Biosafety Unit, CRISP, FHML, MUMC+
Working Instruction 11: Safe handling human material
5.1 Objective and terms of application
Biological material originating from humans is potentially dangerous to people who work wit hit, because of the chance that the material is contaminated with micro organisms that are pathogenic to man, like the hepatitis virus, HIV-virus, etcetera. This applies to a lesser extent to biological material originating from animals.
This procedure was written to draw attention to the possible dangers of working with humane and/or animal material, and, to give guidelines for the best way to control these risks.
The following are considered humane materials:
- blood, lymph, tissues (biopsies) and liquids excreted by the body like urine, saliva, sputum, faeces, sperm, etc.
- materials derived from humane tissues like cells that may or may not have been programmed into cell lines
- micro organisms that have been isolated from the materials listed above.
Micro organisms that are pathogenic to humans are those that can cause infections in humans. Depending on the micro organism, the consequences of such an infection can range from mild to serious illnesses and deadly infections (for example AIDS). For more information on the risks of humane material that is possibly contaminated with pathogenic micro organisms, see addendum 1.
This working instruction is set up base don 5 aspects, being:
1. preparatory activities
2. hygiene and protocols for working safely
3. daily cleaning and disinfection procedures
4. storage and waste
5. incidents, accidents and emergency situations.
5.2.1 Preparatory activities
Risk assessments
· The manager in charge must ensure that risks represented by working with humane materials are assessed and evaluated (an RI&E as required by the law of Occupational Health and Safety). This risk assessment must be evaluated by a certified occupational hygienist. This certified expert will consequently determine the necessary requirements for the room in which the work is being performed. These requirements will match the dictated equipment needed for ML-I or ML-II classification (also refer to instructions for working safely with genetically modified organisms).
· As a manager, fill in the checklist for occupational risks for new employees or in case working conditions or tasks change (arbochecklist). Submit on this form that an employee will regularly come into contact with humane materials.
Education and training of employees
· The manager in charge is responsible for the education and training of his employees with regard to working safely with humane materials. Employees that did not graduate a biologically oriented education must be schooled in working safely in a biological laboratory environment (use of disinfectants, use of items for personal protection, hygiene, use of safety cabinets, etc.). Also, inform employees on the risks of an accident that involves being pricked with a needle and the proper way of handling such an incident. Make sure the employees knowledge and understanding has been evaluated in a test and register the results in the employees files that are being kept at the Human Resources department.
· The departmental manager should make sure that employees who have to draw blood are being trained in this skill to make sure they are licensed to do so ( a possibility is the training module offered by the Department of Education in the academic hospital Maastricht (azM), email contact: ).
Vaccination
· Offer vaccination against hepatitis B to all employees working with humane materials. Hand out the form ‘Participation in hepatitis B vaccination (request for participation in hepatitis B vaccination program). Check addendum 2 for more information on hepatitis B.
· The employee working with humane materials should fill out the form for participation in the hepatitis B vaccination program (request for participation in hepatitis B vaccination program) and send it to Ease Travel Clinic.
· As a health physician, make sure that an employee is being vaccinated, checked for titer and served with a written result of the vaccination procedure. Information on the achieved status should also be sent to the coordinating officer on Occupational Safety of Randwijck
5.2.2 Hygiene and protocols for working safely
· It is prohibited to eat, drink, smoke, apply cosmetics and store food items in the laboratory.
· Rings, bracelets, wrist watches and the like should not be worn during work. Personal belongings like purses should be stored outside the laboratory area.
· Wear a closed lab coat. After finishing work, the coat should be left in the laboratory.
· Wear nitril gloves in case of small wounds or damaged skin.
· In case no gloves are being worn, hands should be frequently washed using water and soap; at least after handling potentially contaminated materials and before leaving the laboratory (after taking off the lab coat). In case gloves are being worn, hands should be washed after taking the gloves off.
· Pipetting by mouth is never allowed; even if no liquid enters the mouth, aerosols can still be inhaled. Use either a pipetting balloon or Pipetman.
· Formation of aerosols should be avoided. This can be done as follows:
- centrifuging and shaking should always be done in closed vials. In case open centrifuging vials are necessary, use closed buckets which are opened only in a safety cabinet.
- wet needles for inoculation should be dried before glowing them out.
- liquid filled pipettes should not be held in flames.
- never blow out a pipette, instead let it flow out against a surface or into another liquid.
- minimize the use of needles as much as possible.
- devices that give rise to formation of aerosols (for example blenders, sonicators) should be placed in a safety cabinet, without disturbing the protective functionality of the cabinet (also see procedure for working safely in a class II safety cabinet)
5.2.3 Storage, internal transportation and waste
· Store humane materials always at a temperature that does not exceed 4o C. In case of prolonged storage freeze materials at –20° C, or, even better, at –70o C (provided this is not damaging to the desired activity of the biological material).
· Place non-disposable, used materials in stainless steel or plastic containers to be autoclaved.
· Place waste (including used cultivating solutions of micro organisms) in bins for microbiological waste or in containers suited for autoclaving. After autoclaving the waste is being disposed of as conventional laboratory waste in orange/red bags. For small amounts, white containers that contain the message ‘potentially contaminated microbiological waste’ in green writing are available. They are collected by the employee who is responsible for potentially dangerous waste and being disposed of as potentially contaminated waste.
· Check each sterilisation cycle in the autoclave by registering pressure and temperature and write down the results. Check the effectiveness of the autoclaving process once a day using a bio-indicator, that is placed in the centre of the product that is to be sterilised, like an ampoule containing Bacillus stearothermophilus spores (Sterikon-bioindicator, Merck, Darmstadt). This ampoule is retrieved after the sterilisation process and incubated in a 60° C water bath. The liquid in the ampoule turning turbid and a change in colour to yellow are both indicators of an ineffective sterilisation process. In that case, the autoclave must be checked and repaired immediately. The contents of previous loads should be sterilized again, if possible.
· When applying suction under vacuum, install a flask that holds a chlorine solution (0.1 % active chlorine and a splash of detergent) followed by a flask filled with a solution of 1M NaOH to collect chlorine fumes. Replace these flasks weekly. Make sure the waste is sitting in the chlorine solution at least overnight. In case of frequent use, autoclaving the waste is to be preferred (do not use the chlorine solution for this).
5.2.4 Cleaning and disinfection procedures
The cleaning of laboratory areas where humane materials are being used, are to be cleaned following procedures for cleaning conventional laboratories. The cleaning is being done by a cleaning firm that is hired by Maastricht University. Benches and sinks must be cleaned by MU employees, not by the cleaning firm.
The purpose of disinfection is to kill as many micro organisms as possible, to a level that represents an acceptable risk. In case total destruction is necessary, for instance for tools used in medical procedures, sterilisation is obligatory. This process also destroys spores of bacteria.
· Disinfect working areas after ending work activities. Use a solution with a 0,1 % chlorine content from an organic compound (1 tablet Stafilex or Suma Tab D4 in 1,5 litres of luke warm water (Stafilex: Johnson/Diversey; Suma Tab D4 en Suma Total D24: Diversey Lever, Maarssen) en let this incubate for at least 5 minutes.
· Disinfect the skin in case of contamination with an amount of alcohol that is enough for keeping the skin wet for at least a full minute.
· Clean contaminated areas or tools with water and detergent before disinfecting. This prevents a diminished efficiency of the chlorine solution caused by organic materials as well as prevents the formation of dangerous substances such as chloroform.
· Treat centrifuge rotors with a solution containing 70% ethanol in a way that they will stay wet for at least 5 minutes (warning: inflammable!)
· Autoclave heat-resistant materials that are non-disposable (laboratory glass, certain plastics)
· Dry-sterilise contaminated metal objects like scissors and tweezers (3 hours at 180 C)
· Disinfect items that are not heat-resistant and will be reused, by incubating them in the aforementioned mentioned chlorine solution for 5 minutes.
5.2.5 Accidents and incidents
· If necessary, call for (first aid) help through phone number 1333.
· Participants of the ‘Company Energency Services’ (Bedrijfs Hulp Verlening / BHV) should always use a ‘Kiss of life’ to prevent ingestion of blood or vomit.
· Contaminated eyes should be rinsed for at least 10 minutes using the available device for eye rinsing (in a bottle or integrated in the tap next to the sink).
· Spit out materials that entered the mouth and immediately rinse with water or a physiological saline solution.
· Participants of the Company Emergency Services should disinfect the skin of contaminated persons by using a chloro-hexidine solution in 70% alcohol. In case of an infection with risk category 1 organisms or blood, clothes should be washed with detergent. In case of contamination with risk category 2 or 3 clothes should be autoclaved prior to washing.
· Cover large quantities of human spoilage material with a 0.1% chlorine solution and leave it for at least 5 minutes. Wearing gloves, absorb this with tissue and dispose of the tissues in a bin for microbiologically contaminated waste (not in an autoclave container because autoclaving would give rise to chlorine fumes). Afterwards, the contaminated area should be cleaned with water and detergent, again while wearing gloves.
· Follow the step program ‘pricking incidents’ in case of pricking or cutting accidents (see addendum 3). Call the Biological Safety Officer if there are any questions or uncertainties.
· In all cutting or pricking accidents, a ‘zero serum’ measurement should be taken in the emergency room of the azM, to be stored in the laboratory of the department of Virology at –20o C. The emergency room bill should be sent to the manager, who will transfer it to the head of the department.
· The company physician should report an infection with hepatitis B or C at the Ministery of Public Health (obligatory) and RIVM (voluntarily).
13
March 2011
Biosafety Unit, CRISP, FHML, MUMC+
Addendum 1
Risks of human material
People can be infected with or carrier of micro organisms that are pathogenic to humans. Micro organisms are classified based on their pathogenic properties for man (Table 1). Micro organisms that have not yet been classified should not automatically be placed under the regime of class 1. An assessment and evaluation of risks represented by working with such a micro organism must result in the proper risk category. The legally binding summary of risk categories is published as Guideline 2000/54/EG as well as in Occupational Health Information Sheet 9 (AI 9). The risk levels listed under 1 (low, medium and high) equal the risk categories of the micro organisms involved. This means that working with risk category 1-micro organisms must be done in an environment listed as risk category low or up. For working with micro organisms classified as class 4, like Ebola virus, extremely high standards are defined, both for the safety of employees and the containment of micro organisms. (Containment consists of building and procedural measures that must ensure that the chance of the micro organism leaving the laboratory is practically zero). Such facilities are not available in the Netherlands.
Table 1. Risk classification of biological agents
Risico category / Pathogenity / Spreading / Treatment/Procedure1 / -
2 / + / - / +
3 / ++ / + / +
4 / ++ / ++ / -
- non-existent
+ present
++ strongly present
Risks do also depend on the porte d’ Entrée of the microorganisms. The Influenza virus will not cause Influenza after ingestion of the virus, but will cause influenza after inhalation. The effect of the exposure is also influenced by the amount of microorganisms and the personal resistance. (vaccinated etc)
Micro-organisms depend on suitable environmental factors for multiplication. Human pathogenic microorganisms are able to multiply under a humid condition between 20 and 40o C and with a sufficient amount of nutrients. For their multiplication, viruses needs living cells. However, many microorganisms can survive under sub-optimal conditions like a low temperature. Traces, cysten and worm-seeds can survive for years extreme circumstances like dry-out and high temperature up to 100o C, disinfectants and lack of food and germinate again under changing environmental conditions. Some pathogens produce toxins, like Clostridium botulinum In this case, contact with the bacteria is not that dangerous, but the Growth media in which the bacteria were multiplied.
Humane material is potentially contaminated with different microorganisms, for example:
- Blood: hepatitus B, hepatitis C, HIV
- Brain- and nerv-tissue: Listeria, arbovirusses, rabies, Creutzfeld-Jacob prion