WEEK 2- MODX311LEC.pdf

MOLECULAR BIOLOGY AND DIAGNOSTICS BIOSAFETY AND WASTE MANAGEMENT IN MOLECULAR LABORATORY JUSTIN KIM C. VERGARA, RMT, MPH COLLEGE OF MEDICAL LABORATORY SCIENCE OUR LADY OF FATIMA UNIVERSITY- VALENZUELA LESSON INTENDED LEARNING OUTCOMES (LILO): At the end of the session, you wil...

MOLECULAR BIOLOGY AND DIAGNOSTICS BIOSAFETY AND WASTE MANAGEMENT IN MOLECULAR LABORATORY JUSTIN KIM C. VERGARA, RMT, MPH COLLEGE OF MEDICAL LABORATORY SCIENCE OUR LADY OF FATIMA UNIVERSITY- VALENZUELA LESSON INTENDED LEARNING OUTCOMES (LILO): At the end of the session, you will be able to: 1. Adhere to personal health and safety practices 2. Maintain a clean and organized workspace 3. Disinfect and dispose of infectious materials 4. Take appropriate actions following accidental exposure to potentially infectious specimen 5. Follow written safety procedures and keep proper safety records OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE  The Laboratory Quality System Organization Personnel Equipment Stock Quality Data Management Control Management SOPs, Occurrence Documents & Assessment Records Management Process Specimen Safety & Waste Improvement Management Management OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Why is Safety Important? Coming in contact with human blood or blood products (plasma, serum, etc.), or with certain chemicals used in the laboratory, is potentially hazardous. Safety involves taking precautions to protect you and coworkers against infection, injury or poisoning. OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE HAND HYGIENE – Persons must wash their hands after working potentially hazardous materials and before leaving the Laboratory. – Lather and Scrub: 20 seconds 20 : 10 – Rinse: 10 seconds – Do not use Hand Dryers! -- – Performance of entire handwashing: 40-60 seconds OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE  DECONTAMINATION I. Decontamination – Process of removing or neutralizing chemical or -- Biological Agents so that they no longer pose a hazard. (https://ncbi.nlm.nih.gov) 2 Sterilization – Removal or Destruction of all forms of life, including. bacterial spores. 3 Disinfection – Removal, inhibition, or killing of microorganisms. including potential pathogens by using chemical agents usually on inanimate objects; does not remove all bacterial spores. Antisepsis – Antimicrobial substances typically applied in the skin to reduce possibility of sepsis formation or putrefaction. OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE  Standard Precautions Minimum infection prevention practices that apply to all patient care, regardless of suspected or confirmed infection status of the patient, in any setting where health care is delivered (CDC) a. Avoid touching eyes, nose, or mouth with gloved or unwashed hands - b. ALL SPECIMENS shall be treated as infectious - c. Avoid wearing jewelry - d. Refrain from using mobile electronic devices inside the Laboratory - - e. Keep work area tidy, clean, and free of cluster and materials not necessary for the work being done OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE DECONTAMINATION/DISINFECTION - Use of decontamination solutions with proven activity against enveloped RNA viruses – 70% Ethyl Alcohol – 10% Sodium Hypochlorite: Spills – 1% Sodium Hypochlorite: General Surface Decontamination - Contact Time of Bleach: 10-15 minutes - All surfaces and equipment must be decontaminated before and after usage. OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE CHEMICAL AGENTS Efficacy is based on different factors: – Organic Load – Microbial Load – Type of Organism – Condition of surface to be decontaminated – Disinfectant concentration, pH, temperature, contact time, environmental humidity OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Resistance of Microorganisms to Disinfectants OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Sodium Hypochlorite Contact Time: 10-15 Minutes Bought in concentrations of 5-8.25%, most common: 5.25% Preparation: 1:10 or 1:100 – At least 5000 ppm, no more than 10,000 ppm Chlorine for decontamination CORROSIVE: Needs to be washed after (Steel) DO NOT AUTOCLAVE BLEACH SOLUTIONS! Effect: Oxidative effect of Hypochlorous acid Avoid the following: –v Exposure to air (↓ free Chlorine concentration due to Chlorine evaporation) – Storage in direct sunlight and varying temperatures (NaOCl solutions can be stored up to 6 months if this is followed!) – Bleach loses 20-50% of its concentration after 6 months OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Different Cleaning Jobs Require Different Bleach Solutions General lab use - Hypochlorite Solutions General Spills Disinfection 10% 1% (1 part + 9 parts) (1 part + 99 parts) * WHO Laboratory Biosafety Manual OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Alcohol Best preparation: 60-90% Alcohol (↑ Water conc while maintaining high Alcohol concentrations ↑ contact time of Alcohol to microorganism) If used as a disinfecting agent: – Without Bleach:2-5minutes – With Bleach: No need for contact time Bactericidal, Fungicidal, Virucidal, non-Sporicidal Allowed to evaporate from the surface to which they were applied to achieve maximum effectivity. Effect: Cause denaturation of protein and dissolution of lipid membrane Should not be used as a lone decontaminant for MOLECULAR e TESTING – causes aggregation of Nucleic Acids not eradicating them OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Ultraviolet Light A type of non-ionizing radiation that causes damage to cellular DNA by producing & Thymine dimers – Most lethal wavelength: 260nm Longer wavelength (>1 nm) and low energy Microorganisms are destroyed when water is passed under the UV Lamps Quality Control Indicator: Bacillus pumilus Disadvantage: Can only destroy those under its direct contact. Wears out overtime. Has little to no effect on RNA. OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Lysol Active Ingredients: Benzalkonium Chloride or Hydrogen Peroxide KNOWN EFFECTIVE AGAINST MANY MICROORGANISMS Effects: – Benzalkonium Chloride: Disruption of cell membrane, resulting in leakage of cell contents. – Hydrogen Peroxide: Degrades Organic compounds by the highly reactive Hydroxyl radical (-OH). OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Inactivation Method for Prions Instrument: – Immerse in 1N NaOH or 2.5% NaOCl for 1 hour; remove and rinse in water, and then transfer to open pan and treat in a gravity displacement (1210C) or porous load (1340C) autoclave for 1 hour. Surfaces: – Spray or pour 1N NaOH or 2.5% on surface and let sit on 1 hour. Ensure surfaces stay wet for entire period. Surfaces should be clean of any gross contamination as organic material can reduce the effectiveness of the solutions. OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Inactivation of Microorganisms: AUTOCLAVE Principle: Steam under pressure Fastest and simplest method of sterilization – all organisms (except Prions) and spores are killed within 15 minutes. Biological Indicator: Geobacillus stearothermophilus Properties: – 1210C, 15 psi, for 15 minutes: Media, Liquids, Utensils, Glass Pipettes, and Instruments for Assay – 1320C, 15 psi, 30-60 minutes: Decontaminating Medical Wastes OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Quality Control for Autoclave Biological Indicator: – No color change if sterilization is complete – Turns color YELLOW if growth happens Autoclave Tape: – Turns black if Sterilization is complete These black diagonal lines will appear on the tape after 10 minutes at 121°C or 2 minutes at 134°C in a steam sterilizer OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Biosafety Cabinet Calibrated and Certified before use Practice proper usage, placement, and decontamination The Biosafety Cabinet is a requirement to process infectious specimens. OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE How Biosafety Cabinets provide protection 1. HEPA Filter – Standard for Biosafety – 99.97% efficiency at 0.3 microns – Made from Pleated borosilicate glass, arranged in random fibers 2. ULPA Filter – 99.999% efficiency at 0.12 microns 3. SULPA Filter – 99.9999% efficiency at 0.12 microns OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE How do Biosafety cabinets provide containment/protection? “Curtain of Air” at the opening – protection of product Laminar flow of filtered air within the BSC – Protection of the personnel Filtration of exhausted air – Protection of the environment OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Biosafety Management 1. Ensure the development and adoption of a biosafety management plan and a safety or operations manual. 2. Ensure that regular training in laboratory safety is provided. 3. Personnel should be advised of special hazards, and required to read the safety or operations manual and follow standard practices and procedures 4. There should be an arthropod and rodent control program. 5. Appropriate medical evaluation, surveillance and treatment should be provided for all personnel in case of need, and adequate medical records should be maintained. OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Health and Medical Surveillance 1. Provision of active or passive immunization where indicated 2. Facilitation of the early detection of laboratory- acquired infections 3. Exclusion of highly susceptible individuals (e.g. pregnant women or immuno-compromised individuals) from highly hazardous laboratory work 4. Provision of effective personal protective equipment and procedures. OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Apply Safety Practices Throughout the Testing Process 1. Before Testing (Pre-analytical) -Specimen collection -Specimen preparation -Specimen transport 2. Testing (Analytical) -Testing 3. After Testing (Post-analytical) -Disposal OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE  Develop Personal Safe Work Habits Wash hands before and after entering the lab Change gloves frequently Wear lab coat or apron Dispose of contaminated sharps and waste immediately after testing Pipetting by mouth is strictly forbidden Never eat, drink or smoke at the test site Keep food out of the laboratory/testing site refrigerator OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Proper Disposal of Biowastes Good Safety Practices – Identify Hazards – Implement safety strategies to contain hazards – Audit existing practices to determine whether new ones are needed OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Biological Waste Exposure: Ingestion, Inoculation, Tactile Contamination, Aerosolization, Inhalation of Infectious materials Blood – HBV, HCV, HIV – Blood-borne pathogens – Inactivated and Autoclaved OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE  Swabs – Respiratory infections/ – Inactivated (Heat, Chemical) and Autoclaved before disposal Tissues – Should be fixed with a fixative – Unfixed tissues must be inactivated and autoclaved Other body fluids – Sputum: Tuberculosis, other respiratory infections – Inactivated and Autoclaved OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Prevention Gloves – Primary barrier protection – Not reused or washed for reusing Masks/Respirators, Protective Eyewear, Face Shield – for exposure from splashes to the mouth, eyes, nose Decontaminate work area regularly OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Chemical Hazards Stored in certain sections of the Laboratory – Not in use, packaging with leakage or corrosion: Replaced – Water reactive chemicals: No contact with water MATERIAL SAFETY DATA SHEET – Document that gives detailed information about a material and about any hazards associated with the material OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Handling Chemicals How to properly handling different kinds of chemicals in the workplace Chemical Hazards in the Genotyping Laboratory Why hazardous? -Ignitable -Corrosive -Reactive -Toxic OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE  Handling Chemicals Check label for substance verification Chemical-resistant gloves Containers held away from the body during transferring Do not heat with direct flame ACID TO WATER Do not touch, taste, or smell chemicals Use a Laboratory Chemical Hood. Clean spills properly and promptly, dispose accordingly. OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE  Carcinogens Any substance that can cause cancer. – Changes the metabolic processes by damaging the genomes Different types & ①– Decontaminating Agents – Dyes g – Probes and Labels OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Chemical Hazards in the Genotyping Laboratory Examples: Guanidinium thiocyanate- commonly used in lysis buffers Ethidium Bromide (mutagen)- DNA staining in agarose gels, buffers Acrylamide (neurotoxin) OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Ethidium Bromide - For Agarose Gel Electrophoresis - Intercalating agent, Nucleic Acid stain - Fluorescence: Orange - Not directly mutagenic: metabolites are mutagenic OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE  SYBR Green – Repacement for Ethidium Bromide – Can still bind to DNA with high affinity = potential Carcinogen Acrylamide – Used in PAGE (Polyacrylamide Gel Electrophoresis) – Cross-linking agent for gel chromatography and electrophoresis – Can cause peripheral neuropathy, possible carcinogen Phenol – Used in Phenol-Acetic Acid-Urea Polyacrylamide Gel Electrophoresis (PAU-PAGE) Chloroform – Extraction of Proteins – Eye damage, fainting, life-threatening OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE In Case of a Spill or Splash 1. Evacuate Room and notify others to leave the room and post a warning sign for No entry 2. Remove all contaminated clothing and /or lab coat and place in a biohazard bag 3. Wash all exposed skin with antiseptic soap and water 4. Inform supervisor 5. Decontaminate the area: Assemble clean-up materials Large spill - Cover with paper towels and soak with 10% household bleach and allow to stand for 10-30 minutes Small spill - Wipe with paper towel soaked in 10% bleach 6. Dispose all contaminated towels in biohazard bag and dispose it properly OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE In Case of an Accident What types of accidents can happen? ❑ Potential Injury, i.e., needlesticks, falls ❑ Environmental, i.e., splashes or spills ❑ Equipment damage What should you do? ❑ Report to supervisor immediately ❑ Assess & take action ❑ Record using form ❑ Monitor situation OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Action Plan for Implementing Safety Practices Identify hazards Establish and implement safety polices and procedures Conduct safety specific training ❑ Must be a priority ❑ Communication is key Perform regular audits or assessments OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE Definitions Many different terms are used for disinfection and sterilization. The following are among the more common in biosafety: Disinfectant – A chemical or mixture of chemicals used to Antimicrobial – An agent that kills microorganisms or kill microorganisms, but not necessarily spores. suppresses their growth and multiplication. Disinfectants are usually applied to inanimate surfaces or Antiseptic – A substance that inhibits the growth and objects. development of microorganisms without necessarily Disinfection – A physical or chemical means of killing killing them. Antiseptics are usually applied to body microorganisms, but not necessarily spores. surfaces. Microbicide – A chemical or mixture of chemicals that Biocide – A general term for any agent that kills kills microorganisms. The term is often used in place of organisms. “biocide”, “chemical germicide” or “antimicrobial”. Chemical germicide – A chemical or a mixture of Sporocide – A chemical or mixture of chemicals used to chemicals used to kill micro-organisms. kill microorganisms and spores. Decontamination – Any process for removing and/or Sterilization – A process that kills and/or removes all killing microorganisms. The same term is also used for classes of microorganisms and spores. removing or neutralizing hazardous chemicals and radio- active materials. OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE REFERENCES 1. www.cdc.gov/od/ohs/pdffiles/4th%20BMBL.pdf 2.www.who.int/csr/resources/publications/biosafety/WHO_CDS_CSR _LYO_2004_11/en/ 3. DEAN MAGDALENA F. NATIVIDAD (FEU – NRMF), PASMETH Webinar on Molecular Biology and Diagnostics OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE NEXT MEETING: Review of DNA, RNA, and PROTEIN structure and function “Strength does not come from physical capacity. It comes from an indomitable will” – Mahatma Gandhi THANK YOU FOR LISTENING! OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE