(For a context to these annexes, see the main page)
1. Sample handling
Knowledge of the origin of the sample and awareness of the potential presence of pathogens (genetically modified or not) are keys for lab scientists' protection.
- All unfixed materials (blood samples, various body fluids, cultured cells and environmental samples) should be a priori considered as biohazardous (the use of a fixative is not always sufficient to eliminate all type of biohazards);
- The use of Class II biological safety cabinet for sample preparation is highly recommended;
- Flow cytometer operators should be experienced and well trained;
- In relation with the Biosafety status of the sample: appropriate containment level, protective equipment, work practices and waste management are adopted after a thorough risk assessment (according to the European Directive 2009/41/EC repealing Directive 90/219/EEC, European Directive 2000/54/EC).
2. Flow-generated aerosols and/or droplets
a. Aerosols
Cell-sorters are equipped with a nozzle to form a jet of microdroplets : this experimental step is likely to generate aerosols (fig. 1).
Biohazardous materials should not be sorted unless suitable containment measures are applied. A droplet containment module should be installed to reduce the risk of lab personnel exposure to generated droplets and aerosols.
Instrument failures such as clogged sort nozzle or air in the fluidic system can drastically increase aerosol formation.
Fig. 1: aerosols
There are different sized aerosols (Table 1) and as a consequence different routes of possible infections (fig. 2, PDF). Inhalation, ingestion and dermal contact are route of human exposure for airborne microorganisms. Inhalation is the main route giving adverse health effects. The average human inhales approximately 10 m3 of air per day. Large airborne particles are lodged in the upper respiratory tract. Generally, particles <6 micrometers in diameter are transported to the lung, but the greatest retention in the alveoli is of 1 to 2 micrometer particles. After penetration, infection also depends on the nature and concentration of the infectious agents present in aerosols.
Once aerosols are generated, settling velocity can vary greatly depending on the particle type. The quicker the settling velocity is, the shorter the FCM operator risky exposure is.
Table 1 shows the size clasification of aerosols (* fpm = foot per minute):
| Particle type | size range (micrometer) | settling velocity (fpm*) |
|---|---|---|
| Droplet | 100 - 400 | 59 - 498 |
| Dust | 10- 100 | 0.59 - 59 |
| Droplet nuclei |
1 -10 0.0 - 0.1 |
0.007 - 0.59 0.00016 - 0.007 |
The larger droplets, greater than 100 micrometers in diameter settle quickly and will contaminate the surfaces on wich they come to rest. The smaller droplets do not settle but evaporate very rapidly. For example : droplets with a diameter of 100 micrometers evaporate in 1.7 s and those with a diameter of 50 micrometer in 0.4 s. A bacteria (or any other micro-organism) in droplets remain in a dried state as "droplet nuclei", also referred to as infected airborne particles. Such particles are moved around rooms and buildings by air currents generated by ventilation and the movement of people. The smaller they are the greater their potential for travelling long distances.
The larger particles of an aerosol drop to the floor (or to the bench), within seconds, where they form an agregate of dust that is unlikely dispersed into the air. Droplets larger than 140 micrometers tend to fall to the ground before they evaporate and those smaller than 140 micrometers would be more likely to evaporate before contacting the ground or any solid surface.
Table 2 summarizes the evaporation times and falling distance of droplets based on size (adapted from Fleming et al., Laboratory Safety, principle and practices, Second edition. 1995):
| Diameter of droplets (micrometer) | Evaporation time (s) | Distance fallen before evaporation (meter) |
|---|---|---|
| 200 | 5.2 | 7.2 |
| 100 | 1.3 | 0.45 |
| 50 | 0.31 | 0.03 |
| 25 | 0.08 | 0.002 |
The particles coming from evaporated droplets are able to remain in the air for very long period. These tiny particles are called "droplet nuclei". If droplet nuclei contain an pathogenic organism (e.g M. tuberculosis), airborne transmission to human or animal is possible. If the micro-organisms (pathogen and/or genetically modified) are contained not in aquous but proteinaceaous fluids (sputum, mucus, serum) evaporation will be much slower as these material tend to retain water. The droplets will settle more rapidly; fewer will remain suspended in air and fewer infected airborne particles, available for wider dispersion, will be produced.
Other potential sources of infectious airborne particles are:
- Lyophilized cultures
- Dried bacterial colonies
- Dried materials on stoppers and caps of culture tubes and bottles
- Dried exudates Fungal and actinomycetes spores
- Dust from animal cages
It is worth mentioning that even the larger particles and droplets, which do not evaporate rapidly, may be a source of infection by contaminating surfaces (direct or indirect contacts). By this way, non glove-protected fingers may be contaminated by pathogenic micro-organisms (genetically modified or not) and then transferred to both mouth and eyes.
b. Pathogens infecting via aersols
Table 3 describes some sources of infection with pathogens (viruses and bacteriae) affecting humans, such as Hepatitis B, C or D virus, or M. tuberculosis and others. The main aerosol route of infection for these micro-organisms are: blood, body fluids and/or various tissues. Some laboratory-acquired infections are documented for these pathogens.
Table 3:
| Biological agent | Laboratory facilities, equipment and work practices |
|---|---|
| Epstein-Barr Virus | L2 |
| N meningitidis | |
| Hepatitis B, C and D virus, HIV-1 and HIV-2 | L2, L3 if large quantities and high concentrations |
| M tuberculosis | L3 |
| Brucella spp. | L2, L3 for tissue culture of infected cells |
c. Containment facilities, equipment and work practices
All containment measures should be adequate for work with pathogens (genetically modified or not) with emphasis on potential spread by aerosols, (micro)droplets and/or contaminated surfaces and objects (see pages on containment level L2, L3).
It is recommended to asses the efficiency of cell-sorters equiped with aerosol containment module. There are two methods to assess the containment of aerosols generated by cell-sorting:
- Plating of T4-susceptible Escherichia coli that are placed IN and OUT the sort area allows the detection of aerosols containing sorted T4-bacteriophage (Schmid et al, Cytometry 1997). This method is sensitive, time-consuming and could contaminate subsequently sorted viable eucaryotic cells.
- Observation of the flow scattering using calibrated fluorescent microspheres called "Glo Germs" (Oberyszyn & Robertson, Cytometry, 2001). This method is rapid, inexpensive and provides good qualitative data
Main rules to contain the formation of aerosols:
- Use of centrifuges equiped with biosafety cups ;
- Do not use a syringe for mixing infectious fluids and check that only the tip of the needle is immersed below the level of fluid in the container avoiding the necessity of excessive force ;
- Prepare bacterial or infected cell plates in a class II biosafety cabinet ;
- Whenever possible aerol-producing operation should be performed in a biosafety cabinet.
d. Other laboratory techniques that produce aerosols
Many common laboratory techniques produce aerosols consisting of various particle sizes.
Laboratory activities releasing particles larger than 5 micrometer:
- Opening containers
- Pipettes (no visible spill)
- Test tube mixers
- Opening lyophilized cultures
- Centrifugation
Laboratory activities releasing particles smaller than 5 micrometer:
- Careful pouring Fixed-volume automatic pipettorsPipette mixing of fluid cultureHarvesteing/dropping of infected eggsHigh-speed blendersShaking machineDropping tubes or flasks of cultures
- Pipette spills
3. Waste management and equipment maintenance
The procedures of waste management and equipment maintenance should be adapted to the biosafety status of the handled biological material.
Waste management: Usually, the effluents are collected from the instrument in a flask containing fresh concentrated bleach solution to achieve a 10% final concentration. The Inactivation method of the effluent must be appropriate and validated. The contents of the flask is disposed according to institutional and local regulations for waste disposal.
Maintenance of fluid lines and instrument parts: Fluid lines desinfection requires regular instrument runs with a 1:10 fresh dilution of a 5.25% sodium hypochlorite solution for at least 10 minutes, followed by distilled water.
Flow cytometer's parts : follow the manufacturer's recommendations for appropiate desinfection procedure.