Practical 5

Chromosomal analysis

Karyotyping

Cytogenetic examination

• Karyotyping

• Analysis of chromosomal abnormalities

– Numerical

• Changes in chromosomal number

– Structural

• Changes in chromosomal structure

• Importance

– Diagnostics of chromosomally conditioned syndromes

– Examination of risk for the offspring

– Diagnostics of tumors associated with chromosomal

abnormalities

Cytogenetic examinations

• Prenatal

– Examinations of the fetus

• Postnatal

– Examination of an individual after the birth

Written test

• 10 minutes

• Don't forget to put down your name, your

group and the test version.

• In multiple choice questions more than 1

statement could be correct.

• Don't write anything on the question sheet!

Cytogenetic examinations

• Prenatal

– Examinations of the fetus

• Postnatal

– Examination of an individual after the birth

fetus

chorion

amnion

umbilical cord

Chorionic villi sampling (CVS)

Amniocentesis

Cordocentesis

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.End of prenatal examinations

Weeks of pregnancy

Prenatal examinations

Chorionic villi

Chorionic villi sampling

since 11th week of pregnancy

Transabdominal CVS Transvaginal (transcervical) CVS

– biopsy from the placenta and examining the baby's chromosomes

Chorionic villi sampling

Tissue of chorionic villi

Examination under the stereomicroscope

Schedule of prenatal examinations

Chorionic villi sampling

Amniocentesis

Cordocentesis

11. 16. 20. Pregnancy (weeks)

12th – 20th weekUltrasound

Biochemical test in 16th weekAFP, hCG, E3, PAPP

24.

End of prenatal examinations

Ultrasound examinationis offered to all pregnant women

An instrument called a transducer emits sound waves that bounce or echo off internal organs.This information is relayed to a computer, which produces an image on a nearby screen.

Ultrasound

• ultrasound scan, sonogram, or

ultrasonography.

• A diagnostic or screening procedure that

uses high-frequency sound waves to

create a picture of internal body structures,

such as a developing fetus.

Ultrasound examination

Nuchal translucency

normal increased

The fetus may be affected with the Down syndrome

•on ultrasound it appears as a black space beneath the fetal skin.

•this black space that you will see measured during the ultrasound scan

between 11-14 weeks of pregnancy

Nuchal Translucency• collection of fluid beneath the fetal skin in the

region of the fetal neck and is present in all fetuses in early pregnancy.

• The fluid collection is increased in many fetuses with Down syndrome and many other chromosomal abnormalities.

• normally less than 2.5mm and when increased (i.e.>2.5mm) may indicate the baby has Down syndrome or another chromosomal abnormality.

• If the nuchal translucency is increased then pregnant woman will be offered chorionic villus sampling or amniocentesis.

Biochemical testTriple screen or quad screen

• a set of tests, which screen for genetic problems

• The test determines, and also measures the levels of: – alpha-fetoprotein (AFP) – estriol (E3)– human chorionic gonadotropin (hCG)– inhibin A (for the quad screen)

• This test is offered to all pregnant women.

Maternal Serum Alpha-Fetoprotein (MSAFP)

• Alpha-fetoprotein (AFP) is a protein that is produced by the fetus' liver.

• Between weeks 15 and 20 of a pregnancy, a maternal serum alpha-fetoprotein (MSAFP) screen will be offered.

• The quantity of AFP that is considered normal depends upon many variables, including age, weight, race, and stage of pregnancy. Insulin-dependent diabetes also influences AFP levels. Of those women whose tests show high or low levels of AFP, only two or three in 100 will have a child with a birth defect.

• Up to 10% of results are positive, meaning you have high- or low-AFP levels. With a positive AFP, additional tests will be suggested to help determine the cause.

Amniocentesis

15th – 16th week of pregnancy

Amniocentesis

• can diagnose or rule out many possible birth defects.

• Most often, it's used to spot common genetic defects (such as Down syndrome) and neural tube defects.

• is usually performed at 15 to 18 weeks gestation, although it can be done as early as 11 or 12 weeks.

Amniocentesis

Amniocentesis is typically offered to women who

• Will be 35 or older when they give birth.

• Have a screening test or exam result that indicates a possible birth defect or other problem.

• Have had birth defects in previous pregnancies.

• Have a family history of genetic disorders.

Risk of amniocentesis• About one woman in every 200-400

women miscarry as a result of amniocentesis.

• Amniocentesis done during the first trimester carries a greater risk for miscarriage than amniocentesis done after the 15th week.

• Less than one woman in every 1,000 women develop a uterine infection after amniocentesis.

Cordocentesis

• percutaneous umbilical blood sampling (PUBS)

• umbilical vein sampling

• fetal blood sampling

since 20th week of pregnancy

Cordocentesis

• diagnostic procedure in which a doctor extracts a sample of fetal blood from the vein in the umbilical cord.

• The fetal blood can be analyzed to detect chromosomal defects or other abnormalities.

Cordocentesis – advantages and risks

• results are usually ready much faster than with amniocentesis. With cordocentesis, the results may be ready within 48 hours. With amniocentesis, results can take about two weeks.

• The miscarriage rate after cordocentesis is about 1 – 2%.

• As with amniocentesis, there is a risk of infection, cramping, and bleeding.

fetus

chorion

amnion

umbilical cord

Chorionic villi sampling (CVS)

Amniocentesis

Cordocentesis

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.End of all prenatal

examinations

Weeks of pregnancy

Prenatal examinations

For all of invasive prenatal samplings written consent of the

mother is necessary.

Cytogenetic examinations

• Prenatal

– Examinations of the fetus

• Postnatal

– Examination of an individual after the birth

Indications for postnatal chromosomal analysis

• Possible chromosomal abnormality

• Multiple anomalies or growth retardation

• Gonadal abnormalities

• Unexplained mental retardation

• Infertility or multiple miscarriages

• Death of a fetus, death of a newborn child

• Occurrence of tumors

Tissues for postnatal karyotyping

• Peripheral blood

• Skin fibroblasts

• Bone marrow

(leukemia)

• Tumor

• Autopsy material (in

case of a death of

patient)

How to take a blood sample for chromosomal analysis?

• Disinfect the site of

injection with alcohol

(not iodine solution)

• The blood should be

taken to heparin tube

(heparin prevents blood

clotting)

Cultivation of peripheral blood lymphocytes

• Add phytohemaglutinin to medium – highly

immunogenic compound - stimulates blood

lymphocytes proliferation

• At the end of cultivation – application of

colcemide (disrupts the mitotic spindle)

• Hypotonization

• Fixation

• Staining

Solid staining of chromosomes

We use only Giemsa-Romanowski solution

6 – 12

13 – 15

19 – 20 21 – 22

4 – 5

G-banding (GTG)Trypsin + Giemsa

Each G-band has concrete number:

Xq27.3

Chromosome X with G-bands

Another methods of chromosome staining

• R-banding (reverse bands – opposite to G-

bands)

• Q-banding (quinacrin banding)

• C-banding (staining of constitutive

heterochromatin

• Ag-NOR (staining of satellites in acrocentric

chromosomes)

HRT

• High resolution technique

• Special cultivation method that allows isolation of prometaphase chromsomes

• Very long chromosomes

• Identification of small rearrangements is possible

Tasks1. Arrange a karyotype (small box with

chromosome photos + table with chromosomes)

2. Observe human G-banded chromosomes (box with slides)

– Find the chromosomes or interphase nuclei on the slide using 10x objective lens.

– Change the objective magnification into 40x and observe chromosomes.

– Compare the picture in the microscope with adjacent photo.

Results will be controlled by Mrs. Tůmová, Dr. Diblík or Dr. Kočárek.

Description of a normal karyotype according to cytogenetic

nomenclature (ISCN 2005)

• Normal karyotype– Male … 46,XY– Female … 46,XX

Total number of all chromosomes, sex chromosomes

46,XY

Put the slides back to boxes, please.

Next practical

• Numerical chromosomal abnormalities

• No test!

See you next week!