FFPE vs Frozen Tissue Samples


Both formalin-fixed paraffin-embedded (FFPE) tissue and frozen tissue are cornerstones in the research field. Medical advancements and research rely on the availability of biospecimens as research material. For example, studying a specific disease, how it progresses, and how it can be cured would require access to high-quality biospecimens that contain the diseased tissue. Since biospecimens are fragile and perish easily, professional care is necessary to prepare and preserve the tissue to ensure that it can be studied by researchers. The two major tissue preparation methods used for preservation are FFPE and frozen tissue. 


FFPE tissue specimens are invaluable to the research industry and have had therapeutic applications for decades. It is a method of preservation that enables biospecimens to be used in experimental research, drug development, and diagnostics. In this method, the tissue is first saturated with formalin or formaldehyde to preserve the vital structures in the tissue. It is then embedded in paraffin wax to make sectioning easier so it can be mounted on a microscope for examination purposes. FFPE specimens can include both normal and diseased tissue as comparison studies are also important. In some cases, both normal and diseased tissue can also be obtained from the same individual. In the field of oncology, a primary tumor sample is often compared to tissues from distant metastatic tumors. 

FFPE Method

Once the tissue is identified and excised, it is immersed in a solution of 10 percent neutral-buffered formalin for 18 to 24 hours to harden the tissue so it can survive the following steps. Once ready, the tissue is dehydrated using increasing concentrates of ethanol. It is then embedded into immunohistochemistry grade paraffin. Fixation time is crucial in FFPE as tissues that are fixed too soon after excision can be useless for molecular studies. Quality control is important as biospecimens that are not maintained properly may exhibit unusual characteristics that result in misleading data and conclusions in research. The preparation method for FFPE may also depend on requests from the researcher as some may have specific instructions regarding the size, purpose, and cut of the tissue. Biospecimens that are completed are stored in biobanks established in research centers or hospitals. Records regarding the time of collection, preservation, demographic information, and legal documents should be kept as it may have a definitive impact on its usability in clinical trials or research. 

Frozen Tissue

Frozen tissue are biospecimens that are preserved using an ultra-low temperature freezer and liquid nitrogen. Compared to FFPE, frozen tissue is generally more important in areas where FFPE samples are not as reliable. One good example is molecular analysis. Tissue preserved using this method is dipped in liquid nitrogen (flash freezing) and stored in a freezer at less than -80 degrees Celsius. Frozen tissue is also important in pathology as it can be used to determine if the margins of a tissue section are clear during tumor removal in surgeries. Frozen tissue is also preferable compared to FFPE in mass spectrometry, next-generation sequencing, and western blotting. Compared to the FFPE method, it is much faster and preserves proteins in their native state. However, this method is disadvantageous as there is rapid deterioration of the samples once it is in room temperature. Tissues need to be frozen as fast as possible and may pose some difficulty as it requires the proper equipment. Once the frozen tissue is ready, storage of the biospecimens is also much more expensive as it requires specialized equipment. Any power outages or mechanical failures may cause the biospecimens to go bad. 

FFPE vs Frozen Tissue

Both FFPE tissue samples and frozen tissue have their pros and cons. Besides the preparation and storage method that was previously mentioned, both samples have different applications. These include:

  • Molecular analysis – Frozen tissue is much more suitable for molecular analysis due to the non-standardized preparation methods for FFPE samples. The use of formalin in FFPE preparation may also result in degraded DNA and non-native configurations of phosphorylated proteins. Frozen tissue is also considered the gold standard for mass spectrometry, Western blotting, quantitative real-time polymerase chain reaction, and next-generation sequencing.

  • Immunostaining and Morphology – In this instance, FFPE specimens are preferable due to the mediocre histomorphological quality when frozen specimens are used. When samples are frozen incorrectly, it can also lead to the formation of vacuoles. FFPE samples are preferable when both tissue structure analyses and immunostaining are simultaneously required.

  • Native Morphology – For studies involving native morphology, frozen specimens are preferable as it has the closest physiological native morphology. This allows IHC to be performed on the native form of protein, epitope, and antigen since the components in frozen tissue are not crosslinked as seen in FFPE specimens due to formalin fixation. Frozen tissue specimens also allow the results from IHC to be repeatable and more reliable compared to using FFPE samples. However, native morphology studies should be done as soon as possible as the specimen quality is highly dependent on ischemia time.


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