Liquid Biomarker

Question 1

what is a liquid biopsy

Answer 1

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.

Question 2

examples of liquids used for liquid biopsy (6 points)

Answer 2

blood, plasma, CSF, urine, amniotic fluid, saliva

Question 3

Types of material we can find circulating in blood (7 points )

Answer 3

Circulating endothelial cells (CEC), Extracellular micro-vesicle (exosomes), metabolites, disseminated tumour cells (DTCs),cell free nucleotide, tumour educated platelets (TEPs), circulating tumour cells (CTCs)

Question 4

what are the 2 types of tube used in liquid biopsy

Answer 4

EDTA,citrate

cell-free DNA tubes

Question 5

properties of EDTA, citrate tubes

Answer 5

contain anticoagulant to prevent clotting

Question 6

Logistics and storage of EDTA, citrate

Answer 6

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

Question 7

Properties of cell free DNA tubes

Answer 7

Contain a stabiliser to prevent release of gDNA from white blood and haemolysis of red blood cells

Question 8

Logistics and storage of cell free DNA tube

Answer 8

Samples can be stored for 6-14 days at 6-37 degree Celsius

Question 9

speed, time and temperature at which you centrifuge for liquid biopsies

Answer 9

15 minutes
2000 xg
4 degrees Celsius

Question 10

order of content from top to bottom after centrifugation for liquid biopsies and percentages of each content

Answer 10

Plasma 55%
less than 1% buffy coat
45% haematocrit

Question 11

Which content contains cfDNA and exosomes

Answer 11

plasma

Question 12

which content contains WBC and CTCs

Answer 12

buffy coat

Question 13

which content contains RBC

Answer 13

Haematocrit

Question 14

what are circulating tumour cells (CTC)

Answer 14

• 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.

Question 15

why can we isolate tumour cells

Answer 15

We can isolate circulating tumour cells because they are CD45 negative EpCAM positive And CK a positive

Question 16

What is Circulating Tumour DNA (ctDNA)

Answer 16

• 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)

Question 17

how do you isolate ctDNA

Answer 17

using magnetic beads, cellulose based or silica based systems

Question 18

what can next generation sequencing (NGS), Digital droplet PCR (ddPCR) and array CGH be used for when analysing ctDNA from liquid biopsy

Answer 18

Amplifications and deletions, Translocations, Point

mutations, Chromosomes abnormalities, epigenetic status (methylation)

Question 19

what can Real Time Quantitative Polymerase Chain reaction (qPCR) be used for when analysing ctDNA from liquid biopsy

Answer 19

ctDNA presence quantification

Question 20

5 advantages of liquid biopsies

Answer 20

Lower invasiveness 
Higher patient compliance 
Higher cost/effectiveness 
Allow repeated access and multiple 
sampling 
No special training required for extraction

Question 21

3 disadvantages of liquid biopsies

Answer 21

Low amount of material
Early diagnosis
Data interpretation

Question 22

why liquid biopsy for detecting cancer

Answer 22

• 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