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By Doug Harbrecht
MANY ARTICLES HAVE BEEN WRITTEN DESCRIBING A variety of sterilization processes in great depth. This article is intended to provide a broad overview of common sterilization technologies and the basic elements of sterility assurance, followed by guidelines for selecting a sterilization process. A brief history of microbiology is provided to remind readers how recently it was discovered that sterility is a critical factor in medical procedures.
Early Discoveries in Microbiology
Until the invention of the compound microscope, in which a second lens further magnifies the image from a first lens, individual microorganisms could not be observed. Simple lenses were not powerful enough to provide the magnification needed. Typical microorganisms vary between one half and several microns in size. A micron is one thousandth of a millimeter. The human eye can detect objects as small as one tenth of a millimeter. The Dutch investigator Anton von Leeuwenhoek (1632--1723), skilled in glassblowing and fine metalwork, was the first to identify the world of microorganisms by use of a compound microscope that he constructed. In the ensuing years, many researchers attempted to determine if life arose from non-living matter and air (spontaneous generation or abiogenesis) or whether living organisms were carried in dust and other materials. Thus, the connection of this life form to human disease was not made for many more years. Louis Pasteur (1822--1895), in the course of investigating fermentation of wine and beer, largely disproved the theory of abiogenesis. He used flasks with long, bent necks to demonstrate that broth boiled in such a flask would not spoil, even though exposed to air, until the flask was tipped, forcing the broth to touch the dust captured in the neck of the flask. During such early investigations, heat was the means used to prevent the development of growth. Insufficient heating, or dependence on boiling, often triggered confusing results due to the unknown existence of heat-resistant spores. John Tyndall (1820--1893) disproved abiogenesis in 1876 by demonstrating that there was a heat-resistant form of life. He boiled broth, allowed growth to develop, then boiled it again, and demonstrated that no further growth would occur. In this process, which became known as tyndallization, the heat-resistant spores had been allowed to germinate and the growing microorganisms were then boiled before they could again form spores. Robert Koch (1843--1910) is known for developing isolation methods and pure culture techniques, and demonstrating the connection between specific microorganisms and human disease (Koch's postulates). Proving the connection between microorganisms and disease or infection led to the development of aseptic surgery by Joseph Lister (1827--1912). Dilute solutions of carbolic acid (phenol) were used to cleanse wound and surgical sites, and the importance of sterilizing surgical instruments was realized.
Handwashing and preoperative surgical scrubbing became part of the system of aseptic surgery introduced in 1882 by Trendelenburg, von Bergmann, and Schimmelbusch in Germany and by Halstad in the US when it was demonstrated that bacteria present on the skin could cause a wound infection. Prior to this, it was not unusual for a patient to survive an operation only to succumb to infection shortly thereafter. As recently as the American Civil War (1861--1865), there were more deaths in the military due to infection than to the wounds themselves. As always, the reason to be concerned with sterility of medical devices is to reduce the risk of causing infection in the patient. Thus, the primary reason to sterilize medical devices is to eliminate human pathogens--organisms that are known to cause human disease. Because patients often are weakened or have compromised or suppressed immune systems due to either disease or drug therapy, it is also important to eliminate organisms that are not normally considered pathogens. The combination of sterile devices, aseptic surgical procedures (attempting to exclude microorganisms), and antibiotics has made possible the wide range of invasive medical procedures used today.
Sterilization means to free an object or substance from all life of any kind. Sterilization should be distinguished from disinfection, which means the killing or removal of organisms capable of causing infection that may not necessarily result in sterilization. Common disinfectants include phenol, formaldehyde, chlorine, and iodine. Sanitization is a form of disinfection, generally as it applies to inanimate objects. Antiseptics are similar to disinfectants, but are generally considered to be substances that kill or inhibit microorganisms in contact with the body without causing extensive damage to the flesh. Asepsis, or aseptic technique, refers to the exclusion of microorganisms from an environment or procedure.
While there are many variations of sterility and microbial count procedures, two of the more common methods are briefly described here. Live organisms on a device are most commonly detected by placing the device in a nutrient solution or rinsing it with a nutrient solution, incubating the solution for a period of time at a specified temperature, and then looking for growth of organisms by the formation of turbidity. This is referred to as a sterility test. The sterility test merely demonstrates the presence or absence of microorganisms. Alternatively, the rinse solution may be filtered through a sub-micron filter before incubation, followed by placing the filter on a nutrient agar (gel) or nutrient pad and incubating it for a period of time at a specified temperature. Each organism trapped on the filter gives rise to a colony, and the number of colonies can be counted. This is commonly referred to as a bioburden test, which demonstrates the total number, or burden, of microorganisms on a device. The accuracy of counting the organisms in this way is affected by how efficiently the rinse procedure removes organisms from the device.
Reliability of both the counting and sterility methods also is affected by the growth conditions used, because some organisms grow only under very specific conditions. Some of the most common variations in growth conditions are the type of nutrient, incubation time, incubation temperature, and presence or absence of oxygen. Obviously, it would be very easy to fail to detect organisms if any of these conditions are incorrect for the growth of the organisms that are present on a device. While this limitation should be acknowledged, in most cases just one or a small number of conditions are used which will detect the majority of types of microorganisms of concern.
The assumption is made that it is highly unlikely for a large number of microorganisms to be present that are unable to grow under these generic conditions.
There have been some noteworthy exceptions, however, and the validity of this assumption should always be considered when working with new or unusual device materials. For reprocessed devices, the environment and conditions to which the device has been exposed, must be considered when selecting growth conditions for sterility testing.
How Sterility Is Assured
There are several factors that contribute to the assurance of sterility of a medical device. They include: the number of organisms on the device prior to sterilization, the resistance of these organisms to the sterilization process, the characteristics of the sterilization process, and the length of time of exposure to the sterilizing conditions.
Sterility, especially when dealing with a large number of devices, is difficult to prove for three reasons. First, it is possible to introduce viable organisms in the process of testing for their presence. Even under well-controlled conditions, a contamination rate of 0.1% is about the best that can be expected without going to extraordinary effort. Second, sterility testing is generally destructive, as an entire individual device can only be tested under one set of growth conditions. Finally, absolute sterility cannot be proven, it is expressed as a mathematical probability of survival.
For example, a population of viable organisms is gradually reduced in numbers over time under a given set of sterilizing conditions until there is a probability of less than one survivor. There is a logarithmic relationship between the number of viable microorganisms in a population and the time under a given set of sterilizing conditions. In other words, the number of viable organisms is reduced by 90% in a given time period. Exposure for another equal time period reduces the population by 90% again, and so on. This is commonly expressed in terms of D-value. For example, for an organism with a D-value of four minutes, the population will be reduced by 90% in four minutes under a specified set of sterilizing conditions. For most types of devices, manufacturers are required to reduce the probability of a surviving microorganism to less than one in a million. That is commonly expressed as a sterility assurance level (SAL) of 10-6. A SAL of 10-6 means that statistically, less than one in every million devices carries a viable organism. The importance of this can be appreciated when tens of thousands of a given type of medical device are produced in a year. Obviously, it is not practical to test enough devices to demonstrate this level of assurance through sterility testing alone, and the possibility of contamination during sterility testing carries the risk of falsely concluding that a sterile product is not sterile. In addition, while the average number of microorganisms on a given type of device can be determined, any one product carries an unknown level of contamination, and the resistance of these organisms to the sterilizing process is difficult to determine. Therefore a simple sterility test of naturally contaminated product cannot be used to establish the required SAL.
To address these problems, a known quantity of a standard organism with a known level of resistance to the sterilization process is used to contaminate the product. The organism of choice has a high resistance to the sterilization process being evaluated. By demonstrating sterility of a product contaminated with 106 of these organisms and then exposed to the sterilizing conditions, a reduction of 106 (six log reduction) is demonstrated. By doubling the exposure time to the sterilizing conditions, an SAL of 10-6 (12 log reduction in this case) can be achieved.
The other component of sterility assurance has to do with the population of viable organisms present on a device prior to sterilization. For example, if the bioburden is routinely controlled to less than 1,000, the exposure time can be reduced to provide a nine log reduction and still achieve an SAL of 10-6 (103- 109= 10-6).
Methods of Sterilization for Medical Devices
Common methods of sterilization include physical methods and chemical methods. Physical methods include dry heat, steam, radiation, and plasmas. Radiation encompasses a variety of types, including gamma radiation, electron beam, X-ray, ultraviolet, microwave, and white (broad spectrum) light. Chemical methods include, for example, ethylene oxide, propylene oxide, chlorine dioxide, ozone gases, and a variety of chemicals in liquid and vapor form, such as glutaraldehyde, hydrogen peroxide, and peracetic acid.
Reprocessing of Medical Devices
Few topics have generated more debates in the healthcare system in recent years than the reprocessing of medical devices. Cleaning is one of the most critical aspects of reprocessing because biological substances, such as tissue and blood, can greatly shield microorganisms from the effects of sterilizing agents. Cleaning typically includes mechanical and chemical removal of debris along with chemical disinfection. Historically, medical devices were often made of metal, and reprocessing was a practical and expected part of using these devices. Simple devices were relatively easy to clean and could be readily sterilized without loss of function.
Reprocessing became more difficult as devices became more complex. Dismantling and reassembly of the device often became necessary to assure cleanliness. Advances in technology brought smaller and more intricate devices, with cavities that were difficult to clean. Materials with superior performance characteristics, such as polymers, adhesives, and coatings, became available. Many of these materials cannot withstand some of the common cleaning and sterilization processes, or multiple exposures to those processes. Cost pressure on the healthcare system has put pressure on medical device manufacturers to design devices for reprocessing. Depending on the device and the desired performance characteristics, this may not always be possible. The appropriate balance between performance and reprocessing continues to stir debate. What must not be compromised is patient safety.
Selecting a Sterilization Process
The sterilization processes most commonly used by medical device manufacturers are ethylene oxide and radiation. Steam also continues to play a significant role. This is due in part to the availability of equipment for large-scale processing and the flexibility of application of these processes to a wide variety of products and packaging. Alternative methods are more common in hospital applications because they can be cost effective on a smaller scale and tend to have fewer operational safety concerns. Such alternatives are also being investigated for use at the end of manufacturing lines rather than in a central batch processing environment.
When selecting a sterilization process, its effects on the product function and package integrity, and the effectiveness of it on each particular product must be considered. The first consideration should be the characteristics of the device to be sterilized. The main device characteristics are materials, design, and dimensions. These characteristics will determine thermal stability (softening, melting, warping, shrinking, taking on a shape set, etc.), absorption of humidity (swelling, tackiness, etc.), radiation stability (yellowing, cracking, crazing, embrittlement, etc.), reaction with chemical sterilants, ability to withstand pressure and vacuum conditions, and ability to be penetrated by the sterilant (permeability, density, long narrow lumens, small cavities, etc.). Packaging needs to be considered as well, particularly temperature stability, permeability and seal strength; however, packaging can often be adapted to the needs of the sterilization process.
Other considerations for selection of a sterilization process include:
Other than this, it is difficult to make general statements about the different methods of sterilization because there can be large differences in these factors depending on the size and location of the sterilizer, as well as the methods employed for product release. For example, required exposure time for gamma irradiation may be just a few hours. However, exposure time and the dose are controlled by the speed of the product carrier on a moving track that winds its way past the gamma source multiple times. Products are usually grouped by their minimum and maximum allowable doses, so it might take days for product to actually be processed through a large facility. Similarly, an ethylene oxide cycle may take less than 12 hours in the sterilizer, but pallets of product often need to be preconditioned (allowed to heat and humidify) and later aerated to remove ethylene oxide residuals, resulting in a total process time of two to four days. Use of biological indicators for release can add another two to seven days to the process. All of these issues will affect selection of the sterilization process and the efficiency with which it operates.
Doug Harbrecht is the senior scientist of sterilization systems for Boston Scientific Corporation. He received his bachelor's and master's degrees in microbiology from the University of Wisconsin--Madison, is active in AAMI and ASTM, and has 20 years of sterilization and research experience in the medical device and biopharmaceutical industries.
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