Autoclave  Quality Assurance Program for bio-hazard decontamination

Autoclaving is an accepted procedure for decontamination of certain bio-hazardous waste. Biological cultures and stocks, contaminated solid waste, liquid waste, and small animals carcass waste can be sterilised through autoclaving. After sterilisation in a steam autoclave,

these materials are considered non-infectious. Except for animal carcasses, this bagged waste can be disposed of as ordinary trash; however, it is recommended that autoclave bags containing sterilised waste be placed in an opaque trash bag prior to disposal. Materials that contain hazardous chemicals or radioisotopes are not to be autoclaved.

To ensure that the bio-hazardous waste is properly decontaminated during autoclaving, the following procedures should be followed.

1. Infectious waste must be treated in an autoclave for a minimum of 30 minutes at 121°C (250°F); however, the total processing time required to decontaminate infectious waste depends on the specific loading factors (container type, water content, quantity, etc.). A total processing time of 60 minutes is recommended for gravity displacement autoclaves and 10 minutes for vacuum-type autoclaves (132°C).

Sterilisation by autoclaving is accomplished through exposure and penetration of the contaminated material by superheated steam for an adequate amount of time. Since steam will not penetrate a sealed plastic autoclave bag, bags containing dry loads must not be tightly sealed (rubber band closures will allow bags to “breath”) or adequate amounts of water must be added to the load. Consult the manufacturer’s instruction manual for sterilizing materials inside plastic autoclave bags.

2. All autoclaved waste must include a steam sterilization indicator (the use of biohazard bags with a “built-in” indicator is recommended).

3. Steam autoclaves used to treat infectious waste must operate at a minimum temperature of 121°C. The operating temperature of the autoclave must be verified for each run by maintaining a record of temperature either as a chart or paper tape recording or a manual recording in a logbook.

4. For autoclaves used for bio-hazardous waste, a monthly basis test should be performed by using ampoules containing heat resistant spores (Bacillus stearothermophilus) placed in the centre of an autoclave load. In conjunction with the Bacillus stearothermophilus testing, measure and record the maximum temperature achieved during the autoclave cycle through the use of a maximum registering (or “holding”) thermometer.

5. Record of Bacillus stearothermophilus and maximum autoclave temperature recording must be maintained for a minimum of one year.

1. Be sure the operator read the Instruction Manual of the cabinet and understand the Panel Instructions

2. Switch on the BSC / UAC 5/15 minutes before to start the activity

3. Check that all functions of the BSC /UAC are regular

4. Dress the clean overall with button on the back

5. Dress sterile gloves, covering the wrists

6. Wipe all inside surfaces of the BSC / UAC with suitable disinfectant (e.g.: sterile 70% alcohol)

7. Wipe all material with sterile 70% alcohol before transfer it inside the bench and before to start the activity

8. Avoid creating air turbulence by fast movement of the arms

9. Avoid speaking, sneezing, and coughing

10. Never move or work over open bottles, flasks, dishes, plates

11. Never block air flow through the ventilation holes

12. No unnecessary equipment and material in the clean working bench area

13. Three separate areas inside the working bench: right “clean”, centre “working”, left “dirty”

14. Immediately remove spilled liquids in the clean bench

15. Sterilize by autoclaving the working surfaces, when necessary

16. Clean and disinfect all the surface of the inside BSC at the end of activity

17. Leave the BSC / UAC on for 15 minutes at the end of activity

18. A regular technical check-up of the BSC should be performed annually.

BSC classifications and standards for the United States are set by NSF International (formerly the National Sanitation Foundation). NSF defines four types of Class II cabinets (A1, A2, B1 and B2) that are distinguished by differences in airflow patterns and velocities, HEPA air filter positions, ventilation rates and exhaust methods.

The classification system has changed throughout the years, and the following overview refers to the 2011 NSF/ANSI 49 2011 manual.

Fonte: Baker

Font: The Baker Company

As an example, though an extreme one, at the State Research Center of Virology and Biotechnology, known as Vector, a former Soviet biological weapons laboratory in Siberia, a Russian scientist died after accidentally sticking herself with a needle laced with Ebola, the deadly virus for which there is no vaccine or treatment.² Since Vector did not report it to the World Health Organization (WHO) for three weeks, the delay meant that scientists at the health agency could not provide prompt advice on treatment that might have saved her life. WHO scientists said that although Vector had isolated the scientist to contain any potential spread of the disease, there was no requirement that accidents involving Ebola be reported. What this points out is that poor BBP practices combined with a nonexistent preventive program can be deadly, which prompted this column.

Preventing exposures to blood-borne pathogens begins with strong implementation of OSHA’s BBP standard 40 CFR 1910.1030.³ This standard applies to all occupational exposures to blood or other potentially infectious materials (OPIM). Blood means human blood, components thereof, or products made from human blood. OPIM is defined as human body fluids such as semen, vaginal secretions, cerebrospinal fluid, synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid, amniotic fluid, or any body fluid that is visibly contaminated with blood. OPIM also includes any unfixed tissue or organ (other than intact skin) from a human (living or dead) and HIV-containing cell cultures or tissues.

Applying the OSHA BBP standard starts with developing an exposure control plan (ECP), a mandated, written document designed to eliminate or minimize employee exposures. The ECP must be reviewed and updated annually and include documented consideration or implementation of new technology or safer medical devices to achieve its purpose. The main elements of the ECP are 1) exposure determinations, 2) communication about hazards to employees, 3) methods of compliance, and 4) recordkeeping. Exposure determination is simply a list of employee classifications, tasks, and procedures in which occupational exposures occur, and it is made without regard to personal protective equipment (PPE). Compliance methods include universal precautions; engineering and work practice controls; PPE; and housekeeping issues, especially waste handling. Other ECP elements address Hepatitis B vaccination and postexposure evaluation and HIV/HBV research laboratories.

Although the heart of OSHA’s BBP standard lies in the Methods of Compliance section, the Safety Guys prefer to begin with training, or communicating the hazards. It is paramount that every employee has a solid understanding of which fluids present infection hazards and which do not. Be concerned with blood and OPIM, which have been defined above. Other fluids typically encountered that are not covered by the BBP standard include vomitus, feces, urine, sweat, tears, sputum, and nasal secretions UNLESS these are visibly contaminated with blood. However, one would obviously still want to avoid direct skin contact with these fluids. So make sure everyone knows which fluids present the greatest risks.

The next piece of the puzzle is to learn the avenues of potential exposure. These are 1) through the skin; 2) through mucous membranes, which are the nose, mouth, and eyes; and 3) parenteral, a medical term for injection or breaking the skin barrier. The last exposure route results from needlesticks, cuts, abrasions, and other such injuries.

The final piece of the training puzzle is to know how to protect against potential exposures, and that leads us back to methods of compliance.

The Golden Rule of preventing exposures is one of the universal precautions. Most scientists who work with biohazards and are reading this have the universal precautions tattooed on the backs of their eyeballs. Refreshing your memory never hurts, so here goes. First and foremost, all blood and OPIM are always considered infectious; therefore, you must prevent contact through the use of appropriate PPE that will create barriers between the fluids of concern and the routes of exposure. PPE is considered “appropriate” only if it does not permit blood or other potentially infectious materials to pass through or reach the employee’s clothes, skin, eyes, mouth, or other mucous membranes during normal conditions of use. Under the OSHA standard, when there is occupational exposure the employer must provide all employees at risk, at no cost to the employees, with appropriate PPE, which includes but is not limited to gloves, gowns, laboratory coats, face shields or masks and eye protection, pocket masks, or other ventilation devices.

For most lab scientists, normal activities always entail the use of gloves as the primary means of preventing contact with blood and OPIM. Gloves are worn when it can be reasonably anticipated that the employee may have hand contact with blood, other potentially infectious materials, mucous membranes, or non-intact skin. Gloves are also worn for handling items or surfaces soiled with blood or body fluids. Recommendations for the use of gloves are presented in detail in the Morbidity and Mortality Weekly Report dated June 24, 1988.⁴ Our advice is to buy goodquality, powder-free, non-latex gloves, and plenty of them.

The next most common piece of PPE used is eye protection. Protective eyewear or face shields are worn to prevent exposure of the mucous membranes of the mouth, nose, and eyes during activities or procedures that are likely to generate droplets of blood or body fluids. These types of activities are commonly encountered in the laboratory when preparing and analyzing specimens. So carry all types—eyeglasses, goggles, and face shields—just in case.

After the Golden Rule of always assuming that fluids are infectious and preventing exposure with appropriate quality barriers, the final part of the universal precautions is avoiding injuries and accidents caused by needles, scalpels, and other sharp instruments or devices. Be extremely careful whenever handling these “sharps.” Never recap needles, and always have sharps disposal containers available.

References

- Blood-borne Infectious Diseases: HIV/AIDS, Hepatitis B, Hepatitis C—Universal Precautions for Preventing Transmission of Bloodborne Infections. Centers for Disease Control and Prevention, Atlanta, GA. December 2011. http://www.cdc.gov/niosh/topics/bbp/universal.html 2
- “Russian Scientist Dies in Ebola Accident at Former Weapons Lab,” Judith Miller, The New York Times. May 25, 2004. http://www.nytimes.com/2004/05/25/world/ russian-scientist-dies-in-ebola-accident-at-formerweapons- lab.html
- Blood-borne Pathogens. Occupational Safety and Health Administration, U.S. Department of Labor, Washington, DC. April 2012. http://www.osha.gov/pls/oshaweb/owadisp. show_document?p_table=STANDARDS&p_id=10051
- Perspectives in Disease Prevention and Health Promotion Update: Universal Precautions for Prevention of Transmission of Human Immunodeficiency Virus, Hepatitis B Virus, and Other Blood-borne Pathogens in Health Care Settings. Centers for Disease Control and Prevention, MMWR Weekly. Atlanta, GA. June 1988. http://www.cdc.gov/mmwr/ preview/mmwrhtml/00000039.htm

By Vince McLeod

Fonte: LabManager

www.lbl.gov/ehs/biosafety/

www.lbl.gov/ehs/biosafety/manual/