September 15, 2011 (Vol. 31, No. 16)

Josh P. Roberts

Tips and Tricks to Optimize Preservation and Monitor Biorepository Specimen Handling

The need to preserve biological material—whether as a reference archive, a repository for future research, for potential therapeutic use, or as the basis of ongoing drug discovery or production—seems to be growing at an exponential pace.

From the collection and storage of umbilical cord blood to preservation of transgenic mouse tails, researchers and clinicians alike are constantly faced with challenges of conflicting, confusing, and ever-changing regulatory oversight, expensive cold storage chain logistics, demanding colleagues, unreliable data collection, and unidentifiable samples.

Yet with challenges come innovations—many of which were discussed at the “World Biobanking Summit,” held as part of the inaugural “European Laboratory Automation” conference.

Identification and validation of targets, identification of drug compounds, and identification and development of biomarkers are all done on biobanked tissue, said Antje Stratmann, Ph.D., global biomarker research at Bayer HealthCare.

The company has a centralized repository where biological samples from animals, disease models, patients, and healthy individuals are housed.

The biobank acts like a bank safe, with a constant supply of samples being deposited and withdrawn. It provides high-quality samples and their associated comprehensive clinical dataset for bioanalytics, both internally and to Bayer’s international network of collaborators.

This all has to be under strict compliance with legal, ethical, biosafety, biotechnology, standard operating procedures (SOPs), data privacy, and IT guidelines, Dr. Stratmann noted. “And the centralization insures best practice and, in particular, compliance.

“It makes no sense for each project leader to take responsibility on his own to get this tissue, because you also need the clinical network.”

To assure that the best use is made of the centrally banked tissue, Bayer has instituted a three-tiered decision tree to determine access. The request value is judged by biobank staff based on defined criteria in order to avoid conflicts.

Green-lighted tissue—the easiest to obtain, such as healthy control blood samples—can be used for just about any project, including optimization of IHC process. Yellow-lighted tissue—“the vast majority of our biobanks”—can be used only for established protocols. And red-lighted tissues—specific studies from the company, for example—are considered the highest value and are restricted to particular projects.

Bayer HealthCare has a centralized repository where biological samples from animals, disease models, patients, and healthy individuals are housed.

For the People

The mandate of public biobanks is to provide access to a variety of different lines grown and maintained under strict quality control standards. They can act as an intermediary between researchers who might otherwise be asked to provide tissues and cells directly to their collaborators—shouldering the responsibilities of preservation, data collection, privacy, regulatory, and IP issues.

“First, we obviously want to preserve pristine, early passage stocks against any potential future accidents where you might lose the cell lines or they might undergo permanent, irreversible changes,” pointed out Glyn Stacey, Ph.D., director of the UK Stem Cell Bank (UKSCB).

Biobanks are charged with guaranteeing the authenticity of reliable, well-characterized, uncontaminated cells that are not mixed up with other cell types or switched with other cell lines.

It’s important to provide a direct link between the stocks of cells preserved in a freezer and the growing body of data associated with them. Information provided in the form of feedback from researchers as well as from periodic literature reviews is added to the cell’s documentation.

It’s vital to have a complete history of cells destined for human application, Dr. Stacey continued, including the method by which they were derived and maintained, all the way back to informed consent, “so they can go forward for clinical application with confidence in low levels of risk.”

The UKSCB can carry out independent verification of the findings of other laboratories. Patent repositories—the UKSCB is one—have for decades maintained the stocks required to check the veracity of a patent that is dependent on a particular culture.

Accepted stem cell lines must fulfill a series of acceptance criteria that cover most of the common features found in cell culture testing. These include being free from mycoplasma, bacteria, and fungi, as well as tests for viability and morphological examinations. And they must be free from common viral pathogens.

In addition, cells are assayed for a series of differentiation markers—“not essential for depositing in the bank,” remarked Dr. Stacey, but it’s useful “to know that these cells that have been sent to us are typical human embryonic stem cells, and they’re not deficient in differentiation capacity.”

Dr. Stacey explained that storage is maintained at three different levels, “which is quite normal for a tissue-banking environment”: quarantine, for new material that comes in untested; in-process, for which a bank of cells has been produced but testing is still under way; and long-term, where the culture is judged to be pathogen-free.

Controlled Collection

Not only is it important for tissue banking to be tightly controlled, but obtaining tissue samples and their associated data should be highly standardized as well. You have to “make sure you collect tissues that are representative of what’s going on in the body, not what’s going on during the collection process,” said Hartmut Juhl, M.D., CEO of Indivumed.

Perhaps 1,000 omics papers have been published, but not a single one defines exactly the same group of markers, Dr. Juhl noted. This is because biobank tissue has a handling history. Tissues change with the environment: they’re affected by oxygen, temperature, and pH.

Patients are given a host of different treatments before and during surgery, and these can be reflected in excised tissue as well. As an example, he pointed to a case in which a sequela of atropine administration was mistaken for a tumor biomarker.

Indivumed focuses on understanding the heterogeneity of tumors, and the differences of how patients respond to treatments, to help guide therapy. To this end, they maintain a biobank with about 14,000 tumor samples—with about 2,000 new cases added per year—each collected under very exacting specifications.

The company goes into the operating room with its own trained staff to collect blood and tumor for fixed, fresh, and frozen tissue samples, allowing for no more than seven minutes cold ischemia time. Three hundred clinical data points per patient—including 112 different compounds that may be seen before processing—are documented.

The company also works on finding protein biomarkers: what and how much is expressed, what post-translational modifications it displays, and the like. In general, it’s not worth bothering if you don’t know the stability of the protein you’re looking at, Dr. Juhl said. Take mitogen-activated protein kinase, for example: “if the sample is older than 10 minutes, you measure rubbish.”

A second goal of Indivumed is to find a way to correct data that was obtained in less-than-optimal ways, Dr. Juhl noted. But, he admitted, “this is still far away.”

Destroy the Matrix

KBioscience isn’t a biobank in the traditional sense: “We destroy the matrix,” explained sales director Niels Kruize. Rather, blood, tissue, saliva, and other biologicals including agricultural samples such as seeds, are processed into DNA as quickly as possible, and it is this that is banked for the CRO’s customers.

Sample collections are often larger than the customer can hold and need to be condensed, normalized, and standardized. Sometimes they come in with labels that are wrong or just can’t be read.

Among the first things KBioscience does is separate these out and apply common barcodes to all the tubes and plates they get in. Samples are then transferred to individual barcoded tubes. “That’s a critical step,” Kruize said. So critical, in fact, that they dedicate two people to the process, “to make sure that you get the right sample into the right tube.”

Samples are mixed with buffer and silica, and as such can remain stable at room temperature for weeks. There is no need for a precipitation step during purification. Eluted samples are tested—including a 220–350 nm scan to test for purity, PCR-based genotyping, and customer-specific assays for quality control—and aliquotted into three portions.

Of the eluted DNA, 80% goes into the 2-D barcoded tube, and 10% goes into plates that are shipped back to the customer. The last 10% is frozen down at -20°C and stored at KBioscience for future SNP genotyping.

The tube with the remaining silica resin, still containing a small percentage of the DNA, is dried and then taped to and stored along with the original sample tube and housed off-site for long-term storage. All fractions remain the property of the customer.

Every step along the way is controlled and documented in the company’s custom LIMS. But rather than automating the entire process, which Kruize explained would have been too expensive and error prone, “we buy simple plastic trays and we label them per project. Individual plates and tubes go in these labeled barcoded trays and can be retrieved at any given time.”

Within days of the conference, it was announced that KBioscience had been acquired by LGC and will join with LGC’s genomics division.

Cohorts of extracted DNA samples used for subsequent SNP genotyping at KBioscience

Extremeophiles Turn Up the Heat

Shadow storage can guard against a catastrophic failure. But that doesn’t change the fact that “it’s very difficult to handle cold-chain logistics,” pointed out Rolf Muller, Ph.D., CSO of Biomatrica. “This goes for the life of the sample from collection, transport, and analysis, to storage.”

Freezers are very expensive to operate, equipment-intense, unreliable, and environmentally unfriendly. Dr. Muller thinks it’s time for a paradigm shift—for scientists to get past the “paranoia” of the last 100 years of thinking that the only way to protect our samples is to stick them in the freezer.

There are extremophiles called anhydrobiotes that can survive in a dry state without refrigeration, preserving their DNA, RNA, proteins, and cell membranes intact. Biomatrica set out to mimic this process in a laboratory setting.

Beginning with large libraries of potentially stabilizing molecules, the company utilizes “screens to define the best way to stabilize these biological materials at room temperature,” Dr. Muller said. Among the company’s offerings are products that essentially turn the solution into glass and “shrink-wrap every single biomolecule in the sample.”

More recently, the company has developed room-temperature liquid stabilization tools based on osmolytes found in organisms around thermal vents—“molecules that prevent the degradation of proteins in hot environments.”

Stabilization in liquid has the advantage that the sample needn’t be dried down or speed-vac’d. The sample is stored or transported just like traditionally frozen samples, only they needn’t be put in the freezer or shipped with dry ice.

Having samples at room temperature can facilitate a lot of projects that are ongoing in Third-World countries where samples can be collected in fairly crude locations and crudely done, explained Rod Westrop, Ph.D., director of European operations for GeneVault (acquired in February by IntegenX).

“Biomatica’s products, we believe, will aid that process by stabilizing the point of collection so that you’re getting good quality material coming through.”

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