FFPE vs Frozen Tissue Samples


Both formalin fixed paraffin embedded (FFPE) and frozen tissues are important in research. They can be used to study the cell biology, morphology, biochemistry, and disease in all living creatures. Both FFPE and frozen tissues have their own pros and cons as both have different applications. Some basic differences seen between FFPE and frozen tissue samples are:

FFPE Samples

FFPE is a method where tissue samples are preserved by using formalin to paralyze cell metabolism while paraffin is used to seal the tissue and decrease oxidation rates. There are many tissue samples that are stored using this method due to its cost efficiency as it can be stored at room temperature. FFPE tissue blocks are crucial as they play a key role in biotech research, drug discovery, and retrospective gene studies. Tissue samples preserved using the FFPE method can be stored for decades and is an invaluable source to allow correlation of clinical outcome, therapy, and molecular findings. It is also easier to section FFPE samples since they are embedded in wax and can be easily mounted on a microscope for examination. 

  • Uses – FFPE samples are important in fields such as immunology, hematology, and oncology. 

  • Advantages – Due to its cost efficiency for storage at room temperature, it is a great resource as a research material as it remains viable without requiring specialized equipment. 

  • Disadvantages – Some main disadvantages of this method include using formalin for fixation of the tissue sample, time consuming process for fixation and embedding, and non-standardized protocols in preparation of the tissue samples. Due to its preparation method, the proteins in FFPE samples become denatured limiting the use of FFPE samples to only certain studies. 

Frozen Tissue

Frozen tissue refers to tissue samples that are preserved and stored using ultra-low temperature freezers and liquid nitrogen.

  • Uses – Frozen tissue is important in areas where FFPE samples are not reliable such as molecular analysis. It is also important to help determine if the margins are clear for tumor removal in surgeries. It is also preferable compared to FFPE in next generation sequencing, western blotting, and mass spectrometry.

  • Advantages – This method is much faster compared to the FFPE process. It also preserves proteins in their native state. 

  • Disadvantages – Some disadvantages of this method include the rapid deterioration rate of the frozen tissue samples once it is in room temperature. Since the samples need to be frozen as fast as possible once the sample is collected, it can pose some difficulty as the equipment required will need to be available. Storage of frozen samples are also expensive as specialized equipment are required to keep the samples frozen. The samples are also vulnerable especially if there are mechanical failures or power outages. 

Molecular Analysis

Frozen samples are better than FFPE samples for molecular analysis. This includes work that involves post translational protein modifications (PTMs), DNA, and RNA. This is due to the non-standardized preparation methods used for FFPE sample preparation. Another reason is because of the involvement of formalin in FFPE preparation. The use of formalin often results in non-native configurations of phosphorylated proteins and degraded RNA.  Frozen samples are also a necessity for procedures such as Western blotting, next generation sequencing, mass spectrometry, and quantitative real time polymerase chain reaction (PCR) as they are considered the gold standard. 

While FFPE samples are not the best option for molecular analysis, it can be used when there are no frozen samples available from a deceased donor. However, it is important to note that the isolation of proteins and DNA from FFPE samples can be difficult with results that are not on par with results obtained from frozen specimens. It is a known issue that DNA obtained from FFPE samples can lead to the accumulation of sequence artifacts resulting in false results in sequencing experiments. This issue has not been encountered with frozen tissue.

Immunostaining and Morphology

FFPE samples are preferable compared to frozen samples for immunostaining and morphology purposes. This is due to the poor or mediocre histomorphological quality when frozen tissues are used. Tissues that are frozen incorrectly can lead to the formation of vacuoles in the tissue. When both immunostaining and tissue structure analyses of the tissue are required simultaneously, FFPE samples are also better.

Native Morphology

For native morphological studies, frozen tissue samples are much more desirable compared to FFPE samples. Frozen specimens allow the closest to physiological native morphology study. Immunohistochemistry can be performed on the native form of antigen, epitope, or protein since these components in frozen tissue are not crosslinked due to formalin fixation. The results from the immunohistochemistry are also repeatable and much more reliable when performed using frozen tissue when compared to those using FFPE samples. However, it is important that studies on native morphology uses specimens where the freezing protocols are done as soon as possible as the quality of the specimen highly depends on the ischemia time. A rapid freezing time allows the PTMs and biomolecules to stay close to the living state. 


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