Frozen Tissue and its use in Bioresearch

Introduction

There has been significant progress in modern medical research such as in diffuse lung disease, cancer, lung cancer, colorectal cancer, and more. This means researchers require access to high-quality specimens of human tissue samples such as bodily fluids. There are now multiple organizations that are involved in the collection, processing, storage, and distribution of these tissues to help the progress of biomedical research. There are general guidelines to help guide individuals new to investigation or research. Due to the limitations of space, it prevents the manuscript from being detailed as the goal is to discuss issues that help those who are new to the industry. 

Biorepositories

Biobanks or biorepositories are organizations that collect, processes, stores, and distributes tissue samples. They also collect the associated data and provide a reliable and organized source of biospecimens for RNA-based analysis. This is important as RNA integrity is crucial for gene expression studies. Since RNA is highly susceptible to degradation, standard and careful handling are required to preserve the quality of RNA. Recently, considerable attention has been given to the impact of preanalytic variables on the quality of biospecimens. This phase summarizes the procedures at different stages ranging from tissue sampling to gene expression analysis as this can affect the sample quality which in turn affects the research results and reproducibility. The quality control of stored tissue is also crucial to avoid embarking on costly, time-consuming, and labor-intensive projects. It is estimated that about 10% of the frozen tissue cannot be used in molecular analyses as there is inadequacy of tissue sampling. Based on the available literature, the most vital parameters are the number of tumor cells and the extent of necrosis. Evidence also shows that RNA integrity from colonic tissue and other parts of the gastrointestinal tract are significantly lower compared to other parts of the body.

Frozen Tissue

Frozen tissue are tissue samples that are fixed and mounted rapidly using a device called a cryostat. It is often used in surgeries to fix tissue samples during the removal of tumors to help surgeons determine if the tumor has been removed completely by studying the margins. Compared to formalin-fixed paraffin-embedded (FFPE) samples, frozen tissues are preferred in analyses such as western blotting for post-translational protein modifications, mass spectrometry, quantitative real-time polymerase chain reaction, and next-generation sequencing. Therefore, frozen tissue is often the gold standard for RNA and DNA sequencing. It is also essential in immunohistochemistry as frozen tissue have proteins that are preserved in their native state. However, frozen tissue rapidly deteriorates once it is in room temperature. This means that the sample will need to be frozen rapidly once collected. This can be challenging as the equipment needed will have to be closer to the donor. Storage of frozen tissue can also be expensive as the biospecimens have to be kept frozen making it vulnerable during mechanical failures or power outages. 

Frozen Tissue in Colorectal Cancer Research

RNA degradation occurs faster if it originates from the gastrointestinal tract. This also means it tends to have lower RNA quality compared to other organ sites such as the prostate, breast, tonsil, or kidneys. However, the RNA quality was higher in tumor tissues compared to normal mucosa. A recent study found that RNA quality is influenced by the location of the colonic adenocarcinoma. This may be due to the difference in the enzymatic environment and colonic bacterial flora between the right and left colon. This was also correlated with findings from animal and human models where tissue from the proximal small intestine was found to degrade faster compared to the distal small intestine, colon, or caecum suggesting a possible influence of bacterial and enzymatic environment on RNA quality. Despite not being statistically significant, the researchers were able to find out that the right colonic adenocarcinoma has distinct molecular characteristics such as BRAF mutation and frequent microsatellite instability compared to left colonic adenocarcinomas. 

Besides the location of the tumor, the researchers were also able to observe that the surgical approach and post-surgical complications also significantly influenced the quality of RNA. It was found that the RNA quality of tumor samples from a laparoscopic colectomy was lower or when there was a complication such as anastomotic leakage. Although a rare complication, it is a very severe complication and tends to occur in individuals with previous abdominal surgery, male patients, elderly, postoperative blood transfusion, etcetera. While unclear why the quality of tumor RNA is lower in those with post-surgery anastomotic leakage, it is postulated that it could be due to underlying medical conditions and location of the tumor. Though RNA degradation measurement is representative of RNA quality, the direct effect of the degradation on gene expression must also be considered. The researchers concluded that the RNA quality is lower in normal colonic tissue compared to colonic tissue with tumors regardless of the location. In tumor tissue, the RNA quality is believed to be influenced by surgical and clinical parameters such as surgical approach, tumor location, and occurrence of suture failure. However, more investigations will be required. 

References:

1)    Galissier T, Schneider C, Nasri S, et al. Biobanking of fresh-frozen human adenocarcinomatous and normal colon tissues: which parameters influence RNA quality? Plos One. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0154326

2)    The pros and cons of FFPE vs frozen tissue samples. Geneticist Inc. Accessed 9/30/2019. https://www.geneticistinc.com/blog/the-pros-and-cons-of-ffpe-vs-frozen-tissue-samples