Various types of contemporary research, including genomics and regenerative medicine, have been supported by biobanks and biorepositories over the last thirty years. Within these sample storage repositories, specimens are placed in a sterile glass or plastic vial and immersed in vapor phase Liquid Nitrogen (LN2) to ensure the integrity and viability of the specimens over prolonged periods.
Sample integrity is extremely important and so leak-free cryogenic vials are essential for ensuring this. It is critical that the sample does not leak from the vial and that no contaminants can enter the vial so the closure should be robust enough to ensure this. Samples will be rendered useless for further research if the specimen is contaminated.
Problems Associated with Leakage of Cryogenic Vials
Sample evaporation can occur if the seal of the cryogenic vial is not hermetic, meaning that the sample may be “lost” or the pH of the sample may be changed, rendering it damaged.
Furthermore, as the vial is placed in ultra-low temperature storage (< -100 °C), if there is a leak around the seal then a vacuum will be created inside the vial. This is because the low temperature causes the contents to contract. This vacuum will cause LN2 to be drawn into the vial if there is a leak around the seal and the vial is accidentally submerged in liquid phase LN2.
The LN2 returns to room temperature once the sample has been taken out of storage. It expands to 3000 times the liquid volume and causes the vial to explode. This causes three problems:
1) It poses a safety hazard to the researcher due to shards of glass or plastic.
2) It creates a potential biohazard as the specimen is thrown around the room.
3) The specimen is lost along with many years of priceless research.
The final problem associated with leakage of cryogenic vials is that contaminants may enter the vial if the seal is not intact, rendering it useless.
Methods of Testing for Leakage of Cryogenic Vials
Before being placed in LN2 storage, the weight of a cryogenic vial containing a specimen is taken. It is then regularly re-weighed over a long period to assess whether there is any loss of sample due to evaporation. This process provides a good indication but it is very time-consuming.
Once the capped vial has been placed inside a capsule it is sealed and the vials are subjected to a pre-set pressure. Measurements are taken over time to detect any pressure leaks and to assess the seal integrity. Below is a typical method used for carrying out this test.
Figure 1: To the left the pressure capsule can be seen, with an adapter for a particular tube size inserted. At the back (right) the lid to the pressure capsule can be seen. In front are other adapters for different tube sizes
Ziath Test Procedure – Pressure Method
FOR TEST Model ET99W
Proprietary algorithms are employed by this instrument to measure the decrease in pressure over time. It provides fast and accurate results and can work in over-pressure or applied vacuum mode with a precision of 1 part in 100000.
Figure 2: FOR TEST Universal Leak Tester Model ET99W
Once the capped CryzoTraq™ Cryogenic Vials have been placed inside a steel pressure capsule, this is sealed, and the vials are subjected to a pre-set pressure (1000 millibars/100kPa).
Compliance with IATA Packing Instruction 650 is ensured with this pressure -“receptacles and packaging filled and closed under an absolute atmospheric pressure lower than 95 kPa”1. Tests are then carried out on the tubes at room temperature, -80 °C and 55 °C. The seal integrity is measured by monitoring pressure leaks from the vial over some time. The maximum acceptable pressure drop is 0.2 mbar.
A record is made of any pressure loss and vials are discarded if they fall outside the acceptable limits.
Figure 3: End of Leak Test result. The display shows the date and time of the test, the pressure loss recorded (Drop -0.064 mbar) and the pressure at the end of the test (625.721 mbar). The green "PASS" light shows the pressure drop value has fallen within acceptable limits (in this case <0.2 mbar)
Figure 4: 2 ml internally threaded cryogenic vial
Figure 5: The caps have an innovative 2 step injection moulding process design
Samples are very precious and a lot of work goes into them. At Ziath, many years’ experience of sample management within the laboratory has taught the company this. As such, its cryo-tubes are rigorously tested for leaks and integrity.
The CryzoTraq™ screw-cap has a compression ring (silicone) moulded into it through a unique two-step injection moulding process. This removes the need for an o-ring. O-rings provide a challenge when it comes to leak-free seals as they can constrict on freezing and potentially fall out of the cap.
Support and expertise comes as standard with Ziath so if clients have any queries about sample storage or sample tracking, then please contact our friendly and knowledgeable team.
1. IATA Packing Instruction 650, applicable to UN 3373 on passenger and cargo aircraft and ICAO
About Ziath Ltd
Ziath specializes in instrumentation control and information management in both the academic and the pharmaceutical/biotech industry sectors with a focus on the application of laboratory automation. In particular, we focus on managing large sample libraries (compound management, biobanking and sample management) using 2D datamatrix tubes.
Founded in 2005 by scientists and engineers; Ziath is over a decade old and is proud to server customers across the world. Ziath develops innovative new products designed to simplify processes in life science organizations. In addition Ziath offers consulting and contracting work to clients.
Ziath has a range of products split into four main categories of 2D barcode scanners, devices for handling tubes, 2D barcoded tubes and sample management software.
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