Practical 5

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Practical 5

• Chromosomal analysis
• Karyotyping

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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

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Cytogenetic examinations

• Prenatal
• Examinations of the fetus
• Postnatal
• Examination of an individual after the birth

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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!

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Cytogenetic examinations

• Prenatal
• Examinations of the fetus
• Postnatal
• Examination of an individual after the birth

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Prenatal examinations
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Chorionic villi
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Chorionic villi sampling
biopsy from the placenta and examining the
baby's chromosomes
Transabdominal CVS
Transvaginal (transcervical) CVS
since 11th week of pregnancy
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Chorionic villi sampling
Tissue of chorionic villi
Examination under the stereomicroscope
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Schedule of prenatal examinations
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Ultrasound examination
is 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.
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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.

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Ultrasound examination
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Nuchal translucency

• on ultrasound it appears as a black space beneath
  the fetal skin.
• this black space that you will see measured
  during the ultrasound scan

normal
increased
between 11-14 weeks of pregnancy
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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.gt2.5mm) 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.

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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.

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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.

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Amniocentesis
15th 16th week of pregnancy
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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.

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Amniocentesis
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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.

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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.

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Cordocentesis

• percutaneous umbilical blood sampling (PUBS)
• umbilical vein sampling
• fetal blood sampling

since 20th week of pregnancy
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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.

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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.

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Prenatal examinations
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For all of invasive prenatal samplings written
consent of the mother is necessary.
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Cytogenetic examinations

• Prenatal
• Examinations of the fetus
• Postnatal
• Examination of an individual after the birth

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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

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Tissues for postnatal karyotyping

• Peripheral blood
• Skin fibroblasts
• Bone marrow (leukemia)
• Tumor
• Autopsy material (in case of a death of patient)

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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)

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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

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Solid staining of chromosomes
We use only Giemsa-Romanowski solution
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G-banding (GTG)
Trypsin Giemsa
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Each G-band has concrete number
Chromosome X with G-bands
Xq27.3
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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)

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HRT

• High resolution technique
• Special cultivation method that allows isolation
  of prometaphase chromsomes
• Very long chromosomes
• Identification of small rearrangements is possible

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Tasks

• Arrange a karyotype (small box with chromosome
  photos table with chromosomes)
• 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. Tumová, Dr.
Diblík or Dr. Kocárek.
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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
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Put the slides back to boxes, please.
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Next practical

• Numerical chromosomal abnormalities
• No test!

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See you next week!