Gold standard method to preserve tissue for research or clinical use called into question

March 15, 2018, Johns Hopkins University
A method currently used by thousands of laboratories across the nation to preserve tissue could render samples useless over time for a common test to assess gene activity. Credit: iStock

A method currently used by thousands of laboratories across the country to preserve tissue could render samples useless over time for a common test to assess gene activity, a study led by Johns Hopkins researchers suggests. The findings, published in the November 2, 2017 American Journal of Clinical Pathology, could eventually lead to significant changes in how tissues are stored for clinical and research purposes.

For more than a century, biomedical scientists have been preserving some harvested from patients and research animals in the same way: by soaking these tissues in the preservative formalin and then embedding them in blocks of a wax called paraffin. More than a billion of these blocks containing formalin-fixed paraffin-embedded (FFPE) tissues are thought to be housed in labs around the world.

"We have a warehouse of these blocks at Johns Hopkins that go back to at least the 1918 flu pandemic," says study leader Angelo M. De Marzo, M.D., Ph.D., professor of pathology at the Johns Hopkins University School of Medicine and associate director of cancer research pathology at the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins.

The gold standard has been to leave these FFPE blocks at room temperature. Then, when laboratories are ready to use the samples, they cut thin slices of the embedded tissue and place them on slides to stain for the presence of various molecules. One test that's become increasingly useful is RNA in situ hybridization (RISH), which assesses the activity of particular genes. Those findings might be able to tell doctors or researchers whether certain viruses are present in tissue or how a patient's cells responded to particular therapies, for example.

Several years ago, De Marzo, who studies prostate cancer, and his colleagues noticed that prostate tissue samples that should have roughly the same amount of activity in specific genes had wide variations in those genes' RISH signals. "It occurred to us that the blocks stored for longer might be producing less signal because their RNA was degrading," he says.

To test that theory, the researchers collected 25 FFPE blocks containing tissue samples taken from radical prostatectomies of prostate cancer patients. When the researchers performed RISH on these slides to look at the activity of four prostate and prostate cancer-specific genes, they found a significantly lower visible signal in the older slides compared to the younger ones.

To confirm these findings, the researchers performed a similar test on tissue microarrays (TMAs)—multiple small cores of tissue embedded in the same block. This time, they used computer image analysis to get an unbiased assessment. Once again, the signal strength for the probed genes declined over time.

The researchers performed additional tests with TMAs containing tissue from human patients grown on mice—a technique called patient-derived xenografts, often used in cancer research—and using tissue samples sent from colleagues at a different medical institution. All showed the same age-weakening results.

However, when De Marzo and his colleagues compared slides that were flash-frozen at -20 degrees Celcius (about the same temperature as most commercial freezers) right when they were harvested from patients 10 years ago to adjacent tissue from the same patients that was embedded in FFPE blocks over the decade, they found a dramatic difference in RISH signal—while those stored in the freezer produced a strong signal for the probed genes, those stored at room temperature showed a markedly weaker signal. Additional tests showed similarly strong RISH signals for sections of tissue that were cut and placed on slides before being frozen for five years compared to PPFE tissues from the same patient that were stored at room temperature.

 Together, these findings suggest that storing tissues at room temperature could weaken RISH signals over time, a finding that could give misleading results. For example, De Marzo explains, RISH results might reveal low or no activity of targeted genes in older tissue samples even though activity of those genes was high at harvest.

"There's no reason to think that this isn't happening around the world with the millions of tissue samples that are harvested every year," he says. "These blocks are thought to be a treasure trove of data, but with every year that goes by, they might lose their effectiveness for this technique."

Although freezing does appear to preserve function for RISH, De Marzo adds, the long-term answer won't be to freeze every tissue —there isn't room or other resources to house these numerous samples. Thus, he says, researchers may want to start freezing select samples they predict to be especially useful rather than storing them at .

Explore further: Study finds no evidence of common herpes type virus in aggressive brain cancer tissue

More information: Javier A Baena-Del Valle et al. Rapid Loss of RNA Detection by In Situ Hybridization in Stored Tissue Blocks and Preservation by Cold Storage of Unstained Slides, American Journal of Clinical Pathology (2017). DOI: 10.1093/ajcp/aqx094

Related Stories

Study finds no evidence of common herpes type virus in aggressive brain cancer tissue

February 28, 2017
In a rigorous study of tumor tissue collected from 125 patients with aggressive brain cancers, researchers at Johns Hopkins say they have found no evidence of cytomegalovirus (CMV) infection and conclude that a link between ...

New method increases life span of donated brain tissue

March 9, 2018
Researchers at Lund University in Sweden have developed a method that enables them to use donated brain tissue from people with epilepsy for 48 hours. Previously, the researchers only had 12 hours to test new treatments before ...

Chronic inflammation linked to 'high-grade' prostate cancer

April 18, 2014
Men who show signs of chronic inflammation in non-cancerous prostate tissue may have nearly twice the risk of actually having prostate cancer than those with no inflammation, according to results of a new study led by researchers ...

BGI achieves next-gen sequencing analysis of FFPE DNA as low as 200 ng

March 8, 2012
BGI, the world's largest genomics organization, reported that it can use next-generation sequencing to analyze DNA as low as 200 ng from formalin-fixed paraffin-embedded (FFPE) samples. This advancement enables researchers ...

Liquid biopsy results differed substantially between two providers

December 14, 2017
Two Johns Hopkins prostate cancer researchers found significant disparities when they submitted identical patient samples to two different commercial liquid biopsy providers. Liquid biopsy is a new and noninvasive alternative ...

KRAS mutations linked to brain arteriovenous malformations

January 5, 2018
(HealthDay)—Many patients with arteriovenous malformations of the brain have somatic activating KRAS mutations, according to a study published online Jan. 3 in the New England Journal of Medicine.

Recommended for you

Study examines disruption of circadian rhythm as risk factor for diseases

December 11, 2018
USC scientists report that a novel time-keeping mechanism within liver cells that helps sustain key organ tasks can contribute to diseases when its natural rhythm is disrupted.

New light-based technology reveals how cells communicate in human disease

December 11, 2018
Scientists at the University of York have developed a new technique that uses light to understand how cells communicate in human disease.

Researchers explore new way of killing malaria in the liver

December 8, 2018
In the ongoing hunt for more effective weapons against malaria, international researchers said Thursday they are exploring a pathway that has until now been little studied—killing parasites in the liver, before the illness ...

Study may offer doctors a more effective way to treat neuroblastoma

December 7, 2018
A very large team of researchers, mostly from multiple institutions across Germany, has found what might be a better way to treat patients with neuroblastoma, a type of cancer. In their paper published in the journal Science, ...

Progress made in transplanting pig hearts into baboons

December 6, 2018
A large team of researchers from several institutions in Germany, Sweden, Switzerland and the U.S. has transplanted pig hearts into baboons and kept them alive for an extended period of time. In their paper published in the ...

'Chemo brain' caused by malfunction in three types of brain cells, study finds

December 6, 2018
More than half of cancer survivors suffer from cognitive impairment from chemotherapy that lingers for months or years after the cancer is gone. In a new study explaining the cellular mechanisms behind this condition, scientists ...

0 comments

Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.