Cytogenetic Techniques

Christeen RJ Pedurupillay

Cytogenetics

The study of the structure ,function, & evolution of chromosomes in a metaphase(usually)/ prophase of mitosis

What is Kayotype?
The characterization of the chromosomal complement of an individual or a species, including number, form, and size of the chromosomes

Cytogenetic Testing

Important for the diagnosis, treatment and prognostication

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

Chromosome Banding Techniques

Cytogenetic Banding Techniques:
G-banding (Giemsa) R-banding (Reverse) C-banding (Centomeric) Q-banding (Quinacrine)

Normal male 46,XY

Normal female 46,XX

Chromosomal Study Methods

Traditional karyotyping method (cytogenetics) Advanced methods of chromosomal analysis( molecular cytogenetics)

Blood

Bone Marrow

Sample Collection and Transport

Peripheral blood: 2-5ml Bone Marrow: 0.5-1.0ml Anticoagulant: Sodium Heparin (sterile vial) Transport: At room temperature (4C, do not freeze) Reach the lab within 3-4 hours (ASAP) Clinical details & suspected diagnosis Consent from the patient

Traditional Karyotyping Method

1. Culture setup 2. Arresting Cell Division at Metaphase 3. Harvesting 4. Slide Preparation 5. Capturing and Analyzing

Methods
Patients Sample

Bone Marrow 24 hrs Culturing

Peripheral Venous Blood 72 hrs culturing

Harvesting Slide preparation Chromosome Analysing & Karyotyping

Culturing

 Growth promoting culture medium

Contain Mitotic agents Nutrient and salts

Sterile laminar flow cabinet

CO2 cell culture incubator

Harvesting

 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

Slide Preparation

 Affected by humidity, Temperature & Air flow Ensure to use Clean Slides - Fixed cell suspension dropped on to slides - Drying of Slides ( Chromosome Aging)

Staining

Air Dried Slides Add- Tripsin Solution Rinse with Water Add Giemsa Solution Rinse with Water

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, more transcriptionally active, appear as light bands due to less condensed chromatin

Normal male 46,XY

Normal female 46,XX

Capturing

Applied Imaging Cytovision Automated Karyotyping System

Analysing

Numerical Abnormalities
Chromosome gain Chromosome loss Ploidy Levels

Structural Abnormalities

Chromosome gain
eg: Trisomy
should be seen in 2 spreads to be clonal

Chromosome loss
eg: Monosomy
should be seen in 3 spreads to be clonal

Ploidy Levels
eg: Hyperploidy, Hypoploidy

Structural chromosome abnormalities

eg: Translocations, Deletions, Inversions, Duplications

47,XY,+21

45,X

86, XXX, -X, -6,-7,-9,-10,-11,-13,-14,-16,-18,+19,+19,20,+22,+marker x 2

46,XX,del(5)(p13)

46,XX,dup(4p)

46,XX,t(14;21)

Disadvantages of Traditional GBanding Techniques

Cytogenetic abberation below the band resolution for visualization - Micro deletions - Cryptic translocation

Exchange of similar size and banding pattern cannot be distinguished

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

Molecular Cytogenetic Techniques

 Fluorescence in situ hybridization Multicolour fluorescent technologies - Spectral Karyotyping - Multiplex-fish (M-FISH) - Comparative genomic hybridization (CGH)

Analysis of Chromosomes and Genes

- Gene location - Micro deletions - Rearrangements - Amplification status