Methods of Verification of Cleaning in CSSD. Dr. Winfried Michels Mechelen Oct.2012
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1 Methods of Verification of Cleaning in CSSD Dr. Winfried Michels Mechelen Oct.2012
2 Excerpts from ISO EN GTZ/PI/Dr. Michels 2
3 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
4 Use of swabs for sampling is limited to good accessible surfaces, i.e. uncritical or semi critical MD s GTZ/PI/Dr. Michels 4
5 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 % recovery Wipe 3x Wipe 6x 1 µl blood 10 µl blood GTZ/PI/Dr. Michels 5
6 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
7 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
8 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 µl/ 3 cm² 12,5 µl/3 cm² 25 µl/3 cm² Series 1 GTZ/PI/Dr. Michels 8
9 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
10 Sampling recoveries after SDS elution of haemostatic forceps and OPA assessment 120 % detection Sample no. OPA GTZ/PI/Dr. Michels 10
11 GTZ/PI/Dr. Michels 11
12 Example for Sampling GTZ/PI
13 Sampling from the inner shaft of an dismountable MIS instrument GTZ/PI
14 Da Vinci EndoWrist Instrument GTZ/PI/Dr. Michels 14
15 Test Kit using the semi-quantitativebiuret/bca detection method Dr. Michels
16 Comparison with the colour chart Dr. Michels
17 Measurement with relectometer RQflex (VWR) Dr. Michels
18 RQflex [µg/ml] BSA calibration curve with with Test Kit and RQflex Standard [µg/ml] Studiendag
19 Additionally to OPA, Biuret-BCA and Ninhydrin a haemoglobin detection method is given in ISO TS Studiendag 19
20 Rust or Blood? Studiendag VSZ_Mechelen_Okt2012_Michels_ENG.ppt 20
21 Hämo-Check Medi-Test Combi V Test for Microhaematurie Studiendag 21
22 Blood dilutions and influences of temperature and alkalinity Studiendag VSZ_Mechelen_Okt2012_Michels_ENG.pp t 22
23 Frequency of BCA and haemoglobin results (540 Instruments) Round Robin 2005 by DGKH, DGSV and AKI Hämoglobin/µl Studiendag µg Protein (BSA)/Instrument 23
24 ATP Bioluminescence Method All living cells use ATP as a central energy transmitter
25 ATP level variation in blood is quite large GTZ/PI/Dr. Michels 25
26 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 Reproduced with permission from reference 14, copyright John Wiley & Sons Ltd., GTZ/PI/Dr. Michels 26
27 Influence of temperature and ph on luminescence Published December 2006, doi: / clinchem Clinical Chemistry February 2007 vol. 53 no GTZ/PI/Dr. Michels 27
28 ATP hydrolysis occurs dependent upon chemical condition and temperature as can be expected with the main wash in WDs GTZ/PI/Dr. Michels 28
29 ATP measurements with blood published in aseptica 2/2007 GTZ/PI
30 Some measurements: 50 µl of each solution were pipetted into the swab GTZ/PI
31 Lecture of Buchrieser et al. at WFHSS Conference 2009 GTZ/PI/Dr. Michels 31
32 GTZ/PI/Dr. Michels 32
33 GTZ/PI/Dr. Michels 33
34 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. 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 RLU. Results can not be confirmed by protein assessment.
35 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 GTZ/PI/Dr. Michels 35
36 A very quick and simple qualitative routine check is the selective staining of protein Ponseau S azo dye GTZ/PI/AWT Dr. Winfried Michels
37 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
38 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
39 Real instrument with wear friction corrosion and residual soil stained GTZ/PI/Dr. Michels 39
40 GTZ/PI/Michels 40
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