Liquid Biopsies Flashcards
When are liquid biopsies used?
In stratification and personalisation of medicine
What is a liquid biopsy?
Sampling and analysis of non-solid biological tissue, primarily blood. (Saliva, CSF, urine)
Minimally invasive technology for detection of molecular bio markers.
Representative of tissues from which it has spread
The future of biopsies is to turn from solid to liquid.
What is a example of a well established liquid biopsy?
Amniotic fluid analysis (fluid surrounding foetus)
What can be detected using blood as a Iiquid biopsies?
Extracellular micro-vesicles (exosomes)
Metabolites
Tumour educated platletes (TEPs)
Disseminated tumour cells (DTCs)
Circulating tumour cells (CTCs)
Circulating endothelial cells (CEC)
Cell free nucleotides
What information can be extracted from a blood biopsy?
Somatic information (more common because germaline information can be extracted from any cell in the body) - able to detected damaged cells from other of the body
Germaine information
How can we construct a blood liquid biopsy?
10ml of blood collected by venipuncture (4-5mL plasma)
After 15 minutes centrifugation at 2000xg speed at degrees 3 layers form in the tube. A plasma layer, huffy coat and a hematocrit
What is a good extraction tube for a liquid biopsy?
- prevent blood clots
- genomic DNA release (from white blood cells)
- haemolysis
Give 2 examples of extraction tubes used for a liquid biopsy?
EDTA, Citrate:
- contain anti coagulant forms prevent clotting
- on site centrifugation within 6 hours of collection
- sample can be stored for up to a week at 4 degrees
Cell-free DNA tubes:
- contain a stabiliser to prevent release of gDNA from WBC and haemolysis of RBC
- stored for 6-14 days at 6-37 degrees
What 3 layers form after centrifugation of a blood biopsy?
55% Plasma
- water, proteins, nutrients, hormones
- cfDNA and exosomes
<1% Buffy coat:
- white blood cells and platelets
45% hematocrit:
- red blood cells
What are circulating tumour cells?
Cells that have detached from a tumour and have travelled through the blood stream to other parts of the body.
Marker for tumour growth and negative cancer prognosis a d treatment response
Extremely rare: 1-10 per ml of blood
Found in buffy coat with wbcs
How are CTCs isolated and charecterised?
- biological properties and/or physical properties
- identified characteristics based on transcripts (PCR done on total RNA extracted from cells)
Have cell markers that are to found on other blood cells (CK, CD-45, EpCAM)
What can we do once CTC are extracted?
- phenotypic studies
- proteomics
- transcriptomics
- genomics
- NGS
- FISH
- flow cytometry
What are ctDNA (tumour DNA)?
Tumour DNA present in urine, plasma, serum, and others.
Of Low concentration
Amount highly variable person to person depending on health status in the same person
Presence of permanent genomic DNA background in plasma
Highly fragmented but with a specific size range (<500bp)
Provides information of current genetic makeup with 80-95% specificity and 60-85% sensitivity
How can ctDNA be isolated after centrifugation?
- Transfer supernatant to a clean polypropylene tube and freeze it if needed.
- Isolation using magnetic bead, cellulose-based or silica based systems
- Can store forever
What type of information does ctDNA provide for us?
- point mutations
- study of epigenetic modifications
- translocations
- amplification and deletions
- NGS
- digital PCR
