28. Direct testing

Direct testing on the fetus is offered when there is an ultrasound marker of anomaly; serum screening (triple markers, quadruple markers) is positive or when one of the parents or siblings is a carrier of chromosomal abnormality. Individuals and relationships are described as a diagram when clinical history is obtained (Figures 11 and 12).

#### 28.1. Clinical indications for prenatal diagnosis

Diagnostic interventions in obstetrics are mainly directed at some form of fetal tissue sampling for genetic, biochemical, hematological and histological processing. Samples of fetal tissues like amniotic fluid, chorionic villi and blood can be obtained by a variety of tissue sampling

8. Multiple soft tissue markers seen on ultrasound in the second trimester

The fetal tissue sample obtained by these invasive procedures can further be processed.

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Amniocentesis is the oldest procedure performed for prenatal diagnosis [27]. As early as 1952, it was found that cells in the amniotic fluid represented fetal tissue and could be used for gender determination. This procedure was performed initially without any guidance. Now, with the help of real time ultrasound and color flow mapping, amniocentesis is performed

Amniocentesis is performed in the second trimester when the uterus has become an abdominal organ and the amniotic fluid is about 200 ml. At any gestation, 10% of amniotic fluid volume can be aspirated for diagnostic procedures. A prerequisite is that there should be an adequate

Improved laboratory techniques have made it possible to culture amniotic fluid cells from as little as 10 ml of amniotic fluid at 12 weeks. However, the volume of 10 ml will account for almost a third of the amniotic fluid volume at this gestational period [28]. This may lead to problems in the fetus like lung hypoplasia and limb deformities like congenital talipes equinovarus. To overcome the loss of fluid volume, a technique of entrapping the cells in a filter and returning the amniotic fluid into the cavity has been tried. Randomized controlled

Under continuous ultrasound guidance and strict aseptic precautions, a 20–22 gauge spinal needle is introduced in a pool of amniotic fluid, which is devoid of fetal parts. It is preferable to avoid the placenta. If the placenta is anterior, the needle is introduced laterally or above the

easily with accuracy. There are minimal complications and the procedure is safe.

9. Positive maternal serum biochemistry

7. Histological studies by light and electron microscopy.

amniotic fluid volume for that gestational age.

trials are required to establish the safety of this procedure.

1. Karyotyping 2. DNA analysis

3. Hematological parameters

4. Biochemical analysis

5. Enzyme assays 6. Microbial assays

29. Amniocentesis

29.1. Early amniocentesis

29.2. Technique of amniocentesis

Figure 12. Diagrammatic representation of relationships in clinical genetics.

methods under ultrasound guidance. Other fetal tissues that can be sampled for prenatal diagnosis are fetal urine, skin, liver and brain.

#### 28.2. Indications of prenatal diagnosis


The fetal tissue sample obtained by these invasive procedures can further be processed.


#### 29. Amniocentesis

Amniocentesis is the oldest procedure performed for prenatal diagnosis [27]. As early as 1952, it was found that cells in the amniotic fluid represented fetal tissue and could be used for gender determination. This procedure was performed initially without any guidance. Now, with the help of real time ultrasound and color flow mapping, amniocentesis is performed easily with accuracy. There are minimal complications and the procedure is safe.

Amniocentesis is performed in the second trimester when the uterus has become an abdominal organ and the amniotic fluid is about 200 ml. At any gestation, 10% of amniotic fluid volume can be aspirated for diagnostic procedures. A prerequisite is that there should be an adequate amniotic fluid volume for that gestational age.

#### 29.1. Early amniocentesis

methods under ultrasound guidance. Other fetal tissues that can be sampled for prenatal

2. History of chromosomal abnormality or single gene disorder in a sibling

7. Increased nuchal translucency in first trimester ultrasound scan

diagnosis are fetal urine, skin, liver and brain.

Figure 12. Diagrammatic representation of relationships in clinical genetics.

Figure 11. Diagrammatic representation of individuals in clinical genetics.

94 Genetic Diversity and Disease Susceptibility

3. Chromosomal translocation in one parent

28.2. Indications of prenatal diagnosis

1. Advanced maternal age

4. Rh isoimmunization 5. Congenital infections 6. Fetal growth disorders Improved laboratory techniques have made it possible to culture amniotic fluid cells from as little as 10 ml of amniotic fluid at 12 weeks. However, the volume of 10 ml will account for almost a third of the amniotic fluid volume at this gestational period [28]. This may lead to problems in the fetus like lung hypoplasia and limb deformities like congenital talipes equinovarus. To overcome the loss of fluid volume, a technique of entrapping the cells in a filter and returning the amniotic fluid into the cavity has been tried. Randomized controlled trials are required to establish the safety of this procedure.

#### 29.2. Technique of amniocentesis

Under continuous ultrasound guidance and strict aseptic precautions, a 20–22 gauge spinal needle is introduced in a pool of amniotic fluid, which is devoid of fetal parts. It is preferable to avoid the placenta. If the placenta is anterior, the needle is introduced laterally or above the placental margin. The amnion is pierced with a sudden controlled force, a jab of needle to prevent tenting of the membranes. The first few ml of fluid is discarded to minimize the contamination with maternal blood. About 20 ml of fluid is withdrawn for analysis. Not more than two insertions should be performed at one sitting.

proximal end of cannula. Suction is applied and a gentle to and fro motion of the cannula will ensure aspiration of villi into the medium in the syringe. The cannula is then withdrawn with continuous suction being maintained. The sample is then examined under a microscope to ensure the presence of branching villi. A minimum of 15 mg of tissue will be required for

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Rodeck transabdominal chorionic villous biopsy forceps can perform transabdominal collection of fetal villi. A 20 Gauge spinal needle can also be used for sampling the villi transabdominally. The needle is advanced under ultrasound guidance and suction is applied as via the transcervical technique. A double lumen technique can also be used. In this, a large lumen outer needle is introduced into the uterus and a smaller gauge needle is passed through the outer needle to sample the villi. The advantage of this technique is that resampling can be done easily if the sample if insufficient in the first passage of needle. Transabdominal approach

Fetal loss can occur in 0.6–2% of cases. Loss rates are greater than 10% if more than two needle insertions are made to collect the chorionic villi. A sub chorionic hematoma may form in 4% cases, which usually resolves spontaneously. Chorioamniotis is a rare complication occurring in less than 0.3% cases. A delayed rupture of membranes can happen weeks to days after

The risk of fetomaternal hemorrhage is dependent on the amount of tissue aspirated and is detected by a rise in maternal serum alpha feto protein [31]. All Rh Negative non-sensitized mothers should receive a prophylactic Anti D immunoglobulin [32]. Perinatal complications like premature rupture of membranes, small for gestational age or intrauterine growth restriction have not been noticed after chorionic villous sampling. Chorionic villous sampling performed prior to 9 weeks of gestation is known to be associated with specific fetal malformations. Oromandibular limb hypo genesis syndrome and terminal transverse limb reduction anomalies have been documented when chorionic villous sampling was performed prior to 9 weeks of gestation. Chorionic villous sampling should not be performed prior to

Fetal blood can be sampled after 18 weeks of gestation. Fetal blood sampling rules out the possibilities of pseudomosaicisms that are more common in chorionic villi and amniotic fluid cultures. Fetal blood can also be used for hematological estimations and enzyme and hormone assays. Direct access to fetal circulation is also used to give intra uterine exchange transfusions

has been found safer with few complications than trans cervical route.

30.4. Complications of chorionic villous sampling

chorionic villous sampling in about 0.3% of cases.

9 weeks of gestation [33, 34].

31. Fetal blood sampling

in cases of Rh isoimmunization.

culture.

30.3. Trans abdominal approach

#### 29.3. Amniocentesis in twins

Individual sampling of both sacs using two different needles under direct ultrasound guidance can perform amniocentesis in twins. A single insertion technique in which needle is advanced to the second sac after aspirating the first sac has been advocated by Jeanty.

#### 29.4. Complications

A blood stained aspirate can occur in 1–2% of the procedures. If the color is red, it indicates fresh blood possibly due to maternal blood contamination. Dark red or dark brown color of the fluid indicates prior intraamniotic bleed that is associated with poor fetal outcome. A brown color amniotic fluid can also indicate fetal aneuploidy. The overall pregnancy loss following amniocentesis is estimated as 0.5%.

The incidence of fetomaternal hemorrhage is 63% in anterior placentas wherein the needle has traversed through the placenta. It reduces to 18% in posterior placentas. Hence, it is essential to determine Rh type of the mother prior to the procedure. Anti D immunoglobulin must always be given in rhesus negative women after any prenatal procedure. Fetal trauma is unlikely if the procedure is done continuous ultrasound monitoring.
