1. Students will be able to demonstrate a microtechnique for reliable chromosomal analysis of leucocytes obtained from peripheral blood.

2. Students will be able to prepare a karyotype from the chromosomes of a normal human male or female.

3. Students will be able to use the karyotyping techniques for diagnosing a chromosomal disorder.

Objectives:

INTRODUCTION

INTRODUCTION

Triploidy

XYY Syndrome: 47, XYY

Detecting Cancer

• A chromosome is divided by its centromer into short arm p (= petite or short) and long arm q (= queue, or long)

p

q

Chromosomes can be classified by the position of their centromer:

• Humans do not possess any telocentric chromosomes.

Chromosomes are arranged into seven groups

AA 1-3 Large metacentric

BB 4,5 Large submetacentric

CC 6-12, X Medium submetacentric

DD 13-15 medium acrocentricDD 13-15 medium acrocentric

EE 16-18 short metacentric

FF 19-20 Short metacentric

• Samples for chromosomal analysis can be prepared relatively easily using skin, bone morrow, chorionic villy or cells from amniotic fluid.

• It is estimated that one in 156 live births have some kind of chromosomal abnormality.

• To create a karyotype, chromosomes from a cell are arranged, stained and photographed.

• 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.

– Heparinzed whole blood– Heparin sodium injection– Peripheral blood Karyotyping medium with PHA (RPMI

1640)– Incubator 5% CO2 at 37 °C

– Colcemide solution 10g/ml– 0.075 M KCl– Fixative solution ( 3x methanol : 1x glacial acetic acid )– Giemsa stain solution– Slides and Microscope

Metirals

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.

Stains and Dyes

• Used to produce a pattern of bands specific to each type of chromosome

• One common method is G-banding– Treated with trypsin– Stained with Giemsa stain

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 37 oC for total of 72 hours.

3- After total of 69 hours from seeding add 100μl of Colcemid Solution to each culture tubes.

4- Incubate the tubes at 37 oC for an additional 20-30 minutes. 5- Spin at 500g (1500 rpm) for 7 minutes.6-Remove the supernatant and re-suspend the cells in 5ml of hypotonic

0.075M KCl prewormed to 37oC.

Karyotyping procedure

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-by- drop (with continous vortexing), 5ml of fresh, ice-cold fixative.

11- Leave at 4ºC for 20 minutes. 12- Repeat steps 9 and 10,until the supernatant is clear.13- Spine 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 ahigh of approximately 30cm onto aclean slide and blow carefully on the drops for spreading them on the slide.

16- Put the slides on a 45ºC heated plate for 2-4 minutes.17- Heat the slides to 60 ºC for overnight or to 90 ºC for 90 minutes.18- Place the slides and flood them with Giemsa stain solution for 8

minutes.19- Gently rinse the slides in distilled water and air dry. 20- Observe the chromsomes under microscope by using 10, 40 and

100x and photograph it and cut each chromosome from the photograph and arrange the chromosomes according to the size and position of centomer.

Staining procedure

Materials:1. Giemsa stain solution - 6ml Giemsa stain in

70ml ddH202. HBSS3. Trypsin X104. PBS without Ca, Mg

Method

1. Incubate the slides in Trypsin solution:2ml Trypsin x10 in 50ml of HBSS at room temperature.

2. After 2.5 min., neutralize the trypsin by immersing the slides in PBS with 5% FCS.

3. Rinse the slides in PBS.4. Place the slides horizontally and flood them with stain

solution for 2.5 minutes.5. Gently rinse the slides in ddH20 and air-dry.• Exact times of Trypsin treatment and staining should be

determined personally by each lab to get optimal results

Chromosomes are arranged by :

• Size.• Position of the centromere.• Specific banding patterns.