Cheng Hong: Cytogenetics

Basically in cytogenetics lab the lab technologist must be able to do karyotyping(sort out the chromosomes 1 to 22 and X & Y.

The lab technologist have to be able to detect any abnormalities in the fetus/ child/ adult so as to be able to help the clinicians to give accurate diagnosis and patients will be able to go for genetic counseling if needed. Some genetic diseases like trisomy 21 which gives rise to a down syndrome child/ a deletion in a certain part of the gene and might affect the heart of the fetus etc

Type of specimens:
1) Amniotic fluid culture(collected around 4months after pregnancy)
2) Chronic villi (collected 10 to 12 weeks after pregnancy so that the vilius is not too thick/ deep into the placenta)
3) Cord blood (collected around 20-22weeks after pregnancy so that the umbilical cord is think enough to be pricked)
4) Bone Marrow to detect Leukemia due to genetics
5) Product of conception(fetus/ fetal eye/fetal gonads/ fetal skin)
6) Tumor
7) Peripheral blood

The General work flow in a Cytogenetics lab:
1) Grow cells in media for harvesting (so that there is enough cells to obtain enough metaphases to study the chromosome)
2) Add mitotic inhibitor so that the cells are not able to proceed into the Mitotic phase of the cell cycle
3) Add hypotonic solution (so that the nucleus will swell up thus the chromosomes can spread out)
4) Add fixative (to “freeze the cell in the swelled up state)
5) Stain the slides (normally with Giemsa stain so that the bands in the chromosome can be seen)
6) Microscopy and Karyotyping

Cell cultures
§ The daily maintenance of the cultures is quite tedious and labour intensive, normally each specimen is cultured for 9 to 11 days. We have to check the cultures daily for any microbial contamination then action must be taken to save the culture and prevent the spreading of the contamination.
§ If the media is used up we have to change fresh media for the cells. And if the cells are very confluent, we must tiff(sub-culture) into a new culture so that the cells will not be so packed
§ Once the culture is enough for harvest(at least 4 colonies)

Amniotic fluid culture setup (to be done in the BSC)
§ The samples are sent in 25ml syringe (the doctors should collect around 20ml of fluid)
§ Prepare 2 tubes for each sample (1 conical base tube, 1 slant tube)
§ Invert the syringe to mix the cells in the fluid so that it is evenly distributed
§ Add to each of the culture tubes (10ml each)
§ Spin down at 1200rpm for 10 mins
§ Prepare culture dish(4 each for each sample in case of any contamination in 1 dish/ low growth)
§ Add a cover slip into the dish
§ Record the pellet size after spin down, colour of supernatant, with or with out feces or RBCs
§ Aspriate and discard the supernatant
§ Add Chang media to the slant tube(0.5ml)
§ Add Bio-AMF medium to the conical tube(0.5ml)
§ Resuspend pellet
§ Fill slide with the cells in the conical tube (slant tube as back up)
§ Incubate tubes in 2 different incubators at 37oC, 5%Co2, 5%O2

*to prevent any incident of unable to obtain cells due to contaminations, the lab uses to separate incubators (2 dish of the 4 dishes in each incubator), in case of event that 1 of the incubator breaks down/ contaminated the other 2 dishes will not be affected. The lab also uses media from different vendors in case the batch has any defects there will sill be back ups. The incubators are also washed and wipe down with alcohol weekly.

Blood culture setup (to be done in BSC)
§ Blood should be sent in sodium heparin tube to prevent coagulation and to preserve the cells
§ In blood culture we want to grow the White cells
§ Spin down the blood tube at 1200rpm for 10mins
§ Use a pasture pipette to aspirate up the layer of buffy coat(whitish layer where the WBC is)
§ Invert the pipette and mix the cells
§ Add into tubes containing RPMI medium and 1 M199 medium
§ Check if blood is clotted (if clotted mesh up clot and place in tube with media as the clot may trap the WBCs)
§ Add PHA to induce the white cells to enter the cell cycle as currently it is in G0 phase
§ Mix well and incubate at 37oC in a Co2 incubator (using a 45o angle rack to facilitate gaseous exchange)
§ On 2nd day mix the tubes and in the afternoon add MTX(to remove the thymidine so that the cells cant go through Sphase)
§ On 3rd day add thymidine, now the cells can proceed into S phase so that we will be able to synchronize the cells to enter metaphase together
§ Proceed to harvesting

Blood Harvesting
§ Warm up hypotonic solution to 37oC in a water bath and add 50µl of colcemid at 20mins and 2hrs interval
§ Spin down the tubes at 1200rpm for 10mins and discard supernatant
§ Dislodge cell pellet and add 8-10ml for hypotonic solution and mix well
§ Incubate for 5mins then spin at 1500rpm for 6mins and discard supernatant
§ Add 8-10ml of hypotonic solution again and incubate for 5mins and spin down again
§ Discard supernatant and add 2ml of fixative(3parts of methanol and 1 part of acetic acid)
§ Mix well and spin down at 1500rpm for 6mins and discard supernatant
§ Resuspend pellet and add 10ml of fixative and incubate for 30mins at room temp
§ Spin down at 1000rpm for 10mins is discard supernatant
§ Add 8ml of fixative and let it stand for 15mins
§ Proceed to slide making

Slide making (dropslide)
§ Warm up beaker containing washed glass slides, to 90oC
§ Depending on the humidity and temperature, height that is will be dropped at, amount of water present on the slide, concentration of cells, it will affect the spreading of the chromosomes, lenghth and colour
§ Spin down at 1000rpm for 10mins
§ Aspirate most of the fixative and transfer to an empty tube and leave some to dislodge the cells
§ Add the fixative to obtain best concentration to drop slide
§ Get slide and flick once/ twice to remove excess water
§ Hold the slide at a 45o angle and on the other hand hold the pipette and space them about 15-30cm apart and drop on the top part of the slide and allow the cells to slide down the slide
§ Wipe off excess water from the sides and let the slide dry
§ Check under microscope if the cells are too pack/ chromosomes are too short/ dark thus we have to adjust the humidity of the slide and height of dropping
§ Proceed to slide staining

Slide staining
The slides are stained with giemsa stain and wright’s stain, the combination of this 2 stains gives good contrast of the dark and light band in the chromosome, thus we will be able to do karyotyping and spot any abnormalities like translocation/ addition/ deletion/ inversion etc.

Any questions do ask thanks,

Cheng Hong TG02