Islamic University of Gaza

Human Karyotype

Training Course Manual

Prepared by:
Mazen Ali Abo Zayed

Table of contents
Title

Page No.

1.

Objectives

2.

Introduction to Karyotyping

3.

Materials and Methods

3.1.

Materials required

3.2.

Methods

3.2.1. Peripheral blood media preparation

3.2.2. Blood preparation

3.2.3. Karyotyping procedure

3.2.4. Staining Procedure

3.2.5. Karyotype analysis

3.2.6. Classification of Chromosomes for Karyotyping

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4.

Examples of chromosomal aberrations

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4.1

Human disorders due to chromosome alterations in

autosomes

12

4.2.

Nondisjunction of the sex chromosomes

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Human Karyotype
1. Objectives:
There are several genetic disorders that involve entire chromosomes. The
objectives of this lab will be:
1. To demonstrate a microtechnique for reliable chromosomal analysis of
leucocytes obtained from peripheral blood.
2. To prepare a karyotype from the chromosomes of human male or
female.
3. To use the karyotyping techniques for diagnosing a chromosomal disorder.

2. Introduction to Karyotyping:
Chromosomal aberrations are abnormalities in the number or microscopically
observable structure of chromosomes. The number of chromosomes in
human cells is 46 with 22 autosomal pairs (one of each type contributed by
the mother and one of each type from the father) and 2 sex chromosomes - 2
X chromosomes for females (one from father and one from mother) or an X
and a Y chromosome for males (the X from the mother and the Y from the
father). The chromosomes visible only at the metaphase stage of mitosis, 22
homologous pairs of autosomes and two sex chromosomes. Each
chromosome has a characteristic size and shape in the normal cell.
During most of the cell cycle, interphase, the chromosomes are somewhat
less condensed and are not visible as individual objects under the light
microscope. Mitosis, or nucleus division, is the first part of M-phase and in
consists of four stages (prophase, metaphase, anaphase and telophase).
However during cell division, mitosis, the chromosomes become highly
condensed and are then visible as dark distinct bodies within the nuclei of
cells. The chromosomes are most easily seen and identified at the metaphase
stage of cell division.

Cell Cycle = Interphase + M phase

Interphase = G1phase: initial growth cycle, S phase: DNA synthesis (replication), G2 phase: second
growth stage.

M phase

= Mitosis + cytokinesis

The banding of chromosomes by using dyes was discovered in the late

1960's and before that cytogeneticists depended on chromosome length and
position of a constriction to identify the individual chromosomes. The band
width and the order of bands is characteristic of a particular chromosome - a
trained cytogeneticist can identify each chromosome (1,2,3...22, X and Y) by
observing its banding pattern under the microscope. chromosomes are
arranged and numbered by size, from largest to smallest. This arrangement
helps scientists quickly identify chromosomal alterations that may result in a
genetic disorder.
Identifying chromosomes has become easier in recent years by using certain
staining techniques. One of the most common staining techniques involves
giemsa stain, which gives the chromosomes a banded appearance (hence
giemsa banding or G-banding)
G-banding is the treatment of chromosomes in the metaphase stage with
trypsin (to partially digest the protein) and stain them with Giemsa. Each
homologous chromosome pair has a unique pattern of G-bands, enabling
recognition of particular chromosomes.
Karyotyping is the process by which doctors and geneticists take pictures of
the chromosomes while the cell are undergoing mitosis. The picture is then
enlarged. The picture of the chromosomes are then cut up so that each
chromosome is removed. The chromosomes are matched up and attached to
a paper according to size. The chromosomes pairs are numbered from largest
to smallest. There are 22 pairs of chromosomes which match up exactly.
Then the sex chromosomes are paired, in the female (XX) the chromosomes
match and in the male (XY) the chromosomes do not match. This technique
can be used to assess the normalcy of an individuals chromosomes and to
assay for various genetic diseases such as Downs syndrome and
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Klinefelters syndrome. It is estimated that one in 156 live births have some
kind of chromosomal abnormality.
A chromosome is divided by its centromer into short arm (p) and long arm (q).
chromosomes can be classified by the position of their centromer:
- Metacentic: If its two arms are equal in length.
- Submetacentric: If arms' lengths are unequal.
- Acrocentric: If the p arm is so short that is hard to observe, but still present
The photograph is enlarged and cut up into individual chromosomes. The
homologous chromosomes can be distinguished by length and by the position
of the centromer so the chromosomes can be arranged in 7 groups (A, B, C,
D, E, F, G).
Karyotypes are arranged with the short arm of the chromosome on top, and
the long arm on the bottom. In addition, the differently stained regions and
sub-regions are given numerical designations from proximal to distal on the
chromosome arms. For example, Cri du chat syndrome involves a deletion on
the short arm of chromosome 5. It is written as 46,XX,5p-. The critical region
for this syndrome is deletion of 15.2, which is written as 46,XX,del(5)(p15.2).
Peripheral Blood Karyotyping Medium With Phytohemagglutinin (PHA) is
intended for use in short-term cultivation of peripheral blood lymphocytes for
chromosome evaluation. It is based on RPMI-1640 basal medium
supplemented with L-Glutamine, fetal bovine serum and antibiotics (penicillin
and streptomycin). Karyotyping Medium is supplied as frozen medium, which
is ready for use after thawing and phytohaemagglutinin supplementation.
The blood cell karyotyping method was developed to provide information
about chromosomal abnormalities. Lymphocyte cells do not normally undergo
subsequent cell divisions. In the presence of a mitogen (PHA), lymphocytes
are stimulated to enter into mitosis by DNA replication. After 48-72 hours, a
mitotic inhibitor (colcemid) is added to the culture to stop mitosis in the
metaphase stage. After treatment by hypotonic solution, fixation and staining,
chromosomes

can

be

microscopically

abnormalities.

observed

and

evaluated

for

Alterations in chromosome number:

Nondisjunction occurs when either homologues fail to separate during
anaphase I of meiosis, or sister chromatids fail to separate during anaphase
II. The result is that one gamete has 2 copies of one chromosome and the
other has no copy of that chromosome (The other chromosomes are
distributed normally).
If either of these gametes unites with another during fertilization, the result is
aneuploidy (abnormal chromosome number)

A trisomic cell has one extra chromosome (2n +1) = example: trisomy
21 ( Down syndrome).

A monosomic cell has one missing chromosome (2n - 1) = usually

lethal except for one known in humans: Turner's syndrome (monosomy
XO).

The frequency of nondisjunction is quite high in humans, but the results are
usually so devastating to the growing zygote that miscarriage occurs very
early in the pregnancy. If the individual survives, he or she usually has a set of
symptoms - a syndrome - caused by the abnormal dose of each gene product
from that chromosome.

Translocation is the result of chromosomal breakage but the broken

segment transfers itself to a broken segment of another chromosome. There
are both balanced and unbalanced translocations.

Deletion - Deletion occurs when a chromosome breaks and a portion of the

chromosome is lost.
Inversion - a section of the chromosome is inverted (reversed) on the same
chromosome.
3. Materials and methods:
3.1 Materials required:

Peripheral human blood

Heparin sodium injection 25000units/vial
Karyotype medium RPMI-1640
Fetal Bovine Serum
Penicillin Streptomycin 10000 U/ml penicillin and 10000 mg/ml
streptomycin
Phytohemagglutinin-M (PHA-M)
L-glutamin 200 mmol/L
Colcemid solution 10mg/ml
Hypotonic solution (0.075M KCl)
3X Absolute Methanol +1X Glacial Acetic Acid
Giemsa stain
Hanks buffer
Trypsine
Slides and microscope

3.2 Methods:
3.2.1. Peripheral blood media preparation:
Blood culture media; 500 ml RPMI 1640 with 100ml fetal bovine serum, 6.5ml
penicillin streptomycin and 7ml glutamine. Dispense 10ml aliquots into
sterile tube and add 2% (0.2ml) PHA to each tube. Store at 4C for along as 2
weeks.

3.2.2. Blood preparation:

1- Wash 10ml syringe with Heparin solution and collect blood
aseptically to the syringe (0.5ml for each tube).
2- Remove the blood to sterile vacuette tubes or to sterile,
Heparin prewashed tube.
3- Store at 4oC until required (upto 7 hours).
4- Before use, invert the tube with blood several times.
.

3.2.3. Karyotyping procedure:

1- Inoculate 0.5ml of heparinized whole blood into tube with 10ml of
karyotyping medium
2- Incubate the tubes in incubator with 5% CO2 at 37C for total of 69-72
hours.
3 -After total of 69 hours from seeding add 100l of Colcemid Solution to
each culture tubes.
4 - Incubate the tubes at 37oC for an additional 20-30 minutes.
5 - Spin at 500g (1500RPM) for 7 minutes.
6 - Remove the supernatant and re-suspend the cells in 5ml of hypotonic
0.075M KCl pre wormed to 37C.
7- Incubate at 37oC for 15 minutes.
8- Add drop-by- drop (with vortexing) 1ml fresh ice cold fixative.
9- Spin at 500g (1500RPM) for 7 minutes.
10- Remove the supernatant, agitate the cellular sediment and add drop-bydrop (with continous vortexing), 5ml of fresh, ice-cold fixative.
11- Leave at 4C for 20 minutes.
12- Repeat steps 9 and 10 ,until the supernatant is clear.
13- Spin at 500g (1500RPM) for 7 minutes.
14- Re-suspend the cell pellet with a 1.5ml of fresh fixative.
15- Drop 4-5 drops, from a high of approximately 50 cm onto a clean slide
and blow carefully on the drops for spreading them on the slide.
16- Heat the slides to 55C for overnight.

3.2.4. Staining Procedure:

The staining procedure should be done in a 37C water bath.
Put the slides in a staining rack (e.g. coplin staining jar) and treat as follows:
1- 2 minutes in 50ml Hanks solution.
2- 1.47 minutes in: 47.5ml Hanks solution + 2.5ml Trypsin-EDTA solution.
3- Wash in: 40ml Hanks solution +10ml Fetal bovine serum.
4- Wash in: 50ml Hanks solution.
5 1.5 minutes in: 47ml Buffer solution pH 6.8 + 3ml Giemsa stain solution.
6- Wash several times with 50ml Buffer solution pH 6.8.
7- Air dry the chromosome slides.
8- Check for chromosome spreads in a phase contrast lab microscope.
3.2.5. Karyotype analysis:
1- Obtain a set of chromosomes.
2- Match the chromosomes with their homologous mate. One
chromosome of each pair is numbered, as you match your chromosomes
number the homologous pairs. You need to be very systematic.The
number one chromosome is the largest. Its corresponding mate should be
of the same size, with the same banding pattern, and have the same
centromere location.
3- Determine the karyotype abnormality using the Chromosome Analysis
Key that is below. Keep in mind there might be some slight
variations. The key below will help guide you.
4- Research your abnormality using the resources available. A
photograph of a real karyotype for the disorder is in the computer
database, for example BandView is Applied Spectral Imaging's fullyautomated image acquisition and analysis system for Karyotyping
(High resolution images with 12-bit digital CCD-camera (1280x1024
pixels).
5- Simple key for diagnosing chromosome abnormalities :

1A 46 chromosomes in karyotype......................................................
GO TO STATEMENT 3

1B Not 46 chromosomes in karyotype...............................................

GO TO STATEMENT 2

2A 47 chromosomes in karyotype (3 of one chromosome).............

Trisomy condition

2B One chromosome missing from pair.............................................

Monosomy condition

3A All chromosomes are matched with its homologue without obvious

missing pieces or additions ...............................................................................................................
......Possible Normal individual

3B Homologous pairs are not identical in size...................................

GO TO STATEMENT 4

4A There are additions pieces attached to a chromosome..............

Translocation

4B There are missing pieces in a chromosome..................................

Deletion

3.2.6. Classification of Chromosomes for Karyotyping:

Chromosomes are arranged into seven groups based on size and centromere location.
The centromeres can be found in the middle of the chromosome (median), near one
end (acrocentric), or in between these first two (submedian)
Group A: chromosomes 1-3 are largest with median centromere.
Group B: chromosomes 4-5 are large with submedian centromere
Group C: chromosomes 6-12 are medium sized with submedian centromere
Group D: chromosomes 13-15 are medium sized with acrocentric centromere
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Group E: chromosomes 16-18 are short with median or submedian centromere

Group F: chromosomes 19-20 are short with median centromere
Group G: chromosomes 21-22 are very short with acrocentric centromere.
Chromosome X is similar to group C.
Chromosome Y is similar to group G

4. Examples of chromosomal aberrations:

4.1. Human disorders due to chromosome alterations in autosomes
(Chromosomes 1-22).
There only 3 trisomies that result in a baby that can survive for a time after birth; the
others are too devastating and the baby usually dies in utero.

Trisomy 13, XX

Patau Syndrome. The karyotype here demonstrates trisomy 13 (47, XX, +13). It is
rare for fetuses with this condition to go to term, so it occurs in only 1 in 15,000 live
births. It is rare for babies to survive for very long if liveborn because of the multitude
of anomalies that are usually present. Forty five percent die within the first month,
90% by six months and less than 5% reach 3 years. There is severely abnormal
cerebral functions and virtually always leads to death in early infancy. This baby has
very pronouced clefts of the lip and palate, broad nose, small cranium, polydactyl (An
extra finger), deafness, and nonfunctional eyes. Heart defects and severe mental
retardation are also part of the clinical picture.
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Trisomy 18 (Edwards Syndrome):

Edwards Syndrome. This karyotype demonstrates trisomy 18 (47, XY, +18). It is

uncommon for fetuses with this condition to survive, so the incidence is only 1 in
8000 live births. Thirty percent of these children die within the first month and only
10% survive one year. It is rare for babies to survive for very long if liveborn because
of the multitude of anomalies that are usually present. There is severe mental
retardation and a highly characteristic pattern of malformations such as elongated
skull, a very narrow pelvis, rocker bottom feet and a grasping of the two central
fingers by the thurm and little finger. In addition, the ears are often low set and the
mouth and teeth are small. Nearly all babies born with this condition die in early
infancy.

Trisomy 21 (Down syndrome)

This is an example of trisomy 21 (47, XY, +21) also known as Down

syndrome. Note the extra chromosome 21. Additions or deletions of genetic
material are generally lethal in utero. Trisomy 21 is an example of one form of
addition in which the fetus may occasionally survive to term and beyond. Of
those born with Down syndrome, 1/6 die within the first year and the average
life span is 16.2 years. The non-dysjunctional event in meiosis that produces
this anomaly increases in incidence with increasing maternal age. Trisomy 21,
one of the most common causes of mental retardation. The child can have an
IQ between 25-74. An average person has an IQ between 90-110. This
results in a number of characteristic features, such as short stature, broad
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hands, stubby fingers and toes, a wide rounded face, a large protruding
tongue that makes speech difficult. Individuals with this syndrome have a high
incidence of respiratory infections, heart defects, and leukemia. The average
risk of having a child with trisomy 21 is 1/750 live births. Mothers in their early
twenties have a risk of 1/1,500 and women over 35 have a risk factor of 1/70,
which jumps top 1/25 for women 45 and over.

Trisomy 16 with Monosomy X (Never survive):

Here is an example of trisomy 16. This is the most common chromosomal

abnormality, but the fetuses NEVER survive past the first trimester. Many first
trimester fetuses are lost in this fashion (many are "silent" abortions). Note in
this case that a sex chromosome is missing as well. Intrauterine demise is
nature's way of eliminating abnormal karyotypes.

4.2. Nondisjunction of the sex chromosomes (X or Y chromosome):

Can be fatal, but many people have these karyotypes and are just fine!

Klinefelter syndrome: 47, XXY males.

Male sex organs; unusually small testes, sterile. Breast enlargement and other
feminine body characteristics. Normal intelligence.

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This is Klinefelter's syndrome with a 47, XXY karyotype. A non-dysjunctional

event in meiosis left two X chromosomes in an ovum. This particular anomaly
is relatively common (about 1 in 500 males), with affected persons being
relatively normal. Characteristics associated with this condition are tall stature
and sterility.

XXXXY Variation of Kleinfelter's Syndrome

49,XXXXY. This karyotype shows a variant of Klinefelter's syndrome.

Individuals with this syndrome are male, typically with the karyotype 47,XXY.
They exhibit a characteristic phenotype including tall stature, infertility,
gynecomastia and hypogonadism. Aneuploidy above one extra chromosome
is usually fatal but because of X-inactivation, which "turns off" all but one X
chromosome per cell, the effects of 3 extra chromosomes are reduced.

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 47, XYY males (Jacobs Syndrome):

Individuals are somewhat taller than average and often have below normal
intelligence. At one time (~1970s), it was thought that these men were likely to be
criminally aggressive, but this hypothesis has been disproven over time.

Jacobs Syndrome. A chromosome aberration which is caused by

nondisjunction of the Y chromosome during the second phase of meiosis
giving a 47 XYY karyotype. Occurence is 1/1000 live male births. Men with
this karyotype are tall and have low mental ability.
Trisomy X: 47, XXX females:
1:1000 live births - healthy and fertile - usually cannot be distinguished from
normal female except by karyotype

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Monosomy X (Turner's syndrome):

This is monosomy X (Turner's syndrome, with karyotype 45, XO). This can
occur in about 1 per 2,700 births. It is not linked to maternal age. Women with
Turner's syndrome can live relatively normal lives, though they are unable to
bear children. The phenotype of this female includes short stature, short
broad neck, and a broad chest. Intelligence does not seem to be affected.
(98% of these fetuses die before birth)

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