WO2002051451A1

WO2002051451A1 - Inspection technique

Info

Publication number: WO2002051451A1
Application number: PCT/GB2001/005609
Authority: WO
Inventors: Frank Prior; Gerry Heneaghan
Original assignee: Trust Sterile Services Limited
Priority date: 2000-12-23
Filing date: 2001-12-21
Publication date: 2002-07-04
Also published as: GB0031595D0

Abstract

The invention relates to a method of and materials for testing for material containing amine groups and therefore for proteins. In particular, the method can be used to detect prion proteins.

Description

Inspection Technique

This invention relates to a production type test to detect the presence of material containing amine groups, and therefor proteins, on surgical instruments and in particular a production type test which is able to detect proteins and prion proteins at concentrations believed to transmit infections between different tissues in the body.

The presence of prion proteins has been linked to a number of Bovine Spongiform Encephalopathy type diseases in animals and humans. These diseases include Bovine Spongiform Encephalopathy (BSE) , Scrapie, Gerstmann- Strausler-Scheinker Syndrome (GSS) , Fatal Familial Insomnia (FFI) , Kuru, Transmissible Mink Encephalopathy (TME) , Chronic Wasting Disease (CWD) , Feline Spongiform Encephalopathy (FSE) and Creutzfelt-Jakob Disease (CJD) , which encompasses Variant Creutzfeldt-Jakob Disease (vCJD) , familial CJD, sporadic CJD and iatrogenic CJD. At the present time vCJD in particular is a major concern in the United Kingdom and Europe and is also a growing concern in Europe and the United States.

CJD is a disease which occurs in patients over 50 years of age which develops into a progressive encephalopathy resulting in dementia and death over 10-30 years. VCJD is also a disease of the brain but differs in affecting much younger patients. It also exhibits as a progressively fatal Encephalopathy, but the dementia progresses to death in about 2 years It is believed that CJD and v CJD are caused by the presence of mutated prion proteins. CJD results from genetic mutation of normal prion in the body. VCJD is believed to result from infection of prion protein from an external source.

Different tissues are believed to have different rates of infectivity, resulting in the situation where muscle and blood have a relatively low level of infectivity, lymph has a higher level and transmission from brain directly to brain carries the highest risk of infection. The exact amount of infected tissue required to transmit the infection between sites is still unclear but believed to be in the range of 1 x 10^"10 to 1 x 10^"15 g between brain to brain, lxlO^-6 to lxlO^-11 in lymph, and substantially more in blood and tissue. Oral transmission has been estimated as lg between members of the same species .

Measures have been introduced to prevent infectious food sources from entering the food chain, principally the removal of brain and spinal chord from meat. Human blood donations are checked for the absence of the disease before samples are pooled for blood transfusion or the preparation of blood fractions for human use. Due to these measures, the greatest risk of the spread of both CJD and vCJD is probably cross-infection during surgery. Surgical procedures pose a problem because most surgical instruments are made out of high quality stainless steel and are therefore of high cost. As a result, they are usually cleaned, sterilised and re-used. Although normal steam sterilisation kills most infectious organisms through damaging the cell wall and denaturing DNA, prions lack cell walls and contain very little DNA and are therefore heat resistant. In view of this there is a significant risk that pieces of infectious tissue that are not removed during the cleaning and sterilisation process could result in transfer of prions between patients with resultant CJD and v CJD infection.

At the present time research is focused towards finding a diagnostic test to detect CJD and vCJD in patients. However, most of this research is high level and too expensive, technical and slow to apply as part of the routine quality control of cleaning and sterilisation of surgical instruments for routine use. Currently no production type tests exist to identify when surgical instruments are free of blood, tissue protein and prion protein at levels which are suspected of transmitting infection. Due to the potential risk of CJD transmission, it would be beneficial if there were a production type test for identifying when instruments used for different surgical procedures are free of protein, including prion protein, to a level safe for their use. The current research suggests safe levels are:

Blood less than 1 x 10^~6g Tissue less than 1 x 10^~9g Lymph less than 1 x 10^"12g Brain less than 1 x 10^"15g

The ideal test would ensure that less than lxl0^~15g of protein remained on any surgical instrument. However, currently the available technology and knowledge does not make this possible. As a first step the object of this invention is to establish a simple test to demonstrate that less than 10^~9 g of protein is remaining on the instruments . This would be appropriate for all instruments trays apart from those used for neurosurgery and lymphatic excision.

This would ensure that the cleaning methods that are being used are working satisfactorily to remove protein to the level of 1 x 10^~9 g.

It is the first object of the present invention to provide a method of extracting protein contamination from surgical instruments and demonstrating that this contains less than 10^~6g of blood or protein.

It is a further object of the invention to provide a method of concentrating the extraction solution to enable the determination of protein down to a level of less than 10^"9g.

It is a further object to provide a method of determining whether surgical instruments are free of prion proteins and therefore are free of CJD, vCJD, BSE, GSS, FFI, Kuru, TME, Scrapie CWD and FSE to a level of less than 10^"9g.

A further object of this invention is to provide a method, which allows the amount of protein present on a surgical instrument to be quantified to a level of less than 10^~9g.

Another object of this invention is to provide a method of validating whether washing and disinfecting procedures are working to suitable standards to a level of less than 10^_9g. A yet further object of this invention is to provide a method of detecting proteins, and in particular prion proteins, in watercourses and waste to a level of less than 10^"9g.

A final objective of this invention is to provide a method of determining whether protein is present on surgical instruments, or in watercourses or waste, and then storing information from the test results using an electronic medium.

According to a first aspect of the present invention, there is provided a combined wetting agent and solvent (wetting/solvent) comprising borax, glycerine, sodium chloride and distilled water, the purpose of which is to render the protein including prion protein into a state which will allows UV quantification, concentration by ultrafiltration or centrifugation, and reaction with the intended chromophore or fluorophore.

Preferably the borax is between 0% to 3% (w/v) of the wetting/solvent.

Preferably, the glycerine is between 0.0% to 15% (v/v) of the wetting/solvent agent.

Preferably the sodium chloride is between 0.0% and 2.0% (w/v) of the wetting/solvent agent.

Preferably the distilled water is between 0.0% and 99.9% (v/v) of the wetting/solvent agent. Preferably the wetting/solvent agent contains sodium carbonate 0-2%w/v, sodium bicarbonate 0-2%w/v or sodium hydroxide 0-2% to adjust the pH to between 9 and 11

Most preferably, the volume of the wetting/solvent agent used is determined according to the size of the instrument being tested.

Most preferably the amount of wetting/solvent agent used is the minimum amount required to cover the instrument which is being tested.

Preferably, the wetting/solvent agent also includes preservative to prevent bacterial growth. I Preferably the wetting/solvent contains a minimum amount of impurities, which absorb ultraviolet light.

Preferably the wetting/solvent contains a minimum amount of impurities which either have a UV spectra or can react with fluorophores and fluoresce under fluorescent light.

According to the second aspect of the invention, there is provided a resealable container large enough to contain the instrument and the wetting/solvent.

Preferably the container is plastic.

Preferably the seal has to be watertight.

Preferably the size of the resealable container is determined by the size of the instrument. Preferably the leaching of plasticiser that absorbs ultraviolet light must be minimal (absorbance less than 0.05 at wavelength at the peak 250-265nm) .

Preferably the leaching of plasticiser must be consistent from bag to bag (absorbance at range 250-265, preferably 256-259 with a known value between 0 and 0.05 absorbance units) .

According to the third aspect of the invention, there is provided a method for removing protein, comprising taking an instrument and the wetting/solvent of the first aspect and placing it on an apparatus which provides agitation of the solution over the surface of the instrument to facilitate the removal of the dissolved protein from the instrument surface.

Preferably the instrument and combined wetting agent and solvent are placed in the container of the second aspect.

Preferably the agitation process will last from 5 to 60 minutes .

Most preferably the agitation process will last 30 minutes.

According to the fourth aspect of the invention there is provided a method of determining levels of contamination on the surgical instrument.

Preferably the method comprises taking a UV spectra of a sample of the solution immediately after wetting solvation over the range 190nm to llOOnm. Preferably the absorbance of any peaks at 206-212, 250- 260, 270-278, 343-348, 405-415, 540-546, 580-585, 912-918, 960-980, 1030-1050, 1055-1075 nm are measured and compared against standard UV spectra measured for blood, serum albumin, and common known contaminants such as povidone iodine, chlorhexidine, mechanical oils and rust.

According to the fifth aspect of the invention there is provided a method of concentrating the volume of the wetting/solvent.

Preferably the method comprises carrying out membrane ultrafiltration.

Alternatively, the method comprises high speed centrifugation.

Preferably the method of concentrating enables reduction of the initial volume of wetting/solvent to between 0.05 to 0.005ml.

According to a sixth aspect of the present invention, there is provided concentrating apparatus comprising an ultrafiltration membrane with a pore diameter between 5,000 to 10,000 Daltons Molecular weight cut off.

Preferably the concentrating apparatus has a means of delivering pressure to the wetting solvent to speed up the filtration process. This pressure will be in the range 1-5 barr.

According to the seventh aspect of the invention there is provided a method of quantifying the amount of protein in ultrafiltered wetting solvent using UV spectrophotometry. Preferably the method can quantify protein down to a level of 10^"9g.

Alternatively, the method can quantify protein down to a level of 10^~δg.

Optionally, quantifiable testing can be carried out by placing the test material which has been exposed to the wetting and testing agent, in a UV spectrometer, the absorbance measured at predetermined peaks and compared with known standards.

Alternatively, chromophores can be added to the wetting solvent and the resultant mixture measured in a UV spectrometer or fluorimeter to measure the quantity of UV absorbent or fluorescent material present and to compare it to known values to determine the quantity of protein material that is present across the complete test piece.

According to an eighth aspect of the present invention, there is provided a method of validating whether a washing or disinfecting machine is processing correctly and to the standards prevailing at the time by determining whether an object is free of protein.

Preferably, objects are soiled with artificial soiling to BS 2745 part 1 and then cleaned before being tested for the presence of protein.

In order to provide a better understanding of the inventions, embodiments will now be described by way of example only and with reference to the following figures, in which:

Figure 1 is a diagram of the extraction bag in which wetting solvent is added to immerse the instrument in a re-sealable plastic bag;

Figure 2 is a diagram of the agitation process in which the protein is dissolved into the wetting solvent solution;

Figure 3 is a diagram of the UV initial quantification process determining the concentration of protein in an aliquot of the wetting soaking solution;

Figure 4 is a diagram of the ultrafiltration concentrating process in which the wetting soaking solution is ultrafiltered to increase the concentration of the protein; and

Figure 5 is a diagram of the UV quantif cation process to determine the levels of protein in the ultrafiltrate.

In order to provide a suitable area where the testing for the presence of prion protein on a surgical instrument 4 can be carried out, there is provided prescriptive bag 1 with double chambers, where the two chambers are separated from each other by a hermetic glass breakable type seal 3.

In a preferred embodiment, the first chamber is filled with a quantity of wetting agent, comprising distilled water, glycerine and sodium chloride mixed with testing agent, comprising nano-orange, CBQCA, fluorescamine and OPA 2. This chamber is fully sealed once it has been filled. The second empty chamber 1 is not sealed initially so that the instrument under test 4 can be placed into the bag 1. Once the instrument under test 4 has been placed into the second chamber of the prescriptive bag 1, the second chamber is also completely sealed.

In order to begin the chemical test, the hermetic seal 3 is broken to release the mix of wetting agent and testing agent 2 into the chamber containing the instrument under test 4. The bag 1 is then mechanically agitated to maximise the flow of the wetting and testing agents 2 across and around the instrument or utensil. After a predetermined time period the instrument 4 can be removed and allowed to dry so that the reacted material is not disturbed. The instrument 4 can then be examined for fluorescence using either a UV light box for qualitative results or using a UV spectrometer 5 (as shown in Figure 3) to gain quantifiable results. The UV spectrometer 5 comprises a source 6, detector 7 and a slice cell 8 which will contain the wetting agent and dissolved protein. If a UV spectrometer 5 is used, the reading of 5.2 to 5.3 will mean there is no protein present and hence no prion protein. This will therefore also mean that there is no CJD causing agents or related Spongiform Encephalopathy causing agents present on the instrument 4.

In an alternative embodiment, chromophores can be added to the wetting agent which does, or has, contained an instrument. The wetting agent can also be concentrated using membrane ultrafiltration with the ultrafiltration having a 5,000 to 10,000 Da molecular weight cut off.

In order to record the results, a simple camera can be used and digital image transfer allows the information to be recorded permanently in an electronic format. It is then possible to securely store and retrieve the information when required.

In an alternative embodiment, the first chamber of the prescriptive bag 1 is filled only with wetting agent which comprises distilled water, sodium and glycerine. As in the preferred embodiment, the object under test 4 is placed in the second compartment of the prescriptive bag 1, which is then sealed. The inner hermetic seal 2 is then broken to release the wetting agent so that it covers the object under test. The bag 1 is then mechanically agitated for a predetermined time period so that any protein present is rendered in a state suitable for reacting with the testing agent. An aliquot of the testing agent, which comprises nano-orange, CBQCA, fluorescamine and OPA is then added to the wetting agent and the UV absorbance is measured. Alternatively, an aliquot of the wetting agent solution, which has had the object soaking in it, is added to a separate aliquot of testing agent and the UV absorbance is measured.

The embodiments disclosed above are merely exemplary of the invention, which may be embodied in different forms. Therefore, the details disclosed herein are not to be interpreted as limiting, but merely as a basis for the Claims and for teaching as to the various uses of the present invention in any appropriate manner.

Claims

1. A combined wetting agent and solvent (wetting/solvent) comprising borax, glycerine, sodium chloride and distilled water.

2. A combined wetting agent and solvent according to Claim 1, wherein the borax is between 0% to 3% (w/v) of the wetting/solvent agent.

3. A combined wetting agent and solvent according to Claims 1 and 2, wherein the glycerine is between 0% and 15% (v/v) of the wetting/solvent agent.

4. A combined wetting agent and solvent according to any of the previous Claims, wherein the sodium chloride is between 0% and 2% (w/v) of the wetting/solvent agent.

5. A combined wetting agent and solvent according to any of the previous Claims, wherein the distilled water is between 0% and 99.9% (v/v) of the wetting/solvent agent .

6. A combined wetting agent and solvent according to any of the previous Claims, wherein the volume of the wetting/solvent agent used is determined according to the size of the instrument being tested.

7. A combined wetting agent and solvent according to any of the previous Claims, wherein the amount of wetting/solvent agent used is the minimum amount required to cover the instrument which is being tested.

8. A combined wetting agent and solvent according to any of the previous Claims, wherein the wetting/solvent agent also includes preservative to prevent bacterial growth.

9. A combined wetting agent and solvent according to any of the previous Claims which contains a minimum amount of impurities which absorb ultraviolet light.

10. A combined wetting agent and solvent which contains a minimum amount of impurities which either have a UV spectra or can react with fluorophores and fluoresce under fluorescent lights.

11. A resealable container large enough to contain the instrument and the combined wetting solution and solvent of Claims 1 to 10.

12. A resealable container as described in Claim 11 which is made out of plastic.

13. A resealable container as described in Claims 11 and 12, wherein the resealable seal is watertight.

14. A resealable container as described in Claims 11 to 13, wherein the size of the container is larger than the size of the instrument to be tested.

15. A resealable container as described in Claims 11 to 14, wherein the container comprises a plasticiser.

16. A resealable container as described in Claiml5, wherein the plasticiser only absorbs a minimal amount of ultraviolet light, that is the absorbance is less than 0.05 at wavelength at the peak 250-265nm) .

17. A resealable container as in Claims 15 or 16, wherein the leaching of a plasticiser is consistent.

18. A method for removing protein, comprising taking an instrument and the wetting/solvent of the first aspect and placing it on an apparatus which provides agitation of the solution over the surface of the instrument to facilitate the removal of the dissolved protein from the instrument surface.

19. A method for removing protein as described in Claim 18, wherein the instrument and combined wetting agent and solvent are placed in the container described in Claims 11 to 17.

20. A method for removing protein as described in Claims 18 or 19, wherein the agitation process last from 5 to 60 minutes.

21. A method for removing protein as described in Claim 20, wherein the agitation process lasts 30 minutes.

22. A method of determining levels of contamination on a surgical instrument, comprising taking a UV spectra of sample of solution which has been produced from the method described in Claims 18 to 21.

23. A method of determining levels of contamination on a surgical instrument, comprising taking a UV spectra immediately after wetting solvation over the range 190nm to llOOnm.

24. A method of determining levels of contamination on a surgical instrument as described in Claims 22 and 23, wherein when taking the UV spectra, the absorbance of any peaks at 206-212, 250-260, 270-278, 343-348, 405- 415, 540-546, 580-585, 912-918, 960-980, 1030-1050, 1055-1075nm are measured and compared against standard UV spectra measured for blood, serum, albumin and common known contaminants, such as povidone, iodine, chlorhexidine, mechanical oils and rust.

25. A method of concentrating the volume of the combined wetting agent and solvent as described in Claims 1 to 10, after it has been used in the method for removing protein as described in Claims 18 to 21 by carrying out membrane ultrafiltration.

26. A method of concentrating the volume of the combined wetting agent and solvent as described in Claims 1 to 10, after it has been used in the method for removing protein as described in Claims 18 to 21 by carrying out high speed centrifugation.

27. A method of concentrating the volume of combined wetting agent and solvent as described in Claims 25 and 26, wherein the method of concentration enables reduction of the initial volume wetting agent and solvent to between 0.05 to 0.005ml.

28. Concentrating apparatus for carrying out the method described in Claims 25 or 27, comprising an ultrafiltration membrane with a pore diameter between 5,000 to 10,000 Daltons Molecular weight cut off.

29. Concentrating apparatus as described in Claim 28 which has a means of delivering pressure to the wetting solvent to speed up the filtration process.

30. Concentrating apparatus as described in Claim 29 wherein the pressure will be in the range 1-5 barr.

31. A method of quantifying the amount of protein in ultrafiltered wetting solvent using UV spectrophotometry.

32. A method of quantifying the amount of protein as described in Claim 31, wherein the method can quantify protein down to a level of 10^~9g.

33. A method of quantifying the amount of protein as described in Claim 31, wherein the method can quantify protein down to a level of 10^~6g.

34. A method of quantifying the amount of protein as described in Claims 31 to 33, wherein the testing can be carried out by placing the test material which has been exposed to the wetting and testing agent in a UV spectrometer, the absorbance measured at predetermined peaks and compared with known standards.

35. A method of quantifying the amount of protein as described in Claims 31 to 33, wherein chromophores can be added to the wetting/solvent and the resulting mixture measured in a UV spectrometer for fluorimeter to measure the quantity of UV absorbent or fluorescent material present and to compare it to known values to determine the quantity of protein material that is present across the complete test piece.

36. A method of validating whether a washing or disinfecting machine is processing correctly and to the standards prevailing by determining whether an object is free of protein using the method described in Claims 22 to 24.

37. A method of validating whether a washing or disinfecting machine is processing correctly as described in Claim 36, wherein objects are soiled with artificial soiling to BS 2745 Part 1 and then cleaned prior to being tested for the presence of protein.