Repository Planning,Design,and Engineering: Part II—Equipment and Costing

Specimen Storage

The bulk of biological and environmental material being preserved for research requires preservation in some type of environmentally controlled facility, with storage below ambient temperature most commonly used. For relatively short-term storage, or if the material has already been stabilized, refrigeration (typically +4°C to 8°C) may be sufficient. For sensitive materials and longer-term storage, lower temperatures are required, usually at −20°C, −40°C, −70°C to −80°C, or cryogenic (<–140°C). Equipment to meet these needs in a variety of configurations and specific applications is available from a wide range of manufacturers.

All refrigerators and freezers must circulate air or refrigerant within the “box” to maintain a uniform temperature throughout, and materials must be stored in configurations that will not impede this circulation. Use of a racking system is key to proper cold-air circulation. While most samples are stored in vials or containers in cardboard or plastic boxes, if the boxes are piled up against each other and the refrigerator/freezer walls, areas of increased temperature (“hot spots”) will occur. These hot spots can be well above the temperature needed for effective preservation of the materials. Racking systems providing space for the needed airflow for any storage environment or configuration are readily available from any refrigeration supplier and from specialty rack suppliers. If a custom configuration is needed, it can be readily developed for a modest cost. Racks are typically aluminum or stainless steel, equipped with locking rods, and can function in either horizontal or vertical configurations.

All sides of a refrigerator or freezer box must be insulated to maintain temperature. The better the insulation, the less a freezer costs to operate. There are two types of insulation commonly employed: a closed cell with sprayed-in foam, or vacuum panels. The vacuum panels are superior but are more costly to produce and are generally only found in ultra-low freezers. Vacuum insulation is commonly employed in liquid nitrogen (LN₂)-powered freezers where much lower temperatures must be maintained.

All low-temperature storage units should be mapped and validated when initially installed, and again after any major maintenance procedure. The US Department of Energy's Energy Star program has published a test method for laboratory grade refrigerators and freezers. ¹

Mechanical Refrigerators and Freezers

When using mechanical refrigerators and freezers in a repository, remember that an electromechanical system is being employed. The system consists of refrigeration units; the heating, ventilation, and air-conditioning (HVAC), which maintains the required environment; the electrical distribution system, which powers the refrigeration and HVAC; and the backup power generation, which provides electrical power when commercial power is interrupted. The temperature monitoring system is also a critical component.

Refrigerators

Refrigerators are designed to hold material at ∼4.5°C (40°F) without freezing [>0°C (32°F)]. If drugs or vaccines are being held, the normal storage requirement for refrigerated storage is +2.0°C to +8.0°C (35.6–46.4°F). A wide variety of configurations of commercial/scientific refrigerators is available for specialized applications. Depending on the application, household refrigerators may be suitable. In any case, the refrigerators should be mapped for temperature consistency and a temperature monitoring methodology applied if critical materials are being held. The amount of effort and cost required here will depend on what material is being stored. Operational and lab supplies may be of nominal value and criticality, while biological samples, vaccines, and drugs may be of high value and have critical storage requirements. For example, if Food and Drug Administration (FDA)-controlled material goes outside its temperature specification, a discrepancy report and possible testing documentation will be required.

Cold Rooms and Walk-In Freezers

Cold rooms and walk-in freezers can be built in virtually any size, and can be single- or multistory in height. Units can range from ∼50 ft² (5 m²) to thousands of ft² (hundreds of m²) in facilities where forklifts are operational and the whole receiving dock is included.

For storage requirements at +2°C to +8°C >30 ft² (∼6′w × 8′d × 8′h), cold rooms can be very cost-effective. They are generally custom-built of standard insulated panels to meet the size required in the available space, and a wide variety of racking systems are available to optimize storage capacity.

Humidity in cold rooms can cause a variety of problems. High humidity can cause water to condense in the refrigerator, which then causes cardboard storage boxes to lose their structural integrity, resulting in the development of mold. Once mold becomes established, a refrigerator must be emptied and totally cleaned with appropriate fungicides. The most common source of mold is the use of brown corrugated cardboard boxes. Mold can be minimized by employing plastic storage containers or coated (“white”) cardboard. Employing dehumidification equipment internal to the cold room may be necessary. Humidity control for the entire large repository is generally unnecessary and expensive.

Refrigerated cold rooms and walk-in freezers have the advantage that the refrigeration equipment can be located external to the building, thus minimizing the internal ambient heat load. Cold rooms should have dual compressors, which electrically alternate while running to improve reliability. If one compressor fails, the other can support the cooling requirements while repairs are performed. Like all mechanical equipment, the units should receive regular preventive maintenance.

Walk-in freezers are also very cost-effective when significant amounts of storage in this temperature range are required. The only difference in construction between a cold room and a walk-in freezer is that the freezer requires the floor to be insulated. Most walk-in freezers are designed to function at −20°C (−4°F). However, units that function at −40°C (−40°F) are also employed. The amount of time personnel can work effectively at −40°C is very limited, however.

Humidity can be an issue in walk-in freezer units. Excess humidity in a walk-in freezer can be a safety hazard if ice forms. However, good operational procedures will minimize this problem. In some instances, the local fire codes may require sprinkler systems inside the walk-in freezers. If required, a pre-action (“dry pipe”) system should be employed. Consult with the Architectural/Engineering (A/E) resource and the local fire marshal if this requirement arises.

Since walk-in freezers and cold rooms are all custom designed/built, it is especially important that this equipment be temperature mapped prior to being placed in service. Because of the size and design, and also depending on the racking employed, hot or cold spots can develop, particularly if the air circulation is impeded or not uniform. Like all low-temperature storage systems, operating temperature should be monitored (see Freezer Monitoring).

Stand-Alone Freezers (−20°C to −40°C)

Stand-alone freezers in this temperature range are frequently used in the lab and for storage of relatively small quantities of material, such as drugs, vaccines, or DNA. Units designed for both commercial and residential use are commonly utilized, but particularly with freezers designed for residential use, care must be taken that the units are not self-defrosting. The heating used around the door in the defrost cycle can cause unacceptable temperature excursions in the storage areas. This problem is especially critical at −20°C, and long-term storage in these units can quickly lead to sample degradation for labile analytes in serum or plasma specimens. Always remember that the eutectic point (where a material is neither liquid nor solid, hence not solidly frozen) of serum or plasma is −20°C.

Ultra-Low Freezers (−70°C to −86°C)

Ultra-low freezers (ULTs) are the most common devices used to store biopharmaceutical research materials. These freezers typically operate in the −70°C to −86°C range (−94°F to −123°F). While some organizations prefer to operate these units at −70°C in the name of energy conservation, the equipment typically operates most efficiently at the lowest possible set point. This lower setting also provides additional time for the stored materials to warm up in the event of a freezer failure.

ULTs are widely available in two configurations: upright and chest. Because of space limitations, most repositories use the upright versions. Upright ULTs typically are configured with 3–5 shelves with internal doors for each shelf. Choice of shelf configuration is based on a combination of choices offered by the freezer manufacturer and the repository's needs and size of containers to be stored. Once a preferred shelf configuration is established, it should be standardized throughout the repository for efficiency in relocation of materials when a freezer fails, and also for consistency in designing a freezer inventory system. If the shelf configuration is uniform, the racks containing materials can be moved from the original freezer to the same shelf and space in a spare freezer. The location data can then be changed at once in the inventory system with minimal effort, and the chance of material being misplaced/mislocated in the data system will be minimized.

The most common type of ULT uses a two-stage compressor. These compressors work in series to provide the cooling, referred to as a cascade system. Both compressors must be working to achieve the desired temperature, usually ∼–86°C. These compressors, located on the bottom of the freezer box, are vapor-compressing devices operating on reverse Carnot cycle thermodynamic principles, ² and their mechanics are relatively complex. The average refrigeration mechanic does have not the training to work on this equipment—a specialist is required.

Coming into the market presently are ULTs that use two independent compressors to provide cooling. Either compressor has the ability to maintain temperature independently. Some manufacturers claim higher energy efficiency and less heat rejection for this configuration. Common refrigerators and freezers use a Freon™ derivative as a heat-transfer medium. One company now offers a freezer based on a Stirling ³ thermodynamic cycle, using gaseous helium as a refrigerant. The mechanically simpler (fewer moving parts) cooling device is mounted on top of the freezer box and does not require an air filter.

ULT freezers have a service life of ∼15 years. For enhanced reliability, a preventive maintenance program should be in place. Typically, compressors may start to fail within 5–7 years of service. Normal service intervals of 6 months are recommended, and the air filters must be cleaned periodically. Ice buildup around the door seals must be removed to ensure full closure integrity. Thorough maintenance records should be kept on all freezers, and replacement should be considered when either unreliability (number and frequency of failure/repairs) increases significantly or the cost of a currently needed repair, such as a compressor, becomes excessive.

The thermocouples that monitor and control the temperature of the freezers are critical devices. Thermocouple function should be checked frequently and calibrated annually. All freezers come with a built-in thermostat and a port to allow installation of an additional thermocouple. The “dry contacts” for the built-in thermocouple should be connected to the facility monitoring system, if one exists. Use of an external temperature monitoring system is strongly recommended. Also recommended is the addition of an additional thermocouple that can be connected to a handheld device as an independent measure of freezer function. The classic example of why these precautions are needed is the incident that occurred at the Harvard McLean Brain Research Center ⁴ in May 2012. The thermostats mounted on the freezers were displaying proper temperature, but the freezers were not functioning, had warmed up, and irreplaceable brain samples collected for research into diseases such as Alzheimer's, amyotrophic lateral sclerosis, autism, and Parkinson's were lost.

All freezer manufacturers advertise the efficiency and advantages of their product. Recently, the US Department of Energy's Energy Star program developed and published standard test methods for laboratory refrigerators and freezers in November 2014, ⁵ and a draft set of specifications for the equipment to be Energy Star compliant was published. ⁶ The Energy Star program addresses a number of critical parameters of interest for the user community. The standards, methods, and measurements include such items as energy consumption while running, temperature cool-down time from ambient to optimal operating temperature, cool-down time to operating temperature after door opening, and temperature distribution inside the units. For the first time, users may obtain real data on how a piece of equipment can be expected to operate, free of marketing hyperbole.

Power costs are a significant part of a repository's operating budget. In January 2015, the average US commercial cost of power was $0.103 per kilowatt hour (KWH). An average freezer consuming 19 KWH/day equates to an annual cost of ∼$714 per freezer for electricity, plus the cost of the supporting HVAC. The current average cost of power is available at www.eia.gov/electricity/monthly/epm_table_grapher.cfm?t=epmt_5_6_a

For an excellent presentation on the measurement of power consumption and comparisons of different units, see http://energy.gov/sites/prod/files/2014/11/f19/ult_demo_report.pdf ⁶

ULT Walk-In Freezers

ULT walk-in freezers have been used where large volumes [i.e., >1,000 ft³ (28 m²)] of material are required to be stored at ≤–70°C, preferably where the storage conditions will be relatively static. It is neither feasible nor safe to have repository staff working in this temperature environment. Therefore, the freezer has two or three components:

• The storage chamber has multiple doors to allow access to the specimens and to operate at the storage temperature.

Only a few of these systems have been produced and installed to date. The up-front capital cost can be expected to exceed US$1 million. However, once in place, operational efficiencies (cost/sample over time) can be substantial. Utilization of floor space is maximized. Because the compressors and heat exchangers can be located externally to the building, HVAC and electrical costs are reduced. The cost to purchase, operate, and maintain the number of conventional individual ULT freezers for the same storage volume would be significantly higher.

Cryogenic Systems

Cryogenic storage is maintained at ≤–150°C. The rationale for utilizing this temperature environment is that all chemical and physical reactions cease below T_g, the glass transition temperature, at which the stored material is a true solid and will not degrade. Extensive testing and experience has shown that biological samples can be maintained almost indefinitely in this thermal environment without degradation. Cells and tissue are commonly stored in this manner. For these products, the upper temperature is the T_g of water (nominally −136°C, but modified by any other material in the solution). The key consideration is that ice crystals do not grow at this temperature, as they can at the ULT freezer temperature of −80°C. Care must be taken not to cycle the temperature above and below T_g, as this has been shown to induce apoptosis in cells. ⁸

Cryogenic storage should also be regarded as a system, consisting of the refrigerant storage, the transport system for moving the LN₂ from the storage vessel into the freezer, and the freezer itself.

Cryogenic Mechanical Chest Freezers

Mechanical freezers operating at −150°C are available from several manufacturers. They are most suitable for use in a lab where considerations preclude the use of LN₂ freezers or Dewars. From a biorepository point of view, however, these freezers suffer several shortcomings. The amount of usable storage compared to the footprint of the freezers is very low—typically no more than 10 ft³ in a freezer occupying >20 ft² of floor space. They are relatively expensive to purchase and operate, and historically, their reliability has been poor. If a unit fails, the time for stored material to warm up to >T_g is relatively brief.

LN₂ Freezers

LN₂ freezers are essentially large vacuum bottles, or Dewars, which hold LN₂ as a coolant in the bottom of the tank. They are available in a wide variety of sizes and capacities, from small portable units used in labs to the 60" (23.6 cm)-diameter freezers used in repositories. Storage capacities range from a few vials to >100,000 cryovials. At standard pressure, the temperature of LN₂ is −196°C, well below T_g. The freezers have an internal and external shell, connected at the neck, with a vacuum pulled between the layers. The inner wall of the freezer is wound with a material that functions as a hydrogen sieve. Hydrogen gas can diffuse through the steel walls of the freezer over time (years) and slowly degrade the vacuum, causing loss of insulating ability.

A few types of sample storage systems, such as straws, require the samples to be immersed in the LN₂. Use of liquid-phase nitrogen storage rather than vapor phase should be minimized for safety reasons. Pulling samples from the liquid phase can be dangerous if not done slowly and properly. Any liquid that splashes out can cause burns and damage flooring and equipment. Thus, use of heavy personal protective equipment (PPE) is mandated. When samples are frozen, a vacuum is created in the vials. If the sample storage containers are not hermetically sealed, the containers can entrain LN₂ and explode upon being exposed to ambient temperature, acting like shotgun shells, sending shards and bits of samples flying at high velocity. There is published evidence of disease transfer from LN₂ becoming ingrained in the samples.^9–11 Sample storage in the gaseous/vapor phase is therefore recommended, since there are no reports of disease transfer when the samples have been stored this way.

There are two common formats of LN₂ freezers: wide mouth and small mouth. Wide-mouth freezers have a lid that is the diameter of the freezer. A large foam insulation plug on the bottom of the lid helps maintain temperature. These freezers may or may not have a tray at the bottom of the freezer for the racks to sit on. The main advantage of these freezers is the ready visibility and access to the racks holding the samples. The significant downside is that these freezers do not hold temperature well when the lid is opened. Typically, with these freezers, the top two rack positions should not be used because of the high temperature gradient when the lid is open. Thus, it is critical to minimize the amount of time the freezer is left open to prevent warm-up of the upper rack positions. If the freezer does not have a shelf on the bottom, the lower one or two rack positions should not be used because they are immersed in the LN₂. Using both considerations, significant amounts of storage space cannot be used in these models.

The last several generations of LN₂ freezers commonly used in repositories are referred to as small-mouth units. The mouth of the freezer is of a size only large enough to allow ready access to the racks. The mouth extends above the top of the Dewar and has a fold-back top with a foam plug for insulation. These freezers have a bottom shelf with a rotating turntable upon which the racks sit. The turntable is manually rotated to bring the desired rack into position for removal. These freezers are excellent at maintaining temperature, including holding their validated temperature at the top of the racks for an extended period of time with the top open. Additionally, this type of freezer will maintain its temperature without recharging the LN₂ for an extended period of time—up to 21 days for some models—a significant advantage in the event of extended electrical power failure in the repository. Units that are validated to the FDA standard are available in configurations designed to hold temperature at −150°C and −190°C.

The largest-capacity [ultra-high density (UHD)] freezers present an advantage in terms of increased storage capacity, but a downside is their height. Most UHD freezers come equipped with 1–2 steps for easy access, but racks can be heavy, and pulling samples may be difficult, especially for a short staff member.

Freezers designed for repository use are equipped with controls that display freezer status, including temperature, LN₂ level, and status alarms. These controllers have standard computer system interfaces. Most freezers are equipped with a solenoid, allowing an operator to add LN₂ at the freezer via the control panel. Control systems are now available to allow freezer monitoring and filling by remote computers and mobile devices.

While LN₂ freezer systems are highly reliable and efficient, they can experience problems. The worst problem is loss of vacuum, which is most likely to occur at the neck of the freezer and can easily distort it, making removal of racks difficult. However, temperature can be maintained simply by adding more LN₂ until the situation can be resolved, which may take days. The major disadvantage is increased LN₂ consumption. A more common problem is for the fill solenoid to freeze, causing a freezer not to fill or to overfill if not monitored. “Ice balls” can occur on joints or valves in the piping that are not covered by insulation. Moisture in the air can condense and freeze in these spots. If the lid on the freezer is left open too long, especially in humid areas, ice will condense out of the air, fall to the bottom, and displace the LN₂, and it will have to be cleaned out eventually. As with mechanical systems, spare LN₂ freezer(s) should be maintained at operating temperature. Given the relative reliability of these systems, a spare ratio of 3%–5% is recommended, with a minimum of one spare with the same capacity as the largest operating unit. Having the spare at operating temperature is necessary, since several days may be required to bring an LN₂ freezer to a stable operating temperature from ambient. With reasonable care, the functional life of an LN₂ freezer can exceed 30 years.

LN₂ Storage and Handling Systems

LN₂ freezers in a repository are normally connected to a LN₂ supply system. Depending on the number and size of cryogenic freezers employed, economics and space should dictate the method to be employed. For the small repository, the use of LN₂ Dewars may be the most feasible refill source. Portable Dewars, up to 240-L capacity, can be hooked to each freezer. These Dewars typically require filling/replacement once or twice a week, and this process can be labor intensive.

For larger repositories, bulk LN₂ storage is needed. Storage tanks (which can be purchased or provided by the LN₂ supplier) of up to 20,000 gallons are readily available, although most repositories will utilize tanks in the 1,500–6,000-gallon range. For the larger tanks, the LN₂ supplier is usually willing to provide the tanks and amortize the cost of the tank by increasing the cost of the gas provided. A caution here: gas supply contracts that include the tank are long term and difficult to terminate, and the total cost can be high. If the storage tank is purchased outright, the cost can generally be recouped in the reduced cost of LN₂ over a 2–3-year period. The cost of bulk LN₂ provided to a repository-owned tank will generally be about a quarter of the cost of refrigerant provided in Dewars. As long as the tank is properly sized and a reasonable minimum supply is maintained in the bulk tank (typically ∼25% of capacity), no outages should occur. Gas suppliers usually provide telemetry devices to monitor the levels in the tank, and schedule resupply accordingly. A well thought-out supply contract will provide for service from several gas production locations so that local conditions do not impact long-term supply. No electrical power is required to move the LN₂ from the tank to the freezers—the gas pressure within the storage tank will push the liquid into the freezers.

The repository should also monitor the LN₂ levels for availability of supply and ensure that gas consumption is normal. Excessive gas use could indicate a problem with the supply tank, the supply system, or the freezers. It is feasible for a repository to start out with a small tank, and trade it in for a larger vessel as the repository capacity grows. Suppliers of bulk tanks readily accommodate trade-ins. If this option is being considered, the mounting pad for the bulk system should be sized initially to accommodate the largest anticipated tank in order to minimize future cost and replacement time.

With a bulk system, LN₂ is delivered to the individual freezers by a vacuum-insulated piping (VIP) system. A few repositories have tried to use insulation-wrapped pipe for this purpose, with less than satisfactory results. Individual freezers can be filled using manual valves near each freezer, or with an automated fill system. The repository's chosen bulk tank and piping supplier should provide piping design and assistance in determining the optimal filling method. Repository managers should be aware that their piping system will contain several over-pressure relief valves.

An intermediate bulk storage solution is referred to as a mini- or micro-bulk system. In this case, a small bulk tank is installed, usually inside the building. The tank size will typically range from a few hundred gallons to ∼1,000 gallons of LN₂. Refrigerant can be provided to the freezers by either Dewar or VIP piping.

Automated Storage and Retrieval Systems

Some of the most exciting, interesting, and expensive developments in repositories are in the area of automated freezers.

For a number of years, vendors have been trying to develop and market automated ULT and cryogenic freezer systems. Most vendors have been small companies who failed, from either inadequate technical innovation or their inability to deploy and market their product successfully. The early developments generally suffered from lack of flexibility, low capacity per storage unit, and high cost. There have been some notable successes in producing large automated systems, particularly for the large pharmaceutical market where millions of biological and chemical samples must be stored for product development and testing. Recently, there has been significant consolidation among the companies developing automated freezer systems, and several companies appear to have viable options for both −80°C and cryogenic applications.

There are a number of reasons to automate sample storage and retrieval. Automated units promise the ultimate in inventory accuracy and accountability, and greater speed in specimen storage and retrieval operations. Each sample is tracked by a unique barcode or radio frequency identification (RFID) on the storage container. A record is created when each sample enters the system, with the temperature conditions during storage, and when the sample was retrieved, thus creating a full audit trail, especially for the FDA's current Good Manufacturing Practice (cGMP). This equipment can also lower the labor cost to store and retrieve samples. The sample retrieval rates of these sophisticated systems are quite high—they retrieve samples in a fraction of the time required for a person to move to a freezer, locate, and pull the samples. The labor and time saved in providing the samples to the requestor can become significant if the repository inventory has a high utilization rate, which is the goal of every repository. There may also be less quantifiable benefits. The samples being retrieved via an automated system have much less exposure to higher temperatures than those being manually recovered. Thus, sample quality may be better preserved.

While automation has become common in the large-scale refrigerator and freezer applications (−20 to −40°C) such as the storage and distribution of food, development of automation for biomedical applications has been much slower. Automated systems that will function reliably at ≤–80°C present special challenges. Materials that function very effectively at room temperature and down to common freezer temperatures frequently fail at the lower temperatures required for optimal storage of research samples. Many materials, including many plastic and load-bearing materials, even some metals, become brittle and disintegrate at the slightest stress at cryogenic temperatures. Parts that are formed at room temperature may not fit at low temperatures, due to the effects of thermal expansion and contraction, and thus may not meet the tolerances required by robotic equipment. The electronics that control and guide the automation must be mounted outside the low temperature areas because most electronic devices, including semiconductors, do not operate <–40°C.

While these automated systems are very attractive, they do come at a significant cost. Currently, they are only suitable for the well-funded, sophisticated, and, typically, large-scale repository. At a time when repository sustainability is a huge issue, a full understanding of the costs—both up front and long term—is mandated. The initial acquisition cost of these systems is quite high compared with deploying individual freezers of comparable storage capacity. The least-expensive system reviewed by the authors has a cost of ∼$150,000. Depending on the storage capacity needed and the amount of automation and flexibility required, plus system operation and maintenance considerations, the ultimate cost can range into millions of dollars. Using acquired capital cost per sample stored as a metric, the cost per sample ranges from ∼$1.15 per sample to $28.00. The highest capacity systems have the lowest per-unit sample cost.

Another factor in employing the automated systems is the cost of maintaining them. These are highly sophisticated mechanical/electronic/computer devices. Without proper service, they are operationally inferior to conventional freezers. Servicing them requires a highly skilled technician with specific training (and located in reasonable proximity to the purchased unit, not overseas). Spare parts can also be expensive, and must be readily attainable to ensure timely repair and continued operation. The manufacturer must normally provide the needed maintenance. Purchasers should expect to pay in the range of 6%–15% of the acquisition cost per year for this assistance.

A decision to procure one of these systems should be preceded by a detailed analysis of all the costs and benefits of the automated systems, with due consideration of the storage alternatives. The advantages are absolute inventory accuracy and security, decreased labor, speed of retrieval, and possible quality benefits in sample handling. Disadvantages are the costs of acquisition and maintenance. Costs for space, power, HVAC, labor, and monitoring for the automation solutions and the non-automated solutions should also be compared.

Other Storage

Besides refrigerated and frozen samples, a repository may archive samples in a variety of other formats. Examples of commonly preserved materials are formalin-fixed paraffin (FFP) blocks, glass slides, environmental samples such as vacuum cleaner bags and water samples, and animal bodies preserved in various ways.

FFP blocks and glass slides are usually stored in cabinets with shallow trays to optimize storage density, and kept in a controlled ambient temperature environment. As the volume of these samples increases, the weight of the storage cabinets can get very high, especially for glass slides. Slides are somewhat temperature sensitive; the lower the temperature, the more brittle the glass becomes, so care in handling is important. Glass slivers from broken slides can be a significant safety hazard when manipulating the inventory.

When archiving tissue blocks and slides, it is just as important to have a good, well-maintained inventory system as it is for low-temperature storage. Otherwise, sample retrieval can be a nightmare. At a minimum, location data should include room, cabinet, tray, grid or box number, and preferably location within the box (i.e., 5 levels of location).

Animal carcasses, body parts, and organs can cause specific challenges. Some are stored in bags or boxes, some may require refrigerated or frozen storage, and some may be preserved in chemicals such as alcohol or formaldehyde. Chemical preservation can be especially bothersome, as many of the chemicals are flammable, requiring storage in fire-rated cabinets. Not only can these cabinets be relatively costly, the interior configurations are often less than optimal for storage efficiency, and the cabinets themselves must be connected to an electrical earth ground.

Freezer Monitoring

Every refrigerator and freezer comes with a basic built-in control that manages the unit and monitors the temperature. Mechanical freezer controllers manage the compressors that cool the storage chamber and also initiate audible alarms from the unit when conditions exceed tolerance set points. LN₂ freezer controllers monitor the liquid level and temperature, and initiate audible alarms. LN₂ controllers also sound alarms for a variety of conditions such as for a valve that is stuck open or closed, supply failure, temperature too high or low, and liquid level too high or low. The primary characteristic to monitor is the temperature of the storage chamber, whether it is a refrigerator or a freezer. Mechanical storage units have an additional output called a “dry contact.” A dry contact is essentially a relay in the unit's controller that either closes or opens when an alarm condition develops. Dry contacts can be energized for a variety of conditions. For example, alarm relays may be set on mechanical storage units to be activated when the temperature is out of the set point range, a filter requires changing, or a door is open for an extended period. Some of these conditions have timing parameters associated with them. LN₂ freezer relay alarms could be activated for stuck-open/-closed solenoid valves, high-/low-level liquid conditions, and temperatures out of range. Manufacturers and distributors recommend that dry contact (relay) alarms be monitored in addition to temperature alarms.

As repository workers know from bitter experience, refrigerators and freezers can fail and alarm systems can fail to actuate. Redundancy in temperature monitoring is therefore required. An independent monitoring system must be employed to ensure sample safety. There are several types of monitoring systems currently available, from sophisticated, complex, integrated systems that provide extensive data to basic, single-function units that sound an alarm when a maximum temperature set point is exceeded.

There are several different approaches to monitoring systems. At the base level, the controlling software system can be in the repository, or only the sensors and a transmitting unit can be at the repository, with the software “in the cloud.” Typically, the systems located within the repository are purchased and the cloud-based systems are fee-for-service. The simplest units connect a single temperature-sensing unit to a device that can provide an alarm to a cell phone, pager, or computer. For large repositories, it is more efficient to combine multiple sensors into a system that can identify which storage unit is failing and provide an alarm and data on the conditions. Signals from multiple sensor units are transmitted to a collector node, processed by a computer, and then transmitted out via a phone or Internet connection. The sensor units may be wired from the storage units to the collector or connected wirelessly. Wired systems tend to be more robust and secure; wireless systems allow freezers to be moved around as needed.

Some freezer manufacturers are now providing built-in monitors, or are providing monitoring systems with the freezer purchase as a package. These systems generally provide an output signal that can be connected to a computer, tablet, or cell phone. In utilizing these systems, remember that at least two independent thermocouples should be employed to ensure redundancy and security. Depending on its function, a single thermocouple to provide a signal for both freezer control and monitoring is insufficient for truly adequate security.

Some monitoring systems will only indicate that there is an alarm, and the repository worker must travel to the facility, identify the problem, and correct it. Other systems will provide all the information needed to know about the condition, but lack the ability to make corrections such as resetting alarms or controls, or making adjustments remotely. Some higher-end systems provide all these features.

Support Areas and Services

Quality management system

As part of planning a repository, the quality management system (QMS) that will be utilized to ensure the integrity of the samples and associated data should be structured. This applies to any scale of repository, from start-up to a large facility. While the repository is being designed and built, a quality policy and a quality program manual should be generated. SOPs should be put in place for all processes and functions. SOPs can be internally generated, or borrowed from other organizations and customized to the new repository. Many repositories are willing to share their SOPs, and many are posted on the web. ¹³

As part of the QMS, calibration and maintenance systems should be structured. Every instrument or piece of equipment that records, measures, or controls must be calibrated on a routine basis, and documentation of that calibration—traceable to national standards—must be maintained. This requirement includes all lab equipment, thermocouples, thermometers, and alarm systems. Calibration may be done in-house by qualified technicians, or contracted out. Similarly, records of all equipment maintenance must be kept up to date.

Considerations for Start-Up/Small Repositories

Start-up and small repositories face a number of challenges due to scale that are avoided in larger operations. Constraints on long-term funding or available space are the most common issues to be considered. When starting or operating a repository, its prime charter is to receive, store, and distribute samples and their associated data with no loss of integrity. In storing the materials, the repository must address the normal risks of operation—equipment failure, power failure, security, and human failure.

The absolute minimum number of storage units of a given temperature is two: one as the functional storage and one as a backup (maintained at operating temperature). A single storage unit is not a repository. At best, it is a curated collection of samples because there is no extended safety net for the material. As previously stated, all equipment fails over a period of time, and at some point, the commercial electrical power supply will fail. Just because equipment is new does not mean it cannot malfunction. While most mechanical freezers and refrigerators can be expected to provide 15 years of service life, early failure is certainly not uncommon. Brand-new units have been known to fail to start, and units have failed after a few weeks or months of operation. Thus, the requirement to have a backup equal freezer with capacity at least equal to the largest size freezer storing samples must be considered an absolute requirement for a repository. If the start-up repository is part of a larger operation that has sufficient backup capacity, this reserve capacity can serve until the repository can obtain its own needed backup equipment.

The same argument applies to the electrical supply for the repository. Without a backup generator (and its reserve fuel supply) or a secondary source of power, the operation is not viable as a repository. If the repository is a part of a larger institution such as a hospital or academic research center, separate backup power is probably not an issue, as the capability is normally in place, and the repository must only ensure that its equipment is tied into the overall system. This situation is a case in which second-hand equipment may be employed at a significant cost savings. The used equipment must have been well maintained without excessive usage, and the unit should be thoroughly tested before being placed in service.

LN₂ freezers are less likely to experience failure than mechanical storage units, but the possibility of failure must still be addressed as an insurance measure. A standby unit must always be in place and maintained at operational temperature. A reliable source of refrigerant resupply must also be in place as the start-up operation is unlikely to have a bulk supply system.

All storage units must have some type of temperature monitoring. If the overall facility has a monitoring system, at a minimum the dry contacts provided on all units should be connected to the plant monitoring system. It may be economically feasible to employ a cloud-based remote monitoring system. Several freezer manufacturers now offer monitoring systems as part of the freezer purchase. An auxiliary thermocouple for monitoring can be easily and cheaply installed. Most storage units come equipped with a port for extra thermocouples. Using these thermocouples and an inexpensive handheld device, the temperature can be checked and recorded regularly (at least once a day, preferably more often) as a check on the sensors built into the storage devices (thermocouples can also fail).

If the available repository staff is very limited, having to be on-call can cause a significant strain. Freezers fail at the most inconvenient times, and usually not during normal working hours. When this happens, the material must be moved to the spare storage unit(s). With mechanical units, this must happen within 4–5 h. Personnel trained in this transfer must be available 24/7/365 to accomplish this. The location data for the relocated material must also be input to the inventory system or at least recorded. If the repository is located with a larger facility that has a round-the-clock security presence, it may be possible to train these people to relocate material properly.

Physical security is usually not a problem in a start-up that is housed in a larger institution, but must be considered up front. Freezers should be located in a controlled access area. If this limited access cannot be accommodated, then all freezers should be locked, except when being directly accessed by the repository staff. The biggest risk to the inventory is access by non-repository staff.

Every repository requires a data system that tracks inventory and sample data. While very small repositories can get by using spreadsheets, this tactic is not a good strategy to save money. As soon as economically possible, a data system should be procured and implemented. Minimum needs should be defined to purchase a system that meets these requirements and can grow and expand with the repository.

Economics, Cost Drivers, and Cost Model

A repository may be “just a warehouse,” but the necessary support and control systems make it a very upscale and expensive warehouse. Costs will vary widely depending on the location and purpose of the repository. Here are some of the high-expense items that will drive up the total start-up cost, assuming that an existing vacant warehouse type building will be “built-out” as a repository:

▪ Lease or acquisition cost: “Dark, cold” warehouse space costs $11–$16+/ft²/year triple net, depending on location. To arrive at final net cost, typically add an additional $2–3/ft²/year.

Cost Model

The presented cost model assumes the repository being designed will accommodate 50 low-temperature freezers, 30 of which will be ULTs of ∼26–28 ft³ capacity and 20 will be large 60" diameter LN₂ units. It is assumed that all storage units will be acquired at repository initiation.

For this model, the minimum floor space required would be ∼4,000 ft². Any repository that is initially constructed and equipped with 50 storage units should be anticipating growth. This does not include any allowance for room-temperature or refrigerated storage. For this growth, it is prudent to assume that the number of storage units will double over the planning horizon. Therefore, the model will assume the leased and built-out facility will be 5,500–8,000 ft², and even this size is fairly minimal. A truly large-scale repository is more like to occupy closer to 20,000 ft² and more.

For the ULTs, sufficient electrical distribution capacity, HVAC capacity, and backup generator capacity is assumed. The LN₂ freezers will be fed from a bulk tank of either 3,000 gallons or 6,000 gallons capacity. The 3,000-gallon tank would require a bulk LN₂ delivery about every 10 days, and a 6,000 tank about every third week.

The cost model presents an expected range of acquisition costs for the large capital items in developing a repository. A range of 5,000–7,000 ft² built out with 50 frozen storage units has been assumed. No design or engineering costs, permit or regulatory costs, or cost of smaller items such as the computers, software, furniture, phones and barcode readers required for operations have been included. The cost model can be seen in Table 3. Appendix l provides a list of Web sites for repository vendors.