ATP Adenosine TriPhosphate Bioluminescence. What do we ... Agenda 1. ATP Bioluminescence Technology

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  • 1. All Rights Reserved.20 March 2018© 3M 3M Confidential.

    ATP Adenosine TriPhosphate Bioluminescence. What do we need to know?

    Decon Safety Prog Network Event

    Dublin

    26th March 2018

    Dr Brian Kirk 3M UK

  • Agenda

    1. ATP Bioluminescence Technology – What it is and what it is not, how it works.

    2. What are its characteristics and what benefits does it bring.

    3. What standards say.

    4. Applications areas

    5. Endoscopy – why do we care about cleanliness. The clinical evidence.

    6. Data reporting.

    7. Questions

  • 3. All Rights Reserved.20 March 2018© 3M 3M Confidential.

    • ATP is present in ALL living cells

    • Microbes

    • Plants

    • Animals

    • Humans - tissue and Bodily Fluids

    • ATP stores energy in the phosphate bonds.

    • Energy is released when the phosphate

    bonds are broken

    • This energy used in cell metabolism

    Adenosine

    Phosphates

    Adenosine Tri-Phosphate

    What is Adenosine Triphosphate (ATP)? The energy molecule for life

    3M Confidential

  • 4. All Rights Reserved.20 March 2018© 3M 3M Confidential.

    The principle of ATP bioluminescence

    ATP:

    Adenosine Triphosphate

    The “energy currency” molecule of all living organisms

    Luciferin/luciferase +

    Indicates

    contamination of

    biological origin

  • Bioluminescence Bioluminescence is where the energy of a biochemical bond (ATP to ADP) is converted to light energy via a biochemical reaction.

    The reaction takes place in the cold and is found in many natural processes

    Origin:

    • Greek bios for “living” • Latin lumen for “light”

  • Bioluminescence in nature

  • ATP - Adenosine triphosphate

    The “battery” of all living cells.

    Found in all living or once living biological cells.

    Source of energy easily stored and used when needed for cellular functions

    Cleavage Cleavage Cleavage Cleavage of phosphate from ATP releases energy of phosphate from ATP releases energy of phosphate from ATP releases energy of phosphate from ATP releases energy

    ATP +HATP +HATP +HATP +H2222O O O O ADP ADP ADP ADP + HPO+ HPO+ HPO+ HPO3333 2222----

    ΔΔΔΔЄ (30kJ/(30kJ/(30kJ/(30kJ/molmolmolmol))))

  • Bioluminescence - mechanism

    The following reaction is catalysed by the enzyme luciferase and yields light:

    *Light emitting bio pigment

    ATP + D-Luciferin* + O2

    Oxyluciferin + ADP + phosphate + CO2 + Light Photons (RLU’s)(560nm)

    Luciferase

  • 9. All Rights Reserved.20 March 2018© 3M 3M Confidential.

    increase in organisms and other biological residuesincrease in organisms and other biological residuesincrease in organisms and other biological residuesincrease in organisms and other biological residues

    increase in ATP levelsincrease in ATP levelsincrease in ATP levelsincrease in ATP levels

    increase in light (RLU)increase in light (RLU)increase in light (RLU)increase in light (RLU)

    Simple Relationship

  • ATP Bioluminescent Intensity vs concentration A highly sensitive method

    Copyright – Dr Virginia Deibel, TRAC Microbiology

    attoM femtoM picoM

  • 11. All Rights Reserved.20 March 2018© 3M 3M Confidential.

    The emitted light is detected by a luminometer

    Light is converted into a number - RLU

    – relative light units.

    The bigger the number, the more ATP detected, the

    “dirtier” the surface sampled

    How the emitted light is measured.

    Technical explanation:

    Photons of light enter a photomultiplier tube and impact on a special crystal which releases electrons as a consequence of the photon excitation. The released

    electrons enter a cascade of charged electrodes which release more electrons. The electron stream creates a current flow which is amplified. The amplified signal

    enters an analogue to digital converter circuit. The digitised signal is further processed and finally presented on screen as a numerical value

  • 12. All Rights Reserved.20 March 2018© 3M 3M Confidential.

    What Can ATP Bioluminescence tell us;

    A very sensitive marker of biological contamination.

    HOWEVER:

  • 13. All Rights Reserved.20 March 2018© 3M 3M Confidential.

    How Do ATP Results Relate to Microbiology Methods?

    ATP + Firefly enzyme = 200 RLU

    CFU

    RLU

    With human residues, RLU does not correlate

    well with CFU

    Non-Microbial ATP

    Microbial ATP

    With these typical proportions of product and microbial

    ATP on a swab RLU does not correlate with CFU but

    with “cleanliness”

    They Don’t except in pure microbial cultureThey Don’t except in pure microbial cultureThey Don’t except in pure microbial cultureThey Don’t except in pure microbial culture

  • ATP - Bioluminescence

    Does not measure residual Does not measure residual Does not measure residual Does not measure residual proteinproteinproteinprotein

    Very sensitive measure of residual tissue debris associated

    with incompletely cleaned devices.

    EN ISO 15883…

  • 15. All Rights Reserved.20 March 2018© 3M 3M Confidential.

    A Log Normal function was found to provide

    the best fit to the data (solid red line, R2=0.72;

    95% confidence intervals shown as dotted red

    lines). The analysis shows that ATP and protein

    levels are linearly correlated (dashed black line,

    R2=0.68) for protein concentrations greater

    than 8 µg/mL and ATP values greater than 20

    RLUs (corresponding to an ATP concentration of

    ~7 fmoles/mL)

    The study measured levels of protein and ATP

    in 89 endoscopes (48 colonoscopes, 38

    gastroscopes, 3 duodenoscopes) at two US

    clinical sites for three points during

    reprocessing: bedside cleaning, post-manual

    cleaning, and after high level disinfection

    (HLD)

    Relationship between residual protein and ATP in complex soils

  • Detection of Whole Blood – a complex soil

    • Ten fold dilutions of whole blood • Determined the analytical limit of detection to be less than 1x10-7 diluted

    whole blood (between 5-50 RBCs/swab)

    Other tissues similarly detected.

  • ATP Sensitivity - Detection of Whole Blood

    y = 0.9571x + 8.9202

    0.00

    1.00

    2.00

    3.00

    4.00

    5.00

    6.00

    7.00

    -9-8-7-6-5-4-3-2

    A T

    P [

    L O

    G (R

    L U

    s) ]

    Whole Blood Dilution [LOG(dilution)]

    Instrument Background

    • Ten fold dilutions of whole blood • Determined the analytical limit of detection to be 1x10-8 diluted whole blood (approximately

    50 RBCs/swab)

  • 18. All Rights Reserved.20 March 2018© 3M 3M Confidential.

    � A sensitive and objective method compared to visual assessment � A sensitive and objective indicator of generic soil (living or once living) – non specific.

    � Results are available in less than one minute.

    �Provides a definition for what “clean” is Provides a definition for what “clean” is Provides a definition for what “clean” is Provides a definition for what “clean” is ---- quantificationquantificationquantificationquantification

    �Implement simple pass / caution / fail levels

    �Gives chance to take corrective action – re-clean

    � Monitor and understand hygiene levels over time – reveals variability and indicates consistency

    ATP bioluminescence

  • What is meant by clean – in the past 3.9 Cleaning:

    Removal of contamination from an item to the extent necessary for its further processing and its intended subsequent use.

    • EN ISO 15883EN ISO 15883EN ISO 15883EN ISO 15883----1:2006 1:2006 1:2006 1:2006 –––– Washer Disinfectors Washer Disinfectors Washer Disinfectors Washer Disinfectors –––– General requirements terms definitions and General requirements terms definitions and General requirements terms definitions and General requirements terms definitions and tests.tests.tests.tests.

    So how clean should a re usable surgical instrument be that will be used in a body So how clean should a re usable surgical instrument be that will be used in a body So how clean should a re usable surgical instrument be that will be used in a body So how clean should a re usable surgical instrument be that will be used in a body cavity?cavity?cavity?cavity?

  • The alert and action level criteria for other analytes are:

    What are the main methods available to assess cleanliness: According to ISO 15883-5 (CD)

    What is “Clean” ?What is “Clean” ?What is “Clean” ?What is “Clean” ?

    The alert and Action level criteria for protein assays are: Alert Level >= 3 microg/ cm2

    Action Level >= 6.4 microg / cm2

    AnalyteAnalyteAnalyteAnalyte Alert Level (per cmAlert Level (per cmAlert Level (per cmAlert Level