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Urinalysis & Body Fluids

Collection & Handling

We’ve explored the intricate physiology of amniotic fluid, understanding it as a dynamic reflection of fetal health. Now, we must turn to the critically important pre-analytical phase. The process of collecting amniotic fluid - amniocentesis - is an invasive procedure with inherent risks to both the fetus and the mother. The sample obtained is, therefore, one of the most precious specimens to enter the laboratory

There is no room for error. A single mistake in collection, labeling, transport, or handling can lead to a catastrophic misdiagnosis. A false-negative result for a genetic disorder could lead to the birth of a child with unexpected, severe medical needs. A false-positive result could, in the most extreme cases, lead to the termination of a healthy pregnancy

Our responsibility as laboratory scientists is to be the unwavering guardians of this specimen’s integrity from the moment the needle enters the amniotic sac until the final result is reported. This requires a deep understanding of the unique vulnerabilities of the analytes we are measuring

Procedure: Amniocentesis

While performed by an obstetrician, we must understand the procedure to appreciate the potential for pre-analytical complications

  • Timing
    • For Genetic Studies: Typically performed between 15 and 18 weeks of gestation. This is the optimal window where there is sufficient fluid volume and a high concentration of viable fetal cells for culture, while still allowing time for decisions to be made based on the results
    • For Fetal Lung Maturity (FLM) or HDFN assessment: Performed later in the third trimester (>32 weeks)
  • Technique
    • The procedure is performed under continuous ultrasound guidance.: This is non-negotiable. The ultrasound is used to locate a safe pocket of fluid, to avoid puncturing the fetus or the placenta, and to guide the needle insertion
    • A long, thin, sterile needle is passed through the mother’s abdominal wall, through the uterine wall, and into the amniotic sac
    • Approximately 10 to 20 mL: of amniotic fluid is gently aspirated into sterile syringes

Pre-Analytical Complications at the Time of Collection

1. Maternal Urine Contamination

  • The Problem: The most common technical challenge is differentiating amniotic fluid from accidentally aspirated maternal urine. They can appear visually identical
  • Why It’s a Disaster: Submitting maternal urine for genetic analysis will result in a normal female karyotype (46,XX) regardless of the fetus’s genetic status. This is a complete diagnostic failure
  • The Solution (Bedside Testing)
    • The laboratory must provide the tools for the clinician to test the fluid at the bedside before sending it to the lab
    • The Fern Test: A drop of fluid is placed on a clean glass slide and allowed to air dry. Amniotic fluid, due to its content of proteins and electrolytes, will crystallize into a beautiful, delicate, fern-like pattern. Urine will not.
    • Chemical Confirmation: Simple dipstick tests can be used. Amniotic fluid will have detectable levels of protein and glucose, whereas normal urine will be negative for both. Creatinine and urea levels are also much lower in amniotic fluid than in urine
  • Lab Responsibility: Our policies must mandate that the fluid is confirmed as amniotic at the point of collection

2. Bloody Contamination

  • The Problem: The amniocentesis needle may traverse a maternal blood vessel or nick the placenta, causing blood to contaminate the amniotic fluid sample
  • Why It’s a Problem for Different Tests
    • For Cytogenetics: Maternal blood contains maternal white blood cells. If these cells are cultured instead of the fetal cells, it will again lead to a normal maternal karyotype and a failed diagnosis
    • For Fetal Hemolysis (ΔA450 scan): The presence of hemoglobin from the contaminating RBCs will cause a massive, false elevation in the spectrophotometric scan, completely invalidating the test for bilirubin. A bloody sample cannot be used for a ΔA450 scan
    • For Alpha-Fetoprotein (AFP): Maternal serum has a low level of AFP, but fetal blood has an extremely high level. If fetal blood contaminates the sample, it will cause a falsely elevated AFP, which could be misinterpreted as a neural tube defect
  • The Solution
    • When a bloody tap occurs, the clinician should attempt to clear the needle or use a new syringe to collect a subsequent, less bloody sample
    • In the lab, a Kleihauer-Betke (KB) stain: can be performed to determine if the contaminating red blood cells are of fetal or maternal origin, which can help in interpreting AFP results

Handling & Transport: Protecting the Analytes

Once collected, the sample begins a race against time and light. Different tests have different, and sometimes conflicting, handling requirements

Specimen #1: Fetal Hemolysis (HDFN) Workup

  • Analyte of Interest: Bilirubin (measured by ΔA450 scan)
  • CRITICAL HANDLING REQUIREMENT: PROTECT FROM LIGHT.
    • Rationale: Bilirubin is a photosensitive molecule. Exposure to ambient or UV light will cause it to rapidly photodegrade, leading to a falsely low result. A falsely low bilirubin could give the clinician a false sense of security, delaying a life-saving intervention for a severely anemic fetus
    • Procedure: The collection syringe or tube must be immediately placed into a light-blocking amber tube, wrapped in aluminum foil, or placed in a light-proof transport container

Specimen #2: Fetal Lung Maturity (FLM) Testing

  • Analytes of Interest: Phospholipids (Lecithin, Sphingomyelin, PG) and Lamellar Bodies.
  • CRITICAL HANDLING REQUIREMENT: TRANSPORT ON ICE.
    • Rationale: The phospholipids are susceptible to enzymatic degradation at room temperature by phospholipases present in the fluid. Chilling the specimen to 4°C halts this enzymatic activity, preserving the true ratio of the different phospholipids
    • Procedure: The specimen must be placed in an ice-water slurry for transport. It should be centrifuged in a refrigerated centrifuge upon arrival
    • Lamellar Bodies: These are counted in the “platelet” channel of a hematology analyzer. A bloody specimen cannot be used, as the platelets will be falsely counted as lamellar bodies

Specimen #3: Cytogenetic Analysis

  • Analyte of Interest: Viable fetal cells (amniocytes).
  • CRITICAL HANDLING REQUIREMENT: MAINTAIN AT ROOM TEMPERATURE.
    • Rationale: The goal here is to keep the cells alive so they can be cultured in the lab. Both freezing and chilling can damage the cell membranes and kill the cells, leading to culture failure
    • Procedure: The specimen should be kept at ambient temperature (20-25°C) and transported to the genetics lab as quickly as possible. It should never be frozen or placed on ice

Summary of Handling Requirements

Because of these conflicting requirements, if multiple tests are ordered, the fluid must be collected into separate, appropriately labeled syringes or tubes, each designated for its specific test and handled accordingly.

  • For ΔA450 (HDFN): Amber tube, protected from light, transport at room temperature
  • For FLM: Sterile tube, transport on ice
  • For Cytogenetics: Sterile tube, transport at room temperature
  • For AFP: Sterile tube, can be transported at room temp; must be separated from cells promptly

Conclusion

The pre-analytical handling of amniotic fluid is a high-stakes protocol that demands precision and communication between the clinical team and the laboratory. There is no “one size fits all” handling procedure. Each test has its own unique vulnerability - bilirubin to light, phospholipids to heat, and fetal cells to cold. By understanding the physiology behind these vulnerabilities and enforcing strict, analyte-specific handling protocols, we ensure that this irreplaceable specimen yields the most accurate and reliable diagnostic information, safeguarding the health of both the mother and her unborn child