Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Peering into human tissues—whether to study morphology, cell biology, biochemistry or disease—requires sections of biopsied tissue that have been specially prepared for long-term preservation. These samples are of two primary types, frozen tissue and formalin-fixed paraffin-embedded (FFPE) tissue. Each has its pros and cons, as well as applications to which it is best suited. The following guidelines can help you better decide which sample type to use and when.
The basics
Both FFPE and frozen samples can preserve specimens well, but which to choose depends on your application.
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FFPE samples can be stored at room temperature, which is convenient and cost-effective, and they work well for immunohistochemical staining and morphology analyses. FFPE is also widespread, and there exist vast archives of such specimens from which users can select samples. That said, formalin is toxic, and the fixation and paraffin-embedding procedures are time-consuming. FFPE samples are generally unsuitable for molecular analysis, and FFPE protocols are not standardized, so there’s no guarantee different samples were prepared in the same way.
Freezing has the advantage that it is fast, compared to FFPE sample preparation, and the resulting samples are well suited for molecular analysis. But as frozen samples deteriorate rapidly at room temperature, they must be frozen as soon after collection as possible, meaning the required materials must be close to the site of surgery. And they must be kept frozen, meaning you’ll need dedicated freezer space.
FFPE vs. frozen
Molecular analysis
For molecular analysis, including work with DNA, RNA and post-translational protein modifications (PTMs), frozen samples are preferable to FFPE samples for several reasons. The variability that nonstandardized preparation methods introduce into FFPE samples affects the integrity of molecular data disproportionately. “For example, some users leave a tissue biopsy in formalin for a longer period of time, which might not affect immunostaining much but is detrimental for any further molecular analysis,” says Michael Kazinski, senior director of molecular preanalytic technologies in sample technologies marketing at QIAGEN. Long DNA fragments, protein phosphorylation states and other PTMs are rarely, if ever, preserved in FFPE samples, for instance. And, because formalin inactivates neither RNases nor phosphatases, FFPE samples often contain degraded RNA and non-native configurations of phosphorylated proteins.
For subsequent molecular analyses, such as mass spectrometry, quantitative real-time PCR, next-generation sequencing and Western blotting for PTMs, frozen samples are a necessity. “Frozen sections are considered the gold standard for most molecular assays, especially [for sequencing DNA or RNA] strands that are longer than 50 base pairs,” says Jennifer Freeland, research and development manager at Thermo Fisher Scientific (which recently acquired Life Technologies). Also, Freeland says, “frozen tissue is best when experimenting with new antibodies and immunohistochemistry results are unknown.”
Still, though FFPE samples are not ideal for molecular analysis, there may be instances in which one might try it anyway—for example, if only FFPE, but no frozen, samples were available from a deceased patient. Also, “the ubiquity of FFPE samples makes them a hugely valuable resource,” says Kazinski. However, isolating DNA or proteins from them is a difficult task, and the resulting samples are not on par with those from frozen specimens. “It is known that DNA in FFPE tissue accumulates sequence artifacts that can lead to false results in sequencing experiments,” says Kazinski. “Those artifacts do not occur in frozen tissue.”
If you are determined to try, begin with an optimized extraction kit. For DNA sequencing, for example, these kits can help remove the fragmented DNA found in FFPE samples that can complicate sequencing. “The extraction kit should include an artifact-removal step like [QIAGEN’s] GeneRead DNA FFPE Tissue Kit,” says Kazinski. Other options include Epicentre’s QuickExtract™ Kit, Life Technologies’ MagMAX™ Kit, MO BIO’s BiOstic® Kit and Promega’s Maxwell® 16 Kit, among many others.
Immunostaining and morphology
For immunostaining and morphology, FFPE samples are your best bet. Frozen tissues have “a poor to mediocre quality of histomorphology” compared to FFPE samples, Freeland says. “If tissues are frozen incorrectly, an artifact can present itself in the formation of vacuoles within the tissue,” she says.
FFPE samples are also best if you need to analyze both tissue structure and immunostaining simultaneously. “Morphological and non-PTM protein analyses, such as special histological stains and IHC (immunohistochemistry),” are best performed in FFPE tissues, says Jared Isaac, a scientist in research and development at Thermo Fisher Scientific.
Native morphology
There is one situation in which FFPE samples lag behind frozen for morphological studies: If you want to study the native morphology of the tissue—or as close to physiological as possible—then frozen samples are best. Using frozen tissue, users can “perform IHC on the native form of the protein, antigen or epitope, since the conformation has not been altered by crosslinking through formalin-fixation,” says Freeland. “The IHC results tend to be more reliable and repeatable, since collection and freezing methods among laboratories [are] more standardized than with formalin fixation.”
For such studies, however, proper freezing protocols performed as soon as possible are essential. Isaac cautions that “native biomolecule status is highly dependent on ischemia time” as is the “detection of all biomolecules and PTMs in as close” to a living state as possible.
Why not try both?
Sometimes it’s better not to choose just one sample type. “If you are interested in both staining and molecular analysis, it could make sense to use both types,” says Kazinski. In fact, in clinical situations such as tumor-removal surgery, surgeons and cancer researchers take advantage of the strengths of both types of samples. They use frozen sections of the removed tissue during the surgery to validate clear margins around the tumor. “The frozen-section method is used because it is relatively quick, 20 minutes or less, and can be completed during surgery,” says Freeland. They then back this up post-operatively with more rigorous morphological verification by preparing FFPE samples of the same tissue. “These are used to verify the original diagnosis made on the frozen section during the surgery,” says Freeland. Using both sample types gives surgeons and patients some peace of mind that the tumor has been completely removed.
Perhaps one day researchers will develop a unified sample preparation method for every situation. Until then, FFPE and frozen samples make a powerful duo when it comes to extracting information from human clinical samples.
Image: Cell Signaling Technology