Prenatal Culture/Analysis

Question 1

What is mosaicism? What causes it?

Answer 1

Two or more cell lines derived from a single cell line.

Caused by:

• postzygotic (mitotic) non-disjunction resulting in normal cells and cells with aneuploidy, the proportion and distribution of these cells depends on where about the error occurred but may include:
  1. majority of all cells aneuploid - present across placenta and fetus
  2. Aneuploidy confined to the placenta, full or mosaic
  3. Aneuploidy confined to the fetus, full or mosaic
• rescue event (anaphase lag) in a fetus that originally started out with trisomy resulting in a proportion of normal cells: levels and distribution will depends on how early the rescue event took place, as above it may result in:
  1. Normal placenta and aneuploid fetus
  2. Aneuploid placenta and normal fetus
  3. Majority of cells aneuploid
• very rarely, mosaicism of a translocation.

Question 2

What question does mosaicism in a CVS pose?

Answer 2

• is the mosaicism confined to the placenta?
• is it present in the fetus?
• or is it generalised?

Question 3

What could the outcome of trisomy rescue be?

Answer 3

Depends on where the rescue event occurred:

• very early = disomic fetus and placenta (all normal)
• trisomy in fetus but disomy in placenta
• trisomy in placenta but disomy in fetus
• trisomy in the placenta but mosaic fetus
• mosaic placenta but trisomy fetus.

Question 4

What is important if there could be trisomy rescue at play? E.g. mosaicism or trisomy seen on CVS.

Answer 4

MUST remember that dependant on which chromosome is ‘kicked out’ we could end up with UPD (e.g. both remaining chromosomes from the same parent).
Important for:
- chromosome 15 (PWS or Angelman’s)
- chromosome 7 and 11 for Russell Silver
- chromosome 7 for CF (parent could be carrier of CF mutation)
- chromosome 14 (maternal or paternal UPD14 is associated with defects, paternal more severe = bell shaped thorax).

Question 5

What would we do if CVS shows a trisomy and subsequent amniotic fluid is normal?

Answer 5

If the trisomy was for a chromosome known to be implicated in UPD we could do parent of origin testing.
Also would suggest detailed US and ?referral to clinical.

Question 6

What physical problems can CPM cause?

Answer 6

Compromised placental function which may lead to IUGR in fetus.

However, be wary if the chromosome implicated in the trisomy is known to be associated with imprinting effects.

Question 7

Name at least 2 commonly observed CPM trisomies.

Answer 7

2, 3, 7, 8, 16 and 22.

Question 8

What is the difference between trophoblast cells and mesenchyme core cells?

Answer 8

Trophoblast cells are 1st cells to differentiate from the morula and are therefore more distantly related to the fetal cells.
The mesenchymal core cells differentiate later and are more closely representative of the extraembryonic mesoderm. They are therefore more likely to represent the fetal karyotype.

Question 9

An amnio has been sent for array and comes back with an abnormal finding, what would we do next?

Answer 9

Firstly we need to confirm sample identity to make sure the result relates to the correct patient.
We have lots of checks in place at all sample transfers however the consequences of an error of this nature would be severe and therefore we would repeat the microarray from a fresh extraction.
Alternatively it could be confirmed with FISH as this is quicker and robust.
Once the result has been confirmed it can be reported.

Question 10

What type of cells does amniotic fluid contain?

Answer 10

• Heterogenous population of cells

- includes fetal skin cells, cells from the respiratory, digestive and urinary tract.

Question 11

What cell predominates in amniotic fluid culture?

Answer 11

Fibroblast cells.

Question 12

What is level 1 mosaicism?

Would we report it?

Answer 12

A single abnormal cell.

No.

Question 13

What is level 2 mosaicism?

Answer 13

Two or more cells with the same abnormality but either in a single colony or a dispersed culture.

Question 14

What is level 3 mosaicism?

Answer 14

Two or more cells with the same abnormality seen in 2 or more independent cultures.

Question 15

What is the minimum number of cells to be analysed and checked on an amnio/CVS according to BPG?
What about if there is mosaicism?

Answer 15

Minimum of 2 fully analysed, this must include all bands.
Extra cells should be used for crossovers.
A minimum of 1 further cell should be checked.

The above applies to each cell line.

Question 16

What prenatal guidelines are used for the confirmation of true mosaicism?

Answer 16

Hsu and Benn 1999

Question 17

Briefly what do the mosaicism guidelines require you to do?

Answer 17

• either a basic, moderate or extensive work-up,

- the work-up depends on what you’ve seen and the culture type you are using e.g. flask v in-situ.

Question 18

What would a basic, moderate and extensive work-up consist of in a suspension culture?

Answer 18

Basic: 20 cells from 2 independent cultures one of which can contain the anomalous metaphase

Moderate: as basic but the 20 cells must be from 2 cultures excluding the one with the anomalous cell.

Extensive: 20 cells from each of the 2 cultures which don’t contain the anomalous cell, totalling 40 cells.

Question 19

What would be a basic, moderate and extensive work-up for an in-situ culture?

Answer 19

Basic: examination of a total of 15 colonies from 2 separate cultures which can include the culture with the anomalous cell.

Moderate: examination of at least 12 colonies from separate cultures excluding the culture that contains the anomalous cell.

Extensive: examination of 24 colonies from at least 2 cultures excluding the one with the anomalous cell.

Question 20

Which level of mosaicism requires a particular level of care?
What could we do if this was found in a CVS?

Answer 20

Level 2
2 or more abnormal cells but from the same colony or culture.
May be appropriate to recommend a follow up amniocentesis if this is found in a CVS.

Question 21

What type of cultures do we use for our CVS samples?

Answer 21

In situ cultures

Question 22

Could UPD be important where mosaicism is involved?

Answer 22

Yes if the chromosome involved is implicated in UPD.
A trisomy rescue event may have occurred which has resulted in the remaining 2 chromosomes being from the same parent.
Same can be said for a structural rearrangement such as a robertsonian.

Question 23

If mosaicism is detected in a CVS what should we include on the report?

Answer 23

A comment to advise follow-up sampling by amniocentesis or fetal blood.
Detailed ultrasound scans.

Question 24

If mosaicism is detected by either CVS or amniocentesis what should we state on the report?

Answer 24

That the levels detected may not necessarily reflect the proportion or tissue distribution in the fetus.

Question 25

What is the relevance of UPD in a prenatal sample which contains a marker chromosome?
What is the mechanism?

Answer 25

The 2 complete chromosomes might be from the same parent - be worried if something like chr15 is involved!
It may have started out as a trisomic conceptus and then one of these chromosomes has been involved in a structural alteration resulting in a small marker e.g. u-loop mechanism. If this mar chromosome is derived from the sole chromosome from a particular parent then the 2 remaining intact ones will be from the same parent. This will cause UPD.

Question 26

Why do we do 30 cells when looking for mosaicism?

Answer 26

It is to do with confidence levels.

30 cells gives you 95% confidence that mosaicism at 10% or higher would be detected in the analysis.

Question 27

Why are CVS samples sorted on receipt and then checked?

Answer 27

The purpose is to wash away and dissect out all recognisable maternal tissue to reduce the chance of the maternal genotype being present in the sample and affecting the result.

Question 28

Why do our culture regimes favour the growth of fetal cells?

Answer 28

• the media is specially designed to nourish the fetal cells
• maternal WBC do not settle and adhere to the coverslip so once fetal cells have settled we do a media change which will remove any maternal cells in suspension

Question 29

What would you do if you received a heavily bloodstained amnio?

Answer 29

• call clinician to inform them sample is bloodstained and that a rapid result won’t be possible
• request a maternal blood for future MCC exclusion (this could be used to speed an abnormal result up as will confirm sample identity)
• set-up cultures, these will preferentially grow the fetal cells
• once cultures are ready we can then do QF-PCR and array as normal

Question 30

If we did an array on cultured cells and it showed a mosaic trisomy, what could we do?

Answer 30

?We would use another culture to confirm the finding e.g. potentially could FISH another culture or do a repeat microarray?