Why is annotation so important for FFPE samples?
FFPE blocks with annotation data that includes important demographic data, as well as common biomarkers and clinically relevant mutations help researchers focus on key molecular targets of interest. Demographic data assists companies seeking to test new therapies or diagnostic tests in the preclinical stage to find specimens from specific patient cohorts for age and other demographic matching purposes. Sometimes, these projects require large volumes of individual donors who are age-matched to a particular range that their particular therapy will target. Drug discovery pre-clinical research proceeds faster and more accurately with deep clinical annotation for each biospecimen. Having access to detailed demographic and pathological data increases the quality of research and the confidence that results are clinically leverageable.
When would a researcher choose FFPE tissue samples versus other options?
By some estimates, there are over one billion FFPE tissue samples stored in archives around the world, making them invaluable resources for many purposes, including histopathology analysis and more recently, the pursuit of molecular studies. Many promising drug discovery programs require hundreds or thousands of samples, multi-year studies, and availability of same specimens for follow-up research.
What diseases are able to be studied using FFPE?
FFPE specimens are useful for studying a wide range of diseases, including many types of cancer, as well as several immune disorders. Commonly requested FFPE specimens include: breast, colorectal, brain, lung, melanoma, liver, pancreatic, gynecologic, and hematologic cancers. Increasing numbers of researchers seek FFPE specimens with known mutation status, particularly for breast, colorectal, and lung cancers.
How is DNA extracted from FFPE tissue?
Today, a variety of commercial products permit the rapid and accurate extraction of DNA from FFPE tissue. These are the most commonly followed steps for DNA extraction: removal of paraffin via manual trimming and solvent dissolution; proteinase K digestion; incubation to reverse formaldehyde modification of nucleic acids; binding of DNA to silica membrane; washing of sample to remove remaining contamination; and elution to obtain DNA.
How long can FFPE samples be stored?
This is dependent in part on what you want to use the specimen for. Because of the wide availability of FFPE specimens, researchers have studied the issue of storage time on a variety of factors, including gene expression, mutation detection and nucleic acid yield and viability. Studies have concluded that given proper fixation and storage conditions, FFPE blocks stored for 10 years, or even longer, can be viable samples. However, it's very important to carefully assess the reliability of the specimen provider.
Can you isolate RNA from your FFPE tissue samples?
The good news is that, yes, it is possible to isolate RNA from FFPE tissues, given the right process and specimens that have been properly fixed and stored. This was once a challenge but now, recovery of RNA is possible with newer lab isolation techniques. Most commercial RNA isolation kits use optimized lysis and incubation conditions to reverse formaldehyde modification of RNA. The lysis buffer releases RNA from FFPE tissue samples, which prevents further RNA degradation.
What sort of quality control should a researcher do with FFPE tissue samples?
It's critical that the quality control process is overseen by U.S. licensed pathologists. FFPE blocks should be evaluated for proper tissue fixation and the presence of necrosis. The block must match the pathology report, i.e. tumor size, tumor percentage, how many 4um sections are available from the tissue present. QC data provided with each block should include the following patient information: diagnosis, resection date, gender, staging, pathology report; and the following block information: tumor type, tumor percentage, biopsy vs. resection, estimated 4 um sections, tissue and tumor size (mm).
How do you take a peripheral blood sample?
Peripheral blood mononuclear cells (PBMCs) are obtained from whole blood samples. When obtaining PBMCs from whole blood, there are a couple of key precautions that should be noted so that the end samples are useful:
What does peripheral blood contain?
PBMCs have a single distinct nucleus (hence mononuclear) and are made up primarily of monocytes and lymphocytes. Lymphocytes are comprised of T and B cells, as well as NK cells. These cells are extremely important in the body’s immune defense mechanism. T cells are in charge of cellular immunity functions. B cells interact with antibodies and help confer what is known as antibody-mediated immunity. NK (natural killer) cells prowl the body looking for antigens with abnormal cell membranes, such as cancer cells.
What diseases should PBMCs be used to research?
Researchers looking into immune responses to vaccines, new immunotherapy modalities, or seeking a better understanding of the molecular basis of immune system function often source PBMC specimens. PBMCs are used to study the role of stem cells in cancer development and metastasis; find biomarkers for a wide range of diseases, including metabolic disorders such as diabetes; develop therapies for various hematologic disorders, including anemia and blood cancers, and classify tumors subtypes.
What is the benefit to comparing human peripheral blood samples of healthy donors to those from patients with cancer or autoimmune/inflammatory disorders?
Access to normal donor samples that can serve as research controls is key for companies seeking to test new therapies or diagnostic tests in the preclinical stage. Normal healthy donors allow for a comparison to disease samples when researchers are seeking specific biomarkers or gene expression profiles linked to disease status.
Are there different types of PBMC samples, and what is the difference between them?
Researchers use both fresh and cryopreserved PBMC samples for studying a wide range of diseases, in particular, cancer and immunity disorders. The choice which of PBMC sample type to use depends to a large degree on the purpose of the research. For example, fresh blood yields more viable PBMCs than do frozen samples, due mainly to the impact of freezing, thawing, storage, temperature and other variables on sample integrity. Certain studies, such as those looking at CD4+ T cells, are more reliable with fresh PBMC specimens.
What are cryopreserved PBMCs? When and why is this method used?
Cryopreserved samples are stored at -190° C. Cryopreservation has been used for many decades to store a wide range of biological specimens, including PBMCs. A huge inventory of cryopreserved specimens is available, making them important samples for today's increasingly large and complex immune studies. In addition, reliance on fresh PBMCs can limit future access to samples researchers needed for additional immunological/oncologic studies or validation tests. This is not a problem with cryopreserved biospecimens.
How are PBMCs isolated?
To obtain PBMCs, they must first be isolated from whole blood samples. One of the most common and reliable methods for isolating PBMCs uses a silica colloid known as Percoll™. First introduced in 1977, this density gradient medium is utilized for many studies because it is non-toxic, has low osmolarity and viscosity, doesn't penetrate biological membranes, will not affect assay procedures, is easily removed from purified isolates, and can form a continuous and discontinuous gradient.
How long is bone marrow fresh?
Bone marrow specimens are invaluable resources that help researchers unlock the source of disorders such as lymphoma and leukemia, anemia, multiple myeloma and even certain autoimmune diseases. Once collected, proper specimen handling is important so that researchers can be assured of the sample viability.
In some cases, fresh bone marrow is preferable; for example, one study of multiple myeloma concluded that CD138+ levels were considerably lower in delayed and frozen samples than in fresh samples. On the other hand, other studies investigating the long-term viability of cryopreserved bone marrow have concluded that these are useful specimens, given certain caveats. In one study, human bone marrow-derived mesenchymal cells containing mesenchymal stem cells were compared after cryopreservation and non-cryopreservation. The viability of the cryopreserved cells was about 90% regardless of the length of storage – ranging from 0.3 to 37 months.
One important variable that can affect viability is temperature fluctuation during storage. Ensure your lab protocol minimizes how many times and for how long the specimen freezer is opened to prevent unnecessary temperature excursions.
What are the components of bone marrow?
Bone marrow is the soft, sponge-like tissue that fills the hollow center of bones. It contains hematopoietic stem cells which are progenitors to the three classes of blood cells: leukocytes (white blood cells), erythrocytes (red blood cells), and thrombocytes (platelets). Bone marrow also contains mesenchymal stem cells (MSCs), multipotent stem cells that can differentiate into other cell types including osteoblasts, chondrocytes, myocytes, adipocytes, and beta-pancreatic islets cells.
The stroma is the bone marrow tissue not directly involved in hematopoiesis (red blood cell formation). It consists mainly of yellow bone marrow and to a lesser extent, stromal cells found in the red bone marrow. The stroma is indirectly involved in hematopoiesis by providing a microenvironment conducive to this process. For example, the stroma produces colony stimulating factors necessary for hematopoiesis. Other cells that constitute the bone marrow stroma include fibroblasts, macrophages, adipocytes, osteoblasts, osteoclasts, endothelial cells, and mesenchymal stem cells.
When does bone marrow produce fresh blood?
The production of red blood cells is called erythropoiesis. It takes about 7 days for a bone marrow stem cell to mature into a fully functional red blood cell. Red blood cells have a limited life span of approximately 120 days and must be continuously replaced by the body. When necessary, the kidneys will produce a hormone known as erythropoietin (EPO). EPO stimulates the red bone marrow cells to produce RBCs. These new red cells migrate into the bloodstream and increase the blood's oxygen-carrying ability.
What is the difference between yellow bone marrow and red bone marrow?
Red Marrow - Consisting mainly of hematopoietic tissue or tissue involved in the formation of red blood cells, all bone marrow is red at birth. About 50% of red marrow converts to yellow marrow over time. Red marrow is found primarily in flat bones such as the pelvis, sternum, cranium, ribs, vertebrae, and in the sponge-like, material at the rounded ends of long bones such as the femur and humerus. Red marrow's main function is to produce red and white blood cells and platelets. Its red color comes from the hemoglobin in the erythroid cells, and is highly vascular.
Yellow Marrow - Consisting mainly of adipocytes (fat cells), yellow marrow is found in the medullary cavity, the hollow interior of the middle portion of long bones. Yellow marrow's main function is to store adipocytes whose triglycerides can serve as a source of energy. In cases of severe blood loss, yellow marrow can be converted back to red marrow to increase blood cell production. It produces fat, cartilage and bone. The yellow color comes from the carotenoids in the fat droplets from the high number of fat cells. Unlike red marrow, it is not highly vascular.