Sunday, July 29, 2007
Cytogenetics
Sample collected: Aminotic Fluid (AF)
Purpose:
Check the fetus’ chromosomes at metaphase to see if it is normal.
A normal set of chromosome has
- a total chromosomal count of 46 consisting of a pair of each chromosome(1-22) and the sex chromosome- either XX or XY.
1) Detect abnormalities in fetus of women with advanced maternal age
With a maternal age of 35 and above, the chances of carrying a fetus with genetic abnormality increases. Common genetic abnormalities include Down’s syndrome (Trisomy 21) and Edward’s syndrome (Trisomy 18).
2) Doctors noticed abnormalities during ultrasound
During ultrasound, doctors notice something amiss about the baby’s condition and with may send AF out for test.
Why Aminotic Fluid?
AF is present all around in the placenta (which holds the baby). As the baby grows, it will shed cells in to the AF. Therefore, the AF contains baby cells that can be cultured and analysis for chromosomal abnormality.
How is it process?
Collection – Processing (Set-up) – Harvest – Drying – Baking – Staining -- Analysis
Firstly, the AF will be equally allocated into 3 tubes, one of which will be kept as a back-up. The tubes will then be centrifuged. After the supernatant is removed, culture media is added to resuspend the cells. Different media e.g. (will usually use) BioAMF and Chang’s Media will be used in the 3 tubes, this is part of quality control.
Secondly, the cell suspension will be aliquot into 4 petri dish containing cover-slips. They will then be incubated and the cells will eventually grow directly on the cover-slips.
After the cells have grown to form about 4 colonies, they will be harvested. Harvesting is done automatically using an auto-harvestor- Tecan. Tecan is able to carry out the entire harvesting process independently and during this period the technologist is able to carry out other duties.
Tecan will carry out the following processes:
-remove the culture media via a pump
-add hypotonic solution (KCl)
-timed an incubation period of 30mins in hypo
-remove hypo
-add fixative- made up of methanol and acetic acid.
-timed an incubation of 30mins in fixative
-change another round of fixative
When cells are exposed to hypotonic solution, they will swell and this allows the chromosomes to spread out and not clump together (which will be difficult for analysis). The addition of fixative removes water and preserves cells in its swelled-up state.
After the harvesting process is done, the Petri dish containing the cover-slip will be transferred into the Thermotron for further removal of water aka Drying. Drying involves the addition and removal of fixatives up to 3 times for complete dehydration of cell and to spreading the cells out. Thermotron is a incubator that facilitates the drying process, it is able control the humidity and temperature thus promoting optimal drying. This process is important as:
- if drying is too fast(low humidity, high temp), chromosomes won't be able to spread out effectively
-if drying is too slow(high humidity, low temp), chromosomes will spread so much that it goes beyond the cover-slip and thus will be washed away.
Next, the cover-slip will be remove from the Petri dish and be mounted on a glass slide. The glass slide will then be baked in the hot plate at 90°C for 11/2 hours; this is to fix the cells on the slide to prevent it from floating away during staining. Baking also causes the A-T bonds to dissociate thus allowing the dye to be taken up by the chromosome.
After which, cells will be stain in either Wright’s or Geimsa to give a visible/prominent light and dark bands on the chromosomes for analysis. The staining process involves the trysinisation of cells. Trypsin hydrolyses protein component of the chromatin thereby allowing the dye to react(dye) with the exposed DNA.
Finally, after staining, it's ANALYSIS time. Real-experienced med techs will study and identify the chromosomes and detect any deletion, addition or presence of extra chromosome with their naked eyes. They're real quick in identifying them!
Alrights, here's the end, if there's any question, just bring it on =)
phuiyuen
Purpose:
Check the fetus’ chromosomes at metaphase to see if it is normal.
A normal set of chromosome has
- a total chromosomal count of 46 consisting of a pair of each chromosome(1-22) and the sex chromosome- either XX or XY.
1) Detect abnormalities in fetus of women with advanced maternal age
With a maternal age of 35 and above, the chances of carrying a fetus with genetic abnormality increases. Common genetic abnormalities include Down’s syndrome (Trisomy 21) and Edward’s syndrome (Trisomy 18).
2) Doctors noticed abnormalities during ultrasound
During ultrasound, doctors notice something amiss about the baby’s condition and with may send AF out for test.
Why Aminotic Fluid?
AF is present all around in the placenta (which holds the baby). As the baby grows, it will shed cells in to the AF. Therefore, the AF contains baby cells that can be cultured and analysis for chromosomal abnormality.
How is it process?
Collection – Processing (Set-up) – Harvest – Drying – Baking – Staining -- Analysis
Firstly, the AF will be equally allocated into 3 tubes, one of which will be kept as a back-up. The tubes will then be centrifuged. After the supernatant is removed, culture media is added to resuspend the cells. Different media e.g. (will usually use) BioAMF and Chang’s Media will be used in the 3 tubes, this is part of quality control.
Secondly, the cell suspension will be aliquot into 4 petri dish containing cover-slips. They will then be incubated and the cells will eventually grow directly on the cover-slips.
After the cells have grown to form about 4 colonies, they will be harvested. Harvesting is done automatically using an auto-harvestor- Tecan. Tecan is able to carry out the entire harvesting process independently and during this period the technologist is able to carry out other duties.
Tecan will carry out the following processes:
-remove the culture media via a pump
-add hypotonic solution (KCl)
-timed an incubation period of 30mins in hypo
-remove hypo
-add fixative- made up of methanol and acetic acid.
-timed an incubation of 30mins in fixative
-change another round of fixative
When cells are exposed to hypotonic solution, they will swell and this allows the chromosomes to spread out and not clump together (which will be difficult for analysis). The addition of fixative removes water and preserves cells in its swelled-up state.
After the harvesting process is done, the Petri dish containing the cover-slip will be transferred into the Thermotron for further removal of water aka Drying. Drying involves the addition and removal of fixatives up to 3 times for complete dehydration of cell and to spreading the cells out. Thermotron is a incubator that facilitates the drying process, it is able control the humidity and temperature thus promoting optimal drying. This process is important as:
- if drying is too fast(low humidity, high temp), chromosomes won't be able to spread out effectively
-if drying is too slow(high humidity, low temp), chromosomes will spread so much that it goes beyond the cover-slip and thus will be washed away.
Next, the cover-slip will be remove from the Petri dish and be mounted on a glass slide. The glass slide will then be baked in the hot plate at 90°C for 11/2 hours; this is to fix the cells on the slide to prevent it from floating away during staining. Baking also causes the A-T bonds to dissociate thus allowing the dye to be taken up by the chromosome.
After which, cells will be stain in either Wright’s or Geimsa to give a visible/prominent light and dark bands on the chromosomes for analysis. The staining process involves the trysinisation of cells. Trypsin hydrolyses protein component of the chromatin thereby allowing the dye to react(dye) with the exposed DNA.
Finally, after staining, it's ANALYSIS time. Real-experienced med techs will study and identify the chromosomes and detect any deletion, addition or presence of extra chromosome with their naked eyes. They're real quick in identifying them!
Alrights, here's the end, if there's any question, just bring it on =)
phuiyuen
# posted by Star team @ 8:53 PM 
Comments:
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Hello =)
As mentioned, the baking will break the A-T bonds right? How come the C-G bonds are not broken? Is it because it has more hydrogen bonds hence they are able withstand a higher temperature?
Chen KangTing
0503331A
TG02
As mentioned, the baking will break the A-T bonds right? How come the C-G bonds are not broken? Is it because it has more hydrogen bonds hence they are able withstand a higher temperature?
Chen KangTing
0503331A
TG02
Hey Phuiyuen,
Is analysis done manually? to label and pair up the individual chromosomes?
Randall
TG02
0503272G
Is analysis done manually? to label and pair up the individual chromosomes?
Randall
TG02
0503272G
hey HI
Im in cyto lab too but haven tried anything on AF, dealing with BMA. SOunds cool. Just one question, You mentioned about TECAN. how long does it take to finish the process?? and wat if something goes wrong with the machine? do u guys practice any manual procedures??
Vinodhini
TGO2
Im in cyto lab too but haven tried anything on AF, dealing with BMA. SOunds cool. Just one question, You mentioned about TECAN. how long does it take to finish the process?? and wat if something goes wrong with the machine? do u guys practice any manual procedures??
Vinodhini
TGO2
hey phuiyuen,
wanna ask.. how long does the whole process from specimen collection to analysis takes? how much sample can u all handle everyday? what other tests do u all handle in cytogenetics lab?
thanks for answering.. :)
doreen (tg01)
wanna ask.. how long does the whole process from specimen collection to analysis takes? how much sample can u all handle everyday? what other tests do u all handle in cytogenetics lab?
thanks for answering.. :)
doreen (tg01)
Hello!
Just want to ask, when is this test done? At which month of pregnancy? =D
Thanks!
Charmaine Tan
TG01
Just want to ask, when is this test done? At which month of pregnancy? =D
Thanks!
Charmaine Tan
TG01
to KangTing,
hey =)
yup, cos C-G has got more hydrogen bonds so denature them would require a higher temperature/longer duration. If all the hydrogen bonds are denatured, then the chromosome will be separated(becoming single-strands, so no product may be used for analysis.
Randall Randall,
analysis are done both manually and automatically. For accurate analysis the medtechs have to analyse 15metaphases (through the microscope) and of the 15, 3 will be captured by cytovision(print it out and be verified by the scientific officer). Cytovision has a program that is able to recognise the bands on the chromosomes and put them in the grp they belong to but sometimes they do make some mistakes in recognising the chromosomes. So, manually, medtech will do the editing.
to Vino:
hello =)
Tecan takes abt an hour plus (on an average) depending on the amount of petri dishes. If Tecan breaks down, the harvesting process will be done manually and the steps will be exactly the same as Tecan, so nothing is pretty much affected.
to doreen:
from AF processing to analysis takes abt 5-7days and for the report to be sent out takes abt 8-11days. The amount of samples depends on how much iit is sent in by the doctors, whatever the amount they collected will be sent into the lab on the very day itself. The lowest ever was only 1 AF sample and highest was 15. The other test done are FISH, Blood, Bone marrow, Villi, Product of Conception.
to charmaine:
this test is if the expecting mother is above 35 years old, usually between 12 to 20 gestational weeks. As the turnaround time is abt 2 weeks so the sample is preferably sent in early cos after the 24th gestational week, no abortion is allowed even the baby is abnormal.
Hope these answers help =)
hey =)
yup, cos C-G has got more hydrogen bonds so denature them would require a higher temperature/longer duration. If all the hydrogen bonds are denatured, then the chromosome will be separated(becoming single-strands, so no product may be used for analysis.
Randall Randall,
analysis are done both manually and automatically. For accurate analysis the medtechs have to analyse 15metaphases (through the microscope) and of the 15, 3 will be captured by cytovision(print it out and be verified by the scientific officer). Cytovision has a program that is able to recognise the bands on the chromosomes and put them in the grp they belong to but sometimes they do make some mistakes in recognising the chromosomes. So, manually, medtech will do the editing.
to Vino:
hello =)
Tecan takes abt an hour plus (on an average) depending on the amount of petri dishes. If Tecan breaks down, the harvesting process will be done manually and the steps will be exactly the same as Tecan, so nothing is pretty much affected.
to doreen:
from AF processing to analysis takes abt 5-7days and for the report to be sent out takes abt 8-11days. The amount of samples depends on how much iit is sent in by the doctors, whatever the amount they collected will be sent into the lab on the very day itself. The lowest ever was only 1 AF sample and highest was 15. The other test done are FISH, Blood, Bone marrow, Villi, Product of Conception.
to charmaine:
this test is if the expecting mother is above 35 years old, usually between 12 to 20 gestational weeks. As the turnaround time is abt 2 weeks so the sample is preferably sent in early cos after the 24th gestational week, no abortion is allowed even the baby is abnormal.
Hope these answers help =)
# posted by 
: July 31, 2007 at 10:58 PM
: July 31, 2007 at 10:58 PM
Hi Phui Yuen
What is Edward Syndrome? If the bady has this particular abnormalities, what are the features the bady will have once he/she is born? See you soon =)
Eugene Wong
TG02
What is Edward Syndrome? If the bady has this particular abnormalities, what are the features the bady will have once he/she is born? See you soon =)
Eugene Wong
TG02
Elooo ThamPY
Would like to know how long does the cells take to form the colonies and ae the colonies visible or a microscope is needed to view it.
Najib (0503217B)
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Would like to know how long does the cells take to form the colonies and ae the colonies visible or a microscope is needed to view it.
Najib (0503217B)
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