FFPE Vs Frozen Samples: Human Clinical Samples

Introduction Human Clinical Samples

The analysis of human tissues requires tissue samples which have been preserved for an extended period of time. This analysis will include studying the:

·         Morphology

·         Cell biology

·         Biochemistry

·         Disease or pathology


There are usually two type of tissue samples, either:

o   Formalin fixed paraffin embedded (FFPE)


o   Frozen tissue

Both tissue samples have their pros and cons and the applications it is best suited for.


FFPE Versus Frozen

Both these methods can preserve tissue samples well but when it comes to picking a sample, it depends on what you are using it for.

a)       General comparison

o   FFPE tissue samples are much more cost effective as they are able to be stored in room temperature. This also makes it much more convenient. FFPE tissue blocks can be used in applications such as immunohistochemical staining and the analysis or morphology. FFPE samples are widely used and there is a large archive of specimens where research teams and scientists can select and share. However, formalin is also toxic while the procedure for fixation and paraffin embedding can be time consuming. Since the procedure and protocols for FFPE are not standardized, there is also no assurance that all the samples selected for analysis are prepared the same way. It is also unsuitable for molecular analysis.

o   Frozen specimens have the advantage of consuming less time and effort when it comes to sample preparation. While FFPE samples are unsuitable for molecular analysis, frozen specimens are well suited for it. However, frozen specimens rapidly deteriorate once it is in room temperature and requires to be frozen as soon as possible once the specimen is collected. This means that the equipment required to freeze specimens have to be close to the site of surgery. Dedicated freezer space is also required as it is necessary that these samples be kept frozen. This increases the space and cost needed to store these specimens.

b)      Molecular Analysis

In molecular analysis, frozen samples are preferred over FFPE tissue samples as the non-standardized protocols in the preparation of FFPE samples are likely to affect molecular data. This includes work with:

o   DNA

o   RNA

o   Post translational protein modifications

As an example, long DNA fragments, post translational protein modifications, and protein phosphorylation states are rarely preserved in FFPE samples making it unsuitable for certain applications. Frozen samples are also a necessity in other analyses such as:

o   Mass spectrometry

o   Quantitative real time polymerase chain reaction

o   Next generation sequencing

o   Western blotting for post translational protein modifications

This is why in most molecular assays frozen specimens are the gold standard especially when it comes to DNA and RNA sequencing where the strands are required to be longer than 50 pairs. It is also the best when research is being conducted on new antibodies where the immunohistochemistry results are indefinite.


FFPE samples are also not used in molecular analysis as the isolation of DNA and proteins can be difficult while the results obtained are not on par compared to those from frozen samples. FFPE tissue can accumulate sequence artifacts that may leads to false results when it comes to sequencing experiments. However, if no frozen samples are available, FFPE samples might be an option. In these instances, an optimized extraction kit can be used. This is why FFPE blocks can be a valuable resource.

c)       Immunostaining and Morphology

In immunostaining, morphology, and tissue structure, FFPE specimens are the best as frozen specimens can have artifacts if not frozen correctly. Hence, frozen samples have poor to mediocre quality in histomorphology.

d)      Native morphology

While FFPE samples may be the best in morphology, it can lag behind frozen specimens in the study of native morphology or physiology. Frozen specimens can be used to perform immunohistochemistry on the native form of:

o   Protein

o   Antigen

o   Epitope

This is due to the alteration of cross linkages in FFPE specimens. Once again, frozen specimens are preferred over FFPE samples as the procedures in the collection and preservation of tissue are much more standardized compared to that of formalin fixation. In these studies, it is essential that freezing protocols are performed as soon as possible as the native biomolecule status depends highly on the rate of tissue ischemia. It is crucial that the biomolecules and post translational protein modifications are as close to a living state.


After the review of the above information, it can be said that it may not be wise to prefer one sample type over the other as both sample types are used in different applications. In clinical situations, many surgeons and cancer researchers optimize the strength of both sample types. Frozen sections are used to validate margins around a tumor while verification of the diagnosis made from the frozen tissue is done using an FFPE sample of the same tissue. When used together and in suitable applications, both FFPE and frozen specimens are key in the extraction of data from human clinical samples.


Smith C. FFPE or frozen? Working with human clinical samples. 2014. Biocompare. Accessed 4/11/2018 from https://www.biocompare.com/Editorial-Articles/168948-FFPE-or-Frozen-Working-with-Human-Clinical-Samples/