Chemical Testing

The physical examination of amniotic fluid gives us a rapid, qualitative snapshot of fetal health. Now, we move to the quantitative and highly specific world of chemical analysis. This is where we measure precise molecular markers to answer the most critical questions of prenatal diagnosis

It is essential to understand that, unlike a serum chemistry panel, we are not performing a broad screen. Each chemical test ordered on amniotic fluid is a targeted investigation, designed to answer a single, profound clinical question:

• Is the fetus suffering from severe, life-threatening anemia?
• Does the fetus have a catastrophic open neural tube defect?
• Are the fetal lungs mature enough to support life outside the womb?

The answers to these questions are found by measuring the unique biochemical fingerprints that the fetus leaves in its amniotic environment

Assessment of Fetal Hemolysis (HDFN)

This is one of the original and most important indications for amniocentesis

Spectrophotometric Scan (ΔA450) for Bilirubin

• The Clinical Question: “How severe is the fetal anemia caused by Hemolytic Disease of the Fetus and Newborn (HDFN)?”
• Physiology: In HDFN, maternal antibodies cross the placenta and destroy fetal red blood cells. This hemolysis releases massive amounts of hemoglobin, which is broken down into bilirubin. This bilirubin floods the fetal circulation and diffuses into the amniotic fluid. The amount of bilirubin in the fluid is a direct measure of the rate of RBC destruction
• Methodology
  • This is not: a standard chemistry bilirubin test. We perform a full spectrophotometric scan of the amniotic fluid supernatant from 365 nm to 550 nm
  • On the resulting graph, a peak from normal oxyhemoglobin is seen at 410 nm
  • In a normal fluid, the absorbance curve is smooth. In a fluid from an HDFN-affected fetus, the bilirubin causes a characteristic “bulge” or “hump” in the curve, with a peak absorbance at 450 nm.
  • We measure the change in absorbance (ΔA): at 450 nm by drawing a straight baseline from 365 nm to 550 nm and measuring the height of the bilirubin peak above this baseline. This value is the ΔA450.
• Pre-analytical Considerations
  • CRITICAL: The specimen must be protected from light (using an amber tube or foil). Light will photodegrade the bilirubin, leading to a falsely low ΔA450 and a catastrophic underestimation of the disease severity
  • Bloody specimens are unacceptable, as hemoglobin interferes with the scan
• Interpretation (The Liley Graph)
  • The ΔA450 value is plotted on a Liley Graph, which correlates the level of bilirubin with the gestational age. The graph is divided into three zones:
    • Zone I (Lower Zone): The fetus is mildly or not affected
    • Zone II (Middle Zone): The fetus is moderately affected and requires close monitoring and possible repeat testing
    • Zone III (Upper Zone): The fetus is severely affected and is at high risk of death. This is a critical result that mandates immediate clinical intervention, such as an intrauterine blood transfusion or emergency delivery

Assessment of Neural Tube Defects (NTDs)

Alpha-Fetoprotein (AFP)

• The Clinical Question: “Does the fetus have an open neural tube defect (like spina bifida or anencephaly)?”
• Physiology: AFP is a major plasma protein produced by the fetal liver and yolk sac. It functions similarly to adult albumin. Levels are very high in the fetal circulation and very low in the amniotic fluid, as it is contained within the fetal body. In an open NTD, there is a lesion on the fetal back or head that allows fetal plasma and CSF to leak directly into the amniotic fluid, causing a massive increase in the amniotic fluid AFP concentration
• Methodology
  • AFP is measured by a quantitative immunoassay
  • CRITICAL: The result is not interpreted as a simple value. It is reported as a Multiple of the Median (MoM). This is because the normal median AFP level changes for every single week of gestation. The patient’s result is compared to the median for their specific gestational age
• Interpretation
  • A value ≥ 2.0 MoM: is considered positive and is highly suspicious for an open NTD
• Pre-analytical Considerations
  • A bloody tap, especially one contaminated with fetal blood, will cause a falsely elevated AFP, as fetal blood has an extremely high concentration of the protein

Acetylcholinesterase (AChE): The Confirmatory Test

• The Clinical Question: “Is the elevated AFP truly from a neural source, or is it from another cause (like an abdominal wall defect or fetal blood)?”
• Physiology: AChE is an enzyme that is normally found only in CSF and neural tissue. It should not be present in amniotic fluid. If an open NTD exists, AChE will leak from the exposed neural tissue into the fluid along with the AFP
• Methodology
  • The test is performed by gel electrophoresis. The presence of a specific AChE band is considered a positive result
• Interpretation
  • The presence of AChE is highly specific for an open neural tube defect.: It is used to confirm a positive AFP result. A positive AFP with a positive AChE is diagnostic for an open NTD

3. Assessment of Fetal Lung Maturity (FLM)

This is the most common reason for amniotic fluid chemical testing in the third trimester

• The Clinical Question: “Are the fetal lungs mature enough to produce surfactant and prevent Respiratory Distress Syndrome (RDS) if the baby is delivered now?”
• Physiology: Surfactant is a complex of phospholipids (primarily lecithin and phosphatidylglycerol) produced by Type II pneumocytes in the fetal lung. It reduces surface tension, preventing the alveoli from collapsing on expiration. This surfactant is secreted into the amniotic fluid, where we can measure it

Lecithin/Sphingomyelin (L/S) Ratio

• The Principle: This is the historical “gold standard.” Early in pregnancy, the concentrations of lecithin and sphingomyelin (another membrane phospholipid) are roughly equal. At about 34-36 weeks, the production of lecithin (the main active component of surfactant) surges dramatically, while sphingomyelin remains constant. The ratio of the two is a powerful indicator of lung maturity
• Methodology: The phospholipids are extracted and separated by Thin-Layer Chromatography (TLC), and the spots are quantified
• Interpretation
  • L/S Ratio ≥ 2.0: This is the classic cutoff indicating fetal lung maturity. The risk of RDS is very low
  • L/S Ratio < 2.0: Indicates immaturity and a high risk of RDS

Phosphatidylglycerol (PG)

• The Principle: PG is another critical phospholipid component of surfactant. It is the last component to appear in significant amounts, usually around 36 weeks. Its presence, therefore, is a final “stamp of approval” for full biochemical maturity
• Methodology: A qualitative immunologic agglutination test or TLC. The result is simply “present” or “absent.”
• Interpretation
  • The presence of PG: is a very strong indicator of fetal lung maturity, even if the L/S ratio is borderline. Its presence is particularly reassuring in samples from diabetic mothers

Lamellar Body Count (LBC)

• The Principle: This is the modern, rapid, and widely used method for FLM testing. Lamellar bodies are the small, layered storage packets of surfactant that are secreted by the pneumocytes into the alveolar space and then into the amniotic fluid. By a fortunate coincidence, lamellar bodies are the same size as platelets.
• Methodology
  • The amniotic fluid sample is run on a standard hematology analyzer: using the platelet channel. The instrument counts the lamellar bodies as if they were platelets
• Interpretation
  • LBC > 50,000 /µL: Strongly correlates with fetal lung maturity.
  • LBC < 15,000 /µL: Strongly correlates with fetal lung immaturity.
• Pre-analytical Considerations: This test is fast and inexpensive but cannot be performed on bloody or meconium-stained specimens, as platelets and debris will interfere

Assessment of General Fetal Maturity

These are older tests, now largely replaced by ultrasound and specific FLM tests, but are important for historical context

• Creatinine: Reflects fetal muscle mass and kidney function. A level > 2.0 mg/dL was historically used as an indicator of fetal maturity (gestational age > 36 weeks)

Conclusion

Amniotic fluid chemistry is a powerful example of targeted diagnostics. Each test is a specific probe into a particular aspect of fetal development and well-being. From the elegant physics of the ΔA450 scan to the clever application of a hematology analyzer for lamellar body counts, these tests provide the obstetrician with the critical, quantitative data needed to make profound, often life-saving, decisions about the management of a high-risk pregnancy