Fluid Analysis – Part 5 – Amniotic fluid Examination (Amniocentesis), Normal and Abnormal Findings

Sample

1. Amniotic fluid is collected by amniocentesis.
2. Amniotic fluid needs to be refrigerated.
3. Avoid amniotic fluid from light for the estimation of bilirubin.
4. Amniocentesis is done during the 14th, 16th, and 18th, weeks of gestation.
5. Chorionic villus sampling is better than amniotic fluid for karyotyping and genetic analysis.

The purpose of the test (Indications):

1. To diagnose the genetic disorder (cytogenetics analysis).
2. To diagnose chromosomal abnormalities.
3. To diagnose inherited metabolic disorders like cystic fibrosis.
4. To diagnose neural tube defect like myelomeningocele, anencephaly, and spina bifida,
5. Measure bilirubin in Rh sensitization for erythroblastosis fetalis.
6. To diagnose chorioamnionitis.
7. To assess fetal lung maturity, a sample was taken after 32 weeks. The lecithin/sphingomyelin ratio is a measure of fetal lung maturity. Phospholipids are measured in the amniotic fluids.
8. To assess postmature pregnancy >40 weeks.
9. To find intrauterine retardation.
10. To find congenital infections like:
    1. Toxoplasmosis.
    2. Cytomegalovirus (CMV).
    3. It can do a culture for bacterial infections.

Procedure for amniotic fluid collection:

1. Amniotic fluid is aspirated by the needle into the amniotic sac is called amniocentesis.
2. This is transabdominal amniocentesis.
3. Another route is transvaginal amniocentesis. This method carries a great risk of infection.
4. This procedure is safe if performed after the 14th week of gestation.
5. Fluid for chromosomal analysis is collected after the 16th week of gestation.
6. For fetal distress and maturity is collected in 3rd trimester.
7. 30 mL of amniotic fluid is collected in the sterile syringes.
   1. The first 2 to 3 ml collected is discarded because this may be contaminated by the maternal blood, tissue fluid, and cells.
   2. In the case of hemolytic disease of the newborn, the sample should be avoided from the light. The sample should be kept in the amber-colored bottle or test tubes.
8. Sample for the fetal lung maturity, the sample should be placed in the ice for delivery to the laboratory and keep the sample in the fridge.
   1. sample for the cytogenetics may be maintained at room temperature (37 °C) prior to analysis.
   2. The sample for the biochemical testing should be separated immediately from the cellular elements and debris to avoid the effect of cellular metabolism.

Complications of the procedure:

1. There may be a miscarriage.
2. There are chances for injury to the fetus.
3. There may be a leak of amniotic fluid.
4. There are chances for abortion.
5. There are chances for premature labor.
6. There are chances for infections.
7. There may be amniotic fluid embolism.
8. There are chances for the maternal Rh isoimmunization.
9. There may be damage to the urinary bladder or intestine.

Precaution and contraindications:

1. Fetal blood contamination can cause a false raised level of AFP.
2. Do not perform this test in case of:
   1. Placenta previa.
   2. Patients with placenta abruptio.
   3. Patients with an incompetent cervix.
   4. Patient with a history of premature labor.

Pathophysiology

1. Amniotic fluid is present in the amnion, a membranous sac that surrounds the fetus.
   1. Amniotic fluid keeps are increasing throughout the pregnancy, reaches a peak of around one liter (1 L) during the third trimester.
   2. It gradually decreases before delivery.
   3. During the first trimester around 30 mL of the amniotic fluid is derived from the maternal circulation. Its composition is similar to the mother plasma with the addition of the fetus cells. These cells may provide the basis for the cytogenetics analysis.
   4. After the first trimester, fetal urine is the major contributor to ther amniotic fluid volume.
   5. Now fetal swallowing of amniotic fluid begins and it regulates the increase in the fluid from the fetal urine.
      1. If there is a failure in the swallowing of amniotic fluid, will lead to the accumulation of the fluid called Hydramnios.
      2. This will lead to fetal distress and this is usually seen in the neural tube defect.
      3. Oligohydramnios is decreased amniotic fluid seen in increased swallowing of fluid, urinary tract abnormalities, and membrane leakage.
   6. Amniotic fluid composition changes when urine formation starts.
      1. Creatinine, urea, and uric acid concentration increase while glucose and protein concentration decreases.
      2. The concentration of the urea and creatinine is much lower in the amniotic fluid than the maternal urine.

2. Amniotic fluid surrounds, protects, and gives nourishment to the fetus during gestation.
   1. A part of the amniotic fluid derived from:
      1. The fetal respiratory system.
      2. amniotic membrane.
      3. Urine.
      4. Umbilical cord.
3. A fetus can move freely in the amniotic fluid of the uterus. This will prevent the compression of the umbilical cord.
4. Amniotic fluid maintains the temperature as well.

5. Amniotic fluid contains protein, hormones, nutrients, and antibodies.
6. There is water transfer between intrauterine compartments are:
   1. The placenta.
   2. The fetus.
   3. Amniotic fluid.
7. As pregnancy (gestation) advances then there are changes as follow:
   1. After 25 weeks there is an increase in amylase, alkaline phosphatase, urea, uric acid, creatinine, and phospholipids.
   2. There is a decrease in chloride, bilirubin, protein, glucose, and sodium.
   3. AFP increased level suggest neural tube defect while decreased AFP level is associated with increased risk for trisomy 21.
8. Amniocentesis help for elective abortion in the defective fetus.
9. The lecithin/sphingomyelin (L/S) ratio is a measure of fetal lung maturity. Lecithin is the major surfactant required for alveolar ventilation. In the case of deficiency of surfactant, alveoli collapse during expiration and lead to respiratory distress syndrome(RSD). This may be a major cause of death in immature babies. As the L/S ratio decreases, the risk of RDS increases.
   1. Phosphatidylglcerol (PG): This is the minor lung surfactant about 10%. This is entirely synthesized by the mature lung alveolar cells, so this is a good indicator of lung maturity.

10. Analysis of the amniotic fluid for bilirubin level (or absorbance at 450)  in the Rh-negative mothers in later weeks of the gestation gives the severity of anemia due to Rh-incompatibility.
11. Lilly plotted the graph to give the severity of the hemolytic anemia.

The mean volume of amniotic fluid during gestation:

Weeks of gestation	Quantity of fluid
10  weeks	30 mL
12 weeks	90 mL
14 weeks	120 mL
16 weeks	230 mL
18 weeks	280 mL
20 weeks	320 mL
40 weeks	1000 mL
Full-term	<1500 mL

Source 2

• 15 weeks of gestation = 450 mL of amniotic fluid
• 25 weeks of gestation = 750 mL of amniotic fluid
• 30 to 35 week of gestation= 1500 mL of amniotic fluid
• Full-term   =   <1500  mL of amniotic fluid

Normal Amniotic Fluid findings:

General test	Result
Appearance	clear, pale to straw yellow
AFP	14 to 16 weeks = <5.2 mg/dL,            22 weeks = <3.0 mg/dL
Bilirubin	< 0.2 mg/dL
Creatinine	>2.0 mg/dL after 37 weeks
Chromosomal abnormality	absent
phosphatidylglycerol	positive                   (negative in immature)
Lecithin/sphingomyelin ratio	mature >2.0 , immature <2.0 , Diabetic mother >3.5
Microscopic examination	There are amniotic epithelial cells from the lining of the sac. Fetal squamous cells originating from fetal skin, oral mucosa, and vagina.

Abnormal amniotic fluid findings:

Lab findings	Findings	Clinical significance
Appearance	Yellow	Erythroblastosis (due to the presence of bilirubin)
	Red-brown  (dark red-brown)	Indicate fetal death
	Amber color	Indicates bilirubin
	Green color	Due to meconium, fetal hypoxia
	Pink-red (blood-streaked)	Blood contamination, traumatic tap, intra-amniotic hemorrhage
Volume	Increased	Hydramnios due to fetal abnormality
	Decrease	Oligohydramnios is associated with rupture membranes and fetal abnormality, urinary tract deformities
Presence of cells	Long bipolar cells, multiple filamentous pseudopodia, large vacuolated cells with inclusions	Neural tube defect
Bilirubin	At 28 weeks = >0.075	Erythroblastosis fetalis, hepatitis
	At 40 weeks	Maternal infection, Sickle cell crises
Saturated phosphatidylcholine	>500 µg/L	Respiratory distress syndrome
AFP	Increased,>2.5 MoM	Open spinal defects (neural tube defect)
Creatinine	>2.0 mg/dL	Indicates fetal maturity and the maternal level is normal

 Result and Clinical finding (amniotic fluid analysis):

1. Hemolytic anemia shows increased bilirubin.
   1. Rh immunization also shows increased bilirubin.
2. Raised AFP shows a neural tube defect. Acetylcholinesterase can confirm the diagnosis.
3. AFP may also be raised in Sacrococcygeal teratoma.
4. Fetal distress: The presence of meconium gives greenish color to amniotic fluid indicating fetal distress.
5. Can diagnose Immature fetal lung.
6. Can diagnose Hereditary metabolic disorders like cystic fibrosis, Tay-Sachs disease, and galactosemia.
7. Can diagnose Sex-linked disorder like hemophilia.
8. Genetic abnormalities like sickle cell anemia, thalassemia, and Down’s syndrome are diagnosed.
9. Polyhydramnios (>2000mL) seen in diabetic patients leads to increase chances of congenital abnormality.
10. Oligohydramnios when the amniotic fluid is less than 300 mL at 25 weeks of gestation. This is associated with fetal renal disease.
11. Genetic disorder identified by the amniotic fluid analysis.

Chromosomal anomalies diagnosis is:

Trisomy 21	Down’s syndrome
Trisomy 13	Patau’s syndrome
XO 45	Turner’s syndrome
XXY 47	Klinefelter’s syndrome
Trisomy 18	Edward’s syndrome

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