Saturday, June 30, 2007

Cytogenetics (Lab technique)

Name of test: in situ culture technique for chorionic villi samples


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.

The next day, flood culture ‘A’ and ‘B’ with α-AM while ‘C’ and ‘D’ with BioAMF2 (another medium). This is to optimise the growth as different cells may favour different medium.


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.

Overall, It takes about 14 days to go through the process of Culturing -> Harvesting -> Banding -> Analysing.

Done by: Yvonne Lau


14 comments:

royal physicians said...

hey Yvonne, ur post was a very detailed and clear one.

Anyway i would like to us, since the process for in situ culture to analysis takes about 14 days(which is quite long), do cytogenetic labs handle emergency samples? if so, what kind of tests are usually done?

Nisha Bte Mohd Rafiq
0503254E
TG02

Star team said...

Hey Yvonne. You stated that Clinical indication: Nuchal Translucency (NT) 6, 7mm. 1:2 (T21). 1:7 (T13:T18). I don know if it's a stupid qns, but what does it actually menas. What do you mean by NT 6,7mm & 1:2 (T21)?

Hope you reply soon

Eugene Wong TG02

royal physicians said...

heya yvonne, a fun experiment u have done there. i have a few qn to ask u...what do u mean by in situ?? and i also would like to clarify sth...you mention that Culture A and C is placed in one incubator and the other two in another incubator to prevent failed culture during power failure, but if got power failure, won't all the incubators be down??...aniwei all the best for ur SIP and hope u njoy wat u r doing :)

by: nur zahirah (tg02)

77th Medical Technologists said...

Hello! ur work sounds really interesting! u're so lucky.. but it's kinda sad if in the end the fetus is positive for the test.. anyway wanna ask why is there a need to wash the tissues with complete �-MEM / Amniomax� - II medium (�-AM)? thanx for sharing ur experience

Distinction in Disaster! said...

Hi there!
Can I know what are the differences in morphology of the stains of the Chorionic villi cells which indicates the different type of fetal abnormalities?
Thanks!

Charmaine Tan~

VASTYJ said...

hihi.. juz to ask a few qns.. how is the banding usually done? and does this helps to interpret and provide the disgnosis required? and my next qn is for the fixation part for the cells b4 staining.. can any other fixatives be used.. especially those that we usually encountered in the histopath lab? thanks..

Jia Hao
TG01

77th Medical Technologists said...

Hello! ur work sounds really interesting! u're so lucky.. but it's kinda sad if in the end the fetus is positive for the test.. anyway wanna ask why is there a need to wash the tissues with complete �-MEM / Amniomax� - II medium (�-AM)? thanx for sharing ur experience!

Joanne
TG01

J.A.M.M.Y.S said...

reply to nisha:
yes, we do handle emergency samples. In the lab, if a neonate or fetal cord blood sample comes in, it means the case is serious and got to be done asap. 3 cultures will be set up. A preliminary result is usually available within 3 days by utilising a 48hrs culture on 1 tube while the other tubes will be cultured for 72hrs to provide longer chromosomes with higher banding resolution, which allow us to confirm the results.

J.A.M.M.Y.S said...

reply to eugene:

Nuchal Translucency (NT) 6, 7mm. 1:2 (T21). 1:7 (T13:T18)

This information is stated on the request form under 'clinical indications'

NT 6, 7mm - nuchal translucency(collection of fluid found beneath the skin at the back of the foetus neck seen via an ultrasound scan) measurement is about 6, 7mm.

1:2(T21) - Having a measurement of NT 6-7mm, the chances of the foetus suffering from T21 (down syndrome) is 1 out of 2.

Distinction in Disaster! said...

Hi, in case u didn't know, cytogenetics is up and rising niche area of SGH!

Anyway, I have a few questions. What is the difference between the banding and analysing processes? Is subculturing the cells or media changing necessary during the 5 day incubation?

Kent

J.A.M.M.Y.S said...

reply to nur zahirah:

in situ - "in place"

in situ harvesting - method of growing cells in coverslips inside the petri dihes.

This way the coverslips are harvested in situ ("in place") in the dishes, removed and later attached to slides for microscopy.

For my department, each incubators are plugged into different power plug, thus if one is down, the other may not be.

J.A.M.M.Y.S said...

reply to joanne:

washing the tissues is neccessary as we want fetal and not the maternal tissues, which is always present and will interfere with our analysis.

J.A.M.M.Y.S said...

reply to charmaine tan:

we identify fetal abnormalities by analysing the chromosomes banding and their arrangement, using giemsa and wright's stain. Dark and light bands will be produced and will be compared with the normal chromosomes banding.

For your information: Dark bands generally replicate their DNA late in S phase, contain A+T rich DNA, appear to contain relatively few active genes and may differ from light bands in terms of protein composition.

reply to kent:
Banding is the process of staining the cells, producing dark and light bands.
Analysing is the time when the cytogenetist will 'capture' the desired chromosomes, arrange and check for abnormalities.

During the 5 day incubation, minimal movement of the culture is recommended. This is to allow the cells to settle and attach onto the coverslips. Thus, changing of media or subculturing is unneccessary.

J.A.M.M.Y.S said...

reply to jia hao:

Banding is done by using giemsa and wright's stain usually. It helps to interpret the results as the dark and light bands are one of the components that cytogenetist look out for during analysis.

Other fixative, such as formalin are not possible in fixing chromosomes. 1:3 methanol – glacial acetic acid is the most favourable fixative as its action is less vigorous, much gentler.