Sorry for the late BLogginG. Today i am going to talk about how Amniotic fluid is being set-up.
Amniotic fluid set up
Purpose: The most common reasons for prenatal diagnosis of chromosome disorders of the fetus are advanced maternal age, family history of a chromosome abnormality, abnormal maternal serum screen or fetal defect identified by ultrasonography.
Reason for using amniotic fluid: Amniotic fluid contains cells derived 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. The in situ culture technique is used where single AF cells are plated onto a sterile glass coverslip in a petri dish where they grow in situ to form discrete colonies.
Steps involved:
1) Usually AF is collected in a tube or sometimes it comes in the syringe.
2) Spin down the tubes containing AF at 1200 rpm for 10 mins
3) Once you have spun down the sample, check the appearance of supernatant and the pellet size
4) After spinning down the tubes, discard the supernatant and dislodge the pellet
5) In one of the tubes, add 1 ml of alpha-AM media and mix it well and in the other tubes add 1 ml of Alpha-bio media. (These are the two media used in my lab for all samples: AF, Chorionic villi etc)
6) There is a need to do 4 cultures. Thus four petri dishes are labeled wit the patients’ name, lab number. (labeled as A,B,C and D)
Note: Each dish has a coverslip attached to the surface of the dish
7) For the dishes A and B, aliquot 0.5 ml of the cell suspension (media used alpha- AM) into each dish
8) As for dishes C and D, 0.5 ml of cell suspension (media used alpha-bio) is added to each dish.
9) Make sure the cell suspension is confined to the coverslips. This is to allow the cell attachment and growth of cells on the surface of the coverslip. (Harvest cells that grow on the coverslipknown as in situ)
10) After which, place the dishes into the respective 37oCCO2 incubator for 4-5days. Culture A and c into incubator A and Culture B and D in incubator B (for backup)
11) And also make sure that you have placed in dishes in the right tray (so that the next day who ever is going to flood the dishes with media knows which dish to flood. )
Diagram to summarize:
Vinodhini
TGO2
12 comments:
hey
u forget to write yr name
elaine
hey thnx for that ! vino =P
hey liu qian here:
you mention abt alpha-AM media and Alpha-bio media. what's the purpose of these two media and what's the difference between them ?
thanks~
hihi vino,
what is amniocentesis and why there is a need to do 4 cultures?
Juexiu
tg02
Hello Vino,
What happened if cells aren't viable, what will you do? Actually, what cells are you targetting for culture here? Skin of fetus?
Thanks.
-Alex Tg02
Hey
You said "And also make sure that you have placed in dishes in the right tray (so that the next day who ever is going to flood the dishes with media knows which dish to flood. )"
Just wanna know what media is used in this step and why do you have to flood the dishes?
Thanks,
Azhar
1) Liu Qian
hey the alpha-AM media and Alpha-bio media are used to grow the AF cells. They contain the essential nutrients for the AF cells to grow. There many different types of media used to culture AF cells, but in our lab we use commercially made Alpha-AM media and home made Alpha BIo. (alpha bio contains extra nutrients that are not found in the media). The difference is that one is commercially prepared thus they contain fixed amounts of the essential nutrients. As for Alpha Bio, we have added extra ingredients to promote the growth of the AF cells.
2)Juexiu
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 (obtained from Medline). Usually samples come either in Two tubes or in syringe as i have mentioned in my blog. Sample from one tube is cultured in one dish. Thus two tubes require Two dishes. The other two dishes are for back up.If one of the dishes has no growth, we still have other dishes to depend on.
3) Alex
AF is composed of cells that are from the skin and the urine of the fetus.If the cells are not viable i think we won be able to see any attachment or growth thus n results can be obtained. But most of the time infact everytime when we receive AF sample, we will have both viable and non viable cells. we are still able to obtain results.
4) Azhar
Oh in my blog i mentioned that Alpha AM is used for culture A and B and alpha bio is used for culture C and D. The next when we flood the dishes, we still use the respective media. The reason for flooding the dishes : rmb in my blog i said that the cell suspension is confined to the coverslip. In order to supply the cell with nutrients, we need to flood it with media so that it can ge sufficient nutrients for survival and growth. We dun flood it on the day we set up coz we want the cells to attach to the coverslip first, then the next day we flood it. Hope i have ansed ur question!
heya,
just wanna ask ya do u encounter any problems in performing this experiment before?
thanks ya
michelle
tg02
hi michelle
No so far i have not encountered any problem. Jus to give an example of the kinda probs u may encounter during the set up.
Lets say the pelet appears bloody after u have spun down the tubes containing the amniotic fluid,there is a need to treat the sample with Sodium citrate. this is becase the red cells are heavier than the amniotic cells and they settle down on the coverslip thus not allowing the attachment of the amniotic cells. The sodium citrate will lyse these red cells and cause the red celld to float once u have spun the tubes again after the treatment.
Vinodhini
Hey Vino,
after culturing on coverslip, how does the analysis goes about?
izit staining the chromosomes? if so, what kind of abnormalities u look for?
Tanx!
Nisha
TG02
Hi nisha..
After u have cultured the cells on the coverslip, the next step is harvesting.. The haresting is different from BM. During harvesting,
1) suck up the media in the dishes and add in Na citrate which is a sweller. it causes the cells to swell so that the chromosomes have more space to spread out and not condensed.
After which u have to do few series of washing by adding fixative..
After washing, u have to blow dry the coverslip and observe under the microscope for metaphases.
Then transfer the coverslip from the dishes to a glass slide( using DPX to stick the coverslip tot he slide). U will have to then bake the slides(place them in a 90 oC oven) for about 1 hour and u can procede on to staining.
Abnormality you look out for : look out for trisomies.. structural abnormalities.
Vino
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