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Preservation of biological samples such as tissues, plasma and forensic and pathology specimens relies on maintaining proper storage temperatures and on the type of sample and analysis to be done. Sample temperatures must be monitored and facilities supported by backup systems and tracking.1 As per the FDA, frozen samples should be kept at –28 to –18 °C and refrigerated samples between –2 and –8 °C; ambient samples should be protected from heat and moisture.2
Biosample preservation monitoring and record-keeping
The importance of record-keeping is stressed in FDA Office of Regulatory Affairs publications. Dr. F. John Mills, chairman of the board of BioStorage Technologies (Indianapolis, Ind.), notes that “Many current systems are not able to comprehensively record the process of handling of samples and provide a complete historical record of the temperature at which the sample has been stored, emphasizing any deviation in temperature, particularly freeze–thaw cycles.”3
Monitoring and record-keeping are addressed by the Centers for Disease Control (CDC) in its recommendations for vaccine storage and handling4 covering what is known as the “cold chain,” from manufacturing to administering vaccines. Losses due to poor vaccine-storage practices run into thousands of dollars annually and include not only vaccine replacement but also the inconvenience of calling back for revaccination those who received compromised vaccine.
This article provides criteria to consider when purchasing equipment for biosample preservation: refrigeration systems including defrosting options, temperature capability/temperature control, alarming and record-keeping.
Refrigeration systems for biosample preservation
Advances in laboratory refrigerators and freezers along with temperature monitoring, alarm and record-keeping devices provide researchers with a wide range of equipment from which to choose. Storage capacity and storage temperature decisions are guided by lab needs and appropriate regulations and recommendations for short- or long-term biosample preservation.
Household and commercial-scale refrigerators and freezers are not recommended for use in laboratories because they lack the ability to maintain strict temperature control, which can include rapid temperature recovery when doors are opened to place or retrieve samples. While scientific refrigerators and freezers are more expensive, the cost of sample degradation due to incorrect temperature storage is far greater. Points to consider when selecting refrigeration equipment include:
Temperature capability
Scientific refrigerators typically operate from 2 °C to 10 °C and freezers from –25 °C to –10 °C. Low-temperature freezers are available for temperatures of –25°, –30° and –40 °C. Ultralow-temperature freezers can be set from –50° to –86 °C (note: lower-temperature units require more power).
Figure 1 – Ultralow-temperature freezer with individual internal storage compartments.Ultralow-temperature upright and chest freezers maintain internal temperatures for long-term sample storage due to efficient insulation and cabinet- and door-mounted gaskets. Equipment with separate internal compartments with individual insulated magnetic catch doors confine ambient air to the compartment being accessed. Labs that work with a large number of samples should look for ultralow-temperature freezers with compartments fitted with individual specimen racks (see Figure 1). These can hold fiberboard containers that accommodate cell dividers. Labeling compartment doors, specimen racks and fiberboard containers makes content removal and return faster and enhances operating efficiency.
Temperature monitoring and control
Key to CDC vaccine storage compliance is temperature monitoring and control. Recommendations can apply to storage of biological samples. Some scientific refrigerators and freezers have built-in capability; others can be brought into compliance with optional equipment.
Internal temperatures for biostorage and biobanking refrigeration units are best monitored with product temperature sensors. These are inserted into a bottle of glycerin or glycol to mimic the temperature of the samples because internal air temperature changes faster than sample temperature when storage unit doors are opened. Since the variation in content temperature is the important factor, product sensors avoid unnecessary temperature alarms triggered by routine door openings. Sensors may be used to record product temperature in two areas of the unit as well as air temperature.
Temperature control options range from simple dial-type thermostats with letters or numbers (cold, colder, coldest) to sophisticated digital LED display microprocessor temperature controllers. The internal temperature display may be located at the bottom or near the top of smaller units. Premium scientific refrigeration systems use programmable logic controls, the best choice to maintain the absolute minimum temperature drift. High-end models also allow setting temperatures to one decimal place.
Temperature excursion alarming
This is an important feature when storing temperature-sensitive biological specimens. Alarming functions take the form of built-in digital audio and visual high/low temperature alarms, some with contacts that alert personnel elsewhere in the laboratory. Otherwise compliant equipment can be fitted with optional digital temperature alarms. These consist of internal sensors placed in bottles as described above. They are connected by wire passing over the hinge-side door gasket or through optional sensor access ports to an external control and display module.
Extremely critical temperature monitoring systems are available to provide local and remote alarming, including contacting offsite personnel by e-mail, text, phone or pager, and can be fitted to any scientific refrigerator or freezer. On-door sample storage is not recommended because opening doors causes immediate exposure to ambient conditions.
Manual or auto-defrost options
Figure 2 – Auto-defrost scientific refrigerator.Most auto-defrost units (see Figure 2) have fans that circulate chilled air throughout the compartment. This has the advantage of creating a uniform internal temperature. Auto-defrost refrigerators have a timed defrost cycle, during which the compressor shuts down and the fans continue to run to remove frost accumulation on the coils. Some models allow users to control the frequency and duration of the defrost cycle. Keeping the unit filled with samples or water bottles helps reduce temperature fluctuations due to compressor cycling and door openings.
Manual-defrost freezers are better able to maintain temperature uniformity than auto-defrost models. It is customary for ice and frost to accumulate inside manual-defrost freezers; a thin layer of frost does not affect cooling performance, but a thick layer limits a unit’s ability to efficiently maintain temperatures and will eventually cause failure. Standalone manual-defrost freezers should be defrosted regularly to maintain temperature stability. A second storage unit that maintains appropriate freezer temperatures will be needed for temporary sample storage while defrosting the primary unit. As with refrigerators, freezer packs should be used to fill empty spaces to minimize temperature fluctuations during door openings or placing or removing samples.
A frost-free unit with an automatic defrost cycle may be preferred. Since automatic defrost cycles cause temperature excursions, a unit capable of maintaining the temperature within the acceptable range should be selected. Before starting a manual defrosting cycle, it should be confirmed that the backup freezer is at the correct temperature, and that the primary unit reaches its set temperature before samples are restocked.
Record-keeping options
Figure 3 – Digital thermometer monitor and alarm.Record-keeping is simplified due to the ability of temperature sensors to collect data within the units for validation purposes. Procedures for monitoring and accessing data vary based on the system. Some examples include:
- For measuring sample storage unit temperatures—using calibrated temperature-monitoring devices with a Certificate of Traceability and Calibration Testing (also known as Report of Calibration)
- Collecting temperature data continuously on a USB device for viewing on a computer
- Using calibrated digital data-loggers as a best practice
- Employing web-based record-keeping systems.
While continuous digital data-recording systems (see Figure 3) are extremely useful, lab personnel should manually record temperatures at the beginning and end of the day.
Backup power systems
Even the best biosample preservation practice can be rendered useless in the event of power failure. On-site backup generators, liquid nitrogen or liquid CO2 systems are an important component of proper storage practices and should be checked regularly. Facilities that are not equipped with backup power or an alternate means of on-site capability for protecting samples should establish a plan to quickly relocate the samples to an alternate storage facility.
References
- http://www.fda.gov/ScienceResearch/FieldScience/
- http://online.liebertpub.com/doi/abs/10.1089/bio.2009.0702.fjm
- http://www.cdc.gov/vaccines/hcp/admin/storage/toolkit/
Robert Sandor, Ph.D., is director, Tovatech LLC, 205 Rutgers St., Maplewood, N.J. 07040, U.S.A.; tel.: 973-821-4400; e-mail: [email protected]; www.tovatech.com