Chromosomal Karyotyping Chromosomal Karyotyping

Chromosomal Karyotyping
Karyology
• Karyotyping - process of pairing and ordering all chromosomes of an
organism, thus providing a genome-wide snapshot of an individual's
chromosomes.
• Karyotypes describe chromosome count of an organism and what these

chromosomes look like (length, position of centromeres, banding pattern,
differences between sex chromosomes, and other physical characteristics)
under a light microscope.
• Preparation and study of karyotypes is part of Cytogenetics.
• Chromosomes are depicted (by rearranging a photomicrograph) in a

standard format (in pairs, ordered by size and position of centromere for
chromosomes of the same size) known as a Karyogram or Idiogram.
• Clinical Cytogeneticists analyze human karyotypes to detect gross genetic

changes (anomalies involving several megabases or more of DNA).
Karyotypes
Karyotypes are prepared using standardized
staining procedures that reveal characteristic
structural features for each chromosome.
Sampling
• Peripheral Blood
• Placental Villi (Second Trimester)
• Amniotic Fluid
• Chorionic Villus
• Skin Biopsy
• Tumor Biopsies
• Bone Marrow
Complication of Sampling Methods

– Rare
– Amniocentesis carries a very minimal risk of miscarriage
– Slight risk of bleeding and infection in bone marrow biopsy
– Chemotherapy can cause breaks in chromosomes leading to skewed
results
Making a Karyotype
Draw 3 to 5 ml blood
Add a few drops of blood Add phytohemagglutinin to
stimulate mitosis
Incubate at 37oC for 3 days
Centrifuge to concentrate
cells. Add low-salt
solution to eliminate RBC
Transfer to tube and swell lymphocytes Add colchicine to culture
Transfer cells to tube
containing fixative for 1 to 2 hours to stop
mitosis in metaphase
Drop cells onto

microscope slide
Stain slide with Giemsa Digitized chromosome

Examine under images processed to
microscope make karyotype
Preparing Karyotypes from
Mitotic Cells
• Karyotypes are prepared from mitotic cells arrested in metaphase or
prometaphase portion of cell cycle, when chromosomes assume most condensed
conformations.
• Begins with the short-term culture of cells derived from specimen

• Allow a period of cell growth and multiplication
• Dividing cells are arrested in metaphase by addition of colchicine, which poisons
the mitotic spindle
• Cells are next treated with hypotonic solution that causes their nuclei to swell and
cells to burst
• Nuclei are then treated with a chemical fixative, dropped on a glass slide, and
treated with various stains that reveal structural features of chromosomes
O'Connor, C. (2008) Karyotyping for chromosomal abnormalities. Nature Education 1(1):27
Banding Patterns Reveal Structural
Details of Chromosomes
• Q-banding - first banding technique (1970)
– Involves use of the fluorescent dye quinacrine, which alkylates DNA and is subject to quenching over
time.
• G-banding/ Giemsa banding – Giemsa dye offers better resolution of individual bands,
produces more stable preparation, & can be analyzed with ordinary bright-field microscopy
– Metaphase chromosomes are first treated briefly with trypsin that degrades proteins before staining
with Giemsa.
– Trypsin relaxes chromatin structure and allows Giemsa dye access to the DNA.
– Heterochromatic regions, AT-rich & relatively gene-poor stain more darkly
– Less condensed chromatin, GC-rich & more transcriptionally active, stain lightly
– Giemsa stain produces ~400-800 bands distributed among 23 pairs of chromosomes thus
representing several million to 10 million base pairs DNA, containing hundreds of genes.
• R-banding - also involves Giemsa stain, but generates reverse pattern from G-banding
– Before Giemsa staining, heat treatment melts DNA helix in AT-rich regions that bind Giemsa stain
most strongly, leaving only GC-rich regions to take up the stain.
– Often used to provide critical details about gene-rich regions located near telomeres.
• C-banding - used to specifically stain constitutive heterochromatin, or genetically inactive

DNA, but it is rarely used for diagnostic purposes these days.

Chromosome Banding Revealed by
Different Staining Techniques
G-/Giemsa banding Q-banding
R-banding
C-banding
2001 Nature Publishing Group Rowley, J.

Chromosome translocations. Nature Reviews
Cancer 1, 246; Stamatoullas, A. et al.
Organizing Chromosomes in
Karyograms for Review
• According to international conventions, human autosomes are numbered from 1
to 22, in descending order by size, with exceptions of chromosomes 21 and 22, the
former actually being smallest autosome.
• Sex chromosomes are placed at end of a karyogram.
• Short p (petite) arms are at top & long q (queue) arms are at bottom.
• Chromosomes are aligned along a horizontal axis shared by their centromeres.
• Centromere placement can also be used to identify the gross morphology, or

shape, of chromosomes. Eg.
– Metacentric chromosomes 1, 3, and 16 have p and q arms of nearly equal lengths
– Submetacentric chromosomes 2, 6, and 10 have centromeres slightly displaced from
center
– Acrocentric chromosomes 13, 14, 15, 21 and 22 have centromeres located near their
ends
• Arranging chromosomes into a karyogram can simplify the identification of

abnormalities.
Using Karyograms to Detect
Chromosomal Abnormalities
Resolution of chromosomal changes detectable by karyotyping is typically a few
megabases
Chromosomal Disorders Detected by Karyotyping –

• Aneuploidy, which is often caused by absence or addition of a chromosome.
– Down syndrome (Trisomy 21) caused by an extra chromosome 21
– Edwards syndrome (Trisomy 18) caused by an extra chromosome 18
– Patau syndrome (Trisomy 13) caused by an extra chromosome 13
• Subtle structural changes, such as chromosomal Deletions (Cri du chat syndrome,
Angelman syndrome, Prader-Willi syndrome), Insertions, Duplications,
Translocations, or Inversions.
• Genetic diseases (eg. Premature Ovarian Failure)
• Some birth defects
– Klinefelter syndrome caused by an extra X chromosome (most common sex
chromosome abnormality in males)
– Turner syndrome caused by missing one X chromosome in females
• Certain haematologic and lymphoid disorders (e.g., Leukaemia, Lymphoma,
Myeloma, Refractory Anaemia)
When to Get Tested?
• When pregnancy screening tests are abnormal
• Bad obstetric history
• Recurrent pregnancy loss
• Infertility
• Primary amenorrhea
• Dismorphic features of newborn
• When signs of a chromosomal abnormality or associated
disorder are present
• When a specific abnormality has been detected in a family
member
• When a person has leukaemia, lymphoma, myeloma,
myelodysplasia or another cancer and an acquired
chromosome abnormality is suspected
Genetic Tests For Non-
obstructive Azoospermia/
Severe Oligospermia
Karyotype is recommended by the American Urological ssociation (AUA) and the European Academy of Andrology (EAA) guidelines
in all men with a total motile sperm count below 5 million who are thought to have non-obstructive azoospermia
J. Hotaling. Andrology, 2014, 2, 339–350

Tests Done in SRL
TEST METHOD CODE
CHROMOSOME ANALYSIS IN HEMATOLOGICAL DISORDERS 5800
ACUTE PROMYELOCYTIC LEUKEMIA (APL) 5840
BLOOD LYMPHO CULTURE 5814B
FANCONI ANEMIA 5812
FRAGILE X CHROMOSOME ANALYSIS 5364
KARYOTYPE
NEONATAL KARYOTYPING (NEWBORN TO ONE MONTH
CELL 5815
OLD CHILD)
CULTURE
PRENATAL AMNIOTIC FLUID KARYOTYPING 5832K
PRENATAL CHORIONIC VILLUS BIOPSY KARYOTYPING 5833K
PRENATAL FETAL CORD BLOOD KARYOTYPING 5831K
COUPLE KARYOTYPING 7535
AUTOGEN (COUPLE KARYOTYPING + ANTIPHOSPHOLIPID) DT5102
Thank You

Chromosomal Karyotyping Chromosomal Karyotyping

Uploaded by

Copyright:

Available Formats

Chromosomal Karyotyping Chromosomal Karyotyping

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Chromosomal Karyotyping Chromosomal Karyotyping

Uploaded by

Copyright:

Available Formats

Chromosomal Karyotyping

• Karyotypes describe chromosome count of an organism and what these

• Preparation and study of karyotypes is part of Cytogenetics.

• Chromosomes are depicted (by rearranging a photomicrograph) in a

• Clinical Cytogeneticists analyze human karyotypes to detect gross genetic

Complication of Sampling Methods

Incubate at 37oC for 3 days

Drop cells onto

Stain slide with Giemsa Digitized chromosome

• Begins with the short-term culture of cells derived from specimen

• C-banding - used to specifically stain constitutive heterochromatin, or genetically inactive

O'Connor, C. (2008) Karyotyping for chromosomal abnormalities. Nature Education 1(1):27

2001 Nature Publishing Group Rowley, J.

• Sex chromosomes are placed at end of a karyogram.

• Chromosomes are aligned along a horizontal axis shared by their centromeres.

• Centromere placement can also be used to identify the gross morphology, or

• Arranging chromosomes into a karyogram can simplify the identification of

Chromosomal Disorders Detected by Karyotyping –

J. Hotaling. Andrology, 2014, 2, 339–350

You might also like