In-situ protein detection ProReveal protein detection system Prof. David Perrett William Harvey Research Institute Barts & the London School of Medicine & Dentistry CSC April 2014 Approaches to Protein Detection on Instruments Off-instrument Desorb (swab) followed by a commercial test Wash off, acid hydrolyse the washings and measure protein as amino acids Wash off with detergent then measure removed whole proteins in washings In-situ Apply a chemical reagent and observe “Sandwich type” immunoassay Exotic methods e.g. DESI-MS Total Organic Carbon (ToC) Surrogate BIOtak AK assay ValiProTag TOSI type tests and observe - some use dyes Tagged proteins e.g. Radiolabelled. © Professor David Perrett Off-instrument testing ISO 15883-1:2009 part C.3.3.1.2 requires that about 10 cm2 of an instrument is swabbed with water wetted swabs for a few minutes prior to testing with recommended chemical tests Swabbing 10 cm2 in itself is a challenge! Swab with what? For how long? Tend to swab visually dirty area not necessarily protein © Professor David Perrett Efficiency of removing hydrophobic proteins using wetted Rayon swabs Water BSA Fibrinogen 0.5% Triton X-100 32 ± 4% 20 ± 3% remaining remaining 61 ± 5% 24 ± 3% remaining remaining Swabbing 10-15 strokes 10-15 strokes n=6 We swabbed where we knew we had placed the protein! © Professor David Perrett Some test kits for residual proteins & their chemistries available in U.K. Chemistry Mode Trade Name Supplier Sensitivity (µg BSA) Copper binding + BCA complexation BCA Pro-Test-Q Medisafe Clean-Trace protein 3-M 1 3 – 50 Depending on temperature and time BCA Dye binding Medi-check residual Hygiena protein test Pyrogallol Red Pyromol Test + Molybdate Coomassie Scope Check Brilliant Blue 1 – 10 Depending on temperature and time Pereg GmbH 1 Valisafe/Medisafe 1 Denta Check Valisafe/Medisafe 1 Ninhydrin Protein Detection Test Haemostik Browne (Steris) (Bradford Assay) Ninhydrin Ninhydrin Haemoglobin Bioluminescence ATP na na na Sensitivity taken from manufacturer’ s publications The methods for residual protein detection contained in ISO, EU, HTM and CFPP-0101 contains fundamental scientific flaws. The desorption (swabbing) methods cannot effectively remove protein from box joints, serrations, small orifices etc. They lack sensitivity for many purposes Most are non-quantitative In-situ Detection of Residual Protein DH (England) Funded Detection Research Ca. 2009 3 groups (Barts, Southampton, Edinburgh) developed fluorescent ‘in situ’ detection methods in conjunction with local SSDs GOSH / UCLH / Barts were to compare instrument storage post-operations In 2011 Barts co-ordinated a comparative study across the 3 groups in order to inform DH calculations on maximum permissible protein levels © Professor David Perrett Fluorescence Compared to colorimetric measurements is more 1000x more sensitive is many fold more specific needs intense light sources often lasers Few compounds are naturally fluorescent but chemical reactions can be used to make fluorophores CFPP-0101 Appendix D calls for new approaches © Professor David Perrett ‘In situ’ detection – my ideal Simple – suitable for the SSD environment: Giving a permanent record Sensitive – 100-1000 fold better than established tests? Protein Specific Fast – capable of high throughput Instrumentation - Readily available, should not use lasers Stable reagents giving stable fluorophores Non-toxic – Safe reagent: Protein products can be readily removed Can reveal proteins residues - on “whole” instruments Low cost - both capital and running Chronology 1999/2000 - DH England established Decontamination Working Group (DECON) with specific reference to vCJD 2000 – OPA/NAC assay for proteins desorbed from instruments 2001 – reagent shown to work on surfaces using a scanning fluorimeter 2008 – Improved OPA/NAC reagent shown to work on instruments using a hand-held mercury lamp 2010 – In-situ imaging using OPA/NAC, G-Box and a mixture of software shown to Syngene Modified OPA / NAC reagent for proteins © Professor David Perrett OPA/NAC fluorescence solution method High sensitivity 60 y = 28.571x - 1.2381 2 R = 0.9804 Fluorescence 50 40 30 20 10 0 0 0.5 1 1.5 2 2.5 BSA Conc (µg/ml) Used in many published studies e.g. Dental papers © Professor David Perrett Could a fluorescent OPA/NAC solution assay be combined with a gel imaging system plus 2-D visualisation from my proteomic work for ‘in situ’ detection? © Professor David Perrett The Proteomic based system G-Box from Syngene Cambridge © Professor David Perrett G-Box (ID-box) system in 2008 Cooled CCD Camera Optimum Emission filters Lamps – White light Mercury 338nm Platform with sheet of black paper © Professor David Perrett Typical BSA protein standard calibrant 8 µg 4 µg 2 µg 10 µg 0.5 µg 0.25 µg 0.125 µg © Professor David Perrett SSD cleaned instruments A supposedly washed instrument sprayed with OPA/NAC reagent showing lots of protein Area 2,570,000 pixels Approx 1ug protein ProReveal System • Developed at Barts with DH funding and in collaboration with Synoptics Cambridge • The system is highly sensitive (<50 ng BSA) • This fluorescent protein detection system can be used in a typical SSD. • Software continuously being modified and improved Improved System May 2012 Cooled CCD camera takes B&W and fluorescent images now internal New Hg lamps and different Emission filter Instrument sprayed in situ with OPA/NAC reagent Sliding tray for up to A4 sized instruments Smaller footprint, portrait, no change in sensitivity, updated software takes B&W image both before and after gathering fluorescent image for ca. 2 minutes © Professor David Perrett The current ProReveal System Improved ProReveal Spray Reagent • OPA/NAC was reformulated with improved chemistries • It is now stable for nine months • It retains its sensitivity • Revealed fluorescent protein residues are stable • The reagent compoments are completely washed off with a 2nd detergent wash • BSA protein calibrant is stable • Studies have been done to prove removal of chemical residues with a re-wash Patented by QMUL © Professor David Perrett A typical inspection image of a washed surgical instrument Total Protein shown = 8.7 µg The ProReveal technology can help improve cleaning of surgical instruments Hospital A Total Protein shown = 158 µg Hospital C Total Protein shown = 0.8 µg © Professor David Perrett Validation Sensitivity <50ng relative to BSA Linearity 0 – ca. 250ug BSA Reproducibility 5.2% n=10 for an instrument 3.1% (n = 10) for tags ProReveal Software The software generates all these different units of measurement automatically. The choice which to display and use is yours All files are automatically uniquely numbered and can be readily accessed for future use It also provides a visual record of the areas of protein residuals on an instrument Raw data is stored on the hard-drive or externally for quality purposes Although the traffic light display is not the default it is still there Most recent change is that the amount of protein in a highlighted spot can be displayed © Professor David Perrett Software output is configurable depending on local needs Timers appear on the screen Result are shown in 2 - 3 minutes Pass Appearance 2-D or 3-D image Quantitative data is obtained Simple Pass/Fail indication is also possible Fail Print label to record result © Professor David Perrett ProReveal Software • 3D vew can be rotated and orientated Setting limits Eye set (dry µg/side) © Professor David Perrett Which data presentation? Craniotomy set (dry, enzymatic wash ) Total protein µg / instrument Max. Protein µg / hotspot Protein / unit area © Professor David Perrett Safety All chemicals should be safe for both the patient and SSD personnel. In addition they should not attack components of instruments All chemicals should be easily removed from surfaces Residual proteins are strongly adhered to stainless steels All detergents in some way chemically change proteins. Chemical changes can also result from other additives such as water retaining gels The pharm industry requires these tests to be at the ppm levels Components of ProReveal Spray Sodium tetraborate* o-Phthaldialdehyde N-acetyl-cysteine* Dithiothretiol Triton-X100 Sodium EDTA* All these are dissolved in water at 1-10 mg/L Even in the neat spray NONE even approach published toxicity values They will only react with proteins if present together and at pH 9-10 Contaminated and OPA/NAC sprayed instruments before and after washing with water Before After hand wash under tap water Removal of majority of fluorescence shown n.b/ The instruments were not re-sprayed after wash Arginine pipetted directly on to sscalpel blades and then sprayed with OPA/NAC Before wash 10 µg Arginine After washing with an alkaline detergent Control showing no residuals After washing then re-sprayed with OPA- free reagent but containing arginine Control Residual OPA levels <LoD e.g. 1ng Washing-off of Fluorescent-labelled proteins (Fibrinogen) Before washing After washing unsprayed (not visibly clean) 26 ng protein 0 2 4 6 8 10 Fibrinogen (µg) After washing then re-sprayed So re. safety All reagents can leave deposits and modified residual proteins OPA/NAC is removal at our limits of detection after a second wash Modified very sticky proteins and brain proteins are also removed to <50ng on instrument after a second wash The reagent works by both spraying and dipping Other sensible but standard precautions are Wash hands after use (gloves prevent false results from fingerprints Spray away from yourself. Do not over spray Dispose of the black paper regularly Clean ProReveal tray frequently We meet FDA Pharma requirements Some operational considerations ProReveal is very sensitive. It was developed for residuals not masses of protein If you can see residues - you do not high sensitivity detection! Haemoglobin i.e. whole blood, at amounts you can see quenches the signal by absorbing the fluorescence emitted It only measures the top of an instrument unless you turn an instrument over! The results on tests pieces can be fully quantitative but Results on instruments are semi-quantitative The ProReveal needs to be correctly calibrated and configured Challenges Orifices and lumens are a problem to all detection methods Some plastics are made to fluoresce – they are usually obvious! Applications • Routine QC and QA in SSDs • Performance testing of AWDs and/or detergents before purchase • Validation of AWDs in routine operation • Manufacturers of AWDs or detergents could use ProReveal for developing improved systems and chemistries • Surgical instrument manufacturers to improve their designs • General research applications © Professor David Perrett Questions to you • How clean should we realistically aim for? • How many instruments should be sampled and tested routinely? • How frequently should tests be performed? • What should the “units of cleanliness” be? These are not addressed any present standards. © Professor David Perrett Conclusions • A simple, high sensitivity in situ system useable in SSDs is possible and is now commercialised • Keeping instruments moist and washing with enzymatic detergent is our best current advice. • However all actual reagents need testing, AWD operation needs better validation, new procedures are not necessarily improvements • Very clean instruments can be achieved © Professor David Perrett More Current DH (England) Research Barts & PHE (Porton Down) are optimising AWD performance using statistially driven Design of Experiment techniques, brain protein tags and ProReveal Novel coatings for instruments with Dundee University are being studies using ProReveal A metanalysis of methods for evaluating novel prion removing detergents Novel decontamination methods e.g. gas plasmas © Professor David Perrett Current DECON thinking Drs Nigel Tomlinson and Andre Chalot have been using new residual protein data to develop new risk benefit calculations CFPP is expected to be revised in 2015 DH is to investigate means EU and ISO bodies can be influenced to reflect the scientific evidence that as been generated by the DECON group and its £10+ million spend on research © Professor David Perrett Acknowledgements Dr Nanda Nayuni Staff of various SSDs for both instruments and AWD time Synoptics Health staff especially Alaisdair Hayden-Wright, who programs ProReveal DH for financial support and advice © Professor David Perrett Sources ProReveal is available in the UK and Ireland via Peskett Solutions Synoptics Health manufacture the imaging system in Cambridge and develop the software Deconsure make the chemistries and tags © Professor David Perrett Thank you. I hope I have left some time for questions
© Copyright 2024 ExpyDoc
![[612] Seminar by Prof. Ramamoorthy at RIKEN Yokohama](http://s3.expydoc.com/store/data/000392580_1-fc50fed5cdc5e7909e2119e43a4e8dc1-250x500.png)
