Purpose: To establish haematopoietic cell cultures grown in culture flasks for cytogenetic analysis of haematological disorders.
BM is ideal specimen for chromosome studies in leukaemia. When BM aspirate is unsuccessful or is a dry tap, bone core or leukaemic blood may be used instead.
Blood may be taken in a sodium heparinized vacutainer .
Overnight culture set up (24- hour culture set up)
1.For marrow collected in sodium heparin vacutainer tube, pipette half the amount of BM aspirate into the Direct harvest Medium for direct harvest.
2.Obtain the recent FBC result from haematological lab and record in the BM culture worksheet.
3.Use the WBC as a guide to determine the number of drops of BM into the culture media.
4.Thaw one flask of complete RPMI 1640 culture media in a 37oC water bath for 20 mins
5.Place the specimen tube in bucket (a container to hold the tubes during centrifugation), screw tight the lid cover and centrifuge at 1500 rpm for 10 mins
6.Pipette the buffy coat out of specimen tube with a sterile 3 ml plastic transfer pipette into a sterile centrifuge tube.
**Buffy Coat > it is a layer of white cells which can be used to determine the cell lineage pathway
7.Sometimes, in order to assess the cellularity of the BM specimen (to determine the number of drops of buffy coat into the centrifuge tube). Smear and staining is also done and observes under the microscope (similar to our BBANK smear and stain). If there are more white cells we call it hyperplastic and low WBCs, we call it as hypoplastic. In case of hyperplastic, we only use ½ or 1 drop of the buffy coat ( > 1 drop will result in increased growth cells leading to insufficient space for growth). Hypoplastic BM, we will add about 3-4 drops.
8.After which, the buffy coat is then transferred to a RPMI 1640 culture media and placed in the CO2 incubator for overnight.
**For the steps are similar for 48-, 72- hour culture too. Interleukins are added before placing the culture flasks into the CO2 incubator.
LMQA
1.Safety of all lab staff is very important especially when handling the samples
2.It is also very important to maintain the temperature of refrigerator, incubator, freezer and other lab equipment as all these will have an effect on the sample.
3.All equipment is calibrated everyday early in the morning and is recorded. All QC forms are reviewed periodically by QC officer to ensure the equipment functions and perform according to specification
4.QC forms must be used for recording the performance check and maintenance on each piece of equipment
5.All documented QC forms are filled
Vinodhini
TGO2
8 comments:
Hey Vino
Just want to ask what is the significance of RPMI 1640 culture media and how is it different from other mammalian cell media. Also, how regular are the QC checks done?
Johanna TG02
HI JOhana
The 1640 RPMI culture media has nutrients and factors that are developed for growth of human normal and neoplastic leukocytes and is generally, supplemented with serum. It provides an in vivo environment. I think u can still consider this media as a Mammalian cell media. I am not sure abt this I will let you abt this asap. sorry abt that.
As for QC checks, i heard from my lab that its done daily for certain equipment e.g. CO2 incubator and freezer. It depends on the equipment. Yup
Hope i have answed ur question
Vinodhini =)
TGO2
Hey,
Besides the RPMI media, do you use any other media? eg. Chang media?
Randall
TG02
Hi vino
lol seen u almost everyday, but din have the chance to see what u're doing. ok here's my qn:
1)Any difference in accuracy of results when BM aspirate, bone core or leukaemic bllod is used?
2)how do u determined the amt of blood add into culture media based on wbc count?
Chaur Lee TG01
hi vino,
what's the purpose of adding interleukin? Also, why is there a need direct, 24hr and 48/72 hr harvest?
Thanks :)
phuiyuen
TG 02
HI Randall
Actually in the RPMI 1640 media contains different types of chang Media. e.g in my lab they call it chang B, chang C. All these contributes to the RPMI media. Yeah
Vinodhini
TGO2
hi Chaur Lee
Actually i would say theres no difference btw BM and BC in results. Because usally we collect BM from patients to analyse them to see if there are any abnormal cells in the BM where all the cells will start to mature and from this we can detect the cell lineage pathway of the cell, meaning whether it is myeloid or lymphoid. If we cant collect the BM from patients due to reasons like the patients BM is a dry tap meaning theres no BM, then we will collect the BC to see why is it that the patient does not have BM and try to identify the cause. The only disadvantage of BC is that sample size is too small thus direct harvest cannot be done. BC can only be cultured.
As for peripheral blood, we will collect it if the patients disgnosed with chronic lymphoid leukaemia have high lymph ( cant rmb the range, will find out for ya). The reason for this is still quite unclear. My senior told me that he will give me resources to explain why. So i will update this asap.
White cells: how do we determine the no of drops of BM? WHen we do a smear to analyze it under the microscope, you will observe both red and white cells. As we all noe red cells appear red and white cells appear blue undert he microscope. We browse the slide and try to judge the ratio bwt red and white cells. If there are more white cells we call it hyperplastic, thus we will add only 1/2 to 1 drop. If the white cells are low in number, we call it hypoplastic, thus we will add more drops (3--4 drops) of BM into the culture
Vinodhini
TGO2
hi phuiyuen
As i had mentioned in my blog, interleukins are mitogens. These are growth factors that are also produced in vivo. Certain diseases need these factors to grow, for e.g. myeloma needs IL-6 as a growth factor. Thus we add IL to provide the growth factors and thus providing in vivo environment.
Cultures are done to grow and stimulate the growth of both normal and abnormal cells/clones. From the cell production, we can identify the abnormality. Direct harvest is done because, we are trying to capture the cells that are already present in the sample at the point of collection of the BM and not allow them to die off. This is because sometimes abnormality can be found in the spotaneously dividing cells then slow growing cells.
Vino
TGO2
Post a Comment