Formalin fixed paraffin embedded tissue (FFPE) processing is a modern method used in clinical diagnostics and archives for the preservation of tissues. Specimens taken represent a wide variety of clinically tested samples stored for long durations at room temperature. Formalin Fixed Paraffin Embedded (FFPE) tissues have been proven to offer a great deal in retrospective analysis of pathological processes as compared to cyropreserved specimens which are in most cases deemed complicated and hugely challenging. However, the overall quality of samples is compromised due to DNA trapping and fragmentation, as well as chemical modification during fixation process. This happens entirely due to nucleic acids and proteins cross-linking.
Extreme RNA degradation, poly-A tail damage, and modification of RNA nucleotide bases through addition of monomethylol are some of the effects of formalin fixation. Chances of these modifications altering gene expression profiling and reversing mRNA to cDNA are high. However, during genomic studies, researchers have used RNA and DNA extracts from FFPE specimens successfully despite all the challenges.
Technicalities associated with formalin fixed paraffin embedded tissue processing
1.Nucleic acids obtained from FFPE blocks are basically of low quality as well as quantity, and that rightly becomes one of the biggest
challenges. The main idea of the FFPE process is not centered majorly on the preservation of DNA or RNA, but rather on the preservation of cellular proteins for evaluation in immunohistochemistry.
2. Formalin fixation causes fragmentation and degradation of nucleic acids. This happens during the handling process of samples, tools involved, which include solvents.
3.The overall quality of the nucleic acids extracted from the FFPE blocks is compromised due to extended storage periods.
The more time it takes for these samples to be stored, they lose their originality.
4. Quantity of DNA and RNA in FFPE specimens is reduced significantly as a result of acid degradation and protein cross-linking.
Factors affecting extraction of Nucleic Acids from FFPE Blocks
1. The period between collection of tissues and the commencement of the setting process is known as pre-fixation. Immediately the tissue gets exposed to hypoxia and other environmental impurities, the material begins degrading. Biochemical processes start taking place after only 10 minutes; therefore, the pre-clamping process needs to be reduced to seconds.
2.The fixation conditions involve temperature and time, as well as the fixation type. The processes involved alter preservation of materials and hence compromise both the quality and quantity of nucleic acid.
3.It has been chemically proven that formaldehyde disrupts bonds in DNA adenine and thymine rich areas, causing unwarranted chemical
It is of paramount importance to sterilize the workstation in order to minimize contamination brought about by handling. Tools used in the processes must also be DNA-RNA free. This can be done through deparaffinization before the extraction process to ensure efficient extraction of nucleic acids. Solvents like xylene are commonly used to get rid of paraffin cuts, after which rehydration takes place using ethanol.
Despite the above mentioned challenges, Formalin Fixed Paraffin Embedded Tissue Processing is cheap and less demanding in terms of storage. Research is still underway to establish whether larger and more pronounced repositories are possible in the near future.