by Jack Hoff, President, Aseptec LLC and Cindy Buckmaster, Director of Public Outreach, National Animal Interest Alliance (NAIA)
According to Richard Harris’ bestselling book, Rigor Mortis, American taxpayers spend about $30 billion annually funding biomedical research, and over half of these studies can't be replicated due to poor experimental design, improper methods, and sloppy statistics. Sloppy science doesn't just delay medical progress, it robs patients currently struggling with serious, life threatening diseases of hope for themselves and their loved ones. Additionally concerning is that only about 5% of the drugs eventually developed and tested actually make it to market. That 5% success rate has still improved countless human and animal lives worldwide, but the amount of time and resources wasted as a consequence of inconsistent study design and practices is unacceptable. And the number of animal lives wasted is, simply, unforgivable. The ultimate source of information for the development of all biomedical treatments comes from research with animals. How much of this replication crisis is due to inconsistent care and treatment of research animals by the laboratory animal science community? Are there gaps and inconsistencies in our own practices that require our attention?
This article addresses the improvements that can be made in steam sterilizer design to best ensure reliable and consistent performance for vivarium use in order to protect the health of the animals on study. These improvements can be specified for new equipment or can be retrofitted on older equipment at a relatively low cost.
Reproducibility and Repeatability have long been a strict design criterion for cGMP pharmaceutical grade steam sterilizers in order to guarantee proper sterilization of the product or goods and to ensure that no damage has occurred to the product since many of the products are for human consumption. This is accomplished through extensive work procedures and validation protocols used to prove machine performance including Installation Qualifications (IQ), Operational Qualifications. (OQ), Software Qualifications (SQ), and Performance Qualifications (PQ). The Validation protocols become the standard to which end users must follow as part of their work procedure.
Work protocols within the vivarium should also include procedures such as chamber loading, cycle parameter verification, a review and confirmation of each parameter in the sterilizer cycle printout to the programmed parameters of the cycle at the end of each sterilization cycle to ensure conformance. Copies of the sterilization cycle data should be filed in the report on the experiment.
To improve reproducibility in animal experiments, researchers should have the same degree of low tolerance for imprecision in their work with animals as in the Pharma industry to the extent of accuracy and performance. Without accurate string data from animals, there is no drug development in pharma.
Steam Sterilizer Design for Vivarium Applications
Most Steam sterilizers used in vivarium applications today have low tolerance accuracy and performance that can affect repeatability of the experiment, possibly degrade animal cages by overshooting in temperature, and cause maintenance shutdowns on a regular basis. Conditions such as under temperature, wet bedding, and lack of sterilization of porous loads, such as red bags, add to the problems experienced by the researcher that can affect the animals environment.
Common Sterilizer Operating Issues include: door seal reliability, poor steam quality, wet bedding or feed, low & high temperature alarms, and degradation of animal cages.
In order to drastically improve the sterilizer’s reproducibility and address the issues listed above, a more accurate and repeatable sterilizer is required that has more robust fail-proof features such as improved temperature control, more accurate instruments, a more reliable door sealing system, a better air removal system for the application, a validation protocol that verifies all the performance improvements, and internal facility work protocols that require adherence on a daily basis.
Higher accuracy protects the goods being sterilized from over-temperature or damage from vacuum conditions such as with animal cages.
Sterilizers for animal applications should be high-vacuum sterilizers that use a vacuum pump or other mechanical means to remove air from the chamber. Air displacement cycles cannot be used with cages or bedding since this method will not remove trapped air in these items. Air displacement cycles should not be used for the sterilization of porous loads such as bedding or feed. Air Displacement cycles should be used for sterilizing liquids in open & vented containers, such as water bottles.
Steam Quality
The steam quality being provided by a building system or steam generator must be validated to prove it is generating saturated steam with sufficient moisture to kill the food supply of bacteria. If the steam is too wet, bedding will be moist and items may not be sterilized. If the steam is superheated (too dry), it does not contain the moisture to break down the food supply any bacteria may be consuming thus leaving the sterilizer load unsterile. Steam supply systems should be properly trapped to eliminate a wet steam condition. Steam quality can be tested with commercially-available steam test kits.
Air Removal System
Any steam sterilizer used in a vivarium application should incorporate a vacuum pump or similar air ejector system as part of its air removal system. Air removal is an extremely important phase of the process since steam penetration into the load is affected by how well air is evacuated from the load.
Vacuum pumps have the ability to remove trapped pockets of air in loads such as red bags and stacked animal cages thus ensuring that steam can penetrate to the center of the bag or cage and guarantee sterility.
A vacuum pump can also be used to dry bedding & cages that become moist from wet steam supplied to the sterilizer chamber.
A standard feature on most steam sterilizers is an automatic leak test which is used to determine the sterilizer chamber is leak-tight and that the vacuum system is capable of attaining a pre-set vacuum level. Vacuum pumps can typically attain 27.5” Hg with seal water temperature of 60o F or less.
Bowie Dick Test & Biological Indicators
Bowie Dick Tests can be used for every sterilization cycle along with quick-read biological indicators in order to guarantee proper air removal and sterilization of the chamber load.
Poor steam quality (wet steam) can cause the bowie dick test to fail even though a pre-set vacuum level was attained in the chamber. Using Air Displacement cycles will cause the bowie dick test to fail since air displacement cannot remove trapped pockets of air in items such as bedding or feed.
Door Seals
A much more reliable method of activating the sterilize door seal, for sliding door seals, is to eliminate the use of steam and switch to either compressed air or instrument air to activate the door gasket. Using steam to activate the door gasket has two (2) inherent problems: degradation of the door gasket over time thus reducing the life of the gasket and problems with unsealing the sterilizer door if wet steam is present and condensate builds up in the door seal cavity.
The use of compressed air seals & unseals the door instantaneously making the system much more reliable. Compressed air should be dry, filtered, and instrument air quality at 90-100 psig.
Life expectancy of door gaskets using compressed air increases by at least factor of 4 as compared to steam-activated door gaskets. In addition, graphite used on some manufacturer’s door gaskets can be eliminated thus eliminating a foreign substance from the process.
In the event of a power failure, using compressed air is an added safety factor since it will keep the door sealed indefinitely thus leaving the sterilizer in a fail-safe condition even if under pressure. The use of steam is unreliable during a power outage since steam, over time, collapses which may cause the sterilizer door to unseal. When steam collapses it will create a vacuum in the door seal cavity (behind the seal) which can suck back the gasket and unseal the door, even if the chamber is under pressure.
Valves & Temperature Control
A steam sterilizer in a vivarium is a critical path instrument that must be as reliable as possible to protect the integrity of the weekly cage-changing routines of any facility. Which means the sterilizer components must be durable and cannot shut down the sterilizer on a regular basis for maintenance. The use of pneumatic piston valves provides a much more reliable device that can operate to over one million cycles and can be used as a control valve to attain +/- 0.5C control. The use of solenoid valves is not recommended due to their inherent short life span, especially for use as a control valve. Autoclavable temperatures for animal cages are listed below.
Sterilizer Instruments
In order to improve the sterilizer’s accuracy and reproducibility, more accurate devices for sensing temperature and pressure should be used.
RTD resolution should be +/- 0.1C. Pressure Transducers should be as accurate as +/- 0.1 psig.
The total loop temperature and pressure accuracy of the sterilizer should be verified:
Total Loop Accuracy = accuracy of RTD + Accuracy of the Analog Input RTD module in the controller. The total loop accuracy should be less than +/- 0.25 C in order to attain +/- 0.5C temperature control.
Sterilizer Loading
In order to guarantee sterilization of various load configurations, the Performance Qualifications allows the end user to validate various load configurations that will be used during normal operation of the sterilizer. This ensures that the sterilizer is loaded the same way every time for a particular type load, such as bedding, cages, or red bags, and will guarantee proper air removal and steam penetration into the load. The Facility’s operational protocols should be based on these validated loads and cycles.
Validation Protocols
The following validation protocols should be the foundation of the sterilizer testing and operation. Any errors or malfunctions will be determined and corrected prior to placing the sterilizer in full operation. All test data shall be collected and filed to support each validated cycle.
Software Qualification (SQ)
A major factor in attaining repeatability and reproducibility of the sterilization process is determined by how well the sterilizer’s software is tested to eliminate bugs and errors that cause a failed operation.
Both the operational program code and the Human Operator Interface (HMI) should be tested to be in compliance with the project specifications. Every temperature control loop should be tested, every trigger, every alarm, every pushbutton. For a touchscreen HMI, every setting or button should be tested for the low setting, the high setting, and out of range parameters to determine the control system only accepts parameters within its program. All testing should be documented with approval signatures. Software validation should be one of the first inspections of the system prior to loading the software on the sterilizer. This can be done off-line on a bench.
Installation Qualification (IQ)
In this phase of the equipment validation, every aspect of the sterilizer design is inspected. This includes dimensions of the equipment to fabricated drawings and site plan, verifying all instruments & devices in the piping, pneumatic, and electrical systems, and verifying all materials of construction. All electrical wiring and pneumatic lines should be verified to be correct. Any deviations must be corrected and verified with QC signatures prior to moving to Operational Qualification testing.
This protocol should include site steam quality testing to confirm a saturated steam condition.
Operational Qualification (OQ)
Operational Testing involves running the sterilizer cycles to prove it runs according to the cycle parameters and attains sterilization of an empty chamber. All cycle parameters are verified to be met based on the cycle data such as sterilization temperature and time, vacuum depth for drying, and drying time. Each type of cycle shall be run three (3) times consecutively and successfully, each attaining sterilization temperature, to successfully pass the protocol inspection. Sterilization cycle data should be inspected to confirm each phase of the cycle met the programmed parameters.
A full chamber temperature distribution is performed using a minimum of 24 thermocouples connected to a calibrated data recorder such as Kaye. Temperature distribution shall be +/- 0.5C from the coldest to hottest location within the chamber. This information can be used to determine the best load configurations when running Performance Qualifications. After the OQ has been passed, the validation can proceed to the Performance Qualifications.
Performance Qualification (PQ)
In this final phase of the validation process, the sterilizer cycles are run with actual loads that the end user plans to run in their everyday operation. Loads can be bags with bedding, stacked animal cages, water bottles, and loading equipment. The end user has the option to validate every cycle on the sterilizer or just certain cycles pertaining to their operations. Each type of load is documented with a diagram for future reproduction in daily use. All parameters of the sterilization cycle & load are documented. Once cycles & respective loads are validated, the facility will use these cycles and load configurations in their everyday operation in order to eliminate failed cycles, lack of sterility, and other failures regarding reproducibility of process.
During daily use of the sterilizer, inspection of the cycle data printout should be confirmed with the programmed cycle parameters at the end of every sterilization cycle to ensure conformance. Cycles determined to be out-of-conformance can be re-run at the discretion of the end user.
Conclusion
There should be a new standard for the Animal Care industry for steam sterilizers that incorporates a lot of the pharma requirements in so far as accuracy & repeatability. Establishing autoclave consistency in animal research is a function of the sterilizer design features, proper testing of all components of the sterilizer to prove performance, and establishing everyday work procedures that must be adhered to in order to guarantee sterility and protection of the goods being sterilized.
Incorporating proper quality assurance, validation procedures, and workflow procedures, will ensure a higher level of consistency and confidence in experimental findings to support improved reproducibility across animal studies. If we can do better, we must. The lives of our animals, and our patients, hang in the balance.