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Cytogenetic Techniques

Christeen RJ Pedurupillay

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Cytogenetics

The study of the structure ,function, &evolution of chromosomes in a

metaphase(usually)/ prophase of 

mitosis

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What is Kayotype?

The characterization of the

chromosomal complement of anindividual or a species, including

number, form, and size of the

chromosomes

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Important for thediagnosis, treatment and prognostication

Cytogenetic Testing

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Objective

To detect the numerical and structural

abnormalities in

� Children with Dysmorphic features

� Children with Congenital defects

� Couple with Recurrent Pregnancy loss

� Couples with subfertility

� Patients with Hematological Malignancies

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Chromosome Banding

Techniques

Cytogenetic Banding Techniques:

� G-banding (Giemsa)

� R-banding (Reverse)

� C-banding (Centomeric)

� Q-banding (Quinacrine)

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Normal male

46,XY

Normal femaleNormal female

46,XX46,XX

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Chromosomal Study Methods

� Traditional karyotyping method

(cytogenetics)

� Advanced methods of chromosomal

analysis( molecular cytogenetics)

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Blood

Bone Marrow

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Sample Collection and Transport �Peripheral blood: 2-5ml

�Bone Marrow: 0.5-1.0ml

�Anticoagulant: Sodium Heparin (sterile vial)

�Transport: At room temperature (4ºC, do not freeze)

�Reach the lab within 3-4 hours (ASAP)

�Clinical details & suspected diagnosis

�Consent from the patient 

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Traditional Karyotyping Method

1. Culture setup

2. Arresting Cell Division at Metaphase

3. Harvesting

4. Slide Preparation

5. Capturing and Analyzing

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Methods

Bone Marrow

24 hrs Culturing

Patient·s Sample

Harvesting

Slide preparation

Chromosome Analysing &

Karyotyping

Peripheral Venous Blood

72 hrs cult uring

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Culturing

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� Growth promoting culture medium

Contain  ² Mitotic agents

 ²  Nutrient and salts

� Sterile laminar flow cabinet 

� CO2 cell culture incubator

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Harvesting

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Mitosis arrest at metaphase ( add Colcimid)

Hypotonic treatment of the cells ( add KCl)

- Swells the cells

- Allow well spread of chromosomes

- Lyse RBCs

Fixation of cells ( methanol and acetic acid)- Stop cell division & Hypertonic Swelling

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Slide Preparation

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� Affected by humidity, Temperature & Air flow

� Ensure to use ² Clean Slides

- Fixed cell suspension dropped on to slides

- Drying of Slides ( Chromosome Aging)

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Staining

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Air Dried Slides

Add- Tripsin Solution

Rinse withWater

Add ² Giemsa Solution

Rinse withWater

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GTG Banding Pattern

� Giemsa stain following digestion of chromosome

with trypsin yields series of lightly and darkly

staining bands

� Heterochromatic regions: AT-rich DNA,

transcriptionally inactive, stain more darkly

� Euchromatic regions: GC-rich DNA, moretranscriptionally active, appear as light bands due

to less condensed chromatin

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Normal male

46,XY

Normal femaleNormal female

46,XX46,XX

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Capturing

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Applied Imaging Cytovision

Automated Karyotyping System

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Analysing

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Chromosome gain

Chromosome loss

Structural Abnormalities

Ploidy Levels

Numerical Abnormalities

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Chromosome gain

eg: Trisomyshould be seen in �2 spreads to be clonal 

Chromosome loss

eg: Monosomyshould be seen in � 3 spreads to be clonal 

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Structural chromosome abnormalities

eg: Translocations, Deletions, Inversions,

Duplications

Ploidy Levels

eg: Hyperploidy, Hypoploidy

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47,XY,+21

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45,X

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86, XXX, -X, -6,-7,-9,-10,-11,-13,-14,-16,-18,+19,+19,-

20,+22,+marker x 2

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46,XX,del(5)(p13)

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46,XX,dup(4p)

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46,XX,t(14;21)

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Disadvantages of Traditional G-

Banding Techniques

� Cytogenetic abberation below the band

resolution for visualization

- Micro deletions- Cryptic translocation

� Exchange of similar size and banding

pattern cannot be distinguished

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Why Culture Failures?

� Careless Handling

� Unsterile equipments, tubes, tips

� Personal Contamination (Lab Coat, Gloves)� Careless timings

� Environmental Conditions ( CO2, Humidity,

temperature)

� Contamination of the reagents

� Rarely reagents conditions & expairy

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Molecular Cytogenetic

Techniques

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� Fluorescence in situ hybridization

� Multicolour fluorescent technologies

- Spectral Karyotyping- Multiplex-fish (M-FISH)

- Comparative genomic hybridization

(CGH)

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Analysis of Chromosomes and Genes

- Gene location

- Micro ² deletions

- Rearrangements

- Amplification status

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