Centre for Flow Cytometry and Microscopy
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Biosafety

Application

Risk Group

Sorters

Analyzers

Primary cells or cell lines from any source other than human or primate

RG1

No aerosol management system (AMS); any sorter

Live or fixed cells

Ex vivo human cells from prescreened group or human cell lines rated BSG 1 from ATCC

RG2

AMS, biosafety cabinet (BSC) recommended

Live or fixed cells

Tissue from animals xenografted with noninfectious human cells

RG2

AMS, BSC recommended

Live or fixed cells

Live yeast or bacteria

RG2

Limited access

Limited access

Primary human tissue retrieved from clinic tested for known pathogens HIV, Hep B, C, EBV, CMV

RG3

AMS, BSC mandatory

Live or fixed cells

Genetically engineered human or animal cell lines with other than third-generation or older lentiviral or adenoviral gene transfer

RG2/3

AMS, BSC mandatory

Fixed cells only

Primary human cells from unscreened group or unknown patient history

RG3/4

Not permitted

Fixed cells only

Primary human cells from infectious patients

RG4

Not permitted

Not permitted


Rationale behind the guidelines

Cell sorters work by encapsulating single cells in saline drops. The drops are created by pinging turbulence with a set frequency onto a nozzle. The stream velocity and the nozzle diameter work together to produce free droplets at a consistent spot. Cells are given an electric charge once they become encapsulated. As cells enter an electrostatic field, they gravitate to an appropriate pole, and are separated from the larger population. The generation of drops, the collection of residual waste and unsorted cells, and the production of side streams from the charged droplets all create aerosol spray, and aerosols can escape into the environment from within these sort chambers. If the aerosol contains a pathogen, then the operator and anyone in the vicinity of the sorter can be infected.

With the advent of genetic manipulations, the risk of converting cells into potentially hazardous samples increased, especially with the use of lentiviral or retroviral inductions. Ex vivo tissue or blood from human patients has always been considered a potential source of pathogen introduction into the environment as those cells undergo sorting. In 2003, an aerosol management system (AMS) was introduced to control forward direction of aerosols generated from sorting. This was viewed as successfully decreasing the amount of escaped aerosol into the environment. In 2007, biosafety cabinets from Baker were introduced to contain a BD FACSAria or equivalent-sized cell sorter, to produce a better containment system for complete elimination of aerosol into the lab space. The computer and operator remained outside.

The biggest drawback is the size of these biosafety cabinets, which are 10 feet wide by four feet deep, and anywhere from eight to 10 feet high. Add on the computer station for the operator, and space becomes an issue. The cabinets are not cheap, and companies such as BD Biosciences and Beckman-Coulter are designing their new-generation sorters with smaller footprints, as well as customized biocontainment shields that produce a sleeker and elegant stand-alone biosafety sorter.

Health Canada is now mandating that all core facilities that sort cells for researchers introduce better containment than AMS alone. The retrofit of our FACSAria II into a biosafety cabinet will occur; however we currently house the Influx cell sorter in a biosafety cabinet. With these units in place, the facility will be able to handle all nonhazardous human cells, along with some of the more commonplace genetically manipulated cell lines. It is imperative, however, to create a better understanding of pathogens for users, and to educate users and operators on how to handle pathogens.

Bioaerosols

A biological aerosol, or bioaerosol, is a biological particle in suspension that contains pathogens to produce an allergic response or infection, or a toxicological response. The smaller the particle, the greater the risk and the finer the aerosol, which increases the possibility of the aerosol escaping containment.

To sort cells from Risk Level 4, one must have a designated lab space and personnel in a contained room that is designated to the appropriate biosafety level:

Biosafety Level 1 (BSL1): well-characterized agents not consistently known to cause disease in healthy adult humans and of minimal potential hazard to laboratory personnel and the environment.

Biosafety Level 2 (BSL2): agents of moderate potential hazard to personnel and the environment.

Biosafety Level 3 (BSL3): indigenous or exotic agents that may cause serious or potentially lethal disease as a result of exposure by the inhalation route (applicable to clinical, diagnostic, teaching, research or production facilities).

Biosafety Level 4 (BSL4): dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections and life-threatening disease.

In 2009, the International Society for Advancement of Cytometry produced a guideline for cell-sorting core facilities to assist with understanding risks and biosafety terms. The guideline focused mainly on the most common types of cells and applications handled by cell-sorting facilities:

BSL1: mouse cells, ex vivo or cell lines, untreated; no toxoplasmosis.

BSL2: mouse cells, third-generation, lentiviral, adenoviral or retroviral gene transfections, and established human cell lines from ATCC.

BSL2/3(new grading): mouse cells, second-generation lentiviral, adenoviral and retroviral gene transfections; human tissue prescreened for HIV, CMV, EBV, and Hepatitis A, B, C, and E.

BSL3: normal human tissue biopsy, normal human blood and normal nonhuman primate blood .

BSL4: infected blood, known pathogens and unknown data.

Summary

Taking all points into considerations, and understanding the cell types that enter our facility, we see an opportunity to encapsulate two of three cell sorters with biosafety containment modules, thereby protecting both operator and user. As the core facility is still open-concept, we cannot classify ourselves as BSL3 or greater. We can accept up to BSL2/3, as this covers all potentially hazardous materials that have been prescreened through preoperative or clinical trial groups.

Contact

Paul Oleynik
paul.oleynik@sri.utoronto.ca
416-480-6100, ext. 7282

Sunnybrook Health Sciences Centre
2075 Bayview Avenue, M7 301
Toronto, ON Canada M4N 3M5