Liquid Biomarker Flashcards
what is a liquid biopsy
Sampling and analysis of non-solid biological tissue, primarily blood. It is a minimally invasive technology for detection Of molecular biomarkers. Representative Of the tissue/s from which it has spread.
examples of liquids used for liquid biopsy (6 points)
blood, plasma, CSF, urine, amniotic fluid, saliva
Types of material we can find circulating in blood (7 points )
Circulating endothelial cells (CEC), Extracellular micro-vesicle (exosomes), metabolites, disseminated tumour cells (DTCs),cell free nucleotide, tumour educated platelets (TEPs), circulating tumour cells (CTCs)
what are the 2 types of tube used in liquid biopsy
EDTA,citrate
cell-free DNA tubes
properties of EDTA, citrate tubes
contain anticoagulant to prevent clotting
Logistics and storage of EDTA, citrate
Onsite centrifugation within six hours of collection to isolate plasma and avoid white cell apoptosis. if not possible sample can be stored at 4 degrees Celsius for up to a week
Properties of cell free DNA tubes
Contain a stabiliser to prevent release of gDNA from white blood and haemolysis of red blood cells
Logistics and storage of cell free DNA tube
Samples can be stored for 6-14 days at 6-37 degree Celsius
speed, time and temperature at which you centrifuge for liquid biopsies
15 minutes
2000 xg
4 degrees Celsius
order of content from top to bottom after centrifugation for liquid biopsies and percentages of each content
Plasma 55%
less than 1% buffy coat
45% haematocrit
Which content contains cfDNA and exosomes
plasma
which content contains WBC and CTCs
buffy coat
which content contains RBC
Haematocrit
what are circulating tumour cells (CTC)
- 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.
why can we isolate tumour cells
We can isolate circulating tumour cells because they are CD45 negative EpCAM positive And CK a positive
What is Circulating Tumour DNA (ctDNA)
- 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.
-Highly fragmented with specific size range (less than 500bp)
how do you isolate ctDNA
using magnetic beads, cellulose based or silica based systems
what can next generation sequencing (NGS), Digital droplet PCR (ddPCR) and array CGH be used for when analysing ctDNA from liquid biopsy
Amplifications and deletions, Translocations, Point
mutations, Chromosomes abnormalities, epigenetic status (methylation)
what can Real Time Quantitative Polymerase Chain reaction (qPCR) be used for when analysing ctDNA from liquid biopsy
ctDNA presence quantification
5 advantages of liquid biopsies
Lower invasiveness Higher patient compliance Higher cost/effectiveness Allow repeated access and multiple sampling No special training required for extraction
3 disadvantages of liquid biopsies
Low amount of material
Early diagnosis
Data interpretation
why liquid biopsy for detecting cancer
- cancer is a heterogenous disease therefore molecular properties with a tumour differ and also between metastatic sites. this means it would difficult to know where the tumour originates and so will make solid biopsy difficult to do
- No need to identify tumour site before taking a biopsy
- allows repeat sampling
- allow analysis of tissues that’s difficult to access
