Principle of Test: The tissue is desegregated into a single cell single cell suspension by means of enzyme or mechanical disruption. These single cells are plated onto a sterile glass coverslip in a petri dish where they grow in situ to form colonies.
Clinical interpretation: Test for foetal abnormality.
When sample comes in, the first thing to be done is requisition. These are the main information stated in the request form:
Samples Collected: Chorionic villi specimens
Clinical indication: Nuchal Translucency (NT) 6, 7mm. 1:2 (T21). 1:7 (T13:T18)
Possible Diagnosis: Foetal Abnormalities - Down’s syndrome (Trisomy 21), Edwards syndrome (Trisomy 18) and Patau syndrome (Trisomy 13).
Given this information, the cytogenetist have to culture, followed by harvest and banding, then lastly, analyse the chromosomes karyotype to confirm the diagnosis.
Before I continue, I would like to explain on certain terms.
Chorionic villi – a small sample of chorion or placental tissue for prenatal diagnosis, usually by aspiration biopsy with a thin plastic catheter inserted trans-cervically or trans-abdominally into the uterus. This tissue can be obtained in early pregnancy, thus allowing early detection of genetic abnormalities. This is usually performed at 10-12 weeks of pregnancy, whereas, amniocentesis (amniotic fluid) is performed after 14-15 weeks.
Nuchal Translucency – small transparent area beneath the skin at the back of the foetus’ neck. An increase in nuchal translucency measurement indicates an increase in the risk of chromosomal abnormalities, such as Down’s syndrome.
Trisomy – extra chromosome in the respective position eg, T13 means 3 copies of chromosome at chromosome 13 instead of usual 2 copies.
Now, I’m going to briefly explain the procedure of handling chorionic villi samples.
Culture
- Upon receiving the tissues, wash it with complete α-MEM / Amniomax™ - II medium (α-AM)
- Dissect under dissecting microscope to choose the appropriate fetal tissue
- Strip the outer layer to remove maternal tissues
- Clean the choronic villi tissues with 3ml of trypsin EDTA (10X concentration).
- Incubate it for 25 minutes, 37°C
- Centrifuge it at 1200rpm for 10 minutes
- Remove the supernatant (trypsin EDTA) and add in 1ml of collagenase into the tube with the pellet. Trypsin EDTA and collagenase aids in the digestion of various proteins to dissociate the tissues and obtain cells.
- Incubate it for 30-90 minutes, 37°C
- Single cells will be form and add in 3ml of α-AM (medium). This is to neutralise and stop the collagenase activity.
- Centrifuge it at 1200rpm for 10 minutes
- Prepare 4 culture dishes and label it as ‘A’, ‘B’, ‘C’ and ‘D’.
- Resuspend the pellet with 2ml of α-AM (medium) and transfer 0.5ml of cell suspension onto each coverslip in each of the 4 dishes.
- Culture ‘A’ and ‘C’ in one incubator while ‘B’ and ‘D’ in another incubator. This is one of CAP’s regulation and also to prevent failed cultures upon power failure.
Incubate the cultures for 5 days and then, check for attachment.
Harvest
Once more than 4 colonies or 50-200 cells can be seen, the culture can be prepared for harvest. During harvest, Colcemid® working solution is added to prevent the synthesis of spindle fibres and this arrest the cells at metaphase stage. Colcemid® is a cytotoxic chemical, therefore, it has to be handled properly and disposed into the designated cytotoxic waste.
Warm 0.8% sodium citrate hypotonic solution is added to increase the cell volume by swelling it, spreading the chromosomes apart. This enlarges the view of chromosomes.
1:3 methanol – glacial acetic acid fixative is needed to fix the cells, in order to preserve the architectural structure of the cells and prepare for staining. Giemsa & Wright’s stain is usually used.
Done by: Yvonne Lau
