Cryogenic pipeline systems with automatic nitrogen supply and distribution systems - Design, installation and testing

1. INTRODUCTION 3

2. SCOPE 3

3. NORMATIVE REFERENCES 4

4. TERMS AND DEFINITIONS 4

5. DESCRIPTION OF THE LIQUID NITROGEN PIPELINE SYSTEM 5

6. RISK MANAGEMENT 6

7. PREREQUISITES FOR THE INSTALLATION SITE 6

7.1. Outdoor areas 6

7.2. *Indoor areas (location, dimensions and volume) used as infrastructure 6

8. CRYOGENIC STORAGE SYSTEM DESIGN 7

8.1. General criteria 7

8.2. Stationary cryogenic tank 8

8.2.1. *Stationary cryogenic tank capacity 8

8.2.2. *Plot/base for stationary cryogenic tank 9

8.2.3. *Stationary cryogenic tank alarms and signals 9

8.3. Safety isolation system 10

8.3.1. Liquid nitrogen inlet solenoid valve on the cryogenic pipeline 10

8.3.2. Backup supply connection point 10

8.3.3. External backup withdrawal point 10

8.4. Cryogenic pipeline 10

8.4.1. General 10

8.4.2. Piping to transfer liquid nitrogen 11

8.4.2.1. Vacuum sealed piping 11

8.4.2.2. Insulated piping 11

8.4.3. Pressure Safety Valves 11

8.4.4. *Thermal safety valves 12

8.4.5. Additional liquid withdrawal point 12

8.4.5.1. Automatic Liquid withdrawal point for manual filling 12

8.4.6. Cooling Unit 13

8.5. Equipment connected to the Cryogenic pipeline 13

8.5.1. Characteristics of the cryogenic containers 13

8.5.2. Connected cryo-mechanical freezers -80 °C 13

8.5.3. Controlled rate freezers 13

8.6. Supervision and automation system 14

8.7. Infrastructure and accessory systems 14

8.7.1. Environmental monitoring 14

8.7.1.1. General 14

8.7.1.2. Oxygen concentration measuring system 14

8.7.1.3. *Ventilation system 15

8.7.1.4. Indoor conditions monitoring system 15

8.7.1.5. Other User safety monitoring system 15

8.7.1.6. Performance of the supervision and automation system for environmental monitoring 16

8.7.2. Electrical systems 16

9. CHECK AND TEST 17

9.1. General 17

9.2. Installation and operational checks 17

9.2.1. Stationary cryogenic storage check 17

9.2.2. Room checks 17

9.2.3. Electrical system check 17

9.2.4. Vacuum insulated pipeline testing 17

9.2.4.1. Tests prior to installation - Mechanical strength tests 17

9.2.4.2. Tests prior to installation - Vacuum sealing 18

9.2.4.3. Installation site tests and checks - Leak tests 18

9.2.5. Insulated pipelines tests 18

9.2.5.1. Mechanical strength test 18

9.2.5.2. Leak tests 18

9.2.6. Cryogenic pipeline checks after installation 18

9.2.7. Pressure and thermal safety valves tests 18

9.2.8. Vent lines check 19

9.2.9. Freezers checks and tests 19

9.2.10. Ventilation system checks and tests 19

9.2.11. Oxygen concentration monitoring system checks and tests 19

9.2.12. Emergency stop buttons checks 19

9.2.13. Supervision and automation system checks 19

9.3. Performance tests 20

9.3.1. Environmental monitoring performance tests 20

9.3.2. Cryogenic pipeline performance tests 20

10. DOCUMENTATION 21

ANNEX A (informative) 22

ANNEX B (informative) 25

Low-temperature cryopreservation, by means of liquid nitrogen, of biological materials and their derivatives is nowadays a well-established practice in clinical and scientific, biomedical and environmental research, and is practiced in a variety of institutions such as hospitals, biological tissue institutes, pharmaceutical workshops, laboratories and research institutions.

Liquid nitrogen or its vapours enable the long-term storage of valuable material such as biological samples and derivatives and/or molecules or other materials that require very low temperature, called cryogenic temperatures, to preserve their characteristics.

Long-term preservation of biological samples is ensured by keeping them at low temperatures in liquid or gaseous phase nitrogen.

For the purpose of process efficiency, samples are stored without interruption at these temperatures in an appropriate level of nitrogen.

Despite the fact that cryogenic freezers dedicated to the storage of biological samples are provided with of effective thermal insulation, a decrease in the level of liquid nitrogen, below the minimum threshold, can lead to an increase in the temperature inside the cryogenic freezers to such an extent that the proper preservation of samples is compromised and their subsequent use is prevented.

Maintaining the correct level of liquid nitrogen in cryogenic freezers can be done by manual or automatic replenishment.

Proper risk management identifies potentially hazardous situations that may compromise the proper storage of samples. Relevant aspects for proper management of the process are:

• the constant maintenance of at least a minimum level of liquid nitrogen in each cryogenic freezers.
• adequate records to provide evidence that the cryopreservation temperature is continuously maintained.

A solution that has long been introduced and used in dedicated facilities consists of one or more stationary cryogenic tanks that feed, via an automated system, several cryogenic freezers in which the samples are stored, by continuously ensuring the correct level of nitrogen in each of them.

For the safe performance of the cryogenic pipeline system in those dedicated facilities, those facilities have to fulfill special requirements in infrastructure, like ventilation, drains, materials used in walls and floor, access and safety control and interactive warning systems.

Given its complexity, the cryogenic storage system requires adequate design, installation, testing and commissioning of all its components, and this standard defines the minimum general principles for its implementation.

The automated nitrogen supply and distribution system supplies a room intended for low-temperature storage of biological material and its derivatives, commonly called a cryobiological room.

Biological material preserved in the cryogenic storage system may be used for clinical, diagnostic, therapeutic activities, biodiversity studies and research purposes.

The standard:

• applies to design, installation, testing for a cryogenic storage system with automated nitrogen supply system used for preservation of biological material for clinical, diagnostic, therapeutic activities;
• defines the minimum requirements for all components and equipment serving and forming an integral part of a cryogenic storage system;
• applies both to newly built, cryogenic storage systems and to modifications, extensions and changes of use of cryogenic storage systems already in operation;
• defines the minimum requirements of the infrastructure (e.g. intended to contain components and installations serving the cryogenic storage system);
• contains requirements relating to the commissioning, acceptance activities and documentation made available by the manufacturer.

This standard does not apply:

• Cryogenic rooms without an automatic freezers filling system cryogenic container are outside the scope of this standard.
• to additional components used within cryogenic rooms that are not permanently connected to the cryogenic pipeline and are therefore not an integral part of the cryogenic storage system;
• to systems intended for the storage of material that require manual filling in order to maintain an adequate level of liquid nitrogen inside the cryogenic freezers;
• the management, identification and traceability of biological samples;
• the management of devices using CO₂, that could be installed in the same room and the associated risks of CO₂ poisoning;
• the responsibilities of managing the biological samples in normal and emergency conditions;
• the installation of the stationary cryogenic tank and its conformity with local requirements.

This standard incorporates by dated or undated reference, provisions from other publications. These normative references are cited at the appropriate places in the text and the publications are listed hereafter. For dated references, subsequent amendments to or revisions of any of these publications apply to this standard only when incorporated in it by amendment or revision. For undated references, the latest edition of the publication referred to applies (including amendments).

For the purposes of this standard, the following terms and definitions apply:

4.1 Additional liquid withdrawal point: it is an indoor point where the liquid nitrogen can be manually or automatically collected.

4.2 Backup supply connection point: outdoor connection point that allows the connection of an emergency source of supply.

4.3 Backup withdrawal point: outdoor point where the liquid nitrogen can be directly collected from the stationary cryogenic tank.

4.4 Controlled rate freezer: Device used for freezing biological material at programmable descending temperatures.

4.5 Cooling unit: assembly of equipment to cool down the pipeline before the freezers filling.

4.6 Cryogenic freezer: A non-pressurized, automatically controlled device for the storage of biological samples designed to maintain low temperatures using cryogenic fluid supplied by the cryogenic pipeline.

4.7 Cryogenic pipeline: Insulated or vacuum insulated piping for transferring cryogenic fluid from the stationary cryogenic tank(s) to the cryogenic freezer(s) including all the necessary valves, fittings and control and safety instruments.

4.8 Cryogenic temperature: Temperature < - 140°C.

NOTE: Definition taken from IUPAC Compendium of Chemical Terminology 2nd, Edition (1997), Chapter 13.2.

4.9 Cryo-mechanical freezer: electrically powered device for the storage of biological samples designed to maintain low temperatures using a refrigeration system based on mechanical compression and equipped with an auxiliary liquid nitrogen cooling system.

4.10 Freezers: equipment used to store biological samples at different low temperatures.

4.11 Infrastructure: Physical infrastructure consisting of the area and rooms designed to contain all the elements of the cryogenic storage system and the systems and installations serving it.

4.12 Automated nitrogen supply and distribution system: a complete system that comprises the liquid nitrogen source of supply, a supervision and automation system, and the cryogenic pipeline.

4.13 Liquid withdrawal point: it is the non-insulated part of the cryogenic pipeline that allows the connection of a served device.

4.14 Mobile cryogenic tank: Pressurized mobile cryogenic liquid nitrogen storage container.

4.15 Normal condition: the normal condition is with an oxygen concentration above 19.5% in a confined space.

4.16 Oxygen sensors: Devices for continuous measurement of the environmental oxygen content.

4.17 Phase separator: Device connected between the stationary cryogenic tank and the utilities of the cryogenic pipeline, capable of constantly supplying nitrogen in the liquid state, separated from the gaseous phase and capable of decreasing the outlet pressure of the stationary cryogenic tank by adapting it to the maximum pressure allowed by the various utilities.

4.18 Pressure safety valve (PSV): to release pressure in the system when internal pressure exceeds a safe level.

4.19 Safety isolation unit: outdoor system to isolate the stationary cryogenic tank from the indoor part of the cryogenic pipeline.

4.20 Stationary cryogenic tank: A liquid nitrogen storage device capable of continuously feeding the cryogenic pipeline.

4.21 Served device: Any device connected and refilled by the cryogenic pipeline.

4.22 Supervision and automation system: A set of hardware and software components capable of ensuring, by means of monitoring and control, the management of the entire cryogenic storage system to guarantee the storage of samples indefinitely and the safety of the operators and users.

4.23 Thermal safety valve (TSV): to relieve pressure caused by thermal expansion of the liquid within the system.

4.24 Vent line: line used to conduct gas from the system to outside of the building where gas can be discharged safely.

An automated nitrogen supply and distribution system consists of:

1. stationary cryogenic tank;
2. safety isolation unit;
3. backup supply connection point;
4. back up withdrawal point;
5. phase separator (optional);
6. liquid withdrawal point;
7. cryogenic pipeline;
8. cooling unit;
9. supervision and automation system.

Systems or devices connected to the automated nitrogen supply and distribution system but not part of the system:

1. cryogenic freezer(s);
2. mechanical cryogenic freezer(s);
3. controlled rate freezer(s);
4. mobile cryogenic tank(s);
5. Ventilation system;
6. Indoor environmental monitoring system;
7. user safety monitoring system;
8. Access control system;
9. Electrical system.

A automated nitrogen supply and distribution system shall be built, extended, modified, set up in order to ensure that risks are reduced to an acceptable level on the basis of an accurate risk assessment and management.

The main risks, although not exhaustive, are:

• Uncontrolled thawing of biological samples;
• Anoxia;
• Cold burns;
• Overpressure.

The stationary cryogenic tank area should be identified as close as reasonably possible to the infrastructure; this helps to minimize cryogenic fluid losses in the distribution cryogenic pipeline.

Note: It is advisable to have the stationary cryogenic tank area at the same level as or slightly higher than the infrastructure.

There shall be sufficient space with an access and restricted access for installing the backup supply connection point and the safety isolation unit.

If it is considerable a confined space additional safety measures may be required.

NOTE: Local regulations may be present for the location of the vent line.

The rooms intended for the installation of cryogenic storage systems shall be chosen considering the following criteria:

• Sizing, the following three conditions shall be met:
  1. the minimal size shall be 6 m² surface and 20m³ volume;
  2. the minimal space of 1.5 m² shall be considered per each served device and the additional liquid withdrawal point(s);
  3. following the size of the cryogenic freezer the distance between each one shall not be less than 0.3 m;
• access doors: shall prevent the spread of gaseous or liquid gases outside. The doors shall be equipped with panic bars and shall open outwards to ensure the rapid evacuation of the rooms if required. The door sizes shall consider the size of the equipment to be installed in the cryogenic room;
• visibility: it shall always be possible to view the rooms from outside through access doors with visual or other systems allowing a view of the inside (e.g. using transparent walls).

NOTE: the general considerations about the location of the room the maximum loads and the type of surfaces of wall and floor, are relevant but out of scope of this standard. Additional information are provided in Annex A.

The design inputs are determined by the customer’s specifications.

These specifications include at least the following elements:

• the purpose of the liquid nitrogen pipeline system;
• the rooms (location, dimensions and volume) used for the infrastructure;
• the external area used to install the stationary cryogenic tank, safety isolation unit and backup supply point;
• the number, the size and the type of freezers that will be permanently connected to the liquid nitrogen pipeline system;
• the number, the size and the type of backup freezer to be permanently connected for maintenance activities and to stay operational in event of power or other equipment failure;
• the desired routing of pipelines;
• if there are plans for future expansions;
• the number of liquid withdrawal points;
• the characteristics of the room including the functionality and the location:
  • the ventilation system;
  • the indoor environment monitoring system;
  • the access control system;
  • the electrical system;
  • the fire detection system;
• constraint in the design due to other services and/or equipment.

The design documentation shall provide evidence that the customer’s specifications have been complied with and shall include:

• checks on the suitability of the available rooms or the modifications required to make them suitable for use as infrastructure; including:
  • the environmental monitoring and ventilation system;
  • the electrical systems;
  • the lighting system;
  • the fire detection system;
  • the access control system;
• the positioning, capacity and the refilling and alarm thresholds of the stationary cryogenic tank;
• the properties, sizing and routing of the cryogenic pipeline; including, where necessary, the possible connection for future extensions;
• the number and the location of the liquid withdrawal points;
• the characteristics of the supervision and automation system;
• the location of other components, according to the customer’s specifications;

The capacity of the stationary cryogenic tank should be such as to ensure both the following conditions:

• 10 (ten) days of autonomy, considering the average daily consumption of the month of maximum consumption;
• 15 (fifteen) days of autonomy, considering the average daily consumption calculated on the basis of the annual consumption.

NOTE: Autonomy refers to both the regular operation of the cryogenic storage system without any refilling of the stationary cryogenic tank.

If it is impossible to comply with the above indicated criteria, alternative solutions shall be put in place to ensure continuity of supply.

To limit the pressure fluctuations at the liquid withdrawal point a device for maintaining the stationary cryogenic tank pressure lower than the calibration value of the pressure-relief valves shall be installed.

NOTE 1: an example of device to maintain pressure is a back pressure regulator.

NOTE 2: it is not good practice to use the pressure safety valves as a pressure regulation system.

The choice of working pressure (PE) value shall consider the length of the cryogenic pipeline and any difference in height between the stationary cryogenic tank and the location of the liquid withdrawal points.

NOTE: the pressure at the liquid withdrawal point is limited by the maximum permitted pressure of the freezers.

The area intended to contain the stationary cryogenic tank/s shall be located outdoors.

If the stationary cryogenic tank is in a confined or semi-confined space the risk of anoxia shall be evaluated, and mitigation measures implemented.

NOTE: examples of semi-confined spaces are areas where the ambient air renewal is limited by three walls, or roof or below the ground level.

A suitable plot/base shall be designed for the correct positioning of the stationary cryogenic tank and any other accessories.

NOTE: local regulations may be present for the installation of stationary cryogenic tank/s.

Sizing shall consider any further increased consumption that could require a greater capacity of the stationary cryogenic tank.

An unloading area shall be set up next to the plot for transfer operations.

The stationary cryogenic tank shall be equipped with systems for measuring, monitoring (7/7, 24/24) and signaling nitrogen level and pressure alarms.

Each alarm signaling system used shall be able to provide the alarm condition through two different channels (for example telephone dialer, dry contact). Both channels shall be used to ensure alarm sending redundancy 7/7, 24/24 in the methods defined by the contractor.

NOTE: the level, pressure and the alarms can also be sent to the liquid nitrogen supplier.

In annex A an example of how to identify reference levels and pressures for alarms is reported.

For remote sensing for nitrogen suppliers/maintenance engineers, a system shall be adopted with the following alarm thresholds:

• pre-alarm level: reports an anomalous situation that requires checking;
• alarm level (2 days): reports an anomalous situation that requires actions and, if the normal situation is not restored, implements a plan of preventive measures;
• alarm pressure (high, low): reports an anomalous situation that requires checking and actions, if the normal situation is not restored, implements a plan of preventive measures.

In annex A examples of preventive measures are reported.

If the stationary cryogenic tank and/or only the remote sensing system is replaced, the above-indicated alarm signals shall always be available and checked.

The stationary cryogenic tank instruments shall ensure the possibility:

• to read locally the pressure and level, independently from supervision system and power supply;
• to read the pressure and level by the supervision system.

In the presence of a supervision and automation system for the cryogenic pipeline the stationary cryogenic tank instruments shall ensure the reading of the pressure and level.

The liquid nitrogen inlet solenoid valve shall have the following characteristics:

• located in a well-ventilated and accessible place;
• located as close as possible to the stationary cryogenic tank;
• clearly identified and labeled to allow identification by the emergency services;
• it shall be manually bypassed through manual valves (to offer a further possibility for disconnection in emergency conditions or during maintenance); The manual valve shall be protected with a lockout-tagout system.
• it shall work in normally closed mode in the event of a power cut or loss of the actuation signal.

The liquid nitrogen inlet solenoid valve to the cryogenic pipeline shall include an automatic system able to stop delivery in the event of severe under-oxygenation (less than or equal to 18%) in the cryogenic rooms or signaling by other installed systems.

A backup supply connection point shall be installed on the line upstream from the safety isolation system outside the cryogenic storage system (see figure 1), to ensure the supply of liquid nitrogen to the cryogenic pipeline from a pressurized cryogenic tank (either mobile cryogenic tank or another stationary cryogenic tank) in the event of the temporary unavailability of nitrogen in the stationary cryogenic tank.

The sizing of the connection branch shall be the same as the main line.

The valve shall be closed and capped. The cap shall have a small hole in case of internal valve leak.

If the cryogenic pipeline is out of service, in order to tap the liquid nitrogen, the availability (direct or by contract) of a mobile cryogenic tank shall be assured to allow the manual filling of each freezer.

An external backup withdrawal point shall be installed on the line upstream from the safety isolation system outside the cryogenic storage system.

A valve shall be installed to allow venting the nitrogen.

The cryogenic pipeline consists of:

• piping to transfer the liquid nitrogen;
• an external sleeve to maintain and insulate the internal piping;
• liquid nitrogen withdrawal points;
• manual withdrawal point, controlled by the automation system and able to enable delivery only when the cryogenic pipeline has reached a suitable temperature (see point 8.4.5.1) and only in the active presence of an operator. If the operator feels sick, the system stops the delivery of liquid nitrogen immediately;
• an internal piping cooling unit, equipped with a solenoid valve, temperature probe, by-pass valve and related pressure-relief valve;
• main external pressure-safety valve downstream the safety insulation system, to protect against overpressure.

The cryogenic pipeline may include one or more phase separators, the position of which shall be specifically designed outside of the scope of this standard.

For complex cryogenic pipelines with more than one branch, the components indicated shall be replicated on each line.

The cryogenic pipeline shall be sized defining the route, number of withdrawals points the type and related simultaneous filling of the cryogenic freezers, in order to ensure the delivery of liquid nitrogen to the served device furthest away.

The piping shall be insulated or vacuum sealed.

Due to their high heat transmission coefficient compared to vacuum sealed piping, insulated piping may be used for short stretches and/or for a limited number of served devices.

The cryogenic pipeline route shall be inspectable, therefore no concealed cryogenic pipelines are permitted.

Where horizontal and long pipelines are present, it is recommended to have between 0,5 - 1% of positive slope in the direction of the flow.

Vacuum sealed piping

Vacuum-insulated lines are double-walled piping systems designed to guarantee maximum thermal insulation between the inner and outer tubes.

Bayonet or welded joints may be used to join the different stretches of the line. It shall be possible to restore the vacuum using specific connectors.

Both the piping and the external sleeve shall be made from AISI 304 or AISI 316 stainless steel.

Considering the variations in internal pipeline temperature, suitable expansion compensation systems shall be installed.

The external sleeve shall carry a label indicating the liquid nitrogen content and the flow direction.

The external sleeve shall be connected to the earthing system.

The parameter for permitting rigid vacuum lines is linear thermal dispersion.

NOTE: For example, the maximum permissible linear thermal dispersion for diameters up to a nominal diameter of 20 mm (DN20) shall not be more than 0,5 W/m for rigid vacuum lines.

How to fix the line considering the weight of the line: the system includes robust support structures- such as clamps, hangers, and brackets engineered to maintain mechanical integrity, ensure proper alignment.

Insulated piping

A conventional pipe with thermal insulation is a single-walled stainless steel or copper pipeline.

If the material used is stainless steel, it shall be AISI 304L or AISI 316L. If the material used is copper, it shall be EN 13348 or equivalent.

It is externally wrapped with thermal insulation material to reduce heat ingress from the surrounding environment.

The insulation material shall be protected from the water accumulation (rain or moisture).

NOTE 1: The risk of oxygen accumulation due to the liquefaction of oxygen in the air close to the pipeline generated by the liquid nitrogen temperature should be taken into consideration when selecting thermal insulation material.

NOTE 2: in some installations the insulated piping is used for small parts of the piping while most of it is made by vacuum sealed piping. For efficiency point of view is more relevant to insulate the part near the tank than the one close to the withdrawal points.

To prevent overpressure caused by liquid gas vaporization or pipeline failures, which can exceed component design limits, the following pressure safety valves are needed:

• pressure safety valves on the tank;
• an outdoor pressure safety valve.

The outdoor pressure safety valve shall be installed outside downstream the safety insulation system and before entering the building and accessible.

If the piping from the stationary cryogenic tank to the cryobiology room is very long or has multiple bends and branches, overpressure risks may vary along the pipeline, in such cases the presence of additional PSVs may be needed.

If indoor pressure safety valves are installed they shall be conveyed outside to a safe location.

NOTE: for details on how to convey outside see annex A.

The pressure safety valves calibration value shall be chosen:

• according to the maximum design pressure of the equipment connected to the system;
• the altitude of the cryogenic room and the stationary cryogenic tank;
• guaranteeing the calibration differential of the pressure relief valve opening pressure, listed below in increasing order from the lowest to the highest:
  • the ones on the tank;
  • the outdoor pressure safety valve;
  • those inside, if present.

NOTE: This calibration mode makes the opening of the pressure safety valves located inside the cryogenic room less probable.

The PRVs shall be easily accessible for inspection, testing, and maintenance.

Thermal safety valves shall be installed on all components in which liquid gas gasification or a fault on the pipeline can generate a pressure higher than the design pressure of the individual components.

For those thermal safety valves located inside the rooms shall be conveyed outside to a safe location.

Each thermal safety valve shall be set at a higher pressure than the pressure safety valves settings described above.

NOTE: for details on how to convey outside see annex A.

The TSV shall be easily accessible for inspection, testing, and maintenance.

The liquid withdrawal point shall be equipped with a manual shut-off valve thermal safety valve and a connection point.

Note: local or regional regulations may apply to the connection point (e.g. DN10).

To the liquid withdrawal point cryogenic freezers and cryo-mechanical freezers shall be connected using a flexible cryogenic connection hose.

The connection hoses shall be in compliance with ISO 21012.

If additional liquid withdrawal points for filling pressurized mobile cryogenic tanks are located indoor, the gaseous nitrogen naturally generated during its filling shall be conveyed outside the room.

If an automatic liquid withdrawal point is designed for manual filling of liquid nitrogen, it

shall be equipped with a solenoid valve, a manual shut-off valve, a pressure-relief valve, a manual vent valve and a connection point.

The solenoid valve shall be kept open with a momentary button.

A cooling unit shall be installed at the end of the line to cool each branch of the internal line before filling the served devices.

The cooling unit shall ensure that each served device is filled only with liquid nitrogen and not with a mixture of gaseous and liquid nitrogen.

The cooling unit shall be equipped with one or more solenoid valves, a thermocouple to measure the gas temperature in the line, a manual valve, pressure-relief valves and if required a pressure gauge or pressure transducer.

Cooling unit operation shall be activated manually, through the supervision system by itself, or on request of one of the freezers.

The solenoid valve is activated and the liquid nitrogen flows through the piping to cool it.

When the thermocouple measures a gas temperature compatible with the liquid phase (indicatively -150 °C), the solenoid valve is closed and the liquid nitrogen can fill the served devices.

Gaseous and liquid nitrogen produced during cooling shall be evacuated to a safe location, away from personnel transit and stopping points with a vent line.

If the length of the vent line is not allowing the vaporization of all the liquid, appropriate means shall be installed on the end of the vent line to prevent liquid release (for example, a vaporizer).

In order to limit the formation of condensation, it may be required to insulate the indoor section of the vent line.

The freezers intended to be connected to a cryogenic pipeline shall be equipped with an automatic filling control unit. The control unit shall be able to:

• measure the level of liquid nitrogen inside the container;
• measure the temperature inside the container;
• manage the filling of the container with liquid nitrogen;
• report the operating state and any anomalies.

These parameters and signals shall be reported to the supervision and automation system.

NOTE: other data can be communicated to the supervision and automation system.

The connected cryo-mechanical freezers -80 °C are connected to the cryogenic pipeline as a reserve source of liquid nitrogen and shall be equipped with automatic devices to avoid the flow of liquid nitrogen inside the compartment containing samples.

Controlled rate freezers require the supply of nitrogen at a constant flow and pressure.

For this reason they cannot be directly connected to the cryogenic pipeline, they should be connected to an independent mobile cryogenic tank.

Through the cryogenic pipeline, it is possible to automatically refill the mobile cryogenic tank manually or automatically with one of the liquid withdrawal points.

Note: the mobile cryogenic tank is not connected at the same time to both the cryogenic pipeline and the controlled rate freezer.

The supervision and automation system in the cryogenic storage system shall ensure:

• the cooling unit management;
• the automatic filling of the freezers with liquid nitrogen;
• the management of equipment connected directly to the line.
• the continuous monitoring and the recording of parameters ensuring the conservation of the stored samples (temperature and level);
• the continuous monitoring and the recording of parameters from the environmental monitoring system sensors (e.g. oxygen sensors, …);
• the reporting and recording of emergency conditions (e.g. under-oxygenation, man down, emergency button pressed);
• the activation of the safety devices, such as the safety isolation unit, the high-speed ventilation and the acoustic/luminous alarms;
• the continuous monitoring and the recording of parameters from the stationary cryogenic tank (level and pressure);
• the remoting of the alarms through two different channels (e.g. telephone dialer, dry contact). Both channels shall be used to ensure alarm sending redundancy 7/7, 24/24 in the methods defined by the contractor;

NOTE: the first channel is used for example for users immediate risks (e.g. anoxia risk, the second channel for sample related risks (e.g. low nitrogen level in freezers);

NOTE: The system can be interfaced with access control devices to the cryogenic storage system.

To ensure that samples are maintained safely the system shall react to the conditions reported as in the following table:

General

During operations, especially during the automatic filling of the freezers and when manually inserting or collecting the biological material, under-oxygenated atmospheres may form in specific areas near the automated nitrogen supply and distribution system..

The under-oxygenation condition derives from a potential and sudden release of liquid nitrogen into the confined environment, with the consequent formation of gaseous nitrogen following its rapid evaporation.

Nitrogen vapor is heavier than air and it accumulates close to the floor and can create an under-oxygenated environment. As part of the risk management process an assessment of the maximum amount of gaseous nitrogen released in both normal and worst-case conditions shall be conducted.

Indoor area shall be subject to a risk assessment individually, considering the intended use, the activities performed inside and its volume in order to avoid anoxia risk for the operator.

For this reason, all rooms shall be continuously ventilated and monitored by systems that identify hazardous conditions for the operators.

Any outdoor areas where stationary cryogenic tanks are located shall also be included in the assessment.

Oxygen concentration measuring system

The oxygen concentration inside the cryogenic storage room shall be controlled by a suitable measuring system.

At least two sensors shall be installed in each room, whatever its volume. The minimum number of sensors installed depends on the volume of the rooms, as reported in the following table:

Above 50 m³ of the room and one additional sensor for every additional 50 m³.

Sensors shall not be positioned near air extraction vents and possibly not near the freezers.

The sensors shall be installed at a height from the ground of no more than 1,00 m.

Obstructions in close proximity to sensors that could reduce their effectiveness shall be minimized. Consideration shall be given to expected air flow patterns and the potential usage of areas around each sensor.

The under-oxygenation alarm thresholds shall be the following:

• Threshold 1 (medium priority alarm ): oxygen concentration 19.5%;
• Threshold 2 ( high priority alarm): oxygen concentration 18%.

NOTE: there may be regional legislative provisions that require stricter thresholds.

The oxygen concentration value measured by the sensors located in the room, shall be always shown inside the cryogenic room and at the access points to the cryogenic storage system.

*Ventilation system

The air extraction grilles shall be located low down in the room, preferably 10-15 cm from the floor. Grilles for fresh air shall be located high up.

The assessment of the maximum amount of gaseous nitrogen released into the environment should lead to the determination of the sufficient number of exchanges/hour to be met both in normal and worst-case conditions.

If all the elements required to ensure a correct assessment are not available, the typical minimum values used are:

• In normal condition 6 exchanges/hour (permanent ventilation);
• In worst case condition 25 exchanges/hour (emergency ventilation).

The ventilation system shall be independent from the air handling unit and it shall be without any recirculation.

The ventilation system shall be dedicated to cryogenic rooms and the risk of nitrogen vapors transfer to another area shall be assessed.

NOTE: the air handling unit would follow the same principle.

Means shall be provided to detect a malfunctioning of the ventilation system.

Indoor conditions monitoring system

Indoor ambient temperature and humidity shall be maintained within the following limits:

• temperature between 18 °C and 25 °C;
• relative humidity no higher than 65%.

NOTE 1: Values are referred to normal conditions of the environmental monitoring. See paragraph 8.7.1.6

NOTE 2: these values reduce the formation of condensation and ice on the coldest parts of the components in the cryogenic storage system.

A monitoring system of these parameters shall be installed.

One or more emergency stop button shall be installed both inside the cryogenic room and outside in front of the access points to allow the operator to report a hazard condition inside the room.

The emergency stop buttons shall always quickly accessible.

NOTE: other systems can be connected like the man down.

Performance of the supervision and automation system for environmental monitoring

To ensure that any operator works safely the system shall react to the environmental conditions reported as in the following table:

The high priority alarms shall be signaled through two different channels (for example: telephone dialer, clean contact). Both channels shall be used to ensure alarm sending redundancy 7/7, 24/24.

The condition shall be reported both inside each monitored room and outside the room at each entrance. No obstacles shall limit the view of the visual indication of the environmental room condition.

Means shall be provided for testing the alarms.

The equipment in the cryogenic storage system shall be powered by one or more general electrical panels located outside the room, equipped with independent and dedicated electrical lines for the following equipment:

• freezers;
• other power equipment (video surveillance system and other accessories, where present);
• ventilation system;
• supervision and automation system, including the environmental monitoring system.

Each line shall be individually protected.

NOTE: The location of terminal electrical sockets is determined by the layout of the different equipment.

Critical electrical equipment or systems in the cryogenic room (for example, supervision and automation systems) shall be constantly powered even in the event of a power cut, through connection to a generator set or UPS (Uninterruptible Power Supply).

Records shall be provided for all installation and operational checks, as well as for performance tests. Where a measured value is required, it shall be recorded.

For the stationary cryogenic tank installation the following checks shall be carried out as a minimum to conform to the approved design:

• The location;
• The capacity;
• The working pressure;
• A suitable fence was installed to prevent access to the stationary cryogenic tank by unauthorized personnel;
• The stationary cryogenic tank allows the remote transmission of its internal level and pressure parameters.

Collect and check all the documents related to the compliance with the relevant local regulation for pressurized equipment, if any applies.

For the room the following checks shall be carried out as a minimum to conform to the approved design:

• Sufficient maneuvering spaces;
• The access doors are equipped with panic bars and open outwards;
• There is visibility of the inside of the room.

For the electrical system the following checks shall be carried out as a minimum to conform to the approved design:

• There are all necessary dedicated lines;
• The lines are equipped with the electrical protection;
• The electrical system is connected to a generator set and/or a UPS (Uninterruptible Power Supply);
• The electrical accessibility of cable tray or conduits, including communication port and electrical sockets;
• The appropriate shielding of electrical cables and communication cables to prevent interference.

The whole line shall be tested after installation.

Tests prior to installation - Mechanical strength tests

The mechanical strength test shall be carried out to highlight any leaks in the internal liquid nitrogen pipeline.

Each stretch of pipeline shall be tested before final installation for mechanical strength and vacuum sealing.

The whole line shall be tested after installation for leaks only.

The pipeline shall be pressurized with gaseous nitrogen at a pressure equal to 1,43 times the maximum permitted pressure for at least 15 minutes.

At the end of the test period, the internal pipeline shall maintain its mechanical integrity.

The results, including the measured pressure value shall be provided for each stretch of pipeline.

Tests prior to installation - Vacuum sealing

The test shall be carried out to highlight any leaks in the vacuum interspace. This test shall be carried out after the positive completion of the mechanical strength and leak tests.

The depressurization (vacuum packing) of the interspace shall be carried out up to a value of 10^-5 mbar, using specific high vacuum production equipment.

After depressurizing the line, the vacuum value produced shall be monitored. After a period of 24 hours, the acceptable vacuum value shall not be less than 10^-4 mbar.

The line manufacturer shall record the results.

Mechanical strength test

If a vacuum sealed pipeline is installed then the mechanical strength test of each stretch of pipeline has already been carried out at the manufacturing site.

For the cryogenic pipeline the following checks shall be carried out as a minimum to conform to the approved design:

• The characteristics (size, development, number of outlets for manual withdrawals, number of cryogenic container filling connections, possibility of cooling, etc.);
• The correct connection to the stationary cryogenic tank;
• The accessibility of all parts of the cryogenic pipeline for inspection and maintenance;
• The clear identification of manual control valves and solenoid valves and the presence of seals where relevant;
• The clear identification of the instrumentation (e.g. thermocouples, pressure gauges, etc.…);
• The electrical connection and communications to the supervision system;
• The identification of the flow direction of the liquid nitrogen;
• The external sleeve has been bound to an earth terminal.

A functionality test of the entire line shall be performed after the leak test.

Installation site tests and checks - Leak tests

The leak test shall be carried out to highlight any leaks in the internal liquid nitrogen pipeline.

The pipeline shall be pressurized with gaseous nitrogen to working pressure for at least two hours.

The pressure and thermal safety valves are installed according to what defined in the approved design:

• The type and characteristics are conform (size, opening pressure, flow rate of the gaseous nitrogen that can be evacuated);
• The indoor thermal relief valves are installed along all the stretches of the cryogenic pipeline in which liquid nitrogen could remain trapped;
• The opening pressure values are recorded and compared to ensure the appropriate cascade.

The vent line conforms to what indicated in the approved design:

• The vent line prevents accidental blockages due to accumulated rainwater and other debris or the entry of insects;
• The vent line of the cooling unit that is indoor has been insulated;
• The vent line is located far from:
  • Work environments;
  • Personnel transit and stopping points;
  • Drainpipes;
  • Entrances to buildings;
  • Tight and confined spaces;
  • Air conditioning system intake points.

For the freezers the following checks shall be carried out as a minimum to conform to the approved design:

• They are fitted with an automatic filling control unit.

For the ventilation system the following checks shall be carried out as a minimum to conform to the approved design:

• The location, number and size of the grills for fresh air and their opening status
• The location, number and size of the air extractions grills and their opening status
• The ventilation system flow rate is able to meet the minimum requirements for both permanent and emergency ventilation.

For the oxygen concentration monitoring system the following checks shall be carried out as a minimum to conform to the approved design:

• The number of sensors, their location and position;
• The activation thresholds.

For the emergency stop buttons the following checks shall be carried out as a minimum to conform to the approved design:

• the number of buttons, their location and position;
• the accessibility (free from obstacles).

For the supervision and automation system the following checks shall be carried out as a minimum to conform to the approved design:

• the location and position;
• the communication of each element:
  • the freezers;
  • the cooling unit;
  • the safety isolation unit;
  • the ventilation system;
  • the automatic liquid withdrawal point;
  • the emergency stop buttons;
  • the stationary cryogenic tank;
  • the remote control of alarms;
  • oxygen monitoring system;
  • other additional systems connected (e.g. man down, access control, temperature and humidity …).

Before starting the performance test the environmental monitoring requirements of paragraph 11.1.6 shall be tested and recorded and approved.

In the normal condition check:

• Activator and sensors status are compliant with the condition;
• Consequences are compliant with the condition.

In the medium priority alarm condition check:

• For each activator and sensors status change that lead to the medium priority condition the appropriate alarm went off;
• For each combination of the previous that that lead to the medium priority condition the appropriate alarm went off;
• That all the consequences are activated;
• the restoring of normal conditions.

In the high priority alarm condition check:

• For each activator and sensors status change that led to the medium priority condition the appropriate alarm went off;
• For each combination of the previous that that lead to the medium priority condition the appropriate alarm went off;
• That all the consequences are activated;
• The restoring of normal conditions.

The following tests shall be performed on the cryogenic pipeline:

• manual cooling down:
  • the cooling time is no longer than the time defined in the design;
  • no visible liquid leaks;
  • no visible ice or condensation in insulated parts;
• automatic request of filling through a freezer:
  • the system starts with a request from each freezer;
  • the system does not allow the filling of the freezers before the pipeline cooling down step is completed;
  • the system allows the filling of the freezer when the pipeline cooling down step is completed;
• automatic request from the supervision and automation system:
  • the system starts with a request from the supervision and automation system;
  • the system does not allow the filling of the freezers before the pipeline cooling down step is completed;
  • the system allows the filling of the freezer when the pipeline cooling down step is completed;
• automatic request from the manual/automatic liquid withdrawal point for manual filling;
• simulation of the alarms activation from freezers (e.g. level, temperature, solenoid valve open);
• simulation of the activation of the alarms on the stationary cryogenic tank;
• stress test for malfunction of the systems (e.g. sensor disconnection, communication failure, freezer disconnection, …);
• power loss and automatic reactivation of the system;
• remoting of alarms;
• every operation has been recorded on the supervision and automation system (alarms, levels in freezers, temperature values, activation of safety isolation unit, …).

The documentation to be collected following the positive completion of the testing of the cryogenic system and prior to commissioning shall include:

• the layouts of the cryogenic storage system rooms with an indication of the installed equipment;
• the diagram and construction drawings of the cryogenic pipeline;
• the wiring diagrams of the supervision and automation system;
• the wiring diagrams and construction drawings
• the electrical wiring diagrams of the interconnection between system components;
• the operating manuals of the installed components and system;
• the maintenance manuals of the installed components and system;
• any declarations of conformity to applicable standards or laws;
• the testing reports.

Guidance on the provisions of this standard.

7.2A indoor areas

For the floor and walls in the cryogenic room the following criteria are suggested:

• The floor made with easily washable materials that withstand low temperatures shocks;
• The floor does not allow spills into drains or technical networks;
• The wall coverings are made of smooth, waterproof and easy-to-clean material;
• The wall coverings reach a height of ≥ 1,80 m off the ground;
• The wall coverings are made with materials that withstand low temperatures;
• The wall coverings are joined to the floor.

Natural and artificial lighting will ensure the correct handling of samples and the use of equipment in the cryogenic room.

An emergency switch outside the cryogenic room to switch on the lighting inside the room.

An emergency lighting system should be activated in the event of a power cut.

8.2.1A Calculation of stationary cryogenic tank capacity

The capacity of the stationary cryogenic tank should be calculated and approved according to:

• the estimation of average consumption based on the design inputs.
• The frequency of liquid nitrogen refilling of the stationary cryogenic tank;
• the characteristics of the pipeline system (pipeline length, insulation and gasification, etc.…);

Capacity = autonomy * estimated average daily consumption.

8.2.2A Plot/base for the stationary cryogenic tank

The sizing of the plot depends on:

• the climate conditions (exposure of the area where the stationary cryogenic tank is positioned);
• the characteristic (number and capacity…) of the cryogenic tank/s to allow the calculation of the maximum load to be borne;
• the characteristics of the ground;
• any seismic activity in the installation area;
• the traction, compression and shearing forces and the maximum total torque that could remove or tip up the cryogenic tank;
• site specific conditions;
• the possibility for rainwater drainage and the availability of collection systems.

The height should allow the correct fixing of the cryogenic tank using suitable anchor systems guaranteeing stability in both normal conditions and in the event of an earthquake.

Unauthorized vehicles should not be permitted to stop, even temporarily, anywhere in the area intended for transfer operations. The sizing of the area and flooring of the unloading area and the access routes shall consider the weight and size of the tankers used for unloading.

No manholes or air vents connected to the mains drains or other bordering rooms shall be in the area.

The plot should be protected by a fence to prevent tampering and accidental knocks.

The access gate, opening outwards and fitted with a lock, shall be positioned near the flanges used to transfer the gas to the cryogenic tank.

A water withdrawal point should be present near the area and should always be available for defrosting any components that so require.

At least two luminaires and an emergency lighting system should be installed to ensure correct lighting.

The area where the stationary cryogenic tank is installed should be equipped with an industrial, type 380V/63 A three-phase electrical power socket with earthing.

The area intended for unloading the mobile tank should also be equipped with at least one 220V/16 A single-phase electrical power socket.

All the metallic components installed should be earthed and there shall be a specific point for earthing the mobile refilling tank.

The stationary cryogenic tank should be equipped with all the required operating and safety systems.

8.2.3A Identification of levels and alarms of the stationary cryogenic tank

To identify the reference levels and pressures, the following parameters should be determined:

CMG = average daily consumption of the cryogenic storage system, calculated considering the consumption of the containers, consumption due to the distribution system and storage plants.

TL = logistic time, defined as the hours required by the nitrogen supplier to carry out emergency refilling.

CMG% = C/(CMG*100) average daily consumption percentage

TL% = Logistic time percentage (TL/24)*CMG%

(a,b) = coefficients defining the optimal operating interval of the tank pressure.

Figure 2 shows the levels and pressures related to the operating thresholds and the start of the operating volume reset cycle.

figure 2 Levels and pressures related to operation

The standard levels and pressures are (see figure 2):

• Level 1: refilling level [TL% + 5 (CMG%)] should be such as to ensure a residual autonomy of at least 5 days. The gas supplier shall define the update of the refilling threshold in their own procedures, according to the design data and subsequently the consumption data measured by the system; when the threshold is reached, the nitrogen delivery procedure shall be activated.

This value should necessarily be higher than the pre-alarm level activation threshold.

• Level 2: pre-alarm level [ 3 (CMG%)]: guarantees three days of autonomy;
• Level 3: alarm level [ 2 (CMG%)]: guarantees two days of autonomy.

The standard pressures are:

• Pressure A: working pressure (PE);
• Pressure B: alarm pressure a* (PE);
• Pressure C: alarm pressure (PE)/b.

The value of the coefficients a, b are agreed between the supplier and the client.

If it is impossible to comply with the indicated criteria, alternative solutions to be adopted should be agreed and documented with the contractor.

Table 1 gives the filling and pressure level thresholds with the consequent actions to be carried out by the contractor and the supplier/maintenance engineer.

table 1 Filling and pressure level thresholds

8.4.4.A How to convey outside the discharge point of gaseous nitrogen

Any external discharge points is used to be positioned far away from:

• work environments;
• personnel transit and stopping points;
• drainpipes;
• entrances to buildings, tight and confined spaces, air conditioning system intake points, etc.

The size of the external pipelines should not compromise the operation of the thermal safety valve, for example causing a loss in flow rate.

The discharge point should be designed to prevent accidental or intentional blockages or the accumulation of rainwater, other debris or the entry of insects.

8.7.1.3.A Environmental and ventilation system

The worst case condition could be, for example:

The simultaneous release of all the liquid nitrogen contained in:

• one or more cryogenic containers,
• one or more of the other equipment permanently connected to the cryogenic pipeline;
• Release in one or more pieces of equipment not permanently connected to the cryogenic pipeline and present in the rooms;
• Release due to leaks or breakage of the cryogenic pipeline.

Room management guidelines

In the absence of standards for the management of the cryogenic room here are reported some recommendations for their management.

B.1 Access controls

Access to the cryogenic storage system shall be limited through suitable procedures and/or devices, exclusively to authorized personnel.

The cryogenic storage system shall be equipped with a system for monitoring and recording accesses, and it is also possible to implement a video surveillance device.

B.2 Safety signage

The cryogenic storage system shall be circumscribed and delimited by signage giving safety warnings, mandatory instructions, warnings and prohibitions; the signage should be placed in a clearly visible position.

The main signs that could be affixed include, but are not limited to:

• Access prohibited to unauthorized persons
• Risk of asphyxia due to under-oxygenated atmosphere
• Mandatory use of personal protective equipment (PPE)
• Leave the premises in the event of an under-oxygenation alarm
• Danger of cold burns.