Assessment of cryogenic sample tubes for biobanking applications

sample integrity - work safety - aging

⇒ Tightness tests according to real conditions of transport and storage

⇒ Differentiated and quantitative results

⇒ Long-term storage simulations

In course of technical progress and increased lab automatization the trend in the cryogenic storage of biological samples is moving towards longer storage periods and an increasing sample count. While modern biobanks were designed for a maximum storage capacity of 10.000 – 20.0000 samples a few years ago, today storage capacities of more than 20 million samples are realized within the scope of major cohort surveys for public health research. Even the banking of autologous stem cells for personalized therapies requires a lifetime storage of vital samples.

The banking of medical samples makes highest demands on the cryogenic sample packages. The quality of the medical samples or the vitality of cryopreserved cells has to be guaranteed even after a long term storage or time-consuming transport operations. This applies to every irretrievable sample and thus, defines specific requirements to every single sample tube.

We can check whether your package will meet these demands. Our test methods enable a quantitative evaluation of the product quality which provides the basis for a comparative assessment by the user. Our tests can also reveal material weaknesses and design problems. The tests can provide important indications for specific limitations in use of cryogenic packages and the possibility of constructive improvements. So we contribute to ensure that your samples withstand storage and transportation save and without a lack of quality:

By tests of transport safety we prove the qualification of cryogenic packages for the transportation by road and air in compliance with the latest guidelines for the transportation of biological samples. Our CO2 tightness test determines the risk of a sample contamination during the shipping on dry ice.

Within the scope of storage ans safety tests we prove the tightness and material durability of the packages under cryogenic storage conditions. The focus is on the physicochemical behavior of the material during the cooling and rewarming process as well as the resistance against the intrusion of liquid nitrogen into the packages which is related to a serious risk of bursting in the thawing process.

By means of aging tests the resistance of the packages to long storage periods is examined. After the simulation of a cryobanking scenario with a storage period of up to 30 years the packages undergo different tests to prove the material aging in a quantitative way. In the simplest case, this could be a leak test or the determination of the burst pressure. Furthermore, tensile strength, compressive strength as well as the permeability and tightness of the closure system can be measured in relation to the temperature to clarify the causes of the aging process.

Leaching studies identify the potential risk of a sample contamination with leachable components. Those components may be released from the plastic of the sample vial and closure. Even technical additives from the production process, like residues of detergents and lubricants, can be absorbed by the sample material.




Transport safety tests

Gravimetric leak-tightness test IATA/ADR

During transportation by road and air, sample packages are exposed to changes of the physical conditions. On the one hand, serious temperature fluctuations may occur. On the other hand, during air transport the samples have to resist a pressure reduction of the cargo space as well as the risk of large pressure drops. Both can lead to a drastic increase of the pressure difference across the wall of the sample package and thus, lead to a loss of tightness. This may either cause sample contamination or a risk for the public safety in case of a release of potentially harmful sample material.

Transportation of biological samples by air and by road (left side) and proof of the biological safety by a gravimetric tightness test in a vacuum chamber.

The gravimetric leak-tightness test is designed to proof the transport safety of biological samples in accordance with the IATA (International Air Transport Association) guidelines and the guidelines of the ADR (European Agreement concerning the International Carriage of Dangerous Goods by Road). The sample vials to be tested are filled with a volatile test substance, sealed according to the manufacturer’s specifications and refrigerated. Subsequently, the vials have to pass a test routine in a vacuum chamber which implies the application of specified changes of pressure and temperature. At a differential pressure of 95 kPa the sample temperature increases from −40 °C to +55 °C. The test simulates the highest physical stress to the sample vials which can be caused by changes of the environmental conditions during air transport. If a loss of test substance can be determined by differential weighting, there is a risk of tightness loss during transportation and the sample package does not receive a transport approval.

Proof of sample contamination during the shipping on dry ice

Despite of different alternatives, even today the transportation of frozen samples is carried out in polystyrene boxes in direct contact with dry ice in many cases. Because of its low costs and high heat capacity even a multi-day shipment in a non-cooled environment is possible with low expenses. On the other side there is a serious risk of an accumulation of carbonic acid in the sample material if the package gets leaky. By sublimation of the dry ice a saturated CO2 atmosphere is formed in the transportation box already after a short time. Contemporaneously, there is the risk of a decreasing sealing efficiency of the sample vials caused by the negative heat expansion during cold transport which leads to a contamination of the sample package with carbon dioxide. After the thawing of the sample this can lead to a drastic pH decrease which may cause serious sample degradation.


Simulation of the transportation process by incubation of a sample rack with 96 sample tubes on dry ice in a saturated cabon dioxide atmosphere (left side) and quantitative measurement of the carbon dioxide accumulation (right side).

Therefore, by use of our CO2 tightness test we determine the CO2 absorption of the sample material after the simulation of real life shipping conditions. As well as the gravimetric leak-tightness test, the CO2 tightness test gives a quantitative test result for each individual sample tube. Based on the gathered data, differentiated statements on the performance of the vials can be made. This provides the manufacturer with the basics for quality assurance and a targeted quality improvement.

Different pattern of carbon dioxide accumulation for each rack with 96 sample tubes of different manufacturers after a shipping simulation on dry ice provide information on the nature of the tightness problem: Type A - vials tight by design, minor variations of quality might be production-related; Type B - significant problem of the sealing behavior.




Quality assurance for cryogenic long term storage

Proof of thightness against liquid nitrogen (LN2)

In many biobanks, the sample packages are stored in the gaseous phase of liquid nitrogen. At a storage temperature of −130 °C and below, this storage method enables an almost unlimited lifetime of the samples without a loss of vitality in case of cells and tissue or a quality degradation of biological sample material. But in many cases, the sample packages are exposed to liquid nitrogen during storage, intentionally or not. Especially in small labs, cryogenic sample vials are often banked in LN2 to enable a rapid cooling of the samples or to use the storage capacity more efficiently, even if they are not specified for the field of application. Any unwanted overfilling of the storage tank or unintended opening of a sample holder during loading or removal of the samples could lead to a direct contact between sample package and liquid nitrogen, too. In those cases the sample tube should be tight because the penetration of LN2 into the package can cause severe problems:

  • Risk of sample contamination in case of permanent leakiness

In case of a permanent failure of the sealing because of the drastic cooling, liquid nitrogen penetrates into the sample vial. Despite of all safety measures, dangerous germs like bacteria, viruses and mycoplasma can intrude into the sample with the LN2. Even the cross-contamination of samples by a transmission of sample material is possible. This may lead to the transmission of infections or – e. g. in case of DNA – to the falsification of sample properties.

  • Occupational safety risk in case of a secondary seal up

In case of slight leakage the rewarming of the sample during removal from the storage tank can completely regenerate the tightness of the sample tubes. In this case, an evaporation of the accumulated nitrogen within the package can result in an extreme pressure rise which causes spontaneous bursting of the tubes during the removal procedure or opening of the vials. This may not only destroy other samples but it can also endanger the personnel.

Cryogenic storage of cell material in the gaseous phase above the liquid nitrogen at the European Stem Cell Bank (left side) and simulation of a direct contact of the sample packages with liquid nitrogen in the test lab (right side).

For the tightness test, the sample vials are filled with two porous microgranules of different color in form of a specific bi-layer arrangement. After several hours of incubation time in LN2 an analysis is done. The combination of the results with the outcome of an additional test for residual pressure allows a precise assessment of the sealing behavior. Thus, a potential safety risk can be evaluated with sufficient reliability.

Different results of the test substances within the sample tube after concact with liquid nitrogen and residual pressure test: only one of the tested tubes is tested as permanently tight (second from right).

The combination of residual pressure and visible blending effects enables a differentiated evaluation of the sealing behavior for each individual package. The statistical evaluation of the observed effects may even help to reveal the causes for existing quality problems.


Comparative assessment of a tightness test for 96 sample tubes of two different manufactures after the incubation with liquid nitrogen: Type A - slight tightness problems for a high sample count, high risk of bursting because of the expansion of the nitrogen within the reclosed sample package; Type B - large shares of either tight or permanently leaking vials, high danger of sample contamination but low risk of bursting.




Assessment of aging effects at long storage periods

Large biobanks already exist since the end of the 20th century. Meanwhile, the first preserved batches achieved a storage time of more than 20 years. Current medical cohort surveys are designed for a storage period that considerably exceeds 30 years in many cases. Even the storage of autologous stem cells (cord blood) for therapeutic use requires a lifelong storage time. Thus, the question of the long-term stability of the packages used and their contribution for the preservation of a long-term sample quality in a cryogenic environment is constantly rising.

Our aging test allows to forecast the aging behavior of the sample package during long storage periods which may have influence on the working safety and the quality of the samples. It is based on the perception, that the cryogenic storage itself is not the key factor for a hidden aging process of plastic material and closure system. At temperatures of −130 °C or below the aging of the sample material and package stops nearly completely. In contrast, there is a critical influence of repeated variations in temperature stress which lead to material fatigue due to phase transition effects. Interruptions of the cryogenic storage for the removal of specific samples means thermal stress for all sample vials of the related rack and thus, can drastically accelerate their material aging.

Test chamber for the simulation of thermal material aging by repeated tehrmal cycling (left side) with liquid nitrogen inlet system for sample cooling with a cooling rate up to 100 K/min (right side).

To reproduce the stress to the material with respect to aging in the best way, our aging test simulates the repeated load and unload cycles of typical biobank scenarios. The test conditions can be adapted to the real conditions of charging and storage as well as the removal scenarios of real biobanks. Heating rates of up to 60 K/min and cooling rates of 100 K/min enable a simulation of hundreds of removal cycles within a short time. Subsequent to the simulated long-term storage potential weaknesses of the cryogenic sample tubes can be safely revealed by comprehensive material examinations and tightness tests.

The comparison of physicochemical properties between brand new and thermally aged sample tubes allows a qualitative and quantitative assessment of the aging of cryogenic sample tubes and other packages. Besides different tightness tests, a range of further test methods is available to generate a differentiated evaluation of cryogenic packages:

  • Thermal analysis

Determination of critical physical states of the packaging material during the passage of the temperature range between sample receipt and cryogenic storage in the biobank. Phase transitions of the plastic have the potential to change physical properties like mechanical stability or the permeability for gases within a certain temperature range in a drastic manner.

  • Temperature-dependent permeation tests

Permeation tests are designed to evaluate changes in the gas tightness of wall material and closure system of the sample vial. The test can be done at different temperatures, e. g. at the loading temperature and storage temperature in the cryobank or in critical temperature ranges with respect to the material (glass temperatures and phase transition temperatures of the relevant polymers).

  • Temperature-dependent tensile testing and evaluation of the bursting pressure

The bursting pressure test determines the influence of the material aging to the pressure resistance of the sample tube. The method enables the detection of the internal pressure which results from the volume expansion of intruded liquid nitrogen or condensed and accumulated air components (oxygen, carbon dioxide) and would destroy the package in the rewarming process. The resulting damage pattern can be evaluated microscopically. The examination of tensile strength and compressive strength at different temperatures allows an exact characterization of the mechanical behavior of the packaging material in the cooling process by means of different characteristic values.




Leaching studies

In the manufacturing process of plastic ware, raw materials, semi-finished and final products get into contact with auxiliary materials in a number of ways. Residuals of lubricants, release agents, detergents and solvents can remain on the material surface and pass over to the sample during storage. Especially in case of sample tubes for cells or medical examination material, this is very problematic. Leachable components of the plastic or chemical residuals from the production process may affect the viability of cryopreserved cells and falsify the examination results of medical samples.

In the course of the leaching analysis soluble residuals are extracted, concentrated by modern chromatographic methods and subsequently analyzed. In this way, even the slightest traces of leachable components and problematic residuals like amides, esters, aldehydes or fatty acids within the material or at the surface of the sample vials can be detected reliably. A negative test result can proof the safety of the package for the cryogenic storage of sensitive samples under the aspects of its chemical purity.