paramount during shipping or inventory - what’s the point of shipping or inventorying samples that aren’t worth saving? • These processes can be accomplished in harmony • Imperative to understand some basics about the freeze/thaw and ice phase transition process • Follow practical tips for incorporation into lab SOP
- 6. 6 Refrigerated Storage: the +4ºC Range • Refrigerated samples need to avoid freezing! • Keep them in the +10ºC to +1ºC range if possible Going back and forth between frozen and thawed states is very bad for refrigerated samples - ice formation and cellular dehydration are very stressful and will cause mechanical damage to biological samples. Some guidelines to follow: • Guard against samples getting too warm • Avoid dry ice for non-frozen samples • A water/ice slurry is the best option for holding samples during the inventory process • Minimize the duration and frequency of door openings - this will help the samples that are not actively being handled to maintain their integrity and keep the samples in the right temperature range
- 7. 7 Frozen Storage: the -20ºC Range • Frozen samples need to avoid thawing! • Keep them in the -20ºC to -30ºC range if possible The key here is to keep samples from thawing into the liquid state. The phase change from solid ice to liquid and back again is highly stressful for biological samples - ice formation can perforate cell walls or change the structure of compounds. Some guidelines to follow: • With samples in -20º freezers you have some options, but speed is the key • Dry ice or bench-top coolers around -20ºC are better than just an ice bucket • If using dry ice, be aware that enzymes in glycerol are not recommended to be frozen, so work quickly • If an ice bucket is your only option, use a water and ice slurry for better temperature transfer than ice alone
- 8. 8 Ultra Cold Storage: the -80ºC Range • Frozen samples need to avoid thawing! • Keep them in the -60ºC to -90ºC range if possible Thawing refers to not only the change from ice to liquid but also the change from ice phase to ice phase. Just because it “looks frozen” doesn’t mean changes aren’t happening inside the sample. Some guidelines to follow: • Samples in the ultra cold storage range are riding the line between two forms of ice. As much as possible, these samples need to be handled using dry ice which has a temperature of about -70ºC. This will keep the samples from warming into a higher temperature range and will reduce undue stresses on the samples • If these samples thaw, make sure you use a controlled freezing technique or equipment designed to control the rate of freezing (Mr. Frosty or a mechanical controlled rate freezer) to bring them back to the cryogenic range
- 9. 9 Cryogenic Storage: Frozen Means Frozen, Right? • Frozen samples need to avoid thawing! • Keep them below -130ºC if possible Thawing not only refers to the change from solid ice to liquid but also the change from ice phase to ice phase. Just because it “looks frozen” doesn’t mean things aren’t happening or changing. Some guidelines to follow: • Samples stored below -130ºC are in a “glassy state”. They have crossed the glass transition phase and have taken on the properties of glass. Below this threshold, all known mechanical and metabolic activity within the cells stops. • It is imperative to keep samples that are stored below the glass transition phase under this threshold at all times. Samples that toggle back and forth across this threshold can experience instantaneous ice nuclei formation which can seriously damage or destroy samples. • If these samples thaw, make sure you use a controlled freezing technique or equipment designed to control the rate of freezing (Mr. Frosty or a mechanical controlled rate freezer) to bring them back to the cryogenic range.
- 10. 10 Summary • Frozen samples need to avoid thawing! Follow the suggested techniques • Refrigerated samples need to avoid freezing and thawing! • If thawing occurs, be prepared with a suitable re-freeze plan that accounts for the needs of the samples • Know where to find the tools you need Useful Links and Resources: Ice formation and Biological Impact: http://www.crcpress.com/product/i sbn/9780415247009 Cryopreservation theory and practice: Thermo Scientific Cryogenic Preservation Guide
- 12. Preparing for an Inventory Event Product Management Team Thermo Fisher Scientific
- 13. 13 Proprietary & Confidential It’s Time to Inventory…What Do I Need to Know? • Be aware of and familiarize yourself with the Biological Safety Level (BSL) of your samples and make sure you are in compliance with your organizational safety requirements • http://www.ors.od.nih.gov/sr/dohs/BioSafety/Pages/bio_chem _safety.aspx • Understand what you will need to inventory your samples in various environments • Have a plan for maintaining your log - Excel™, LIMS, logbook, database, sample tracking documents, etc • Have a plan for “unidentified sample vials”
- 14. 14 Proprietary & Confidential Refrigerated Storage: +1ºC to +10ºC Remember: refrigerated samples need to avoid freezing! • Avoid dry ice for non-frozen samples • What you need to have ready: • Ice bucket/cooler with dry ice • Tube storage boxes • Conical tube storage racks • Absorbent pads • Markers for wet surfaces • Labels for wet surfaces • Logbook or database • If you silence your local alarm during the inventory, remember to turn it back on when you are done • If you have alarms at a centralized location, make sure you advise facilities what you are doing • Try to limit door openings to 60 seconds or less, and space them out by at least ten minutes • Post a copy of the log you generate on the outside of the refrigerator • When returning tubes to the refrigerator, try to avoid door storage – consider storing only your daily solutions and plates in the door (if applicable)
- 15. 15 Proprietary & Confidential Frozen Storage: -30ºC to -10ºC Remember: frozen samples need to avoid thawing! • Dry ice or bench-top coolers are better than just an ice bucket • What you need to have ready: • Dry ice containers or bench-top coolers • Tube storage boxes • Absorbent pads • Markers for wet surfaces • Labels for wet surfaces • Liquid transfer devices (manual or automatic) • Log book or database • If you have a local alarm on your freezer, make sure you silence it • If you have alarms at a centralized location, advise those facilities what you are doing • Try to limit door openings to 30 seconds or less, and space them out by at least ten minutes • Post a copy of the log you generate on the outside of the freezer • When returning tubes to the freezer, try to avoid door storage
- 16. 16 Proprietary & Confidential Ultra Cold Storage: -40ºC to -80ºC Remember: frozen samples need to avoid thawing! • Dry ice is best; ice buckets not recommended • What you need to have ready: • Two (2) dry ice containers • Tube storage boxes • CryoCradle • Freezer racks • Absorbent pads • Cryo markers • Labels for wet surfaces • Liquid transfer devices (manual or automatic) • Log book or database • Consider adjusting your local alarms during the inventory • If you have alarms at a centralized location, advise those facilities what you are doing • Try to limit door openings to 30 seconds or less, and space them out by at least ten minutes • Post a copy of the log you generate on the outside of the ULT unit
- 17. 17 Proprietary & Confidential Cryogenic Storage: Below -130ºC Remember: frozen samples need to avoid thawing! • These samples need to spend a minimum of time away from cryogenic temperatures • What you need to have ready: • Two (2) bench-top LN2 containers • CryoCart (if available) • Cryo tongs or long forceps • Cryo tubes • Tube storage boxes • Liquid transfer devices (manual or automatic) • Heat block/water bath • Cryo markers rated for -200ºC • Log book or database • Deactivate your LN2 level alarm during the inventory, but remember to turn it back on when completed • Inventory one box at a time • Stage your samples: cryo storage bench-top LN2 container #1 log information/re-label bench-top LN2 container #2 cryo storage system (this will allow you to keep the samples in LN2 for most of the process)
- 18. 18 Proprietary & Confidential Summary • Take a deep breath – this process can be accomplished! • Make sure you have the tools you need for each situation • Logging is key to risk mitigation • Know where to find the tools you need • Fisher BioServices offers a complete range of biobanking services in support of clinical research and cohort studies Useful Links: www.fishersci.com www.fisherbioservices.com www.thermoscientific.com/content/ tfs/en/products/cold-storage.html www.thermoscientific.com/en/prod ucts/lab-consumables.html Sample Tracking Documents
- 19. 19 The world leader in serving science How to Maintain Sample Integrity During Sample Transfer Thermo Fisher Scientific Product Management Team
- 20. 20 Why Might You Need to Transfer Samples? Sample Unit Transfer • Consolidation of sample types, reagents, or other custom categories • Maximizing storage density during re-inventory and organization • Replacing old sample boxes • Disposing unknown samples (temporary storage location) Temperature Ranges: •Ambient, refrigerated, -20ºC, -80ºC, < -130ºC (vapor phase LN2) Sample Content Transfer • Compromised sample container • Aliquot to improved storage format • Tube size • Improved writing space or label compatibility • Desirable format for future inventory requirements Temperature Range Ambient, refrigerated, -20ºC, -80ºC, < -130ºC (vapor phase LN2)
- 21. 21 Sample Unit Transfer - to a New Box Transferring samples to a new box • Inspect for mold, moisture buildup, aging, structural integrity • Label box before pre-conditioning • Pre-condition box +1 to +10ºC: Use wet/slurry ice, cold packs, or walk-in refrigerators -10 to -30ºC: Use dry ice (DI) station - 40 to -80ºC: Use dry ice station < -130ºC: Use Cryocart/LN2 stations or DI (box by box) • Transfer tubes using picker/tweezer tool; don’t touch the tube body Maintaining Temperature • Use a metal sleeve to hold the boxes, for example CryoCradleTM • Acts as a heat sink to remove heat from the specimen • Helps to maintain temperature closer to that of the storage temp • 12ºC colder vs without a sleeve • Serves as a physical barrier between box and dry ice • Stop by and see us for a free CryoCradle Avoid freezing refrigerated samples and thawing frozen samples!
- 22. 22 Sample Unit Transfer - to New Storage • Transport vessels: +1 to +10ºC: Insulated cooler with ice or cold packs -10 to -30ºC: Dry ice (DI) cart or insulated cooler with DI - 40 to -80ºC: DI cart or insulated cooler with DI < -130ºC: Cryocart or dry shippers with temperature monitor (for temporary hold time) • Depending on the process, if you are transporting right after re-inventory, then transport samples rack by rack Limit time of exposure and frequency of door opens Avoid freezing refrigerated samples and thawing frozen samples!
- 23. 23 Sample Unit Transfer - to an Off-site Facility LN2 Trucks • Cooled by LN2 pipes - units not powered • Multiple temperature in one trip • Design has exemption for infectious material? • Multiple solenoid for control and monitoring • Back-up & redundancies Generator Trucks • Mechanically powered - units have power • No cryogenic temp • Mechanical power risks on hot days • Stability of storage unit parts • Not recommended to run during transport (oil, motor, etc). • Shipping via LN2 vs. Generator Trucks: Transporting refrigerator, freezer, or LN2 tanks • Service provider-assisted pre-qualified shippers +1 to 10ºC: Gel pack shipper -10 to 30ºC: Dry ice (DI) or phase change shipper - 40 to -80ºC: DI or phase change shipper < -130oC: DI shippers of various sizes • Things to note if shipping without a service provider • Are samples category A or B? • What is the shipper hold temperature? • What is the IATA/DOT biohazard policy Avoid freezing refrigerated samples and thawing frozen samples!
- 24. 24 Sample Unit Transfer - to an Off-site Facility http://connect.fisherbioservices.com/10-things-about-dry-shippers http://blog.fisherbioservices.com/ Avoid freezing refrigerated samples and thawing frozen samples! • Shipping via LN2 vs. Generator Trucks: Transporting refrigerator, freezer, or LN2 tanks • Service provider-assisted pre-qualified shippers +1 to 10ºC: Gel pack shipper -10 to 30ºC: Dry ice (DI) or phase change shipper - 40 to -80ºC: DI or phase change shipper < -130oC: DI shippers of various sizes • Things to note if shipping without a service provider Are samples category A or B? What is the shipper hold temperature? What is the IATA/DOT biohazard policy Dry Shipper (Vapor phase LN2) Selection of dry shipper Canister or Racking options for boxes Hold times for the shipper Temperature monitoring Orientation and labeling factors
- 25. 25 Fisher BioServices Blog—an excellent resource
- 26. 26 Sample Content Transfer - When Thawing is Required Why Thaw quickly? Thawing and re-freezing is the most stressful series of events a frozen biological sample can undergo. Thawing quickly provides the best chance to “outrun” the formation of harmful ice nuclei during the warming process. Generally speaking, The faster the better. Sample Thawing Recommendations • Biological samples stored at cryogenic or ultra-cold temperatures need to be thawed as rapidly as possible • Place sample container in a water bath at 37ºC • Gently agitate if needed to accelerate thawing • When sample is thawed, gently wipe exterior of container with disinfectant such as alcohol to prevent contamination during sample transfer process Carefully following good thawing practices will help you maintain sample quality! Resources for thawing best practices: • Thermo Scientific Cryopreservation Guide • ATCC http://www.atcc.org/ • NCI http://biospecimens.cancer.gov/bestpractice s/2011-NCIBe • YouTube Video: Thawing Cells
- 27. 27 Sample Content Transfer - Sample Transfer Sample Transfer Recommendations • Sample transfer procedures should be performed only in an environment that is in accordance with safety practices and procedures • Ensure that the liquid transfer device is properly calibrated and ancillary products are within reach at the workspace • Sterile or filtered pipette tips • Sample containers • After sample is transferred, properly secure the container with compatible cap or seal It is critical to maintain sample quality during the transfer process! Resources for sample transfer best practices: • Biological Safety & Compliance http://www.ors.od.nih.gov/sr/dohs/BioSafet y/Pages/bio_chem_safety.aspx • Cell Culture Cafe Increase Pipette Efficiency Webinar
- 28. 28 Sample Content Transfer - Sample Identification Sample Identification • Ensure the new sample container is appropriately labeled for future identification • Remember to log that the sample was thawed and transferred to a new sample container • Cool your sample to refrigerated, frozen, or cryogenic storage temperature using appropriate methods for the sample • Controlled rate freezing • Make sure to incorporate the appropriate rate of cooling for your sample type into your protocol Log not only the sample identification information, but transfer history Resources for sample identification best practices: • Thermo Scientific Cryopreservation Guide • ATCC http://www.atcc.org/
- 29. 29 Summary Avoid freezing refrigerated samples and thawing frozen samples! • Sample unit transfer – to a new box or new location • Sample content transfer • Thaw samples as quickly as possible using a warming bath at 37ºC and gently agitate if necessary • Disinfect the container before the sample transfer process to prevent contamination • Transfer samples using calibrated pipettes and appropriate plastic products • Securely cap or seal new sample containers and ensure labeling for future identification is complete and logged • Properly control freezing rate appropriate for sample type • Record sample transfer details, source location, and new container identification and location
- 31. 31 A full team is here to support you—please stop by! We are here to help improve your bio-banking experience Richard Tsai Kiara Biagioni Alex Esmon Jeffrey GoldmanDave Wilson Robert Jones Kate Enright Erik Steinfelder
- 32. 32 Content Presented Today • Come visit us, Thermo Fisher Scientific, Booth #202 • Stop by so we can discuss your needs and how we are helping scientists to Bank on Personalized Medicine http://acceleratingscience.com/biobanking/