HANDLING  SPECIMENS


 
   
 

GUIDANCE TO INFLUENZA LABORATORIES
DIAGNOSING SWINE INFLUENZA A/H1N1 AND AVIAN INFLUENZA
 

Diagnosing Swine Influenza A/H1N1 Infections of current concern
 

WHO laboratory biosafety guidelines for handling specimens suspected of containing avian influenza A virus

12 January 2005

General recommendations

The possibility that an influenza infection in humans caused by avian influenza viruses A viruses could occur following a laboratory accident is a risk to which it is crucial to be constantly alert. Efforts to minimize transmission of infection in humans will be compromised by breaches in laboratory biosafety.

Responsibility for developing a comprehensive safety policy, including a safety manual, and supporting programmes for its implementation normally rests with the director or head of an institute or laboratory. However, laboratory safety is also the responsibility of all supervisors and laboratory employees, and individual workers are responsible for their own safety and that of their colleagues.

Good microbiological technique is fundamental to laboratory safety. The use of safety equipment, combined with good procedures and practices, will help to reduce the risks involved in dealing with biosafety hazards. The most important concepts are outlined below.

  • Standard precautions should always be followed; barrier protection (gowns, gloves) should be used whenever samples are obtained from patients. In addition to these standard precautions, eyes should be protected

     

  • Basic containment – Biosafety Level 2 (BSL2) – practices and procedures should be the minimum requirement for handling specimens (see WHO laboratory biosafety manual, 3rd edition).

     

  • Examples of routine laboratory procedures that require BSL2 include:
    - routine diagnostic testing of serum and blood samples (including haematology and clinical chemistry);
    - manipulations involving neutralized or inactivated (lysed, fixed, or otherwise treated) virus particles and/or incomplete, non-infectious portions of the viral genome;
    - final packaging of specimens for transport to diagnostic laboratories for additional testing; specimens should already be in a sealed, decontaminated primary container.

     

  • Good laboratory practices should be followed. Eating, drinking, smoking, applying cosmetics, and handling contact lenses are prohibited in the laboratory working areas.

     

  • Personal protective equipment (gown, gloves, eye protection) should be worn in the laboratory when handling and processing specimens and performing diagnostic testing.

     

  • All technical procedures should be performed in a way that minimizes the formation of aerosols and droplets.

     

  • Biological safety cabinets or other physical containment devices should be used for all manipulations that may cause splashes, droplets, or aerosols of infectious materials (e.g. centrifugation, grinding, blending, vigorous shaking or mixing, sonic disruption, opening of containers of infectious materials whose internal pressure may be different from the ambient pressure).

     

  • The use of hypodermic needles and syringes should be limited. They must not be used as substitutes for pipetting devices or for any purpose other than parenteral injection or aspiration of fluids from laboratory animals. Mouth pipetting must be strictly forbidden.

     

  • Adequate and conveniently located biohazard containers should be available for disposal of contaminated materials.

     

  • Work surfaces must be decontaminated after any spill of potentially dangerous material and at the end of the working day. Generally, freshly prepared bleach solutions1 are appropriate for dealing with bio-hazardous spillage. More information on disinfection and sterilization is provided in the WHO laboratory biosafety manual.

     

  • Personnel must wash their hands often – especially after handling infectious materials and animals, before leaving the laboratory working areas, and before eating.

     

  • Personal protective equipment must be removed before leaving the laboratory.
     

WHO biosafety guidelines for handling specimens that may contain avian influenza A virus

Laboratories must meet basic BSL2 standards and use BSL3 work practices to be able to safely:

- aliquot and/or dilute specimens
- perform diagnostic testing that does not involve propagation of viral agents in vitro or in vivo
- perform nucleic acid extractions that involve untreated specimens
- prepare smears using heat or chemical fixation.

BSL3 practices cover the following areas:

  • Any procedure that may generate aerosols or droplets should be performed in a biological safety cabinet (e.g. sonication, vortexing).

     

  • Laboratory workers should wear protective equipment, including disposable gloves, solid-front or wrap-around gowns, scrub suits, or coveralls with sleeves that fully cover the forearms, head coverings and, where appropriate, shoe covers or dedicated shoes, eye protection and a surgical mask, or full-face shield, because of the risk of aerosol or droplet exposure when performing specific manipulations.

     

  • Centrifugation of specimens should be performed using sealed centrifuge rotors or sample cups. These rotors or cups should be unloaded in a biological safety cabinet.

     

  • Work surfaces and equipment should be decontaminated after specimens are processed. Standard decontamination agents that are effective against non-enveloped viruses should be adequate if used according to the manufacturer’s recommendations. Generally, freshly prepared bleach solutions1 are appropriate for dealing with biohazardous spillage. More information on disinfection and sterilization is provided in the WHO laboratory biosafety manual.

     

  • Biological waste contaminated with suspect or confirmed influenza A/H5 specimens, should be treated as outlined in the WHO laboratory biosafety manual.

When a procedure or process cannot be conducted within a biological safety cabinet, appropriate combinations of personal protective equipment (e.g. respirators, face shields) and physical containment devices (e.g. centrifuge safety cups or sealed rotors) must be used.

WHO strongly recommends that the BSL3 precautions described above are adopted and followed for work in BSL2 laboratories with influenza A/H5 virus specimens.

Where laboratory facilities do not meet at least basic BSL2 containment conditions, specimens should be referred to suitably equipped reference laboratories for primary diagnostic tests.

For laboratories that meet BSL3 containment standards and are operated by staff trained in the use of appropriate BSL3 work practices, the following procedures can be undertaken:

- diagnostic tests that involve propagation of viral agents in vitro or in vivo
- work involving the replication of influenza A/H5 virus in cell culture and/or storage of cell culture isolates
- recovery of viral agents from cultures of influenza A/H5 specimens
- manipulations involving growth or concentration of influenza A/H5 virus.

1Work surfaces must be decontaminated after any spill of potentially dangerous material and at the end of the working day. A general all-purpose laboratory disinfectant should have a concentration of 1 g/l available chlorine (0.1%). A stronger solution, containing 5 g/l available chlorine (0.5%), is recommended for dealing with biohazardous spillage and in the presence of large amounts of organic matter. Sodium hypochlorite solutions, as domestic bleach, contain 50g/l available chlorine and should therefore be diluted 1:50 or 1:10 to obtain final concentrations of 1 g/l or 5 g/l, respectively. Bleach dilutions should be freshly prepared and allowed a contact time of at least 10 min. Chlorine is corrosive and cannot be used on all surfaces. Alternative compounds for disinfection and sterilization are provided in the WHO laboratory biosafety manual

WHO guidelines for the storage and transport of human and animal specimens for laboratory diagnosis of suspected avian influenza A infection

12 January 2005

Specimen storage

Specimens in viral transport medium for viral isolation should be kept at 4 °C and transported to the laboratory promptly. If specimens are transported to the laboratory within 2 days, they may be kept at 4 °C; otherwise they should be frozen at or below –70 °C until they can be transported to the laboratory. Repeated freezing and thawing must be avoided to prevent loss of infectivity. Sera may be stored at 4 °C for approximately one week, but thereafter should be frozen at –20 °C.

Specimens should be collected and transported in a suitable transport medium on ice or in liquid nitrogen. Standard precautions should always be followed, and barrier protections applied whenever samples are obtained from patients. Specimens for influenza should not be stored or shipped in dry ice (solid carbon dioxide) unless they are sealed in glass or sealed, taped and double plastic-bagged. Carbon dioxide can rapidly inactivate influenza viruses if it gains access to the specimens through shrinkage of tubes during freezing.

Specimen transport

Transport of specimens should comply with theWHO guidelines for the safe transport of infectious substances and diagnostic specimens (WHO, 1997).

The receiving laboratory should be notified before shipment of specimens in order to arrange for an import license for the specimens.

Transport of specimens within national borders should comply with the procedures detailed within each country’s regulations.

International air transport of human specimens known or suspected to contain the avian influenza agent, or of specimens from avian influenza infected animals must follow the current edition of the International Air Transport Association (IATA) Dangerous Goods Regulations.

The IATA Regulations, Consignment of Diagnostic Specimens, 2003 allow specimens known or suspected to contain the avian influenza agent to be transported as UN 3373 “diagnostic specimens” when they are transported for diagnostic or investigational purposes.

Specimens transported for any other purposes, and cultures (as defined in the IATA Regulations) prepared for the deliberate generation of pathogens, must be transported as UN 2814 or UN 2900, as appropriate.

All specimens to be transported (UN 3373, UN 2900, or UN 2814) must be packaged in triple packaging consisting of three packaging layers as indicated in the Dangerous Goods Index.

UN 3373, Diagnostic Specimens, shall be packed in good quality packaging, which shall be strong enough to withstand the shocks and loads normally encountered during transport. Packaging shall be constructed and closed so as to prevent any loss of contents that might be caused under normal conditions of transport, by vibration or by changes in temperature, humidity or pressure.

Primary receptacles shall be packed in secondary packaging in such a way that, under normal conditions of transport, they cannot break, be punctured or leak their contents into the secondary packaging. Secondary packaging shall be placed in a final outer package with suitable cushioning material. Any leakage of the contents shall not substantially impair the protective properties of the cushioning material or of the outer packaging.

For liquids

The primary receptacle(s) shall be leakproof and shall not contain more than 500 ml. There shall be absorbent material placed between the primary receptacle and the secondary packaging; if several fragile primary receptacles are placed in a single secondary packaging, they shall be either individually wrapped or separated so as to prevent contact between them. The absorbent material shall be in sufficient quantity to absorb the entire contents of the primary receptacles and there shall be a secondary packaging that shall be leakproof. The primary receptacle or the secondary packaging shall be capable of withstanding without leakage an internal pressure producing a pressure differential of not less than 95 kPa (0.95 bar). The outer packaging shall not contain more than 4 litres.

For solids

The primary receptacle(s) shall be sift-proof and shall not contain more than 500 g. If several fragile primary receptacles are placed in a single secondary packaging, they shall be either individually wrapped or separated so as to prevent contact between them and there shall be a secondary packaging which shall be leak proof. The outer packaging shall not contain more than 4 kg.

For air transport, the smallest overall external dimension of a completed package must be at least 10 cm.

Packaging must conform to certain performance standards.

For further information about definitions, packaging requirements, markings and labels, accompanying documentation, and refrigerants, please refer to the competent authority, current IATA shipping guidelines, commercial packaging suppliers, or available courier companies

 

WHO laboratory biosafety guidelines for handling specimens suspected of containing avian influenza A virus

12 January 2005

General recommendations

The possibility that an influenza infection in humans caused by avian influenza viruses A viruses could occur following a laboratory accident is a risk to which it is crucial to be constantly alert. Efforts to minimize transmission of infection in humans will be compromised by breaches in laboratory biosafety.

Responsibility for developing a comprehensive safety policy, including a safety manual, and supporting programmes for its implementation normally rests with the director or head of an institute or laboratory. However, laboratory safety is also the responsibility of all supervisors and laboratory employees, and individual workers are responsible for their own safety and that of their colleagues.

Good microbiological technique is fundamental to laboratory safety. The use of safety equipment, combined with good procedures and practices, will help to reduce the risks involved in dealing with biosafety hazards. The most important concepts are outlined below.

  • Standard precautions should always be followed; barrier protection (gowns, gloves) should be used whenever samples are obtained from patients. In addition to these standard precautions, eyes should be protected

     

  • Basic containment – Biosafety Level 2 (BSL2) – practices and procedures should be the minimum requirement for handling specimens (see WHO laboratory biosafety manual, 3rd edition).

     

  • Examples of routine laboratory procedures that require BSL2 include:
    - routine diagnostic testing of serum and blood samples (including haematology and clinical chemistry);
    - manipulations involving neutralized or inactivated (lysed, fixed, or otherwise treated) virus particles and/or incomplete, non-infectious portions of the viral genome;
    - final packaging of specimens for transport to diagnostic laboratories for additional testing; specimens should already be in a sealed, decontaminated primary container.

     

  • Good laboratory practices should be followed. Eating, drinking, smoking, applying cosmetics, and handling contact lenses are prohibited in the laboratory working areas.

     

  • Personal protective equipment (gown, gloves, eye protection) should be worn in the laboratory when handling and processing specimens and performing diagnostic testing.

     

  • All technical procedures should be performed in a way that minimizes the formation of aerosols and droplets.

     

  • Biological safety cabinets or other physical containment devices should be used for all manipulations that may cause splashes, droplets, or aerosols of infectious materials (e.g. centrifugation, grinding, blending, vigorous shaking or mixing, sonic disruption, opening of containers of infectious materials whose internal pressure may be different from the ambient pressure).

     

  • The use of hypodermic needles and syringes should be limited. They must not be used as substitutes for pipetting devices or for any purpose other than parenteral injection or aspiration of fluids from laboratory animals. Mouth pipetting must be strictly forbidden.

     

  • Adequate and conveniently located biohazard containers should be available for disposal of contaminated materials.

     

  • Work surfaces must be decontaminated after any spill of potentially dangerous material and at the end of the working day. Generally, freshly prepared bleach solutions1 are appropriate for dealing with bio-hazardous spillage. More information on disinfection and sterilization is provided in the WHO laboratory biosafety manual.

     

  • Personnel must wash their hands often – especially after handling infectious materials and animals, before leaving the laboratory working areas, and before eating.

     

  • Personal protective equipment must be removed before leaving the laboratory.
     

WHO biosafety guidelines for handling specimens that may contain avian influenza A virus

Laboratories must meet basic BSL2 standards and use BSL3 work practices to be able to safely:

- aliquot and/or dilute specimens
- perform diagnostic testing that does not involve propagation of viral agents in vitro or in vivo
- perform nucleic acid extractions that involve untreated specimens
- prepare smears using heat or chemical fixation.

BSL3 practices cover the following areas:

  • Any procedure that may generate aerosols or droplets should be performed in a biological safety cabinet (e.g. sonication, vortexing).

     

  • Laboratory workers should wear protective equipment, including disposable gloves, solid-front or wrap-around gowns, scrub suits, or coveralls with sleeves that fully cover the forearms, head coverings and, where appropriate, shoe covers or dedicated shoes, eye protection and a surgical mask, or full-face shield, because of the risk of aerosol or droplet exposure when performing specific manipulations.

     

  • Centrifugation of specimens should be performed using sealed centrifuge rotors or sample cups. These rotors or cups should be unloaded in a biological safety cabinet.

     

  • Work surfaces and equipment should be decontaminated after specimens are processed. Standard decontamination agents that are effective against non-enveloped viruses should be adequate if used according to the manufacturer’s recommendations. Generally, freshly prepared bleach solutions1 are appropriate for dealing with biohazardous spillage. More information on disinfection and sterilization is provided in the WHO laboratory biosafety manual.

     

  • Biological waste contaminated with suspect or confirmed influenza A/H5 specimens, should be treated as outlined in the WHO laboratory biosafety manual.

When a procedure or process cannot be conducted within a biological safety cabinet, appropriate combinations of personal protective equipment (e.g. respirators, face shields) and physical containment devices (e.g. centrifuge safety cups or sealed rotors) must be used.

WHO strongly recommends that the BSL3 precautions described above are adopted and followed for work in BSL2 laboratories with influenza A/H5 virus specimens.

Where laboratory facilities do not meet at least basic BSL2 containment conditions, specimens should be referred to suitably equipped reference laboratories for primary diagnostic tests.

For laboratories that meet BSL3 containment standards and are operated by staff trained in the use of appropriate BSL3 work practices, the following procedures can be undertaken:

- diagnostic tests that involve propagation of viral agents in vitro or in vivo
- work involving the replication of influenza A/H5 virus in cell culture and/or storage of cell culture isolates
- recovery of viral agents from cultures of influenza A/H5 specimens
- manipulations involving growth or concentration of influenza A/H5 virus.

1Work surfaces must be decontaminated after any spill of potentially dangerous material and at the end of the working day. A general all-purpose laboratory disinfectant should have a concentration of 1 g/l available chlorine (0.1%). A stronger solution, containing 5 g/l available chlorine (0.5%), is recommended for dealing with biohazardous spillage and in the presence of large amounts of organic matter. Sodium hypochlorite solutions, as domestic bleach, contain 50g/l available chlorine and should therefore be diluted 1:50 or 1:10 to obtain final concentrations of 1 g/l or 5 g/l, respectively. Bleach dilutions should be freshly prepared and allowed a contact time of at least 10 min. Chlorine is corrosive and cannot be used on all surfaces. Alternative compounds for disinfection and sterilization are provided in the WHO laboratory biosafety manual.

 


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