At the recent Institute of Decontamination Sciences (IDSc) annual conference, held in Blackpool, the problematic issue of protein detection and removal was high on the agenda. LOUISE FRAMPTON reports.
Just how ‘clean’ should we be aiming for, when reprocessing instruments? How should this be measured and are current testing methods, for detecting residual proteins, capable of providing the assurances that are required? These were some of the questions addressed by Professor David Perrett, Professor of Bio-analytical Science, William Harvey Research Institute, Barts & the London School of Medicine & Dentistry, Queen Mary University of London. He reminded the audience of the potential risks posed by failure to remove protein on reusable instruments. Despite the perception that vCJD fears have now passed, the latest data highlights the continued threat of patient-to-patient transmission. There are 220 cases of vCJD worldwide and 176 in the UK. However, this may be the tip of the ‘iceberg’ – the most recent appendix study (of 32,441 appendix samples, collected during surgery on patients born between 1941 and 1985 in the UK) showed that one person in every 2,000 is carrying the prion associated with vCJD. The release of this worrying data by the Department of Health (England) in August did not receive widespread coverage – just one UK newspaper reported the news at the time (The Independent, 11 August 2012). In fact, the estimated number of people with the suspect prion has doubled compared to an earlier survey, conducted in 2004. Prof. Perrett pointed out that the infective dose for transmission is extremely low (possibly only 1,000 prion molecules) and epidemiological calculations suggest that vCJD could still be considered a self-perpetuating disease in the UK population. Experimentally, vCJD is readily transmissible via stainless steel instruments, although there have been very few reported cases of infection via medical devices. The most well-known transmission incidence involved brain electrodes, he explained.
Problem of protein removal
Sterilisation and decontamination are not the same thing, he pointed out. “We need to remove prions. However, the prion protein is highly hydrophobic and has a strong ‘affinity’ with stainless steel. Animal experiments have shown that when a stainless steel wire or ball bearing, contaminated with the infective prion protein, is implanted into the brain, even for just a few seconds, the animal will go on to develop vCJD,” he commented, adding: “Unfortunately there is no sensitive assay for vCJD. Therefore, the Department of Health (England) and its advisors have taken the view that we need to study total protein as a surrogate – if you can reduce total protein you should be able to reduce prion protein,” suggested Prof. Perrett. There is a significant body of DH funded research underway into tackling the issue of protein contamination on reusable surgical devices, at various institutions throughout the UK. Projects highlighted in the presentation included research to evaluate the use of high sensitivity fluorescent methods for detecting proteins, instrument storage post-operation, as well as a programme to optimise washer-disinfector cycles. There are also projects being funded to progress and explore decontamination methods such as gas plasmas, for example, and novel coatings for surgical instruments to reduce protein binding. Prof. Perrett went on to describe the processes currently available for protein detection. Many sterile services departments (SSDs) use the desorb (wipe) and test method. Water wetted swabs are used to remove as much of the protein as possible. Protein quantities are then measured. Prof. Perrett explained that Ninhydrin testing is currently recommended in guidelines. The chemical reacts with amines and amino acids, forming a deep blue or purple colour (known as Ruhemann’s purple). However, research carried out by Prof. Perrett and colleagues showed that negative results for protein, using Ninhydrin, did not correlate to (positive) visual scores or protein measured by other tests. Prof. Perrett emphasised that the Ninhydrin test reacts positively with arginine, the supplied standard, but this is not the same as a protein – it is an amino acid. Tests further evaluated the efficiency of removing hydrophobic proteins (fibrinogen) by ‘wiping off’ the protein. They found that two-thirds of the fibrinogen protein remained stuck to the surface when water was used, after 15 strokes of scrubbing. Even when using a detergent solution, 25% of the protein remained. “The swabbing method is not efficient at removing protein,” he concluded. “We have two problems – Ninhydrin doesn’t detect and swabbing doesn’t remove. Therefore, this test, in my opinion, should not be in the guidelines.”
ATP: testing for protein?
He went on to discuss the use of ATP (adenosine triphosphate) testing for detecting protein. There is a body of manufacturer’s literature that claims ATP measures protein, he explained, adding that, in fact, ATP is ‘an energy molecule found in all living cells’. “ATP relates to purine nucleotide – it does not indicate residual protein levels,” he asserted. “ATP is good at detecting live organisms – whether they are bacteria or biofilms… But it is not a good method for protein detection on washed surgical instruments.” He commented that each of these test methods has its limitations and advantages. Many tests used in SSDs have been adapted from the laboratory, for use with the swabbing procedure, but often show a poor understanding of the underlying chemistry, in his view.
Biuret method
Prof. Perrett went on to describe the Biuret method – based on the reaction of proteins and peptides involving the use of copper and an alkaline solution. The blue solution turns violet to indicate a positive result. A spectrometer is required to evaluate the degree of colour change from light blue to violet. Another very common method is the Lowry assay, which measures protein by Folin reaction – producing a red colour change. He commented that the test is relatively sensitive in the laboratory, but there can be various interferences – such as detergents. Prof. Perrett added that a variation on this test is the bicinchoninic acid assay (BCA), developed by the Pierce Chemical Company, which also results in the development of a colour change and involves the use of copper. It is more sensitive than the Lowry assay, although it is also subject to interferences – such as detergents, he explained. Other tests include the Bradford assay – a Coomassie dye binding method. This provides a very quick reaction and is best performed using a spectrometer, according to Prof. Perrett. However, he added that there is a reasonable colour change that can be seen by the human eye. Different proteins respond differently to the dye in this test, he explained – in much the same way different cloth types respond to dye, depending on their composition. Concluding, he summarised:
• Swabbing using cotton or rayon and water is not effective at removing proteins – especially hydrophobic proteins.
• The Ninhydrin test does not work with any sensitivity in detecting protein.
• The Biuret method is insensitive.
• The BCA method needs incubation for a true colour formation and careful monitoring.
• The Bradford type methods are relatively insensitive and users need to look at the tip of the swab for the colour change, contrary to the instructions.
In situ detection of proteins on instruments
The second part of his presentation looked at in situ methods of detection of proteins on surfaces. This requires the application of the reagent to the surface, observation and measurement. The Choice Framework for Local Policy and Procedures (CFPP-0101) mentions new approaches including research around fluorescence detection, which is much more sensitive than colorimetric methods. Prof. Perrett commented that fluorescence is at least 100 times more sensitive and much more specific. “Intense light sources are required, often involving laser beams; however, few compounds are naturally fluorescent,” he continued. “The ideal solution would be a simple system that is suitable for the SSD environment, which provides a permanent record. Sensitivity needs to be at least 100 times better than current methods; it needs to be protein specific; it should be fast and capable of high throughput; the instrumentation should be readily available; it should not use laser beams as they are very expensive; a stable reagent producing stable fluorophores would be welcome; the solution should be non-toxic and safe; capable of revealing protein on the entire instrument; and lowcost in terms of capital outlay and running costs.”
Detection using fluorescence
There are various reagents that show promise, according to Prof. Perrett, including o-phthaldialdehyde (OPA) in the presence of a thiol. OPA produces a reaction with proteins and amino acids, yielding an ultraviolet colour change. He explained that the OPA derivative is not only UV-absorbing, but also a fluorescent compound. In addition, low reagent concentrations are required; it is relatively inexpensive and offers the benefit of very high sensitivity. Prof. Perrett and his colleagues have investigated the use of this approach for in situ protein detection on instrument surfaces, using a gel imaging system for fluorescence applications – G-BOX from Syngene (a division of Synoptics) based in Cambridge. Using the G-BOX, they were able to obtain images, which clearly captured the location and quantity of protein on the instrument in situ. Red indicated the highest levels of protein, yellow indicated significant protein and black areas indicated no protein at all. “We subsequently developed an experimental procedure using the reagent with the G-BOX and spoke to Syngene about taking this forward,” Prof. Perrett revealed. He explained that research was carried out which involved drying protein onto stainless steel material and processing items through validated washers. The captured images, using OPA/ G-BOX, showed that alkaline detergent offered poor protein removal. If the stainless steel was kept moist for 48 hours before being washed, these results showed some improvement. The results for the enzymatic detergent showed that some protein still remained when protein was dried onto the surface of the stainless steel. However, when kept moist, the enzymatic detergent performed very efficiently. The enzymatic detergent was found to be around 10 times better at removing protein than the alkaline detergent. In addition, the test found that cleaning was more effective on the middle shelves of the washer, compared to the bottom shelves. By 2012, the protein detection system had been developed and improved, while further research was carried out with GOSH and UCLH, using laboratory contaminated neurosurgical stainless steel instruments. Once again, keeping instruments moist improved the efficiency of cleaning, while enzymatic detergent proved very effective at removing protein compared to alkaline. Imaging indicated that areas such as screws and ‘hooks’ on the instruments retained some protein even when cleaned with enzymatic detergent. The researchers went on to look at ‘real’ instruments that had been processed by decontamination facilities, in the previous 10 days. Prof. Perrett presented some images of a reprocessed orthopaedic set which showed areas of fluorescent yellow, indicating a covering of protein of around 10 nanograms per mm2, with some bands on surfaces that proved very difficult to wash. A washed ophthalmic set was also found to have around 10 micrograms per mm2 of protein contamination. Prof. Perrett concluded by presenting some captured images of the results of testing of unopened, packed, sterile, single-use instruments, from various suppliers – which showed worrying levels of protein contamination. Prof. Perrett reported that the system has been successfully used in two SSDs. He commented that this approach has highlighted:
• The importance of keeping instruments moist before cleaning.
• Very clean instruments can be achieved – it is not difficult with a proper optimisation system.
• The cleanliness of new single-use instruments is very suspect.
Prof. Perrett concluded by raising some key questions that need to be addressed: How clean should we be aiming for? How many instruments should we be testing routinely? “The Department of Health (England) will now be considering what are the risks and the tests that should be performed,” he commented. “Such tests are standard in other industries. When travelling on an aeroplane, passengers can feel confident that the nuts and bolts have been tested and are extremely unlikely to fail while they are in flight, because there are standards that outline exactly what tests should be done, and how many are to be done. We need to adopt the same approach.”
About the protein testing system
The system described by Prof. Perrett is now marketed under the name of ProReveal. It is built in Cambridge, by Synoptics Health, and is commercially available in the UK through Peskett Solutions. Users lightly cover a reprocessed surgical instrument with the ProReveal spray and then place it in the ProReveal imaging system. At the touch of an on-screen button, the system automatically shows an image of contaminating proteins on the instrument and measures the amount of residual proteins. The built-in ProReveal software indicates via an on-screen green tick or red cross if this is a pass or fail of the decontamination process. This simple process takes less than five minutes, enabling users in SSDs to rapidly perform sensitive in situ detection of proteins on reprocessed surgical instruments.
