In veterinary practice, surgical instruments and kits are routinely prepacked and sterilised ready for use, but then may remain in storage for a period prior to being used. There is currently no published veterinary research to support an optimal storage method and duration of storage to maintain sterility of surgical instrumentation (SI) when using self-seal autoclave bags (SSABs). However, there is a small body of research in human healthcare (Sattayasanskul et al, 1990; Schwartz and Davis, 1990; Butt et al, 1991; Bhumisirikul et al, 2000; Rosa et al, 2001; Bhumisirikul et al, 2003; Brusca et al, 2004; Barker et al, 2011), which could be used as a guide in clinical practice. Some research recommended storage duration, whereas others expressed inconclusive evidence. There are several recommendations from manufacturers of veterinary SSABs and in existing veterinary literature. For example, Millpledge (2023) suggested a 3–6 month shelf life of their SSABs, post sterilisation, depending on the handling and storage conditions In addition, KRUUSE stated that SI will remain sterile for up to 6 months post sterilisation when using their SSABs (https://tinyurl.com/2y2mb3mf).
Sustainability in the veterinary practice is currently an important topic. The recently created Greener Veterinary Practice Checklist by Vet Sustain (2021), outlines the points that a veterinary practice should consider to ensure good sustainability practices. One of the points under the ‘practice responsible resource use’ section advises veterinary practices to reduce their disposable materials usage, including single-use plastics where possible. Furthermore, this section also advises to review and optimise waste management, for example, reduce, re-use and recycle (Figure 1). The use of SSABs should be considered in line with this checklist, including storage duration and the requirement to repackage and resterilise SI once the stated expiry date has passed.
The clinical element of the research aimed to determine an ideal storage duration and method of single SI, packaged in SSABs. The questionnaire element aimed to determine the current practice of sterilisation and storage methods and durations using SSABs in small animal practice.
Literature review
Multiple studies in the field of human healthcare have demonstrated the ability of sterilisation packaging materials in maintaining barrier integrity for several months post sterilisation. For example, a non-randomised control study by Puangsa-Ard et al (2018) discovered that all 1680 sterilised autoclave pouches remained sterile following a 1–6 month closed storage duration (n=240 in each of the seven negative control groups). The positive control group consisted of 280 pouches which were intentionally damaged at the beginning of the study, all of which were found to be contaminated following their storage durations as stated above. An additional element discovered that pouches in the negative control groups that were resterilised once, three times and five times remained sterile following a 6 month, closed storage duration, which concluded the strength of the pouches and their ability to maintain barrier integrity and sterility for up to 6 months. However, study limitations include the absence of SI in the pouches as the researchers wanted to prevent the possibility of sharp objects causing damage. This is not realistic to real life practice therefore results should not be extrapolated to clinical practice as they may have been different results if test objects were used in the pouches. Furthermore, there is a lack of evidence to suggest whether the pouches were undisturbed during storage. Current human and veterinary guidance suggest a loss of sterility is associated with event-related factors rather than a specific storage duration (Widmer et al, 1992; Hamilton, 2012; Morton and Conner, 2014). Therefore, further information would have been beneficial when interpreting the results.
Findings from a similar human, non-randomised control study by Klumdeth et al (2020) suggested the reusing of paper/plastic sterilisation pouches to be a safe infection control procedure. A total of 320 pouches containing dental examination mirrors underwent clinical use three times, including subsequent cleaning and sterilisation in the original pouch. On the fourth occasion, the mirrors were replaced with a strip of filter paper prior to being sterilised again and put into a closed storage for 6 months. A total of 291 pouches completed the study and following microbial cultivation, the internal environment of all pouches was sterile. As the 291 pouches remained sterile, this may explain why the 1680 pouches that underwent one sterilisation process in the study by Puangsa-Ard et al (2018) remained sterile. If pouches that have been processed multiple times can retain a sterile internal environment, pouches undergoing one process stand a good chance of retaining sterility. However, a similar study by Palananthana et al (2006) identified a 1.33% contamination rate of surgical wires following a 4 month storage duration (n=12 out of 900 pouches). These pouches underwent one to five resterilisation processes prior to storage and the 12 contaminated samples were in the same group (resterilised once). The researchers were unsure as to whether contamination occurred as a result of deterioration of the packages or inadvertent contamination during experimental procedures.
The association between loss of sterility and event-related factors, rather than association with a specific storage duration is supported by a human, non-randomised control study by Butt et al (1991). The researchers did not identify a trend for an increased contamination rate over time for any sterilisation pack type (paper envelopes, nylon sleeves, peel pouches) when examined for contamination at monthly intervals for one year (n=100 per group, per month). However, conflicting results were identified in a non-randomised control study by Webster et al (2003) whereby 131 sterilisation packs were found to be sterile following a 24 month storage, which involved the handling and movement of a sample of packs every 3 months. This result challenges the current belief that there is an association between loss of sterility and event-related factors during storage.
Another human, non-randomised control study by Bhumisirikul et al (2000) found comparable results to those in the study by Puangsa-Ard et al (2018) as 150 individually packaged orthopaedic screws remained sterile following a 52 week storage in open conditions (open shelving). Furthermore, a subsequent study by Bhumisirikul et al (2003) followed an identical methodology to that in the preliminary study, except it spanned for 96 weeks. The same results were observed, and the screws remained sterile for 96 weeks further, supporting a longer storage duration under these conditions. However, orthopaedic screws make up a small proportion of SI used in the veterinary practice. Therefore, these conditions should not be followed for all veterinary SI until further research has been conducted.
Many studies in this review hold clinical significance in the veterinary practice but because of multiple limitations, the use of dissimilar sterilisation packaging materials and the differing field of healthcare, many findings cannot be safely extrapolated to clinical practice until further veterinary research has been conducted.
Methods
Online questionnaire
Jisc Online Surveys was used to create an online questionnaire aimed at members of the veterinary team in the UK. This was conducted to determine the current practice of sterilisation and storage methods and durations using SSABs in small animal practice. Social media platforms were used to disseminate the questionnaire link and posts were made shareable to reach a wider population.
Laboratory study
This was a non-randomised control study consisting of six study groups (1 open, 1 closed, 3 open, 3 closed, 6 open, 6 closed). The number indicates the storage duration of the packages following sterilisation in months. The words ‘open’ and ‘closed’ indicate the storage method. Open packages were stored on an open shelf and closed packages were stored in a closed plastic box, both in a non-clinical room. Packages were positioned horizontally, in contact with each other. Blade holders were used and ten were included in each group, totalling 60. They were packaged individually in medium SSABs prior to sterilisation in a steam autoclave. Each instrument was packaged by inserting the handle first followed by a time, steam, temperature (TST) strip as this is one of the recommended sterility monitoring methods in small animal practice (Packer and Devaney, 2010) (Figure 2). Bowie Dick tape, sterility indicators on the SSAB and spore tests were also used for sterility monitoring.
Instruments were exposed to a temperature of 134°C and adequate steam penetration for at least three and a half minutes during sterilisation as indicated by the TST strips. Packages were positioned in the autoclave following standard guidelines used in small animal practice (Hamilton, 2012; Kerrigan, 2013) and the cycle included drying. Packages were undisturbed during storage. For each storage duration category, two additional instruments were processed using the same methods described in this section. Unlike the other packages, they were processed in the laboratory as soon as they were cool following sterilisation. This was conducted for quality assurance, to establish whether they were sterile prior to continuing and following laboratory analysis, they were confirmed sterile.
Following storage, packages were assessed for damage. Instruments were removed using aseptic technique and tipped into individual sterile glass jars containing sterile nutrient broth (NB). NB No.2 was used because of its ability to cultivate fastidious bacteria and other microorganisms (ThermoFisher Scientific, 2023). Following a 48 hour incubation at 37.5°C, jars were gently inverted to ensure any particles were evenly distributed. A sample from each was withdrawn using a disposable sterile pipette and analysed in a spectrophotometer. The spectrophotometer provided an objective, numerical value for the optical density of the samples. Optical density measurements are considered the standard approach in microbiology for characterising concentrations of bacteria in growth media (McBirney et al, 2016). Optical density correlates directly with the cell concentration of a sample for both dead and living bacterial cells (McBirney et al, 2016). A light wavelength of 600 nanometres (OD600) was used as this is a common wavelength for determining the concentration of an inoculum (Matlock, 2019). For each storage category, a separate sample of sterile NB was firstly used to calibrate the spectrophotometer. Samples with an OD600 of ≤0.099 were deemed sterile. Samples with an OD600 of ≥0.100 were deemed contaminated as this indicates increased turbidity, most likely associated with contamination. Visual assessment of turbidity was also used as a robust outcome measure as the studies by Bhumisirikul et al (2000), Bhumisirikul et al (2003), Webster et al (2003) and Puangsa-Ard et al (2018) used this simple approach in their research (Figure 3). Odour assessment of the NB was also conducted with malodorous samples being indicative of contamination.
For both methodologies, ethical approval was granted by the Harper Adams University (HAU) Research Ethics Committee and piloting was conducted.
Results
Questionnaire results
A total of 59 participants took part. Single bagging was the most selected method for packaging and sterilising single SI as selected by 89.8% (n=53) of participants. Storage duration of single bagged SI ranged from less than one month up to more than 12 months (Figure 4). The most common storage duration of sterile single SI was between one and three months (n=40, 75.5%), followed by 4–6 months (n=11, 20.8%).
Participants undertaking single bagging were asked what happens to SI if they are not used once the expiry date of the package has been reached. Most participants reported rebagging and resterilising of the instrument (n=37, 69.8%). Only 5.7% (n=3) stated they resterilise the original bag containing SI (Figure 5).
The preparation room was the most frequently used storage location for the majority of sterilised SI (n=38, 64.4%), followed by the operating theatre (n=11, 18.6%).
A large proportion of participants stated their practice had a standard operating procedure (SOP) for the processing, sterilisation and management of SI (89.8%, n=53). Equal numbers of participants stated they did not have or were unsure about whether their practice had a SOP (n=3 in each group).
Laboratory results
Data varied across the storage categories (Figure 6). As a result of leakage of one jar in group 3 closed, a total of 59 samples were analysed. Data in all but one category (three closed) were skewed therefore non-paramet-ric tests were mostly conducted to assess for statistical significance. A P value of ≤0.05 was set as the level of statistical significance.
A Mann-Whitney U (Wilcoxon rank-sum) test was performed to compare the OD600 values in the 1 month open and 1 month closed groups and no significant difference was identified (U=43.0, sample sizes, 10,10, P=0.617). The median (interquartile range (IQR)) OD600 in group 1 open was −0.0115 (0.01625) and −0.0095 (0.01375) in group 1 closed (Figure 6). The same statistical test was performed to separately compare the OD600 values in the 3 month open and 3 month closed groups and the 6 month open and 6 month closed groups. Again, there was no significant difference (3 month group, U=43.5, sample sizes 10, 9, P=0.918, 6 month group, U=49.5, sample sizes, 10, 10, P=0.987). The median (IQR) OD600 in group 3 open was 0.006 (0.06025) and 0.006 (0.001) in group 3 closed. The median (IQR) OD600 in group 6 open was 0.002 (0.00575) and 0.002 (0.001) in group 6 closed (Figure 6).
As a statistically significant difference between the OD600 values of the open and closed groups (1, 3 and 6) was not identified, values were combined per group for further analysis. Residual plots were not acceptable therefore a Kruskal-Wallis ANOVA was performed to analyse these three data sets. A statistically significant difference was identified (H=19.35, d,f=2, P=<0.001). To assess where this difference occurred, multiple post hoc testing using Mann-Whitney U (Wilcoxon rank-sum) tests were performed and statistically significant differences were identified (Table 1). A total of five out of the 59 samples were identified as contaminated (8.5%). The mean (standard deviation (±)) OD600 of the contaminated samples was 0.2174 (± 0.0698). The NB of these samples had increased turbidity in contrast to the 54 sterile samples. Additionally, these samples were notably malodorous and thick and sticky in consistency, indicating contamination (Figure 3). In contrast, the median (IQR) OD600 of the sterile samples was 0.002 (0.0085) and the NB of these samples were identical to when they were originally formulated (clear) with no evidence of malodour. A Mann-Whitney U (Wilcoxon rank-sum) test was conducted, and a statistically significant difference was identified between the 54 sterile values and the five contaminated values (U=0.0, sample sizes, 54, 5, P=<0.001). The overall contamination rate was 8.5% (n=5 out of 59). The rates per group (open and closed combined) are displayed in Table 2.
| Data comparison groups | Results |
|---|---|
| 1 and 3 | U=64.0, sample sizes, 20, 19, P=<0.001 |
| 1 and 6 | U=72.0, sample sizes, 20, 20, P=<0.001 |
| 3 and 6 | U=102.5, samples sizes, 19, 20, P=0.012 |
| Group | Contamination rate |
|---|---|
| 1 month | 5% (n=1 out of 20) |
| 3 months | 10.5% (n=2 out of 19) |
| 6 months | 10% (n=2 out of 20) |
Discussion
No significant difference was identified between the OD600 values of the open and closed groups across each storage duration (P=0.617, P=0.918, P=0.987). This suggests a superior storage method to maintain sterility of SI does not exist and either open or closed could be used for effective storage in undisturbed conditions. The absence of a difference could be due to undisturbed conditions throughout the study. Additionally, regardless of the method, previous research identified the ability of autoclave pouches to maintain sterility of SI for up to 96 weeks in open storage (Bhumisirikul et al, 2003). Therefore, storage method may not be a significant factor. There is a lack of research on storage method in the human and veterinary field as the main body of research focuses on storage duration.
Results from the questionnaire revealed that 75.5% (n=40) of practices store sterile SI for between one and three months which implies they are adopting a cautious approach to infection control and prevention. Furthermore, as 89.8% (n=53) of participants stated their practice had an SOP on the processing, sterilisation and management of SI, evidence-based storage durations could be written in an updated SOP to ensure consistency and evidence-based practice. A further sensible suggestion is to sterilise rarely used SI when required to prevent unnecessary prolonged storage, wastage of valuable storage space and wastage of disposable materials, especially if instruments are rebagged and resterilised if their expiry date has been reached. This would help to improve the practice’s sustainability performance. As identified in the study by Klumdeth et al (2020), the reusing of paper/plastic sterilisation pouches was deemed a safe infection control procedure as the internal environment of 291 pouches remained sterile following multiple sterilisation processes. This raises the question of whether the veterinary practice could begin to move away from disposing of single use SSABs after one use?
When comparing the OD600 values of each storage duration group (1, 3 and 6 (open and closed combined)), a statistically significant difference was identified (P=<0.001). Further analysis revealed that there was a significant difference between all groups (P=<0.001, P=<0.001, P=0.012). This suggests that storage duration did influence the OD600 values across the three groups. The clinical significance of these results are questionable as it is highly probable that most of the samples were sterile at the end of their storage duration, as observed in several other studies (Butt et al, 1991; Bhumisirikul et al, 2000; Bhumisirikul et al, 2003; Puangsa-Ard et al, 2018; Klumdeth et al, 2020). Additionally, there was no evidence of contamination in 54 samples in contrast to the five samples that were identified as contaminated. Interestingly, the NB of the contaminated samples were of an increased turbidity and notably malodorous and thicker and stickier in consistency, clearly indicating the presence of contamination. Furthermore, the OD600 values of the contaminated samples were significantly higher than those of the sterile samples indicating an increase in turbidity because of microbial contamination. These were higher than the value set as the level of contamination (≥0.100) as stated in the methods.
The contamination rates per storage duration group suggest that sterile SI stored for up to one month are at a reduced risk of contamination in contrast to those stored for a longer period. Palananthana et al (2006) reported a considerably lower contamination rate of 1.33% following multiple resterilisation procedures and a 4 month storage duration. The researchers were unsure as to whether contamination occurred due to deterioration of the packages during storage or inadvertent contamination due to experimental procedures. A suggestion for the current 8.5% contamination rate is inadvertent contamination during transfer of the blade holders from the SSABs into the NB jars, as opposed to package deterioration. The authors consider this to be the most likely cause due to the difficulties experienced during the transfer of some instruments into the jars containing NB. Additionally, several studies conclude the strength of autoclave pouches and their ability to maintain barrier integrity and sterility for several months (Butt et al, 1991; Bhumisirikul et al, 2000; Bhumisirikul et al, 2003; Puangsa-Ard et al, 2018; Klumdeth et al, 2020), therefore, inadvertent contamination during transfer is the most probable cause. Furthermore, the loss of sterility during storage is said to be associated with event-related factors rather than a specific storage duration (Butt et al, 1991; Hamilton, 2012; Morton and Conner, 2014). Thus, the greater contamination rates in groups 3 and 6 are unlikely to be associated with longer storage.
Limitations
A small sample size was obtained for the questionnaire, and this undermined the validity and representativeness of the results. Limitations in the laboratory element include the small sample size. Additionally, spectrophotometry is not a perfect technique and technical variation and issues may occur, particularly in low microbial concentrations (Tom, 2021). Due to microbial diversity, no single medium can be used to culture all microorganisms (Klumdeth et al, 2020). In an ideal world, two broth types could be used to assess for contamination (Klumdeth et al, 2020). The storage methods used are unlike those used in real-life practice as sterile SI were stored in a non-clinical room. The questionnaire revealed that 83% (n=49) of participants stored the majority of sterile SI in a clinical room. The results may have been different if sterile SI was stored under similar conditions to that of clinical practice. However, this is solely a prediction and it highlights a relevant study area because of a lack of existing research.
Suggestions for further research
Further research could be conducted including single versus double bagging of SI, the effect of frequent handling and events on the sterility of stored SI, the impact of further storage durations in a clinical environment, whether early loss of sterility is associated with the weight and/or size of the SI contained in the SSAB and the current state of sustainability practices regarding the use of SSABs in clinical practice.
Conclusions
Research shows consistent findings, suggesting longer storage durations do not impact the sterility of SI. The durations examined in the literature are significantly longer than those used in current practice which implies veterinary professionals are adopting a cautious approach to infection control and prevention procedures. The laboratory results are mostly consistent with those in existing research. If practices adhere to current evidence, significant savings could possibly be achieved in supply and labour costs without impacting patient care, in addition to improving sustainability performance. A prevention is better than cure approach is often adopted and as sterilisation of SI is a major factor in the prevention of disease transmission, further research would be beneficial to enable the implementation of evidence-based practice.