Liquid Biopsies Flashcards
What is a liquid biopsy
Sampling and analysis of non-solid biological tissue, primarily blood. It is a minimally invasive technology for the detection of molecular biomarkers. Representative of the tissue/s from which it has spread.
What type of biopsy do we have
- Solid biopsy
- liquid biopsy
What can we use as samples for liquid biopsies
- Saliva
- CSF
- Serum
- Urine
How can blood be used a a liquid biopsy
Cells in blood undergo apoptosis or necrosis and release their genetic material into the blood.
Cells can actively secrete genetic material Ito the blood while alive
What do we detect in blood
- Extracellular micro-vesicles (exosomes)
- Metabolites
- Cell-free nucleotides
- Tumour educated platelets
- Circulating endothelial cells
- Disseminated tumour cells
- Circulating tumour cells
What type of info do we get from liquid biopsies
- We get both germline and somatic information
- More of a snapshot of what was happening at the time of drawing the blood sample
How is a blood sample collected
10ml collected by venipuncture and stored in tubes that prevent clotting and haemolytic
How is blood samples stored
- EDTA, citrate tubes: contains anticoagulants and used for onsite centrifugation within 6hrs
- Cell free DNA tubes: contains stabilisers to prevent DNA release, can be stored for up to 2 weeks at 6-37 degrees C
After centrifuging what does the blood separate into
55% plasma:
- Water
- Nutrients
- Hormones
- ctDNA
-Exosomes
1% Buffy coat:
- WBC
- Platelets
- Circulating tumour cells
45% Hematocrit: - RBC
What can we detect in liquid biopsies
- Circulating Tumour DNA
- Circulating Tumour cells
- MicroRNAs
- Exosomes
- Metabolites
What are circulating tumour cells
- Cells that have detached from a tumour and travel through the bloodstream to other parts of the body- single cells or clusters.
- Marker for tumour growth and negative cancer prognosis and treatment response.
- Extremely rare: 1-10 per 1ml of blood.
- Found in a high background of normal cells! - sensitive and specific methods are needed to study them.
How can we isolate and characterise CTCS
- Based on their biological and physical properties
- Identified/characterised based on transcripts- PCR done on total RNA extracted from the cells
What are circulating tumour DNA
- Present in different fluids: plasma, serum, urine and others.
- Low concentration (1-50ng DNA/mL plasma).
- Amount highly variable for person to person and depending on health status in the same person (increase in cancer, trauma, etc.).
- Presence of permanent genomic DNA background in plasma.
How can we test for ctDNA
- Highly fragmented but with specific size range (<500bp)
- Provides information of current genetic make-up (including irregularities/mutations) with 80-95% specificity and 60-85% sensitivity.
How can we isolate ctDNA
- Transfer supernatant to a clean polypropylene tube and freeze it if needed
- Isolation using magnetic bead, cellulose-based or silica-based systems.
