Methods of Verification of Cleaning in CSSD Dr. Winfried Michels Mechelen Oct.2012
Excerpts from ISO EN 15883-1 GTZ/PI/Dr. Michels 2
Before we consider which tracer to be monitored as a criterion for cleanliness we will have to think about sampling Samples must allow detection of >80% of existing proteins within the specified range. This applies in particular to complex instruments (crevices) and lumens. These should be checked in each case. Instrument surfaces should not be subjected to damage through chemical action. Sampling and methods of analysis must be well matched. GTZ/PI/AWT
Use of swabs for sampling is limited to good accessible surfaces, i.e. uncritical or semi critical MD s GTZ/PI/Dr. Michels 4
Swabbing The recovery of proteins is critical. The chart shows the recovery of heparinised blood not reactivated after drying on stainless steel by swabbing. Determination after swabbing was performed by extracting the swab with 1% SDS solution and protein assessment with the modified OPA method. 80 60 40 20 0 % recovery Wipe 3x Wipe 6x 1 µl blood 10 µl blood GTZ/PI/Dr. Michels 5
Swabbing methods for sampling are widely used in pharmaceutic productions for orientational checks and a limited recovery of 40 to 60 % reported in some literature. Example: GTZ/PI/AWT
Validation guidelines jointly compiled by: AKI - Instrument reprocessing working group DGKH - German Society for Health-Care Hygiene DGSV - German Society for Sterile Supply For cleaning assessment of critical MD s sampling shall be performed using 1% SDS solution and if the process under investigation had temperatures above 60 C the SDS solution shall be adjusted to ph 11
Recovery as a function of layer thickness Recovery % 5, 12.5, 25 µl of protamine reactivated, heparinised sheep s blood was applied to five 3 cm² stainless steel plates and allowed to dry for 1 hour at 70 C. This was followed by elution using 2 ml 1% SDS (ph 11) each for 1 hour at room temperature. The recovery (mean value from 5 metal plates each) is shown in the chart on the right. 120 100 80 60 40 20 0 5 µl/ 3 cm² 12,5 µl/3 cm² 25 µl/3 cm² Series 1 GTZ/PI/Dr. Michels 8
Test challenge... Dilute heparinised sheep's blood with 10% Aqua Bidestillata. Reinstate ability to coagulate by adding 1.5 international units of protamine sulphate per ml blood. Apply 100 µl to fulcrum of each instrument using a pipette. GTZ/PI/Dr. Michels 9
Sampling recoveries after SDS elution of haemostatic forceps and OPA assessment 120 % detection 100 80 60 40 20 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Sample no. OPA GTZ/PI/Dr. Michels 10
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Example for Sampling GTZ/PI
Sampling from the inner shaft of an dismountable MIS instrument GTZ/PI
Da Vinci EndoWrist Instrument GTZ/PI/Dr. Michels 14
Test Kit using the semi-quantitativebiuret/bca detection method Dr. Michels 2009 15
Comparison with the colour chart Dr. Michels 2009 16
Measurement with relectometer RQflex (VWR) Dr. Michels 2009 17
RQflex [µg/ml] 60 50 40 30 20 10 BSA calibration curve with with Test Kit and RQflex 0 0 10 20 30 40 50 60 Standard [µg/ml] Studiendag
Additionally to OPA, Biuret-BCA and Ninhydrin a haemoglobin detection method is given in ISO TS 15883-5 Studiendag 19
Rust or Blood? Studiendag VSZ_Mechelen_Okt2012_Michels_ENG.ppt 20
Hämo-Check Medi-Test Combi V Test for Microhaematurie Studiendag 21
Blood dilutions and influences of temperature and alkalinity Studiendag VSZ_Mechelen_Okt2012_Michels_ENG.pp t 22
Frequency of BCA and haemoglobin results (540 Instruments) Round Robin 2005 by DGKH, DGSV and AKI 300 250 200 Hämoglobin/µl 150 100 50-20 0 Studiendag -50 40 80 100 120 160 200 240 µg Protein (BSA)/Instrument 23
ATP Bioluminescence Method All living cells use ATP as a central energy transmitter
ATP level variation in blood is quite large GTZ/PI/Dr. Michels 25
ATP measurement Figure 1. (A), effect of sample temperature on bioluminescence at a constant ATP concentration (10 nmol/l). Maximum light output and minimum temperature dependence are achieved near room temperature. (B), effect of sample ph on bioluminescence. We added 300 µl of stabilizing solution at various ph values to 100 µl of 100 nmol/l ATP in distilled water; 25 µl of 177 mmol/l MgCl 2 and 100 µl of luciferase reagent were added by automatic injection in the luminometer. The ph and light output of the resulting mixtures are plotted. Maximum light output with minimum ph dependence was obtained in the ph range 7.75 7.95. Reproduced with permission from reference 14, copyright John Wiley & Sons Ltd., 2003. GTZ/PI/Dr. Michels 26
Influence of temperature and ph on luminescence Published December 2006, doi: 10.1373/ clinchem.2006.076364 Clinical Chemistry February 2007 vol. 53 no. 2 318-325 GTZ/PI/Dr. Michels 27
ATP hydrolysis occurs dependent upon chemical condition and temperature as can be expected with the main wash in WDs GTZ/PI/Dr. Michels 28
ATP measurements with blood published in aseptica 2/2007 GTZ/PI
Some measurements: 50 µl of each solution were pipetted into the swab GTZ/PI
Lecture of Buchrieser et al. at WFHSS Conference 2009 GTZ/PI/Dr. Michels 31
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Other reports Measuring of ATP bioluminescence as a means of assessing washer disinfector performance and potentially as a means of validating the decontamination process Raymond Heathcote A C, Brett Stadelmann B A Epworth Freemasons Hospital, Clarendon Street, East Melbourne, Vic. 3002, Australia. B Central Sterile Supply Department, Epworth Freemasons Hospital, Clarendon Street, East Melbourne, Vic. 3002, Australia. C Corresponding author. Email: raymond.heathcote@epworth.org.au No method validation. Comparison of manual and automatic washing (incl. thermal disinfection). Instruments processed in WD gave mostly 0 RLU and those processed manually mostly 8000 9000 RLU. Results can not be confirmed by protein assessment.
ATP bioluminescence measurements as a method for assessment of cleaning has been discussed at ISO TC 198 WG 13 and CEN TC 102 WG 8 meetings It has been decided not to include this method in EN ISO 15883-1 GTZ/PI/Dr. Michels 35
A very quick and simple qualitative routine check is the selective staining of protein Ponseau S azo dye GTZ/PI/AWT Dr. Winfried Michels
Test percedure: - Wet the surface area to bechecked with dye solution - Wait three minutes for staining protein - Rinse the area with flowing tap water for one to two seconds - Remaining color indicates proteinacous residue GTZ/PI/AWT Dr. Winfried Michels
1 cm² of each metal plate had been wetted with solution containing different amounts of BSA, dryed, thermally denaturated, stained and after rinsing a photo was taken. 20 µg 10 µg 5 µg BSA GTZ/PI/AWT Dr. Winfried Michels
Real instrument with wear friction corrosion and residual soil stained GTZ/PI/Dr. Michels 39
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