Prenatal Screening & Diagnosis

Question 1

What is the purpose of screening tests?

Answer 1

• screening tests are used to look for an abnormality / certain condition
• they are NOT definitive and another test is required to confirm the diagnosis
• they are used to dictate the clinical pathway and determine whether further testing is required

Question 2

What do screening tests rely on?

Answer 2

• they rely on factors produced by the foetus / placenta that diffuse across the placenta and enter the maternal circulation
• increased or decreased levels of these factors may indicate an abnormality
• increased / decreased levels of the same factor can be indicative of different conditions, so the change from normal is measured

Question 3

What is meant by diagnostic testing?

Answer 3

• diagnostic tests are used to confirm a suspicion of an abnormality / condition
• they have a high degree of specificity but there are also associated risks

Question 4

What are the 2 main reasons why screening tests are important?

Answer 4

• to determine the health / condition of the unborn foetus
• to avoid / manage potentially untoward outcomes for the foetus, mother or both
• it allows for parents to make informed choices about their pregnancy, depending on whether it is deemed to be high-risk or not

Question 5

What is the NICE recommendation for prenatal screening in England?

Answer 5

• all pregnant women are offered Down’s syndrome screening in the first trimester
• this can be performed via 2 different methods, depending on when the woman presents:

1. the combined test
2. the quadruple serum test

Question 6

What is involved in the combined test for Down’s syndrome?

What is the detection rate?

Answer 6

• this involves a nuchal translucency measurement and maternal serum markers at 12 weeks
• the serum markers measured are:

1. human chorionic gonadotrophin (hCG)
2. pregnancy-associated plasma protein A (PAPP-A)

• it has an 80% detection rate and gives an early indication, but is also associated with 5% false positive rate

Question 7

What is involved in the quadruple serum test and when can this be performed?

Answer 7

• this involves a blood test to measure 4 serum factors:

1. human chorionic gonadotropin (hCG)
2. alpha-fetoprotein (AFP)
3. inhibin-A
4. estriol (E3)

• it can be performed up to 24 weeks, but is most accurate between 15-18 weeks

Question 8

When is the quadruple serum test used?

What is its drawback over the combined test?

Answer 8

• the quadruple test is less accurate than the combined test
• it can be used for women who present later
• if the results of the combined test are positive, the woman will still be sent for a quadruple serum test at 15-18 weeks

Question 9

What are both the quadruple serum and combined test followed up by?

Answer 9

• both tests are followed up by a foetal anomaly USS at approx 20 weeks (18-21)

Question 10

During which period can the combined test be performed?

What 2 conditions can the results raise a suspicion of?

Answer 10

• usually performed at 12 weeks, but can be performed between 10-14 weeks
• can be used to raise suspicion of Down’s syndrome (trisomy 21) and Edward’s syndrome (trisomy 18)
• PAPP-A is decreased from the norm in both trisomy 21 and 18
• hCG is elevated in trisomy 21
• hCG is depressed in trisomy 18

Question 11

What factors are measured in the quadruple serum test and what conditions does this screen for?

Answer 11

• performed in 2nd trimester between 15-22 weeks to look for levels of:
• hCG
• estriol (E3)
• alpha-fetoprotein (AFP)
• + / - Inhibin A
• elevated levels of AFP indicate neural tube defects (NTDs)
• AFP, E3 and hCG are all depressed in trisomy 18
• AFP and E3 are depressed in trisomy 21, but hCG and inhibin A are elevated

Question 12

Why is accurate dating of pregnancy vital for interpreting screening tests?

Answer 12

• accurate dating of pregnancy is vital as levels of serum markers are dependent on foetal age
• there are peaks and troughs of serum markers throughout pregnancy, so values need to be compared to what is expected at that point in pregnancy

Question 13

What are the 3 major benefits of performing USS?

Answer 13

1. safe as it is non-ionising
2. cheap to perform
3. image quality and detail have improved massively recently

• X-ray and CT scans may be more helpful in diagnosis, but they are radiating and carry a risk of harm to the unborn foetus

Question 14

How is gestational age estimated using USS?

Why is this clinically important?

Answer 14

• the most accurate way to date a pregnancy is by measuring the crown-rump length (CRL)
• bi-parietal diameter (BPD) and femoral length (FL) are less accurate when used alone
• usually CRL, BPD and FL are combined with other clinical factors to estimate gestational age
• this is important in dating a pregnancy** and **interpreting serum markers

Question 15

How is nuchal translucency measured?

When is it measured and why is it significant?

Answer 15

• this measures the thickness of fluid present in the subcutaneous tissue in the nuchal region
• it is performed between 11-14 weeks, but usually at 12 weeks alongside the combined test
• nuchal translucency > 3mm** is strongly associated with **cardiovascular system defects and chromosomal abnormalities
• it is particularly indicative of Down’s syndrome

Question 16

What are the 2 types of diagnostic tests and why are they performed?

What must the mother be aware of before agreeing to these tests?

Answer 16

1. chorionic villus sampling (CVS)
2. amniocentesis

• they are performed to confirm any suspected abnormality
• diagnostic tests are INVASIVE procedures and carry a risk of damage to the foetus and foetal death

Question 17

When are diagnostic tests routinely offered?

Answer 17

• diagnostic tests are routinely offered regardless of screening tests when:

1. mother is >35 years of age
2. previous child with a congenital abnormality
3. one of the parents has a chromosomal disorder

• they are also offered when a screening test is suggestive of a congenital abnormality being present

Question 19

What is involved in chorionic villus sampling?

When is this performed?

Answer 19

• a needle is inserted transabdominally or transvaginally to obtain a small sample of villus tissue from the placenta
• the placental cells are cultured and karyotyped for chromosomal, genetic and molecular analysis
• CVS is performed at 10-12 weeks

Question 20

What is the benefit of using CVS over amniocentesis as a diagnostic test?

Answer 20

• it is performed earlier on in the pregnancy, allowing for earlier diagnosis of genetic / chromosomal abnormalities
• more options are available to the parents if a diagnosis is made earlier on in the pregnancy

Question 21

What are the 3 major drawbacks of using CVS as a diagnostic test over amniocentesis?

Answer 21

• it is less accurate than amniocentesis and may have to be repeated

this is due to placental mosaicism or contamination with maternal cells

• there is a higher risk of pregnancy loss (1-2%)
  
  • the risk is higher when the transcervical method is used
• it cannot be used to sample AFP

Question 22

Why is CVS considered to be less accurate than amniocentesis?

Answer 22

Contamination of sample:

• the sample of placental cells may be contaminated with maternal cells, reducing the diagnostic value

Placental mosaicism:

• this describes a subset of cells with chromosomal defects being present in the placenta
• these cells are localised to the placenta, so sampling them will not be representative of the condition of the foetus

Question 23

How is amniocentesis performed?

When is it performed and why can it not be performed earlier on?

Answer 23

• a needle is inserted through the abdominal wall, uterine wall and membranes to take a sample of amniotic fluid
• it is performed at 15-16 weeks
• it cannot be performed prior to this as insufficient amounts of amniotic fluid have developed
• removal of amniotic fluid prior to 15 weeks could be detrimental to the health of the foetus

Question 24

What types of cells will be present in the amniotic fluid sample obtained through amniocentesis?

What is done with these cells?

Answer 24

• amniotic fluid contains cells from:

1. the amnion
2. foetal skin
3. foetal lungs
4. foetal urinary tract

this is due to the foetus swallowing and excreting amniotic fluid during development

• the cells are cultured and karyotyped for chromosomal, genetic and molecular analysis

Question 25

Why might amniocentesis be performed in the 3rd trimester?

Answer 25

* it can be used to test for **markers of _lung maturity_** * this is a **good prognostic factor** in determining whether a foetus will survive or there will be failure to thrive

Question 26

What are the risks associated with amniocentesis?

Answer 26

* risk of **_Rhesus sensitisation_** if there is mixing of foetal and maternal blood * *Rh prophylaxis is given where appropriate* * small risk of **pregnancy loss** (0.06%) * risk of **injury to foetal organs** if USS guidance is not used during the procedure

Question 27

Why does the risk of pregnancy loss increase if amniocentesis is performed prior to 14 weeks?

Answer 27

* risk of pregnancy loss increases to 7% as amniotic fluid is being withdrawn during a cruical period of development

Question 28

Why is karyotyping performed? How many cells are needed?

Answer 28

* examination of chromosomes from **_only 1 cell_** can reveal **chromosomal abnormalities**, including: 1. aneuploidy 2. deletions 3. duplications 4. translocations

Question 29

How is karyotyping performed?

Answer 29

* cells are cultured for **2-3 days** * **_colchicine_** is added, which **prevents spindle formation** the cells are arrested in **_metaphase_**, allowing genetic material to be karyotyped and sequenced * **_hypotonic solution_** is added to **rupture the nuclear membrane** * the genetic material is withdrawn and placed on a slide and **stained with _Giesma_** * a **light microscope** is used to take a photomicrograph, allowing the chromosomes to be visualised

Question 30

What are the 2 different types of chromatin that are present within a chromosome?

Answer 30

**_Heterochromatin:_** * represented by the **_dark bands_** on the chromosomes * it is **highly condensed**, contains **A-T rich bonds** between nucleotide bases and **_few genes_** **_Euchromatin:_** * represented by the **_light bands_** on the chromosomes * it is **less condensed**, contains **G-C rich bonds** between nucleotide bases and **_many genes_**

Question 31

How is fluoresence in situ hybridisation (FISH) performed?

Answer 31

* this method uses **small sections of cloned DNA** called **_probes_** * probes are labelled with a **fluorescent marker** or antibody * the probe will then **anneal (bind) to a _matching region of DNA_** (hybridisation)

Question 32

What are the benefits and drawbacks of using FISH over karyotyping?

Answer 32

* it is **more sensitive** than karyotyping and can detect **_smaller chromosomal abnormalities_** * it can be used in **interphase and metaphase** * however gene-specific FISH is limited as you need to know which area of the genome to target

Question 33

How is chromosome painting used during FISH and what is the benefit of this?

Answer 33

* several probes are used for each chromosome, allowing for quick and easy detection of structural abnormalities * during the cloning DNA process, it is easy to see where fluorescent tags may be missing

Question 34

What is meant by pre-implantation genetic diagnosis? When is this used and why?

Answer 34

* used in couples **_undergoing IVF_** that are at a **high risk of transmitting an inherited disorder** * a **_blastomere_** is removed from the embryo during the **_cleavage stage_** (6-10 cells) * **_FISH_** is performed to examine the chromosome structure * FISH allows for identification of any **disorders inherited by the embryo**, allowing for decisions about its **viability** to be made

Question 35

Why does pre-implantation genetic diagnosis have no effects on development?

Answer 35

* during this procedure, a **blastomere** is removed from the embryo during the **cleavage stage** * blastomeres are **_totipotent_** at the cleavage stage, so removal of a cell should have **no impact on development**

Question 36

What is the benefit of non-invasive prenatal screening and diagnostic tests? What are the 2 different types?

Answer 36

* non-invasive tests can be divided into: 1. ***screening tests (NIPT)*** 2. ***diagnostic tests (NIPD)*** * they do not present any risk to the unborn foetus

Question 37

How are non-invasive screening and diagnostic tests performed?

Answer 37

* **short strands of foetal DNA** were discovered in **maternal blood plasma** these foetal cells crossing the placental membrane are **sparse**, so it is **difficult to obtain sufficient amounts** of genetic material for analysis * **_cell-free DNA strands_** also cross the placenta and are far **more abundant** these come from **foetal trophoblast cells** that have passed into the maternal circulation and **undergone apoptosis** * cell-free DNA is **abundant** so is **_easy to sample_** and can be detected as early as **_4-5 weeks_**

Question 38

What can NIPT be used to detect privately? What are the drawbacks of using this method?

Answer 38

1. Down's syndrome 2. Edward's syndrome 3. Patau's syndrome 4. Turner's syndrome * it is **98% accurate**, but an **_invasive diagnostic test is still required_** to confirm the abnormal result * it may have to be **_conducted several times_** if an **insufficient amount of cell-free DNA** is obtained * *this makes it relatively inaccessible as it is already expensive and may need to be repeated several times*

Question 39

When is NIPD used? How is it performed and what conditions does it look for?

Answer 39

* it is used in the UK for **_determination of foetal sex from 7/40_** * it looks for **Y chromosome DNA sequences** and is 99.5% accurate * a blood sample is taken and cultured to obtain and entire genomic screening, which can be used to detect conditions such as: * duchenne muscular dystrophy * congenital adrenal hyperplasia * haemophilia * achondroplasia * foetal rhesus typing in Rh D -ve mothers

Question 40

When screening for Down's syndrome, what is the normal outcome? When may further testing be performed?

Answer 40

* 95% of screening tests are classed as ***"lower risk"*** and **_no further testing_** is offered * further testing may be offered in **specific circumstances** (e.g. family history of chromosomal disorders, mother \>35) * 5% of screening tests are classed as ***"higher risk"*** and offered a **_diagnostic test_** * *this could be either amniocentesis or CVS*

Question 41

What are the benefits of performing NIPT over traditional screening tests for Down's syndrome? When is this performed?

Answer 41

* NIPT performed at **_10 weeks_** and is **98% accurate** * **_higher sensitivity_** - able to detect **placental mosaicisms** * **_fewer false positives_** (0.3% compared to 5%) * **higher prognostic predictive value (PPV)** than standard screening * however, **_invasive diagnostic tests are still required_** to confirm an abnormal result

Question 42

When is foetal anomaly screening performed and why?

Answer 42

* foetal anomaly screening is performed at **_20/40 weeks_** * at this stage the embryo has gone through the **organogenesis period** and some of these structures are **beginning to mature**

Question 43

What conditions are screened for through the foetal anomaly screening at 20 weeks?

Answer 43

* anencephaly * myelomeningocele * gastroschisis * cleft lip * bilateral renal agenesis * diaphragmatic hernia * serious cardiac abnormalities

Question 44

What are the results of the foetal anomaly screening test combined with?

Answer 44

* the results of the test are combined with further maternal serum analysis, which measures: 1. alpha-fetoprotein (AFP) 2. human chorionic gonadotrophin (hCG) 3. estriol (E3)