SMILE
Johns Hopkins University
Baltimore, MD USA
Anne Sholander
Document Number:
Effective (or Post) Date:
Fac12-03
5-Feb-08
Review History Date of last review: 26-Jan-12
Reviewed by: Heidi Hanes
SMILE Comments: This document is provided as an example only. It must be revised to accurately reflect your lab’s specific processes and/or specific protocol requirements. Users are directed to countercheck facts when considering their use in other applications. If you have any questions contact SMILE.
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SMILE
Johns Hopkins University
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Liquid Nitrogen Monitoring and Safety Manual
Subject
Liquid Nitrogen Monitoring and Safety Guidelines
Guideline Number
Effective Date
Page
Supersedes
Fac12-03-G
5-Feb-08
1 of 1
New
SMILE Comments: This document is provided as an example only. It must be revised to accurately reflect your lab’s specific processes and/or specific protocol requirements. Users are directed to counter check facts when considering their use in other applications. If you have any questions contact SMILE.
Questions pertaining to Liquid Nitrogen can be found in the audit section for Equipment..
The CAP Accreditation Checklist addresses Liquid Nitrogen safety in the Laboratory Safety section in the General Laboratory Checklist.
Liquid nitrogen is a colorless, odorless, extremely cold liquid and gas under pressure. Contact with liquid or cold vapors can cause severe frostbite. Cold vapors in the air will appear as a white fog due to condensation of moisture. Nitrogen gas is colorless, odorless and nonflammable. The primary health hazard is asphyxiation due to displacement of oxygen. CAP and OSHA requirements state that the laboratory must have adequate policies and procedures for the safe handling of liquid nitrogen.
1. Material Safety Data Sheet, Liquid Nitrogen.
2. College of American Pathologists (CAP) 2006. Commission on Laboratory
Accreditation, Laboratory Accreditation Program; Laboratory General Checklist Revised
4/6/2006.
3. NIOSH/OSHA Health Guideline, Nitrogen gas. CAS Number 7727-37-9; US Department of Labor.
4. Clinical Laboratory Standards Institute (CLSI). Clinical Laboratory Technical Procedure
Manuals; Fourth Edition . CLSI Document GP17-A2 (ISBN 1-56238-458-9). Clinical and
Laboratory Standards Institute, Wayne, PA
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Johns Hopkins University
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Liquid Nitrogen Monitoring and Safety Policy
Approval Signature: Laboratory Director
Policy Number
Effective Date
Review Cycle
Supersedes
Fac12-03 -P
5-Feb-08
1 year
New
Policy
Purpose
The laboratory has processes and procedures to effectively ensure that liquid nitrogen is monitored and handled in a safe manner.
This policy provides direction for the processes and procedures to effectively ensure that liquid nitrogen is monitored and handled in a safe manner.
Responsibility The Laboratory Medical Director is responsible for
The Laboratory Manager is responsible for
The Lead Technologists are responsible for
Additional Information as required
Include as many other policy topics related to the subject. Refer to CLSI for guidance.
Supporting
Documents
The following processes support this policy:

Laboratory Safety

Liquid Nitrogen Resupply procedure
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Liquid Nitrogen Monitoring and Safety Process Policy Number
Approval Signature: Laboratory Director
Effective Date
Review Cycle
Supersedes
Liquid nitrogen level and temperatures are monitored and documented daily
Room where liquid nitrogen is stored is monitored for oxygen levels (required only if LN2 is stored in room without adequate ventilation)
Safety precautions are followed to avoid injury when handling liquid nitrogen
Transfer of liquid nitrogen follows safety instructions
Safety equipment and first aid are available to personnel handling liquid nitrogen.

Technicians/Technologists

Technicians/Technologists

Technicians/Technologists

Technicians/Technologists

Laboratory supervisor
Fac12-03 -Pro
5-Feb-08
1 year
New
Liquid Nitrogen Monitoring and Safety SOP
Liquid Nitrogen Monitoring and Safety SOP
Liquid Nitrogen Monitoring and Safety SOP
Liquid Nitrogen Monitoring and Safety SOP
Liquid Nitrogen Monitoring and Safety SOP
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SMILE
Johns Hopkins University
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Author(s), Name &
Title
Anne Sholander Document
Number
Effective
Date
International QA/QC Coordinator Fac12-03-SOP 5-Feb-08
SMILE Comments: This document is provided as an example only. It must be revised to accurately reflect your lab’s specific processes and/or specific protocol requirements. Users are directed to counter check facts when considering their use in other applications. If you have any questions contact SMILE.
Approved
By
SOP
Annual
Review
Name, Title
Name, Title
Signature
Signature
Date
Date
Revision
History
Version # [0.0]
1.1
Distributed
Copies to
Name (or location)
Revision Date
[dd/mm/yy]
Description (notes)
10 June 2009 Additional of definitions, Hazards and General
Handling
# of copies Name (or location) # of copies
Associated Forms:
Appendix A: Liquid Nitrogen Monitoring and Maintenance Log
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I acknowledge that I have read, understand and agree to follow this SOP.
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This Standard Operating Procedure establishes procedures for safe handling and monitoring of liquid nitrogen in c ryogenic refrigerators and Dewar’s flasks. This procedure applies to all
Laboratory personnel who will be handling liquid nitrogen.
LN2 – Liquid Nitrogen
NCCLS – National Council of Clinical Laboratory Standards
Cryogenic liquid: liquid with a normal boiling point below -130oF (-90oC). Common industrial gases transported, handled and stored in the liquid state at cryogenic temperatures are Argon,
Helium, Hydrogen, Nitrogen, and Oxygen
Dewars: Liquid dewar flasks are non-pressurized, vacuum-jacketed vessels, similar to a
Thermos bottle. Dewars are designed with either loose-fitting caps or pressure relief valves, that prevents air and moisture from entering, yet allows excess pressure to vent
A. Frostbite/Tissue Damage - Cryogens are extremely cold and can cause instant, severe frostbite and tissue damage. Cryogen vapors can freeze the skin or eyes faster than liquid contact, and even faster than metal contact. Direct contact with cryogenic liquids, un-insulated cryogenic pipes or equipment can cause freeze burns and tissue damage. The fluid in eyes will freeze in contact with a cryogen, causing permanent eye damage.
B. Asphyxiation - On vaporization, cryogenic liquids will expand at least two orders of magnitude. Liquid nitrogen, the most commonly used cryogenic liquid, will expand over 700 times. Liquid oxygen will expand almost 900 times. If a cryogenic liquid is vaporized so as to reduce the oxygen percentage below 19.5%, there is a risk of oxygen deficiency and asyphyxiation. In confined space or poorly ventilated areas (such as cold rooms, elevators, or storage rooms), the expanding gas can displace oxygen, presenting an asphyxiation hazard to staff working in the area. Simple asphyxiants such as nitrogen do not have good warning properties. To prevent asphyxiation hazards, cryogenic liquids must be stored and used in wellventilated areas.
C. Pressure Buildup and Explosions – Because of their high liquid to gas expansion ration, cryogenic liquids present a potential explosion hazard when they evaporate. Cryogens will boil as they sit in their storage vessels by absorbing heat energy from the warmer surroundings. The vessels used to store cryogenic liquids must have a pressure relief valve or venting lid to allow for the release of evaporated gas from the container.
D. Oxygen Enrichment - When transferring liquid nitrogen through un-insulated metal pipes, the air surrounding a cryogen containment system can condense. Nitrogen, which has a lower boiling point than oxygen, will evaporate first. This can leave an oxygen-enriched condensate on the surface that can increase the flammability (combustibility) of materials near the system, creating potentially explosive conditions. In order to minimize the fire hazard potential, equipment containing cryogenic fluids must be kept clear of combustible materials.
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E. Material Brittleness – Cryogenic liquids cause many common materials such as carbon steel, plastic and rubber to become brittle or possibly fracture under stress.
A. Remove metal jewelry/watches on your hands and wrists before working with cryogens. If exposed to cryogenic liquids or boil-off gases, jewelry can freeze to the skin.
B. Wear protective clothing. Cover all exposed skin by wearing long sleeve shirts, long pants
(cuff-less), a long sleeve lab coat, well-fitted leather shoes (no sneakers or sandals) and gloves.
Gloves should be loose-fitting, lightweight, flexible, and insulated to allow for quick removal if cryogenic fluids are spilled on them. Wear a cryogen apron when a splash potential exists or when large quantities of cryogens are handled.
C. Protect your eyes by wearing safety goggles whenever working with cryogen fluids. Full face shields shall be used in the following situations: when a cryogen is poured; for open transfers; or if fluid in an open container may bubble.
D. Handle cryogenic liquids carefully. Do not allow unprotected areas of skin touch objects cooled by cryogenic liquids. Use tongs to withdraw objects immersed in the liquid, and handle the object carefully.
E. Transfer or pour cryogens slowly to minimize boiling, splashing and spilling. Use proper transfer equipment, such as a phase separator or special filling funnel (the top of the funnel should be partly covered to reduce splashing). If the liquid cannot be poured, use a cryogenic liquid withdrawal device for the transfer (be sure to follow all instructions provided with the device).
F. Do not overfill containers. Do not use hollow rods or tubes as dipsticks since liquid could be release from the top of the tube. Instead, use wooden or solid metal dipsticks.
G. Store and use cryogenics in a well-ventilated area. In closed areas, gases can reduce the oxygen concentration and can result in asphyxiation. To avoid asphyxiation, an oxygen monitor is recommended when working with a cryogen in a confined space.
Dewars
A. Use containers specifically designed for low-temperature liquids, such as a dewar. Liquid dewar flasks are non-pressurized, vacuum-jacketed vessels, similar to a Thermos bottle.
Dewars are designed with either loose-fitting caps or pressure relief valves, that prevents air and moisture from entering, yet allows excess pressure to vent. Do not connect the tank and the dewar tightly to avoid pressure build up in the dewar. Do not use any stopper or other device that would interfere with venting of gas.
B. Cryogenic containers are designed and made of materials that can withstand rapid changes and extreme temperature differences encountered in working with cryogenics. Fill containers slowly to minimize internal stresses that occur when any material is cooled.
C. When hand-carrying cryogen-containing dewar, ensure the dewa r is your only load (don’t carry anything else). Watch for people who may run into you, and ensure that the dewar is carried with both hands and as far away from your face as comfortably possible.
D. Ensure dewars are properly labeled with the identity of the cryogen. Do not mix different cryogens in the same Dewar
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E. Use care when filling portable dewars and do not overfill them.
F. Do not cover or plug the entrance opening of any dewar. Do not use any stopper or other device that would interfere with venting of gas.
G. Keep containers upright at all times except when pouring liquids from dewars specifically designed for that purpose. Handle containers gently; rough handling can cause serious damage to dewars and refrigerators. Dropping the container can cause partial or complete loss of vacuum. Do not walk, roll or drag these containers across a floor; uses a dolly or handcart.
H. Keep containers clean and dry. Moisture, chemicals, strong cleaning agents may promote corrosion which should be removed promptly. Use water or mild detergent for cleaning and dry the surface thoroughly. Do not use strong alkaline or acid cleaners that could damage the finish and corrode the metal shell.
Refer to the following procedures:

Enter other basic procedures needed for your lab .
The exceptions to these procedures are listed below:

Enter exceptions to the procedures listed above that are needed for this procedure.

Or NA
Specimen Information: NA
Reagents/Media: NA
Supplies:

Cryogenic liquid handling gloves
 Face shield

Oxygen monitor with alarm (required only if LN2 is stored in room without adequate ventilation)

Cryogenic containers
 Dewar’s flasks
Equipment Calibration/Maintenance:
I. Daily Checks:
Step
1
Action
Record Liquid Nitrogen Levels daily
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2
3
If
Level is < 25%
Then
Initiate and document Liquid
Nitrogen resupply procedure
Record Liquid Nitrogen Temperature daily
If Then
Outside acceptable ranges Notify section supervisor
Record Oxygen level daily (required only if LN2 stored in room with inadequate ventilation)
If Then
Outside acceptable ranges Notify section supervisor
4 Perform weekly cleaning procedure
If Then
Any problems or issues Record in comments section of daily log
5 If there are any issues, report all problems to the supervisor.
Quality Control:
A .
LN2 Level and temperatures must be monitored and documented daily.
B .
List the minimum acceptable LN2 level to determine and initiate the re-supply
process.
Test Method Instructions:
A. Safety Warnings and Precautions-The safety precautions in this SOP must be followed to avoid potential injury or damage. Do not attempt to handle liquid nitrogen until you read and fully understand the potential hazards, their consequences, and the related safety precautions.
Step
1
2
3
4
Action
Notify another staff member before working with liquid nitrogen or in a
LN2 storage room.
Use cryogenic containers that are specifically designed for liquid nitrogen and made of materials that can withstand extreme temperature differences. These containers should be filled slowly to minimize internal stresses that occur when any material is cooled.
Do not cover or plug any opening on liquid nitrogen refrigerators or
Dewar’s flask. Do not use any stopper or other device that could interfere with the venting of gas.
Cryogenic liquid containers are designed to operate with minimal or no internal pressure. Inadequate venting can result in excessive pressure,
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5
6
7
8
9
10
11 which could damage or burst the container. Use only the loose-fitting neck tube core supplied or one of the approved accessories for closing the neck tube. Check the unit periodically to be sure VENTING is not restricted by accumulated ice or frost.
Use a phase separator or special filling funnel to prevent SPLASHING and SPILLING when transferring liquid nitrogen into or from a Dewar flask or refrigerator. Use only small, easily handled Dewar’s flasks for pouring liquid. For larger, heavier containers, use a cryogenic liquid withdrawal device to transfer liquid from one container to another. Be sure to follow all instructions supplied.
Do Not Over Fill Containers .
Filling above the bottom of the neck tube (or specified maximum level ) can result in OVERFLOW and SPILLAGE upon closing.
Never Use Hollow Rods or Tubes as Dipsticks. When a warm tube is inserted into liquid nitrogen, liquid will spout from the top of the rod or tube. This can result in a risk of facial exposure due to gasification and rapid expansion of the liquid inside the tube.
Nitrogen Gas Can Cause Suffocation without Warning
Store and use only in a well ventilated area. As liquid evaporates, nitrogen gas is released and displaces normal air. This reduces the concentration of oxygen and can result in asphyxiation.
Nitrogen gas is colorless, odorless and tasteless. It cannot be detected by human senses and may be breathed as if it were normal air.
Breathing atmospheric air that contains less than 18% oxygen causes dizziness and quickly results in unconsciousness and death.
The cloudy vapor that appears when liquid nitrogen is exposed to air is condensed moisture ; not the gas itself. The issuing liquid nitrogen gas is invisible .
Never dispose of liquid nitrogen in confined areas or places that may expose others to the gas. Dispose only outside and in a safe place.
Pour the liquid slowly on gravel or bare earth where it can evaporate without causing damage. Do not pour liquid on pavement. Do not inhale the vapors.
Protective Clothing – Always wear protective face covering, insulated gloves and long sleeved clothing to help prevent unnecessary exposure to liquid nitrogen.
Contamination - If containers stored in liquid nitrogen contain hazardous biological materials, they must be opened in appropriate biological safety cabinets only and in accordance with ( your lab’s ) safety policy for handling potentially infectious substances. Broken containers within storage freezers pose a risk of contamination due to potential survival of the released contents. Handle all broken containers in accordance with ( your lab ) policy.
Explosion - Liquid nitrogen is a cryogen with a boiling point of –196C
(- 320F). When removed from a liquid nitrogen atmosphere, improperly
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6 sealed sample containers may explode. Placing containers in vapor phase nitrogen for several hours before immersing them in liquid nitrogen minimizes the risk of explosion.
If
Exposure to LN2 causes any adverse effects
Then
Follow steps below to administer first aid
B. First Aid
1 . If a person working with liquid nitrogen appears dizzy or loses consciousness: a. Move to a well-ventilated area immediately. b. If breathing has stopped, apply artificial respiration. c. If breathing is difficult, give oxygen. d. Call a physician. e. Keep warm and at rest.
2 . If exposed to liquid or cold gas: a. Restore tissue to normal body temperature (37 o C) as rapidly as possible. b. Protect from further damage and infection. c. Remove or loosen clothing that may constrict blood circulation to the frozen area. d. Call a physician. e. Rapidly warm the affected part using water at (108 o F) and NEVER over
(112 o F). f. Do not rub affected part either before or after re-warming.
Calculations: N/A
Expected Values: N/A
Interpretation of Results: N/A
Method Limitations: N/A
How are Results Prioritized: N/A
Result Entry into Lab Reporting System: N/A
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Notification of Result to Appropriate Personnel: N/A
Archiving Results and Report Documents: N/A
Specimen Retention: N/A
: List sources of information such as instrument manuals that may assist with the procedure.
1. Forma Scientific, Inc. P.O. Box 649, Marietta, Ohio 45750 USA. TEL (740) 373-4763
Telefax (740) 373-4189. Manual No. 7007400.
2. Clinical Laboratory Standards Institute (CLSI). Clinical Laboratory Technical Procedure
Manuals; Fourth Edition . CLSI Document GP2-A4 (ISBN 1-56238-458-9). Clinical and
Laboratory Standards Institute, Wayne, PA
:
Appendix A: LN2 Monitoring and Maintenance Log
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