Safety rules for utilizing and working in laboratory "Genteknologisk Klasse II"

The purpose of the guide

To provide guidelines for safe working conditions of all laboratory work in the area of the laboratories in Department of Clinical Research. The entrance to the relevant laboratories is marked with "Genteknologisk område – klasse 2" and a biohazard sign.

The GMO-class II (GMO-2) laboratories can accommodate research with human viruses and biological material containing virus (cell culture, tissue- and blood samples). All methods of handling biological active material must be done so following this instruction.  

Using and working in any GMO-class II laboratory carries a real, albeit low, health risk, which means, that additional safety precautions and guidelines can apply in the specific projects. It is the head of safety's responsibility that specific project guidelines are updated and trained before commencing any individual work. 

Area of application

This guide applies to the Department of Clinical Research's laboratories, classified for gen-technological work class II (GMO 2).

The guide is supplemented by the mentioned project-specific precautions that are defined by each projects' risk classification as stated by the Danish Working Environment Authority, see e.g. specific guidelines for COVID-19 related projects. Therefore, this guideline reflects the minimum standard for safety precautions, which all projects and procedures in GMO 2 must abide by.

Responsibilities

These instructions apply to everyone who works in genetic engineering laboratories class II, laboratory no. 232, 242, 252 and 262 with limited access for employees via the locks 400.21.402 and 400.23.404. Note that for work with coronavirus in rooms no. 400.23.401 and 400.23.402, an extended set of safety rules must be used, which is attached to this guide as an appendix - Bilag Klasse II.

It is the safety manager and chief of laboratories responsibility that the guide is followed. The general responsibility for the GMO 2 area is the head of the Department of Clinical Research. The local Work Evironment Organization has the responsibility to maintain the guide and control safety procedures.

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Procedure

1. ACCESS RULES

The access to the GMO 2 areas is restricted. Access requires a mandatory safety introduction to the content of this guideline and instruction/training in any specific precautions, which relevant staff will be working with. Only hereafter, access must be given via an e-mail to the keycard office, Nøglekortkontoret, cc the laboratory manager.

It is the safety manager's responsibility to inform the chief of laboratories about new staff and is the latter's responsibility to provide for the mandatory safety introduction, including documentation hereof. It is the individual project manager's/supervisor's responsibility to give instructions/training to specific precautions and procedures, which exceeds this guideline.

Access is not allowed for persons:

• With open wounds, inflammation, eczema, and the like, on hands or face
• Who is being treated with drugs and/or have deficiencies that may lower their resilience to infections
• Under the age of 18 years 

1.1 ACCESS FOR ANY OTHER PERSONS

See specific safety guidelines for external technicians and cleaning staff in VIP.  

1.2 ACCESS THROUGH THE LOCK
The doors between the unclassified corridor area and the lock room 'slusen' are the only entrances to the classified areas. The pressure system in the lock rooms ensures that the doors to the corridor area and the classified areas cannot be opened at the same time. The safety mechanism can be deactivated in an emergency by pressing the red emergency button next to the entrance to the corridor area.

1.2.1 WORKWEAR FOR GMO 2
Special workwear is required for GMO 2 work over the uniforms required for regular laboratory work. All staff must change to workwear in the lock room. For work with GMO 2 protocols the special workwear consists of:

• Disposable lab coat
• Underarm clothing shields
• Disposable gloves - double gloves when working in LAF bench
• Shoe covers or designated work shoes for GMO 2 use only 

The workwear must be dressed in the order mentioned, before the doors to the GMO 2 area are opened and the work begins. Before exiting the GMO 2 area, the outer pair of gloves, clothing shields, and disposable lab coat is to be removed, while the inner pair of gloves and shoe covers/work shoes are removed in the lock room. All clothing is discarded in the marked container for clinical hazardous waste after use.

Persons wearing the special workwear for GMO 2 work must not leave the GMO 2 classified area.

As a rule, permanent staff should use work shoes and not shoe covers. Work shoes are stored in the lock room and must not be used outside the areas covered by this guideline. If shoe covers are used, they should be removed with gloved hands together with the other special workwear and disposed of as clinical hazardous waste. Ordinary white coats must not be used under a disposable lab coat.

As a minimum, staff who needs to enter the GMO 2 area for other tasks than GMO 2 procedures must wear a disposable lab coat, gloves and shoe covers/work shoes, e.g. for a task not involving practical laboratory work (instruction, demonstration, etc.). Bags, mobile phones, outerwear, jewelry, etc. are not allowed in the GMO 2 areas or lock room. 

 

2 PROHIBITIONS

2.1 FOOD AND BEVERAGES

Food and beverages are not allowed in areas covered by this guideline.

2.2 LIMITATIONS OF WASTE

All materials from the GMO 2 laboratories must be handles as class 2 waste, meaning as potentially genetically engineered. To limit the amount of waste, considerations must be taken to limit the entry of extraneous materials such as cardboard boxes, wrappings, etc.

2.3 DRAINS

Never pour active biological material into sinks, drains, or sewers. All biological waste must be autoclaved in a closed container before existing the laboratories as clinical hazardous waste. 

2.4 WASTEBASKETS

There are no wastebaskets in the GMO 2 area because all waste is classified as GMO 2 waste and thus clinical hazardous waste.

2.5 POSTERS

Necessary notices and posters on walls and the like must be laminated or otherwise covered with plastic.

2.6 WINDOWS

Windows must always be kept closed, as the efficiency of the ventilation system depends on this. The windows are closed with a breakable seal for emergencies only.    
 

3 GENERAL WORK PROCEDURES

3.1 DEFINITIONS

LAF bench (LAF stands for Laminar Air Flow) is a laboratory bench where a clean and/or sterile work zone is maintained though a unidirectional flow of air filtered through a HEPA filter. A LAF bench protects both the environment and the user.

HEPA filter (HEPA stands for High-Efficiency Particles Air) removes small particles from the air before the air exits the ventilation system in the GMO 2 areas.The fume cupboard/hoods is a laboratory bench with suction. A fume cupboard protects the user.

3.1.1 PRECAUTIONS FOR WORK IN A FUME CUPBOARD

The fume cupboards are under regular inspection and maintenance via the centralized technical department. The date for the latest maintenance can be seen on labels on the fume cupboard.

The individual user of the fume cupboard is responsible for checking suction status, before and during the work and for reporting all faults to the technical department. The fume cupboard must not be used if an error has been reported.

3.1.2 PROCEDURES IN FUME CUPBOARD

All laboratory work with a risk of releasing toxic fumes or chemicals must take place in a fume cupboard.

3.1.3 PRECAUTIONS FOR WORK IN A LAF-BENCH
The LAF benches in the GMO 2 area are HERAsafeKS12, a model that meets all safety requirements and guidelines described in the current standard EN 12469/2000.

The LAF benches are under regular inspection and maintenance via the centralized technical department. The date for the latest maintenance can be seen on labels on the LAF bench.

The individual user of the LAF bench cupboard is responsible for checking the functionality of the bench (see 3.1.8), before and during the work and for reporting all faults to the technical department. The LAF bench must not be used if an error has been reported.

3.1.4 PROCEDURES IN LAF-BENCHES

All open handling of biologically active material must take place in the LAF bench, in particular with regards:

• All open handling of active virus and virus-containing material until inactivation has been completed.
• All open handling of cell cultures, regardless of whether they contain viruses or not.

3.1.5 EXTRA WORKWEAR FOR SAFE WORK IN LAF BENCHES

The GMO 2 required workwear described in 1.2.1 must be supplemented before starting work in the LAF benches, with surgical gloves pulled up over the edge of the disposable lab coat. In connection with work that may cause aerosol formation, such as sonication and homogenization, additional safety clothing (e.g. cover hood, mask and hairnet) must be – see the specific requirements for the individual project. After completing the work on the LAF bench, e.g. to work elsewhere in the GMO 2 laboratory, the outer surgical gloves must be removed and discarded as waste.

3.1.6 CONTAMINATION OF AND/OR DAMAGE TO WORKWEAR

If contamination with viral material occurs, e.g. in the event of splashes or sprays on gloves or lab coat, the following procedure must be executed immediately in the stated order:

• Disinfect outer disposable gloves and disposable lab in the LAF bench and discard these as waste
• Remove inner gloves and discard these as waste
• Perform hand washing thoroughly with disinfectant soap
• Put on new inner gloves, a new disposable lab coat, and new surgical gloves

In the event of damage to external surgical gloves, these must be replaced immediately. In the event of damage to the outer and inner gloves, stop work immediately and follow the procedure for incidence, including informing the safety manager.

For all open handling of active virus or virus-containing material, two sets of undamaged gloves are always required.

3.1.7 GENERAL WORK INSTRUCTIONS FOR LAF BENCHES

• Avoid crowding the workstation within the LAF bench
• When working with virus-containing material, all utensils must, to the extent possible, be disposable materials
• Only use plastic pipettes and, to the extent possible, bottles made of plastic
• The use of glassware, needles, and scalps must be limited and only used with utmost precaution and in agreement with the project manager)
• Avoid aerosol formation - spills and sprays within the LAF bench must be disinfected and cleaned immediately
• The LAF benches are cleared for utensils, cleaned and disinfected at the end of the work
• Remember to wash your hands after removing your gloves - every time - after working with viruses and other biological material

Cleaned work areas in the laboratory must not be touched with gloves that have been used for work in LAF benches.

3.1.8 USER CONTROL OF LAF BENCHES

The HERAsafeKS12 LAF bench is fitted with a control panel and an acoustic alarm. For a complete overview of the control panel's functions, see Operating Instructions (Safety Cabinet HERAsafe KS / KSP), which can be found in the lock room.

The LAF benches are working properly and may be used if the two green lamps on the benches' display light up. Green lamp no. 12 indicates whether the air exhaust is working, and green lamp no. 13 indicates that the front window is in the working position (lowered).

The LAF benches operate at maximum speed and air exhaust around the clock and are tested according to instructions from the Technical Department via periodic inspections, see VIP.

3.1.9   TROUBLESHOOTING 

If the green lights/indicators are switched off, or the alarm sounds at the same time as the red lights on the control panel are switched on, this means that:

• The front window is not in the working position, see red lamp no. 10
• Or a technical error has occurred

In the event of a type no. 1 fault, the work in the LAF bench can be resumed when the front window is returned to the working position. All types of error no. 2 mean that work must be stopped in the LAF bench in question, including emptying and cleaning the LAF bench. When the LAF bench is emptied, the bench must be switched off, and the manual damper closed, after which the laboratory manager is notified.

Do not work in LAF benches with faults.

In the event of a power failure, the display goes out after 10 seconds and the acoustic alarm signal sounds for 30 seconds as an indication that ventilation is stopped. Follow the procedure for type no. 2 errors.
In the event of a power failure to all LAF benches, all staff must leave the GMO 2 area after closing open containers of biological material.

Do not work in the laboratory when there are faults on all LAF benches.

4 CENTRIFUGATIONS

All centrifugation must take place in plastic centrifuge tubes that are securely sealed. The tubes must be closed and disinfected on the outer surface before exiting the LAF bench. Centrifugation of biologically active material must always take place with special safety centrifuge cups. Centrifuge cups are always emptied in the LAF bench and sprayed before new usages. Concentration/multiplication of a virus is only allowed by express agreement with the safety manager.

4.1 SUCTION

The suction of supernatants must only take place in the LAF bench. The suction flask must be made of plastic and contain one packet of Virkon S with a final concentration of 1%.

It is prohibited to discard active biological material into laboratory sinks.

4.1.1 VACUUM CONTAINERS

Use of Virkon S must be limited, including the use of vacuum containers. Disposure via the sink must unly be used for non-biological fluids. If Virkos S is used, the solution must incubate for a minimum of 24 hours, after which the waste can be disposed of in a drain. The solution will have a faint pink color when the Virkon is active. When the solution becomes colorless, the disinfection properties of Virkon S is no longer active but can be reactivated with a new letter Virkon S.

Do not mix ethanol in the solution as it neutralizes the effect of Virkon S.

4.2 CELL COUNTING

To the extent possible, use disposable counting chambers of plastic. Handling of biological material and counting chambers must follow this procedure:

• The clean counting chamber is prepared for use outside the LAF bench
• The chamber is placed in a plastic petri dish, which is then placed in the LAF bench
• The sample of biological material is applied without the chamber leaving the petri dish
• A lid is applied to the petri dish, which is then wiped with ethanol, and removed from the LAF bench for microscopic analysis
• Petri dishes with counting chambers are disposed of as solid waste

To the extent possible, recyclable counting chambers of glass must not be used, and if so, only in agreement with the safety manager. After use, the chambers are disinfected with 70% ethanol or similar and packed in autoclave bags for autoclaving.

4.3 PIPPETING

Instruments and utensils for pipetting are located within the GMO 2 areas. Hand pipettes used for infected material should be disinfected after use, either by 'dry' autoclaving or ethanol. When possible, use pipette tips with filters.

4.4 PAPER AND NOTE TAKING

Only take notes in the laboratory if it is strictly necessary and use a designated workstation for the purpose. Papers transported to the GMO 2 area must be in a plastic folder. Suspend any laboratory procedure and use – with gloves – the writing utensils present in the laboratory. Writing utensils and paper remain in the laboratory until discarded via autoclaving.

Under no circumstances should note-taking take place on workstations where laboratory procedures are carried out.

4.5 HAND HYGIENE

When leaving the GMO 2 areas, take off inner gloves in the lock room and perform hand wash before exiting. Hands are dried in disposable wipes to be discarded as clinical hazardous waste. The tap works with by sensor and must not be touched with or without gloves.

It is strictly forbidden to touch unprotected skin areas with gloves on.

Hand cream is not available after hand washing, as hand cream must sit for 15-20 minutes to work. 

 

5 CLEANING

5.1 RESPONSIBILITY FOR CLEANING TASKS

• The cleaning staff is solely responsible for floor washing and removal of waste located outside the GMO 2 classified areas
• All other cleaning tasks, as well as all autoclaving, are handled by the laboratory staff
• When possible, the use of disposable utensils and equipment is mandatory

The cleaning staff is not allowed to touch or handle technical equipment and waste bins in the GMO 2 areas.

5.1.1 FLOOR WASH

The floors are washed weekly with disposable wet wipes. An appropriate time for floor washing has been arranged with the laboratory manager, and under no circumstances must other activities take place in the laboratory outside the LAF benches during floor washing. The cleaning staff must be able to work unhindered, which in practice means no-entry for other people, including the regular laboratory staff during floor washing.

The following precautions apply:

• Floor wipes/or other materials used are discarded after use as clinical hazardous waste
• Do not use the same wipe/cloth on both sides of the marking on the floor in the lock rooms
• Cleaning trolleys are not allowed to enter the GMO 2 areas

5.1.2 MAJOR CLEANING

A major cleaning is carried out by the laboratory staff once a year.

5.1.3 WINDOW POLISHING

The windows must always be kept closed to secure the efficiency of the ventilation system. Window cleaning (on the inside) can be arranged with the laboratory manager.

6 WASTE MANAGEMENT

6.1 WASTE

All forms of waste from the GMO 2 areas that can be autoclaved must be so followed by disposal as clinical hazardous waste.

Disposable pipettes and tips that have been used inside the LAF bench are discarded in tall yellow 'needle' boxes, which are subsequently closed and autoclaved in suited waste containers. After autoclaving, the container and its contents are discarded via the standard cardboard boxes for clinical hazardous waste. These boxes are closed and placed in the hallway outside the lock room, and subsequently removed by the cleaning staff.

Make sure that a Serological pipette and other long objects that can perforate plastic sealings do not protrude the 'needle' box.

Other kinds of solid waste that has been inside the LAF bench must be sprayed off when taken out of the bench and discarded in containers for clinical hazardous waste placed in the GMO 2 areas, to be followed by autoclaving.

Media and other liquid biological waste must not be mixed with solid waste but collected separately. 

All types of papers, including towel paper, are considered clinical hazardous waste and must also be autoclaved.

Glass containers and other glass items must be autoclaved after use. After autoclaving, the items can be placed for regular washing in laboratory no. 201 on a designated roller table.

The waste containers are marked with a hazard symbol for the biologically active material.

Lager objects such as boxes for pipette tips and containers for cellculture, where the content is inactivated, can be waste managed as described in 7.1. before being transported outside the laboratory.

6.1.1 AUTOCLAVED WASTE

Only autoclaved waste in tightly and properly closed yellow sacks and/or yellow containers must be removed from the GMO 2 areas and lock rooms to be disposed of via the hospital's standard waste management. All other types of waste and materials must remain in the classified areas.
All waste is marked with autoclave tape. Before autoclaving, the tape has a faint light oblique striping, which changes to clear brown after adequate autoclaving. This makes it easy to distinguish between autoclaved and non-autoclaved waste.

• Authorized cleaning staff – working with the removal of waste from the hallway – must be instructed in the difference between marked autoclaved and non-autoclaved tape.
• Only waste with tape that indicates that the waste is autoclaved can be discarded

6.1.2 DRAINS

Never pour active biological material into drains, sinks, or sewers. Biological active material must be inactivated by autoclavation.
 
7 TRANSPORT AND STORAGE

7.1 TRANSPORT

All transportation of viruses and virus-containing material must take place in tightly closed containers, which are marked with its contents. The containers can be plastic trays, bottles, centrifuge tubes, cryotubes, etc. During transport, the containers must be handled in a stable rack or similar for safe handling. If the containers are to exit the GMO 2 areas, they must be disinfected with a spray containing 70% ethanol or Virkon S solution before exiting.

7.2 STORAGE

Samples containing biologically active material must be stored in closed plastic bottles or freezer tubes suited for safe freezing. The material must – when possible – be stored on freezers or refrigerators in the GMO 2 areas.

If this is not viable, the surface of the containers must be disinfected with 70% ethanol, Virkon S solution, or similar before taken out of the GMO 2 areas for storage under approved conditions.

8 ACCIDENTS, SPILLS, POWER FAILURE, AND FIRE

In case of first aid:

• Stop accidents
• Call for help
• Always handle personal injuries first
• Inactivate biologically active material
• Always read and comply with the instructions for handling of toxic chemicals or solvents
• Wear appropriate personal protective equipment

8.1 ACCIDENTS

If somebody puncture stings or cuts themselves (penetrating lesion) on materials that either contain or may be contaminated with biologically active material (e.g. infectious viruses), seek immediate help from the laboratory manager and the safety manager and/or a medic.

The first response is always to remove gloves and immediately rinse the lesion with warm water to promote bleeding as much as possible. The lesion is then washed at least three times with disinfectant soap. Further measures are decided by the summoned laboratory manager and safety manager and/or summoned medic after assessing the exposure to the relevant.

It is mandatory, to report the incident as an occupational injury.

8.2 SPILLS

All spills should be covered with absorbent material and deactivated by Virkon S solution.

• Minor spills - defined as less than 100 ml: Cover the spill with absorbent paper soaked in 1% Virkon S and leave it to incubate for a minimum of 10 minutes. After further wiping and cleaning with 1% Virkon S, spray with 70% ethanol.
• Larger spills - defined as more than 100 ml: Cover the spill with granules and subsequently with absorbent paper soaked in 1% Virkon S corresponding to the volume of the spill (1: 1). The spill site is disinfected for 30 minutes before further wiping with 1% Virkon S and subsequently sprayed with 70% ethanol.

All types of papers used for absorption and wiping are disposed of as clinical hazardous waste to be autoclaved.

When cleaning after major spills, use a mask and heavy surgical long-handled gloves over the regular gloves as well as double disposable shoe covers. If GMO 2-designated shoes are worn without covers during the cleanup, the shoes must be discarded as clinical hazardous waste.
Only personnel directly involved in the cleaning procedure may occupy the laboratory during the cleaning -all other staff must exit.

Larger spills must be entered in the waste log stored in the lock room followed with a notification to the laboratory manager who decides if working in the lab can continue.

If there is a risk of heavy aerosol formation and spraying (e.g. in the case of fall from high altitude), the disinfection is supplemented by wiping the floor, tables, walls, and equipment at an appropriate distance from the spill site with 1% Virkon S followed by spraying with 70% ethanol.

8.3 POWER FAILURE, FIRE ETC.

If the situation is deemed critical, e.g. with a risk of personal injury, the laboratory must be evacuated immediately by all staff. This is especially relevant in case of power failures affecting larger sections of the GMO 2 area, with interruptions to the flow in the LAF benches and dangers of direct contact with biologically active materials.

When possible, close any open containers with biologically active material before exiting the laboratory. If the situation allows it, follow the standard procedure for removing safety workwear.

9 CONTACTS

A list with contact details can be found at the entry door to the lock rooms.

After an accident, necessary work such as shutdown of ongoing activities can only be resumed after agreement with the responsible safety manager.

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Bekendtgørelse om genteknologi og arbejdsmiljø, nr. 910 af 11/09/2008.

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Bilag Klasse II.pdf

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