KARYOTYPING

Chromosomal analysis

Tri Indah Winarni, MD, PhD

Faculty of Medicine, Diponegoro University
Case-1
• Young couples came to your clinic with their 1 month old
(boy? Girl?)
• They want to know what is the sex of their baby
• What are you going to do?
Study Objectives
• Part 1: Understand how the procedure of
cell culture-slide preparation-G-
banding technique
• Part 2: Understand how to analyze the
chromosome/karyotyping
• Part 3: What is the implication of
karyotype result to the patient
Karyotype is a process in which chromosomes
are cut out from metaphase “picture”,
arranged in order of size (and group), and
pairing to identify TO provide an important
information for clinical phenotype
The study of structure and function of chromosome
• Chroma: colour, soma: body  colored body that visualized by light
microscope
• In 1956, 46 chromosome were systematically counted in human cell
 maleness is determined by the presence of Y
• Visualized during mitosis (usually in somatic cells (convenience)
• During interphase, the chromosomes are not visible under the light
microscope.
• During cell division, the chromosomes become highly condensed and
visible, mostly identified at the metaphase stage
• The number of chromosomes in human cells is 46 with 22 autosomal
pairs and 2 sex chromosomes - 2 X chromosomes for females or an X
and a Y chromosome for males
Chromatin
DNA, RNA and primary
protein (histone)
complex inside nucleus
cell that makes up
chromosomes.
Function: transcription
regulation (strengthen
>< extended)
Types of chromatin based on G-banding technique
1. Extended/loose chromatin can conduct the process of
transcription
• Euchromatin, which consists of CG-rich DNA that is active, e.g.,
expressed as protein  light band
2. Strengthened/tight chromatin restricts access to the
transcription initiation  no transcription occurs
• Heterochromatin, which consists of AT-rich DNA mostly inactive DNA
 dark band
Chromatid, a single DNA double helix connected by centromere
• Normally exist as a single chromatid  during cell division
becoming two sister chromatids joined at centromere
• Centromere divided the short arm (p) and long arm (q) and
responsible for the movement of chromosomes to different poles
at cell division
• During metaphase each chromosome can be seen consist of two
identical strands known as sister chromatids  duplicated
chromosome contains two identical daughter DNA molecules
called a sister chromatid
• The tip of each chromosome arm is known as telomere which
play a crucial role sealing the ends of chromosomes  provide
cap
 Chromosome Visualization
• Since 1970s, various methods to visualize human
chromosomes were discovered and become an important
diagnostic tool for clinical practice
• The length and arm ratio (centromeric index) are rarely
sufficient to confirm an ambiguous identification of the
chromosome  can be used for grouping
• Banding techniques are extremely useful for the detection of
numerical and structural abnormality
• Each of human chromosome has characteristic in
arm length and the position of the
centromere/arm length ratio  classified into A
to G group
• Arms:
• p arm = short arm
• q arm = long arm

prophase
 Technique to visualize chromosome

1. GTG/G banding: (trypsine induced Giemsa stain):

differentiates light and dark bands
• The most common technique for chromosomal
analysis/karyotyping
2. C-banding: (centromere stain) to demonstrate constitutive
heterochromatin  centromere region darkly stained
3. Silver-banding/Ag-NOR banding: to identify satellites/short
arms of all acrocentric chromosomes (D and G group
except Y chromosome)
 G-banding Technique
• G-banding technique were introduced in 1970s is a distinct,
permanent, easy to photograph, and can be visualized using light
microscope
• The aim of this method is to produce G-bands based on proteolytic
digestion of the chromosome by trypsine followed by staining with
Giemsa
• The G-light bands and G-dark bands forms unique pattern 
landmark for chromosomes identification  used to pair and
identify each chromosomes and conclude using ISCN (International
System for Human Cytogenetic Nomenclature)
• G-bands patterns can be used to pair and identify each of the
human chromosomes accurately, called karyotype
 Conventional Cytogenetics only
can detect abnormality above 4 Mb
The higher the band size, the
better/smaller can be detected
Chromosome 4
• Ambiguous Genitalia
Basic cell culture is mitosis division

Prophase

Metaphase
Telophase

Anaphase
 CELL CULTURE (Blood lymphocyte)
• 5-10 ml blood collected in heparin tube  care must be taken to avoid
extremes temperature (stored at room or refrigerated > 4°C)
• Heparin  red cells in the bottom, while the white cells and platelets
forming thin layer on the top (buffy coat)
• 5-6 drops with a sterile pasteur pipette added to the medium
• Medium (MEM, TC 199, RPMI) added with at least:
1. FCS/FBS  promote cells growth
2. PHA  mitotic stimulant
3. Antibiotic  to prevent contamination
 Incubate for 72 hours in CO2 incubator
• Add Colcemid (about 45 min prior harvesting)  arresting in metaphase
HARVESTING
3 Steps of harvesting procedures:
1. Stopping the cells division in metaphase by adding
colcemid, prevents the cells from entering anaphase
2. Exposing the cells to a hypotonic solution (KCL) 
uptake the water through semi-permeable cell
membrane  liquid-filled balloon
3. Fixative (Carnoy’s/Methanol + Acetic acid) removes
water from the cells & enhance chromosome
morphology and their ability to take up the stain
SLIDE PREPARATION
• Is the most critical steps in visualizing a good
metaphase spreads:
• Over spread  lost (miss diagnosis)
• Under spread  too many crossing (challenging)
• The cells membrane should remain intact, if not the
chromosome may spill out causing chromosome loss.
• Next step is making slide old enough in order to stain
(manipulative or natural 3 days in room temperature)
• G-banding (Giemsa)  to see bands (dark and light)
The best spread Common spread
Seven groups of chromosomes based on size and
centromere location
• Group A: chromosome 1-3, large, metacentric
• Group B: chromosome 4-5, large, submetacentric
• Group C: chromosome 6-12 and X, medium, submetacentric
• Group D: chromosome 13-15, medium, acrocentric
• Group E: chromosome 16-18, short, submetacentric
• Group F: chromosome 19-20, short, metacentric
• Group G: chromosome 21-22 and Y, short, acrocentric
Chromosome spread with chromosomes shown by bright field G-banding
Karyotype shown by bright field G-banding of chromosomes
Karyotype shown by bright field G-banding of chromosomes
IDEOGRAM “ a TEMPLATE” TO ANALYZE
ABERRATION
 Molecular cytogenetic:
FISH (Fluorescent in situ Hybridization) is a cytogenetic technique which
can be used to detect and localize DNA sequences on chromosomes
TO determine the origin of small (<4Mb/submicroscopic)
deletion/duplication/translocation
Spectral Karyotype is cytogenetic technique to detect numbers of
complex aberrations (solid cancer)
“the higher the resolution the bigger the change”
 75°

37°
Spectral Karyotype
Case 2
• A 7 years old girl referred by pediatricians to our
laboratory with Down Syndrome as a presumptive
diagnosis
• Clinical features: Upslanting palpebrae, flat nasal
bridge, macroglossy, low set ear (striking DS
phenotype), learning disabilities
• She is the only child of young couple (mother: 28 yo,
father:29yo)
• They want to have another child
Patient’s karyotype
 • What is her diagnosis?

• What is your plan?

46,XX,t(14;21)(q10;q10),+21
Mother’s karyotype
45,XX,t(14:21)(q10:q10)
Father’s karyotype
46,XY
What is the conclusion of case 2 ?
• De Novo ?
• Inherited ?

What should you tell to the parent about the

recurrent risk ? (if the parent still want to have
a baby)
Case 3
• A 13 years old boy came to our laboratory with
learning disabilities, and hypogonadism(no
secondary sexual signs, small testis, undescensus
testis)
• He has 10 years younger sister with normal
phenotype
• His mother (33yo) did not consumed contraceptive
pills or implant or other family planning program
• They want to have another child (they want to have
normal boy)
 46, XY / 48, XXXY (60%)

• What is the diagnosis?

• What is consequences for
proband?
• Additional information: both parent have
normal karyotypes
• What is the possible cause of this
abnormality?
1. De Novo or not?
2. Mosaicism?
• What is your suggestion for the parent
regarding having another child?
Post zygotic mitotic non-disjunction

46,XX/47,XX,+21 (20%)

Mosaic: 45/47