ATP+AMP assay. A most reliable indicator of surface cleanliness and cleaning efficacy
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- Horatio Miller
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1 +AMP assay A most reliable indicator of surface cleanliness and cleaning efficacy 1
2 Easy, Rapid, Expressing numerically +AMP assay which can detect invisible soil or dirt A portable luminometer Lumitester PD 10N A swabbing and dipping device LuciPac W 2
3 Cleaning is the most important at hospitals from different points of view Hand hygiene Environmental hygiene Reusable medical instrument and devices 3
4 Everyone knows that cleaning is the most important So everyone dose cleaning But after cleaning, how do you know whether the cleaning has been really done??? They looks clean.. But from the beginning, some of them were already clean visually.. Visually clean means really clean??? 4
5 Yes, +AMP assay can show cleaning efficacy detects all kinds of soil including microorganism contamination ultra sensitive no comparison with visual inspection rapid easy expressing numerically 5
6 Why +AMP assay can detect soil? First of all, what is +AMP assay? quantitative analysis of (adenosine triphosphate and AMP(adenosine monophosphate) AMP 6
7 and AMP are included in all kinds of soil of hospital are not included in clean water and AMP is the most practical index for cleaning +AMP assay reveals that your cleaning has been done (or not). Why can the quantitative analysis of and AMP indicate cleaning efficacy? Soils of hospital : blood, lymph, digestive juice, saliva, sweat, dirt, 7 tissue, excrement, microorganisms, etc.
8 In case of hand hygiene AMP AMP AMP after washing AMP AMP many soils, dirt or microorganisms fewer soils, dirt or microorganisms many and AMP fewer and AMP (washed properly) 8
9 How to use Anyone can does it easily anywhere anytime swab the surface of a test object measure with Lumitester (The result comes within 10 sec. ) 9
10 How to use 10
11 +AMP assay is useful and effective in many cases 1. Education of hand hygiene 2. Environmental inspection 3. Checking cleanness of surgical instruments 4. Checking cleanness of endoscope 5. Checking cleanness of cooking instruments 11
12 1. Education of hand hygiene microorganisms dirt There are a lot of dirt and microorganisms on the surface of hands First, infection control starts from hand hygiene 12
13 1. as an effective tool for education measure the surface of hands before hand washing let them know their hands on duty are not so clean hand washing lesson measure again (the value should be lower) let them know hand hygiene is very important the hand hygiene lesson is done in about 15 min. (stamp method takes 2 days at least) 13
14 2. Environmental inspection Environment cleansing is very important to prevent person to person infection Anyone can does it easily anywhere anytime 14
15 3. Checking cleanness of surgical instruments assay is filed on the guideline for cleaning of surgical instrument, published by Japanese society of medical instrumentation 15
16 3. Checking cleanness of surgical instruments Evaluation methods Direct method visual protein assay + AMP assay advantage: easy high sensitive rapid expressing numerically re-washing is not needed Indirect method indicator (test soil plate) 16
17 3. Checking cleanness of surgical instruments A question : How we set a benchmark? Study 1 with a digital microscope A digital microscope / KEYENCE corp. product No. VH
18 Method 1. Cleaned stainless steel plate photo by microscope +AMP assay 2. Apply blood on the stainless steel plate ditto 3. Cleaning with 10 sec. ultrasonication ditto 4. Cleaning with furthermore10 sec.(total 20 sec.) ultrasonication ditto 5. Cleaning with furthermore20 sec.(total 40 sec.) ultrasonication ditto microscope : X 3,000 swabbing area 15mm 55mm (back and forth 5 times) 18
19 original stainless steel after blood applying 13 RLU over 999,999 RLU after 10 sec. ultrasonication after 20 sec. ultrasonication 21,881 RLU 375 RLU 35 RLU after 40 sec. ultrasonication visually it is cleaned. but it is not clean actually 19
20 3. Checking cleanness of surgical instruments Study 2 with a washer disinfector Method After a surgery We washed a variety of surgical instruments with a washer disinfector at a hospital ( an indirect indicator <test soil plate> showed the washer disinfector worked properly ) We did +AMP assay 20
21 Result All of the surgical instruments showed less than 100 RLU. Some other studies are on going. At the moment, we suggest 100 RLU as the benchmark for surgical instruments. Value (R LU) 抜糸剪刀抜糸剪刀 expressing numerically It helps to make a standard operation procedure The cleaning history can be recorded 抜糸剪刀剪刀鑷子 a variety of surgical instruments 鑷子鑷子持針器持針器鉗子鉗子鉗子鉗子鉗子 鉗子鉗子鉗子鉗子筋鉤筋鉤吸引管 21
22 4. Checking cleanness of endoscope The results come within a minute. even if the result shows that the endoscope is not clean, the operator can decide to wash it again, and it minimizes a risk of infection. 22
23 5. Checking cleanness of cocking instruments assay is filed on the guideline for food hygiene test methods, supervised by the Japanese Ministry of Health and Welfare. Kikkoman PD-10N and LuciPac W has already been adopted by Japanese health centers, Japan food hygiene association, Japan school meal association, food processing factories, restaurants, hotels, etc. 23
24 appendix 24
25 is decomposed by heat treatment into AMP Heat (washer disinfector) +PPi AMP Detecting both and AMP is very important from technical point of view Yes, Kikkoman can No, other competitors cannot 25
26 Principle of +AMP assay and AMP are measured by two enzymes, Luciferase and PPDK. and AMP are converted into light finally. The Lumitester detects an intensity of light. Luciferase Luciferin O 2 Mg 2+ Oxyluciferin CO 2 Light AMP LuciPac W Pi Pyruvate Mg 2+ PPi PEP Patented technology Pyruvate orthophosphate dikinase(ppdk) Lumitester 26
27 Other competitors Theirs detect only Luciferase Luciferin O 2 Mg 2+ Oxyluciferin CO 2 Light AMP 27
28 Cycling assay system by two enzymes It leads stable light emission reliable value Luciferase light Relative Light Intensity (%) PPDK AMP Reaction time (min) 28
29 Standard curve Relative Light Unit (21.8) (4.3) (7.8) (5.3) (CV value) (7.1) good linearity precise assay wide dynamic range good adaptability assay superior sensitivity Amount of (mol/swab) 29
30 Lumitester PD-10N Auto-judgement(A, B or C ) Result (cleanness index) Preset benchmark Assay results can be transferred to PC. 30
31 Relationship between amount of +AMP and amount of microorganisms Assay results at a hospital CFU Relative Light Unit=Amount of +AMP dirt dirt= nutrient for microorganisms CFU dose not shows its cleaning efficacy 31
32 Relationship between amount of +AMP and amount of microorganisms No relationship between amount of +AMP and amount of microorganisms assay objects are different CFU dose not shows cleaning efficacy Fewer CFU dose not mean cleanliness CFU Relative Light Unit=Amount of +AMP Fewer +AMP means fewer CFU Microorganisms can not germ in the fewer +AMP condition ( CFU takes 1 or 2 days to get results. +AMP assay takes only a minute ) 32
33 Relative 発光量 Light (RLU) Units 1E E E E E E 検量線 standard curve 1 X10,000 万倍希釈液 dilution ( 平均 ) X100,000 万倍希釈液 dilution ( 平均 ) 管理基準値 benchmark (example) 検出限界 detection (blank+3sd) limit +AMP assay versus Protein assay from sensitivity point of view Sample : blood 1E (blank+3sd) 0.1 1E E E E E E E AMP(M) Protein タンパク質濃度 conc.(μg/ml) (μg/ml) (M) 吸光度 595nm タンパク質検量線 standard curve (BSA) 1 X10,000 万倍希釈液 dilution ( 平均 ) 10 X100,000 万倍希釈液 dilution ( 平均 ) 検出限界 detection ( カタログ limit (specification ) 検出限界 sheet) (blank+3sd) detection limit (blank+3sd) +AMP assay has 69 times higher sensitivity rather than Protein assay ( blank+3sd ) Abs. 595nm 33
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