Karyotyping - Procedure and

interpretation of reports

Presented by – Dr. Kashmiri Lal Sharma

Moderated by – Dr. Anshu Palta
 History
• Presence of 46 chromosomes in normal human cells was
first demonstrated in 1956
• Associations between abnormal numbers of
chromosomes and congenital anomalies were initially
reported in 1959 in Down syndrome
• In 1960 Nowell and Hungerford reported the first
chromosomal anomaly associated with cancer
(Philadelphia chromosome)
• In late 1960 Q-banding was done
• Chromosome nomenclature was established in 1971
 Karyotyping
• Karyotyping is the process by which pictures of the
chromosomes are taken while the cells are
undergoing mitosis
• The picture is then enlarged
• The picture of the chromosomes are cut up so that
each chromosome is removed
• The chromosomes are matched up and attached to a
paper according to size and location of centromere
Clinical indications for karyotyping
• Problems during early growth and development
• Stillbirth and neonatal death
• Fertility problems
• Family history
• Neoplasia
• Pregnancy in advanced age
Preparing a karyotype
• Harvest cells
• Culture cells 1-2 days
• Arrest cells in metaphase with colchicine

• “Spread” cells on slide and stain

• Count chromosomes in 20
representative cells
• Capture image of 5 “best” cells
and construct karyotypes for each

metaphase
 Harvesting cells
• Tissue should be source of dividing cells
Antenatal diagnostic karyotyping
• Amniotic fluid
• Chorionic villous sampling
Post natal diagnostic karyotype
• Tumour cells
• Bone marrow (MDSs and MPDs)
• Peripheral blood
• Lymph nodes
 Sample collection
• 1 to 2 ml of bone marrow is generally adequate
• Sample must be aspirated into a sterile syringe coated
with preservative free sodium heparin to prevent
clotting
• Transferred into a sterile tube containing preservative
free heparin or a medium such as RPMI 1640,
McCoy’s 5A or Hanks’ balanced salt solution with
preservative free heparin
• Antibiotics (penicillin, streptomycin) may be added to
transport medium
 Sample collection contd.
• 10ml of blood should be drawn aseptically into a
sterile tube containing heparin
• The WBCs are removed from the blood
• If a fetus is being karyotyped amniotic fluid is
removed from the amniotic sac
• Living cells are removed from the amniotic fluid
 Antenatal diagnosis:
amniocentesis

• sampling cells from amniotic fluid

• usually done ~ 15-18 weeks
Antenatal diagnosis: Chorionic villi sampling (CVS)

• Sampling cells from placenta

• Usually done 10-12 weeks
 Sample collection contd.
• Lymph nodes are used for analysis of lymphomas
• Lymph nodes are obtained aseptically
• Transported in sterile tube containing medium and
antibiotics
• Transferred to sterile Petri dishes with medium, then
minced using sterile scissors
• Triturated using a Pasteur pipette to create a cell
suspension
• Processing of this cell suspension is similar to that of
bone marrow and blood
Sample processing and cell culture
• Marrow, blood or lymph node cells are centrifuged
and then resuspended in medium
• Cells are then counted with a automatic counter or a
hemocytometer
• Resuspended at 1×106 cells/ml, which is optimal
concentration for cell culture
• Analysis can be performed
- direct preparation (bone marrow)
- indirect (cultured) preparation
 Sample processing ctd.
• Direct preparations
- allow rapid Karyotyping of cells that are undergoing
division in vivo
- mitotic index is low
- poor chromosome morphology
- yield normal rather than abnormal metaphases
• Indirect preparations
- increases mitotic rate
- improves chromosome morphology
- generally promotes proliferation of malignant cells
 chromosomes
MITOSIS condense

DNA
replication

nuclear
envelope
breaks down

metaphase

chromosomes
aligned on
spindle fibers
 Sample processing contd.
• Colchicine is used to arrest cells in metaphase
• In direct preparations - added to cell suspension
without prior cell culture and cells are harvested after
incubation at 37°C for 1hr.
• In overnight colchicine treated cultures cells are
cultured overnight in medium with colchicine and
harvested next morning
• In short term cultures cells are cultured in medium
without colchicine at 37°C for 24 to 72 hrs, before
harvesting cells colchicine is added at 0.01 to
0.02µg/ml for 30mins to 1 hr.
 Sample processing contd.
Cell cycle synchronization
• In early stages of cell division
- to increase mitotic index
- yields elongated chromosomes allowing high resolution
chromosome banding
- detection of subtle abnormalities
• Cell division is blocked by incubation with an antimetabolite
(MTX)
• Block is then released (thymidine)
• Also duration of colchicine treatment can also be reduced
resulting in better chromosome morphology
 Sample processing contd.
• Hypotonic treatment
• Used to produce cell swelling, allowing
chromosomes to spread within cells
• A dilute solution of KCl (0.075M) is most commonly
used
• Cells are centrifuged after colchicine treatment and
resuspended in prewarmed hypotonic solution and
incubated at 37°C for 10 to 45 mins
 Fixation
• Cells are fixed after hypotonic treatment in a
modified Carnoy fixative (3:1= methanol:acetic acid)
• Fixation causes denaturation and precipitation of
proteins and nucleic acids resulting in hardening of
chromatin which improves morphology
• Cells are centrifuged, gently resuspended in fixative,
incubated at room temperature, then centrifuged again
• These steps are repeated until a cell suspension that is
only slightly cloudy is obtained
 Slide preparation
• Slides can be prepared immediately or at a later time
• Fixed cells are dropped onto cleaned glass slides
under several different conditions
• The angle at which the pipette and slide are held is
varied
• Slides may be wet, cold or dry
• Slides prepared are examined for cell density and
quality of metaphase spreads by phase contrast
microscopy
 Staining
• Several banding techniques yield staining of chromosome
regions with variable intensity based on their nucleotide
and protein composition
• Staining with dyes produces a unique banding pattern on
each chromosome that is specific for a chromosome with
a dye
• Q-banding, uses quinacrine mustard or quinacrine
dihydrochloride to create fluorescent transverse bands
• It requires fluorescence microscopy and Q bands fade
over time
• G-banding
- pretreatment with and enzyme (trypsin)
- staining with Giemsa to produce G-bands
- identical to Q-bands
- dark G bands contain more condensed chromatin
- light G bands contain most of genes
• It is preferred over Q-banding
• The most widely used G-banding technique GTG banding
(G-bands by trypsin using Giemsa)
• Other G-banding techniques utilize Wright and Leishman
stains
G-banding
• R-banding
- treatment of chromosomes with a hot alkaline
solution before Giemsa staining
- bands are reverse of G-band and called R-bands
- correspond to light G-bands
• R-banding is useful for analyzing rearrangements
involving the terminal ends of chromosomes
• C-banding
- constitutive heterochromatin, mainly at centromere
- chromosomes are exposed to barium hydroxide
prior to giemsa staining
• T-banding
- subsets of R-bands concentrated at telomeric
regions
- involves heat treatment of chromosomes prior to
giemsa staining
C-banding
 Microscopy
• Slides are scanned under a light microscope to locate
chromosome spreads of good quality
• Adequate analysis requires analysis of at least 20
chromosome spreads
• Two morphologically different populations of metaphases
may be present
• The population with better spreading and morphology often
consists of cells with normal karyotypes
• Chromosomes are counted in each chosen metaphase spread
to determine whether they are present in normal numbers
 Microscopy contd.
• Chromosomes are then identified and characterized as
structurally normal or abnormal
• Chromosomes from each metaphase spread are
arranged in an order of size and position of
centromere
• In recent years automated karyotyping systems
utilizing computerized image analysis have increased
efficiency of karyotyping
 Factors determining karyotyping
• Adequate numbers of cells must be available
• Cells must be viable and undergoing division
• Sufficient no. of cells must be present in metaphase
• Chromosomes must be separated from one another
within the cell so that each chromosome can be
resolved as a distinct entity
• For analysis of malignancies, it is critical that
malignant cells be analyzed, rather than coexisting
normal cells
 Interpretation
• Karyotypes are described according to the International
System for Human Cytogenetic Nomenclature
• The most recent guidelines were published in 2005
• Description of a karyotype starts with the total number
of chromosomes, including sex chromosome
• Total number of chromosomes is followed by a list of
the sex chromosomes present in the cell
• Then a list of abnormalities of autosomes listed in
ascending numerical order
• Each chromosome is visualized as a chromatid that
are joined at a central constriction called the
centromere
• Centomere is the region where the chromosomes
attaches to the spindle during mitosis
• By convention chromosomes are always shown with
the p-arm on top and q-arm on bottom
• Chromosomes are divided into 7 groups (A-G)
• Group-A = chromosomes 1,2&3 (large, metacentric)
• Group-B = chromosomes 4&5 (large, submetacentric)
• Group-C = chromosomes 6,7,8,9,10,11,12&X (medium
sized, metacentric or submetacentric)
• Group-D = chromosomes 13,14&15 (medium sized,
acrocentric chromosomes with satellites
• Group-E = chromosomes 16,17&18 (small, metacentric
and submetacentric)
• Group-F = chromosomes 19&20 (small and metacentric)
• Group-G = chromosomes 21,22&Y (small and acrocentric)
• Nomenclature also describes the structure of each
chromosome
• Landmarks are distinct morphologic feature important
in identifying chromosomes
- centromere
- telomeres
- prominent chromosome bands
• Chromosomes regions are defined as areas lying
between adjacent landmarks
• Regions in each arm are numbered sequentially
moving outward from the centromere
• Bands are defined as chromosome segments that are
clearly distinguishable from adjacent segments by
virtue of appearing lighter or darker with one or more
banding techniques
• Bands within regions are also numbered sequentially
moving outward
• eg 1p31, 1p32
• High-resolution banding divides chromosome bands
into sub-bands which are light and dark regions that
constitute finer structure within chromosome bands
• Sub-bands are also numbered from the centromere
toward the telomere
• Sub-band nos. are given after band no. separated by
decimal points (eg 1p31.1,1p31.2 etc)
• Sub-sub-band nos. are given after sub-band no (eg
1p31.11,1p31.12 etc)
 11.32
11.3
11.31
11.23
11.22
p 11.2
11.21

11.1 11.1

11.2 11.2

12.1
12 12.2
12.3
q
21.1
21 21.2
21.31
21.32
21.33
22.1
22 22.2
22.3

23 23
 Normal female karyotype
• 46,XX
 Normal male karyotype
• 46,XY
 Chromosomal abnormalities
• Numerical and structural
• Constitutional or acquired
• Constitutional abnormalities are present in all or
almost all cells in body and exist in the earliest stage
of embryogenesis
• Constitutional abnormality is designated by c in a
karyotype
• 47,XX,+21c designates a constitutional abnormality
and 47,XX,+21 designates an acquired abnormality
• Numerical chromosomal abnormalities
• Chromosome loss or gain
• Indicated in the karyotype by a minus or a plus sign
• Followed by the number of the missing of additional
chromosome
• 45,XX,+8
• 48,XY,+13,+13
 Down syndrome
• 47,XY,+21
 Edward syndrome
• 47,XY,+18
 Trisomy 13 (Patau syndrome)
• 47,XY,+13
 Trisomy 16 with monosomy X
• Most common chromosomal abnormality but fetuses
do not survive beyond 1st trimester
• 46,X,+13
 Klinefelter’s syndrome
• 47,XXY
 Turner syndrome
• 45,X
• Structural chromosomal abnormalities
• Deletion (del) is loss of a chromosome segment may
be either interstitial or terminal
• 46,XX,del(5)(q13q33)
• 46,XX,del(7)(q22)
 Cri-du-chat syndrome
• 46,XY,del(5)(p14.2)
• Isochromosome (i) is a structurally abnormal
chromosome consisting of two identical chromosome
arms positioned as mirror images of each other (i(17q))
• Can be monocentric or dicentric
• Isodicentric chromosome (idic) is an isochromosome in
which there are two copies of centromere (idic(7p))
• Inversion (inv) is180 degree rotation of a chromosome
segment
• Can be pericentric or paracentric
• inv(16)(p13q22) ; inv(3)(q21q26)
• Ring chromosome (r)
- breaks have occurred in both the short and the long
chromosome arms
- ends have joined together, creating a closed circle or ring
- 46,X,r(X)
• Translocation (t)
- relocation of material from one chromosome usually to a
different chromosome
- is most often reciprocal
- 46,XX,t(8;14)(q24;q32)
 Philadelphia chromosome
• 46,XX,t(9;22)(q34;q11) in CML
 References
• Chromosome structure and function. Strachan T,
Read A.P. eds Human molecular genetics 3rd ed. New
Delhi, Ajanta offset packaging. 2004: 34-52
• Principles of clinical cytogenetics. Nussbaum R.L,
McInnes R.R, Willard H.F, Boerkoel III C.F. eds.
Thompson & Thompson Genetics in medicine. 6th ed.
Philadelphia, Saunders. 2004:135-155.
• Sait S.N.J, Baer M.R. Cytogenetics in Greer J.P,
Paraskevas F, Foerster J, Glader B, Rodgers G.M,
Arber D.A. eds. Wintrobe’s Clinical Hematology. 12th
ed. Philadelphia: Lippincott Williams and Wilkins,
2009; 50-69.
• Vermeesch J.R, Fiegler H, de Leeuw N, Szuhai K,
Schoumans J, Ciccone R. et al. Guidelines for
molecular karyotyping in constitutional genetic
diagnosis. European journal of Human genetics.
2007; 15:1105-1114.
Thank you…