New approach to prion protein removal

Following the outbreak of bovine spongiform encephalopathy (BSE) – commonly known as “mad cow disease” – in the 1990s, research demonstrated that the transmissible spongiform encephalopathy’s (TSEs) could be transmitted between laboratory animals via contaminated surgical instruments. Not surprisingly this caused serious shock waves throughout all central sterile services departments in the UK. The alarm was further heightened by the discovery that neither disinfectants nor autoclaving removed prion.1 The Health Protection Agency moved quickly to ensure that all potentially contaminated instruments were removed and quarantined.2 However, there are ongoing fears of how much prion disease remains quiescent in the community. TSEs, of which BSE is one, are believed to be caused by infectious proteins (prions) rather than bacteria or viruses. Investigations into scrapie in sheep in the 1960s led to the hypothesis that certain proteins could be infectious. Research in the USA by Prusiner suggested that these infective proteins exist in two forms. The normal coiled form (PrPc) exists as a marker on the outside of cells. However, the infectious form (PrPsc) is folded into beta pleated sheets (i.e. folded like old continuous computer paper). Prusiner suggested that the pleated form could “flip” the coiled form into the infectious pleated configuration – a hypothesis that won him a Nobel prize. Build up of prion leads to the formation of amyloid plaques and vacuoles in the brains of infected persons. The TSEs are highly infectious with transmission being achieved with tiny amounts of infected nervous tissue. Synergy’s research in this area started with the development of a quantitative instrument cleaning test that would identify residual protein at a nanogram level. Present Quality Control tests used to validate instrument cleaning processes are described in HTM 2030. These tests recommend that visible soils (Edinburgh soil or Brownes soil) are painted onto a number of test instruments which are then allowed to dry. The instruments are put through the cleaning and disinfection processes, then visually inspected. Any visible trace of soil remaining on the instruments is considered a “fail”. Our tests on these soils yielded disturbing results – the Edinburgh soil showed a variation of 240% in haemoglobin concentration between batches. Tests on Brownes soil showed that it failed to mimic the chemistry of the normal contaminants found on surgical instruments. Both tests were far from ideal and only enabled approximately 1mg of residual protein to be identified. Ideally tests should at least identify nanogram amounts of residual protein. These investigatory studies showed that a better instrument cleaning test was required. The test should be quantitative, reproducible and much more sensitive than those listed in HTM 2030. To meet these ideals we developed the “cleaning disk test”.

Cleaning disk test

The two most difficult to clean parts of surgical instruments are catheter grooves and box joints. We therefore designed a stainless steel test disk containing three catheter grooves and a box joint (see Fig. 1). This disk is used as the standardised test instrument. The next steps were to select a test soil and an analytical method. After a wide literature search and wide discussion with colleagues, it was decided that the best test soil was porcine blood. UV spectrometry was selected as a tried and tested method of lab analysis. The disk test is carried out by pipetting a known volume of blood onto the lower half of the disk and allowing this to dry. The lid of the disk is then attached and the disk put through the cleaning process. The disk is then removed, opened and the lower half placed in an extraction dish. Solvent is added and the dish disk and solvent agitated on a laboratory rocker. A sample of the extract is then taken and placed in a scanning UV spectrophotometer. The peaks and their corresponding absorbencies are recorded. The normal spectra of porcine blood is shown in Figure 2. The absorbance (the height) of the major peak at 415 nm is used to quantify the amount of blood remaining on the test disk. The amount extracted into the solvent is compared with the amount in a control sample containing the same amount of blood in 100 ml of the solvent. This test is now used routinely as a quality control test to check the efficiency of the chamber washers at Synergy’s Bellshill site. In the lab, the cleaning disk test has enabled us to identify factors that complicate instrument cleaning – the two major ones being alcohol and temperature fixation. Alcohol reacts with blood to cause an insoluble layer on the surface which complicates and slows its removal.3 Seventy-five per cent alcohol, the concentration most commonly used as a disinfectant in wards and theatres, causes the greatest amount of protein fixation to steel. Increased temperature also results in fixation. Exposure to as little as 50°C for two minutes results in 10% fixation. Increasing the temperature to 60°C for two minutes results in 20% fixation. Temperature fixation complicates cleaning. Alcohol and temperature binding both have significant implications on instrument reprocessing. The use of alcohol containing disinfectants and skin preps in theatres is widespread. Alcohol fixed blood is virtually inevitable on, at least, some instruments returned from theatre. Temperature fixation is also likely during instrument reprocessing. Most automated decontamination processes have a cold prewash cycle that lasts for about seven minutes. At the end of this cycle the temperature is increased to 50°C. Measurable temperature fixation occurs within minutes at this temperature. To avoid this problem, blood and soil have to be removed from the instruments during the cold prewash. Furthermore, cleaning blood and protein must be removed during the first seven minutes of the cold prewash. We further investigated which detergents could achieve complete cleaning in seven minutes. The most efficient detergent achieved complete removal of blood from external surfaces in seven minutes. However, the same detergent only achieved 95% cleaning of the catheter groves and the box joint in this time. Alcohol fixed blood was much more difficult to remove. Detergents achieved only 90% removal of alcohol fixed blood from external surfaces and 85% removal from internal surfaces. Enzymatics were particularly disappointing removing less than 30%. This study suggested that blood soiled instruments which have come in contact with either alcohol or heat will not be completely cleaned during the first cycle of the normal chamber washer process. Having demonstrated that current cleaning processes are challenged by alcohol and temperature fixation, a better method of instrument cleaning was sought. Ideally the cleaning process should: • Remove blood and alcohol fixed blood from internal and external surfaces. • Remove prion from instruments. • Destroy prion and digest protein to enable safe disposal of waste. • Be safe and simple to use. • Be economically viable for use. Current cleaning methods concentrate on the removal of soil by chemically dissolving the soil from the outside of the soil down to the metal surface (a top down approach). The alternative would be to repel the soil from directly from the instrument surface (a bottom up approach). Figure 3 illustrates these two different approaches. Most research has investigated the top down approach, but there has been little investigation of the bottom up concept. Hence, this was targeted as the focus of the initial studies. We investigated a range of surface coatings with the aim of preventing protein sticking to the metal surfaces. However, technical difficulties encountered in the application and removal of these coatings led to the abandonment of this idea and another approach was required. Inspiration came a few years later during a meeting with colleagues from the Neuropathogenesis Unit in Edinburgh, which specialises in animal prion research. As a young pharmacist at Guy’s hospital in the 70s, I was given the challenge of removing mercury from a gold wedding ring. A tearful nurse had broken a mercury thermometer, and the mercury had “silvered” her ring. My challenge was how to remove the mercury plating without damaging the ring. It was a challenge that stuck in my mind. In a “eureka moment”, it occurred to me: could it be possible for a protein to cold electroplate onto steel? If so, it should be possible to reverse the process by electrolysis. Proteins are charged molecules – if you put the same electrical charge on the metal surface as on the protein, then the protein should be repelled from the surface. If this worked it could offer a simple method of removing prion. Working with research partner, Dr Bob Smith, the hypothesis was put to the test – using a 12 volt car battery, two metres of wire and a large plastic tank to provide the basis for the electrolytic cleaning apparatus. To our amazement and delight, it removed alcohol fixed blood in around 45 seconds. Along with our colleagues from the Neuropathogenesis Unit, we christened the process “electro-elution”.