Formalin-fixed paraffin-embedded (FFPE) tissue is a widely used method for biological sample preservation, mainly for histological analysis, including immunohistochemistry, in situ hybridization, and sequencing. FFPE tissue samples are often utilized in retrospective and longitudinal studies, which allow researchers to investigate the molecular mechanisms behind different pathological conditions and study disease progression over an extended period.
FFPE tissue preparation
FFPE tissue starts with fixation of the tissue sample in formalin, which results in crosslinking of the proteins and nucleic acids, fixating the cellular morphology and preserving the antigenicity of the sample. The sample is then dehydrated and embedded in paraffin wax, which protects the tissue from degradation and makes it easier to section the tissue for analysis.
a) Tissue acquisition
First, fresh tissue must be obtained. It is essential that the tissue be handled with great care as it is easily be damaged after removal.
b) Fixation
Fixation is the first step in FFPE tissue processing. Key factors to consider include the solution to be used, the length of time allowed for fixation, and the thickness and histological properties of the tissue sample to be fixed. The tissue must remain in the fixative long enough to penetrate through every part of the specimen, which typically takes 6-24 hours, depending on the size and type of tissue sample.
c) Dehydration
Next, in order for the tissue to be properly infiltrated with melted paraffin wax, it must be dehydrated and cleared. A series of alcohol (usually ethanol) solutions, frequently beginning with 70% and working up to 100% alcohol, will be used to remove nearly all of the water from the tissue. Using fresh solutions or replacing used solutions frequently is required for optimum dehydration results, as the alcohol is gradually diluted with use.
d) Clearing
Since paraffin is actually immiscible with alcohol, either, the next step is to remove the alcohol with a paraffin-miscible substance. This step is known as clearing, and xylene is a commonly used clearing agent and gives the tissue a transparent look.
e) Paraffin embedding
The final step in processing FFPE samples is paraffin embedding. At this point, the tissue can be infiltrated with paraffin wax at about 60°C, and then cooled to make it solidify, which can be easily cut into sections.
The applications of FFPE tissue
FFPE tissues have multiple applications in biomedical research and clinical diagnostics, as they provide a valuable resource for examining the cellular and molecular changes that occur during disease progression.
a) The primary use of FFPE tissues is for histological examination, where they are used to analyze the morphology and structure of tissue samples.
b) Moreover, FFPE tissue samples are utilized in retrospective studies to investigate the molecular mechanisms behind different pathological conditions and study disease progression over an extended period.
c) FFPE tissues are also used in longitudinal studies as they can be stored at room temperature for long periods without significant deterioration, making them a valuable resource for analyzing samples over time.
d) The preserved tissue samples are also used to investigate the genetic basis of various diseases such as cancer, genetic disorders, and infections. The compatibility of FFPE tissue samples with various molecular analysis techniques, including sequencing, immunohistochemistry, and in situ hybridization, has significantly contributed to the advancement of medical research.