Rai's SIP.

For the past 4 weeks, im attached to a Cytogenetics Laboratory. Written below is what i have learnt so far, enjoy =)

In a nutshell, the Cytogenetics lab processes patients sample to have their chromosomes analysed so as to conclude if patient has abnormalities in their chromosomes or not.

Basically, what I have learnt in the 4 weeks of my attachment to the Cytogenetics laboratory can be summarised into 5 parts.

Why perform cytogenetics test?
Why that certain sample is collected?
Summary of processes
Journey of my blood
Identification and karyotyping of chromosomes

Why perform cytogenetics test?

The few reasons include prenatal diagnosis where parents would want to find out if the baby in the womb is abnormal, diagnosis of neonates with abnormalities, diagnosing children with dysmorphic features/ developmental delay and couples who have frequent miscarriages who might want to know if they have any chromosomal abnormalities and patients with haematological malignant diseases.

Why that certain sample is collected?

For adults, peripheral blood is withdrawn from patient and used as a sample.
However, for pregnant women who wants to have a prenatal diagnosis, different type of sample is withdrawn for women of different gestation week due to several reasons.
For pregnant women between 10-12 weeks gestation, the sample ‘’chorionic villi’’ is extracted. U can know what is the ‘’chorionic villi’’ from the diagram below.

image taken from http://www.nwabr.org/studentbiotech/winners/studentwork/2007/WB_BA_TRONGTHAM/piccvs.jpg

Why this is extracted is because during this time, the chorionic villi are still new and small and would not cause serious bleeding to the mother when extracted. Also, the amniotic fluid cannot be withdrawn at this stage because the amniotic fluid is too little and if withdrawn, might cause dehydration to the fetus.

For pregnant women between 16-20 weeks gestation, amniocentesis is carried out. This is the withdrawing of amniotic fluid. At this stage, the amount of aminotic fluid is sufficient and this is confirmed by ultrasound.

For pregnant women of 22weeks gestation, fetal cord blood is extracted because by this time, the baby in the womb is already huge and taking up a lot of space in the womb, making it difficult to extract the amniotic fluid as there is a high risk of injuring the baby with the syringe.

Also, cord blood takes up to 7 days to obtain results, which is considered a short period of time compared to other samples, it is important that the lab technicians does not make a mistake in handling the sample tat might lead to wasting the sample because abortion can only be done up to 24th week gestation. This is especially critical for cases where the fetus is found to be abnormal and parents would want to consider aborting the child.
Here, we can see the importance of handling the samples with care, accuracy and precision. That is why the cytogenetics laboratory is a highly critical laboratory which could not afford to make mistakes and waste their samples and so, I am only allowed to observe the processes or try out the processes once or twice in cases where the samples are abundant.

Summary of processes

For each sample that comes into the laboratory, it will go through a common procedure that is

Cultured with cultured and colcemid is added. Colcemid is a mitotic inhibitor which causes the cells not to divide at metaphase. This is so that when the cells are analysed later on, the chromosomes would be seen and can be analysed.
Centrifugation and removal of growth medium. Changing of medium and cells are resuspend.
Cells are checked under microscope to check for growth. If sufficient amount of colonies are present, cells are ready for ‘’Harvest’’. If overcrowding os observed, cells are ‘tiffed’ (redistributed) and checked a few days later for growth.
Harvesting. Cells treated with hypotonic solution which increases the cell volume to allow the chromosomes to spread.
Cells fixed with fixative that draws out water out of cells so as to preserve them.
Cells transferred onto glass slide and allowed to dry.
Cells stained with Trypsin and Giemsa stain.
Slide is viewed under the microscope.
Using a microscope that is connected to the computer, an image of the desired metaphase can be captured and shown on the computer.
A specific programme is used to analyse the chromosome, karyotype, and analysed for any chromosomal abonormalities.

Journey of my Blood sample

Tuesday, 8th July
4pm:
Peripheral blood was withdrawn from my right arm and collected into a sodium heparin tube (anti-coagulant).

Blood set-up was done. This procedure includes centrifuging my blood sample for 5 minutes so that the buffy coat layer can be obtained. This is shown in the diagram below.

image taken from http://www.nsbri.org/HumanPhysSpace/index.html

The buffy coat layer is extracted from the tube and reconstituted into 2 separate tubes containing M199 media. 5 microliters of ‘PHA’ is added into each tube to stimulate the division of my T-lymphocytes present in the buffy coat layer.

Thursday 10th July
4pm:
50 microliters of MTX was added into each tube to synchronise the cells.

Friday 11th July
10am: 50 microliters of Thymidine added into each tube.

12pm: 50 microliters of Colcemid added to tube 1.

1.40pm: 50 microliters of Colcemid added into tube 2

*Colcemid, as mentioned earlier will allow chromosomes to be seen under the microscope later on. Exposure to colcemid for 2 hours will give rise to many metaphases with short chromosome fragments. Exposure to colcemid for 20 minutes will give rise to few metaphases with long chromosomes.

4pm: Harvesting. Addition of hypotonic solution and subsequently, fixative.

Monday 13th July

10am: Cells prepared on glass slide, stained and ready to be analysed using the computer system.

Below is the pictures of my chromosomes in a metaphase.

(u can click on the image to enlarge it, if u want to see my chromosomes =))

Then, I identified the chromosomes and place them in their respective groups, this is called "karyotyping" shown below. It is then analysed for any chromosomal abnormalities such as translocation, deletion and others.

It looks like my chromosomes are normal and I have no chromosomal abnormalities, Thank God. =) (I was a little nervous to analyse them)

Identification and Karyotyping

So how did I learn to identify the chromosomes one by one? =) That was really something. It couldn’t be learnt overnight and I had nightmares about chromosomes during the first week.
Altogether, it took me 3 weeks to practice everyday identifying chromosomes until I could get them all correct although sometimes there is still human error =).
During the 3 weeks, when I was struggling really hard to recognize the chromosomes, I prepared for myself a table which I could refer to during identification of chromosomes which can be seen below.

A little tedious, but hey, no pain no gain. I am happy now that I could identify chromosomes and I’m proud to say I’ve learnt something.

Hope all of u like my post.
Byebye. Take care everyone.