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2. Flow
Cytometry - Flow
generated aerosols and/or droplets
(Author: P. Herman) (Last revised:
July 10, 2006
)
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 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 exist different sized aerosols (Table 1) and as a consequence different routes of possible infections (fig. 2, 
).
Inhalation, ingestion and dermal contact are route of human exposure for
airborne microorganisms. Inhalation is the main route giving averse 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.
Table 1 : classification of aerosols
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 |
*fpm is foot per minute
The larger droplets, greater than 100 micrometers in diameter settled quickly and will contaminate the surfaces on wich they come to rest. The smaller droplets did not settle but evaporated very rapidly. For example : droplets with a diameter of 100 micrometers evaporated in 1.7 s and those with a diameter of 50 micrometer in 0.4 s. The bacteria (or any other biological agent) 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 generatd by ventilation and the movement of people. The smaller they are the greater their potential for travelling long distances.
The Table2 describes some sources of infection with pathogens (viruses and bacteriae) affecting human as Hepatitis B, C or D virus as well as Mycobacterium tuberculosis and others. The main aerosol route of infection for these infectious agents are : blood, body fluids and/or various tissues. Some laboratory-acquired infections were documented for these pathogens.
Table 2 : Some biological agents with known source and aerosol route of infection and recommended containment level to adopt
| Biological agent | Laboratory facilities, equipment and work practices |
|---|---|
Epstein-Barr Virus |
L2 |
Neisseria meningitidis |
L2 L3 if large quantities and high concentrations |
| Hepatitis B, C and D virus, HIV-1 and HIV-2 | L2 L3 if large quantities and high concentrations |
Mycobacterium tuberculosis |
L3 |
Brucella spp. |
L2 L3 for tissue culture of infected cells |
A description of L2 and L3 facilities, equipment and work practices may be found respectively in the following pages : containment level L2, containment level L3. All
containment measures should be adequate for work with viral or bacterial
agents with emphasis on potential spread by aerosols, (micro)droplets and/or
contaminated surfaces and objects.
Laboratory techniques, other than Flow cytometry that produce aerosols
Many common laboratory techniques produce aerosols consisting of various sizes of particles.
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
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
- Wherever possible aerol-producing operation should be performed in a biosafety cabinet
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 provide good qualitative data
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