Cold Chain Best Laboratory Practices for Biobanking

Cold Chain Best Laboratory Practices for Biobanking
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I always understood that a biobank is not an investigation laboratory practice but rather an investigative support service. For this reason, process control is a requirement for any installations where biological samples are collected, processed, stored, and distributed.

To have a system that controls and audits products, processes, and equipments is the basis in our labs to offer our clients, who are investigators and biological researchers. Our fundamental target is to achieve reliability, uniformity in research projects, reproducibility and data consistency, and quality in every test conducted with biobanked samples.

Sometimes diseases are associated with very small changes in the nucleic acids and other metabolites, and using reliable samples to study such biomarkers becomes crucial. Without proper protocols, the integrity and viability of these samples can often be at risk. Small variations in sample handling and temperature can affect the reproducibility of basic and clinical research, particularly in rapidly advancing fields like metabolomics and next-generation sequencing (NGS), etc.

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At biobanking, the sample life cycle does not start when the sample comes to our labs but when we develop the sample recruitment plan. Harvesting technique, stabilizers, and the rule of the three T (Time, Temperature, Technique) when transport, process and store them play a crucial role in determining the final quality of distributed samples. You can imagine that, in the hake manufacturing chain, the process is not controlled from the moment, the raw material is caught in the biological biobank of Terranova until it reaches the supermarket shelves.

Sometimes, one of the most forgotten steps could occur at that time when samples are into our facilities and become manipulatable during processing, aliquoting, and storing. During sample processing, as far as possible without unnecessary holding/wait times to obtain the best “instant photo” of the analytes more labile to temperature, or other effects due to natural degradation processes with deleterious effects like a cellular response to mechanical and thermal stress or metabolite light exposure, and oxidizing or chemical degradation.

The more we advance in technology, the more challenges we present to have to preserve the sample integrity during all stages of the complete life cycle of a biological sample: collection, transport, process, storage, selection and distribution and even when researchers use our product must observe the same controls to have a good “raw material.”

Although, that is not to say that to freeze is not synonymous with stabilizing: many enzymes could stay working at lower temperatures: RNAse A (-90°C), Ribonuclease A (-56°C), or B-glucosidase (-70°C), amongst others. Perhaps the only way to stabilize a sample is to keep below glass water transition temperature: -136°C, but it may not be cost-effective for some samples. It is very important to understand because when we store a sample in our freezers, the control must be even more precise than audit trails, verifications, or calibration of freezers. I refer to what took place in all-day events, somehow so-called “24/7 events”.

These are what happens in our routine work: door openings, sample-box extraction, sample storage of fresh samples together with things already completely frozen, sample pick-up to send to clients. These manipulations have a real impact on the quality of samples shipped that could be hard to evaluate. I do not wish to comment further on some samples stored during years with control of processes and quality.

It looks like having the Diogenes Syndrome, a disorder of compulsive harvesting thinks without a plan to use, and recalls non-compliant products. Diogenes syndrome is a very widespread “illness” in some biobanks, whose need to fulfill stakeholder requirements and are slaves of indicators of their quality management systems, forgetting that they cost a lot of money with any returns for anybody.

One of the most prevalent “24/7 events” could be opening doors to pull out a sample box and put in new fresh samples together with other frozen samples. Quite often every day, this routine forces the ones neither used nor touched, going along for the warming ride. These samples may take this ride countless times throughout their storage lives, and our goal must be to ensure excessive temperature excursions do not damage them in the process. But, how extend these deviations are?

Inspired by a publication of Brooks Automation LTD at the ISBER 2016 conference about these concerns, I designed similar experiments related to -80°C freezers. In a box of 2-D microtubes of a well know supplier. I placed only two tubes of 0,75 ml filled with 0,4ml of fetal bovine serum (positions 1 and 44) with a thin wire thermocouple submerged into and connected to a datalogger (Testo 175-T3). The sample box was placed in the front position of the rack, closest to the door of the freezer. When it was stabilized at those of the freezer temperatures, I extracted them from the freezer. I placed them in the tabletop without any protection, lasting for one minute until I returned.

Results are very frustrating: while the freezer reaches the set point in a few minutes, samples go warm-up several degrees for a long time (figure 1). Their thermic inertia was greater than expected, more than 10-15°C for 30 minutes depending on the position of sample probes. When the cycle is repeated continuously, then the deviation shows accumulative. This transient warming-up is a serious concern in the life sample cycle and could be detrimental to its quality.

Cool Chain Best Laboratory Practices
Figure 1: image shows how front located samples warms up when open the doors until they are again closed (yellow arrow). ULT probe was submerged in a falcon tube filled with glycerol and rear located.

Temperature related problems were not stopped here. The next step was to study how to draw out a box to manipulate, pick up, or store samples on a daily basis cycle. At this point, I used a 96 well box with 64 positions occupied with filled tubes and samples with probes located at positions 44 and 49. This configuration provides some protection for the controlled tubes and more inertial mass into the box. For testing purposes, the box was pulled out for one minute and lasted for ten minutes into the freezer for several cycles. The box was in the tabletop without any protection as a foam lid with dry ice and was not manipulated in any way (touched, opened).

Even worse, temperature spikes were moved away 30°C from the set point in the “un-protected” box. But when dry-ice was used in the cycle, only deviations were 8°C at the first cycle and no more than 5°C in the second and third cycle; the temperature was maintained in a shorter interval of 15ºC below the set point (figure 2).

  • Key findings suggest that the qualification and temperature logging of freezers is not the most informative way of trace the life cycle of a sample.
  • Samples exposed to room temperature never should be re-exposed until their warm-down and always must be considered the use of “cryoprotection” (dry-ice) when manipulates.
  • Specific programs should be implemented to facilitate the control of all the steps in the sample life cycle, also during processes and storage, and above all on the maintenance of the cold chain.

Taking the cold chain process into account can minimize production deviations and final quality assessment, and therefore minimize product rejection and overall costs.

Cool Chain Best Laboratory Practices
Figure 2: Temperature excursion of samples with external dry-ice protection or without protection.


To Banco Nacional de ADN-Carlos III (Spain), where experiments took place.


(1) Malm et al., “Developments in biobanking workflow standardization providing sample integrity and stability.” Proteomics. 2013(16):38-45.

(2) Hubel et al.,” State of the art in preservation in a fluid biospecimen.” Biopreservation and Biobanking, 2011(3):237-44.

(3) John Fink et al., (2016).” Sample warming during innocent exposures from an LN2 freezer: Comparing temperature, time and workflow using manual vs. automated systems”. Conference papers at International Society for Biological and Environmental Repositories (ISBER)

Francisco was the manager at the Cellular Culture and verification and control of equipment departments, Banco Nacional de ADN-Carlos III, Salamanca University in Spain (2005-2020). He has spent his career working in Molecular Biology, providing research support services (biobanking area), designing, implementing, auditing and working at Salamanca University research laboratories. He actively participated in the National DNA Bank since it was created, performing validation, control and calibration tasks on all biobank processing equipments. Currently, he is working on combining his specialization as certified professional on Biosafety Management with laboratory integrated management (LIMS), sample database management and monitoring of processes in accordance with the standard ISO 9001:2015.