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Path 19.02 notes > 20.02.21 Cell culture and chromosome staining techniques > Flashcards

20.02.21 Cell culture and chromosome staining techniques Flashcards

1
Q

What clinical indications may warrant chromosome analysis follow up after an array

A
  • Neonates with features consistent with trisomy
  • Patients with an imbalance that may indicate the presence of a derivative chromosome
  • Looking for chromosome instability syndromes (ataxia telangiectasia, fanconi anaemia, bloom syndrome)
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2
Q

What blood cells are manipulated in postnatal samples

A

Lymphocytes

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3
Q

What growth media is used

A
  • RPMI 1640.
  • Balanced salt solution containing Hepes buffer, energy source, amino acids, hormones, proteins, peptides and fatty acids.
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4
Q

What happens during culturing

A

Lymphocyte suspension culutres are set up and incubated.

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5
Q

What are synchronised cultures

A
  • Chemical manipulation of the cell cycle with the aim of producing as many cells as possible at the same point of the cell cycle (pro-metaphase).
  • Gives the highest number of metaphases possible for downstream staining.
  • often 72 hours
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6
Q

What is used as a chemical block for synchronised cultures

A
  • Thymidine
  • Pauses progression of cell cycle at the beginning of S-phase.
  • Thymidine inhibits the uptake of deoxycytidine, slowing DNA synthesis.
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7
Q

What is used to release the chemical block in synchronised cultures

A

Deoxycytidine (DOC)

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8
Q

WHat does deoxycytidine do in synchronised cultures

A

-Lets cells progress to metaphase

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9
Q

What are unsynchronized cultures

A

-For urgent referrals, 48 hours.

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10
Q

How are cell cultures harvested

A
  • Mitotic arrest agent is added (colcemid), to stop cell cycle
  • The longer the colcemid is added the shorter the chromosomes and the higher the yield of metaphases
  • Hypotonic is added (Ohnuki). This increases the volume of cells, giving chromosomes more space to spread
  • Fixative added (Carnoy’s fixative), to kill cells, prepare for banding and remove biohazardous risk.
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11
Q

How are slides made

A
  • Aim is to make a slide with well spread metaphases that will band well.
  • Drop of cell suspension is dropped onto slide and allowed to dry.
  • As the fix evaporates it becomes thinner and pushed down on top of the cell. This enlarges the area of the cell and flattens them out.
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12
Q

What is chromosome banding

A

-Main method is G-banding using a combination of trypsin and Leishman’s stain.
-G-banding stains chromosomes with bands that make chromosomes distinguishable based on their banding patterns and makes structural rearrangements detectable
-Euchromatin is pale and gene rich, GC rich.
Heterochromatin is dark, gene poor and AT rich.
-Trypsin is a proteolytic enzyme that partially digests the chromosome at pale regions allowing staining.

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13
Q

Uses of cell culture and staining for prenatal genetic testing

A

-Confirm results of an abnormal screening test, e.g. high risk of trisomy.

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14
Q

What is chorionic villus sampling

A
  • Sampling part of the placental tissue.
  • Usually carried out between 11 and 12 weeks (before 15 weeks).
  • Spontaneous abortion risk is 0.5-1%
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15
Q

What is amniotic fluid sampling

A
  • Obtained from amniotic sac surrouding the detus via amniocentesis.
  • Performed between 16-20 weeks.
  • Spontaneous abortion risk is 0.5-1%
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16
Q

What is fetal blood sampling

A
  • Fetal blood is obtained from umbilical cord or fetus. Also known as cordocentesis.
  • Generally only used when CVS/Amniocentesis indicated an abnormality requiring further investigation
  • Spontaneous abortion risk is 2%
  • Carried out from week 17 onwards
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17
Q

What is cell-free fetal DNA (cffDNA)

A
  • Harvested from maternal bloodstream.
  • DNA in cffDNA originates from placental trophoblast and is fragmented when placental microparticles are shed into maternal blood.
  • CffDNA present in maternal blood from 5-7 weeks
  • No risk of spontaenous abortion
18
Q

What considerations are made for prenatal samples

A
  • Quality= is there blood staining, test could fail due to MCC
  • Quantity= may lead to PCR failure
19
Q

Why are long term cultures established for prenatal samples

A

-used to obtain additional DNA and metaphase chromosomes for analysis if needed.

20
Q

What considerations should be made when culturing prenatal samples

A
  • Independent cultures to ensure high success rate and minimise risk of contamination
  • Handled separately to non-prenatal cultures (to minimise microbial cross contamination)
  • Keep cultures until after the final report is issued.
21
Q

What do chorionic villi consist of

A
  • Cytotrophoblast= more distantly related to fetus

- mesodermal cells.

22
Q

Problems of using cytotrophoblast cells for prenatal testing

A

Cytotrophoblast cells are less representative of the fetus, so abnormalities detected are more likely to be the result of confined placental mosaicism (CPM)

23
Q

What is confined placental mosaicism (CPM)

A
  • CPM is the presence of chromosomal abnormalities in the extra-embryonic tissue which are absent from the fetal tissue
  • These chromosomal abnormalities are observed in about 1 to 2% of CVS carried out between the 9th and 12th weeks
24
Q

How does culturing overcome contamination of sample with cytotrophoblasts

A
  • Digested CV samples have a mix of cytotrophoblast and mesodermal cells
  • Culturing after digestion will result in the selection of more actively replicating mesodermal cells.
25
Q

How are CVS processed

A
  • Cleaned= removes blood clots, non-villi and maternal decidua
  • Morphology and weight assessed
  • Dipase added to digest it
  • Collagenase suspends cells
  • Some suspension used for DNA extraction
  • Remaining suspension used to establish cultures
26
Q

What do best practice guidelines recommend for CVS processing

A
  • Should be dissected to determine best material for processing
  • Use of direct and long term cultures
  • Three independent cultures set up, in different incubators and media to minimise contamination.
27
Q

How are amniotic fluids processed

A
  • Apportioned according to testing indicated
  • Aliquots are centrifuged
  • Presence of pellet checked
  • Supernatant removed
  • Pellet resuspended and transferred for DNA extraction (if required)
  • For culture, pellet resuspended in Amniomax media
  • Cell suspension examined under microscope to check for amniocytes and put in incubator.
28
Q

How are cultures maintained

A
  • Assessed after 6-7 days using a stereomicroscope.
  • If there are colonies then the media is replaced.
  • 5 or more active colonies likely enough metaphases for harvesting.
29
Q

Is overgrowth of a culture good

A
  • No, overgrowth of cells limits the number of actively dividing cells, so fewer metaphases.
  • Make subcultures, spreads etc to increase free space to increase mitotic index of sample
30
Q

How are cultures harvested

A
  • Done in a class 2 hood (due to mutagenic BrdU use)
  • Cultures have B-ONC added (BrdU and colcemid mixture) afternoon before harvesting to increase number of mitotic cells
  • Next morning, media is decanted and trypsin/versene is added to suspend the cells
  • Cells pelleted by centrifugation, supernatant removed
  • Pellet resuspended in hypotonic solution
  • Fresh fixative added and tube agitated to prevent clumps forming
31
Q

What is the purpose of BrdU in harvesting cultures

A

Gives longer chromosomes

32
Q

What is the purpose of colcemid in harvesting cultures

A

Halts mitotic spindle

33
Q

How are slides made

A
  • Temperature and humidity is important in producing consistently high quality slides.
  • slides are made (first is a test)
  • Breath on slide and 1 drop of sample suspension is rolled across slide, followed by a drop of fix
  • Left to dry and checked under the microscope
  • More than 5 good metaphases are required to ensure successful analysis
34
Q

How are cells counted

A
  • Coulter counter

- Used for appropriate seeding of culture

35
Q

Considerations for culturing neoplastic cells

A
  • Already dividing.
  • Healthy= white blood cells are non-dividing. De-differentiate into immature cells and divide in presence of PHA/antigen
  • Neoplastic white blood cells= already immature and dividing.
  • Can’t synchronise
36
Q

What is G banding

A
  • Giemsa banding
  • Trypsin added first, followed by a Romanovsky dye (Giemsa, Leishman’s stain)
  • Dark bands= A-T rich, heterochromatin (late replicating)
  • Light bands= C-G rich, euchromatin, early replicating
37
Q

What is Q banding

A
  • Quinacrine banding
  • Quinacrine mustard is a fluorescent dye that forms bands when interacting with A and T bases (DAPI also produces a similar banding)
  • Used for visualising polymorphisms on Y chromosome and variable regions including centromeres of chromosomes 3 and 4.
38
Q

What is R banding

A
  • Reverse banding
  • Reverse of Q and R banding
  • denatures AT rich DNA.
  • End of chromosomes are darkly stained
  • Good to look at abnormalities involving chromosome ends.
39
Q

What is C banding

A
  • Constitutive heterochromatin banding
  • Used to identify heterochromaitc segments
  • Uses restriction endoncuelases (Alu1)
  • Centromeres are visible
40
Q

What is NOR staining

A
  • Silver staining of NORs (nucleolar organising region)
  • NORs are located in stalk region of chromosomes (13, 14, 15, 21, 22), where ribosomal gene clusters are located.
  • Only NORs which were active in previous cell cycle will stain positively (functional staining)
41
Q

What is solid staining

A
  • Staining of chromsomes directly with Giemsa to show fragile sites (Fragile X or chromosome breakage disorders).
  • Used to assess chromsome morphology for roberts syndrome or mosaic variegated aneuploidy referrals.
  • Visualises sister chromatid separation
42
Q

What is differential replication stainging

A
  • BrdU is an analogue of thymidine and is incorporated into DNA during DNA synthesis.
  • If BrdU is added late in S phase, areas which replicate later will incorporate BrdU instead of Thymidine.
  • BrdU containing strands are more sensitive to damage from UV exposure and will not stain.
  • Used for indicating late replicating (inactivated) X chromosomes and assess X-autosome translocations.
  • Late replicating X will incorporate BrdU and be pale.

Path 19.02 notes (26 decks)

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