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Polymath pharmacist beats prion to a pulp

Pharmacists frequently forget that they have a wonderful rainbow of experience in chemistry, physics, pathology, pharmacology, therapeutics and business. In these days of increasing specialisation, it is essential that we retain a few polymaths, writes Frank Prior of the Osmosis Unit, Edinburgh. Here he tells how he used his experience to come up with a solution to the transmission of Creutzfeld-Jakob disease via surgical instruments

by Frank Prior

Pharmacists frequently forget that they have a wonderful rainbow of experience in chemistry, physics, pathology, pharmacology, therapeutics and business. In these days of increasing specialisation, it is essential that we retain a few polymaths, writes Frank Prior of the Osmosis Unit, Edinburgh. Here he tells how he used his experience to come up with a solution to the transmission of Creutzfeld-Jakob disease via surgical instruments

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It had been a quiet day in the quality control laboratory. I then got a call asking if I could come to the hatch. There was a young nurse in tears. She had broken a mercury thermometer (this was in the early 1970s) and the mercury had plated her wedding ring. Could I do anything to help?

When I set up the QC lab at Guy’s Hospital in London it was to provide a routine check on the intravenous infusions that we used to make in-house. However, every now and then, something out of the ordinary popped up that was a good chemical challenge.

After half an hour thumbing through the reference books I came across two methods of removing the mercury: dissolution with aqua forte (a mixture of concentrated sulphuric and nitric acid) and electrolysis. I did not have the apparatus for electrolysis so an acid bath was the only option.

Fortunately this worked well and there was great relief (on both sides) when I handed back a shiny, clean gold wedding ring. It was a small incident but it stuck in my memory.

Dreams of inventions

At the turn of the millennium, after 11 of my 12 east of Edinburgh hospital pharmacies had been closed, I decided it was time to leave the NHS. I had always wished to follow my dream of being a medical inventor and in my new role as director of the Centre for Innovation in Healthcare Technology at Glasgow Caledonian University, I got one step closer.

One of my requests while there, was to help Trust Sterile Services, a local firm (now part of Synergy Health) that was having problems in reprocessing surgical instruments.

At the time, I was preparing to leave Caledonian and set myself up as a freelance medical inventor. Here was my first fascinating chemical challenge.

I set up a research and QC laboratory for them in Bellshill, developed a quantitative blood extraction test and invented the Cleaning Disk, a device that enabled quantification of the removal of blood protein from the inside of catheter grooves and box joints (the bit around the hinge in forceps and scissors). These were duly patented.

(Before this, the testing of cleaning processes involved visual judgement of removal of coloured soils, which only detects down to about 1mg.)

The next challenge was to address the iatrogenic transmission of the transmissible spongiform encephalopathies (TSEs) via surgical instruments.

Although “mad cow disease” (one of the TSEs) had hit the headlines in 1996 it was only in the late 1990s that evidence became available that it could be transmitted from infected surgical instruments. Worse than this, there was mounting evidence that the infectious protein was not destroyed by normal instrument cleaning processes or by autoclaving.

Prions

The TSEs are believed to be transmitted not by bacteria or viruses, but by proteins. The concept of infectious proteins was first put forward by Tikvah Alper and John Stanley Griffith in the 1960s.

Stanley Prusiner experimented with these infective proteins (prions) and published his prion hypothesis in Nature in 1982. He proposed that prion protein existed in two forms: a non-infective normal form (prion protein cellular; PrPc) and a highly infectious prion protein scrapie (PrPsc).

PrPc has a helical structure whereas PrPsc is folded like old computer printer paper and the form is known as a beta pleated sheet. Both prions contain the same amino acid sequence. The difference is in the three-dimensional folding of the molecule.

Prusiner’s hypothesis suggested that adding PrPsc protein to PrPc protein could flip the healthy form into the beta pleated sheet form. More recently, other workers have suggested that a small amount of DNA must be involved. Others suggest that the three-dimensional conformation is dependent on a balance between copper and aluminium in the environment.

Whichever is the case, PrPsc aggregates into amyloid fibres that implant into nervous tissue. These amyloid plaques cause vacuoles and serious damage to neurological tissue. Other histological changes caused by amyloid include astrogliosis and absence of an inflammatory reaction.

Although the incubation period for most prion diseases is generally 10 to 20 years, the incubation period for mad cow disease (more formally, new variant Creutzfeld-Jakob disease [nvCJD]) is just a few months. Once symptoms have appeared the disease progresses rapidly, resulting in brain damage and death. Neurodegenerative symptoms include convulsions, dementia, ataxia and behavioural or personality changes.

Shortly after the outbreak, nvCJD was shown to be transmissible from both infected meat and blood. All UK hospital sterile services departments were under the spotlight. How were they going to avoid transmission of the disease via surgical instruments?

Guidance was quickly issued that all instruments used on patients with known TSEs were to be removed, boxed and sent to the Health Protection Agency’s Porton Down site for storage or destruction. On the ground, this raised two practical problems.

The first was that diagnosis of nvCJD can only conclusively be made at post mortem, and the second was the practicalities of tracing all the instruments used in any particular surgical case.

From the chemical point of view the challenge was to work out how a protein could resist the detergents used in the washing process. The normal chamber washer process generally consists of a 20-minute dowse in hot alkali detergent (pH 10–11, 20C for seven minutes raising to 50–60C for 13 minutes) followed by ultrasonics and an acid bath in phosphoric acid to neutralise. After that everything is autoclaved.

How can a protein hang on to a high grade steel surface throughout this process? My initial little study grew into a rather large project. Through one of my sailing pals, Graham Bulfield, former director of the Roslin Institute, University of Edinburgh, I was put in contact with the institute’s neuropathogenesis unit.

Here I met two wonderful researchers, Karen Fernie and Chris Plinston who are not only at the cutting edge of TSE research but also have the facilities to experiment with the prion protein.

Eureka moment

We met for lunch every few months and puzzled how protein could bind onto steel. I arranged to get electron micrographs of instrument surfaces in order to see if there was anything obvious on the surface onto which a protein could stick. The scans clearly showed that steel is a crystal but there were no lumps or bumps which looked as though they could trap a protein on the surface.

At our next lunch we knocked about a few ideas as usual and at one point I clunked my wedding ring against the wine glass. I had a Eureka moment.

If mercury could cold electroplate onto a gold wedding ring, was it possible for a protein to cold electroplate onto stainless steel? If so it should be easy to remove.

Concentrated acid was out of the question as it would just dissolve the instruments. However, reverse electrolysis was definitely worth a go.

Bucket chemistry

The next day Bob Smith, my research partner, and I nipped off to B&Q and bought a 12 volt car battery, six feet of wire and some crocodile clips. We spread a scalpel handle with pig’s blood, fixed it with 75 per cent alcohol and let it dry. We selected another, put it into a 100ml beaker and clipped the top of it to the negative wire with a pair of forceps.

We then popped the blood stained handle into the beaker and, making sure the two handles did not touch, connected this one to the positive wire.

The choice of electrolyte was a bit more complex. We needed something that would conduct but not release any noxious or explosive gasses. Our previous experiments demonstrated that aqueous sodium carbonate solution was an excellent solvent for blood. This should electrolyse into carbon dioxide and hydrogen, which should be relatively safe. We gave it a try.

As soon as we added the electrolyte, there was a strong fizz from the electrodes and the blood quickly got welded into a black mess. Either the process didn’t work or we had the polarity around the wrong way. We tried again making the blood stained instrument the cathode.

It was amazing — the fixed blood was blown off the electrode in about 45 seconds. We repeated the experiment lots of times just to make sure it wasn’t a fluke. It worked as regularly as clockwork.

Our previous experiments demonstrated that normal detergents can remove dried blood from instruments in about 10-15 minutes. However, if the blood comes into contact with alcohol, the blood becomes water insoluble in seconds. As a result, normal detergents take about 10–15 minutes to penetrate and remove fixed blood.

Fixation is greatest with 75 per cent alcohol, the strength most commonly used in theatre. Our excitement was that we had discovered a process that could remove alcohol fixed blood 10 to 15 times faster than the best detergent.

We were down at the Patent Office before the end of the week.

Scale up

The next step was to scale up the process and produce a prototype large enough to decontaminate a full load

The next step was to scale up the process and produce a prototype large enough to decontaminate a full load of instruments. We raided the storeroom and acquired a range of discarded plastic boxes, stainless steel trays and stuff.

We added a car battery charger to our 12 volt battery, built a control panel with a car ammeter and voltmeter, and started testing out our new electro-elution device with our standard test disks.

The results continued to demonstrate that the process was highly reliable and repeatable. In addition, it showed that not only did it rapidly repel blood protein from instruments but it also denatured protein in the body of the electrolyte — after only five minutes the initial pink colour of the eluted blood changed to colourless as the blood was denatured.

This corresponded to a shift in UV peak from 416nm to less than 400nm in five minutes. If this process did the same to brain tissue, we may have cracked the prion problem.

We sent details of our device to our pals at the neuropathogenesis unit. They built a device and tried it. The first experiments showed that it removed ME7 prion from stainless steel disks in less than a minute and destroyed it in the body of the electrolyte in five minutes. This they worked up into a larger experiment the results of which were published in the Journal of Hospital Infection in 2007.

Since then we have worked the process up to a usable prototype. We have been across to Germany to discuss the process with our industrial partner Getinge, presented to the Department of Health decontamination group in London, the Deputy Chief Medical Officer of Scotland, Health Protection Scotland, the Health Protection Agency at Porton Down, the Central Sterilising Club and the Institute of Decontamination Sciences.

We were particularly pleased to win a prize in the 2008 Medical Futures Innovation Awards. Life is getting very exciting.

The development of electro-elution required the experience of a mercuric accident, setting up and running a QC lab, practical UV spectroscopy, knowledge of what goes on in theatre, intellectual property protection and commercialisation.

(It also required having the confidence of a company that believed we could solve the problem — many thanks Synergy Health!)

Invention is the process of providing a practical solution to a problem. Pharmacists are in the rare position of having a wide enough knowledge base to be able to invent.

Is there any pharmacist out there who would like to help me develop pulse reverse osmosis into a fluid balance monitor, and overcome the problems of orthostatic intolerance in space flight? If so, get in touch.

Citation: The Pharmaceutical Journal URI: 10043778

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Supplementary images

  • Prion proteins, and not bacteria or viruses, are believed to transmit spongiform encephalopathies (Medi-Mation/ Science Photo Library)

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