Comprehensive assessment of cryogenic storage risk and ...

Storage tank filling

All manual-fill LN2 storage tanks (i.e., standard low-capacity dewar tanks; <'60'L) should be filled at least weekly using appropriate safety precautions per SOP. The safety measures routinely used include wearing insulated thermal gloves and/or liners, protective eyewear, and other personal protective equipment (PPE). Measurements of tank LN2 levels should be performed at least weekly, prior to all manual filling events. The purpose of these measurements is to assess the usage/evaporation rate of each tank and determine whether the liquid levels are within acceptable range limits. In addition, personnel should routinely perform spot-check assessments, possibly measurements, that the LN2 levels are above canister/sample device levels whenever a sample is removed from or placed into a tank.

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The value of weekly measurements could be compromised under standard IVF lab cryogenic handling conditions when LN2 is removed from dewar storage tanks for other lab purposes, such as to fill flasks or baths for cryopreservation procedures. Alternatively, the bulk supply source (e.g., 160-L tank) can be used for these purposes, but is inefficient due to increased vapor loss and time consumption. LN2 removal can be recorded on a daily log and the residual proportion returned estimated; however, over time the estimated amounts would be erroneous. To minimize recording errors, including failed QC entries, it is more accurate and less laborious in terms of QC paperwork to simply use the spare emergency back-up tank as an LN2 reservoir. This functional use of a back-up tank facilitates a more reliable weekly assessment of primary tank evaporation rates.

Assessing the evaporation rate of a tank, in conjunction with physical inspection of welds, seals, or other visual anomalies, as detailed below, is the best gauge of a tanks' integrity. Any tank experiencing out-of-range evaporation rates, unexplained by excessive daily usage, must be ear-marked for limited use and daily measurement monitoring performed for at least a week. If the tank remains out-of-range revealing abnormal evaporation rates, it should be retired immediately and its contents moved to the back-up reserve tank. A new tank should be purchased and OQ tested before placing into use.

In addition to a reserve storage tank, a secondary supply tank should always be maintained on-site and/or attached to a multi-port auto-fill supply manifold. Typically, IVF labs use 160- to 240-L supply tanks. Arrangements with the gas supply vendor should accommodate same-day and/or emergency deliveries, otherwise it is advisable to maintain a back-up supply tank(s) on-site. Under large cryostorage situations, if the property is suitable, the use of a large refillable LN2 silo supply tank (e.g., to 10,000'L) may be a safer, more practical, and cost-effective approach. Silo tanks can also be connected in series to supply LN2 into large cryostorage facilities. The low-pressure passage of LN2 from supply to storage tanks over an appreciable distance (e.g., 10' to 100') is best accommodated in vacuum-jacketed stainless steel piping. The latter specialized piping comes in different sizes and styles including flexible hoses. Although it is expensive, the cost of LN2 vapor loss to non-insulated hoses or piping must be factored into any budget.

The use of automatic filling systems has been predominately applied to high-capacity LN2 bulk tanks and LNv tanks. These on-off fill systems are triggered by LN2 level sensors and use solenoids to activate the passage of LN2 into the tanks through check valves. In turn, these auto-fill systems are susceptible to computer malfunction of the sensors and mechanical failure of solenoids and valves. Excessive icing and frosting of hose connections reveals problems warranting attention, as does the jack-hammering sound of a solenoid needing replacement. A routine equipment maintenance program that involves daily testing, frequent calibration, and periodic part replacement is critically important to stay ahead of unexpected failures. Test the auto-fill supply daily by opening the insulated lid and confirming an auto-fill response. Additionally, the manual override should be activated to conduct a fill test. As mentioned above, auto-fill tanks require a back-up LN2 supply at all times.

Manual tank monitoring and physical assessments

As we discussed above, manual measurements of LN2 levels should be performed daily upon opening a tank in use or at least weekly prior to filling it. In addition, each tank and the component parts of the fill system must be visually inspected and physically touched daily to thoroughly inspect each tank. Bulk tanks encased by a decorative case are visually pleasing but less functional in terms of preventive maintenance practices. The physical inspection of a tank should involve a multifaceted inspection approach that follows the 'LIFES' acronym. LIFES stands for the detection of the following:

1. 1)

   Leakage'LN2 escaping from hoses or a tank warrants immediate attention and resolution;

2. 2)

   Ice'its presence on a tank, hose connection, valves, or electronic connection should never be ignored, and efforts should be made to determine the root cause of 'icing';

3. 3)

   Frost'its unusual presence on tank, hoses, and lids (i.e., all connections) warrants investigation;

4. 4)

   Evaporation and other suspicious conditions considered odd or unusual, like condensation, should be recorded and further observed for possible resolution if the problem persists. Condensation can be detected as 'sweating' outer surfaces of a tank and/or water pooling on the floor, which may indicate loss of tank vacuum pressure; and

5. 5)

   Sounds'any odd/unusual sound (e.g., hissing, jack-hammering) should be identified and resolved.

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Note that the neck region of aluminum LN2 dewar and LNv shipper tanks are particularly vulnerable to cracks in the welds that could compromise the vacuum. Therefore, close inspection of condensation, outer welds, and inner neck integrity, as possible, is vital to daily tank QC.

Any occurrence of the above possible events would then mandate the implementation of a 'Corrective Action-Preventive Action' (CAPA) plan. The immediacy of the response is dependent on the severity of the impending problem. Overall, the daily physical inspection of storage tanks should focus on welds, especially around the neck, lid attachment, hoses, display panel, and all other connections. As part of the QM program, it is critically important to create a CAPA report to proactively address any potential problem(s) as part of standard QC practice. The previously discussed visual LIFES cues should never be ignored. These observations coupled with LN2 level measurement trends and/or other remote monitoring measurements will provide invaluable information to help determine when to retire a storage tank and move all samples to a new qualified tank, and when to replace dry LNv tanks from routine shipment use.

Remote monitoring systems

The most common form of monitoring system for LN2 storage tanks is temperature, both for dewar tanks and high-capacity tanks. Continuous monitoring of temperature is performed in association with a remote alarm system activated by out-of-range measurements. Consistency of temperature sustainability below a threshold set-point (e.g., '185 °C, '180 °C, '175 °C) is an excellent QC/QA measure which documents tank stability; however, temperature measurements by themselves are a poor gauge of LN2 levels, except at critically low levels requiring immediate action (i.e., LN2 filling). Surprisingly low levels of LN2, in a tank with an intact vacuum, can maintain a supercool vapor environment which safely preserves samples; however, in very short order, perhaps <'24 h, safe temperature below -150 °C can rise to ambient temperatures once dry. The latter condition occurs much more rapidly in a tank whose vacuum has been compromised, which is accelerated in smaller capacity dewar tanks. Low level LNv tanks are particularly susceptible to catastrophic failure if a critical level of LN2 is not maintained by its auto-fill system, as previously mentioned. Therefore, temperature monitoring alone is not considered a particularly effective gauge to avert a crisis.

To optimize the sensitivity of the temperature monitoring system, it is important to secure the probe high in the tank and a low sub-zero temperature set as the threshold (e.g., '185 °C). Although the latter setting will potentially increase your emergency response time, it will likely also increase the occurrence of false alarm responses, e.g., if a lid is removed too long and ambient air settles in over the vapor phase. Daily control temperature measurements should be recorded, and calibrations routinely performed when out-of-range greater than 2 consecutive days. Furthermore, routinely test the alarm systems, as discussed below.

A common alternative or secondary monitoring system should sense LN2 levels. In dewar tanks, these sensors are typically a single central probe through the lid to alert users when liquid levels go below a predetermined level (e.g., top of canisters), indicating a need to refill. In bulk tanks, level sensors (e.g., float switches, ultrasonic device) are commonly used to activate and de-activate the auto-fill system to maintain a consistent LN2 level. Some level sensors (e.g., capacitive and ultrasonic) are now used to remotely measure changes in LN2 levels to evaluate tank evaporation rates.

The use of weight to determine LN2 fill capacity has been a standard QM practice for LNv dry shipper tanks, typically by commercial third party cryogenic companies. Comparing pre-ship and post-return weights are a routinely applied QC practice that provides valuable secondary information to the maintenance of sub-zero temperature logging for PQ determinations. Static LN2 holding capacity (max days) should be correlated to daily weight measurements and internal chamber temperature measurements. Applying the concept of weight measurements, or perhaps pressure sensors, to dewar tanks (20'73'L) could represent a practical, safe, and accurate way to externally monitor evaporation rates. Through properly conducted OQ testing (i.e., accounting for empty to full capacity canisters), it is possible to correlate fluid levels to changes in tank weight or pressure. In combination with a remote monitoring system, real-time evaporation rate determinations could be an invaluable resource for (1) anticipating tank failures and objectively justifying tank retirements and (2) sensing problems and optimizing response-time intervals under emergent conditions. This type of system would require semi-annual calibration of the weight scale(s). The scale itself could rest between the roller base and the tank or be adapted as its own independent roller base. This innovative system is currently under development, with the intent to remotely network to a continuous, cloud-based data recording/alarm system.

An additional source of valuable information is real-time and time-lapsed video documentation. The use of multiple wireless cameras around the cryogenic storage area creates a vital source of continuous data that can be viewed actively as well as used as a library resource. Real-time viewing can allow an individual to periodically audit daily activities, e.g., visualize digital control panels, and staff performing QC procedures, and perhaps more importantly to safely survey the area in the event of an emergency O2 alarm and/or LN2 spillage situation. Alternatively, reviewing saved video footage provides for precise determination and documentation when possible incidents occur. Therefore, video monitoring is a critical component to facilitate root cause assessments (RCA) in an optimized total quality management (TQM) plan [17].

Remote alarm systems

A -based alarm call-out system (i.e., Sensaphone) has been effectively used in the IVF industry for decades in the monitoring of incubator function and to alert staff of low-level LN2 conditions. This is a wire-driven monitor/alarm system with physical space and line limitations. Typically, it has 4 'alert zones' that alarm sensors hard-wired to lab equipment. When an alarm activates the sensor attached to an alert zone, the specific piece of equipment causing the alarm is identified by the alert zone it is connected to. If more than four pieces of equipment need monitoring, multiple sensors can be connected to each alert zone. The group of equipment causing the alarm would be identified, instead of an individual piece of equipment, by the alert zone they were connected to. The latter daisy chain of sensors potentially slows emergency response time as the actual tank in distress still needs to be identified onsite.

Newer wireless monitoring/alarm-based units offer a more versatile solution with the ability to gather and store large amounts of data while delineating normal from abnormal conditions among individual tanks. In the event of an alarm, the Sensaphone, or other web-based information gathering QC applications, can automatically call preprogrammed numbers in a directed order, e.g., IVF lab number, on-call number, and lab personnel numbers. These alarm notification systems, as well as the newer Wi-Fi and e-cloud texting and -linked systems, will continue to call and send messages in a set order until someone cancels the alarm either directly in the lab, or by communicating by or computer directly with the alarm system. The Sensaphone is a hard-wired system that has battery back-up, thus allowing it to function if the building loses power.

Although the wireless monitoring systems can also function during black-out conditions, their reliance on batteries makes them vulnerable to the strict adherence to a QM policy of battery replacement. Furthermore, wireless alarm systems are vulnerable to Internet disruptions. It is important to have a reliable, dedicated Wi-Fi connection or ensure notice is provided when IT personnel service shared computer systems potentially triggering false alarms/disabling the system temporarily. Test all alarm systems, particularly O2 monitoring of personnel safety, regularly (e.g., daily to weekly) as standard QC practice to ensure proper functioning. Additionally, the call-out system should be tested regularly (i.e., weekly, monthly) to confirm their effectiveness and personnel responsiveness. Such testing can help identify software issues and equipment faults. Also, routinely clear alarm logs to ensure that sufficient storage exists, whereas exceeding maximum storage space could stop current event recording.

Alarm response

Once the tanks alarms are connected and SOP is being followed for monitoring, filling, and regular alarm testing, laboratories are prepared to handle an emergency situation. If this occurs during the middle of the day, when IVF staff are present, the situation can be handled expediently: the source of the alarm can be located and, if the tank in question is determined to be in failure, the frozen specimens can be moved to one of the fully-charged backup tanks. The failed tank is removed from service and an order is placed for a replacement tank. However, if the alarm is triggered at a time when IVF staff are not on-site, the situation becomes more critical. The alarm activates the automatic preprogrammed call system and, ideally, the responsible staff member on-call will respond to the alarm within 15 to 60 min, depending on how far they live from the laboratory. The situation is then handled similarly to the response to a daytime alarm. What if the embryologist on-call lives an hour away? By the time he or she reaches the lab, the frozen samples could be compromised. This is something that needs to be well thought out in advance of an actual emergency situation and included in the SOP. Often, call lists are prioritized by ranking individuals whom live closest to the laboratory. If some of the embryologists live a great distance from the lab, a plan should be developed to decrease their response time to the lab in case such an event occurs. For instance, the on-call embryologist could contact another embryologist or designated, trained individual who could respond more rapidly before their arrival.

Identifying the tank causing the alarm is imperative to optimizing response time. As mentioned earlier, multiple sensors connected to one 'alert zone' on an older alarm monitoring system is potentially problematic. In the latter situation, each piece of equipment should be fitted with its own alarm that sounds and lights up for quick identification in an alarm situation.

Sample management and inventory

As part of standard cryopreservation procedures for the labeling and accessioning of gametes, embryos, and reproductive tissues, it is imperative to verify and witness all sample-labels prior to storage. As mentioned earlier, labels should possess two unique identifiers (e.g., patient name, cycle ID number), date of cryopreservation, and sample description (e.g., embryo number, stage, quantity if >'1). In the accessioning of samples, the information should be accurately recorded by at least two separate methods, e.g., written forms, log books, and electronic files input. The storage location must be identified and recorded, indicating the sample ID (i.e., cane, visotube, or box) and its location (i.e., tank number, canister number, level).

A reliable inventory is based on an accurate recording of cryostorage location for each sample cryopreserved. Regular thawing events performed in an IVF laboratory provides an excellent source of spot check verifications when organized as a QC/QA process. Furthermore, confirmation of sample locations relative to the routine process of discarding patient consented samples is another valuable spot check inventory resource that should be integrated into a QM plan. In addition to maintaining a daily QC log of sample disposition relative to their accuracy of location, random sampling (ID verification) of a given number of samples on a regular basis (monthly, quarterly) is a reassuring QC step in a comprehensive QM plan. The latter verification procedure is less desirable to thawing /discarding procedures, as it may unnecessarily expose viable samples to brief but possible changes in temperature. Perform all verifications in tandem with a witnessing staff member. Any sample confirmed to have been misidentified or incorrectly located warrants a CAPA report. The latter situation, especially if replicated, may mandate the implementation of a complete inventory check.

Complete inventory verifications can be extremely time consuming and is one of the most labor-intensive laboratory QC processes. The complete process requires a preliminary organization of records to be verified by tank, and a QC measure implemented by at least two staff members participating in a 'call out' confirmation QA process of sample ID, location and number of straws, vials, or cryo-devices present. An effective way of managing such a task is the performance of a 'rolling inventory' of tanks. This involves surveying the entire contents of one or more tanks on a monthly basis, as to not overwhelm staff time over a block of time. Rolling inventories on a monthly or quarterly basis are an alternative to a labor intensive annual inventory. Evaluation of the risk-benefits of performing complete or partial inventories must be seriously assessed, especially in regard to open vitrification devices. An efficient 'spot check' inventory QM practice, assuring 100% accuracy on a monthly basis, is a far more cost-effective approach to verify the cryo-inventory overtime.

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