Observership at

Jaslok Hospital

By Kush PuriCathedral & John Connon School

About the Observership• Location: Genetics Laboratory, Department of Assisted Reproduction and

Genetics at Jaslok Hospital

Tasks Completed

• Observed routine Cytogenetic diagnostic techniques on various human tissues such as peripheral blood, bone marrow, amniotic fluid and chronic villi from products of conception.

• Hands on experience of karyotyping blood sample from the stage of culture set-up to chromosome analysis

• Observed FISH (Fluorescence in situ hybridisation) set up, hybridisation, post hybridisation washes and analysis.

Techniques

• Cytogenetics- Study of chromosomes and their abnormalities

• Karyotyping and FISH- used to diagnose genetic diseases.

Karyotyping

• Duration: 12 day process• Took my own blood sample and carried out the entire

process.

Planting Harvesting Washing Dropping Banding Analysin

g

Method for Karyotyping• Planting: Culture the tissue for appropriate amount of time (usually 48 to 72 hours). • Harvesting: Add a spindle poison (we used colchicine) to “arrest” the cells in

metaphase. The is because chromosomes are maximally condensed in metaphase and thus easiest to see. Then add a hypotonic solution to swell up the cells.

• Washing: Wash the cells• Dropping: Drop the sample onto the slide from a height• Banding: Stain with designated nuclear stain. This helps to identify individual

Chromosomes. (Giemsa banding/G banding done in this case)• Analysing: Capture images of slides under microscope and using software arrange

the chromosomes into a karyotype.

Karyotyping

Day 1: Planting Process

Withdrew blood from

vein

Added 5ml of blood to a

growth medium

Left this in the pre-heated

incubator for 3 days

Karyotyping

• Day 4: Harvesting

Add 50 micro litres

of colchicine

to the blood sample and leave it in

the incubator for 1 hour.

Then centrifuge for 10 min

at 1300 rpm.

Remove the supernatant liquid and mix the

sample with a pipette.

Add KCl upto 8 mL and mix.

Then leave it in the

incubator for 15 min

Centrifuge for 10 min

againAdd fixative upto 0.8 mL

Leave in fridge

overnight

Karyotyping

• Day 5: Washing

Centrifuge the sample for 10 minutes and remove the supernatant

fluid.

Add fixative upto 8 ml and

mix thoroughly.

Repeat this process two more times

Karyotyping

• Day 7: Dropping

Pipette the

solution onto the slide by dropping

the sample from a

height of ~1m.

Only 4 to 5 drops on

a slide. Make sure drops do

not overlap.

Put the slide over a water bath to

allow the vapour to dry it for

~30 seconds.

Then dry it on a hot plate for ~1 min

Then label the slide

with name,

date and number of sample.

Place it on a hot plate

overnight.

Karyotyping

• Day 8: Banding3 solutions are used

for chromoso

me banding: Trypsin (with an

additive), saline and

stain.

Blood sample is dripped in trypsin for 10 seconds, then saline for 10

seconds and then rinse it

with saline.

Then drip the slide in the stain

for 8 mins. (Giemsa stain is used)

Then rinse the slides

(with saline) and

air dry.

Then observe under a

microscope.

Karyotyping

• Day 9: AnalysingUsing a light microscope, metaphases

were identified with a 10x

lens. A 100x lens was used to capture an

individual metaphase.

Using the microscope image, the

chromosomes were identified and arranged according to

their shape and size onto a karyotype.

Now you can see if there is

anything wrong with any of the chromosomes.

Sample of My Chromosome Analysis

FISH• Fluorescence In Situ Hybridisation• Take DNA probe that is complementary to the

selected region on the chromosome. Denature the probe and chromosome sample.

• Hybridization takes place for 3 hours, or overnight. Washing is then done. Observe coloured signals under fluorescent microscope. Capture image. Chromosome deletions, aneuploidies (duplications) and translocations (rearrangements) can be detected.

• A specific chromosome is looked at, which gives the observer some idea of which disease they’re testing for.

DNA extraction

• Some diseases can only be diagnosed on a molecular level (not via chromosomes). Thus using the patient’s DNA, one can test for microdeletions.

• PCR- polymer chain reaction. A specific gene is located, and that gene is replicated.

Letter from Jaslok Hospital