Chromosomal Aberrations 19 4 23

Chromosomal Aberrations
Presenter : Shalini Dhiman

PhD Scholar
Cell
Nucleic acid was discovered in 1868 by Friedrich Miescher, who called the material 'nuclein' since it was found in the
nucleus. Nucleic acids are long chain like molecules composed of a series of nearly identical building blocks called
nucleotides. Each nucleotide consists of a nitrogen-containing aromatic base attached to a pentose (five-carbon) sugar,
which is in turn attached to a phosphate group.
Chromosome
KARYOTYPING
Karyotyping is a Genetic test to examine

chromosomes in a sample of cells.
This test can help identify genetic problems as
the cause of a disorder or disease.
Karyotyping describe the chromosome count of
an organism and what these chromosomes look
like under a light microscope.
 Length
 Position of centromers
 Banding pattern
 Difference between the sex chromosomes
 Any other physical characterstics
History of Karyotype
 Chromosomes were first observed in plant cells by Carl

Wilhelm Von Nageli in 1842.
 In animal salamander cells was described by Walther
Flemming .
 The normal human karyotypes contain
 22 pairs of autosomal chromosomes
 One pair of sex chromosomes.
Idiogram of Chromosome
This can be detected by
1. Sanger sequencing
2. MLPA Multiplex ligation
probe amplification
3. Microarray CMA
Down syndrome
Clinical Feature:
A flattened face, especially the bridge of
the nose.
•Almond-shaped eyes that slant up.
•A short neck.
•Small ears.
•A tongue that tends to stick out of the
mouth.
•Tiny white spots on the iris (colored
part) of the eye.
•Small hands and feet.
How the test is performed?
Sample can be Blood cells, Bone Marrow cells, Amniotic fluid

Instruments for Karyotyping
Pipette set
Microscope
CO2 Incubator
Laminar
Falcons
Hood
Centrifuge
Water bath
Apparatus and reagents
 Beaker , falcons, pipet 200ul, 1000ul

 Tips 200ul, 1000ul.
 Hybridiser Thermobite
 Slide plate heater.
 Slides ALPHA- CHEM Microscope slides, DK LAB.
 Gimesa stain gibco for banding
 Trypsin, KCl , PBS, Acid alcohol .
Protocol
 Mix the blood with media.
 Incubate the sample falcons at 37Co , CO2 Incubator (keep sure the falcon cap is loose for
exchange of gaseous) for 72hr.
 Add colcimide 50μl and 25μl Etbr (72hr) and mix well, place the falcon in co2 for 1hr.
 Harvesting step:
 Mix the sample properly and centrifuge for 10 mins. at 1000 RPM.
 Prepare fresh KCl and Fixative (Methanol 3part and 1part acidic acid)
 Discard the supernatant, add 10ml KCl drop by drop and shake the falcon / mix the solution
properly.
 Place the falcons in water bath ( 37C ) without wastage of time.
 Now, Add 1mL fixative in to the falcons mix well and centrifuge 10 min at 1000 RPM
 Discard the supernatant without disturbing pellet, Add 1ml fixative into the falcon and
discard the supernatant 1min.
Protocol
 Add again 1ml fixative and discard the supernatant.
 Shake the falcon, add 10ml fixative again
 Centrifuge at 1000 rpm for 10 mins
 Discard the supernatant directly in one tap.
 Add 10ml of fixative , centrifuge for 10 mins.
 Place the falcons in the freeze.
 Cells are ready for slide preparation.
Slide Preparation
 Slides are cleaned with acid alcohol overnight, then ready for use.
 Take the falcons from freeze, add 10ml fixative into the falcon.
 Dip slide into ethanol than chilled water, place the sample on slide by drop wise
(135microlitre).
 Add fixative on the slide.
 Clear the bottom side of slide.
 Place the slide on slide heater plate.
 Dry the slide for15-20 mins.
Slide Preparation
For Metaphase slides:
 Prepare PBS buffer sodium dihydrogen phosphate +sodium hydrogen phosphate (NaH2PO4
{5ML} + Na2HPO4 {5ML}
 Make 10 ml + 90 ml of distilled water then buffer composition is 100ml.
 Take 45ml of PBS + 5ml Gimesa.
 Place slide in Gimesa for 10mins.
 Wash (stir) the stained slide in Distilled water, 2 times.
 Place slide on slide rack.
 Air dry the slide, then visualise slide under microscope.
Slide Preparation
 Slide formation with Trypsin.
 Make slide and dry on slide heater.
 Place the slides in hybridiser for 20 mins. at 90C programme for aging.
 Take 50ml PBS ,Place them in water bath add 26mg Trypsin in to the PBS.
 Take outside the PBS container then dip slide for 4sec.
 Then place the slide in to the chilled PBS 1sec. , stain slide with Gimsa stain 3mins.
 Rinse the slide in Distilled water.
 Dry the slides on slide heater, then visualise under microscope.
Procedure of Karyotyping Form Amniotic Fluid
AMNIOTIC FLUID
COLLECTION BY AMNIOCENTESIS
 Amniocentesis is a procedure in which amniotic fluid
 Is extracted from the uterus for testing or treatment.
Amniotic fluid is the fluid that surrounds and protects
a baby during pregnancy. Most amniocentesis procedures
happen between 15 and 20 weeks gestation (during the 2ND Trimester of pregnancy).
 This fluid contains fetal cells and various proteins.
 Approximately 15-20 mL of amniotic fluid - sterile, clearly labelled
 with patient details. If fluid appears blood-stained, place the first few
 blood-stained ml into one universal and the remainder into a second.
 Send to laboratory in "Prenatal Transport" pack as soon as possible.

AMNIOTIC FLUID CULTURE
 It consist of heterogeneous cell population displaying a range of morphologies and their behaiviour.
 Flask method culture of amniotic fluid cells:
 1. Using sterile technique, dispense the amniotic fluid into two 15 ml centrifuge tubes.
 2. Centrifuge the tubes at 1,000 rpm for 10 minutes.
 3. Transfer the supernatant from each tube into separate T- 75 flasks. Leave approximately 0.7 ml of
supernatant above each pellet.
 4. Add 5 ml of Amniocyte Medium to the T-75 flasks containing the supernatant. Place the caps loosely on the
flasks and incubate at 37 deg C and 5% CO2.
 5. Add 0.7 ml of culture medium to the pellets and resuspend by gentle mixing.
 6. Add your cell suspension from each tube into separate T- 25 flasks. Add 5 ml of culture medium to each
flask.
 7. Place the caps loosely on the flasks and incubate at 37oC and 5% CO2.
 8. Check all flasks for growth in approximately 5-6 days.
 9. When approximately 10-12 medium sized colonies are present, process the flasks for harvesting.
Flask culture
 Add 100 μl of Demecolcine (10 μg/ml,) to each flask and incubate for 1 hour.
 Tap the flasks to get the cells in metaphase to float. Remove medium and save in 15 ml centrifuge tubes.
 Rinse cells quickly with 2 ml of trypsin-EDTA . Add 4 ml of Trypsin-EDTA to the flasks and incubate for 6 minutes.
 Following incubation, add 5 ml of culture medium to the flasks to inhibit the trypsin action. Triturate the medium over the
surface of the flasks to gently dislodge the cells.
 Remove the cell suspension mixture and place into separate 15 ml centrifuge tubes.
 Centrifuge the tubes at 1,000 rpm for 10 minutes.
 Resuspend the cell pellets from each tube with 1ml of the supernatant and combine the pellets into one tube. Fill the
tube with 10 ml of pre-warmed Hypotonic Solution.
 Immediately centrifuge the tubes at 1,000 rpm for 10 minutes.
 Resuspend the cells in 10 ml of hypotonic solution and incubate at 37o C for 10 minutes.
 Following incubation, add drops of Carnoy’s fixative , mix by inverting tubes, and centrifuge at 1,000 rpm for 10 minutes.
 Discard the supernatant re-suspend the pellets in 10 ml of fresh fixative. Let cultures stand for 30 minutes .
 Again centrifuge tubes at 1,000 rpm for 10 minutes. Repeat this step for 2-3 times
Step 3: Slide preparation:-
 Next day 3 – 4 washes given with fresh carnoy’s fixative until the cell pellet
is clear.
 Cell is suspended in appropriate volume of carnoy’s fixative and drop onto a
oil free chilled slide, and allow to air dry.
 The slides should be washed with hot water and then with
methanol.
 The slides should be stored in cold water or chilled carnoy’s
fixative.
 After air dried the slide , cell density and metaphase are checked by using a
phase contrast microscope, slide is kept at 80°C for aging.
 Step 4: GTG-Banding :-
 Most commonly used in the analysis of structural chromosomal aberration. It’s

obtained by digesting the chromosome with trypsin followed by Giemsa staining.
 Three coplin jar is taken and placed in a sequence.
First coplin jar 1% Trypsin in 0.05% PBS
Second coplin jar Distilled water.
Third coplin jar 40 ml d/w, 5 ml PBS and 5ml Giemsa stain.
 Dried slides are used in GTG banding.

 After the five min, slide rinsed with tap water and allowed to air dry.
 After the air dry slide observed under the microscope. At least 20 metaphases are
analysis by cytogenetic work station.
 Metaphase karyotyped on the basis of ISCN 2016.
Fig: Chromosomal G-banding image under the microscope
The image of Giemsa banded chromosomes directly taken from the microscope
Metaphase Karyogram of under the cytogenetic investigation showing a normal chromosomal
constitution using GTG – banding (band resolution 350).
Application
The most common things look for karyotype tests include:
 Down syndrome (trisomy 21). A baby has an

extra, or third, chromosome 21
 Edwards syndrome (trisomy 18). A baby has an
extra 18th chromosome.
 Patau syndrome (trisomy 13). A baby has an
extra 13th chromosome.
 Klinefelter syndrome XXY.
 Turner syndrome. X0
Evolution in Cytogenetics
1970- 1986-
1992-
Chromosome banding technique FISH developed by
CGH developed by
developed Pinkel
Kallioniemi
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Chromosomal Aberrations

Presenter : Shalini Dhiman

Karyotyping is a Genetic test to examine

 Chromosomes were first observed in plant cells by Carl

Sample can be Blood cells, Bone Marrow cells, Amniotic fluid

 Beaker , falcons, pipet 200ul, 1000ul

 Amniocentesis is a procedure in which amniotic fluid

 Is extracted from the uterus for testing or treatment.

Amniotic fluid is the fluid that surrounds and protects

a baby during pregnancy. Most amniocentesis procedures

 This fluid contains fetal cells and various proteins.

 Approximately 15-20 mL of amniotic fluid - sterile, clearly labelled

 blood-stained ml into one universal and the remainder into a second.

 Send to laboratory in "Prenatal Transport" pack as soon as possible.

 Most commonly used in the analysis of structural chromosomal aberration. It’s

 Dried slides are used in GTG banding.

The most common things look for karyotype tests include:

 Down syndrome (trisomy 21). A baby has an

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