Name: Hardina Bte Hamzah
Topic: Cytogenetics
Title: Amniotic Fluid Set-up
Amniotic fluid is a clear, slightly yellowish liquid that surrounds the unborn baby (fetus) during pregnancy. It is contained in the amniotic sac. The fluid protects the fetus from injury and helps to regulate the temperature of the fetus. It allows the baby to move around so that the muscles and bones develop properly. It also helps the digestive and respiratory system to develop as the baby swallows and inhales and exhales it from his lungs. In addition to various enzymes, proteins, hormones, and other substances, the amniotic fluid contains cells shed by the fetus. These cells contain genetic information that can be used to diagnose chromosomal disorders. The fluid is sent to cytogenetics lab so that the cells can grow and be analyzed.
To collect the amniotic fluid, the obstetrician will insert a very fine needle through the woman's abdomen into the uterus and amniotic sac and withdraws approximately 20ml of amniotic fluid for testing. A local anesthesia will be given to the patient. The obstetrician uses ultrasound images to guide needle placement and collect the sample, thereby minimizing the risk of fetal injury and the need for repeated needle insertions.
Before starting the set-up, the medical technologist has to ensure that the labeling on all petri dishes and tubes is done correctly. The set-up must be done in the Biological Safety Cabinet (BSC) as all specimens must be processed in a sterile environment.
One conical test tube and one slant tissue culture tube are used for each specimen. Before transferring the amniotic fluid from the syringe to the tubes, invert the syringe to ensure that there is adequate mixing of the amniotic fluid. After mixing, equal amount of amniotic fluid is transferred to each tube. The tubes will then undergo centrifugation at 1200 rpm for 10 minutes. The buckets in the centrifuge must be closed before centrifugation starts.
Each specimen will have a record sheet where the medical technologist will record down the volume and appearance of fluid, size and appearance of pellet and the presence of RBCs after centrifugation are done.
A sterile coverslip will be placed in each petri dish using a pair of sterile forceps. The dishes will then be divided into 2 sets. One of the set will have the culture tube. Each set will use a different kind of media. The first set with the culture tube will use complete Chang media and the second set will use the complete Bio-AMF-2 media. Both media consists of antibiotics to prevent any bacteria from growing and contaminating the whole culture. The supernatant will be aspirated out leaving around 0.5ml in the tubes. The Chang media will be added into the culture tube and the Bio-AMF-2 media will be added to the conical tube. The amount of media to be added is depending to the number of dishes used. One dish needs 0.5 ml of media. For example, if the first set consists of 2 dishes and 1 culture tube, 1.5ml is needed to be added into the tube.
After adding the media, the pellet will be gently re-suspended and the cell mixture will be transferred onto the coverslip in the dish. The cell mixture must be confined to the coverslip. A drop of cell mixture will be placed from the conical flask to the last dish of that set. This will act as a backup. 0.5ml of cell mixture will be left in the culture tube. The culture tube will also acts as a backup.
The first set of dishes and culture tube will be placed on one tray and the other set on a separate tray. The two trays will be placed in two separate incubators with independent electrical circuits and gas sources. The cultures will be left inside the incubators and on day 3, the medical technologist will check for cells attachment. Other than that, 1.5ml of media will be added to the dishes and tube. If there is no cell attachment, media will not be added and put on hold for another day. On day 5, the media will be changed and be replaced with a fresh media. After enough cells are grown, the cells will then proceed to tissue culture.
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8 comments:
Hey dina =)
You mentioned in order to retrieve the amniotic fluid, a very fine needle will be injected into the mother's abdomen into the uterus and amniotic sac, and local anesthesia will be given.
Just being curious, will anesthesia affect the growth of the unborn?
Thanks.
Lyn
TG02
hi hardina
you said that the bio-AMF2 and chang media consist of antibiotics. are the antibiotics originally present in both media or are they added in? and what type of antibiotics are used?
thanks
nur azeimah
0607060A
tg 02
hi
How come use 2 different media and different incubation conditions?
Xin Ni
Hi dina!
We have to discuss here, heh.
Anyway, could you tell me more about the media that you all use to culture. I've not heard of any before even I am in Micro. HAHAHA!
Thanks!
LESLIE
hey hardina!
You mentioned that 'the medical technologist will record down the volume and appearance of fluid, size and appearance of pellet and the presence of RBCs after centrifugation are done'
What is the purpose of it? Does it aid in diagnosing whether the baby is suffering from chromosomal disorder?
Thanks!
Cheers,
Huimin
tg01=)
HI!
when the amniotic fluid arrive at your lab, it is a fresh specimen or does it contain preservative?
does your lab do it as soon as it arrives to the lab or do it by batch?
and have you encounter porblem when performing the setup? like insufficient samples or something else?
Thanks!
YuMei
TG01
Hi Dina
I know the amniotic fluid contains proteins, electrolytes, lipids etc. But does it contain any cells? Because the endometrium is what is holding the amniotic fluid right? So it might contain endometrial cells.
In other words, what cells do u see in the amniotic fluid?
Thanks
Ernest
0606330i
Lyn:
Q: You mentioned in order to retrieve the amniotic fluid, a very fine needle will be injected into the mother's abdomen into the uterus and amniotic sac, and local anesthesia will be given.
Just being curious, will anesthesia affect the growth of the unborn?
A: There will be minimal or no effect at all. As the anesthesia is localized at one area, it does not spread out. However, it may numb the fetus in some patients but it is just for a short while. Adverse reaction may occur when the baby or mother may be allergic to anesthesia.
Azeimah:
Q: you said that the bio-AMF2 and chang media consist of antibiotics. are the antibiotics originally present in both media or are they added in? and what type of antibiotics are used?
A: Antibiotics are already in the media. It is being added by the manufacturer. However, due to the duration of the shipment and the condition of media during the delivery, the effectiveness of the antibiotics may decrease. As a preventive measures or should I say, being cautious, an adequate amount of antibiotics will be added. Too much antibiotics can become toxic.
Antibiotics used: Penicillin and Streptomycin
Xin Ni:
Q: How come use 2 different media and different incubation conditions?
A: The purpose of both media is the same. To provide nutrients and allow cells to multiply. 2 types of media are used as a preventive measures. If the other media does not work well, there is still another media to rely on. Due to delivery conditions, it may affect the effectiveness of the media. In cytogenetics lab, we must be caution in every step we take, especially handling amniotic fluid. We can’t afford to damage the sample, as it is very risky to collect another tube of amniotic fluid.
They have the same incubation conditions but placed in separate incubators. If one incubator is contaminated, another incubator acts as a back-up. It is another preventive measure.
Leslie:
Q: Anyway, could you tell me more about the media that you all use to culture. I've not heard of any before even I am in Micro.
A: The thing is, these media is not used in Micro (I think). I have only come across with them when I’m attached to cytogenetics lab.
Chang Medium C may be used for the primary culture of amniotic fluid cells, growing passaged amniotic fluid cells, the culture of bone marrow cells and solid amnion tissue from chorionic villi sampling. Chang Medium is developed for the primary culture of human amniotic fluid cells for use in karyotyping and other antenatal genetic testing.
Bio-AMF-2 Medium is designed for the primary culture of human amniotic fluid cells and chorionic villi samples in open systems (5% CO2). The medium allows rapid growth of amniocytes or chorionic villi for use in karyotyping.
To make a complete Chang Medium and Bio-AMF-2 Medium, L-glutamine (amino acid) and antibiotics (such as penicillin and streptomycin) are added. L-glutamine is added for the stability of the culture. Antibiotics are needed to prevent contamination of culture.
Hui Min:
Q: You mentioned that 'the medical technologist will record down the volume and appearance of fluid, size and appearance of pellet and the presence of RBCs after centrifugation are done'
What is the purpose of it? Does it aid in diagnosing whether the baby is suffering from chromosomal disorder?
A: It does aids in the diagnosis. It really helps when the chromosomes are found to have some abnormalities. From the appearance, we can see whether it is purely fetal cells or mixture of fetal and mother’s cells. For example, if there’s a presence of blood in the fluid, it shows that mother’s cells can be present. So, the fluid collected and the results obtained are invalid as we can’t differentiate which cells belong to the fetus or mother. In this case, the abnormal chromosomes can belong to the mother instead of the fetus.
Yu Mei:
Q: when the amniotic fluid arrive at your lab, it is a fresh specimen or does it contain preservative?
A: It is a fresh specimen. It is delivered in a syringe used during collection of amniotic fluid.
Q: does your lab do it as soon as it arrives to the lab or do it by batch?
A: It is done by batch.
Q: and have you encounter problem when performing the setup? like insufficient samples or something else?
A: I never got a chance to practice the set-up as the procedure is very crucial. If insufficient amount of sample is collected, the doctor will not send it to the lab as the lab will surely reject it. That applies to a very little amount collected. However, if the sample is slightly lesser than 20ml, the lab will reduce the number of cultures. For example, instead of preparing 6 cultures, it is reduced to 4 cultures. Other problem may be the temperature of the incubator. It may be lower or higher than 37°C. Fortunately, there are around 4 incubators. If one of them malfunctions, there are other incubators.
Ernest:
Q: I know the amniotic fluid contains proteins, electrolytes, lipids etc. But does it contain any cells? Because the endometrium is what is holding the amniotic fluid right? So it might contain endometrial cells.
In other words, what cells do u see in the amniotic fluid?
A: We can see fibroblast cells and epithelial cells. The lab prefers taking fibroblast cells as it gives better metaphases than epithelial cells. Chromosomes taken from fibroblast cells are longer and the bands are clearer. Chromosomes from epithelial cells appear short and difficult to analyze, as the bands are not clear. When there is a deletion on any chromosomes, it can’t be spotted as the chromosomes are too short.
Hardina =)
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