Preparing a Safe Handling Plan for Liquid Nitrogen and Cryogenic Materials

Recently the Safety Guys were asked to help evaluate a stem cell laboratory for possible safety concerns. Though calling it a lab may be a stretch. It was a converted storage room, now housing about a dozen ultra-low temperature freezers and a half dozen large dewars that contained liquid nitrogen used to fill the freezers. As you might imagine, cryopreservation is fraught with dangers and serious potential hazards.

Cryopreservation has become one of the fastest-growing areas in research, especially in the medical and health care arenas. The purpose is to store stocks of living cells and cell lines in order to access the genetic material when needed. In fact, technological advances have enabled the development of techniques for the preservation of a wide variety of biological material including microorganisms, tissues, primary cells, established cell lines, small multicellular organisms, and complex cellular structures such as embryos, as well as nucleic acid and proteins. The biological material or cell lines can be cryopreserved in a suspended state for indefinite periods, essentially stopping time, provided a temperature of less than -135°C is maintained.¹

Related Article: Safe Cryogenics Practices and Procedures

The primary methods of cryopreservation in use today are electric freezers, liquid phase nitrogen freezers, and vapor phase nitrogen freezers. The method most used is liquid nitrogen vapor storage. Electric freezers are mechanically complex, provide the highest storage temperature, and still require a liquid nitrogen backup. Liquid phase nitrogen freezers provide the lowest and most stable temperatures along with simplicity and mechanical reliability. But the risks of cross-contamination and potential for exploding cryovials² (more on this in a minute) are serious drawbacks, which leave the vapor phase liquid nitrogen freezers as the method of choice. Although these are simple and reliable and eliminate the risk of cross-contamination, they produce vertical temperature gradients that necessitate careful and diligent monitoring of the liquid nitrogen levels and temperature variations, especially in the upper regions.

Nitrogen characteristics and hazards

Nitrogen seems harmless enough. After all, if we recall our high school science, 78 percent of the earth’s atmosphere is nitrogen. It is a colorless, odorless, tasteless, and nonflammable gas, slightly lighter than air (molecular weight of 28 compared to 28.96 for dry air, give or take). But it is precisely these properties that present the dangers, as we will see shortly.

Nitrogen boils at -320°F; thus, liquid nitrogen is extremely cold. Contact can cause severe frostbite and may freeze and shatter certain substances. When it vaporizes, it expands 700-fold. This may cause explosions of sealed containers as the pressure rises and exceeds the strength of the container material (remember cryovials). More importantly, the tremendous expansion may displace oxygen in enclosed, confined, or poorly ventilated spaces, generating a potentially life-threatening asphyxiation hazard. Combined with its colorless, odorless, tasteless properties, you might not realize you are walking into a death trap.

Safe handling of liquid nitrogen (LN₂)

Now we understand the primary hazards of liquid nitrogen are direct contact, explosion due to pressurization, and oxygen displacement. The best way to protect against these and reduce the chance of injury is with training and proper protective equipment (personal and facility). The Safety Guys strongly encourage the development of written hazard analyses and standard operating procedures wherever liquid nitrogen is used. Below, we will provide a few guidelines and suggestions to get you started down the right path.

When it comes to cryopreservation and the use of liquid nitrogen, the most serious hazard is potential asphyxiation. Liquid nitrogen is released and vaporized when transferring between containers (e.g., from dewars to freezers or secondary containers, etc.), from liquid tanks (dewars) venting through pressure relief valves, from leaking valves, and from opening containers (accessing freezers). Ensure that your facility or cryopreservation area has adequate mechanical ventilation. Have oxygen sensors installed near breathing zone height, and establish a strict maintenance schedule (oxygen sensors must be calibrated and usually wear out every two years). An alarm set point of 18 percent is recommended, with a return to safe conditions set to 20 percent or more. Hard-wiring the alarms is preferred to prevent possible tampering or placing into inoperable status.

Related Article: Helium or Nitrogen, Tank or Generator?

Preventing direct contact is the next most important issue to address. Start by having only trained personnel working with liquid nitrogen. Ensure proper personal protective equipment is always available and used. At a minimum, this should include goggles and face shield, heavy leather or cryogenic gloves, lab coat, pants, and closed-toe shoes. When thawing cryotubes or vials, place them in a heavy walled container or behind a safety shield. Avoid breathing vapors. Use only approved containers for transferring and transporting liquid nitrogen. Carry containers away from your body and face. Do not leave containers open or unattended.

Finally, transporting and transferring liquid nitrogen requires special equipment. Here are a few more guidelines for these operations:

• Ensure fittings on regulators, adaptors, and containers match.
• Open valves slowly to minimize splashes, thermal effects, pressure buildup, and vapor release.
• Do not fill containers to more than 80 percent of capacity.
• Use specialized devices such as LN₂ pumps instead of pouring.
• LN₂ dewars vary in size and shape, but all are very heavy (a 160L dewar can weigh 200 pounds empty and 500 pounds full).
• Use special carts for moving cylinders and always store upright.
• Ensure cylinders are equipped with proper and functioning pressure relief devices, vent valves, pressure gauges, and contents gauges.
• Always store in a very well-ventilated area.
• Remove any ice or frost buildup on the pressure relief with a damp cloth.

The points discussed and listed above should help you prepare your written safe handling plan for liquid nitrogen and cryogenic materials. Always start with a good, comprehensive plan. Train your employees and ensure that proper PPE is used every time. Should you have any questions, bring in an expert to help in hazard evaluation and assessment.

References

1. Fundamental Techniques in Cell Culture Laboratory Handbook, Sigma-Aldrich. 2015. http://www.sigmaaldrich.com/technical-documents/protocols/biology/cryopreservation-and.html 

2. Hazards of Liquid Nitrogen Cryovials, Occupational Health & Safety Unit, University College London Medical School, London, UK. 2006. http://www.ucl.ac.uk/medicalschool/msa/safety/docs/cryovials.pdf