SIP sharing week 5

Subject: Cytogenetics laboratory technique

Hi Friends!!

I am TING-JIE here! 5 weeks had already passed, time flies right? I do have fun at my attachment, all of the staffs are very nice to me, and I learnt a lot of new things from them, by the way, I am attached to Cytogenetics laboratory.

Basically there are 3 different sections in cytogenetics lab which include prenatal lab, bone marrow lab and FISH (fluorescence in-situ hybridization) lab. Currently I am under prenatal lab for the past 4 weeks.

And now I will be sharing with you all my experience in cytogenetics lab-prenatal section!
Firstly, what is cytogenetics?

Cytogenetics actually is the study of chromosome number and morphology, particularly as it relates to a normal or pathological state, the cells being studied are best at late prophase or metaphase of mitosis for analysis.

What is chromosome? It is a long, stringy aggregate of genes that carry heredity information and are formed from condensed chromatin.

Why late prophase or metaphase? For this we will have to refer back to Molecular Genetics (MGEN) and Molecular Biology (MBIO).

In the late prophase, the chromatin condenses into discrete chromosomes. The nuclear envelope breaks down and spindles form at opposite "poles" of the cell. The chromosomes begin to migrate toward the cell center.

picture taken from http://iweb.tntech.edu/mcaprio/late_prophase.jpg

In metaphase, the spindle fully develops and the chromosomes align at the metaphase plate (a plane that is equally distant from the two spindle poles). At this stage the nuclear membrane disappears completely and chromosomes are held at the metaphase plate by the equal forces of the polar fibers pushing on the centromeres of the chromosomes. This is also the stage when the chromosomes are at their most contracted state that is why chromosomes are best study at metaphase.

picture taken from http://iweb.tntech.edu/mcaprio/metaphase1.jpg

Only in those 2 phase, chromosomes are ready to be study under light microscope, whereas in anaphase, the paired chromosomes (sister chromatids) separate and begin moving to opposite ends (poles) of the cell. Spindle fibers not connected to chromatids lengthen and elongate the cell. And in telophase, which is the final stage of mitosis, the chromosomes uncoil, the nucleolus reappears the nuclear envelope reappears, the spindle fibers disappear, and results in two daughter cells. Therefore there are no complete chromosomes for us to study at anaphase and telophase due to the progression into daughter cells.

picture taken from http://iweb.tntech.edu/mcaprio/anaphase2.jpg

http://iweb.tntech.edu/mcaprio/Telophase1.jpg

Prenatal diagnosis is requested by obstetricians and gynaecologists, through chromosome analysis, it helped in the clinical management of pregnancies by detecting possible constitutional abnormalities of the fetus. The specimens used for prenatal cytogenetic diagnosis include amniotic fluid (AF), chorionic villi (CV) and fetal cord blood (FB).

Amniotic Fluid (AF) contains cells derive from the amnion, from the gastrointestinal tract and from the skin of the fetus. Some of these cells are viable and are capable of undergoing cell division in vitro. Through amniocentesis, a sample is collected for the purpose of culturing the cells to obtain chromosomes that reflect the fetal karyotype.

Amniocentesis is a diagnostic procedure performed by inserting a hollow needle through the abdominal wall into the uterus and withdrawing a small amount of fluid from the sac surrounding the fetus.



illustration of the amniocentesis procedure

picture taken from http://atlasgeneticsoncology.org/Educ/PrenatID30055ES.html


Chorionic Villi (CV) sample is collected through the removal of a small amount of chorion of placental tissue. Chorionic villi are usually collected by aspiration biopsy with a thin plastic catheter inserted trans-cervically or trans-abdominally into the uterus. Chorionic villi consists of coral like projections that surround the embryonic sac in early pregnancy.

illustration of the chorionic villus sampling procedure

picture taken from http://atlasgeneticsoncology.org/Educ/PrenatID30055ES.html

Fetal cord blood (FB) is obtained from the umbilical cord, it is constitutional and is able to reflect the chromosomal constitution of the fetal.

The prenatal chromosome analysis is usually done in:
-Advanced maternal age, 35 years or older
-Recurrent miscarriages
-Abnormal ultrasound findings
-Previous child with a chromosome disorder such as Down syndrome
-One member of the couple is known to carry a chromosomal rearrangement or relatives of a child with a chromosome disorder
-Parental anxiety
-And other indications

Many women who have any above indications choose to undergo prenatal test, to reassure the normality of the foetus.

The whole flow of cytogenetics procedure is as followed:
Cell culture set up --> Harvest --> Slide making --> chromosome Staining
-->Analysis -->Karyotype

And now I will be sharing with you the procedure of amniotic fluid (AF) culture set up. For harvest, slide making, chromosome staining, analysis and karyotype I will post in another blog.

Amniotic Fluid (AF) culture set up

1.Usually the specimen will arrives in a 25 ml syringe, and we have to aliquot the specimen equally into 2x15ml pre-labeled V-shaped tubes(labelled with the lab number and patient’s name)

2.Spin the 2 tubes at 1200 rpm for 10 minutes in centrifuge.

3.After centrifugation, record the pellet size (small, medium, large) and appearance (clear or blood-stained) for future reference.

4.Use a sterile pipet to aspirate the supernatant to just above the pellet and discard it.

5.Break the pellet up by gently flicking the bottom of the tube.

6.Resuspend one of the tube with 1.0ml alpha-AM medium. Mix well and pipet 0.5ml of the suspension onto the sterile 22mm X 22mm coverslips which is pre-prepared in the 35mm petri dish. Labelled the cultures as culture “A” and culture “B”.

7.Resuspend 2nd tube with 1.0ml of alpha-BIO medium. Set up culture “C” and “D” with 0.5ml suspension each. It is important to ensure that the suspensions are confined to the coverslips. This is because only the coverslips will be mounted onto the glass slides for analysis and not the petri dish.

8.Place culture “A” and “C” in one incubator , and place culture “B” and “D” in another incubator.

That is basically what we will do when we receive AF sample.

For the alpha-AM media and Alpha-bio media I mention above in the steps are actually used to grow the AF cells. They contain the essential nutrients for the AF cells to grow. Actually there many different types of media used to culture AF cells, but in our lab we use Alpha-AM media and Alpha-BIo. The difference between Alpha-AM and Alpha-BIO is that Alpha-AM is commercially made while Alpha-BIO is homemade which contains extra nutrients that are not found in the Alpha-AM media. Furthermore Alpha-AM is commercially prepared thus they contain fixed amounts of the essential nutrient whereas for Alpha Bio, we have added extra ingredients to promote the growth of the AF cells.

Hope my blog is clear, if there is any doubt, feel free to ask me, I will try my best to answer your queries. =)

Ting-Jie
(0608495h)
TG02