lipid biopsies

Question 1

what is a liquid biopsy?

Answer 1

sampling and analysis of non solid biological tissue,, primarily blood. it is minimally invasive technology for detection of molecular biomarkers. representative of the tissues from which it has spread

Question 2

example of liquid biopsy

Answer 2

amniotic fluid = established liquid biopsy

Question 3

what do liquid biopsies do?

Answer 3

prevent:

• blood clots
• genomic DNA release
  haemolysis

Question 4

describe EDTA, citrate

Answer 4

contain anticoagulant to prevent clotting

on-site centrifugation within 6 hours of collection to isolate plasma and avoid white cells apoptosis if not possible sample can be stored at 4 degrees for up to a week

Question 5

describe cell-free DNA tubes

Answer 5

contain stabiliser to prevent release of gDNA from white blood cells and haemolysis of RBC

Samples stored for 6-14 days at 6-37 degrees

Question 6

describe circulating tumour cells

Answer 6

cells that have detached from tumour and travel through bloodstream to other parts of body, single cell or clusters

marker for tumour growth and negative cancer prognosis and treatment response

extremely rare = 1:10 per 1ml of blood

found in high background of normal cells. sensitive and specific methods needed to study them

Question 7

circulating tumour cells: isolation and characterisation

Answer 7

biological properties and or physical properties

identified based on transcripts, PCR done on total RNA extracted from cells

Question 8

describe ctDNA

Answer 8

present in different fluids: plasma, serum, urine and others

low conc

amount highly variable for person to person and depends on health status in same person

presence of permanent genomic DNA background in plasma

highly fragmented but with specific size range (<500bp)

provides info of current genetic makeup with 80-95% specificity and 60-85% sensitivity

transfer supernatant to clean polypropylene tube and freeze if needed
isolation using magnetic bead, cellulose based or silica based systems

Question 9

ctDNA 2

Answer 9

next gen sequencing, digital droplet PCR, array CGH = amplification and deletions, translations, point mutations, chromosomes abnormalities, epigenetic status.

real time quantitative polymerase chain reaction = ctDNA presence quantification

Question 10

advantages of liquid biopsies vs soli/traditional biopsies

Answer 10

lower invasiveness
higher patient compliance
higher cost/effectiveness
allow repeated access and multiple sampling
no special training required for extraction

Question 11

disadvantages of liquid biopsies vs solid biopsy

Answer 11

low amount of material
early diagnosis
data interpretation

Question 12

clinical application: cancer biomarkers

Answer 12

why liquid biopsies?

• cancer = heterogenous disease
• molecular properties within a tumour differ and also between metastatic sites
• primary tumour info may not reflect the current disease condition

no need to identify the tumour site before taking a biopsy and allow repeat sampling

allow analysis tissues difficult to access

Question 13

Non invasive pre natal test

Answer 13

screening of Down syndrome, Edwards and Patau’s

replacing amniocentesis:

• detection of trisomies
• sex determination through Y chromosomes detection
• detection of markers for various X linked conditions
• detection of Rh factor
• identification of pre-eclampsia
• single nucleotide polymorphism analysis
• paternity
• 1-5% failure rate

Question 14

transplant rejection diagnosis

Answer 14

cell free DNA = promising biomarker for monitoring health of solid green organ transplantations, heart, lung, kidney and liver

heart transplants = 25-30% rejection in first year. endomycardial biopsy = current gold standard

donor cfDNA increases and rejection risk increases

SNPs can distinguish donor from recipient.