This document describes research conducted on detecting Edward syndrome (trisomy 18) using karyotyping and fluorescence in situ hybridization (FISH). It provides background on both techniques, limitations of karyotyping, principles and applications of FISH including for detecting structural abnormalities. Details are given on Edward syndrome including symptoms, diagnosis, genetics, tests used and epidemiology. Limitations of both techniques and differential diagnosis are also summarized.

1. DETECTION OF EDWARD SYNDROME BY
KARYOTYPING AND FLUORESCENCE IN SITU HYBRIDISATION
By
M. Jerry Angel
SATHYABAMA UNIVERSITY
Submitted to
Department of Human Genetics
Faculty of Biomedical Science, Research and Technology
Sri Ramachandra University
Porur, Chennai-600116

2. BONA- FIDE CERTIFICATE
This is to certify that this short term project “Detection of Edward syndrome by Karyotyping
and Fluorescence in situ Hybridisation” is the bona-fide work carried out by Ms. M. Jerry
Angel, under my guidance during January 2015
Supervisor Professor and Head
Ms. Teena Koshy
Lecturer
Dept. of Human Genetics
Sri Ramachandra Medical University
Dr. Solomon Paul
Dept. of Human Genetics
Sri Ramachandra Medical University

3. Chapter
No.
INDEX Page
No.
1. INTRODUCTION
1.1 Karyotyping
1.2 Fluorescent In Situ Hybridization
1.3 Edward syndrome
1.3.1 Symptoms
1.32 Diagnosis
1.3.3 Treatment
2. PROCEDURE
2.1 Fluorescence In Situ Hybridization
2.2 Preparation of metaphase chromosomes from peripheral blood
2.3 Giemsa banding for karyotyping
3. RESULTS
4. DISCUSSION
5. REFERENCES

4. INTRODUCTION
Conventional cytogenetic testing or routine chromosome analysis, sometimes referred to as
karyotyping, is provided for a variety of clinical applications. These types of studies are utilized
to detect numerical and/or structural chromosome abnormalities in metaphase cells. Banding
karyotype is a routine technique used for the identification of numerous aneusomoy and/or
aneuploidy in congenital diseases and cancers. However this fails to detect simple or complex
chromosome rearrangements. (Belaud-Rotureau et al; 2003)
Limitations of karyotyping

5. Depending on the application (i.e., high-resolution vs. cancer studies), detection of structural
chromosome changes, resulting in a loss or gain of genetic material by these methods, has been
estimated to be limited to those of approximately 4 - 6 mb in size.
These types of studies do not rule-out other forms of genetic abnormalities, such as
submicroscopic or molecular defects (i.e., gene mutations), uniparental disomies or subtelomeric
rearrangements. Additionally, the detection of low-level or tissue-specific mosaicisms is limited.
Molecular cytogenetics technique like Fluorescent In Situ Hybridization can be used to
overcome these limitations.
Fluorescent In Situ Hybridization (FISH)
FISH (Fluorescent in situ hybridization) is a cytogenetic technique that can be used to detect and
localize the presence or absence of specific DNA sequences on chromosomes. It uses
fluorescent probes that bind to only those parts of the chromosome with which they show a high
degree of sequence similarity. FISH is often used for finding specific features in DNA. These
features can be used in genetic counselling, medicine, and species identification.
The earliest histochemistry techniques consisted of the use of different sorts of natural and
synthetic dyes to stain cellular structures and sub-cellular accumulations. These compounds were
generally non-specific. The ability to detect specific molecular identities was first demonstrated
using antigen-antibody interactions. The earliest in situ hybridizations, performed in the late
1960s, were not fluorescent at all, but rather utilized probes labeled with radioisotopes. (Jeffrey.
M. Levsky et al., 2003). The first application of fluorescent in situ detection came in 1980, when
RNA that was directly labeled on the 3′ end with fluorophore was used as a probe for specific
DNA sequences (Bauman et al., 1980).Principle of FISH
FISH is based on DNA probes annealing to specific target sequence of sample DNA. Attached to
the probes are fluorescent reporter molecules which under fluorescence microscopy confirm the
presence or absence of a particular genetic aberration when viewed under fluorescence
microscopy. The main advantage of FISH over other in situ hybridization methods is mainly due
to improved speed and spatial resolution. A particular advantage of FISH technique is the
possibility to study chromosomal aberrations in non-dividing cells, which is useful for the

6. visualization of chromosomal aberrations in cytological preparations and tissue sections. (Clare
O’ Connor et al; 2008)
Fig 1: Principle of FISH
The microscope setup
The light is first directed through the excitation filter. This filter can be mounted directly in front
of the light source or within a filter cube as illustrated here. The beam splitter is an additional
filter and like a mirror directs the light on to the specimen. The fluorescent light emitted from the
specimen is then directed through the beam splitter and will be even more selective after passing
the emission filter. The fluorescent signal is then analyzed by eye at the microscope or recorded
by a camera.

7. Fig 2: Setup of a fluorescent microscope
Applications of FISH in clinical genetics
FISH can be used to test chromosomal abnormalities that are difficult or impossible to recognise
using G banding or HR banding. It can be applied to detect genetic abnormalities such as
characteristic gene fusions, aneuploidy, loss of a chromosomal region or whole chromosome or
to monitor the progression of an aberration serving as a technique that can help in both diagnosis
of a genetic disease or suggesting prognostic outcomes. For chromosome testing on embryos,
FISH is the only option because there is not enough time to culture the cells. The diagnosis
therefore has to be made from one or at the most two cells. (Ryan Bishop et al; 2010)
FISH using interphase chromosomes
FISH in interphase is used to identify numerical chromosomal abnormalities. The probes are
designed so that they bind to (large portions of) DNA in interphase. We can also simultaneously
use multiple different probes with different colours to visualise two or more different
chromosomes. This allows us not only to determine the gender (XX or XY) but also to see
whether there is an incorrect number of specific chromosomes present. FISH can be used to
detect abnormalities in chromosome 21 as a diagnosis of Down’s syndrome. In this case
microscopy reveals three copies of chromosome 21 fluorescing in each cell. Charcot-Marie-
Tooth disease (CMT) type 1A is a relatively common neurological condition caused by

8. duplication in a gene on chromosome 17 that encodes one of the proteins in the myelin sheath
that surrounds nerve axons. FISH can be used to detect the duplication in chromosome
17. Because interphase chromatin is about 10,000 times less compacted than mitotic chromatin,
it is possible to resolve the duplicated regions as discrete points. This small duplication would
have been difficult to resolve in mitotic chromosomes. (Svetlana G Vorsanova et al; 2010)
FISH using metaphase chromosomes
A probe can be designed so that it binds specifically to a (small) region of a single chromosome.
This technique can be used to look for structural abnormalities such as deletions, duplications or
translocations of a specific region in a specific chromosome in metaphase. For example, for
patients who have DiGeorge syndrome, in which 22q11 deletions are characteristically found
this technique can be used. On the other hand to detect translocations, the two colours used for
two different chromosomes will appear to be separate (in the case of normal chromosomes) or
close to each other (in the case of a chromosome with a translocation).
FISH can also be used for the identification of novel oncogenes or genetic aberrations that
contribute towards various cancers. The power of its ability to identify specific genetic
aberrations has propelled FISH-based techniques to the forefront of screening procedures for
prenatal, paediatric and adult cases in a wide variety of cell types, including paraffin-embedded
tissue, making FISH analysis data a useful tool in the decision of therapy to combat cancer. The
applications of FISH are not limited to gene mapping or the study of genetic rearrangements in
human diseases. Indeed, FISH is increasingly used to explore the genome organization in various
organisms and extends to the study of animal and plant biology.
A major advantage of this technique resides in its ability to provide an intermediate degree of
resolution between DNA analysis and chromosomal investigations, while also maintaining the
cell structure.
Limitations of FISH
FISH can only detect deletions or duplications of regions specifically targeted by the probe used
and which are larger than the probe used. It is possible that rare very small deletions may not be
detected by FISH.

9. Edwards Syndrome
Trisomy 18 is named after John Hilton Edwards, who first described the syndrome in 1960. It
is the second-most common autosomal trisomy,
This chromosomal disorder caused by the presence of all, or part of, an extra 18th chromosome.
This genetic condition almost always results from non disjunction during meiosis
OCCURANCE: Occurs in around one in 6000 live births, and around 80% of those affected are
female.
SIGNS and SYMPTOMS:
• Kidney Malformations,
• Structural heart defects at birth – ventricular septal defect, artrial septal defect, patent
ductus arteriosus,
• The majority of fetuses with this syndrome die before birth. The incidence increases as
mother’s age increases
• arteriosus
• Growth deficiency and developmental delay,
• Intestines protruding outside the body ( omphalocele ),
• Esophageal atresia,
• Intellectual difficulties,
• Breathing difficulties,
• Anthrogryposis ( A muscle disorder that causes multiple joint contractures at birth),

10. • Physical malformation – Microcephaly ( small need) ,
>Occiput ( accompanied by a prominent back portion of the head),
 Micrognathia ( small Jaw),
 Palpebral fissures (narrow eyelid folds),
 Ocular hypertelorism (widely spaced eyes),
 Ptosis (drooping of the upper eyelid),
 Webbing of 2nd
and 3rd
toes. ( clubfoot / rocker bottom feet),
 Choroid plexus cysts ; radial limb defects cryptorchidism. Hypertonia, exomphalos
( pockets of fluid on brain),
 In male – undescended testicles.
GENETICS:
Trisomy 18 ( extra copy of whole chromosome ) – ( 47, XX, + 18)
NON DISJUNCTION: Numerical errors arise at either of two meiotic divisions ( in 23 pairs)
and cause the failure of a chromosome to segregate into the daughter cells.
This result in extra chromosome ( making the haploid no.24 rather 23)
Types
• Regular Edward's S.: 47XX, +18
• Mosaic type: 46XY / 47XY +18(Mosaics: are individuals who have a mixture of cells in their
body of different proportions of two or more different karyotpyes .)
Situations where analysis is strongly recommended
 Problems with early growth & development

11.  Fertility problems
 Neoplasia
 Pregnancy in older women
TESTS: ( Pre-Natal Diagnosis)
• Fetoscopy• Amniocentesis
• Chorionic villus sampling• Fetal blood cells in maternal blood
• Maternal serum (alpha-fetoprotein,• Inhibin-A
beta-HCG, and estriol)• Gross Examination
• Karyotyping
• Radiography
•FISH (on fresh tissue or paraffin blocks)• Microscopic Examination
• Flow Cytometry
• Microbiologic Culture & Serology
EPIDEMIOLOGY:
Freguency in U.S :
1. Mortality/Morbidity: Increase death rate is due to the presence of carbide and renal
malformations, feeding difficulties, sepsis, apnea and CNS defects.
 Severe psychomotor and growth retardation are invariably present in those who
survive beyond infancy (baby hood).
 Long term survival upto 27years has been reported.
2. SEX: ~ 80% of trisomy occur in females.
3. AGE: It is detectable during prenatal and newborn periods.
Fig 2: Characteristic features of Edward syndrome

12. DIFFERENTIAL DIAGNOSIS
“Pseudo-trisomy 18 syndrome” was formerly diagnosed in infants with some signs of Edwards
syndrome, such as prominent occiput, dysmorphic pinnae, blepharophimosis, distal limb
contractures, and profound developmental retardation, who had a normal karyotype. Pseudo-
trisomy 18 is practically never diagnosed nowadays, but syndrome delineation in such infants
remains difficult.
EPIDOMIOLOGY
Frequency
United States
• Prevalence is approximately 1 in 6,000-8,000 live births.

13. • At the time of first trimester screening, the incidence of trisomy 18 is 1 in 400, but due to
high spontaneous loss, the birth prevalence is 1 in 6,500.
Mortality/Morbidity
• Approximately 95% of conceptuses with trisomy 18 die as embryos or fetuses; 5-10% of
affected children survive beyond the first year of life.
• For liveborn infants with trisomy 18, the estimated probability of survival to age 1 month
was 38.6% and to age 1 year was 8.4%. Median survival time was 14.5 days (population
based study).
• The high mortality rate is usually due to the presence of cardiac and renal malformations,
feeding difficulties, sepsis, and apnea caused by CNS defects.
• Severe psychomotor and growth retardation are invariably present in those who survive
beyond infancy.
• Long-term survival up to age 27 years has been reported.
Race
• Trisomy 18 has no racial predilection.
Sex
• Approximately 80% of cases occur in females.
• The preponderance of females with trisomy 18 among liveborn infants (sex ratio, 0.63)
compared with fetuses with prenatal diagnoses (sex ratio, 0.90) indicates a prenatal
selection against males with trisomy 18 after the time of amniocentesis.
Age
• Trisomy 18 is detectable during the prenatal and newborn periods.
MOSAIC TRISOMY 18

14. The phenotypes displayed by cases of trisomy 18 mosaicism ranges from full trisomy 18
syndrome through a milder, nonspecific, dysmorphic phenotype often but not always associated
with growth deficiency to a normal phenotype in cases ascertained serendipitously.
CYTOGENETICS OF TRISOMY 18
Edwards syndrome is almost due to three copies of chromosome 18, and like trisomy 21, trisomy
18 is a maternal age-related autosomal trisomy with the rate of prenatal diagnosis or birth rising
until 43 years and then leveling off. Studies employing polymorphic DNA markers indicated that
the extra chromosome in trisomy 18 is usually of maternal origin; however, in contrast to trisomy
21, wherein maternal meiosis I errors are most frequent, maternal meiosis II errors predominate
ion trisomy 18. chromosome specific factors complicate the simple model of susceptible chiasma
distributions interacting with age-dependent deterioration of the meiotic mechanism.
Partial Trisomy 18 by translocation
Trisomy 18 non disjunction

15. LABORATORY STUDIES
• Hematological studies in patients with trisomy 18 during the first week of life
o Thrombocytopenia: This is the most common hematological abnormality
detected, occurring in 83% of those with trisomy 18; some patients need platelet
transfusions.
o Neutropenia: This is the second most commonly detected abnormality. Neutrophil
concentrations exceeding the reference range for age were reported in 42% of
patients with trisomy 18.
o Abnormal erythrocyte values: This is the third most common hematological
abnormality detected. Only 43% of patients with trisomy 18 had normal
erythrocyte values; anemia was detected in 40%, and polycythemia was detected
in 17%.
• Conventional cytogenetic studies
o Full trisomy 18 (about 95% of cases)
o Trisomy 18 mosaicism (about 5% of cases)
o Translocation type trisomy 18 syndrome (very rare)
IMAGING STUDIES
• Echocardiography is indicated for cardiac anomalies: In a study from Lin et al, the
anomalies identified included ventricular septal defect (94%), patent ductus arteriosus
(77%), atrial septal defect (68%), and complex congenital heart defects (32%).
• A barium swallow is indicated for GI anomalies.
• In Lin et al's study, using brain ultrasonography, the most common brain lesion revealed
was cerebellar hypoplasia (32%), followed by brain edema (29%), enlarged cisterna
magna (26%), and choroid plexus cysts (19%).[12]
Ultrasonography is also indicated for
genitourinary anomalies.

16. • Skeletal radiography is used to discern phocomelia, absent radius, tight flexion of the
fingers with the second over the third and the fifth over the fourth, talipes equinovarus,
short sternum, hemivertebrae, fused vertebrae, short neck, scoliosis, rib anomaly, and
dislocated hip.
MEDICAL CARE
• Medical care in trisomy 18 is supportive.
• Treat infections as appropriate. These are usually secondary to otitis media, upper
respiratory tract infections (eg, bronchitis, pneumonia), and urinary tract infection.
• Sepsis is a continuous concern.
• Provide nasogastric and gastrostomy supplementation for feeding problems.
• Orthopedic management of scoliosis may be needed secondary to hemivertebrae.
• Cardiac management is primarily medical. Most of these children require a diuretic and
digoxin for congestive heart failure. Optional treatment for cardiac lesions includes the
following:
o Intensive cardiac management with pharmacological intervention for ductal
patency (indomethacin and/or mefenamic acid for closure, and prostaglandin E1
for maintenance) and palliative and corrective cardiac surgery was demonstrated
to improve survival in patients with trisomy 18.
o In a study of patients with trisomy 18 who had cardiac lesions, 82% of patients
undergoing heart surgery were discharged home with alleviated cardiac
symptoms; congenital heart defect–related death occurred in only one patient,
suggesting that cardiac surgery is effective in preventing congenital heart defect–
related death; and initial palliative surgery was associated with longer survival
than intracardiac repair.
• Neonatal intensive care management

17. o Management of neonates with trisomy 18 is controversial, supposedly because of
the prognosis and the lack of precise clinical information concerning efficacy of
treatment.
o Improved survival (survival rate at age 1 wk, 1 mo, and 1 y was 88%, 83%, and
25%, respectively; median survival time was 152.5 d) through neonatal intensive
treatment such as cesarean delivery, resuscitation, respiratory support, and
surgical procedures are helpful for clinicians to offer the best information on
treatment options to families of patients with trisomy 18.
• Genetic counseling
o Recurrence risk is 1% or less for full trisomy 18. If a parent is a balanced carrier
of a structural rearrangement, the risk is substantially high.
o The risk should be assessed based on the type of structural rearrangement and its
segregation pattern.
o The wide phenotypic variation and lack of correlation with the percentage of
trisomic cells in mosaic trisomy 18 makes informative prediction of natural
history and genetic counseling challenging.
• Psychosocial management
o During the neonatal period, issues of diagnosis and survival are paramount.
Parents need information about the syndrome, including its cause, implications,
and possible outcomes.
o Support services within the hospital and in the community should be made
available to the family.
o The presence of a disabled child in any family is a source of stress and anxiety.
o Families also undergo a complex grieving process that combines both the reactive
grief predominant in chronic illness and the preparatory grief associated with
impending death.

18. TRISOMY 18 RECURRENCE RISK
Recurrence of trisomy 18 is exceptionally rare, but over 40 years, Hecht and colleagues
suggested increased risk of Down Syndrome in families in which the index case had trisomy 18.
recurrence of heterotrisomy cannot be accounted for by gonadal mosaicism in one parent. If the
Edwards syndrome phenotype results from a chromosome 18 balanced structural rearrangement
present in one parent, the risk of recurrence will almost always be significantly higher, but a
more precise risk estimate will depend on the type of rearrangement and the pattern of its
segregation in the extended family tree, if this is known.
In general, the likelihood of having a second child with Trisomy 18 is low. Studies suggest that
the probability is 1% or less. However, as women age, this figure may increase slightly. This is
because we know that older women are more likely to have a baby with a chromosome change
than younger women.
CASE STUDY

19. A new born baby was referred for genetic evaluation because of dysmorphic
features which included cleft palate, clenched hands, rocker bottom feet and
congenital heart defects.
Karyotyping of the baby’s peripheral blood sample and a control sample was
performed

20. TECHNIQUES

21. The techniques adopted to detect Edward syndrome are either karyotyping or fluorescence in situ
hybridization (FISH) or both.
KARYOTYPING:
Routine chromosome analysis refers to analysis of metaphase chromosomes which have been
banded using trypsin followed by Giemsa,. This creates unique banding patterns on the
chromosomes. The molecular mechanism and reason for these patterns is unknown, although it
likely related to replication timing and chromatin packing.
SLIDE PREPARATION:
Metaphase cells are required to prepare a standard karyotype, and virtually any population of
dividing cells could be used. Blood is easily the most frequently sampled tissue, but at times,
karyotypes are prepared from bone marrow, blood, amniotic fluid, cord blood, tumor, and tissues
(including skin, umbilical cord, liver, and many other organs) can be cultured using standard cell
culture techniques in order to increase their number.
A mitotic inhibitor (colchicine, colcemid) is then added to the culture. This stops cell division at
mitosis which allows an increased yield of mitotic cells for analysis. The cells are then
centrifuged and media and mitotic inhibitor is removed, and replaced with a hypotonic solution.
This causes the cells to swell so that the chromosomes will spread when added to a slide. After
the cells have been allowed to sit in hypotonic, Carnoy's fixative (3:1 methanol to glacial acetic
acid) is added. This kills the cells, lyses the red blood cells, and hardens the nuclei of the
remaining white blood cells. The cells are generally fixed repeatedly to remove any debris or
remaining red blood cells. The cell suspension is then dropped onto specimen slides. After aging
the slides in an oven or waiting a few days they are ready for banding and analysis.

22. ANALYSIS
Analysis of banded chromosomes is done at a microscope by a clinical laboratory specialist in
cytogenetics. Generally 20 cells are analyzed which is enough to rule out mosaicism to an
acceptable level. The results are summarized and then given out reported in an International
System for Human Cytogenetic Nomenclature 2005 (ISCN2005
A karyotype is the characteristic chromosome complement of a eukaryote species. The
preparation and study of karyotypes is part of cytogenetics
In normal diploid organisms, autosomal chromosomes are present in two identical copies. There
may, or may not, be sex chromosomes. Polyploid cells have multiple copies of chromosomes and
haploid cells have single copies. The study of whole sets of chromosomes is sometimes known
as karyology. The chromosomes are depicted (by rearranging a microphotograph) in a standard
format known as a karyogram or idiogram: in pairs, ordered by size and position of centromere
for chromosomes of the same size
The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex
chromosomes. Normal karyotypes for women contain two X chromosomes and are denoted
46,XX; men have both an X and a Y chromosome denoted 46,XY.
Each chromosome has two "arms," or sections. The top section is called the "p" arm, because it
is the small arm, and the bottom section is called the "q" arm, because q follows p in the alphabet
and the scientists who chose these names were apparently not very creative. The structure that
separates the two arms is called the centromere.
Standard nomenclature been developed for human chromosomes:
ISCN( International system for cytogenetic nomenclature)
• Total number of chromosomes
• Sex chromosome constitution
• Description of abnormality i.e. 46,XX (normal female ) 46,XY ( normal male)

23. Human chromosomes are divided into 7 groups
• Group A 1-3 Large metacentric 1,2 or submetacentric
• Group B 4,5 Large submetacentric, all similar
• Group C 6-12, X Medium sized, submetacentric -
• Group D 13-15 medium-sized acrocentric plus satellites
• Group E 16-18 short metacentric 16 or submetacentric 17,18
• Group F 19-20 Short metacentric
• Group 21, 22, Y Short acrocentric with satellites. Y no satellites.
Given below are karyotypes of a normal male and a normal female.
Images courtesy : Department of Human Genetics, Sri Ramachandra University
Normal Female: 46,XX
Normal Male: 46,XY

24. FLUORESCENT IN SITU HYBRIDIZATION:
FISH testing, or FISH analysis as it is sometimes referred to, is a relatively new cytogenetic
technique that allows a cytogeneticist to determine how many copies of a particular chromosome
are present without having to go through all of the steps involved in producing a karyotype. For
example, FISH analysis can quickly tell you how many number 21 chromosomes are present, but
it cannot tell you anything about the structure of those chromosomes
FISH testing is usually done on the same samples as a karyotype - blood, amniocytes or a
chorionic villi sample. A FISH test is done using a fluorescent probe that binds to certain specific
chromosomes. These fluorescent probes are made of DNA specific to certain chromosomes and
are tagged with fluorescent dye. The cells used in FISH analysis don’t have to be grown or
cultured (which can take 7 to 10 days), so the results of a FISH analysis are available much faster
than the results of a karyotype.
Typically, a sample is obtained and sent to the laboratory and the chromosomes are isolated on a
slide. The probes are then placed on the slide and allowed to hybridize (or find their match) for
about 12 hours. Because the probes are made of DNA, they will bind to the “matching” DNA of
their specific chromosome. For example, a probe made of DNA specific to chromosome 21 will
bind to any number 21 chromosome that is present.
After hybridization (or sticking), the slide is examined under a special microscope that can see
fluorescent images. By counting the number of fluorescent signals, a cytogeneticist can
determine how many of a specific chromosomes are present. For example, a person without
Down syndrome will have two fluorescent signals corresponding to their two number 21
chromosomes. A person with trisomy 21 will have three fluorescent signals corresponding to
their three number 21 chromosomes. Typically, a cytogeneticists will use probes for the 13, 18,
21, X an Y chromosomes. These are the chromosomes that can result in trisomies for humans.

25. Although it doesn’t look at the actual structure of the chromosomes analyzed, FISH analysis can
tell you how many copies of a particular chromosome are present. In Down syndrome, the
cytogeneticist uses probes for the number 21 chromosome. If there are three fluorescent signals
seen under the microscope, then the diagnosis of Down syndrome is made.
The main advantages to FISH analysis is that it can provide information about certain
chromosomes quickly. For example, in three to four days, it can tell how many copies of a
number 21 chromosome a particular person may have. In contrast, a traditional karyotype can
take up to two weeks.
The main disadvantage of FISH analysis compared to karyotyping is that FISH analysis gives
you less information about all of the chromosomes being studied. For example, a typical prenatal
FISH test will tell you how many number 13, 18, 21, X and Y chromosomes are present (i.e.,
whether there are two copies or three) but will not give you any information about any of the
other chromosomes or any information about the actual structure of chromosomes
IMAGE ANALYSIS:
Metaphase finding is a fully automated scanning application customized for high throughout
processing of karyotypes & metaphases. Cytovisions©
customized imaging modular are
incorporated into one software applications thereby rendering it unnecessary to convert or
export cells found during the scan in order to karyotype or analyze them.
Cytovision’s unique method of scanning and applying classifiers provides a powerful method to
select cell populations. They can be applied before a scan to provide the criteria for automatic
high-power capture. They can also be applied after a scan to change the type of cell under study.

26. GENERAL FEATURES:
 Fully automated, unattended, scanning – to – capture workflow, scan settings specify the
number of types of cells for capture and the result is cells ready for karyotyping or
analysis.
 Fast scanning of slides for high throughput workflow.
 Easy to use inference guides you through scanning and capture set up.
 Accurate user- defined scanning classifiers to find different types of cells based on
morphology easily created in minutes.
Figure 3 showing an Image Analyzer

27. MATERIALS

28. MATERIALS REQUIRED FOR KARYOTYPING:
1. RPMI 1640 – culture medium. Commercially available
2. FBS (Fetal bovine serum). Commercially available
3. PHA (Phytohaemogglutinin). Commercially available
4. Ethidium bromide Concentration: 1mg/ml
5. Colchicine Concentration: 1mg/ml
6. Potassium chloride Concentration: 0.56g of KCl and 100ml of distilled water
7. Carnoy’s fixative Methanol: Acetic acid : 3:1.
8. Trypsin Concentration8mg/50ml of PBS
9. Giemsa stain :5ml of giemsa stain + 43ml of water
MATERIALS REQUIRED FOR FISH:
1. Commercially available probes
2. 20X SSC
3. DAPI
INSTRUMENTATION:
1. Water jacketed carbon dioxide incubator.
2. Centrifuge.
3. Laminar air flow.
4. Phase contrast Microscope.
5. Hybridisation chamber or hybrite
6. Image analyzer.
METHODOLOGY

29. CULTURE INITIATION:
• Prepare culture vials by placing the 8ml of RPMI 1640, 2ml FBS, 400µl of PHA and 1ml of
blood sample in each vial.
• Mix the contents of each culture tube gentle and incubate the cultures for 72 hrs at 37 c, 5%
CO2
• EtBr is added at 66 1/2
hr.
• Colchicine is added at 67 hr which stops the cell division at metaphase.
• Harvest the culture after 72 hrs.
HARVESTING:
• It is then followed by the hypotonic treatment for 20 minutes.
• Then fixation overnight at 4 c will strengthen the cells.
• Multiple fixations will strengthen the cell membrane and improve the chromosomal
morphology.
• Then centrifuge the tubes with the fixative and collect the pellet, repeat the steps till the
pellet becomes white without any RBCs.
• After final centrifugation suspend the pellet in small volume of fixative.
SLIDE PREPARATION:
Drop 2 or 3 drops of the cell suspension from a height of 30cm on chilled slides that an even thin
film of water.
Label the slides and allow it to dry.
STAINING:
• Trypsin treatment: 8mg in 50ml of PBS. Agitate for approx 20 sec
• Washing : wash with PBS followed by distilled water
• Giesma staining : 5ml of giemsa + 45 dw 2 – 5 minutes
• wash with distilled water and air dry

30. METAPHASE SCREENING:
• The G-banding slides are scanned for metaphases under 10x.
• The metaphases are analyzed in detail under 100x oil immersion objective.
• A minimum of 20 – 30 banded metaphases are captured using image analysis through CCD
camera.
• The metaphases are karyotyped using software cytovision.
FLUORESCENCE IN SITU HYBRIDISATION
• Slides were prepared by concentrating and dropping the cells from a height of approx 10
cm onto prechilled clean slides.
• the slides were kept on a slide warmer maintained at a temperature of 40 c for drying
• slides were checked under a phase contrast microscope for cell concentration, height and
drying optimization.
• area with maximum number of cytoplasm free cells was marked using a glass marker
• The hydridization probe - 10µl was added to the slide, and sealed with the rubber solution
• The denaturation step is set for 730
c for 5 minutes, followed by hybridization for 370
c for
16 – 24 hrs.
• It is then carried on for the washing technique, wherein two buffers are utilized :
 0.4x SSC /0.3% np 40 at 730
c for 2min.
 2x SSC/0.1% np 40 at 370
c for 2min
• The counter stain DAPI is then added ( 10µl ) and then sealed with nail enamel.
It is stored for a while and then viewed under fluorescence light microscope using
appropriate filters

31. RESULTS

32. RESULTS OF KARYOTYPING:
25 metaphases analyzed in the control case showed no numerical or structural abnormalities and
was assigned a normal female karyotype designated as 46,XX according to the ISCN. The
karyotype image is presented in Fig 4.
25 metaphases analyzed in the case study showed a numerical abnormality and was assigned a
Jacob syndrome karyotype designated as 47,XX,+18 according to the ISCN. The karyotype
image is presented in Fig 5.
RESULTS OF FISH:
50 metaphases and 50 interphase cells were screened for the18, X and Y chromosomes. In the
control sample all the cells analyzed showed two red signals implying the presence of two X
chromosomes in all the cells and two aqua signals indicating the presence of two copies of
chromosome 18 while in the case study sample two red signals and three aqua signals was
observed in all the cells implying Trisomy 18. A representative FISH image for the control
sample is presented in Fig 6 and for the case study is presented in Fig 7

33. Fig 2 (a): A raw metaphase plate
Fig 2 (b): My karyotype 46,XX

34. Fig 3 (a): A raw metaphase plate of Edward syndrome
Fig 3 (b): A karyotype of Edward syndrome 47,XX,+18

35. Fig 4: FISH image showing presence of one X(red) and one Y chromosome (green)
Fig 5: FISH image showing presence of three chromosome 18s indicated by the red arrows

36. DISCUSSION

37. Many approaches aimed to gain a better knowledge of chromosomal structure, rearrangements,
identification of the chromosomes were developed: autoradiography, banding techniques,
electronic microscopy. Since 1980, new developments in clinical cytogenetic and molecular
biology have occurred. FISH is a relatively new cytogenetic technique that allows a
cytogeneticist to determine how many copies of a particular chromosome are present without
having to go through all the steps involved in producing a karyotype.
The karyotyping of metaphase chromosomes takes about a week whereas FISH using interphase
chromosomes can be done within two days. Moreover cells of eukaryotes are more likely to be in
interphase. The duration it takes to do FISH and the less time to obtain the results has been very
useful in prenatal diagnosis and cancer diagnosis. Molecular and cytogenetic approaches are
routinely used for diagnostic and prognostic purposes and give accurate results. However, care
should be taken to consider the limitations of these approaches.
Based on the results of karyotyping and FISH it can be confirmed that the patient with female
phenotype has a male genotype and has Swyers syndrome. The treatment of Swyer syndrome
may require the coordinated efforts of a team of specialists. Pediatricians, pediatric
endocrinologists, geneticists, urologists or gynecologists, psychologists or psychiatrists, social
workers and other healthcare professionals may need to systematically and comprehensively plan
an affect child’s treatment. Genetic counseling may be of benefit for affected individuals and
their families.
In conclusion, cytogenetic analysis using both karyotyping and FISH either in conjungion
or individually provides important diagnostic and prognostic in,formation for patients.
.

38. REFERENCES

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